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High Strength Chitosan Hydrogels with Biocompatibility via New Avenue Based on Constructing Nanofibrous Architecture Jiangjiang Duan, Xichao Liang, Yan Cao, Sen Wang, and Lina Zhang* College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: Breaking the limitation of traditional acid dissolving methods for chitosan by creating an alkali/urea hydrogen-bonded chitosan complex, a new solvent (4.5 wt % LiOH/7 wt % KOH/8 wt % urea aqueous solution) was used to successfully dissolve chitosan via the freezing−thawing process, for the first time. Subsequently, high strength hydrogels with unique nanofibrous architecture were constructed from the chitosan alkaline solution. The results from 13C NMR, laser light scattering, atomic force microscopy, transmission electron microscopy, and scanning electron microscopy confirmed that chitosan easily aggregated in the solution and could self-assemble in parallel to form perfect regenerated nanofibers induced by heating. At elevated temperature and concentration, the regenerated chitosan nanofibers could entangle and cross-link with each other through hydrogen bonds to form hydrogels. The novel chitosan hydrogels exhibited homogeneous architecture and high strength as a result of the strong networks woven with the compact nanofibers. The compression fracture stress of the chitosan hydrogels was nearly 100 times that of the chitosan hydrogels prepared by the traditional acid dissolving method, revealing that the nanofibrous network microstructures contributed greatly to the reinforcement of the hydrogels. Furthermore, the chitosan hydrogels exhibited excellent biocompatibility and safety as well as a smart controlled drug release behavior triggered by acid. Therefore, we opened up a completely new avenue to construct high strength chitosan hydrogels for applications in biomedicine.

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worthwhile endeavor would be to find a new strategy of constructing high strength chitosan hydrogel without depressing its desirable properties. In our laboratory, a class of novel solvents, alkali/urea aqueous solutions, for directly dissolving cellulose and chitin at low temperature have been developed, and cellulose and chitin hydrogels with excellent mechanical properties have been simply constructed from their solutions by heating or introducing poor solvents.21−26 Chitosan is consisted of β(1−4)-linked D-glucosamine and N-acetyl-D-glucosamine units, with the similar molecular structure as cellulose and chitin. Moreover, it has been confirmed all of the cellulose and/or chitin exist as an extended wormlike chain conformation in the solution, leading to easy parallel aggregation.27,28 Therefore, a bold idea of dissolving of chitosan in alkali/urea solutions at low temperature rather than in acidic systems emerged. In the present work, breaking away from the limitation of conventional acid dissolving methods of chitosan, a new solvent system (4.5 wt % LiOH/7 wt % KOH/8 wt % urea aqueous solution) was chosen to be used for the dissolution of chitosan via the freezing−thawing process, for the first time. Subsequently, robust chitosan physical hydrogels were

INTRODUCTION Hydrogels are composed of three-dimensional polymer networks that contain abundant water in the porous structures, and their soft and rubbery consistency and low interfacial tension with water or biological fluids are common to human tissues.1−3 Recently, the potential of polysaccharide-based hydrogels as biomaterials has been widely recognized due to their excellent biocompatibility, biological activity, safety, and biodegradability.4−6 Chitosan, the unique alkaline polysaccharide derived from the deacetylation of chitin, is readily soluble in dilute acidic solutions, and chitosan hydrogels can be regenerated by using alkaline coagulating bath. Thanks to their intrinsic biocompatibility, nontoxicity, biodegradability, strong affinity, antimicrobial activity, and low immunogenicity, chitosan hydrogels are considered to have potential applications in a wide variety of fields such as water treatment, food industry, catalysis, agriculture, and biomedicine.7−12 However, the poor mechanical properties, the “Achilles’ heel” of chitosan hydrogels, are serious impediments for their practical applications.13,14 To date, several methods such as chemical cross-linking, nanofillers reinforcement (e.g., nanoclay, silica nanoparticles, carbon nanotube, and graphene), and blending with other polymers have been used to enhance their mechanical strength. However, these techniques resulted in only a moderate enhancement and sometimes even partly sacrificed the intrinsic properties of chitosan.15−20 Therefore, a © XXXX American Chemical Society

Received: January 19, 2015 Revised: March 24, 2015

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Characterization. The morphological change of chitosan before and after being dissolved in the alkaline aqueous solvent system was observed by optical microscopy (EX20, Sunny, China). The chitosan solution dissolved in the alkaline solvent system was characterized by using liquid-state 13C NMR spectrometer on a Mercury 600 MHz NMR spectrometer (Varian, Inc.) at 0 °C. For comparison, the crosspolarization/magnetic angle spinning (CP/MAS) solid-state 13C NMR spectra of chitosan powders were recorded on a Varian Infinity Plus 400 spectrometer (13C frequency = 100.12 MHz) with a CP/MAS unit at ambient temperature. The stability measurement of the chitosan alkaline aqueous solution was carried out on an ARES-RFS III rheometer (TA Instruments, USA). A double-concentric cylinder geometry with a gap of 2 mm was used to measure the dynamic viscoelastic parameters such as the shear storage modulus (G′) and the loss modulus (G″) as functions of shearing rate (γ) or temperature (T). To understand the chitosan aggregation behavior, dynamic laser light scattering measurements were performed on a commercial light scattering spectrometer (ALV/SP-125, ALV, Germany) equipped with an ALV-5000/E multidigital time correlator and a He−Ne laser (at 632.8 nm). In order to further clarify the fibrous aggregates of chitosan in the solution, visualized nanofibers were observed by using atomic force microscopy (AFM) and transmission electron microscopy (TEM). AFM observations of the diluted chitosan alkaline solution were performed on a Picoscan AFM (Molecular Imaging, Tempe, AZ) in a MAC mode with commercial MAC lever II tips (Molecular Imaging, USA), with a spring constant of 0.95 N/m. TEM images were obtained by using a JEM-2010 (HT) transmission electron microscope (JEOL TEM, Japan). The structure and morphology of the chitosan hydrogel were characterized by scanning electron microscopy (SEM), UV−vis spectrometry, and wide-angle X-ray diffraction (XRD). The optical transmittance (Tr) of the chitosan hydrogels was observed with a UV−vis (UV-6, Shanghai Meipuda instrument Co., Ltd., Shanghai, China) spectrometer at a wavelength from 200 to 800 nm. SEM observations of the inner structure of chitosan hydrogel were made on a Hitachi S-4000 microscope. XRD measurement was carried out on an XRD diffractometer (D8-Advance, Bruker). The XRD patterns with Cu Kα radiation (λ = 0.154 06 nm) at 40 kV and 30 mA were recorded in the region of 2θ from 5° to 45°. The samples were ground into powder and dried in a vacuum oven at 60 °C for 48 h. The crystallinity index (CrI) of the chitosan samples was determined using the equation

constructed by regenerating the chitosan solution in hot water, which can destroy the alkali/urea hydrogen-bonded chitosan complexes, leading to the aggregation of the chitosan chain in parallel to form regenerated chitosan nanofibers. Additionally, chitosan chemical hydrogels cross-linked by epichlorohydrin (ECH) were also prepared, and their mechanical properties and pH-sensitive properties were researched. Furthermore, the drug release properties as well as the adhesion and spreading of C2C12 and Raw 264.7 on the chitosan hydrogels were carried out to evaluate their applications in the biomedicine field. This work would open up a completely new avenue for the construction of high strength chitosan materials.

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EXPERIMENTAL SECTION

Materials. Commercial grade chitosan (CS) from shrimp shell was purchased from Ruji Biotechnology Co., Ltd. (Shanghai, China). The degree of deacetylation (DD = 89%) of CS was determined by twoabrupt-change potentiometric titration method and calculated using the equation

α=

Δν × C NaOH × 10−3 × 16 × 100% m × 0.0994

(1)

where CNaOH and Δv stand for the concentration and volume of NaOH consumption between the two abrupt changes of pH, respectively, m is the dry weight of a chitosan sample, and α is the degree of deacetylation of the chitosan sample. Other chemical reagents from commercial sources in China were of analytical grade and used without further purifications. Preparation of the Chitosan Hydrogels. The aqueous solution containing LiOH/KOH/urea/H2O in the ratio of 4.5:7:8:80.5 by weight was used as an alkaline solvent of chitosan. To prepare the solutions, chitosan powders were dispersed into the alkaline aqueous solvent with stirring for 5 min and then were stored under refrigeration (−30 °C) until completely frozen. Then, the frozen solid was fully thawed and stirred extensively at room temperature. After removing air bubbles by centrifugation at 7000 rpm for 10 min at 5 °C, a transparent chitosan solution with the concentration of 4 wt % was obtained. The resultant chitosan alkaline solution was cast on a glass plate with a 1 mm thick layer and then immersed into a hot-water (T ⩾ 40 °C) coagulating bath for 30 min to transform into a chitosan physical hydrogel, coded as PGEL-B. After thoroughly washing with distilled water to remove any residuals, the PGEL-B was stored in deionized water at 5 °C for characterization. A chitosan chemical hydrogel was prepared by using epichlorhydrine (ECH) as cross-linker in the chitosan alkaline solvent system. In details, the desired volume of ECH was added into 100 g of the chitosan solution and stirred at low temperature (−20 °C) for 2 h to obtain a homogeneous pregel solution. After removing air bubbles by centrifugation at 7000 rpm for 5 min at 0 °C, the transparent chitosan pregel solution was poured into mold and kept at room temperature for overnight to form a “raw” chemical hydrogel with weak mechanical properties. Subsequently, the “raw” chitosan hydrogel was “cured” by immersion into distilled water for 3 days to remove the residual alkali and urea. Finally, a series of chitosan chemical hydrogels (CGEL-B) were fabricated, labeled as CGEL1-B, CGEL3-B, and CGEL5-B, corresponding to the volume of ECH of 1, 3, and 5 mL, respectively. As control, common chitosan hydrogels were prepared from acidic solvent system. A chitosan acidic solution with a concentration of 4 wt % was first prepared by dissolving the chitosan powder in 2 wt % acetic acid (HAc). A chitosan physical hydrogel (PGEL-A) was fabricated from the chitosan acidic solution by coagulating with 1 mol/L NaOH aqueous solution. In addition, a common chitosan chemical hydrogel (CGEL-A) was prepared by cross-linking a 100 g 4 wt % chitosan acidic solution with 5 mL of 2.5 wt % glutaraldehyde (GA) aqueous solution, and then neutralized by 1 mol/L NaOH aqueous solution. All the common chitosan hydrogel were thoroughly washed until all alkali residuals were removed.

CrI =

I110 − Iam × 100% I110

(2)

where I110 is the maximum intensity (2θ = 20°) of the (110) lattice diffraction and Iam is the intensity of amorphous diffraction at 2θ = 16° of chitosan. The mechanical properties of the chitosan hydrogels were characterized by tension and compression tests, which were performed on a universal testing machine (CMT6350, Shenzhen SANS, China) according to ISO527-3-1995 (E) at a speed of 1 mm/min. The size of the hydrogel specimens for tension and compression tests were 60 mm × 10 mm × 1 mm and 10 mm × 10 mm × 10 mm, respectively. C2C12 and Raw Cell Culture. Mouse myoblast cells (C2C12 cells) with common proliferation ability and murine macrophage cells (Raw 264.7 cells) with a strong proliferation ability were obtained from China Center for Typical Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco). The materials were sterilized by soaking with ethanol, UV irradiation. RAW 264.7 cells (50 × 104 cells/well), and C2C12 cells (10 × 104 cells/well) were seeded onto the material, which were put into 24-well plates or seeded into plates directly as control. After incubation for 24 h, the cells on material or plates were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and then observed by using a fluorescence microscope. pH-Sensitive Swelling Behaviors. The gravimetric method was employed to measure the swelling ratios of the hydrogels with different cross-linking density in the 2 wt % acetic acid aqueous solution or hydrochloric acid aqueous solution with different pH values (ionic B

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Macromolecules strength was controlled to be 0.1 M by adjusting the NaCl content). The swelling ratio (SR) was calculated as SR =

Ws − Wd × 100% Wd

(3)

where Ws is the weight of the swollen hydrogel and Wd is the weight of the dried gel. In Vitro Drug Loading and Release Studies. 30 mg of dried CGEL3-B hydrogen particles was incubated in a serious of 10 mL of tetracycline solution with different concentrations for 48 h. The drug entrapment efficiency and loading capacity of CGEL3-B were determined by subtracting the amount of the drug in the supernatant from the total amount added. Drug loaded hydrogel particles were incubated in 10 mL of hydrochloric acid aqueous medium with different pH values in a shaking water bath at 37 °C. The ionic strength was controlled to be 0.1 M by adjusting the NaCl content. At each time point, 2.5 mL of supernatant was withdrawn, and the same volume of the fresh medium was added. The release amount was obtained by accumulating the drug amount in each of release medium. The amount of tetracycline was assayed using a UV−vis spectrometer at 350 nm, and the actual values were calculated based on a calibration curve.

Figure 1. Liquid 13C NMR spectra of the chitosan solution in LiOH− KOH/urea/D2O (a) and acetic acid/D2O (b).

electron densities.21 However, the small chemical shift changes for C2, C3, C5, and C6 could be referred to that the intramolecular hydrogen bonds were either maintained or reformed because of their relatively immobilized architecture and position, which could sustain the chain stiffness of chitosan. The data of the chemical shifts in 13C NMR for the chitosan in alkaline solvent were close to that of cellulose in LiOH/urea system.32 On the basis of the above results, it can be inferred that the original intermolecular hydrogen bonds of chitosan were broken through formation of new hydrogen-bonded complexes between the macromolecules and the solvent, leading to the good dissolution. It is notable that the chemical shifts of all carbon of chitosan in acidic solution were clearly lower than that of chitosan in alkaline system, indicating that the higher electron density of carbons of chitosan appeared in acidic solution due to the high solvent−polymer adhesive force, namely strong solvation, leading to the highly efficient dissolution of chitosan in acidic solution. It was not hard to imagine that it was difficult for chitosan to maintain a stiff chain conformation in acidic solution due to the destruction of intramolecular hydrogen with relatively difficult recovery, resulting in semiflexible conformation in the acidic solution.33,34 To determine the stability of the chitosan solution in alkali/ urea aqueous system, the rheological properties and gelation behavior of the concentrated chitosan solution were investigated with dynamic viscoelastic measurements. Figure 2 shows the effects of the concentrations of the chitosan alkaline solution on the G′ and G″ values. The temperature at point of

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RESULTS AND DISCUSSION Dissolution and Stability of Chitosan in Alkali/Urea Aqueous Solution. Chitosan was hardly dissolved in the alkali/urea aqueous solvent before the freezing−thawing (F− T) process (Figure S1a). However, a transparent chitosan solution was obtained via the F−T process (Figure S1b). Optical microscopy images further revealed that a large number of the swollen chitosan powders existed in the alkali/urea aqueous solvent before the F−T process (Figure S1c), whereas transparent chitosan solution appeared after the F−T process (Figure S1d), indicating that low temperature was essential for the chitosan dissolution. It is worth nothing that the dissolution of chitosan was similar to that of cellulose and chitin in alkali/ urea aqueous solution, in which these polysaccharides are associated with solvent molecules through the formation of new hydrogen bonds complexes between the macromolecules and alkali/urea, which are relatively stable at low temperatures.27,28 It was not hard to image that the fact of the chitosan dissolution at low temperature was relative to the formation of new hydrogen bonding between chitosan and solvent, leading to the good dispersion of the chitosan macromolecules in the aqueous system. It has been reported that chitosan is easily dissolved in acidic aqueous solution due to strong electrostatic attractions between protonated chitosan chains and acid, speeding up the diffusion of solvent into chitosan and leading to the good dissolution of chitosan.29,30 Undoubtedly, the dissolution mechanism of chitosan in alkali/urea aqueous solution at low temperature is very different with the tradition dissolution method. NMR spectra can provide information about the molecular interaction and motions of polymers.31 To further clarify the dissolution of chitosan in alkaline solvent system, the 13C NMR experiments of the chitosan powder and chitosan in alkali/ urea/D2O and acetic acid/D2O were performed, respectively. The liquid 13C NMR spectra of the chitosan solution in LiOH− KOH/urea/D2O and acetic acid/D2O are shown in Figure 1, and their chemical shifts are summarized in Table S1. The chemical shifts of C1 and C4 in the chitosan alkaline solution moved to higher magnetic field than those of the solid state by 3.8−4.8 ppm, suggesting intermolecular hydrogen bonds of chitosan themselves were destroyed, leading to changes of the

Figure 2. Temperature dependence of the storage modulus G′ and loss modulus G″ for chitosan alkaline solutions with different concentrations. C

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Macromolecules intersection of G′ and G″ was the gelation temperature. Apparently, the gelation temperature of chitosan solution decreased from 49 to 37 °C with an increase of chitosan concentration from 2 to 4 wt %. This suggested that aggregation and entanglement among the chitosan chains significantly occurred in a relatively concentrated solution due to the progressively increased number of the hydrogen-bonded junction zones between the chitosan chains. Meanwhile, when the 4 wt % chitosan solution was stored at 5 °C for 5 days, it was stable without gel formation. Furthermore, the degree of deacetylation (DD) and zero shear viscosity of chitosan solution was also observed (Figure S2), indicating that during the storage process the chitosan solution was stable at relative low room temperature. This could be helpful for facilely constructing chitosan materials from its solution. Construction and Structure of the Chitosan Hydrogels. Novel chitosan physical hydrogel (PGEL-B) were facilely constructed from the chitosan alkaline aqueous solution by regenerating with hot water (T ≥ 40 °C). In our finding, the chitosan physical hydrogels regenerated at different temperature (from 40 to 80 °C) of hot water exhibited the similar structures and mechanical properties (Figure S7). In this work, the PGEL-B regenerated from 60 °C was chose as representative samples. The normal chitosan physical hydrogel (PGEL-A) prepared from chitosan acetic acid aqueous solution was used as control. Figure 3a,c shows the photographs of the

hydrodynamic radius distribution curves, corresponding to the isolated LiOH−KOH−urea hydrogen-bonded chitosan complexes and their aggregates. This indicated that the chitosan easily formed aggregates in the alkaline solution, even in dilute solutions, and giving only the apparent weight-average molecular weight value (Mw = 36.0 × 104). Figure 4a shows

Figure 4. A transparent dilute chitosan alkaline aqueous solution (1 × 10−3 g/mL) (a), TEM image of the dilute chitosan alkaline solution (b), AFM images of the extremely dilute chitosan solution (1 × 10−6 g/mL) (c), and chitosan aggregates (nanofibers) regenerated by hot water from the chitosan alkaline solution with a concentration of 1 × 10−4 g/mL (d).

a transparent dilute chitosan alkaline aqueous solution, indicating a good dissolution of chitosan in the alkali solvent system. The TEM images (Figure 4b) of the dilute chitosan alkaline solution displayed that the fibrous aggregates with the mean diameter of 12.4 nm were dispersed in the chitosan alkaline solution, further confirming that the chitosan chains easily aggregated in parallel in this case. It could be explained that once the alkali/urea hydrogen-bonded chitosan complex was destroyed, the chitosan chains aggregated in parallel (as maxima contact area) to form compact bundles. To provide direct evidence on the formation of the chitosan nanofibrous aggregates in the solution, AFM images of the diluted chitosan alkaline solution before and after aggregation induced by hot water were visualized, as shown in Figure 4c, d. In an extremely low concentration (1 × 10−6 g/mL), after drying on mica, the chitosan solution exhibited fibrous patterns (Figure 4c) as a result of easy aggregation of the chitosan chain complexes in the alkaline solution, which was consistent with the results from dynamic laser light scattering and TEM. Interestingly, with an increase of the chitosan concentration, the regenerated chitosan formed perfect nanofibers with compact structures at elevated temperature (Figure 4d). The mean diameter of the nanofibers aggregates calculated from AFM was 25 nm, which was consistent with that from SEM. On the contrary, unordered aggregates of chitosan appeared in the acid solution, as shown in the AFM and TEM images (Figure S5). These results from SEM, TEM, AFM, and laser light scattering supported strongly that the chitosan chains were selfassembled in parallel in the alkaline solution to form nanofibers by heat inducing and then rapidly aggregated to construct the physical hydrogels through entanglement and cross-linking, leading to the highly homogeneous architecture. In view of these results mentioned above, a schematic illustration for the formation of the hydrogels weaved from the chitosan nanofibers is proposed, as shown in Scheme 1. The extended chitosan chains and their aggregates coexisted in the dilute solution (Scheme 1a), supported by results in Figures 1, 4, and S4. The chitosan chains self-assembled in parallel to form regenerated nanofibers induced by heating (Scheme 1b), supported by results in Figures 3, 4, and S4. Subsequently, the PGEL-B hydrogels with nanofibrous architecture were constructed at an elevated temperature and concentration through physical entanglement and cross-linking junctions of

Figure 3. Pictures of PGEL-B (a) and PGEL-A (c). SEM images of cross-sectional structures of PGEL-B (b) and PGEL-A (d).

PGEL-B and PGEL-A hydrogels. Clearly, the PGEL-B exhibited better optical transmittance than the PGEL-A. Usually, optical transmittance is an auxiliary method to evaluate the homogeneity and porosity of materials.35 The light transmittance curves (Figure S3a) of the two kind of chitosan hydrogels in the range of visible light further revealed that the homogeneity of PGEL-B was better than PGEL-A. Furthermore, the optical scattering and refraction in hydrogels dramatically occurred about 700 nm for PGEL-A and 350 nm for PGEL-B (Figure S3b), revealing that a serious phase separation with larger size existed in PGEL-A. Figure 3b,d shows the SEM images of cross-sectional structures of PGEL-B and PGEL-A. As expected, the PGEL-B exhibited homogeneous and compact network architecture woven by nanofibers with a mean diameter of 24 nm, whereas an inhomogeneous and loose structure appeared in PGEL-A. To understand the formation mechanism of the chitosan nanofibers in the alkaline solution, the chitosan alkaline solution properties were studied by static and dynamic laser light scattering (Figure S4). There were two peaks in the D

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Scheme 1. Schematic Illustration of the Formation of Chitosan Hydrogel in Alkaline Aqueous Solution by Heating: Chitosan Dilute Solution (a), Chitosan Nanofibers (b), and Chitosan Hydrogel (c)

than that of CGEL-B (91%), reaching to 99%. The results indicated that CGEL-A had high swelling state and loose structures. In the XRD patterns of chitosan powders and chitosan hydrogel (Figure S6), three crystalline diffraction peaks were observed in the chitosan powder, indexed as (020), (110), and (130) lattice diffraction of chitosan.38 However, only two reflection peaks, without (130), were observed in all chitosan hydrogel samples, indicating that original chitosan chain packing was restructured during the regeneration process. As it is well-known, crystallinity index (CrI) reflects the regularity of the aggregation state of polymer chains. In this work, CrI was determined according to the method of Focher et al.,39 as listed in Table 1. It was noted that the CrI of the chitosan powder was lower than that of the chitosan hydrogel from alkaline solvent, strongly confirming that chitosan chains were restructured to form more regular aggregate architecture during the regeneration process, leading to a high CrI. Moreover, the CrI of PGEL-B was higher than that of PGEL-A, indicating that regular nanofibers of chitosan occurred in the alkaline solvent system, which further supported the model in Scheme 1. In addition, the CrI of CGEL-B was high and nearly equal to that of PGEL-B at a low cross-linking density, whereas it was significantly reduced with an increase of ECH as a result of chemical cross-linking partially restricting the regular arrangement of the chitosan chains. On the contrary, the CrI of CGELA was very low, suggesting that CGEL-A exhibited a disordered and loose state. Mechanical Properties of Hydrogels. As it is wellknown, the mechanical properties of hydrogels are very important for their applications. Figure 6 shows the typical tensile, compressive stress−strain curves and photographs of two kinds of chitosan hydrogels obtained from alkaline and acidic solvent systems, respectively. The detailed mechanical parameters are summarized in Table 1. As expected, the PGELA and CGEL-A were very weak, and their compressive fracture stress and strain were (0.05 MPa, 48.5%) and (0.06 MPa,

nanofibers (Scheme 1c), supported by results in Figures 3 and S7. Thus, a completely new avenue to construct chitosan hydrogels by the thermally induced self-assembly method in alkali/urea aqueous solution was opened up, drastically different from that of the traditional acid dissolving chitosan. Chemical Cross-Linked Chitosan Hydrogels in Alkali System. Sometimes, the chitosan hydrogel cross-linked by chemical bonds is necessary to improve its acidic resistance in a harsh environment. The etherification between chitosan and epichlorhydrine (ECH) occurred easily in the alkaline system.36,37 Hence, ECH was used to cross-link chitosan to prepare chemical hydrogel. As a control, a common chitosan chemical hydrogel was prepared by using glutaraldehyde (GA) as cross-linking agent in acidic solvent system because the reaction between ECH and chitosan hardly occurred in the acidic solvent system. Figure 5 shows the SEM images of cross

Figure 5. SEM images of cross-sectional structures of CGEL1-B (a), CGEL5-B (b), and CGEL-A (c).

sections of the chitosan chemical hydrogels from alkaline solvent system (CGEL-B) and from acidic solvent system (CGEL-A), respectively. As expected, the CGEL-B all exhibited homogeneous nanoporous network structures, and the pore size was reduced with the increase of the volume of ECH added in CGEL-B, as a result of the increase of cross-linking density. Moreover, the CGEL-B networks consisted of nanofibers with a mean diameter of 23 nm, which was consistent with that of PGEL-B. On the contrary, the CGEL-A exhibited a microporous structure, and its water content was significantly higher

Table 1. Physical and Mechanical Properties of Chitosan Hydrogel from Alkaline and Acidic Solvent Systems tensile properties

a

compressive properties

sample

water content (%)

CrIa (%)

σ (MPa)

ε (%)

E (MPa)

σ (MPa)

ε (%)

E (MPa)

PGEL-B PGEL-A CGEL1-B CGEL3-B CGEL5-B CGEL-A

94.5 94.6 94.4 91.0 92.0 98.9

32.88 27.37 32.11 23.44 22.52 11.73

0.21

83.9

0.60

0.37 0.35 0.37

92.2 107.8 117.3

0.44 0.24 0.20

3.31 0.05 3.31 4.25 4.83 0.06

85.9 48.5 86.3 82.8 77.7 12.2

0.60 0.10 0.34 0.34 0.43 0.42

The crystallinity index (CrI) of the chitosan samples was determined using eq 2. E

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Figure 6. Typical compressive and tensile stress−strain curves of chitosan chemical and physical hydrogels from two solvent systems (a, b). The pictures of PGEL-B (c) and PGEL-A (d) loaded with a 250 g weight. The pictures of CGEL3-B (e) and CGEL-A (f) compressed by a thumb. The picture of PGEL-B was stretched (g); CGEL3-B was twisted (h) and knotted (i).

12.2%), respectively. Interestingly, the compressive fracture stress and strain of PGEL-B and CGEL-B were unexpectedly high, reaching 3.3 MPa and 85.9% for PGEL-B and 4.8 MPa and 77.7% for CGEL5-B, respectively. Apparently, PGEL-B was robust and could support the weight without any deformation (Figure 6c). Moreover, the CGEL-B was tough enough without being broken (Figure 6e), whereas the PGEL-A was very soft and easily crushed by a weight (Figure 6d). It was noted that the CGEL-A was brittle and completely collapsed by the compression of a thumb (Figure 6f). Furthermore, the PGEL-B and CGEL-B hydrogels all exhibited excellent tensile properties, and their tensile fracture stress and strain were over 0.2 MPa and 80%, respectively (Figure 6b). It was worth noting that the PGEL-B and CGEL-B could be severely stretched, twisted, and knotted (Figure 6g−i). The mechanical properties of CGEL-B were better than that of PGEL-B, and their mechanical properties moved close to each other with a decrease of ECH added in CGEL-B. These results suggested that chemical and physical cross-linking networks weaved with nanofibers coexisted in the CGEL-B, greatly contributing to its better mechanical properties. Therefore, the novel chitosan physical and chemical hydrogels constructed from the alkaline solution system possessed extremely excellent mechanical properties, and their compressive fracture stress was nearly 100 times that of the chitosan hydrogels prepared by traditional acid dissolution method. In addition, compared with other high

strength hydrogels, the chitosan hydrogels from an alkali solvent system exhibited higher or similar level compressive mechanical properties (Table S2). This could be explained that the CGEL-B hydrogels composed of the compact regenerated nanofibers had homogeneous architecture and strong networks, which greatly contributed to the reinforcement of the hydrogels. In contrast, the CGEL-A hydrogels prepared by the traditional acid dissolving method were consisted of random aggregates, leading to the imperfect and loose architecture (see Figures 3, 5, and S5), resulting in the poor mechanical properties. Biocompatibility and pH Sensitivity of the Novel Chitosan Hydrogel. The in vitro cytotoxicity tests on the novel chitosan hydrogels (Figure S8) showed that the cell viability values on all gels were higher than 100%, indicating no cytotoxicity to the mouse myoblast cells (C2C12 cells). To further evaluate the biocompatibility of the chitosan hydrogels, C2C12 cells and Raw 264.7 cells were used to culture on the hydrogels. Figure 7 shows the fluorescent images of C2C12 cells stained with DiO and the optical microscopy images of Raw 264.7 cells without staining treatment, respectively, cultured on the surface of the culture plates, PGEL-B and CGEL3-B for 1 day. Clearly, the seeded C2C12 cells could adhere, spread, and proliferate well on PGEL-B and CGEL3-B. Their weaker clarity compared with the control group was as a result of a strong green fluorescent background because F

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about 150. It could be due to the greatly protonated −NH2 groups in the chitosan chain in low pH medium, leading to the expansion of the hydrogel networks as a result of the strong electrostatic repulsion. Therefore, CGEL-B exhibited high pHresponsiveness, which could rapidly swell in an acidic medium. As it is well-known, stomach is a unique organ in the human body with an acidic environment (pH range from 1 to 3).41 Thus, the CGEL-B hydrogel can be used for a stomach-specific drug delivery system. As shown in Figure 9a, the changes of entrapment efficiency and loading capacity of CGEL3-B for tetracycline depended on the drug concentration, and the maximum of entrapment efficiency was about 86% when the drug concentration was 1 g/L. Meanwhile, drug-loading capacity increased with the increase of drug concentration and reached the saturated capacity about 290 mg/g for a concentration of 2 g/L. The drug release kinetics curves in different pH mediums (Figure 9b) indicated that the drug was hardly released from the hydrogels at pH = 7, whereas the drug burst out at low pH mediums, displaying a smart controlled release swelling behavior and drug carrier triggered by an acidic medium. Therefore, CGEL3-B was a kind of smart hydrogel and would have great potential applications in the biomedical field.

Figure 7. Fluorescent images of a kind of mouse myoblast cells (C2C12 cells) stained with DiO cultured on the surface of culture plates (a), PGEL-B (b), and CGEL3-B (c) and optical microscopy images of Raw 264.7 cells without staining treatment cultured on the surface of culture plates (d), PGEL-B (e), and CGEL3-B (f) for 1 day. The scale bar is 50 μm.

chitosan with amino groups was easily stained with DiO. Moreover, Raw 264.7 cells also could adhere, spread, and proliferate on the surface of PGEL-B and CGEL3-B well. Compared with the control group, the Raw 264.7 cells on the chitosan hydrogels exhibited a similar cell density and morphology. These results were consistent with that from the C2C12 cells culture, further confirming that the either the chitosan physical or chemical hydrogels could be used as an excellent matrix having biocompatibilities, showing great potential applications in the field of tissue engineering. In all polyelectrolyte gels, pH-responsive swelling behavior is resulted from the competition between the Donnan osmotic pressure and the elasticity of the gel network.40 Interestingly, the CGEL-B hydrogels exhibited significant pH-responsive swelling behavior. Figure 8a shows the swelling kinetics of the CGEL-B in a 2% acetic acid aqueous solution at 37 °C and the photographs of CGEL3-B before and after swelling in 2% acetic acid aqueous solution. Obviously, the circle-shaped CGEL3-B dyed with methyl orange was significantly swollen at low pH, and its diameter was increased to 4 times that of its initial size. Moreover, all of the CGEL-B tended to swell, once transferred into acetic acid aqueous solution, and reached the swelling equilibrium after 6 h. Figure 8b shows the effects of pH on the swelling behaviors of the chitosan hydrogels. When pH < 4, the CGEL5-B exhibited significant swelling behavior with ESR of

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CONCLUSION A new solvent system composed of 4.5 wt % LiOH/7 wt % KOH/8 wt % urea aqueous solution with cooling was developed successfully to dissolve chitosan. Furthermore, high strength chitosan hydrogels with unique structure were constructed from the chitosan alkaline solution, for the first time. The chitosan in the alkaline solution easily self-assembled to form compact regenerated nanofibers induced by heating, which then facilely entangled and cross-linked with each other through hydrogen bonds to form the hydrogels at elevated temperature and concentration. The novel chitosan hydrogels had homogeneous architecture and excellent mechanical properties, and their compression fracture stress was nearly 100 times that of the chitosan hydrogels prepared by traditional acid dissolving method. This was as a result of the strong networks woven with the perfect chitin nanofibers, which greatly contributed to the reinforcement of the hydrogels. Moreover, the novel chitosan hydrogels exhibited excellent biocompatibility, pH sensitivity, and smart controlled drug release behavior. Therefore, this work would be very important

Figure 8. Swelling kinetics of the CGEL-B in 2% acetic acid aqueous solution at 37 °C and photographs of CGEL3-B before and after swelling in 2% acetic acid aqueous solution (a). The equilibrium swelling ratio of CGEL3-B in hydrochloric acid aqueous solution with different pH values (b), and the ionic strength of various hydrochloric acid aqueous solution with different pH values was controlled to be 0.1 M by adjusting NaCl content. G

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to construct smart chitosan hydrogels with high strength and biocompatibilities for the potential applications in the fields of tissue engineering and drug controlled release.

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ASSOCIATED CONTENT

S Supporting Information *

Photographs of chitosan powder dispersed in alkali/urea aqueous solution before and after freezing−thawing process; detailed chemical shifts of carbons of chitosan; dependence of zero-shear viscosity and DD of chitosan dissolved in alkaline solution on its storage time; light transmittance curves and their differential curves of the two kinds of chitosan hydrogels in the range of visible light of light transmittance; hydrodynamic radius distributions and Zimm plots of chitosan alkaline solution at 10 °C; AFM image and TEM image of chitosan aggregation regenerated by sodium hydroxide from dilute chitosan acidic solution with a concentration of 1 × 10−4 g/mL; SEM images and compressive stress−strain curves of chitosan hydrogels regenerated from hot water with different temperature; detailed data of different high strength hydrogels; in vitro cytotoxicity tests on different chitosan hydrogel samples; XRD patterns of chitosan powder, chitosan physical and chemical hydrogel constructed form the two solvent systems. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(L.Z.) E-mail [email protected]; Ph +86-27-87219274. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, 2010CB732203), the Major Program of National Natural Science Foundation of China (21334005), and the National Natural Science Foundation of China (20874079, 81171480). We gratefully acknowledge Prof. Chi Wu and Prof. Fan Jin in University of Science & Technology of China for their kind favors in light scattering.

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DOI: 10.1021/acs.macromol.5b00117 Macromolecules XXXX, XXX, XXX−XXX