Injectable microspheres gel of a performance-controllable silk fibroin and a preparation method thereof

Abstract

The application discloses an injectable microsphere gel of controllable performance silk protein and a preparation method thereof, and belongs to the field of biomaterials.The preparation method comprises the following steps: preparing a regenerated silk protein solution, including degumming, dissolving and purifying of mulberry silk, and obtaining a regenerated silk protein solution with a target molecular weight through molecular weight screening; preparing a silk protein microsphere aqueous solution by adding ethanol to the regenerated silk protein solution and performing freezing and thawing; obtaining a silk protein microsphere redispersible powder by drying two solutions prepared in the above steps; and preparing a silk protein microsphere gel by dispersing the silk protein microsphere redispersible powder in a solvent and adding a gel inducer.The whole preparation process does not involve toxic chemical reagents, and thus the excellent mechanical properties, ideal biocompatibility and biodegradability of the material are ensured; the silk protein microsphere gel has thixotropy, can be injected into subcutaneous tissue through a syringe, has a certain self-supporting property after injection, has controllable mechanical properties due to the molecular weight screening, and can be used in different application scenarios.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetics and plastic surgery technology, and specifically relates to an injectable microsphere gel of silk protein with adjustable properties and its preparation method. Background Technology

[0002] In the field of cosmetic surgery, the purpose of using implants is to reconstruct structures or improve appearance. Filler hydrogels used in facial cosmetic procedures can correct wrinkles, improve facial contours, fill in sunken scars or lips, and repair damaged tissue.

[0003] Hydrogel materials are increasingly widely used in dermal fillers. Hydrogels are polymers with a three-dimensional cross-linked network structure and high water absorption and retention capacity. They are generally classified into chemically cross-linked hydrogels and physically cross-linked hydrogels. Chemically cross-linked hydrogels are formed through cross-linking agents, radiation cross-linking, and photo-initiated cross-linking. Physically cross-linked hydrogels do not involve chemical reactions; the cross-linking between molecular chains is formed through intermolecular forces (van der Waals forces, hydrogen bonds, etc.). Injecting hydrogels, which are biomedical materials with a certain degree of fluidity, can largely avoid the risks of surgery and reduce patient discomfort. Collagen-based hydrogels and hyaluronic acid-based hydrogels are the most widely used products on the market. Collagen fillers are mainly extracted from organisms and then prepared through purification and sterilization processes. They can be used to restore lip lines, repair facial wrinkles, and address other soft tissue contour deficiencies. However, due to the rapid degradation of collagen, the short-lived filling effect, low mechanical strength, and poor shapeability, it is generally used for areas such as neck wrinkles where shaping is not required. If a certain degree of shaping is required, filler formulations made from hyaluronic acid, polylactic acid, and other synthetic materials are usually chosen. Sodium hyaluronate is a glycosaminoglycan, a high-molecular-weight polysaccharide. When used as a dermal filler, butylene glycol glycidyl ether or divinyl sulfone is typically used as a cross-linking agent to slow down its degradation. However, these cross-linking agents are slowly absorbed by the body as the gel degrades, posing a potential risk to the use of sodium hyaluronate in the human body. Furthermore, sodium hyaluronate with a low degree of cross-linking degrades faster, maintaining its effect for 2-3 months after implantation in the face, while sodium hyaluronate with a high degree of cross-linking lacks elasticity, has higher hardness, and feels lumpy and solid after implantation in the face.

[0004] When hydrogel formulations are applied in various dermal filler applications, we desire a gel material that is both durable and resistant to degradation over a longer period, possesses mechanical strength similar to that of human tissue, exhibits thixotropy (displaying fluid rheological behavior under shear stress), can be injected subcutaneously using a syringe, and provides a degree of self-support after injection. To improve this self-support, we have found that polymer microspheres, as microstructural units of a material, are often used to improve or enhance its properties. Polymer microspheres refer to polymeric materials or high-molecular-weight materials with diameters ranging from nanometers to micrometers and shapes that are spherical or other similar geometric forms. Preparing polymer microspheres to improve the structural properties of the material, and then using this process to create a gel for dermal filler applications, significantly broadens the application prospects of gels.

[0005] Silk fibroin (the remaining part of silk after the sericin is removed, also known as fibroin protein) is a biomaterial with good biocompatibility, no immunogenicity, and high safety in biodegradable products, and is widely used in product research in the field of biomedical materials. Chinese invention patent CN202010659791.4 describes the preparation of a gel by blending silk fibroin and gelatin, but it does not consider different application scenarios to prepare a gel material suitable for the target filling site. The reason for this is that it is currently impossible to screen and classify silk fibroin by molecular weight, making it impossible to accurately measure the molecular weight. Because large silk fibroin molecules tend to aggregate under an electric field, traditional gel electrophoresis methods for testing the molecular weight of large silk fibroin molecules cannot accurately provide molecular weight values ​​and molecular weight distribution. Figure 1 As shown, the composition and conformation of silk fibroin vary with its molecular weight. Furthermore, silk fibroin has the advantage of forming both physical and chemical gels without the need for chemical cross-linking agents. By controlling the molecular weight of silk fibroin, the gel inducing agent, and the gelation mechanism, the mechanical properties of silk fibroin gels can be adjusted, thus achieving suitable mechanical properties while maintaining a longer degradation time. Therefore, screening silk fibroin at different molecular weights through the preparation process is crucial for preparing gel materials with different mechanical properties suitable for different parts of the body. Summary of the Invention

[0006] To address the problem of the inability to simultaneously achieve the mechanical properties and degradation rate of existing filled gels, as well as the potential risks associated with crosslinking agents in the field of injectable hydrogels, this invention proposes an injectable microsphere gel with adjustable performance based on the thixotropic properties of silk fibroin microsphere gels. This gel exhibits fluid rheological behavior under shear stress and possesses a certain degree of self-support when at rest.

[0007] The present invention provides a method for preparing an injectable microsphere gel of silk fibroin with adjustable performance, comprising the following steps:

[0008] S1: Preparation of regenerated silk protein solution, including degumming, dissolving and purifying steps of mulberry silk, and obtaining regenerated silk protein solution with target molecular weight through molecular weight screening;

[0009] S2: Prepare an aqueous solution of silk fibroin microspheres based on the regenerated silk fibroin solution with a specific molecular weight obtained in step S1;

[0010] S3: The silk protein solution prepared in step S1 and the silk protein microsphere aqueous solution prepared in step S2 are dried to obtain a redispersible powder of silk protein microspheres.

[0011] S4: The silk protein microsphere redispersed powder obtained in step S3 is used to prepare a silk protein microsphere gel with adjustable performance.

[0012] Preferably, in step S1, the preparation step of the regenerated silk protein solution includes: a) degumming: placing silkworm silk in an aqueous solution of sodium carbonate and sodium bicarbonate or a mixed solution of sodium carbonate and sodium bicarbonate, heating to boiling, removing and washing with purified water to remove sericin, leaving silk protein, drying the silk protein to obtain dried silk protein for later use; b) dissolving: dissolving the dried silk protein in an aqueous solution of lithium bromide to obtain a mixture containing silk protein and a small amount of insoluble particles; c) purification and screening: diluting the mixed solution with purified water, passing it through an ultrafiltration system for desalination, screening regenerated silk protein solutions of corresponding molecular weights according to the specific application scenario of the gel, and concentrating the silk protein concentration of the final solution to 5-40 wt%.

[0013] Furthermore, in step S1, during the preparation of the regenerated silk protein solution, the screening method involves selecting different pore sizes in the ultrafiltration system to screen out regenerated silk protein solutions with different molecular weights.

[0014] Preferably, the weight-average molecular weight range of the regenerated silk protein solution screened in step S1 is 80-100kDa, 100-150kDa, 150-200kDa, or 200-250kDa.

[0015] Preferably, the specific preparation method of the silk protein microsphere aqueous solution in step S2 is as follows: anhydrous ethanol is added to the silk protein solution prepared in step S1 and mixed evenly. The volume of ethanol added is 0.01-2 times the volume of the silk protein solution. After mixing, the solid content of silk protein in the solution is 1-10 wt%. Then, the mixed solution is placed in an environment of -10℃ to -40℃ and thawed after 1-48 hours to obtain the silk protein microsphere aqueous solution. The silk protein microsphere aqueous solution is then concentrated to a solid content of 10-50 wt%.

[0016] Furthermore, the diameter of the silk protein microspheres in step S2 is 30-800 nm.

[0017] Preferably, the specific method for drying the silk protein microspheres into redispersible powder in step S3 is as follows: the silk protein solution prepared in step S1 and the silk protein microsphere aqueous solution prepared in step S2 are mixed evenly, wherein the volume of the silk protein solution is 0.01-100 times the volume of the silk protein microsphere aqueous solution; then the mixed solution is passed into a spray drying system for spray drying, or freeze-dried by freeze drying.

[0018] Furthermore, the silk fibroin microspheres prepared in step S3 are dried into a redispersible powder, which facilitates storage and transportation. This prevents denaturation of the regenerated silk fibroin solution during long-term storage and avoids silk fibroin precipitation caused by vigorous shaking during transportation. Moreover, the dried powder, a mixture of regenerated silk fibroin and silk fibroin microspheres, dissolves under gentler conditions, requiring no vigorous mechanical stirring and preventing aggregation and precipitation. If pure regenerated silk fibroin is directly dried into powder, the silk fibroin chains tend to aggregate during redissolution, causing β-sheet formation and ultimately leading to significant precipitation. The presence of silk fibroin microspheres acts as a solubilizer. They increase the distance between the regenerated silk fibroin molecular chains, making the redissolution process easier.

[0019] Preferably, the specific preparation method of the silk protein microsphere gel in step S4 is as follows: the redispersible powder prepared in step S3 is added to deionized water and mechanically stirred to disperse and dissolve the powder, and then a gel inducing agent is added and mixed evenly; the mixed solution is placed in an environment of 25-80℃ and kept warm for 0.5-3 hours to form a uniform and stable silk protein microsphere gel.

[0020] Furthermore, in step S4, the gel inducing agent is a compound or mixture that can induce silk fibroin to gel, including cellulose-based gel inducing agents, bentonite-based gel inducing agents, or enzyme-based inducing agents. The cellulose-based inducing agents include one or more of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose. The bentonite-based gel inducing agents include lithium bentonite, and the enzyme inducing agents include horseradish peroxidase. Pure silk fibroin microsphere gels have a relatively low elastic modulus; by adding a gel inducing agent, the elastic modulus of the gel can be adjusted to a higher level, making it more suitable for shaping.

[0021] Preferably, in step S4, when using a cellulose-based gel inducer to prepare silk fibroin microsphere gel, the solid content of silk fibroin in the solution is 1-40%, and the solid content of cellulose is 1-40%.

[0022] Preferably, in step S4, when using an enzyme gel inducer to prepare silk fibroin microsphere gel, the solid content of silk fibroin in the solution is 1-40 wt%, preferably 7-15 wt%, and the enzyme content in the mixed solution is 1-100 U / mL, preferably 15-75 U / mL.

[0023] Preferably, in step S4, when preparing silk fibroin microsphere gel using lithium saponin as an inducer, the solid content of silk fibroin in the solution is 1-40 wt%, preferably 5-15 wt%, and the concentration of lithium saponin is 1-20 wt%.

[0024] The beneficial effects of this invention are:

[0025] 1) High safety: The silk protein microsphere gel product prepared by this invention does not involve toxic chemical reagents in the entire preparation process, ensuring the material's excellent mechanical properties and ideal biocompatibility and biodegradability.

[0026] 2) Injectability: The silk protein microsphere gel prepared by this invention is thixotropic and exhibits fluid rheological behavior when subjected to shear force. It can be injected into subcutaneous tissue by syringe and needs to have a certain degree of self-support after injection.

[0027] 3) Adjustable mechanical properties: The silk protein microsphere gel prepared by this invention has adjustable mechanical properties and can be applied to scenarios with different mechanical property requirements. Attached Figure Description

[0028] Figure 1 This is a banding diagram for determining the molecular weight of macromolecular silk proteins using gel electrophoresis.

[0029] Figure 2 This is a flowchart illustrating the preparation process of the silk fibroin-based injectable microsphere gel in this embodiment;

[0030] Figure 3 The mechanical properties of the silk fibroin microsphere gels prepared in Examples 1-4 are shown in the figure.

[0031] Figure 4 The silk protein microsphere gel prepared by the present invention;

[0032] Figure 5 The experimental results for the silk fibroin microsphere gels prepared in Examples 1-4 were obtained using the three-stage thixotropic test method.

[0033] Figure 6 Scanning electron microscope images of the silk fibroin microspheres prepared in Examples 1-4;

[0034] Figure 7 The diagram shows the cell compatibility experiment of the silk protein microsphere gels prepared in Examples 1-4. Detailed Implementation

[0035] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the disclosure of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope of protection defined by this invention.

[0036] Example 1

[0037] This embodiment proposes a method for preparing injectable microsphere gels for neck wrinkle filling, such as... Figure 2 As shown, it includes the following steps:

[0038] S1: Preparation of regenerated silk fibroin solution: a) Degumming: Place silkworm silk in an aqueous solution of sodium carbonate and sodium bicarbonate or a mixed solution of sodium carbonate and sodium bicarbonate, heat to boiling, remove and wash with purified water to remove sericin, leaving silk fibroin. Dry the silk fibroin to obtain dried silk fibroin for later use; b) Dissolving: Dissolve the dried silk fibroin in an aqueous solution of lithium bromide to obtain a mixture containing silk fibroin and a small amount of insoluble particles; c) Purification and screening: Dilute the mixed solution with purified water, pass it through an ultrafiltration system for desalination, select a system with a pore size of 80-100 kDa, prepare a regenerated silk fibroin solution with a molecular weight of 80-100 kDa, and concentrate the silk fibroin concentration of the final solution to 5-40 wt%.

[0039] S2: Preparation of silk fibroin microsphere solution:

[0040] Anhydrous ethanol was added to the regenerated silk protein solution prepared in step S1 and mixed evenly. The volume ratio of silk protein solution to ethanol was 5:1. After mixing, the solid content of silk protein in the solution was 3 wt%. The mixed solution was then placed in a -40°C environment for 48 hours to thaw, and an aqueous solution of silk protein microspheres was obtained and concentrated to a solid content of 10 wt%.

[0041] S3: Drying the silk protein microspheres into redispersible powder: Mix the silk protein solution (solid content of 10 wt%) prepared in step S1 and the silk protein microsphere aqueous solution (solid content of 10 wt%) prepared in step S2 at a volume ratio of 1:1, and then pass the mixed solution into a spray drying system to dry it into powder.

[0042] S4: The specific preparation method of silk protein microsphere gel is as follows: the redispersible powder prepared in step S3 is added to deionized water and mechanically stirred to disperse and dissolve, forming a dispersion with a solid content of 30wt%. Then, hydroxypropyl methylcellulose solution is added and mixed evenly. The total solid content in the solution after mixing is 20%, and the mass ratio of silk protein to hydroxypropyl methylcellulose in the solution is 20:1. Then, the mixed solution is placed in a 70℃ water bath for 1 hour to obtain silk protein microsphere gel.

[0043] like Figure 3 As shown, the silk protein microsphere gel prepared in Example 1 has a modulus of 5.5 kPa, which is suitable for filling neck wrinkles.

[0044] Example 2

[0045] This embodiment proposes a method for preparing injectable microsphere gel for filling the temple area, including the following steps:

[0046] S1: Preparation of regenerated silk protein solution: Same as step S1 in Example 1, but select an ultrafiltration system with a pore size of 100-150kDa to prepare a regenerated silk protein solution with a molecular weight of 100-150kDa.

[0047] S2 Preparation of silk fibroin microsphere solution: Same as step S2 in Example 1;

[0048] S3: Dry the silk protein microspheres into a redispersible powder: Same as step S3 in Example 1, to obtain a redispersible powder;

[0049] S4: The specific preparation method of silk fibroin microsphere gel is as follows: Add the redispersible powder prepared in step S3 to deionized water and stir mechanically to disperse and dissolve, forming a dispersion with a solid content of 15wt%; add horseradish peroxidase (HRP enzyme) and 0.5% H2O2 (90-360μL / mL) to the dispersion, and the enzyme content in the solution after mixing is 18-72U / mL. Then place it in a 37℃ environment for 2 hours to form microgels. Then soak the microgels in an ethanol solution for 1 hour to form silk fibroin microsphere gels.

[0050] like Figure 3 As shown, the silk protein microsphere gel prepared in Example 1 has a modulus of 34.1 kPa, making it suitable for filling the temple area.

[0051] Example 3

[0052] This embodiment proposes a method for preparing injectable microsphere gel for cheek augmentation, comprising the following steps:

[0053] S1: Preparation of regenerated silk protein solution: Same as step S1 in Example 1; Select an ultrafiltration system with a pore size of 150-200kDa to prepare a regenerated silk protein solution with a molecular weight of 150-200kDa.

[0054] S2: Preparation of silk fibroin microsphere solution: Same as step S2 in Example 1;

[0055] S3: Dry the silk protein microspheres into a redispersible powder: Same as step S3 in Example 1, to obtain a redispersible powder;

[0056] S4: The specific preparation method of silk fibroin microsphere gel is as follows:

[0057] a) Add the redispersible powder prepared in step S3 to deionized water and mechanically stir to disperse and dissolve, forming a dispersion with a solid content of 15 wt%.

[0058] b) Disperse lithium bentonite powder in deionized water for 24 hours, then add 0.06 wt% sodium polyacrylate (molecular weight 2000 Da), and continue stirring for 2 hours to obtain a uniformly dispersed lithium bentonite aqueous solution.

[0059] c) Then add the lithium saponin aqueous solution to the silk protein microsphere aqueous solution prepared in step a, mix well, add horseradish peroxidase (HRP enzyme) and 0.5% H2O2 (90-360 μL / mL) to the dispersion, and the enzyme content in the solution after mixing is 18-72 U / mL. Then place it in a 37°C environment for 1 hour to form a microgel. Then soak the microgel in an ethanol solution for 1 hour to form a silk protein microsphere gel.

[0060] like Figure 3 As shown, the silk protein microsphere gel prepared in Example 3 has a modulus of 57.3 kPa, making it suitable for filling the cheek area.

[0061] Example 4

[0062] This embodiment proposes a method for preparing injectable microsphere gel for filling the nasal bridge cartilage area, including the following steps:

[0063] S1: Preparation of regenerated silk protein solution: Same as step S1 in Example 1; Select an ultrafiltration system with a pore size of 200-250kDa to prepare a regenerated silk protein solution with a molecular weight of 200-250kDa.

[0064] S2: Preparation of silk fibroin microsphere solution: Same as step S2 in Example 1;

[0065] S3: Dry the silk protein microspheres into a redispersible powder: Same as step S3 in Example 1, to obtain a redispersible powder;

[0066] S4: The specific preparation method of silk fibroin microsphere gel is as follows:

[0067] a) Add the redispersible powder prepared in step S3 to deionized water and mechanically stir to disperse and dissolve, forming a dispersion with a solid content of 15 wt%.

[0068] b) Disperse lithium bentonite powder in deionized water for 24 hours, then add 0.06 wt% sodium polyacrylate (molecular weight 2000 Da), and continue stirring for 2 hours to obtain a uniformly dispersed lithium bentonite aqueous solution.

[0069] c) Then, the lithium saponin aqueous solution is added to the silk protein microsphere aqueous solution prepared in step a, and mixed evenly. Horseradish peroxidase (HRP enzyme) and 0.5% H2O2 (90-360 μL / mL) are added to the dispersion. After mixing, the enzyme content in the solution is 18-72 U / mL. Then, it is placed in a 37°C environment for 1 hour to form a microgel. Then, the microgel is soaked in an ethanol solution for 1 hour to form a silk protein microsphere gel.

[0070] like Figure 3 As shown, the silk protein microsphere gel prepared in Example 4 has a modulus of 152 kPa, making it suitable for filling the nasal cartilage area.

[0071] from Figure 3 As can be seen, by adjusting parameters such as the molecular weight of silk fibroin and gel inducing agents, fillers with different mechanical strengths can be prepared for different applications.

[0072] The silk protein microsphere gels prepared in Examples 1-4 of this invention, such as Figure 4 As shown, an oscillation mode was adopted with a fixed frequency of 1Hz. During the structural failure stage, a large amplitude of 1000% was selected and lasted for 100s. To monitor structural recovery, an amplitude of 1% was selected. This was repeated multiple times, and the changes of G' and G” over time were observed. Figure 5 As shown, the results indicate that all four gels have good injectability and exhibit a certain degree of self-support.

[0073] The silk fibroin microspheres prepared in Examples 1-4 of this invention were observed using a scanning electron microscope, such as... Figure 6 As shown, the higher the molecular weight of silk fibroin, the larger the size of the prepared silk fibroin microspheres.

[0074] Primary fibroblasts were seeded onto the surface of the silk protein microsphere gels prepared in Examples 1-4, and the spread of cells on the gel surface was then observed under a microscope. Figure 7 As shown, the cells spread out on the gel surface, indicating that the prepared gel is conducive to cell adhesion and spreading.

Claims

1. A method for preparing an injectable microsphere gel of silk fibroin with tunable performance, characterized in that, Includes the following steps: S1: Preparation of regenerated silk protein solution, including degumming, dissolving, and purifying steps of mulberry silk, and obtaining the solution through molecular weight screening. A regenerated silk protein solution with a target molecular weight; wherein the weight-average molecular weight of the regenerated silk protein solution is in the range of 80-100 kDa, 100-150 kDa, 150-200 kDa, or 200-250 kDa. S2: Prepare an aqueous solution of silk fibroin microspheres based on the regenerated silk fibroin solution with a weight-average molecular weight range of 80-100 kDa obtained in step S1; S3: The 100-150kDa, 150-200kDa, or 200-250kDa silk protein solution prepared in step S1 and the silk protein microsphere aqueous solution prepared in step S2 are dried to obtain a redispersible powder of silk protein microspheres; wherein, the specific method for drying the silk protein microspheres into a redispersible powder is as follows: the silk protein solution prepared in step S1 and the silk protein microsphere aqueous solution prepared in step S2 are mixed evenly, wherein the volume of the silk protein solution is 0.01-100 times the volume of the silk protein microsphere aqueous solution; then the mixed solution is spray-dried in a spray drying system or freeze-dried by lyophilization; S4: The redispersible powder of silk fibroin microspheres obtained in step S3 is used to prepare a silk fibroin microsphere gel with adjustable performance. Specifically, the redispersible powder prepared in step S3 is added to deionized water and mechanically stirred to disperse and dissolve the powder. Then, a gel inducing agent is added and mixed evenly. The mixed solution is placed in an environment of 25-80℃ and kept at that temperature for 0.5-3 hours to form a uniform and stable silk fibroin microsphere gel. The gel inducing agent is a compound or mixture that can induce silk fibroin to form a gel, including cellulose gel inducing agents, bentonite gel inducing agents, or enzyme inducing agents. Cellulose inducing agents include one or more of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose. Benzoate gel inducing agents include lithium bentonite. Enzyme inducing agents include horseradish peroxidase.

2. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to claim 1, characterized in that, In step S1, the preparation steps of the regenerated silk protein solution include: a) degumming: placing silkworm silk in an aqueous solution of sodium carbonate and sodium bicarbonate or a mixed solution of sodium carbonate and sodium bicarbonate, heating to boiling, removing and washing with purified water to remove sericin, leaving silk protein, drying the silk protein to obtain dried silk protein for later use; b) dissolving: dissolving the dried silk protein in an aqueous solution of lithium bromide to obtain a mixture containing silk protein and a small amount of insoluble particles; c) purification and screening: diluting the mixture with purified water, passing it through an ultrafiltration system for desalination, screening regenerated silk protein solutions of corresponding molecular weights according to the specific application scenario of the gel, and concentrating the silk protein concentration of the final solution to 5-40 wt%.

3. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to claim 2, characterized in that, In step S1, the screening method for the regenerated silk protein solution is to select different pore sizes in the ultrafiltration system, thereby screening out regenerated silk protein solutions with different molecular weights.

4. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, The specific preparation method of the silk protein microsphere aqueous solution in step S2 is as follows: add anhydrous ethanol to the silk protein solution prepared in step S1 and mix evenly. The volume of ethanol added is 0.01-2 times the volume of the silk protein solution. After mixing, the solid content of silk protein in the solution is 1-10 wt%. Then, place the mixed solution in an environment of -10℃ to -40℃ for 1-48 hours to thaw, and obtain the silk protein microsphere aqueous solution. Concentrate the silk protein microsphere aqueous solution to a solid content of 10-50 wt%.

5. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, In step S4, when using a cellulose-based gel inducer to prepare silk fibroin microsphere gel, the solid content of silk fibroin in the solution is 1-40%, and the solid content of cellulose is 1-40%.

6. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, In step S4, when using an enzyme gel inducer to prepare silk fibroin microsphere gel, the solid content of silk fibroin in the solution is 1-40 wt%, and the enzyme content in the mixed solution is 1-100 U / mL.

7. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, In step S4, when using an enzyme gel inducer to prepare silk fibroin microsphere gel, the solid content of silk fibroin in the solution is 7-15 wt%, and the enzyme content in the mixed solution is 15-75 U / mL.

8. The method for preparing the performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, In step S4, when preparing silk fibroin microsphere gel using lithium saponin as an inducer, the solid content of silk fibroin in the solution is 1-40 wt%, and the concentration of lithium saponin is 1-20 wt%.

9. A method for preparing a performance-tunable injectable microsphere gel of silk fibroin according to any one of claims 1-3, characterized in that, In step S4, when preparing silk fibroin microsphere gel using lithium saponin as an inducer, the solid content of silk fibroin in the solution is 5-15 wt%.

10. An injectable microsphere gel of silk fibroin with tunable performance, characterized in that, It is prepared by any one of claims 1-9.

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