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Tensile reinforcement of silk films by the addition of telechelic-type polyalanine Kousuke Tsuchiya, Hiroyasu Masunaga, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01891 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Tensile reinforcement of silk films by the addition of telechelic-type polyalanine Kousuke Tsuchiya,*,† Hiroyasu Masunaga,‡ and Keiji Numata*,† †

Enzyme Research Team, Biomass Engineering Research Division, RIKEN Center for

Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ‡

Japan Synchrotron Radiation Research Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-

5198, Japan

E-mail: [email protected], [email protected]

KEYWORDS silk, polyalanine, telechelic, composite, tensile deformation, -sheet structure

1 ACS Paragon Plus Environment

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ABSTRACT

An appropriate modification technique for silk materials is needed to effectively improve their physical properties for specific applications. A telechelic-type polyalanine (T-polyA) was synthesized by papain-catalyzed polymerization as a novel reinforcing agent for silk materials. A silk fibroin obtained from Bombyx mori was homogeneously doped with T-polyA, and casting a solution of silk fibroin and T-polyA in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) resulted in a robust and transparent film. Tensile deformation studies of the silk composite film containing TpolyA with pre-stretching revealed that the tensile strength and toughness were enhanced relative to those of a silk-only film. To determine the capability of T-polyA to reinforce the tensile property of silk films, the secondary structure in the silk composite film was characterized by wide-angle X-ray diffraction (WAXD) analysis. Antiparallel -sheet structures of T-polyA and GAGAGS motifs of silk fibroin formed independently in the pre-stretched composite film. In addition, measuring the tensile deformation and performing WAXD analysis simultaneously demonstrated that the -sheet structures of both T-polyA and the silk fibroin were aligned along the stretching direction and that T-polyA had no significant effect on the final morphology of the silk crystal domains. The silk film was toughened by the addition of T-polyA because of the generation of the T-polyA -sheet in the amorphous region of the composite film. This work provides novel insight into the design and development of tough silk materials with controlled and aligned -sheet structures.


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Introduction Silk fibroin obtained from silkworm cocoons is a structural protein that exhibits biocompatibility, biodegradability, and excellent mechanical properties.1 Using special processes established over decades of research, this silk can be regenerated into various forms, such as nanofibers, films, tubes, sponges, and gels, indicating that this silk has broad applicability in numerous fields beyond its conventional use in the textile industry.2,3 Each application requires the silk material to have specific physical properties. Therefore, appropriate modification of the silk fibroin is essential for its practical use. Several techniques for the modification of silk materials have been reported. It was reported that the mechanical properties of silk films can be reinforced by unique processing techniques.4,5 Introduction of functional groups into the primary structure of the silk via chemical reactions is one facile approach for changing the physical properties of silk or adding novel functions, such as a high affinity for living cells,6,7 the ability to bind biomedical materials (drugs) for controlled release,8 and water repellency.9 In addition, blending silk with other materials, such as polymers10-13 and inorganic fillers,14-16 can produce the desired property. Polymeric materials based on natural sources that exhibit biodegradability and biocompatibility are good candidates for blending with silk to maintain the biodegradable and biocompatible properties this material. However, appropriate molecular designs of the dopant polymers and/or special processing techniques for blending are required to achieve homogeneous dispersion in the silk matrix because a heterogeneous morphology resulting from the segregation of the components would deteriorate the properties of the silk. The high mechanical strength of silk materials is attributed to their unique structure, which consists of -sheet crystal domains and an amorphous matrix.17-19 The -sheet structure is formed by an oligopeptide motif (i.e., GAGAGS) that is frequently observed in the repetitive


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sequence of the silk fibroin from the Bombyx mori silkworm.20,21 In fact, the formation of this higher-order structure is crucial for establishing high mechanical strength and toughness, even in silk blend films. Therefore, the filler material must fulfill the following three requirements: i) adequate compatibility with silk fibroin to prevent large phase separation in the blend film, ii) low affinity to the motifs of silk fibroin to prevent the structure from collapsing, and iii) functionality to enhance the physical properties of the blend film. Polypeptide materials are expected to represent one of the best candidates for use as a filler for silk materials because they are miscible with the silk fibroin, and their properties can be tuned by selecting the appropriate sequence.22 In this study, a telechelic-type polyalanine (T-polyA) was used as a filler to reinforce silk fibroin films. Polyalanines are frequently observed in the sequences of spider silks and silkworm silks23, except those of B. mori, and exist as -sheet crystals, resulting in robust and tough fibers.24-26 Our previous study demonstrated that T-polyA assembles into nanofibers with a high aspect ratio that are morphologically different from conventional linear polyalanines with similar chain lengths.27 This unique crystallization feature should enable the T-polyA dopant to assemble into an antiparallel -sheet structure in the silk composite films with a crystal lattice different from that of the silk fibroin. Additionally, this structure should coexist with the intact higher-order structures of the silk fibroin. The influence of T-polyA on the mechanical properties of the silk blend films was investigated using a tensile deformation test. The relationship between the physical properties and the secondary structure was characterized by wide-angle Xray diffraction (WAXD) analysis, especially during the tensile deformation of the silk composite films. Experimental


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Materials. Silk fibroin was obtained from B. mori silk cocoons, and its aqueous solution was prepared according to a previously reported protocol.2 The degummed silk fibroin sponge was obtained by lyophilization of the silk solution prior to its experimental use. Papain (EC No. 3.4.22.2) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and used as received. The activity was approximately 0.5 U g−1, where one unit hydrolyzes 1 mmol of Nbenzoyl-DL-arginine p-nitroanilide per minute at pH 7.5 and 25 °C. The other chemicals were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and used as received without purification, unless otherwise noted. Synthesis of bis(alanine ethyl ester) initiator (1). To a 200-mL flask equipped with a stir bar and an addition funnel, alanine ethyl ester hydrochloride (4.76 g, 31 mmol), triethylamine (9.2 mL, 66 mmol), and ethyl acetate (100 mL) were added. To this mixture, a solution of succinyl chloride (1.7 mL, 15 mmol) in ethyl acetate (50 mL) was added dropwise at 0 °C under nitrogen. The resulting solution was stirred at 0 °C for 2 h, and the reaction was quenched by the addition of water. The mixture was washed with water, sodium bicarbonate aq. (1 M), and brine. The organic layer was dried with sodium sulfate and concentrated using a rotary evaporator. The crude product was recrystallized from hexane/ethyl acetate to afford a pale-yellow, needleshaped crystal. The yield was 1.65 g (35%). Infrared (IR) (neat):  = 3301, 2991, 1729, 1639, 1545, 1356, 1238, 1204, 1168, 1020 cm-1. 1H nuclear magnetic resonance (NMR) (500 MHz, CDCl3, 25 °C, ppm):  = 6.66 (s, 2H), 4.53 (m, 2H) 4.20 (q, J = 7.1 Hz, 4H), 2.57 (m, 4H), 1.40 (d, J = 7.1 Hz, 6H), 1.28 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, CDCl3, 25 °C):  = 173.16, 171.73, 61.37, 48.18, 31.55, 17.97, 14.04. Anal. Calcd for C14H24N2O6: C 53.15, H 7.65, N 8.86; Found: C 53.08, H 7.61, N 8.85.


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Synthesis of T-polyA by chemoenzymatic polymerization. To a 10-mL glass tube equipped with a stir bar, alanine ethyl ester hydrochloride (0.922 g, 6.0 mmol), 1 (0.190 g, 0.60 mmol), phosphate buffer (2.0 mL, 1 M, pH 8.0), and methanol (1.0 mL) were added, and the mixture was stirred at 40 °C until all substrates were completely dissolved. Then, a solution of papain (0.300 g) in phosphate buffer (2.0 mL) was added in one portion. The final concentrations of alanine ethyl ester and papain were 1 M and 50 mg mL−1, respectively. The mixture was stirred at 40 °C and 800 rpm for 6 h using an EYELA ChemiStation (Tokyo, Japan). After cooling to room temperature, the precipitate was collected by centrifugation at 7000 rpm and 4 °C for 10 min. The crude product was washed twice with deionized water and methanol and lyophilized to afford a white powder. The yield was 0.150 g (35%). Linear polyalanine (L-polyA) was also synthesized using a similar procedure without initiator 1. Fabrication of a silk composite film doped with T-polyA. Degummed silk fibroin was obtained from B. mori silkworm cocoons according to a previously reported procedure.9 The lyophilized silk fibroin (0.765 g) and T-polyA (0.0383 g, 5 wt% of the total components) were completely dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (15.2 mL, 5 w/v%) under stirring. The solution was gently poured into a Teflon petri dish with a diameter of 100 mm and dried in a fume hood at room temperature overnight. The film was peeled off of the dish and further dried in a desiccator under vacuum for 4 h. The final thickness of the film was ca. 100 m. A silk composite film containing L-polyA (5 wt% of the total components) was also fabricated using the same procedure. Tensile deformation test of the silk composite films. Three types of silk films (silk only, doped with T-polyA, and doped with L-polyA) were cut into small plates (3 mm x 15 mm). The plate samples were immersed in methanol for 5 min and slowly pre-stretched using a uniaxial film


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stretcher (IMC-1A11, Imoto Machinery Co., Ltd., Tokyo, Japan) until the samples reached a 1.25-, 1.5-, 1.75-, or 2-fold length (stretching ratio was 25, 50, 75, or 100%, respectively). The pre-stretched films were fixed on a glass petri dish using double-sided tape and dried in a desiccator at room temperature under vacuum for 3 h. Each pre-stretched film was placed on a load cell (500 N) by clamping at both edges, and the tensile properties were measured using an EZ-LX HS universal/tensile tester (Shimadzu Corporation, Kyoto, Japan) at a stretching rate of 0.5 mm min−1 (strain rate: 1.65 x 10−3 s−1) at 25 °C and 35 to 45% relative humidity. The tensile properties of the silk composite films (i.e., Young's modulus, maximum tensile strength, elongation at break, and toughness) were calculated from the obtained stress-strain curves. Five replicates were performed for each measurement, and the values of the mechanical properties were averaged to determine the standard deviation. Simultaneous tensile deformation measurements and WAXD. The silk-only films and composite films containing T-polyA (5 wt%) with pre-stretching ratios of 75 and 100% were cut into small plates (1 mm x 15 mm) and placed on a load cell (20 N) by clamping at both edges using super glue. The tensile deformation was conducted on a lab-made uniaxial tensile testing machine on the BL45XU beamline (SPring-8, Harima, Japan) at a stretching rate of 2 mm min−1. The synchrotron WAXD measurement was initiated at the threshold of the stress-strain curve and repeated at 5-second intervals until the silk films fractured. To perform the same measurement using the methanol-swollen silk composite film, the small plate sample of the silk composite film containing T-polyA was immersed in methanol for 5 min. After wiping off the residual methanol on the surface, the film was immediately subjected to simultaneous tensile deformation measurement and WAXD under the same conditions.


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Analyses. The IR spectra of the bulk samples were recorded on an IRPrestige-21 Fourier transform IR spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with a MIRacle A single-reflection attenuated total reflectance (ATR) unit using a Ge prism. The 1H and 13C NMR spectra were recorded on a Varian NMR System 500 (Varian Medical Systems, Palo Alto, CA, USA) at 25 °C and frequencies of 500 and 125 MHz, respectively. Chloroform-d or deuterated dimethylsulfoxide (DMSO-d6)/trifluoroacetic acid (TFA-d) (5/1 volume ratio) was used as the solvent, and tetramethylsilane (TMS) served as an internal standard for initiator 1 and T-polyA. Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometric analysis was conducted using an ultrafleXtreme MALDI-TOF spectrophotometer (Bruker Daltonics, Billerica, MA, USA) operating in reflection mode at an accelerating voltage of 15 kV. The sample was dissolved in water/acetonitrile (0.8 mg mL−1) containing 0.1% TFA mixed with a solution of a-cyano-4-hydroxycinnamic acid (CHCA) in water/acetonitrile (10 mg mL−1) and deposited on an MTP 384 ground steel BC target plate. The synchrotron WAXD measurements of the silk composite films were performed on the BL45XU beamline (SPring-8, Harima, Japan) using an X-ray energy of 12.4 keV (wavelength: 0.1 nm). The obtained two-dimensional (2D) diffraction patterns were converted to one-dimensional (1D) profiles by azimuthal integration using Fit2D.28 The degree of crystallinity was estimated by comparing the areas between the crystal peaks and the amorphous halo in the 1D profiles, which were deconvoluted by fitting the curves using Gaussian functions. Scanning electron microscopy (SEM) images of the silk composite films were recorded on a JCM-6000 (JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV. The film samples were coated with gold prior to SEM.

Results


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Synthesis of T-polyA. As a dopant for silk composite films, T-polyA was synthesized by chemoenzymatic polymerization using papain in the presence of a telechelic-type initiator (1) possessing two alanine ethyl ester moieties as described in a previously published study.27 The structure of TpolyA was confirmed by 1H NMR and MALDI-TOF mass spectrometric analyses (Figure 1a). The signal assigned to the methylene spacer of the initiator unit was observed at 3.2 ppm in the 1

H NMR spectrum (Figure S1), indicating that 1 was successfully involved in the polymerization

of alanine ethyl ester to form T-polyA. The number average molecular weight (Mn) of T-polyA was estimated to be 800 based on a comparison of the integral ratio of the -methine proton of the repeating units and the methyl protons of the terminal ethyl groups. The formation of TpolyA was also confirmed by MALDI-TOF mass spectrometry, which revealed a series of peaks corresponding to the oligoalanines with an initiator unit (Figure S2).


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Figure 1. (a) Chemical structures of telechelic-type initiator 1, T-polyA, and L-polyA; the appearance of the silk solution in HFIP (5 w/v%) with (b) T-polyA (5 wt%) and (c) L-polyA (5 wt%); and (d) the silk composite films cast from the HFIP solutions (left: T-polyA, right: LpolyA).

Tensile deformation tests of the silk composite films The silk composite films that were doped with T-polyA (5 wt% to silk fibroin) were prepared by casting the HFIP solution (5 w/v%) of the silk fibroin and T-polyA. Other acidic solvents, such as trifluoroacetic acid and dichloroacetic acid, can also dissolve T-polyA as well as the silk fibroin. However, we did not use these solvents because of their corrosiveness. For comparison, the L-polyA (Mn = 750) was prepared by chemoenzymatic polymerization under conditions


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similar to those employed for T-polyA and was used as an additional dopant.29 As a control, a silk-only film was also fabricated from the HFIP solution. Because polyalanines tend to strongly aggregate via hydrophobic interactions via -sheet formation, L-polyA was barely soluble in HFIP, producing a turbid dispersion (Figure 1b). Additionally, the resulting silk film that contained L-polyA was slightly opaque (Figure 1d, right). By contrast, the transparent HFIP solution consisting of silk mixed with T-polyA afforded a transparent film (Figures 1b and 1d left), indicating that T-polyA has better miscibility with the silk fibroin. Therefore, a homogeneous dispersion of T-polyA was obtained. Because HFIP is known to induce a helical conformation, the obtained films are likely to be amorphous and enriched in -helices.30-31 The silk films were immersed in methanol and then subjected to uniaxial pre-stretching to convert the amorphous structure to a -sheet structure. The pre-stretching ratio ranged from 25 to 100%, which was a maximum before the breakage occurred during pre-stretching. Unfortunately, the silk film containing L-polyA was intolerant of pre-stretching ratios exceeding 50%, likely because L-polyA induces heterogeneous aggregations in the film. Higher doping amount of TpolyA than 5 wt% provided also transparent composite films. However, the silk composite film became too brittle to be subjected to the tensile deformation tests. Therefore, the doping amount of T-polyA was fixed at 5 wt% against the silk for the measurement of mechanical properties. To examine the effect of the dopants on the mechanical properties of the silk films, the composite films were subjected to uniaxial tensile deformation tests. Figure 2 shows representative stress-strain curves for the silk-only and silk composite films containing T-polyA or L-polyA, and the averaged mechanical properties are summarized in Figure 3 (all of the stress-strain curves obtained for these silk films are shown in Figures S3-S5). The Young's modulus and maximum tensile strength (max) of the as-prepared silk-only film were


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approximately 1 GPa and 40 MPa, respectively, and these values gradually increased as the prestretching ratio increased. A similar tendency was observed for the silk composite film with TpolyA, except its max was higher than that of the silk-only film. The yielding point became distinct, and the elongation at break also increased after pre-stretching was applied, which resulted in improved toughness. By contrast, the composite film containing L-polyA exhibited mechanical properties similar to those of the silk-only film up to a pre-stretching ratio of 25%. The ductility and strength of the silk film are attributed to secondary structures with appropriate orientations, and the antiparallel -sheet derived from the GAGAGS motifs plays a key role in improving the mechanical properties.32 The addition of T-polyA to the silk film did not influence the Young's modulus or elongation at break but significantly reinforced the tensile strength. These results indicate that T-polyA does not significantly affect the -sheet crystal structure of silk and contributes to enhancing the strength without sacrificing the elasticity and extensibility of the silk. For all the films, the elongation at break decreased at higher pre-stretching ratio than 75% because the polymer chains in the amorphous region became more extended by the prestretching.

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Figure 2. Representative stress-strain curves for the uniaxial tensile deformation of (a) the silkonly films, (b) the silk composite films containing T-polyA (5 wt%), and (c) the silk composite films containing L-polyA (5 wt%) with pre-stretching ratios ranging from 0 to 100%.

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Structural characterization of the silk composite films using WAXD analysis.


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To elucidate the reinforcing behavior of the silk composite film containing T-polyA, the secondary structure of the silk molecules and T-polyA was characterized by WAXD analysis. The 1D WAXD profiles of the silk-only and T-polyA-doped composite films with different prestretching ratios are shown in Figure 4. The as-prepared silk-only film exhibited broadened peaks because of the -helix,33 and pre-stretching the film changed the secondary structure from an helix structure to an antiparallel -sheet structure, which resulted in two major peaks at 0.45 and 0.37 nm. The Miller indices were assigned as (020) and (021), respectively.34 However, a peak appeared at 0.52 nm in addition to those corresponding to the -sheet structure when the TpolyA-doped film was pre-stretched. The WAXD profile of the powdery sample of T-polyA (broken line in Figure 4) contained three major peaks at 0.52, 0.43, and 0.37 nm, which correspond to the antiparallel -sheet formed by the polyalanine sequence.35-36 The strongest peak was consistent with a newly observed peak in the WAXD profiles of the silk composite film with T-polyA. Because the crystal lattice of the -sheet structure was identical to that described in the literature,34 we assumed that the GAGAGS motifs in the silk fibroin and TpolyA independently formed the antiparallel -sheet crystal in the silk composite film. The WAXD profiles of the silk composite film containing L-polyA exhibited behavior similar to that of the silk-only film upon pre-stretching (Figure S6). No characteristic peak assignable to the sheet structure of polyalanine was observed, even at higher pre-stretching ratios. This result suggests that both T-polyA and L-polyA existed in amorphous forms prior to stretching the film and that the pre-stretching only induced the crystallization of T-polyA. Furthermore, the peaks derived from the -sheet structure of the GAGAGS motifs became sharper when T-polyA was doped. This indicates that the size of GAGAGS crystallite became larger and/or more spatially aligned in the presence of T-polyA. As demonstrated in previous studies, T-polyA most likely


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assembled into nanofibrils based on a -sheet structure, whereas L-polyA, which has a similar molecular weight, formed a granule-like morphology.27, 37 Therefore, the specific crystallization of T-polyA was most likely triggered by the pre-stretching of the silk composite films.

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Simultaneous tensile measurements and WAXD analysis. To further investigate the effect of T-polyA on the mechanical properties of the silk composite films, tensile measurements and WAXD analyses were conducted simultaneously. A uniaxial tensile testing machine was installed on the beamline with synchrotron X-rays, and time-course WAXD profiles were collected at 5-second intervals during the tensile deformation. Figure 5 shows the resulting stress-strain curves with the 2D patterns at specific time points: (i) elastic, (ii) yielding, and (iii) plateau regions. As the films were stretched, the 2D patterns changed slightly although no substantial structural transition was observed during the tensile deformation. The orientation of the -sheet crystal differed in the silk composite films subjected to prestretching ratios of 75% and 100%. When the composite film was pre-stretched at 75%, the orientation of the -sheet crystal domains derived from the silk GAGAGS motifs was relatively low and aligned slightly perpendicular to the stretching direction. The-sheet structure of TpolyA was aligned parallel to the stretching direction, indicating that the -sheet of T-polyA was more easily crystallized during the pre-stretching process than the GAGAGS motifs in the composite film. Increasing the pre-stretching ratio to 100% substantially changed the structural alignment. The -sheet structures of both the GAGAGS motifs and T-polyA were aligned along the stretching direction, and the intensity of the (020) reflection increased, resulting in a higher tensile strength compared to that obtained at a stretching ratio of 75% (Figure 5B). By contrast, the silk-only film exhibited parallel alignment of the -sheet, even at lower pre-stretching ratios (i.e., less than 75%), and the 2D profiles indicated no significant change between the 75% and 100% pre-stretched films (Figure S7). The azimuthal intensity profiles of the WAXD patterns of


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the (020) reflection peak of the -sheet structures from the GAGAGS motifs (Figures S8 and S9) also indicated the formation of a -sheet crystal aligned along the stretching direction (the equatorial direction on the 2D patterns, i.e., approximately 90° and 270°). The orientation of the (020) reflection relative to the stretching direction was previously observed at a pre-stretching ratio of 25% in the silk-only film. By contrast, the orientation in the silk composite film was initiated at a pre-stretching ratio of 75%. In addition, the azimuthal intensity profiles of the (020) reflection derived from the -sheet of T-polyA in the silk composite film indicated that the orientation of the -sheet of T-polyA was parallel to the stretching direction in the 75% prestretched film (Figure S10). Based on these results, the -sheet crystal of T-polyA readily aligned along the pre-stretching direction, hampering the -sheet formation of the GAGAGS motifs in the silk composite films.


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Figure 5. Stress-strain curves with 2D WAXD profiles collected at specific time points for the silk composite film containing T-polyA with pre-stretching ratios of (a) 75% and (b) 100%.

Figure 6 shows the SEM images of the fracture surface of the silk composite films containing TpolyA after tensile deformation. A crazing-like structure was observed at the surface near the edge region for the film subjected to a pre-stretching ratio of 75%, and this structure occurred vertical to the stretching direction (Figure 6a). By contrast, the surface of the film formed with a 100% pre-stretching ratio was smooth, and cracking occurred along the stretching direction (Figure 6b). This difference in the fractured structure is assumed to reflect the orientation of the silk fibroin chains in the composite films. The silk polymer chains are better aligned along the stretching direction in the composite film subjected to a pre-stretching ratio of 100%, as determined via the WAXD analyses. No large aggregation was observed at either the surface or cross-section of the composite films, indicating that T-polyA was well-dispersed in the silk because of their high compatibility. The SEM images of the silk-only film exhibited little difference between 75% and 100% pre-stretching (Figure S11).


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Figure 6. SEM images of the fracture surface obtained by the simultaneous tensile deformation and WAXD analyses of a silk composite film containing T-polyA with pre-stretching ratios of (a) 75% and (b) 100%.

The -sheet crystals of both silk fibroin and T-polyA were aligned along the stretching direction during pre-stretching. Therefore, tensile testing and WAXD analysis were performed simultaneously during the pre-stretching of a methanol-swollen silk composite film. The silk composite film containing T-polyA was immersed in methanol for 5 min, gently wiped to remove the residual methanol, and subjected to tensile deformation with WAXD measurement. The stress-strain curve for the tensile deformation of the methanol-swollen silk composite film and its 1D WAXD profiles at specific time points are shown in Figure 7. In the stress-strain curve, plateau and hardening regions were observed after the yielding point, and the maximum


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strain reached 130%. The (020) reflection peak derived from the antiparallel -sheet of T-polyA gradually appeared after yielding occurred at pre-stretching ratios ranging from 75% to 100%, indicating that T-polyA formed a crystal domain in the methanol-swollen silk film during the pre-stretching process. The lack of a change in the d-spacings of the -sheet structure of the GAGAGS motifs revealed that the generation of the -sheet crystal of T-polyA did not influence the crystal lattice of the silk’s -sheet structure.

Figure 7. (a) Stress-strain curve of the methanol-swollen silk composite film containing T-polyA and (b) the WAXD profiles obtained at specific time points.


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Discussion The great enhancement of both the strength and extensibility of the silk films after pre-stretching was achieved, which is originated from the formation of the antiparallel -sheet structure, and further improvement on the strength was observed when T-polyA was doped in the silk films. By contrast, doping with L-polyA did not affect the tensile properties of the silk films, and the films became brittle (i.e., the extensibility was less than 50%). Recently, we revealed that T-polyA most likely assembles into nanofibrils via -sheet formation with a high aspect ratio, which differs from the granule-like morphology observed for L-polyA.27 This difference was observed in the higher-order structures of T-polyA and L-polyA, despite their similar molecular weights. This specific crystallization feature of T-polyA is attributable to its unique primary structure (i.e., two polyalanine chains propagating in the opposite direction from the initiating unit). In general, the antiparallel -sheet structure is formed by folding the polypeptide chain via turn structures in natural proteins. However, telechelic-type polypeptides can form intramolecular hydrogen bonds in an antiparallel manner without folding of the chains. Therefore, T-polyA efficiently crystallized to form an antiparallel -sheet structure during pre-stretching, even in the silk matrix. The -sheet structure of T-polyA readily aligned along the stretching direction, most likely because of its preference to form long fibrous structures; however, the size and distribution of the T-polyA crystals in silk composite films have not been clarified. By contrast, L-polyA barely assembled into a -sheet structure in the silk composite films, even during pre-stretching, because of its restricted mobility in the elastic silk matrix. Collectively, the experimental data from the simultaneous tensile test and WAXD measurements indicate that the formation of the -sheet crystal of T-polyA in the silk composite films is


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independent of the -sheet structure of the silk fibroin, resulting in the coexistence of two types of -sheet crystals (Figure 8). The structure in the as-prepared silk composite film was-helix rich, and no remarkable crystal orientation was achieved, as described in a previous study.38 Pretreatment by stretching under methanol-swollen conditions facilitated the formation of an antiparallel -sheet derived from both silk and T-polyA during the early stage (i.e., a prestretching ratio of less than 75%) and a parallel-oriented -sheet of T-polyA along the stretching direction. Because T-polyA hampered the crystallization of the silk GAGAGS motifs, no alignment of the silk -sheet occurred at this stage. Finally, further stretching (i.e., a prestretching ratio of 100%) allowed the -sheets of both silk and T-polyA to align along the stretching direction. In addition, the degrees of crystallinity of the silk-only film and the silk composite film subjected to pre-stretching ratios of 100% were nearly the same (i.e., approximately 30%). These results indicate that the addition of T-polyA did not influence the final morphology of the -sheet crystal domains. Therefore, the Young's modulus of the initial elastic region in the stress-strain curves did not differ significantly between the silk-only and composite films because the elastic modulus is typically attributed to the stiffness of the crystal domains. However, the tensile strength and toughness of the silk composite film were reinforced by doping with T-polyA. T-polyA and the silk GAGAGS motifs independently form -sheet crystals, and the crystal domains of T-polyA are most likely located in the amorphous region in the composite films. Therefore, the additional existence of the -sheet crystal of T-polyA in the amorphous domains toughened the silk films, resulting in a higher tensile strength. The slight increase in the degree of crystallinity (32.5%) in the 100% pre-stretched silk composite films compared to that of the silk-only film (28.7%) also supports this mechanism of tensile reinforcement using T-polyA.


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Figure 8. A plausible model of the structural change in the pre-stretched silk composite films with T-polyA.

Conclusion The novel T-polyA, which was used as a highly miscible dopant for silk film, was successfully synthesized by chemoenzymatic polymerization using papain. The tensile deformation study of T-polyA-doped silk composite films demonstrated that T-polyA can function as a tensilereinforcing agent in silk composite films, enhancing these films’ tensile strength and toughness. The WAXD measurements revealed that the crystallization of the antiparallel -sheets derived from T-polyA and the silk GAGAGS motifs occurred independently and aligned along the stretching direction. The -sheet crystal of T-polyA was formed in the amorphous region and toughened the silk composite film without disturbing the crystallization of the silk. Our methodology for modifying silk materials using polypeptide additives offers a new perspective regarding the utilization of silk materials for novel applications in various fields. Because the chemoenzymatic polymerization can afford various polypeptide primary structures, novel telechelic polypeptides which is soluble in aqueous buffers can also lead more environmentally


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benign process for the preparation of the silk composite materials without using organic solvents such as HFIP. ASSOCIATED CONTENT Supporting Information. 1H NMR and MALDI-TOF mass spectra of T-polyA, stress-strain curves for tensile deformation tests, WAXD profiles (2D and azimuthal intensity profile data), and SEM images. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (K.T.); [email protected] (K.N.). Tel: +81-48-467-4064 (K.T.); +8148-467-9525 (K.N.). Author Contributions All authors have given approval to the final version of the manuscript. Funding Sources This work was financially supported by the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). ACKNOWLEDGMENT The authors would like to acknowledge the Materials Characterization Support Unit, RIKEN, for performing the elemental analysis.

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Insert Table of Contents Graphic and Synopsis Here

A telechelic-type polyalanine (T-polyA) was synthesized by papain-catalyzed polymerization as a novel reinforcing agent for silk materials. A silk fibroin obtained from Bombyx mori was homogeneously doped with T-polyA, and casting a solution of silk fibroin and T-polyA in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) resulted in a robust and transparent film. Tensile deformation studies of the silk composite film containing T-polyA with pre-stretching revealed that the tensile strength and toughness were enhanced relative to those of a silk-only film. To determine the capability of T-polyA to reinforce the tensile property of silk films, the secondary structure in the silk composite film was characterized by wide-angle X-ray diffraction (WAXD) analysis. Antiparallel -sheet structures of T-polyA and GAGAGS motifs of silk fibroin formed independently in the pre-stretched composite film. In addition, measuring the tensile deformation and performing WAXD analysis simultaneously demonstrated that the -sheet structures of both T-polyA and the silk fibroin were aligned along the stretching direction and that T-polyA had no significant effect on the final morphology of the silk crystal domains. The silk film was toughened by the addition of T-polyA because of the generation of the T-polyA -sheet in the amorphous region of the composite film. This work provides novel insight into the design and development of tough silk materials with controlled and aligned -sheet structures.


Tensile Reinforcement of Silk Films by the Addition ... - ACS Publications

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