A growth factor-containing silk fibroin-based biomaterial for injection and a preparation method thereof

Abstract

The application relates to the technical field of biomaterials, in particular to a silk fibroin-based biomaterial containing growth factors for injection and a preparation method. The silk fibroin and sodium hyaluronate are cross-linked, the gel is freeze-dried to ensure the stability of the product, the SF-HA freeze-dried powder is compounded with cross-linked sodium hyaluronate as a dispersion medium to form a composite gel particle system, the composite gel is sterilized, and then is blended with growth factors to obtain the silk fibroin-based biomaterial, in which the silk fibroin particles are wrapped by the hyaluronic acid cross-linked lattices. The silk fibroin-based biomaterial is prepared, the degradation period of the product is prolonged, and meanwhile, the uniformity, biocompatibility and active ingredient reservation of the material are considered.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to an injectable silk fibroin-based biomaterial containing growth factors and its preparation method. Background Technology

[0002] With the rapid development of medical aesthetics and regenerative medicine, the market demand for soft tissue fillers has increased significantly. Local injection, as a minimally invasive treatment, effectively improves local morphology and restores function by injecting drugs or chemical agents into loose soft tissue. However, existing filler materials still face several key technological bottlenecks, such as insufficient controllable degradation. Traditional hyaluronic acid (HA) gel degrades too quickly, resulting in short-lasting filling effects and requiring frequent touch-ups. Limitations in mechanical properties mean that while silk fibroin materials possess excellent biocompatibility, their water absorption and lubrication are insufficient, making it difficult to meet clinical requirements for filler materials in terms of shaping and comfort. Furthermore, existing materials have limited functionality and lack bioactive components that actively promote tissue regeneration, failing to achieve a synergistic effect of "filling-repair-regeneration." To address these shortcomings, this product, based on the synergistic innovation of silk fibroin and hyaluronic acid, incorporates different growth factors to meet the filling needs of various areas in medical aesthetics.

[0003] Silk fibroin is a natural high-molecular-weight fibrous protein extracted from silkworm silk. Its core advantages include structural stability: the crystal structure composed of β-sheets endows it with excellent mechanical strength and resistance to enzymatic degradation; biocompatibility: it is homologous to human epidermal proteins, rich in 18 essential amino acids, and its degradation products can be directly absorbed and utilized by cells; and functional modifiability: its degradation rate and surface properties can be controlled through physical cross-linking or chemical modification. Hyaluronic acid, as a major component of the extracellular matrix, has the following advantages: high water absorption: it can absorb 1000 times its own weight in water, maintaining a moist tissue environment; lubrication: it reduces inter-tissue friction and improves injection comfort; and it acts as a biological signal carrier: it regulates cell behavior. Growth factors, as core signaling molecules regulating cell proliferation, differentiation, and migration, play an irreplaceable role in soft tissue repair. They promote angiogenesis: VEGF can stimulate angiogenesis and improve local blood supply; regulate collagen synthesis: TGF-β1 can activate fibroblasts and promote the secretion of type I / III collagen; and have anti-inflammatory effects: EGF can downregulate the expression of pro-inflammatory factors such as TNF-α, reducing postoperative swelling.

[0004] Existing technologies have achieved physical compounding of silk fibroin and hyaluronic acid, but they suffer from the following drawbacks: poor mixing uniformity: simple physical blending easily leads to the sedimentation of silk fibroin particles, triggering local inflammatory reactions; low cross-linking efficiency: traditional chemical cross-linking agents (such as glutaraldehyde) may leave residual toxicity and are difficult to control precisely; insufficient functional integration: the effective loading of bioactive components such as growth factors is not achieved, limiting tissue regeneration capacity. Free growth factors also have limitations such as short half-life and susceptibility to protease degradation.

[0005] To address the aforementioned issues, we propose an injectable silk fibroin-based biomaterial containing growth factors and its preparation method. Summary of the Invention

[0006] The purpose of this invention is to provide an injectable silk fibroin-based biomaterial containing growth factors and its preparation method, so as to solve the problems raised in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, comprising the following steps:

[0009] Step 1: Take degummed silk fibers and dissolve them in lithium bromide solution to obtain dissolved silk fibroin; cool the dissolved silk fibroin, and then dialyze, filter, and centrifuge to obtain silk fibroin solution;

[0010] Step 2: Mix the silk fibroin solution, sodium hyaluronate solution and water for injection evenly, add 1,4-butanediol diglycidyl ether, stir thoroughly for 30-60 minutes, keep warm at 70-90℃ for 1-3 hours, freeze dry, pulverize and sieve to obtain SF-HA freeze-dried powder;

[0011] Step 3: Mix 1,4-butanediol diglycidyl ether and sodium hydroxide solution evenly, add sodium hyaluronate and mix evenly, then add SF-HA lyophilized powder, crosslink in a water bath at 30-60℃ for 3-5 hours, and then let stand at 25-35℃ for 60-80 hours to obtain the crosslinked gel; cut the crosslinked gel into small pieces, soak in phosphate buffer solution for 2-4 days, change the solution every 12 hours, sieve and granulate to obtain the composite gel;

[0012] Step 4: Fill the composite gel into a pre-filled syringe, perform moist heat sterilization, add growth factors under sterile conditions, mix evenly, and then perform aseptic filling to obtain silk fibroin-based biomaterial.

[0013] Further, in step one, the bath ratio of the degummed filament to lithium bromide is 1:(4-10), the concentration of the lithium bromide solution is 9.3mol / L, the dissolution temperature is 58-62℃, and the concentration of the silk fibroin solution is 3-6wt%.

[0014] Further, in step two, the mass ratio of the silk fibroin solution, sodium hyaluronate solution, water for injection, and 1,4-butanediol diglycidyl ether is 1:(1.0-1.2):(3.4-3.6):(0.01-0.02), and the concentration of the sodium hyaluronate solution is 1-5wt%.

[0015] Furthermore, the water for injection is physiological saline or a phosphate buffer solution with a pH of 6.8-7.2.

[0016] Further, in step three, the mass ratio of 1,4-butanediol diglycidyl ether, sodium hydroxide solution, sodium hyaluronate and SF-HA lyophilized powder is 1:(50-60):(4-6):(3-5), and the concentration of sodium hydroxide solution is 1.0wt%.

[0017] Further, in step four, the mass ratio of the composite gel to the growth factor is 1000:(0.002-0.008).

[0018] Furthermore, the growth factor is at least one of fibroblast growth factor, epidermal growth factor, and keratinocyte growth factor.

[0019] Furthermore, the growth factor undergoes a coating modification process, the specific process of which is as follows:

[0020] Step A: Disperse the growth factor in an aqueous solution of sodium alginate to obtain a mixed solution, add calcium chloride solution dropwise over 1-2 hours, stir for 30-40 minutes, filter, wash, and freeze-dry to obtain CA microspheres;

[0021] Step B: Disperse CA microspheres in a modified chitosan solution, stir for 20-30 min, filter, wash, and freeze-dry to obtain CA-CS composite microspheres;

[0022] Step C: Disperse CA-CS composite microspheres in an aqueous solution containing double-bonded hyaluronic acid to obtain a mixed solution containing double bonds. Add a mixed solution of thiolized collagen, thiolized silk fibroin and deionized water, and then add lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. After stirring thoroughly, cure under ultraviolet light, dialyze, and freeze-dry to obtain the coated modified growth factor.

[0023] Further, in step A, the concentration of sodium alginate in the aqueous solution is 0.02 g / mL to 0.04 g / mL.

[0024] Further, in step A, the Ca in the calcium chloride solution... 2+ The concentration is 0.1 mol / L to 1 mol / L.

[0025] Further, in step B, the concentration of chitosan in the modified chitosan solution is 0.02 g / mL to 0.04 g / mL, and the solvent is 2 wt% acetic acid.

[0026] Furthermore, the method for preparing the modified chitosan solution is as follows:

[0027] Sodium polyglutamate was aldehyde-treated to obtain aldehyde-modified polyglutamate; methacrylamide chitosan, aldehyde-modified polyglutamate and phosphate buffer solution were mixed evenly and reacted at 30-50℃ for 20-30 min to obtain modified chitosan solution.

[0028] Further, the preparation steps of the aldehyde-modified polyglutamic acid are as follows: sodium polyglutamate and deionized water are mixed evenly, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and 3-amino-1,2-propanediol are added, the reaction is carried out for 12-24 hours, dialyzed and freeze-dried to obtain hydroxylated polyglutamic acid; the hydroxylated polyglutamic acid is dissolved in deionized water, sodium periodate is added, oxidized in the dark for 30-60 minutes, dialyzed and freeze-dried to obtain aldehyde-modified polyglutamic acid.

[0029] Further, the mass ratio of the sodium polyglutamate, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and 3-amino-1,2-propanediol is 1:(50-70):(4-5):(1.8-2.5):(0.06-0.08).

[0030] Further, the mass ratio of the hydroxylated polyglutamic acid, deionized water and sodium periodate is 1:(20-40):(1.2-1.5).

[0031] Furthermore, the molar ratio of the amino group of the methacrylamide chitosan to the aldehyde group of the aldehyde-modified polyglutamic acid is 1:(0.5-0.7).

[0032] Furthermore, the mass of the phosphate buffer solution is 3-5 times the total mass of methacrylamide chitosan and aldehyde-modified polyglutamic acid.

[0033] Furthermore, in step C, the concentration of the aqueous solution containing double-bonded hyaluronic acid is 3-5 wt%.

[0034] Further, in step C, the mass ratio of thiolated collagen, thiolated silk fibroin, deionized water and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 1:(5-10):(30-50):(0.01-0.03), and the mass of thiolated collagen is 0.2-0.4 times the mass of hyaluronic acid containing double bonds.

[0035] Furthermore, the method for preparing the thiolized collagen is as follows:

[0036] Collagen was added to an acetic acid solution and stirred to dissolve, thus obtaining a collagen solution. The collagen solution was transferred to a dialysis bag and dialyzed at 4-6°C with an alkaline phosphate buffer solution. Dialysis was performed until the pH reached 9-10. DL-N-acetylhomocysteine ​​and ethylenediaminetetraacetic acid were added, and the reaction was carried out under nitrogen protection for 22-24 hours. Dithiothreitol was then added, and stirring was continued for 10-12 hours. The mixture was then dialyzed and freeze-dried to obtain thiolized collagen.

[0037] Furthermore, the concentration of the acetic acid solution is 0.4-0.6M.

[0038] Further, the mass ratio of collagen, acetic acid solution, DL-N-acetylhomocysteine ​​thiolactone, ethylenediaminetetraacetic acid, and dithiothreitol is 0.4:(100-200):(0.3-0.6):(0.02-0.04):(0.01-0.03).

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] 1. This invention ensures product stability by cross-linking silk fibroin with sodium hyaluronate to form a gel, followed by freeze-drying. SF-HA freeze-dried powder is then compounded with cross-linked sodium hyaluronate as a dispersion medium to form a composite gel particle system. After sterilization, the composite gel is aseptically blended with growth factors, and the silk fibroin particles are encapsulated using a hyaluronic acid cross-linked network to obtain silk fibroin-based biomaterials. This invention, through the preparation of silk fibroin-based biomaterials, extends the product's degradation cycle while simultaneously ensuring material uniformity, biocompatibility, and retention of active ingredients.

[0041] 2. Based on the above technical solution, this invention achieves a long-lasting sustained-release effect by coating and modifying the growth factor. First, under the action of calcium ions, the growth factor and sodium alginate cross-link, and sodium alginate forms a uniform coating layer on the surface of the growth factor, thus producing sodium alginate loaded with growth factor, i.e., CA microspheres. Then, CA microspheres and modified chitosan cross-link with each other to obtain CA-CS composite microspheres. The cross-linking effect ensures that the microspheres are uniformly dispersed and avoids agglomeration. The modified chitosan is prepared by aldehyde-modified polyglutamic acid and methacryloxylated chitosan through Schiff base reaction. In this invention, by controlling the molar ratio of amino groups of methacryloxylated chitosan to aldehyde groups of aldehyde-modified polyglutamic acid to be 1:(0.5-0.7), not only can sufficient polyglutamic acid be grafted onto chitosan, enhancing the functionality of the material, but also prolonging the maintenance time of tissue filling. Furthermore, the unreacted amino groups can cross-link with the carboxyl groups of sodium alginate, further enhancing the stability and mechanical strength of the material, making it more reliable in biological applications.

[0042] Finally, under photoinitiator and ultraviolet light conditions, CA-CS microspheres were embedded into a hyaluronic acid (HA) / collagen / silk fibroin (SF) cross-linked network via a thiol-olefin click reaction, forming a sustained-release barrier. This achieved long-term sustained release of growth factors, which is beneficial for cell proliferation and regeneration. The addition of hyaluronic acid and silk fibroin to the outer coating layer improved the compatibility between the growth factors and the composite gel, reduced the rejection response of organisms to the material, and enhanced its application potential in the biomedical field.

[0043] Currently, there are no products that have established a stable binding mechanism between silk fibroin, hyaluronic acid, and growth factors. This technological innovation effectively fills the gap in the existing market. Attached Figure Description

[0044] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0045] Figure 1 This is a schematic diagram of the composite gel in this invention;

[0046] Figure 2 This is a schematic diagram of the injectable silk fibroin-sodium hyaluronate composite gel containing growth factors in this invention. Detailed Implementation

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] It should be noted that there are no special restrictions on the suppliers of all raw materials involved in this invention. Exemplary examples include (in this embodiment) degummed filaments: 6A raw filaments, sourced from Haian Suhao Silk Manufacturing Co., Ltd., which obtains degummed filaments after degumming the raw filaments; sodium hyaluronate: sourced from Bloomage Biotechnology Co., Ltd.; growth factor: acidic fibroblast growth factor, model NRPA15, sourced from Hangzhou Newlong Biotechnology Co., Ltd.; methacrylamide chitosan: model Q-0291683, sourced from Xi'an Qiyue Biotechnology Co., Ltd.; sodium polyglutamate: Bloomage Biotechnology Hyafactor™-PGA; collagen: recombinant type III humanized collagen, sourced from Shanxi Jinbo Biopharmaceutical Co., Ltd.; double-bond hyaluronic acid: methacrylamide hyaluronic acid, model FS1424, sourced from Shanghai Fushen Biotechnology Co., Ltd.; thiolated silk fibroin: sourced from Shenzhen Meiluo Technology Co., Ltd.

[0049] Example 1: A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, comprising the following processes:

[0050] Step 1: Take degummed silk fibers and dissolve them in 9.3 mol / L lithium bromide solution at a bath ratio of 1:4 at a dissolution temperature of 58℃ to obtain dissolved silk fibroin; cool the dissolved silk fibroin and put it into a dialysis bag with a molecular weight cutoff of 12 kDa. Dialyze it with flowing purified water for three days and then take it out. Filter the solution through gauze and centrifuge it at 4℃ and 9000 rpm for 20 min to obtain a clear 3 wt% silk fibroin solution;

[0051] Step 2: Mix 61.76g of 3wt% silk fibroin solution, 70g of 2wt% sodium hyaluronate solution and 217g of water for injection until homogeneous. Add 1.05g of 1,4-butanediol diglycidyl ether and stir thoroughly for 40 minutes. Seal and keep warm at 80℃ for 2 hours. After freeze-drying, pulverizing and passing through a 200-mesh sieve, SF-HA freeze-dried powder is obtained.

[0052] Step 3: Mix 0.6g of 1,4-butanediol diglycidyl ether and 30g of 1.0wt% sodium hydroxide solution evenly, add 3g of sodium hyaluronate and mix evenly, then add 2.4g of SF-HA lyophilized powder, crosslink in a 45℃ water bath for 4h, and then let stand at 30℃ for 72h to obtain the crosslinked gel; cut the crosslinked gel into small pieces, soak in phosphate buffer solution for 2 days, change the solution every 12h, sieve and granulate to obtain the composite gel;

[0053] Step 4: Fill 5g of composite gel into a pre-filled syringe and perform moist heat sterilization. Under sterile conditions, add 25μg of growth factor and mix evenly. Then perform aseptic filling to obtain silk fibroin-based biomaterial.

[0054] Example 2: A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, comprising the following processes:

[0055] Step 1: Take degummed silk fibers and dissolve them in 9.3 mol / L lithium bromide solution at a bath ratio of 1:6 at a dissolution temperature of 60℃ to obtain dissolved silk fibroin; cool the dissolved silk fibroin and put it into a dialysis bag with a molecular weight cutoff of 13 kDa. Dialyze it with flowing purified water for three days and then take it out. Filter the solution through gauze and centrifuge it at 4℃ and 9000 rpm for 20 min to obtain a clear 4 wt% silk fibroin solution;

[0056] Step 2: Mix 70 parts of 4wt% silk fibroin solution, 70g of 1wt% sodium hyaluronate solution and 238g of water for injection evenly, add 0.7g of 1,4-butanediol diglycidyl ether, stir thoroughly for 30min, keep warm at 70℃ for 3h, freeze dry, pulverize and pass through a 200-mesh sieve to obtain SF-HA freeze-dried powder;

[0057] Step 3: Mix 0.8g of 1,4-butanediol diglycidyl ether and 40g of 1wt% sodium hydroxide solution evenly, add 3.2g of sodium hyaluronate and mix evenly, then add 2.4g of SF-HA lyophilized powder, crosslink in a 30℃ water bath for 5h, and then let stand at 25℃ for 80h to obtain the crosslinked gel; cut the crosslinked gel into small pieces, soak in phosphate buffer solution for 2 days, change the solution every 12h, sieve and granulate to obtain the composite gel;

[0058] Step 4: Fill 5g of composite gel into a pre-filled syringe, perform moist heat sterilization, add 10μg of growth factor under sterile conditions, mix evenly, and then perform aseptic filling to obtain silk fibroin-based biomaterial.

[0059] The growth factors underwent coating modification treatment, the specific process of which is as follows:

[0060] Step A: Disperse 10 μg of growth factor in 100 mL of 0.02 g / mL sodium alginate aqueous solution to obtain a mixed solution. Add 150 mL of 0.1 mol / L calcium chloride solution dropwise over 1 hour. Stir for 30 minutes, filter, wash, and freeze-dry to obtain CA microspheres.

[0061] Step B: Disperse 10 μg of CA microspheres in 100 mL of 0.02 g / mL modified chitosan solution, stir for 20 min, filter, wash, and freeze dry to obtain CA-CS composite microspheres;

[0062] Step C: Disperse 10 μg of CA-CS composite microspheres in 100 mL of 3 wt% aqueous solution containing double-bonded hyaluronic acid to obtain a mixed solution containing double bonds. Add a mixed solution of 0.6 g of thiolated collagen, 3 g of thiolated silk fibroin and 18 g of deionized water, and then add 0.06 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. After stirring thoroughly, cure under ultraviolet light (365 nm), dialyze, and freeze-dry to obtain the coated modified growth factor.

[0063] The preparation method of modified chitosan solution is as follows:

[0064] Sodium polyglutamate, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 3-amino-1,2-propanediol were mixed evenly in a mass ratio of 1:50:4:1.8:0.06 and reacted for 12 h. After dialyzing and freeze-drying, hydroxylated polyglutamic acid was obtained. Hydroxylated polyglutamic acid, deionized water, and sodium periodate were mixed evenly in a mass ratio of 1:20:1.2 and oxidized in the dark for 30 min. After dialyzing and freeze-drying, aldehyde-modified polyglutamic acid was obtained.

[0065] Methacrylamide chitosan, aldehyde-modified polyglutamic acid, and phosphate buffer solution were mixed evenly and reacted at 30°C for 20 min to obtain a modified chitosan solution. The molar ratio of amino groups in methacrylamide chitosan to aldehyde groups in aldehyde-modified polyglutamic acid was 1:0.5. The mass of the phosphate buffer solution was three times the total mass of methacrylamide chitosan and aldehyde-modified polyglutamic acid.

[0066] The preparation method of thiolized collagen is as follows:

[0067] 1g of collagen was added to 250g of 0.4M acetic acid solution and stirred to dissolve, thus obtaining a collagen solution. The collagen solution was transferred to a dialysis bag and dialyzed at 4°C with an alkaline phosphate buffer solution. Dialysis was continued until the pH reached 9. 0.75g of DL-N-acetylhomocysteine ​​thiolactone and 0.05g of ethylenediaminetetraacetic acid were added, and the reaction was carried out under nitrogen protection for 22h. Then, 0.025g of dithiothreitol was added, and stirring was continued for 10h. Dialysis and freeze-drying were performed to obtain thiolized collagen.

[0068] Example 3: A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, comprising the following processes:

[0069] Step 1: Take degummed silk fibers and dissolve them in 9.3 mol / L lithium bromide solution at a bath ratio of 1:8 at a dissolution temperature of 60℃ to obtain dissolved silk fibroin; cool the dissolved silk fibroin and put it into a dialysis bag with a molecular weight cutoff of 14 kDa. Dialyze it with flowing purified water for three days and then take it out. Filter the solution through gauze and centrifuge it at 4℃ and 9000 rpm for 20 min to obtain a clear 5 wt% silk fibroin solution;

[0070] Step 2: Mix 63.6g of silk fibroin solution, 70g of 3wt% sodium hyaluronate solution and 220g of water for injection evenly, add 0.9g of 1,4-butanediol diglycidyl ether, stir thoroughly for 50min, keep warm at 80℃ for 2h, freeze dry, pulverize and pass through a 200-mesh sieve to obtain SF-HA freeze-dried powder;

[0071] Step 3: Mix 0.7g of 1,4-butanediol diglycidyl ether and 38.5g of 1.0wt% sodium hydroxide solution evenly, add 3.5g of sodium hyaluronate and mix evenly, then add 2.8g of SF-HA lyophilized powder. Crosslink the mixture in a 50℃ water bath for 4 hours, and then let it stand at 30℃ for 70 hours to obtain the crosslinked gel. Cut the crosslinked gel into small pieces, soak them in phosphate buffer solution for 3 days, changing the solution every 12 hours, and granulate by sieving to obtain the composite gel.

[0072] Step 4: Fill 5g of composite gel into a pre-filled syringe and perform moist heat sterilization. Under sterile conditions, add 30μg of growth factor and mix evenly. Then perform aseptic filling to obtain silk fibroin-based biomaterial.

[0073] The growth factors underwent coating modification treatment, the specific process of which is as follows:

[0074] Step A: Disperse 30 μg of growth factor in 120 mL of 0.03 g / mL sodium alginate aqueous solution to obtain a mixed solution. Add 160 mL of 0.5 mol / L calcium chloride solution dropwise over 1.5 h. Stir for 35 min, filter, wash, and freeze-dry to obtain CA microspheres.

[0075] Step B: Disperse 30 μg of CA microspheres in 120 mL of 0.03 g / mL modified chitosan solution, stir for 20 min, filter, wash, and freeze dry to obtain CA-CS composite microspheres;

[0076] Step C: Disperse 30 μg of CA-CS composite microspheres in 120 mL of 4 wt% aqueous solution containing double-bonded hyaluronic acid to obtain a mixed solution containing double bonds. Add a mixed solution of 1.4 g of thiolated collagen, 10 g of thiolated silk fibroin and 56 g of deionized water, and then add 0.03 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. After stirring thoroughly, cure under ultraviolet light, dialyze, and freeze-dry to obtain the coated modified growth factor.

[0077] The preparation method of modified chitosan solution is as follows:

[0078] Sodium polyglutamate, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 3-amino-1,2-propanediol were mixed evenly in a mass ratio of 1:60:4.5:2.0:0.07 and reacted for 12 h. After dialyzing and freeze-drying, hydroxylated polyglutamic acid was obtained. Hydroxylated polyglutamic acid, deionized water, and sodium periodate were mixed evenly in a mass ratio of 1:30:1.4 and oxidized in the dark for 30 min. After dialyzing and freeze-drying, aldehyde-modified polyglutamic acid was obtained.

[0079] Methacrylamide chitosan, aldehyde-modified polyglutamic acid, and phosphate buffer solution were mixed evenly and reacted at 40°C for 25 min to obtain a modified chitosan solution. The molar ratio of amino groups in methacrylamide chitosan to aldehyde groups in aldehyde-modified polyglutamic acid was 1:0.6. The mass of the phosphate buffer solution was 4 times the total mass of methacrylamide chitosan and aldehyde-modified polyglutamic acid.

[0080] The preparation method of thiolized collagen is as follows:

[0081] 2g of collagen was added to 750g of 0.5M acetic acid solution and stirred to dissolve, thus obtaining a collagen solution. The collagen solution was transferred to a dialysis bag and dialyzed at 5°C with alkaline phosphate buffer solution. Dialysis was performed until the pH reached 9.5. 2g of DL-N-acetylhomocysteine ​​thiolactone and 0.15g of ethylenediaminetetraacetic acid were added, and the reaction was carried out under nitrogen protection for 23h. Then, 0.1g of dithiothreitol was added, and stirring was continued for 11h. Dialysis and freeze-drying were performed to obtain thiolized collagen.

[0082] Example 4: A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, comprising the following processes:

[0083] Step 1: Take degummed silk fibers and dissolve them in 9.3 mol / L lithium bromide solution at a bath ratio of 1:10 at a dissolution temperature of 62℃ to obtain dissolved silk fibroin; cool the dissolved silk fibroin and put it into a dialysis bag with a molecular weight cutoff of 14 kDa. Dialyze it with flowing purified water for three days and then take it out. Filter the solution through gauze and centrifuge it at 4℃ and 9000 rpm for 20 min to obtain a clear 6 wt% silk fibroin solution;

[0084] Step 2: Mix 50g of 6wt% silk fibroin solution, 60g of 5wt% sodium hyaluronate solution and 180g of water for injection evenly, add 1g of 1,4-butanediol diglycidyl ether, stir thoroughly for 60min, keep warm at 90℃ for 1h, freeze dry, pulverize and pass through a 200-mesh sieve to obtain SF-HA freeze-dried powder.

[0085] Step 3: Mix 0.5g of 1,4-butanediol diglycidyl ether and 30g of 1wt% sodium hydroxide solution evenly, add 3g of sodium hyaluronate and mix evenly, then add 2.5g of SF-HA lyophilized powder, crosslink in a 60℃ water bath for 3h, and then let stand at 35℃ for 60h to obtain the crosslinked gel; cut the crosslinked gel into small pieces, soak in phosphate buffer solution for 4 days, change the solution every 12h, sieve and granulate to obtain the composite gel;

[0086] Step 4: Fill 5g of composite gel into a pre-filled syringe and perform moist heat sterilization. Under sterile conditions, add 40μg of growth factor and mix evenly. Then perform aseptic filling to obtain silk fibroin-based biomaterial.

[0087] The growth factors underwent coating modification treatment, the specific process of which is as follows:

[0088] Step A: Disperse 40 μg of growth factor in 150 mL of 0.04 g / mL sodium alginate aqueous solution to obtain a mixed solution. Add 200 mL of 1 mol / L calcium chloride solution dropwise over 2 hours. Stir for 40 minutes, filter, wash, and freeze-dry to obtain CA microspheres.

[0089] Step B: Disperse 40 μg of CA microspheres in 150 mL of 0.04 g / mL modified chitosan solution, stir for 30 min, filter, wash, and freeze dry to obtain CA-CS composite microspheres;

[0090] Step C: Disperse 40 μg of CA-CS composite microspheres in 150 mL of 3 wt% aqueous solution containing double-bonded hyaluronic acid to obtain a mixed solution containing double bonds. Add a mixed solution of 1.8 g of thiolated collagen, 18 g of thiolated silk fibroin and 90 g of deionized water, and then add 0.054 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. After stirring thoroughly, cure under ultraviolet light (380 nm), dialyze, and freeze-dry to obtain the coated modified growth factor.

[0091] The preparation method of modified chitosan solution is as follows:

[0092] Sodium polyglutamate, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 3-amino-1,2-propanediol were mixed evenly in a mass ratio of 1:70:5:2.5:0.08 and reacted for 24 h. After dialyzing and freeze-drying, hydroxylated polyglutamic acid was obtained. Hydroxylated polyglutamic acid, deionized water, and sodium periodate were mixed evenly in a mass ratio of 1:40:1.5 and oxidized in the dark for 60 min. After dialyzing and freeze-drying, aldehyde-modified polyglutamic acid was obtained.

[0093] Methacrylamide chitosan, aldehyde-modified polyglutamic acid, and phosphate buffer solution were mixed evenly and reacted at 50°C for 30 min to obtain a modified chitosan solution. The molar ratio of amino groups in methacrylamide chitosan to aldehyde groups in aldehyde-modified polyglutamic acid was 1:0.7. The mass of the phosphate buffer solution was 5 times the total mass of methacrylamide chitosan and aldehyde-modified polyglutamic acid.

[0094] The preparation method of thiolized collagen is as follows:

[0095] 2g of collagen was added to 1000g of 0.6M acetic acid solution and stirred to dissolve, thus obtaining a collagen solution. The collagen solution was transferred to a dialysis bag and dialyzed at 6°C with alkaline phosphate buffer solution. Dialysis was performed until the pH reached 10. 3g of DL-N-acetylhomocysteine ​​thiolactone and 0.2g of ethylenediaminetetraacetic acid were added, and the reaction was carried out under nitrogen protection for 24h. Then, 0.15g of dithiothreitol was added, and stirring was continued for 12h. Dialysis and freeze-drying were performed to obtain thiolized collagen.

[0096] Comparative Example 1: Comparative Example 1 is based on Example 2. In Comparative Example 2, the modified chitosan was replaced with the same mass of chitosan, and the remaining process steps and reaction parameters were the same as in Example 2.

[0097] Comparative Example 2: Comparative Example 2 is based on Example 2. Comparative Example 3 does not introduce thiolized silk fibroin. The remaining process steps and reaction parameters are the same as in Example 2.

[0098] experiment:

[0099] Injectability test: The silk fibroin-based biomaterials obtained in Examples 1-4 were loaded into a 1 mL syringe, and a 27G injection needle was attached to simulate actual use. The push rod was pushed at a constant speed of 30 mm / min, with a pushing displacement of 20 mm, and the sample in the syringe was extruded through the needle. Within a certain compression displacement, the compressive load of the injectable gel varied between 15 and 17 N.

[0100] Swelling degree test: The silk fibroin-based biomaterials obtained in Examples 1-4 were dried in a vacuum drying oven at 40°C until the weight of the gel remained constant. The weight was recorded as WD. The gel was then placed in distilled water at 34°C for 24 hours. After swelling equilibrium, the gel was quickly removed, dried, and weighed, which was recorded as WT. The swelling degree SR was calculated as (WT-WD) / WD. The test results are shown in Table 1.

[0101] Table 1. Performance test results of silk fibroin-based biomaterials containing growth factors for injection.

[0102]

[0103] In vitro release rate test of growth factors: The silk fibroin-based biomaterials obtained in Examples 1-4 and Comparative Examples 1-2 were soaked in sterile PBS buffer. After swelling equilibrium, they were taken out, washed three times with sterile PBS buffer solution, and then placed in centrifuge tubes containing sterile PBS buffer solution. The tubes were kept at a constant temperature of 34°C in air atmosphere and centrifuged at 2, 8, 16, 24, 72, 144, and 240 h. The same amount of sterile phosphate buffer solution was added. The amount of growth factors released from the gel was determined by ELISA method, and the cumulative release rate was calculated. The test results are shown in Table 2.

[0104] Table 2 Results of in vitro release rate test of growth factors

[0105]

[0106] Based on the data in the table above, the following conclusions can be clearly drawn:

[0107] This invention extends the degradation cycle of products by preparing silk fibroin-based biomaterials, while simultaneously ensuring material uniformity, biocompatibility, and retention of active ingredients. In Examples 2-4, the cumulative release rate of growth factors increased slowly over time; however, in Example 1, the cumulative release rate of growth factors increased rapidly over time and ultimately could not be completely released. This indicates that the coating modification treatment effectively controls the slow release of growth factors and improves the compatibility between growth factors and the composite gel.

[0108] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a silk fibroin-based biomaterial containing growth factors for injection, characterized in that: Includes the following steps: Step 1: Take degummed silk fibers and dissolve them in lithium bromide solution to obtain dissolved silk fibroin; cool the dissolved silk fibroin, and then dialyze, filter, and centrifuge to obtain silk fibroin solution; Step 2: Mix the silk fibroin solution, sodium hyaluronate solution and water for injection evenly, add 1,4-butanediol diglycidyl ether, stir thoroughly for 30-60 minutes, keep warm at 70-90℃ for 1-3 hours, freeze dry, pulverize and sieve to obtain SF-HA freeze-dried powder; Step 3: Mix 1,4-butanediol diglycidyl ether and sodium hydroxide solution evenly, add sodium hyaluronate and mix evenly, then add SF-HA lyophilized powder, crosslink in a water bath at 30-60℃ for 3-5 hours, and then let stand at 25-35℃ for 60-80 hours to obtain the crosslinked gel; cut the crosslinked gel into small pieces, soak in phosphate buffer solution for 2-4 days, change the solution every 12 hours, sieve and granulate to obtain the composite gel; Step 4: Fill the composite gel into a pre-filled syringe, perform moist heat sterilization, add growth factors under sterile conditions, mix evenly, and then perform aseptic filling to obtain silk fibroin-based biomaterial. The growth factor undergoes a coating modification process, the specific process of which is as follows: Step A: Disperse the growth factor in an aqueous solution of sodium alginate to obtain a mixed solution, add calcium chloride solution dropwise over 1-2 hours, stir for 30-40 minutes, filter, wash, and freeze-dry to obtain CA microspheres; Step B: Disperse CA microspheres in a modified chitosan solution, stir for 20-30 min, filter, wash, and freeze-dry to obtain CA-CS composite microspheres; Step C: Disperse CA-CS composite microspheres in an aqueous solution containing double-bonded hyaluronic acid to obtain a mixed solution containing double bonds. Add a mixed solution of thiolized collagen, thiolized silk fibroin and deionized water, then add lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. After stirring thoroughly, cure under ultraviolet light, dialyze, and freeze-dry to obtain the coated modified growth factor. The modified chitosan solution is prepared as follows: Sodium polyglutamate was aldehyde-treated to obtain aldehyde-modified polyglutamate; methacrylamide chitosan, aldehyde-modified polyglutamate and phosphate buffer solution were mixed evenly and reacted at 30-50℃ for 20-30 min to obtain modified chitosan solution. The molar ratio of the amino group of the methacrylated chitosan to the aldehyde group of the aldehyde-modified polyglutamic acid is 1:(0.5-0.7). The hyaluronic acid containing double bonds is methacrylamide hyaluronic acid.

2. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: In step one, the bath ratio of the degummed filament to lithium bromide is 1:(4-10), the concentration of the lithium bromide solution is 9.3mol / L, the dissolution temperature is 58-62℃, and the concentration of the silk fibroin solution is 3-6wt%.

3. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: In step two, the mass ratio of the silk fibroin solution, sodium hyaluronate solution, water for injection, and 1,4-butanediol diglycidyl ether is 1:(1.0-1.2):(3.4-3.6):(0.01-0.02), and the concentration of the sodium hyaluronate solution is 1-5 wt%.

4. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: In step three, the mass ratio of 1,4-butanediol diglycidyl ether, sodium hydroxide solution, sodium hyaluronate and SF-HA lyophilized powder is 1:(50-60):(4-6):(3-5), and the concentration of sodium hydroxide solution is 1.0wt%.

5. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: In step B, the concentration of modified chitosan in the modified chitosan solution is 0.02 g / mL to 0.04 g / mL.

6. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: The specific steps for preparing the aldehyde-modified polyglutamic acid are as follows: Sodium polyglutamate and deionized water are mixed evenly, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and 3-amino-1,2-propanediol are added, the reaction is carried out for 12-24 hours, dialyzed and freeze-dried to obtain hydroxylated polyglutamic acid; the hydroxylated polyglutamic acid is dissolved in deionized water, sodium periodate is added, oxidized in the dark for 30-60 minutes, dialyzed and freeze-dried to obtain aldehyde-modified polyglutamic acid.

7. The method for preparing an injectable silk fibroin-based biomaterial containing growth factors according to claim 1, characterized in that: The method for preparing the thiolized collagen is as follows: Collagen was added to an acetic acid solution and stirred to dissolve, thus obtaining a collagen solution. The collagen solution was transferred to a dialysis bag and dialyzed at 4-6°C with an alkaline phosphate buffer solution. Dialysis was performed until the pH reached 9-10. DL-N-acetylhomocysteine ​​and ethylenediaminetetraacetic acid were added, and the reaction was carried out under nitrogen protection for 22-24 hours. Dithiothreitol was then added, and stirring was continued for 10-12 hours. The mixture was then dialyzed and freeze-dried to obtain thiolized collagen.

8. A silk fibroin-based biomaterial containing growth factors for injection, prepared by the method according to any one of claims 1-7.

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