Spider Silk Research Articles

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties.

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.

Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture.

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by-products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non-toxic, xeno-free, and has good mechanical properties. A recombinant spider silk protein-derived hydrogel that rapidly forms at 37°C is recently developed. The temperature and gelation times are well-suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE-19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

In vitro Blood–Brain barrier model based on recombinant spider silk protein nanomembranes for evaluation of transcytosis capability of biomolecules

The blood–brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engineered shuttle proteins facilitates ranking and the selection of promising candidates during development. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody.

Helium Ion Microscopy and Sectioning of Spider Silk.

Focused ion beams have recently emerged as a powerful tool for ultrastructural imaging of biological samples. In this article, we show that helium ion microscopy (HIM), in combination with ion milling, can be used to visualize the inner structure of both major and minor ampullate silk fibers of the orb-web weaving spider Nephila madagascariensis. The internal nanofibrils were imaged in pristine silk fibers, with little or no damage to the sample structure observed. Furthermore, a method to cut/rupture the fibers using He+ ions combined with internal sample tension is presented. This showed that the stretching and rupturing of spider silk is a highly dynamic process with considerable material reorganization.

Spider silk erectile nerve reconstruction in robot-assisted radical prostatectomy: a first-in-men feasibility analysis.

To investigate the safety and feasibility of spider silk interposition for erectile nerve reconstruction in patients undergoing robotic radical prostatectomy (RARP). The major-ampullate-dragline from Nephila edulis was used for spider silk nerve reconstruction (SSNR). After removal of the prostate with either uni- or bilateral nerve-sparing, the spider silk was laid out on the site of the neurovascular bundles. Data analysis included inflammatory markers and patient reported outcomes. Six patients underwent RARP with SSNR. In 50% of the cases, only a unilateral nerve-sparing was performed, bilateral nerve-sparing could be performed in three patients. Placement of the spider silk conduit was uneventful, contact of the spider silk with the surrounding tissue was mostly sufficient for a stable connection with the proximal and distal ends of the dissected bundles. Inflammatory markers peaked until postoperative day 1 but stabilized until discharge without any need for antibiotic treatment throughout the hospital stay. One patient was readmitted due to a urinary tract infection. Three patients reported about erections sufficient for penetration after three months with a continuous improvement of erectile function both after bi- and unilateral nerve-sparing with SSNR up to the last follow-up after 18months. In this analysis of the first RARP with SSNR, a simple intraoperative handling without major complications was demonstrated. While the series provides evidence that SSNR is safe and feasible, a prospective randomized trial with long-term follow-up is needed to identify further improvement in postoperative erectile function due to the spider silk-directed nerve regeneration.

Bioinspired ultra-tough, exceptionally stretchable, and self-recoverable bio-based coating for visual damage detection and self-healing

Developing intelligent materials with integrated ultra-high-performance damage monitoring and repair functions is of paramount significance for broadening the application of the latest generation of smart materials. Here, inspired by spider silk and biological skin, we embedded multiple hydrogen bonding motifs and rigid alicyclic groups with attached damage monitoring sensors in bio-based polyurethanes to create bio-based elastomers with exceptional mechanical properties combined with repair and damage monitoring functionalities. Benefiting from the abundance of multiple hydrogen bonding elements within the polyester backbone for fracture and remodeling as external energy dissipation, and the contribution of asymmetric alicyclic groups embedded in hard domains. The elastomer possesses exceptional toughness (583 MJ·m−3) and tensile strength (43 MPa), the elongation at break point is 2600 %, and self-healing (86 % at 60 °C for 6 h), environmental adaptability, and recyclability, thereby achieving the preparation of ultra-tough self-healing elastomeric materials. Simultaneously, the internally embedded sensors enable the localization and monitoring of damage through visually observable means, such as quenching and discoloration. The elastomer recovers well in 3.5 wt% NaCl solution and provides corrosion protection (9.23 × 107 Ω·cm2 even after 50 days of immersion), achieving the integration of ultra-high mechanical performance coating materials with damage warning and repair functions. Therefore, the molecular structure design scheme of the biomimetic elastomer provides constructive guidance for surface protection application of engineering equipment in service under harsh environments.

Startle Responses of Jorō Spiders (Trichonephila clavata) to Artificial Disturbance

The jorō spider (Trichonephila clavata, originally from east Asia) has been introduced in the southeastern United States, and is rapidly expanding this range, leading to questions about what facilitates this spread. Meanwhile, its cousin, the golden silk spider (T. clavipes), already has a range that covers most of the southeast. In an ongoing effort to understand the behavior of jorō spiders in their introduced range, we undertook the current project to evaluate how they react to perceived threats, which can inform us on how a species interacts with conspecifics, or how well it can tolerate anthropogenic disturbances. We collected mature females of both Trichonephila species, plus three locally common orb-weaving species in Georgia, and we evaluated the time spent immobile after experiencing a mild disturbance (a brief puff of air). We also collected similar “air puff response” data for five other North American species from the published literature. Collectively, the dataset totaled 453 observations of freezing behavior across 10 spider species. Comparing these data across species revealed that most spiders remained immobile for under a minute after the stimulus. Meanwhile, both Trichonephila spiders remained immobile for over an hour, which appears to be unprecedented, and suggests that spiders in this genus are the “shyest” ever documented. This reaction could also allow Trichonephila spiders to tolerate urban environments by remaining motionless throughout each disturbance instead of fleeing.

Development and characterization of a recombinant silk network for 3D culture of immortalized and fresh tumor-derived breast cancer cells.

Traditional cancer models rely on 2D cell cultures or 3D spheroids, which fail to recapitulate cell-extracellular matrix (ECM) interactions, a key element of tumor development. Existing hydrogel-based 3D alternatives lack mechanical support for cell growth and often suffer from low reproducibility. Here we report a novel strategy to make 3D models of breast cancer using a tissue-like, well-defined network environment based on recombinant spider silk, functionalized with a cell adhesion motif from fibronectin (FN-silk). With this approach, the canonical cancer cells SK-BR-3, MCF-7, and MDA-MB-231, maintain their characteristic expression of markers (i.e., ERα, HER2, and PGR) while developing distinct morphology. Transcriptomic analyses demonstrate how culture in the FN-silk networks modulates the biological processes of cell adhesion and migration while affecting physiological events involved in malignancy, such as inflammation, remodeling of the ECM, and resistance to anticancer drugs. Finally, we show that integration in FN-silk networks promotes the viability of cells obtained from the superficial scraping of patients' breast tumors.

PH-dependent self-assembly mechanism of a single repetitive domain from a spider silk protein

Spider silk is self-assembled from full-length silk proteins, and some silk protein fragments can also form silk-like fibers in vitro. However, the mechanism underlying the silk fiber formation is not understood well. In this study, we investigated the fiber formation of a single repetitive domain (RP) from a minor ampullate silk protein (MiSp). Our findings revealed that pH and salt concentration affect not only the stability of MiSp-RP but also its self-assembly into fibers and aggregates. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the three-dimensional (3D) structure of MiSp RP in aqueous solution. On the basis of the structure and mutagenesis, we revealed that charge-dipole interactions are responsible for the pH- and salt-dependent properties of MiSp-RP. Our results indicate that fiber formation is regulated by a delicate balance between intermolecular and intramolecular interactions, rather than by the protein stability alone. These findings have implications for the design of silk proteins for mass production of spider silk.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

Dry-Spinning of Artificial Spider Silk Ribbons from Regenerated Natural Spidroin in an Organic Medium.

Natural spider silks with striking performances have achieved extensive investigations. Nonetheless, a lack of consensus over the mechanism of the natural spinning hinders the development of artificial spinning methods where the regenerated spider silks generally show poor performances compared with the natural fibers. As is known, the Plateau-Rayleigh instability tends to break solution column into droplets and has been considered as a main challenge during fiber-spinning. Here in this study, by harnessing the viscoelastic properties of the regenerated spidroin dope solution via organic salt-zinc acetate (ZA), we avoid this outcome and successfully realize dry-spinning of long and mechanically robust regenerated spider silk ribbons. The as obtained dry-spun spider silk ribbons show an enhanced modulus up to 14±4GPa and a toughness of around 51±9 MJm-3 after the post-stretching treatment, which is even better than the pristine spider silk fibers. Our facile and flexible strategy enriches the spinning methodologies which bypasses the bottleneck of precisely mimicking the complex natural environment of the glands in spiders, shining a light to the spider-silk-based textile industrial applications. This article is protected by copyright. All rights reserved.

Bioinspired artificial spider silk photocatalyst for the high-efficiency capture and inactivation of bacteria aerosols

Bioaerosol can cause the spread of disease, and therefore, capture and inactivation of bioaerosols is desirable. However, filtration systems can easily become blocked, and are often unable to inactivate the bioaerosol once it is captured. Herein, we reported a bioinspired artificial spider silk (ASS) photocatalyst, consisting of a periodic spindle structure of TiO2 on nylon fiber that can efficiently capture and concentrate airborne bacteria, followed by photocatalytic inactivation in situ, without a power-supply exhaust system. The ASS photocatalyst exhibits a higher capture capacity than the nylon fiber substrate and a photocatalytic inactivation efficiency of 99.99% obtained under 4 h irradiation. We found that the capture capacity of the ASS photocatalyst can be mainly attributed to the synergistic effects of hydrophilicity, Laplace pressure differences caused by the size of the spindle knots and surface energy gradients induced by surface roughness. The bacteria captured by the ASS photocatalyst are inactivated by photocatalysis within droplets or at the air/photocatalyst interfaces. This strategy paves the way for constructing materials for bioaerosol purification.

Nanoscale imaging of major and minor ampullate silk from the orb-web spider Nephila Madagascariensis

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Variation in the Elastic Modulus and Increased Energy Dissipation Induced by Cyclic Straining of Argiope bruennichi Major Ampullate Gland Silk

The trends exhibited by the parameters that describe the mechanical behaviour of major ampullate gland silk fibers spun by Argiope bruennichi spiders is explored by performing a series of loading-unloading tests at increasing values of strain, and by the subsequent analysis of the true stress-true strain curves obtained from these cycles. The elastic modulus, yields stress, energy absorbed, and energy dissipated in each cycle are computed in order to evaluate the evolution of these mechanical parameters with this cyclic straining. The elastic modulus is observed to increase steadily under these loading conditions, while only a moderate variation is found in the yield stress. It is also observed that a significant proportion of the energy initially absorbed in each cycle is not only dissipated, but that the material may recover partially from the associated irreversible deformation. This variation in the mechanical performance of spider silk is accounted for through a combination of irreversible and reversible deformation micromechanisms in which the viscoelasticity of the material plays a leading role.

Self-Healing of Recombinant Spider Silk Gel and Coating.

Self-healing properties, originating from the natural healing process, are highly desirable for the fitness-enhancing functionality of biomimetic materials. Herein, we fabricated the biomimetic recombinant spider silk by genetic engineering, in which Escherichia coli (E. coli) was employed as a heterologous expression host. The self-assembled recombinant spider silk hydrogel was obtained through the dialysis process (purity > 85%). The recombinant spider silk hydrogel with a storage modulus of ~250 Pa demonstrated autonomous self-healing and high strain-sensitive properties (critical strain ~50%) at 25 °C. The in situ small-angle X-ray scattering (in situ SAXS) analyses revealed that the self-healing mechanism was associated with the stick-slip behavior of the β-sheet nanocrystals (each of ~2-4 nm) based on the slope variation (i.e., ~-0.4 at 100%/200% strains, and ~-0.9 at 1% strain) of SAXS curves in the high q-range. The self-healing phenomenon may occur through the rupture and reformation of the reversible hydrogen bonding within the β-sheet nanocrystals. Furthermore, the recombinant spider silk as a dry coating material demonstrated self-healing under humidity as well as cell affinity. The electrical conductivity of the dry silk coating was ~0.4 mS/m. Neural stem cells (NSCs) proliferated on the coated surface and showed a 2.3-fold number expansion after 3 days of culture. The biomimetic self-healing recombinant spider silk gel and thinly coated surface may have good potential in biomedical applications.

Design of Recombinant Spider Silk Proteins for Cell Type Specific Binding (Adv. Healthcare Mater. 9/2023)

Cell-Selective Spider Silk Proteins In article number 2202660, Vanessa Tanja Trossmann and Thomas Scheibel design recombinant spider silk proteins to guide cell-specific materials interactions. The fusion of spider silk proteins with short peptides from extracellular matrix proteins such as KGD yield cell type selective binding, which enables the development of hierarchically organized tissues.

Silk‐in‐Silk Nerve Guidance Conduits Enhance Regeneration in a Rat Sciatic Nerve Injury Model (Adv. Healthcare Mater. 11/2023)

Silk Fibers Silk has emerged as a versatile biomaterial for regenerative medicine. In article 2203237, Christine Radtke and co-workers use a silk-based nerve conduit filled with spider silk fibers to treat a rat sciatic nerve defect. The silk-in-silk conduit achieves a similar regenerative performance as the current gold standard, the autograft, and represents a promising approach for nerve reconstruction.

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins.

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

Spider silk-inspired dynamic covalent polyurethane with fast repairing, shape memory, and strong dynamic adhesion via lignin enhanced microphase separation

Introducing microphase separation and dynamic bonds into the polymeric materials is an effective strategy to enhance the performance of polyurethane. Herein, a modified lignin-based polyol (MLOH) was developed via a facile one-step approach which greatly improved its reactivity and dispersibility compared to that of virgin lignin. Subsequently, inspired by the biphasic structure of natural spider silk, a high-performance modified lignin-based dynamic covalent polyurethane (MLPU) with lignin enhanced microphase separation was fabricated, exploiting the synthetic MLOH as the crosslinker and the dynamic oxime-carbamate bonds as the linkage. Besides possessing high repairing efficiency (3 min for 100%, 80 °C without pressure), and outstanding shape-memory capability, the optimized MLPU-2 demonstrated robust adhesive performance when applied as a polyurethane hot-melt adhesive, including a fast curing rate and high adhesive strength (10.50 MPa). Meanwhile, it also manifested remarkable solvent resistance, temperature tolerance from −30 °C to 60 °C, long-term durability for at least 3 months, on-demand debonding ability, and excellent reusability. The synergistic effect of lignin enhanced microphase separation and adjustable dynamic crosslinked structure endows the remarkable performance of this resultant dynamic covalent MLPU, which provides another way to develop green, sustainable polyurethane simultaneously with highly comprehensive performances for the potential practical application.