Water-soluble silk fibroin based on random crimp / Silk I structure as well as preparation method and application of water-soluble silk fibroin

Abstract

The invention discloses a water-soluble silk fibroin with a random crimp / Silk I structure as a main structure as well as a preparation method and application of the water-soluble silk fibroin. The silk fibroin product is characterized in that in a protein secondary structure, the total content of random curls and Silk I structures exceeds 85%, and the proportion of Silk I in the whole crystal structure is not lower than 75%; the product can be directly dissolved in water at room temperature to form a stable clarified solution with the concentration not lower than 5%, and is not gelatinized after being stored at 4 DEG C for 90 days. The preparation method comprises the following steps: degumming and cleaning the mulberry silk raw material; the method comprises the following steps: carrying out short-time (less than or equal to 20 minutes) high-temperature dissolution by adopting a multi-element solution dissolution system with a specific ratio, and then carrying out dialysis purification at a low temperature by taking the conductivity (lt, 10 [mu] S / cm) of a dialysis external liquid and the concentration (lt, 50 ppm) of calcium ions as double end-point standards. The directional regulation and control of the silk fibroin structure are realized through the accurate control of the whole process, and the obtained product has excellent water solubility, storage stability and biological safety, and can be widely applied to the fields of drug delivery, 3D biological printing, medical beauty filling, tissue engineering and the like.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a method for preparing water-soluble silk fibroin with a specific secondary structure, as well as the silk fibroin product obtained by this method and its application in the biomedical field. Background Technology

[0002] Silk fibroin (SF) is a natural high-molecular-weight protein extracted from silkworm silk. Due to its good biocompatibility, degradability and low immunogenicity, it has shown great application potential in fields such as drug delivery, tissue engineering and regenerative medicine.

[0003] However, existing technologies have failed to provide a silk fibroin product with a "specific stable structure" and "excellent solubility and storage properties," and this is not merely a methodological issue. Traditional silk fibroin preparation methods (such as using high-concentration lithium salts, lithium bromide, and other solvents) typically result in the formation of a stable β-sheet (Silk II) crystalline structure in the silk fibroin molecular chains. This structure makes silk fibroin poorly soluble in water, often requiring redissolution in strong acids, high-concentration organic solvents, or ionic liquids before processing. This not only complicates the process but may also leave toxic solvent residues, limiting its application in precision biomedical fields.

[0004] While existing technologies have reported the use of a ternary solvent of calcium chloride-water-ethanol, and Chinese patent CN104672316A uses calcium chloride as a ternary solvent for silk fibroin dissolution, the solvent ratio, dissolution time, and final silk fibroin properties (such as structural stability) are fundamentally different from the technical solution of this invention. A comprehensive analysis of existing literature reports on silk fibroin preparation methods reveals shortcomings such as crude process control, poor batch-to-batch reproducibility, unstable silk fibroin structure, and a tendency to transform into an insoluble Silk II structure during storage or subsequent processing, leading to loss of water solubility. These deficiencies make it difficult to meet the extremely high requirements for material stability and reproducibility in applications such as drug carriers and bio-inks.

[0005] Therefore, there is an urgent need in this field for an innovative method to obtain structurally stable, highly water-soluble silk fibroin with low impurity residue, mainly composed of random coil / Silk I structure, through a reproducible preparation process with precise parameter control. Summary of the Invention

[0006] (a) Purpose of the invention

[0007] The primary objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing water-soluble silk fibroin with precise and controllable process parameters and high repeatability.

[0008] Another objective of this invention is to provide a water-soluble silk fibroin product prepared by this method, which has a stable secondary structure dominated by random coils and Silk I structure, can be directly dissolved in water without the need for a solubilizer, and has good long-term storage stability.

[0009] Another objective of this invention is to expand the application of the aforementioned silk fibroin products in high-end biomedical materials, such as drug carriers, bio-inks, medical aesthetic fillers, and tissue engineering scaffolds.

[0010] (II) Technical Solution

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a water-soluble silk fibroin product primarily composed of random coil and Silk I structures: in the secondary protein structure of the silk fibroin product, the combined content of random coil and Silk I structures exceeds 85%, and the proportion of Silk I structures in all crystalline structures is not less than 75%. The silk fibroin product can be directly dissolved in water at room temperature to form a stable, clear solution with a mass-to-volume ratio of not less than 5%, and this solution shows no gelation after being stored at 4°C for 90 days. Preferably, its intrinsic viscosity at 25°C is 3.5–4.5 dL / g, and the Zeta potential of its 1% (w / v) aqueous dispersion is -25 mV to -35 mV.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned silk fibroin product, the core of which lies in the synergistic effect of the following steps: S1. Degumming: At least one high-temperature alkaline degumming process combined with multiple washings is used to ensure that the sericin protein is completely and evenly removed, providing pure raw materials for subsequent dissolution.

[0014] Further, in step S1, the alkaline degumming solution is a sodium carbonate solution with a mass-to-volume ratio of 0.1~10 g / L; the mass-to-volume ratio (g / mL) of the mulberry silk raw material to the alkaline degumming solution is 1:50 to 1:200.

[0015] Furthermore, in step S1, the degumming process is performed twice, with a degumming temperature of 90°C to 100°C and a degumming time of 20 to 40 minutes each time.

[0016] S2. Dissolution: Using a multi-component solution dissolution system with a specific ratio, under strictly controlled short time (≤20 minutes) and high temperature (above 85℃) conditions, combined with shearing treatment, the silk fibroin fiber is rapidly and uniformly dissolved, while minimizing the hydrolytic degradation of macromolecular chains and effectively inhibiting the formation of Silk II structure.

[0017] Further, in step S2, the mass-to-volume ratio (g / mL) of the degummed silk fibroin fiber to the multi-component solution dissolution system is 1:10 to 1:20.

[0018] Furthermore, in step S2, the temperature of the dissolution step is 90°C to 100°C, and / or the dissolution time is 5 to 12 minutes.

[0019] Further, in step S2, in the multi-component solution dissolution system, the volume fraction of formic acid is 0%-10%, the volume fraction of ethanol is 0%-35%, and the concentration of the salt is 0.5-4.5 mol / L, wherein the salt is one or more of calcium chloride, calcium nitrate, calcium thiocyanate, zinc chloride, zinc sulfate, and strontium chloride.

[0020] S3. Purification: Dialysis is performed at low temperature, and innovatively, "dialysis fluid conductivity" and "calcium ion concentration" are used as dual endpoint criteria to ensure that small molecule salts and calcium ions are completely removed, thereby stabilizing the random coil / Silk I structure of silk fibroin and preventing structural transformation and gelation induced by ion residues.

[0021] Furthermore, in step S3, the molecular rejection capacity of the dialysis device is 3.5 kDa to 20 kDa.

[0022] S4. Post-processing: The purified solution is filtered and can be flexibly processed into various end forms such as lyophilized powder, gel, and scaffold.

[0023] Through precise parameter control and synergistic effects between each step of the above-mentioned process, the present invention successfully "locks" the secondary structure of silk fibroin in a state dominated by random coil / Silk I structure, and its water solubility, structural stability and long-term storage stability are significantly better than those of silk fibroin prepared by traditional methods.

[0024] Thirdly, this invention provides the application of the water-soluble silk fibroin product in the preparation of biomedical materials. These biomedical materials include, but are not limited to, drug carriers, bio-inks, cosmetic injection fillers, cell culture scaffolds, tissue engineering scaffold materials, or medical dressings.

[0025] (III) Beneficial effects of the present invention:

[0026] (1) Structural novelty and controllability: This invention is the first to achieve targeted regulation of the secondary structure of silk fibroin by combining a "multi-component solution dissolution system with specific ratio", "short-time high-temperature dissolution" and "low-temperature dual-standard purification" process, and obtains a high proportion (>85%) of random coil / Silk I structure, which is fundamentally different from the products obtained by traditional methods that are mainly based on the Silk II structure.

[0027] (2) Excellent product performance: The obtained silk fibroin product has excellent water solubility (can be directly dissolved in water to form a clear solution of ≥5%) and extraordinary stability (does not gel after 90 days of storage at 4℃), which solves the technical problems of traditional silk fibroin solutions being easy to gel and having a short shelf life.

[0028] (3) High purity and safety: Strict degumming and dialysis purification with clearly defined endpoints ensure low sericin residue and extremely low inorganic salt (especially calcium ion) residue in the product, resulting in higher biocompatibility and making it more suitable for high-standard in vivo applications. The entire preparation process of the silk fibroin in this invention is mild and environmentally friendly, avoiding toxic solvent residues and ensuring its safety in biomedical applications.

[0029] (4) Stable and repeatable process: All key steps have quantified process parameters and clear endpoint judgment criteria, which are highly operable, have good batch repeatability, and are easy to achieve standardization and large-scale production.

[0030] (5) Broad application prospects: Based on its excellent water solubility, stability and biosafety, this product is an ideal material for preparing high-end drug delivery systems, high-precision 3D bioprinting, injectable medical aesthetic fillers and tissue engineering scaffolds. Attached Figure Description

[0032] Figure 1 Fourier transform infrared (FTIR) spectrum of the lyophilized silk fibroin powder prepared in Example 1 of this invention.

[0033] Figure 2 X-ray diffraction (XRD) pattern of the lyophilized silk fibroin powder prepared in Example 1 of this invention.

[0034] Figure 3 Raman spectrum of the lyophilized silk fibroin powder prepared in Example 1 of this invention.

[0035] Figure 4 The images show the solution state of the silk fibroin lyophilized powder prepared in Example 1 of this invention after reconstitution (the left image is a photo taken at 0°C after reconstitution, and the right image is a photo taken after storage at 4°C for 90 days after reconstitution; all images show clear and transparent solutions).

[0036] Figure 5 Scanning electron microscope (SEM) image of the three-dimensional porous scaffold of silk fibroin prepared by Example 2 of the present invention (illustrating its uniformly interconnected porous structure).

[0037] Figure 6 Three-dimensional morphology photograph (FE-SEM) of silk fibroin nanoparticles prepared by Example 3 of the present invention. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples, so that those skilled in the art can better understand and implement the present invention. However, the examples given are not intended to limit the present invention. The described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: Preparation of Lyophilized Silk Fibroin Powder

[0041] Degumming: Take 10g of mulberry silk and place it in 1L of a solution containing 2.5g / L anhydrous sodium carbonate. Degumme in a 100℃ water bath for 30 minutes. After removal, wash with purified water by shaking 4 times, 5 minutes each time. Place it again in fresh degumming solution and repeat the above degumming process once. After degumming, dehydrate and dry to obtain approximately 7.5g of degummed silk fibroin fibers.

[0042] Dissolution: Take 5g of degummed silk fibroin fiber and add it to 100 mL of a multi-component solution preheated to 90℃ (composition: 30% ethanol, 3.5 mol / L CaCl2, and 5% formic acid). Stir vigorously for 12 minutes until the silk fibroin is completely dissolved. After cooling the solution to room temperature, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant.

[0043] Dialysis: The supernatant was injected into a dialysis bag with a molecular weight cutoff of 10 kDa and placed in purified water at 4°C for dialysis. The water was changed every 4 hours for the first 12 hours, and then every 12 hours thereafter, for a total dialysis time of 72 hours. At the end of dialysis, the conductivity of the external fluid was measured to be 3 μS / cm, and the calcium ion concentration was 25 ppm.

[0044] Lyophilization: The dialysate was filtered through a 0.22 μm microporous membrane and then transferred to a lyophilizer. After pre-freezing, it was lyophilized for 48 hours to obtain approximately 3.8 g of white, loose silk fibroin lyophilized powder.

[0045] The lyophilized silk fibroin powder prepared by the above method, upon testing, showed that the combined content of random coils and Silk I structures in its protein secondary structure exceeded 85%, and Silk I accounted for more than 75% of the crystalline structure. This lyophilized powder can be directly dissolved in water to form a clear solution with a concentration higher than 5%, and the solution remains clear and gel-free after storage at 4°C for 90 days. This product meets the characteristics defined in claim 1 of this invention.

[0046] Performance testing:

[0047] FTIR analysis: Appendix Figure 1The characteristic absorption peaks are located at 1652 cm⁻¹ (amide I region) and 1541 cm⁻¹ (amide II region), confirming that the structure of silk fibroin is mainly composed of random coils and Silk I.

[0048] XRD analysis: Appendix Figure 2 The presence of a distinct peak at 24°–27° confirms that silk fibroin is composed of non-corrugated and SilkI crystal forms.

[0049] Raman spectroscopy analysis: (attached) Figure 3 Displayed at 1678cm -1 1455cm -1 1256cm -1 The peaks at these locations correspond to the characteristic peaks of amide I, amide II, and amide III of silk fibroin, respectively.

[0050] Solubility test: Add 50 mg of lyophilized powder to 1 mL of deionized water, gently shake at room temperature, and it should completely dissolve within 5 minutes, producing a clear and transparent solution, as shown in the attached image. Figure 4 As shown.

[0051] Stability test: After the above solution was stored in a refrigerator at 4°C for 30 days, it remained clear without gel or precipitation, and no significant changes were observed in the FTIR structure.

[0052] Intrinsic viscosity and Zeta potential: The intrinsic viscosity at 25°C was measured to be 4.1 dL / g using an Ubbelohde viscometer. The Zeta potential of the 1% (w / v) aqueous dispersion was measured to be -30 mV using a nanoparticle size and Zeta potential analyzer.

[0053] Example 2: Fabrication of a three-dimensional porous scaffold

[0054] The lyophilized silk fibroin powder obtained in Example 1 was prepared into a 6% (w / v) aqueous solution. 2 mL of this solution was injected into a mold and pre-frozen at -80°C for 4 hours, and then transferred to a freeze dryer for 48 hours to obtain a three-dimensional porous scaffold.

[0055] Performance testing:

[0056] Structural characterization: as shown in the appendix Figure 5 As shown, SEM observation revealed that the scaffold has a uniform interconnected pore structure of 100-200 μm.

[0057] In vitro osteogenic induction assay: The scaffold was used to culture mouse osteoblasts (MC3T3-E1). On day 7 of culture, CCK-8 assay showed that the absorbance of the experimental group was 1.8 times that of the control group (TCP culture plate), indicating that the scaffold could significantly promote cell proliferation. After 14 days of culture, ALP activity was detected. The ALP activity of the experimental group was 3.5 times that of the uninduced group, indicating that the cells were actively undergoing osteogenic differentiation. After 21 days of culture, alizarin red staining showed obvious formation of red calcium nodules, confirming efficient in vitro mineralization.

[0058] Example 3: Preparation of drug-loaded nanoparticles

[0059] The lyophilized silk fibroin powder prepared in Example 1 was dissolved in deionized water to prepare a 1% (w / v) solution. A model drug (e.g., doxorubicin hydrochloride, DOX) was dissolved in water to prepare a 1 mg / mL solution. Under magnetic stirring, the DOX solution was slowly added dropwise to the silk fibroin solution at a specific volume ratio, ensuring a silk fibroin to drug mass ratio of 10:1. After stirring for 30 minutes, the mixture was filtered through a 0.22 μm filter membrane for sterilization, yielding a DOX-loaded silk fibroin nanoparticle suspension.

[0060] Performance testing:

[0061] Structural characterization: as shown in the appendix Figure 6 As shown, FE-SEM observation reveals the three-dimensional morphology of the nanoparticles.

[0062] Encapsulation efficiency and drug loading: Take an appropriate amount of drug-loaded nanoparticle suspension, centrifuge at high speed, take the supernatant, and use HPLC to determine the concentration of unencapsulated free drug, and calculate the encapsulation efficiency (>85%) and drug loading (approximately 8.5%).

[0063] Particle size and Zeta potential: Dynamic light scattering (DLS) measurements showed that the average hydrated particle size of the drug-loaded nanoparticles was 150±20 nm, the polydispersity index (PDI) was <0.2, and the Zeta potential was -28±2 mV, indicating that the system was stable and suitable for intravenous delivery.

[0064] In vitro drug release study: Drug-loaded nanoparticles were placed in PBS buffer (pH 7.4) and shaken at 37°C. Samples were taken at predetermined time points, and an equal volume of fresh release medium was added. The results showed that the drug exhibited a mild burst release (approximately 30%) within 24 hours, followed by a sustained slow release phase that lasted for over 120 hours, demonstrating excellent sustained-release properties.

[0065] Example 4: Application of silk fibroin as a filler in medical aesthetics (preparation of injectable hydrogel)

[0066] The lyophilized silk fibroin powder prepared in Example 1 was dissolved in phosphate-buffered saline (PBS) to prepare a 15% (w / v) high-concentration solution. This solution remained liquid at room temperature with a viscosity suitable for injection. An equal volume of the silk fibroin solution was mixed with 200 mM K3PO4 solution and gently pipetted several times. After incubating the mixture at 37°C for 5-10 minutes, a transparent hydrogel with a certain strength was formed.

[0067] Performance testing:

[0068] Gel time: The cross-linking gel time was approximately 8 minutes, as determined by the inverted test tube method.

[0069] Rheological properties: The cross-linked hydrogel was tested using a rheometer. Its storage modulus (G') was significantly higher than its loss modulus (G''), and it was stable in the frequency scan of 1-100 Hz. The G' value was in the range of 2000-5000 Pa, showing excellent elasticity, which matches the mechanical properties required for soft tissue filling.

[0070] In vitro degradation: A known weight of the hydrogel was immersed in PBS solution containing collagenase and shaken at 37°C. It was periodically removed and weighed. The results showed that the gel lost approximately 50% of its mass within 4 weeks, demonstrating a controllable degradation rate.

[0071] Cell compatibility: Co-culturing the hydrogel extract with L929 fibroblasts and detecting the relative cell growth rate (RGR) by CCK-8 assay showed that the material was not cytotoxic.

[0072] Example 5: Application of Bio-ink

[0073] The lyophilized silk fibroin powder obtained in Example 1 was dissolved in PBS buffer to prepare an 8% (w / v) solution, which was then mixed with human mesenchymal stem cells (hMSCs) at a volume ratio of 9:1, resulting in a final silk fibroin concentration of 7.5% (w / v) and a cell density of 5 × 10⁻⁶ cells / mL. 6 The mixture, with cells / mL, is the bio-ink of this invention. Using a laboratory-grade extrusion 3D bioprinter, printing parameters were set, and a pre-designed digital model (such as a mesh structure) was used to print layer by layer in a culture dish, successfully constructing a three-dimensional cell-scaffold structure.

[0074] Performance testing:

[0075] Printing performance: The printing process is continuous and smooth, with uniform lines and no broken lines or blockages. The printed structure has high fidelity, clear grid, regular holes, and the dimensional deviation between the first and last layers is less than 5%.

[0076] Cellular behavior: The printed structure was placed in a cell culture incubator for further culture. After 7 days of culture, observation using live / dead staining and cytoskeleton staining (such as phalloidin) showed that cells extended and spread within the silk fibroin scaffold, maintaining good morphology and activity, indicating that the bio-ink can provide a favorable microenvironment for cells.

[0077] Example 6: Application of medical dressings (Preparation of porous sponge dressings)

[0078] The lyophilized silk fibroin powder prepared in Example 1 was dissolved in PBS buffer and the concentration was adjusted to 4% (w / v). A small amount of glycerol and sodium carboxymethyl cellulose were added to increase the flexibility of the dressing. The prepared solution was poured into a mold and pre-frozen in an ultra-low temperature freezer at -80°C for 4 hours to allow ice crystals to grow fully. Then it was quickly transferred to a freeze dryer and freeze-dried for 48 hours to obtain a white porous silk fibroin sponge.

[0079] Performance testing:

[0080] Macroscopic morphology: The resulting sponge is soft and elastic, and can conform well to the skin.

[0081] Air permeability: Tested using a water vapor transmission rate tester, it shows that it has good water vapor transmission rate and can maintain a suitable moist environment for the wound.

[0082] Comparative Example 1: Traditional calcium chloride aqueous solution dissolution method

[0083] Preparation method:

[0084] The degumming process is exactly the same as in Example 1 of this invention.

[0085] Dissolution: Instead of using the multi-component solution dissolution system of this invention, a 4.5 M calcium chloride aqueous solution commonly used in the prior art was employed. Degummed silk fibroin fibers (mass-volume ratio 1:20) were added to a preheated 100°C calcium chloride aqueous solution and stirred to dissolve for 60 minutes.

[0086] Purification and post-processing: Subsequent centrifugation, dialysis (dialysis for 72 hours based on experience only, without using dual endpoint standards), filtration and lyophilization steps were kept as consistent as possible with those in Example 1.

[0087] Results and comparisons:

[0088] Dissolution process: Slow dissolution rate, dark solution color, and high viscosity indicate that more severe hydrolysis may be present.

[0089] Product structure: FTIR analysis shows that its secondary structure is mainly β-sheet (Silk II) (content is about 65%), while random coil / Silk I structure accounts for less than 35% in total.

[0090] Core performance: The resulting lyophilized powder is almost insoluble in water; attempts to prepare a 5% aqueous solution failed, forming a flocculent precipitate. This proves that it completely lacks the water solubility that is the core of this invention.

[0091] Conclusion: Based on the structural characteristics of this comparative example, it is proven that the product structure is dominated by Silk II, completely failing to meet the core characteristics of random coil / Silk I structure content and water solubility as stated in claim 1 of this invention. Simply changing the solvent system from a "multi-component solution dissolution system" to a traditional "calcium chloride dissolution system" and employing conventional long-term dissolution methods cannot obtain the structure and properties defined by this invention. This underscores that the synergistic combination of a "multi-component solution dissolution system with a specific ratio" and "short-time high-temperature shearing" is crucial and not obvious for obtaining a high proportion of random coil / Silk I structure.

[0092] Comparative Example 2: Preparation method based on the closest prior art (CN104672316A)

[0093] Preparation method:

[0094] Strictly follow the parameters described in the example of cited document CN104672316A: use a solvent with CaCl2:EtOH:H2O = 1:2:8 (molar ratio), and stir to dissolve for 60 minutes at 70-75°C.

[0095] Other steps (degumming, dialysis, etc.) were performed in accordance with the description in the patent document, but the dual endpoint determination criteria of this invention were not adopted.

[0096] Results and comparisons:

[0097] Product structure: FTIR shows that its structure is a mixture of random coil / Silk I and Silk II, wherein the total content of random coil / Silk I is about 50%, which is lower than the 85% required by the present invention.

[0098] Stability (Key Difference): When the comparative product was formulated into a 5% aqueous solution and stored at 4°C, the solution began to become cloudy around day 3 and completely gelled within 7 days.

[0099] Calcium ion residue: The calcium ion residue in the lyophilized powder was measured to be >100 ppm, which is much higher than that in the product of this invention (<50 ppm).

[0100] Conclusion: Based on the stability data of this comparative example, it is demonstrated that the product exhibits poor structural stability and fails to meet the stability requirement of claim 1 of this invention regarding 'no gelation after 90 days of storage at 4°C'. This comparative example directly simulates the closest prior art, and the results show that even when using a ternary solvent, without the key technical features of this invention—"a multi-component solution dissolution system with a specific ratio" (promoting Silk I formation), "short-time high temperature" (inhibiting Silk II formation), and "low-temperature dual-endpoint dialysis" (completely removing calcium ions to stabilize the structure)—the structural purity and long-term stability of the resulting product are far inferior to those of this invention. This clearly demonstrates the significant progress and superior technical effects of this invention compared to the prior art.

[0101] Comparative Example 3: Comparison with commercially available silk fibroin

[0102] Reference standard: Commercially available lyophilized silk fibroin powder (prepared by the traditional lithium bromide method according to the test report).

[0103] Comparative Test:

[0104] Solubility: According to the instructions, the commercially available product needs to be dissolved in 9.3 M LiBr solution and then dialyzed before use, a cumbersome process that takes several days. In contrast, the product of this invention can be directly dissolved in water or PBS, a simple process that only takes tens of minutes.

[0105] Stability: Even after dissolving using standard procedures, the silk fibroin solution formed by the commercially available product typically has a stable storage period of no more than one week at 4°C, which is much shorter than the more than three months of the product of this invention.

[0106] Direct applicability: An attempt was made to directly mix the commercially available product with cells for bioprinting according to the method in Example 5 of this invention, but the attempt failed due to its poor water solubility.

[0107] Conclusion: Comparative data with mainstream commercially available products demonstrates a fundamental difference between these products and those in terms of the two core performance characteristics defined in claim 1 of this invention: 'direct water solubility' and 'storage stability'. This invention not only provides a novel preparation method but also delivers a new product with disruptive advantages in 'ease of use', 'efficiency', and 'product stability,' fully demonstrating its significant practical value and commercial potential.

Claims

1. A water-soluble silk fibroin product primarily composed of random coils and Silk I structures, characterized in that: In the secondary protein structure of the silk fibroin product, the total content of random coil structure and Silk I structure exceeds 85%, and the proportion of Silk I structure in all crystalline structures is not less than 75%; the silk fibroin product can be directly dissolved in water at room temperature to form a stable clear solution with a mass-volume ratio of not less than 5%, and the solution does not exhibit gelation after being stored at 4°C for 90 days.

2. The silk fibroin product according to claim 1, characterized in that: Its intrinsic viscosity at 25℃ is 3.5–4.5 dL / g.

3. The silk fibroin product according to claim 1 or 2, characterized in that: The zeta potential of its 1% (w / v) aqueous dispersion is -25 mV to -35 mV.

4. A method for preparing a silk fibroin product as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Degumming: Mix the raw silk with an alkaline degumming solution and degumming at least once at a temperature above 80°C. After each degumming, wash with water to obtain degummed fibroin fibers. S2. Dissolution: The degummed silk fibroin fibers are mixed with a multi-component solution dissolution system preheated to above 85°C, and sheared. The total dissolution time shall not exceed 20 minutes to obtain a silk fibroin protein solution. S3. Purification: Dialyze the silk fibroin solution at 2°C to 10°C until the conductivity of the dialysate drops below 10 μS / cm and the calcium ion concentration is below 50 ppm; S4. Post-processing: The purified silk fibroin solution is filtered or further processed into forms such as solution, gel, lyophilized powder, microspheres, film, fiber or porous scaffold.

5. The preparation method according to claim 4, characterized in that, In step S1, the alkaline degumming solution is a sodium carbonate solution with a mass-to-volume ratio of 0.1 to 10 g / L; the mass-to-volume ratio (g / mL) of the mulberry silk raw material to the alkaline degumming solution is 1:50 to 1:

200.

6. The preparation method according to claim 4, characterized in that, In step S1, the degumming process is performed twice, with a degumming temperature of 90°C to 100°C and a degumming time of 20 to 40 minutes each time.

7. The preparation method according to claim 4, characterized in that, In step S2, the mass-to-volume ratio (g / mL) of the degummed silk fibroin fiber to the multi-component solution dissolution system is 1:10 to 1:

20.

8. The preparation method according to claim 4, characterized in that, In step S2, the temperature of the dissolution step is 90°C to 100°C, and / or the dissolution time is 5 to 12 minutes.

9. The preparation method according to claim 4, characterized in that, In step S2, in the multi-component solution dissolution system, the volume fraction of formic acid is 0%-10%, the volume fraction of ethanol is 0%-35%, and the concentration of the salt is 0.5-4.5 mol / L, wherein the salt is one or more of calcium chloride, calcium nitrate, calcium thiocyanate, zinc chloride, zinc sulfate, and strontium chloride.

10. The preparation method according to claim 4, characterized in that, In step S3, the molecular cutoff of the dialysis device is 3.5 kDa to 20 kDa.

11. The use of the silk fibroin product according to any one of claims 1 to 3 in the preparation of biomedical materials.

12. The application according to claim 11, characterized in that, The biomedical materials mentioned are drug carriers, bio-inks, medical aesthetic injection fillers, cell culture scaffolds, tissue engineering scaffold materials, or medical dressings.

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