Facile preparation of biocompatible macroporous chitosan hydrogel by hydrothermal reaction of a mixture of chitosan-succinic acid-urea

Materials Science & Engineering C 104 (2019) 109845

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Facile preparation of biocompatible macroporous chitosan hydrogel by hydrothermal reaction of a mixture of chitosan-succinic acid-urea

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Prabha Govindaraj, Narayanan Abathodharanan, Kartik Ravishankar, ⁎ Dhamodharan Raghavachari Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Macroporous chitosan Hydrogel Super water absorption Herschel-Bulkley gel Copper (+2) adsorption

The facile preparation of macroporous, super water absorbing, biocompatible hydrogels of chitosan involving the hydrothermal reaction of a mixture of chitosan (CH), succinic acid (SA) and urea (UR), all of which are sustainable materials, is reported. The structure of the dry CHSAUR was ascertained by CP MAS-SS NMR spectroscopy, Fourier transform infrared (FTIR) spectroscopy, powder x-ray diffraction analysis (PXRD), and thermogravimetric analysis (TGA). The principle role of UR in the synthesis was identified as the source of ammonia, which increased the pH of the acidic chitosan solution with reaction time, leading to the formation of the insoluble hydrogel of chitosan accompanied by the formation of pores of different sizes and volumes. In addition, a small fraction of urea participated in chemical reaction with the primary hydroxyl groups in the sixth position of the glucosamine repeat units of chitosan resulting in carbamate linkages. The as-prepared hydrogel, following workup and methanol extraction, was found to be chitosan crosslinked with succinic acid through electrostatic interaction. It was macroporous with percentage porosity varying between 49.4% to 64.2%. It also exhibited different extents of water uptake with the maximum of 760 ± 20 g/g being for the one prepared with the weight ratio of 1: 4: 4 of chitosan: succinic acid: urea. The absorption of water is found to arise out of the porosity as well as presence of water attracting chitosan ammonium cation-succinate electrovalent bonds that are formed by the reaction between SA and ammonium cation of the chitosan backbone. The absorption of saline water was relatively poor suggesting that the saline water absorption might be arising largely due to the presence of micropores and specific interaction. The hydrogels exhibited Herschel-Bulkley rheological behavior. The extraction of CHSAUR with 0.1 N NaOH in methanol resulted in the removal of the physical crosslinks, consisting of succinate anions; the presence of chitosan with porous morphology was confirmed additionally by copper (+2) adsorption. In contrast to the widely reported method of preparing microporous chitosan scaffold of cylindrical shape that takes several days to a week, the present method offers a simple means of preparing macroporous chitosan of any shape and size in very large scale with soft foam-like morphology. With its biocompatibility towards mouse fibroblast cells it could find applications in drug delivery, biodegradable super water absorbency and haemostatic applications.

1. Introduction Chitosan is the partially deacetylated form of chitin that is obtained by the hot alkaline hydrolysis of chitin (when the extent of deacetylation is 50 or more mole% it is classified as chitosan) [1–4]. Chitin is a linear random copolymer consisting almost exclusively β-(1,4)-2-acetamido-2-deoxy β-D-glucopyranose repeat units and relatively smaller extent of β-(1,4)-2-amino-2-deoxy-β-D-glucopyranose repeat units. It is present in crustaceans (as one of the components of the exoskeleton), insects, algae, fungi and yeast and is bio-synthesized to the extent of

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1011 tons per annum [5,6]. Chitin is separated from the exoskeletons of marine crustaceans (such as crab, shrimp, and prawn) by the chemical method [7] among others [8]. Chitosan is well known for haemostatic activity, non-toxicity, biodegradability, biocompatibility, antimicrobial and antifungal activities, good adhesion to surfaces, flocculation promotion, stability over a wide range of pH (1 to 5.5), low immune-stimulating activity and adsorption of toxic metal ions such as cadmium, lead, mercury, etc. Hence, chitosan is studied widely. The unique properties of chitosan make it an ideal polymer for variety of industrial and biomedical applications in the form of film, fiber, nanoparticles and

Corresponding author. E-mail addresses: [email protected] (N. Abathodharanan), [email protected] (D. Raghavachari).

https://doi.org/10.1016/j.msec.2019.109845 Received 18 March 2019; Received in revised form 13 May 2019; Accepted 30 May 2019 Available online 31 May 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

hydrogel [9–13]. However, there are certain inherent limitations associated with chitosan such as, poor solubility in aqueous media at neutral and alkaline conditions as well as in most organic solvents; relatively poor mechanical properties; inability to soften upon the application of heat; and limited availability in the porous form. Chitosan is characterized by its solubility in dilute organic acids such as acetic acid. The solubility is also affected by the distribution of acetyl groups (for example, blocky versus random), extent of acetylation, pH, ionic strength and extent of intramolecular hydrogen bonding. To improve the mechanical properties of chitosan-based materials several methods have been established. One of the simple methods is crosslinking [14]. It has been established that the primary amino group of chitosan can be used to crosslink the polymer by using a variety of crosslinkers such as glutaraldehyde, genipin and multifunctional carboxylic acids such as malonic, maleic, succinic acid, etc. However, certain applications such as chelation of metal ions, scaffold for tissue engineering, personal hygiene, agriculture, construction, water purification, drug delivery, biomedical applications and controlled release require chitosan in the form of a super absorbing hydrogel and preferably in the porous form [15–18]. The synthesis of super absorbing porous chitosan gel using ternary solvents [19] as well as the advances made in chitosan-based superabsorbent hydrogels has been reviewed [15]. We have also reported, recently, on the preparation of porous chitosan gels by the hydrothermal synthetic route using a mixture of chitosan, EDTA [20] or citric acid [21] and urea. In these works, we could not quantitate the extent of porosity of the chitosan gels and their rheological properties. Further, citric acid (with pKa1 of 3.1), a stronger acid that could, in principle, result in partial depolymerization of chitosan under the hydrothermal synthetic conditions was employed. Here we explore the hydrothermal synthesis of chitosan-succinic acid-urea mixture in the preparation of super absorbing porous hydrogels. Succinic acid (SA) was chosen for the following reasons. It is a weaker acid (pKa1 of 4.2) compared to citric acid that occurs naturally in plant and animal tissues and is highly soluble in water (58–100 mg/ mL at 20–25 °C). Being a weaker acid it is less likely to depolymerize chitosan under the hydrothermal synthetic conditions reported earlier by us. Films of chitosan crosslinked with succinic acid have been reported to enhance the strength of the chitosan film formed due to the formation of physical crosslinks and these films in turn are proposed to be good wound dressing as well as tissue engineering material [22,23] without the risk of accumulation in the body [23]. Thus being a relatively weaker acid compared to citric acid with better thermal stability it can facilitate reaction with chitosan under hydrothermal conditions without depolymerization of chitosan while simultaneously acting as a physical crosslinking agent enabling the preparation of succinic acid containing hydrogels of chitosan. The choice of urea as another reactant was due to its known tendency to decompose and release ammonia under the experimental conditions and enable the preparation of porous material as reported earlier by us [20,21]. The established non-toxicity of succinic acid and suitability in external applications in chitosan gel matrix was additional reason for selecting succinic acid in this work. The preparation of physically crosslinked macroporous chitosan by the reaction of chitosan in the presence of succinic acid and urea under hydrothermal conditions is reported here. Further the structural characterization, water absorption, extent of porosity and rheological properties are also reported. We also show that the physical crosslinks formed in the synthetic process between chitosan and succinic acid contribute to a fairly significant extent to the water absorption and also show that they can be removed by extraction with 0.1 N NaOH in methanol resulting in porous chitosan hydrogels with relatively lesser water absorption. These hydrogels are shown to be Herschel-Bulkley in nature and biocompatible for the growth of 3T3 L1 mouse fibroblasts cells in vitro.

2.1. Materials Chitosan (CH, ≈85% degree of deacetylation as ascertained by FTIR spectroscopy and PXRD; number average molecular weight 43,000) was purchased from the Kerala State Cooperative Federation for Fisheries Development Ltd. (Matsyafed, Cochin). Aqueous ammonium hydroxide solution was purchased from Rankem, India. Methanol, glacial acetic acid, succinic acid (SA), sodium chloride, and urea (UR) were purchased from Merck, India. methylthiazolyl diphenyl- tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich. 3T3 L1 mouse fibroblasts cell lines were obtained from National Center for Cell Sciences (NCCS), Pune, India. The chemicals and all the solvents used in this study were of reagent grade and used as received. 2.2. Synthesis of porous chitosan by the hydrothermal reaction with succinic acid and urea (CHSAUR) The procedure reported previously by us [20] was followed in the preparation of CHSAUR hydrogel. Briefly, SA and UR of a particular weight ratio (one of the following SA:UR weight ratios 0.5:0.5, 1:1, 2:2, 3:3, and 4:4 with respect to unit mass of chitosan) was dissolved in 60 mL of distilled water and taken in a 250 mL poly(propylene) bottle. To this solution, calculated amount of CH (3 g; representing the weight ratio of 1) was added and shaken physically for 5 min upon which it turned viscous due to the dissolution of CH. Then the poly(propylene) bottle was placed in an air oven (this was part of a GC and enabled controlled heating) equipped with a temperature controller. The reaction mixture was heated from room temperature (~30 °C) to 100 °C at the heating rate of 10 °C/min and then maintained at this temperature for 8 h. After that it was allowed to cool at room temperature. The product was pale brown to brown in color and was either a strong or loose gel depending on the weight ratio of the reactants. It was transferred to a beaker containing four to five-fold excess methanol (by volume; to remove unreacted and unbound urea and succinic acid that are soluble in methanol). The light color gel thus formed was rinsed with acetic acid and then with aqueous ammonium hydroxide solution. Finally, the gel was extracted (Soxhlet) for 2 to 3 h with methanol and dried at 50 °C for 5 h in an air oven to obtain a light brown colored solid (CHSAUR). Each one of the preparation was done thrice to ensure consistency of the process and reproducibility of the results. Yield: 4.2 g. CP-MAS SS NMR (100 MHz, δ, ppm): 23.2 (NHCOCH3; repeat units of chitosan that are acetylated), 55.7 to 57.2 (C2 of CH), 60.3 to 61.2 (C6 of CH), 74.7 to 75 (C3 and C5 of CH), 81.8 to 83.4 (C4 of CH), 102.7 to 104.9 (C1 of CH), 160.6 to 161.8 (-CH2O-CONH2; carbamate), 174.4 to 175.9 (-NH-CO-CH3 from CH), 180.1 to 181.3 (O=C-O– from SA) ppm, respectively. 2.3. Characterization techniques CP MAS-SS NMR spectra of solid CHSAUR samples were recorded using a Bruker Avance 400 spectrometer (400 MHz for 1H and 100 MHz for 13C) under the following conditions: probe diameter = 4 mm; spinning rate = 10,000 kHz; contact time = 2000 μm, repetition time = 5 s; number of scans = 10,000. Prior to the measurement, the instrument was calibrated for all the nucleus, using bromine signal from KBr. NMR spectra (1H NMR and 13C NMR) of chitosan, products of control reaction between chitosan and succinic acid, chitosan and urea as well as succinic acid, in solution, were obtained using a Bruker Avance 500 spectrometer (500 MHz for 1H and 125 MHz for 13C). For the purpose of recording solution NMR spectrum, chitosan was dissolved in minimum quantity of concentrated HCl. Adequate quantity of D2O was then added to this solution to get good signal to noise ratio. Fourier Transform-infrared spectra (FT-IR) were recorded using JASCO 2

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FTIR-4100 spectrometer (resolution 16 cm−1). The sample in the pellet form (in KBr) was used. Inductively coupled plasma - optical emission spectroscopy (ICP-OES) was performed on Perkin Elmer Optima 5300 DV. MALDI–MASS measurements were performed on Bruker UltrafleXtreme using DHB (2,5-dihydroxybenzoic acid) matrix. Powder X-ray diffraction patterns were recorded with a Bruker D8 Advance diffractometer equipped with Cu anode and a Cu Kα (wavelength 1.5406 Å). Thermogravimetric analyses were conducted using a TA instruments Q-500 Hi-Res-TGA under N2 atmosphere. The samples were heated at a heating rate of 10 °C/min. Differential scanning calorimetry (DSC) measurements were made using a TA Instruments Q200 modulated differential scanning calorimeter (MDSC). Nitrogen was used as the carrier gas and the flow rate was 50 mL/min. Ramp rate was 10 °C/min for both heating and cooling. The measurement range was between −85 to 90 °C. The SEM images were recorded using an (Hitachi S-4800) field emission scanning electron microscope (electron acceleration voltage was 3 kV; analysis of sample was done under ultralow voltage mode). Rheological response of the CHSAUR gels (200 mg in 9.8 mL water; ~2 wt%) were analyzed using steady and oscillatory shear rheological measurements on an Anton Paar Modular Compact Rheometer (MCR 102) equipped with a P-PTD 200/AIR Peltier temperature device. Parallel Plate 20 (PP 20) geometry was utilized for all the measurements, which were performed with a measurement gap of 1 mm. The gel samples (200 mg in 9.8 mL water) were equilibrated for 2 min prior to the measurements. Gels were analyzed by both steady and oscillatory shear rheology. Steady shear rheological measurements were carried at room temperature between a shear rate of 0.01 to 100 s−1. In dynamic shear rheological measurements, strain sweep was carried out at room temperature between 1 and 100%, at a constant linear frequency of 10 Hz. Frequency sweep, on the other hand, was carried out at room temperature between 6 and 100 rad/s, at a constant strain of 1%. Temperature sweep (Room temperature to 65 °C) was carried out by steady shear rheology by maintaining the shear rate at 0.1 s−1. Under oscillatory shear, both the frequency and strain sweeps were performed (these results are presented in Figs. 9, 10). All these measurements were carried out at room temperature. In addition, temperature sweep has also been performed to determine the thermal stability of the gel. This temperature sweep was carried out between room temperature and 65 °C. 3D x-ray microtomography imaging of representative dry samples were carried out with Zeiss Xradia Versa 510 equipped with Dragonfly Pro 3.5 image processing software. The spatial resolution of this instrument was 1 μm. During microCT imaging, sample was scanned at 80 kV energy with 1601 projections acquired at one second exposure per projection. Projections were subjected to Feldkamp back-projection algorithm to get virtual cross-sectional images. The specimens were placed on a rotation table which was rotated 360° during which X-ray radiographs were recorded at every angular position. Three-dimensional (3D) reconstruction of the data was performed using Zeiss Xradia Versa 510 equipped with Dragonfly Pro 3.5 image processing software. Image enhancement and segmentation were performed with Dragonfly Pro (Version 3.5) software package. Optimum threshold values between the local maximum were selected for image segmentation. Porosity and pore-volume distribution were calculated by the software after segmentation. The surface area of the dry samples was assessed through nitrogen adsorption using Micromeritics ASAP 2020 V4.03.

water was allowed to drain over a 5 min interval. The mass of the sample and filter cone was then weighed (w2). The water uptake per g of sample was calculated using following equation (w2 − w1) / w1 in units of g of water absorbed per g of sample. The procedure was repeated until the gel reached its equilibrium absorption. Each experiment was performed in triplicate and the values reported are the mean values. The absorption of saline water of CHSAUR was also determined for three different aqueous solutions of sodium chloride (0.01, 0.05 and 0.1 w/w %) according to the procedure described above.

2.5. Cell culture Biocompatibility/toxicity studies were carried out using 3T3 L1 mouse fibroblasts. The 3T3 L1 cells were procured from NCCS, Pune, and the cells were grown and maintained in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), 2.5 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (antibiotics). The cells were handled in sterile environment and maintained at 37 °C in a 5% CO2 humidified incubator. The cells were passaged at 70–80% confluency for propagation and assay. The cells were counted post trypsinization and suitable number of cells were seeded in 96 well plates for cell viability assays.

2.6. Cell viability assay The toxicity studies were carried out with all the new material prepared CHSAUR0.5, CHSAUR1, CHSAUR2, CHSAUR3 and CHSAUR4. Among these the results from CHSAUR4, the material with the highest extent of modification that would be expected to show the highest toxicity, should toxicity arise out of the modification of chitosan, is presented. MTT assay was carried out for determining cell viability in the following way: The cells were trypsinized from culture flasks to form single cell suspension and counted. 5000 cells were seeded per well in 96 well plate in 100 μL of growth media with all supplements. Blank wells (only media), and control (positive and negative controls) were taken in duplicate. The well plate was incubated overnight for cells to adhere and form colonies. 100 mg of CHSAUR4 was dispersed in 1 mL of Dulbecco's Phosphate-Buffered Saline (DPBS) in a centrifuge tube, diluted further 20 times in serum-free Dulbecco's Modified Eagle Medium (DMEM) to obtain a final concentration of 5 mg/mL. 100 μL of the CHSAUR4 dispersion was added to test wells. Serum free DMEM was added in blank and negative control, whereas 5fluorouracil, a known anti-cancer agent and DNA binding drug (at 56 μM concentration), was added to cells as positive control. Separate well plates were used for different time points and the plates were left undisturbed for the incubation period. MTT was added to the wells at 24 h and 48 h [24] post-addition of the materials and incubated at 37 °C for 3 h. The reaction was stopped using stop-solution (DMSO) and the purple color solution was read using Berthold spectrophotometer at 590 nm wavelength and 640 nm as background. Media with cells alone was used as control while plain DPBS without any cells was used as blank and 5-fluorouracil, a known anti-cancer agent and DNA binding drug (at 56 μM concentration), was added to cells as positive control. The experiment was repeated thrice with technical duplicates. Significance was calculated between test and positive control.

2.4. Water absorption studies [20] 2.7. Statistical analysis Initially, a known weight (30 mg) of dried powder sample (CHSAUR) was weighed into a water filter cone of known mass. The initial mass of the sample plus filter cone was (w1). It was placed in a 100 mL beaker filled with water such that the sample is immersed entirely in water. After the desired time interval, the filter cone was removed from the top of the beaker and the excess and loosely bound

All data on cell viability are presented as mean ± standard deviation (SD). Statistical analysis was performed based on unpaired t-test using graph pad prism and the significant differences were considered where **** P ≤ 0.0001 and ***P = 0.0002 (for CHUR control alone).

3

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Fig. 1. Process flow chart representing the preparation of CHSAUR.

3. Results and discussion

reported for chitin [25,26], chitosan [27], succinic acid and urea solids and the solid state NMR spectrum of chitosan-blank (chitosan subjected to the same reaction conditions and workup without the use of succinic acid and urea). In CHSAUR samples, in addition to the standard peaks that arise from chitosan, two new peaks were observed. Among the two peaks the one observed between 180.1 and 181.3 ppm arises from the carboxylate group of SA, which in turn is probably formed due to the different extents of protonation of amine groups of CH by SA. The second new peak observed between 160.6 and 161.8 ppm could arise out of the carbamate linkage (-CH2-O-CO-NH2) formed by the reaction between urea and the primary hydroxyl groups present in the repeat unit of chitosan at the C6 position [21,28,29]. Thus CHSAURs appear to be predominantly CH with relatively smaller quantities of SA and UR (in the carbamate form). The SA component could be present as physical crosslinks. While the presence of these functional groups were evident by the solid state NMR data, the extent of the same could not be quantitated, by solid state NMR.

3.1. Synthesis of porous hydrogels The preparation of porous hydrogel material, CHSAUR, using chitosan (CH), succinic acid (SA), and urea (UR) is represented in Fig. 1. Different weight ratios of CH, SA and UR (1:0.5:0.5, 1:1:1, 1:2:2, 1:3:3, and 1:4:4) were used in these preparations. The hydrogels thus prepared were labeled as CHSAUR0.5 to CHSAUR4 in which the last digit indicates the weight ratio of succinic acid:urea used in the preparation with respect to chitosan. The wet products were pale-brown to brown colored gels, were macroporous (to the naked eye) with the pore size of the order of several mms, with the bigger pores at the bottom and relatively smaller pores at the top, as shown by an example in Fig. 2. The gels (photographs of other gels are shown in Supporting Information Fig. S1) were worked up to obtain a powder as detailed in the experimental section. All of them were insoluble in a number of common organic solvents (such as methanol, a known solvent for SA as well as UR), water (solvent for UR) and aqueous acetic acid (solvent for CH) and therefore it was inferred that they might be crosslinked. Hence to assess the structure of the product a number of characterization tools such as solid state NMR, FTIR, PXRD, and TGA were used.

3.2.2. Fourier transmission infrared spectroscopy (FTIR) analysis The FTIR spectra of CHSAUR0.5 to CHSAUR4 are presented in Supporting Information (Fig. S2). The significant peaks observed in these samples and their assignments [26,30] are also given in the Supporting Information. The new observations were: the peak at 1711 cm−1, arising from carbonyl stretching peaks of carboxylic acid group associated with SA and observed as a shoulder, increased with increasing composition of SA in the reaction mixture, suggesting its presence in increasing proportion to its use in the preparations; the peak at 1116 cm−1 suggested the presence of UR as carbamate in CHSAUR [27,28]. Further, the intensity of the peaks at around 1559

3.2. Characterization of prepared porous hydrogels 3.2.1. CP MAS-SS NMR spectroscopy The 13C cross polarization magic angle spinning solid state [CPMAS] NMR spectrum of different CHSAUR powders is presented in Fig. 3. The peaks were assigned by comparing the spectrum with that 4

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Fig. 2. Photograph of as-prepared CHSAUR4 (CH: SA: UR = 1:4:4) gel and analysis of pore size and distribution.

and 1403 cm−1 were observed to increase with increasing composition of SA in the reaction mixture suggesting the increasing presence of SA in the product. It is clear from FTIR spectroscopy that the different samples of CHSAUR are predominantly CH with relatively smaller quantity of SA. The characteristic peaks of free UR were not observed in any of the CHSAURs. To ascertain the structure of CHSAUR and especially the nature of bonding, control experiments involving the reaction between any two of the three components (CH and SA; SA and UR; as well as CH and UR; the products obtained after workup from these reactions were labeled as CHSA, CHUR and SAUR, respectively) were carried out. The photographs associated with these reactions are presented in Supporting Information Fig. S3. The FTIR spectrum of the reactants and products

obtained in control reactions are displayed in Supporting Information as Figs. S4 to S6. More detailed characterization data are given in Supporting Information as Figs. S7 to S10. From the spectroscopic data associated with the control experiments it could be inferred that only the mixture of SA and UR undergo reaction to form SAUR (no reaction between CH and SA as well as CH and UR). The product was different from SA and UR (the starting materials) and that it was soluble in methanol. The reaction between SA and UR was not surprising since multifunctional carboxylic acids have been reported to react with urea [31,32]. Based on this control experiment, it is unlikely that free SAUR would be present in CHSAUR after the methanol workup in view of its solubility. Thus CHSAUR samples were identified to be essentially CH physically crosslinked by SA through electrovalent bonds.

Fig. 3. Solid state NMR spectrum of CH and CHSAUR0.5 (1:0.5:0.5) to CHSAUR4 (1:4:4). 5

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3.2.4. Thermogravimetric analysis (TGA) The thermal decomposition patterns of all the CHSAUR samples were investigated by TGA. The TGA and DTG patterns are presented in Supporting Information Fig. S12. The initial weight loss for all the CHSAUR samples occurred in the temperature range between 170 and 240 °C and this might arise predominantly from the UR component (which was estimated to lose 87% of its mass in this temperature window as assessed from the TGA of pure UR) and SA (SA decomposes entirely in this window as assessed from the TGA of pure SA). The next significant weight loss occurs in the temperature range 280 and 410 °C and this arises predominantly from CH. The residue at 900 °C (in nitrogen atmosphere) for CHSAUR0.5 to CHSAUR4 were 25.62, 23.70, 24.89, 24.44, and 24.88%, respectively. Under these condition pure samples of CH, SA and UR leave a residue of 30.14%, 0.25% and 0% residue, respectively. This enabled the rough estimation of CH, SA and UR components in CHSAUR samples to be ~ 77 to 85% by weight of chitosan with the rest 23 to 15% by weight arising from SA and UR (as carbamate). Thus TGA suggests that CHSAUR samples could consist of all the three molecules CH, SA and UR. However, TGA is not as sensitive to structural features and hence the composition determined can at best be a rough estimate. The results from TGA of the products of control reactions are presented as Supporting Information in Fig. S10.

Table 1 Variation in [020] and [110] plane spacing in CHSAUR samples. Sample label (Composition of CH:SA:UR used in the preparation) Chitosan (control) CHSAUR0.5 (1:0.5:0.5) CHSAUR1 (1:1:1) CHSAUR2 (1:2:2) CHSAUR3 (1:3:3) CHSAUR4 (1:4:4)

[020]

[110]

2θ

d (Å)

2θ

d (Å)

9.41 8.97

9.39 9.87

19.79 20.04

4.48 4.43

8.87

10.0

20.09

4.42

7.87

11.22

20.05

4.42

7.65

11.54

20.26

4.38

7.57

11.66

20.28

4.37

3.2.3. X-ray diffraction (XRD) analysis The PXRD patterns for CHSAUR (prepared with different compositions of SA and UR) are shown in Supporting Information Fig. S11. The PXRD patterns of chitosan displayed strong diffraction peaks at 2θ = 9.41 [hkl value of (020), d = 9.39°A)] and 2θ =19.79 (wide at the base) [hkl value of (110), d = 4.48°A]. CHSAUR exhibited a composition dependent variation in the d-spacing of the [020] plane as shown in Table 1. The d-spacing along the [020] plane is observed to increase from 9.39 Å to 11.66 Å with increasing composition of SA and UR. The increase along the [020] plane might be due to the presence of SA along the “ab” plane, which in turn is probably due to the replacement of water of hydration as well as intermolecular H-bonds within CH molecules by those with SA [33]. The d-spacing in the case of [110] plane is marginally decreased and this may be due to non-reproducible setting of the sample before measurement. The PXRD pattern did not show any peak(s) characteristic of unreacted SA, UR and SAUR. It could be concluded from the PXRD studies that the semi-crystalline nature of chitosan is not affected to a significant extent as a consequence of hydrothermal reaction.

3.2.5. Analysis of macro- and micro- pores The as synthesized samples (wet) of CHSAUR were porous as evident to the naked eye (Fig. 2). To get some idea about the pore size and volume, all the dried samples were assessed for nitrogen adsorption at 77 K. The surface area of CHSAUR3 was 0.8134 m2/g while that of CHSAUR4 was 0.7578 m2/g (the samples were degassed overnight at 80C before BET analysis). Similar results were observed for other CHSAUR samples (surface area observed was between 0.7 and 0.8 m2/ g) and these results suggested that the samples did not carry adequate micropores to cause significant nitrogen adsorption. This result compares very poorly with that reported earlier for chitosan hydrogel prepared by neutralization of aqueous acid solution of chitosan by 3 M aqueous NaOH followed by evaporative drying or supercritical CO2

Fig. 4. Micro-CT X-ray images 2D, 3D and volume histogram of CHSAUR1 (1:1:1) (A, A1, A2) and CHSAUR4 (1:4:4) (B, B1, B2). 6

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Fig. 5. SEM image of dried CHSAUR4 (1:4:4).

drying, which results in surface area ranging from 5 to 142 m2/g with a pore volume of 33 cm3/g [34]. It is also one order of magnitude lower than that reported for hierarchical porous sponges prepared by freezedrying method where specific surface area in the range 2.36 to 5.98 m2/ g [35]. The analysis of the CHSAUR samples by x-ray micro-CT enabled the quantitative assessment of porosity (Fig. 4). The porosity of CHSAUR1 was 49.4% while that of CHSAUR4 was 64.2% indicating that the extent of porosity increased with increasing concentration of succinic acid and urea. The porosity of the samples prepared here is much less than that reported for high-strength pristine porous chitosan scaffolds prepared by freeze drying where porosity between 86.1 and 94.5% is reported [36]. However, given that the porosity arises due to the formation of ammonia from the decomposition of urea during the synthesis and given that this increases the viscosity of the mixture (due to increasing pH and decreasing solubility), the presence of very small pores was also expected, especially towards the end of the reaction. To assess the presence of micropores, the dry samples were also analyzed by SEM (an example is shown in Fig. 1). The SEM analysis (smaller sampling area) of one of the dried samples is presented in Fig. 5. This suggested the presence of pores of size 47 ± 22 nm. The SEM image of CHSAUR4, post-water absorption followed by freeze-drying, showed the presence of fibrous backbone (Fig. 6). The average size of pores in this sample was 34 ± 20 mm (length) and 21 ± 13 mm (width). Thus it can be concluded that CHSAUR consists largely of macropores with a very wide pore size distribution and a relatively smaller fraction of micropores. The formation of pores of such wide distribution is related to the rate of formation of ammonia in the polymer solution/gel whose viscosity changes as the pH of the reaction mixture changes from its initial value to 7.2 in the course of the reaction.

The absorption of double distilled water by CHSAUR samples, at room temperature, after the extraction of sodium hydroxide soluble component, was determined and the results are summarized in Fig. 8. While the details are presented in Table S2. The results indicate clearly that the extent of absorption decreases after the extraction of the sodium hydroxide soluble component from CHSAUR. However, the percent porosity increases (CHSAUR1 52.1% and CHSAUR4 76.3%) suggesting that the water absorption could be arising principally from the chitosan ammonium cation-succinate anion electrovalent bonds. The decrease in water absorption thus appears to be due to the removal of the physical crosslinks consisting of succinate-chitosan ammonium cation electrovalent crosslinks, which in turn were substituted by hydroxide anions. The results from the absorption of saline water by CHSAUR samples at room temperature are summarized in Fig. 7. The corresponding data (various concentrations of salt solutions 0.01%, 0.05% and 0.1%) are given in Figs. S13B, 13C, and 13D. At 0.1 wt% of NaCl the absorption was poor for all the CHSAUR samples. The samples did not disintegrate in 0.1 wt% solution suggesting that succinate ions were not displaced by chloride ions, which would have led to disappearance of the crosslinking leading to dissolution of chitosan. At this high concentration of the sodium chloride solution the contribution to water absorption can be expected to be low as pores are prone to collapse due to screening of the repulsive interactions between the positively charged backbone of chitosan. The relatively larger reduction in the absorption in 0.1 wt% aqueous sodium chloride solution compared to pure water suggests that absorption under high salt concentration might arise due to smaller pores and might reflect the extent of smaller pores present in the sample. The reduction in water absorption could also arise out of the stronger attraction of water to sodium cation than the chitosan polycation backbone and hence at this stage we do not have clear evidence for the possible causes proposed above.

3.2.6. Water absorption studies The results from the absorption of double distilled water by CHSAUR samples at room temperature are summarized in Fig. 7 while the details are presented, in Figs. S13A to S13D and Table S1. The results indicate that the extent of absorption increased with increasing extent of SA and UR weight ratio used in the preparation. The increase in water absorption could arise due to different factors such as increased porosity accompanied by the presence of higher density of polar surface functional groups. As discussed earlier, the formation of CHSAUR is accompanied by the release of ammonia gas whose volume increases with increasing weight ratio of UR. Thus the increase in water absorption with increasing weight ratio of SA and UR may arise out of increase in pore volume per unit mass as one of the factors. The maximum water absorption 760 ± 20 g/g was observed for the CH: SA: UR mole ratio 1:4:4 with 64.2% porosity in contrast to the 1:1:1 preparation with 49.4% porosity (as ascertained by X-ray micro-CT analysis).

3.2.7. Rheological properties of CHSAUR hydrogels The steady shear rheological measurements indicated that all the CHSAUR gels were “Herschel-Bulkley” in nature, whereby a non-linear pseudoplastic flow occurred along with a yield stress. Interestingly, the mechanical properties closely depended on the composition of the gel. The CHSAUR0.5 (1:0.5:0.5) gel gave the highest yield stress among all the compositions. This might be due to greater degree of crosslinking in the polymer network (the molar ratio of free amine to carboxylic acid for 1:0.5 ratio is 1:1.35). However, at higher ratios of SA and UR, the gel properties deteriorated. This could be because of a far greater concentration of the carboxylic acid (COOH) crosslinkers, relative to the reactive amine (NH2) groups on chitosan. As a result, the degree of crosslinking decreases, resulting in poorer mechanical properties. The shear stress (τ) vs. shear rate (dγ/dt) and viscosity ((η) vs. shear rate (dγ/dt) plots for the CHSAUR gels are presented in Fig. 9(A) and (B), 7

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Fig. 6. SEM images of freeze-dried CHSAUR4 (1:4:4) hydrogel at four different magnifications.

were stable at all frequency, when strained at 1%, which was in the linear regime. The strain and the frequency sweeps are presented in the Fig. 10(A–D). The effect of temperature on the steady shear rheological response was determined using CHSAUR4 as the model. At temperatures between 25 and 65 °C, this gel showed exceptional stability. This stability probably arises out of adequate crosslinking of chitosan chains, which prevents any gel to sol transition. The shear stress (τ), viscosity (η) vs. temperature plots are given in Fig. 11.

respectively. Stability of the gels under varying strain and frequency was determined using oscillatory shear rheological experiments. At low strains, the storage modulus (G') dominated the loss modulus (G") by almost an order of magnitude, portraying the viscoelastic nature of the gel. At shear strains between 15 and 25%, the gels lost their stability, evident by the crossover between the storage and the loss modulus. Among the gels studied, CHSAUR0.5 had the highest modulus and shear stability (23%, under a linear frequency of 10 Hz). Also, these gels

Fig. 7. Bar chart from equilibrium liquid uptake (g/g) data for CHSAUR4 (1:4:4) to CHSAUR0.5 (1:0.5:0.5) (left) and viscosity of the corresponding gel (~ 2 wt%) (right). 8

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groups in porous chitosan compared to the starting material. The lower value observed in the case of CHSAUR4 with respect to CH is due to the lower concentration of free amino groups that are otherwise bonded to succinic acid. 3.2.9. Cell viability assay CHSAUR4 was non-toxic to mouse fibroblast cells (3T3 L1) compared to that of positive control viewed for 48 h. In fact there was a significant increase in cell viability compared to control with viability reaching 125% and above at 48 h as inferred from the metabolic activity measured using MTT. Thus CHSAUR4 is biocompatible and nontoxic to control cells (fibroblast) based on cell proliferation, which is evident from the MTT assay summarized in Fig. 12. A novel and simple means of fabricating macroporous chitosan hydrogel scaffold by heating aqueous acidic solution of chitosan is introduced. In contrast the methods reported in literature enable the preparation of microporous chitosan but require controlled freezedrying that can take days [37,38]. The present method offers unique scope to prepare macroporous scaffolds of desired shape containing essentially chitosan while those prepared by reported methods such as freeze-drying, salt leaching and phase separation restrict the preparation to cylindrical scaffolds. The limitation of the present method, at this stage, is the inability to prepare microporous chitosan with higher surface area and porosity and it would be idea if nanoporous chitosan could be prepared through control of process parameters. However with the ability to prepare hard to soft foam (spongy) as well as soft hydrogel material and with the non-toxicity towards mouse fibroblast cells it offers scope for drug delivery [39] as well as in haemostatic applications.

Fig. 8. Normal water (double-distilled) uptake of CHSAUR0.5 (1:0.5:0.5) to CHSAUR4(1:4:4) samples after extraction with NaOH.

3.2.8. Preparation of porous chitosan from CHSAUR by the extraction of physically crosslinked succinic acid To test if the succinic acid component that is present as physical crosslinks in CHSAUR samples could be removed by extraction, they were treated with 0.1 N NaOH in 1:1 methanol: water mixture. The solid product obtained in this process was characterized by PXRD, FTIR and TGA (presented for one of the samples CHSAUR4 in Supporting Information Figs. S15 to S17). The PXRD of this product suggested that it was essentially CH while the FTIR of the solid showed the characteristic features of CH; especially the disappearance of the peaks due to the carboxylic acid around 1710 and 1400 cm−1 was evident. The TGA also suggested that SA could be displaced and extracted from the product although the initial decomposition temperature was lowered possibly due to the replacement of succinate anion by hydroxide anion. The CHSAUR4 samples, post-extraction with NaOH, was essentially porous chitosan (porosity by micro CT x-ray was 64.2% before and 76.3% after) with a small fraction of carbamate linkages (arising out the reaction between urea and the primary hydroxyl groups in chitosan as detailed earlier). The equilibrium adsorption of copper (+2) from an aqueous solution of CuSO4 (0.2 M, 35 °C for 24 h) by the porous chitosan gel prepared as described above was 65 ± 0.6 mg/g while that of chitosan and CHSAUR4 (not treated with NaOH) were 43 ± 0.9 mg/g and 22 ± 0.4 mg/g suggesting the greater accessibility to free amino

4. Conclusions Novel, macroporous hydrogels, with soft-spongy morphology, are synthesized by a simple and rapid hydrothermal reaction from sustainable materials, namely, chitosan, succinic acid and urea. This is in contrast to the present methods of preparing microporous chitosan through well-controlled freeze-drying process that takes several days. The structure of the hydrogel is established to consist of electrovalent crosslinks between chitosan (CH) and succinic acid (SA) that are removed upon extraction with 0.1 N NaOH in 1:1 methanol:water mixture offering macroporous chitosan framework. The as-prepared material (hydrogels) and dried material were macroporous with a wide variation in pore size. The maximum equilibrium absorption of 760 ± 20 g/g double distilled water was obtained for CHSAUR4 hydrogel and this also exhibited saline water uptake of 145 ± 7 g/g in 0.1 w % sodium

Fig. 9. (A) Shear Stress (τ) vs. Shear Rate (dγ/dt) and (B) Viscosity (η) vs. Shear Rate (dγ/dt) plot for the CHSAUR0.5 (1:0.5:0.5) to CHSAUR4 (1:4:4) gels. 9

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Fig. 10. (A) Modulus (G', G") vs. Shear Strain (ε) plots (strain sweeps); (B) Loss Factor vs. Shear Strain (ε) plots (strain sweeps); (C) Modulus (G', G") vs. Angular Frequency (ω) plots (frequency sweeps); and (D) Loss Factor vs. Angular Frequency (ω) plots (frequency sweeps) for the CHSAUR0.5 (1:0.5:0.5) to CHSAUR4 (1:4:4) gels.

Abbreviations CH SA UR CHSA

chitosan succinic acid urea product of control reaction between chitosan and succinic acid CHUR product of control reaction between chitosan and urea SAUR product of control reaction between succinic acid and urea CHSAUR product of hydrothermal reaction between chitosan, succinic acid and urea Acknowledgments One of the authors G. Prabha is grateful to the Science and Engineering Research Board (SERB) of the Department of Science & Technology (DST), Government of India, for providing funds under National Post-Doctoral Fellowship (PDF/2016/002403). The authors thank Prof. Ramesh Gardas of the Department of Chemistry for extending the rheology measurement facilities. Special thanks to Dr. Manohar Badiger and Mr. T. Arun of National Chemical Laboratory, Pune, India for the timely help in X-ray microscopy (micro-CT) imaging and analysis.

Fig. 11. Shear Stress (τ), Viscosity (η) vs. Temperature for the CHSAUR4 (1:4:4) gel.

chloride solution. The hydrogels exhibited “Herschel-Buckley” rheological behavior. The material was compatible for the growth of 3T3 L1 mouse fibroblast cells and could be useful in drug delivery as well as in haemostatic application.

Notes The authors declare no competing financial interest. 10

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Fig. 12. Cell viability of CHSAUR4 (1: 4: 4) with SD bars where **** P ≤ 0.0001 and ***P = 0.0002 denotes statistically same and statistically different mean, respectively.

Appendix A. Supplementary data

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Photographs of poly(propylene) bottle containing the mixture of starting materials (before reaction) and products obtained (after the preparations of CHSAUR); FT-IR spectrum of chitosan (CH), CHSAUR0.5 to CHSAUR4 and SAUR (control); Photographs of poly (propylene) bottles containing the reaction mixtures used in control reactions (before – above and after - below). CH – chitosan; SA – succinic acid; UR – urea; FT-IR spectrum of urea (UR) and succinic acid (SA); FT-IR spectrum of chitosan (CH), CHSA and CHUR (control reactions); FT-IR spectrum of SA, UR and SAUR (control reaction); MALDI-MASS analysis of SAUR; Variable temperature 1H NMR of SAUR; DSC of SAUR over two cycles of heating and cooling (bottom– first cycle heating and top – first cycle cooling); TGA of CH, CHSAUR3 (1:3:3), and products of control reactions (CHSA, CHUR, SAUR); PXRD pattern of chitosan and CHSAUR0.5 to CHSAUR4; TGA and DTG of CH, SA, UR, and CHSAUR0.5 to CHSAUR4; Water uptake versus time (A) and saline water uptake versus time for CHSAUR0.5 to CHSAUR4 samples (B, C, D); Equilibrium liquid absorption values for CHSAUR0.5 to CHSAUR4; Extent of liquid absorption by CHSAUR samples after extraction with NaOH; Uptake of saline water of different concentrations by CHSAUR after treatment with NaOH; PXRD pattern of CHSAUR4 and NaOH/MeOH treated CHSAUR4; FT-IR spectrum of CHSAUR4 and NaOH/MeOH treated CHSAUR4; TGA and DTGA of CHSAUR4 and NaOH/MeOH treated CHSAUR4. Supplementary data to this article can be found online at doi:https://doi.org/10.1016/j.msec. 2019.109845. References [1] R.A.A. Muzzarelli, Natural Chelating Polymers: Alginic Acid, Chitin, and Chitosan. International Series of Monographs in Analytical Chemistry, 1st ed, Pergamon Press, United Kingdom, 1973. [2] R.A.A. Muzzarelli, Chitin, 1st ed, Pergamon Press, United Kingdom, 1977. [3] C.K.S. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (2009) 641–678. [4] M.N.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1–27. [5] K.H. Prashanth, R.N. Tharanathan, Chitin/chitosan: modifications and their unlimited application potential – an overview, Trends Food Sci. Technol. 18 (2007) 117–131. [6] N. Yan, X. Chen, Don't waste seafood waste: turning cast-off shells into nitrogen-rich chemicals would benefit economies and the environment, Nature 524 (2015) 155–158. [7] A. Percot, C. Viton, A. Domard, Optimization of chitin extraction from shrimp shells, Biomacromolecules 4 (2003) 12–18. [8] R. Devi, R. Dhamodharan, Pretreatment in hot glycerol for facile and green separation of chitin from prawn shell waste, ACS Sustain. Chem. Eng. 6 (2018) 846–853. [9] A. Bhatnagar, M. Sillanpää, Applications of chitin-and chitosan-derivatives for the detoxification of water and wastewater—a short review, Adv. Colloid Interf. Sci.

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