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Artificial superstrong silkworm silk surpasses natural spider silks

 1 year ago
source link: https://www.cell.com/matter/fulltext/S2590-2385(22)00517-3
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Artificial superstrong silkworm silk surpasses natural spider silks

Introduction

Natural protein-based silks are primarily spun by spiders, silkworms, and other insects and mussels., They have long attracted our interest for thousands of years owing to their extraordinary physical properties, excellent biocompatibility, and long-term biodegradability. The natural silks exhibit various physical properties due to their unique amino acid sequences and spinning conditions, leading to distinct hierarchical 3D structures. Silkworm silk and its derivatives have been most widely studied and produced to date, including various regenerated silk fibroins (RSF)., ,  Over the past 20 years, researchers have been putting considerable efforts into genetic, structural, and functional studies on different types of spider silks as well as development of artificial silk spinning methods since spider silks significantly outperform silkworm silk in physical properties., , , , , ,  Among six types of spider silk and one silk glue, dragline silk has the highest breaking strength and is widely well studied. Unlike silkworms, spiders cannot be farmed due to their territorial and aggressive nature. Therefore, recombinant production of spider fibroins (spidroins) becomes a feasible solution for spinning artificial spider silk with desirable mechanical properties., Nevertheless, only limited success was achieved. Although artificial spider silk fibers spun from recombinant spidroins with high molecular weights (MWs) of >200 kDa could show comparable strength to the natural dragline silk, the harsh condition used in the coagulation bath and/or low yield of purified high-MW recombinant spidroins make it difficult for large-scale production and applications., In fact, most artificial spider silk fibers are still inferior to their native counterparts in one or multiple mechanical properties.
Instead of purifying and spinning recombinant silk proteins, obtaining proteinaceous silk or fibroin from commercialized mulberry silkworm Bombyx mori is the most economical and practical way for the needs in the textile and medical industries. The basic unit of B. mori fibroin consists of a heavy chain of ∼390 kDa, a light chain of ∼26 kDa, and a glycoprotein P25 of ∼25 kDa. The disulfide-bonded heavy and light chains associate with P25 through noncovalent interactions at a molar ratio of 6:6:1., In water solution, multiple fibroin units can interact with each other and form nanometer micelle-like structures with hydrophilic terminal domains on the surface. These micelles further assemble into micrometer protein globules before shear-induced structural transition and silk fiber formation. Similar to spider dragline silks, silkworm silk has high glycine and alanine content of >70%. These two types of amino acids are the major components of highly repetitive sequences responsible for the formation of β sheet and nano-crystallite structures in silk fibers. However, silkworm silk shows significantly weaker strength or toughness than all types of spider silks. For example, the tensile strength of most spider dragline silks can reach 0.9–1.4 GPa, while raw silkworm silk and artificial silk fibers spun from RSFs only exhibit breaking tenacities of 400–600 MPa., ,  Alternatively, transgenic silkworms stably expressing chimeric spider and silkworm fibroins could provide another way for mass production of high-performance silks, which typically show mechanical properties between silkworm and spider silks under appropriate testing speed., ,  In this report, we present a simple strategy for producing artificial silkworm silk using undegraded RSFs and an inorganic coagulation bath. The resulting artificial silk fibers exhibit superstrong physical properties with an average tensile strength of ∼2.0 GPa and an average Young’s modulus of ∼43 GPa. To our knowledge, this is the first report ever on the spinning of artificial silk that significantly surpasses natural spider silks in strength and stiffness.

Results and discussion

In order to produce artificial silk fibers, silkworm fibroins were first regenerated through degumming and dissolution processes. Previous studies have demonstrated that the degumming process can easily cause breakage of peptide and intermolecular disulfide bonds, leading to more chain ends in the fibers., , ,  Therefore, artificial fibers spun from the degraded RSFs could not have a high tensile strength. Through proper control of degumming temperature and time using specific chemical agents or proteolytic enzymes, an optimal degumming ratio with minimal degradation can be achieved (Figure 1). The degumming ratios using SDS and Na2CO3 (RSF-SN) and papain (RSF-Pa) at 80°C –85°C are both around ∼28% (Figure 1A), indicating removal of most of sericins from the silk fibers. The higher degumming ratio of ∼31% using Na2CO3 (RSF-N60) at 100°C could be due to the weight loss resulting from fibroin degradation and dissolution. The RSF-SN and the RSF-Pa fibroins show a low degree of protein degradation (Figure 1B). The MWs of major bands of the RSF-SN and the RSF-Pa fibroins estimated from the gradient gel are >350 kDa, in agreement with the theoretical MW of the heavy chain (∼390 kDa). However, the RSF-N60 is severely degraded into smaller proteins with MWs ranging from ∼40 to ∼200 kDa, consistent with previous reports. Dynamic light scattering shows that the RSF-SN can assemble into large complexes with hydrodynamic radii (Rh) of 20–30 and 200–300 nm after refolding from LiBr solution, although low-MW fibroins with radii between 2 and 3 nm also exist (Figure 1C). This measurement is in line with a previous observation indicating that heavy and light chains can interact with P25 to form a large complex of >2000 kDa in water solution. The RSF-Pa fibroins form slightly smaller complexes with a similar size distribution, while the radii of the RSF-N60 molecules are decreased to <200 nm. Circular dichroism (CD) spectra indicate that the RSF-SN and the RSF-Pa fibroins aggregate into β-rich structures,, which are drastically diminished in the RSF-N60 since protein chain degradation may disrupt refolding of silk fibroins in water solution (Figure 1D). These β structures are further confirmed by Congo red (CR) staining showing redshifts and significant increases in absorbance between 500 and 540 nm when CR interacts with the RSF-SN and the RSF-Pa (Figure 1E). In contrast, the RSF-N60, which lacks β-like structures, shows nearly no binding to CR. These results highlight the importance of intact molecular chains, especially the heavy chain, for the folding and pre-assembly of fibroins prior to silk fiber formation.
The RSF-SN and the RSF-Pa in deionized water were further concentrated to prepare spinning dopes for artificial fiber formation. However, we found that either the RSF-SN or the RSF-Pa at a concentration of ≥1% quickly transforms into protein hydrogel within 1–2 days at 4°C. Higher temperatures can lead to faster gelation. The instability of these spinning dopes in vitro could be largely due to intact high-MW fibroins, which have already folded into β-rich structures. Very differently, the RSF-N60 is much more stable at a high concentration of ≥10% due to its lower MW and β sheet-deficient structures in water solution. It is, therefore, not practical to use pure water as a solvent for preparing concentrated fibroins with high MWs. In this work, thus, all the RSFs were lyophilized and then dissolved in hexafluoro-isopropanol (HFIP) for silk fiber spinning. CD spectra of the RSFs in HFIP also show similar β sheet-rich structures (Figure S1).
Design of a coagulation bath is another critical step that may significantly affect silk fiber quality. Harsh and toxic organic solutions, like methanol and isopropanol, are not suitable for large-scale production. In previous research, ammonium sulfate solution was often used as an inorganic coagulation bath of low toxicity for spinning degraded RSF. Nevertheless, both the RSF-SN and the RSF-Pa fibroins are challenging to form continuous fibers in ammonium sulfate solution due to fast solidification of silk fibroins. Inspired by the fact that silkworm silk has a similar function to spider eggcase silk and both amino acid sequences contain high content of Ala (>30%) and Ser (>10%) residues, we tested the fiber formation of RSFs in different metal-ion coagulation baths that were previously investigated for spinning artificial eggcase silk. Interestingly, we found both the RSF-SN and the RSF-Pa at a concentration of 10%–11% undergo moderate solidification and form continuous fibers during the extrusion process in a coagulation bath containing two metal ions Zn2+ and Fe3+ at room temperature. Optimal fiber formation rate was achieved when the metal-ion coagulation bath was supplemented with ∼6% sucrose. Sucrose may increase the density and viscosity of the coagulation bath, which consequently affects the initiation and elongation of the fibers. In sharp contrast, the RSF-N60 only forms short and broken fibers with lengths of <0.5 cm in this coagulation bath (Figure S2). Therefore, a mechanical test on the RSF-N60 fiber was not feasible. Since the RSF-N60 sample mainly contains degraded fibroins, it is possible that low-MW fibroins that harbor more chain ends and unfolded structures prevent continuous fiber formation in the metal-ion coagulation bath.
Similar to other artificial silk fibers, the as-spun RSF-SN and RSF-Pa fibers typically exhibit a low tensile strength of <300 MPa. After post-spinning drawing in the metal-ion solution and 70% ethanol, the diameters of artificial fibers decrease from ∼15 μm to 4–6 μm, smaller than that of the degummed B. mori silk (9–11 μm) but close to spider dragline silk’s (3–5 μm). Scanning electron microscopies (SEMs) of the cross-sections of the RSF-SN and the RSF-Pa fibers show globular-like nano-structure morphology, indicating that RSFs were initially assembled into micelle-like structures (Figure 2A and 2B). This globular-like morphology was also observed in natural B. mori silk degummed in Na2CO3 (Figure 2C). The smooth surfaces and strong birefringence under polarizing light indicate that the post-drawn artificial fibers contain well-aligned fibroin chains in the direction of the applied force (Figures 2D–2F and S3).
Figure 2Comparison of morphology and mechanical properties of artificial and natural silk fibers
Mechanical tests for both post-drawn artificial silks are shown in Figures 2G–2K and Table S1 (supplemental information), including stress-strain curves and the derived mechanical properties (Young’s modulus and toughness). Surprisingly, the RSF-SN fiber after two-step post-spinning drawing exhibits an exceptionally high tensile strength of 2,054 ± 177 MPa, which is more than two times stronger than that of the degummed natural B. mori silk (610 ± 84 MPa) and more than 70% stronger than the average tenacity (1,117 ± 275 MPa) of spider dragline silks. The RSF-SN fiber also exhibits an average Young’s modulus of 43 ± 6 GPa, significantly higher than that of any known natural silks (<20 GPa). The extraordinarily high stiffness indicates that the RSF-SN fiber is unsusceptible to deformation caused by an external force. Different from the RSF-SN, the RSF-Pa fiber shows high toughness of 215.8 ± 29.6 MJ m−3, which is approximately 60% tougher than those of dragline silks (Figure 2J), although its tensile strength (816 ± 124 MPa) is close to dragline silks.
To understand the relationship between the mechanical properties and structure of the artificial fibers, Fourier transform infrared spectroscopy (FTIR) and wide-angle X-ray diffraction (WAXD) were conducted. Post-spinning drawing significantly increases β structure and decreases random coil (Table 1, Figures S4, and S5). The β sheet contents of the post-drawn final RSF-SN and RSF-Pa silk fibers were estimated to be ∼56% and ∼51%, respectively, which are higher than that of the natural B. mori silk (∼44%) and close to the natural dragline silk’s (∼51%). WAXD profiles reveal that the crystallinity of the RSF-SN fiber is more than 50%, noticeably higher than those of the natural B. mori silk (∼40%–42%) and spider dragline silk (∼22%–31%)., Furthermore, the nano-crystallite sizes of the RSF-SN and RSF-Pa fibers are similar to spider dragline silk but significantly smaller than the B. mori silk (Table 2 and Figure S6)., Previous studies have highlighted the importance of small crystallite size, which could distribute strain more uniformly, leading to better mechanical performance. High β sheet contents and crystallinity with a small size of nano-crystallite observed in the RSF-SN silk fiber may explain the dramatic increase in its tensile strength and stiffness. Interestingly, WAXD diffraction pattern of the RSF-Pa fiber has an apparent shoulder near 2θ = 8.8°, suggesting lateral packing of helices. Higher content of helical structure and random coil may contribute to better extensibility of the RSF-Pa fiber when compared with the RSF-SN fiber (Figure S6).
Table 1Secondary structure content of silk fibers characterized by FTIR
Ratio (%)β sheetβ turnHelixRandom coil
RSF-SN55.8 ± 1.614.9 ± 1.714.6 ± 1.014.8 ± 2.2
RSF-Pa51.3 ± 1.114.0 ± 1.517.3 ± 0.317.3 ± 0.8
B. mori silk44.2 ± 1.117.6 ± 0.716.1 ± 0.322.1 ± 0.6
Data are shown as means ± SD from three replicates.
Table 2Crystallinity and crystallite size of silk fibers characterized by WAXD
Crystallite size (nm)Crystallinity (%)
(020)(200)/(210)
RSF-SN2.2 ± 0.12.4 ± 0.252.5 ± 0.6
RSF-Pa2.2 ± 0.12.4 ± 0.144.9 ± 0.9
B. mori silk2.3 ± 0.23.9 ± 0.340.5 ± 1.2
Data are shown as means ± SD from 3 replicates.
We previously showed that Zn2+ ions were incorporated into the artificial eggcase fiber (approximate 7 mg/g) using a similar metal-ion coagulation bath and may contribute to the high tenacity of artificial eggcase silk fiber. The recombinant eggcase silk spidroin contains ∼23% Ser residues, which could use their sidechain oxygen to bind Zn2+ ions. Since metal ions (Zn2+ and Fe3+) were also used in this study, they could be incorporated into artificial silkworm silk fibers. Inductively coupled plasma mass spectrometry (ICP-MS) shows relatively weak Zn2+ signals of approximately 2 mg/g and undetectable Fe3+ signals from both RSF-SN and RSF-Pa fibers (Table S2). On the contrary, we did not find any Zn2+ or Fe3+ ion in degummed natural B. mori silk. Amino acid sequence analysis reveals that silkworm fibroin is composed of ∼12% Ser residues. Compared with eggcase spidroin, a lower abundance of Ser residues in silkworm fibroin could result in a smaller amount of Zn2+ ions binding to the RSF. Nevertheless, incorporation of Zn2+ metal ions into artificial fibers during spinning and post-spinning drawing processes may contribute to the excellent mechanical properties of the RSF-SN and the RSF-Pa fibers (Figure 3).
Figure 3Models of RSF-SN, RSF-Pa, and degummed natural B. mori silk fibers

Conclusion

In summary, we report a facile strategy to spin superstrong artificial silkworm silk using cost-effective undegraded RSFs and a metal-ion coagulation bath. The tensile strength of the RSF silk fiber (∼2.0 GPa) can significantly surpass the value of the strongest natural silk, spider dragline silk. The method developed here thereby opens a promising door to produce profitable, high-performance silk-based materials on a large scale.

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