Silk fibroin fibers decorated with urchin-like Au/Ag nanoalloys: a flexible hygroscopic SERS sensor for monitoring of folic acid in human sweat.

Surface-enhanced Raman scattering (SERS) spectroscopy has become a powerful and sensitive analytical tool for the detection and assessment of chemical/biological molecules in special scenarios. Herein we propose a flexible hygroscopic SERS biocompatible sensor based on the silk fibroin fibers (SFF) decorated with urchin-like Au/Ag nanoalloys (NAs). The hybrid SFF-Au/Ag NAs with a stronger absorbance capacity (500∼1100 nm) and excellent hygroscopicity provide a remarkable higher near-infrared (NIR)-SERS activity than that of bare urchin-like Au/Ag NAs. The interesting NIR-SERS sensor enables the limit of detection (LOD) of folic acid (FA) to be achieved at nanomolar (nM, 10-9 M) level, facilitating the ultrasensitive monitoring of FA in human sweat and offering reliable real-time personal health management in the near future.

Silk Fibroin as a Functional Biomaterial for Tissue Engineering.

Tissue engineering (TE) is the approach to combine cells with scaffold materials and appropriate growth factors to regenerate or replace damaged or degenerated tissue or organs. The scaffold material as a template for tissue formation plays the most important role in TE. Among scaffold materials, silk fibroin (SF), a natural protein with outstanding mechanical properties, biodegradability, biocompatibility, and bioresorbability has attracted significant attention for TE applications. SF is commonly dissolved into an aqueous solution and can be easily reconstructed into different material formats, including films, mats, hydrogels, and sponges via various fabrication techniques. These include spin coating, electrospinning, freeze drying, physical, and chemical crosslinking techniques. Furthermore, to facilitate fabrication of more complex SF-based scaffolds with high precision techniques including micro-patterning and bio-printing have recently been explored. This review introduces the physicochemical and mechanical properties of SF and looks into a range of SF-based scaffolds that have been recently developed. The typical TE applications of SF-based scaffolds including bone, cartilage, ligament, tendon, skin, wound healing, and tympanic membrane, will be highlighted and discussed, followed by future prospects and challenges needing to be addressed.

Direct Femtosecond Laser Printing of Silk Fibroin Microstructures.

Fabrication of functional silk fibroin microstructures has extensive applications in biotechnology and photonics. Considerable progress has been made based on lithographic methods and self-assembly approaches. However, most methods require chemical modification of silk fibroin, which restricts the functionalities of the designed materials. At the same time, femtosecond laser-induced forward transfer (fs-LIFT) has been explored as a simple and attractive processing tool for microprinting of high-resolution structures. In this paper, we propose the use of LIFT with fs-pulses for creating high-resolution structures of regenerated silk fibroin (SF). Furthermore, upon adding Eu3+/Tb3+ complexes to SF, we have been able to demonstrate the printing by LIFT of luminescent SF structures with a resolution on the order of 2 µm and without material degradation. This approach provides a facile method for printing well-defined two-dimensional (2D) micropatterns of pure and functionalized SF, which can be used in a wide range of optical and biomedical applications.

Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield.

Spider silk has been a hotspot in the study of biomaterials for more than two decades due to its outstanding mechanical properties. Given that spiders cannot be farmed, and their low silk productivity, many attempts have been made to produce recombinant spidroins as an alternative. Herein, we present novel chimeric recombinant spidroins composed of 1 to 4 repetitive units of aciniform spidroin (AcSp) flanked by the nonrepetitive N- and C-terminal domains of the minor ampullate spidroin (MiSp), all from Araneus ventricosus. The spidroins were expressed in the form of inclusion body in E. coli with high yield. Remarkably, the aqueous solubility of the four spidroins ranged from 13.4% to over 50% (m/v). The four spidroins could self-assemble into silk-like fibers by hand-drawing. The secondary structures of these proteins, determined by circular dichroism spectrum (CD) and Fourier transform infrared spectrum (FTIR), indicated a prominent transformation from α-helix to ß-sheet after fiber formation. The mechanical properties of the hand-drawn fibers showed a positive correlation with the spidroin molecular weight. In summary, this study describes promising biomaterials for further study and wide application.

Silk fibroin and silk-based biomaterial derivatives for ideal wound dressings.

Silk fibroin (SF) is derived from Bombyx mori silkworm cocoons and has been used in textiles and as a suture material for decades. More recently, SF has been used for various new biomedical applications, including as a wound dressing, owing to its excellent biological and mechanical properties. Specifically, the mechanical stiffness, versatility, biocompatibility, biodegradability, water vapour permeability and slight bactericidal properties make SF an excellent candidate biomaterial for wound dressing applications. The effectiveness of SF as a wound dressing has been tested and well-documented in vitro as well as in-vivo, as described here. Dressings based on SF are currently used for treating a wide variety of chronic and acute (e.g. burn) wounds. SF and its derivatives prepared as biomaterials are available as sponges, hydrogels, nanofibrous matrices, scaffolds, micro/nanoparticles, and films. The present review discusses the potential role of SF in wound dressing and its modulation for wound dressing applications. The comparison of SF based dressings with other natural polymers understands the readers, the scope and limitation of the subject in-depth.

A marine photosynthetic microbial cell factory as a platform for spider silk production.

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

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.

Tailored monoclonal antibody as recognition probe of immunosensor for ultrasensitive detection of silk fibroin and use in the study of archaeological samples.

The ultrasensitive detection of fibroin in unearthed silk relics has great significance for investigating the origin and transmission of silk. In this study, an anti-fibroin monoclonal antibody was successfully prepared through animal immunization. Next, a label-free electrochemical immunosensor was fabricated using layer-by-layer self-assembly technology, and an indirect enzyme-linked immunosorbent assay (ELISA) was proposed. The two methods exhibited excellent sensitivity and specificity in the detection of silk fibroin, while the immunosensor showed a wider quantitative detection range (0.1-100 ng mL-1) and a lower detection limit (0.051 ng mL-1) than ELISA (10-100 ng mL-1 and 8.71 ng mL-1). Furthermore, the performance of the immunosensor was superior in archaeological sample detection. Taking advantage of the well-prepared monoclonal antibody, the two proposed immunological assays demonstrate tremendous potential for the ultrasensitive detection of silk fibroin, which can make great contributions to exploring the origin and transmission routes of ancient silks.

A heparin-functionalized woven stent graft for endovascular exclusion.

With the increase of vascular diseases in recent years, it is of importance to develop an anti-occlusion stent graft, which can meet the requirements of transplants for a long term. In this paper, we describe a silk fibroin (SF)/heparin-functionalized bifurcated stent graft (BSG) using textile forming technology. The BSGs were prototyped based on seamless weaving technology, and the surface was modified with SF-loaded heparin under steam/air treatment to improve their patency. The physical properties such as thickness, water permeability, contact angle, mechanical properties, and in vitro drug release and coagulation time of the BSGs were examined. The results showed that heparin modification can improve its coagulation time, and the water permeability resistance of the BSGs reached 1.154 ± 0.854 mL/(cm2×min), while their thicknesses were just 0.085 ± 0.004 mm. The heparin release of the BSGs showed that the release time was prolonged upon steam treatment by means of the increase in the ß-sheet structure and crystallinity of SF. The viability and attachment of human vascular smooth muscle (HVSM) cells cultured in the release of modified BSGs demonstrated that the modified BSGs could significantly inhibit the proliferation of HVSM cells. The heparin-functionalized BSG with satisfactory thickness, water permeability resistance and anti-occlusion function, which has potential applications in the treatment of vascular diseases.

Silk protein nanowires patterned using electron beam lithography.

Nanofabrication approaches to pattern proteins at the nanoscale are useful in applications ranging from organic bioelectronics to cellular engineering. Specifically, functional materials based on natural polymers offer sustainable and environment-friendly substitutes to synthetic polymers. Silk proteins (fibroin and sericin) have emerged as an important class of biomaterials for next generation applications owing to excellent optical and mechanical properties, inherent biocompatibility, and biodegradability. However, the ability to precisely control their spatial positioning at the nanoscale via high throughput tools continues to remain a challenge. In this study electron beam lithography (EBL) is used to provide nanoscale patterning using methacrylate conjugated silk proteins that are photoreactive 'photoresists' materials. Very low energy electron beam radiation can be used to pattern silk proteins at the nanoscale and over large areas, whereby such nanostructure fabrication can be performed without specialized EBL tools. Significantly, using conducting polymers in conjunction with these silk proteins, the formation of protein nanowires down to 100 nm is shown. These wires can be easily degraded using enzymatic degradation. Thus, proteins can be precisely and scalably patterned and doped with conducting polymers and enzymes to form degradable, organic bioelectronic devices.

Delivery of chemotherapeutics using spheres made of bioengineered spider silks derived from MaSp1 and MaSp2 proteins.

AIM: Analysis of the properties and chemotherapeutics delivery potential of spheres made of bioengineered spider silks MS1 and MS2. MATERIALS & METHODS: MS1 and MS2 derived from Nephila clavipes dragline silks - MaSp1 and MaSp2, respectively - formed spheres that were compared in terms of physicochemical properties, cytotoxicity and loading/release of chemotherapeutics. RESULTS: MS2 spheres were more dispersed, smaller, of solid core, of higher beta-sheet structure content, and of opposite (negative) charge than MS1 spheres. Preloaded MS2 showed greater applicability for mitoxantrone, while postloaded for etoposide delivery compared with MS1 spheres. However, MS1 spheres were a better choice for doxorubicin delivery than MS2. CONCLUSION: Bioengineered silks can be tailored to develop a system with optimal drug loading and release properties.

Silk Fibroin Based Drug Delivery Applications: Promises and Challenges.

BACKGROUND: Silk Fibroin (SF), a natural source material obtained from Bombyx mori, has been widely enlisted as biomaterial having outstanding mechanical properties. SF has been reported as one of the propitious bio-polymers for various drug delivery systems, as well as drug delivery vehicle. OBJECTIVE: This review is a summary of comprehensive applications of silk fibroin in various drug delivery systems, and also to present the current opportunities and requirements by furnishing a definitive assessment on silk fibroin as a polymer. RESULTS: SF has been reported as one of the propitious bio-polymers for various drug delivery systems, as well as drug delivery vehicle. SF is inestimable owing to its non-toxic and non-antigenic character, except for the firmness formation whilst being stored at lower temperature. Unlike other polymeric biomaterials, SF is regenerated in aqueous systems in defined temperature, pressure and pH, which is one of its major advantages in formulation. SF nanoparticles are also used to deliver proteins and peptides. Recently, SF has been used to deliver anti-cancer agents like paclitaxel, doxorubicin, floxuridine, and methotrexate, and including the natural product curcumin, has shown to elicit significant biological activity when compared to their conventional form. Interestingly, SF has shown to be a promising biomaterial for implantables and injectable drug delivery applications. CONCLUSION: In the present review, we have summarized the physical and chemical properties, biocompatibility and non-immunogenic characters of SF and its applications in various drug delivery systems.

Molecular cloning and analysis of the full-length aciniform spidroin gene from Araneus ventricosus.

Orb-web spiders produce more than seven different protein-based silks/glues by specialized abdominal glands for different uses. Prey-wrapping silk is secreted by aciniform glands for wrapping prey and forming the inner layer of egg case, and is almost twice as tough as other silks because of high strength and extensibility. So far, only two complete gene sequences have been obtained for aciniform spidroins (AcSp1). Here we describe the AcSp1 full-length gene sequence from the spider species Araneus ventricosus, using a long-distance PCR (LD-PCR) approach. The full-length AcSp1 gene is a single enormous exon of 10,338 bp in size, and the predicted protein sequence is 3445 amino acids long and consists of a conserved nonrepetitive N-terminal domain, a central predominantly repetitive region composed of fifteen repeat units, and a conserved nonrepetitive C-terminal domain.

Treatment with solubilized Silk-Derived Protein (SDP) enhances rabbit corneal epithelial wound healing.

There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP) on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day. Post-injury treatment with SDP formulations was found to accelerate the acute healing phase of the injured rabbit corneal epithelium. In addition, the use of SDP corresponded with an enhanced tissue healing profile through the formation of a multi-layered epithelial surface with increased tight junction formation. Additional biological effects were also revealed that included increased epithelial proliferation, and increased focal adhesion formation with a corresponding reduction in the presence of MMP-9 enzyme. These in vivo findings demonstrate for the first time that the presence of SDP on the injured ocular surface may aid to improve various steps of rabbit corneal wound healing, and provides evidence that SDP may have applicability as an ingredient in therapeutic ophthalmic formulations.

Colored and fluorescent nanofibrous silk as a physically transient chemosensor and vitamin deliverer.

Biodegradable and physically transient optics represent an emerging paradigm in healthcare devices by harnessing optically active system and obviating issues with chronic uses. Light emitting components that can efficiently interact with their environments have advantages of high sensitivity, visibility, and wireless operation. Here, we report a novel combination of silk biopolymer and optically active organic dyes resulting in versatile fluorescent silk nanofibers (FSNs). FSNs generated by the electrospinning method exhibit attractive functions of the doped organic dyes along with programming the system that physically disappear at prescribed time. Red-green-blue (RGB) fluorescent nanofibrous mats, eco-friendly and transient fluorescent chemosensors for acid vapor detection, and disposable membranes for nutrition delivery were successfully demonstrated using FSNs. These functions introduced using four water soluble dyes: rhodamine B, sodium fluorescein, stilbene 420, and riboflavin. The FSN with sodium fluorescein especially, showed a sensing capability for hazardous and volatile hydrochloric acid vapors. Delivering riboflavin (vitamin B2, an important nutrient for skin care) in the FSN to a biological tissue could be observed by tracing the fluorescence of riboflavin.

Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations.

Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cß dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three 13C NMR peaks observed in the ß-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different ß-sheet structures.

Composite Electrospun Scaffold Derived from Recombinant Fibroin of Weaver Ant (Oecophylla smaragdina) as Cell-Substratum.

Unlike silkworm (Bombyx mori) fibroin (SF), weaver ant (Oecophylla smaragdina) fibroin (WAF) is much less studied. Due to differences in amino acid composition and protein structure, this work aimed to produce the recombinant WAF protein, designated as WAF1, and investigated on its potential application as a biomaterial for producing a cell-substratum. The composite electrospun scaffolds derived from poly(vinyl alcohol) (PVA), WAF1, and extracted SF were produced by electrospinning. SEM images revealed non-woven and smooth fibers of PVA, PVA-WAF1, and PVA-SF scaffolds with the average diameters of 204.1 ± 59.9, 206.5 ± 71.5, and 238.4 ± 77.9 nm, respectively. ATR-FTIR spectra indicated characteristic absorption peaks related to the chemical structure of PVA and protein. The PVA-WAF1 scaffold demonstrated a higher water uptake, a slightly higher rate of degradation, and a similar low cytotoxicity as compared with the PVA-SF scaffold. Although the adhesion and proliferation of cells on the PVA-WAF1 scaffold were lower than those on the PVA-SF scaffold, it showed significantly greater values of adhering and proliferating cells than the PVA scaffold. The results of this work suggested that WAF1 could be used as a biomaterial for producing a cell-substratum that supports cell adhesion and growth.

Fabrication and Optimization of Stable, Optically Transparent, and Reusable pH-Responsive Silk Membranes.

The fabrication of silk-based membranes that are stable, optically transparent and reusable is yet to be achieved. To address this bottleneck we have developed a method to produce transparent chromogenic silk patches that are optically responsive to pH. The patches were produced by blending regenerated silk fibroin (RSF), Laponite RD (nano clay) and the organic dyes neutral red and Thionine acetate. The Laponite RD played a central role in the patch mechanical integrity and prevention of dye leaching. The process was optimized using a factorial design to maximize the patch response to pH by UV absorbance and fluorescence emission. New patches of the optimized protocol, made from solutions containing 125 µM neutral red or 250 µM of Thionine and 15 mg/mL silk, were further tested for operational stability over several cycles of pH altering. Stability, performance, and reusability were achieved over the tested cycles. The approach could be extended to other reporting molecules or enzymes able to bind to Laponite.

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

Thin films of silk fibroin and its blend with chitosan strongly promote biofilm growth of Synechococcus sp. BDU 140432.

The activating role of different polymer thin films coated over polystyrene support on the Synechococcus sp. biofilm growth was examined concurrently by measuring biofilm florescence using a dye and by measuring cell density in the isolated biofilm. Compared to blank (no coating), the increase in biofilm formation (%) on silk, chitosan, silk-chitosan (3:2) blend, polyaniline, osmium, and Nafion films were 27.73 (31.16), 21.55 (23.74), 37.21 (38.34), 5.35 (8.96), 6.70 (6.55) and (nil), respectively with corresponding cell density (%) shown in the parentheses. This trend of biofilm formation on the films did not significantly vary for Escherichia coli and Lactobacillus plantarum strains. The films of 20 residues long each of glycine-alanine repeat peptide, which mimics a silk fibroin motif, and a hydrophobic glycine-valine repeat peptide, increased the biofilm growth by 13.53 % and 26.08 %, respectively. Silk and blend films showed highest adhesion unit (0.48-0.49), adhesion rate ((4.2-4.8)×10(-6), m/s) and Gibbs energy of adhesion (-8.5 to -8.6kT) with Synechococcus sp. The results confirmed interplay of electrostatic and hydrophobic interaction between cell-surface and polymer films for promoting rapid biofilm growth. This study established that the thin films of silk and the blend (3:2) promote rapid biofilm growth for all the tested microorganisms.