Filling silk fibroin hyaluronic acid gel and preparation method thereof

Abstract

The invention provides a composition for filling as well as a preparation method and application thereof. The composition comprises gel particles and an aqueous medium hyaluronate solution, the gel particles comprise silk fibroin, hyaluronic acid or a salt thereof, and a cross-linking agent, and the gel particles comprise a porous three-dimensional body; the mass ratio of the silk fibroin to the hyaluronic acid or the salt thereof to the cross-linking agent is (0.1-10): (1-20): 1.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a silk fibroin hyaluronic acid gel for filling and its preparation method. Background Technology

[0002] Cosmetic medicine is divided into surgical and non-surgical categories. Non-surgical cosmetic medicine, also known as minimally invasive cosmetic medicine, refers to using non-invasive or minimally invasive medical treatments to meet aesthetic needs. Compared with surgical cosmetic medicine, minimally invasive cosmetic medicine has become the main growth point in the cosmetic medicine market due to its advantages such as simple operation, less trauma, high safety, and short recovery period.

[0003] However, currently available filler materials for minimally invasive cosmetic procedures have drawbacks, such as easily inducing adverse reactions like inflammation, and the space left after rapid degradation of the filler material cannot be effectively filled due to a mismatch between the degradation rate of the filler material and the cell ingrowth rate. Therefore, there is an urgent need in the market for safe, effective, and side-effect-free cosmetic filler products. Summary of the Invention

[0004] The purpose of this invention is to provide a silk fibroin hyaluronic acid gel for filling and its preparation method. The gel has the dual functions of immediate filling effect and promoting collagen regeneration, and is suitable for facial injection filling with good biocompatibility.

[0005] The present invention adopts the following technical solution:

[0006] In one aspect, the present invention provides a composition for filling, characterized in that the composition comprises gel particles and an aqueous medium; wherein the gel particles comprise silk fibroin, hyaluronic acid or a salt thereof and a crosslinking agent, and the gel particles comprise a porous three-dimensional body.

[0007] In a preferred embodiment, the gel particles are prepared by adding the porous three-dimensional body to a solution containing hyaluronic acid or a salt thereof, silk fibroin, and a crosslinking agent.

[0008] In a preferred embodiment, the porous three-dimensional body is selected from one or more of porous microspheres, hollow conduits, porous microparticles, and porous irregular blocks; preferably, it is a porous microsphere; more preferably, it is a silk fibroin porous microsphere; even more preferably, it is a silk fibroin porous microsphere wetted with hyaluronic acid or its salt; preferably, the particle size of the porous three-dimensional body is less than 600 mm, more preferably less than 250 mm, even more preferably less than 180 mm, and most preferably less than 120 mm.

[0009] In a preferred embodiment, the mass ratio of silk fibroin: hyaluronic acid or its salt: crosslinking agent in the gel particles is (0.1-10):(1-20):1, preferably (1-5):(5-15):1, more preferably (1-5):10:1, and most preferably 2:10:1.

[0010] In a preferred embodiment, the mass ratio of the gel particles to the aqueous medium is (0.1 to 10):1, preferably 1:1.

[0011] In a preferred embodiment, the aqueous medium is a solution of hyaluronic acid or its salt, preferably, the concentration of hyaluronic acid or its salt in the aqueous medium is 2wt%-8wt%, and more preferably 5wt%.

[0012] In a preferred embodiment, the hyaluronic acid or its salt has a molecular weight of 200-4000 kDa, preferably 400-1600 kDa, and more preferably 800-2000 kDa.

[0013] In a preferred embodiment, the silk fibroin has a molecular weight of 2-500 kDa, preferably 10-250 kDa, and more preferably 50-100 kDa.

[0014] In a preferred embodiment, the elastic modulus of the gel particles is 300-800 Pa, preferably 400-700 Pa.

[0015] In a preferred embodiment, the viscous modulus of the gel particles is 100-250 Pa, preferably 150-250 Pa.

[0016] In a preferred embodiment, the pushing force of the gel particles is 10-25N, preferably 15-25N.

[0017] In a preferred embodiment, the salt of the hyaluronic acid is selected from sodium hyaluronate, zinc hyaluronate, and potassium hyaluronate, preferably sodium hyaluronate.

[0018] In a preferred embodiment, the crosslinking agent is selected from 1,4-bisglycidoxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipamide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, or any combination thereof, preferably BDDE.

[0019] In a preferred embodiment, based on the total mass of the gel particles, the content of porous three-dimensional bodies in the gel particles is 0.01wt%-50wt%, preferably 0.1wt%-20wt%, more preferably 0.1wt%-10wt%, more preferably 0.2wt%-5wt%, and most preferably 0.2wt%.

[0020] In a preferred embodiment, the porous three-dimensional body is prepared by freeze-drying, preferably by freeze-drying an aqueous solution of silk fibroin, and more preferably by the following method: preparing a concentrated solution of silk fibroin, then freeze-drying the concentrated solution, fumigating the resulting freeze-dried silk fibroin block with an organic solvent at 37°C for a period of time, pulverizing it and passing it through a 120-mesh sieve, preferably, the organic solvent is selected from one or more of methanol, ethanol, acetone and isopropanol.

[0021] In a preferred embodiment, the porous three-dimensional body is added to a solution of hyaluronic acid or a salt thereof to obtain a hyaluronic acid-infiltrated porous three-dimensional body; preferably, a mixture of the porous three-dimensional body and hyaluronic acid or a salt thereof is placed in an oil phase to obtain a hyaluronic acid-infiltrated porous three-dimensional body; even more preferably, the hyaluronic acid-infiltrated crosslinked porous three-dimensional body is prepared according to the following method:

[0022] a. Prepare an alkaline solution containing BDDE for later use;

[0023] b. Weigh an appropriate amount of sodium hyaluronate and silk fibroin porous microspheres, add an alkaline solution containing BDDE, stir evenly, degas under vacuum, and then slowly drop the mixture into the oil phase and react at -20℃ for 3 days.

[0024] c. Remove and place at room temperature, adjust pH to neutral, and dialyze in phosphate buffer;

[0025] Preferably, the oil phase is selected from soybean oil, liquid paraffin, glycerin, silicone oil, etc.

[0026] Preferably, the volume ratio of the mixture to the oil phase is 1:(1-20).

[0027] In a preferred embodiment, the composition is a solution, gel, lyophilized powder, emulsion, or cream; preferably, the composition is an injectable filler.

[0028] In a preferred embodiment, the composition is used to fill into the face, neck, or torso, preferably into the face.

[0029] In a second aspect, the present invention provides a kit comprising the aforementioned gel particles and an aqueous medium.

[0030] In a third aspect, the present invention provides the use of the aforementioned composition or kit in the preparation of a product for improving the skin condition of a subject in need, preferably, the skin condition being selected from skin dehydration, lack of skin elasticity, rough skin, lack of skin tightness, skin stretch lines, skin stretch marks, pale skin, dermal pitting, sunken cheeks, thin lips, retro-orbital defects, facial folds and wrinkles.

[0031] In a preferred embodiment, the composition is applied to the dermal region of the subject.

[0032] In a fourth aspect, the present invention provides a non-therapeutic method for improving the skin condition of a subject in need, comprising administering the aforementioned composition or kit to the subject; preferably, the skin condition is selected from skin dehydration, lack of skin elasticity, rough skin, lack of skin tightness, skin stretch lines, skin stretch marks, pale skin, dermal pitting, sunken cheeks, thin lips, retro-orbital defects, facial folds and wrinkles.

[0033] In a preferred embodiment, the composition is applied to the face of a subject; preferably, the composition is applied to the dermal region of the subject.

[0034] In a fifth aspect, the present invention provides a method for preparing the aforementioned composition, characterized by comprising the following steps:

[0035] 1) A solution of hyaluronic acid or its salt, silk fibroin, a cross-linking agent, and a porous three-dimensional body are mixed, allowed to stand, and then sieved to obtain gel particles; preferably, the porous three-dimensional body is selected from one or more of porous microspheres, hollow conduits, porous microparticles, and porous irregular blocks; preferably, porous microspheres; more preferably, silk fibroin porous microspheres; even more preferably, silk fibroin porous microspheres wetted with hyaluronic acid or its salt; preferably, the particle size of the porous three-dimensional body is less than 600 mm, more preferably less than 250 mm, even more preferably less than 180 mm, and most preferably less than 120 mm;

[0036] 2) Add the gel particles obtained in step 1) to an aqueous medium.

[0037] In a preferred embodiment, the porous three-dimensional body is prepared by freeze-drying, preferably by freeze-drying an aqueous solution of silk fibroin, and more preferably by the following method: preparing a concentrated solution of silk fibroin, then freeze-drying the concentrated solution, fumigating the resulting freeze-dried silk fibroin block with an organic solvent at 37°C for a period of time, pulverizing it and passing it through a 120-mesh sieve, preferably, the organic solvent is selected from one or more of methanol, ethanol, acetone and isopropanol.

[0038] In a preferred embodiment, the porous three-dimensional body is added to a solution of hyaluronic acid or a salt thereof to obtain a hyaluronic acid-infiltrated porous three-dimensional body; preferably, a mixture of the porous three-dimensional body and hyaluronic acid or a salt thereof is placed in an oil phase to obtain a hyaluronic acid-infiltrated porous three-dimensional body; even more preferably, the hyaluronic acid-infiltrated crosslinked porous three-dimensional body is prepared according to the following method:

[0039] a. Prepare an alkaline solution containing BDDE for later use;

[0040] b. Weigh an appropriate amount of sodium hyaluronate and silk fibroin porous microspheres, add an alkaline solution containing BDDE, stir evenly, degas under vacuum, and then slowly drop the mixture into the oil phase and react at -20℃ for 3 days.

[0041] c. Remove and place at room temperature, adjust pH to neutral, and dialyze in phosphate buffer;

[0042] Preferably, the oil phase is selected from soybean oil, liquid paraffin, glycerin, silicone oil, etc.

[0043] Preferably, the volume ratio of the mixture to the oil phase is 1:(1-20).

[0044] In a sixth aspect, the present invention provides a method for preparing the aforementioned composition, characterized by comprising the following steps:

[0045] 1) Prepare hyaluronic acid salt solution and BDDE-containing alkaline solution separately for later use;

[0046] 2) Weigh an appropriate amount of raw material and dissolve it in an alkaline solution containing BDDE, stir evenly, and let it stand at 0-10℃ for 10-72h, preferably at 4℃ for 48h; then place it at 10-60℃ to deepen cross-linking for 1-10h, preferably at 40℃ to deepen cross-linking for 3h, to obtain a co-crosslinked gel.

[0047] 3) The above co-crosslinked gel was dialyzed in phosphate buffer for a period of time, and then granulated through a 60-80 mesh sieve to obtain gel particles;

[0048] 4) Add an appropriate amount of hyaluronic acid salt solution to the above gel particles, sterilize by moist heat, and the product is obtained.

[0049] Step 2) The raw material is hyaluronic acid or its salt and silk fibroin; or hyaluronic acid or its salt, silk fibroin and silk fibroin porous microspheres, or hyaluronic acid or its salt, silk fibroin and hyaluronic acid-infiltrated and cross-linked silk fibroin porous microspheres.

[0050] Beneficial effects

[0051] Compared to single hyaluronic acid gels, the gel provided by this invention contains two filler materials: silk fibroin and hyaluronic acid. It has good biocompatibility and low immunogenicity, avoiding adverse reactions such as subclinical inflammatory reactions and fibrosis caused by chemical filling methods. The gel prepared by this invention has good mechanical properties. After intradermal injection, it can support depressions in soft tissues, making the face fuller and rounder. It has good injectability, can promote the growth of skin fibroblasts, and has good cell adhesion after filling. It can promote skin collagen regeneration and skin tissue regeneration, and has the effects of immediate filling and long-term tissue repair.

[0052] This invention utilizes silk fibroin microspheres as a porous, sponge-like scaffold material, providing excellent support, facilitating cell adhesion, and promoting collagen regeneration. This invention combines a rigid porous silk fibroin microsphere framework with a flexible, continuous gel matrix, exhibiting programmed gradient degradation and a tissue-stimulating regenerative effect. This slows down the degradation rate of the filler material, prolongs its effectiveness in facial soft tissue repair and filling, and improves the material's efficacy, achieving long-term filling results. Simultaneously, it avoids adverse facial reactions such as granulomas caused by excessive stimulation after injection of commercially available microsphere materials like PLLA, demonstrating good safety. This invention, as a filler, possesses anti-aging and rejuvenating effects. Brief description of the attached diagram

[0053] Figure 1 This is a schematic diagram of the gel structure and mechanism of action in Example 3.

[0054] Figure 2 In the examples, A represents the silk fibroin microspheres in Comparative Example 3, and B represents the porous silk fibroin microspheres in Example 2.

[0055] Figure 3 The effect of the gel sample of the present invention on cell proliferation was shown (compared with the normal control (CTRL) group, **P<0.01, ***P<0.001, ****P<0.0001).

[0056] Figure 4 This study investigates the effects of HA-SF gels with different SF molecular weights on collagen regeneration.

[0057] Figure 5 The effect of the gel of the present invention on collagen regeneration was shown (***P<0.001, ****P<0.0001 compared with the CTRL group).

[0058] Figure 6 This is an HE-stained section of mouse back skin after gel injection in Example 3 (A in the figure is the normal group, B is the Example 3 group, C is the control group 1, and D is the control group 2). Invention Details

[0060] definition

[0061] As used herein, the term "silk fibroin" includes silk fibroin, insect or spider silk fibroin, or recombinant silk fibroin. In one embodiment, the silk fibroin is obtained from domestic silkworms.

[0062] As used herein, the term "hyaluronic acid" is a biodegradable polymeric component composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. This water-soluble polymer is naturally found in almost all tissues, particularly in the extracellular matrix, synovial fluid of the eye, and joints. HA is commercially available in pure form. Small gel-particle HA fillers can be used to stimulate the production of natural collagen, which is believed to be induced by the mechanical stretching of the dermis and the activation of dermal fibroblasts.

[0063] As used herein, the term "porous three-dimensional body" includes porous microspheres, hollow vessels, porous microparticles, and porous irregular blocks. It is a functional polymeric material with numerous micropores, typically prepared from polymeric or inorganic materials. It possesses a three-dimensional spatial pore structure with relatively large pore size and specific surface area. Porous three-dimensional bodies exhibit many unique characteristics. Due to the numerous micropores and defects within them, these micropores provide additional surface area, thereby increasing the loading capacity of active substances. Furthermore, porous three-dimensional body materials also possess excellent mesoporous properties. The varying sizes, shapes, and connectivity of the pore structure allow for a tunable pore size distribution.

[0064] As used herein, the term "crosslinking" refers to the intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into a more stable structure such as a gel. The term "crosslinking agent" refers to a substance that links individual polymer molecules, macromolecules, and / or monomer chains into intermolecular bonds. Representative crosslinking agents include 1,4-bisglycidoxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), dicarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic hydrazide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, etc.

[0065] As used herein, the term "gel" refers to a thick liquid or semi-solid formulation in the form of a solution, suspension, or emulsion. Gel matrices are single-phase dispersion systems and can be aqueous or oil-based. Aqueous gel matrices are generally composed of water, glycerol or propylene glycol with cellulose derivatives, carbomer and alginate, tragacanth gum, gelatin, starch, etc.; oil-based gel matrices are composed of liquid paraffin with polyethylene or fatty oils with colloidal silica or aluminum soaps, zinc soaps, etc.

[0066] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts may include hydrochlorides, phosphates, pyrophosphates, hydrobroms, sulfates, sulfinates, nitrates, malates, maleates, fumarates, tartrates, succinates, citrates, acetates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, and alkylates (e.g., acetates, HOOC-(CH2)). n -COOH, where n is 0-4). Furthermore, if the compound herein is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the compound herein is a free base, the addition salt (particularly a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with acid, according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts.

[0067] As used herein, the term "subject" refers to an animal, such as a mammal (including a human), which has been or will be the subject of treatment, observation, or experimentation. The methods described herein can be used for therapeutic and / or veterinary applications in humans. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0068] As used herein, the term "kit" may include two or more single-dose or multi-dose pharmaceutical agents, each individually packaged or formulated; or two or more single-dose or multi-dose pharmaceutical agents packaged or formulated in combination. Thus, one or more pharmaceutical agents may be present in a first container, and the kit may optionally include one or more pharmaceutical agents in a second container. One or more containers are housed within a package, and the package may optionally include instructions for administration or dosage. The kit may include additional components, such as syringes or other components for administering the pharmaceutical agents and diluents, or other components for formulation. Detailed Implementation

[0069] Injectable fillers involve injecting natural or synthetic biological materials into the dermis or subcutaneous layer to improve skin laxity, depressions, and delay skin aging by filling tissue or stimulating collagen regeneration. The filling effect depends on the properties of the filler material. Based on their bioabsorption properties and mechanisms of action, injectable filler materials are mainly divided into three categories: (1) endogenous biological materials, whose composition is similar to the extracellular matrix of human cells, such as hyaluronic acid (HA) and collagen; (2) synthetic polymers, which have good bioabsorbability and degradability, and can stimulate tissue to generate collagen and other tissue structures, thereby increasing tissue volume to a limited extent. These are also known as semi-permanent fillers, such as poly-L-lactic acid (PLLA), polycaprolactone (PCL), and fillers containing hydroxyapatite (CaHA); (3) non-absorbable and non-degradable polymer materials, which permanently and irreversibly provide tissue space and cause an immune response in the body, such as polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), and polyacrylic acid (PAA). Ideal injectable fillers should integrate "filling, repairing, and anti-aging," first quickly filling the skin and then stimulating collagen regeneration to achieve a "safer, more natural, and longer-lasting" effect.

[0070] Hyaluronic acid is an endogenous component of the human body, possessing advantages such as good biocompatibility and biodegradability. An exemplary injectable filler product based on a hyaluronic acid composite system may include a cross-linked sodium hyaluronate gel containing L-lactic acid-ethylene glycol copolymer microspheres. This gel is composed of cross-linked sodium hyaluronate, L-lactic acid-ethylene glycol copolymer microspheres, lidocaine hydrochloride, and a phosphate buffer system, and possesses the dual functions of "filling and stimulating regeneration."

[0071] Silk fibroin (SF) is a natural high-molecular-weight fibrous protein extracted from silkworm silk, composed of 18 amino acids. It possesses excellent biocompatibility, biodegradability, low immunogenicity, tissue cell adhesion ability, and mechanical properties. In one embodiment of this invention, a gel for injection filling is provided by combining a rigid porous silk fibroin microsphere framework with a flexible continuous gel matrix.

[0072] Example

[0073] The technical solution of the present invention will be described in detail below with reference to the embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0074] The main materials used in the following embodiments are sourced from the following sources:

[0075] Hyaluronic acid (HA): Bloomage Biotechnology Co., Ltd., molecular weight 800-2000kDa, batch number: 22061041;

[0076] Silk fibroin (SF): Shenzhen Huasi Biotechnology Co., Ltd., molecular weight <50kDa, batch number: 23041101; molecular weight 50-100kDa, batch number: SPSDK2F005; molecular weight >100kDa, batch number: 231012;

[0077] Comparative Example 1: Commercially available product Restylane 2 (modified sodium hyaluronate gel for injection), manufactured by Galderm, batch number 21771;

[0078] Comparative Example 2: Commercially available product Aivilan (crosslinked sodium hyaluronate gel containing L-lactic acid-ethylene glycol copolymer microspheres), manufactured by Changchun Shengboma Company, batch number T23043014;

[0079] Comparative Example 3: Prepared according to the following steps.

[0080] (1) Preparation of silk fibroin microspheres: 40g of silkworm silk was added to 2700ml of 0.2% sodium carbonate aqueous solution, and the mixture was heated in a water bath at 96-100℃ for 60min. After filtration, the filter cake was taken and the water bath was repeated 3 times. The filter cake from the last filtration was dried at 60℃ and then slowly dissolved in 200ml of 9mol / L lithium bromide aqueous solution. The mixture was heated in a water bath at 60℃ for 6h, and then centrifuged at 8000rpm for 5min. The supernatant was collected and the supernatant after centrifugation was placed in a dialysis bag (MW 8,000-14,000). Purified water was used as the dialysis solution, and the mixture was magnetically stirred and dialyzed for three days. The retentate was collected and prepared into a 5% silk fibroin solution with purified water.

[0081] At 25°C, the above 5% silk fibroin solution was allowed to stand to form 5g of white gel material. The gel material was homogenized in a homogenizer (T25, IKA) at 24000 rpm / min for 5 min to obtain 5g of silk fibroin gel particles of different diameters. After filtration, 3.5g of silk fibroin particles that passed through a 200-mesh sieve were collected. A small amount of the collected silk fibroin particles were weighed and dried to constant weight to determine the water content of the silk fibroin particles and to determine the mass concentration of the silk fibroin particles as 3.23%.

[0082] (2) Dissolve 0.7g of sodium hyaluronate in 7ml of 1% sodium hydroxide solution to prepare a 0.1g / ml sodium hyaluronate solution, and add 2.167g (0.07 / 0.0323=2.167g) of silk fibroin particles to the sodium hyaluronate solution. After mixing thoroughly, add 56μl of BDDE, mix evenly, and place at 40℃ for 5 hours to crosslink and form a silk fibroin hyaluronic acid composite gel material.

[0083] The silk fibroin-hyaluronic acid composite gel material was added to a dialysis bag with a MW 8,000-14,000 capacity. Using PBS buffer (pH 7.4) as the dialyzing solution, the gel was dialyzed under magnetic stirring. The dialyzed composite gel material was then homogenized at 24,000 rpm for 10 minutes. The gel was then squeezed through a 60-mesh sieve in a syringe, and the composite gel particles were collected. The gel was then sterilized by high-temperature autoclaving at 120°C for 15 minutes, followed by aseptic aliquoting into disposable syringes. This yielded 35g of injectable silk fibroin-hyaluronic acid composite gel.

[0084] Example 1: Preparation of Hyaluronic Acid-Silk Fibroin Gel (HA-SF Gel)

[0085] Preparation of HA-SF gel particles: Prepare a 1% sodium hydroxide aqueous solution and add 1 wt% 1,4-butanediol diglycidyl ether (BDDE) and mix well. Accurately weigh 0.5 g of HA (molecular weight 800-2000 kDa) and 0.05 g of SF (molecular weight 50-100 kDa), add 5 mL of the above BDDE solution, stir well, and let stand at 4℃ for 48 h for initial cross-linking; then place in a 40℃ water bath for 3 h to deepen cross-linking, thus forming HA-SF co-crosslinked gel. Take 1 g of HA-SF co-crosslinked gel and add it to phosphate buffer, dialyze and swell for 10 h. After dialyzing, take it out and pass it through a 60-mesh and 80-mesh sieve to obtain HA-SF gel particles (HA 10 wt%, SF 1 wt%).

[0086] Preparation of 5% sodium hyaluronate solution: Weigh 0.5g of HA (molecular weight 800-2000kDa) and dissolve it in 10ml of PBS buffer. Stir and let it stand until it swells for later use.

[0087] Preparation of HA-SF gel: Accurately weigh HA-SF gel particles, add the same weight of 5% sodium hyaluronate solution, mix well, and then load into a sterile syringe. Sterilize by moist heat to obtain HA-SF gel. The mass ratio of SF, HA and BDDE in the gel is 1:5:0.5.

[0088] Example 2: Preparation of HA-SF gel containing silk fibroin porous microspheres

[0089] 1) Preparation of porous silk fibroin microspheres

[0090] Weigh 0.3g of silk fibroin and dissolve it in 3mL of purified water to obtain a silk fibroin concentrate with a concentration of 10wt%. Freeze-dry the silk fibroin concentrate, then fumigate it overnight with ethanol or methanol vapor (37℃), and then pulverize it with a multi-functional pulverizer and pass it through a 120-mesh sieve to obtain silk fibroin porous microsphere powder.

[0091] 2) Preparation of HA-SF gel containing silk fibroin porous microspheres

[0092] Accurately weigh 0.5 g of hyaluronic acid (800-2000 kDa), 0.1 g of silk fibroin (50-100 kDa), and 0.01 g of silk fibroin porous microspheres. Mix thoroughly, add 5 mL of a solution containing 1 w / v% sodium hydroxide and 1 wt% BDDE, stir well, and allow to crosslink at 4°C for 48 h. Then, crosslink at 40°C for 3 h. Take 1 g of gel, cut it into small pieces, and dialyze with phosphate buffer to swell for 7 h. After dialyzing, remove the gel and pass it through a 60-mesh and 80-mesh sieve to obtain HA-SF gel particles containing silk fibroin porous microspheres (SF porous microspheres 0.2 wt%, SF 2 wt%, HA 10 wt%).

[0093] Add 5% sodium hyaluronate solution in equal proportion, mix well, and then load into a sterile syringe. Sterilize by moist heat to obtain HA-SF gel containing porous microspheres of silk fibroin. The mass ratio of SF, HA and BDDE in the gel is 1:5:0.5.

[0094] Example 3: Preparation of HA-SF composite gel

[0095] (1) Preparation of hyaluronic acid-infused cross-linked silk fibroin porous microspheres

[0096] 20 mL of 1 w / v sodium hydroxide aqueous solution and 0.3 mL of 1.5 wt% BDDE solution were mixed thoroughly. 1 g of sodium hyaluronate (800-2000 kDa) and 0.5 g of the silk fibroin porous microspheres from Example 2 were added, stirred until homogeneous, and degassed under vacuum. The mixture was then slowly added dropwise to soybean oil, stirred until homogeneous, and reacted at -20°C for 3 days. The mixture was then removed and allowed to stand at room temperature until the ice crystals completely melted. The pH was adjusted to neutral with hydrochloric acid solution, and the microspheres were purified by dialyzing in PBS buffer to obtain hyaluronic acid-infiltrated cross-linked silk fibroin porous microspheres.

[0097] (2) Preparation of HA-SF composite gel containing hyaluronic acid-infused cross-linked silk fibroin porous microspheres

[0098] Accurately weigh 0.5 g of hyaluronic acid (800-2000 kDa), 0.1 g of silk fibroin (50-100 kDa), and 0.01 g of hyaluronic acid-infiltrated cross-linked silk fibroin porous microspheres. Mix thoroughly, add 5 mL of an aqueous solution containing 1 w / v% sodium hydroxide and 1 wt% BDDE, stir well, and allow to cross-link at 4°C for 48 h. Then, place at 40°C for 3 h of cross-linking. Take 1 g of gel, cut it into small pieces, and dialyze with PBS buffer to swell for 7 h. After dialyzing, remove the gel and pass it through a 60-mesh and an 80-mesh sieve to obtain HA-SF gel particles containing hyaluronic acid-infiltrated cross-linked silk fibroin porous microspheres.

[0099] Add 5% sodium hyaluronate solution in equal proportion, mix well, and then load into a sterile syringe. Sterilize by moist heat to obtain the HA-SF composite gel containing hyaluronic acid-infiltrated cross-linked silk fibroin porous microspheres of the present invention (HA-infiltrated cross-linked SF porous microspheres 0.2wt%, SF 2wt%, HA 10wt%, the mass ratio of SF, HA and BDDE in the gel is 1:5:0.5).

[0100] like Figure 1 As shown, the HA-SF composite gel containing hyaluronic acid-infiltrated cross-linked silk fibroin porous microspheres is prepared through a two-step chemical cross-linking process. Specifically, hyaluronic acid-infiltrated silk fibroin porous microspheres are first prepared, and then these microspheres are added to a gel system co-cross-linked with hyaluronic acid and silk fibroin to prepare the HA-SF composite gel containing the microspheres. After injection into the dermis, it undergoes a triple degradation process: firstly, the outermost layer of the HA-SF composite gel has an immediate effect, filling soft tissue depressions; secondly, over time, the outer composite gel undergoes a degradation process... The gel is gradually degraded by HA enzymes in the skin, which is the first layer of degradation. Subsequently, some hyaluronic acid-infiltrated microspheres are exposed, which also have a supporting effect, and the microsphere structure has a stimulating effect on promoting skin tissue regeneration, which is the second layer of degradation. As time goes on, HA enzymes degrade the HA in the microspheres, exposing porous SF microspheres. At this time, the porous microspheres exert the unique properties of silk fibroin material, promote the adhesion and cell infiltration of surrounding tissues, and allow cells to gradually grow into the porous microspheres, promoting skin collagen regeneration, which is the third layer of degradation.

[0101] Example 4: Performance Measurement

[0102] HA-SF gels with different HA and SF concentrations were prepared according to the method in Example 1, and their rheological properties and extrusion force were measured. The formulation composition and results are shown in Table 1.

[0103] The rheological properties and extrusion force of Examples 2, 3 and Comparative Examples 1-2 were measured, and the results are shown in Table 2.

[0104] The determination method is as follows:

[0105] 1. Elastic modulus and viscous modulus

[0106] The elastic and viscous moduli of the samples were determined using a Kinexus Lab+ rotational rheometer. The parameters were set as follows: rotor: P40 Ti L; gap value: 1.00 mm; temperature: 25℃; measurement mode: oscillation frequency scan; stress 1%; frequency range: 0.05–10 Hz. The elastic and viscous moduli of the samples were recorded at 1 Hz. Each sample was tested three times, and the results are shown in Table 1.

[0107] 2. Pushing force

[0108] The pushing force was determined using a universal testing machine. The sample was filled into a 1.0 mL syringe, fitted with a 30G needle, and a small amount of air was expelled from the syringe tip. The syringe was then mounted on the universal testing machine (Jinan Zhongzheng ZDW-T100 model). The testing parameters were set, and the machine was allowed to equilibrate to room temperature 1 hour in advance. The pushing speed was set to 30 mm / min. The test was then started, and the average force at full scale was read. Each sample was repeated 5 times. The results are shown in Table 1.

[0109] Table 1. Elastic modulus, viscous modulus, and extrusion force of HA-SF gel (mean ± SD)

[0110]

[0111] It is believed that the injectability of the facial filler composition is good when the extrusion force is in the range of 10-25 N, the elastic modulus is in the range of 400-800 Pa, and the viscous modulus is in the range of 100-300 Pa. Table 1 shows that the mechanical properties of the samples are good when the SF concentration is in the range of 1 wt% to 5 wt%. Samples 6, 7, and 4, with different SF molecular weights, all have elastic and viscous moduli within suitable ranges. Samples 1-7 have good mechanical properties and extrusion force (injectability), making them suitable for facial filling.

[0112] Table 2. Elastic modulus, viscous modulus, and extrusion force of gels containing SF porous microspheres (mean ± SD)

[0113] Group Elastic modulus (Pa) Viscous modulus (Pa) Pushing force (N) Example 2 560.90±21.22 176.40±6.70 34.82±1.62 Example 3 633.07±2.03 188.93±2.04 40.84±0.36 Comparative Example 2 757.17±13.98 165.37±2.57 43.26±0.09

[0114] The results in Table 2 show that the gels prepared in Examples 2 and 3 have good mechanical properties, moderate viscosity and extrusion force, and can be used for facial filling.

[0115] Example 5: Characterization of the porous microsphere structure of silk fibroin

[0116] The porous silk fibroin microspheres prepared in Example 2 and the silk fibroin microspheres prepared in Comparative Example 3 were freeze-dried for 35 hours, then cut into small pieces of about 2-3 mm in cross-section. These pieces were then subjected to two 30-second gold sputtering processes and subsequently observed under a scanning electron microscope. The results are shown below. Figure 2 .

[0117] The results showed that Comparative Example 3 had a solid microsphere structure with no pores; the SF porous microspheres prepared in Example 2 had obvious pores and were porous and sponge-like, which was more conducive to cell entry and adhesion and tissue regeneration.

[0118] Example 6: Resistance to enzymatic hydrolysis

[0119] When hyaluronic acid is used directly for facial filling, it is rapidly degraded by enzymes into glucuronic acid. Glucuronic acid reacts with carbazole reagent to produce a reddish-purple color, and the intensity of the color is directly proportional to the content of glucuronic acid. Therefore, by measuring the absorbance value to determine the glucuronic acid content, the anti-enzymatic ability of hyaluronic acid-containing gel can be indirectly reflected.

[0120] Examples 2, 3, and Comparative Examples 1-3 were used to determine their resistance to enzymatic hydrolysis. The experimental methods are as follows:

[0121] 1) Solution preparation

[0122] Carbazole ethanol solution with a volume fraction of 0.125%: Weigh 0.125g of carbazole, dissolve it in 100ml of anhydrous ethanol, transfer it to a dark brown bottle, store it in a dark place, and the shelf life is 15 days.

[0123] Glucuronic acid standard solution: Accurately weigh 0.100 g of D-glucuronic acid, dissolve in water, and dilute to 100.0 g. Mix well to prepare a stock solution. Store at 4℃±2℃ for 3 hours. Accurately measure 5.0 ml of the stock solution into a 100 ml volumetric flask, add water to prepare a solution containing 50 μg per 1 g, shake well, and store at 2-8℃.

[0124] 0.025 mol / L sodium tetraborate-sulfuric acid solution: Weigh 9.54 g of sodium tetraborate (Na₂B₄O₇·10H₂O), add it to 1 L of concentrated sulfuric acid, cover, and shake intermittently until the sodium tetraborate is completely dissolved. Store at room temperature; shelf life is 12 months.

[0125] 0.5 mol / L sulfuric acid solution: Take 5 ml of 98% sulfuric acid and add it to a beaker containing 179 ml of water. Mix well.

[0126] 1 mol / L sodium hydroxide solution: Take 10 g of sodium hydroxide, add 250 ml of water, and stir to dissolve.

[0127] 2) Method for determining sodium hyaluronate content

[0128] 2.1 Take 1 ml of each sample and place it in a 50 ml volumetric flask. Add 2 mg of HA enzyme, place the volumetric flask in a 37℃ water bath and shake to degrade for 72 h, and then take samples for measurement.

[0129] 2.2 Preparation of reference solution: Accurately measure 0, 0.2, 0.4, 0.6, 0.8, ...

[0130] Add 1.0 ml of water to each of the 20 ml stoppered test tubes, and then add water to each tube until the volume reaches 1.0 ml. Prepare three parallel aliquots. One blank tube (0) is sufficient.

[0131] 2.3 Sample preparation: Weigh approximately 0.1 g of sample (accurate to 0.1 mg), add 10 ml of 0.5 mol / L sulfuric acid solution, place in a 95±5℃ constant temperature oven, heat to completely dissolve, add 10 ml of 1 mol / L sodium hydroxide solution, dilute with water to 50 ml, shake thoroughly to mix, and then take 1 ml from the solution and place it in a test tube.

[0132] 2.4 Methods

[0133] Place the reference solution and blank solution (2.1) and the sample solution (2.2) tubes in an ice-water bath. Add 5 ml of 0.025 mmol / L sodium tetraborate-sulfuric acid solution (pre-cooled in an ice bath and stored at 2-8°C for at least 2 hours before use) to each test tube, shaking constantly while adding. Place in a boiling water bath and time precisely for 10 minutes, then cool to room temperature. Next, add 0.2 ml of 0.125% carbazole ethanol solution to each test tube, seal, shake, and place in a boiling water bath for precisely 15 minutes, then cool to room temperature. Use the blank tube for zeroing. Measure the absorbance of each tube at 530 nm using a calibrated UV spectrophotometer. Plot a standard curve using the average of the concentrations of each reference solution and their corresponding absorbance values. Calculate the average concentration of D-glucuronic acid in the sample solution based on the standard curve and the corresponding absorbance values. (n=3)

[0134] 2.5 Calculation

[0135] Calculate the mass concentration C (mg / ml) of sodium hyaluronate in the sample using the following formula.

[0136] content

[0137] Where m1 is the sample mass, in grams;

[0138] m2 — Mass of sample and purified water, mg;

[0139] d1 — Sample density, g / ml;

[0140] d2—Density of the sample and purified water after mixing, in g / ml;

[0141] ρ1—The content of D-glucuronic acid in the sample test solution, in μg / ml.

[0142] Table 3. Enzymatic resistance of HA-SF gel containing SF microspheres (mean ± SD)

[0143]

[0144] Table 3 shows that, compared with Comparative Example 1, Examples 2, 3, 2 and 3 all have a certain resistance to enzymatic hydrolysis. Among them, Example 3 has a stronger resistance to enzymatic hydrolysis, which is significantly better than that of Comparative Examples 1-3.

[0145] Example 7: Cell Proliferation Experiment

[0146] Cell proliferation experiments were performed on Examples 1-3 and Comparative Examples 1-2. Logarithmic growth phase mouse fibroblast L929 cells were digested and seeded in 24-well plates. 0.3 mL of each sample was placed in a Transwell chamber and co-cultured with the cells for 7 days. Cell viability was then observed by measuring absorbance at 450 nm. Results are shown below. Figure 3 .

[0147] The results showed that the gels of Examples 1-3 could significantly promote the proliferation of fibroblasts, indicating good safety and significantly better performance than Comparative Examples 1 and 2.

[0148] Example 8: Cell Adhesion

[0149] 1 mL of samples from Examples 2, 3, and Comparative Example 3 were added to DMEM medium. The resulting sterile extracts were co-cultured with mouse fibroblast L929 cells for 3 days, followed by washing with PBS to remove non-adherent cells. The morphology, number, and distribution of adherent cells were observed and recorded, and the cell count was statistically analyzed (n=5). The results are shown in Table 4.

[0150] Table 4 Cell adhesion properties of the filler of the present invention

[0151] Group Adhesive cells (cells / ml) Blank group 2,608.22±239.76 Example 1 Sample 4 3,028.89±167.22 Example 2 5,423.60±196.67 Example 3 4,998.74±74.52 Comparative Example 1 2,591.37±137.38 Comparative Example 2 2,191.51±199.66 Comparative Example 3 3,259.92±243.48

[0152] The results showed that, compared with the blank group, the number of adhering cells in samples 4 of Example 1, Examples 2 and 3 of the present invention was significantly increased, and was better than that in comparative examples 1 and 2; indicating that the cell adhesion of the gels prepared in Examples 1-3 of the present invention was good.

[0153] Example 9: Promoting Collagen Regeneration

[0154] 1. HA-SF gels with different SF molecular weights

[0155] 1 g of samples 4, 6, and 7 from Example 1 were dissolved in DMEM medium to prepare sterile extracts. L929 cells in logarithmic growth phase were digested, seeded in 96-well plates, and cultured with the aforementioned sterile extracts. After 14 days, the collagen content in the cell supernatant was detected using an ELISA kit. Results are shown below. Figure 4 .

[0156] The results showed that, compared with the blank control group which was cultured in DMEM complete medium without any sample, all three molecular weights of SF significantly promoted collagen regeneration in fibroblasts.

[0157] 2. Gel containing porous microspheres of silk fibroin

[0158] L929 cells in logarithmic growth phase were digested and seeded in 24-well plates. 0.3 mL of samples from Examples 1-4, 2-3, 1, 2, and 3 (Comparative Examples) were placed in TRANSWELL chambers and cultured for 10 days. Collagen in the fibroblast supernatant was then detected using an ELISA kit. Results are shown below. Figure 5 .

[0159] The results showed that, compared with the blank group (CTRL), the gel of the present invention promoted collagen regeneration, and Examples 2 and 3 were superior to Comparative Examples 1, 2 and 3.

[0160] Example 10: Biocompatibility – ICR Mouse Injection Test

[0161] 0.3 mL of each of Example 3, Comparative Example 1, and Comparative Example 2 was injected into the dorsal dermis of ICR mice. After 14 days, the presence of subcutaneous and muscle abnormalities at the injection sites was observed. Tissue reactions (presence of hemorrhage, edema, cysts, and hyperplasia) were observed under low magnification. Good biocompatibility resulted in minimal tissue inflammation; conversely, poor biocompatibility resulted in significant tissue inflammation. The tissue health was also observed through HE-stained pathological sections. Results are shown below. Figure 6 .

[0162] The results showed that the dorsal skin tissue of normal mice exhibited basket-like keratinization, a granular layer with regular arrangement, a normal spinous layer, a regular basal layer with a clear junction with the papillary dermis, abundant collagen fibers in the dermis with normal morphology and distribution, and a generally normal subcutaneous fat layer. No inflammatory infiltration was observed in the dermis or subcutaneous fat layer, indicating a high level of skin health. In Comparative Example 2, mice treated with this method showed a higher number of neutrophils in the dermis and subcutaneous fat layer. In Example 3 and Comparative Example 1, mice treated with this method showed a smaller number of neutrophils in the dermis and subcutaneous fat layer. Compared to Comparative Example 2, mice treated with this method showed a higher level of skin health. Compared to Comparative Example 1, mice treated with this method showed a higher level of skin health.

Claims

1. A composition for filling, characterized by, The composition comprises gel particles and an aqueous medium, wherein, The gel particles comprise silk fibroin, hyaluronic acid or a salt thereof and a crosslinking agent, and the gel particles comprise porous three-dimensional bodies.

2. The composition of claim 1, wherein, The gel particles are prepared by adding the porous three-dimensional bodies into a solution comprising hyaluronic acid or a salt thereof, silk fibroin and a crosslinking agent.

3. The composition according to claim 1 or 2, characterized in that, The porous three-dimensional bodies are selected from one or more of porous microspheres, hollow conduits, porous microparticles, porous irregular blocks; preferably porous microspheres; more preferably silk fibroin porous microspheres; still more preferably silk fibroin porous microspheres infiltrated with hyaluronic acid or a salt thereof; preferably, the porous three-dimensional bodies have a particle size of less than 600 mm, more preferably less than 250 mm, still more preferably less than 180 mm, most preferably less than 120 mm.

4. The composition according to any one of claims 1 to 3, characterized in that, The mass ratio of silk fibroin:hyaluronic acid or a salt thereof:crosslinking agent in the gel particles is (0.1-10):(1-20):1, preferably (1-5):(5-15):1, more preferably (1-5):10:1, most preferably 2:10:

1.

5. The composition according to any one of claims 1-4, characterized in that, The mass ratio of the gel particles to the aqueous medium is (0.1-10):1, preferably 1:

1.

6. The composition according to any one of claims 1-5, characterized in that, The aqueous medium is a solution of hyaluronic acid or a salt thereof, preferably the concentration of hyaluronic acid or a salt thereof in the aqueous medium is 2wt%-8wt%, preferably 5wt%.

7. The composition according to any one of claims 1-6, characterized in that, The molecular weight of the hyaluronic acid or a salt thereof is 200-4000kDa, preferably 400-1600kDa, more preferably 800-2000kDa.

8. The composition according to any one of claims 1-7, characterized in that, The molecular weight of the silk fibroin is 2-500kDa, preferably 10-250kDa, more preferably 50-100kDa.

9. The composition according to any one of claims 1-8, characterized in that, The elastic modulus of the gel particles is 300-800Pa, preferably 400-700Pa.

10. The composition according to any one of claims 1-9, characterized in that, The viscous modulus of the gel particles is 100-250Pa, preferably 150-250Pa.

11. The composition according to any one of claims 1-10, characterized in that, The push force of the gel particles is 10-25N, preferably 15-25N.

12. The composition according to any one of claims 1-11, characterized in that, The salt of the hyaluronic acid is selected from one or any combination of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

13. The composition according to any one of claims 1-12, characterized in that, The crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis-carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, preferably BDDE.

14. The composition of any one of claims 1-13, wherein, The content of the porous three-dimensional body in the gel particles is 0.01wt%-50wt%, preferably 0.1wt%-20wt%, more preferably 0.1wt%-10wt%, more preferably 0.2wt%-5wt%, most preferably 0.2wt%, based on the total mass of the gel particles.

15. The composition of any one of claims 1-14, wherein, The porous three-dimensional body is prepared by a freeze-drying method, preferably by freeze-drying a silk fibroin aqueous solution, more preferably by the following method: preparing a silk fibroin concentrate, then freeze-drying the concentrate, and pulverizing the obtained silk fibroin freeze-dried block after fumigating it with an organic solvent at 37°C for a period of time, preferably, the organic solvent is selected from one or more of methanol, ethanol, acetone, isopropanol.

16. The composition of claim 15, wherein, The porous three-dimensional body is added to a solution of hyaluronic acid or a salt thereof to obtain a hyaluronic acid-infiltrated porous three-dimensional body; preferably, the porous three-dimensional body is placed in an oil phase with a mixture of hyaluronic acid or a salt thereof to obtain a hyaluronic acid-infiltrated porous three-dimensional body; still more preferably, the hyaluronic acid-infiltrated cross-linked porous three-dimensional body is prepared according to the following method: a. Prepare an alkali solution containing BDDE for use; b. Weigh an appropriate amount of sodium hyaluronate and silk fibroin porous microspheres, add the alkali solution containing BDDE, stir uniformly, vacuum degas, then slowly drop the mixture into the oil phase, and place it at -20°C for 3 days; c. Take out and place at room temperature, adjust the pH value to neutral, and place it in a phosphate buffer for dialysis; Preferably, the oil phase is selected from one of soybean oil, liquid paraffin, glycerol, silicone oil, etc. Preferably, the volume ratio of the mixture to the oil phase is 1:(1-20).

17. The composition according to any one of claims 1-16, wherein the composition is a solution, a gel, a lyophilized powder, an emulsion or a cream, preferably a gel, more preferably, the composition is an injectable filler.

18. The composition according to any one of claims 1-17, wherein the composition is used to fill facial, neck, joint or torso sites, preferably for filling the face.

19. A kit comprising the gel particles as defined in any one of claims 1-18 and an aqueous medium.

20. Use of the composition according to any one of claims 1-18 or the kit of claim 19 in the manufacture of a product for improving the skin condition of a subject in need thereof, preferably the skin condition is selected from the group consisting of skin dehydration, lack of skin elasticity, skin roughness, lack of skin tightness, skin stretch lines, skin stretch marks, skin pallor, dermal dents, cheek hollows, thin lips, post-orbital defects, facial folds and wrinkles.

21. The use according to claim 20, wherein the composition is administered into the dermal region of the subject.

22. A non-therapeutic method of improving skin condition in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 1-18 or a kit according to claim 19; preferably, the skin condition is selected from the group consisting of skin dehydration, skin lack of elasticity, skin roughness, skin lack of tightness, skin stretch lines, skin stretch marks, skin pallor, dermal dents, cheek hollows, thin lips, post-orbital defects, facial folds and wrinkles.

23. The method of claim 22, wherein the composition is applied to the face of the subject; preferably, the composition is applied into the dermal region of the subject.

24. A method of preparing the composition according to any one of claims 1-18, characterized in that, comprising the following steps: 1) mixing a solution of hyaluronic acid or its salt, silk fibroin, crosslinking agent with porous three-dimensional bodies, then standing and sieving to obtain gel particles; preferably, the porous three-dimensional bodies are selected from one or more of porous microspheres, hollow conduits, porous microparticles, porous irregular blocks; preferably, porous microspheres; more preferably, silk fibroin porous microspheres; still more preferably, silk fibroin porous microspheres infiltrated with hyaluronic acid or its salt; preferably, the porous three-dimensional bodies have a particle size of less than 600 mm, more preferably less than 250 mm, still more preferably less than 180 mm, most preferably less than 120 mm; 2) adding the gel particles obtained in step 1) into an aqueous medium.

25. The method of claim 24, wherein the porous three-dimensional bodies are prepared by freeze-drying method, preferably by freeze-drying a silk fibroin aqueous solution, more preferably by the following method: preparing a silk fibroin concentrate, then freeze-drying the concentrate, crushing the obtained silk fibroin freeze-dried block and sieving through a 120-mesh sieve after fumigating at 37°C with an organic solvent for a period of time; preferably, the organic solvent is selected from one or more of methanol, ethanol, acetone, isopropanol.

26. The method of claim 25, wherein the porous three-dimensional bodies are added into a solution of hyaluronic acid or its salt to obtain hyaluronic acid-infiltrated porous three-dimensional bodies; preferably, the porous three-dimensional bodies are mixed with a solution of hyaluronic acid or its salt in an oil phase to obtain hyaluronic acid-infiltrated porous three-dimensional bodies; still more preferably, the hyaluronic acid-infiltrated crosslinked porous three-dimensional bodies are prepared according to the following method: a. preparing a BDDE-containing alkali solution for use; b. weighing an appropriate amount of sodium hyaluronate and silk fibroin porous microspheres, adding into the BDDE-containing alkali solution, stirring uniformly, vacuum degassing, then slowly dropping the mixture into an oil phase, placing at -20°C for 3 days; c. taking out, placing at room temperature, adjusting the pH value to neutral, placing into a phosphate buffer for dialysis; preferably, the oil phase is selected from one of soybean oil, liquid paraffin, glycerol, silicone oil, etc.; preferably, the volume ratio of the mixture to the oil phase is 1:(1-20).

27. A method of preparing a composition according to any one of claims 1 to 18, characterised in that, comprising the following steps: 1) respectively preparing a hyaluronic acid salt solution and a BDDE-containing alkali solution for use; 2) take an appropriate amount of raw materials dissolved in the alkali solution containing BDDE, stirring evenly, placed in 0-10℃ for 10-72h, preferably in 4℃ for 48h; then placed in 10-60℃ for 1-10h to deepen cross-linking, preferably 40℃ for 3h to deepen cross-linking, to get co-crosslinked gel; 3) the above co-crosslinked gel is placed in phosphate buffer for a period of time, then granulated through 60-80 mesh sieve to get gel particles; 4) add an appropriate amount of hyaluronate solution to the above gel particles, wet heat sterilization, and get it; The raw materials in step 2) are hyaluronic acid or its salt and silk fibroin; or hyaluronic acid or its salt, silk fibroin and silk fibroin porous microspheres, or hyaluronic acid or its salt, silk fibroin and hyaluronic acid infiltrated cross-linked silk fibroin porous microspheres.

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