Fabrication of nanostructured and microstructured chitin materials through gelation with suitable dispersion media

Abstract

Recent developments in the fabrication of nano- and microstructured chitin materials are reviewed, specifically focusing on approaches through gelation with suitable dispersion media. Although chitin is one of the most abundant natural polysaccharides, it is under-used as a result of its poor solubility and difficulties in processing. The dissolution of chitin in different solvent systems, including ionic liquids, has been investigated for the production of various materials. For example, the ionic liquid 1-allyl-3-methylimidazolium bromide dissolved chitin at concentrations up to 5% w/w and formed ion gels at higher concentrations of chitin. A highly concentrated solution of CaBr₂·2H₂O/methanol also induced the gelation of chitin. As one of the most efficient methods of production of nanomaterials from chitin, self-assembled nanofibers have been fabricated by regeneration from solutions or gels of chitin with the appropriate solvents and dispersion media using a bottom-up approach. For example, a chitin ion gel with 1-allyl-3-methylimidazolium bromide was regenerated using methanol to produce a chitin nanofiber dispersion, which was then used to construct a film with a highly entangled nanofiber morphology by filtration. Physical and chemical approaches have been investigated for the fabrication of composite materials of self-assembled chitin nanofibers with other polymeric components. Poly(vinyl alcohol) and carboxymethyl cellulose were made compatible with chitin nanofibers by co-regeneration and electrostatic interaction procedures, respectively. Surface-initiated graft polymerization of some monomers from chitin nanofiber films with the appropriate initiating groups have been conducted using the latter approach to obtain composite films covalently linked to graft chains on the nanofibers. Regeneration from gels with CaBr₂·2H₂O/methanol resulted in the efficient production of microporous materials.

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Jun-ichi Kadokawa

Jun-ichi Kadokawa studied applied and materials chemistries at Tohoku University, where he received his PhD in 1992. He then joined Yamagata University as a Research Associate. From 1996 to 1997 he worked as a visiting scientist at the Max-Planck-Institute for Polymer Research in Germany. In 1999 he became an Associate Professor at Yamagata University and moved to Tohoku University in 2002. He was appointed as a Professor of Kagoshima University in 2004. His research interests focus on nanostructured polysaccharide materials. He received the Award for Encouragement of Research in Polymer Science (1997) and the Cellulose Society of Japan Award (2009).

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Introduction

Natural polysaccharides are widely distributed in nature and are vital materials for in vivo functions.¹ Of the many kinds of polysaccharides, cellulose and chitin are the most well-known and abundant organic substances on Earth. Cellulose is used as a structural material in the cell walls of plants and chitin is used in the exoskeletons of crustaceans, shellfish and insects.^2–8 Cellulose and chitin have similar structures: cellulose is composed of β-(1 → 4)-linked D-glucose residues and chitin is an aminopolysaccharide consisting of N-acetyl-D-glucosamine units linked through β-(1 → 4)-glycosidic linkages (Fig. 1). Cellulose is used in a number of traditional applications, including furniture, clothing and medical products. Chitin remains an under-utilized biomass resource despite its high abundance in nature and easy accessibility. The difficulty in the practical applications of chitin is mainly a result of its bulk structure and its insolubility in water and common organic solvents. In addition, the production of chitin from natural resources involves complicated and harmful procedures, including treatment with strong acids and bases. This is because the native chitin from crustaceans has a fibrous structure of microfibrils with proteins in chitin–protein complexes; these assemble with minerals to construct exoskeletons of crustaceans.^9–11 The isolation of chitin requires the removal of the mineral and protein constituents from the raw materials by treatment with acids and bases.

Fig. 1 Chemical structures of cellulose and chitin and schematic diagram of the crystalline structures of different forms of chitin.

As there is much interest in the conversion of chitin into various materials with controlled nano- and microstructures after its isolation from natural resources and the disentanglement of these fibrils, considerable effort has been devoted to finding solvent systems and dispersal media to dissolve chitin and form gels.^7,12 Ionic liquids, which are low melting point salts that form liquids at temperatures below the boiling point of water, are regarded as good solvents for polysaccharides such as cellulose.^13–20 For example, Swatloski et al.²¹ reported that the ionic liquid 1-butyl-3-methylimidazolium chloride (BMIMCl) dissolved cellulose at relatively high concentrations. Only limited investigations have been reported on the dissolution of chitin with ionic liquids,^22–26 although these are attracting much attention in the fabrication of new chitin-based functional materials.

The fabrication of nanoscaled polymeric assemblies, e.g. nanofibers and nanowhiskers, is an important method for the practical utilization of polymeric compounds such as cellulose as material components.^27,28 For example, cellulose nanowhiskers have been used as reinforcing fillers in natural polymeric matrices.²⁹ Efficient approaches have also been developed for the fabrication of chitin nanofibers and nanowhiskers (nanocrystals).^7,12 Conventional approaches to the production of chitin nanofibers mainly use top-down procedures that break down the starting bulk material from native chitin resources.^30,31 This is because native chitin microfibrils consist of nanofibers 2–5 nm in width formed by crystalline structures among the chitin chains resulting from numerous hydrogen bonds between hydroxy and acetamido groups (Fig. 2 and 3).^32,33 Three crystalline forms of chitin are known based on the arrangement of polymeric chains: α-, β-, and γ-chitin (Fig. 1). The most abundant form is α-chitin (e.g. crab and shrimp shells), in which the polymeric chains are aligned in an antiparallel fashion.³⁴ This arrangement is favorable for the formation of strong intermolecular hydrogen bonds and is the most stable of these three crystalline structures. In β-chitin (e.g. squid pens), the polymeric chains are packed in a parallel arrangement, resulting in weaker intermolecular forces³⁵ and β-chitin is considered to be less stable than α-chitin. The γ-chitin structure may be a mixture of the α- and β-forms.

Fig. 2 Hierarchically ordered assemblies of native chitin.

Fig. 3 Top-down and bottom-up approaches for the fabrication of chitin nanofibers.

The formation of nanoscaled chitin assemblies, in a representative top-down approach for the fabrication of nanostructured chitin materials, has been achieved by acid treatment of a native chitin resource.^36–39 Microfiller fragments of crab and shrimp chitins were prepared by hydrolysis in an aqueous solution of HCl (3 mol L⁻¹) at its boiling point. After removal of the acid by centrifugation, washing and dialysis, ultrasonication converted the residual product into a colloidal suspension of crystallites (chitin nanowhiskers). In another top-down approach, the radical-mediated oxidation of chitin with 2,2,6,6-tetramethylpiperidine-1-oxyl in water at pH 10 under specific conditions and subsequent ultrasonication was performed to fabricate chitin nanowhiskers and nanofibers dispersed in water.^40,41 A simple grinding technique has also been developed for the preparation of chitin nanofibers from chitin microfibrils.^42–45 I recently reported the facile nanofibrillation of a native chitin powder by bubbling with N₂ gas under conventional ultrasonication in water.⁴⁶

Another method for the fabrication of chitin nanostructures is based on a self-assembling generative (bottom-up) route, in which fibrillar nanostructures are produced by regeneration from chitin solutions or gels via processes such as electrospinning^47,48 or simple precipitation (Fig. 3).^49–53 It has also been reported that self-assembled chitin nanofibers can be fabricated by facile regeneration from an ionic gel of chitin with an ion liquid using methanol followed by ultrasonication.^12,26,31

Porous materials with controlled porosity on the nano- and microscales have been used practically in applications such as adsorption and extraction.⁵⁴ These porous structures are generally constructed by reconstitution methods, such as lyophilization after the dissolution or gelation of the polymeric components. Chitin microporous materials have also been prepared by regeneration from chitin solutions or gels with dispersion media.^55,56

This paper reviews recent development in the fabrication of nano- and microstructured chitin materials based on the regenerative bottom-up approach, specifically focusing on approaches using gelation with suitable dispersion media. Various solvent systems and dispersion media for chitin, including ionic liquids, are briefly reviewed, followed by a discussion of the fabrication of self-assembled chitin nanofibers by the regeneration technique, mainly from a chitin ion gel with an ionic liquid. Nanostructured chitin has been combined with other polymeric components to produce chitin nanofiber composite materials. The facile production of chitin microporous materials by regeneration from gels with dispersion media is reviewed.

Dissolution and gelation of chitin

Chitin shows a limited affinity toward common solvents as a result of strong intermolecular hydrogen bonding and is not dissolved by conventional organic solvents and water. The most widely used solvent systems for chitin are 5–7% LiCl/N,N-dimethyladcetamide (DMAc)⁵⁷ and CaCl₂·2H₂O-saturated methanol.^58–61 Because LiCl forms a complex with chitin that is soluble in DMAc, 5–7% LiCl/DMAc dissolves chitin. Tamura et al. reported that methanol saturated with CaCl₂·2H₂O dissolved a few percentage concentrations of chitin at reflux temperatures,^58–61 although anhydrous CaCl₂ was not suitable for dissolution. It has also been reported that 0.9–3.5% w/v mixtures of chitin with highly concentrated CaBr₂·2H₂O/methanol (3.85 mol L⁻¹) formed gels on stirring at room temperature, whereas the chloride analogue of this solution system did not induce gelation with chitin at room temperature.⁶²

The dissolution of chitin using highly polar fluorinated solvents such as hexafluoroisopropyl alcohol (HFIP) and hexafluoroacetone sesquihydrate has also been reported.^63,64 Strong protic acids, such as trichloroacetic acid and dichloroacetic acid, have been reported to dissolve chitin^65–67 and methanesulfonic acid formed a colloidal chitin.⁶⁸ Cold aqueous NaOH (16% w/w) also dissolves chitin^69,70 and the alkaline chitin produced had a lower critical solution temperature.⁷¹

Some ionic liquids have been found to dissolve chitin (Fig. 4).^22–26 Over the past decade, ionic liquids have attracted much attention as a result of their excellent thermal stability, negligible vapor pressure and controllable physical and chemical properties.^72,73 Research on ionic liquids has been extended to biological macromolecules such as polysaccharides as a result of their good affinities with these molecules.¹³ The dissolution behavior of chitin in a series of alkylimidazolium chloride, dimethyl phosphate and 1-allyl-3-methylimidazolium acetate ionic liquids has been investigated.²² The former two series of ionic liquids did not dissolve chitin very well (<1.5 wt%), but 1-allyl-3-methylimidazolium acetate dissolved 5 wt% chitin. The dissolution behavior was affected by the degree of deacetylation, the degree of crystallinity and the molecular weight. 1-Butyl- and 1-ethyl-3-methylimidazolium acetate (BMIMOAc and EMIMOAc, respectively) have also been found to dissolve chitin at certain concentrations.^74,75 BMIMOAc dissolved both α- and β-chitins with different molecular weights at relatively lower temperatures. Cooling the chitin/BMIMOAc solutions to ambient temperature resulted in the formation of the corresponding chitin/BMIMOAc gels, which further formed chitin sponge and film materials by regeneration with water or methanol coagulant. The extraction of chitin from raw crustacean shells, such as shrimp shells, was demonstrated using EMIMOAc.

Fig. 4 Representative ionic liquids that dissolve chitin.

The dissolution of chitin with ionic liquids has also been studied on the basis of previous studies that considered the synthesis of polyamides and polyimides using ionic liquids with the imidazolium bromide structure as the reaction medium.⁷⁶ This previous investigation inspired the use of the same kind of ionic liquids for the dissolution of chitin, which demonstrates the same solubility problem as polyamides and polyimides with the formation of strong hydrogen bonds by the –N–C=O groups of the acetamido groups. Three imidazolium-type ionic liquids with a bromide counter anion were prepared for the dissolution study: 1-allyl-3methylimidazolium bromide (AMIMBr), 1-methyl-3-propylimidazolium bromide (MPIMBr) and 1-butyl-3-methylimidazolium bromide (BMIMBr).

For the dissolution study, a mixture of chitin with each ionic liquid was heated at 100 °C and the dissolution process was simply followed by observation using a charge-coupled device (CCD) camera with 200× magnification (Fig. 5a). When AMIMBr was used, a clear solution of chitin was obtained at concentrations up to about 5% w/w (Fig. 5b), whereas chitin powders remained after heating chitin with MPIMBr or BMIMBr, even at 3% w/w concentrations (Fig. 5c and d).⁷⁷ These results indicated that, of the three ionic liquids with a bromide counter anion, only AMIMBr dissolved chitin. The dissolution of chitin with AMIMBr was further confirmed by SEM measurements. The SEM image of the mixture of chitin with AMIMBr (5% w/w) after heating at 100 °C for 48 h did not show any solids, unlike that observed in the SEM image before heating (Fig. 6a and b), suggesting that 5% w/w chitin was dissolved by AMIMBr.

Fig. 5 (a) Procedure for the dissolution of chitin with ionic liquids. Charge-coupled device (CCD) camera view of dissolution experiment mixtures after heating at 100 °C for 48 h with (b) 5% w/w AMIMBr, (c) 3% w/w MPIMBr and (d) 3% w/w BMIMBr.

Fig. 6 SEM images of mixtures of chitin with 5% w/w AMIMBr (a) before and (b) after dissolution experiment and (c) photographs of 5% w/w liquid and (d) 7% w/w gel chitin with AMIMBr after heating at 100 °C for 48 h.

Gel-like materials (ion gels) with a higher viscosity were formed when 7–12% w/w chitin was immersed in AMIMBr followed by heating at 100 °C for 24 h and cooling to room temperature. The resulting 7% w/w chitin with AMIMBr did not flow when the test-tube was leaned over, whereas the 5% w/w solution of chitin with AMIMBr started to flow with leaning (Fig. 6c and d). Although dynamic rheological measurements showed that both 5 and 7% w/w chitin with AMIMBr behaved as weak gels, the much lower yield stress of 5% w/w chitin with AMIMBr compared with the 7% w/w system supported the result that the former flowed under gravity.

Chitin/cellulose composite ion gels using two ionic liquids (AMIMBr and BMIMCl) were prepared (Fig. 7)^78–80 based on a previous report of the formation of an ion gel of cellulose with BMIMCl.⁸¹ Chitin (5% w/w) and cellulose (10% w/w) were dissolved in AMIMBr and BMIMCl, respectively. The two solutions were then mixed in the desired ratios at 100 °C to give homogeneous mixtures. Gels were obtained by standing the mixtures at room temperature for 4 days. The resulting ion gels were characterized by powder X-ray diffraction (XRD) and thermal gravimetrical analysis (TGA), which showed relatively good miscibility among the polysaccharides and ionic liquids in the materials. The mechanical properties of the gels varied depending on the ratios of chitin to cellulose in the materials. The composite ion gels were further converted into chitin/cellulose composite films as follows (Fig. 7).^78,82 The prepared chitin/cellulose homogeneous mixtures with AMIMBr/BMIMCl were cast on a glass plate and left to stand at room temperature for 2 h; the gel-like materials obtained were then subjected to successive Soxhlet extractions with ethanol for 12 h and with water for 12 h before drying to give the composite films.

Fig. 7 Preparation procedures of chitin/cellulose composite gel and film.

Chitin/cellulose composite ion gels have been used as novel electrolytes in electric double-layer capacitors.^83–85 The ion gel was first treated with an aqueous solution of 2.0 mol L⁻¹ H₂SO₄ for 3 h until conversion into an acidic gel. The electrochemical characteristics of the acidic chitin/cellulose composite gel electrolyte were investigated by galvanostatic charge–discharge measurements. The test cell using the composite gel electrolyte had a specific capacitance of 162 F g⁻¹ at room temperature, which was higher than that of a cell with an H₂SO₄ electrolyte (155 F g⁻¹). The discharge capacitance of the test cell retained >80% of its initial value over 10⁵ cycles, even at a high current density of 5000 mA g⁻¹. These results indicated that the acidic chitin/cellulose composite gel electrolyte could be applied to a practical advanced electric double-layer capacitor with excellent stability and working performance.

Fabrication of self-assembled chitin nanofibers by regeneration from ion gels

Self-assembled chitin nanofibers were fabricated from solutions of chitin such as HFIP and LiCl/DMAc.^49–52 The self-assembly process was initiated through either solvent evaporation in the HFIP system or the addition of water in the LiCl/DMAc system. The self-assembly of chitin was achieved simply by slowly drying the appropriate concentrations of HFIP solutions to give long (10–100 μm) nanofibers with a small diameter (ca. 2.8 nm). A simple drying approach was not suitable for the low volatility LiCl/DMAc solutions, from which fibers were easily precipitated out with the addition of water. Nanofibers fabricated in this way had a larger diameter (ca. 10 nm) than those obtained from the HFIP system, whereas the lengths of the nanofibers from both systems were comparable. The nanofibers had α-chitin crystalline structures.

An electrospinning method was used to fabricate self-assembled nanofibers.^47,48 Chitin was first depolymerized by γ-irradiation to improve its solubility. Electrospinning of the resulting chitin was then carried out with HFIP as the spinning solvent. SEM images of the nanofibers were obtained from the electrospun samples. At concentrations of HFIP solutions <3 wt%, large irregular beads or beaded fibers were generated by electrospinning. Continuous nanofibers were fabricated at concentrations >4 wt%, indicating that extensive chain entanglements were necessary to produce continuous fibers of chitin by this approach. At a concentration of 6 wt%, however, the continuous and uniform process of electrospinning was inhibited because the solution had a very high viscosity. The resulting fibrous structure contained small, irregular beads. Although the as-spun nanofibers had a broad distribution of diameters, most of the fibers had diameters <100 nm.

The ionic liquid AMIMBr was also used as the medium for the fabrication of self-assembled chitin nanofibers by the regeneration approach (Fig. 8a).⁸⁶ A commercially available chitin powder from crab shells was first swollen with AMIMBr according to the previously reported procedure to give chitin ion gels (10–12% w/w).⁷⁷ Dispersions were obtained when the gels were soaked in methanol at room temperature for 24 h to slowly regenerate chitin, followed by sonication. The resulting dispersions were further diluted with methanol for SEM measurements to evaluate the morphology of the regenerated chitin. The SEM images of samples from the dispersion showed nanofibers about 20–60 nm in width and several hundred nanometers in length (Fig. 8a), indicating the self-assembly of chitin nanofibers by regeneration from the ion gel. When the dispersion was filtered to isolate the nanofibers, the residue formed a film, which was further purified by Soxhlet extraction with methanol. The SEM image of the resulting film showed a pattern of highly entangled nanofibers (Fig. 8a). Such an entangled structure probably contributed to the formation of the film. The XRD pattern of the nanofiber film showed four diffraction peaks at around 9.5, 19.5, 20.9 and 23.4°, which corresponded to the crystalline structure of α-chitin⁸⁷ and was identical with the pattern of an original chitin powder from crab shells. This indicated that the α-chitin crystalline structure has been reconstructed during the formation of the nanofibers by the regeneration procedure.

Fig. 8 Preparation procedures of (a) chitin nanofiber dispersion and film and (b) chitin nanofiber/PVA composite film and their SEM images.

I have reported previously that the morphologies of self-assembled chitin nanofibers were affected when regenerated from chitin ion gels using Ca halide·2H₂O/methanol solutions.⁸⁸ Regeneration from ion gels using CaCl₂·2H₂O or CaBr₂·2H₂O/methanol solutions at high concentrations did not induce the assembly of nanofibers. Compared with regeneration using methanol alone, nanofibers with a higher aspect ratio were produced by regeneration with a CaBr₂·2H₂O/methanol solution at lower concentrations. The mechanical properties of the film formed by the filtration of the resulting dispersion was enhanced compared with those of chitin nanofiber films obtained using other methanolic solutions.

Preparation of composite materials from self-assembled chitin nanofibers

Attempts have been made to fabricate composite materials of self-assembled chitin nanofibers with other polymers by both physical and chemical approaches (Fig. 9).¹² In the physical approaches, the nanofibers and polymer components are made compatible by appropriate physical interactions, whereas the latter approach results in the formation of covalent linkages between the nanofibers and polymers.

Fig. 9 Physical and chemical approaches for the preparation of composite materials of chitin with another polymeric component.

Self-assembled chitin nanofiber/poly(vinyl alcohol) (PVA) composite films were fabricated by the physical approach (Fig. 8b).⁸⁶ A 10% w/w chitin ion gel with AMIMBr was prepared and a solution of PVA (DP = ca. 4300) with a small amount of hot water was added to the gel (feed weight ratio chitin to PVA 1 : 0.3). Because methanol is a poor solvent for PVA, the two polymers were co-regenerated by soaking the mixture in methanol, followed by filtration and Soxhlet extraction with methanol to produce a self-assembled chitin nanofiber/PVA composite film. The SEM image of the composite film showed the retained nanofiber morphology (Fig. 8b). This result suggested that the two polymer components were relatively immiscible in the composite and the PVA components probably filled in the spaces among the fibers. The DSC profile of the film showed an endothermic peak that could be assigned to the melting point of PVA, indicating the formation of a crystalline structure separate from the chitin nanofibers as a result of this immiscibility. However, the peak melting point of PVA in the DSC curve of the film was broadened as a result of the decreasing crystallinity. These data indicate that chitin and PVA might be partially miscible at the interfacial area of the fibers by hydrogen bonding between the two polymer components. Both the tensile strength and the elongation at break under tension of the composite film were larger than those of the chitin nanofiber film. This supported the suggestion that the presence of the PVA components in the composite film contributed to an enhancement of the mechanical properties.

Because chitin can be considered as a basic polysaccharide because of the presence of several per cent of free amino groups in the repeating units as a result of the deacetylation of acetamido groups, the self-assembled chitin nanofibers were used as reinforcing agents in an acidic polysaccharide [carboxymethyl cellulose (CMC)] by electrostatic interaction.⁸⁹ CMC is one of the most widely applied cellulose derivatives in the detergent, food, paper and textile industries and can be easily processed, e.g. for the formation of films.⁹⁰ To induce their compatibility, the CMC films, prepared by a casting technique, were soaked in different concentrations of the self-assembled chitin nanofiber dispersions obtained by regeneration from ion gels (Fig. 10). After centrifugation, the resulting composite films were washed with methanol and dried. The amount of chitin nanofibers in the films increased with increasing amounts of the nanofibers in the dispersions. The SEM images of the films showed the nanofiber morphology of the films (Fig. 10). The reinforcing effect of the nanofibers on the films was confirmed by tensile testing and the amount of the nanofibers strongly affected the enhancement in the mechanical properties.

Fig. 10 Preparation procedure for chitin nanofiber/CMC composite film and its SEM image.

Chitin-based eco-friendly composite materials have previously been prepared with aliphatic biodegradable and biocompatible polyesters,^91–93 such as poly(L-lactide) and poly(ε-caprolactone), by their physical compatibility.^94–97 Because such polyesters are easily synthesized by ring-opening polymerization of the corresponding cyclic monomers [L-lactide (LA) and ε-caprolactone (CL)] using alcohols as initiators in the presence of Lewis acid catalysts,^98,99 chemical grafting of the biodegradable polyesters by covalent linkages has also been performed by a ring-opening polymerization approach using chitin as a multifunctional initiator with a number of hydroxy (alcohol) groups.^100–103

To obtain chitin nanofiber/biodegradable composite materials by a chemical approach, the surface-initiated ring-opening graft copolymerization of LA/CL initiated from hydroxy groups on self-assembled chitin nanofiber films has been investigated (Fig. 11).¹⁰⁴ To efficiently initiate copolymerization on the surface of the nanofibers, spaces were made among the nanofibers by aqueous treatment of the chitin nanofiber film. After the pre-treated film had been immersed in a solution of the monomers LA/CL (molar ratio 20 : 80) in toluene, the surface-initiated ring-opening graft copolymerization was carried out in the presence of a Lewis acid catalyst [tin(II) 2-ethylhexanoate] by heating the system at 80 °C for 48 h to give the chitin nanofiber-graft-poly(LA-co-CL) film. The IR spectrum of the resulting film supported the presence of the polyester in the product, which was bound to the nanofibers by covalent linkages. The amount and compositional ratio of the grafted LA/CL polyester were evaluated by the weight difference of the films before and after grafting and by ¹H-NMR analysis of the graft chains separated from the nanofibers by alkaline treatment to be 12.2% and 40/60, respectively. The LA/CL compositional ratio was higher than that in the feed solution as a result of the higher polymerizability of LA compared with CL.¹⁰⁵ The SEM image of the resulting composite film retained the nanofiber morphology, but the fibers were increased in width (60–100 nm) (Fig. 12a). Some nanofibers were merged at the interfacial areas, which was probably caused by the grafted polyesters. The stress–strain curve of the composite film in tension supported the enhancement of the mechanical properties compared with the original chitin nanofiber film.

Fig. 11 Surface-initiated ring-opening graft (co)polymerization of LA/CL and BLG-NCA from chitin nanofiber film.

Fig. 12 SEM images of (a) chitin nanofiber-graft-poly(LA-co-CL) film, (b) chitin nanofiber-graft-polyBLG film and (c) chitin nanofiber-graft-polyGA network film.

To provide further useful composite materials based on self-assembled chitin nanofibers, the surface-initiated grafting technique from the chitin nanofiber film was extended to use a synthetic polypeptide as a biocompatible polymer. As it is well-known that synthetic polypeptides with well-defined structures are synthesized by the ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs) accompanied by decarboxylation initiated from the amino groups,^106,107 the surface-initiated graft polymerization of a NCA monomer from the chitin nanofiber film with amino initiating groups to give the chitin nanofiber-graft-polypeptide film was investigated.¹⁰⁸ The monomer γ-benzyl L-glutamate-NCA (BLG-NCA) was selected because its ring-opening polymerization and subsequent hydrolysis of ester linkages had previously been used to produce poly(L-glutamic acid) (polyGA) with free carboxylic acid groups. Because the ring-opening polymerization of the NCA monomer had been initiated with an amino group, deacetylation of the acetamido groups in the chitin nanofiber film was achieved by treatment with 40% w/v aqueous NaOH at 80 °C for 7 h to produce a partially deacetylated chitin (PDA-chitin) nanofiber film.¹⁰⁹ The degree of deacetylation of the product was ca. 24%, as estimated by the ratio of the absorption due to the amido II group at 1560 cm⁻¹ to that due to the C–O stretching vibration at 1070 cm⁻¹ in the IR spectrum. The SEM image and the XRD pattern of the resulting film were comparable with those before alkaline treatment, suggesting the retention of the nanofiber morphology and the crystalline structure, respectively. The resulting PDA-chitin nanofiber film was immersed in a solution of BLG-NCA (20 equiv. for an amino group) in ethyl acetate at 0 °C for 24 h for surface-initiated graft polymerization from the amino groups. The IR spectrum of the chitin nanofiber-graft-polyBLG film obtained showed the C=O absorption of an ester linkage at 1735 cm⁻¹, strongly suggesting the presence of polyBLG in the product, bound to the nanofibers by covalent linkages. The amount of grafting of the polyBLG chains was evaluated to be 18 wt% by the weight difference of the films before and after graft polymerization. The SEM image of the composite film indicated that the nanofiber morphology was still retained, although some fibers were merged at the interfacial areas (Fig. 12b), as well as the chitin nanofiber-graft-poly(LA-co-CL) film.

A highly flexible chitin nanofiber-graft-polyGA network film was obtained by alkaline hydrolysis of ester linkages in the polyBLG chains on the composite film, followed by condensation of the sodium carboxylate groups with the amino groups present in the film. The composite film was first immersed in 1.0 mol L⁻¹ aqueous NaOH at 60 °C for 5 h for the alkaline hydrolysis of benzyl esters to convert into the chitin nanofiber-graft-polyGA film with sodium carboxylate groups. The IR spectrum of the film after alkaline treatment supported the complete cleavage of the benzyl esters into sodium carboxylate groups. Condensation of the resulting carboxylate groups with the amino groups at the terminal end of the polyGA chains or those remaining on the nanofibers (which had not participated in the initiation of the graft polymerization) was conducted using a N-hydroxysuccinimide/water-soluble carbodiimide condensing agent in water at room temperature for 12 h to construct polyGA/chitin networks in the film. The intensity ratio of the amido II absorption to the C–O stretching absorption in the IR spectrum of the product was 22% larger than that of the film before condensation. This result indicated the progress of the desired condensation reaction to yield the chitin nanofiber-graft-polyGA network film. The nanofiber morphology was seen in the SEM image of the film produced (Fig. 12c). The stress–strain curve of the resulting network film under tension showed a larger elongation value at break than that of the original chitin nanofiber film with comparable tensile strength values, indicating the more elastic nature of the former film. The chitin nanofiber-graft-polyGA network film had a highly flexible nature, which could be bent without breaking.

Surface-initiated atom transfer radical polymerization (ATRP) from a chitin nanofiber macroinitiator film was also investigated. ATRP is a versatile technique to control the chain length and polydispersity of the resulting polymers and has been used to synthesize a wide range of polymeric materials with designed structures.^110,111 Because ATPR is initiated from α-haloalkylacyl groups, the chitin macroinitiator was synthesized with these initiating groups by esterification of the hydroxy groups in chitin with the α-haloalkylacyl bromide, which was further used for the grafting of styrene by ATRP.¹¹²

On the basis of this study, the chitin nanofiber macroinitiator film for ATRP was synthesized by the reaction of the hydroxy groups on the surface of the nanofibers with α-bromoisobutyryl bromide (30 equiv. for a repeating unit of chitin) in DMAc (Fig. 13).¹¹³ The degree of substitution was calculated by SEM-EDX measurements to be 0.61 for a repeating unit. The surface-initiated ATRP of 2-hydroxyethyl acrylate (HEA) (20 eqiuv. for an initiating site) from the macroinitiator film was then performed in the presence of CuBr/2,2′-bipyridine in 3 wt% LiCl/DMAc at 60 °C (Fig. 13).¹¹² The conversion of HEA was estimated by the weight difference of the films before and after ATRP, which increased with increasing reaction times. The intensity ratio of the ester C=O absorption to the amido C=O absorption in the IR spectra of the products increased compared with those of the macroinitiator film, suggesting the progress of the graft polymerization. For gel permeation chromatography (GPC) measurements, the grafted polyHEA chains were separated from the produced composite films by alkaline hydrolysis to give poly(acrylic acid), which was further converted into poly(methyl acrylate) by methyl esterification. The GPC peaks of the obtained poly(methyl acrylate)s shifted to a higher molecular weight region with increasing reaction time and their polydispersities were relatively narrow. These GPC results strongly supported the view that the longer polyHEA chains were grafted onto the chitin nanofiber films by a gradual ATRP process with prolonged reaction times. The nanofiber morphology was seen in the SEM image of the composite film resulting from the lower monomer conversion (6%, polymerization time 3 h), but the average fiber width increased compared with that of the macroinitiator film (20 and 40 nm, Fig. 14a and b, respectively). The SEM image of the composite film obtained with a higher monomer conversion (62%, polymerization time 12 h) did not show the nanofiber morphology (Fig. 14c), indicating that the nanofibers were covered by the longer grafted polyHEA chains. The stress–strain curves of the composite films under tension showed larger values for elongation at break compared with the original chitin nanofiber film. The elongation at break increased with increasing monomer conversion, whereas the tensile strength decreased. These data suggested an enhancement of flexibility by grafting longer polyHEA chains onto the chitin nanofiber films.

Fig. 13 Synthesis of chitin nanofiber macroinitiator film and surface-initiated graft ATRP of HEA.

Fig. 14 SEM images of (a) chitin nanofiber macroinitiator film and chitin nanofiber-graft-polyHEA films obtained by the reaction times of (b) 3 and (c) 12 h.

Fabrication of microporous chitins through gelation with suitable dispersion media

Microporous chitins have previously been fabricated by multi-step regeneration from chitin solutions in solvent systems. A series of microporous chitins was obtained by the freezing and lyophilization of chitin hydrogels prepared by soaking 0.2–2.0% chitin solutions in a 5% LiCl/DMAc solvent system.⁵⁵ By subjecting the chitin hydrogels to dry ice/acetone cooling, the resulting porous chitins had pore sizes measuring 200–500 μm. The smaller pore sizes (100–200 μm) were obtained when the chitin gels were frozen with liquid nitrogen and 10 μm pores were produced when the gels were placed in a freezer for 20 min.

Microporous chitins were also fabricated by lyophilization of the hydrogels, which were prepared by dialysis of the chitin solutions with a saturated CaCl₂·2H₂O/methanol solution.^114,115 Microporous chitins were obtained by regeneration from solutions of chitin with BMIMOAc using ethanol, followed by Soxhlet extraction with ethanol and successive steps of supercritical drying (CO₂).¹¹⁶

The use of moderate concentrations of a CaBr₂·2H₂O/methanol solution affected the regeneration process from the chitin ion gel, resulting in the production of chitin nanofibers with higher aspect ratios. This result suggested the specific affinity of CaBr₂·2H₂O/methanol solutions toward chitin. It was found that chitin gels were easily obtained by stirring mixtures of chitin with highly concentrated CaBr₂·2H₂O/methanol solutions at room temperature, which were subsequently converted into microporous chitins (Fig. 15).⁶² Because chitin was not swollen by the same procedure when using a saturated CaCl₂·2H₂O/methanol solution at room temperature, the swelling of chitin with the CaBr₂·2H₂O/methanol solution appears to be a specific behavior. In a typical gelation procedure, when mixtures of chitin (0.9–3.5% w/v) with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol solution were stirred at room temperature, chitin was gradually swollen, leading to the formation of a gel after 48 h. The mixtures of chitin (1.8 w/v) with different concentrations of CaBr₂·2H₂O/methanol solutions also resulted in the formation of gels by the same procedure. The dynamic viscoelastic measurement of the resulting chitin gels suggested a gelling state. Microporous chitins were efficiently fabricated by removing methanol from the gels under reduced pressure, followed by washing out the CaBr₂ with water and subsequent lyophilization (Fig. 15).⁶² The XRD profiles of the products indicated the construction of an α-chitin crystalline structure during the regeneration procedure. SEM measurements of the products supported the microporous morphology. It was found from the SEM results that the porosities depended on the concentration of chitin in the gel (Fig. 16). The mechanical properties of the microporous chitins under compression were affected by the pore size; however, the amount of CaBr₂·2H₂O in the gel did not affect the porosity and mechanical properties of the microporous chitins.

Fig. 15 Preparation procedures of chitin gel with CaBr₂·2H₂O/methanol media and microporous chitin.

Fig. 16 SEM images of microporous chitins obtained from (a) 0.9, (b) 1.8, (c) 2.4 and (d) 3.5% w/v chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media.

Conclusions

Recent developments in research on the fabrication of chitin nano- and micromaterials through gelation with suitable dispersion media have been reviewed. Much of the review has been concerned with the author's own work in this area. A variety of solvent systems and dispersion media for chitin have been found, leading to the efficient production of materials from chitin. Chitin nanofibers and microporous materials have been fabricated by regeneration from gels in suitable dispersion media. Ionic liquids have good potential as solvents and dispersion media for chitin and self-assembled chitin nanofibers can be obtained by regeneration from ion gels. Composite materials of chitin nanofibers with other polymeric components have been constructed by both physical and chemical approaches.

Chitin is an abundant natural polysaccharide and has good biocompatibility. Practical chitin materials are likely to be fabricated and applied in fields related to medicine, pharmaceutics and the environmental industries in the future.

Acknowledgements

The author is indebted to his co-workers, whose names are found in the references for his papers, for their enthusiastic collaboration.

Notes and references

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