Conformational Transition Of Silk Fibroin Research Articles

Dynamic Adaptive Wrinkle‐Structured Silk Fibroin/MXene Composite Fibers for Switchable Electromagnetic Interference Shielding

AbstractFlexible and dynamic adaptive conductive composite fibers are demanded for functional fabrics and wearable devices, yet the weak interfaces often impair the critical performances for complicated application scenarios. Here, a general and scalable strategy is proposed to construct robust and reversible wrinkle structures on regenerated silk fibroin (RSF) fiber with 2D nanomaterials (i.e., MXene, graphene) by wet‐spinning technique. The dynamic conformation transition of silk fibroin and the core‐shell modulus mismatching together are responsible for the formation of wrinkled structures. Then, it effectively optimizes the interfaces between the assembled nanomaterials and fiber substrate, which significantly reinforce the fibers, complete conductive pathways, and endow exceptional robustness and durability. The MXene‐based fiber shows a high conductivity of ≈1125 S cm−1, far superior tensile strength of 214.3 ± 6.8 MPa and toughness of 15.2 ± 1.5 MJ m−3 as compared to RSF fiber. More interestingly, the transformable wrinkled structure further modulates surface behaviors and properties of the fibers, such as the switchable shielding performances from “off‐state” to “on‐state” induced by humidity. Additionally, the integration of mechanical flexibility and outstanding capacitance (≈829 F cm−3) further expects the great promise of MXene‐based fibers for self‐powered wearable electronics, and intelligent fabrics.

Dynamic Changes and Characterization of the Metal Ions in the Silk Glands and Silk Fibers of Silkworm

Metal ions are involved in the conformational transition of silk fibroin and influence the structure and mechanical properties of silk fibers. However, the dynamic characteristics of metal ions during the formation of silk fibers remain unclear. In this study, we found that the silk glands of silkworms contain various metal elements, with varying levels of the metal elements in different zones of the glands and higher levels in the anterior silk glands. Additionally, the content of various metallic elements in the silk glands varied greatly before and after spinning, similar to their content in different cocoon layers, thus, indicating that the anterior silk glands maintain a certain metal ion environment for the transport and conformational transformation of the silk proteins. Most of the metallic elements located in fibroin were confirmed using degumming experiments. For the first time, a scanning electron microscope energy spectrometry system was used to characterize the metal elements in the cross-section of silk and cocoons. These findings have deepened our understanding of the relationship between the overall metal ion environment and silk fiber formation and help us further conceptualize the utilization of metal ions as targets to improve the mechanical properties of the silk fibers.

Instantaneous Formation of Silk Protein Aerosols and Fibers with a Portable Spray Device Under Ambient Conditions.

A variety of artificial silk spinning approaches have been attempted to mimic the natural spinning process found in silkworms and spiders, yet instantaneous silk fiber formation with hierarchical structure under physiological and ambient conditions without post-treatment procedures remains unaddressed. Here, we report a new strategy to fabricate silk protein-based aerosols and silk fibers instantaneously (< 1 s) in situ using a simple, portable, spray device, avoiding complicated and costly advanced manufacturing techniques. The key to success is the instantaneous conformational transition of silk fibroin from random coil to β-sheet right before spraying by mixing silk and polyethylene glycol (PEG) solutions in the spray device, allowing aerosols and silk fibers to be sprayed in situ, with further control achieved via the molecular weight of silk. The spinning process of the spray device is based on the use of green solvents, i.e., all steps of instant conformational transition of silk fibroin are carried out in aqueous conditions or with buffers at ambient conditions, in combination with shear and elongational flow caused by the hydraulic pressure generated in the spray container. The system supports a portable and user-friendly system that could be used for drug delivery carriers, wound coating materials and rapid silk fiber conformal coatings on surfaces.

Effect of pH on the Conformational Transition of Silk Fibroin in Aqueous Solution Monitored by Thioflavin-T Fluorescence.

In this work, the potential application of the fluorescence dye Thioflavin-T (ThT), which can specifically bind to amyloid, as a powerful tool for monitoring secondary structural transitions of silk fibroin (SF) induced by pH in low solution concentrations was examined. Results showed that ThT emission intensities substantially increased when pH decreased from 6.8 to 4.8. This increase may be ascribed to conformational transitions from random coil to β-sheet. The morphology and secondary structure of SF were also investigated via TEM, AFM and circular dichroism spectroscopy. The information obtained herein can be utilized not only for the development of convenient and efficient noninvasive method for monitoring the assembly behavior of SF in aqueous solution but also for in vitro fluorescence imaging.

Silk fibroin hydrogels induced and reinforced by acidic calcium phosphate – A simple way of producing bioactive and drug-loadable composites for biomedical applications

Silk fibroin (SF) hydrogels have attracted extensive interest in biomedical applications due to their biocompatibility and wide availability. However, their generally poor mechanical properties limit their utility. Here, injectable, ready-to-use SF-based composites, simultaneously induced and reinforced by acidic calcium phosphates, were prepared via a dual-paste system requiring no complex chemical/physical treatment. The composite was formed by mixing a monocalcium phosphate monohydrate paste with a β-tricalcium phosphate/SF paste. The conformational transition of SF in an acidic environment forms continuous networks, and the acidic calcium phosphate, brushite and monetite, formed simultaneously in the networks during mixing. The composites displayed a partly elastomeric compression behavior, with mechanical properties increasing with an increasing calcium phosphate and β-sheet content at the lower calcium phosphate contents evaluated (22.2–36.4 wt%). While the stiffness was still relatively low, the materials presented a high elasticity and ductility, and no failure at stresses in the range of failure stresses of trabecular bone. Furthermore, the calcium phosphate confers bioactivity to the material, and the composites with a promising in vitro cell response also showed potential as drug vehicles, using vancomycin as a model drug. These dual-paste systems exhibit potential utility in biomedical applications, such as bone void fillers and drug vehicles.

Polydopamine-assisted immobilization of silk fibroin and its derived peptide on chemically oxidized titanium to enhance biological activity in vitro

Biochemical modification can endow the surface of implants with superior biological activity. Herein, silk fibroin (SF) protein and its anionic derivative peptides (Cs) were covalently immobilized onto a titanium implant surface via a polydopamine layer. The successful conjugation of SF and Cs was revealed by X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and contact angle measurements. The addition of Cs prevented the conformational transition of silk fibroin to silk II. The deposition of apatite on its surface was significantly accelerated, and the bioactive composite coating was observed to enhance protein adsorption and cell proliferation. More importantly, it also promoted the osteogenic differentiation of bone marrow stem cells (BMSCs) for the quantitative and qualitative detection of alkaline phosphatase (ALP) and alizarin red (ARS). Overall, the stable performance and enhanced osteogenic property of the composite coating promote an extensive application for clinical titanium-based implants.

Enzymatic degradability and release properties of graphene oxide/silk fibroin nanocomposite films

AbstractThe structure evolution of silk fibroin (SF) in the nanocomposite films with graphene oxide (GO) was investigated and related to the enzymatic degradability and release property. The interaction with GO was found to induce conformation transition of SF from random coil to β‐sheet. However, the surface binding constrained the rearrangement of the silk chains, leading to a decrease of β‐sheet when GO content was more than 1.0%. The crystal structure of SF played a key role in the degradation of GO/SF composites. The preferential degradation of the hydrophilic blocks resulted in a faster degradation of SF films with higher β‐sheet content. The addition of GO to SF matrix led to a slower release and a reduction of the burst release of RhB, the model compound. The release profile was well fitted to the Rigter–Peppas equation, from which the characteristic constant decreased and the diffusional exponent increased with increasing GO content but quickly leveled off when GO content was more than 1.0%. Degradation of the composites had little influence on the characteristic constant of RhB release, however, led to an increased diffusional exponent, which was more evident for the composites with higher β‐sheet content.

Получение нерастворимых в воде пленок из фиброина шелка, исследование их структуры и свойств

Silk fibroin is one of the promising natural polymers that already being used in medicine to forming of suture materials. The possibility of forming scaffolds in the form of films, sponges or gels for cell cultivation and transplantation will significantly expand the scope of application of this material in tissue engineering. The conditions for obtaining silk fibroin insoluble in water in the form of films were worked out. The structural transition on the surface of the films to a coarsely fragmented state after treatment of the films with methanol was demonstrated using optical microscopy. Using FTIR spectroscopy, the conformational transition of silk fibroin from the disordered structure of the untreated film to the β-folded structure was confirmed. Using the method of differential scanning calorimetry, the glass transition temperature of the native film was determined as 216 °C. The study of the mechanical properties of silk fibroin films in a liquid medium showed that their strength decreases and their elasticity increases by almost 15 times compared to tests of the same films in a dry state.

Direct Observation of Native Silk Fibroin Conformation in Silk Gland of Bombyx mori Silkworm.

To understand the natural silk spinning mechanism, synchrotron Fourier transform infrared (S-FTIR) microspectroscopy was employed in this study to monitor the conformation changes of silk protein in the silk gland of Bombyx mori silkworm. The ultrahigh brightness of S-FTIR microspectroscopy allowed the imaging of the silk gland with micrometer-scale spatial resolution. Herein, tissue sections of a silk gland, including cross-section slices and longitudinal-section slices, were characterized. The results obtained clearly confirm that the conformation of the silk fibroin changes gradually along the silk gland from the tail to the spinneret. In the middle silk gland, silk fibroin mainly contains random coil/helix conformation. When it comes to the spinneret through the anterior silk gland, the content of β-sheet increases, but the content of random coil/helix instead reduces gradually. Further, the β-sheet distribution in the cross-section of the anterior silk gland was imaged using S-FTIR mapping technique. The results show that the structural distribution of the silk fibroin in cross-section is uniform without significant shell-core structure, which implies that the primary driving force to induce the conformation transition of silk fibroin from random coil/helix to β-sheet during the spinning process is elongational flow of silk fibroin in the silk gland and not the shear force between the silk fibroin and the lumen wall of silk gland. These direct pieces of evidence of silk fibroin structure in the silk gland would definitely promote a deeper understanding of the natural spinning process.

Microstructure Transitions and Dry-Wet Spinnability of Silk Fibroin Protein from Waste Silk Quilt.

With excellent biocompatibility and biodegradability, silk fibroin has been developed into many protein materials. For producing regenerated silk fibroin (RSF) fibers, the conformation transition of silk fibroin needs to be thoroughly studied during the spinning process. Since the many silk fabrics that are discarded comprise an increasing waste of resources and increase the pressure on the environment, in this paper, waste silk fiber was recycled in an attempt to prepare regenerated fibroin fiber by dry-wet spinning. Ethanol was the coagulation bath. The rheological properties of all the RSF solutions were investigated to acquire rheology curves and non-Newtonian indexes for spinnability analysis. Four stages of the spinning process were carried out to obtain RSF samples and study their conformation transitions, crystallization, and thermal properties by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, and differential scanning calorimetry. Quantitative analysis of the FTIR results was performed to obtain specific data regarding the contents of the secondary structures. The results showed that higher concentration spinning solutions had better spinnability. As the spinning process progressed, random coils were gradually converted into β-sheets and crystallization increased. Among the different influencing factors, the ethanol coagulation bath played a leading role in the conformation transitions of silk fibroin.

Disruption of the Metal Ion Environment by EDTA for Silk Formation Affects the Mechanical Properties of Silkworm Silk.

Silk fiber has become a research focus because of its comprehensive mechanical properties. Metal ions can influence the conformational transition of silk fibroin. Current research is mainly focused on the role of a single ion, rather than the whole metal ion environment. Here, we report the effects of the overall metal ion environment on the secondary structure and mechanical properties of silk fibers after direct injection and feeding of silkworms with EDTA. The metal composition of the hemolymph, silk gland, and silk fiber changed significantly post EDTA treatment. Synchrotron FTIR analysis indicated that the secondary structure of silk fiber after EDTA treatment changed dramatically; particularly, the β-sheets decreased and the β-turns increased. Post EDTA treatment, the silk fiber had significantly decreased strength, Young’s modulus, and toughness as compared with the control groups, while the strain exhibited no obvious change. These changes can be attributed to the change in the metal ion environment in the silk fibroin and sericin in the silk gland. Our investigation provides a new theoretical basis for the natural silk spinning process, and our findings could help develop a method to modify the mechanical properties of silk fiber using metal ions.

Understanding Secondary Structures of Silk Materials via Micro- and Nano-Infrared Spectroscopies.

The secondary structures (also termed conformations) of silk fibroin (SF) in animal silk fibers and regenerated SF materials are critical in determining mechanical performance and function of the materials. In order to understand the structure-mechanics-function relationships of silk materials, a variety of advanced infrared spectroscopic techniques, such as micro-infrared spectroscopies (micro-IR spectroscopies for short), synchrotron micro-IR spectroscopy, and nano-infrared spectroscopies (nano-IR spectroscopies for short), have been used to determine the conformations of SF in silk materials. These IR spectroscopic methods provide a useful toolkit to understand conformations and conformational transitions of SF in various silk materials with spatial resolution from the nano-scale to the micro-scale. In this Review, we first summarize progress in understanding the structure and structure-mechanics relationships of silk materials. We then discuss the state-of-the-art micro- and nano-IR spectroscopic techniques used for silk materials characterization. We also provide a systematic discussion of the strategies to collect high-quality spectra and the methods to analyze these spectra. Finally, we demonstrate the challenges and directions for future exploration of silk-based materials with IR spectroscopies.

Modification of electrospun silk fibroin nanofiber mats: using an EDC/NHS ethanol solvent

The application of electrospun silk fibroin nanofiber mats ware restricted for its poor mechanical property. The goal of the present study was to improve the mechanical property of silk fibroin nanofiber mats. EDC/NHS ethanol system was employed to treat silk fibroin nanofiber mats to modify the property of silk fibroin nanofiber mats. The as-spun, ethanol treated, and EDC/NHS ethanol treated silk fibroin nanofiber mats were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry-Differential thermal analysis, and Instron tester. After ethanol and EDC/NHS treatments, the conformation transition of silk fibroin was detected by FTIR and WAXD. The crosslinker EDC/NHS will form abundant “amide” bonds within silk fibroin which was confirmed by FTIR and tensile mechanical test. Compared to as-spun and ethanol treated silk fibroin nanofiber mats, the EDC/NHS ethanol treated one demonstrated significantly enhanced breaking elongation by 136%. The significant increase in breaking elongation made the electrospun silk fibroin nanofiber mats more promising scaffold materials for practical tissue engineering application.

Intrinsically reinforced silks obtained by incorporation of graphene quantum dots into silkworms

Silkworm silks have been widely used in a variety of fields due to their sensuousness, luster and excellent mechanical properties. Researchers have paid special attention in improving the mechanical properties of silks. In this work, Bombyx mori larval silkworms are injected with graphene quantum dots (GQDs) through a vascular injection to enhance mechanical properties of the silkworm silks. The GQDs can be incorporated into the silkworm silk gland easily due to hemolymph circulation and influence the spinning process of silkworm. The breaking strength, elongation at break and toughness modulus of the silks increase by 2.74, 1.33 and 3.62 times, respectively, by injecting per individual with 0.6 μg GQDs. Wide-angle X-ray scattering indicates that the size of β-sheet nanocrystals in GQDs-silks is smaller than that in control-silks. Infrared spectra suggest that GQDs confine the conformation transition of silk fibroin to β-sheet from random coil/α-helix, and the change of the size and content of β-sheet may be the reason for the improvement of the mechanical properties. The toxicity and safety limit of GQDs incorporated into each silkworm is also evaluated, and the results show that the upmost dose of GQDs per silkworm is 30.0 μg. The successful obtainment of reinforced silks by in vivo uptake of GQDs provides a promising route to produce high-strength silks.

Conformational Transitions of Silk Fibroin in Solutions under the Action of Ultrasound

The influence of ultrasonic treatment on the fibroin conformation in solutions and films was studied by dynamic light scattering, FTIR spectroscopy, and X-ray diffraction. Ultrasonic treatment of fibroin solutions leads to a decrease in the fraction of the α-helical conformation and to an increase in the fraction of β-turns and β-structure in the fibroin structure in the films regenerated from solutions.

High-Strength, Durable All-Silk Fibroin Hydrogels with Versatile Processability toward Multifunctional Applications.

Hydrogels have been the focus of extensive research due to their potential use in fields including biomedical, pharmaceutical, biosensors, and cosmetics. However, the general weak mechanical properties of hydrogels limit their utility. Here, we generate pristine silk fibroin (SF) hydrogels with excellent mechanical properties via a binary solvent induced conformation transition (BSICT) strategy. In this method, the conformational transition of SF is regulated by moderate binary solvent diffusion and SF/solvent interactions. β-sheet formation serves as the physical crosslinks that connect disparate protein chains to form continuous 3D hydrogel networks, avoiding complex chemical and/or physical treatments. The Young's modulus of these new BSICT-silk fibroin hydrogels can reach up to 6.5±0.2 MPa, tens to hundreds of times higher than that of conventional hydrogels (0.01-0.1 MPa). These new materials filled the "empty soft materials space" in the elastic modulus/strain Ashby plot. More remarkably, the BSICT-SF hydrogels can be processed into different constructions through different polymer and/or metal based processing techniques, such as molding, laser cutting, and machining. Thus, these new hydrogel systems exhibit potential utility in many biomedical and engineering fields.

Nanofibers of silk fibroin controlled by the crystallization of polyethylene glycol in frozen solution

The conformation of silk fibroin (SF) frozen with polyethylene glycol (PEG) at a molecular weight from 2 kDa to 20 kDa and a mass ratio of PEG:SF from 1:5 to 10:1 was studied by spectral and microscopic methods. It is found that the conformation transition of SF from random coil to β-sheet could be induced by the stress resulting from PEG crystallization at −20 °C, and greatly depended on the cooling rate, PEG:SF mass ratio and PEG molecular weight. These findings provide a new method for the preparation of desired SF nanofibers.

In vivo effects of metal ions on conformation and mechanical performance of silkworm silks

BackgroundThe mechanism of silk fiber formation is of particular interest. Although in vitro evidence has shown that metal ions affect conformational transitions of silks, the in vivo effects of metal ions on silk conformations and mechanical performance are still unclear. MethodsThis study explored the effects of metal ions on silk conformations and mechanical properties of silk fibers by adding K+ and Cu2+ into the silk fibroin solutions or injecting them into the silkworms. Aimed by CD analysis, FTIR analysis, and mechanical testing, the conformational and mechanical changes of the silks were estimated. By using BION Web Server, the interactions of K+ and N-terminal of silk fibroin were also simulated. ResultsWe presented that K+ and Cu2+ induced the conformational transitions of silk fibroin by forming β-sheet structures. Moreover, the mechanical parameters of silk fibers, such as strength, toughness and Young's modulus, were also improved after K+ or Cu2+ injection. Using BION Web Server, we found that potassium ions may have strong electrostatic interactions with the negatively charged residues. ConclusionWe suggest that K+ and Cu2+ play crucial roles in the conformation and mechanical performances of silks and they are involved in the silk fiber formation in vivo. General significanceOur results are helpful for clarifying the mechanism of silk fiber formation, and provide insights for modifying the mechanical properties of silk fibers.

Feeding Single-Walled Carbon Nanotubes or Graphene to Silkworms for Reinforced Silk Fibers.

Silkworm silk is gaining significant attention from both the textile industry and research society because of its outstanding mechanical properties and lustrous appearance. The possibility of creating tougher silks attracts particular research interest. Carbon nanotubes and graphene are widely studied for their use as reinforcement. In this work, we report mechanically enhanced silk directly collected by feeding Bombyx mori larval silkworms with single-walled carbon nanotubes (SWNTs) and graphene. We found that parts of the fed carbon nanomaterials were incorporated into the as-spun silk fibers, whereas the others went into the excrement of silkworms. Spectroscopy study indicated that nanocarbon additions hindered the conformation transition of silk fibroin from random coil and α-helix to β-sheet, which may contribute to increased elongation at break and toughness modules. We further investigated the pyrolysis of modified silk, and a highly developed graphitic structure with obviously enhanced electrical conductivity was obtained through the introduction of SWNTs and graphene. The successful generation of these SWNT- or graphene-embedded silks by in vivo feeding is expected to open up possibilities for the large-scale production of high-strength silk fibers.

Effect of EGCG On Fe(III)‐induced conformational transition of silk fibroin, a model of protein related to neurodegenerative diseases

The abnormal aggregation of amyloid proteins is reported to play a critical role in the etiology of neurodegenerative disorders. Studies have shown that excessive ferric irons are associated with the misfolding of amyloid proteins, and that (-)-epigallocatechin gallate (EGCG) is a good metallic ion chelator with inhibitory effect on the aggregation of amyloid proteins. EGCG has been thus considered as a potential drug candidate for the treatment of neurodegenerative diseases. However, the mechanism of action for EGCG in inhibition of aggregation of amyloid proteins is still remaining unclear. Silk fibroin (SF) shares similarities with amyloid proteins in some amino acid sequences and fibrillation kinetics. In this work, therefore, we used SF as a model of protein to investigate the effects of Fe(III) and EGCG on conformational transition by using turbidity assay, thioflavin T (ThT) fluorescence spectroscopy, Raman spectroscopy, and atomic force microscope (AFM). We demonstrated that low concentration of Fe(III) ions promoted the formation of β-sheet conformers, while high concentration of Fe(III) ions inhibited further aggregation of SF. EGCG could significantly inhibit the conformational transition of SF when induced by Fe(III), and decrease the amount of β-sheet conformers dose-dependently. The findings provide important information regarding to EGCG as a potential agent for the prevention and treatment of neurodegenerative diseases. Fe(III) can accelerate the conformation transition of silk fibrion (SF) from random coil into β-sheet, while (-)-epigallocatechin gallate (EGCG) inhibits Fe(III)-induced β-sheet aggregation of SF., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 100-107, 2016.