Silk fibroin composite microsphere, gel, preparation method and application

Abstract

The invention relates to silk fibroin composite microspheres, gel, a preparation method and application. The preparation method has the advantages that the accelerant and the zero-length cross-linking agent are used for realizing self-crosslinking among molecular chains to construct a chemical interpenetrating network, so that introduction of a small-molecule chemical cross-linking agent can be effectively avoided, accumulation of the chemical cross-linking agent in a living body is avoided, and potential biotoxicity is reduced; the silk fibroin composite microsphere is a chemical interpenetrating network, natural polysaccharide / protein contained in the silk fibroin composite microsphere can effectively reduce the crystallinity of silk fibroin, the problems of hardness, brittleness and hydrophilicity reduction caused by too high crystallinity are avoided, and the elasticity, hydrophilicity and biocompatibility are improved; after a chemical interpenetrating network of the silk fibroin composite microspheres is formed, the silk fibroin composite microspheres are induced to be properly crystallized by utilizing a folding promoting agent, so that the degradation resistance and the surface smoothness of the silk fibroin composite microspheres are improved, and a composite material with a long-term filling effect is obtained; the microspheres prepared by the tubular emulsification method are good in uniformity and high in yield.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a silk fibroin composite microsphere, a silk fibroin composite microsphere gel, its preparation method, and its applications. Background Technology

[0002] Silk fibroin (SF) is a natural protein derived from silkworm silk. It is a high-purity protein secreted by the epithelial cells of the silk glands in silkworms. It contains no organelles, poses no risk of cross-infection with human pathogens, and exhibits good biocompatibility. Therefore, it was used to make sutures for the medical industry more than a century ago. Later, with further research, scientists discovered that the arginine-glycine-aspartic acid (RGD) sequence in silk fibroin can be recognized by integrin receptors in cell membranes, promoting cell adhesion and proliferation. Furthermore, silk fibroin itself is composed of 18 amino acids, some of which have whitening and moisturizing functions. Therefore, silk fibroin is now a highly sought-after material in the regenerative medicine, medical aesthetics, and cosmetic industries.

[0003] Compared to other natural biopolymers, silk fibroin has unique advantages in terms of excellent mechanical properties and readily modifiable chemical groups. The silk fibroin molecular chain is composed of highly ordered glycine-alanine-glycine-alanine-serine, exhibiting a relatively good crystalline structure. However, this crystallinity can undergo crystalline / amorphous and amorphous / crystalline transformations under the influence of external solvents. Therefore, controlling the crystallinity of silk fibroin can effectively control the mechanical properties of silk fibroin materials.

[0004] Hyaluronic acid (HA), a naturally occurring linear polysaccharide, is widely used in soft tissue fillers due to its excellent biocompatibility, moisturizing properties, and non-immunogenicity. However, natural hyaluronic acid is readily degraded by hyaluronidase in the human body, has a short half-life, and pure hyaluronic acid gel has weak mechanical strength, is prone to displacement, and is difficult to maintain long-term filling effects. Collagen shares the common problem of rapid degradation and difficulty in maintaining long-term effects in the human body with hyaluronic acid. Therefore, by combining silk fibroin with hyaluronic acid or collagen, it is possible to avoid the hardening and brittleness of silk fibroin due to excessive crystallinity, and obtain a composite material with better elasticity, hydrophilicity, degradation resistance, high moisturizing properties, and biocompatibility.

[0005] Common methods for preparing silk fibroin microspheres include spray drying, freeze crystallization, emulsification, and microfluidics. Chinese invention patent application CN114874466A discloses a method for preparing silk fibroin microspheres via spray drying and its applications. The preparation steps are as follows: silk fibroin is mixed with a spheroidizing agent and then coagulated at low temperature to obtain a silk fibroin microsphere solution. The crystallinity of the solution is then increased through hydrothermal treatment, and finally, solid silk fibroin microspheres are obtained through oven drying. The mechanism involves first stimulating silk fibroin crystallization with a spheroidizing agent to form low-crystallinity nanocrystals, then obtaining high-crystallinity nanocrystals through hydrothermal treatment, and finally assembling the nanocrystals into micron-sized microspheres through spray drying. However, this method can only prepare pure silk fibroin microspheres, and the particle size is small (~2-5 μm) with a rough surface.

[0006] The common problem between the freeze-crystallization method and the spray drying method for preparing silk fibroin microspheres is that they cannot be blended with raw materials such as collagen and hyaluronic acid that do not undergo low-temperature phase transitions. This results in only pure silk fibroin microspheres with nanoscale dimensions or smaller than 10 μm (Chinese invention patent application CN119751919A). While microfluidic methods can produce uniform microspheres with controllable particle size, their low production efficiency and difficulties in cleaning and sterilizing equipment and accessories limit their current industrial application.

[0007] Chinese invention patent application CN120204480A discloses an injectable sodium hyaluronate-silk fibroin composite hydrogel, its preparation method, and its applications. This patent involves combining physically cross-linked silk fibroin sponge microspheres of a specific particle size with chemically cross-linked sodium hyaluronate gel particles, and then compounding with a sodium hyaluronate solution to prepare the hydrogel. It utilizes the preferential degradation of sodium hyaluronate and the degradation-resistant properties of silk fibroin, which stimulates collagen production, to achieve in-situ fixation of the particles, resulting in excellent extrusion properties, low cytotoxicity, and timely and lasting filling of facial wrinkles. However, it suffers from drawbacks such as complex preparation processes that are difficult to industrialize, high costs for particle size quality control, lack of clear control over reagent residues, poor adaptability of fixed formulation ratios, limited application scenarios, and a lack of methods for controlling degradation rates.

[0008] Chinese invention patent application CN120501932A discloses a silk fibroin composite gel and its preparation method. This patent modifies silk fibroin by amylation, then uses enzyme catalysis to form Schiff base bonds for dynamic cross-linking with oxidized sodium alginate. A temperature-sensitive component is added to construct a temperature-sensitive-dynamic dual network. Active ingredients are loaded and reinforced with disulfide bonds, followed by sterilization. The resulting gel exhibits rapid temperature-sensitive gelation, secondary shaping capability, and good self-healing properties, while also possessing multiple functions such as promoting regeneration, antibacterial, anti-inflammatory, and antioxidant effects, achieving an integrated injection-gelation-shaping effect. However, it suffers from drawbacks such as cumbersome preparation steps, high cost due to the delicate quality control process for active ingredients, the risk of residues from raw materials like chloroform without clear testing standards, the potential for gamma-ray sterilization to damage active ingredients, unclear long-term compatibility between the temperature-sensitive component and the gel system, and high energy consumption and equipment requirements, making industrialization difficult.

[0009] To address the problems associated with the aforementioned preparation methods, emulsification is currently one of the most widely used methods in industry for preparing microspheres. However, traditional methods of emulsification using mechanical stirring have drawbacks such as wide particle size distribution, low microsphere yield, and high oil-to-water ratio.

[0010] To address the shortcomings of mechanical stirring emulsification methods, some manufacturers employ membrane emulsification. For example, Chinese invention patent application CN121021874A discloses a uniform-size silk fibroin microsphere, its preparation method, and its application. The microsphere preparation steps are as follows: modified silk fibroin precipitate is obtained by trypsin enzymatic digestion, then dissolved in an aqueous solution of a β-sheet inhibitor to prepare an aqueous phase of silk fibroin; subsequently, the aqueous phase is pressed into an oil phase using an SPG external pressure membrane emulsification device to obtain an emulsion; a crosslinking agent is added to the emulsion for crosslinking, and an inducing agent is added to induce crystallization to obtain silk fibroin microspheres. The patent shows that the microspheres obtained by membrane emulsification have a uniform particle size (10~30 μm) and a smooth surface. However, the microspheres prepared by this method contain toxic chemical crosslinking agents. These chemical crosslinking agents accumulate in the human body after the microspheres degrade and cannot be metabolized, posing a certain degree of biotoxicity and cumulative use risk. In addition, SPG membranes are currently only produced by SPG Corporation of Japan. The emulsification equipment for this type of membrane can only be imported from Japan. There are no ceramic membranes of this type in China, and SPG membranes have a short service life and need to be replaced frequently. Therefore, the application of membrane emulsification in the microsphere preparation industry is severely limited.

[0011] In conclusion, developing a universal and efficient method to prepare silk fibroin composite microspheres with uniform particle size, good sphericity, smooth surface, high water content, good elasticity, simple preparation method, high yield, and no chemical cross-linking agents is of great significance for promoting the application of silk fibroin in the medical industry.

[0012] In summary, no effective solutions have yet been proposed to address the existing problems of uneven particle size, poor sphericity, rough surface, low water content, poor elasticity, and biotoxicity. Summary of the Invention

[0013] The purpose of this invention is to address the shortcomings of existing technologies by providing a silk fibroin composite microsphere, a silk fibroin composite microsphere gel, a preparation method, and applications, in order to solve problems such as uneven particle size, poor sphericity, rough surface, low water content, poor elasticity, and biotoxicity in related technologies.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In one aspect, a silk fibroin composite microsphere is provided, wherein the silk fibroin composite microsphere is an interpenetrating network structure of silk fibroin network and natural polysaccharide network and / or protein network; Silk fibroin composite microspheres are chemically cross-linked microspheres without cross-linking agents; The silk fibroin composite microspheres are spherical, and the surface of the silk fibroin composite microspheres is a smooth, non-porous surface. The particle size distribution of the silk fibroin composite microspheres is: D50: 35~50 μm, D90: 55~75 μm; The crystallinity of the silk fibroin composite microspheres is 25-55%.

[0015] In a second aspect, a method for preparing silk fibroin composite microspheres is provided, for preparing silk fibroin composite microspheres as described in the first aspect, comprising: Soluble silk fibroin, natural polysaccharides and / or proteins, and promoters are added to an aqueous solution to obtain a silk fibroin composite solution as the dispersed phase. Emulsifiers and zero-length crosslinking agents are added to the organic phase to obtain a continuous phase; Emulsification was performed using a tubular emulsifier to disperse the dispersed phase in the continuous phase, resulting in a water-in-oil emulsion containing droplets of silk fibroin composite solution. The emulsion was then subjected to a solidification reaction under low-speed stirring to obtain a silk fibroin composite microsphere system. Add the folding promoter to the silk fibroin composite microsphere system, stir and mix, let stand and settle, discard the supernatant, and wash the silk fibroin composite microspheres with organic solvent; The silk fibroin composite microspheres were sieved under water flow using a sieve to obtain a uniform silk fibroin composite microsphere slurry. The silk fibroin composite microsphere material solution was freeze-dried to obtain silk fibroin composite microspheres.

[0016] In some of these embodiments, the molecular weight of the soluble silk fibroin is 100-200 kDa.

[0017] In some of these embodiments, the natural polysaccharide is any one or a combination of sodium hyaluronate, chitosan, agarose, and xanthan gum.

[0018] In some of these embodiments, the protein is any one or a combination of type I collagen, type III collagen, recombinant type III collagen, recombinant type XVII humanized collagen, and gelatin.

[0019] In some of these embodiments, the accelerator is any one or a combination of hydrogen peroxide, N-hydroxysuccinimide (NHS), ethanol, n-hexane, and sodium hydroxide.

[0020] In some of these embodiments, the mass ratio of soluble silk fibroin: natural polysaccharide and / or protein: water is (5~9):(5~1):90.

[0021] In some of these embodiments, the amount of accelerator used is 0.2 to 0.6% of the mass of the dispersed phase.

[0022] In some of these embodiments, the concentration of the promoter is 10 wt%.

[0023] In some of these embodiments, the organic phase is any one of liquid paraffin, soybean oil, ethyl acetate, petroleum ether, and silicone oil.

[0024] In some of the embodiments, the emulsifier is a nonionic emulsifier, which is any one or a combination of polysorbate 20, polysorbate 40, sorbitan oleate, and sucrose fatty acid ester.

[0025] In some of these embodiments, the zero-length crosslinking agent is any one or a combination of horseradish peroxidase, 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0026] In some of these embodiments, the amount of emulsifier used is 1 to 6%.

[0027] In some of these embodiments, the amount of zero-length crosslinking agent is 0.2 to 2% of the mass of the dispersed phase.

[0028] In some of these embodiments, the concentration of the zero-length crosslinking agent is 10 wt%.

[0029] In some of these embodiments, the mass ratio of dispersed phase to continuous phase is 1:(5~10).

[0030] In some of these embodiments, the emulsification conditions are: an emulsification speed of 500-1000 rpm and 1-3 emulsification cycles.

[0031] In some of these embodiments, the curing conditions are: a stirring speed of 30~200 rpm, a curing temperature of 20~37℃, and a curing time of 1.5~24 h.

[0032] In some of these embodiments, the folding accelerator is any one or a combination of methanol, 75% ethanol, glycerol, and polyethylene glycol.

[0033] In some of these embodiments, the stirring conditions are: stirring speed of 50-100 rpm, stirring time of 1-24 h, and settling time of 0.5-2 h.

[0034] In some of the embodiments, the organic solvent is any one or a combination of anhydrous ethanol, 75% ethanol, 50% ethanol, and n-hexane.

[0035] In some of these embodiments, the cleaning conditions are: cleaning 2 to 3 times with any organic solvent.

[0036] In some of these embodiments, the screening machine is a vibrating screening machine.

[0037] In some of these embodiments, the mobile phase is purified water or water for injection.

[0038] In some embodiments, the sieve mesh size of the screening machine is any one or more combinations of 100 mesh, 200 mesh, 500 mesh, 800 mesh, and 1000 mesh.

[0039] In some of these embodiments, the freeze-drying conditions are as follows: freezing temperature is -20 to -40°C, freezing time is 1 to 5 hours, freeze-drying temperature is -10 to -8°C, freeze-drying time is 24 to 28 hours, and desorption temperature is 5 to 8°C with a desorption time of 10 to 12 hours.

[0040] Thirdly, a silk fibroin composite microsphere gel is provided, the silk fibroin composite microsphere gel comprising: Silk fibroin composite microspheres as described in the first aspect or prepared by the preparation method described in the second aspect; The compound material is any one or more of the following: sodium hyaluronate, hydroxypropyl cellulose, sodium carboxymethyl cellulose, water, cross-linked sodium hyaluronate gel, collagen, and recombinant collagen.

[0041] In some of these embodiments, the mass ratio of silk fibroin composite microspheres to composite materials is (1~9):(9~1).

[0042] Fourthly, an application of a silk fibroin composite microsphere product is provided. The product is an injectable product, including silk fibroin composite microspheres as described in the first aspect, or silk fibroin composite microspheres prepared by the preparation method described in the second aspect, or silk fibroin composite microsphere gel as described in the third aspect. Applications include skin filling, lacrimal canaliculus filling, and vocal cord filling.

[0043] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: 1) By using promoters and zero-length crosslinking agents to achieve self-crosslinking between molecular chains and constructing chemical interpenetrating networks, the introduction of small molecule chemical crosslinking agents can be effectively avoided, further preventing the accumulation of chemical crosslinking agents in organisms and reducing potential biotoxicity. 2) Silk fibroin composite microspheres are chemically interpenetrating networks. The natural polysaccharides / proteins contained in the silk fibroin composite microspheres can effectively reduce the crystallinity of silk fibroin, avoiding the problems of hardening, brittleness and decreased hydrophilicity caused by excessive crystallinity. At the same time, they can improve the elasticity and hydrophilicity of the silk fibroin composite microspheres and improve their biocompatibility. 3) After the formation of the chemical interpenetrating network of silk fibroin composite microspheres, the method of inducing appropriate crystallization of silk fibroin composite microspheres by using a folding promoter can further improve the degradation resistance and surface smoothness of silk fibroin composite microspheres, and obtain a composite material with long-term filling effect. 4) The preparation method of silk fibroin composite microspheres is tubular emulsification, which is a common continuous production equipment in the emulsification industry. It has the capability for large-scale industrial application, and the microspheres prepared by this method have good uniformity and high yield. Attached Figure Description

[0044] Figure 1 This is a scanning electron microscope (SEM) image of the silk fibroin composite microspheres according to Example 2 of the present invention; Figure 2 This is a scanning electron microscope (SEM) magnified image of the silk fibroin composite microspheres according to Example 2 of the present invention; Figure 3 This is a particle size distribution curve of the silk fibroin composite microspheres according to Example 2 of the present invention. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0046] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0048] Example 1 This embodiment relates to the silk fibroin composite microspheres, silk fibroin composite microsphere gels, preparation methods, and applications of the present invention.

[0049] An illustrative embodiment of the present invention provides a silk fibroin composite microsphere, which is an interpenetrating network structure of silk fibroin network and natural polysaccharide network, or an interpenetrating network structure of silk fibroin network and protein network, or an interpenetrating network structure of silk fibroin network, natural polysaccharide network and protein network.

[0050] Among them, the silk fibroin composite microspheres are chemically cross-linked microspheres without cross-linking agents.

[0051] The silk fibroin composite microspheres are spherical, and the surface of the silk fibroin composite microspheres is smooth and non-porous.

[0052] The particle size distribution of the silk fibroin composite microspheres is as follows: D50: 35~50 μm, D90: 55~75 μm.

[0053] The crystallinity of the silk fibroin composite microspheres is 25-55%.

[0054] The preparation method of the silk fibroin composite microspheres of the present invention is as follows: Step S1: Add soluble silk fibroin, natural polysaccharides and / or proteins, and promoters to an aqueous solution to obtain a silk fibroin composite solution as the dispersed phase. Step S2: Add the emulsifier and zero-length crosslinking agent to the organic phase to obtain a continuous phase; Step S3: Emulsify the dispersed phase in the continuous phase using a tubular emulsifier to obtain a water-in-oil emulsion containing silk fibroin composite solution droplets. Then, solidify the emulsion under low-speed stirring to obtain a silk fibroin composite microsphere system. Step S4: Add the folding accelerator to the silk fibroin composite microsphere system, stir and mix, let it stand to settle, discard the supernatant, and wash the silk fibroin composite microspheres with an organic solvent. Step S5: Use a sieve to sieve the silk fibroin composite microspheres under water flow to obtain a uniform silk fibroin composite microsphere slurry. Step S6: Freeze-dry the silk fibroin composite microsphere solution to obtain silk fibroin composite microspheres.

[0055] In step S1, the soluble silk fibroin is prepared as follows: silk is degummed by alkali washing, washed, dried, dissolved, dialyzed, impurity removed, concentrated, and freeze-dried to obtain soluble silk fibroin.

[0056] Soluble silk fibroin needs to be dissolved before use, and its solubility rate is 95-99%.

[0057] In step S1, the molecular weight of the soluble silk fibroin is 100~200 kDa.

[0058] In step S1, the natural polysaccharide is any one or a combination of sodium hyaluronate, chitosan, agarose, and xanthan gum.

[0059] Sodium hyaluronate has a molecular weight of 1000~2000 kDa.

[0060] The degree of deacetylation of chitosan is 85%~90%.

[0061] In step S1, the protein is any one or a combination of type I collagen, type III collagen, recombinant type III collagen, recombinant type XVII humanized collagen, and gelatin.

[0062] Among them, type I collagen is collagen extracted from bovine Achilles tendon.

[0063] Type III collagen is collagen extracted from pig skin.

[0064] In step S1, the accelerator is any one or a combination of hydrogen peroxide, N-hydroxysuccinimide (NHS), ethanol, n-hexane, and sodium hydroxide.

[0065] In step S1, the mass ratio of soluble silk fibroin: natural polysaccharide and / or protein: water is (5~9):(5~1):90.

[0066] In step S1, the amount of accelerator is 0.2-0.6% of the mass of the dispersed phase. It should be noted that the mass of the dispersed phase here is the sum of the masses of soluble silk fibroin, natural polysaccharides and / or proteins, and water. That is, the mass of the accelerator is 0.2-0.6% of the sum of the masses of soluble silk fibroin, natural polysaccharides and / or proteins, and water.

[0067] In step S1, the concentration of the promoter is 10 wt%.

[0068] In step S2, the organic phase is any one of liquid paraffin, soybean oil, ethyl acetate, petroleum ether, and silicone oil.

[0069] Preferably, the organic phase is any one of liquid paraffin, soybean oil, or silicone oil.

[0070] In step S2, the emulsifier is a nonionic emulsifier, which is any one or a combination of polysorbate 20, polysorbate 40, sorbitan oleate, and sucrose fatty acid ester.

[0071] In step S2, the zero-length crosslinking agent is any one or a combination of horseradish peroxidase and 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0072] In step S2, the amount of emulsifier used is 1~6%.

[0073] In step S2, the amount of zero-length crosslinking agent is 0.2-2% of the mass of the dispersed phase. It should be noted that the mass of the dispersed phase here is the sum of the masses of soluble silk fibroin, natural polysaccharides and / or proteins, and water (i.e., excluding the mass of the accelerator). That is, the amount of zero-length crosslinking agent is 0.2-2% of the sum of the masses of soluble silk fibroin, natural polysaccharides and / or proteins, and water.

[0074] In step S2, the concentration of the zero-length crosslinking agent is 10 wt%.

[0075] In step S3, the mass ratio of dispersed phase to continuous phase is 1:(5~10). It should be noted that the mass of the dispersed phase here is the sum of the masses of soluble silk fibroin, natural polysaccharides and / or proteins, and water (i.e., the mass excluding accelerators), and the mass of the continuous phase here is the mass of the organic phase (i.e., the mass excluding emulsifiers and zero-length crosslinking agents).

[0076] Preferably, the mass ratio of the dispersed phase to the continuous phase is 1:(5~7).

[0077] In step S3, the emulsification conditions are: emulsification speed of 500-1000 rpm and emulsification times of 1-3 times.

[0078] Preferably, the emulsification conditions are: an emulsification speed of 500~700 rpm and an emulsification cycle of 1~2 times.

[0079] In step S3, the curing conditions are: stirring speed of 30~200 rpm, curing temperature of 20~37℃, and curing time of 1.5~24 h.

[0080] Preferably, the curing conditions are: a stirring speed of 50~150 rpm and a curing time of 3~6 h.

[0081] It should be noted that in this invention, based on the combined action of the accelerator and the zero-length crosslinking agent, soluble silk fibroin and natural polysaccharides and / or proteins undergo self-crosslinking to form a chemical crosslinking network without crosslinking agent. By controlling the ratio of soluble silk fibroin, natural polysaccharides and / or proteins to accelerator and zero-length crosslinking agent, as well as the curing temperature and curing time, hydrophilic silk fibroin composite microspheres with adjustable elasticity can be obtained.

[0082] In step S4, the folding accelerator is any one or a combination of methanol, 75% ethanol, glycerol, and polyethylene glycol.

[0083] Preferably, the folding accelerator is any one or a combination of 75% ethanol, glycerin, and polyethylene glycol.

[0084] In step S4, the stirring conditions are as follows: stirring speed is 50~100 rpm, stirring time is 1~24 h, and settling time is 0.5~2 h.

[0085] Preferably, the stirring conditions are: stirring time of 20-24 h and standing time of 0.5-1 h.

[0086] In step S4, the organic solvent is any one or a combination of anhydrous ethanol, 75% ethanol, 50% ethanol, and n-hexane.

[0087] In step S4, the cleaning conditions are: cleaning 2-3 times with any one organic solvent. That is, if multiple organic solvents are used, each organic solvent is used for 2-3 cleanings. The total number of cleaning times is the number of organic solvents multiplied by (2-3).

[0088] Furthermore, in step S4, the system is left to stand after each cleaning. The standing time is 0.5 to 1 hour.

[0089] Furthermore, in step S4, if multiple organic solvents are used for cleaning, the multiple organic solvents are used alternately for cleaning.

[0090] In step S5, the screening machine is a vibrating screening machine.

[0091] In step S5, the mesh size of the sieve of the screening machine is any one or more combinations of 100 mesh, 200 mesh, 500 mesh, 800 mesh, and 1000 mesh.

[0092] Preferably, the sieve mesh size is 100 mesh, 500 mesh, or 800 mesh.

[0093] In step S5, the mobile phase is purified water or water for injection.

[0094] In step S6, the freeze-drying conditions are as follows: freezing temperature is -20~-40℃, freezing time is 1~5 h, freeze-drying temperature is -10~-8℃, freeze-drying time is 24~28 h, desorption temperature is 5~8℃, and desorption time is 10~12 h.

[0095] Preferably, the freeze-drying vacuum degree is 0~10 Pa.

[0096] The yield of silk fibroin composite microspheres obtained by the preparation method of this invention is 77-88%. It should be noted that the yield is the yield after sieving using a sieving machine.

[0097] In the preparation method of the present invention, the particle size distribution of the silk fibroin composite microspheres is the same as the particle size of the silk fibroin composite microspheres after being sieved by a sieve.

[0098] The silk fibroin composite microspheres described above can be combined with compound materials to form silk fibroin composite microsphere gels.

[0099] The compound material is any one or more of the following: sodium hyaluronate, hydroxypropyl cellulose, sodium carboxymethyl cellulose, water, cross-linked sodium hyaluronate gel, collagen, and recombinant collagen.

[0100] In the silk fibroin composite microsphere gel, the mass ratio of silk fibroin composite microspheres to composite materials is (1~9):(9~1).

[0101] The silk fibroin composite microspheres or silk fibroin composite microsphere gels described above can be used to prepare injectable products. These injectable products are filler materials, such as skin fillers, lacrimal canaliculus fillers, and vocal cord fillers.

[0102] As described above, injectable products typically come in gel form, known as injectable gels.

[0103] As described above, the skin filler material can be injected into the dermis or between the periosteum and dermis to address wrinkles, volume defects, etc., caused by natural or unnatural factors.

[0104] The lacrimal canaliculus filling material described above can be injected into the lacrimal canaliculi to embolize them for the treatment of dry eye.

[0105] The vocal cord filling material described above can be injected into the vocal cords to treat vocal cord insufficiency.

[0106] For the injectable products described above, once they are released from the tissue, the silk fibroin composite microspheres can promote cell migration and adhesion, fix the silk fibroin in place to prevent diffusion, and achieve a three-in-one effect of "structural support + nutrient supply + regeneration induction" in vivo through the structure, thus achieving a long-lasting regenerative filling effect.

[0107] The silk fibroin composite microspheres and related products of the present invention have the following technical effects: 1) By using promoters and zero-length crosslinking agents to achieve self-crosslinking between molecular chains and constructing chemical interpenetrating networks, the introduction of small molecule chemical crosslinking agents can be effectively avoided, further preventing the accumulation of chemical crosslinking agents in organisms and reducing potential biotoxicity. 2) Silk fibroin composite microspheres are chemically interpenetrating networks. The natural polysaccharides / proteins contained in the silk fibroin composite microspheres can effectively reduce the crystallinity of silk fibroin, avoiding the problems of hardening, brittleness and decreased hydrophilicity caused by excessive crystallinity. At the same time, they can improve the elasticity and hydrophilicity of the silk fibroin composite microspheres and improve their biocompatibility. 3) After the formation of the chemical interpenetrating network of silk fibroin composite microspheres, the method of inducing appropriate crystallization of silk fibroin composite microspheres by using a folding promoter can further improve the degradation resistance and surface smoothness of silk fibroin composite microspheres, and obtain a composite material with long-term filling effect. 4) The preparation method of silk fibroin composite microspheres is tubular emulsification, which is a common continuous production equipment in the emulsification industry. It has the capability for large-scale industrial application, and the microspheres prepared by this method have good uniformity (D50: 35~50 μm, D90: 55~75 μm) and high yield (77~88%).

[0108] Furthermore, the silk fibroin composite microspheres and related products of the present invention differ from those of the prior art. Specifically: (a) Compared with Chinese invention patent application CN120204480A (1) Differences in microsphere preparation methods: Chinese invention patent application CN120204480A uses silk fibroin solution + organic solvent to freeze and dry to make block sponge, and then prepares sponge microsphere particles by crushing and sieving. These particles are non-spherical, physically cross-linked porous sponge particles. In contrast, this invention prepares spherical, dense particles through reverse emulsion polymerization. The particles are composed of chemical cross-linking and physical cross-linking.

[0109] (2) Differences in gel preparation methods: Chinese invention patent application CN120204480A uses sponge microspheres, hyaluronic acid, and cross-linking agent to prepare block gels. After dialysis and granulation, an injectable composite hydrogel is obtained. The composite gel particles are composed of sponge microspheres and cross-linked sodium hyaluronate, and the particle size is relatively large. This invention uses composite microspheres, sodium hyaluronate, and purified water to prepare injectable gels. No further dialysis or granulation is required, and it does not contain cross-linked sodium hyaluronate. Therefore, from the perspective of preparation methods, this invention and Chinese invention patent application CN120204480A have significant differences in both microsphere preparation and gel preparation.

[0110] (ii) Compared with Chinese invention patent application CN120501932A Differences in crosslinking processes: Chinese invention patent application CN120501932A uses amino-containing silk fibroin and sodium oxidized alginate (containing aldehyde groups) to crosslink through Schiff base bonds (dynamic covalent bonds), and forms a reversible dynamic network through the catalytic reaction of horseradish peroxidase and hydrogen peroxide. This avoids the problem that gels formed by traditional chemical crosslinking (such as covalent bonds) cannot be delivered through syringes due to irreversibility. Its principle is to construct a dynamic network by utilizing the Schiff base reaction of amino and aldehyde groups.

[0111] The technology disclosed in this invention utilizes a promoter and a zero-length crosslinking agent to achieve self-crosslinking between molecular chains to construct a chemical interpenetrating network, while simultaneously utilizing the self-folding of silk fibroin to construct a physical crosslinking network. It cleverly utilizes the polysaccharides in the composite microspheres to inhibit excessive crystallization of silk fibroin, resulting in composite microspheres with a dual physical / chemical network structure. Furthermore, the composite microspheres exhibit good elasticity and hydrophilicity. The preparation technology of this invention employs a "three-in-one" approach of zero-length crosslinking agent + polysaccharide and silk fibroin composite + self-folding to construct composite microspheres that possess both elasticity and hydrophilicity, rather than solely relying on enzymatic crosslinking to obtain composite microspheres. Therefore, this invention differs significantly from Chinese invention patent application CN120501932A.

[0112] Example 2 This embodiment relates to a specific implementation of the silk fibroin composite microspheres of the present invention.

[0113] In this embodiment, the preparation method of silk fibroin composite microspheres is as follows: Preparation of soluble silk fibroin: After degumming silk, it is dissolved in lithium bromide solution, dialyzed to remove salt to obtain silk fibroin solution, and then freeze-dried to obtain regenerated silk fibroin with a molecular weight of 150 kDa.

[0114] Preparation of the dispersed phase: 45 g of regenerated silk fibroin and 5 g of sodium hyaluronate were dissolved in water to obtain 450 g of 10% silk fibroin solution and 50 g of 10% sodium hyaluronate solution. The silk fibroin solution and sodium hyaluronate solution were mixed to obtain 500 g of blend (the concentration of regenerated silk fibroin was 9% and the concentration of sodium hyaluronate was 1%). 100 g of the blend was dissolved in 100 g of water to prepare 200 g of mixed solution (the concentration of regenerated silk fibroin was 4.5% and the concentration of sodium hyaluronate was 0.5%). 1.2 g of 10 wt% hydrogen peroxide (the mass ratio of hydrogen peroxide to dispersed phase was 0.6%) was added and mixed evenly to prepare the dispersed phase for later use.

[0115] Organic phase preparation: Take 1000 g of liquid paraffin as the organic phase, add 5 g of Tween-20 and 5 g of Span-80 as emulsifiers (the amount of emulsifier is 1%), add 1.2 g of horseradish peroxidase solution (enzyme concentration: 700 U) as a zero-length crosslinking agent (the mass ratio of horseradish peroxidase solution to dispersed phase is 0.6%), stir and mix evenly to prepare the organic phase for later use.

[0116] Emulsification: Connect the tubular emulsifier to the mixing tank, mix the dispersed phase and the organic phase at a mass ratio of 1:5, and then add them to the tubular emulsifier. Set the emulsification speed to 500 rpm, the stirring speed to 50 rpm, and the emulsification process to 2 times. After emulsification, set the stirring speed to 5 h and the stirring temperature to room temperature.

[0117] Folding promotion: After stirring, add 400 mL of 75% ethanol, start stirring at 100 rpm for 24 h, and allow to stand for 60 min. After standing, discard the supernatant and wash alternately with 1000 g n-hexane and 1000 g anhydrous ethanol for a total of 4 times. During the washing process, the stirring speed is 100 rpm and the stirring time is 30 min per wash. After each washing, allow to stand for 30 min and discard the supernatant. Finally, soak the microspheres in water for injection for 1 day, changing the water 3 times.

[0118] Sieving: Use 100-mesh and 800-mesh sieves and water for injection as the mobile phase for cleaning. After sieving, take the silk fibroin composite microspheres between the 100-mesh and 800-mesh sieves and freeze them.

[0119] Freeze-drying: The silk fibroin composite microspheres were placed in a freeze dryer, and the freezing temperature was set to 35℃ for 3 hours, the drying temperature to -3℃ for 24 hours, the desorption temperature to 6℃ for 12 hours.

[0120] The silk fibroin composite microspheres of this embodiment were subjected to relevant tests, and the test results are as follows: Figures 1-3 As shown.

[0121] Depend on Figures 1-2 As can be seen, the silk fibroin composite microspheres of this embodiment have a good spherical shape, and the surface of the microspheres is smooth, dense, and without microporous structure.

[0122] Depend on Figure 3 It can be seen that the silk fibroin composite microspheres in this embodiment have good uniformity and a relatively concentrated microsphere size distribution. The microsphere sizes are: D10: ~ 30 μm, D50: ~ 43 μm, D90: ~ 70 μm.

[0123] Example 3 This embodiment relates to a specific implementation of the silk fibroin composite microspheres of the present invention.

[0124] In this embodiment, the preparation method of silk fibroin composite microspheres is as follows: Preparation of soluble silk fibroin: After degumming silk, it is dissolved in lithium bromide solution, dialyzed to remove salt to obtain silk fibroin solution, and then freeze-dried to obtain regenerated silk fibroin with a molecular weight of 150 kDa.

[0125] Preparation of the dispersed phase: 45 g of regenerated silk fibroin and 5 g of sodium hyaluronate were dissolved in water to obtain 450 g of 10% silk fibroin solution and 50 g of 10% sodium hyaluronate solution. The silk fibroin solution and sodium hyaluronate solution were mixed to obtain 500 g of blend (the concentration of regenerated silk fibroin was 9% and the concentration of sodium hyaluronate was 1%). 100 g of the blend was dissolved in 100 g of water to prepare 200 g of mixed solution (the concentration of regenerated silk fibroin was 4.5% and the concentration of sodium hyaluronate was 0.5%). 0.4 g of 10 wt% hydrogen peroxide (the mass ratio of hydrogen peroxide to dispersed phase was 0.2%) was added and mixed evenly to prepare the dispersed phase for later use.

[0126] Organic phase preparation: Take 1000 g of liquid paraffin as the organic phase, add 5 g of Tween-20 and 5 g of Span-80 as emulsifiers (the amount of emulsifier is 1%), add 0.4 g of horseradish peroxidase solution (enzyme concentration: 700 U) as a zero-length crosslinking agent (the mass ratio of horseradish peroxidase solution to dispersed phase is 0.2%), stir and mix evenly to prepare the organic phase for later use.

[0127] Emulsification: Connect the tubular emulsifier to the mixing tank, mix the dispersed phase and the organic phase at a mass ratio of 1:5, and then add them to the tubular emulsifier. Start the emulsification speed at 800 rpm and the stirring speed at 50 rpm. Emulsify twice. After emulsification, set the stirring speed to 5 h and the stirring temperature to room temperature.

[0128] Folding promotion: After stirring, add 400 mL of 75% ethanol, start stirring at 100 rpm for 24 h, and allow to stand for 60 min. After standing, discard the supernatant and wash alternately with 1000 g n-hexane and 1000 g anhydrous ethanol for a total of 4 times. During the washing process, the stirring speed is 100 rpm and the stirring time is 30 min per wash. After each washing, allow to stand for 30 min and discard the supernatant. Finally, soak the microspheres in water for injection for 1 day, changing the water 3 times.

[0129] Sieving: Use 100-mesh and 800-mesh sieves and water for injection as the mobile phase for cleaning. After sieving, take the silk fibroin composite microspheres between the 100-mesh and 800-mesh sieves and freeze them.

[0130] Freeze-drying: The silk fibroin composite microspheres were placed in a freeze dryer, and the freezing temperature was set to 35℃ for 3 hours, the drying temperature to -3℃ for 24 hours, the desorption temperature to 6℃ for 12 hours.

[0131] Example 4 This embodiment relates to a specific implementation of the silk fibroin composite microspheres of the present invention.

[0132] In this embodiment, the preparation method of silk fibroin composite microspheres is as follows: Preparation of soluble silk fibroin: After degumming silk, it is dissolved in lithium bromide solution, dialyzed to remove salt to obtain silk fibroin solution, and then freeze-dried to obtain regenerated silk fibroin with a molecular weight of 150 kDa.

[0133] Preparation of the dispersed phase: 45 g of regenerated silk fibroin and 5 g of sodium hyaluronate were dissolved in water to obtain 450 g of 10% silk fibroin solution and 50 g of 10% sodium hyaluronate solution. The silk fibroin solution and sodium hyaluronate solution were mixed to obtain 500 g of blend (the concentration of regenerated silk fibroin was 9% and the concentration of sodium hyaluronate was 1%). 100 g of the blend was dissolved in 100 g of water to prepare 200 g of mixed solution (the concentration of regenerated silk fibroin was 4.5% and the concentration of sodium hyaluronate was 0.5%). 1.2 g of NHS was added as an accelerator (the mass ratio of NHS to the dispersed phase was 0.6%). After mixing evenly, it was used as the dispersed phase for later use.

[0134] Organic phase preparation: Take 1000 g of liquid paraffin as the organic phase, add 50 g of Tween-20 and 50 g of Span-80 as emulsifiers (the amount of emulsifier is 10%), add 12 g of EDC aqueous solution (EDC concentration is 10 wt%, containing 1.2 g EDC + 10.8 g water) as zero-length crosslinking agent (the mass ratio of EDC to dispersed phase is 0.6%), stir and mix evenly to prepare the organic phase for later use.

[0135] Emulsification: Connect the tubular emulsifier to the mixing tank, mix the dispersed phase and the organic phase at a mass ratio of 1:5, and then add them to the tubular emulsifier. Set the emulsification speed to 500 rpm, the stirring speed to 50 rpm, and the emulsification process to 2 times. After emulsification, set the stirring speed to 5 h and the stirring temperature to room temperature.

[0136] Folding promotion: After stirring, add 400 mL of 75% ethanol, start stirring at 100 rpm for 24 h, and allow to stand for 60 min. After standing, discard the supernatant and wash alternately with 1000 g n-hexane and 1000 g anhydrous ethanol for a total of 4 times. During the washing process, the stirring speed is 100 rpm and the stirring time is 30 min per wash. After each washing, allow to stand for 30 min and discard the supernatant. Finally, soak the microspheres in water for injection for 1 day, changing the water 3 times.

[0137] Sieving: Use 100-mesh and 800-mesh sieves and water for injection as the mobile phase for cleaning. After sieving, take the silk fibroin composite microspheres between the 100-mesh and 800-mesh sieves and freeze them.

[0138] Freeze-drying: The silk fibroin composite microspheres were placed in a freeze dryer, and the freezing temperature was set to 35℃ for 3 hours, the drying temperature to -3℃ for 24 hours, the desorption temperature to 6℃ for 12 hours.

[0139] Example 5 This embodiment relates to a specific implementation of the silk fibroin composite microspheres of the present invention.

[0140] In this embodiment, the preparation method of silk fibroin composite microspheres is as follows: Preparation of soluble silk fibroin: After degumming silk, it is dissolved in lithium bromide solution, dialyzed to remove salt to obtain silk fibroin solution, and then freeze-dried to obtain regenerated silk fibroin with a molecular weight of 150 kDa.

[0141] Preparation of the dispersed phase: 10 g of regenerated silk fibroin and 10 g of sodium hyaluronate were dissolved in water to obtain 100 g of 10% silk fibroin solution and 100 g of 10% sodium hyaluronate solution. The silk fibroin solution and sodium hyaluronate solution were mixed to obtain 200 g of blend (the concentration of regenerated silk fibroin was 5% and the concentration of sodium hyaluronate was 5%). 100 g of the blend was dissolved in 100 g of water to prepare 200 g of mixed solution (the concentration of regenerated silk fibroin was 2.5% and the concentration of sodium hyaluronate was 2.5%). 1.2 g of NHS was added as an accelerator (the mass ratio of NHS to the dispersed phase was 0.6%). After mixing evenly, it was used as the dispersed phase for later use.

[0142] Organic phase preparation: Take 1000 g of liquid paraffin as the organic phase, add 50 g of Tween-20 and 50 g of Span-80 as emulsifiers (the amount of emulsifier is 10%), add 12 g of EDC aqueous solution (EDC concentration is 10 wt%, containing 1.2 g EDC + 10.8 g water) as zero-length crosslinking agent (the mass ratio of EDC to dispersed phase is 0.6%), stir and mix evenly to prepare the organic phase for later use.

[0143] Emulsification: Connect the tubular emulsifier to the mixing tank, mix the dispersed phase and the organic phase at a mass ratio of 1:5, and then add them to the tubular emulsifier. Set the emulsification speed to 500 rpm, the stirring speed to 50 rpm, and the emulsification process to 2 times. After emulsification, set the stirring speed to 5 h and the stirring temperature to room temperature.

[0144] Folding promotion: After stirring, add 400 mL of 75% ethanol, start stirring at 100 rpm for 24 h, and allow to stand for 60 min. After standing, discard the supernatant and wash alternately with 1000 g n-hexane and 1000 g anhydrous ethanol for a total of 4 times. During the washing process, the stirring speed is 100 rpm and the stirring time is 30 min per wash. After each washing, allow to stand for 30 min and discard the supernatant. Finally, soak the microspheres in water for injection for 1 day, changing the water 3 times.

[0145] Sieving: Use 100-mesh and 800-mesh sieves and water for injection as the mobile phase for cleaning. After sieving, take the silk fibroin composite microspheres between the 100-mesh and 800-mesh sieves and freeze them.

[0146] Freeze-drying: The silk fibroin composite microspheres were placed in a freeze dryer, and the freezing temperature was set to 35℃ for 3 hours, the drying temperature to -3℃ for 24 hours, the desorption temperature to 6℃ for 12 hours.

[0147] Example 6 This embodiment relates to a specific implementation of the silk fibroin composite microspheres of the present invention.

[0148] In this embodiment, the preparation method of silk fibroin composite microspheres is as follows: Preparation of soluble silk fibroin: After degumming silk, it is dissolved in lithium bromide solution, dialyzed to remove salt to obtain silk fibroin solution, and then freeze-dried to obtain regenerated silk fibroin with a molecular weight of 150 kDa.

[0149] Preparation of the dispersed phase: 40 g of regenerated silk fibroin, 5 g of sodium hyaluronate, and 5 g of recombinant collagen were dissolved in water to obtain 400 g of 10% silk fibroin solution, 50 g of 10% sodium hyaluronate solution, and 50 g of 10% recombinant collagen solution. The silk fibroin solution, sodium hyaluronate solution, and recombinant collagen solution were then mixed to obtain 500 g of a blend (the concentration of regenerated silk fibroin was 8%, the concentration of sodium hyaluronate was 1%, and the concentration of recombinant collagen was 1%). 100 g of the blend was dissolved in 100 g of water to prepare 200 g of a mixed solution (the concentration of regenerated silk fibroin was 4%, the concentration of sodium hyaluronate was 0.5%, and the concentration of recombinant collagen was 0.5%). 1.2 g of NHS was added as a promoter (the mass ratio of NHS to the dispersed phase was 0.6%). After mixing evenly, the mixture was used as the dispersed phase for later use.

[0150] Organic phase preparation: Take 1000 g of liquid paraffin as the organic phase, add 50 g of Tween-20 and 50 g of Span-80 as emulsifiers (the amount of emulsifier is 10%), add 12 g of EDC aqueous solution (EDC concentration is 10 wt%, containing 1.2 g EDC + 10.8 g water) as zero-length crosslinking agent (the mass ratio of EDC to dispersed phase is 0.6%), stir and mix evenly to prepare the organic phase for later use.

[0151] Emulsification: Connect the tubular emulsifier to the mixing tank, mix the dispersed phase and the organic phase at a mass ratio of 1:5, and then add them to the tubular emulsifier. Set the emulsification speed to 500 rpm, the stirring speed to 50 rpm, and the emulsification process to 2 times. After emulsification, set the stirring speed to 5 h and the stirring temperature to room temperature.

[0152] Folding promotion: After stirring, add 400 mL of 75% ethanol, start stirring at 100 rpm for 24 h, and allow to stand for 60 min. After standing, discard the supernatant and wash alternately with 1000 g n-hexane and 1000 g anhydrous ethanol for a total of 4 times. During the washing process, the stirring speed is 100 rpm and the stirring time is 30 min per wash. After each washing, allow to stand for 30 min and discard the supernatant. Finally, soak the microspheres in water for injection for 1 day, changing the water 3 times.

[0153] Sieving: Use 100-mesh and 800-mesh sieves and water for injection as the mobile phase for cleaning. After sieving, take the silk fibroin composite microspheres between the 100-mesh and 800-mesh sieves and freeze them.

[0154] Freeze-drying: The silk fibroin composite microspheres were placed in a freeze dryer, and the freezing temperature was set to 35℃ for 3 hours, the drying temperature to -3℃ for 24 hours, the desorption temperature to 6℃ for 12 hours.

[0155] Example 7 This embodiment relates to a specific implementation of the silk fibroin composite microsphere gel of the present invention.

[0156] The preparation method of the silk fibroin composite microspheres used in this embodiment is the same as that in Example 2, and will not be repeated here.

[0157] In this embodiment, the preparation method of silk fibroin composite microsphere gel is as follows: Preparation of sodium hyaluronate gel: Dissolve 2 L of water for injection and 20 g of sodium hyaluronate to obtain sodium hyaluronate gel.

[0158] Preparation of silk fibroin composite microsphere gel: Take 20 g of composite microspheres and mix them with sodium hyaluronate gel to obtain sodium hyaluronate gel containing silk fibroin composite microspheres.

[0159] Preparation of injectable product: Sodium hyaluronate gel containing silk fibroin composite microspheres is filled and sterilized by moist heat to obtain injectable sodium hyaluronate gel containing silk fibroin composite microspheres.

[0160] In this embodiment, the injectable product is used for skin filling.

[0161] Comparative Example 1 The difference between this comparative example and Example 2 is that no accelerator is used, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), and the dispersed phase does not contain natural polysaccharides or proteins.

[0162] Comparative Example 2 The difference between this comparative example and Example 2 is that the emulsification method is ordinary mechanical emulsification, and the emulsification speed is 350 rpm.

[0163] In this comparative example, the product obtained was silk fibroin microspheres.

[0164] Comparative Example 3 The difference between this comparative example and Example 7 is that silk fibroin composite microspheres are not used.

[0165] Comparative Example 4 The difference between this comparative example and Example 7 is that the silk fibroin microspheres prepared in Comparative Example 2 are mixed with sodium hyaluronate gel.

[0166] Example 8 This embodiment is a performance comparison test between Embodiments 2 to 6 and Comparative Examples 1 to 2. The test results are shown in the table below.

[0167] The particle size distribution is calculated as (D90-D10) / D50.

[0168] As can be seen from the table above, compared with Comparative Example 2 which uses mechanical emulsification, the Span values ​​(1.01~1.10) of Examples 2~6 of the present invention which use tubular emulsification are much smaller than the Span value (1.32) of Comparative Example 2, indicating that the particle size uniformity of the present invention is good.

[0169] As shown in the table above, compared with Examples 2 and 4 to 6, the increased emulsification speed in Example 3 (from 500 rpm to 800 rpm) results in a significantly smaller D50 than that in Examples 2 and 4 to 6, indicating that the above results conform to the fluid dynamics shear law.

[0170] As shown in the table above, compared with Comparative Example 2 which uses mechanical emulsification, the yields of Examples 2 to 6 of the present invention, which use tubular emulsification (80.5 to 85.2%), are significantly higher than the yield of Comparative Example 2 (62.4%).

[0171] As shown in the table above, compared with the silk fibroin microspheres of Comparative Example 1, the microspheres prepared in Examples 2 to 6 of the present invention are composite microspheres. Since the same emulsification method was used, the differences in particle size and yield were not significant. However, Comparative Example 1 contained a chemical cross-linking agent, while Examples 2 to 6 did not, resulting in lower biotoxicity.

[0172] Example 9 This embodiment is a performance comparison test between Example 7 and Comparative Examples 3 to 4. The test results are shown in the table below.

[0173] As shown in the table above, compared with Comparative Example 3 without silk fibroin composite microspheres, the elasticity of Example 7 of the present invention (680.37) is significantly higher than that of Comparative Example 3 (166.92), which proves that the silk fibroin composite microspheres of the present invention have better elasticity. By adding the silk fibroin composite microspheres of the present invention, the elasticity of injectable products (such as dermal fillers) can be effectively improved, and the support of injectable products can be enhanced.

[0174] As shown in the table above, compared with Comparative Example 3 (without silk fibroin composite microspheres) and Comparative Example 4 (with silk fibroin microspheres added to Comparative Example 2), the pushing force of Example 7 of the present invention (13.87 & 14.14) is greater than that of Comparative Example 3 (9.92 & 10.32) and less than that of Comparative Example 4 (20.13 & 23.83). This proves that if silk fibroin microspheres with uneven particle size are used, it is easy to cause the injection product to clog the injection tool (such as the injection needle), which has a great risk.

[0175] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A silk fibroin composite microsphere, characterized in that, Silk fibroin composite microspheres are interpenetrating network structures of silk fibroin network, natural polysaccharide network and / or protein network; Silk fibroin composite microspheres are chemically cross-linked microspheres without cross-linking agents; The silk fibroin composite microspheres are spherical, and the surface of the silk fibroin composite microspheres is a smooth, non-porous surface. The particle size distribution of the silk fibroin composite microspheres is: D50: 35~50 μm, D90: 55~75 μm; The crystallinity of the silk fibroin composite microspheres is 25-55%.

2. A method for preparing silk fibroin composite microspheres, used to prepare the silk fibroin composite microspheres as described in claim 1, characterized in that, include: Soluble silk fibroin, natural polysaccharides and / or proteins, and promoters are added to an aqueous solution to obtain a silk fibroin composite solution as the dispersed phase. Emulsifiers and zero-length crosslinking agents are added to the organic phase to obtain a continuous phase; Emulsification was performed using a tubular emulsifier to disperse the dispersed phase in the continuous phase, resulting in a water-in-oil emulsion containing droplets of silk fibroin composite solution. The emulsion was then subjected to a solidification reaction under low-speed stirring to obtain a silk fibroin composite microsphere system. Add the folding promoter to the silk fibroin composite microsphere system, stir and mix, let stand and settle, discard the supernatant, and wash the silk fibroin composite microspheres with organic solvent; The silk fibroin composite microspheres were sieved under water flow using a sieve to obtain a uniform silk fibroin composite microsphere slurry. The silk fibroin composite microsphere material solution was freeze-dried to obtain silk fibroin composite microspheres.

3. The preparation method according to claim 2, characterized in that, Soluble silk fibroin has a molecular weight of 100-200 kDa; and / or Natural polysaccharides are any one or more combinations of sodium hyaluronate, chitosan, agarose, and xanthan gum; and / or The protein is any one or more combinations of type I collagen, type III collagen, recombinant type III collagen, recombinant type XVII humanized collagen, and gelatin; and / or The accelerator is any one or a combination of hydrogen peroxide, N-hydroxysuccinimide, ethanol, n-hexane, and sodium hydroxide; and / or Soluble silk fibroin: natural polysaccharides and / or proteins: water in a mass ratio of (5~9):(5~1):90; and / or The amount of accelerator used is 0.2~0.6% of the mass of the dispersed phase; and / or The concentration of the accelerator is 10 wt%.

4. The preparation method according to claim 2, characterized in that, The organic phase is any one of liquid paraffin, soybean oil, ethyl acetate, petroleum ether, and silicone oil; and / or The emulsifier is a nonionic emulsifier, and is any one or a combination of polysorbate 20, polysorbate 40, sorbitan oleate, and sucrose fatty acid ester; and / or The zero-length crosslinking agent is any one or more combinations of horseradish peroxidase, 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide; and / or The amount of emulsifier used is 1-6%; and / or The amount of zero-length crosslinking agent is 0.2~2% of the mass of the dispersed phase; and / or The concentration of the zero-length crosslinking agent is 10 wt%.

5. The preparation method according to claim 2, characterized in that, The mass ratio of dispersed phase to continuous phase is 1:(5~10); and / or Emulsification conditions: emulsification speed of 500-1000 rpm, emulsification times of 1-3 times; and / or Curing conditions: stirring speed 30~200 rpm, curing temperature 20~37℃, curing time 1.5~24 h.

6. The preparation method according to claim 2, characterized in that, The folding accelerator is any one or more combinations of methanol, 75% ethanol, glycerol, and polyethylene glycol; and / or Mixing conditions: mixing speed 50~100 rpm, mixing time 1~24 h, settling time 0.5~2 h; and / or The organic solvent is any one or a combination of anhydrous ethanol, 75% ethanol, 50% ethanol, and n-hexane; and / or Cleaning conditions: Clean 2-3 times with any organic solvent.

7. The preparation method according to claim 2, characterized in that, The screening machine is a vibrating screen; and / or The mobile phase is purified water or water for injection; and / or The screen mesh size of the screening machine is any one or more combinations of 100 mesh, 200 mesh, 500 mesh, 800 mesh, and 1000 mesh.

8. The preparation method according to claim 2, characterized in that, Freeze-drying conditions: freezing temperature -20~-40℃, freezing time 1~5 h, freeze-drying temperature -10~-8℃, freeze-drying time 24~28 h, desorption temperature 5~8℃, desorption time 10~12 h.

9. A silk fibroin composite microsphere gel, characterized in that, Silk fibroin composite microsphere gel include: Silk fibroin composite microspheres as described in claim 1 or silk fibroin composite microspheres prepared by any of the preparation methods described in claims 2 to 8; The compound material is any one or more of the following: sodium hyaluronate, hydroxypropyl cellulose, sodium carboxymethyl cellulose, water, cross-linked sodium hyaluronate gel, collagen, and recombinant collagen.

10. An application of a silk fibroin-containing composite microsphere product, characterized in that, The product is an injectable product, including the silk fibroin composite microspheres as described in claim 1, or the silk fibroin composite microspheres prepared by any of the preparation methods described in claims 2 to 8, or the silk fibroin composite microsphere gel as described in claim 9. Applications include skin filling, lacrimal canaliculus filling, and vocal cord filling.

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