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Fabrication scheme for obtaining transparent, flexible, and water-insoluble silk films from apparently dissolved silk-gland fibroin of Bombyx mori silkworm Taiyo YOSHIOKA, Tamako Hata, Katsura Kojima, Yasumoto Nakazawa, and Tsunenori Kameda ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00602 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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ACS Biomaterials Science & Engineering

Fabrication scheme for obtaining transparent, flexible, and water-insoluble silk films from apparently dissolved silk-gland fibroin of Bombyx mori silkworm

Taiyo YOSHIOKA1, Tamako HATA1,*, Katsura KOJIMA1, Yasumoto NAKAZAWA2, Tsunenori KAMEDA1

1

Silk Materials Research Unit, National Agriculture and Food Research Organization (NARO), 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan

2

Division of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

Corresponding Author e-mail:[email protected]

Abstract Films from silk fibroin-protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water soluble, and therefore require post-production treatment inducing β-sheet formation, to render them insoluble in water. Many kinds of post-production treatments, using alcohol-water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less-flexible or even brittle due to the

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formation of an over content of β-sheet. In order to overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel-state of the silk glands with an imperfect β-sheet structure, by treating them with an ethanol/water mixture. Such fibroin gel was found to dissolve in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The SGF film cast from the HFIP solution shows a flexible and water-insoluble nature with high reproducibility. In addition to this improvement, the SGF film produced by this method contains a significantly low level of residual HFIP molecules compared to the traditional RSF films prepared from an HFIP solution. The mechanism underlying these advantageous characteristics was investigated from the structural viewpoint, by using techniques such as

13

C solid-state NMR, differential scanning calorimetry, and wide-

angle X-ray diffraction.

Keywords: silk, biomaterials,β-sheet conformation, α-helix conformation, HFIP, ethanol treatment

1. Introduction Bombyx-mori silkworm silk has outstanding applications in various fields such as biomedicine, cosmetics, and structural materials, as well as textiles.1 Particularly in tissue engineering, silk-fibroin protein is one of the most prominent biomaterials because of its exquisite balance between mechanical properties and biocompatibility.2 There are two ways to obtain silk-fibroin proteins. One is from native silk fiber and the other is directly from the silk glands of live silkworms. In the former, native silk fiber is dissolved and the product is referred as regenerated silk fibroin (RSF), whereas in the 2


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latter, live silkworms are used and the product is called liquid silk fibroin (LSF). Both these methods have advantages and disadvantages. RSF can be obtained easily by dissolving degummed silk fibers, from which sericin has already been removed. In the degumming process, aqueous solutions of acids or alkalis at high temperatures are generally used. An alkaline reaction at pH > 9 or acid reaction at pH < 2.5 ensures rapid elimination of sericin.3 As a result, the molecular weight is essentially reduced during degumming.4 Moreover, aqueous solutions of highly concentrated salts such as calcium chloride or calcium nitrate at high temperatures could also decrease the molecular weight during the dissolution process.5,6 The RSF aqueous solution (RSFaq) is dialyzed before it is turned into various products. Among various products, a film prepared by casting the solution on a plate is the conventional form. Because of the difficulty of long-term storage, RSFaq is often used after it is lyophilized and then dissolved again.7 The lyophilized RSF is no longer insoluble in water. Therefore, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is widely used because it is known to be a good solvent for silk at room temperature and does not cause significant reduction of molecular weight.8 Moreover, HFIP is a volatile solvent and the fibroin film can be readily cast from its solution. Both the fibroin films fabricated from RSFaq and HFIP solutions of RSF (RSFHFIP) are water-soluble, and therefore, a subsequent process is required to induce βsheet formation which leads to water insolubility.9-11 For this purpose, treatment with an alcohol (including an aqueous alcohol solution) is widely used. However, it is difficult to control the β-sheet content in such alcohol treatment and frequently induces an over content of β-sheet, which alters the mechanical properties of the fibroin products into less flexible or even brittle.9,12-15 Instead of the alcohol treatment, other post-production 3



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treatments, such as water-vapor annealing14,16,17 or controlled-temperature treatment,18 have been developed for inducing a more moderate formation of β-sheet structure. Fine tuning of the water-vapor annealing conditions makes it possible to also induce the water insoluble silk I structure.9,11 Such the water insoluble RSF films with a low content of β-sheet structure, however, show significantly low tensile modulus and strength compared to those treated with alcohol.9,11,14,16,17 The issues associated with the reduction of the molecular weight of RSF during degumming and dissolution can be resolved by utilizing LSF, which can be obtained directly from silk glands. Moreover, the risk of contamination of endotoxin is reduced by utilizing LSF. However, to utilize LSF, the fibroin protein should be dissolved in water immediately after extraction of the silk glands from a living silkworm. In other words, LSF is only accessible to those who have access to live silkworms. Another difficulty related to the utilization of LSF is the separation of sericin from fibroin. Controlling the concentration of LSFaq is also difficult. Furthermore, similar to the case of RSF, LSF films fabricated from LSFaq are water-soluble. The fine tuning of postproduct-treatment to induce moderate β-sheet formation for altering the LSF-film into being water insoluble is required to avoid rendering it less flexible. Here, we report a novel scheme to process fibroin cast films to overcome the disadvantages recognized in the conventional processing approaches, as mentioned above. In this method, silk gland fibroin (SGF) extracted from living silkworms is gelated with an alcohol/water mixture and then dissolved in HFIP. Short-term dissolution of SGF in HFIP allows the fabrication of water-insoluble and flexible fibroin films. The resultant films show significantly less residue of HFIP compared to those fabricated from conventional RSFHFIP. The mechanism leading to these features was 4


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investigated by Fourier transform infrared (FTIR) spectroscopy, wide-angle X-ray diffraction (WAXD), thermal analysis, and 13C solid-state NMR spectroscopy.

2. Experimental 2.1. Materials 2.1.1. Silk gland fibroin The silkworms (Bombyx mori, Ariake) were reared on an artificial diet in the laboratory. The living fifth-instar silkworms were rinsed with 70% ethanol/water mixture to restrict the propagation of bacteria and endotoxin contamination. Subsequently, silk glands were extracted from them and rinsed quickly with 40% ethanol/water (40%-EtOH), to enable solidification and removal of sericin. The silk glands were kept immersed in fresh 40%-EtOH and refrigerated.

2.1.2. Regenerated silk fibroin The cocoons (Bombyx mori, Ariake) used in this study were produced in our Institute. The cocoon fiber was degummed by boiling for 30 min in an aqueous solution of 0.02 M sodium carbonate (Na2CO3). The degummed fiber was dissolved in 9.3 M aqueous lithium bromide (LiBr) at 37 °C, overnight, and then the solution was dialyzed for 4 days using a cellulose tube (cut-off molecular weight of 14000; Viskase Sales, USA) at 25 °C.

2.2. Characterization 2.2.1. Water contact angle test The wettability of the fibroin cast films was evaluated using a contact angle and surface

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tension analyzer, FTA 188 (JASCO International Co. Ltd., Japan). A 3.5 µL water droplet was set on the fibroin film surface cast on a glass slide, and the change in the contact angle was measured every 2 s during the first 5 min.

2.2.2 FTIR analysis The structural information of the fibroin cast films was investigated by transmissiontype FTIR spectroscopy performed with FTIR-620 (JASCO International Co. Ltd., Japan). For each measurement, 32 scans were accumulated with a spectral resolution of 2 cm-1 and then background spectrum measured under the same scan condition was subtracted from the resultant spectrum. The wavenumbers of conformation sensitive bands in random coil, α-helix, and

β-sheet for the Bombyx mori silk and those relating to the HFIP solvent, used in this report, are listed in Table 1. For the determination of protein conformation including Bombyx mori silk, vibrational bands in the amide I and II regions are quite often utilized.19,20 However, in amide I and II regions, these conformation sensitive bands overlap each other and therefore not easy to distinguish definitively. Thus, in addition to them, we also utilized the weak but independent conformation sensitive bands listed in Table 1, whose assignment was well established in combination with X-ray analysis in a previous report.21

2.2.3. 13C solid-state NMR analysis High-resolution solid-state 13C NMR spectra were recorded on Avance 600 WB (Bruker, Karlsruhe, Germany), with a magnetic field of 14.1 T. The spectrometers were operated at the

13

C NMR frequency of 150.94 MHz. The samples were placed in a solid-state

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probe and spun at the magic-angle spinning (MAS) frequency of 10.0 kHz in a 4.0-mm-

φ zirconia rotor. 1H 90° pulse length of 3.5 µs and 1H-13C cross-polarization (CP) contact of 70 kHz were employed for the CP experiments. High-power 1H decoupling using the SPINAL-64 method was employed. The repetition time for the CP experiments was set at 3.0 s. All the spectra were calibrated using adamantane as the standard, and the chemical shift of the adamantane CH2 peak appearing at 29.5 ppm was referenced to the tetramethylsilane (TMS) peak appearing at 0 ppm.

2.2.4. WAXD analysis The structure of SGF-gels was investigated by an R-axis Rapid II X-ray diffractometer (Mo-Kα) (Rigaku Co., Japan) equipped with a cylindrical-type imaging plate camera. Cerium oxide was used to calibrate the camera distance. A free software FIT2D (developed by A. P. Hammersley of European Synchrotron Radiation Facility, Grenoble) was used for subtracting the air-scattering and for extracting the 2θ-profiles from the 2dimensional WAXD patterns. The peak assignments of WAXD 2θ-profiles were performed based on the unit cell parameters of silk II (β-sheet) crystal modification (a = 9.38 Å, b = 9.49 Å, and c (fiber axis) = 6.98 Å and the space group P21) proposed by Takahashi et al.22

2.2.5. Differential scanning calorimetry (DSC) Thermal properties of the fibroin cast films were measured using DSC Q200 (TA Instruments, USA). For the measurements, a sample of about 5 mg was encapsulated in an aluminum pan and heated in the temperature range of –30 to 300 °C at a heating rate

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of 5 °C/min under a nitrogen flow of 50 mL/min. The instrument was calibrated in advance with indium for heat flow and temperature. The standard references of aluminum and sapphire were used for calibration of heat capacity.

2.2.6. Dynamical mechanical thermal analysis (DMTA) Mechanical properties (storage modulus (Er) and loss modulus (Ei)) of the fibroin cast films were measured by DMTA (DVA205 IT Keisoku Seigyo, Japan) in the temperature range of –30 to 300 °C at a heating rate of 6 °C/min. The frequency of the sinusoidal oscillation was 10 Hz and the dynamic strain was 0.01%. The sample length, width, and thickness were 20 mm, 5 mm, and 0.005 mm, respectively. The loss tangent, defined as the ratio of Er and Ei (tan δ = Ei/Er) was also evaluated.

2.2.7. Tensile test The tensile properties of the SGFHFIP cast films, the RSFHFIP cast films, and the 80%EtOH (for 12 h) post-production treated RSFHFIP cast films were measured at room temperature using a mechanical tensile stage EZ Test/CE (Shimadzu Co., Japan) equipped with a 50 N load cell. A cross-head speed at a 10 mm / min was used. The dimensions of the specimen films with a rectangular shape were roughly as follows: length (gauge distance): 10 mm, width: 1.5 mm, and thickness: 50 – 150 µm. A pair of sandpapers with fine grains was sandwiched between the sample and grips to prevent the sample from sliding. To estimate the sectional area of each specimen film, the specimen width and thickness were measured using a vernier caliper and a micrometer, respectively. The Young’s modulus was estimated from the tangent of the initial slope of the stress-strain curve. Each tensile property reported in this article was averaged from 8


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the 5 measurements (n = 5).

3. Results and discussion 3.1. Structure of alcohol-treated SGF Figure 1(a) shows a photograph of the silk gland that had been immersed in 40%-EtOH at 4 °C for 2 months. The gelation of SGF occurred within 24 h after immersion in 40%EtOH, and subsequently, the gel state was maintained for at least 6 months under visual observation. The sericin and skin layer could be removed relatively easily from the SGF gel, as shown in Figures 1(b) and S1(a). A comparison of surface morphologies, observed by SEM, of the SGF gels before and after hand peeling of sericin layer shows it’s clear removal (see Figure S1(a)). The removal of sericin protein was confirmed also by SDSPAGE analysis (see Figure S1(b)). For WAXD and NMR measurements, two types of samples were prepared, viz., SGF-gel in the immersed state and the one dried in room humidity. The former is referred as a wet gel and the latter as a dried gel. The WAXD 2θ profiles of both the wet and dried gels of SGF, which were treated with 40%-EtOH for 6 days, are shown in Figure 2. Both profiles show the β-sheet pattern, and the crystallinity of the dried sample was significantly higher than that of the wet one. However, the intensity balance of the β200 and β211 reflections in the wet-gel sample was reversed owing to the overlapping of the scattering from the absorbed bulk water (see Figure S2). The low crystallinity of the gel state was maintained at least during the immersion period of 240 d (Figure 2). NMR spectra in the range of 0 to 80 ppm, obtained from the wet- and dried-gel SGF samples treated with 40%-EtOH at 4 °C for 6 days, are shown in Figure 3. Similar 9



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to WAXD results, both the NMR spectra revealed the formation of a β-sheet structure. Each peak of wet-gel SGF is observed to be sharper than that of the dried one. A sharper peak implies higher molecular mobility and weaker intermolecular interactions involved in β-sheet formation. The structure observed in the wet sample is referred as ‘imperfect

β-sheet structure’. In this structure, many water molecules and EtOH molecules remain.

3.2. Two stages (types) of the dissolution of alcohol-treated SGF gel in HFIP solvent 3.2.1. Apparent dissolution It is empirically known that, once the silk fibroin crystallizes in the β-sheet form, it can no longer dissolve in HFIP.23 In fact, the dried-gel SGF samples exhibiting the β-sheet structure, as ascertained from WAXD and NMR results, did not dissolve in HFIP. In contrast, as shown in Figure 4, the wet-gel SGF sample dissolved in HFIP within several hours with mild agitation at 25 °C, and as a result, a transparent SGFHFIP solution was obtained (Figure 4a, right). Such phenomenon is considered to occur due to low β-sheet crystallinity and/or imperfect β-sheet structure. By casting the solution on a polystyrene petri-dish at room temperature, a transparent and flexible film was obtained (see Figure 4b). To obtain the smooth surface of the films, a slow evaporation rate (namely, the drying rate) was achieved by covering the petri-dish with paired polystyrene cover. By changing the concentration and casting volume, the film thickness can be controlled from few micrometers to several tenth micrometers, keeping the transparency and flexibility. HFIP solvent is known to induce the formation of helical conformation in various kinds of proteins,24-27 including the silk proteins.28-31 The induced helical conformation remains unchanged during the cast film formation

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process. In fact, the FTIR spectrum of the film cast from the HFIP solution of lyophilized RSF shows typical features assigned to the α-helical conformation21 (Figure 5). Here, it is also noted that the RSFHFIP spectrum shown in Figure 5 does not show a characteristic absorption band around 1690 cm-1, which was related to the β-turn conformation.32 The film cast from SGFHFIP solution obtained by dissolving wet-gel SGF in HFIP for 10 days, however, exhibited a β-sheet spectrum. Here, we hypothesize that the dissolution of SGF at the molecular level had not occurred within the dissolution duration of 10 days, and as a result, the structural memory of the β-sheet conformation remained. In order to confirm this hypothesis, the dissolution duration was extended and the conformation of the resultant cast film was investigated.

3.2.2. Perfect dissolution The FTIR spectra obtained from three types of SGFHFIP cast films, fabricated from different HFIP solutions obtained under different dissolution durations of 10, 60, and 90 days, are shown in Figure 6. Because the dissolution test was carried out using sealed screw tubes at 25 °C with mild agitation, the evaporation of the solvent during the test was negligibly small. Whereas the film cast from SGF dissolved for 10 days (SGFHFIP (10 days)) (same data as shown in Figure 5) presented a typical β-sheet-type spectrum, the spectrum of SGFHFIP (60 days) revealed a mixture of β-sheet and α-helix types, and SGFHFIP (90 days) provided an almost pure α-helix spectrum. These results are consistent with the hypothesis proposed in section 3.2.1., viz., the dissolution duration of 10 days is not sufficient to erase the β-sheet memory. A significant increase in the helical conformation upon extending the dissolution duration was also confirmed

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by DSC and DMTA measurements. The DSC and DMTA curves for the SGFHFIP cast films, prepared from SGFHFIP solutions obtained after different dissolution duration of 10 days and 60 days are shown in Figure 7. The DSC curve of the SGFHFIP (60 days) film shows a continuous endothermic peak at 154 °C and an exothermic peak at 168 °C. The continuous thermal change corresponds to the structural transition from α-helix to β-sheet structure (the former peak corresponds to the structural deformation of the helical conformation and the latter the formation of the β-sheet crystal).21,33 Simultaneously, according to the deformation of the helical conformation and formation of the β-sheet crystal, the DMTA curve shows a decrease and subsequent increase in Er as well as an increase and subsequent decrease in tan δ, respectively. In contrast, both the DSC and DMTA curves of the SGFHFIP (10 days) film show no specific peaks within this temperature range. This demonstrates that the dominant structure of the cast film prepared from the 10 daysdissolved solution is not the helical conformation but the β-sheet structure. The experimental results of FTIR (Figure 6), DSC, and DMTA (Figure 7) indicate that (i) the apparent dissolution of SGF-gel in HFIP occurs quickly within several hours, (ii) the structural memory of the β-sheet structure still remained, and (iii) the dissolution at the molecular level occurs quite slowly. The critical dissolution duration, during which the dominant structure changed from β-sheet to α-helix, varied widely between different experiments (the process at least takes more than 10 days and mostly over 60 days). This is mainly attributed to the differences in the initial structures of SGF-gels. Determining the influence of other factors such as concentration, temperature, and agitation level remains to be investigated.

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It was also found that the structural difference between the SGFHFIP cast films (10 and 60 days) leads to different mechanical properties. The storage moduli Er of the SGFHFIP cast films (10 and 60 days) at room temperature (20 °C) are 3.9 and 2.2 GPa, respectively (see the DMTA data in Figure 7). The fact that the modulus of SGFHFIP (10 days) film is higher than that of SGFHFIP (60 days) is interesting, because the SGFHFIP (10 days) film was cast from an incompletely dissolved solution. Moreover, the SGFHFIP (10 days) film was extremely flexible, as described above. Thus, the SGFHFIP (10 days) film is highly resistant against loading and extremely tough against bending.

3.3. Advantages of using the apparently dissolved SGF 3.3.1. Reduction of the residual HFIP solvent As mentioned in the previous section, there are two stages in the SGF-gel dissolution in HFIP. In the first stage, the SGF-gel dissolves apparently but not perfectly at the molecular level. As a result, the structural memory of the originally formed imperfect βsheet conformation remained. In contrast, with increasing dissolution duration, further dissolution of the remaining β-sheet memory is induced in the subsequent stage. The isolated molecular chains are induced to achieve helical conformation by the HFIP molecules. Note that in the FTIR spectra shown in Figure 5, the intensity of the bands assigned to HFIP21 corresponding to the SGF HFIP (10 days) film is significantly lower than that of the RSFHFIP film. This clear difference in the intensities of HFIP bands originates from the structural difference between the films, depending on whether the film is β-sheet rich or α-helix rich, which may be reasonably explained on the basis of the following findings. Drummy et al. found out that the α-helices of fibroin in the 13



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HFIP solution are packed hexagonally in the cast film as a result of self-assembly.33 Yoshioka et al. clarified that the hexagonal packing is realized via the formation of a complex between the α-helices and HFIP molecules.21 Due to the helical fibroin-HFIP complex formation, the HFIP molecules, whose boiling point is 58.2 °C, get stably trapped in the fibroin products up to ~140 °C.21 This explains the strong interaction between the silk-fibroin and HFIP molecules. Owing to this strong interaction, large amounts of HFIP molecules get trapped in the SGFHFIP (60 days). In contrast, further heating over 140 °C induced the rupture of the helix-HFIP complex, and hence the structural transition from the aggregated state of the hexagonally packed α-helices into the β-sheet structure.21 This implies that the β-sheet structure is energetically more stable compared to the hexagonally packed aggregation state of α-helices formed via the fibroin-HFIP complex. This well-explains the significantly lower intensities of the HFIP bands in the FTIR spectra of the β-sheet rich films compared to those in the FTIR spectra of the α-helix rich films (Figure 5). Because HFIP is a harmful organic solvent, residual HFIP in the final fibroin products must be removed thoroughly, particularly from those used in biomedical applications. In this aspect, using the apparent dissolution condition of SGF provides considerable advantages in fibroin processing, especially for biomedical applications. Moreover, it was found that the amount of residual HFIP in the SGFHFIP cast film is affected by the EtOH content of the solvent mixture in which the silk gland was immersed. The FTIR spectra of the SGFHFIP cast films, prepared from four types of SGFs that were treated for 3 h in different EtOH/water mixtures: 10, 40, 70, and 90% ethanol/water, each of which was dissolved for 6 h in HFIP, are shown in Figure 8(a).

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For comparison, the spectrum obtained from SGFHFIP (40%-EtOH, 90 days) is also shown (This spectrum is same as that shown in Figure 6). The normalized absorbance of the HFIP band near 894 cm-1 in each spectrum was evaluated and plotted in Figure 8(b). For normalization, the spectral intensity of C-H stretching band at 2880 cm-1, which was confirmed to be not sensitive to the morphological change, was used.21 It is observed that lower EtOH concentration (or higher water content) results in lower amount of residual HFIP. When the concentration was lower than 10%, the fibroin could not be solidified, but dissolved. The mechanism of the alcohol concentration effect is not clarified.

3.3.2. Water Solubility In contrast with the water-soluble as-cast films of RSF or LSF prepared by the conventional processing schemes,9,14,16,18 the β-sheet SGFHFIP cast film obtained from the apparently dissolved solution is insoluble in water. A comparison of the water solubility of the conventional RSFHFIP cast film and the β-sheet SGFHFIP cast film, was performed by floating them on the water surface. The films used were approximately 10 mm in length, 10 mm in width, and 10 µm in thickness. Whereas the conventional RSFHFIP film dispersed into small pieces immediately after floating on the water surface (see Figure 9(a)), the β-sheet SGFHFIP film floated stably on the water surface (see Figure 9(b)). Moreover, the water-insoluble SGFHFIP film did not show brittleness after the water floating test. The water contact angle and its time-dependent change during the first 5 min were investigated for the β-sheet SGFHFIP and RSFHFIP cast films (see the top and middle plots in Figure 10, respectively). Whereas the initial contact angle of the RSFHFIP 15



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film was ~65°, the SGFHFIP film showed much higher water repellency with a contact angle of approximately 100°. While this high contact angle of the SGFHFIP film remained almost unchanged during the first 30 s, that of the RSFHFIP film decreased significantly up to 50°. This indicates the surface dissolution of the film, and therefore, high surface wettability of the RSFHFIP film. The contact angle of the RSFHFIP film decreased up to approximately 25° during 5 min (i.e., decreased by ~40°). In contrast, the contact angle of the SGFHFIP film decreased by only 13° during the 5 min test duration. These contact angle measurements indicate the lower wettability of the β-sheet SGFHFIP film, which supports the results of the water floating test. The high waterrepellency of the β-sheet SGFHFIP cast film without brittleness is another significant advantage to use the dissolution condition to achieve the apparent dissolution of SGF in fibroin processing. It should be noted that, the decrease in the contact angle during the dynamic measurement is caused not only by the increase of surface wettability, but also due to the evaporation of the water droplet.34 Judging from the essentially similar behavior of decreasing contact angle with the one observed on the glass slide (see the bottom plot in Figure 10), the decrease in the contact angle observed on the SGFHFIP cast film is mainly attributed to water evaporation.

3.3.3. Mechanical Property The tensile property of SGFHFIP (10 days) cast film was evaluated and compared with the ones of the RSFHFIP cast film and of the 80%-EtOH (for 12 h) treated RSFHFIP cast film. Their typical stress-strain curves are shown in Figure 11, and their

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averaged Young’s modulus, strength, and elongation are listed in Table 2. As reported previously, the post-production treatment of RSFHFIP cast film using alcohol enhanced the modulus and strength but lowered the elongation significantly. On the other hand, the SGFHFIP (10 days) cast film shows comparable modulus and strength with the alcohol treated RSFHFIP film as well as comparable elongation with the intact one. Such good balance of mechanical property is not achieved even by moderate water-vapor annealing as well as mixing with plasticizers.35,36

4. Conclusion We present a novel scheme for processing silk gland fibroin (SGF). The essence of this scheme is to create a fibroin gel-state consisting of an imperfect β-sheet structure by ethanol/water mixture treatment. The gel state was confirmed to be stable in the ethanol/water mixture for at least several months. It was found that the fibroin gel dissolves in HFIP in two stages. In the first stage, the fibroin gel did not dissolve at the molecular level, although the resultant solution was transparent. In such imperfectly dissolved state, namely, apparent dissolution state, the imperfect β-sheet structure remained. The imperfect β-sheet dissolved perfectly in the subsequent stage, over a quite long dissolution duration of several months. The SGF cast film obtained from the apparently dissolved HFIP solution has a β-sheet structure, and is therefore waterinsoluble and interestingly presents the additional advantage of not being brittle with a good balance of Young’s modulus, strength, and elongation. Furthermore, FTIR analyses revealed that the SGF film obtained from the apparently dissolved state contains a significantly low level of residual HFIP molecules.

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Acknowledgements This project was entrusted to us by MAFF as part of a scientific technique research promotion program from the Agriculture, Forestry, Fishers and Food Industry (26051A). We thank Prof. Kohji TASHIRO (Toyota Technological Institute, Japan) for his kind support with the WAXD measurements. We are grateful to K. Hashimoto, Y. Takeda, Y. Inami (NARO) for rearing silkworms regularly.

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Supporting Information

Surface morphologies and SDS-PAGE analysis of the SGF before and after removing the sericin layer; WAXD 2θ profiles of the wet-gels of the silk gland fibroin

Tables Table 1. Assignments of the conformation sensitive bands and the ones relating to the HFIP solvent Wavenumber / cm-1 ~1014 ~922 ~955 ~1548 1656-1662 ~975 ~1520 ~1628 ~842 ~894

Conformation Random coil α-helix α-helix α-helix α-helix β-sheet β-sheet β-sheet HFIP HFIP

Reference 15 15 15 15 37 15 15 15 15 15

Table 2. Mechanical properties of the SGFHFIP, RSFHFIP and 80%EtOH-treated RSFHFIP films (n=5). Sample SGFHFIP RSFHFIP 80%EtOH-RSFHFIP

Young’s modulus / GPa 2.6 ± 0.6 1.7 ± 0.3 2.3 ± 0.5

Strength / MPa 84.6 ± 10.1 55.9 ± 5.1 72.5 ± 7.0

Elongation / % 8.3 ± 1.5 9.5 ± 1.1 4.4 ± 1.5

Figure captions Figure 1 Photographs of the silk gland (SG) immersed in 40% EtOH/water for 2 months (a) and silk gland fibroin (SGF) in the gel state (b). Cell wall and sericin layer

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of SG could be removed easily after immersion in 40% EtOH/water for 24 h. After their removal, a translucent SGF is exposed.

Figure 2 WAXD 2θ profiles of the wet- and dried-gels of SGF. SGF was gelated by immersing it in 40%-EtOH for 6 and 240 days.

Figure 3 13C solid-state NMR spectra of wet- (green) and dried- (red) gel SGF samples treated with 40%-EtOH for 6 days.

Figure 4 Photographs of SGF-gel in HFIP solvent before dissolution (left-side in (a)) and HFIP solution of SGF after 24 h dissolution (right-side in (a)), and a transparent SGF film cast from the HFIP solution obtained by dissolving SGF-gel in HFIP solvent for 2 d (b).

Figure 5 FTIR spectra of the films cast from the HFIP solution of regenerated silk fibroin (RSFHFIP) and the solution obtained by dissolving wet-gel SGF in HFIP for 10 days (SGFHFIP 10d).

Figure 6 FTIR spectra of SGFHFIP films cast from different HFIP solutions prepared by different dissolution durations of 10, 60, and 90 days.

Figure 7 DSC and DMTA curves for the SGFHFIP films cast from SGFHFIP solutions obtained by different dissolution durations of 10 and 60 days.

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Figure 8 (a) FTIR spectra of the SGFHFIP cast films prepared from four types of SGFs treated for 3 h in different EtOH concentrations, viz., 10, 40, 70, and 90 % ethanol/water mixtures. Each of these was dissolved for 6 h in HFIP. (b) Plots of the normalized absorbance of HFIP observed near 894 cm-1 in each spectrum shown in (a).

Figure 9 Water solubility test for as-cast films of RSFHFIP and SGFHFIP.

Figure 10 Time-dependent change in the water contact angle for the β-sheet SGFHFIP cast film and RSFHFIP one, together with that on glass slide shown for comparison.

Figure 11 Typical stress-strain curves of the SGFHFIP, RSFHFIP, and 80%EtOH-treated RSFHFIP films.

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Ceｌｌ wall + Sericin

(a)

SGF

Figure 1


(b)

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Figure 2



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dry wet

Figure 3


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(a)

(b)

Figure 4



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Figure 5


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Figure 6



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(a) (b)

Figure 8



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(a) 1 min

RSFHFIP

(b) 10 min

SGFHFIP (10 days)

Figure 9


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