Combination of amorphous silk fiber spinning and post-spinning

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Combination of amorphous silk fiber spinning and postspinning crystallization for tough regenerated silk fibers Kenjiro Yazawa, Ali D Malay, Nao Ifuku, Takaoki Ishii, Hiroyasu Masunaga, Takaaki Hikima, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00232 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Combination of amorphous silk fiber spinning and post-spinning crystallization for tough regenerated silk fibers Kenjiro Yazawa,†,§ Ali D. Malay,†,§ Nao Ifuku,† Takaoki Ishii,† Hiroyasu Masunaga,‡,# Takaaki Hikima,# and Keiji Numata*,† †

Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

‡

Japan Synchrotron Radiation Research Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 6795198, Japan.

*Corresponding author Keiji Numata Tel.: +81-48-467-9525, Fax: +81-48-462-4664, E-Mail: [email protected] Author Contributions §(K.Y. and A.D.M.) These authors contributed equally to this work.

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ABSTRACT

An artificial spinning system using regenerated silk fibroin solutions is adopted to produce highperformance silk fibers. In previous studies, alcohol-based agents, such as methanol or ethanol, were used to coagulate silk dope solutions, producing silk fiber with poor mechanical properties compared with those of native silk fibers. The alcohol-based coagulation agents induce rapid !sheet crystallization of the silk molecules, which inhibits subsequent alignment of the !-sheet crystals. Here, we induce gradual !-sheet formation to afford adequate !-sheet alignment similar to that of native silk fiber. To this aim, we developed an amorphous silk fiber spinning process that prevents fast !-sheet formation in silk molecules by using tetrahydrofuran (THF) as a coagulation solvent. In addition, we apply postdrawing to the predominantly amorphous silk fibers to induce !-sheet formation and orientation. The resultant silk fibers showed a 2.5-fold higher extensibility, resulting in 1.5-fold tougher silk fibers compared with native Bombyx mori silk fiber. The amorphous silk fiber spinning process developed here will pave the way to the production of silk fibers with desired mechanical properties.


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INTRODUCTION Silk is a lightweight and tough structural material with excellent strength and ductility, which plays a structural role in silkworm cocoons and spider dragline and web.1, 2 In addition, silk is naturally derived, biocompatible with low cytotoxicity, and biodegradable,3, 4 suggesting that silk has the potential to substitute petroleum-based polymeric materials.5 Although silk is difficult to dissolve in commonly used organic solvents due to the high density of hydrogen bonds, silk is soluble in concentrated chaotropic agents, such as lithium bromide or lithium thiocyanate; hexafluoroisopropanol; and CaCl2-formic acid mixed solvent, which results in regenerated silk fibroin solutions.6, 7 To produce high-performance silk fibers from such regenerated silk solutions, artificial spinning systems have been studied by several groups.7-14 In previous studies, the regenerated silk fibroin solution was spun using an alcohol coagulation bath with, for example, methanol or ethanol. However, the production of silk fibers with both strength and extensibility was still difficult.10-14 This is because alcohol-based coagulants induce the fast !-sheet formation of silk molecules. The rate of the !-sheet formation is too rapid to attain subsequent alignment of the !-sheet crystals, judging from the mechanical properties of the resultant silk fibers, which have strength but lack extensibility.13, 15 In addition to the solidification of silk solution in an alcohol bath, postdrawing is an essential process to enhance the strength of silk fibers, enabling the production of silk fibers with higher tensile strength.10-14 The extensibility of the resultant silk fibers is generally low, resulting in poorer overall toughness compared with native silk fibers.10 Alternatively, biomimetic spinning systems have been developed by using recombinant silkworm silk or spider silk-like protein solutions as the starting material.9, 16, 17 In nature, silkworms and spiders spin silk fibers from concentrated silk fibroin solutions, which are stored in specialized glands under physiological conditions.18 Such natural spinning systems are



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sophisticated and can be finely tuned by pH, salt concentration, water content, spinning rate, and shear forces through self-assembly of silk fibroins.18, 19 Biomimetic silk spinning has been performed using aqueous coagulation baths with various pH values and ionic compositions as well as different reeling speeds.16, 17 Though the tensile strength of biomimetic silk fiber is enhanced, it is still difficult to obtain the biomimetic silk fiber with high extensibility, probably due to the difficulty in controlling gradual changes in pH and ion composition as well as shear forces that are used in native spinning systems.16, 17 By using alcohols and their solutions as a coagulation baths, several artificial tough silk fibers were produced.20-23 In particular, the biomimetic fibers of recombinant spidroins containing the N- and C-terminus motifs demonstrated the same toughness as natural spider draglines.21 Artificial silk spinning systems using regenerate silk fibroin or recombinant silk protein solution have been developed, as mentioned above. However, the produced fibers do not show both strength and extensibility as native silk fibers do. To produce regenerated silk fibers with both strength and extensibility, we prepared predominantly amorphous silk fibers with a trace amount of !-sheet structures and then postdrew the silk fibers to align the !-sheet crystals. In the present study, we found that tetrahydrofuran (THF), which is a heterocyclic ether, can solidify the regenerated silk fibroin solution and effectively prevent crystallization of the silk molecules. We used a spinning setup containing regenerated silk solutions derived from Bombyx mori (B. mori) silk with THF as the coagulation solvent, followed by postdrawing to induce the crystallization and alignment of the silk fibers. As a post-treatment, we applied vapor annealing in organic solvent and a heating process to induce further !-sheet formation in the produced silk fibers. The combined process of amorphous silk fiber spinning and postdrawing provided silk fibers with higher extensibility and toughness than natural B. mori silk fibers. This spinning


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process based on our material design contributes to the formation of new types of regenerated silk fibers with desired mechanical properties.

EXPERIMENTAL SECTION Preparation of regenerated silk fibroin aqueous solution. B. mori silkworm silk fibers were first degummed by boiling in 0.02 M Na2CO3 solution for 30 min.6 After thorough washing in deionized water, a degummed silk fibroin solution in aqueous 9.3 M LiBr was prepared by heating to 60°C for 4 h with continuous stirring.6 This solution was concentrated by reverse dialysis using SnakeSkin Dialysis Tubing (MWCO: 3.5 kDa, ThermoFisher, Waltham, MA) against a 20 wt% aqueous solution of polyethylene glycol (20 kDa, Sigma) at 25°C for 12 h to yield a 29 wt% regenerated silk fibroin solution.13 The silk solution of 1 mL was dried at 60°C for 24 h, and subsequently the resultant dried silk protein was weighed to determine the concentration of the silk solution according to a previous report.24 Preparation of silk film. The degummed silk fibroin solution in aqueous 9.3 M LiBr was dialyzed against water and then cast on a plastic Petri dish. After drying, a silk film with a thickness of approximately 10 µm was obtained. !-sheet formation in silk film induced by organic solvents. The silk film was immersed in

seven kinds of organic solvents, namely, tetrahydrofuran (THF), dioxane, methanol (MeOH), isopropanol

(iPrOH),

dimethyl

sulfoxide

(DMSO),

dimethylformamide

(DMF),

and

dimethylacetamide (DMAc), for 72 h, and then, the silk film was dried under ambient conditions. Spinning of regenerated silk fibroin solution. Regenerated silk fibroin solutions were spun at 25°C using a micro spin tester (KISCO, Tokyo, Japan) equipped with two coagulation baths for



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solidification and four rollers to postdraw the solidified silk. The regenerated silk fibroin solution was extruded directly into a THF coagulation bath at 0.1 mL/min through a nozzle with a 0.2 mm diameter at 25°C. The air gap between the spinneret and the coagulation bath was 1 mm. The solidified silk was postdrawn in the second coagulation bath (water or THF). The postdrawing rate of the first roller was set at 2.5 m/min. The draw-down ratio was defined as the ratio of the rate of the third roller to that of the first roller. Three sets of draw-down ratios were applied in this study. At a draw-down ratio of 1, all four rollers were reeled at 2.7 m/min. At a draw-down ratio of 3, the first roller was reeled at 2.7 m/min, and the following three rollers were reeled at 8.1 m/min. At a draw-down ratio of 4.5, the first and second rollers were reeled at 2.7 and 8.1 m/min, respectively, and the following two rollers were reeled at 12.2 m/min. The first, second, and fourth rollers were set at 25°C, whereas the third roller was set at 60°C to evaporate any THF that remained in the postdrawn fiber. The resultant silk fibers were finally wound on a bobbin. Preliminary spinning experiment using additive solvents in silk dope. The 29 wt% regenerated silk fibroin solution was mixed with an additive solvent, either 1 wt% dioxane or 1 or 3 wt% DMSO, and then spun directly into a 30 mL THF coagulation bath at 6 mL/min through a 0.7 mm-diameter syringe needle. Then, the silk fiber was immersed in THF for 1 min and dried under ambient conditions. Preparation of silk fibers with different water contents. The silk fibers were incubated at 25°C for 48 h under various relative humidities (RHs), controlled by the saturated water vapor of seven types of salts, according to a previous method.25 The seven types of salts used in this study were lithium bromide (RH6%), lithium chloride (RH11%), potassium acetate (RH23%),


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magnesium chloride (RH33%), potassium carbonate (RH43%), sodium bromide (RH58%), sodium chloride (RH75%), and potassium sulfate (RH97%). Vapor annealing of silk fibers in organic solvents. The silk fibers were incubated for 30 min under organic solvent vapor (methanol, acetone, or acetonitrile) at 75°C, 66°C, and 92°C; these temperatures are 10°C higher than the boiling temperature of the respective solvent at ambient pressure. Heating of silk fibers. The silk fibers were incubated at 220°C, which is the crystallization temperature of silk,26 in a vacuum oven for 5 min. Wide-angle X-ray scattering (WAXS) measurement. Synchrotron WAXS measurements were conducted at the BL45XU beamline of SPring-8, Harima, Japan, according to a previous report.27 The X-ray energy was 12.4 keV at a wavelength of 0.1 nm, the sample-to-detector distance for the WAXS measurements was 258 mm, and the exposure time for each diffraction pattern was 10 s. The measurements were performed under a controlled RH. The obtained diffraction data were converted into one-dimensional profiles using the software Fit2D.28 The data were corrected for the background diffraction, and the crystallinity was calculated from the area of the crystal peaks divided by the total area of the crystal peaks and the amorphous halo by fitting the Gaussian function using Igor Pro 6.3 (WaveMetrics, Inc., Portland, OR). The current scattering profiles seemed to contain various peaks in terms of sizes and distributions. Thus, we selected the Gaussian function instead of the Lorenz function for the peak separation in this study due to the polydisperse scatterings. The crystallite sizes, determined from the (020) diffraction peak, were evaluated by Scherrer’s equation, as shown in equation 1:29



D=

!" !"cos#

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(1)

where D is the crystallite size, K is the shape factor, " is the wavelength of the X-ray beam, ! is the full width at half maximum (FWHM) of the diffraction peak and # is the corresponding Bragg angle. We used a value of 0.9 for the shape factor based on a previous report on the crystallite size of native B. mori silk.30 Scanning electron microscopy (SEM) measurement. The surface morphology and cross section of the silk fibers were assessed by SEM (JCM 6000, JEOL Ltd., Tokyo, Japan). The samples were mounted on an aluminum stub with conductive tape and sputter-coated with gold for 1 min using a Smart Coater (JEOL, Tokyo, Japan) prior to SEM visualization at 5 kV. Tensile tests. Tensile tests of the silk fibers were conducted by using a mechanical testing apparatus (EZ-LX/TRAPEZIUM X, Shimadzu, Kyoto, Japan). The initial distance between jigs to fix a silk fiber was set at 5 mm and was confirmed by a precise caliper. The extension speed was set at 10 mm/min using a load cell with a 1 N load. When the RH for the tensile test was not controlled, the condition was at approximately 25!°C and RH45%. The cross section was imaged by SEM. The cross-sectional area was calculated by tracing the edge contours of the silk fiber using ImageJ (NIH, Bethesda, MD). The tensile strength, Young’s modulus, elongation at break, and toughness were obtained from the resultant stress-strain curves. Statistical analysis. Tukey’s Honest Significant Difference (HSD) test was used in conjunction with one-way analysis of variance (ANOVA) for single step multiple comparisons to analyze the results using IBM SPSS Statistics for Macintosh v. 22 (IBM, Armonk, NY). Data in experiments are expressed as the means ± standard deviation (n = 3).


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RESULTS AND DISCUSSION

Effect of organic solvents on !-sheet formation. A B. mori silkworm silk film was prepared to assess the effect of organic solvents on the !-sheet formation of silk molecules. The silk film was immersed in seven kinds of organic solvents using the experimental setup. The silk films turned cloudy after immersion in the organic solvents, except after immersion in THF and dioxane. The silk film immersed in THF or dioxane retained its transparent appearance (Figure S1). The silk films immersed in organic solvents were examined by WAXS to confirm structural changes in the silk films (Figure 1a and Figure S2). In the WAXS profiles, several peaks appeared after immersion of the silk films in organic solvents, consistent with the !-sheet formation of silk molecules.31 The crystallinity was calculated by fitting the resultant WAXS profiles with Gaussian functions (Figure S3), which showed an increase in crystallinity after immersion in organic solvents (Figure 1b). The crystallinity of the silk film immersed in THF did not significantly change compared with that of the untreated silk film. The crystallization of silk can be mostly prevented by using THF as the coagulation agent, among the seven organic solvents used in this study. Based on the chemical structure, THF and dioxane are heterocyclic ethers with lower polarity than the other five organic solvents used in this study.32 The !-sheet formation of silk is induced by newly formed intermolecular hydrogen bonds between silk molecules.33 This rearrangement of hydrogen bonds is driven by the interaction between water molecules and the organic solvent. The hydrogen bonds between water molecules and silk are rearranged to intermolecular hydrogen bonds between silk chains by the interaction of water and



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Figure 2. Schematic illustration of the spinning device for the regenerated silk fibroin solution: a, 29 wt% silk dope; b, air gap (1 mm); c, first coagulation bath (THF); d, second coagulation bath (water or THF); e, first draw roller (25°C); f, second draw roller (25°C); g, third draw roller (60°C); h, fourth draw roller (25°C); i, bobbin.

The postdrawn silk fibers were analyzed by tensile tests (Figures 3a and S5a–c). The silk fiber postdrawn at a draw-down ratio of 1 showed relatively lower strength and elongation, possessing a tensile strength of 0.11 ± 0.01 GPa and an elongation at break of 7 ± 2%. The tensile strength and elongation at break increased after postdrawing. The silk fiber postdrawn at a draw-down ratio of 3 showed a tensile stress of 0.18 ± 0.01 GPa and an elongation at break of 67 ± 12%. The silk fiber postdrawn at a draw-down ratio of 3 had a slightly lower tensile strength but higher elongation at break than the fiber spun at a draw-down ratio of 4.5. The tensile strength and elongation at break of the silk fibers prepared at a draw-down ratio of 4.5 were 0.22 ± 0.01 GPa and 44 ± 7%, respectively. As a control, the native B. mori fiber was subjected to tensile testing (Figures 3a and S5d), which agreed to a previous report.34 The mechanical properties of the silk fibers prepared in this section and the native silk fiber are summarized in Figure 3b–e. The tensile strength of the postdrawn silk fiber was still poorer than that of the native silk fiber (Figure 3b). The elongation at break was the highest when the draw-down ratio was 3, showing an approximately 2.5-fold increase over that of native silk (Figure 3c). The Young’s modulus showed the same tendency to increase as the draw-down ratio increased (Figure 3d). Consequently, the toughness of the postdrawn silk was the highest at the draw-down ratio of 3, which exceeded the toughness of the native silk fiber (Figure 3e).


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two-dimensional profiles and are plotted in Figure 5a. The postdrawn silk fibers and native B. mori fiber contained similar !-sheet crystals. The azimuthal intensities of the WAXS profiles of the radially integrated (020) peak of the postdrawn silk fibers are plotted in Figure 5b and indicate that the FWHM of the postdrawn fibers decreased as the draw-down ratio increased. A smaller FWHM means a higher degree of orientation, and hence the smaller FWHM and interference fringes in the WAXS profile of the native B. mori fibers demonstrated a higher degree of orientation than the postdrawn silk fibers at a draw-down ratio of 4.5 (Figures S6d and 5b-iv). The postdrawn silk fibers were evaluated in terms of the degree of crystallinity based on the peak separations of the WAXS profiles (Figure S7). The crystallinity of the postdrawn fibers was not dependent on the draw-down ratio and was significantly lower than that of the native B. mori fiber (Figure 5c). The crystallite sizes of the postdrawn fibers and native B. mori fiber were evaluated using the FWHMs from the one-dimensional radial integration WAXS profiles according to Scherrer’s equation (Figure 5d).29 The crystallite size of the native B. mori fiber was similar to that given in a previous report35 and was significantly higher than those of the postdrawn silk fibers. Structural comparison between the postdrawn silk fibers and B. mori fiber indicated that the native B. mori fiber has an adequate molecular chain alignment, resulting in both high strength and extensibility. However, the postdrawn fiber spun at a draw-down ratio of 3 possesses a comparable crystalline orientation but relatively low crystallinity, which contributes to the higher toughness compared with the native B. mori fiber (Figure 3e). The toughness was enhanced by an increase in elongation at break, even though the tensile strength of the postdrawn fiber spun at a draw-down ratio of 3 was lower than the postdrawn fiber spun at draw-down ratios of 1 and 4.5.




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Effect of second coagulation bath. As mentioned above, water was used as a second coagulation bath to prevent further crystallization and to remove water-soluble impurities in the silk dope. As a result, the tensile strength of the resultant silk fiber was lower than that of the native silk fiber. In this section, we used THF instead of water as the second coagulation bath to induce further solidification of silk molecules to enhance the tensile strength of the silk fibers to a comparable level to that of native silk fiber. Tensile tests were performed on the postdrawn silk fibers using THF as a second coagulation bath (Figure S8), and the tensile strength and Young’s modulus were found to increase as the draw-down ratio increased (Figure S9). Interestingly, the elongation at break and toughness were the highest at the moderate draw-down ratio of 3. The crystallinity and crystallite size were evaluated from the WAXS data (Figures S10–S12) and were not influenced by the draw-down ratios. The degree of orientation increased as the drawdown ratio increased. The SEM images showed that the cross sections of the postdrawn silk fibers were homogeneous with a smooth surface (Figure S13). The mechanical properties of the postdrawn silk fibers were not influenced by either the water or THF second coagulation bath but were dependent on the draw-down ratio. On the other hand, the crystallinity was significantly higher when THF, rather than water, was used as the second coagulation bath. In our spinning system, THF is necessary as the first coagulation bath to solidify the silk solutions and prevent fast crystallization, whereas both water and THF can be used as the second coagulation bath. The water coagulation bath can remove impurities in the silk dope solutions, such as short protein fragments from highly degraded proteins, or entrapped air-bubbles during processing, while THF can induce further !-sheet formation of the resultant silk fibers.



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Effect of additive solvent in silk dope solutions. We applied additive solvents in the silk dope solutions to avoid fast !-sheet formation in the fast coagulation step and investigated the effect of additive solvents in silk dope solutions on the mechanical and structural features of the resultant silk fibers. We selected dioxane and DMSO as the additive solvents among the seven organic solvents used in this study because the 29 wt% concentrated silk dope solution was rapidly solidified and precipitated when mixed with 1 wt% of the other five organic solvents (THF, MeOH, iPrOH, DMF, or DMAc). Based on the visual appearance of the resultant silk fibers (Figure S14), the silk fibers spun using silk dope solution with additive solvents (dioxane or DMSO) were more transparent than the silk fibers spun without any additive solvent in the silk dope. We performed WAXS analyses of the silk fibers that were spun using silk dope solutions with additive solvents (Figures 6a and S15). The crystallinity of the silk fibers was calculated by deconvoluting the WAXS profiles (Figure S16). The crystallinity was decreased by the addition of dioxane or DMSO to the silk dope solution (Figure 6b). To prevent crystallization of the resultant silk fibers, 1 wt% dioxane and 1 and 3 wt% DMSO were effective (Figure 6b).


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(v) 2.5 wt% dioxane, and (III) silk dope with (i) 0.5 wt% to (ix) 15 wt% DMSO. The dashed lines represent the positions of the WAXS peaks from the crystal regions. (b) Effect of dioxane and DMSO as additive solvents in silk dope on the crystallinity of the resultant silk fibers using a THF coagulation bath.

Accordingly, we evaluated the effect of the additive solvents (1 wt% dioxane, 1 and 3 wt% DMSO) on the mechanical and structural properties of the resultant silk fibers. Tensile tests were performed on the postdrawn silk fibers using silk dope solutions with additive solvents of 1 wt% dioxane (Figure S17), 1 wt% DMSO (Figure S18), and 3 wt% DMSO (Figure S19). The effects of additive solvents in the silk dope solutions under different second coagulation baths (water or THF) and draw-down ratios (1, 3, or 4.5) on the mechanical properties are shown in Figure 7. Necking behavior was detected in the stress-strain curves of the silk fibers spun at a draw-down ratio of 3 (Figure 7a). The addition of dioxane or DMSO in the silk dope solutions did not significantly change the tensile strength (Figure 7b). The tensile strength was influenced by the postdrawing process and increased with a higher draw-down ratio (Figure 7b). Similarly, additive solvents in the silk dope solutions did not influence the elongation at break, Young’s modulus, or toughness of the resultant silk fibers. The moderate draw-down ratio of 3 was effective for enhancing the elongation at break (Figure 7c). As the draw-down ratio increased, the Young’s modulus increased, hardening the resultant silk fiber. Consequently, the toughness of the silk fibers was the highest with the moderate draw-down ratio of 3.


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Figure 7. Effects of different additive solvents (dioxane1%, DMSO1%, or DMSO3%) in the silk dope solutions, different second coagulation baths (water or THF), and different draw-down ratios (1, 3, or 4.5) on the mechanical properties: (a) stress-strain curves, (b) tensile strength, (c) elongation at break, (d) Young’s modulus, and (e) toughness of the postdrawn silk fibers spun with the listed additive solvents. Broken lines in (b) and (d) show the tensile strength and Young’s modulus of the postdrawn silk fiber spun at draw-down ratio of 4.5, respectively. Broken lines in (c) and (e) demonstrate the elongation at break and toughness of the postdrawn silk fiber spun at draw-down ratio of 3, respectively. *Significant differences between groups at p < 0.05.

The WAXS profiles of the silk fibers spun from silk dope solutions with additive solvents were measured to evaluate the structural features of the resultant silk fibers (2D profiles, Figure S20; 1D profiles, Figures S21–S23; the peak separations of the WAXS profiles, Figures S24– S26). The crystallinity of the postdrawn silk fibers was not influenced by additive solvents in the silk dope solution or the draw-down ratio. However, the crystallinity was significantly influenced by the second coagulation bath (Figure 8a). The use of water as the second coagulation bath was more effective at preventing crystallization of the resultant postdrawn silk fibers. The crystallite size was not dependent on the draw-down ratio, additive solvent, or second coagulation bath (Figure 8b). The silk fibers spun from silk dope solutions with additive solvents were observed by SEM (Figure S27), and the fibers showed smooth and homogeneous surfaces without microvoids in the cross section, regardless of the spinning conditions.


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molecules still have mobility for molecular chain rearrangement in the fiber,36, 37 allowing posttreatment processing.

!-sheet formation induced by vapor annealing in organic solvents and heating as post-

treatment. The current spinning method successfully produced predominantly amorphous silk fibers. As a next step, we applied a post-treatment, such as vapor annealing or solvent treatment, to the resultant silk fibers to induce !-sheet formation and enhance the tensile strength. Previous studies showed that vapor annealing in organic solvents induced !-sheet formation in silk films, affording high tensile strength.38 In addition, heat treatment can be used to induce !-sheet formation in silk films by incubation at above the crystallization temperature.26 Therefore, we used vapor annealing and heat treatment as post-treatments to induce further !-sheet formation to increase the tensile strength of silk fibers produced in this study. Among the 24 types of silk fibers produced in this study, we selected the silk fiber that showed highest toughness, namely, the silk fiber spun using silk dope solution with 3 wt% DMSO, with a second coagulation bath of THF, and a draw-down ratio of 3 (Figure 7e). The tensile strength and Young’s modulus of the silk fiber significantly increased after vapor annealing in organic solvent (methanol, acetone, and acetonitrile) but decreased after heating at 220°C (Figures 9 and S28). The decrease in tensile strength and Young’s modulus after heating was probably due to partial thermal degradation of the amorphous regions of the silk fibers.26 In contrast, the elongation at break decreased after vapor annealing and heating, resulting in a decrease in toughness (Figure 9c and e). The crystallinity of the silk fibers did not change after vapor annealing in acetone or acetonitrile but increased after vapor annealing in methanol and after heating (Figure 9f). Although the crystallinity was not changed after acetone vapor annealing, the elongation at break drastically


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Figure 9. The !-sheet formation of silk fibers induced by vapor annealing in organic solvent or heating. (a) Stress-strain curves, (b) tensile strength, (c) elongation at break, (d) Young’s modulus, (e) toughness, and (f) crystallinity of the postdrawn silk fibers, showing the effects of vapor annealing in organic solvent or heating. The silk fibers used in this experiment were spun using silk dope solution supplemented with 3 wt% DMSO, a second coagulation bath of THF, and a draw-down ratio of 3. *Significant differences between groups at p < 0.05.

Effect of humidity on the mechanical properties and !-sheet formation of postdrawn silk fibers. To investigate the physical properties in addition to the mechanical properties of the silk fibers spun in this study, we further studied the water sensitivity of the resultant silk fibers. For instance, native silk fiber has affinity to water molecules owing to the polarity of the dense peptide bonds in silk.39 For this water sensitivity assay, we used the toughest silk fiber, which was spun from a silk dope solution with 3 wt% DMSO using a second coagulation bath of THF and a draw-down ratio of 3. The tensile tests of the silk fibers were performed at various RHs (Figures 10a and S32). The stress-strain curves were dependent on the RH, and the mechanical properties were calculated based on the stress-strain curves (Figure 10b–e). The tensile strength and Young’s modulus decreased at high RH (Figure 10b and d). Meanwhile, the elongation at break was highest under 43% RH, contributing to the highest toughness at 43% RH (Figure 10c and e). Based on the WAXS profiles of the silk fibers after incubation under various RHs (Figures S33–S35), the crystallinity was found to decrease as the RH increased (Figure 10f).


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under high RH, which is called supercontraction.40 Supercontraction is triggered by relaxation of the amorphous regions of the spider dragline.40 In addition, the elastic modulus of Antheraea pernyi silk exhibits an abrupt drop over a narrow range of RHs due to hydration of the amorphous regions of Antheraea pernyi silk.41 Both spider dragline silk and silkworm silk are composed of alanine-rich crystalline region and glycine-rich soft segments. The soft and amorphous regions are easily affected by water plasticization, whereas the beta-sheet formation of the alanine-rich sequence is induced by water vapor. Although the effects of water molecules on spider dragline silk and silkworm silk are different, the interaction of water and silk molecules is important to determine the physical properties under humid conditions. We previously reported that the silk film is more affected at high RH compared with the natural silk fibers.31 This is because the amorphous phase in fibers is more aligned than that in silk films. In addition, the Young’s modulus was less affected than the elongation at break, even in the case of the regenerated silk films.31 Thus, it is reasonable that the RH effect on the Young’s modulus of the regenerated fibers was less compared with the elongation at break and toughness. The mechanical properties of the silk fibers produced in this study were influenced by water molecules in a similar manner to silk hydrogels and resins with network structures.42 The silk fibers produced in this study most likely have network structures, as !-sheet crystals function as cross-linking points for the silk molecules in the fiber.

CONCLUSIONS In this study, we developed a spinning system using THF as the first coagulation bath to prevent fast !-sheet formation. The tensile strength and Young’s modulus of the resultant silk fibers increased as the draw-down ratio in the postdrawing process increased. The silk fibers spun at


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the moderate draw-down ratio of 3 showed a 2.5-fold increase in extensibility, resulting in a higher toughness than that of the natural B. mori silk fiber. However, the fibers produced in this study still did not show comparable strength to the native fibers, presumably due to relatively low crystalline content. Vapor annealing in organic solvent as a post-treatment induced further !sheet formation and enhanced the strength and modulus of the silk fibers. Thus, we can apply post-treatment to tune the mechanical and structural properties of the regenerated silk fibers to a comparable level to those of native silk fiber. The spinning system demonstrated in this study will lead to the production of high-performance silk fibers with desired mechanical properties.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Figures S1-S35 (PDF)

AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed (Tel.: +81-48-467-9525, Fax: +81-48-462-4664, e-mail: [email protected]) Notes The authors declare no competing financial interests. Author Contributions i

(K.Y. and A.D.M.) These authors contributed equally to this work.



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ACKNOWLEDGMENT K.N. acknowledges support from the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT).

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Combination of amorphous silk fiber spinning and post-spinning

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