Preparation method of silk fibroin / sodium hyaluronate cross-linked gel particles

Abstract

The present invention discloses a method for preparing silk fibroin / sodium hyaluronate cross-linked gel particles, wherein the gel is cross-linked between silk fibroin, silk fibroin, and sodium hyaluronate macromolecules by a diglycidyl ether cross-linking agent under neutral conditions. The silk fibroin solution is subjected to a heat treatment-quick freezing process, and the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is subjected to a quick freezing, repeated thawing-freezing treatment, and freeze-drying process. This not only effectively cross-links the macromolecules inside the gel, significantly enhances the ability to resist biodegradation, but also improves the cell compatibility of the gel particles. The gel particles can be used for soft tissue filling and guided soft tissue regeneration in the medical and plastic surgery fields.

Description

Technical Field

[0001] The present invention relates to a method for preparing a polymer gel for medical plastic surgery filling, and in particular to a method for preparing silk fibroin / sodium hyaluronate cross-linked gel particles. Background Art

[0002] Sodium hyaluronate gel has been widely used for cosmetic skin augmentation on the face and other parts of the body. Its advantages include strong moisture retention, good biocompatibility, and complete biodegradability. It effectively reduces wrinkles, improves skin appearance and structure, and alleviates aging of the intradermal and subcutaneous tissues. However, sodium hyaluronate gel biodegrades rapidly in the body and cannot regenerate lost intradermal or subcutaneous soft tissue.

[0003] Silk fibroin has excellent biocompatibility, can guide tissue regeneration, and has a relatively slow biodegradation rate. Silk surgical sutures have been widely used clinically for decades. Gels, porous sponges, and three-dimensional scaffolds prepared with silk fibroin have demonstrated strong adhesion to tissue repair cells and the ability to guide tissue regeneration before degradation. Silk fibroin / sodium hyaluronate composite gels, prepared by blending silk fibroin with sodium hyaluronate, are used for intradermal and subcutaneous soft tissue augmentation, leveraging the advantages of silk fibroin to offset the shortcomings of sodium hyaluronate gels.

[0004] Cross-linking sodium hyaluronate is a key technical challenge in preparing sodium hyaluronate gels for soft tissue augmentation. Common cross-linking agents used for sodium hyaluronate cross-linking include 1,4-butanediol diglycidyl ether, divinyl sulfone, and carbodiimide. Currently, most sodium hyaluronate gel products on the market use 1,4-butanediol diglycidyl ether (BDDE), which offers the advantages of high cross-linking efficiency and low toxicity of the cross-linked products. However, BDDE cross-linking of sodium hyaluronate typically requires alkaline or acidic conditions. Under alkaline conditions, silk fibroin is easily hydrolyzed into peptides or free amino acids. Under weakly acidic conditions, silk fibroin tends to aggregate and precipitate due to its low net surface charge. Under strongly acidic conditions, silk fibroin is easily hydrolyzed into peptides or free amino acids. Therefore, developing new cross-linking technologies is crucial for preparing silk fibroin / sodium hyaluronate composite gels.

[0005] Prior to the present invention, patent CN 102836465 A prepared hyaluronate with a 1% sodium hydroxide aqueous solution to prepare a 0.1 g / mL hyaluronate solution. Silk fibroin microparticles were then added to the hyaluronate solution, followed by the addition of one or more cross-linking agents, including divinyl sulfone, ethylene glycol diglycidyl ether, butanediol diglycidyl ether, or polypropylene glycol diglycidyl ether. The mixture was then allowed to stand at 25-60°C for 2-8 hours to produce a silk fibroin / hyaluronic acid composite gel. The strongly alkaline reaction environment used in this method will cause significant hydrolysis of the silk fibroin, making it difficult for the silk fibroin inside the gel to achieve the desired effect.

[0006] Patent CN 110527116 A first grafts hyaluronic acid with tyramine, then uses laccase to crosslink silk fibroin and the tyramine-grafted hyaluronic acid to produce a silk / hyaluronic acid composite hydrogel. Patent CN114601958 A first modifies aldehydated hyaluronic acid with dopamine, then mixes it with silk fibroin and gelatin for a cross-linking reaction, while simultaneously utilizing the coordination of trivalent iron ions to prepare a hyaluronic acid / silk fibroin double-crosslinked injectable hydrogel. These methods all require chemical modification of silk fibroin or hyaluronic acid with various chemical reagents. The numerous chemical reagents and chemical reaction steps used in the gel preparation process increase the risk of cytotoxicity of the resulting gel due to the use of multiple chemical reagents and the generation and residue of chemical reaction byproducts. On the other hand, because silk fibroin has a tendency to spontaneously aggregate and agglomerate in aqueous solution, it is difficult for the macromolecular chains to fully extend. Directly mixing the silk fibroin aqueous solution with the hyaluronic acid aqueous solution tends to cause the silk fibroin to aggregate and embed in the gaps of the hyaluronic acid macromolecular network, making it difficult to effectively form an interpenetrating network or double network structure between the two macromolecular components. When such a gel is implanted in the body, when the hyaluronic acid network is degraded, the gel as a whole tends to collapse, and its ability to resist biodegradation is insufficient. Summary of the Invention

[0007] In view of the shortcomings of the existing silk fibroin / sodium hyaluronate (or hyaluronic acid) composite gel preparation technology, the purpose of the present invention is to provide a method for preparing silk fibroin / sodium hyaluronate cross-linked gel particles.

[0008] The technical solution adopted by the present invention to solve the above problems is: a method for preparing silk fibroin / sodium hyaluronate cross-linked gel particles, which comprises degumming, dissolving and dialyzing silk to obtain a silk fibroin aqueous solution. The method is characterized in that the following steps are further performed:

[0009] P1. Heat the silk fibroin aqueous solution for a certain period of time and then rapidly freeze it to obtain a frozen silk fibroin body;

[0010] P2. After thawing the frozen silk fibroin, a denatured silk fibroin aqueous solution was obtained;

[0011] P3. The thawed denatured silk fibroin aqueous solution was immediately mixed with a sodium hyaluronate solution, and a diglycidyl ether cross-linking agent was added to the solution to obtain a silk fibroin / sodium hyaluronate / cross-linking agent mixed solution;

[0012] P4. Rapidly freezing the silk fibroin / sodium hyaluronate / crosslinker mixed solution to obtain a frozen mixed solution;

[0013] P5. The frozen silk fibroin / sodium hyaluronate / crosslinker mixed solution was placed in a melting temperature environment for a certain period of time, and then rapidly frozen. After repeating the thawing-freezing process several times, a frozen silk fibroin / sodium hyaluronate complex was obtained;

[0014] P6. freeze-drying the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid;

[0015] P7. The silk fibroin / sodium hyaluronate complex solid was frozen and granulated, and sieved to obtain silk fibroin / sodium hyaluronate complex solid particles;

[0016] P8. Immersing the silk fibroin / sodium hyaluronate composite solid particles in hot water for a certain period of time to obtain a crude silk fibroin / sodium hyaluronate cross-linked gel particle product;

[0017] P9. The crude silk fibroin / sodium hyaluronate cross-linked gel product was first washed with water at room temperature, then balanced with phosphate-buffered saline, and excess liquid was filtered out to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0018] Preferably, in step P1, the temperature of the silk fibroin aqueous solution during heating treatment is 95-100° C., and the treatment time is 10-120 minutes; the rapid freezing temperature is -196--20° C., and the freezing time is 2-6 hours.

[0019] Preferably, in step P2, the thawing temperature is 2-25°C.

[0020] Preferably, in step P3, the average molecular weight of the sodium hyaluronate used is 500-3000 kDa; the mass ratio of silk fibroin / sodium hyaluronate in the mixed solution is 90 / 10-10 / 90, and the total mass fraction of silk fibroin and sodium hyaluronate in the mixed solution is 0.5-2.5%; the diglycidyl ether cross-linking agent is one or a combination of two of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, and neopentyl glycol diglycidyl ether; the ratio of the added diglycidyl ether cross-linking agent to the total mass of silk fibroin and sodium hyaluronate in the mixed solution is 0.1 / 1.0-0.4 / 1.0.

[0021] Preferably, in step P4, the temperature of rapidly freezing the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is -196 to -20°C, and the freezing time is 2 to 6 hours.

[0022] Preferably, in step P5, the melting temperature is 2-8° C., the storage time at the melting temperature is 0.5-2.0 hours, and the number of repeated thawing-freezing times is 2-5 times.

[0023] Preferably, in step P7, the particle size of the silk fibroin / sodium hyaluronate complex solid particles is 50 to 500 μm.

[0024] Preferably, in step P8, the temperature of the hot water used to treat the silk fibroin / sodium hyaluronate composite solid particles is 40-60° C., the treatment time is 12-24 hours, and the hot water is replaced every 3 hours or so.

[0025] The principle of the present invention is:

[0026] (1) The silk fibroin solution is subjected to a heating treatment-rapid freezing process to disassemble the interactions between the silk fibroin macromolecules. More importantly, the secondary bonds such as hydrogen bonds, hydrophobic bonds, and ionic bonds within the silk fibroin macromolecules are disassembled, allowing the silk fibroin macromolecule chains to fully stretch. Subsequently, rapid freezing and fixation are performed to prevent the silk fibroin from quickly returning to its original agglomerated state. When this denatured silk fibroin is immediately blended with sodium hyaluronate after thawing, the number of interaction sites between the silk fibroin and sodium hyaluronate increases, enabling a homogeneous composite between the two and forming an interpenetrating network structure of the two macromolecules.

[0027] (2) By rapidly freezing the silk fibroin / sodium hyaluronate / crosslinker mixed solution and then repeatedly thawing and freezing, the action of ice crystals and the driving force of chemical potential shorten the distance between the solute molecules in the system. On the other hand, the chemically active groups on the side chains of silk fibroin and sodium hyaluronate are exposed, inducing the crosslinking reaction between the diglycidyl ether crosslinker and the silk fibroin and sodium hyaluronate macromolecules. The subsequent freeze-drying process further enhances the crosslinking reaction. Therefore, the crosslinking reaction of the diglycidyl ether crosslinker with silk fibroin and sodium hyaluronate can be achieved under neutral conditions without the need for alkaline or acidic conditions, thereby preparing a silk fibroin / sodium hyaluronate crosslinked gel.

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] (1) Due to the heating treatment-rapid freezing process of the silk fibroin solution, the silk fibroin macromolecular chain is fully stretched, and when mixed with sodium hyaluronate, the interaction sites between the two increase to form an interpenetrating network structure, which prevents the silk fibroin from being embedded in the gaps of the sodium hyaluronate macromolecular network in the form of aggregates. After the hyaluronic acid component in the prepared silk fibroin / sodium hyaluronate gel is degraded, the gel as a whole is not easy to collapse, and the ability to resist biodegradation is enhanced.

[0030] (2) Due to the rapid freezing, repeated thawing-freezing and freeze-drying of the silk fibroin / sodium hyaluronate / cross-linker mixed solution, the cross-linking reaction of the diglycidyl ether cross-linker on the silk fibroin and sodium hyaluronate can be achieved under neutral conditions, avoiding the hydrolysis and destruction of the silk fibroin under alkaline or acidic conditions, so that the silk fibroin component in the silk fibroin / sodium hyaluronate gel can continue to play the role of resisting biodegradation and guiding soft tissue regeneration.

[0031] (3) Instead of using chemical means, physical means are used to induce and enhance the cross-linking reaction of diglycidyl ether cross-linkers on silk fibroin and sodium hyaluronate macromolecules. No other chemical reagents need to be added, which can effectively reduce the cytotoxicity of the gel.

[0032] (4) Diglycidyl ether cross-linkers can not only cross-link silk fibroin and sodium hyaluronate macromolecules, but also cross-link silk fibroin macromolecules and sodium hyaluronate macromolecules with high cross-linking efficiency, which is beneficial to further delay the biodegradation of silk fibroin / sodium hyaluronate gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the preparation process of silk fibroin / sodium hyaluronate cross-linked gel particles of the present invention.

[0034] Figure 2 Schematic diagram of the effect of heating-rapid freezing treatment on the spatial structure of silk fibroin in aqueous solution.

[0035] Figure 3 Schematic diagram of the macromolecular network structure within the silk fibroin / sodium hyaluronate cross-linked gel particles of the present invention.

[0036] Figure 4 is the weight loss rate of the silk fibroin / sodium hyaluronate complex solid in PBS.

[0037] Figure 5 is the biodegradation rate of the silk fibroin / sodium hyaluronate cross-linked gel particles of the present invention.

[0038] Figure 6 These are the in vitro cytotoxicity test results of the silk fibroin / sodium hyaluronate cross-linked gel particles of the present invention. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings.

[0040] The preparation process of the silk fibroin / sodium hyaluronate cross-linked gel particles of the present invention is as follows: Figure 1 shown.

[0041] Example 1

[0042] A1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolve the degummed silk in a ternary solvent of calcium chloride / ethanol / water (molar ratio of 1 / 2 / 8). Dialysis yields a silk fibroin aqueous solution. Adjust the mass fraction of the silk fibroin solution to 1.5% with purified water.

[0043] A2. Place 200 mL of silk fibroin solution in a flask and heat in a 98°C oil bath for 60 minutes while stirring slowly.

[0044] A3. Transfer the silk fibroin solution in the flask to a stainless steel dish and immediately freeze it in a -50°C freezer for 4 hours to obtain a frozen silk fibroin solution.

[0045] A4. Dissolve 3.00 g of sodium hyaluronate (average molecular weight 1000 kDa) in purified water to obtain 200 mL of a 1.5% sodium hyaluronate solution.

[0046] A5. Take the frozen silk fibroin product from step A3 and the stainless steel plate out of the freezer and thaw it at 10°C until it becomes fluid, to obtain a denatured silk fibroin aqueous solution. After processing in steps A2 to A5, the spatial structure of the silk fibroin changes as shown below. Figure 2 shown.

[0047] A6. Immediately mix 200 mL of thawed denatured silk fibroin solution with 200 mL of sodium hyaluronate solution to a silk fibroin / sodium hyaluronate mass ratio of 50 / 50. Add 1.80 g of 1,4-butanediol diglycidyl ether to the solution while stirring, to a 1,4-butanediol diglycidyl ether / (silk fibroin + sodium hyaluronate) mass ratio of 0.3 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0048] A7. Place the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into a stainless steel dish and immediately freeze it in a -50°C freezer for 4 hours to obtain a frozen mixture.

[0049] A8. Remove the stainless steel dish with the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer and store it in a 5°C refrigerator for 1 hour. Then, freeze it in a -50°C freezer for 4 hours.

[0050] A9. Repeat step A8 twice to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0051] A10. Freeze-dry the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0052] A11. The silk fibroin / sodium hyaluronate complex solid is freeze-granulated, and after screening, the silk fibroin / sodium hyaluronate complex solid particles with a particle size of 150 to 300 μm are obtained.

[0053] A12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 50°C hot water and shake for 18 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0054] A13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature, shake and wash for 48 hours, and replace the purified water every 3 hours.

[0055] A14. The crude silk fibroin / sodium hyaluronate cross-linked gel particles were then immersed in phosphate-buffered saline (pH 7.4) and oscillated for 12 hours, with the phosphate-buffered saline replaced every 3 hours. Excess liquid was filtered off to obtain silk fibroin / sodium hyaluronate cross-linked gel particles. The network structure of silk fibroin-sodium hyaluronate macromolecules within the gel is as follows: Figure 3 shown.

[0056] Example 2

[0057] B1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolved the degummed silk in a 9.3 mol / L LiBr solution at 60°C. Dialyze to obtain a silk fibroin aqueous solution. Adjust the mass fraction of the silk fibroin solution to 0.5% with purified water.

[0058] B2. Pour 40 mL of silk fibroin solution into a flask and heat it in an oil bath at 100°C for 10 minutes while stirring slowly.

[0059] B3. Transfer the silk fibroin solution in the flask into an aluminum box and immediately freeze it in liquid nitrogen at -196°C for 2 hours to obtain a frozen silk fibroin body.

[0060] B4. Dissolve 1.80 g of sodium hyaluronate with an average molecular weight of 2800 kDa in purified water to obtain 360 mL of a 0.5% sodium hyaluronate solution.

[0061] B5. Take out the frozen silk fibroin product from step B3 together with the aluminum box from liquid nitrogen, place it in an environment at a temperature of 25°C and thaw it until it becomes flowable, to obtain a denatured silk fibroin aqueous solution.

[0062] B6. Immediately mix 40 mL of the thawed denatured silk fibroin solution with 360 mL of sodium hyaluronate solution to achieve a silk fibroin / sodium hyaluronate mass ratio of 10 / 90. Add 0.40 g of ethylene glycol diglycidyl ether to the solution while stirring to achieve a ethylene glycol diglycidyl ether / (silk fibroin + sodium hyaluronate) mass ratio of 0.2 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0063] B7. Place the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into an aluminum box and immediately freeze it in liquid nitrogen at -196°C for 2 hours to obtain a frozen body of the mixed solution.

[0064] B8. Remove the aluminum box and the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer and store it in an 8°C refrigerator for 1 hour. Then, freeze it in liquid nitrogen at -196°C for 2 hours.

[0065] B9. Repeat step B8 once more to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0066] B10. freeze-dry the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0067] B11. freeze-granulate the silk fibroin / sodium hyaluronate complex solid, and obtain silk fibroin / sodium hyaluronate complex solid particles with a particle size of 50 to 150 μm after sieving.

[0068] B12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 40° C. hot water and shake for 12 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0069] B13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature and shake and wash for 48 hours, replacing the purified water every 3 hours.

[0070] B14. Then, immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in phosphate-buffered saline (pH 7.4) with shaking for 12 hours, replacing the phosphate-buffered saline every 3 hours. Filter out excess liquid to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0071] Example 3

[0072] C1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolve the degummed silk in a ternary solvent of calcium chloride / ethanol / water (molar ratio of 1 / 2 / 8). Dialyze to obtain a silk fibroin aqueous solution. Adjust the mass fraction of the silk fibroin solution to 2.5% using purified water.

[0073] C2. Pour 360 mL of silk fibroin solution into a flask and heat it in a 95°C oil bath for 120 minutes while stirring slowly during heating.

[0074] C3. Transfer the silk fibroin solution in the flask to a stainless steel dish and immediately freeze it in a -20°C freezer for 6 hours to obtain a frozen silk fibroin solution.

[0075] C4. Dissolve 1.00 g of sodium hyaluronate with an average molecular weight of 500 kDa in purified water to obtain 40 mL of a 2.5% sodium hyaluronate solution.

[0076] C5. Take the frozen silk fibroin product from step C3 together with the stainless steel plate out of the low-temperature refrigerator and thaw it at 2°C until it becomes flowable to obtain a denatured silk fibroin aqueous solution.

[0077] C6. Immediately mix 360 mL of the thawed denatured silk fibroin solution with 40 mL of the sodium hyaluronate solution to achieve a silk fibroin / sodium hyaluronate mass ratio of 90 / 10. Add 4.00 g of polyethylene glycol diglycidyl ether to the solution while stirring to achieve a polyethylene glycol diglycidyl ether / (silk fibroin + sodium hyaluronate) mass ratio of 0.4 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0078] C7. Pour the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into a stainless steel dish and immediately freeze it in a -20°C freezer for 6 hours to obtain a frozen body of the mixed solution.

[0079] C8. Remove the stainless steel dish along with the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer and store it in a 2°C refrigerator for 2 hours. Then, freeze it in a -20°C freezer for 6 hours.

[0080] C9. Repeat step C8 three more times to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0081] C10. freeze-drying the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0082] C11. The silk fibroin / sodium hyaluronate complex solid is frozen and granulated, and after screening, the silk fibroin / sodium hyaluronate complex solid particles with a particle size of 300 to 500 μm are obtained.

[0083] C12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 60° C. hot water and shake for 24 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0084] C13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature, shake and wash for 48 hours, and replace the purified water every 3 hours.

[0085] C14. Then, immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in phosphate-buffered saline (pH 7.4) with shaking for 12 hours, replacing the phosphate-buffered saline every 3 hours. Filter out excess liquid to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0086] Example 4

[0087] D1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolved the degummed silk using a 9.3 mol / L LiBr solution at 60°C. Dialyzed the solution to obtain a silk fibroin solution. Adjust the concentration of the silk fibroin solution to 1.0% using purified water.

[0088] D2. Pour 120 mL of silk fibroin solution into a flask and heat it in a 95°C oil bath for 100 minutes while stirring slowly.

[0089] D3. Transfer the silk fibroin solution in the flask to a stainless steel dish and immediately freeze it in a -80°C freezer for 2 hours to obtain a frozen silk fibroin solution.

[0090] D4. Dissolve 2.80 g of sodium hyaluronate with an average molecular weight of 1800 kDa in purified water to obtain 280 mL of a 1.0% sodium hyaluronate solution.

[0091] D5. Take the frozen silk fibroin product from step D3 and the stainless steel plate out of the freezer and thaw them at 10°C until they become flowable, thereby obtaining a denatured silk fibroin aqueous solution.

[0092] D6. Immediately mix 120 mL of the thawed denatured silk fibroin solution with 280 mL of sodium hyaluronate solution to achieve a silk fibroin / sodium hyaluronate mass ratio of 30 / 70. Add 0.50 g of neopentyl glycol diglycidyl ether to the solution while stirring to achieve a neopentyl glycol diglycidyl ether / (silk fibroin + sodium hyaluronate) mass ratio of 0.125 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0093] D7. Pour the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into a stainless steel dish and immediately freeze it in a -80°C freezer for 4 hours to obtain a frozen body of the mixed solution.

[0094] D8. Remove the stainless steel dish and the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer and store it in a 5°C refrigerator for 0.5 hours. Then, freeze it in a -80°C freezer for 4 hours.

[0095] D9. Repeat step D8 4 more times to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0096] D10. freeze-dry the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0097] D11. The silk fibroin / sodium hyaluronate complex solid is frozen and granulated, and after screening, the silk fibroin / sodium hyaluronate complex solid particles with a particle size of 150 to 300 μm are obtained.

[0098] D12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 40° C. hot water and shake for 18 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0099] D13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature and shake and wash for 48 hours, replacing the purified water every 3 hours.

[0100] D14. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in phosphate-buffered saline (pH 7.4) with shaking for 12 hours, replacing the phosphate-buffered saline every 3 hours. Filter out excess liquid to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0101] Example 5

[0102] E1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolve the degummed silk in a ternary solvent of calcium chloride / ethanol / water (molar ratio of 1 / 2 / 8). Dialyze to obtain a silk fibroin aqueous solution. Adjust the mass fraction of the silk fibroin solution to 1.0% with purified water.

[0103] E2. Pour 280 mL of silk fibroin solution into a flask and heat it in a 99°C oil bath for 10 minutes while stirring slowly.

[0104] E3. Transfer the silk fibroin solution in the flask to a stainless steel dish and immediately freeze it in a -30°C freezer for 3 hours to obtain a frozen silk fibroin solution.

[0105] E4. Dissolve 1.20 g of sodium hyaluronate with an average molecular weight of 1500 kDa in purified water to obtain 120 mL of a 1.0% sodium hyaluronate solution.

[0106] E5. Take the frozen silk fibroin product from step E3 together with the stainless steel plate out of the freezer and thaw it at 20°C until it becomes flowable to obtain a denatured silk fibroin aqueous solution.

[0107] E6. Immediately mix 280 mL of thawed denatured silk fibroin solution with 120 mL of sodium hyaluronate solution to a silk fibroin / sodium hyaluronate mass ratio of 70 / 30. Add 0.50 g of 1,4-butanediol diglycidyl ether and 0.50 g of polyethylene glycol diglycidyl ether to the solution while stirring, to a (1,4-butanediol diglycidyl ether + polyethylene glycol diglycidyl ether) / (silk fibroin + sodium hyaluronate) mass ratio of 0.25 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0108] E7. Place the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into a stainless steel dish and immediately freeze it in a -30°C freezer for 3 hours to obtain a frozen mixed solution.

[0109] E8. Remove the stainless steel dish with the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer and store it in a 4°C refrigerator for 1 hour. Then, freeze it in a -30°C freezer for 3 hours.

[0110] E9. Repeat step E8 4 more times to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0111] E10. Freeze-dry the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0112] E11. The silk fibroin / sodium hyaluronate complex solid is freeze-granulated, and after screening, the silk fibroin / sodium hyaluronate complex solid particles with a particle size of 150 to 300 μm are obtained.

[0113] E12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 55°C hot water and shake for 18 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0114] E13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature and wash with shaking for 48 hours, replacing the purified water every 3 hours.

[0115] E14. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in phosphate-buffered saline (pH 7.4) with shaking for 12 hours, replacing the phosphate-buffered saline every 3 hours. Filter out excess liquid to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0116] Example 6

[0117] F1. Degummed mulberry silk using a 0.5 g / L Na2CO3 solution at 98-100°C. Dissolved the degummed silk in a 9.3 mol / L LiBr solution at 60°C. Dialyzed the solution to obtain a silk fibroin solution. Adjust the concentration of the silk fibroin solution to 1.5% using purified water.

[0118] F2. Pour 240 mL of silk fibroin solution into a flask and heat it in a 97°C oil bath for 40 minutes while stirring slowly.

[0119] F3. Transfer the silk fibroin solution in the flask into an aluminum box and immediately freeze it in liquid nitrogen at -196°C for 3 hours to obtain a frozen silk fibroin body.

[0120] F4. Dissolve 2.40 g of sodium hyaluronate with an average molecular weight of 1200 kDa in purified water to obtain 160 mL of a 1.5% sodium hyaluronate solution.

[0121] F5. Take out the frozen silk fibroin product from step F3 together with the aluminum box from liquid nitrogen, place it in an environment at a temperature of 15°C and thaw it until it becomes flowable, to obtain a denatured silk fibroin aqueous solution.

[0122] F6. Immediately mix 240 mL of the thawed denatured silk fibroin solution with 160 mL of sodium hyaluronate solution to achieve a silk fibroin / sodium hyaluronate mass ratio of 60 / 40. Add 1.05 g of ethylene glycol diglycidyl ether and 1.05 g of neopentyl glycol diglycidyl ether to the solution while stirring to achieve a (ethylene glycol diglycidyl ether + neopentyl glycol diglycidyl ether) / (silk fibroin + sodium hyaluronate) mass ratio of 0.35 / 1.0, to obtain a silk fibroin / sodium hyaluronate / cross-linker mixed solution.

[0123] F7. Place the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution into an aluminum box and immediately freeze it in liquid nitrogen at -196°C for 3 hours to obtain a frozen body of the mixed solution.

[0124] F8. Remove the aluminum box and the frozen silk fibroin / sodium hyaluronate / cross-linker mixed solution from the freezer, store it in a 6°C refrigerator for 45 minutes, and then freeze it in liquid nitrogen at -196°C for 3 hours.

[0125] F9. Repeat step F8 twice to obtain a frozen silk fibroin / sodium hyaluronate complex.

[0126] F10. Freeze-dry the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid.

[0127] F11. The silk fibroin / sodium hyaluronate complex solid is freeze-granulated, and after sieving, solid particles of the silk fibroin / sodium hyaluronate complex with a particle size of 300 to 500 μm are obtained.

[0128] F12. Immerse the silk fibroin / sodium hyaluronate complex solid particles in 45°C hot water and shake for 16 hours, replacing the hot water every 3 hours to obtain a crude product of silk fibroin / sodium hyaluronate cross-linked gel particles.

[0129] F13. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in purified water at room temperature and shake and wash for 48 hours, replacing the purified water every 3 hours.

[0130] F14. Immerse the crude silk fibroin / sodium hyaluronate cross-linked gel particles in phosphate-buffered saline (pH 7.4) with shaking for 12 hours, replacing the phosphate-buffered saline every 3 hours. Filter out excess liquid to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

[0131] Comparative Example 1

[0132] The difference from Example 1 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps A2, A3, and A5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps A8 and A9 are omitted, and the process is carried out until step A10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0133] Comparative Example 2

[0134] The difference from Example 2 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps B2, B3, and B5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps B8 and B9 are omitted, and the process is carried out until step B10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0135] Comparative Example 3

[0136] The difference from Example 3 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps C2, C3, and C5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps C8 and C9 are omitted, and the process is continued until step C10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0137] Comparative Example 4

[0138] The difference from Example 4 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps D2, D3, and D5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps D8 and D9 are omitted, and the process is carried out until step D10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0139] Comparative Example 5

[0140] The difference from Example 5 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps E2, E3, and E5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps E8 and E9 are omitted, and the process is carried out until step E10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0141] Comparative Example 6

[0142] The difference from Example 6 is that the heating treatment-rapid freezing process of the silk fibroin solution is omitted, that is, steps F2, F3, and F5 are omitted; at the same time, the repeated thawing-freezing process of the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is omitted, that is, steps F8 and F9 are omitted, and the process is carried out until step F10 is performed to obtain a silk fibroin / sodium hyaluronate composite solid after freeze-drying.

[0143] Gel cross-linking degree test

[0144] The silk fibroin / sodium hyaluronate composite solids prepared in Examples 1 to 6 and Comparative Examples 1 to 6 were tested for weight loss in phosphate buffered saline (PBS). The specific testing method is as follows:

[0145] Take approximately 0.5 g of the silk fibroin / sodium hyaluronate composite solid obtained after freeze-drying, i.e., the samples obtained by completing steps A10, B10, C10, D10, E10, and F10 in Examples 1, 2, 3, 4, 5, and 6, respectively, and the samples obtained by completing all steps of Comparative Examples 1 to 6, and weigh them using an analytical balance. Cut into small pieces, place them in a metal mesh bag, immerse them in 100 mL of phosphate-buffered saline (PBS) (pH 7.4), and dissolve them at 37°C with constant temperature and shaking for 24 hours. Filter, freeze-dry the remaining silk fibroin / sodium hyaluronate composite, and weigh them using an analytical balance. Calculate the weight loss (%) of the silk fibroin / sodium hyaluronate composite solid in PBS according to formula (1). The smaller the weight loss rate, the stronger the ability of the silk fibroin / sodium hyaluronate complex to resist water dissolution, which is used to evaluate the effective cross-linking degree of sodium hyaluronate and silk fibroin in the silk fibroin / sodium hyaluronate complex.

[0146]

[0147] Depend on Figure 4 As can be seen, the weight loss rates of the silk fibroin / sodium hyaluronate composite solids prepared in Examples 1 to 6 in PBS were all less than 13%, while the weight loss rates of the silk fibroin / sodium hyaluronate composite solids prepared in Comparative Examples 1 to 6 in PBS were all greater than 48%. The weight loss rates of the silk fibroin / sodium hyaluronate composite solids in PBS of each group of Examples were significantly lower than those of the Comparative Examples. This demonstrates that due to the present invention's use of a heating treatment-rapid freezing process for the silk fibroin solution, and a rapid freezing, repeated thawing-freezing, and freeze-drying process for the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution, effective cross-linking is achieved between the silk fibroin-silk fibroin, silk fibroin-sodium hyaluronate, and sodium hyaluronate-sodium hyaluronate macromolecules in the silk fibroin / sodium hyaluronate gel.

[0148] Gel biodegradation test

[0149] The silk fibroin / sodium hyaluronate cross-linked gel particles prepared in Examples 1 to 6, and the silk fibroin / sodium hyaluronate composite solids prepared in Comparative Examples 1 to 6, were subjected to in vitro biodegradation rate tests in simulated body fluids. The specific test method is as follows:

[0150] A PBS mixed solution containing 10.0 U / mL hyaluronidase IS and 2.0 U / mL collagenase IA was prepared as a simulated body fluid for in vitro biodegradation of the gel. Approximately 1 g of the freeze-dried silk fibroin / sodium hyaluronate cross-linked gel particles prepared in Examples 1 to 6, and the silk fibroin / sodium hyaluronate composite solids prepared in Comparative Example 1 to Comparative Example 6, were taken and weighed using an analytical balance. The gel particles were placed in a metal mesh bag and immersed in 100 mL of simulated body fluid. The fluid was then degraded at 37°C under constant temperature and oscillation for 7 days. Fresh simulated body fluid was replaced every 48 hours. After degradation was complete, the remaining gel was freeze-dried and its residual mass M2 (g) was measured. The biodegradation rate (%) of the gel in the simulated body fluid was calculated according to formula (2). This was used to evaluate the gel's resistance to biodegradation.

[0151]

[0152] The silk fibroin / sodium hyaluronate cross-linked gel particles of Examples 1 to 6 were able to basically maintain their original morphology after 7 days of degradation, without obvious collapse; while the silk fibroin / sodium hyaluronate composite solids prepared in Comparative Examples 1 to 6 were all disintegrated after 7 days of degradation, with only a small amount of fragments remaining. Figure 5 It can be seen that the biodegradation rates of the silk fibroin / sodium hyaluronate cross-linked gel particles prepared in Examples 1 to 6 were all less than 27%, while the biodegradation rates of the silk fibroin / sodium hyaluronate composite solids prepared in Comparative Examples 1 to 6 were all greater than 90%. The biodegradation rates of the silk fibroin / sodium hyaluronate cross-linked gel particles in each group of Examples were significantly lower than those in the Comparative Examples. This demonstrates that due to the present invention's use of a heating treatment-quick freezing process for the silk fibroin solution, and a quick freezing, repeated thawing-freezing, and freeze-drying process for the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution, the silk fibroin-silk fibroin, silk fibroin-sodium hyaluronate, and sodium hyaluronate-sodium hyaluronate macromolecules in the silk fibroin / sodium hyaluronate gel are effectively cross-linked, significantly enhancing the ability to resist biodegradation.

[0153] Cytotoxicity test of gel

[0154] In accordance with the national standard GB / T16886.5-2017 "Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Test," in vitro cytotoxicity testing was performed on the silk fibroin / sodium hyaluronate cross-linked gel particles prepared in Examples 1 to 6 after steam sterilization to determine relative cell survival. The extraction medium consisted of 89% MEM medium, 10% fetal bovine serum, and 1% penicillin-streptomycin solution. The blank control group consisted of the extract. The negative control group consisted of high-density polyethylene (HDPE). The positive control group consisted of dimethyl sulfoxide (DMSO). Cells were mouse fibroblast L-929 cells. The relative cell survival rate was determined using the MTT assay at a 100% extract concentration in each experimental group after 24 hours of in vitro culture.

[0155] Depend on Figure 6 It can be seen that the cell relative survival rates of the silk fibroin / sodium hyaluronate cross-linked gel particles prepared in Examples 1 to 6 were all greater than 95%, and there was no cytotoxicity, which proves that the silk fibroin / sodium hyaluronate cross-linked gel particles prepared in the present invention have good cell compatibility.

[0156] In addition to the above embodiments, the present invention also includes other implementation methods. Any technical solutions formed by equivalent transformation or equivalent replacement should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing silk fibroin / sodium hyaluronate cross-linked gel particles, comprising degumming, dissolving, and dialyzing silk to obtain a silk fibroin aqueous solution, characterized in that: Then proceed with the following steps: P1. Heat the silk fibroin aqueous solution for a certain period of time and then rapidly freeze it to obtain a frozen silk fibroin body; P2. After thawing the frozen silk fibroin, a denatured silk fibroin aqueous solution was obtained; P3. The thawed denatured silk fibroin aqueous solution was immediately mixed with a sodium hyaluronate solution, and a diglycidyl ether cross-linking agent was added to the solution to obtain a silk fibroin / sodium hyaluronate / cross-linking agent mixed solution; P4. Rapidly freezing the silk fibroin / sodium hyaluronate / crosslinker mixed solution to obtain a frozen mixed solution; P5. The frozen silk fibroin / sodium hyaluronate / crosslinker mixed solution was placed in a melting temperature environment for a certain period of time, and then rapidly frozen. After repeating the thawing-freezing process several times, a frozen silk fibroin / sodium hyaluronate complex was obtained; P6. freeze-drying the silk fibroin / sodium hyaluronate complex to obtain a silk fibroin / sodium hyaluronate complex solid; P7. The silk fibroin / sodium hyaluronate complex solid was frozen and granulated, and sieved to obtain silk fibroin / sodium hyaluronate complex solid particles; P8. Immersing the silk fibroin / sodium hyaluronate composite solid particles in hot water for a certain period of time to obtain a crude silk fibroin / sodium hyaluronate cross-linked gel particle product; P9. The crude silk fibroin / sodium hyaluronate cross-linked gel product was first washed with water at room temperature, then balanced with phosphate-buffered saline, and excess liquid was filtered out to obtain silk fibroin / sodium hyaluronate cross-linked gel particles.

2. The method according to claim 1, characterized in that In the step P1, the temperature of the silk fibroin aqueous solution is heated to 95-100° C. for 10-120 minutes; the rapid freezing temperature is -196--20° C. for 2-6 hours.

3. The method according to claim 1, characterized in that In step P2, the thawing temperature is 2-25°C.

4. The method according to claim 1, characterized in that In step P3, the average molecular weight of the sodium hyaluronate used is 500 to 3000 kDa; the mass ratio of silk fibroin / sodium hyaluronate in the mixed solution is 90 / 10 to 10 / 90, and the total mass fraction of silk fibroin and sodium hyaluronate in the mixed solution is 0.5 to 2.5%; the diglycidyl ether cross-linking agent is one or a combination of two of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, and neopentyl glycol diglycidyl ether; the ratio of the added diglycidyl ether cross-linking agent to the total mass of silk fibroin and sodium hyaluronate in the mixed solution is 0.1 / 1.0 to 0.4 / 1.

0.

5. The method according to claim 1, characterized in that In step P4, the silk fibroin / sodium hyaluronate / cross-linking agent mixed solution is rapidly frozen at a temperature of -196 to -20°C for a period of 2 to 6 hours.

6. The method according to claim 1, characterized in that In step P5, the melting temperature is 2-8° C., the storage time in the melting temperature environment is 0.5-2.0 hours, and the number of repeated thawing-freezing times is 2-5 times.

7. The method according to claim 1, characterized in that In step P7, the particle size of the silk fibroin / sodium hyaluronate composite solid particles is 50-500 μm.

8. The method according to claim 1, characterized in that In step P8, the temperature of the hot water used to treat the silk fibroin / sodium hyaluronate composite solid particles is 40-60° C., the treatment time is 12-24 hours, and the hot water is replaced every 3 hours or so.

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