Silk as an innovative biomaterial for cancer therapy

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.     

Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles

We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.     

Silk Spinning Behavior and Domicile Construction in Webspinners

Analysis of spinning and of silk domiciles revealed similarities and differences for three species of embiids: Antipaluria urichi (Saussure), Pararhagadochir trinitatis (Saussure), and Oligotoma saundersii (Westwood). Each exhibited similar routines while spinning; they positioned the silk by touching the substrate with either front tarsus and by moving the leg to the next position, with many silk strands issuing forth at each step. Reinforced pathways developed as they spun while traveling from diurnal retreats and nocturnal foraging zones. Frass from the interior of their domiciles was spun into the silk. O. saundersii was unique in that it plastered its silk with gathered materials. Analysis of field colonies of P. trinitatis and O. saundersii showed that, like A. urichi, adult females share their silk with offspring and, often, with other females.     

基因工程功能化丝蛋白生物材料及其在生物医学中的应用

丝蛋白生物材料具有优异的力学性能、良好的生物相容性及可降解性,在生物医学领域具有巨大的应用潜力。现有丝蛋白生物材料在结构和功能方面的相关知识,为设计合成新型丝蛋白生物材料提供了理论基础。此外,利用基因工程技术可将编码新肽或结构域的基因序列添加到编码丝蛋白的基因序列中,以获得具有新功能的丝蛋白生物材料,并更好地满足现代生物医学的需求。文中总结了基因工程功能化的丝蛋白生物材料在生物医学领域中的应用现状和发展前景。     

New materials from proteins and peptides

In this review we highlight recent accomplishments in the design of materials from proteins and peptides. Examples include hydrogels made from aggregating designed β-hairpin peptides, whose physical properties respond to small changes in the amino acid composition of the peptide; materials that combine different segments of natural elastomeric proteins - such as elastin, resilin, silk fibroin whose bulk properties are dictated in unanticipated ways by their composition; and hydrogels formed by strings or arrays of protein modules, which are cross-linked by multivalent versions of their peptide ligands, and which may exhibit exquisite stimuli-responsive behavior. The suitability of the unique properties of such new materials for practical applications is also considered.     

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.     

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.     

Transglutamination allows production and characterization of native‐sized ELPylated spider silk proteins from transgenic plants

In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant‐based production technology especially for protein‐based products, for technical use as fibres or biodegradable plastics and also for medical applications. High‐performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM‐based nanoindentation. The layered multimers formed by mixing lysine‐ and glutamine‐tagged monomers were associated with the highest elastic penetration modulus.     

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na₂CO₃-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.     

Tunable self-assembly of genetically engineered silk--elastin-like protein polymers

Silk--elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk-to-elastin blocks in a SELP's repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels, and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering, and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.     

Characterization and assembly of a GFP‐tagged cylindriform silk into hexameric complexes

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP‐silk fusion protein is predominantly α‐helical, and that pH can trigger a α‐ to β‐transition resulting in aggregation. Structural analysis by small angle X‐ray scattering suggests that the GFP‐Silk exists in the form of a hexamer in solution. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 378–390, 2014.     

Ultrastructure of spider thread anchorages

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.     

Durability of Simuliid Silk Pads (Simuliidae,Diptera)

A central aspect of simuliid adaption to life in moving water is a silky secretion which they produce in their salivary glands. This secretion is spread on the substrate surface thereby enabling adhesion of the posterior abdominal hooks of the larvae to the substrate surface. Therefore, silk pads are a prerequisit for simuliid filter‐feeding as well as any kind of locomotive activity. If silk pad adhesion is weak, larvae risk drifting off, either directly or during locomotion. Properties of the adhesive (=silk) as well as the substrate surface may cause weak adhesion. A specialist such as S. noelleri which has little chance of surviving after drifting off its lake outlet habitat, should have adaptations to reduce this risk. Such an adaptation could be very durable silk pads, giving larvae the chance to be safely attached for a relatively long time. In this study larval silk pads of S. ornatum and S. noelleri were stained using Giemsa's staining procedures. Changes in silk pad structure and traces of rot after 2–33 days of exposure to stream water were recorded and compared. Silk pads of S. ornatum and S. noelleri showed differences which indicate differences in ageing processes and biochemistry of this secretion.     

Controlled hydrogel formation of a recombinant spider silk protein

Due to their biocompatibility, biodegradability, and low immunogenicity, recombinant spider silk proteins have a high potential for a variety of applications when processed into morphologies such as films, capsules, beads, or hydrogels. Here, hydrogels made of the engineered and recombinantly produced spider silk protein eADF4(C16) were analyzed in detail. It has previously been shown that eADF4(C16) nanofibrils self-assemble by a mechanism of nucleation-aggregation, providing the basis of silk hydrogels. We focused on establishing a reproducible gelation process by employing different protein concentrations, chemical crosslinking, and functionalization of eADF4(C16) with fluorescein. Fluorescein strongly influenced assembly as well as the properties of the hydrogels, such as pore sizes and mechanical behavior, possibly due to its interference with packing of silk nanofibrils during hydrogel formation.     

Spider silk as a resource for future biotechnologies

Insect silks have been used by mankind for millennia to produce textiles and in particular, the cocoon silk of Bombyx mori was the base of one of the most important industries in history. In fact, B. mori is probably the only domesticated insect if not invertebrate in its true and strict sense, comparable to cattle and other livestock that humans have known and bred since the Neolithic period. In contrast, reports regarding the use of spider silk throughout history have the character of travellers’ tales or anecdotes, and serious attempts to exploit these biomaterials on a large scale have not been undertaken until recently. Indeed, the cannibalism of these carnivores makes their farming difficult and the production of significant yields of spider silk virtually impossible. Only today, with recombinant technologies available, does this problem seem to have been overcome. But why use spider silk at all – if we have the infrastructure to produce significant yields of silk from Bombyx? In contrast to most insects, spiders do not spin from labial glands, and many spiders possess different types of gland, most of them active throughout the whole lifespan. Typical orb‐weavers (Araneoidea) for instance possess up to seven different types of silk gland to produce different silk fibers and glues. Each of these products has evolved for a particular use and the respective material properties are highly adapted to that use. As the group of Araneae is about 400 million years old, the oldest fossil orb‐weaver is dated about 150 million years, and the use of silk is crucial to a spider's survival, we can expect that evolution will have “squeezed out every iota” to achieve optimum performance at minimum cost. Indeed, some dragline silks such as the major ampullate silks of some Nephila species show amazing mechanical properties that, in terms of toughness, are far superior to Bombyx silk. Labels like “stronger than steel” or “even better than Kevlar” were attached to them, and the Canadian‐based biotech company Nexia created the trademark “bio‐steel” for their prospective product. The discovery of these exceptional mechanical properties of those protein fibers triggered intense research on spider silk, with the goal of their commercial exploitation. But there is more to Arachne's weave and science is beginning to pick up those threads.     

Materials fabrication from Bombyx mori silk fibroin

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.     

Analysis of nuclear proteins from silk glands ofBombyx mori

A gentle method for the isolation of nuclei from developing silk glands ofBombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.     

Fine structure of sheet-webs of Linyphia triangularis (Clerck) and Microlinyphia pusilla (Sundevall), with remarks on the presence of viscid silk

We examined the webs of Linyphia triangularis (Clerck) and Microlinyphia pusilla (Sundevall) using light and scanning electronic microscopic techniques and compared them with the better known orb‐webs. The linyphiid sheet‐web consists of an unordered meshwork of fibres of different thicknesses. The sheet is connected to the scaffolding by means of attachment discs. Thin threads with globules, which appear similar to the viscid silk droplets of orb‐webs, are present in most webs examined. Webs of M. pusilla had a higher density of these globules than did webs of L. triangularis. Webs of both species possess five types of thread connections and contain no aqueous glue for prey capture. Instead, unlike orb‐webs, the sticky substances produced by the linyphiid aggregate glands cement the different layers and threads of the sheet by drying up after being produced. Due to their function, sheet webs may not require viscid silk, thereby leading to a more economic web. The assumption made in most previous studies, that the globules in linyphiid webs have the same properties and function as viscid silk in orb‐webs, is unfounded.     

A structural view on spider silk proteins and their role in fiber assembly

Spider silk is the toughest known biomaterial and even outrivals modern synthetic high‐performance materials. The question of understanding fiber formation is how the spider can prevent premature and fatal aggregation processes inside its own body and how the chemical and mechanical stimuli used to induce the fiber formation process translate into structural changes of the silk material, finally leading to controlled and irreversible aggregation. Here, the focus will be on the structure and function of the highly conserved N‐domains and C‐terminal domains of spider dragline silk which, unlike the very long repetitive sequence elements, adopt a folded conformation in solution and are therefore able to control intermolecular interactions and aggregation between other spider silk molecules. The structures of these domains add valuable details for the construction of a molecular picture of the complicated and highly optimized silk assembly process that might be beneficial for large‐scale in vitro fiber formation attempts with recombinant silk material. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.