Silk Protein Research Articles

Flow Analysis of Regenerated Silk Fibroin/Cellulose Nanofiber Suspensions via a Bioinspired Microfluidic Chip

AbstractMicrofluidic spinning has been used to mimic and discover the natural spinning process of silk. However, it is still challenging to understand the orientation and alignment of silk‐spinning through microfluidic chips. Here, flow analysis is performed for a bioinspired microfluidic chip mimicking the shape of a spider's major ampullate gland and spinning duct by in situ small angle X‐ray scattering and simulations using the finite element method. A composite suspension of regenerated silk fibroin and cellulose nanofibers show higher orientation and alignment after flowing through the microfluidic chip. This confinement can be attributed to the drop in the extrusion pressure of the silk proteins and the shear and elongation forces from the anisotropic microchannel. This work reveals the microstructure−property relationship of fibers obtained through the microfluidic chip. Hence, it paves the way to uncover the mystery behind the natural spinning process and further provides comprehensive and systematic insights into preparing highly oriented artificial fibers for biomedical applications.

Fibrillar Nanomembranes of Recombinant Spider Silk Protein Support Cell Co-culture in an In Vitro Blood Vessel Wall Model.

Basement membrane is a thin but dense network of self-assembled extracellular matrix (ECM) protein fibrils that anchors and physically separates epithelial/endothelial cells from the underlying connective tissue. Current replicas of the basement membrane utilize either synthetic or biological polymers but have not yet recapitulated its geometric and functional complexity highly enough to yield representative in vitro co-culture tissue models. In an attempt to model the vessel wall, we seeded endothelial and smooth muscle cells on either side of 470 ± 110 nm thin, mechanically robust, and nanofibrillar membranes of recombinant spider silk protein. On the apical side, a confluent endothelium formed within 4 days, with the ability to regulate the permeation of representative molecules (3 and 10 kDa dextran and IgG). On the basolateral side, smooth muscle cells produced a thicker ECM with enhanced barrier properties compared to conventional tissue culture inserts. The membranes withstood 520 ± 80 Pa pressure difference, which is of the same magnitude as capillary blood pressure in vivo. This use of protein nanomembranes with relevant properties for co-culture opens up for developing advanced in vitro tissue models for drug screening and potent substrates in organ-on-a-chip systems.

Interactive exploration of a hierarchical spider web structure with sound

3D spider webs exhibit highly intricate fiber architectures and owe their outstanding performance to a hierarchical organization that spans orders of magnitude in length scale from the molecular silk protein, to micrometer-sized fibers, and up to cm-scale web. Similarly, but in a completely different physical manifestation, music has a hierarchical structure composed of elementary sine wave building blocks that can be combined with other waveforms to create complex timbres, which are then arranged within larger-scale musical compositions. Although apparently different, spider webs and music have many similarities, as we point out in this work. Here, we propose an intuitive and interactive way to explore and visualize a 3D Cyrtophora citricola spider web geometry that has been digitally modeled with micron-scale details from full-scale laboratory experiments. We use model-based sonification to translate the web architecture into sound, allowing for aural perception and interpretation of its essential topological features. We implement this sonification using Unity3D and Max/MSP to create an interactive spider web environment in which a user travels through a virtual spider web. Each silk fiber in their field of view is sonified using different sine waves. Together, the sonified fibers create new and more complex timbres that reflects the architecture of 3D spider webs. These concepts are implemented into a spider web-based instrument for live performances, art installations and data exploration. It provides an unprecedented and creative way to immerse the composer, audience and user in an immersive multimedia experience generated by the complexity of a 3D spider web.

Nature-derived bionanomaterials for sustained release of 5-fluorouracil to inhibit subconjunctival fibrosis

Effectively inhibiting subconjunctival fibrosis remains a challenge in pterygium and antiglaucoma surgery. As one of the superior first-line clinical drugs, 5-fluorouracil (5-FU) possesses certain disadvantages, such as fast drug metabolism and poor dose controllability. The emergence of appropriate pharmaceutical formulation and administration routes provides attractive solutions. In this work, we report the development of a multilevel drug release strategy using two types of nature-derived biomaterials (biocompatible chitosan and silk protein) processed into nanofibers of different size ranges, which was shown to achieve sustained release of 5-FU, toward the unique application of inhibiting subconjunctival fibrosis. In vitro data demonstrated that this system achieved fast 5-FU release during the first 25 days, where the release became relatively stable and lengthy (3 months) afterward. More importantly, the in vivo outcomes also suggested a continuous long-lasting inhibitory effect on subconjunctival myofibroblasts. These results indicated that our nature-derived bionanomaterials served as a promising drug-carrying platform for inhibiting subconjunctival fibrosis by providing sustained release of pharmaceutical compounds thus reducing the administration frequency, which may find broad utility in the treatment of ocular diseases and possibly other biomedical applications.

Design, Development and Evaluation of Silk Based Film Forming Spray for Wound Healing

The objective of the present study is to formulate and evaluate Silk based film forming spray for wound healing. On the wound surface the solution solidifies into a film which can deliver the active moiety on site of action. The spray solution was prepared by simple mixing of active extract of Centella Asiatica, Silk Protein and various film forming polymers. Silk protein form scaffold for active fibroblast movement and Asiaticosides from Centella Asiatica extract improve and fasten collagen synthesis. A clear yellowish solution was obtained. The formulations (F1-F8) had a pH range between 5.5–6.5, which was close to the pH of skin. The viscosity of formulation in range of 25–50 cps, completely dry film formed within 5 min in open environment. The Effects of polymers, plasticizers and solvents on spreadability. Surface tension and Spray angle were studied. The high content of ethanol in the formulation fastens the drying time. The results indicated that formulation (F8) showed good spreadability and less drying time.
 Keywords: Film forming spray, Wound healing, Silk protein, Asiaticoside, Scaffold

Nondestructive discrimination of red silk single fibers using total reflection X-ray fluorescence spectrometry and synchrotron radiation X-ray fluorescence spectrometry.

In a strangulation case, when a necktie is used as a murder weapon, the dyed silk single fiber becomes an important evidence sample to solve the crime. Dyed silk single fibers contain elements, such as Cr and Co, which are obtained from dyeing using metal mordants. Currently, there are no nondestructive and sufficiently sensitive elementary analytical methods for the forensic analysis of single fibers. Therefore, in this study, eight commercially available red silk samples were collected and used for total reflection X-ray fluorescence (TXRF) and synchrotron radiation X-ray fluorescence (SR-XRF) spectrometry. Benchtop TXRF detected both S in the silk protein and Cl and Ca, which are elements absorbed from the environment by silkworms, but also Cr, which is a dyeing derivative for metal mordants. The presence of Cr and Zn, in addition to the Zn/Cr signal intensity ratios, was reported to be particularly useful identifiers. In SR-XRF, the presence of Cr, Co, Zn, and Br and the Zn/Cr signal intensity ratios were reported to be useful discriminating indicators. In this study, the nondestructive discrimination capabilities of TXRF and SR-XRF measurements for the samples were found to be 85.7% and 100%, respectively. Therefore, we propose a combination of TXRF and SR-XRF as a new nondestructive single fiber identification method for forensic science. Moreover, if partial destruction of a single fiber is allowed, the observation of the cross section and micro-Fourier-transform infrared spectroscopy measurements is useful for identifying red silk fibers.

CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells.

Simple SummaryBased on a CRISPRa approach, activating endogenous fibroin heavy chain (FibH) gene expression in Bombyx mori embryonic (BmE) cells, which was driven by a combination of the dCas9-VPR (a tripartite activator, composed of VP64, p65, and Rta) and the sgRNA targeting to the promoter of FibH gene, was performed for investigating the biological roles of FibH in the development of silk gland cells. The activation of the endogenous FibH gene lead to up-regulation of cellular stress responses-related genes, which suggested a significant positive correlation between activated FibH gene expression and cellular stress responses. Moreover, the present findings might provide a potential model for studying the cellular stress responses caused by silk secretion disorder and lay a foundation for the understanding of silk gland development in silk-spinning insects.The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.

Self‐Powered and Imperceptible Electronic Tattoos Based on Silk Protein Nanofiber and Carbon Nanotubes for Human–Machine Interfaces

AbstractTriboelectric electronic skins (E‐skins) can be used as primary interactive devices for human–machine interfaces (HMIs). However, devices for seamless on‐skin operations must be soft and deformable, and attachable to and compatible with the skin. In this paper, a substrate‐free, skin‐compatible, skin‐attachable, mechanically deformable, and self‐powered E‐tattoo sticker consisting of carbon nanotubes (CNTs) and silk nanofibers (SNFs) is presented. The E‐tattoo can be imperceptibly tattooed on the skin and removed with water. When the device touches naked skin, triboelectric signals are generated. Because of the micro‐to‐nano hierarchical pores (with high surface‐to‐volume ratios), contact‐induced electrification generates a power density of ≈6 mW m−2 and pressure sensitivity of ≈0.069 kPa−1. The power generated on the tattooed skin can activate small electronic devices. Moreover, whole‐body joint movements can be monitored and tactile movements can be mapped by reading the generated electrical signals. The presented E‐tattoo system can promote the development of flexible energy and sensor systems for self‐powered, energy‐autonomous, imperceptible E‐skin devices for HMIs and artificial intelligent robotics and prostheses.

Identification and location of sericin in silkworm with anti-sericin antibodies

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.

Amino Acid Profile and Biological Properties of Silk Cocoon as Affected by Water and Enzyme Extraction.

We compared the efficacy for protein extraction of water versus enzymatic extraction. The amino-acid composition, inhibitory activity against enzymes α-amylase and α-glucosidase, and anti-glycation activities of silk protein extract (SPE) were determined. We used water extraction (100 °C, six hours) and protease-enzymatic extraction. The microstructure of silk fibers was obviously different after extraction. The results showed that enzymatic extraction gave the greater values of protein content, amino acids, total phenolic content (TPC), and total flavonoid content (TFC), as well as all biological activities parameters tested, but it also provided a more bitter taste in the extract—contributing amino acids of 51% (arginine, phenylalanine, histidine, valine, tryptophan, isoleucine, and leucine) and less sweet and umami taste contributing amino acids than did water extraction, which could be more suitable to be used as concentrated nutraceuticals.

Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.

Bioinspired construction of carbonized poly(tannic acid)/g-C3N4 nanorod photocatalysts for organics degradation

Solar energy-driven photocatalysis has become a promising green technology to degrade organic pollutants discharged into water environment in recent years. In this study, a kind of novel cPTA/CN nanorod photocatalysts were prepared by using poly(tannic acid) as both template and adhesive, in which the g-C3N4 (CN) nanolayer formed on the surface of carbonized poly(tannic acid) (cPTA) nanorods, similar to the adhesion of marine mussel foot silk protein on the solid surface. The porous cPTA/CN nanorods with about 400 nm in width and 2–4 μm in length can degrade 97% Rhodamine B within 80 min and 78% tetracycline within 3 h under visible light irradiation. Porous cPTA nanorods play an important role for promoting the photocatalytic activity, which can not only accelerate the separation of photogenerated carriers, but also afford more active sites and molecule diffusion channels. This kind of nanocomposite photocatalysts may find broad application potential in the treatment of organic pollutants in aqueous solution.

Gene expression profiling reveals candidate genes for defining spider silk gland types

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3’ tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

Spinning Beta Silks Requires Both pH Activation and Extensional Stress

AbstractSynthetic silk production has undergone significant technological and commercial advances over the past 5 years, with fibers from most labs and companies now regularly matching the properties of natural silk by one metric or another. Yet the fundamental links between silk protein processing and performance remain largely unresolved and fiber optimization is commonly achieved through non‐natural methods. In an effort to address this challenge, data that closes this loop of processing and performance is presented by spinning a native silk feedstock ex vivo into a near‐native fiber using just two naturally occurring parameters; pH activation and extensional flow (i.e., spinning rate). This allows us to link previous experimental and modelling hypothesis surrounding silk's pH responsiveness directly to multiscale hierarchical structure development during spinning. Finally, fibers that match, and then exceed, natural silk's mechanical properties are spun and understood by rate of work input. This approach not only provides energetic insights into natural silk spinning and controlled protein denaturation, but is believed will help interpret and improve synthetic silk processing. Ultimately, it is hoped that these results will contribute towards novel bioinspired energy‐efficient processing strategies that are driven by work input optimization and where excellent mechanical properties are self‐emergent.

The Power of Silk Technology for Energy Applications

AbstractSilk fibers are a remarkable material made of proteins possessing excellent mechanical properties that match or even outperform, in some aspects, high performance fibers such as Kevlar and steel. Silk proteins can be further produced recombinantly, allowing the possibility for genetic modification, enhancing silks’ already impressive range of benefits. Thus far, little research has explored the possibility of incorporating silk‐based materials in electronic or energy systems. With an increasing global concern for climate change and the dwindling reserves of fossil fuels, silk (or silk‐derived) hybrid materials are a promising avenue of scientific exploration in energy storage and conversion devices, flexible and wearable electronics and even as photovoltaic devices, which will be reviewed here within. Despite this, silk has seen only little interest for applications in hybrid energy devices in the recent years. Here within, some of the applications and benefits of silk‐based materials in several systems including: flexible electronics, thermal and thermoelectric devices, mechanical energy devices, sensors, and photovoltaic solar cells are examined. In addition to biocompatibility, high tensile strength, and renewability, silk also adds many benefits to hybrid energy systems such as tunability, multifunctionality, and versatility, making silk one of the most all‐encompassing materials for use in hybrid devices.

Designing of spider silk proteins for human induced pluripotent stem cell-based cardiac tissue engineering.

Materials made of recombinant spider silk proteins are promising candidates for cardiac tissue engineering, and their suitability has so far been investigated utilizing primary rat cardiomyocytes. Herein, we expanded the tool box of available spider silk variants and demonstrated for the first time that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes attach, contract, and respond to pharmacological treatment using phenylephrine and verapamil on explicit spider silk films. The hiPSC-cardiomyocytes contracted for at least 14 days on films made of positively charged engineered Araneus diadematus fibroin 4 (eADF4(κ16)) and three different arginyl-glycyl-aspartic acid (RGD)-tagged spider silk variants (positively or negatively charged and uncharged). Notably, hiPSC-cardiomyocytes exhibited different morphologies depending on the spider silk variant used, with less spreading and being smaller on films made of eADF4(κ16) than on RGD-tagged spider silk films. These results indicate that spider silk engineering is a powerful tool to provide new materials suitable for hiPSC-based cardiac tissue engineering.

RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

Spider silk: A natural marvel of mechanical and structural strength

The spider silk fibers have unique high performance properties that make it a desirable model for artificial fibers and its performance under benign conditions has important implications for biomimicry. It has tensile strengths comparable to steel and some are nearly as elastic as rubber on a weight to weight basis. The spider spins its silk at ambient temperatures, low pressures and with water as solvent. Spiders are ectotherms and the ambient temperature affects the spinning speed and the mechanical and structural properties of the silk spun. The high cytocompatibility and low immunogenicity of spider silk fibers make them well suited for biomaterial products such as nerve conduits. Spider silk proteins have been shown to be soluble in ionic liquids, thus once soluble, they can be processed into new biomaterials such as films, gels, porous sponges, bone tissue engineering. The spider silk chains with a fixed molecular weight decreases exponentially with the UV irradiation time, since UV irradiation causes the chemical bonds in the protein chains to undergo cleavage. This paper reviews related literature on the spider silk spinning process, conditions and their effects on structure, mechanical properties of spider silk and its resistance to UV degradation. As a bonus, a brief review of the biotechnological production of recombinant spider silk us presented.

Lateral Photovoltaic Effect in Silk-Protein-Based Nanocomposite Structure for Physically Transient Position-Sensitive Detectors

Silk protein is considered as an attractive transient biomaterial with fantastic properties applied to biophotoelectric devices. Combined with noble-metal silver nanoparticles, a transient composite structure of $\mathrm{Ag}$ nanoparticle-embedded silk protein ($\mathrm{Ag}$/silk protein/$\mathrm{Si}$) is constructed. We obtain a lateral photovoltaic effect with a maximum sensitivity of 308.3 mV/mm, which is a very large sensitivity value in the field of organic materials, even comparable to the reported sensitivities of most inorganic materials. We attribute this to the increase of photogenerated carriers at the illuminated spot caused by the local surface-plasmon resonance of silver particles and the effective regulation of the diffusion (or resistivity of the structure) of photogenerated carriers by a silk wrapper. Interestingly, both silk protein and silver have good biocompatibility, and the combination of the two might be an alternative for the future development of biomaterial-related transient photovoltaic devices.

Multifunctional and Ultrathin Electronic Tattoo for On-Skin Diagnostic and Therapeutic Applications.

Epidermal electronic systems for detecting electrophysiological signals, sensing, therapy, and drug delivery are at the frontier in man-machine interfacing for healthcare. However, it is still a challenge to develop multifunctional bioapplications with minimal invasiveness, biocompatibility, and stable electrical performance under various mechanical deformations of biological tissues. In this study, a natural silk protein with carbon nanotubes (CNTs) is utilized to realize an epidermal electronic tattoo (E-tattoo) system for multifunctional applications that address these challenging issues through dispersing highly conductive CNTs onto the biocompatible silk nanofibrous networks with porous nature to construct skin-adhesive ultrathin electronic patches. Individual components that incorporate electrically and optically active heaters, a temperature sensor (temperature coefficient of resistance of 5.2 × 10-3 °C-1 ), a stimulator for drug delivery (>500µm penetration depth in skin), and real-time electrophysiological signal detectors are described. This strategy of E-tattoos integrated onto human skin can open a new route to a next-generation electronic platform for wearable and epidermal bioapplications.