Insights into Silk Formation Process: Correlation of Mechanical

Sep 30, 2016 - ... Properties and Structural Evolution during Artificial Spinning of Silk ... mechanical properties of animal silk (especially spider ...
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Article pubs.acs.org/journal/abseba

Insights into Silk Formation Process: Correlation of Mechanical Properties and Structural Evolution during Artificial Spinning of Silk Fibers Guangqiang Fang,† Yufang Huang,‡ Yuzhao Tang,§ Zeming Qi,# Jinrong Yao,† Zhengzhong Shao,† and Xin Chen*,† †

State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Laboratory of Advanced Materials, and ‡Department of Materials Science, Fudan University, Shanghai 200433, People’s Republic of China § National Centre for Protein Science−Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, People’s Republic of China # National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029, People’s Republic of China S Supporting Information *

ABSTRACT: The extraordinary comprehensive mechanical properties of animal silk (especially spider and silkworm silk) have led to extensive research on the underlying mechanisms involved. Herein, we selected various regenerated silk fibroin (RSF) fibers by choosing different postdraw conditions in a wet-spinning process developed in this laboratory to study their structure−property relationship. We use synchrotron radiation infrared and X-ray diffraction techniques to monitor the structural differences in these RSF fibers and correlate them with their mechanical properties. The results show that with the increase of post draw-down ratio, the β-sheet content, crystallinity, and molecular orientation in these RSF fibers increase while the crystalline size decreases. The relationship between structural changes and the draw-down ratio reflects the corresponding variation in mechanical properties, namely, an increase in breaking stress with a decline in breaking strain in relation to increases in draw-down ratio. Therefore, these results provide solid and direct evidence on the evolution of structure during the artificial spinning process and on how structure determines the final mechanical performance of silk fibers. We believe this study provides a good background on the relationship between microscopic structure and macroscopic properties in polymer science and may prove useful in the production of high performance materials, not only for silk fibers but also for other natural and synthetic polymeric materials. KEYWORDS: silk fibroin, wet-spinning, structure−property relationship, synchrotron radiation techniques

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INTRODUCTION

The methods of artificial spinning for silk protein have typically involved electrospinning,6−8 dry spinning,9−12 and wet spinning.13−17 Despite each method having its own advantages, wet spinning seems the one that is the most suitable for commercial use, and therefore many research groups have done a great deal of excellent work on it.18−21 Indeed, in our previous work, RSF fibers with improved mechanical properties to those of native silkworm cocoon silks have been successfully produced through an environmentally friendly wet-spinning process.13,16,17

Spider dragline silk, considered a biosteel, combines a high elasticity with high strength, outperforming most of the synthetic polymeric fibers available.1−4 Unfortunately, these spider dragline silk is difficult to obtain in a commercial scale because spiders are territorial and very hard to raise in captivity. Alternatively, commercial Bombyx mori (B. mori) silkworm silk is considered as a substitution because we have proved that the silkworm silk reeled directly from silkworm can achieve excellent mechanical properties like those of Nephila spider dragline silk;5 however, the silk obtained from silkworm cocoons is much worse than spider dragline silks. Therefore, the present study focuses on the production of artificial/ regenerated silk fibroin (RSF) fibers with mechanical properties similar or even superior to those of natural spider dragline silk. © XXXX American Chemical Society

Received: July 12, 2016 Accepted: September 30, 2016

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DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Information. The final RSF aqueous spinning dope was prepared by adjusting the protein concentration to 15% (w/w) with deionized water. Wet-Spinning of RSF Fibers. The spinning of RSF fibers followed the established procedure reported in our previous work.13,16,17 In brief, we used a custom-built wet-spinning apparatus with a 0.2 mm spinneret to extrude RSF fibers at room temperature. The 15% (w/w) RSF spinning dope was extruded directly into a 35% (w/w) (NH4)2SO4 coagulant bath at 60 °C. The take-up rate of the first roller was 60 r min−1, equivalent to a spinning rate of 9.4 m min−1. The draw-down ratio was defined as the rate ratio of the second roller to the first one. We used four rollers for postdraw in such a spinning process. The rotation rates of the three remaining rollers were set as 18.8, 37.6, and 56.4 m min−1 in the first series, equivalent to drawdown ratios of 2, 4, and 6, respectively. We labeled the samples as RSF-1X, RSF-2X, RSF-4X, and RSF-6X-1 to represent draw-down ratios of 1, 2, 4, and 6, respectively. In a second series of experiments, we set the rotation rates of the remaining three rollers at 18.8, 56.4, and 84.6 m min−1, equivalent to draw-down ratios of 1, 2, 6, and 9 and labeled the samples as RSF-1X, RSF-2X, RSF-6X-2, and RSF-9X, respectively. The difference in the preparation of RSF-6X-1 and RSF6X-2 fibers is illustrated in Figure S1 to clarify the procedure. The resulting RSF fibers were wound onto the spools. Then, they were immersed in an (NH4)2SO4 coagulant bath at room temperature for 6 h to further solidify the RSF fibers. After immersing in deionized water to wash out (NH4)2SO4 for another 6 h, the RSF fibers were finally dried at room temperature. Mechanical Testing. The mechanical properties of the RSF fibers were tested with an Instron 5565 mechanical testing instrument. The experimental parameters and the method for the calculation of the cross-sectional area can be found in the Supporting Information. For both cross-sectional area calculation and mechanical testing, at least 12 RSF fibers per treatment (every draw-down ratio) were used. S-FTIR Spectroscopy. The experiment were performed on both Beamline U4 at the National Synchrotron Radiation Laboratory, Hefei and Beamline BL01B1 at the National Centre for Protein Science Shanghai in the Shanghai Synchrotron Radiation Facility, China. The description of Beamline U4 and BL01B1can be found in our previous papers.40,41 The detailed experimental conditions can be found in the Supporting Information. We used PeakFit 4.12 to deconvolute the amide III band as we reported in our previous work.49 At least 16 spectra were evaluated to estimate the β-sheet content per treatment (every draw-down ratio). S-WAXD Measurements. The experiments were performed on Beamline BL16B1 at the Shanghai Synchrotron Radiation Facility with a wavelength (λ) of 0.124 nm and a spot size of 80 × 80 μm2 using a MARCCD165 detector. The detailed experimental conditions can be found in the Supporting Information. One-dimensional (1D) WAXD profiles were radially integrated with a region of 360° on the twodimensional (2D) WAXD patterns. We used PeakFit 4.12 to deconvolute 1D WAXD profiles as reported in the literature.50 The definition and calculation of crystallinity (Xc), orientation degree (Π), and crystal size (L) (by the Scherrer equation51) can be also found in the Supporting Information.

The structure−property relationship is considered of great importance in polymer science. It is well accepted that animal silk is a semicrystalline biopolymer with highly oriented antiparallel β-sheet crystals embedded in an amorphous matrix.22 In most cases, natural animal silks and RSF fibers show different properties and microscopic structures in the solid state, yet the underlying mechanism of how the secondary structure of the silk protein determines the final mechanical properties of silks are still not fully understood. Generally, in biomaterials, the correlation between the mechanical properties and the corresponding structure is very important. Evolution always accomplishes excellent performance by hierarchical structures and self-assembly at the molecular and mesoscopic levels.23−28 Therefore, B. mori silk fibroin is thought to be an outstanding model protein to understand how silk protein spins into high-performance fibers because both natural and regenerated silk fibers are relatively easy to be obtained. Several studies have addressed the relationship between the macroscopic properties and the microscopic structure,29−33 with many having achieved a fundamental understanding of the correlation between the structure and function in various kinds of silk. For instance, solid 13C NMR spectroscopy34−36 and Raman spectroscopy37−39 have been applied to determine the secondary structure of silk fibers and obtain much useful information. Further, FTIR spectroscopy and X-ray diffraction (XRD) are two major techniques frequently used to study protein conformation and crystalline structure in silk fibers. FTIR spectroscopy is a very old and well-established experimental method to analyze the conformation of proteins and polypeptides, whereas synchrotron radiation FTIR (SFTIR) microspectroscopy combines the ultrahigh brightness of a synchrotron radiation light source and a high magnification microscope, which is particularly suitable for micrometer-sized samples, like single silk fibers. In previous work, we demonstrated the successful application of S-FTIR microspectroscopy on the investigation of silk fibroin conformation in single natural silk fibers40 and RSF/carbon nanotube hybrid fibers.41 On the other hand, XRD was also extensively applied to study the crystalline structure in silk fibers.42−46 Finally, the crystallinity, orientation of the crystallite, and crystallite size in silk fibers have all been assessed through wide-angle XRD (WAXD).9,10,12,43,47,48 Despite several studies on both natural and regenerated B. mori silk fibers (and other animal silks) providing useful information concerning the protein conformation/crystalline states in silks, the silk samples used were mostly individual and lack of connection from each other. Few studies have shown the evolution of structure during the spinning/extrusion process, nor correlated this to the mechanical properties of silk fibers. Herein, we combine our previous advances in artificial spinning13,16,17 and structure characterization40,41 to study the structure of RSF silk fibers at different stages during the spinning process using S-FTIR and synchrotron radiation WAXD (S-WAXD). We attempt to provide information on the change of protein conformation as well as crystalline structure in various RSF fibers at different spinning conditions and correlate these structural changes to their mechanical properties.

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RESULTS AND DISCUSSION Mechanical Properties of RSF Fibers. Figure 1 is the representative stress−strain curves for the degummed natural B. mori silk and RSF fibers with various draw-down ratios. Their related mechanical properties are listed in Table 1. The data shown represent the different stages of RSF fibers during the spinning process, and therefore its comparison may help to assess the evolution of structure in silk formation. Indeed, increases in the draw-down ratio allows a more obvious alignment of the protein molecular chains along the fiber axis, thus leading to the formation of more β-sheet structure or βsheet nanocrystals, with a subsequent effect on the final mechanical properties of the RSF fibers. RSF fibers show a

EXPERIMENTAL SECTION

Preparation of RSF Aqueous Spinning Dope. The RSF aqueous solution was prepared according to our previous work. 13,16,17,41 The details can be found in the Supporting B

DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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obtained from three postdraw steps, whereas RSF-6X-2 from two (Figure S1). The former showed a relatively large breaking stress but low breaking strain compared to the latter. The highest draw-down ratio sample, RSF-9X, showed the largest breaking stress (0.45 GPa), but the breaking strain decreased quite significantly (27.3%), leading to a low breaking energy (approximately 90 MJ m−3) compared to RSF-6X samples. Moreover, it should be mentioned that, at times, the RSF-9X fiber was hard to obtain because it is really easy to break during the postdraw process. In addition, we demonstrated that the cross-sectional area of RSF fibers decreased with the increase in draw-down ratio. The value of the final products RSF-6X or RSF-9X was similar to that of the natural B. mori fiber, and therefore the comparison of the mechanical properties between them is meaningful. Tensile tests confirmed that RSF fibers with a higher drawdown ratio show higher values of breaking stress but at the expense of lower values of breaking strain, which is not too surprising for a polymeric material. In the meantime, the difference between the two kinds of RSF-6X fibers that have the same draw-down ratio but the different postdraw process adds more importance of spinning process on the final mechanical performance of the RSF fibers. Thus, since these mechanical properties represent a macroscopic parameter, we are more interested in how the microstructure changes in RSF fibers with the various draw-down ratios, i.e., the influence of the spinning process. Therefore, we used S-FTIR and S-WAXD to extensively analyze the protein conformation and crystalline structure in RSF fibers. S-FTIR Characterization of RSF Fibers. It is widely accepted that silk fibers can be considered a composite material in which β-sheet crystallites are embedded in the amorphous protein matrix, and therefore the mechanical properties of silk fibers are significantly dependent on the β-sheet content of the silk fibers.52 Here, we first determine the β-sheet content in different RSF fibers. In our previous work, 40,41,53 we successfully established a method to analyze the protein conformation in single silk fibers by S-FTIR microspectroscopy. This method was used herein to semiquantitatively calculate the β-sheet content in those RSF fibers with various draw-down ratios. Figure 2A shows a typical S-FTIR spectrum of the RSF fiber (RSF-9X), in which the major characteristic absorption bands of silk fibroin are well resolved, as in other single animal silks previously reported.40,41 By deconvoluting the amide III bands of FTIR spectra of RSF fibers (Figure 2B and Figure S2),40,41,53 we obtained the β-sheet content in those RSF fibers (Table 2). It demonstrated that with the increase of the drawdown ratio, the β-sheet content in RSF fiber increased. The RSF-9X fiber had the largest β-sheet content, which was almost

Figure 1. Stress−strain curves of the degummed natural B. mori silkworm cocoon silk and the RSF fibers with different draw-down ratio.

distinct yield point followed by an obvious strain hardening (Figure 1), in contrast to the naturally spun B. mori silk, which has no yield point.5 The stress−strain shape changes with an increase in draw-down ratio, i.e., the yield strength and the slope of the curve following the yield point both increase. The RSF-4X fiber behaved like an elastic material, with a large breaking strain but a relatively low breaking stress, showing a slight strain hardening trend after the yield point. However, the RSF-9X fiber showed a prominent strain hardening trend, indicating a high breaking stress with a relatively low breaking strain. The mechanical properties of these RSF fibers with various draw-down ratios using two different postdraw strategies were similar to those obtained in our previous work.13,16,17 The as-spun RSF fiber (RSF-1X) and the RSF-2X fiber were very weak and brittle, without any significant mechanical properties. When the draw-down ratio increased to 4, the RSF-4X fiber became a practical material, although with a large breaking strain (84.3%) but a low breaking stress (0.29 GPa), yielding a considerably high breaking energy of 185.7 MJ m−3. It should be noted that, following upgrading of the spinning apparatus and continuous improvement of our spinning skills, more uniform and homogeneous RSF fibers were produced, thus leading to a much larger breaking strain for the RSF-4X fiber than previously published.17 Nevertheless, such a RSF-4X fiber still had some distance from the natural silk fiber since the excellent toughness (breaking energy) of natural animal silk comes arises through the balance between breaking stress and breaking strain. Similar to our previous work,13,16,17 RSF-6X (both RSF-6X-1 and RSF-6X-2) fibers exhibited a good balance of breaking stress (approximately 0.4 GPa) and breaking strain (40−50%). As a result, their breaking energy was above 150 MJ m−3, and much higher than that of native B. mori silkworm cocoon silk. Interestingly, there was some difference between two types of RSF-6X fiber. RSF-6X-1 was

Table 1. Comparison of Mechanical Properties of the Degummed Natural B. mori Silkworm Cocoon Silk and RSF Fibers with Various Draw-down Ratio (n = 12)a samples RSF-1X RSF-2X RSF-4X RSF-6X-1 RSF-6X-2 RSF-9X degummed B. mori silk a

cross-sectional area (μm2) 804 514 348 223 236 175 183

± ± ± ± ± ± ±

26 22 21 18 20 17 15

Young’s modulus (GPa) 6.8 14.2 17.5 16.8 18.9 11.8

± ± ± ± ± ±

breaking stress (GPa)

too weak to measure 1.2 0.09 ± 0.02 1.4 0.29 ± 0.02 1.2 0.42 ± 0.03 1.3 0.37 ± 0.02 1.1 0.45 ± 0.03 0.9 0.40 ± 0.02

breaking strain (%) 3.1 84.3 41.7 48.1 27.3 19.7

± ± ± ± ± ±

0.9 5.7 5.1 6.2 4.6 1.3

breaking energy (MJ m−3) 2.3 185.7 154.8 151.3 91.0 57.3

± ± ± ± ± ±

1.08 17.7 17.6 14.3 7.4 4.7

All values measured were expressed as mean ± standard deviations (SD). C

DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 2. Characterizations of RSF-9X fiber. (A) S-FTIR spectrum of RSF-9X, (B) deconvolution of the corresponding amide III band, (C) SWAXD 1D profile, and (D) azimuthal intensity profiles of the radially integrated (200) peak.

Table 2. β-Sheet Content in the Degummed Natural B. mori Silkworm Cocoon Silk and RSF Fibers with Different Drawdown Ratio Determined by S-FTIR Spectroscopy (n = 16)a samples RSF-1X RSF-2X RSF-4X RSF-6X-1 RSF-6X-2 RSF-9X degummed B. mori silk40

1X) showed only diffraction rings (Figure 3A), indicating a random orientation of the crystallites. There was a concentration of diffraction intensity in the meridianal direction (perpendicular to fiber axis) in RSF-2X (Figure 3B), which became more significant in RSF-4X, though there were still some diffraction rings observed (Figure 3C). For the high draw-down ratio samples (RSF-6X and RSF-9X), the diffraction arcs were significant (Figure 3D−F), with those in RSF-9X being very close to the natural B. mori silk (Figure S3). Major diffractions assigned to the (200)/(020), (021)/(201), and (002) lattice planes56 showed obviously oriented characteristics in RSF-6X and RSF-9X. For RSF-9X, the (300)/(030) lattice planes were also clearly apparent. Therefore, from the 2D WAXD patterns, it is intuitively demonstrated that the crystalline orientation along the RSF fiber is enhanced with the increase in draw-down ratio; however, the crystallinity of these RSF fibers needs to be further calculated. According to the method applied in this and other laboratories, crystallinity and crystallite size can be obtained from the deconvolution of the 1D WAXD profile of RSF fibers.9,10,41,50 The 1D WAXD profiles of different RSF fibers and natural B. mori silk are shown in Figure 2C and Figure S3. The shape of the 1D WAXD profiles of the different RSF fibers and natural B. mori silk is very similar, showing a main crystalline peak at approximately d = 4.4 Å (Figure 2C), which can be resolved into two peaks of d = 4.30 and 4.48 Å. The former is the d-spacing of the (200) lattice plane and the latter is that of the (020) lattice plane, corresponding to the interchain and intersheet distance in the β-sheet crystallite, respectively.9,10,55 There are also several other lattice planes in the 1D WAXD profile, such as (023)/(420) (d = 2.09 Å), (040) (d = 2.26 Å), (022)/(130) (d = 2.81 Å), and (021) (d = 3.70 Å), all of which contribute to the crystallinity of the silk fibers.55−57 According to the method reported by Zhang and his co-workers,9,10,50 we separated these Bragg reflections from

β-sheet content (%) 22.8 23.1 24.2 26.6 25.9 28.9 28.0

± ± ± ± ± ± ±

0.6 0.4 0.4 0.3 0.4 0.6 0.3

All values measured were expressed as mean ± standard deviations (SD).

a

the same as that in natural B. mori silk (approximately 28.0%).40 Such a result confirms that the postdraw is able to further induce the conformation transition from random coil/helix to β-sheet, which is consistent with the results reported both in our39 and other laboratories.50 Such a phenomenon can be also found in the natural spinning process of Nephila pilipes spider silk51 and B. mori silkworm silk.42 In addition, there was a difference between the two RSF-6X fibers, the β-sheet content in RSF-6X-1 was larger than that in RSF-6X-2. One-way ANOVA analysis showed that the difference was highly significant (p < 0.05). S-WAXD Characterizations of RSF Fibers. S-WAXD diffraction has been widely used to analyze the structure of animal silks43,45,47,48,54,55 and artificial silk fibers,9,10,20,41 from which the crystallite size, crystallinity, and orientation about the fiber axis can been obtained.9,47 Figure 3 shows the 2D WAXD patterns from RSF fibers at various draw-down ratios. These WAXD patterns clearly show the evolution of the diffraction ring to diffraction arcs, indicating the progress of the molecular orientation along the fiber axis. The as-spun RSF fiber (RSFD

DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Table 3. Crystallinity, Crystal Size, and Orientation of the Degummed Natural B. mori Silkworm Cocoon Silk and RSF Fibers with Different Draw-down Ratio crystallite sizes (nm) samples

crystallinity (%)

La

Lb

Lc

orientation degree (%)

RSF-1X RSF-2X RSF-4X RSF-6X-1 RSF-6X-2 RSF-9X degummed B. mori silk

44.3 46.4 48.9 52.5 51.3 54.7 52.4

3.9 3.4 3.3 3.0 3.2 3.0 3.4

5.9 5.4 5.2 4.8 5.0 3.8 3.8

9.2 7.9 7.1 6.5 6.9 6.1 8.8

74.7 80.3 84.0 82.4 85.0 92.8

In addition to crystallinity, the size and orientation degree of the crystallites in RSF fibers were also calculated (Table 3). Generally, the crystallite size decreased while the orientation degree of RSF fibers increased with the increase in draw-down ratio. In particular, for the two RSF-6X fibers, RSF-6X-1 had a smaller crystalline size but the higher orientation degree compared to RSF-6X-2. Discussion on the Structural Evolution during the Artificial Spinning Process of RSF Fibers. The results presented above demonstrate that the RSF fibers show different mechanical properties and structural features at the various wetspinning stages (represented by the draw-down ratio). A more quantitative comparison of the changes in mechanical properties with relation to changes in structure against draw-down ratio can be seen through a combination of the variation of structure (β-sheet content) and mechanical properties (Young’s modulus, breaking stress, breaking stain, and breaking energy) in x−y scatter graphs (Figure 4). The as-spun RSF fiber (RSF1X) was very weak, and showed the lowest β-sheet content (or crystallinity, which is closely related to the β-sheet content in silk fibers), the largest crystalline size, and the worst degree of orientation (almost impossible to determine) among all the RSF fibers obtained. Through increases in the draw-down ratio, the structure of the RSF fibers changed with a concomitant improvement of the mechanical properties (Figure 4B−E). Generally, with the increase in draw-down ratio, the β-sheet content in RSF fibers also increased (Figure 4A). The crystallinity of these fibers showed the same tendency (Figure 5), i.e., the crystallinity and β-sheet content in the RSF fibers are positively correlated. It is well-known that different regions in silk fibers play different mechanical roles; namely, the β-sheet nanocrystals are thought to serve as molecular cross-links that provide silk fibers with their great stiffness, while the amorphous regions are responsible for their superb elasticity.61 Therefore, the β-sheet content and crystallinity obtained from S-FTIR and S-WAXD measurements (Tables 2 and 3) provide a reasonable explanation to the various mechanical properties of the RSF fibers with different draw-down ratios (Figure 1). When the as-spun RSF fiber was subjected to two rounds of postdraw, the resulting RSF-4X fiber showed acceptable mechanical properties, and in particular excellent elasticity (breaking strain of 84.3%), which resulted in the highest breaking energy (185.7 MJ m−3) among all of the RSF fibers. However, RSF-4X was not very strong (breaking stress of 0.29 GPa) because the β-sheet content (24.2%) or crystallinity (48.9%) in the fiber was not sufficiently high. When the drawdown ratio was further increased to 6 or 9, the breaking stress

Figure 3. S-WAXD patterns of the RSF fibers. (A) RSF-1X, (B) RSF2X, (C) RSF-4X, (D) RSF-6X-1, (E) RSF-6X-2, and (F) RSF-9X.

the broad amorphous background (d = 4.10 Å) to estimate the crystallinity (Xc) of the fiber, which was measured as a ratio of crystalline area to the total area (Figure S4). We know that two crystalline forms (silk I and silk II) are in B. mori silk fibroin.58 The reflections of silk II (β-sheet) reported in the literature55 were all included, as well as a reflection of silk I at d = 7.20 Å.59 It is well-known that natural B. mori silk fiber is highly crystalline, with a typical β-sheet structure;20 the measured crystallinity according the method used herein was of approximately 52.7%, which was close to the reported values in the literature.50,51,60 Table 3 shows the crystallinity calculated from the 1D WAXD profile of RSF fibers with different drawdown ratios. The results show that the crystallinity of the RSF fibers increases with the increase in draw-down ratio. The crystallinity of RSF-6X-1 (52.3%) was larger than that of RSF6X-2 (51.2%), in line with the S-FTIR results reported above, i.e., the β-sheet content of the RSF fibers increased with the increase in draw-down ratio. In addition, we found that the contribution of silk I structure to the crystallinity in the natural silk fiber can be negligible (0.1−0.2%), but its contribution in RSF fibers seems more obvious. The silk I content was 3.6% in the as-spun (RSF-1X) fiber, and decreased to 2.1−2.6% with the postdraw. There was no significant difference of silk I content among the postdraw fibers, in which RSF-9X still had approximately 2.1% of silk I structure. E

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Figure 4. (A) β-sheet content, (B) Young’s modulus, (C) breaking stress, (D) breaking strain, and (E) breaking energy of degummed natural B. mori silkworm silk and RSF fibers with different draw-down ratio.

RSF-9X was the highest among all of the RSF fibers (Figure 5) and even larger than that of the native B. mori silkworm silk (28.0% of β-sheet content and 52.4% of crystallinity).40 Further, the orientation degree of RSF fibers was also found to increase with the increase in draw-down ratio, but with a decrease in crystallite size; these results are consistent with those of previous studies.18−20 Undoubtedly, molecular chains are more difficult to move in RSF fibers with a higher level of molecular orientation, which in turn leads to an increase in stiffness and a decrease in elasticity. Therefore, the more ordered molecular orientation in the RSF fibers obtained at a high draw-down ratio also contributes to the high breaking stress and low breaking strain observed. In addition, silk is thought to be basically made up by highly ordered β-sheet nanocrystals embedded in an amorphous silk fibroin matrix from many models at the nanometer level.22 According to these models, the size of the nanocrystals plays a very important role in determining the mechanical properties of silk fibers, since it allows the conformation of silk fibroin in the matrix to unfold almost completely and thus yields maximum toughness.22 It has been reported by Keten et al. that “smaller β-nanocrystals provide a greater stiffness and fracture resistance as they are predominantly loaded in uniform shear, which leads to

Figure 5. Relationship between the crystallinity and β-sheet content in degummed natural B. mori silkworm silk the RSF fibers with different draw-down ratio.

increased and the breaking strain decreased accordingly (Figure 4C, D). For example, the breaking stress of RSF-9X reached 0.45 GPa (which is more than 10% higher than the natural B. mori silkworm silk), but its breaking strain also decreased significantly (only 27.3%). This phenomenon can be attributed to the increase in β-sheet content or crystallinity in the RSF fibers. The β-sheet content (28.9%) or crystallinity (54.7%) of F

DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

fibers, such as crystallinity, β-sheet content, molecular orientation, and crystal size, were also found to be obviously dependent on draw-down ratio variations. With the increase in draw-down ratio, the β-sheet content (determined by S-FTIR), the crystallinity, and the molecular orientation (determined by S-WAXD) in RSF fibers increased, whereas the crystalline size (also determined by S-WAXD) decreased. As a result, the Young’s modulus and breaking stress of RSF fibers increased and the breaking strain decreased accordingly. Thus, structure evolution during the spinning process firmly determines the final mechanical performance of the resulting RSF fibers. The difference in structure and mechanical properties of the two types of RSF-6X fiber further support this conclusion. Postdraw during the spinning process helps the polypeptide chains rearrange in the RSF fibers; therefore, tailoring of conditions to encourage the alignment of polypeptide chains along the fiber axis may produce an RSF fiber with a relatively “perfect” structure and excellent mechanical properties. In addition, although the structure of the final RSF fibers produced herein is similar to that of natural silkworm silk, some differences, for example, the relatively poor orientation degree and the existence of some silk I structure, still remain. These results provide a process−morphology−property connection that is necessary for the reproducible industrial production for RSF fibers with outstanding mechanical properties. We believe that the microscopic structure−macroscopic property relationship established herein is able to provide useful guidance for the design and preparation of the materials with high performance, not only for silk fibers but also for other natural and synthetic polymeric materials.63,64

cooperative rupture of hydrogen bonds and stick−slip energy dissipation mechanisms”.62 Our results support such an assumption quite well; crystallite size became smaller and smaller with the increase in draw-down ratio, likely as a result from defect reduction in the crystallites.12 We assume that when the as-spun fiber is subjected to more postdraw treatment, the molecular chains have more opportunity to rearrange, forming relatively “perfect” β-sheet nanocrystals.16 Thus, the smaller crystalline size in RSF fibers with a high drawdown ratio contributes to their improved mechanical performance as well, as is observed in spider dragline silk.51 Similar results were also found in dry-spun RSF fibers. Zhang and his co-workers proved that the post-treated dry-spun RSF fiber showed a smaller crystalline size, higher crystalline and orientation crystallinity, and better mechanical performance.9−12 Herein, we designed two types of RSF-6X fiber. The RSF6X-1 fiber was obtained from the three-stage postdraw (2X → 4X → 6X), while the RSF-6X-2 fiber was obtained from the two-stage postdraw (2X → 6X) (Figure S1). Thus, given the above reasoning, the molecular chains in RSF-6X-1 had greater adjustment than those in RSF-6X-2. The results from S-WAXD and S-FTIR measurements clearly corroborate such an assumption. The crystallinity or β-sheet content in RSF-6X-1 was higher than that in RSF-6X-2. In addition, RSF-6X-1 had a smaller crystalline size but a higher degree of molecular orientation compared to RSF-6X-2. Such differences in structural features were also accordingly reflected in the mechanical properties observed. The RSF-6X-1 fiber exhibited the higher breaking stress but lower breaking strain than the RSF-6X-2 fiber (Figure 4C, D). Moreover, it is very interesting to find that, despite there being some difference in breaking stress and strain between RSF-6X-1 and RSF-6X-2, their breaking energies were very similar (Figure 4E). This indicates that the molecular arrangement in RSF fibers with the same draw-down ratio but different postdraw procedures may differ to some extent, with the influence of the β-sheet nanocrystals (which determine the strength of the silk fiber) and the amorphous matrix (which determines the extensibility of the silk fiber) compensating one another to provide a similar material toughness. Finally, it is noted that according to the S-WAXD results, the RSF fiber produced may still retain some silk I structure that did not totally convert to the silk II structure during the spinning process. Therefore, the crystallinity shown in Table 3 is rather the “apparent” crystallinity, including both silk I and silk II contributions. If we exclude the silk I contribution, the “real” crystallinity from silk II is lower, and thus only RSF-9X is close to the natural silkworm silk. This implies that the spinning technique still requires improvement to produce a stronger and tougher RSF fiber of ideal structure.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00392. Experimental details, schematic for the collection of RSF6X-1 and RSF-6X-2 fibers, deconvolution of amide III band in S-FTIR spectra of RSF fibers, 2D S-WAXD pattern, and 1D S-WAXD profile of natural B. mori silkworm silk, deconvolution of 1D S-WAXD profiles of RSF fibers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 21 5163 0300. Tel: +86 21 6564 2866. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (21274028, 21574023, and 21574024). We thank Ms. Jiajia Zhong at NCPSS-SSRF, Prof. Min Chen, Dr. Feng Tian, and Dr. Fenggang Bian at SSRF for their technical support during data collection. We also thank Dr. Yuhong Yang at Fudan University for her valuable suggestions and discussions.

CONCLUSION In this article, we provide qualitative evidence to demonstrate that the structure of silk fiber has a significant impact on its mechanical properties. We first designed a series of RSF fiber samples by choosing the different post draw-down ratios during the wet-spinning process. Then, we tested the mechanical properties these RSF fibers and characterized their structure with advanced S-FTIR and S-WAXD techniques. The results showed that these RSF fibers exhibited significantly different mechanical properties strongly dependent on the draw-down ratio. On the other hand, the structural features of these RSF

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REFERENCES

(1) Vollrath, F.; Knight, D. P. Liquid crystalline spinning of spider silk. Nature 2001, 410, 541−548.

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Article

ACS Biomaterials Science & Engineering (2) Lewis, R. V. Spider silk: Ancient ideas for new biomaterials. Chem. Rev. 2006, 106, 3762−3774. (3) Heim, M.; Romer, L.; Scheibel, T. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins. Chem. Soc. Rev. 2010, 39, 156−164. (4) Heim, M.; Keerl, D.; Scheibel, T. Spider Silk: From Soluble Protein to Extraordinary Fiber. Angew. Chem., Int. Ed. 2009, 48, 3584− 3596. (5) Shao, Z. Z.; Vollrath, F. Materials: Surprising strength of silkworm silk. Nature 2002, 418, 741−741. (6) Kim, S. H.; Nam, Y. S.; Lee, T. S.; Park, W. H. Silk fibroin nanofiber. Electrospinning, properties, and structure. Polym. J. 2003, 35, 185−190. (7) Wang, M.; Jin, H. J.; Kaplan, D. L.; Rutledge, G. C. Mechanical properties of electrospun silk fibers. Macromolecules 2004, 37, 6856− 6864. (8) Cao, H.; Chen, X.; Huang, L.; Shao, Z. Z. Electrospinning of reconstituted silk fiber from aqueous silk fibroin solution. Mater. Sci. Eng., C 2009, 29, 2270−2274. (9) Zhang, C.; Zhang, Y.; Shao, H.; Hu, X. Hybrid Silk Fibers DrySpun from Regenerated Silk Fibroin/Graphene Oxide Aqueous Solutions. ACS Appl. Mater. Interfaces 2016, 8, 3349−58. (10) Pan, H.; Zhang, Y. P.; Shao, H. L.; Hu, X. C.; Li, X. H.; Tian, F.; Wang, J. Nanoconfined crystallites toughen artificial silk. J. Mater. Chem. B 2014, 2, 1408−1414. (11) Wei, W.; Zhang, Y. P.; Zhao, Y. M.; Luo, J.; Shao, H. L.; Hu, X. C. Bio-inspired capillary dry spinning of regenerated silk fibroin aqueous solution. Mater. Sci. Eng., C 2011, 31, 1602−1608. (12) Sun, M. J.; Zhang, Y. P.; Zhao, Y. M.; Shao, H. L.; Hu, X. C. The structure-property relationships of artificial silk fabricated by dryspinning process. J. Mater. Chem. 2012, 22, 18372−18379. (13) Zhou, G. Q.; Shao, Z. Z.; Knight, D. P.; Yan, J. P.; Chen, X. Silk Fibers Extruded Artificially from Aqueous Solutions of Regenerated Bombyx mori Silk Fibroin are Tougher than their Natural Counterparts. Adv. Mater. 2009, 21, 366−370. (14) Yao, J. M.; Masuda, H.; Zhao, C. H.; Asakura, T. Artificial spinning and characterization of silk fiber from Bombyx mori silk fibroin in hexafluoroacetone hydrate. Macromolecules 2002, 35, 6−9. (15) Phillips, D. M.; Drummy, L. F.; Naik, R. R.; De Long, H. C.; Fox, D. M.; Trulove, P. C.; Mantz, R. A. Regenerated silk fiber wet spinning from an ionic liquid solution. J. Mater. Chem. 2005, 15, 4206−4208. (16) Zhou, H.; Shao, Z. Z.; Chen, X. Wet-spinning of regenerated silk fiber from aqueous silk fibroin solutions: Influence of calcium ion addition in spinning dope on the performance of regenerated silk fiber. Chin. J. Polym. Sci. 2014, 32, 29−34. (17) Yan, J. P.; Zhou, G. Q.; Knight, D. P.; Shao, Z. Z.; Chen, X. Wet-Spinning of Regenerated Silk Fiber from Aqueous Silk Fibroin Solution: Discussion of Spinning Parameters. Biomacromolecules 2010, 11, 1−5. (18) Trabbic, K. A.; Yager, P. Comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Macromolecules 1998, 31, 462−471. (19) Marsano, E.; Corsini, P.; Arosio, C.; Boschi, A.; Mormino, M.; Freddi, G. Wet spinning of Bombyx mori silk fibroin dissolved in Nmethyl morpholine N-oxide and properties of regenerated fibres. Int. J. Biol. Macromol. 2005, 37, 179−188. (20) Um, I. C.; Ki, C. S.; Kweon, H. Y.; Lee, K. G.; Ihm, D. W.; Park, Y. H. Wet spinning of silk polymer - II. Effect of drawing on the structural characteristics and properties of filament. Int. J. Biol. Macromol. 2004, 34, 107−119. (21) Corsini, P.; Perez-Rigueiro, J.; Guinea, G. V.; Plaza, G. R.; Elices, M.; Marsano, E.; Carnasciali, M. M.; Freddi, G. Influence of the draw ratio on the tensile and fracture behavior of NMMO regenerated silk fibers. J. Polym. Sci., Part B: Polym. Phys. 2007, 45, 2568−2579. (22) Nova, A.; Keten, S.; Pugno, N. M.; Redaelli, A.; Buehler, M. J. Molecular and Nanostructural Mechanisms of Deformation, Strength and Toughness of Spider Silk Fibrils. Nano Lett. 2010, 10, 2626−2634.

(23) Du, N.; Yang, Z.; Liu, X. Y.; Li, Y.; Xu, H. Y. Structural Origin of the Strain-Hardening of Spider Silk. Adv. Funct. Mater. 2011, 21, 772− 778. (24) Nguyen, A. T.; Huang, Q. L.; Yang, Z.; Lin, N. B.; Xu, G. Q.; Liu, X. Y. Crystal Networks in Silk Fibrous Materials: From Hierarchical Structure to Ultra Performance. Small 2015, 11, 1039− 1054. (25) Porter, D.; Vollrath, F. Silk as a Biomimetic Ideal for Structural Polymers. Adv. Mater. 2009, 21, 487−492. (26) Fu, C. J.; Porter, D.; Chen, X.; Vollrath, F.; Shao, Z. Z. Understanding the Mechanical Properties of Antheraea pernyi SilkFrom Primary Structure to Condensed Structure of the Protein. Adv. Funct. Mater. 2011, 21, 729−737. (27) Vollrath, F.; Porter, D. Spider silk as archetypal protein elastomer. Soft Matter 2006, 2, 377−385. (28) Xu, G. Q.; Gong, L.; Yang, Z.; Liu, X. Y. What makes spider silk fibers so strong? From molecular-crystallite network to hierarchical network structures. Soft Matter 2014, 10, 2116−2123. (29) Glisovic, A.; Vehoff, T.; Davies, R. J.; Salditt, T. Strain dependent structural changes of spider dragline silk. Macromolecules 2008, 41, 390−398. (30) Grubb, D. T.; Jelinski, L. W. Fiber morphology of spider silk: The effects of tensile deformation. Macromolecules 1997, 30, 2860− 2867. (31) Krywka, C.; Krasnov, I.; Figuli, R.; Burghammer, M.; Muller, M. Determination of Silkworm Silk Fibroin Compressibility Using High Hydrostatic Pressure with in Situ X-ray Microdiffraction. Macromolecules 2014, 47, 7187−7193. (32) Plaza, G. R.; Perez-Rigueiro, J.; Riekel, C.; Perea, G. B.; AgulloRueda, F.; Burghammer, M.; Guinea, G. V.; Elices, M. Relationship between microstructure and mechanical properties in spider silk fibers: identification of two regimes in the micro-structural changes. Soft Matter 2012, 8, 6015−6026. (33) Wang, Y.; Guan, J.; Hawkins, N.; Porter, D.; Shao, Z. Z. Understanding the variability of properties in Antheraea pernyi silk fibres. Soft Matter 2014, 10, 6321−6331. (34) Asakura, T.; Sugino, R.; Yao, J. M.; Takashima, H.; Kishore, R. Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state 13C, 15N, and 2H NMR. Biochemistry 2002, 41, 4415−4424. (35) Zhao, C. H.; Yao, J. M.; Masuda, H.; Kishore, R.; Asakura, T. Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system. Biopolymers 2003, 69, 253−259. (36) Asakura, T.; Ohgo, K.; Komatsu, K.; Kanenari, M.; Okuyama, K. Refinement of repeated beta-turn structure for silk I conformation of Bombyx mori silk fibroin using C13 solid-state NMR and X-ray diffraction methods. Macromolecules 2005, 38, 7397−7403. (37) Zhou, W.; Chen, X.; Shao, Z. Z. Conformation studies of silk proteins with infrared and Raman spectroscopy. Prog. Chem. 2006, 18, 1514−1522. (38) Sirichaisit, J.; Brookes, V. L.; Young, R. J.; Vollrath, F. Analysis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman Spectroscopy. Biomacromolecules 2003, 4, 387−394. (39) Colomban, P.; Dinh, H. M. Origin of the variability of the mechanical properties of silk fibres: 2 The nanomechanics of single silkworm and spider fibres. J. Raman Spectrosc. 2012, 43, 1035−1041. (40) Ling, S. J.; Qi, Z. M.; Knight, D. P.; Shao, Z. Z.; Chen, X. Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers. Biomacromolecules 2011, 12, 3344−3349. (41) Fang, G. Q.; Zheng, Z. K.; Yao, J. R.; Chen, M.; Tang, Y. Z.; Zhong, J. J.; Qi, Z. M.; Li, Z.; Shao, Z. Z.; Chen, X. Tough proteincarbon nanotube hybrid fibers comparable to natural spider silks. J. Mater. Chem. B 2015, 3, 3940−3947. (42) Drummy, L. F.; Farmer, B. L.; Naik, R. R. Correlation of the beta-sheet crystal size in silk fibers with the protein amino acid sequence. Soft Matter 2007, 3, 877−882. H

DOI: 10.1021/acsbiomaterials.6b00392 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering (43) Fang, G. Q.; Sapru, S.; Behera, S.; Yao, J. R.; Shao, Z. Z.; Kundu, S. C.; Chen, X. Exploration of the tight structural-mechanical relationship in mulberry and non-mulberry silkworm silks. J. Mater. Chem. B 2016, 4, 4337−4347. (44) Numata, K.; Masunaga, H.; Hikima, T.; Sasaki, S.; Sekiyama, K.; Takata, M. Use of extension-deformation-based crystallisation of silk fibres to differentiate their functions in nature. Soft Matter 2015, 11, 6335−6342. (45) Blamires, S. J.; Liao, C. P.; Chang, C. K.; Chuang, Y. C.; Wu, C. L.; Blackledge, T. A.; Sheu, H. S.; Tso, I. M. Mechanical Performance of Spider Silk is Robust to Nutrient-Mediated Changes in Protein Composition. Biomacromolecules 2015, 16, 1218−1225. (46) Addison, J. B.; Osborn Popp, T. M.; Weber, W. S.; Edgerly, J. S.; Holland, G. P.; Yarger, J. L. Structural characterization of nanofiber silk produced by embiopterans (webspinners). RSC Adv. 2014, 4, 41301− 41313. (47) Sampath, S.; Isdebski, T.; Jenkins, J. E.; Ayon, J. V.; Henning, R. W.; Orgel, J.; Antipoa, O.; Yarger, J. L. X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks. Soft Matter 2012, 8, 6713−6722. (48) Jenkins, J. E.; Sampath, S.; Butler, E.; Kim, J.; Henning, R. W.; Holland, G. P.; Yarger, J. L. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR and X-ray Diffraction. Biomacromolecules 2013, 14, 3472−3483. (49) Chen, X.; Shao, Z. Z.; Knight, D. P.; Vollrath, F. Conformation transition kinetics of Bombyx mori silk protein. Proteins: Struct., Funct., Genet. 2007, 68, 223−231. (50) Luo, J.; Zhang, L. L.; Peng, Q. F.; Sun, M. J.; Zhang, Y. P.; Shao, H. L.; Hu, X. C. Tough silk fibers prepared in air using a biomimetic microfluidic chip. Int. J. Biol. Macromol. 2014, 66, 319−324. (51) Du, N.; Liu, X. Y.; Narayanan, J.; Li, L. A.; Lim, M. L. M.; Li, D. Q. Design of superior spider silk: From nanostructure to mechanical properties. Biophys. J. 2006, 91, 4528−4535. (52) Hayashi, C. Y.; Shipley, N. H.; Lewis, R. V. Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. Int. J. Biol. Macromol. 1999, 24, 271−275. (53) Ling, S. J.; Qi, Z. M.; Knight, D. P.; Huang, Y. F.; Huang, L.; Zhou, H.; Shao, Z. Z.; Chen, X. Insight into the Structure of Single Antheraea pernyi Silkworm Fibers Using Synchrotron FTIR Microspectroscopy. Biomacromolecules 2013, 14, 1885−1892. (54) Mamedov, A. A.; Kotov, N. A.; Prato, M.; Guldi, D. M.; Wicksted, J. P.; Hirsch, A. Molecular design of strong single-wall carbon nanotube/polyelectrolyte multilayer composites. Nat. Mater. 2002, 1, 190−194. (55) Shen, Y.; Johnson, M. A.; Martin, D. C. Microstructural characterization of Bombyx mori silk fibers. Macromolecules 1998, 31, 8857−8864. (56) Takahashi, Y.; Gehoh, M.; Yuzuriha, K. Structure refinement and diffuse streak scattering of silk (Bombyx mori). Int. J. Biol. Macromol. 1999, 24, 127−138. (57) Drummy, L. F.; Phillips, D. M.; Stone, M. O.; Farmer, B. L.; Naik, R. R. Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films. Biomacromolecules 2005, 6, 3328− 3333. (58) Asakura, T.; Suzuki, Y.; Yazawa, K.; Aoki, A.; Nishiyama, Y.; Nishimura, K.; Suzuki, F.; Kaji, H. Determination of Accurate H-1 Positions of (Ala-Gly)n as a Sequential Peptide Model of Bombyx mori Silk Fibroin before Spinning (Silk I). Macromolecules 2013, 46, 8046− 8050. (59) Asakura, T.; Kuzuhara, A.; Tabeta, R.; Saito, H. Conformation characterization of Bombyx-mori silk fibroin in the solid-state by highfrequency 13C cross polarization magic angle spinning NMR, X-ray diffraction, and infrared-spectroscopy. Macromolecules 1985, 18, 1841−1845. (60) Martel, A.; Burghammer, M.; Davies, R. J.; Riekel, C. Thermal Behavior of Bombyx mori silk: Evolution of crystalline parameters, molecular structure, and mechanical properties. Biomacromolecules 2007, 8, 3548−3556.

(61) Lawrence, B. A.; Vierra, C. A.; Moore, A. M. F. Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus. Biomacromolecules 2004, 5, 689−695. (62) Keten, S.; Xu, Z. P.; Ihle, B.; Buehler, M. J. Nanoconfinement controls stiffness, strength and mechanical toughness of beta-sheet crystals in silk. Nat. Mater. 2010, 9, 359−367. (63) Cheng, J.; Lee, S. H. Development of New Smart Materials and Spinning Systems Inspired by Natural Silks and Their Applications. Front. Mater. 2016, 2, 74. (64) Liu, L.; Yang, X. G.; Yu, H. Y.; Ma, C.; Yao, J. M. Biomimicking the structure of silk fibers via cellulose nanocrystal as beta-sheet crystallite. RSC Adv. 2014, 4, 14304−14313.

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