A stable silk fibroin liquid condensate, gel, its preparation method and application

Abstract

This invention relates to a stable silk fibroin liquid aggregate, gel, its preparation method, and its application, belonging to the field of silk fibroin technology. The preparation method of this invention includes the following steps: S1, dissolving silk fibroin in a lithium bromide solution, then adding trypsin for incubation to obtain an enzymatically digested silk fibroin solution; S2, subjecting the enzymatically digested silk fibroin solution to low-temperature programmed centrifugation, including an induction stage, a nucleation stage, and a liquid-liquid phase separation stage, followed by washing to obtain a stable silk fibroin liquid aggregate. This method can reduce aggregate droplet aggregation and improve droplet particle size uniformity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of silk fibroin, and particularly relates to a stable silk fibroin liquid condensate, a gel and a preparation method and application thereof. BACKGROUND

[0002] In recent years, biomolecular condensates, as a newly discovered subcellular structure, have important significance in the formation of membrane-free organelles, intercellular signal transmission, extracellular matrix assembly and disease occurrence. Biomolecular condensates are droplets formed by biological macromolecules such as proteins and nucleic acids through liquid-liquid phase separation (LLPS), also known as liquid condensates. However, these condensates are usually in an unstable state, which can further mature and solidify, complete the transformation from liquid to solid, or cause aggregation; and abnormal solidification and aggregation of liquid condensates are considered to be the cause of Alzheimer's disease, Parkinson's disease, and other diseases.

[0003] Extracellular matrix (ECM) is a complex three-dimensional network structure composed of various proteins and polysaccharides in the extracellular space, which has physiological functions such as providing physical support for cells, transmitting biochemical signals, and regulating cell behavior; elastin, as one of the most important components, has good ductility and elasticity, and can maintain skin elasticity and keep skin young. The sequence contains a highly repetitive VPGVG hydrophobic fragment, which can be assembled into a stable elastic fiber network through liquid-liquid phase separation and covalent cross-linking.

[0004] Silk fibroin is a natural high molecular weight fibrous protein derived from silk, and its molecular structure contains a hydrophobic ordered beta-sheet crystalline region composed of highly repetitive GAGAGS polypeptide fragments, and a hydrophilic disordered non-crystalline region with a high content of hydrophilic amino acids, which can adjust the hydrophobicity of silk fibroin and inhibit the assembly of the beta-sheet crystalline region. This unique structure makes it have excellent mechanical strength, good supportability and controllable water-soluble properties, and it becomes the best ECM elastin mimetic biomacromolecule of natural origin.

[0005] However, research on the preparation of silk fibroin aggregates is currently limited, especially methods for preparing stable liquid silk fibroin aggregates are rarely reported. Chinese invention patent CN118256007A discloses a method for preparing silk fibroin aggregates and their application. This method uses an ionic surfactant to drive liquid-liquid phase separation of the silk fibroin solution through hydrophobic interactions to form silk fibroin aggregates. The aggregates are then solidified to obtain an aggregate gel. However, this method has two shortcomings: first, the ionic surfactant sodium dodecylbenzenesulfonate used has poor biocompatibility and is difficult to remove from the aggregates due to its amphiphilic nature; second, the prepared aggregates are composed of nanodroplets, which have poor droplet stability and are prone to fusion. Chinese invention patent CN116874815A discloses a method for preparing and applying a high-concentration silk fibroin regenerated aqueous solution. The method involves mixing a polyvalent anionic inorganic salt with silk fibroin, and after liquid-liquid phase separation occurs, the mixture is allowed to stand or centrifuged to separate into layers and the supernatant is discarded to obtain a high-concentration silk fibroin regenerated aqueous solution. This method can prepare nanodroplets with diameters of 100nm-1000nm, but it also suffers from the problem of poor stability of liquid condensates and easy fusion between them.

[0006] Therefore, it is urgent to develop a method for preparing stable silk fibroin liquid condensates. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the problems of poor stability and easy droplet fusion of silk fibroin liquid condensates in the prior art.

[0008] To address the aforementioned technical problems, this invention provides a stable silk fibroin liquid condensate, gel, and its preparation method and applications. First, a more stable silk fibroin liquid condensate is prepared using an enzymatic digestion-low-temperature programmed centrifugation method. Then, a physically interlocked enhanced micro-crosslinked nanocage technology is further developed to prepare a stable silk fibroin gel. This technology can prevent the silk fibroin liquid condensate from agglomerating and precipitating during long-term storage and moist heat sterilization. Furthermore, the prepared silk fibroin gel exhibits excellent ECM biomimetic properties, demonstrating promising application potential in cosmetics, drug delivery, and tissue regeneration.

[0009] The first objective of this invention is to provide a method for preparing stable silk fibroin liquid aggregates, comprising the following steps:

[0010] S1. Dissolve silk fibroin in lithium bromide solution, then add trypsin and incubate to obtain enzymatically digested silk fibroin solution;

[0011] S2. The enzyme-digested silk fibroin solution described in S1 is subjected to low-temperature programmed centrifugation, and after washing, the stable silk fibroin liquid aggregate is obtained.

[0012] The low-temperature programmed centrifugation process includes an induction stage, a nucleation stage, and a liquid-liquid phase separation stage.

[0013] The induction phase involves standing at 3℃-5℃ for 1-2 hours. After the silk fibroin is excised by enzymes to remove fragments containing highly hydrophilic amino acids, the hydrophobicity of the molecular chains is enhanced, making it easier to form β-sheets between the chains. Low-temperature incubation can reduce the irregular movement of the silk fibroin molecular chains, thereby inducing the formation of β-sheet sites between the chains and causing physical micro-crosslinking.

[0014] The nucleation stage is carried out at 3℃-5℃, centrifuged at 20rpm-60rpm for 10min-15min. Low-speed centrifugation can bring the silk fibroin molecular chains closer together by applying centrifugal force, promoting the formation of partially dense β-sheet structures between the chains. These sheet structures accumulate, entangle, and nucleate to form microdroplets, causing the solution to become turbid.

[0015] The liquid-liquid phase separation stage is carried out at 14℃-16℃, centrifuged at 200rpm-800rpm for 1min-3min; after high-speed centrifugation, the micro-droplets in the solution will further assemble and aggregate to form larger droplets; because the density of these larger droplets is significantly higher than that of the aqueous phase, liquid-liquid phase separation will occur, and finally silk fibroin liquid aggregates will be formed.

[0016] In one embodiment of the present invention, in S1, the concentration of the lithium bromide solution is 0.1 mol / L-4 mol / L, preferably 0.3 mol / L-2 mol / L;

[0017] The amount of silk fibroin used is 0.5wt%-10wt%, preferably 1wt%-8wt%;

[0018] The mass ratio of trypsin to silk fibroin is 1:(5-20).

[0019] In one embodiment of the present invention, in S1, the incubation is carried out at 25°C-37°C for 24-48 hours.

[0020] A second objective of this invention is to provide a stable silk fibroin liquid condensate prepared by the method described above.

[0021] A third objective of this invention is to provide a method for preparing a stable silk fibroin gel, comprising the following steps:

[0022] S1. Dissolve the stable silk fibroin liquid aggregate in a polysaccharide solution, then add a cross-linking agent to react and obtain a micro-cross-linked silk fibroin gel.

[0023] S2. The micro-crosslinked silk fibroin gel described in S1 is subjected to heating-annealing treatment to obtain the stable silk fibroin gel.

[0024] The heating-annealing process includes a heating stage, a holding stage, and an annealing stage.

[0025] The heating stage involves raising the temperature to 58℃-62℃ at a rate of 0.9℃ / min-1.3℃ / min.

[0026] The maintenance phase involves keeping the temperature at 58℃-62℃ for 12h-24h.

[0027] The annealing stage involves cooling the temperature to 22℃-28℃ at a rate of 0.3℃ / min-0.5℃ / min.

[0028] In one embodiment of the present invention, in S1, the polysaccharide in the polysaccharide solution is selected from one or more of sodium hyaluronate (HA), carboxymethyl cellulose (CMC) and hydroxypropyl methyl cellulose (HPMC), preferably sodium hyaluronate;

[0029] The crosslinking agent is selected from 1,4-butanediol diglycidyl ether (BDDE) and / or diepoxy polyethylene glycol, and the amount of the crosslinking agent is 0.5wt%-1.5wt%.

[0030] In one embodiment of the present invention, in S1, the content of silk fibroin in the micro-crosslinked silk fibroin gel is 0.1wt%-3.0wt%, and the content of polysaccharide is 0.5wt%-2.0wt%.

[0031] In one embodiment of the present invention, in S1, the reaction is carried out at 30°C-40°C for 4-6 hours.

[0032] A fourth object of the present invention is to provide a stable silk fibroin gel prepared by the method described.

[0033] The fifth objective of this invention is to provide an application of the aforementioned stable silk fibroin gel in medical and cosmetic products.

[0034] The technical solution of the present invention has the following advantages compared with the prior art:

[0035] (1) The preparation method described in this invention first uses an enzymatic digestion method to specifically remove hydrophilic disordered non-crystalline regions of silk fibroin to enhance its intermolecular β-sheet and improve cohesion. Then, it promotes liquid-liquid phase separation by low-temperature programmed centrifugation to finally obtain silk fibroin liquid aggregates with higher stability. This method can reduce the aggregation of aggregate droplets and improve the uniformity of droplet size.

[0036] (2) This invention develops a physically interlocked enhanced micro-crosslinked nanocage technology for preparing stable silk fibroin gels. The preparation method is as follows: First, high molecular weight polysaccharides are added to the liquid fibroin aggregates, and then a small amount of crosslinking agent is added to fix the aggregate droplets into the nanocages formed by the crosslinked polysaccharide network to reduce droplet contact and fusion; then, through heating-annealing treatment, the polysaccharide chains are fixed on the surface of the liquid fibroin aggregate droplets by physical interlocking, forming more crosslinking sites, thereby significantly enhancing the mechanical properties of the heterogeneous gel. Attached Figure Description

[0037] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0038] Figure 1 This is a photograph of the solid precipitate formed after the preparation of silk fibroin liquid aggregates in Comparative Example 4 of the present invention.

[0039] Figure 2 These are physical images of the solutions after each stage of the low-temperature programmed centrifugation process in Example 1 of the present invention; wherein, (a) is after the induction stage, (b) is after the nucleation stage, and (c) is after the liquid-liquid phase separation stage;

[0040] Figure 3 This is a photograph of the silk fibroin gel prepared in Example 5 of the present invention.

[0041] Figure 4 This is a SEM image of the silk fibroin gel prepared in Example 5 of this invention after lyophilization.

[0042] Figure 5 This is a SEM image of the silk fibroin gel prepared in Comparative Example 6 of this invention after lyophilization.

[0043] Figure 6 This is a SEM image of the silk fibroin solution prepared in Comparative Example 8 of this invention after lyophilization. Detailed Implementation

[0044] 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.

[0045] In this invention, unless otherwise stated, the trypsin used in the embodiments of this invention was purchased from Aladdin, catalog number T274333.

[0046] In this invention, unless otherwise stated, the CAS number of sodium hyaluronate used in the embodiments of this invention is 9004-61-9.

[0047] In this invention, unless otherwise stated, the CAS number of 1,4-butanediol glycidyl ether used in the embodiments of this invention is 2425-79-8.

[0048] In this invention, unless otherwise stated, the CAS number of the bis-epoxy polyethylene glycol used in the embodiments of this invention is 72207-80-8.

[0049] Example 1

[0050] The silk fibroin liquid condensate and its preparation method in this embodiment specifically include the following steps:

[0051] S1. Dissolve 6g of degummed silk fibroin in 80g of 9.3mol / L lithium bromide aqueous solution, dilute with water to 2mol / L lithium bromide and 1.6wt% silk fibroin, add 1.2g of trypsin, and incubate at 37℃ for 24h to obtain enzymatically digested silk fibroin solution.

[0052] S2. Transfer the enzyme-digested silk fibroin solution to a centrifuge tube and centrifuge it in a low-temperature refrigerated centrifuge. After centrifugation, remove the centrifuge tube, discard the supernatant, add 20 mL of purified water and shake to wash. Centrifuge at 300 rpm for 30 seconds and discard the supernatant. Repeat the washing 3 times to prepare silk fibroin liquid aggregate.

[0053] The low-temperature programmed centrifugation process includes an induction stage, a nucleation stage, and a liquid-liquid phase separation stage.

[0054] The induction phase involves cooling from room temperature to 4°C and then allowing it to stand for 1 hour.

[0055] The nucleation stage was carried out at 4°C and centrifuged at 30 rpm for 10 minutes.

[0056] The liquid-liquid phase separation stage involves heating to 15°C and then centrifuging at 300 rpm for 2 minutes.

[0057] Example 2

[0058] The silk fibroin liquid condensate and its preparation method in this embodiment specifically include the following steps:

[0059] S1. Dissolve 24.8g of degummed silk fibroin in 80g of 9.3mol / L lithium bromide aqueous solution, dilute with water to a lithium bromide concentration of 0.3mol / L and a silk fibroin concentration of 1wt%, then add 1.24g of trypsin and incubate at 37℃ for 24h to obtain an enzymatically digested silk fibroin solution.

[0060] S2. Transfer the enzyme-digested silk fibroin solution to a centrifuge tube and centrifuge it in a low-temperature refrigerated centrifuge. After centrifugation, remove the centrifuge tube, discard the supernatant, add 20 mL of purified water and shake to wash. Centrifuge at 300 rpm for 30 seconds and discard the supernatant. Repeat the washing 3 times to prepare silk fibroin liquid aggregate.

[0061] The low-temperature programmed centrifugation process includes an induction stage, a nucleation stage, and a liquid-liquid phase separation stage.

[0062] The induction phase involves cooling from room temperature to 4°C and then allowing it to stand for 2 hours.

[0063] The nucleation stage was carried out at 4°C and centrifuged at 60 rpm for 15 minutes.

[0064] The liquid-liquid phase separation stage involves heating to 15°C and then centrifuging at 800 rpm for 3 minutes.

[0065] Example 3

[0066] The silk fibroin liquid condensate and its preparation method in this embodiment specifically include the following steps:

[0067] S1. Dissolve 29.76g of degummed silk fibroin in 80g of 9.3mol / L lithium bromide aqueous solution, dilute with water to 2mol / L lithium bromide and 8wt% silk fibroin, add 5.95g of trypsin, and incubate at 37℃ for 24h to obtain enzymatically digested silk fibroin solution.

[0068] S2. Transfer the enzyme-digested silk fibroin solution to a centrifuge tube and centrifuge it in a low-temperature refrigerated centrifuge. After centrifugation, remove the centrifuge tube, discard the supernatant, add 20 mL of purified water and shake to wash. Centrifuge at 300 rpm for 30 seconds and discard the supernatant. Repeat the washing 3 times to prepare silk fibroin liquid aggregate.

[0069] The low-temperature programmed centrifugation process includes an induction stage, a nucleation stage, and a liquid-liquid phase separation stage.

[0070] The induction phase involves cooling from room temperature to 4°C and then allowing it to stand for 1 hour.

[0071] The nucleation stage was carried out at 4°C and centrifuged at 20 rpm for 10 minutes.

[0072] The liquid-liquid phase separation stage involves heating to 15°C and then centrifuging at 200 rpm for 1 min.

[0073] Comparative Example 1

[0074] Take 10 mL of 6% silk fibroin solution into a centrifuge tube, add 10 mL of 0.1 mol / L sodium dodecylbenzenesulfonate solution at a 1:1 volume ratio, shake and mix until the solution becomes turbid to obtain a mixed solution; after the mixed solution is placed at room temperature for 1 min, centrifuge at 300 rpm for 10 s, discard the supernatant, and obtain liquid-liquid phase-separated silk fibroin liquid condensate.

[0075] Comparative Example 2

[0076] The process is basically the same as in Example 2, except that trypsin is not added in S1.

[0077] The results showed that silk fibroin liquid aggregates could not be successfully prepared. This is because trypsin cleaves amorphous fragments containing highly hydrophilic amino acids in silk fibroin, a process that increases the hydrophobicity of the polypeptide chain. Increased hydrophobicity thins the hydration layer on the surface of the silk fibroin polypeptide chain, which inhibits the formation of β-sheet structures. This reduces the energy required to break through the hydration layer to form β-sheet structures, ultimately making silk fibroin more susceptible to induction into forming interchain β-sheet structures, which are crucial for the formation of silk fibroin liquid aggregates.

[0078] Comparative Example 3

[0079] The process is basically the same as in Example 1, except that in S2, no nucleation stage is performed.

[0080] Comparative Example 4

[0081] The process is basically the same as in Example 1, except that in S2, the rotation speed of the liquid-liquid phase separation stage is adjusted to 2500 rpm.

[0082] The results showed that after pouring out the upper clear liquid and the lower liquid condensate, a layer of white solid precipitate appeared at the bottom. Figure 1 This is because high-speed centrifugation during the liquid-liquid phase separation stage can cause some of the silk fibroin liquid aggregates to further aggregate, eventually forming a solid precipitate.

[0083] Test Example 1

[0084] Based on Example 1, the physical images of the solutions after each stage of the low-temperature programmed centrifugation process are as follows: Figure 2 As shown. From Figure 2 It can be seen that the solution is clear after the induction stage. During this stage, the physical micro-crosslinking between silk fibroin chains is promoted by low temperature induction. After low-speed centrifugation induction during the nucleation stage, the silk fibroin molecular chains form some dense coils, and the solution begins to become turbid. After further heating and increasing the centrifugation speed to enter the liquid-liquid phase separation stage, the silk fibroin in the solution undergoes liquid-liquid phase separation, forming silk fibroin liquid aggregates.

[0085] Test Example 2

[0086] The solid content of the silk fibroin liquid aggregates prepared in Examples 1-3 and Comparative Examples 1 and 3 was calculated by the drying method, and the results are shown in Table 1.

[0087] Table 1

[0088]

[0089] As can be seen from Table 1, the solid content of the silk fibroin liquid aggregates prepared in the examples is significantly higher than that in Comparative Example 1. This is because Comparative Example 1 uses sodium dodecylbenzenesulfonate to disrupt the hydration layer on the surface of the silk fibroin molecular chains, forcing them to form β-sheets between the chains. However, the presence of hydrophilic fragments in the silk fibroin fragments results in a loose folded structure, leading to a more dispersed liquid aggregate structure with a lower solid content. In contrast, the present invention first removes the hydrophilic fragments in the silk fibroin using trypsin, and then applies mechanical centrifugal force to shorten the distance between the silk fibroin chains to promote β-sheet formation. The resulting liquid aggregate particles are more compact and have a higher solid content.

[0090] Comparing Example 1 and Comparative Example 3, it can be seen that Comparative Example 3, which omits the nucleation stage in the low-temperature programmed centrifugation process, also ultimately produces liquid condensates, but its solid content is significantly lower than that of Example 1. This is because, inside the silk fibroin liquid condensate droplets formed without induced nucleation, there are significantly more β-sheet point-like cross-linked structures than dense lamellar structures, resulting in lower droplet cross-linking density and higher water content. This indicates that the nucleation stage is of great significance in improving the density of silk fibroin liquid condensates.

[0091] Test Example 3

[0092] The particle size and distribution of the silk fibroin liquid aggregates prepared in Examples 1-3 and Comparative Examples 1 and 3 were determined by a Malvern laser particle size analyzer. The results are shown in Table 2.

[0093] Table 2

[0094]

[0095] As can be seen from Table 2, the particle size of the silk fibroin liquid aggregates prepared in the examples is between 476 nm and 589 nm; while the particle size and Span value of the silk fibroin liquid aggregates prepared in Comparative Examples 1 and 3 are significantly larger than those in the examples, and the particle size is larger and the distribution is uneven.

[0096] Test Example 4

[0097] The silk fibroin liquid aggregates prepared in Examples 1-3 and Comparative Examples 1 and 3 were placed at 60°C for 6 hours, and then their particle size and distribution were detected by a Malvern laser particle size analyzer. The results are shown in Table 3.

[0098] Table 3

[0099]

[0100] As can be seen from Tables 2 and 3, the silk fibroin liquid condensate droplets prepared in the examples exhibit excellent particle size stability after heating. In contrast, the silk fibroin liquid condensates obtained in Comparative Examples 1 and 3 show a significant increase in particle size after heating, and are significantly larger than those in the examples.

[0101] Example 4

[0102] The method for preparing a stable silk fibroin gel in this embodiment specifically includes the following steps:

[0103] S1. Add the silk fibroin liquid aggregate prepared in Example 1 to 50g of sodium hyaluronate solution, stir at 250rpm for 15min using an electronic stirrer, then add 250mg of 1,4-butanediol diglycidyl ether and mix well. Then react the reaction system at 40℃ for 4h to obtain micro-crosslinked gel (the mass fraction of silk fibroin is 3% and the mass fraction of sodium hyaluronate is 0.5%).

[0104] S2. The obtained micro-crosslinked gel is subjected to heating-annealing treatment to obtain silk fibroin gel:

[0105] The heating-annealing process includes a heating stage, a holding stage, and an annealing stage.

[0106] The rapid heating phase involves raising the temperature from room temperature to 60°C at a rate of 1.0°C / min.

[0107] The maintenance phase involves keeping the temperature at 60°C for 24 hours.

[0108] The annealing stage involves cooling from 60℃ to 25℃ at a rate of 0.3℃ / min.

[0109] Example 5

[0110] The method for preparing a stable silk fibroin gel in this embodiment specifically includes the following steps:

[0111] S1. Add the silk fibroin liquid aggregate prepared in Example 1 to 50g sodium hyaluronate solution, stir at 250rpm for 15min using an electronic stirrer, then add 500mg of diepoxy polyethylene glycol and mix evenly. React the reaction system at 40℃ for 4h to obtain micro-crosslinked gel (the mass fraction of silk fibroin is 1% and the mass fraction of sodium hyaluronate is 1.0%).

[0112] S2. The obtained micro-crosslinked gel is subjected to heating-annealing treatment to obtain silk fibroin gel ( Figure 3 ):

[0113] The heating-annealing process includes a heating stage, a holding stage, and an annealing stage.

[0114] The rapid heating phase involves raising the temperature from room temperature to 60°C at a rate of 1.0°C / min.

[0115] The maintenance phase involves keeping the temperature at 60°C for 12 hours.

[0116] The annealing stage involves cooling from 60℃ to 25℃ at a rate of 0.5℃ / min.

[0117] Example 6

[0118] The method for preparing a stable silk fibroin gel in this embodiment specifically includes the following steps:

[0119] S1. Add the silk fibroin liquid aggregate prepared in Example 1 to 50g sodium hyaluronate solution, stir at 250rpm for 15min using an electronic stirrer, then add 750mg of diepoxy polyethylene glycol and mix evenly. React the reaction system at 40℃ for 4h to obtain micro-crosslinked gel (silk fibroin mass fraction of 0.1%, sodium hyaluronate mass fraction of 2.0%).

[0120] S2. The obtained micro-crosslinked gel is subjected to heating-annealing treatment to obtain silk fibroin gel:

[0121] The heating-annealing process includes a heating stage, a holding stage, and an annealing stage.

[0122] The rapid heating phase involves raising the temperature from room temperature to 60°C at a rate of 1.3°C / min.

[0123] The maintenance phase involves keeping the temperature at 60°C for 12 hours.

[0124] The annealing stage involves cooling from 60℃ to 25℃ at a rate of 0.5℃ / min.

[0125] Comparative Example 5

[0126] The basic formula is the same as in Example 5, except that in S1, bis(epoxy) polyethylene glycol is not added.

[0127] The results showed that silk fibroin gel could not be successfully prepared due to the lack of the cross-linking agent diepoxy polyethylene glycol.

[0128] Comparative Example 6

[0129] It is basically the same as Example 5, except that no heating-annealing treatment is performed.

[0130] Comparative Example 7

[0131] The process is basically the same as in Example 5, except that: sodium hyaluronate gel and silk fibroin liquid aggregate are first prepared and then physically mixed before being heated and annealed.

[0132] Comparative Example 8

[0133] The process is basically the same as in Example 5, except that the silk fibroin liquid condensate is directly subjected to heating and annealing treatment.

[0134] Test Example 5

[0135] Based on Examples 4-6 and Comparative Example 7, the rheological characteristics of the gels before and after the heating-annealing treatment were performed, and their elastic moduli are shown in Table 4:

[0136] Table 4

[0137]

[0138] As shown in Table 4, the elastic modulus of the micro-crosslinked gel in the examples was significantly improved after heating and annealing. This is because the micro-crosslinking has fixed the silk fibroin liquid aggregates within the three-dimensional mesh of the polysaccharide backbone. Further heating and annealing allows more β-sheets to form between the exposed silk fibroin chain segments on the aggregate surface, creating new crosslinking points. These crosslinking points lock the adjacent hyaluronic acid chains to the surface of the aggregate particles. This physical interlocking effect between the silk fibroin liquid aggregates and the polysaccharide molecular backbone macroscopically manifests as an increase in the gel's elastic modulus. The higher the content of the silk fibroin liquid aggregates and the backbone polysaccharide, the more significant the percentage increase in modulus.

[0139] Comparing Example 5 and Comparative Example 7, it can be seen that the gel prepared by pre-crosslinking hyaluronic acid into a microgel and then mixing it with silk fibroin liquid aggregates has almost no change in elastic modulus before and after heating and annealing; this result shows that the physical interlocking method used in the examples can effectively enhance the performance of heterogeneous gels.

[0140] Test Example 6

[0141] The products prepared in Examples 5, 6, and 8 were frozen in liquid nitrogen, freeze-dried under vacuum, and their morphology was observed using SEM. The average particle size was then calculated. The results are as follows: Figures 4-6 As shown in Table 5:

[0142] Table 5

[0143]

[0144] from Figures 4-6As shown in Table 5, the average particle size of the silk fibroin gel prepared in the examples did not change much before heating-annealing (Comparative Example 6) and after heating-annealing (Example 5), and was significantly lower than that of Comparative Example 8, which was not stabilized by micro-crosslinked gel. This is because the three-dimensional crosslinked network formed by the polysaccharide and the crosslinking agent first divides the silk fibroin liquid aggregate droplets into multiple "nanocages", reducing the contact and collision between droplets, thereby significantly reducing droplet coalescence and improving stability. Further heating-annealing treatment can form crosslinking sites between the silk fibroin aggregate droplets and the polysaccharide backbone molecules through physical interlocking to enhance the mechanical strength of the heterogeneous gel. Moreover, when this operation is carried out in "nanocages", the effect on the particle size of the aggregate droplets is significantly lower than that in the conventional solution state. This technology is the physically interlocked enhanced micro-crosslinked nanocage technology.

[0145] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a stable liquid aggregate of silk fibroin, characterized by, The method comprises the following steps: S1, dissolving the degummed silk fibroin in a lithium bromide solution, then adding trypsin for incubation to obtain an enzyme-cut silk fibroin solution; S2, performing low-temperature programmed centrifugal treatment on the enzyme-cut silk fibroin solution in S1, and obtaining the stable silk fibroin liquid aggregate through washing; The low-temperature programmed centrifugal treatment comprises an induction stage, a nucleation stage and a liquid-liquid phase separation stage; The induction stage is static standing at 3-5°C for 1-2h; The nucleation stage is centrifugation at 20-60rpm for 10-15min at 3-5°C; The liquid-liquid phase separation stage is centrifugation at 200-800rpm for 1-3min at 14-16°C.

2. The method for preparing stable silk fibroin liquid condensates according to claim 1, characterized in that, In S1, the concentration of the lithium bromide solution is 0.1-4mol / L; The amount of the silk fibroin is 0.5-10wt%; The mass ratio of the trypsin to the silk fibroin is 1: (5-20).

3. The method for preparing stable silk fibroin liquid aggregates according to claim 1, characterized in that, In S1, the incubation is at 25-37°C for 24-48h.

4. A stable silk fibroin liquid aggregate prepared by the method in any one of claims 1-3.

5. A method for preparing a stable silk fibroin gel, characterized by, The method comprises the following steps: S1, dissolving the stable silk fibroin liquid aggregate in claim 4 in a polysaccharide solution, then adding a crosslinking agent for reaction to obtain a micro-crosslinked silk fibroin gel; the polysaccharide in the polysaccharide solution is sodium hyaluronate, and the crosslinking agent is bisepoxy polyethylene glycol; S2, performing heating-annealing treatment on the micro-crosslinked silk fibroin gel in S1 to obtain the stable silk fibroin gel; The heating-annealing treatment comprises a heating stage, a maintaining stage and an annealing stage; The heating stage is heating at a rate of 0.9-1.3°C / min to 58-62°C; The maintaining stage is incubation at 58-62°C for 12-24h; The annealing stage is cooling at a rate of 0.3-0.5°C / min to 22-28°C.

6. The method of claim 5, wherein the stable silk fibroin gel is prepared by the steps of: In S1, the amount of the crosslinking agent is 0.5-1.5wt%.

7. The method of claim 5, wherein the stable silk fibroin gel is prepared by the steps of: In S1, the content of the silk fibroin in the micro-crosslinked silk fibroin gel is 0.1-3.0wt%, and the content of the polysaccharide is 0.5-2.0wt%.

8. The method of claim 5, wherein the stable silk fibroin gel is prepared by the steps of: In S1, the reaction is at 30-40°C for 4-6h.

9. A stable silk fibroin gel prepared by the method in any one of claims 5-8.

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