A controllable β-sheet size silk fibroin aggregate, its preparation method and application

Abstract

This invention discloses a controllable β-sheet size silk fibroin aggregate, its preparation method, and its applications. The preparation method includes: adding trypsin to a regenerated silk fibroin solution to form an enzyme reaction mixture; after the enzymatic reaction, separating the solid and liquid phases and collecting the crystalline precipitate of silk fibroin; dissolving the crystalline precipitate of silk fibroin with a hydrogen bond inhibitor; purifying it by dialysis to obtain a crystalline solution of silk fibroin; and then subjecting it to high-pressure shearing via microfluidic jet to obtain silk fibroin aggregates. This invention utilizes specific enzymatic digestion to regulate the self-assembly behavior of silk fibroin, producing silk fibroin aggregates with a β-sheet size controlled at 1.6-2.0 nm. This aggregate can mimic the natural nanotopology of the extracellular matrix, significantly improving cell adhesion, migration, and proliferation efficiency. Simultaneously, this structure, while ensuring the mechanical stability of the material, can also form channels for delivering cytokines and nutrients, making it suitable for tissue engineering, medical aesthetics, and skincare fields.

Description

Technical Field

[0001] This invention relates to the field of silk fibroin materials technology, specifically to a silk fibroin aggregate with controllable β-sheet size, its preparation method, and its application. Background Technology

[0002] Silk fibroin, as a core component of natural silk, has become a natural biomaterial with great application potential in regenerative medicine, tissue engineering, and functional skincare due to its excellent biocompatibility, biodegradability, and low immunogenicity. Its molecular chain consists of alternating blocks of hydrophobic glycine-alanine-serine repeating sequences forming crystalline regions and hydrophilic random coil amorphous regions. Among them, the β-sheet structure is the core unit that determines the mechanical strength, structural stability, and bioactivity of silk fibroin materials.

[0003] The β-sheet spacing of fibroin in natural silk is approximately 0.7 nm, with tightly packed intermolecular hydrogen bonds, giving silk a tensile strength approaching that of steel wire. However, this natural structure has significant application limitations: firstly, the tightly packed β-sheets are difficult for human tissues to recognize and utilize, with cell adhesion efficiency only 30%-40% that of collagen, failing to meet the needs of tissue repair and regeneration; secondly, traditional physical or chemical induction methods struggle to precisely control the formation process of β-sheets, easily generating aggregates with uneven size distributions, leading to significant differences in mechanical properties and bioactivity between batches, thus limiting its standardized application.

[0004] In the fields of medical aesthetics and skin care, most existing silk fibroin products are mainly in the water-soluble random coil state. This structure is easily lost quickly on the skin surface and is difficult to form a long-lasting repair barrier. β-sheet silk fibroin prepared by conventional methods such as ethanol soaking and heat induction cannot penetrate the stratum corneum of the skin due to its large sheet size. It can only achieve surface physical moisturization and is difficult to exert deep repair and endogenous skin regeneration effects.

[0005] In the field of tissue engineering, the β-sheets of traditional silk fibroin scaffolds are randomly and disordered, failing to mimic the nanoscale ordered topology of the human extracellular matrix (ECM), resulting in low cell adhesion, migration, and proliferation efficiency. Although electrospinning technology can fabricate nanofiber scaffolds, the size of the β-sheets within the fibers still lacks precise control, making it difficult to simultaneously achieve both mechanical support performance and bioactivity of the scaffold, and thus failing to provide an ideal three-dimensional microenvironment for cell growth.

[0006] In summary, existing technologies cannot achieve uniformity and precise control of the β-sheet size of silk fibroin, making it difficult to obtain silk fibroin materials that match the size of human collagen fibrils and possess both high mechanical stability and high bioactivity. This limits their high-end applications in tissue engineering, medical aesthetic repair, and functional skincare. Therefore, developing a method to precisely control the β-sheet size and prepare uniform, stable, and highly bioactive silk fibroin aggregates has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] To address the above technical problems, this invention provides a silk fibroin aggregate with controllable β-sheet size, its preparation method, and its applications. This invention employs a specific enzymatic digestion process to precisely control the self-assembly behavior of silk fibroin, obtaining silk fibroin aggregates with a β-sheet size controlled at 1.6-2.0 nm. This size highly matches the diameter of human collagen fibrils (1.5-2.0 nm), mimicking the natural nanotopology of the extracellular matrix and significantly improving cell adhesion, migration, and proliferation efficiency. Simultaneously, the 1.6-2.0 nm β-sheet size preserves the mechanical stability of silk fibroin while providing a suitable channel for the transport of cytokines and nutrients.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] The first aspect of this invention provides a method for preparing silk fibroin aggregates, comprising the following steps:

[0010] S1. Add trypsin to the regenerated silk fibroin solution to form an enzyme reaction mixture. After the enzymatic hydrolysis reaction is completed, separate the solid and liquid phases and collect the precipitate from the crystalline area of ​​the silk fibroin. The activity of trypsin in the enzyme reaction mixture is 2500-50000 U / mL.

[0011] S2. Dissolve the precipitate of the silk fibroin crystal region with a hydrogen bond inhibitor, and obtain a silk fibroin liquid crystal solution after dialysis purification.

[0012] S3. The silk fibroin liquid crystal solution is subjected to microfluidic high-pressure shearing to obtain the silk fibroin aggregates; the β-sheet size of the silk fibroin aggregates is 1.6-2.0 nm.

[0013] In this invention, trypsin is a specific endopeptidase that can precisely identify and cleave the connecting peptides between the crystalline region and the C-terminal and N-terminal amorphous regions of silk fibroin, avoiding damage to the crystalline region structure by non-specific proteases. If the activity of trypsin is too low, silk fibroin cannot be effectively cleaved, and the crystalline region cannot be obtained; if the activity of trypsin is too high, the precipitation rate is too fast, which can easily cause unreacted peptide chains to co-precipitate in the crystalline region, affecting the purity of the crystalline region.

[0014] This invention assumes that the β-fold structure is a spherical particle model. The scattering signal generated by this structure in the small scattering angle region follows the Guinier law. Therefore, the calculated value of the law is used as the size reference of the β-fold structure.

[0015] Further, in step S1, the concentration of silk fibroin in the regenerated silk fibroin solution is 1-5 wt%.

[0016] Further, in step S1, the preparation method of the regenerated silk fibroin solution includes the following steps: adding silk to sodium carbonate solution and boiling to degumme it, washing it with water, drying it, dissolving it in lithium bromide solution, and dialysis to purify it to obtain the regenerated silk fibroin solution.

[0017] Specifically, the preparation method of the regenerated silk fibroin solution includes the following steps: adding silk to sodium carbonate solution and boiling, removing degummed silk and washing and drying it, then dissolving it in lithium bromide solution and dialysis purification to obtain the regenerated silk fibroin solution.

[0018] Furthermore, in step S1, the temperature of the enzymatic hydrolysis reaction is 35-39 °C, and the time is 20-25 h.

[0019] Furthermore, in step S1, after the enzymatic hydrolysis reaction is completed, a step of inactivating trypsin is also included.

[0020] Furthermore, in step S1, the mass fraction of arginine (R) and lysine (K) in the precipitate of the silk fibroin crystallization zone is no higher than 0.3% of the total amino acids. Arginine and lysine are the cleavage sites of trypsin, and the efficiency of enzymatic cleavage and the purity of the precipitate can be determined by monitoring the content of these two amino acids.

[0021] Further, in step S2, the hydrogen bond inhibitor is selected from one or more of lithium bromide solution, sodium thiocyanate solution, zinc chloride solution, and calcium chloride-ethanol-water ternary solution. The function of the hydrogen bond inhibitor is to disrupt the hydrogen bond network inside the crystalline region of silk fibroin, allowing it to dissolve uniformly and form a solution.

[0022] Furthermore, in step S2, the dialysis purification adopts forward dialysis, reverse dialysis, or tangential flow ultrafiltration purification, with tangential flow ultrafiltration purification being preferred.

[0023] Further, in step S2, the concentration of silk fibroin in the silk fibroin liquid crystal solution is 10-30 wt%. At this concentration, silk fibroin molecules will self-assemble to form a liquid crystal state, which is characterized by highly oriented molecular chains along a certain direction, exhibiting an anisotropic structure.

[0024] Furthermore, in step S3, the pressure of the microjet high-pressure shear is 1000-2000 bar.

[0025] Furthermore, in step S3, the flow rate of the microjet high-pressure shear is 40-100 mL / min.

[0026] Further, in step S3, the diameter of the high-shear zone channel in the microjets is 50-200 μm. When the anisotropic liquid crystal silk fibroin solution passes through the high-shear zone of the microjets, the drastically reduced inner diameter causes the silk fibroin molecules to adopt an extended conformation and align along the flow direction. Subsequently, the silk fibroin molecules further approach each other and combine through hydrophobic interactions and hydrogen bonds, transforming into a β-sheet structure, thereby achieving phase separation.

[0027] Furthermore, in step S3, the sequence of the silk fibroin aggregate comprises 4461 amino acid residues.

[0028] The second aspect of the present invention provides a silk fibroin aggregate prepared by the preparation method described in the first aspect.

[0029] The third aspect of this invention provides the application of the silk fibroin aggregate described in the second aspect in the preparation of tissue engineering scaffolds, medical aesthetic repair agents, or functional skin care products.

[0030] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0031] 1. This invention induces silk fibroin molecules to assemble in an orderly manner into aggregates with a specific β-sheet size through specific enzymatic digestion technology, silk fibroin self-assembly, and microfluidic high-pressure shearing technology. This achieves uniformity and precise control of the β-sheet size, and solves the technical problems of random distribution of β-sheet size and significant differences in mechanical properties and biological activity between batches in conventional physical / chemical induction methods.

[0032] 2. This invention utilizes the site-specific enzymatic cleavage of trypsin to selectively remove the amorphous segments at both ends of the silk fibroin molecular chain, maximizing the preservation of the crystalline core structure with high β-sheet content. This allows the material to degrade slowly and controllably in vivo, providing long-term mechanical support for the tissue regeneration process.

[0033] 3. The β-sheet size of the silk fibroin aggregates provided by this invention is 1.6-2.0 nm, which is highly matched with the diameter of human collagen fibrils (1.5-2.0 nm). It can biomimetically simulate the natural nanotopological structure of the extracellular matrix, significantly improve the efficiency of cell adhesion, migration and proliferation, and at the same time provide suitable molecular channels for the transport of cytokines and nutrients. Attached Figure Description

[0034] Figure 1 This is a photograph of the silk fibroin aggregates prepared in Example 1.

[0035] Figure 2 The images are scanning electron microscope (SEM) images of silk fibroin aggregates prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3. Detailed Implementation

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] This invention provides a method for preparing silk fibroin aggregates, comprising the following steps:

[0038] S1. Add trypsin to the regenerated silk fibroin solution to form an enzyme reaction mixture. After the enzymatic hydrolysis reaction is completed, separate the solid and liquid phases and collect the precipitate from the crystalline area of ​​the silk fibroin. The activity of trypsin in the enzyme reaction mixture is 2500-50000 U / mL.

[0039] S2. Dissolve the precipitate of the silk fibroin crystal region with a hydrogen bond inhibitor, and obtain a silk fibroin liquid crystal solution after dialysis purification.

[0040] S3. The silk fibroin liquid crystal solution is subjected to microfluidic high-pressure shearing to obtain the silk fibroin aggregates; the β-sheet size of the silk fibroin aggregates is 1.6-2.0 nm.

[0041] This invention utilizes specific enzymatic digestion to obtain crystalline precipitates of silk fibroin. After reconstitution and purification, the high-concentration liquid crystal silk fibroin undergoes anisotropic self-assembly. Microfluidic high-pressure shearing technology is employed to induce the formation of aggregates with specific β-sheet sizes, overcoming the problems of random β-sheet size distribution and low bioactivity in traditional methods. This technology maximizes the preservation of the β-sheet structure of silk fibroin, which is a key factor in its excellent mechanical properties. Therefore, it enables precise control of the mechanical properties and bioactivity of silk fibroin materials, providing a new technical pathway for the preparation of high-performance tissue engineering scaffolds, medical aesthetic repair agents, and functional skincare products.

[0042] The technical principle of this invention is as follows: Silk fibroin is composed of 18 amino acid residues, and its molecular structure consists of highly ordered crystalline regions and loosely ordered amorphous regions. The highly repeating hexapeptide amino acid repeating units (GAGAGS) form an antiparallel β-sheet structure, which is the main component of the crystalline region and a major factor contributing to the excellent mechanical properties of silk fibroin. Trypsin is a specific endopeptidase with arginine and lysine residues as cleavage sites. By specifically digesting the regenerated silk fibroin solution with trypsin, the connecting peptides between the crystalline region and the C-terminal and N-terminal amorphous regions of silk fibroin can be precisely identified and cleaved, thereby obtaining a crystalline precipitate rich in highly repeating GAGAGS fragments with a high β-sheet content, providing a structural basis for the subsequent liquid crystal state.

[0043] The enzymatically digested silk fibroin crystals precipitate is isotropic, with randomly distributed β-sheet sizes. The precipitate is reconstituted with a hydrogen bond inhibitor and purified by dialysis to obtain a high-concentration silk fibroin solution. At this high concentration, the silk fibroin exhibits a "prefolded" liquid crystal state, displaying an anisotropic structure with highly oriented molecular chains along a specific direction.

[0044] Anisotropic silk fibroin liquid crystal solutions are subjected to high-pressure shearing via microfluidics. The structure of the high-shear zone in the microfluidic device, which drastically reduces the inner diameter, causes the silk fibroin molecules to adopt an extended conformation and align along the flow direction. The compressed silk fibroin molecules are brought closer together and bonded through hydrophobic interactions and hydrogen bonds, transforming into a β-sheet structure. This achieves phase separation and size homogenization, resulting in silk fibroin aggregates with specific β-sheet sizes.

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0047] In the examples described below, sodium carbonate (Na2CO3) was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10019260; lithium bromide (LiBr) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number L108934; sodium thiocyanate (NaSCN) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S140873; calcium chloride (CaCl2) was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number JJ2974; ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 100092683; and trypsin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number T274333.

[0048] Example 1

[0049] A method for preparing silk fibroin aggregates includes the following steps:

[0050] S1. After boiling the silk in a 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in a 9.3 M lithium bromide solution, and purified by dialysis to obtain a 5 wt% regenerated silk fibroin solution. Trypsin was added to the regenerated silk fibroin solution to form an enzyme reaction mixture with a trypsin activity of 5000 U / mL. The enzyme reaction was carried out at 37 ℃ for 24 h, and the enzyme was inactivated at 95 ℃ for 2 h. After solid-liquid separation, the crystalline precipitate of silk fibroin was collected by centrifugation at 8000 rpm for 10 min.

[0051] S2. Dissolve the precipitated silk fibroin crystals in 9.3 M lithium bromide solution. Following the method described in patent CN202110171751X, purify the fibroin using a tangential flow ultrafiltration membrane with a regenerated cellulose membrane, controlling the tangential flow rate at 5.0 L / min / m. 2 The transmembrane pressure was 1 bar, forming a 10 wt% silk fibroin liquid crystal solution.

[0052] S3. The silk fibroin liquid crystal solution was subjected to micro-jet high-pressure shearing treatment at a pressure of 1500 bar, a flow rate of 40 mL / min, and a channel diameter of 50 μm in the high-shear region to obtain silk fibroin aggregates with a β-sheet size of 1.8 nm, denoted as Favorsun-01.

[0053] Example 2

[0054] A method for preparing silk fibroin aggregates includes the following steps:

[0055] S1. Add silk to a 0.5 g / L Na2CO3 solution and boil for 1 h. Remove the degummed silk, wash and dry it, dissolve it in a 9.3 M lithium bromide solution, and dialysis to purify it, obtaining a 1 wt% regenerated silk fibroin solution. Add trypsin to the regenerated silk fibroin solution to form an enzyme reaction mixture with a trypsin activity of 2500 U / mL. Perform enzymatic hydrolysis at 37 ℃ for 24 h, inactivate the enzyme at 95 ℃ for 2 h, and then centrifuge at 10000 rpm for 5 min to separate the solid and liquid components, collecting the crystalline precipitate of silk fibroin.

[0056] S2. The precipitate of the silk fibroin crystal zone is dissolved in a ternary system solution of calcium chloride-ethanol-water (molar ratio 1:2:8), and purified by positive dialysis with pure water to form a silk fibroin liquid crystal solution with a concentration of 30 wt%.

[0057] S3. The silk fibroin liquid crystal solution was subjected to micro-jet high-pressure shearing treatment at a pressure of 1000 bar, a flow rate of 80 mL / min, and a channel diameter of 100 μm in the high-shear region to obtain silk fibroin aggregates with a β-sheet size of 1.6 nm, denoted as Favorsun-02.

[0058] Example 3

[0059] A method for preparing silk fibroin aggregates includes the following steps:

[0060] S1. After boiling the silk in a 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in a 9.3 M lithium bromide solution, and purified by dialysis to obtain a 4 wt% regenerated silk fibroin solution. Trypsin was added to the regenerated silk fibroin solution to form an enzyme reaction mixture with a trypsin activity of 50000 U / mL. The enzyme reaction was carried out at 37 ℃ for 24 h, and the enzyme was inactivated at 95 ℃ for 2 h. After solid-liquid separation, the crystalline precipitate of silk fibroin was collected by centrifugation at 5000 rpm for 10 min.

[0061] S2. The precipitate of the silk fibroin crystal zone was dissolved in 6 mol / L sodium thiocyanate solution, and purified by reverse dialysis with polyethylene glycol to form a 20 wt% silk fibroin liquid crystal solution.

[0062] S3. The silk fibroin liquid crystal solution was subjected to micro-jet high-pressure shearing treatment at a pressure of 2000 bar, a flow rate of 100 mL / min, and a channel diameter of 200 μm in the high-shear region to obtain silk fibroin aggregates with a β-sheet size of 2.0 nm, denoted as Favorsun-03.

[0063] Comparative Example 1

[0064] A method for preparing silk fibroin aggregates includes the following steps:

[0065] Silk was boiled in 0.5 g / L Na2CO3 solution for 1 h, then degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 5 wt% regenerated silk fibroin solution. The regenerated silk fibroin solution was subjected to microfluidic high-pressure shearing treatment at a pressure of 1500 bar, a flow rate of 40 mL / min, and a channel diameter of 50 μm in the high-shear zone to obtain silk fibroin aggregates.

[0066] Comparative Example 2

[0067] A method for preparing silk fibroin aggregates includes the following steps:

[0068] S1. After boiling the silk in a 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in a 9.3 M lithium bromide solution, and purified by dialysis to obtain a 5 wt% regenerated silk fibroin solution. Trypsin was added to the regenerated silk fibroin solution to form an enzyme reaction mixture with a trypsin activity of 5000 U / mL. The enzyme reaction was carried out at 37 ℃ for 24 h, and the enzyme was inactivated at 95 ℃ for 2 h. After solid-liquid separation, the crystalline precipitate of silk fibroin was collected by centrifugation at 8000 rpm for 10 min.

[0069] S2. After dispersing the precipitate of the silk fibroin crystal zone with pure water, micro-jet high-pressure shearing treatment was carried out at a pressure of 1500 bar, a flow rate of 40 mL / min, and a channel diameter of 50 μm in the high-shear zone to obtain silk fibroin aggregates.

[0070] Comparative Example 3

[0071] A method for preparing silk fibroin aggregates includes the following steps:

[0072] Silk was boiled in 0.5 g / L Na2CO3 solution for 1 h, then degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 5 wt% regenerated silk fibroin solution. Trypsin was added to the regenerated silk fibroin solution to form an enzyme reaction mixture with an activity of 5000 U / mL. The enzyme reaction was carried out at 37 ℃ for 24 h, followed by enzyme inactivation at 95 ℃ for 2 h. After solid-liquid separation, the mixture was centrifuged at 8000 rpm for 10 min to obtain the crystalline precipitate of silk fibroin, which is the silk fibroin aggregate of Comparative Example 3.

[0073] Test Example 1

[0074] The performance of the silk fibroin aggregates prepared in Examples 1-3 and Comparative Examples 1-3 was characterized and tested. The specific test items and methods are as follows:

[0075] (1) Amino acid content test: According to GB / T 32016-2015 "Determination of Amino Acids in Silk", an amino acid analyzer was used for detection. The sample was diluted with concentrated hydrochloric acid and placed in a sealed tube, hydrolyzed at 110 ℃ for 22 h, and then reconstituted in 0.01 M hydrochloric acid solution after nitrogen blowing treatment. After filtration, the sample was tested, and the mass percentage of arginine and lysine in the total amino acids was calculated.

[0076] (2) β-sheet size determination: The β-sheet size of the samples was determined according to Chinese Patent CN119741964A, "A method for quantitative analysis of β-sheet structure in silk fibroin materials based on machine learning". Each group of samples was prepared and tested in parallel three times to verify the stability of the preparation method and the accuracy of β-sheet size control.

[0077] (3) Amino acid sequence analysis: The amino acid sequence was determined and analyzed according to the 0431 mass spectrometry method in the 2025 edition of the Chinese Pharmacopoeia.

[0078] The test results are shown in Table 1:

[0079] Table 1

[0080]

[0081] Trypsin is a specific endonuclease with arginine and lysine sites. Under the action of trypsin, the C-terminus and N-terminus of the whole-chain silk fibroin are cleaved, dissolving in the supernatant, and a highly β-sheeted crystalline region with a sequence of 4461 amino acids precipitates. Because arginine and lysine are cleaved, their content as a percentage of the total amino acids is no higher than 0.3% (Examples 1-3 and Comparative Examples 2 and 3). Comparative Example 1, which was not treated with trypsin, has a whole-chain length of 5263 amino acids containing both amorphous and crystalline regions, and the content of arginine and lysine as a percentage of the total amino acids is much greater than 0.3% (1.24%).

[0082] Figure 1 The image shows the actual silk fibroin aggregates prepared in Example 1. The silk fibroin aggregates are uniformly dispersed in the solution, appearing as a milky white gel. Figure 2 As can be seen from the SEM image of Example 1, the silk fibroin aggregates are basically uniformly spherical. The silk fibroin aggregates with specific β-sheet sizes prepared in Examples 1-3 have β-sheet sizes that are stably in the range of 1.6-2.0 nm. The relative standard deviation (RSD) of the β-sheet size in three repeated sample measurements is less than 5%, which reflects the uniformity of the β-sheet size and the precision of process control.

[0083] The regenerated silk fibroin solution in Comparative Example 1 was subjected to microfluidic high-pressure shearing without trypsin treatment, and its SEM image showed microfibers of varying sizes. Regenerated silk fibroin includes amorphous regions at the C- and N-terminuses and a crystalline region in the middle. During microfluidic high-pressure shearing, not only was the transition of anisotropic prefolding in a liquid crystal state lacked, but disordered physical cross-linking also occurred between the amorphous and crystalline regions. Specifically, the silk fibroin structure exhibited isotropic characteristics. The relative standard deviation (RSD) of the β-sheet size measured in three replicates was 23.7%, far exceeding 5%, indicating a random distribution of β-sheet size and a severe lack of precision and uniformity in control.

[0084] The silk fibroin crystalline precipitate of Comparative Example 2, after being appropriately dispersed in pure water, was directly subjected to high-pressure shearing via microjets. Its SEM image showed irregularly structured microfibers. Although the silk fibroin crystalline precipitate did not include the amorphous regions at the C- and N-termini, the precipitate itself still contained a certain degree of isotropic structure. Lacking the prefolding mechanism for self-assembly to form a liquid crystal state, directly subjecting the silk fibroin crystalline precipitate to high-pressure shearing did not induce hydrogen bond dissociation or rearrangement; it was essentially a physical-mechanical process. Therefore, the relative standard deviation (RSD) of the β-sheet size in three replicate measurements was 40.6%, far exceeding 5%, indicating that the β-sheet size still exhibited a random distribution, lacking precise control and uniformity.

[0085] Similar to Comparative Example 2, Comparative Example 3 did not undergo prefolding in the liquid crystal state or microfluidic high-pressure shearing. Its SEM images showed irregularly structured microfibers. Due to the lack of mechanical fragmentation by microfluidics, its size was even less uniform and regular. The relative standard deviation (RSD) of the β-sheet size measured in three replicates was 34.6%, much greater than 5%, indicating that the β-sheet size still exhibited a random distribution, lacking precision and uniformity in control.

[0086] Test Example 2

[0087] The silk fibroin aggregates prepared in Examples 1-3 and Comparative Examples 1-3 were freeze-dried under vacuum (vacuum degree 20 Pa, temperature -60 ℃, time 24 h) to prepare porous scaffold powder. The powder was dispersed in serum-containing culture medium to prepare a suspension with a concentration of 5 mg / mL. L929 fibroblasts were then inoculated and the cell seeding density was adjusted to 10-1. 4 / well, with a positive control group (dimethyl sulfoxide) and a negative control group (physiological saline) set up simultaneously. Cell proliferation toxicity assay (CCK-8 assay) was used. After continuous culture at 37 ℃ and 5% CO2 for 3 days, 10 μL of CCK-8 solution was added directly to each well, and the cells were incubated at 37 ℃ in the dark for 4 h. OD values ​​were read at 450 nm using a microplate reader, and the cell proliferation level of each group was calculated.

[0088] The test results are shown in Table 2:

[0089] Table 2 Results of cell proliferation rate experiment

[0090]

[0091] The cell proliferation rate was 5.3% in the positive control group and 100% in the negative control group, demonstrating the stability of the experimental system and the reliability of the detection results. The silk fibroin aggregates with a β-sheet size of 1.6-2.0 nm prepared in Examples 1-3 exhibited the highest cell proliferation rate of 156%, demonstrating excellent cell proliferation-promoting effects. This is because the β-sheet size of the silk fibroin aggregates in Examples 1-3 highly matches the diameter (1.5-2.0 nm) of human collagen fibrils, mimicking the natural nanotopology of the extracellular matrix, providing contact guidance, promoting cell extension, and activating focal adhesion kinase and extracellular regulated protein kinase / mitogen-activated protein kinase signaling pathways, thus promoting cell spreading and proliferation. Simultaneously, amino acids themselves provide energy to the cells as nutrients. In contrast, the silk fibroin aggregates prepared in Comparative Examples 1-3 not only had a larger β-sheet size but also exhibited a random distribution, resulting in a lower specific surface area, which is unfavorable for cell extension, and therefore their proliferation effect was not as good as that of Examples 1-3.

[0092] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing silk fibroin aggregates, characterized in that, Includes the following steps: S1. Add trypsin to the regenerated silk fibroin solution to form an enzyme reaction mixture. After the enzymatic hydrolysis reaction is completed, separate the solid and liquid phases and collect the precipitate of the silk fibroin crystal zone. The activity of trypsin in the enzyme reaction mixture is 2500-50000 U / mL. The temperature of the enzymatic hydrolysis reaction is 35-39 ℃ and the time is 20-25 h. S2. Dissolve the precipitate of the silk fibroin crystal region with a hydrogen bond inhibitor, and obtain a silk fibroin liquid crystal solution after dialysis purification. S3. The silk fibroin liquid crystal solution is subjected to microfluidic high-pressure shearing to obtain the silk fibroin aggregates; the β-sheet size of the silk fibroin aggregates is 1.6-2.0 nm; the pressure of the microfluidic high-pressure shearing is 1000-2000 bar; and the flow rate of the microfluidic high-pressure shearing is 40-100 mL / min.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of silk fibroin in the regenerated silk fibroin solution is 1-5 wt%.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass fraction of arginine and lysine in the precipitate of the silk fibroin crystal zone is no higher than 0.3% of the total amino acids.

4. The preparation method according to claim 1, characterized in that, In step S2, the hydrogen bond inhibitor is selected from one or more of lithium bromide solution, sodium thiocyanate solution, zinc chloride solution, and calcium chloride-ethanol-water ternary solution; the dialysis purification adopts forward dialysis, reverse dialysis, or tangential flow ultrafiltration purification.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of silk fibroin in the silk fibroin liquid crystal solution is 10-30 wt%.

6. The preparation method according to claim 1, characterized in that, In step S3, the diameter of the high-shear zone channel of the microjet high-pressure shear is 50-200 μm.

7. A silk fibroin aggregate prepared by the preparation method according to any one of claims 1-6.

8. The use of the silk fibroin aggregate according to claim 7 in the preparation of tissue engineering scaffolds, medical aesthetic repair agents or functional skin care products.

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