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Ultrathin #-carrageenan/chitosan hydrogel films with high toughness and anti-adhesion property Hai Chao Yu, He Zhang, Ke-Feng Ren, Zhimin Ying, Fengbo Zhu, Jin Qian, Jian Ji, Zi Liang Wu, and Qiang Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18343 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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ACS Applied Materials & Interfaces

Ultrathin κ-carrageenan/chitosan hydrogel films with high toughness and anti-adhesion property Hai Chao Yu,1 He Zhang,1 Kefeng Ren,1 Zhimin Ying,2 Fengbo Zhu,3 Jin Qian,3 Jian Ji,1 Zi Liang Wu,1,* Qiang Zheng1 1

Ministry of Education Key Laboratory of Macromolecular Synthesis and Functionalization,

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China; 2Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China; 3Department of Engineering Mechanics, Zhejiang University, Hangzhou 310027, China. *Corresponding author. [email protected] Abstract: Designing tough biopolymer-based hydrogels as structural biomaterials has both scientific and practical significances. We report a facile approach to prepare polysaccharide-based hydrogel films with remarkable mechanical performances and anti-adhesion property. The hydrogel films with thickness of 40-60 µm were prepared by mixing aqueous solutions of κ-carrageenan (κ-CG) and protonated chitosan (CS), evaporating the solvent, and then swelling the casted film in water to achieve the equilibrium state. The obtained κ-CG/CS gel films with water content of 48-88 wt% possessed excellent mechanical properties superior to most existing biopolymer-based hydrogels, with breaking stress of 2-6.7 MPa and breaking strain of 80-120%. The extraordinary mechanical properties of gel films obtained over a wide range of mass ratio of κ-CG to CS should be rooted in the synergistic effect of ionic and hydrogen bonds between the κ-CG and CS molecules. In addition, the tough gel films showed good self-recovery ability, biocompatibility, and cell anti-adhesion property, making them promising as artificial dura mater and diaphragm materials in the surgery. The design principle by incorporating multiple noncovalent bonds to toughen biopolymer-based hydrogels should be applicable to other systems toward structural biomaterials with versatile properties. Keywords: chitosan; κ-carrageenan; hydrogel films; toughness; anti-adhesion 1 ACS Paragon Plus Environment

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1. INTRODUCTION Substantial thin membranes are distributed in the body of organs and tissues, such as dura mater, periosteum, and fascia.1-5 Besides the physiological functions, these membranes usually have an elaborate combination of mechanical performances including stiffness, strength, and toughness. For example, dura mater possesses tensile breaking stress σb of 5.3 ± 1.1 MPa and breaking strain εb of 116 ± 3%, as well as good anti-adhesion property.2,6 In biomedical field, substitute membranes with comparable properties are usually required. At present, bovine pericardium or autologous tissues such as fascias are used as the substitute for dura mater in surgery. However, the bovine pericardium is opaque and only for small wounds, and the obtaining of autologous membranes will cause secondary damage.7 It is really desired to develop artificial substitute membranes with aforementioned properties suitable for biomedical applications. Since the biological membranes are in a gel state with thickness of 70-150 µm and water content of 50-75 wt%,2-4 synthesized hydrogels should be an ideal candidate for substitute membranes. Over the past two decades, various tough hydrogels with distinct network structure and energy dissipation mechanism have been developed, which greatly expand the applications of hydrogels as load-bearing materials.8-23 However, most tough gels contain synthetic polymers as the major and indispensable component, which usually results in poor biocompatibility and degradability, and thus limits their applications as implant substitutes. Compared with synthetic polymers, natural biopolymers are environment-friendly and renewable materials with better biocompatibility and degradability.24 However, it is more challenging to design tough biopolymer-based hydrogels due to relatively poor designability of network structures. In recent years, several groups have prepared tough biopolymer-based hydrogels by applying the similar toughening strategies.25-35 For example, Zhang et al.27 have developed dual-crosslink cellulose hydrogels by sequential chemical and physical cross-linking of cellulose molecules; the resulting tough hydrogels showed σb of 2.7 MPa, εb of 80%, and Young's modulus E of 2 MPa. Mano et al.30 have prepared tough chitosan/alginate hydrogels with σb of 0.5-1.9 MPa and εb of 30-80% by forming ionic bonds between the oppositely charged polysaccharides. These tough hydrogels with good 2 ACS Paragon Plus Environment

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biocompatibility can enhance the proliferation and differentiation of human mesenchymal stem cells.31 However, there is still a gap of mechanical properties between the biopolymer-based hydrogels and biological membranes such as the native dura mater. To design other biopolymer-based hydrogels with better mechanical performances has both scientific significance and biomedical application values. Chitosan (CS) and κ-carrageenan (κ-CG) were selected in this work to prepare tough hydrogels due to their abundant resources. Chitosan becomes dissolvable in acidic solution due to the protonation of amine group, which will be gelled in neutral or alkaline condition because of the deprotonation and intermolecular hydrogen bonding.36 κ-carrageenan has alternating α(1-3)-D-galactose-4-sulfated and β(1-4)-3,6-anhydro-D-galactose units, containing one sulfate group per disaccharide unit.37 At room temperature, κ-CG molecules form helicoidal secondary structure so as to gelate the concentrated solution of κ-CG. It should be rational that ionic bonds will be formed between the oppositely charged κ-CG and CS molecules.38,39 Therefore, we envision to develop κ-CG/CS hydrogels with improved mechanical properties by incorporating multiple noncovalent bonds as physical crosslinks of the interpenetrating biopolymers.8,9,40 We report here a kind of ultratough polysaccharide-based hydrogel films prepared by complexation of κ-carrageenan (κ-CG) and chitosan (CS) in a wide range of weight/charge ratios. The obtained hydrogel films with water content of 48-88 wt% and thickness of 40-60 µm showed remarkable mechanical performances with σb of 2-6.7 MPa, εb of 80-120%, and E of 1.2-25 MPa. These tough hydrogel films also exhibited good biocompatibility and cell anti-adhesion property, which are crucial for the applications as artificial dura mater and diaphragm materials during the surgery. This work should merit the development of other tough biopolymer-based hydrogels and promote their applications as structural biomaterials. 2. EXPERIMENTAL SECTION Materials. Chitosan (CS; medium molecular weight, 75-85% of deacetylation degree) and κ-carrageenan (κ-CG) were used as received from Sigma-Aldrich. The molecular structures of κ-CG and CS are shown in Figure 1a. Acetic acid and NaCl were received from 3 ACS Paragon Plus Environment


Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer saline (PBS) and bovine serum albumin (BSA) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Dulbecco's modified eagle medium (DMEM), penicillin and streptomycin (P/S), and 0.25% trypsin-ethylene diamine tetraacetic acid (trypsin-EDTA) were obtained from Genom Biomedical-tech (Hangzhou, China). Fetal bovine serum (FBS) was purchased from Hyclone (Utah, USA). NIH/3T3 fibroblasts were purchased from Boster Bio-engineering (Wuhan, China). Millipore deionized water was used in all the experiments.

Figure 1. (a) Molecular structures of κ-CG and CS. (b) Schematic for the preparation of κ-CG/CS hydrogel films. Aqueous solutions of κ-CG and CS were mixed in dilute acetic acid solution (i). The mixture was transferred into a petri dish and dried at 70 ºC (ii). The casted film (iii) was swelled in water to obtain κ-CG/CS hydrogel film (iv). Photos of casted film (iii') and hydrogel film together with an illustration of network structure (iv') were shown above the schematic.

Preparation of κ-CG/CS hydrogel films. The protocol of preparing κ-CG/CS hydrogel films is shown in Figure 1b. The total mass of κ-CG and CS was kept constant, and the weight ratio of κ-CG to CS, wr, was varied from 1:9 to 9:1. Prescribed amounts of κ-CG and CS were dissolved in 1 wt% dilute acetic acid solution to prepare solutions with different concentrations. Then, 100 mL κ-CG solution and 100 mL CS solution were slowly added into a beaker containing 50 mL dilute acetic acid solution (1 wt%) under vigorous stirring. The mixture was transferred to a petri dish (polystyrene; 150 mm diameter) and dried at 70 4 ACS Paragon Plus Environment

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ºC. The obtained dry film was immersed in a large amount of water to remove the residual acetic acid. The water was changed every day for one week until the hydrogel film achieved the equilibrium state. Characterizations. Zeta (ζ)-potentials of the solutions of acetic acid, κ-CG, CS, and their mixtures were measured on a Malvern ZET-3000HS apparatus. Fourier transform infrared spectroscopy (FTIR) of κ-CG, CS, and κ-CG/CS hydrogel films were performed at room temperature by using a Nicolet iS10 FTIR spectrometer (Thermo Scientific, USA). All the spectra were obtained with 32 scans and a resolution of 2 cm-1 in the range of 4000-400 cm-1. The microstructure of hydrogel films was observed by Hitachi S4800 field emission scanning electron microscopy (SEM). The samples were prepared by freeze-drying and then cryogenically fractured in liquid nitrogen. Before SEM observation, the fractured surface was coated with a thin layer of gold by the sputtering method. The accelerating voltage for SEM observation was 3 kV. The mechanical properties of hydrogel films were measured by tensile tests (Instron 3343 Tester). The hydrogel films were cut into dumbbell-shaped samples with gauge length of 12 mm and width of 2 mm. The thickness of samples was measured using an optical microscope (Nikon, ECLIPSE LV100N POL). The tests were performed at room temperature with a stretch rate of 10 mm/min. The Young’s modulus, E, was calculated from the slope of the stress-strain curve within the strain of 5%. Cyclic tensile tests were also performed to the sample by sequentially loading and unloading after different waiting time. The loading/unloading rate was 10 mm/min, and the maximum strain was 30%. The hysteresis ratio was calculated by the area ratio of the following loop to the first. The stability of κ-CG/CS hydrogel films in PBS solution was characterized by its mechanical properties. The dumbbell-shaped samples were kept in a large amount of PBS solution at room temperature, which was changed once a week. The mechanical properties of gel films were tested every 5 days up to 40 days; corresponding σb, εb, and E were obtained to evaluate the stability of gel films. Cell culture and initial cell adhesion assay. NIH/3T3 fibroblasts were cultured in DMEM 5 ACS Paragon Plus Environment


containing 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin in Petri dishes at 37 ºC in a humidified atmosphere with 5% CO2. The culture medium was changed every 3 days. Cells at 80-90% confluence were used for further experiments. For initial cell adhesion assay, hydrogel films were coated on the bottom of each well in 24-well plates by evaporating the solvent of the κ-CG/CS mixture at 70 ºC and then swelling it in water to equilibrium state. The plates were then sterilized by UV light irradiation for 30 min. Cells were harvested by using trypsin-EDTA, resuspended in DMEM, and seeded onto the hydrogels at a density of 15000 cells/cm2. After 24 h culture, the samples were washed with PBS three times and fixed with 4% paraformaldehyde in PBS. For fluorescence staining, the samples were permeabilized with 0.1% Triton X-100 (Sigma, USA) for 10 min, and blocked with 0.1% BSA for 1 h. The samples were incubated with fluorescein isothiocyanate labeled phalloidin (FITC-phalloidin, 1:200, Sigma) for F-actin staining. Nuclei were counterstained with 4,6-diamidina-2-phenylin (DAPI, Sigma). Then the samples were immediately imaged with a fluorescence microscope (Olympus DP72, Japan). The fluorescent images were analyzed with ImageJ software (NIH, V1.44p). Cell density was determined using the cell counter plugin. The viability of cells with different hydrogels was characterized by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.41,42 κ-CG/CS hydrogel was coated on a glass coverslip (14 mm diameter), which was placed at the bottom of the petri dish. NIH/3T3 fibroblasts were seeded into 24-well plates at a density of 30000 cells/cm2 and allowed to grow for 24 h. Then, the medium in each well was replaced by fresh one. The hydrogel-coated coverslip was sterilized by UV light irradiation for 30 min and gently placed into the well with the side of hydrogel onto the cultured cells. Bare glass coverslip was also placed on the cultured cells and used as the control. The cells were further incubated with the materials for 48 h, and then treated with the mixture of 500 µL culture medium and 100 µL MTT (5 mg/mL in PBS) at 37 ºC for 3 h. Then, the medium was removed and 1 mL dimethyl sulfoxide (DMSO) was added to each well. The plates were incubated at 37 ºC for 5 min, and then the absorbance of the DMSO solutions at 570 nm was measured by a microplate reader (MODEL 550, Bio Rad). The relative cell viability was 6 ACS Paragon Plus Environment

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calculated by the absorption ratio of the sample to the control.

3. RESULTS AND DISCUSSION Preparation of κ-CG/CS hydrogel films. As shown in Figure 1b, κ-CG/CS hydrogel films were prepared by polyion complexation of oppositely charged κ-CG and CS in dilute acetic acid solution, evaporating the solvent at a relatively high temperature, and swelling the casted film in water to achieve the equilibrium state. The appearance of resulting mixtures varied with the weight ratio (wr) of κ-CG to CS (Figure 2a). When wr =1:9 or 9:1, the mixtures were transparent and stable solutions. However, when wr was in the range from 2:8 to 8:2, some precipitates formed and settled down during the storage. As wr increased, the amount of precipitate increased first and then decreased, with the maximum at wr of 6:4, indicating that this mixture was near the isoelectric point. This was confirmed by zeta (ζ) potential measurements.

Figure 2. Photo (a) and zeta-potential (b) of the mixtures of κ-CG and CS solutions with different weight ratio, wr, of κ-CG to CS. The samples were stored at room temperature without shaking for 24 h.

The ζ-potential of 1 wt% acetic acid solution was 11.7 mV. As shown in Figure S1, the ζ-potential of κ-CG solutions decreased from -50.2 to -65.9 mV as the concentration of κ-CG 7 ACS Paragon Plus Environment


increased from 0.4 to 3.6 mg/mL. In contrast, the ζ-potential of CS solutions increased from 60 to 81.1 mV as the concentration of CS increased from 0.4 to 3.6 mg/mL. After mixing κ-CG and CS solutions, ζ-potential of the mixture decreased from 76.1 to -50.4 mV when wr changed from 1:9 to 9:1 (Figure 2b). The ζ-potential changed from positive value to negative one as wr increased from 6:4 to 7:3; therefore, the charge balance point should be in this narrow range of wr. These results are consistent with the appearance variation of the mixtures showed in Figure 2a. The mixture of κ-CG and CS solutions was used to fabricate hydrogel film according to the procedure schemed in Figure 1b. The obtained gel films were transparent with the thickness of 40-60 µm (Figure S2). The water content of κ-CG/CS gel films was 48-88 wt% (Figure 3), and that of pure κ-CG and CS gel films prepared by a similar way was 97 wt% and 84 wt%, respectively. The κ-CG/CS gel with wr of 6:4, which was close to the charge balance point, had the lowest water content; deviation from this point led to increase in water content, except the gel with wr of 1:9 (relatively low water content might be an artifact during the measurements). 100

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Figure 3. Water content of κ-CG /CS hydrogel films with different wr. Error bars represent the standard deviation of the mean.

The formation of hydrogel films should be closely related to the noncovalent interaction between κ-CG and CS, which was characterized by FTIR (Figure 4a). In the spectrum of κ-CG gel film, the peak at 1220 cm-1 was the stretching vibration band of S=O (SO4). In the 8 ACS Paragon Plus Environment

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spectrum of CS gel film, the peaks at 1635 and 1550 cm-1 corresponded to amide I (CONH2) and amide II (NH2), respectively. In comparison, in the spectrum of κ-CG/CS gel film (wr = 6:4), the peak of NH2 shifted to 1536 cm-1, indicating the formation of ionic bonds between the sulfate group of κ-CG and the protonated amine group of CS.43,44 As shown in Figure 4b, the spectra of κ-CG/CS gel films also varied with the composition. With the increase in wr, the peak of S=O (SO4) did not shift, whereas the peak of amide II shifted from 1550 to 1525 cm-1.45

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Figure 4. (a) FTIR spectra of CS, κ-CG, and κ-CG/CS gel films. wr of κ-CG/CS was 6:4. (b) FTIR spectra of κ-CG/CS gel films with different wr.

The microstructure of κ-CG/CS gel films was observed by SEM, although the freeze-drying influenced the internal structure of gels to some extent. As shown in Figure 5a, a large number of nano-sized grains were embedded in the porous gel matrix with wr of 3:7. The grain size increased in the sample with wr of 6:4 that was closed to the charge balance point (Figure 5b). Similar dense network embedded with nano-sized grains were observed in other samples with different wr. The compact grains should be formed during the complexation process, indicating the strong ionic bonding between oppositely charged κ-CG and CS molecules. Besides the ionic bonds, hydrogen bonds should also be formed between κ-CG molecules to form helicoidal structure and CS molecules to form fibrous structure at room temperature and neutral condition.36,37 The cooperative ionic and hydrogen bonding between the interpenetrating biopolymers should result in the formation of these tough hydrogel films. 9 ACS Paragon Plus Environment


Figure 5. SEM images of the cross-section of hydrogel films with wr of 3:7 (a) and 6:4 (b).

Mechanical properties of gel films. Although we can obtain κ-CG/CS hydrogel films over a wide range of wr, the gels with wr of 9:1 and 1:9 were too weak to measure the mechanical properties by tensile tests. As shown in Figures 6a and 6b, the other gel films showed excellent mechanical performances, with σb, εb, and E being 2-6.7 MPa, 80-120%, and 1.2-25 MPa, respectively, which were superior to the values of most existing biopolymer-based hydrogels (Figure 6c).26-35

Figure 6. (a,b) Tensile stress-strain curves (a) and measured mechanical properties (b) of hydrogel membranes with different wr. Error bars represent the standard deviation of the mean. (c) Material property chart for native dura mater and various biopolymer-based 10 ACS Paragon Plus Environment

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hydrogels. Materials include native dura maters,6 κ-CG/CS gels in this work, CS gels,35 cellulose gels,27 gelatin gels,26 sacran gels,34 silk-fibroin gels,29 bacterial cellulose/gelatin gels,26 CS/alginate gels,30 and CS/chondroitin sacran gels.31

As expected, wr significantly influenced the mechanical properties of the gels. The gel film with wr of 5:5 had the maximum σb, whereas the gel film with wr of 7:3 possessed the highest E. When the amount of κ-CG increased (i.e. wr increased), E of the gels gradually increased up to the maximum value of 25 MPa and then decreased slightly. It was rational that the maximum σb was achieved when the system was close to the charge balance point and the gel had the lowest water content. However, it was interesting that the gel with slight excess of κ-CG had the highest E. κ-CG molecules formed double-helix structure at a low temperature, resulting in a rigid and brittle physical hydrogel. Pronounced yielding was observed in the gels containing a large fraction of κ-CG (wr of 7:3 and 8:2), which was also found in κ-CG/polyacrylamide double-network hydrogels.37,46 A balanced combination of ionic bonds between κ-CG and CS and hydrogen bonds between κ-CGs may account for the highest E of the gels. It was remarkable that the gels possessed excellent mechanical performances over a wide range of wr, despite of large deviations from the charge balance point, which was different from other polyion complex hydrogels.10,43,47 This typical feature should be rooted in the multiple noncovalent interactions and special network structure of the gels as described above. At relatively high wr, the excess κ-CG molecules can form secondary structure or even integrated matrix via inter-chain hydrogen bonds; at relatively low wr, the excess, deprotonated CS molecules will form fibrous structure via intermolecular hydrogen bonding.35,36 The cooperation of ionic bonds and hydrogen bonds afforded the excellent mechanical properties of the gels over a wide range of wr. The gel film with wr of 5:5 showed σb of 6.7 MPa and εb of 100%, which were comparable to those of native dura mater and should have promising applications as dura substitutes. The κ-CG/CS hydrogel films also showed good self-recovery property, which was characterized by cyclic tensile tests. As shown in Figure 7a, large hysteresis was observed from the first loading-unloading curve of the gel film (wr = 5:5) with a maximum strain of 30%, indicating large energy dissipation by the destruction of noncovalent bonds. 11 ACS Paragon Plus Environment


Subsequent loading-unloading cycle without waiting produced a loop with a small hysteresis and a large residual strain. However, as the waiting time increased, the hysteresis loop gradually approached the first cycle. After 120 min, the residual strain disappeared and hysteresis ratio (determined by the area ratio of the subsequent loop to the first one) was as high as 90% (Figure 7b), indicating the good self-recovery involving reformation of noncovalent bonds, which is important for the applications of gels under dynamic, cyclic loading.

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Figure 7. Cyclic tensile stress-strain curves of the gel with wr of 5:5 after different waiting time (a) and corresponding hysteresis ratio and residual strain (b). Error bars represent the standard deviation of the mean.

The synergistic effect of ionic bonds and hydrogen bonds resulted in κ-CG/CS gels with much better mechanical performances than either of their parents (Figure S3). Due to the dynamic nature of noncovalent interactions, the mechanical properties of gel films should be influenced by the incubation conditions. As shown in Figures 8a and 8b, incubation of the gel film (with wr = 5:5) in acetic acid solutions led to slight inflation of gel volume and dramatic decrease in σb and E. This was because of the protonation of amine groups of uncomplexed CS molecules in the acidic condition, which increased the electrostatic repulsion. Incubation of the gel in saline solutions also led to decrease in mechanical properties due to the shielding effect that weakened the ionic interactions (Figures 8c and 8d). We should note that the gel still possessed considerable mechanical strength in acetic acid solution or concentrated saline solution, suggesting the coexistence of multiple noncovalent interactions in the gels. When one type of noncovalent bond was destroyed, the 12 ACS Paragon Plus Environment

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Figure 8. (a,b) Tensile stress-strain curves of gel film (wr = 5:5) swelled in acidic solution with different concentrations of acetic acid, Cacid (a) and corresponding mechanical properties (b). (c,d) Tensile stress-strain curves Tensile stress-strain curves of gel film (wr = 5:5) swelled in saline solution with different concentrations of NaCl, CNaCl (d) and corresponding mechanical properties (d). Error bars represent the standard deviation of the mean.

The κ-CG/CS gel films also had good mechanical stability. There was no evident change of mechanical properties after incubating the gel (wr = 5:5) in PBS solution at room temperature for 40 days (Figure S4). However, good biodegradability should be expected of this gel composed of κ-CG and CS, which can be degraded by enzymatic hydrolysis.21,36,48 These features endow the κ-CG/CS hydrogels with promising applications as structural biomaterials. Anti-adhesion property and biocompatibility. The practical applications of gel films as artificial dura mater and diaphragm material in the surgery require the κ-CG/CS hydrogels to have good anti-adhesion property and biocompatibility.49 NIH/3T3 fibroblast cells were cultured on κ-CG/CS gel films to investigate these properties. As shown in Figure 9a, the 13 ACS Paragon Plus Environment


cells showed less spread morphology on the gel film with wr of 1:9 when compared with that on tissue culture polystyrene (TCPS). For the gel films with wr of 9:1 and 8:2, cells hardly adhered on the surface and poorly spread with a round morphology. For other samples, only sporadic cells still adhered on the gel films, indicating their excellent anti-adhesion against cells. The anti-adhesion property of κ-CG/CS gel films was further confirmed by quantitative analysis of cell density (Figure 9b). The excellent cell anti-adhesion property of κ-CG/CS gels might be related to the hydration layer with water molecules strongly bonded to the gel matrix via hydrogen bonds and ionic solvation,49 which impeded protein adsorption and cell attachment, although the essential reason is not clear.

Figure 9. (a,b) Fluorescent images of NIH/3T3 fibroblast morphology cultured on κ-CG/CS gel films and TCPS for 24 h (a) and corresponding cell density (b). The F-actin of cells were selectively stained. (c) Cell viability of NIH/3T3 fibroblasts cultivated with κ-CG/CS gel films for 2 days. Error bars represent the standard deviation of the mean.

The biocompatibility of κ-CG/CS hydrogel films was assessed in terms of cytotoxicity. After two days of in vitro cytotoxicity test, the viability of NIH/3T3 fibroblast cells had no obvious difference between the test groups and the control (Figure 9c). The viability of cells with κ-CG/CS gel films exceeded 90%. In addition, the κ-CG/CS gel films with a large 14 ACS Paragon Plus Environment

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faction of CS (i.e. relatively low wr) showed moderate antibacterial properties due to the antimicrobial activity of CS (Figure S5).39 The good biocompatibility and cell anti-adhesion properties make the robust κ-CG/CS hydrogel films as promising diaphragm materials in surgeries.

4. CONCLUSIONS We have developed a series of polysaccharide-based hydrogel films with remarkable mechanical performances and good cell anti-adhesion property. The gel films were facilely prepared by casting the mixture of oppositely charged κ-CG and protonated CS solutions and subsequent swelling the dry film in water to achieve the equilibrium state. Transparent hydrogel films with thickness of 40-60 µm were obtained over a wide range of mass ratio of κ-CG to CS, which possessed excellent mechanical properties with σb of 2-6.7 MPa, εb of 80-120%, and E of 1.2-25 MPa, superior to most existing biopolymer-based hydrogels. The excellent mechanical properties should be related to the synergistic effect of ionic bonds and hydrogen bonds between κ-CG and CS molecules. Furthermore, the gels showed good self-recovery, biocompatibility, and cell anti-adhesion properties, making them promising candidate as artificial dura mater and diaphragm materials in surgery. The strategy by incorporating multiple noncovalent interactions to toughen biopolymer-based hydrogels with an appropriate toughening mechanism should be applicable to other systems for designing versatile structural materials with diverse applications.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51773179, 51403184), Fundamental Research Funds for the Central Universities of China, and Thousand Young Talents Program of China.

Supporting Information Available: supplementary figures of the mechanical properties of κ-CG and CS hydrogels and κ-CG/CS gel films after long time storage. This material is available free of charge via the Internet at http://pubs.acs.org. 15 ACS Paragon Plus Environment


REFERENCES

1. Gartner, L. P.; Hiatt, J. L. Color Textbook of Histology, 2nd ed.; Saunders: Philadelphia, 2001. 2. Vandenabeele, F.; Creemers, J.; Lambrichts, I. Ultrastructure of the human spinal arachnoid mater and dura mater. J. Anat. 1996, 189, 417–430. 3. Burrand, D. B.; Guillot, G. M. Almost invisible, often ignored: periosteum, the living lace of bone. Medicographia. 2012, 34, 221–227. 4. Crawford, J. S. Repair of ptosis using frontalis muscle and fascia lata: a 20-year review. Ophthalmic. Surg. 1977, 8, 31–40. 5. Butler, D. L.; Grood, E. S.; Noyes, F. R.; Zernicke, R. F.; Brackett, K. Effects of structure and strain measurement technique on the material properties of young human tendons and fascia. J. Biomech. 1984, 17, 579–596. 6. Zhu, X.; Shi, D.; Dong, X.; Guo, S. Experimental study on mechanical properties of human dura mater encephali falx cerebri and tentorium cerebelli. J. Jilin Univ. (Eng. Tech. Ed.) 1988, 1, 51‒60. 7. Caroli, E.; Rocchi, G.; Salvati, M.; Delfini, R. Duraplasty: our current experience. Surg. Neurol. 2004, 61,55–59. 8. Zhao, X. Multi-scale multi-mechanism design of tough hydrogels: building dissipation into stretchy networks. Soft Matter 2014, 10, 672–687. 9. Wang, W.; Zhang, Y.; Liu, W. Bioinspired fabrication of high strength hydrogels from non-covalent interactions. Prog. Polym. Sci. 2017, 71,1–25. 10. Sun, T. L.; Kurokawa, T.; Kuroda, S.; Bin Ihsan, A.; Akasaki, T.; Sato, K.; Haque, M. A.; Nakajima, T.; Gong, J. P. Physical hydrogels composed of polyampholytes demonstrate high toughness and viscoelasticity. Nat. Mater. 2013, 12, 932–937. 11. Guo, M.; Pitet, L. M.; Wyss, H. M.; Vos, M.; Dankers, P. Y. W.; Meijer, E. W. Tough stimuli-responsive supramolecular hydrogels with hydrogen-bonding network junctions. J. Am. Chem. Soc. 2014, 136, 6969–6977. 12. Dai, X.; Zhang, Y.; Gao, L.; Bai, T.; Wang, W.; Cui, Y.; Liu, W. A mechanically strong, highly stable, thermoplastic, and self-healable supramolecular polymer hydrogel. Adv. Mater. 2015, 27, 3566–3571. 13. Lin, P.; Ma, S.; Wang, X.; Zhou, F. Molecularly engineered dual-crosslinked hydrogel with ultrahigh mechanical strength, toughness, and good self-recovery. Adv. Mater. 2015, 27, 2054–2059. 14. Hu, X.; Vatankhah-Varnoosfaderani, M.; Zhou, J.; Li, Q.; Sheiko, S. S. Weak hydrogen bonding enables hard, strong, tough, and elastic hydrogels. Adv. Mater. 2016, 27, 6899–6905. 15. Sun, J.-Y.; Zhao, X.; Illeperuma, W. R. K.; Chaudhuri, O.; Oh, K. H.; Mooney, D. J.; Vlassak, J. J.; Suo, Z. Highly stretchable and tough hydrogels. Nature 2012, 489, 133–136. 16. Du, G.; Wu, F.; Cong, Y.; Nie, L.; Liu, S.; Gao, G.; Fu, J. Versatile controlled ion release for synthesis of recoverable hybrid hydrogels with high stretchability and 16 ACS Paragon Plus Environment

Page 16 of 20

Page 17 of 20 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


17.

18.

19.

20.

21.

22.

23.

24. 25. 26.

27.

28.

29. 30.

31.

notch-insensitivity. Chem. Commun. 2015, 51, 15534–15537. Chen, Q.; Zhu, L.; Zhao, C.; Wang, Q.; Zheng, J. A robust, one-pot synthesis of highly mechanical and recoverable double network hydrogels using thermoreversible sol-gel polysaccharide. Adv. Mater. 2013, 25, 4171–4176. Sun, Y.; Gao, G.; Du, G.; Cheng, Y.; Fu, J. Super tough, ultrastretchable, and thermoresponsive hydrogels with functionalized triblock copolymer micelles as macro-cross-linkers. ACS Macro Lett. 2014, 3, 496–500. Du, G.; Gao, G.; Hou, R.; Cheng, Y.; Chen, T.; Fu, J. Tough and fatigue resistant biomimetic hydrogels of interlaced self-assembled conjugated polymer belts with a polyelectrolyte network. Chem. Mater. 2014, 26, 3522–3529. Mayumi, K.; Marcellan, A.; Ducouret, G.; Creton, C.; Narita, T. Stress–strain relationship of highly stretchable dual cross-link gels: separability of strain and time effect. ACS Macro Lett. 2013, 2, 1065–1068. Yang, Y.; Wang, X.; Yang, F.; Shen, H.; Wu, D. A universal soaking strategy to convert composite hydrogels into extremely tough and rapidly recoverable double-network hydrogels. Adv. Mater. 2016, 28, 7178–7184. Zheng, S. Y.; Ding, H.; Qian, J.; Yin, J.; Wu, Z. L.; Song, Y.; Zheng, Q. Metal-coordination complexes mediated physical hydrogels with high toughness, stick-slip tearing behavior, and good processability. Macromolecules 2016, 49, 9637–9646. Tian Y.; Wei, X.; Wang, Z. J.; Pan, P.; Li, F.; Ling, D.; Wu, Z. L.; Zheng, Q. A facile approach to prepare tough and responsive ultrathin physical hydrogel films as artificial muscles. ACS Appl. Mater. Interfaces 2017, 9, 34349–34355. Zhao, W.; Jin, X.; Cong, Y.; Liu, Y.; Fu, J. Degradable natural polymer hydrogels for articular cartilage tissue engineering. J. Chem. Technol. Biotechnol. 2013, 88, 327–339. Costa, A. M. S.; Mano, J. F. Extremely strong and tough hydrogels as prospective candidates for tissue repair – a review. Eur. Polym. J. 2015, 72, 344–364. Nakayama, A.; Kakugo, A.; Gong, J. P.; Osada, Y.; Takai, M.; Erata, T.; Kawano, S. High mechanical strength double-network hydrogel with bacterial cellulose. Adv. Funct. Mater. 2004, 14, 1124–1128. Zhao, D.; Huang, J.; Zhong, Y.; Li, K.; Zhang, L.; Cai, J. High-strength and high-toughness double-cross-linked cellulose hydrogels: a new strategy using sequential chemical and physical cross-linking. Adv. Funct. Mater. 2016, 26, 6279–6287. Wu, S.; Dong, H.; Li, Q.; Wang, G.; Cao, X. High strength, biocompatible hydrogels with designable shapes and special hollow-formed character using chitosan and gelatin. Carbohyd. Polym. 2017, 168, 147–152. Luo, K.; Yang, Y.; Shao, Z. Physically crosslinked biocompatible silk-fibroin-based hydrogels with high mechanical performance. Adv. Funct. Mater. 2016, 26, 872–880. Costa, R. R.; Costa, A. M. S.; Caridade, S. G.; Mano, J. F. Compact saloplastic membranes of natural polysaccharides for soft tissue engineering. Chem. Mater. 2015, 27, 7490–7502. Rodrigues, M. N.; Oliveira, M. B.; Costa, R. R.; Mano, J. F. Chitosan/chondroitin sulfate membranes produced by polyelectrolyte complexation for cartilage engineering. Biomacromolecules 2016, 17, 2178–2188. 17 ACS Paragon Plus Environment


32. Shen, Z.-S.; Cui, X.; Hou, R.-X.; Li, Q.; Deng, H.-X.; Fu, J. Tough biodegradable chitosan-gelatin hydrogels via in situ precipitation for potential cartilage tissue engineering. RSC Adv. 2015, 5, 55640–55647. 33. Zhu, F.; Yu, H. C.; Lei, W.-X.; Ren, K.-F.; Qian, J.; Wu, Z. L.; Zheng, Q. Tough polyion complex hydrogel films of natural polysaccharides. Chinese. J. Polym. Sci. 2017, 35, 1276–1285. 34. Sornkamnerd, S.; Okajima, M. K.; Kanoko, T. Tough and porous hydrogels prepared by simple lyophilization of LC gels. ACS Omega 2017, 2, 5304–5314. 35. Duan, J.; Liang, X.; Cao, Y.; Wang, S.; Zhang, L. High strength chitosan hydrogels with biocompatibility via new avenue based on constructing nanofibrous architecture. Macromolecules 2015, 48, 2706–2714. 36. Suh, J. K. F.; Matthew, H. W. T. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000, 21, 2589–2598. 37. Liu, S.; Li, L. Recoverable and self-healing double network hydrogel based on κ-carrageenan. ACS Appl. Mater. Interfaces 2016, 8, 29749–29758. 38. Fu, J.; Fares, H. M.; Schlenoff, J. B. Ion-pairing strength in polyelectrolyte complexes. Macromolecules 2017, 50, 1066–1074. 39. Bratskaya, S.; Marinin, D.; Simon F.; Synytska, A.; Zschoche, S.; Busscher, H. J.; Jager, D.; van der Mei, H. C. Adhesion and viability of two enterococcal strains on covalently grafted chitosan and chitosan/κ-carrageenan multilayers. Biomacromolecules 2007, 8, 2960–2968. 40. Zhao, X. Designing toughness and strength for soft materials. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 8138–8140. 41. Chang, H.; Hu, M.; Zhang, H.; Ren, K.-F.; Li, B. C.; Li, H.; Wang, L.-M.; Lei, W.-X.; Ji, J. Improved endothelial function of endothelial cell monolayer on the soft polyelectrolyte multilayer film with matrix-bound vascular endothelial growth factor. ACS Appl. Mater. Interfaces 2016, 8, 14357–14366. 42. Chang, H.; Zhang, H.; Hu, M.; Chen, X.-C.; Ren, K.-F.; Wang, J.-L.; Ji, J. Surface modulation of complex stiffness via layer-by-layer assembly as a facile strategy for selective cell adhesion. Biomater. Sci. 2015, 3, 352–360. 43. Zhu, F.; Lin, X. Y.; Wu, Z. L.; Cheng, L.; Yin, J.; Song, Y.; Qian, J.; Zheng, Q. Processing tough supramolecular hydrogels with tunable strength of polyion complex. Polymer 2016, 95, 9–17. 44. Wang, X.-S.; Ji, Y.-L.; Zheng, P.-Y.; An, Q.-F.; Zhao, Q.; Lee, K.-R.; Qian, J.-W.; Gao, C.-J. Engineering novel polyelectrolyte complex membranes with improved mechanical properties and separation performances. J. Mater. Chem. A 2015, 3, 7296–7303. 45. Silva, J. M.; Georgi, N.; Costa, R.; Sher, P.; Reis, R. L.; Van Blitterswijk, C. A.; Karperien, M.; Mano, J. F. Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering. Plos One 2013, 8, e55451. 46. Lu, X.; Chan, C. Y.; Lee, K. I.; Ng, P. F.; Fei, B. Xin, J. H.; Fu, J. Super-tough and thermo-healable hydrogel – promising for shape-memory absorbent fiber. J. Mater. Chem. B, 2014, 2, 7631–7638. 47. Luo, F.; Sun, T. L.; Nakajima, T.; Kurokawa, T.; Zhao, Y.; Sato, K.; Ihsan, A. B.; Li, 18 ACS Paragon Plus Environment

Page 18 of 20

Page 19 of 20 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


X.; Guo, H.; Gong, J. P. Oppositely charged polyelectrolytes form tough, self-healing, and rebuildable hydrogels. Adv. Mater. 2015, 27, 2722–2727. 48. Collén, P. N.; Lemoine, M.; Daniellou, R.; Guégan, J. P.; Paoletti, S.; Helbert, W. Enzymatic degradation of κ-carrageenan in aqueous solution. Biomacromolecules 2009, 10, 1757–1767. 49. Lih, E.; Oh, S. H.; Joung, Y. K.; Lee, J. H.; Han, D. K. Polymers for cell/tissue anti-adhesion. Prog. Polym. Sci. 2015, 44, 28–61.

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