Natural Silk Protein Research Articles

Phloretin@cyclodextrin/natural silk protein/polycaprolactone nanofiber wound dressing with antioxidant and antibacterial activities promotes diabetic wound healing

In patients with diabetes, chronic hyperglycemia impairs immune function at wound sites, increasing susceptibility to infections, prolonging inflammation, and delaying healing. This study aimed to develop wound dressings that control bacterial infections and accelerate healing. Phloretin (PHL), which has antibacterial and anti-inflammatory properties, was encapsulated with γ-cyclodextrin (γ-CD) to form a PHL@CD complex with enhanced bioavailability. This complex was incorporated into nanofiber wound dressings composed of polycaprolactone and natural silk protein. The resulting dressings exhibited favorable physical and chemical properties, including nutrient transport and gas exchange, which are essential for wound healing. The nanofiber membranes exhibited antibacterial activity against Staphylococcus aureus (90.31 ± 4.41 % inhibition), with high antioxidant capacity (91.48 ± 0.33 % ABTS scavenging) and blood compatibility. The membranes also promoted cell viability. Importantly, the nanofiber dressings accelerated wound healing in a diabetic mouse model by reducing the duration of inflammation. The novel nanofiber wound dressing can significantly improve the treatment of diabetic wounds.

Transgenic silkworm expressing bioactive human ciliary neurotrophic factor for biomedical application.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in neuronal survival and regeneration, and can also induce the differentiation of several stem cells into neurons, which highlights the broad application of CNTF in biomedicine. However, large-scale production of bioactive recombinant human CNTF protein remains to be explored. Herein, this study aims to express a bioactive human CNTF protein on a large scale by genetically engineering a silk gland bioreactor of silkworm. Our results showed that CNTF protein was successfully expressed in the middle silk gland (MSG) of silkworm, which can be secreted into the silks with the amount of 3.2mg/g cocoons. The fabrication of human CNTF-functionalized silk material was able to promote proliferation and migration of neural cells when compared to the natural silk protein. Importantly, this functional silk material could also facilitate neurite outgrowth of mouse retinal ganglion cell (RGC-5) cells. All these data demonstrated a high bioactivity of the recombinant human CNTF protein expressed in the MSG of silkworm. The further fabrication of different silk materials with CNTF bioactivity will give biomedical applications in tissue engineering and neuroregeneration.

Multifunctional finishing and dyeing of fabric with natural silk protein sericin

Multifunctional finishing and dyeing of fabric with natural silk protein sericin

Mutation in the Bombyx mori BmGMC2 gene impacts silk production and silk protein synthesis

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers.

Constructing amidoxime adsorption sites on the core-shell structured natural silk protein for uranium capture

Amidoxime-based fiber adsorbents hold significant promise for uranium extraction. However, a notable issue is that these adsorbents primarily originate from synthetic polymer materials, which, aside from providing good mechanical support, have no other functions. In recent study, we shifted our focus to silk fiber (SF), a natural protein fiber known for its unique core-shell structure and rich amino acids. The shell layer, due to its abundant functional groups, makes it easily modifiable, while the core layer provides excellent mechanical strength. Leveraging these inherent properties, an amidoxime-based fiber adsorbent was developed. This adsorbent utilizes amino and carboxyl groups for enhanced performance synergistically. This method involves establishing uranium affinity sites on the outer sericin layer of SF via chemical initiation of graft polymerization (CIGP) and amidoximation (SF-g-PAO). The water absorption ratio of SF-g-PAO is as high as 601.16 % (DG = 97.17 %). Besides, SF-g-PAO demonstrates an exceptional adsorption capacity of 15.69 mg/g in simulated seawater, achieving a remarkable removal rate of uranyl ions at 95.06 %. It can withstand a minimum of five adsorption-elution cycles. Over a 4-week period in natural seawater, SF-g-PAO displayed an adsorption capacity of 4.95 mg/g. Furthermore, SF-g-PAO also exhibits impressive uranium removal efficiency in real nuclear wastewater, with a removal rate of 63 % in just 15 min and a final removal rate of 90 %. It is hoped that this SF-g-PAO, prepared through this straightforward method and characterized by the synergistic action of amino and carboxyl groups, can offer innovative insights into the development of uranium extraction adsorbents.

Unraveling the Mechanical Property Decrease of Electrospun Spider Silk: A Molecular Dynamics Simulation Study.

This study investigated the impact of electric fields on Nephila clavipes spider silk using molecular dynamics modeling. Electric fields with varying amplitudes and directions were observed to disrupt the β sheet structure of spider silk and reduce its mechanical properties. However, a notable exception was observed when a 0.1 V/nm electric field was applied in the antiparallel direction, resulting in improvements in Young's modulus and ultimate tensile strength. The antiparallel direction was observed to be particularly sensitive to electric fields, causing disruptions in beta sheets and hydrogen bonds, which significantly influence the mechanical properties. This study demonstrates that spider silk maintains its structural integrity at 0.1 V/nm. Possibly, lowering the power levels of typical electrospinning machines can prevent secondary structural disruption. These findings provide valuable insights for enhancing silk fiber production and applications using natural silk proteins while shedding light on the impact of electric fields on other silk proteins. Finally, this study opens up possibilities for optimizing electrospinning processes to enhance performance in various silk electrospinning applications.

Engineering biomimetic scaffolds by combining silk protein nanofibrils and hyaluronic acid

Nanofibrous scaffolds mimicking important features of the native extracellular matrix (ECM) provide a promising strategy for tissue regeneration. However, 3D scaffolds mimicking natural protein nanofibers and bioactive glycosaminoglycans remain poorly developed. In this study, a biomimetic nanofibrous scaffold composed of natural silk protein nanofibers and glycosaminoglycan hyaluronic acid (HA) was developed. HA functionalization significantly improved the hydrophilicity and bioactivity of silk nanofibers (SNFs). SNFs can be assembled into nanofibrous aerogel scaffolds with low density and desirable shapes on a large scale. More importantly, with the assistance of HA, the silk nanofibrous aerogel scaffolds with ultra-high porosity, natural bioactivity, and structural stability in aqueous environment can be fabricated. In the protease/hyaluronidase solution, the SNF scaffolds with 10.0 % HA can maintain their monolithic shape for >3 weeks. The silk nanofibrous scaffolds not only imitate the composition of ECM but also mimic the hierarchical structure of ECM, providing a favorable microenvironment for cell adhesion and proliferation. These results indicate that this structurally and functionally biomimetic system is a promising tissue engineering scaffold.

Ice Recrystallization Inhibition Activity of Silk Proteins.

The cryopreservation of cells, tissue, and organs is essential in both fundamental research and practical applications, such as modern regenerative medicine and technological applications. However, the formation of ice crystals during ice recrystallization can have harmful or even fatal effects on biological systems. To address this challenge, we explore the ice recrystallization inhibition (IRI) activity of two natural silk proteins of Bombyx mori, fibroin and sericin. We found that silk fibroin (SF) had higher ice recrystallization inhibition activity than silk sericin (SS). Moreover, SF aqueous solutions perform better in inhibiting ice recrystallization than SF phosphate-buffered saline solutions. Sum-frequency generation spectroscopy shows that stronger electrostatic interactions are responsible for the higher IRI ability of SF. This work is significant for broadening the applications of silk proteins in biomedical fields.

Bioengineered silkworm model for expressing human neurotrophin-4 with potential biomedical application.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays important roles in maintaining nerve cell survival, regulating neuronal differentiation and apoptosis, and promoting nerve injury repair. However, the source of sufficient NT-4 protein and efficient delivery of NT-4 remain a challenge. This study aims to express an activated human NT-4 protein in a large scale by genetically engineering silk gland bioreactor of silkworm as a host. We showed that the expression of human NT-4-functionalized silk material could promote proliferation of mouse HT22 cells when compared to the natural silk protein, and no obvious cytotoxicity was observed under the conditions of different silk materials. Importantly, this functional silk material was able to induce the potential differentiation of HT22 cells, promote peripheral neural cell migration and neurite outgrowth of chicken embryo dorsal root ganglion (DRG). All these results demonstrated a high bioactivity of human NT-4 protein produced in silk gland. Therefore, based on the silkworm model, the further fabrication of different silk materials-carrying active NT-4 protein with good mechanical properties and great biocompatibility will give promising applications in tissue engineering and neurons regeneration.

Bioengineered and functionalized silk proteins accelerate wound healing in rat and human dermal fibroblasts.

Wound healing is an intrinsic process directed towards the restoration of damaged or lost tissue. The development of a dressing material having the ability to control the multiple aspects of the wound environment would be an ideal strategy to improve wound healing. Though natural silk proteins, fibroin, and sericin have demonstrated tissue regenerative properties, the efficacy of bioengineered silk proteins on wound healing is seldom assessed. Furthermore, silk proteins sans contaminants, having low molecular masses, and combining with other bioactive factors can hasten the wound healing process. Herein, recombinant silk proteins, fibroin and sericin, and their fusions with cecropin B were evaluated for their wound-healing effects using in vivo rat model. The recombinant silk proteins demonstrated accelerated wound closure in comparison to untreated wounds and treatment with Povidone. Among all groups, the treatment with recombinant sericin-cecropin B (RSC) showed significantly faster healing, greater than 90% wound closure by Day 12 followed by recombinant fibroin-cecropin B (RFC) (88.86%). Furthermore, histological analysis and estimation of hydroxyproline showed complete epithelialization, neovascularization, and collagenisation in groups treated with recombinant silk proteins. The wound healing activity was further verified by in vitro scratch assay using HADF cells, where the recombinant silk proteins induced cell proliferation and cell migration to the wound area. Additionally, wound healing-related gene expression showed recombinant silk proteins stimulated the upregulation of EGF and VEGF and regulated the expression of TGF-β1 and TGF-β3. Our results demonstrated the enhanced healing effects of the recombinant silk fusion proteins in facilitating complete tissue regeneration with scar-free healing. Therefore, the recombinant silks and their fusion proteins have great potential to be developed as smart bandages for wound healing.

Synergistic enhancement on flexible solid-state supercapacitor based on redox-active Fe3+ions/natural spidroin modified vertically aligned carbon nanotube arrays

The conductive skeleton and aligned carbon nanotube array (CNTA) structure can greatly shorten the ion transfer path and promote the charge transfer speed, which makes the CNTA an ideal electrode material for energy storage application. However, poor mechanical stability and low specific capacitance greatly impede its practical utilization. Here, we introduce a promising flexible electrode material based on the natural spider silk protein (SSP) modified CNTA(SSP/CNTA) with improved hydrophilicity and mechanical flexibility. The redox-active Fe3+ doped SSP/CNTA flexible solid-state supercapacitor (FSSC) device with superior energy storage performance was assembled in a symmetric ‘sandwich-type’ structure. The synergetic interaction between Fe3+ ions and the SSP are proved to greatly enhance the electrochemical performance especially the long-term cyclic stability. The Fe3+ doped SSP/CNTA FSSCs device achieves an ultra-high volumetric capacitance of 4.92 F cm−3 at a sweep speed of 1 mV s−1. Meanwhile it exhibited an excellent cycling stability with an increased capacitance by 10% after 10 000 charge–discharge cycles. As a control, a Fe3+ doped CNTA composite device without SSP will lose over 74% of the capacitance after 10 000 cycles. The energy storage mechanism analysis confirms the dominated capacitive behavior of the device, which explained a considerable power density and rate performance. Our method thus provides a promising strategy to build up highly-efficient redox-enhanced FSSCs for next generation of wearable and implantable electronics.

Fabrication of neuroprotective silk-sericin hydrogel: potential neuronal carrier for the treatment and care of ischemic stroke

Ischemic stroke results in severe disabilities due to extensive cellular loss and the resulting impairment of brain functions. Current methods for regenerating brain tissue are ineffective. Stroke treatment requires innovative therapeutic techniques that are both safe and effective. For neuronal repair, a promising alternative is using a hydrogel-based tissue engineering technique that delivers neurotrophic cytokines and cells to injured sites. However, the limited encapsulation effectiveness, less in vivo cell survival ratio and cytokine loss make this strategy difficult to implement. We aim to design a biomaterial that can efficiently construct a matrix enriching the survival of cells and minimizing loss in vivo cytokines to overcome these constraints. We report the development of genipin conjugated sericin hydrogels (Gen-SH) with a high porous morphology and a moderate swelling rate utilizing sericin, a natural silk protein. In vitro, Gen-SH aids in the attachment and development of neurons. Our results indicate that sericin is inherently neuroprotective and neurotrophic, branching and publicizing axon extension and avoiding hypoxia-induced cell death in primary neurons. Notably, the breakdown products of Gen-SH inherit these capabilities, saving the expense of cytokines. Furthermore, we show that the Lkb1–Nuak1 pathway is required for this neurotrophic impact, whereas the Bcl-2/Bax protein ratio is necessary for the neuroprotective effect. Transplanted in vivo, Gen-SH has a high percentage of cell survival and promotes cell proliferation. Taking all this information into account, it's clear that Gen-SH can serve as a viable carrier for treatment and care for ischemic stroke healing, both in terms of delivering neuronal cells and protecting them from oxidative damage.

Silk protein/polyvinylpyrrolidone nanofiber membranes loaded with puerarin accelerate wound healing in mice by reducing the inflammatory response.

In modern clinical applications, wound healing remains a considerable challenge. Excessive inflammatory response is associated with delayed wound healing. In this study, we prepared composite nanofibrous membranes by mixing the Chinese herbal extract puerarin (PUE) with natural silk protein (SF) and synthetic polymer polyvinylpyrrolidone (PVP) using electrostatic spinning technique, and conducted a series of studies on the structural and biological properties of the fibrous membranes. The results showed that the loading of PUE increased the diameter, porosity and hydrophilicity of nanofibers, which were more favorable for cell adhesion and proliferation. ABTS radical scavenging assay also showed that the loading of PUE enhanced the antioxidant properties of the fibrous membranes. In addition, SF/PVP/PUE nanofibers are non-toxic and can be used as wound dressings. In vitro experiments showed that SF/PVP/PUE nanofibers could effectively alleviate lipopolysaccharide (LPS)-induced inflammation in Immortalized human keratinocytes (HaCaT) cells and down-regulate pro-inflammatory cytokine expression in cells. In vivo studies further showed that the SF/PVP/PUE nanofibers could effectively accelerate wound repair. The mechanism is that SF/PVP/PUE nanofibers can inhibit the activation and transduction of toll-like receptor 4/myeloid differentiation factor88/nuclear factor kappa B (TLR4/MyD88/NF-κB) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathways, thereby reducing the inflammatory response and achieving wound healing.

The conversion of castor oil to a series of functional polyamides inspired by natural silks

The design of functional polyamides for specialized applications has attracted increasing attention during recent years. Herein, a facile and efficient method towards mechanical strong functional polyamides with varied side groups is developed. An α,ω-diene functional amide monomer was prepared through the amidation reaction of methyl 10-undecenoate (a kind of castor oil derivative) and 1,3-diamino-2-propanol. The pendant hydroxyl group was converted to ester groups to afford six other monomers. High molecular weight functional polyamides with varied pendant groups, including hydroxyl, acetate, butyrate, isobutyrate, benzoate, acrylate, and imidazole, were prepared via thiol-ene addition polymerization. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) results indicate that the melting temperature, degree of crystallization, and crystalline size depend on the pendant groups. Inspired by natural silk proteins, functional polyamides with controllable mechanical properties can be achieved by copolymerizing of these monomers.

Facile synthesis of sericin modified graphene oxide nanocomposites for treating ischemic diseases

Ischemic heart disease (IHD) is the major reason for death worldwide. Therapeutic angiogenesis serves as an effective approach to treat IHD. Sericin (S), a natural silk protein is widely used in regenerative medicine due to its excellent bioactive properties. Graphene oxide (GO) is extensively used in the field of biomedicine due to its amazing capacity to interact with biomolecules. The main objectives of the present study are to synthesize sericin functionalized graphene oxide (SGO) nanocomposites to treat diseases associated with deficient angiogenesis. Carbodiimide induced cross-linking strategy was employed to functionalize graphene oxide using sericin. The SGO nanocomposites had wrinkled flake like structure with good blood biocompatibility. In vivo chick embryo angiogenesis (CEA) assay was performed to prove the angiogenic potency of SGO nanocomposites. CEA assay results clearly indicated the development of new blood vessels in SGO treated chick embryos when compared with the control.

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.

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Spinning Methods Used for Construction of One- and Two-Dimensional Fibrous Protein Materials.

Natural silk protein fibers have shown a great attraction to the researchers due to the extraordinary mechanical property, biocompatibility, and functional diversity. Unfortunately, the low yield and unevenness have hampered the scale use of the natural silk fibers. Herein, the appearance of the bioinspired artificial spinning strategy offers an effective way to fabricate silk fibers with controllable structures and functionality. This chapter describes an experimental method to prepare silk protein fibers on a large scale and summarizes the method to investigate the effects of the structure-property relationship of the recombinant protein fibers.

Molecular Dynamics Investigation of Self- Association of Synthetic Collagen and Spider Silk Composite System for Biomaterial Applications

ABSTRACTBio-composite materials with optimal mechanical and structural properties and capability of cell differentiation are crucial for tissue engineering. Synthetic collagen proteins with lengths of approximately 10 nanometers, along with natural spider silk proteins provide an opportunity for development of an optimal biomaterial for scaffolding applications in tissue engineering. This combines unique mechanical, structural and biological properties of two of the nature’s best polymer proteins, albeit the collagen used in the present work is of the synthetic nature, it mimics the properties and advantages of natural collagen itself. In the present work, we study the binding capability of these proteins at a molecular level via molecular dynamics modeling. Spider silk and synthetic collagen protein molecular models in biophysical saline conditions under standard pressure and temperature are investigated to understand if natural binding occurs between the two without any other external factors. An initial minimum separation of 10 angstroms between the proteins was used. Binding was observed between the two proteins throughout the dynamic simulation of 100 nanoseconds. The radius of gyration and minimum distance between the proteins shows a decreasing separation between the two proteins until a stable distance of 2.5 nanometers and 0.2 nanometers respectively, is achieved. Binding is further observed between the proteins via formation of strong and stable hydrogen bonds. A hydrogen bond between collagen Proline-31 and silk Serine-96 was observed to be the most stable and frequent bond between the single collagen and silk system. Results clearly indicate a self-assembly behavior of these two systems illustrating their potential as a biomaterial for tissue engineering.

Silk: A bio-derived coating for optical fiber sensing applications

Optical fiber chemical sensing is generally achieved through attachment of sensor molecules to the fiber, a process that involves chemicals that are not biologically compatible or are limited to thin monolayer coatings. To address these limitations and enable in-vivo biosensing, we report here, for the first time, silk fibroin coating of optical fibers for encapsulating fluorescent sensor molecules. Silica exposed core fiber (ECF) samples were coated with a thin layer of silk – a naturally derived biopolymer composed entirely of proteins and amino acids. The silk was doped with the fluorophore 5,6-carboxynapthofluorescein (CNF), which allows optical measurement of pH by a robust ratiometric fluorescence method. The fluorescent signal from the doped-silk layer is coupled into the core of the ECF, enabling remote measurement of pH along the length of the fiber. We have demonstrated real time in vivo pH sensing measurements in a mouse model of hypoxia. Our results showed a continuous drop in the subcutaneous pH in the mouse lumbar area as hypoxia developed. The work explores, for the first time, the potential of a natural silk protein coating to perform fiber sensing inside the body.