Wet-Spinning of Regenerated Silk Fiber from Aqueous Silk Fibroin

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Biomacromolecules 2010, 11, 1–5

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Articles Wet-Spinning of Regenerated Silk Fiber from Aqueous Silk Fibroin Solution: Discussion of Spinning Parameters Jiaping Yan,† Guanqiang Zhou,† David P. Knight,‡ Zhengzhong Shao,† and Xin Chen*,† The Key Laboratory of Molecular Engineering of Polymers of MOE, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, People’s Republic of China, and Oxford Biomaterials Ltd., Unit 4 Galaxy House, New Greenham Park, Newbury, RG19 6HR, United Kingdom Received July 24, 2009; Revised Manuscript Received October 8, 2009

Regenerated silk fibroin (RSF) fibers were obtained by extruding a concentrated aqueous silk fibroin solution into an ammonium sulfate coagulation bath. A custom-made simplified industrial-type wet-spinning device with continuous mechanical postdraw was used. The effect of dope concentration, coagulation bath, extrusion rate, and postdraw treatment on the morphology of RSF fiber was examined. The results showed that although RSF fiber could be formed with dope concentration between 13 and 19% (w/w), the ones spun from 15% RSF solution showed the most regular morphology being dense and homogeneous in cross-section with a smooth surface and a uniform cylindrical shape. Though it had little effect on morphology, postdraw treatment especially under steam, significantly improved the mechanical properties of the RSF fibers.

Introduction Although commercial Bombyx mori silk is considerably weaker and less tough than the best spider dragline silks, silk fibers with mechanical properties approaching that of Nephila spider dragline silk can be directly reeled from silkworms.1,2 Both spider and silkworm silks are spun naturally from aqueous protein solutions at very low hydraulic pressures and ambient temperatures without using noxious chemicals as solvents or in the coagulation bath.3 Moreover, regenerated silk fibroin (RSF) solutions are relatively cheap to prepare and can be made from waste silk materials, including unreelable dupion cocoons. These considerations have lead researchers to seek methods to extrude strong and tough silk fibers from RSF solutions, but most of these have failed to produce materials with useful mechanical properties.4 The biomimetic spinning of RSF fibers has at least two prerequisites: (i) a RSF spinning dope with a high concentration of protein with an average molecular weight close to that of the natural heavy chain fibroin; and (ii) an effective spinning process capable of commercialization and yielding lustrous RSF fibers with a uniform diameter, circular profile, and tensile properties close to or better than the natural ones. Several solvents have been used to dissolve silk fibers.5-10 Among these, concentrated LiBr aqueous solution and CaCl2-EtOH-H2O system have been the most studied. The strong ionic force of CaCl2 is known to produce some cleavage of the silk fibroin molecular chains11 and in consequence LiBr aqueous solution are most widely used to prepare RSF solution for artificial spinning. However, the classical method of dissolving silk fiber in concentrated aqueous LiBr and then dialyzing the resulting solution against deionized water yields * To whom correspondence should be addressed. Tel.: +86-21-65642866. Fax: +86-21-6564-0293. E-mail: [email protected]. † Fudan University. ‡ Oxford Biomaterials Ltd.

a dilute fibroin solution (4.0-5.0%) unsuitable for wetspinning because of its low viscosity. To solve this problem, researchers have lyophilized dilute aqueous RSF solutions and subsequently dissolved them in other solvents, including hexafluoroisoopropanol,12-16 hexafluoroacetone,17 or trifluoroacetic acid9 to increase the protein concentration to meet the requirements of wet-spinning. However, such a spinning dope preparation process is rather complicated and the solvents used are expensive and highly toxic. To overcome this difficulty several research groups have used reverse dialysis to prepare concentrated aqueous RSF solutions.18,19 In most previous reports, methanol or ethanol has been used as the coagulant for wet-spinning of RSF fibers,9,12-17,20-25 but these coagulants do not produce usable silk fibers, probably because methanol or ethanol induce a too rapid conformation transition from random coil/helix to β-sheet structure26 that preventing adequate molecular chain adjustment. Thus, these solvents induce isotropic formation of crystallites without proper orientation together with numerous protein molecular chains entanglements both resulting in fibers with poor mechanical properties. In addition, the toxicity of methanol makes it unsuitable for industrial spinning. To solve these problems, in previous work we used an aqueous inorganic salt or polyol solution coagulation bath. These produced better RSF fibers compared to those produced using a methanol coagulation bath.27,28 Experimental wet-spinning of RSF has mostly been carried out by injection from a hypodermic syringe6,12-14,20,21 or with a simplified industrial wet-spinning device.9,15,17 In addition, a dry-jet wet-spinning line has been used in an attempt to obtain some preorientation of the fibroin molecules in the air gap.29-32 As with traditional industrial extrusion of synthetic polymers or cellulosics, postdrawing to improve molecular orientation and packing is important for the mechanical properties of RSF fibers.33 However, few attempts have been made to use a steady

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Figure 1. Schematic showing the custom-made device used for trial spinning of regenerated silk fibroin: A, nitrogen gas cylinder; B, pressure regulator; C, dope storage cylinder; D, spinning dope; E, extrusion die; F, heated coagulation bath; G, draw rollers; H, take-up roller.

and continuous mechanical postdrawing. Instead, workers have generally chosen to postdraw extruded RSF fibers by extending them to a fixed length overnight, probably in air.9,10,29,30,34 In our previous article we outlined the development of a new method of extruding concentrated RSF solutions, which was able to produce fibers which were equal or slightly stronger or tougher than the natural silk fibers taken directly from the cocoon.35 We used an ammonium sulfate solution coagulation bath and a custom-made simplified industrial wet-spinning device with continuous mechanical postdraw. Here we extend this work by describing in detail the effects of spinning dope concentration, coagulant concentration, and the extent of postdrawing on the morphology and tensile properties of the resultant RSF fibers.

Experimental Section Preparation of RSF Aqueous Solution. Bombyx mori silkworm silk fibers were first degummed by boiling in two 30 min changes of 0.5% (w/w) NaHCO3 solution. After thorough washing in deionized water, a 10% (w/v) solution of degummed silk fibroin in aqueous 9.3 mol L-1 LiBr solution was prepared by heating to 40 °C for 1 h with continuous stirring. This solution was concentrated by reverse dialysis in Visking dialysis tube (MWCO: 10-12 kDa) against a 10% (w/v) aqueous solution of polyethylene glycol (20 kDa, from Sigma) at 5 °C for 3 days to yield RSF solutions that had protein concentration ranging from 10-30% (w/w). The pH of those RSF solutions was 6.5-7.0. Wet-Spinning of RSF Fibers. RSF fibers were spun at room temperature using a custom-made wet-spinning device (Figure 1). A nitrogen pressure of 0-0.3 MPa controlled by a pressure regulator was used to extrude the RSF aqueous solutions (13-19% (w/w)) at 1.0 mL/min through a commercial tantalum die with six 0.2 mm diameter orifices. The RSF spinning dope was extruded directly into an aqueous ammonium sulfate (30-40% (w/v)) coagulant solution at 25 °C. The effective length of the coagulation bath was 60 cm. The take-up rate of the first roller (spinning rate) was 30-70 rpm (equivalent to 4.7-11.0 m/min) and depended on the dope concentration. The draw-down ratio was defined as the rate ratio of the second roller to the previous one. The fibers did not slip on the rollers which were constructed from PVC. The resulting RSF fibers were wound onto spools, which were immersed at 25 °C overnight in the same solution used as the coagulation bath for further solidification. After rinsing in deionized water, the fibers were finally dried at 45 °C. Characterization of RSF Fibers. The surface and the cross-section of the RSF fibers were observed with a TS-5136MM SEM at 20 kV after sputtering with gold. The cross-section of the fibers was obtained by fracturing them perpendicular to the fiber axis under liquid nitrogen. The diameters of the RSF fibers were calculated from the sectioned fibers using the software provided with the SEM. We used an Instron 5565 mechanical testing instrument (at 25 ( 0.5 °C; 60 ( 5% R.H.;

gauge length: 30 mm; cross-head speed: 50% strain min-1) with a load cell of 2.5 N to test the mechanical properties of RSF single fibers. At least 12 replicates from individual fibers were used for the mechanical testing.

Results and Discussion Effect of Dope Concentration on the Spinnability and Morphology of the Fibers. Generally, the spinnability of a polymer depends on its viscoelasticity, but spinnability is difficult to define and measure precisely for a RSF aqueous solution. We therefore simply investigated the effect of RSF concentration in the spinning dope on the tenacity and morphology of the resulting fibers formed using the apparatus shown in Figure 1 and a coagulation bath at 25 °C containing a fixed concentration of ammonium sulfate (30%), and at constant drawing conditions (50 rpm on the first roller, i.e., 7.8 m/min) and a draw-down ratio of 2. When the dope concentration was lower than 11%, no fibers were formed, only dispersed floccules. At concentrations between 11 and 13% fibers could be formed but their cross-section was irregular and the as-spun fibers were very weak. Between 13 and 19%, fibers were moderately strong after postdrawing and collection. However, when the dope concentration was higher than 19%, the resulting fibers were very fragile. The latter observation may result from premature β-sheet formation or increased die swell resulting from the increased extrusion pressure required to force the more viscous dope through the spinneret. The effect of RSF concentration in the dope on the morphology of the as-spun and postdrawn fibers is shown in Figures 2 and 3. At 13% RSF concentration, the cross-section of the as-spun fiber showed numerous small voids and the surface of the fiber had irregular longitudinal pleats, which were only partially removed by postdrawing. At 15% RSF concentration, the as-spun fibers were smoothest, had the most regularly circular profile, and the cross-section appeared homogeneous and without voids. At 19% RSF concentration, the as-spun fibers were irregular in shape and surface morphology while the cross-section was markedly irregular, showing signs of macrophase separation. The appearance of fibers formed at 17% RSF concentration was generally intermediate between those formed at 15 and 19%. The surface morphology of fibers formed from 13% dope solution was similar to that seen in previously published micrographs of fibers formed from regenerated spidroin at low concentrations (0.0836 and 1.0%37), while the surface morphologies from 17 and 19% dope solution were similar to that of

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Figure 4. Morphology of as-spun RSF fibers formed in ammonium sulfate coagulation bath with different concentration under standardized condition (see text): A, 30%; B, 40%. Figure 2. Surface morphology of as-spun (A-D) and postdrawn (E-H) RSF fibers spun from different dope solution. Dope concentration: A and E, 13%; B and F, 15%; C and G, 17%; D and H, 19%.

Figure 5. Morphology of as-spun RSF fibers formed with different spinning rate under standardized condition (see text): A, 4.7 m/min (30 rpm); B, 7.8 m/min (50 rpm); C, 11.0 m/min (70 rpm).

Figure 3. Cross-section morphology of as-spun (A-D) and postdrawn (E-H) RSF fibers spun from different dope solution. Dope concentration: A and E, 13%; B and F, 15%; C and G, 17%; D and H, 19%.

recombinant spidroin fibers extruded from high-concentration dopes (2238 and 21.5%39). The highly folded surface of the fibers formed with an RSF concentration of 13% compared with that formed at 15-19% may have resulted from the formation of a fiber with a low protein packing density in the coagulation bath which subsequently irregularly folded as it shrunk during drying. The indication of macrophase separation seen at high protein concentration (19%) may result from faster coagulation of the dope at the high concentration preventing the gradual realignment of the molecules required to form a homogeneous material. These observations indicate that the optimal dope concentration as judged by the strength of the fibers (see Table 1 below) lay between 13 and 19%, while the concentration for optimal surface morphology was close to 15%. Effect of the Coagulation Bath. Figure 4 illustrates the effect of ammonium sulfate concentration in the coagulation bath on the morphology of the as-spun fibers under standardized conditions (dope concentration, 16%; spinning rate, 7.8 m/min; temperature, 25 °C). The morphology of the fiber was worse at the higher ammonium sulfate concentration (40%) compared with the 30% one in the coagulation bath, and could not be improved by postdrawing (see discussion below). In addition, clear microvoids were seen in RSF fibers formed in the 40% ammonium sulfate coagulation bath. Similar behavior was found in RSF fibers extruded from other dope concentrations. This may due to the quick conformation transition of silk fibroin and fast water removal from the fiber in high ammonium sulfate concentration coagulation bath. Effect of Spinning Rate. We investigated the effect of spinning rate on the morphology of the as-spun RSF fibers

formed under otherwise standardized conditions (coagulation bath concentration, 30%; temperature, 25 °C). For this experiment, we selected a dope concentration of 13% as this dope concentration is within the optimal range for the mechanical performance of the fibers and gave fibers with numerous microvoids making morphological comparison easy. Figure 5 shows the effect of spinning rate on the morphology of fibers. The number of voids per unit cross-sectional area decreased with spinning rates over the range 4.7-11.0 m/min. When dope concentration was increased to 15%, voids were absent and changes in the spinning rate over the range 4.7-11.0 m/min had no observable effect on morphology of the fibers. This is consistent with other reports,33,40 and it may be due to the compensatory effect of a reduced retention time in the coagulation bath and an increased extensional flow at higher spinning rates, as the natural spinning process for fibroin is thought to depend on the combined effects of gelation and shearing and/or extensional flow.41 Effect of Postdrawing. We investigated the effect of postdrawing to different draw-down ratios on the morphology of RSF fibers. Standardized conditions were used for this (dope concentration, 16%; spinning rate, 7.8 m/min; temperature, 25 °C). The results showed that the irregular shape of the as-spun fiber could hardly be improved by postdrawing either in air (Figure 6) or under steam; sometimes the irregular shape even worsened. On the other hand, postdrawing especially under steam did improve the structure of those fibers that were already fairly regular in cross-section as-drawn. In general, the surface of the fibers became smoother and the skin-core structure seen in the as-spun almost disappeared after postdrawing in steam to a draw-down ratio of 4.0 (Figure 7). This improvement could be attributed to improved and more uniform molecular chain alignment during the postdraw process resulting from plasticization by steam. The improvement of mechanical properties of RSF fiber after postdrawing in air was reported in our previous paper.35 Table 1 illustrates the effects on the mechanical properties of RSF fibers of steam postdrawing to different draw-down ratios. It

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Table 1. Effect of Draw-Down Ratio under Steam on the Mechanical Properties of RSF Fibers (n ) 12)a draw-down ratio

breaking strength (GPa)

breaking strain (%)

Young’s module (GPa)

2.0 3.0 4.0 6.0

0.12 ( 0.01 0.19 ( 0.03 0.20 ( 0.02 0.39 ( 0.05

4.8 ( 1.2 4.7 ( 2.2 15.7 ( 4.2 32.1 ( 5.8

6.7 ( 0.7 10.4 ( 2.1 13.2 ( 3.2 15.2 ( 3.3

a Under standardized extrusion conditions, as described in the text, and with a coagulation bath concentration of 30%.

Figure 6. Morphology of RSF fibers with different draw-down ratio in air under standardized condition (see text and coagulation bath concentration, 40%): A, 1.0 (as-spun); B, 2.0; C, 3.0.

Figure 7. Morphology of RSF fibers with different draw-down ratio under steam under standardized condition (see text and coagulation bath concentration, 30%): A, 1.0 (as-spun); B, 2.0; C, 3.0; D, 4.0.

shows the same tendency as postdrawing in air but it was not possible to draw RSF fibers with a draw-down ratio of 6.0 in air without breaking. The mechanical properties of RSF fibers significantly improved with the increase in draw-down ratio. The use of our industrial-type spinning device with continuous mechanized in-line drawing enabled fibers with highly consistent mechanical properties evidenced by low standard deviations. It was also observed that the RSF fibers produced by steam postdrawing showed the typical ductile-stable behavior even when a low draw-down ratio (2.0) was used. In contrast, fibers formed by postdrawing in air at the same draw-down ratio showed brittle fracture behavior. After continuous postdrawing under steam to a draw-down ratio of 6.0, RSF fibers showed considerably high breaking strength, strain, and energy to break (0.39 ( 0.05 GPa, 32.1 ( 5.8%, and 80.8 ( 13.9 kJ/kg, respectively), comparable to the data reported earlier35 (0.45 ( 0.02 GPa, 27.7 ( 4.2%, and 74.5 ( 13.4 kJ/kg, respectively) for fibers hand drawn to a draw-down ratio of 6.0 under steam. Thus, we conclude that postdrawing to a draw-down ratio of 6.0 under steam greatly improved the mechanical properties of RSF fibers. This is in line with the evidence reported by another group that postdrawing in air or under steam improved the mechanical properties of RSF fibers, but the latter showed more effect.42

Conclusions In the present communication we describe the effects of the silk fibroin concentration in the spinning dope, the ammonium sulfate concentration in the coagulation bath, the spinning rate and the postdrawing conditions on the morphology and in some cases the mechanical properties of RSF

fibers spun on a custom-made wet-spinning apparatus. A 15% dope concentration gave the smoothest and most regularly shaped fibers under our experimental conditions. RSF fibers after postdrawing under steam to a ratio of 6.0 showed good mechanical properties (breaking strength of 0.39 GPa, breaking stain of 32.1% and breaking energy of 80.8 kJ/kg) comparable or even better than those of the natural cocoon silk fibers (0.40 GPa, 19.7% and 42.3 kJ/kg, respectively). Other parameters, such as the concentration of coagulation bath and the spinning rate also affected the morphology of RSF fibers. Although more work is needed to consider the interactions between the parameters considered separately in this paper, the work reported here starts to optimize the spinning conditions that could be used for forming useful threads artificially from redissolved silk fibroin solutions. Acknowledgment. This work is supported by the National Natural Science Foundation of China (Grant 20974025, 20434010, and 20525414), the Program for New Century Excellent Talents in University of MOE of China (Grant NCET-06-0354), and the Program for Changjiang Scholars and Innovative Research Team in University of MOE of China (Grant IRT-06-12). We thank Dr. Jinrong Yao and Dr. Lei Huang at Fudan University and Prof. Fritz Vollrath at the University of Oxford for their valuable suggestions and discussions.

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