A preparation method of silk fibroin-hyaluronic acid filler for injection

Abstract

The present invention relates to the field of biomedicine technology, specifically a preparation method of a silk fibroin hyaluronic acid filler for injection. The present invention concentrates a silk fibroin solution to obtain a concentrated silk fibroin solution, and then dilutes it to obtain a diluted silk fibroin solution; the diluted silk fibroin solution is heated to form a silk fibroin gel, granulated to obtain silk fibroin gel particles; 1,4-butanediol diglycidyl ether and a sodium hydroxide solution are mixed uniformly, hyaluronic acid and 1 / 3 of the mass fraction of hydroxyapatite are added, after stirring uniformly, swelling, purification and granulation are carried out, and the remaining mass fraction of hydroxyapatite is added to obtain composite gel particles; the composite gel particles and the silk fibroin gel particles are mixed uniformly, added to the gel to mix, and a silk fibroin hyaluronic acid filler is obtained, which effectively prolongs its degradation time.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a silk fibroin-hyaluronic acid filler for injection. Background Art

[0002] With the improvement of living standards, people's understanding of beauty and their requirements for image and face are gradually increasing. The development of materials and technologies in the medical beauty industry has made it possible to delay natural physiological aging, and it has also provided more options, which has attracted more and more attention. With the increase of age, facial hyaluronic acid (HA) is lost, and collagen in the dermis is reduced, leading to physiological aging problems such as facial sagging and wrinkles. These problems can be improved and solved to a certain extent by injectable soft tissue filling materials. Facial filling materials have been introduced as early as a hundred years ago. In 1893, autologous fat filling was considered the most ideal alternative material for soft tissue expansion. Although medical silicone has the same effect, it is no longer recommended for soft tissue filling due to too many serious complications. By the 1970s, scholars at Stanford University discovered the earliest injectable collagen, and decades later collagen was certified by the US FDA for medical applications. The market for cosmetic filling materials has undergone revolutionary progress. At present, the filling materials on the market are mainly divided into two categories: degradable biomaterials (such as collagen, HA, etc.) and non-degradable biomaterials (such as polyacrylamide, etc.). Although existing products are widely used and show good results, some are potentially carcinogenic, some are immunogenic, and some degrade too quickly, limiting their widespread application.

[0003] Injectable cross-linked HA products have become one of the most widely used soft tissue filling materials on the market due to their excellent hydrophilicity, biocompatibility, non-immunogenicity and biodegradability. The strong hydrophilicity of HA can bring hydration to the skin. The cross-linked HA prepared with 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent can prolong the retention time of the material in the body to a certain extent (about 6 months). However, the cross-linked HA will still gradually disappear (over time, the polymer is reabsorbed in the tissue), so the cross-linked HA must be injected repeatedly regularly (usually every 6-12 months) to maintain the therapeutic effect. As the main component of human bones, hydroxyapatite has excellent biocompatibility and is a commonly used bone tissue engineering material. Hydroxyapatite can be used as a human filling material, often used in facial filling, fat atrophy filling and other aspects. After the injection of hydroxyapatite, a human cell growth scaffold can be formed in the body, which not only provides a platform for cell growth, but also stimulates the human body to accelerate the generation of filling cells, thereby achieving a long-term filling effect. As a natural protein material, silk fibroin has good biocompatibility, adjustable biodegradability, low immunogenicity, and abundant raw material supply. In 1993, silk fibroin (SF) was certified by the U.S. Food and Drug Administration (FDA) as a biomaterial that can be used clinically. While filling facial tissue depressions, it is conducive to the adhesion of connective tissue cells around the implant site and gradually penetrates into the mesh structure of cross-linked sodium hyaluronate, thus forming a bionic structure similar to normal tissue.

[0004] Hydroxyapatite fillers currently on the market are usually pure hydroxyapatite components or hyaluronic acid, carboxymethyl cellulose, etc. as carriers. After filling into facial tissue, the carrier plays a role first, and after degradation, hydroxyapatite then stimulates collagen regeneration. However, hyaluronic acid usually degrades quickly, and the progressive effects of the two may not be accurately connected. The present invention, by adding silk fibroin components, can not only promote tissue regeneration during filling, but also prolong the degradation time, providing a scaffold space for tissue regeneration.

[0005] Therefore, we propose a method for preparing an injectable silk fibroin-hyaluronic acid filler. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a silk fibroin-hyaluronic acid filler for injection, so as to solve the problems raised in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a silk fibroin-hyaluronic acid filler for injection comprises the following steps:

[0009] Step S1: mixing the degummed silk and lithium bromide solution at 40-60° C., cooling to room temperature, dialyzing, filtering and centrifuging to obtain a silk fibroin solution;

[0010] Step S2: Concentrating the silk fibroin solution obtained in step S1 at 50-60° C. to obtain a concentrated silk fibroin solution, adding injection water to dilute it, and obtaining a diluted silk fibroin solution; heating the diluted silk fibroin solution to 50-60° C. to form a silk fibroin gel, and granulating it through a screen to obtain silk fibroin gel particles;

[0011] Step S3: 1,4-butanediol diglycidyl ether and sodium hydroxide solution are mixed evenly, hyaluronic acid and 1 / 3 of hydroxyapatite by weight are added, and after stirring evenly, swelling, purification and granulation are performed, and then the remaining hydroxyapatite by weight is added to obtain composite gel particles;

[0012] Step S4: evenly mix the composite gel particles and the silk fibroin gel particles, add them into the gel and mix them to obtain a silk fibroin-hyaluronic acid filler.

[0013] Furthermore, in step S1, the bath ratio of the degummed silk and the lithium bromide solution is (1:4)-(1:10), and the concentration of the lithium bromide solution is 9.3 mol / L.

[0014] Furthermore, in step S1, the concentration of the silk fibroin solution is 3-6wt%.

[0015] Furthermore, in step S2, the concentration of the concentrated silk fibroin solution is 15-20wt%.

[0016] Furthermore, in step S2, the concentration of the diluted silk fibroin solution is 2-6wt%.

[0017] Furthermore, in step S3, the composite gel particles include the following raw materials, calculated by weight: 1-2 parts of 1,4-butanediol diglycidyl ether, 80-200 parts of sodium hydroxide solution, 10-25 parts of hyaluronic acid, and 12-60 parts of hydroxyapatite.

[0018] Furthermore, the concentration of the sodium hydroxide solution is 0.6-1.0wt%.

[0019] Furthermore, in step S3, the swelling process condition is standing at 30-60° C. for 16-24 hours.

[0020] Furthermore, in step S4, the mass ratio of the composite gel particles to the silk fibroin gel particles is 1:(0.5-2.0).

[0021] Furthermore, in step S4, the gel is formed by mixing sodium carboxymethyl cellulose and physiological saline, and the mass concentration of sodium carboxymethyl cellulose is 1-1.5%.

[0022] Furthermore, in step S4, the volume of the gel is 40-90% of the total volume of the composite gel particles and the silk fibroin gel particles.

[0023] Furthermore, the composite gel particles are subjected to modification treatment, and the specific process is as follows:

[0024] Step (1): 2,2,6,6-tetramethylpiperidinyl oxide, sodium bromide and deionized water are mixed evenly, composite gel particles and sodium hypochlorite solution are added and mixed evenly, a NaOH solution is used to adjust the pH of the system to 10-11, the reaction is carried out for 3-5 hours, anhydrous ethanol is added to terminate the reaction, and after dialysis and freeze-drying, carboxylated composite gel particles are obtained;

[0025] Step (2): the carboxylated composite gel particles and deionized water are mixed evenly, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and 4-morpholineethanesulfonic acid are added, activation reaction is carried out for 0.5-1.0h, and then reduced glutathione is added, reaction is carried out for 22-24h, and after dialysis and freeze-drying, thiolated composite gel particles are obtained;

[0026] Step (3): The thiol-modified composite gel particles and deionized water are mixed evenly, the pH value of the system is adjusted to 8-9 using a NaOH solution, the silk fibroin solution is added, and the mixture is mixed evenly in an ice bath, horseradish peroxidase and hydrogen peroxide are added, and the mixture is incubated at 37° C. for 20-50 min. After dialyzing and freeze-drying, the modified composite gel particles are obtained.

[0027] Furthermore, in the step (1), the mass ratio of 2,2,6,6-tetramethylpiperidinyl oxide, sodium bromide and deionized water is (0.03-0.05):0.5:100.

[0028] Furthermore, in step (1), the mass of the composite gel particles is 2-4% of the mass of deionized water.

[0029] Furthermore, in step (1), the mass ratio of the composite gel particles, the sodium hypochlorite solution and the anhydrous ethanol is 1:(0.7-0.9):(1.0-1.2).

[0030] Furthermore, the sodium hypochlorite solution contains 6-14wt% active chlorine.

[0031] Furthermore, in step (2), the mass of the carboxylated composite gel particles is 2-4% of the mass of deionized water.

[0032] Furthermore, in the step (2), the mass ratio of the carboxylated composite gel particles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and 4-morpholineethanesulfonic acid is 1:(1-2):(0.5-1.0):(1.0-1.5).

[0033] Furthermore, in the step (2), the mass of the reduced glutathione is 1-2 times the mass of the carboxylated composite gel particles.

[0034] Furthermore, in step (3), the mass of the thiolated composite gel particles is 2-5% of the mass of deionized water.

[0035] Furthermore, in step (3), the concentration of the silk fibroin solution is 3-6wt%, and the amount thereof is 20-25 times the mass of the thiol-modified composite gel particles.

[0036] Furthermore, in step (3), the concentration of horseradish peroxidase is 10-12 U / mL, and the concentration of hydrogen peroxide is 3-4 mmol / L.

[0037] Furthermore, the molecular weight of the hyaluronic acid is 100-240 w.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. A preparation method of a silk fibroin-hyaluronic acid filler for injection of the present invention uses 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent to cross-link hyaluronic acid under alkaline conditions to form a three-dimensional network structure, thereby uniformly wrapping hydroxyapatite (HAP) particles, which not only improves the stability and durability of the filler, but also effectively combines HAP with hyaluronic acid to form composite gel particles; hydroxyapatite can prolong the cycle of stimulating collagen regeneration through two combinations; at the same time, the silk fibroin component can immediately promote tissue regeneration during filling; the use of a three-material composite system enables the filler to play a comprehensive role, enhance the filling effect, biocompatibility and tissue regeneration ability, and prolong the degradation time, providing a good scaffold space for tissue regeneration.

[0040] 2. A method for preparing a silk fibroin-hyaluronic acid filler for injection of the present invention, wherein carboxylated composite gel particles are prepared by oxidatively modifying composite gel particles using a 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO) system;

[0041] The amino groups in the reduced glutathione were cross-linked with the carboxylated composite gel particles by using the 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) cross-linking system to introduce thiol groups to obtain thiol composite gel particles. The cross-linking reaction improves the mechanical strength and stability of the material. Among them, glutathione has an antioxidant effect, can remove free radicals, and reduce the phenomenon of skin aging; at the same time, it can also promote the synthesis of collagen, increase skin elasticity and firmness, thereby enhancing the overall effect of the filler;

[0042] Through the dual mediation of horseradish peroxidase (HRP), the thiol groups on the thiol-modified composite gel particles and the tyrosine residues on the side chains of silk fibroin were cross-linked respectively to construct a covalently cross-linked double-network thiol-modified composite gel / silk fibroin blended gel. This double-network structure further increased the cross-linking degree of the gel. When part of the sodium hyaluronate component was degraded first, the overall three-dimensional network structure of the gel was maintained, avoiding the collapse and disintegration of the gel. The gel has a strong anti-biodegradation ability, thereby extending the degradation cycle of the filler. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0044] Figure 1 This is a diagram showing the state of the silk fibroin-hyaluronic acid filler of the present invention being injected through a 27G needle. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The degummed silk in this embodiment: 6A raw silk, sourced from Haian Suhao Silk Co., Ltd., the degummed silk is obtained by the company after degumming the raw silk; hyaluronic acid: molecular weight 150w, sourced from Huaxi Biotechnology Co., Ltd.; hydroxyapatite: particle size 25-45μm, sourced from Nanjing Junzhuo Biotechnology Co., Ltd.; horseradish peroxidase: CAS No. 9003-99-0, sourced from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] The following parts are by mass unless otherwise specified.

[0048] Example 1: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0049] Step S1: the degummed silk and 9.3 mol / L lithium bromide solution were mixed uniformly at 40° C. in a bath ratio of 1:4, cooled to room temperature, loaded into a dialysis bag with a molecular weight cutoff of 14 kDa, dialyzed with flowing purified water for three days, taken out, filtered through gauze, and centrifuged at 4° C. and 9000 rpm for 20 min to obtain a 3 wt% silk solution;

[0050] Step S2: Concentrating the silk fibroin solution obtained in step S1 at 50° C. to obtain a 15 wt% concentrated silk fibroin solution, adding injection water for dilution to obtain a 2 wt% diluted silk fibroin solution; heating the diluted silk fibroin solution to 50° C. to form a silk fibroin gel, and granulating the solution through a sieve to obtain silk fibroin gel particles;

[0051] Step S3: 1 part of 1,4-butanediol diglycidyl ether and 80 parts of 0.6wt% sodium hydroxide solution are mixed evenly, 10 parts of hyaluronic acid and 4 parts of hydroxyapatite are added, and after stirring evenly, swelling (standing at 30°C for 24h), purification and granulation are performed, and then 8 parts of hydroxyapatite are added to obtain composite gel particles;

[0052] Step S4: Evenly mix 12 parts of composite gel particles and 6 parts of silk fibroin gel particles, add into a gel containing 1 wt% sodium carboxymethyl cellulose (the volume of the gel is 40% of the total volume of the composite gel particles and the silk fibroin gel particles), and obtain a silk fibroin-hyaluronic acid filler.

[0053] Example 2: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0054] Step S1: the degummed silk and 9.3 mol / L lithium bromide solution were mixed uniformly at 50° C. in a bath ratio of 1:6, cooled to room temperature, loaded into a dialysis bag with a molecular weight cutoff of 14 kDa, dialyzed with flowing purified water for three days, taken out, filtered through gauze, and centrifuged at 4° C. and 9000 rpm for 20 min to obtain a 4 wt% silk fibroin solution;

[0055] Step S2: Concentrating the silk fibroin solution obtained in step S1 at 55° C. to obtain a 16 wt % concentrated silk fibroin solution, adding injection water for dilution to obtain a 4 wt % diluted silk fibroin solution; heating the diluted silk fibroin solution to 55° C. to form a silk fibroin gel, and granulating the solution through a sieve to obtain silk fibroin gel particles;

[0056] Step S3: 1.5 parts of 1,4-butanediol diglycidyl ether and 100 parts of 0.8 wt% sodium hydroxide solution are mixed evenly, 15 parts of hyaluronic acid and 10 parts of hydroxyapatite are added, and after stirring evenly, swelling (standing at 40° C. for 22 hours), purification and granulation are performed, and then 20 parts of hydroxyapatite are added to obtain composite gel particles;

[0057] Step S4: 20 parts of the composite gel particles and 20 parts of the silk fibroin gel particles are mixed evenly, and added into a gel containing 1.2 wt % of sodium carboxymethyl cellulose (the volume of the gel is 60% of the total volume of the composite gel particles and the silk fibroin gel particles) to obtain a silk fibroin-hyaluronic acid filler;

[0058] The composite gel particles are modified, and the specific process is as follows:

[0059] Step (1): 0.3 parts of 2,2,6,6-tetramethylpiperidinyl oxide, 5 parts of sodium bromide and 1000 parts of deionized water are mixed evenly, 20 parts of composite gel particles and 14 parts of sodium hypochlorite solution are added and mixed evenly, 1 mol / L NaOH solution is used to adjust the pH of the system to 10, the reaction is carried out for 3 hours, 20 parts of anhydrous ethanol is added to terminate the reaction, and after dialysis and freeze-drying, carboxylated composite gel particles are obtained;

[0060] Step (2): 20 parts of carboxylated composite gel particles and 1000 parts of deionized water are mixed evenly, 20 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 10 parts of N-hydroxysuccinimide and 20 parts of 4-morpholineethanesulfonic acid are added, activation reaction is carried out for 0.5 hours, and then 20 parts of reduced glutathione are added, reaction is carried out for 22 hours, and after dialysis and freeze-drying, thiolated composite gel particles are obtained;

[0061] Step (3): 20 parts of thiolated composite gel particles and 1000 parts of deionized water are mixed evenly, 1 mol / L NaOH solution is used to adjust the pH of the system to 8, 400 parts of 3 wt% silk solution are added, and the mixture is mixed evenly in an ice bath, 10 U / mL horseradish peroxidase and 3 mmol / L hydrogen peroxide are added, and the mixture is incubated at 37°C for 20 min. After dialyzation and freeze-drying, modified composite gel particles are obtained.

[0062] Example 3: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0063] Step S1: the degummed silk and 9.3 mol / L lithium bromide solution were mixed uniformly at 55° C. in a bath ratio of 1:8, cooled to room temperature, loaded into a dialysis bag with a molecular weight cutoff of 14 kDa, dialyzed with flowing purified water for three days, taken out, filtered through gauze, and centrifuged at 4° C. and 9000 rpm for 20 min to obtain a 5 wt% silk fibroin solution;

[0064] Step S2: Concentrating the silk fibroin solution obtained in step S1 at 60° C. to obtain an 18 wt % concentrated silk fibroin solution, adding injection water for dilution to obtain a 5 wt % diluted silk fibroin solution; heating the diluted silk fibroin solution to 60° C. to form a silk fibroin gel, and granulating the solution through a sieve to obtain silk fibroin gel particles;

[0065] Step S3: 1.8 parts of 1,4-butanediol diglycidyl ether and 150 parts of 0.9wt% sodium hydroxide solution were mixed evenly, 20 parts of hyaluronic acid and 15 parts of hydroxyapatite were added, and after stirring evenly, swelling (standing at 50°C for 20h), purification and granulation were performed, and 30 parts of hydroxyapatite were added to obtain composite gel particles;

[0066] Step S4: 20 parts of the composite gel particles and 30 parts of the silk fibroin gel particles are mixed evenly, and added into a gel containing 1.4 wt % of sodium carboxymethyl cellulose (the volume of the gel is 80% of the total volume of the composite gel particles and the silk fibroin gel particles) to obtain a silk fibroin-hyaluronic acid filler;

[0067] The composite gel particles are modified, and the specific process is as follows:

[0068] Step (1): 0.32 parts of 2,2,6,6-tetramethylpiperidinyl oxide, 4 parts of sodium bromide and 800 parts of deionized water are mixed evenly, 20 parts of composite gel particles and 16 parts of sodium hypochlorite solution are added and mixed evenly, 1 mol / L NaOH solution is used to adjust the pH of the system to 10.5, the reaction is carried out for 4 hours, 22 parts of anhydrous ethanol is added to terminate the reaction, and after dialysis and freeze-drying, carboxylated composite gel particles are obtained;

[0069] Step (2): 20 parts of carboxylated composite gel particles and 800 parts of deionized water were mixed evenly, 30 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 16 parts of N-hydroxysuccinimide and 24 parts of 4-morpholineethanesulfonic acid were added, activation reaction was carried out for 0.8 hours, and then 30 parts of reduced glutathione were added, reaction was carried out for 23 hours, and after dialysis and freeze-drying, thiolated composite gel particles were obtained;

[0070] Step (3): 20 parts of thiolated composite gel particles and 800 parts of deionized water were mixed evenly, 1 mol / L NaOH solution was used to adjust the pH value of the system to 8.5, 440 parts of 5 wt% silk solution were added, and the mixture was mixed evenly in an ice bath, 11 U / mL horseradish peroxidase and 3.5 mmol / L hydrogen peroxide were added, and the mixture was incubated at 37°C for 30 min. After dialyzing and freeze-drying, modified composite gel particles were obtained.

[0071] Example 4: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0072] Step S1: the degummed silk and 9.3 mol / L lithium bromide solution were mixed uniformly at 60° C. in a bath ratio of 1:10, cooled to room temperature, loaded into a dialysis bag with a molecular weight cutoff of 14 kDa, dialyzed with flowing purified water for three days, taken out, filtered through gauze, and centrifuged at 4° C. and 9000 rpm for 20 min to obtain a 6 wt% silk fibroin solution;

[0073] Step S2: Concentrating the silk fibroin solution obtained in step S1 at 60° C. to obtain a 20 wt % concentrated silk fibroin solution, adding injection water for dilution to obtain a 6 wt % diluted silk fibroin solution; heating the diluted silk fibroin solution to 60° C. to form a silk fibroin gel, and granulating the solution through a sieve to obtain silk fibroin gel particles;

[0074] Step S3: 2 parts of 1,4-butanediol diglycidyl ether and 200 parts of 1.0 wt% sodium hydroxide solution are mixed evenly, 25 parts of hyaluronic acid and 20 parts of hydroxyapatite are added, and after stirring evenly, swelling (standing at 60° C. for 16 h), purification and granulation are performed, and then 40 parts of hydroxyapatite are added to obtain composite gel particles;

[0075] Step S4: 20 parts of the composite gel particles and 40 parts of the silk fibroin gel particles are mixed evenly, and added into a gel containing 1.5 wt % of sodium carboxymethyl cellulose (the volume of the gel is 90% of the total volume of the composite gel particles and the silk fibroin gel particles) to obtain a silk fibroin-hyaluronic acid filler;

[0076] The composite gel particles are modified, and the specific process is as follows:

[0077] Step (1): 0.25 parts of 2,2,6,6-tetramethylpiperidinyl oxide, 2.5 parts of sodium bromide and 500 parts of deionized water are mixed evenly, 20 parts of composite gel particles and 18 parts of sodium hypochlorite solution are added and mixed evenly, 1 mol / L NaOH solution is used to adjust the pH of the system to 11, the reaction is carried out for 5 hours, 24 parts of anhydrous ethanol is added to terminate the reaction, and after dialysis and freeze-drying, carboxylated composite gel particles are obtained;

[0078] Step (2): 20 parts of carboxylated composite gel particles and 500 parts of deionized water were mixed evenly, 40 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 20 parts of N-hydroxysuccinimide and 30 parts of 4-morpholineethanesulfonic acid were added, and the activation reaction was carried out for 1.0 h, and then 40 parts of reduced glutathione were added, and the reaction was carried out for 24 h. After dialysis and freeze-drying, the thiol composite gel particles were obtained;

[0079] Step (3): 20 parts of thiolated composite gel particles and 500 parts of deionized water were mixed evenly, 1 mol / L NaOH solution was used to adjust the pH value of the system to 9, 500 parts of 6 wt% silk solution were added, and the mixture was mixed evenly in an ice bath, 12 U / mL horseradish peroxidase and 4 mmol / L hydrogen peroxide were added, and the mixture was incubated at 37°C for 50 min. After dialyzing and freeze-drying, the modified composite gel particles were obtained.

[0080] Comparative Example 1: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0081] Compared with Example 1, in step S4 of Comparative Example 1, the mass ratio of the composite gel particles to the silk fibroin gel particles is 1:0.2, and the other steps are the same as those in Example 1.

[0082] Comparative Example 2: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0083] Compared with Example 2, Comparative Example 2 did not add reduced glutathione, and the other steps were the same as those of Example 2.

[0084] Comparative Example 3: A method for preparing a silk fibroin-hyaluronic acid filler for injection, comprising the following process:

[0085] Compared with Example 2, Comparative Example 3 does not include step (3), and the modified composite gel particles are replaced with thiolated composite gel particles of the same mass. The other steps are the same as those of Example 2.

[0086] experiment:

[0087] Experiment 1: In vitro degradation performance test: Take the silk fibroin-hyaluronic acid filler obtained in Examples 1-4 and Comparative Examples 1-3, weigh it after freeze-drying, record the initial weight as m0, transfer it and immerse it in phosphate buffered saline (PBS) solution containing 20U / mL collagenase, and place it in an air bath shaker at a temperature of 37°C and a shaking speed of 150rpm for the experiment; take out the sample at regular intervals, freeze-dry it, weigh it as m1, and replace it with a new degradation solution until the condition (m0-m1) / m0>0.99 is met, it is considered to be completely degraded, and its complete degradation time is recorded.

[0088] Experiment 2: Injectability test: The composite gel fillers obtained in Examples 1-4 and Comparative Examples 1-3 were loaded into a 1 mL syringe and equipped with a 27G injection needle to simulate actual use. The push rod was pushed at a constant pushing speed of 30 mm / min, with a pushing displacement of 20 mm. The sample in the syringe was pushed out through the needle. Figure 1 As shown in Figure 2, within a certain compression displacement, the compression load of the injectable gel varies between 8 and 16N.

[0089] The test results are as follows:

[0090]

[0091] According to the data in the above table, we can clearly draw the following conclusions:

[0092] 1. Compared with Example 1, the degradation time of the products obtained in Examples 2-4 increases. It can be seen that the present invention further prolongs the degradation period of the material by modifying the composite gel particles. At the same time, the pushing force is relatively moderate, which is suitable for injection.

[0093] 2. Compared with Example 1, the degradation time of the product obtained in Comparative Example 1 is reduced, indicating that the filler prepared by the present invention is affected by its component ratio. Reducing the amount of silk fibroin gel particles added will affect the performance of the filler, thereby reducing its durability.

[0094] 3. Compared with Examples 2-4, the degradation time of the products obtained in Comparative Examples 2 and 3 is reduced, which indicates that the present invention can cross-link with the carboxylated composite gel particles by adding reduced glutathione, providing more reaction sites for the subsequent cross-linking reaction, thereby effectively prolonging the degradation time of the filler; at the same time, the present invention prolongs the degradation time by cross-linking the thiol groups on the thiol-composite gel particles with the tyrosine residues on the side chains of the silk fibroin.

[0095] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing a silk fibroin-hyaluronic acid filler for injection, characterized in that: The steps include: S1: concentrating the silk fibroin solution to obtain a concentrated silk fibroin solution, adding injection water to dilute it, and obtaining a diluted silk fibroin solution; heating the diluted silk fibroin solution to 50-60° C. to form a silk fibroin gel, and granulating it through a sieve to obtain silk fibroin gel particles; S2: 1,4-butanediol diglycidyl ether and sodium hydroxide solution are mixed evenly, hyaluronic acid and 1 / 3 of hydroxyapatite by weight are added, and after stirring evenly, swelling, purification and granulation are performed, and then the remaining hydroxyapatite by weight is added to obtain composite gel particles; S3: evenly mixing the composite gel particles and the silk fibroin gel particles, adding the particles into the gel and mixing, thereby obtaining a silk fibroin-hyaluronic acid filler; The composite gel particles are subjected to modification treatment, and the specific process is as follows: Step (1): 2,2,6,6-tetramethylpiperidinyl oxide, sodium bromide and deionized water are mixed evenly, composite gel particles and sodium hypochlorite solution are added and mixed evenly, a NaOH solution is used to adjust the pH of the system to 10-11, the reaction is carried out for 3-5 hours, anhydrous ethanol is added to terminate the reaction, and after dialysis and freeze-drying, carboxylated composite gel particles are obtained; Step (2): the carboxylated composite gel particles and deionized water are mixed evenly, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and 4-morpholineethanesulfonic acid are added, activation reaction is carried out for 0.5-1.0h, and then reduced glutathione is added, reaction is carried out for 22-24h, and after dialysis and freeze drying, thiolated composite gel particles are obtained; Step (3): the thiol-modified composite gel particles and deionized water are mixed evenly, the pH value of the system is adjusted to 8-9 using a NaOH solution, the silk fibroin solution is added, and the mixture is mixed evenly in an ice bath, horseradish peroxidase and hydrogen peroxide are added, and the mixture is incubated at 37° C. for 20-50 min, and the modified composite gel particles are obtained after dialyzing and freeze-drying; In the step S3, the mass ratio of the composite gel particles to the silk fibroin gel particles is 1:(0.5-2.0).

2. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: The preparation method of the silk fibroin solution is as follows: the degummed silk and the lithium bromide solution are mixed uniformly at 40-60° C., cooled to room temperature, dialyzed, filtered, and centrifuged to obtain the silk fibroin solution; The bath ratio of the degummed silk and the lithium bromide solution is (1:4)-(1:10), and the concentration of the lithium bromide solution is 9.3 mol / L.

3. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: The concentration of the silk fibroin solution is 3-6 wt %.

4. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: In the step S1, the concentration of the concentrated silk fibroin solution is 15-20wt%; The concentration of the diluted silk fibroin solution is 2-6 wt %.

5. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: In step S2, the composite gel particles include the following raw materials, calculated by weight: 1-2 parts of 1,4-butanediol diglycidyl ether, 80-200 parts of sodium hydroxide solution, 10-25 parts of hyaluronic acid, and 12-60 parts of hydroxyapatite.

6. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: In the step (2), the mass of the reduced glutathione is 1-2 times the mass of the carboxylated composite gel particles.

7. The method for preparing a silk fibroin-hyaluronic acid filler for injection according to claim 1, characterized in that: In the step (3), the concentration of horseradish peroxidase is 10-12 U / mL, and the concentration of hydrogen peroxide is 3-4 mmol / L.

8. A silk fibroin-hyaluronic acid filler for injection prepared according to the preparation method according to any one of claims 1 to 7.

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