Preparation method of a polymer filler containing silk fibroin particles

Abstract

The present invention discloses a preparation method of a polymer filler containing silk fibroin particles, which relates to the technical field of polymer fillers. In the present invention, silk fibroin gel particles and concentrated silk fibroin solution are added simultaneously. The self-reinforced assembly of concentrated silk fibroin and silk fibroin gel improves the mechanical properties of the silk fibroin particles. The present invention also adds sheep collagen fibers and thiolated hyaluronic acid, which can prolong the degradation time and provide a scaffold space for tissue regeneration. At the same time, hydrogen peroxide is used for crosslinking. By controlling the concentration of hydrogen peroxide to be 5-5.5 mmol / L, the stability of the material is increased and its biodegradation rate is reduced, thereby further prolonging the degradation time.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer fillers, and specifically to a preparation method of a polymer filler containing silk fibroin particles. Background Art

[0002] Silk fibroin is a natural and ancient protein material that can be extracted from artificially cultivated silkworm cocoons. It has good biocompatibility, adjustable biodegradability, and non-immunogenicity. It can be used as a suture material in the biomedical field and is a clinical biocompatible material. Silk fibroin materials are easy to process but are thermodynamically unstable and can be regulated by physical and chemical methods to increase the content of β-sheet structures in silk fibroin, gradually transforming it into a thermodynamically stable structure. This structural controllability and rapid transformation ability enable silk fibroin materials to be flexibly regulated in terms of their morphological appearance, mechanical properties, biodegradation properties, etc. when used as tissue engineering materials.

[0003] On the other hand, silk fibroin is rich in 18 kinds of amino acids. Not only is it non-toxic to the tissue body itself, but its degradation products also do not cause adverse reactions. At the same time, there are already many dressing products containing silk fibroin components on the market, which proves the biosafety of silk fibroin. Therefore, silk fibroin can be used as a raw material for implantable fillers. Different tissue sites have different requirements for the size of the filler particles. Silk fibroin has good morphological processability, enabling silk fibroin particles to be compounded with other polymers such as collagen and hyaluronic acid.

[0004] To solve the above problems, the present invention provides a preparation method of a polymer filler containing silk fibroin particles. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a polymer filler containing silk fibroin particles to solve the problems raised in the prior art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A preparation method of a polymer filler containing silk fibroin particles, comprising the following steps:

[0008] Step 1: Take degummed silk, dissolve it with a lithium bromide solution to obtain dissolved silk fibroin; cool, dialyze, and take out the dissolved silk fibroin to obtain a silk fibroin solution;

[0009] Step 2: Take the silk fibroin solution, filter and centrifuge it, concentrate it at 60 - 65 °C, and dilute it with injection water to obtain a diluted silk fibroin solution;

[0010] Step 3: Place the diluted silk fibroin solution at 60 - 65 °C until a silk fibroin gel is formed;

[0011] Step 4: Take the silk fibroin gel, granulate it, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, mix evenly, place it at -40 °C to -80 °C and freeze for 24 - 48 h, then lyophilize it, place it at 25 - 30 °C to absorb moisture for 1 - 3 d, and pulverize it to obtain silk fibroin freeze-dried particles;

[0012] Step 5: Mix the silk fibroin freeze-dried particles, mercapto-modified hyaluronic acid, and deionized water evenly, adjust the pH value of the system to 8 - 8.5 using a sodium hydroxide solution, add mercapto-modified sheep collagen fiber particles, mix evenly under the condition of 0 - 2 °C, add hydrogen peroxide, react at 35 - 40 °C for 20 - 30 min, dialyze and freeze-dry to obtain Particle A; Take Particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin protein particles.

[0013] More optimally, in Step 1, the bath ratio of degummed silk to lithium bromide is 1:(4 - 10).

[0014] More optimally, in Step 2, the concentration of the diluted silk fibroin solution is 2 wt% - 6 wt%.

[0015] More optimally, in Step 4, the mass ratio of silk in the granulated silk fibroin gel particles to the concentrated silk fibroin solution is 1:(0.2 - 2).

[0016] More optimally, in Step 2, the centrifugation conditions are: centrifuge at 9000 rpm for 20 - 30 min.

[0017] More optimally, the preparation method of the mercapto-modified sheep collagen fiber particles is: Mix sheep collagen fibers and acetic acid evenly to obtain a sheep collagen fiber solution, cool it to 2 - 3 °C, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, mix evenly, react for 30 - 40 min, then dropwise add a cysteamine hydrochloride solution, add a 2-(N-morpholino)ethanesulfonic acid solution, adjust the pH value of the solution to 5.5 - 6, stir for 4 - 5 h, react at 4 - 5 °C for 10 - 14 h, dialyze to obtain a mercapto-modified sheep collagen fiber solution, and lyophilize it to obtain mercapto-modified sheep collagen fiber particles.

[0018] More optimally, the preparation method of the sheep collagen fibers is: Dissolve sheep collagen powder in acetic acid, dialyze to neutral to obtain a sheep collagen solution, add a phosphate buffer solution, mix evenly, centrifuge, then resuspend the precipitate in the phosphate buffer solution, centrifuge, and dry to obtain sheep collagen fibers.

[0019] Preferably, the method for preparing the sheep collagen powder is as follows: take sheepskin, break it up, wash it, perform degreasing treatment with n-butanol, centrifuge and wash with water, then extract collagen using an acetic acid solution containing pepsin, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain the sheep collagen powder.

[0020] Preferably, the concentration of the hydrogen peroxide is 5 - 5.5 mmol / L.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. When silk fibroin particles are compounded with substances such as collagen and hyaluronic acid and used as fillers for various tissues, they can possess the advantages of multiple materials. At this time, the polymer filler can play a role in supporting and filling. As the degradation process progresses, the silk fibroin particles are gradually released and play a role in stimulating tissue regeneration again.

[0023] 2. The present invention simultaneously adds silk fibroin gel particles and concentrated silk fibroin solution. The self-reinforcing assembly of concentrated silk fibroin and silk fibroin gel improves the mechanical properties of the silk fibroin particles.

[0024] 3. The present invention adds sheep collagen fibers and mercapto-modified hyaluronic acid, which can extend the degradation time and provide a scaffold space for tissue regeneration. The present invention also uses N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2-(N-morpholino)ethanesulfonic acid to perform mercapto-modification on the sheep collagen fibers, so that the sheep collagen fibers contain mercapto groups. There are also mercapto groups on the mercapto-modified hyaluronic acid and freeze-dried silk fibroin particles. The three have good compatibility, better extending the degradation time. At the same time, the present invention also uses hydrogen peroxide to crosslink them. By controlling the concentration of hydrogen peroxide at 5 - 5.5 mmol / L, the stability of the material is increased and its biodegradation rate is reduced, thereby further extending the degradation time. Specific embodiments

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] There are no special restrictions on the source and model of the substances involved in the present invention. Exemplarily, they include: mercapto-modified hyaluronic acid: model: SH-HA-372, which can be purchased from Feifan Standard Technology Service Co., Ltd.; 6A raw silk, sourced from Hai'an Suhao Silk Co., Ltd., and degummed silk is obtained after the degumming process of raw silk by this company; pepsin: model: S10027, which can be purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; sodium hyaluronate gel: model: 20200701, which can be purchased from Shandong Baolijia Biotechnology Co., Ltd.

[0027] Example 1: A preparation method of a polymer filler containing silk fibroin particles, comprising the following steps:

[0028] Step 1: Preparation of freeze-dried silk fibroin particles:

[0029] Take degummed silk, dissolve it with 9.3 mol / L lithium bromide solution at 60 °C, and the bath ratio of degummed silk to lithium bromide is 1:5 to obtain dissolved silk fibroin;

[0030] Cool the dissolved silk fibroin, load it into a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days to obtain a silk fibroin solution;

[0031] Take the silk fibroin solution, filter it, centrifuge it at 4 °C and 9000 rpm for 25 min to obtain a 5 wt% silk fibroin solution; concentrate it at 62 °C until it reaches 17 wt%; add injection water to the concentrated silk fibroin solution and dilute it to 4 wt% to obtain a diluted silk fibroin solution;

[0032] Place the diluted silk fibroin solution at 60 °C until a silk fibroin gel is formed;

[0033] Granulate the silk fibroin gel through a sieve, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, and the mass ratio of silk fibroin in the granulated silk fibroin gel particles to the concentrated silk fibroin solution is 1:1; mix evenly, freeze at -60 °C for 36 h, freeze-dry, place it at room temperature to absorb moisture for 2 d, and pulverize to obtain freeze-dried silk fibroin particles;

[0034] Step 2: Preparation of a polymer filler containing silk fibroin particles:

[0035] Mix 18 g of freeze-dried silk fibroin particles, 5 g of mercapto-modified hyaluronic acid, and 1000 mL of deionized water evenly, adjust the pH value of the system to 8 using 1 mol / L sodium hydroxide solution, mix evenly under the condition of 1 °C, add 5 mmol / L hydrogen peroxide, react at 38 °C for 25 min, dialyze and freeze-dry to obtain particle A; take 16 parts of particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin particles.

[0036] Example 2: A method for preparing a polymer filler containing silk fibroin particles, comprising the following steps:

[0037] Step 1: Preparation of freeze-dried silk fibroin particles:

[0038] Take degummed silk, dissolve it in a 9.3 mol / L lithium bromide solution at 60 °C, with a bath ratio of degummed silk to lithium bromide of 1:5, to obtain dissolved silk fibroin;

[0039] Cool the dissolved silk fibroin, load it into a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days, to obtain a silk fibroin solution;

[0040] Take the silk fibroin solution, filter it, centrifuge it at 4 °C and 9000 rpm for 25 min to obtain a silk fibroin solution with a concentration of 5 wt%; concentrate it at 62 °C until it reaches 17 wt%; add injection water to the concentrated silk fibroin solution and dilute it to 4 wt% to obtain a diluted silk fibroin solution;

[0041] Place the diluted silk fibroin solution at 60 °C until a silk fibroin gel is formed;

[0042] Granulate the silk fibroin gel through a sieve, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, with a mass ratio of silk fibroin in the granulated silk fibroin gel particles to the concentrated silk fibroin solution of 1:1; mix evenly, freeze at -60 °C for 36 h, lyophilize, place at room temperature to absorb moisture for 2 d, and pulverize to obtain freeze-dried silk fibroin particles;

[0043] Step 2: Preparation of sheep collagen fibers:

[0044] Take sheepskin, break it up, wash it, degrease it using 10% n-butanol, centrifuge and wash with water, and then extract collagen using a 0.5 M acetic acid solution containing 1 g / L pepsin, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain sheep collagen powder;

[0045] Preparation of sheep collagen fibers:

[0046] Take the sheep collagen powder and dissolve it in 0.5 M acetic acid, dialyze it to neutral to obtain a 3.5 mg / mL sheep collagen solution, add 20 mM phosphate buffer, mix evenly, centrifuge, and then resuspend the precipitate in the phosphate buffer at a concentration of 3 mg / mL, centrifuge, and dry to obtain sheep collagen fibers;

[0047] Step 3: Preparation of mercapto-functionalized sheep collagen fibers:

[0048] Mix 3 g of sheep collagen fibers, 50 mL of 0.5 M acetic acid evenly to obtain a sheep collagen fiber solution. Cool down to 2 °C, add 0.2 g of N-hydroxysuccinimide and 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, mix evenly, react for 35 min, then dropwise add a 50 mg / mL cysteamine hydrochloride solution to make the final concentration of cysteamine hydrochloride in the solution 55 mmol / L. Add 2-(N-morpholino)ethanesulfonic acid solution to adjust the pH value of the solution to 5.5, stir for 4.5 h, react at 4 °C for 12 h, dialyze to obtain a thiolated sheep collagen fiber solution, and freeze-dry to obtain thiolated sheep collagen fiber particles;

[0049] Step 4: Preparation of a polymer filler containing silk fibroin particles:

[0050] Mix 18 g of freeze-dried silk fibroin particles, 5 g of thiolated hyaluronic acid, and 1000 mL of deionized water evenly. Use 1 mol / L sodium hydroxide solution to adjust the pH value of the system to 8, add 3 g of thiolated sheep collagen fiber particles, mix evenly under the condition of 1 °C, add 5 mmol / L hydrogen peroxide, react at 38 °C for 25 min, dialyze and freeze-dry to obtain Particle A; Take 16 parts of Particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin particles.

[0051] Example 3: A preparation method of a polymer filler containing silk fibroin particles, comprising the following steps:

[0052] Step 1: Preparation of freeze-dried silk fibroin particles:

[0053] Take degummed silk, dissolve it with 9.3 mol / L lithium bromide solution at 60 °C, and the bath ratio of degummed silk to lithium bromide is 1:4 to obtain dissolved silk fibroin;

[0054] Cool the dissolved silk fibroin, load it into a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days to obtain a silk fibroin solution;

[0055] Take the silk fibroin solution, filter it, centrifuge it at 5 °C and 9000 rpm for 20 min to obtain a silk fibroin solution with a concentration of 3 wt%; concentrate it at 60 °C to 15 wt%; add injection water to the concentrated silk fibroin solution and dilute it to 2 wt% to obtain a diluted silk fibroin solution;

[0056] Place the diluted silk fibroin solution at 60 °C again until a silk fibroin gel is formed;

[0057] The silk fibroin gel is granulated through a sieve. A concentrated silk fibroin solution is added to the granulated silk fibroin gel particles. The mass ratio of silk fibroin in the granulated silk fibroin gel particles to that in the concentrated silk fibroin solution is 1:0.5. After mixing evenly, it is frozen at -40°C for 24 h, freeze-dried, placed at room temperature to absorb moisture for 1 d, and then pulverized to obtain silk fibroin freeze-dried particles.

[0058] Step 2: Preparation of sheep collagen fibers:

[0059] Take sheepskin, break it up, wash it, and perform degreasing treatment with 10% n-butanol. Centrifuge and wash with water. Then, use a 0.5 M acetic acid solution containing 1 g / L pepsin for collagen extraction, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain sheep collagen powder.

[0060] Dissolve the sheep collagen powder in 0.5 M acetic acid, dialyze until neutral to obtain a 3.5 mg / mL sheep collagen solution. Add 20 mM phosphate buffer, mix evenly, centrifuge, and then resuspend the precipitate in the phosphate buffer at a concentration of 3 mg / mL. Centrifuge and dry to obtain sheep collagen fibers.

[0061] Step 3: Preparation of mercapto-modified sheep collagen fibers:

[0062] Mix 3 g of sheep collagen fibers, 50 mL of 0.5 M acetic acid evenly to obtain a sheep collagen fiber solution. Cool the temperature to 2°C, add 0.2 g of N-hydroxysuccinimide and 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, mix evenly, and react for 30 min. Then, dropwise add a 50 mg / mL cysteamine hydrochloride solution to make the final concentration of cysteamine hydrochloride in the solution 55 mmol / L. Add 2-(N-morpholino)ethanesulfonic acid solution to adjust the pH value of the solution to 5.5, stir for 4 h, react at 4°C for 10 h, dialyze to obtain a mercapto-modified sheep collagen fiber solution, and freeze-dry to obtain mercapto-modified sheep collagen fiber particles.

[0063] Step 4: Preparation of a polymer filler containing silk fibroin particles:

[0064] Mix 18 g of silk fibroin freeze-dried particles, 5 g of mercapto-modified hyaluronic acid, and 1000 mL of deionized water evenly. Use 1 mol / L sodium hydroxide solution to adjust the pH value of the system to 8. Add 3 g of mercapto-modified sheep collagen fiber particles, mix evenly under the condition of 0°C, add 5 mmol / L hydrogen peroxide, react at 35°C for 20 min, dialyze and freeze-dry to obtain particle A. Take 16 parts of particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin particles.

[0065] Example 4: A preparation method of a polymer filler containing silk fibroin particles, comprising the following steps:

[0066] Step 1: Preparation of freeze-dried silk fibroin particles:

[0067] Take degummed silk, dissolve it in 9.3 mol / L lithium bromide solution at 60 °C, and the bath ratio of degummed silk to lithium bromide is 1:10 to obtain dissolved silk fibroin;

[0068] Cool the dissolved silk fibroin, put it into a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days to obtain a silk fibroin solution;

[0069] Take the silk fibroin solution, filter it, centrifuge it at 5 °C and 9000 rpm for 30 min to obtain a silk fibroin solution with a concentration of 6 wt%; concentrate it at 65 °C until it reaches 20 wt%; add injection water to the concentrated silk fibroin solution and dilute it to 6 wt% to obtain a diluted silk fibroin solution;

[0070] Place the diluted silk fibroin solution at 60 °C until a silk fibroin gel is formed;

[0071] Granulate the silk fibroin gel through a sieve, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, and the mass ratio of silk fibroin in the granulated silk fibroin gel particles to the concentrated silk fibroin solution is 1:2; mix evenly, freeze at -80 °C for 48 h, freeze-dry, place it at room temperature to absorb moisture for 3 d, and pulverize to obtain freeze-dried silk fibroin particles;

[0072] Step 2: Preparation of sheep collagen fibers:

[0073] Take sheepskin, break it up, wash it, degrease it with 10% n-butanol, centrifuge and wash with water, and then extract collagen with 0.5 M acetic acid solution containing 1 g / L pepsin, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain sheep collagen powder;

[0074] Preparation of sheep collagen fibers:

[0075] Take the sheep collagen powder and dissolve it in 0.5 M acetic acid, dialyze it to neutral to obtain a sheep collagen solution with a concentration of 3.5 mg / mL, add 20 mM phosphate buffer, mix evenly, centrifuge, and then resuspend the precipitate in the phosphate buffer with a concentration of 3 mg / mL, centrifuge, and dry to obtain sheep collagen fibers;

[0076] Step 3: Preparation of mercapto-modified sheep collagen fibers:

[0077] Mix 3 g of sheep collagen fibers, 50 mL of 0.5 M acetic acid evenly to obtain a sheep collagen fiber solution. Cool the solution to 3 °C, add 0.2 g of N-hydroxysuccinimide and 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, mix evenly, and react for 40 min. Then, add a 50 mg / mL cysteamine hydrochloride solution to make the final concentration of cysteamine hydrochloride in the solution 55 mmol / L. Add a 2-(N-morpholino)ethanesulfonic acid solution to adjust the pH value of the solution to 6, stir for 5 h, react at 5 °C for 14 h, dialyze to obtain a thiolated sheep collagen fiber solution, and lyophilize to obtain thiolated sheep collagen fiber particles;

[0078] Step 4: Preparation of a polymer filler containing silk fibroin particles:

[0079] Mix 18 g of freeze-dried silk fibroin particles, 5 g of thiolated hyaluronic acid, and 1000 mL of deionized water evenly. Use a 1 mol / L sodium hydroxide solution to adjust the pH value of the system to 8, add 3 g of thiolated sheep collagen fiber particles, mix evenly under the condition of 2 °C, add 5 mmol / L hydrogen peroxide, react at 40 °C for 30 min, dialyze and freeze-dry to obtain Particle A; Take 16 parts of Particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin particles.

[0080] Comparative Example 1: Do not perform thiolation modification on sheep collagen fibers, and the rest is the same as in Example 2:

[0081] Step 1: Preparation of freeze-dried silk fibroin particles:

[0082] Take degummed silk, dissolve it with 9.3 mol / L lithium bromide solution at 60 °C, and the bath ratio of degummed silk to lithium bromide is 1:5 to obtain dissolved silk fibroin;

[0083] Cool the dissolved silk fibroin, put it into a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days to obtain a silk fibroin solution;

[0084] Take the silk fibroin solution, filter it, and centrifuge it at 4 °C and 9000 rpm for 25 min to obtain a 5 wt% silk fibroin solution; Concentrate it at 62 °C to 17 wt%; Add injection water to the concentrated silk fibroin solution and dilute it to 4 wt% to obtain a diluted silk fibroin solution;

[0085] Place the diluted silk fibroin solution at 60 °C again until a silk fibroin gel is formed;

[0086] The silk fibroin gel is granulated through a sieve. A concentrated silk fibroin solution is added to the granulated silk fibroin gel particles. The mass ratio of silk in the granulated silk fibroin gel particles to that in the concentrated silk fibroin solution is 1:1. After mixing evenly, it is frozen at -60°C for 36 h, freeze-dried, placed at room temperature to absorb moisture for 2 d, and then pulverized to obtain silk fibroin freeze-dried particles.

[0087] Step two: Preparation of sheep collagen fibers:

[0088] Take sheepskin, break it up, wash it, and perform degreasing treatment with 10% n-butanol. After centrifugation and washing with water, collagen is extracted using a 0.5 M acetic acid solution containing 1 g / L pepsin. The pH value is adjusted to neutral, the enzyme is inactivated, dialyzed, and freeze-dried to obtain sheep collagen powder.

[0089] Dissolve the sheep collagen powder in 0.5 M acetic acid, dialyze until neutral to obtain a 3.5 mg / mL sheep collagen solution. Add 20 mM phosphate buffer, mix evenly, centrifuge, and then resuspend the precipitate in the phosphate buffer at a concentration of 3 mg / mL. After centrifugation and drying, sheep collagen fibers are obtained.

[0090] Step three: Preparation of sheep collagen fiber particles:

[0091] Mix 3 g of sheep collagen fibers, 50 mL of 0.5 M acetic acid evenly to obtain a sheep collagen fiber solution, and then freeze-dry it to obtain sheep collagen fiber particles.

[0092] Step four: Preparation of a polymer filler containing silk fibroin particles:

[0093] Mix 18 g of silk fibroin freeze-dried particles, 5 g of thiolated hyaluronic acid, and 1000 mL of deionized water evenly. Use 1 mol / L sodium hydroxide solution to adjust the pH value of the system to 8. Add 3 g of sheep collagen fiber particles, mix evenly at 1°C, add 5 mmol / L hydrogen peroxide, react at 38°C for 25 min, dialyze, and then freeze-dry to obtain particle A. Take 16 parts of particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin particles.

[0094] Comparative example 2: Without adding thiolated hyaluronic acid, the rest is the same as in Example 2:

[0095] Step one: Preparation of silk fibroin freeze-dried particles:

[0096] Take degummed silk, dissolve it with 9.3 mol / L lithium bromide solution at 60°C. The bath ratio of degummed silk to lithium bromide is 1:5 to obtain the dissolved silk fibroin.

[0097] Cool the dissolved silk fibroin, place it in a dialysis bag with a molecular weight cut-off of 14 kDa, take it out after dialysis with purified water for three days to obtain a silk fibroin solution;

[0098] Take the silk fibroin solution, filter it, centrifuge it at 4 °C and 9000 rpm for 25 min to obtain a silk fibroin solution with a concentration of 5 wt%; concentrate it at 62 °C to 17 wt%; add water for injection to the concentrated silk fibroin solution and dilute it to 4 wt% to obtain a diluted silk fibroin solution;

[0099] Place the diluted silk fibroin solution at 60 °C until a silk fibroin gel is formed;

[0100] Granulate the silk fibroin gel through a sieve, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, and the mass ratio of silk fibroin in the granulated silk fibroin gel particles to the concentrated silk fibroin solution is 1:1; mix evenly, freeze at -60 °C for 36 h, lyophilize, place at room temperature to absorb moisture for 2 d, and pulverize to obtain silk fibroin protein lyophilized particles;

[0101] Step 2: Preparation of sheep collagen fibers:

[0102] Take sheepskin, break it up, wash it, degrease it with 10% n-butanol, centrifuge and wash with water, and then extract collagen with a 0.5 M acetic acid solution containing 1 g / L pepsin, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain sheep collagen powder;

[0103] Preparation of sheep collagen fibers:

[0104] Dissolve the sheep collagen powder in 0.5 M acetic acid, dialyze to neutral to obtain a 3.5 mg / mL sheep collagen solution, add 20 mM phosphate buffer, mix evenly, centrifuge, and then resuspend the precipitate in the phosphate buffer at a concentration of 3 mg / mL, centrifuge and dry to obtain sheep collagen fibers;

[0105] Step 3: Preparation of mercapto-modified sheep collagen fibers:

[0106] Mix 3 g of sheep collagen fibers, 50 mL of 0.5 M acetic acid evenly to obtain a sheep collagen fiber solution, cool it to 2 °C, add 0.2 g of N-hydroxysuccinimide and 0.4 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, mix evenly, react for 35 min, then add a 50 mg / mL cysteamine hydrochloride solution to make the final concentration of cysteamine hydrochloride in the solution 55 mmol / L, add 2-(N-morpholino)ethanesulfonic acid solution, adjust the pH value of the solution to 5.5, stir for 4.5 h, react at 4 °C for 12 h, dialyze to obtain a mercapto-modified sheep collagen fiber solution, lyophilize to obtain mercapto-modified sheep collagen fiber particles;

[0107] Step 4: Preparation of the polymer filler containing silk fibroin particles:

[0108] Mix 18 g of freeze-dried silk fibroin particles and 1000 mL of deionized water evenly, adjust the pH value of the system to 8 using 1 mol / L sodium hydroxide solution, add 3 g of mercapto-group modified sheep collagen fiber particles, mix evenly under the condition of 1 °C, add 5 mmol / L hydrogen peroxide, react at 38 °C for 25 min, dialyze and freeze-dry to obtain Particle A; Take 16 parts of Particle A and sodium hyaluronate gel, mix evenly to obtain the polymer filler containing silk fibroin particles.

[0109] Comparative Example 3: In Step 4, the concentration of hydrogen peroxide is 3 mmol / L, and the rest is the same as in Example 2.

[0110] Experiment:

[0111] Experiment 1: In vitro degradation performance test: Take the polymer fillers containing silk fibroin particles obtained in Examples 1-4 and Comparative Examples 1-3, weigh them, record the initial weight as m0, transfer and soak them in a phosphate buffer solution containing 20 U / mL collagenase, and conduct the experiment in a gas bath shaker at a temperature of 37 °C and a shaking speed of 150 rpm; At regular intervals, take out the samples, freeze-dry them, weigh the weight as m1, replace the fresh degradation solution until the condition (m0 - m1) / m0 > 0.99 is met, consider it completely degraded, and record its complete degradation time.

[0112] Experiment 2: Injectability test: Take the polymer fillers containing silk fibroin particles obtained in Examples 1-4 and Comparative Examples 1-3, load them into a 1 mL syringe, install a 27G injection needle, and simulate the actual use situation. Push the plunger at a constant pushing speed of 30 mm / min, the pushing displacement is 20 mm, the sample in the syringe is pushed out through the needle, and within a certain compression displacement, the compression load of the injectable gel varies in the range of 8 to 16 N.

[0113] Degradation time / d Average pushing force / N Example 1 217 10.4 Example 2 267 13.7 Example 3 264 13.5 Example 4 268 13.8 Comparative example 1 244 13.2 Comparative example 2 235 12.1 Comparative example 3 252 11.4

[0114] Conclusion: It can be seen from the data comparison in the table that, compared with Example 1, the average pushing force of Examples 2-4 and Comparative Examples 1-3 has increased slightly, but the pushing force is between 8 and 16 N, which is still suitable for injection use. In Comparative Example 1, the sheep collagen fibers are not modified with mercapto groups, resulting in a decrease in crosslinking and compatibility and a decrease in degradation time. In Comparative Example 2, mercapto-modified hyaluronic acid is not added, resulting in a decrease in anti-degradation performance. In Comparative Example 3, the concentration of hydrogen peroxide is 3 mmol / L. At this time, the concentration of hydrogen peroxide is low, the stability of the material decreases, the biodegradation rate increases, and the degradation time becomes shorter. In Examples 2-4 of the present application, sheep collagen fibers and mercapto-modified hyaluronic acid are added, which can prolong the degradation time and provide a scaffold space for tissue regeneration. The sheep collagen fibers are modified with mercapto groups, so that the sheep collagen fibers contain mercapto groups, and mercapto groups also exist on the mercapto-modified hyaluronic acid and silk fibroin freeze-dried particles. The three have good compatibility and better prolong the degradation time. By controlling the concentration of hydrogen peroxide at 5-5.5 mmol / L, the present invention increases the stability of the material and reduces its biodegradation rate, thereby further prolonging the degradation time.

[0115] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. A preparation method of a polymer filler containing silk fibroin particles, characterized in that: It includes the following steps: Step 1: Take degummed silk, dissolve it with a lithium bromide solution to obtain silk fibroin after dissolution; cool, dialyze, and take out the silk fibroin after dissolution to obtain a silk fibroin solution; Step 2: Take the silk fibroin solution, filter and centrifuge it, concentrate it at 60 - 65 °C, and dilute it with injection water to obtain a diluted silk fibroin solution; Step 3: Place the diluted silk fibroin solution at 60 - 65 °C until a silk fibroin gel is formed; Step 4: Take the silk fibroin gel, granulate it, add the concentrated silk fibroin solution to the granulated silk fibroin gel particles, mix evenly, place it at -40 °C to -80 °C for freezing for 24 - 48 h, freeze-dry it, place it at 25 - 30 °C for moisture absorption for 1 - 3 d, and pulverize it to obtain silk fibroin freeze-dried particles; Step 5: Mix the silk fibroin freeze-dried particles, mercapto-modified hyaluronic acid, and deionized water evenly, adjust the pH value of the system to 8 - 8.5 with a sodium hydroxide solution, add mercapto-modified sheep collagen fiber particles, mix evenly under the condition of 0 - 2 °C, add hydrogen peroxide, react at 35 - 40 °C for 20 - 30 min, dialyze and freeze-dry to obtain particle A; take particle A and sodium hyaluronate gel, mix evenly to obtain a polymer filler containing silk fibroin protein particles.

2. The preparation method of a polymer filler containing silk fibroin particles according to claim 1, characterized in that: In Step 1, the bath ratio of degummed silk to lithium bromide is 1:(4 - 10).

3. The preparation method of a polymer filler containing fibroin protein particles according to claim 1, characterized in that: In Step 2, the concentration of the diluted silk fibroin solution is 2 wt% - 6 wt%.

4. The preparation method of a polymer filler containing silk fibroin particles according to claim 1, characterized in that: In Step 4, the mass ratio of silk fibroin in the granulated silk fibroin gel particles to the concentrated silk fibroin solution is 1:(0.2 - 2).

5. The preparation method of a polymer filler containing fibroin protein particles according to claim 1, characterized in that: In Step 2, the centrifugation conditions are: centrifuge at 9000 rpm for 20 - 30 min.

6. The preparation method of a polymer filler containing silk fibroin particles according to claim 1, characterized in that: The preparation method of the mercapto-modified sheep collagen fiber particles is: mix sheep collagen fibers and acetic acid evenly to obtain a sheep collagen fiber solution, cool it to 2 - 3 °C, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, mix evenly, react for 30 - 40 min, then dropwise add a cysteamine hydrochloride solution, add a 2-(N-morpholino)ethanesulfonic acid solution, adjust the pH value of the solution to 5.5 - 6, stir for 4 - 5 h, react at 4 - 5 °C for 10 - 14 h, dialyze to obtain a mercapto-modified sheep collagen fiber solution, and freeze-dry to obtain mercapto-modified sheep collagen fiber particles.

7. The preparation method of a polymer filler containing fibroin protein particles according to claim 6, characterized in that: The preparation method of the sheep collagen fibers is: take sheep collagen powder, dissolve it in acetic acid, dialyze it to neutral to obtain a sheep collagen solution, add a phosphate buffer solution, mix evenly, centrifuge, then resuspend the precipitate in the phosphate buffer solution, centrifuge, and dry to obtain sheep collagen fibers.

8. The preparation method of a polymer filler containing silk fibroin particles according to claim 7, characterized in that: The preparation method of the sheep collagen powder is: take sheepskin, break it up, wash it, perform degreasing treatment with n-butanol, centrifuge and wash with water, then perform collagen extraction with an acetic acid solution containing pepsin, adjust the pH value to neutral, inactivate the enzyme, dialyze, and freeze-dry to obtain sheep collagen powder.

9. The preparation method of a polymer filler containing silk fibroin particles according to claim 1, characterized in that: The concentration of the hydrogen peroxide is 5 - 5.5 mmol / L.