Chiral nematic self-assembly of minimally surface damaged chitin nanofibrils and its load bearing functions

Chitin is one of the most abundant biomaterials in nature, with 1010 tons produced annually as hierarchically organized nanofibril fillers to reinforce the exoskeletons of arthropods. This green and cheap biomaterial has attracted great attention due to its potential application to reinforce biomedical materials. Despite that, its practical use is limited since the extraction of chitin nanofibrils requires surface modification involving harsh chemical treatments, leading to difficulties in reproducing their natural prototypal hierarchical structure, i.e. chiral nematic phase. Here, we develop a chemical etching-free approach using calcium ions, called “natural way”, to disintegrate the chitin nanofibrils while keeping the essential moiety for the self-assembly, ultimately resulting in the reproduction of chitin’s natural chiral structure in a polymeric matrix. This chiral chitin nanostructure exceptionally toughens the composite. Our resultant chiral nematic phase of chitin materials can contribute to the understanding and use of the reinforcing strategy in nature.


Physical disintegration of chitin to chitin nanowire: Ca-methanol gel
Ca-methanol was prepared as follows 10 . Calcium chloride di-hydrate (428 g) and DI water (30 ml) were added to methanol (470 ml). The mixture was refluxed at 150 °C for 6 h. To produce Ca-methanol gel, chitin powder (8 g) was added to the Ca-methanol (400 ml), then the mixture was refluxed in at 150 °C for 6 h and cooled down at room temperature.

Solvent exchange of Ca-methanol gel to produce hydrogel, methanol gel, and IPA gel
Ca 2+ ions were removed from the Ca-methanol gel via solvent-exchange with three different solvents: DI water, methanol, and IPA 10 . The Ca-methanol gel was immersed in excess volume of each exchange solvent with vigorous stirring for 12 h, then filtrated by nylon cloth to collect the chitin gel precipitate. The chitin gel precipitates were centrifuged at 8000 rpm for 15 min to remove excess of solvent. Consequently, hydrogel (=chiral chitin nanowire hydrogel), methanol gel, and IPA gel were obtained.

Chitin and calcium concentration
Chitin concentration (weight per volume; g/L) of each gel was evaluated as follows. Ten millilitre of each gel was incubated in EDTA buffer at 37 °C for 48 h with shaking and vigorously washed with DI water. The insoluble part was dried at 100 °C in a vacuum oven for 24 h. And then, the dry weight of the insoluble part was measured.
Calcium concentration (weight per volume; g/L) of each gel was evaluated as follows. Ten millilitre of each gel was dried at 100 °C in a vacuum oven for 24 h. And then, the weight of the dried sample was measured. Calcium weight in the dry sample was investigated using an inductively coupled plasma atomic emission spectroscopy (ICP-AES) on an IRIS Advantage 1000.

Preparation of chitin nanowhisker and nanofiber
Chitin nanowhisker was prepared as follows. 17 Chitin powder (20 g) was added into 3 N HCl (400 ml). And then, the mixture was refluxed at 150 °C for 6 h. The solution was purified 3 times with DI water using a series of dilution-centrifugation-decantation process. The suspension was dialyzed overnight in DI water, then lyophilized. The lyophilized chitin nanowhisker was re-dispersed in 50 mM acetate buffer (pH 4) via ultra-sonication.
Chitin nanofiber was prepared as follows [16][17][18] . Chitin powder (20 g) was suspended in 20 wt% NaOH (aq) (400 ml), then refluxed at 150 °C for 6 h. The wet sample was vigorously washed with DI water, then dispersed in DI water to be ~1.3 wt %. Drops of acetic acid was added into the chitin suspension to adjust the pH value to ~4. For disintegration, the chitin suspension passed through a high performance grinder (MKCA6−3; Masuko Sangyo Co., Ltd.) with the rotation speed of 1500 rpm and a clearance gauge of −1.5. The chitin nanofiber suspension was dialyzed overnight in DI water, then lyophilized. The lyophilized chitin nanofiber was re-dispersed in 50 mM acetate buffer (pH 4) by ultra-sonication.

Cryo-TEM
Each sample was placed on gold-plated membrane carriers 29, 31 . The sample-containing carrier was loaded into a Leica EM HPM100 (Germany) and rapidly frozen with liquid nitrogen under high pressure of 2100 bar. The sample was freeze-substituted with 0.2% uranyl acetatecontaining dry acetone at −80 °C for 3 days. The sample was warmed from −80 °C to −20 °C, immersed in pure acetone for 3 day, infiltrated with acetone/epoxy resin precursor (epoxy embedding medium kit) mixtures (4:1 for 75 min and 2:1 for 2 h) at −20 °C, and infiltrated with the pure epoxy resin precursor for 12 h at room temperature. The sample was cured at 70 °C for 24 h. The resin was cut into 200 nm-sections using Leica EM UC7 (Germany) and applied onto copper grids. All TEM images were recorded using a JEOL JEM 1011 transmission electron microscope. The resin precursor contains epoxy embedding medium (20 ml), 2-dodecenylsuccinic anhydride (9 ml), methyl nadic anhydride (12 ml), and 2,4,6tris(dimethylaminomethyl)phenol (1 ml).

Equilibrium water content (EWC) and equilibrium alcohol content (EAC)
To characterize the degree of water and alcohol absorption, the EWC and EAC were measured 32 . Dry chitin (or regenerated chitin) was weighed to an accuracy of 10 −4 g and immersed in water (or isopropanol) for 1 day. And then, the immersed film was weighed 4 again after removal of excess liquid. EWC or EAC were calculated using the following equation: − 0 100 W0 is the weight of the sample before immersion and Wt is the weight of the sample after immersion at time t when the weight of the sample reaches the equilibrium state.
The regenerated chitin was prepared as follows. One gram of chitin powder was dissolved in nine gram of 1-ethyl-3-methylimidazolium acetate ([C2mim] [OAc], an ionic liquid) at 100 °C for 6 h with stirring. The solution was cooled down in ambient conditions for 1 day to form a gel. The gel was soaked in excess of water for 6 h and, then washed with distilled water.

Chitin nanowire-embedded epoxy composites
Ca-methanol gel, IPA gel, and hydrogel were rapidly frozen with liquid nitrogen 29, 31 . Each sample was freeze-substituted with pure acetone at −80 °C for 3 days and at −20 °C for 3 days. The sample was infiltrated with acetone-epoxy resin precursor mixtures (4:1 for 1 day and 2:1 for 1 day) at −20 °C, and infiltrated with the pure epoxy resin precursor at room temperature for 12 h. The chitin/epoxy resin precursor suspension was centrifuged at 8000 rpm for 20 min. The desire amount of the epoxy resin precursor was added into the precipitate-containing bottle to adjust chitin contration. And then, the mixture was homogenized with a domestic blender and an ultra-sonicator. The suspension was cured at 70 °C for 24 h. The epoxy resin precursor contains epoxy embedding medium (20 ml), 2dodecenylsuccinic anhydride (22 ml), methylnadic anhydride (2 ml), and 2,4,6tris(dimethylaminomethyl)phenol (0.6 ml).

Nanofiber-and nanowhisker-embedded epoxy composites
Nanofiber (or nanowhisker)-dispersed solution at the chitin concentration of ~20 g/L was rapidly frozen with liquid nitrogen. The sample was freeze-substituted with pure acetone at −80 °C for 3 days and at −20 °C for 3 days. The sample was infiltrated with acetone-epoxy resin precursor mixtures (4:1 for 1 day and 2:1 for 1 day) at −20 °C, and infiltrated with the pure epoxy resin precursor at room temperature for 12 h. The chitin/epoxy resin precursor suspension was centrifuged at 800 rpm for 20 min. The desire amount of the epoxy resin precursor was added into the precipitate-containing bottle to adjust chitin concentration. And then, the mixture was homogenized with a domestic blender and an ultra-sonicator. The suspension was cured at 70 °C for 24 h. The epoxy resin precursor contains epoxy embedding 5 medium (20 ml), 2-dodecenylsuccinic anhydride (22 ml), methylnadic anhydride (2 ml), and 2,4,6-tris(dimethylaminomethyl)phenol (0.6 ml).
PEGDA is a precursor of PEG hydrogel, and PI is an UV-responsive cross-liking agent of the PEGDA. The chiral chitin nanowire hydrogel has chitin concentration of ~60 g/L. Desired amount of water, PEGDA, and PI were added into the chiral chitin nanowire hydrogel: PEGDA (150 g/L), PI (1.5 g/L), and chitin concentration (0, 5, 10, or 20 g/L). And then, the mixtures were vigorously homogenized using a domestic blender and an ultra-sonicator. The homogenized suspensions were incubated at 4 °C for 3 days for PEGDA infiltration, then cured by 15 min UV irradiation.

Nanowhisker-and nanofiber-embedded PEG hydrogel composites
Nanowhisker and nanofiber-embedded PEG hydrogels were prepared as follows 33

XRD
The crystal structure of chitin and chiral chitin nanowire was studied using wide-angle X-ray diffraction experiment. The experiment was conducted on an X-ray diffractometer (XRD) (D/MAX-2500/PC, Rigaku, Japan), in which a 40 kV/100 mA Ni-filtered Cu Kα radiation was employed. The WRD patterns were recorded in the region of a scattering angle of 5° to 40° with a scanning speed of 4°/min.

1) Chitin/epoxy composites
The tensile properties of the chitin nanowire/epoxy composites were measured on a universal tensile tester (UTS, Instron, Norwood, UK). All samples were cut into the following dog-bone shape.
6 Each sample was clamped onto the grips and loaded with a constant strain rate of 5 mm/min till failure. To calculate the tensile stress (σ, MPa, load per unit cross-sectional area) on the films, thickness and width of the films were determined using a micrometer before the test. In the strain-stress curve, Young's modulus was determined as the initial slope of the curve at 0.2% strain and toughness was determined as the area under the curve until its failure.

2) Chitin PEG hydrogel composites
The tensile properties of the chitin/PEG hydrogel composites were measured on a universal tensile tester (UTS, Instron, Norwood, UK). All PEG hydrogels were cut into 30 mm × 5 mm × 4 mm cuboid shape. Each sample was clamped onto the grips with 10 mm distance and loaded with a constant strain rate of 5 mm/min till failure. To calculate the tensile stress (σ, MPa, load per unit cross-sectional area) on the films, thickness and width of the films were determined using a micrometer before the test. In the strain-stress curve, Young's modulus was determined as the initial slope of the curve at 0.2% strain and toughness was determined as the area under the curve until its failure.

EWC and EAC of pure chitin and regenerated chitin
Chitin is neither meltable nor soluble in most solvents. Only solvents, such as NaOH/urea (aq) and ionic liquids, have been found to successfully dissolve chitin at molecular level 32 .
Re-solidified chitin from those of chitin solutions is called "regenerated chitin", which have low crystallinity, in other word high content of amorphous region. Thus, the EWC data ( Fig.S4) suggests that the crystalline region of chitin is less hydrated than the amorphous domain of chitin. However, the difference between EAC values of pure chitin and regenerated chitin was much lower than the difference between EWC values.
Tensile properties of the chitin nanowire-embedded PEG hydrogel.
The chiral chitin nanowire was impregnated into the PEG hydrogel (Fig. S5). To investigate how the morphology of the chitin nanomaterials affects their reinforcing property, nanowhisker and chitin nanofiber were also impregnated into the PEGDA 33     Each value and error bar represents the mean of quadruplicate samples and its standard deviation.