Guanidinylated nanochitins: guanidinylated chitin nanocrystals are dispersible at neutral pH

Abstract

Despite nanochitins showing favorable biological effects, the colloid stability of positively charged nanochitins by virtue of the amino group is limited to acidic pH, which is different from biological conditions. Here, we show that guanidinylated chitin nanocrystals (GChNCs) are dispersible at neutral pH. The GChNCs are prepared by guanidinylation of partially deacetylated chitin nanocrystals (ChNCs) with 1-amidinopyrazole hydrochloride. The degrees of guanidinylation and acetylation of the GChNCs are 4.6% and 75.7%, respectively. A 1.0 wt% GChNC dispersion is prepared with 0.5 wt% acetic acid solution by sonication treatment. Although slight white turbidity is observed due to scattering, no visible macroscopic precipitates are observed. The average diameter of the GChNCs estimated by DLS analysis is 327.2 nm. When the GChNC dispersion is neutralized by adding 0.1 M NaOH solution, the transmittance of the GChNC dispersion is decreased by aggregation. However, the transmittance of the GChNC dispersion is higher than that of the ChNC dispersion, suggesting that the GChNC particles are less aggregated than the ChNC particles due to the positive charge by virtue of the high basicity of the guanidino group. Interestingly, we find that the GChNCs homogeneously disperse in 0.1 M HEPES buffer (pH 7.4) up to 0.5 wt% by sonication treatment, even though the average diameter of the GChNCs in the solution is 3.4-fold higher (1115.1 nm) than that prepared at pH 3.0. We additionally find no observation of this improved dispersibility of guanidinylated chitin nanofibers due to the guanidino group. This result indicates that the guanidinylation is effective in improving the dispersion of nanochitins with smaller aspect ratios, like ChNCs. Furthermore, we demonstrate that the dispersibility of GChNCs at neutral pH can be utilized for material development, where a gelatin–GChNF composite hydrogel displaying enhanced mechanical properties is successfully prepared by adding 10% (w/w) GChNCs.

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1. Introduction

Chitin is the second most abundant organic compound in nature, and it can be found in the exoskeletons of shellfish and insects, squid pens, mushrooms, etc.^1–4 Structurally, chitin is a linear polysaccharide composed of N-acetyl-D-glucosamine repeating units linked via β-1,4-linkages. Due to their linear and regular primary structure, 18–25 chitin chains form a semi-crystalline structure due to inter- and intramolecular hydrogen bonds, van der Waals forces, hydrophobic interactions, and electrostatic attraction, and form nanofibrils consisting of crystalline and amorphous regions, with a diameter of 2–5 nm.⁵ Although favorable biological effects of chitins have been discovered,^6,7 poor solubility/dispersibility for water and common organic solvents have limited their social implementation. Many researchers have attempted to overcome this problem and have provided innovative progress.^2,8–10 As one of the innovations, homogeneous chitin nanofiber (ChNF) dispersions were developed, where the chitin nanofibrils could be defibrated by mechanical treatments with a grinder and/or high-pressure homogenizer in acidic aqueous media.^2,11 A more convenient defibration process with a cylindrical or ultrasonic homogenizer was achieved by the use of partially deacetylated chitin with amino groups on the nanofibril surfaces, because electrostatic repulsion between the positively charged nanofibrils promoted defibration in acidic media.^12,13 In addition, short nanofibrils with a lower aspect ratio produced by acid-catalyzed hydrolysis of the amorphous region are called chitin nanocrystals (ChNCs) or nanowhiskers.^14,15 These nanoscopic chitins are called nanochitins, and they have accelerated the commercial use of chitins and the development of chitin-based materials. However, the colloid stability of nanochitins is limited to acidic pH, because the pKa of the ammonium group (–NH₃⁺) contributing to stable dispersion by virtue of the electrostatic repulsion is 6.0–6.5.⁵ We have recently reported an N-trimethylated cationic nanochitin, obtained by N-methylation of partially deacetylated chitin and subsequent defibration with a cylindrical homogenizer, which demonstrated improved dispersibility even in basic solution and pH-independent antibacterial activity.¹⁶ Thus, surface modification of nanochitin, imparting a cationic nature at neutral pH, improves dispersibility and antibacterial activity.

We previously reported guanidinylation of chitosan with 1-amidinopyrazole hydrochloride (AP) in the presence of triethylamine (TEA).^17,18 It is well-known that the pKa of the guanidinium group in arginine is 12.5.¹⁹ Therefore, the guanidino group of guanidinylated chitosan should have a positive charge by protonation at around neutral pH. Indeed, guanidinylated chitosan shows an enhanced electrostatic interaction with bovine serum albumin at pH 7.4 due to the lower acidity of the guanidino group compared to the amino group, along with an enhanced cellular internalization of bovine serum albumin.²⁰ In addition, guanidinylated chitosan with a ca. 50% degree of guanidinylation (DG) and ca. 30% degree of acetylation (DA) showed water solubility, where the control of DA was a key factor in developing water solubility. Therefore, we expected that guanidinylated ChNCs (GChNCs) and guanidinylated ChNFs (GChNFs) with appropriate quantities of the guanidino, amino, and acetyl groups on their surface should homogeneously disperse at neutral pH. Note that GChNCs and GChNFs have been reported for developing bioadhesives.²¹ However, their dispersibility was out of the scope of that report.

Here, we show GChNCs dispersible at around neutral pH by virtue of the positively charged guanidino groups. The partially deacetylated ChNCs are guanidinylated with AP. The guanidinylation is confirmed by infrared (IR) and elemental analyses, and the GChNCs are additionally analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and scanning probe microscopy (SPM) measurements. The dispersibility of the GChNCs and ChNCs in acidic and neutral media is investigated with transmittance and dynamic light scattering (DLS) analyses. In addition, we investigate the dispersibility of the GChNFs in acidic and neutral media to evaluate the effect of the guanidinylation on the dispersibility of the nanofiber morphology. Furthermore, a gelatin–GChNC composite hydrogel has been prepared to demonstrate the usefulness of GChNCs for material development.

2. Materials and methods

2.1. Materials

α-Chitin (powder) was purchased from Koyo Chemical Industry Co., Ltd (Tottori, Japan). The DA and molecular weight estimated from elemental analysis and viscosity methods were 96.8% (C/N = 6.80) and 65 kDa, respectively. AP was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). TEA, acetic acid, and gelatin were purchased from Fujifilm-Wako Co., Ltd (Tokyo, Japan). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Other reagents were commercial grade and used without further purification.

2.2. Instrumentation

IR spectra of the samples were recorded using a Nicolet 6700 (Thermo Fisher Scientific, Waltham, MA) equipped with an ATR attachment. SEM micrographs were recorded by an S-5500 (Hitachi, Tokyo, Japan). For SEM analysis, the samples were fixed on an SEM stage with conductive tape. The samples were coated with an approximately 2 nm layer of Pt by an ion sputter coater. DLS analysis was performed with a nanoPartica SZ-100V2 (HORIBA Scientific, Japan). The transmittance spectra were recorded on a Multiskan GO (Thermo Fischer Scientific). Elemental analysis data were measured on a 2400 II CHNS/O (PerkinElmer, Waltham, MA, USA). XRD profiles were obtained with Ni-filtered CuKα from an X-ray generator (RINT Ultima, Rigaku, Japan) operating at 40 kV and 30 mA. SPM analysis in dynamic force mode (DFM) was performed with an AFM5200S (AFM5200S, Hitachi High-Tech, Tokyo, Japan). For DFM analysis, the samples were fixed on a mica substrate via the following procedure. The 0.001% GChNC or ChNC diluted solution (20 µL) was dropped on a mica substrate (1 cm × 1 cm), followed by drying at 60 °C. The mica surface with GChNC or ChNC particles was gently washed with ultrapure water. Sonication treatment was performed with an SLPe40 (BRANSON Ultrasonics, CT, USA). Stress–strain curves were measured with an MCT-2150 (A&D Co., Ltd, Tokyo, Japan) at a cross-head speed of 10 mm min⁻¹. Viscosities were recorded at 25 °C by an LV DV-E (Brookfield Engineering, Middleboro, MA, USA). ¹³C NMR spectra were acquired on a Bruker AV-400M (Bruker, MA, USA).

2.3. Deacetylation of chitin

Partially deacetylated chitin was prepared in accordance with a previous report.¹² Chitin (15.0 g) was stirred in 30 wt% NaOH (500 mL) for 6 h at 90 °C under an argon atmosphere. After cooling to room temperature, the precipitate was collected by vacuum filtration, followed by washing with water until the filtrate became neutral pH. Wet partially deacetylated chitin, 46.8 g (solid content: 23.4 wt%, C/N = 6.26), was obtained.

2.4. Hydrolysis of partially deacetylated chitin

ChNCs were prepared in accordance with a previous report.²² Wet partially deacetylated chitin powder (23.3 g containing 5.5 g of solid content) was added to 3 M HCl solution (150 mL) and stirred for 1.5 h at 100 °C. After cooling to room temperature, the precipitate was collected by centrifugation, followed by washing with water until the supernatant reached neutral pH. A ChNC suspension, 47.0 g (solid content: 10.2 wt%, C/N = 6.44), was obtained.

2.5. Guanidinylation of ChNCs

The ChNCs were guanidinylated in accordance with a previous report.²⁰ AP (3.1 g, 21.1 mmol) was added to the ChNC suspension (28.0 g containing 2.9 g of solid content), and the mixture was stirred for 10 min. TEA (2.16 g, 21.3 mmol) was added to the suspension, and the mixture was stirred for 7 days at room temperature. The reaction mixture was purified by dialysis with a Visking tube (molecular weight cutoff: 12 000) in a large volume of water. The precipitate was collected by centrifugation. A GChNC suspension, 17.5 g (solid content: 15.0 wt%, C/N = 5.93), was obtained.

2.6. Guanidinylation of the partially deacetylated chitin powder

The wet partially deacetylated chitin powder (20.0 g containing 4.6 g of solid content) was added to 1 M acetic acid solution (500 mL) and stirred for 1 h at room temperature to remove a soluble fraction. The precipitate was collected by centrifugation, and subsequently washed with water by centrifugation until the supernatant reached neutral pH. Wet partially deacetylated chitin, 5.2 g (solid content: 21.5 wt%, C/N = 6.40), was obtained.

AP (2.7 g, 18.6 mmol) and water (5 mL) were added to the above wet partially deacetylated chitin powder (4.7 g containing 1.0 g of solid content), and the mixture was stirred for 10 min. TEA (1.88 g, 18.6 mmol) was added, and the mixture was stirred for 7 days at room temperature. The reaction mixture was purified by dialysis with a Visking tube (molecular weight cutoff: 12 000) in a large volume of water. The precipitate was collected by centrifugation. Wet guanidinylated chitin, 6.2 g (solid content: 15.2 wt%, C/N = 5.81), was obtained.

2.7. Preparation of ChNC, GChNC, ChNF, and GChNF dispersions

The ChNC or GChNC suspension, the wet partially deacetylated chitin before guanidinylation in 2.6, or the wet guanidinylated chitin was diluted with 0.5 wt% acetic acid solution to 1.0 wt%. The suspension was mixed by vortexing, followed by sonication for 10 min at a 70% amplitude in an ice bath.

2.8. Preparation of the gelatin–GChNC composite hydrogel

Gelatin (0.50 g) was added to the 0.5% GChNC dispersion prepared with 0.1 M HEPES (pH 7.4) (9.5 mL), and the solution was heated at 60 °C for 10 min with stirring. After dissolution of the gelatin, the gelatin–GChNC solution was poured into a cylindrical mold (φ = 16 mm) and cooled to 25 °C to obtain a gel state. The cryogel for SEM analysis was prepared by sequential solvent exchange of the hydrogel with methanol and t-butyl alcohol, followed by lyophilization.

3. Results and discussion

3.1. Preparation and characterization of GChNCs

The GChNC powder was prepared by the guanidinylation of the ChNCs (DA 75.7%) with AP and TEA (Fig. 1A). Fig. 1B shows the IR spectra of the GChNC and ChNC powders. The characteristic shoulder peak attributed to the C=N stretching vibration of the guanidino group was detected at around 1710 cm⁻¹,^17,18 suggesting the guanidinylation of the ChNCs. The ¹³C NMR analysis of the chitosan/chitin fraction on the GChNC surface also supported the guanidinylation (Fig. S1). The C/N ratio for the product was 5.93, which is smaller than that of the ChNCs, whose C/N ratio was 6.44, indicating that guanidinylation was achieved with AP. The DG estimated by the C/N value was 4.6%. If we can assume that the ChNCs were composed of 25 chains (cuboid: 5 × 5 chains), and the quantity of amino and acetamide groups exposed on the ChNC surface is 32.0% (=16 outer chains/25 total chains × 0.5 alternative inversion of monomer unit × 100) of the total amino and acetamide groups in the ChNCs. Therefore, we estimate that 75.9% (=19.7/32.0 × 100) of the acetamide groups on the ChNC surfaces were deacetylated to be amino groups. Namely, we estimate that the composition of the guanidino, amino, and acetamide groups at the 2-positions exposed on the GChNC surface was 14.3%, 61.6%, and 24.1%, respectively. Thus, we considered that the quantity of acetamide groups on the surface was successfully controlled to a similar value to previously reported DA of water-soluble guanidinylated chitosan.²⁰Fig. 1C shows the XRD spectra of the ChNCs and GChNCs. Although the ChNC surface was deacetylated, characteristic peaks attributed to α-chitin were observed at 9°, 13°, 19°, 21°, 23°, and 26°.²³ The crystallinity indices estimated from the diffraction peaks at 19° of the ChNCs and GChNCs were 89.7% and 90.3%, respectively, which were comparable to previously reported ChNCs with a highly crystallinity index.²⁴Fig. 1D shows SEM images of the dried GChNCs and ChNCs. There is no morphological difference between the ChNCs and GChNCs. In both cases, nanocrystals with ca. 10 nm width are observed. These results indicate that the semi-crystalline structure of the GChNCs was maintained after the guanidinylation process.

Fig. 1 Preparation of GChNCs by guanidinylation of ChNCs using the AP-TEA system (A). Characterization of the GChNCs: IR (B) and XRD (C) spectra of the dried GChNCs and ChNC, and SEM images (D).

3.2. Dispersibility of the GChNCs

Fig. 2A shows photographs of 1.0 wt% GChNC and ChNC dispersions prepared with 0.5 wt% acetic acid solution by an ultrasonic homogenizer. Although slight white turbidity due to scattering was observed, no visible macroscopic precipitate was observed in either case. The transmittance at 600 nm for the GNC solution was 81.9%, which is slightly lower than that of the ChNCs (85.7%) (Fig. 2B), suggesting that the GChNC particles are more aggregated compared to the ChNC particles. Indeed, the GChNCs had a slightly wider distribution on the larger diameter side in the DLS profile (Fig. 2C). The average diameters of the GChNCs and ChNCs were 327.2 nm and 274.7 nm, respectively. In our previous report, guanidinylated chitosan with a lower DA was found to be insoluble.²⁰ Therefore, we supposed that guanidino groups acted as hydrogen bond donors and/or acceptors to promote insolubilization in the absence of DA. Namely, a similar, worse effect of the guanidino group was observed on the GChNCs. In order to confirm the morphology of the GChNCs after dispersion by the sonication treatment, SPM analysis in DFM mode was performed (Fig. 2D). The nanocrystal particle lengths and widths of the GChNCs and ChNCs were 190.6 ± 59.4 nm and 6.1 ± 2.1 nm, and 184.5 ± 75.4 nm and 5.5 ± 2.3 nm, respectively. These values were comparable to those of the previously reported partially deacetylated ChNCs.²⁵ This result indicates that the single particle morphology was preserved after the guanidinylation reaction, and the aggregation of the GChNCs was more advanced than that of the ChNCs in the acidic solution.

Fig. 2 Photograph (A), transmittance spectra (B), and DLS profile (C) of the 1.0 wt% GChNC and ChNC dispersions. DFM images of GChNC and ChNC particles (D) and the detailed characterization of their length and width (E). Error bars show the standard deviation.

In order to evaluate the effect of the guanidino group on the dispersibility of the GChNCs at neutral pH, the GChNC and ChNC dispersions were adjusted to pH 7 by adding a 0.1 M NaOH solution with vigorous stirring, and the concentrations of the GChNC and ChNC dispersions were adjusted to 0.5 wt% by adding water. In the case of the ChNCs, strong aggregation with gelation and water separation was observed (Fig. 3A). However, in the case of the GChNCs, although the transmittance was decreased by the aggregation caused by contact with the alkaline solution (Fig. 3B), the gelation and water separation shown in the ChNCs were not observed. Indeed, no macroscopic aggregates were observed under green laser light irradiation (Fig. 3C). We suggest that this is due to the positive charge by virtue of the guanidino group. The zeta potential of the 0.1 wt% ChNC dispersion at pH 3 was +41.8 mV, which is comparable to that of the 0.1 wt% GChNC dispersion (+42.4 mV). However, the zeta potential of the ChNC dispersion was significantly decreased to +4.5 mV by the neutralization. In contrast, the zeta potential of the GChNCs was ca. 4-fold higher than that of the ChNCs (+17.0 mV). This result suggests that the GChNCs show improved dispersibility at around neutral pH by virtue of the guanidinylation.

Fig. 3 Photograph (A) and transmittance spectra (B) of the neutralized 0.5 wt% GChNC and ChNC dispersions. Photograph of the neutralized 0.5 wt% GChNC and ChNC dispersions under green laser light irradiation (C).

3.3. Preparation of a GChNC dispersion at biological pH

Direct dispersion of GChNCs at biological pH expands the possibility for biomedical applications. Therefore, we investigated the dispersion of the GChNCs in 0.1 M HEPES buffer (pH 7.4). Interestingly, the GChNCs were capable of dispersion by a sonication treatment to form a uniform colloid solution at 0.5 wt% (Fig. 4A and B). In contrast, visible macroscopic aggregates were observed in the case of the ChNCs. This result indicates that the guanidino groups on the GChNC surface contributed to the enhancement of the dispersion of the GChNCs at around neutral pH. However, the transmittance of the GChNC solution prepared at pH 7.4 at 600 nm was lower than that of the solution prepared at pH 3.0, as shown in Fig. 2B. The average diameter of the GChNCs in the solution was 1115.1 nm, which was 3.4-fold higher than that prepared at pH 3.0. Indeed, aggregated GChNCs were observed in the DFM image. These results indicate that GChNCs were dispersed as aggregates in the solution at pH 7.4. We additionally investigated the preparation of higher concentration GChNC solutions. Although homogenous 0.1 wt%, 0.5 wt%, and 0.7 wt% GChNC dispersions were obtained by the sonication treatment (Fig. 4F), lower colloid stability was observed for the 0.7 wt% solution. Fig. 4E shows the change in transmittance of the 0.1 wt%, 0.5 wt%, and 0.7% GChNC dispersions after 5 min, 1 h, 2 h, 24 h, and 48 h. In the case of the 0.1 wt% and 0.5 wt% solutions, no significant changes were observed after 1-24 h. Indeed, there was no visible difference after 48 h (Fig. 4H). However, in the case of the 0.7 wt% solution, the transmittance decreased after 1 h due to the precipitation of the GChNCs (Fig. 4E and G). These results indicate that the GChNCs were stably dispersed up to 0.5 wt% in 0.1 M HEPES buffer (pH 7.4) by the sonication treatment. Note that it was difficult to achieve dispersion of the GChNCs in simulated body fluid, likely due to electrostatic interactions with multivalent anions (Fig. S2).

Fig. 4 Photograph of the 0.5 wt% GChNC and ChNC dispersions prepared in 0.1 M HEPES buffer (pH 7.4) (A) and their observation under green laser irradiation (B). Transmittance spectra of the GChNC and ChNC dispersions (C). DLS profile and DFM image of the GCN dispersion (D). Transmittance of the 0.1 wt%, 0.5 wt%, and 0.7 wt% GChNC dispersions at 600 nm measured after 5 min, 1 h, 2 h, 24 h, and 48 h (E). Photograph of the 0.1 wt%, 0.5 wt%, and 0.7 wt% GChNC dispersions after 5 min (F), the 0.7 wt% dispersion after 1 h (G), and the 0.5% dispersion after 48 h (H).

3.4. Dispersibility of the GCNFs

In order to evaluate the effect of the guanidinylation on the dispersion of GCNFs, the guanidinylation of the partially deacetylated chitin (C/N = 6.40, DA 73.3%) with AP and TEA was performed. The DG of the guanidinylated chitin estimated from the C/N ratio (C/N = 5.81) was 5.5%. This value was comparable to that of the GChNCs. The guanidinylated chitin and the partially deacetylated chitin were defibrated in a 0.5 wt% acetic acid solution by sonication treatment to form 0.5 wt% GCNF and CNF dispersions. Fig. 5A shows the transmittance spectra of the 0.5 wt% GCNFs and CNFs and their neutralized solutions. Although the transmittance of the GChNCs was comparable to that of the ChNCs, the GChNFs showed lower transmittances than the CNFs. In addition, the viscosity of the 0.5% GChNC dispersion (12 239 mPa S) was lower than that of the ChNFs (20 846 mPa S). These results suggest that the GChNFs underwent insufficient defibration. In the DLS analysis of the GCNFs, the less defibrated fraction was found in the 1000–10 000 nm range. This unfibrated fraction was observed in the SEM analysis of the GCNF cast film (Fig. 5C). These results indicate that GCNFs are more difficult to fibrate than CNFs. Such prevention, on defibration was also observed in the GChNCs as described above. We supposed that this difficulty in defibration was caused by the higher number of hydrogen bonds between the GCNFs via the guanidino groups, because a single GCNF has a higher surface area than a single GChNC, and the guanidino groups can promote hydrogen bonding as both a hydrogen bond donor and acceptor to promote aggregation. Therefore, in the case of GCNFs, the guanidino group did not provide any benefit for dispersion. In addition, we confirmed that a favorable effect toward neutralization was not observed. Fig. 5D shows the GCNF dispersions before and after neutralization. The neutralized GCNF dispersion was completely aggregated to form a brittle gel state. We suppose that, in the case of GCNFs, the guanidino and acetyl groups were inhomogenously distributed on the fiber surface, and the nanofibers had a higher surface area than the nanocrystals. Therefore, aggregation occurs at multiple sites on a single fiber upon contact with the alkaline solution, which leads to gelation. Note that although GCNFs are more difficult to defibrate than CNFs, a uniform dispersion was obtained by a high-pressure homogenization treatment (Fig. S3). Therefore, we expect that the GCNFs are also promising nanomaterials with guanidino groups, applicable for material development.

Fig. 5 Transmittance spectra of the 0.5% GChNF and ChNF dispersions or the neutralized GChNF and ChNF dispersions (A). DLS profiles of the 0.5% GChNFs and ChNFs (B). SEM images of the GChNF and ChNF cast films (C). Photograph of the 0.5% GChNF dispersion before and after neutralization (D).

3.5. Preparation of a gelatin–GChNC hydrogel and its mechanical properties

The dispersibility of the cationic GChNCs at neutral pH is an advantage in material design. In order to demonstrate the usefulness, we prepared a 5.0 wt% gelatin–0.5 wt% GChNC composite hydrogel. It is well known that, at neutral pH, gelatin dissolves upon heating and forms a hydrogel upon cooling, due to the formation of triple helices during the cooling process.²⁶ However, no gelation occurs at acidic pH because gelatin is soluble in acidic water. Therefore, the preparation of a gelatin–ChNC composite hydrogel by adding gelatin to acidic ChNC dispersions was impossible. We expected a gelatin–GChNC composite hydrogel reinforced by GChNC to be obtained by a heating–cooling process of the gelatin–GChNC dispersion. Fig. 6A shows a photo image of the 5.0 wt% gelatin–0.5 wt% GChNC composite hydrogel and 5.0 wt% gelatin hydrogel prepared with 0.1 M HEPES buffer (pH 7.4). The gelatin hydrogel was distorted vertically by gravity because it was too soft. However, the gelatin–GChNC composite hydrogel was self-standing while maintaining the size of the mold. In their stress–strain curves, the slope of the gelatin–GChNC composite hydrogel corresponding to the elastic modulus was 6.6-fold higher than that of gelatin (Fig. 6B). The average fracture stress of the gelatin–GChNC composite hydrogel and the gelatin hydrogel was 2.87 ± 0.57 kPa and 0.57 ± 0.19 kPa, respectively. The gelatin–GChNC composite hydrogel showed 5.0-fold higher fracture stress than that of the gelatin hydrogel. In order to perform SEM observation, a cryogel was prepared via the solvent replacement of the composite hydrogel to t-butyl alcohol and lyophilization.²⁷ In the SEM image of the cryo-composite hydrogel, a homogeneous network structure was observed (Fig. 6D). However, GChNC particles were not observed, indicating that the GChNC particles were completely complexed with the gelatin matrix. Namely, the GChNC successfully reinforced the gelatin hydrogel matrix by virtue of the electrostatic interaction. In addition, we found that this composite hydrogel suppressed the collapse during swelling. Fig. 6E shows the swelling behavior of the gelatin–GChNC composite hydrogel in 0.1 M PBS (pH 7.4) at 25 °C. The gelatin hydrogel rapidly swelled to ca. 1.5-fold in weight in 20 min, and it gradually dissolved from the surface thereafter. In contrast, the gelatin–GChNC composite hydrogel gradually swelled up to ca. 3-fold in weight until 4 h, and gradually dissolved from the surface thereafter. This swelling behavior is applicable for the sustained release of drugs.²⁸ In addition, cationic polymers bearing a guanidino group enhance the intestinal absorption of drugs.^29–31 Furthermore, the injectability of the gelatin–GChNC composite hydrogel was demonstrated, as shown in Fig. S4. Thus, we expect that GChNCs will be a promising additive for controlled release and promoting intestinal absorption of drugs.

Fig. 6 Photo image of the 5.0 wt% gelatin–0.5 wt% GChNC composite hydrogel (left) and 5.0 wt% gelatin hydrogel (right) prepared with 0.1 M HEPES buffer (pH 7.4) (A) and their representative compressive stress–strain curves (B). Average fracture stress (left) and strain (right) (C). SEM image of the cryo-gelatin–GChNC composite gel (D). Swelling behavior of the gelatin–GChNC hydrogel and gelatin hydrogel in PBS (pH 7.4) (E). Error bars show standard deviation.

4. Conclusions

We investigated the dispersibility of GChNCs and GCNFs in acidic and neutral conditions. The GChNCs were prepared by the guanidinylation of the ChNCs (DA 75.7%) with the AP-TEA system. We confirmed the guanidinylation with IR and elemental analysis. The DG estimated from the C/N ratio was 4.6%. In addition, XRD analysis showed that the semi-crystalline structure of the GChNCs was maintained after the guanidinylation. The 1.0 wt% GChNC dispersion was prepared with a 0.5 wt% acetic acid solution by sonication treatment. Although slight white turbidity due to scattering was observed in the GChNC solution, no visible macroscopic powder was observed. The average diameter of the GChNCs was 327.2 nm, which was slightly larger than that of the ChNCs. However, the nanocrystal particle lengths and widths of the GChNCs and ChNCs were comparable, indicating that the morphology of the ChNCs was preserved after the guanidinylation reaction, and the aggregation of the GChNCs was more advanced than that of the ChNCs in the acidic solution. However, the effect of the neutralization on the change in the dispersion state of the GChNCs was less than that of the ChNCs. This was due to positive charge by virtue of the guanidino group at neutral pH 7. Interestingly, GChNCs were directly dispersed in 0.1 M HEPES buffer (pH 7.4) up to 0.5 wt% by sonication treatment. However, the average diameter of the GChNCs in solution was 1115.1 nm, which is 3.4-fold higher than that prepared at pH 3.0. This indicates that the GChNC particles were dispersed as aggregates in the solution of pH 7.4. Furthermore, we investigated the effect of the guanidinylation on the dispersion of the GCNFs. However, the guanidinylation did not provide a favorable effect toward the neutralization. The neutralized GCNF dispersion was completely aggregated to be a brittle gel state. We suppose that, in the case of GCNFs, the guanidino and acetyl groups were inhomogenously distributed on the nanofiber surface, and a single GCNF has a higher surface area than a single GChNC. Therefore, aggregation occurs in multiple sites on a single fiber by contact with the alkaline solution, which leads to gelation. This result indicated that the guanidinylation is effective in improving the dispersion of ChNC particles with a smaller aspect ratio. Furthermore, we demonstrated that the dispersibility of the GChNCs at neutral pH can be utilized for material the development where the 5.0 wt% gelatin–0.5 wt% GChNC composite hydrogel displayed 5.0-fold higher fracture stress than the gelatin hydrogel. Although the effect of DA on dispersibility is yet unclear, we proved that GChNCs were able to directly disperse in neutral media. This is the first report of direct dispersion of cationic nanochitins in a neutral medium. In addition, previously reported N-trimethylated nanochitins were highly substituted, and, therefore, there is a concern that this will result in worse biocompatibility.¹⁶ However, the GChNCs displayed high biocompatibility applicable for bioadhesives.²¹ These GChNCs will provide innovative nanochitin-based materials for biomedical applications. Clarification of the effect of DA and the development of GChNC-based biomaterials are now in progress.

Author contributions

H. I. and S. F. conceived the experiments H. I., W. T., D. S., S. A., D. A. Z., and Y. Y. performed experiments. H. I. and S. F. wrote the manuscript. All authors have given approval to the final version of the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary information (SI). The SI contains ¹³C NMR analysis of the surface chitosan on GChNC, dispersibility of GChNC in simulated body fluid, preparation of homogeneous GChNF dispersion by a high pressure homogenization treatment, and injectability of gelatin-GCNC composite hydrogel. See DOI: https://doi.org/10.1039/d5tb01771h.

Acknowledgements

Professor Tatsuya Oshima and Professor Asuka Inada at University of Miyazaki are acknowledged for their provision of nanoPartica SZ-100V2 and their assistance with the measurements. This paper is based on results obtained from a project, JPNP20004, subsidized by New Energy and Industrial Technology Development Organization (NEDO).

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