Decoding Silk Protein: Properties, Extraction Methods, and Applications in Depth

Introduction

Silk protein, derived from the cocoons of silkworms, has been utilized for centuries in textiles due to its remarkable properties. However, recent advancements in biotechnology and material science have uncovered a vast array of applications beyond traditional textiles.

What is Silk Protein?

Silk protein, primarily composed of fibroin and sericin, is a natural polymer produced by silkworms (Bombyx mori) and some spiders. Fibroin, the main structural component, is responsible for silk's strength and flexibility, while sericin acts as a natural gum that binds fibroin fibers together.

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Properties of Silk Protein

Mechanical Strength: Silk protein exhibits exceptional tensile strength, comparable to steel, making it one of the strongest natural fibers.

Biocompatibility: It is non-toxic and non-irritating, making it suitable for biomedical applications.

Biodegradability: Silk protein is environmentally friendly as it can be broken down by natural processes.

Moisture Absorption: It has good moisture-wicking properties, keeping skin dry and comfortable.

UV Protection: Silk protein offers natural protection against harmful ultraviolet rays.

Sources of Silk Protein

The primary source of silk protein is the silkworm (Bombyx mori), which produces silk during its metamorphosis. Spider silk is another source, though it is more challenging to harvest due to the territorial nature of spiders.

Fig1. Development of Silk Glands in Silkworm (Bombyx mori), Synthesis of Silk Proteins, and Their Regulatory Mechanisms.

Extraction Methods

The extraction of silk proteins, primarily fibroin and sericin, is a critical step in harnessing their valuable properties for various applications.

Degumming Process:

This is the most common method for extracting silk proteins. It involves the separation of sericin from silk fibroin fibers. The process typically involves hydrolyzing or enzymatically cleaving the peptide bonds of sericin to facilitate its extraction.

Soap Solution Degumming: Using soap solutions, such as those containing Marseille soap derived from olive oil, has been practiced for over two centuries. The soap's hydrolysis produces alkali, which helps break down sericin from the silk thread. The sericin is then dissolved in water through the emulsifying action of the soap. A degumming time of 90–120 minutes at boiling temperature is usually sufficient. However, Marseille soap is expensive and may cause water quality issues affecting silk quality. Mixtures of soap and alkali have been explored to accelerate degumming while reducing pollution.

Alkali Solution Degumming: Various alkalis, including sodium silicate, sodium carbonate, and sodium phosphate, are used to enhance the degumming process and maintain pH levels. However, sericin recovery from the soap solution is challenging, and the resulting wastewater contains sericin residues, salts, and soap, requiring comprehensive purification.

Chemical Extraction Methods:

Acid or Base Extraction: Acids (e.g., citric, tartaric, or succinic) or bases (e.g., sodium carbonate, sodium phosphate, sodium silicate, or sodium hydrosulfite) are used to extract sericin from silk. These chemicals hydrolyze sericin by breaking the peptide bonds between amino acids, releasing sericin into an alkaline or acidic solution where it is highly soluble. However, this method can significantly degrade the protein. The urea degradation extraction method, often supplemented with 2-mercaptoethanol, has a reduced degradative impact on sericin. It allows for the extraction of approximately 95% of the total sericin content within the fiber without inducing damage. However, this method is costly and time-consuming, and urea-extracted sericin exhibits high toxicity toward cells.

Fig2. Sericin purification process overview.

Biological Extraction Methods:

Enzymatic Extraction: Enzymatic approaches are favored for their energy efficiency. Proteolytic enzymes such as trypsin, papain, bacterial enzymes (e.g., alcalase), and fungal protease enzymes are used for the degumming process. Trypsin targets the peptide bonds between the carboxyl group of lysine or arginine and adjacent amino acids. Papain, with its broad specificity toward polypeptides, also serves as an effective agent for cocoon degumming. The concentration of enzymes and treatment duration significantly impact the process kinetics. Although slightly more expensive than other techniques, this method requires less energy and is more environmentally sustainable. The use of enzymes like savinase and alcalase, along with ultrasound, has also been explored for sericin extraction from silk fibers. However, the integrity of the isolated sericin has not been thoroughly examined. Protease enzymes from Bacillus species can selectively degrade sericin into peptides measuring 10–12 kDa in size under mildly alkaline pH conditions.

Physical Extraction Methods:

Hot Water Extraction: This method involves using hot water between 82–100 °C to extract sericin. However, the purity of sericin obtained is low, and there is limited information about the composition.

High Pressure Technique: A lab-scale autoclave machine is used to explode cocoon shells, allowing for the extraction of sericin.

Infrared Heating: This innovative approach leverages radiation heating to directly transfer energy to the material through electromagnetic waves, facilitating sericin detachment and enhancing solubility in water. Infrared extraction results in minimal denaturation and degradation of sericin molecules compared to conventional autoclave extraction techniques.

Microwave-Assisted Extraction: Microwave degumming offers advantages such as short extraction time, low energy consumption, and absence of chemical pollution. It is one of the most effective techniques alongside infrared heating. It eliminates the need for additional chemicals in the degumming process, making it conducive to achieving high-purity and cost-effective extraction of sericin. Microwave heating can extract sericin of variable molecular weights depending on the temperature and extraction time.

Steam Treatment: This method uses pressurized steam to remove sericin from silk fibers without any chemicals. It minimizes water pollution and consumption, maintains desirable physicochemical properties, and is more energy-efficient and economical compared to conventional methods.

Supercritical Fluid Extraction: The carbon dioxide supercritical fluid method offers an environmentally stable alternative to conventional extraction methods, effectively and cleanly removing sericin protein.

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Applications of Silk Protein

Fig3. Modification and Production of Silk Proteins – Addressing New Demands for Biomedical Applications.

Applications in Drug Delivery Systems

Advantages as a Drug Carrier: Silk protein exhibits excellent compatibility with human tissues and can avoid severe immune responses. It can gradually degrade in the body, reducing the long-term burden on the organism. By precisely controlling its molecular structure, cross-linking degree, and composition with other materials, the degradation rate of silk protein can be accurately regulated to match the specific release requirements of different drugs. Additionally, its outstanding mechanical properties, such as toughness and elasticity, are crucial for maintaining the structural integrity of drug carriers and preventing drug damage during transport and release. For example, Song et al. developed a novel, strong silk protein-based tissue adhesive, PHT-SP-Cu2+, specifically designed for transdermal drug delivery system patches. Experimental results showed good adhesion, drug-loading capacity, and drug-release efficiency. When silk protein is prepared into micro-needle dressings with specific structures using the Kirigami method, its ductility and tensile strength are significantly improved, making it highly adaptable to dynamic wound changes and showing great potential in smart wound management applications.

Precision Regulation and Innovative Applications: Silk protein-based drug delivery systems are renowned for their sustained and stable drug release, prolonging drug action duration and reducing dosing frequency and adverse drug reactions. By finely adjusting the pore structure and cross-linking density of silk proteins, drug diffusion pathways and rates can be influenced to achieve initial control over drug release. Incorporating intelligent responsive elements like temperature- and pH-sensitive groups allows silk proteins to sense environmental changes and dynamically adjust drug release behavior for more precise control. For instance, silk protein films doped with Pluronic polymers not only enhance mechanical properties, hydrophilicity, and light transmittance but also enable sustained release of antibacterial agents such as curcumin, silver nanoparticles, and the antimicrobial peptide KR-12. Modified silk protein nanoparticles offer new possibilities for controlled delivery of anticancer drugs like doxorubicin. In transdermal drug delivery, silk protein-based microneedle technology demonstrates unique advantages, providing painless and efficient solutions for sustained-release applications, such as reducing scar formation and promoting wound healing.

Enhancing Drug Bioavailability: As a drug carrier, silk protein can improve drug bioavailability by encapsulating or adsorbing drugs to reduce degradation and first-pass metabolism in the gastrointestinal tract; enhancing drug solubility and stability to increase solubility and biomembrane permeability in the body; and adjusting carrier size and surface properties to optimize drug distribution and targeting in vivo. Studies have shown that using pH-responsive silk protein-polymethacrylate copolymers as coatings for oral dosage forms can encapsulate novel silk protein drug formulations in capsules. This effectively resists the acidic gastric environment, ensures direct delivery of drugs to the intestine, and facilitates the release of pancreas-dependent drugs. To address low bioavailability of paclitaxel, self-assembly technology was used to conjugate paclitaxel with silk fibroin to form nanoparticles, which were further coated with Escherichia coli outer membrane vesicles. The resulting biomembrane-coated paclitaxel-silk fibroin nanoparticles exhibit good antitumor effects and improve paclitaxel bioavailability.

Targeted Delivery: Silk protein, with its hydrophobic and hydrophilic domains, can bind hydrophobic therapeutic agents and ligands to achieve targeted drug delivery. Silk protein-based drug delivery systems can accomplish targeted delivery through multiple approaches: leveraging the inherent bioadhesive properties of silk protein to adhere drug carriers to specific tissues or cell surfaces; introducing targeting molecules such as antibodies or ligands onto silk protein carriers via chemical modification or physical adsorption for active targeting; and combining external stimuli like light, magnetism, or ultrasound to trigger directional movement and drug release from carriers for remote-controlled and precise delivery. For example, an injectable silk protein hydrogel has been developed as a carrier for loading BMP7. Studies confirm that delivering BMP7 directly to adipose tissue via this silk protein scaffold system constitutes a novel strategy to promote adipose browning and enhance energy expenditure, offering a potential therapeutic option for achieving precise delivery of target substances to fat storage sites and exploring new avenues for metabolic disease treatment. Targeted peptide-transduced silk protein-coated adenoviruses expressing the ING4-IL-24 dual gene have been used to achieve targeted gene therapy for lung cancer, effectively inhibiting lung tumor cell proliferation, promoting cell apoptosis, and blocking tumor angiogenesis.

Fig4. Application Prospects and Advantages of Silk Protein-Based Drug Delivery Systems.

Applications in Tissue Engineering and Regenerative Medicine

Genetically engineered silk protein, as a naturally derived polymer material that has undergone precise design and regulation, demonstrates immense potential in tissue engineering and regenerative medicine due to its exceptional biocompatibility, degradability, mechanical properties, and good cell affinity. Through genetic engineering techniques, the structure and function of silk protein can be customized to meet diverse biomaterial needs in tissue engineering and regenerative medicine. As a biological scaffold material, genetically engineered silk protein can mimic the microenvironment of natural tissues, providing a platform for cell attachment, growth, and differentiation. Its unique three-dimensional structure and porosity facilitate cell migration, nutrient transport, and waste removal, thereby promoting the repair and regeneration of damaged tissues. Notably, genetically engineered silk protein has shown outstanding performance in the repair and regeneration of critical tissues such as skin, bone, and cartilage.

Skin Repair: Silk protein films and fibrous scaffolds, with their excellent breathability and moisture retention properties, serve as ideal substrates for skin cell growth. Studies have shown that such scaffolds significantly promote skin cell proliferation and migration, accelerate wound healing, and effectively inhibit scar formation. Silk protein scaffolds, as delivery systems, can reduce stress peaks around wound sites when loaded with cytokines, bioactive components, cells, and tissues. They provide necessary physical support while optimizing wound care efficiency. For example, collagen/silk protein composite scaffolds loaded with bone mesenchymal stem cells exhibit outstanding skin affinity, breathability, and water permeability, further enhancing wound healing potential. Chitosan-based silk protein topical hydrogels have demonstrated significant efficacy in controlling acute bleeding and promoting wound healing. Additionally, the antibacterial properties of silk protein provide robust protection against wound infections. Near-infrared-responsive silk protein films prepared via electrospinning technology exhibit excellent mechanical properties and blood compatibility. Under near-infrared irradiation, they can rapidly generate reactive oxygen species, effectively killing Staphylococcus aureus, promoting M2 polarization of macrophages, and accelerating wound healing, offering a new strategy for wound infection treatment.

Bone Repair: In bone tissue engineering, silk proteins show significant potential as bone substitute materials and bone-guiding regeneration membranes. Their unique three-dimensional porous structure provides an ideal environment for bone cell attachment, proliferation, and differentiation, promoting vascular network construction to ensure adequate nutrient and oxygen supply for newly formed bone tissue. Due to their excellent mechanical strength and biocompatibility, silk proteins have become ideal materials for 3D-printed bone tissue engineering scaffolds. They can promote osteogenic gene expression, collagen accumulation, and mineralization, supporting bone tissue regeneration and modulating related immune responses. For instance, poly-L-lactic acid/silk fibroin composite nanofiber scaffolds coated with osteoblast extracellular matrix significantly enhance the osteogenic differentiation ability of bone marrow mesenchymal stem cells. Silk fibroin porous bone scaffolds reinforced with short-cut fibers and nano-hydroxyapatite, prepared by freeze-drying, not only optimize mechanical properties but also reduce foreign body reactions, promote anti-inflammatory responses, and accelerate the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Combined with growth factors like BMPs, silk proteins further enhance bone repair efficacy, providing new therapeutic strategies in bone tissue engineering. Chitosan/silk protein composite double-layered PCL nanofiber mats combine enhanced antibacterial properties with osteogenic potential. Their osteogenic mechanism involves complexing Runx2 plasmids to enhance osteogenic-related gene expression and mineralized nodule formation.

Cartilage Repair: Addressing the unique avascular and low cellular metabolic rate characteristics of cartilage tissue, silk proteins demonstrate significant repair potential. By precisely controlling mechanical properties and microstructure, silk protein scaffolds have successfully mimicked the natural cartilage matrix, effectively guiding chondrocyte proliferation and differentiation to promote cartilage regeneration. Studies have shown that silk protein hydrogel scaffolds combined with chitosan nanoparticles exhibit excellent biocompatibility and significantly enhance the chondrogenic capacity of bone marrow stromal cells by precisely regulating the release of TGF-β1 and BMP-2. Silk fibroin scaffolds delivering tanshinone IIA significantly promote cartilage matrix synthesis and regeneration by activating chondrocyte activity, inhibiting apoptosis, and reducing oxidative stress. The silk protein-gelatin-chondroitin sulfate-hyaluronic acid-aloe vera composite three-dimensional scaffold, with its high porosity, large pore size, excellent water absorbency, and mechanical strength, combined with anti-inflammatory properties, significantly promotes the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells and effectively inhibits IL-1β expression, making it an ideal choice for cartilage tissue engineering.

Vascular Tissue Regeneration: Leveraging its excellent biocompatibility and structural tunability, silk protein has significantly optimized the growth, adhesion, survival, and proliferation of vascular tissue cells. This positions silk protein as a highly effective and safe material with potential applications in vascular repair and regeneration, providing a solid foundation for vascular tissue engineering. Studies have confirmed that silk protein/fibroin composite vascular scaffolds prepared using electrospinning technology exhibit outstanding mechanical properties, hydrophilicity, blood compatibility, biodegradability, and cell compatibility. Most importantly, they enhance the proliferation and adhesion of mesenchymal stem cells, making them ideal candidate materials for artificial blood vessels in vascular tissue engineering. Small-diameter silk protein artificial blood vessel implants have shown high patency rates, good endothelial cell coverage, and remodeling capabilities after implantation in dogs, validating their clinical application potential as vascular substitutes for diameters under 6mm. A double-layered microneedle composed of chitosan and a silk protein complex containing the angiogenic drug deferoxamine significantly promotes angiogenesis, accelerates wound contraction, and effectively promotes cell proliferation and fibroblast migration by modulating cytokine expression, demonstrating remarkable wound healing effects. Silk protein gels loaded with thymoproteasome-10 and ZIF-8 also exhibit good ability to promote angiogenesis and bone regeneration.

Applications in Antibacterial and Immune Strategies

Silk protein-based antibacterial wound dressings have demonstrated immense potential and value in the biomedical field. For example, silk protein films prepared via electrospinning technology exhibit excellent mechanical properties and blood compatibility. Under near-infrared irradiation, they can rapidly generate reactive oxygen species to effectively kill Staphylococcus aureus, promote M2 polarization of macrophages, and accelerate wound healing. Additionally, silk protein's antibacterial properties provide strong protection against wound infections. Modified silk proteins expressed in transgenic silkworms show significant advantages in enhancing drug bioavailability and promoting cell proliferation and differentiation. They play an innovative role in antibacterial and immune strategies.

Other Applications

Sensors and Biosensors: Silk protein's excellent biocompatibility and mechanical properties make it suitable for use in sensors and biosensors. For instance, silk protein-based biosensors can detect biomarkers in the body, offering non-invasive monitoring of health conditions.

Gene Therapy: Silk protein can be used as a gene carrier to deliver genetic material into cells, enabling gene therapy for diseases. Its biocompatibility and controllable degradation properties ensure the safe and effective delivery of genes.

Fig5. Processing and types of regenerated sericin biomaterials.

Benefits of Using Silk Protein

• Sustainability: As a natural and renewable resource, silk protein offers an eco-friendly alternative to synthetic materials.
• Versatility: Its diverse properties allow it to be adapted for numerous applications across different industries.
• Health Benefits: In cosmetics and textiles, silk protein promotes skin health and provides a comfortable wearing experience.

Challenges and Considerations

Production Costs: The extraction and processing of silk protein can be labor-intensive and expensive.

Scalability: Meeting the demand for silk protein in various industries requires efficient production methods and sustainable farming practices.

Future Trends and Research

Ongoing research focuses on improving silk protein production techniques, enhancing its properties through genetic engineering, and discovering new applications in emerging fields such as regenerative medicine and flexible electronics.

Conclusion

Silk protein stands as a versatile and valuable material with a rich history and a promising future. Its unique combination of strength, biocompatibility, and sustainability makes it a key player in multiple industries. As technology advances, we can expect even more innovative uses of silk protein to emerge, continuing to benefit both consumers and the environment.

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