Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks.

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

Multi-wavelength coherent random laser in bio-microfibers.

In this paper, pure silk protein was extracted from Bombyx mori silks and fabricated into a new kind of disordered bio-microfiber structure using electrospinning technology. Coherent random lasing emission with low threshold was achieved in the silk fibroin fibers. The random lasing emission wavelength can be tuned in the range of 33 nm by controlling the pump location with different scattering strengths. Therefore, the bio-microfiber random lasers can be a wide spectral light source when the system is doped with a gain or energy transfer medium with a large fluorescence emission band. Application of the random lasers of the bio-microfibers as a low-coherence light source in speckle-free imaging had also been studied.

A Cuboid Spider Silk: Structure-Function Relationship and Polypeptide Signature.

A unique cuboid spider silk from the outer egg sac of Nephila pilipes, with an unusual square cross-section, is disclosed. The structure-function relationships within this silk are first studied through structural characterization, mechanical measurement, protein conformation, and polypeptide signature of silk proteins. This silk maintains the higher stiffness property of egg sac silks, and also shows a species difference. Environmental response of the mechanical properties within this silk are observed. Synchrotron FTIR microspectroscopy is used to monitor the silk protein conformation in a single natural silk. The ß-sheet structure aligns parallel to the fiber axis with a content of 22% ± 2.6%. The de novo resulting polypeptide from the solid silk fibers are novel, and an abundant polar amino acid insertion is observed. Short polyalanine (An , n ≤ 3), alternating serine and alanine (S/A)X, and alternating glycine and alanine (G/A)X, GGX, and SSX dominates in the resulting de novo polypeptide. This accords with the composition pattern of other egg sac silk proteins, besides the rarely observed GGX. This study broadens the library of egg sac spider silks and provides a new perspective to uncover structure-function relationships in spider silk.

Effect of Silk Fibroin on Neuroregeneration After Traumatic Brain Injury.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1 day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

Aggregation State of Residual α-Helices and Their Influence on Physical Properties of S. c. ricini Native Fiber.

Formation of the α-helical conformation in the poly-l-alanine (PA) sequence regions, subsequent structural transition to ß-sheet during natural spinning, and presence of residual α-helices in Samia cynthia ricini (S. c. ricini) native silk fiber have been experimentally proven. However, the aggregation state of the residual α-helices, and their influence on the mechanical deformation behavior in native fiber remain unclear. Here we show that the α-helices form an ordered aggregation state with a hexagonal packing in the aqueous solution, some of which remain during natural spinning. X-ray scattering and differential scanning calorimetry (DSC) analyses revealed occurrence of a structural transition of the residual α-helices to the ß-sheet structure, accompanied by disappearance of the plateau region in the force-strain curve, due to heat-treatment at ~220 °C. On the basis of X-ray scattering before and after tensile stretching of S. c. ricini native silk, a direct connection between the plateau region and the α-helix to ß-sheet structural transition was confirmed. Our findings demonstrate the importance of the PA sequence regions in fiber structure formation and their influence on the tensile deformation behavior of S. c. ricini silk, features believed to be essentially similar in other saturniid silks. We strongly believe the residual ordered α-helices to be strategically and systematically designed by S. c. ricini silkworms to impart flexibility in native silk fiber. We anticipate that these knowledge forms a basis for fruitful strategies in the design and development of amino acid sequences for artificial silks with desired mechanical properties.

Au nanoparticles-ZnO composite nanotubes using natural silk fibroin fiber as template for electrochemical non-enzymatic sensing of hydrogen peroxide.

A novel electrochemical sensor based on the composite of gold nanoparticles/zinc oxide nanotube (AuNPs/ZnO-NTs) was constructed and its application as hydrogen peroxide (H2O2) non-enzymatic sensor was investigated. ZnO-NTs were prepared by a biomineralization strategy in which silk fibroin fiber (SFF) was used as template, and thus the ZnO-NTs inherited the advantages of SFF such as mechanical stability, flexible biomimetic morphology and biocompatibility. The AuNPs/ZnO-NTs was further prepared by the electrostatic absorption of AuNPs onto the surface of ZnO-NTs, and found to be capable to catalyze the reduction of H2O2. The working potential was 0.05 V, which was far higher than those in literatures, indicating the strong anti-interference ability in the real application. The catalytic current was linearly proportional in the concentration range of 1 µM-3.0 mM with a sensitivity of 1336.7 µA mM-1 cm-2. The detection limit was estimated to be 0.1 µM (S/N = 3). Such a high sensitivity was attributed to the electrocatalytic property of ZnO and high electron transfer ability of AuNPs/ZnO-NTs structure. Moreover, the final detection results of H2O2 in real samples showed the acceptable accuracy compared with the traditional potassium permanganate titration, exhibiting the prospect to be used as an applicable sensor in actual detections.

Liquid Crystalline Granules Align in a Hierarchical Structure To Produce Spider Dragline Microfibrils.

The spider silk spinning process converts spidroins from an aqueous form to a tough fiber. This spinning process has been investigated by numerous researchers, and micelles or liquid crystals of spidroins have been reported to form silk fibers, which are bundles of silk microfibrils. However, the formation process of silk microfibrils has not been clarified previously. Here, we report that silk microfibrils are generated through the formation, homogenization, and linkage of liquid crystalline granules without micelle-like structures. Heterogeneous granules on the submicron to micron scale were observed in the storage sac, whereas homogeneous granules with diameters of approximately 100 nm were aligned along the tapering duct. In the spun fibers, the homogeneous granules were connected along the fiber axis. This is the first clear description of the formation of granule-based microfibrils in the spinning process, which is the key conversion process leading to the unique hierarchical structure of spider dragline.

Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk.

Spider silks are outstanding biomaterials with mechanical properties that outperform synthetic materials. Of the six fibrillar spider silks, aciniform (or wrapping) silk is the toughest through a unique combination of strength and extensibility. In this study, a wet-spinning method for recombinant Argiope trifasciata aciniform spidroin (AcSp1) is introduced. Recombinant AcSp1 comprising three 200 amino acid repeat units was solubilized in a 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/water mixture, forming a viscous α-helix-enriched spinning dope, and wet-spun into an ethanol/water coagulation bath allowing continuous fiber production. Post-spin stretching of the resulting wet-spun fibers in water significantly improved fiber strength, enriched ß-sheet conformation without complete α-helix depletion, and enhanced birefringence. These methods allow reproducible aciniform silk fiber formation, albeit with lower extensibility than native silk, requiring conditions and methods distinct from those previously reported for other silk proteins. This provides an essential starting point for tailoring wet-spinning of aciniform silk to achieve desired properties.

Silk Fibroin-Sophorolipid Gelation: Deciphering the Underlying Mechanism.

Silk fibroin (SF) protein, produced by silkworm Bombyx mori, is a promising biomaterial, while sophorolipid (SL) is an amphiphilic functional biosurfactant synthesized by nonpathogenic yeast Candida bombicola. SL is a mixture of two forms, acidic (ASL) and lactonic (LSL), which when added to SF results in accelerated gelation of silk fibroin. LSL is known to have multiple biological functionalities and hence hydrogels of these green molecules have promising applications in the biomedical sector. In this work, SANS, NMR, and rheology are employed to examine the assembling properties of individual and mixed SLs and their interactions with SF to understand the mechanism that leads to rapid gelation. SANS and NMR studies show that ASL assembles to form charged micelles, while LSL forms micellar assemblies and aggregates of a mass fractal nature. ASL and LSL together form larger mixed micelles, all of which interact differently with SF. It is shown that preferential binding of LSL to SF causes rapid unfolding of the SF chain leading to the formation of intermolecular beta sheets, which trigger fast gelation. Based on the observations, a mechanism for gelation of SF in the presence of different sophorolipids is proposed.

Comparative Study of Ultrasonication-Induced and Naturally Self-Assembled Silk Fibroin-Wool Keratin Hydrogel Biomaterials.

This study reports the formation of biocompatible hydrogels using protein polymers from natural silk cocoon fibroins and sheep wool keratins. Silk fibroin protein contains ß-sheet secondary structures, allowing for the formation of physical cross-linkers in the hydrogels. Comparative studies were performed on two groups of samples. In the first group, ultrasonication was used to induce a quick gelation of a protein aqueous solution, enhancing the ability of Bombyx mori silk fibroin chains to quickly entrap the wool keratin protein molecules homogenously. In the second group, silk/keratin mixtures were left at room temperature for days, resulting in naturally-assembled gelled solutions. It was found that silk/wool blended solutions can form hydrogels at different mixing ratios, with perfectly interconnected gel structure when the wool content was less than 30 weight percent (wt %) for the first group (ultrasonication), and 10 wt % for the second group (natural gel). Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) were used to confirm that the fibroin/keratin hydrogel system was well-blended without phase separation. Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structures of blended protein gels. It was found that intermolecular ß-sheet contents significantly increase as the system contains more silk for both groups of samples, resulting in stable crystalline cross-linkers in the blended hydrogel structures. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to analyze the samples' characteristic morphology on both micro- and nanoscales, which showed that ultrasonic waves can significantly enhance the cross-linker formation and avoid phase separation between silk and keratin molecules in the blended systems. With the ability to form cross-linkages non-chemically, these silk/wool hydrogels may be economically useful for various biomedical applications, thanks to the good biocompatibility of protein molecules and the various characteristics of hydrogel systems.

Self-assembly of regenerated silk fibroin from random coil nanostructures to antiparallel ß-sheet nanostructures.

In this work, we studied the effects of incubation concentration and time on the self-assembly behaviors of regenerated silk fibroin (RSF). Our results showed the assembly ways of RSF were concentration-dependent and there were four self-assembly ways of RSF: (i) At relatively low concentration (≤0.015%), RSF molecules assembled into protofilaments (random coil), and then the thickness decreased and the secondary conformation changed to antiparallel ß-sheet; (ii) at the concentration of 0.015%, RSF molecules assembled into protofilaments (random coil), and then assembled into protofibrils (antiparallel ß-sheet). The protofibrils experienced the appearance and disappearance of phase periodic intervals in turn; (iii) at the concentration of 0.03%, RSF molecules assembled into bead-like oligomers (random coil), and then assembled into protofibrils (antiparallel ß-sheet), and finally the height and phase periodic intervals of RSF protofibrils disappeared in turn; and (iv) at the relatively high concentration (≥0.15%), RSF molecules assembled into protofilaments (random coil), then aggregated into blurry cuboid-like micelles (random coil), and finally self-arranged to form smooth and clear cuboid-like micelles (antiparallel ß-sheet). These results provide useful insights into the process by which the RSF molecules self-assemble into protofilaments, protofibrils and micelles. Furthermore, our work will be beneficial to basic understanding of the nanoscale structure formations in different silk-based biomaterials.

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms.

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk-based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials.

Evaluation of stretched electrospun silk fibroin matrices seeded with urothelial cells for urethra reconstruction.

BACKGROUND: We investigated the feasibility of urethral reconstruction using stretched electrospun silk fibroin matrices. MATERIALS AND METHODS: A novel electrospun silk fibroin matrix was prepared. The structure of the material was assessed by scanning electron microscopy and a porosity test. Canine urothelial cells were isolated, expanded, and seeded onto the material for 1 wk to obtain a tissue-engineered graft. The tissue-engineered graft was assessed using hematoxylin and eosin staining and scanning electron microscopy. A dorsal urethral mucosal defect was created in nine female beagle dogs. In the experimental group, tissue-engineered mucosa was used to repair urethra mucosa defects in six dogs. No substitute was used in the three dogs of the control group. Retrograde urethrography was performed at 1, 2, and 6 mo after grafting. The urethral grafts were analyzed grossly and histologically. RESULTS: Scanning electron microscope and a porosity test revealed that the material had a three-dimensional porous structure. Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices. Canines implanted with tissue-engineered mucosa voided without difficulty. Retrograde urethrography revealed no signs of stricture. Histologic staining showed gradual epithelial cell development and stratified epithelial layers at 1, 2, and 6 mo. The canines in the control group showed difficulty in voiding. Retrograde urethrography showed urethra stricture. Histologic staining showed that no or only one layer of epithelial cells developed. A severe inflammatory reaction was also observed in the control group. CONCLUSIONS: Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells, which could prove to be a potential material for use in urethra reconstruction.

Silk layering as studied with neutron reflectivity.

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial ß-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface.

Structure and biodegradation mechanism of milled Bombyx mori silk particles.

The aim of this study was to understand the structure and biodegradation relationships of silk particles intended for targeted biomedical applications. Such a study is also useful in understanding structural remodelling of silk debris that may be generated from silk-based implants. Ultrafine silk particles were prepared using a combination of efficient wet-milling and spray-drying processes with no addition of chemicals other than those used in degumming. Milling reduced the intermolecular stacking forces within the ß-sheet crystallites without changing the intramolecular binding energy. Because of the rough morphology and the ultrafine size of the particles, degradation of silk particles by protease XIV was increased by about 3-fold compared to silk fibers. Upon biodegradation, the thermal degradation temperature of silk increased, which was attributed to the formation of tight aggregates by the hydrolyzed residual macromolecules. A model of the biodegradation mechanism of silk particles was developed based on the experimental data. The model explains the process of disintegration of ß-sheets, supported by quantitative secondary structural analysis and microscopic images.

Flexibility regeneration of silk fibroin in vitro.

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.

Solution structure of eggcase silk protein and its implications for silk fiber formation.

Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with alpha-helical and random coil structures in silk glands into insoluble fibers with mainly beta-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a approximately 366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N- and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at > or = 37 degrees C and a protein concentration of > 0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks.

Protein families, natural history and biotechnological aspects of spider silk.

Spiders are exceptionally diverse and abundant organisms in terrestrial ecosystems and their evolutionary success is certainly related to their capacity to produce different types of silks during their life cycle, making a specialized use on each of them. Presenting particularly tandemly arranged amino acid repeats, silk proteins (spidroins) have mechanical properties superior to most synthetic or natural high-performance fibers, which makes them very promising for biotechnology industry, with putative applications in the production of new biomaterials. During the evolution of spider species, complex behaviors of web production and usage have been coupled with anatomical specialization of spinning glands. Spiders retaining ancestral characters, such as the ones belonging to the Mygalomorph group, present simpler sorts of webs used mainly to build burrows and egg sacs, and their silks are produced by globular undifferentiated spinning glands. In contrast, Araneomorphae spiders have a complex spinning apparatus, presenting up to seven morphologically distinct glands, capable to produce a more complex set of silk polymers with different degrees of rigidness and elasticity associated with distinct behaviors. Aiming to provide a discussion involving a number of spider silks' biological aspects, in this review we present descriptions of members from each family of spidroin identified from five spider species of the Brazilian biodiversity, and an evolutionary study of them in correlation with the anatomical specialization of glands and spider's spinning behaviors. Due to the biotechnological importance of spider silks for the production of new biomaterials, we also discuss about the new possible technical and biomedical applications of spider silks and the current status of it.

Surface analysis of novel fibroin films based on well-preserved crystalline structures.

We recently reported that a highly homogeneous aqueous suspension of fibroin nanofiber (FNF) can be simply obtained by mechanical water-grinding a heterogeneous aqueous fibroin slurry and that the FNF in the suspension preserves the native ß-sheet secondary structure during this mechanical treatment. The current study reports the surface properties of well-preserved crystalline structure novel FNF film from water-grinding preparation as compared with those of typical, conventionally prepared regenerated fibroin (RF) film. RF film was not treated with alcoholic solutions and was verified to be amorphous from a WAXD diffraction diagram. The air-side surfaces of the FNF semi-crystalline and RF amorphous films were studied to clarify differences using scanning electron microscopy (SEM), atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), static water contact angle, and X-ray photoelectron spectroscopy (XPS). The well-preserved crystalline in the FNF film was found to exist near a slightly deep surface region and to act as a physically cross-linking domain, governing the molecular motions of the amorphous polypeptide chains at the very shallow surface region.