Silk fibroin hyaluronic acid gel for filling and preparation method therefor

Abstract

Provided in the present invention are a composition for filling, and a preparation method therefor and the use thereof. The composition comprises gel particles and an aqueous medium hyaluronate solution, wherein 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 structure; and the mass ratio of the silk fibroin to the hyaluronic acid or a salt thereof to the cross-linking agent is (0.1-10):(1-20):1.

Description

A silk fibroin hyaluronic acid gel for filling and a preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to a silk fibroin hyaluronic acid gel for filling and a preparation method thereof. BACKGROUND

[0002] With the rapid development of the national economy and the rapid rise of the appearance value economy and the net red economy in recent years, the domestic medical beauty penetration rate has increased significantly, and the medical beauty market has developed rapidly. Medical beauty is divided into surgical and non-surgical categories. Non-surgical medical beauty, also known as light medical beauty, refers to using non-invasive or minimally invasive medical treatment to meet the beauty demand. Compared with surgical medical beauty, light medical beauty has the advantages of simple operation, small trauma, high safety, short recovery period, etc., and has become the main growth point of the medical beauty market.

[0003] However, the currently marketed filling materials for light medical beauty have the problems of easily inducing inflammation and other adverse reactions, and the defect that the space left after the rapid degradation of the filling material cannot be effectively made up due to the mismatch between the degradation rate of the filling material and the cell ingrowth rate. Therefore, there is an urgent need in the market for safe and effective medical beauty filling products without side effects. SUMMARY

[0004] The purpose of the present application is to provide a silk fibroin hyaluronic acid gel for filling and a preparation method thereof, which has the dual functions of instant filling effect and collagen regeneration promotion, and is suitable for facial injection filling and has good biocompatibility.

[0005] The present application adopts the following technical solutions:

[0006] In one aspect, the present application 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 into a solution comprising 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; still more preferably, it is a silk fibroin porous microsphere infiltrated with hyaluronic acid or a salt thereof; preferably, the porous three-dimensional body has a particle size of less than 600 mm, more preferably less than 250 mm, still more preferably less than 180 mm, and most preferably less than 120 mm.

[0009] In a preferred embodiment, the mass ratio of fibroin: hyaluronic acid or 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.

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

[0011] In a preferred embodiment, 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%.

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

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

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

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

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

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

[0018] In a preferred embodiment, the cross-linking 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)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3- epoxypropyl)-2,3-epoxycyclohexane, a carbodiimide, preferably BDDE.

[0019] In a preferred embodiment, 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.

[0020] In a preferred embodiment, 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, fumigating the obtained silk fibroin freeze-dried block with an organic solvent at 37°C for a period of time, crushing and then passing through a 120-mesh sieve, preferably, the organic solvent is selected from one or more of methanol, ethanol, acetone, 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, the 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; still more preferably, the hyaluronic acid-infiltrated cross-linked porous three-dimensional body is prepared according to the following method:

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

[0023] 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 an oil phase, and place it at -20°C for 3 days;

[0024] c. Take out and place at room temperature, adjust the pH value to neutral, and dialyze in a phosphate buffer;

[0025] Preferably, the oil phase is selected from one of soybean oil, liquid paraffin, glycerol, 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, a gel, a lyophilized powder, an emulsion or a cream, preferably, the composition is an injectable filler.

[0028] In a preferred embodiment, the composition is for filling to a facial, neck or torso site, preferably for filling to a facial site.

[0029] In a second aspect, the present application provides a kit comprising the gel particles and the aqueous medium as defined above.

[0030] In a third aspect, the present application provides use of the composition or the kit as defined above in the manufacture of a product for improving a skin condition in 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 tautness, skin stretch marks, skin stretch lines, skin pallor, dermal indentation, cheek hollow, thin lips, post-orbital defect, facial folds and wrinkles.

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

[0032] In a fourth aspect, the present application provides a non-therapeutic method of improving a skin condition in a subject in need thereof, comprising administering to the subject the composition or the kit as defined above; preferably, the skin condition is selected from the group consisting of skin dehydration, lack of skin elasticity, skin roughness, lack of skin tautness, skin stretch marks, skin stretch lines, skin pallor, dermal indentation, cheek hollow, thin lips, post-orbital defect, facial folds and wrinkles.

[0033] In a preferred embodiment, the composition is administered to a facial site of the subject; preferably, the composition is administered into a dermal region of the subject.

[0034] In a fifth aspect, the present application provides a method of preparing the composition as defined above, characterized by comprising the following steps:

[0035] 1) mixing a solution of hyaluronic acid or a salt thereof, silk fibroin, a crosslinking agent with a porous three-dimensional body, followed by standing and sieving to obtain gel particles; preferably, the porous three-dimensional body is selected from one or more of the group consisting of porous microspheres, hollow conduits, porous microparticles, porous irregular blocks; preferably, the porous three-dimensional body is a porous microsphere; more preferably, the porous three-dimensional body is a silk fibroin porous microsphere; still more preferably, the porous three-dimensional body is a silk fibroin porous microsphere infiltrated with hyaluronic acid or a salt thereof; preferably, the porous three-dimensional body has 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;

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

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

[0038] In a preferred embodiment, the porous three-dimensional body is added into 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 mixed with the hyaluronic acid or a salt thereof in an oil phase 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:

[0039] a. preparing an alkali solution containing BDDE for use;

[0040] b. weighing an appropriate amount of sodium hyaluronate and silk fibroin porous microspheres, adding the alkali solution containing BDDE, stirring uniformly, vacuum degassing, then slowly dropping the mixture into an oil phase, and placing it at -20°C for 3 days;

[0041] c. taking it out and placing it at room temperature, adjusting the pH value to neutral, and placing it in a phosphate buffer for dialysis;

[0042] Preferably, the oil phase is selected from one of soybean oil, liquid paraffin, glycerol, 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 application provides a preparation method of the aforementioned composition, characterized in that it comprises the following steps:

[0045] 1) respectively preparing a hyaluronic acid salt solution and an alkali solution containing BDDE for use;

[0046] 2) weighing an appropriate amount of raw materials and dissolving them in the alkali solution containing BDDE, stirring uniformly, placing it at 0-10°C for 10-72h, preferably at 4°C for 48h, then deep cross-linking at 10-60°C for 1-10h, preferably at 40°C for 3h, to obtain a co-cross-linked gel;

[0047] 3) placing the co-cross-linked gel in a phosphate buffer for dialysis for a period of time, then granulating by sieving through a 60-80-mesh sieve to obtain gel particles;

[0048] 4) Add the hyaluronic acid salt solution to the above gel particles, and sterilize by moist heat, to obtain the product.

[0049] Step 2) the raw materials are hyaluronic acid or a salt thereof and silk fibroin; or hyaluronic acid or a salt thereof, silk fibroin and silk fibroin porous microspheres, or hyaluronic acid or a salt thereof, silk fibroin and hyaluronic acid infiltrated crosslinked silk fibroin porous microspheres. Beneficial effects

[0050] Compared with a single hyaluronic acid gel, the gel provided by the present application contains two filling materials, silk fibroin and hyaluronic acid, has good biocompatibility and low immunogenicity, avoids adverse reactions such as subclinical inflammatory reactions and fibrous hyperplasia caused by chemical filling methods, has good mechanical properties, can support the soft tissue after being injected into the skin, makes the face full and round, has good injectability, can promote the growth of skin fibroblasts, has good cell adhesion effect after filling, can promote the regeneration of skin collagen, promotes the regeneration of skin tissue, has the effects of immediate filling and long-term tissue repair.

[0051] The silk fibroin microspheres of the present application are porous sponge-like scaffold materials, have good support, are conducive to cell adhesion, and promote collagen regeneration. The present application combines the rigid porous silk fibroin microsphere skeleton and the flexible continuous gel matrix, has a programmed gradient degradation and stimulates tissue response regeneration effect, slows down the degradation rate of the filling material, prolongs the action time of the filling material in the repair and filling of the facial soft tissue, improves the effectiveness of the material, and realizes long-term filling effect. At the same time, it avoids the adverse reactions such as excessive stimulation of granuloma after injection of commercially available microsphere materials such as PLLA, and has good safety. The present application has the effect of anti-aging as a filler.

[0052] Brief description of the drawings

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

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

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

[0056] Figure 4 is the effect of HA-SF gels with different SF molecular weights on collagen regeneration.

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

[0058] Figure 6 is a HE-stained section of a gel-injected mouse back skin of Example 3 (Figure A is normal group, Figure B is Example 3 group, Figure C is Comparative Example 1 group, and Figure D is Comparative Example 2 group).

[0059] DETAILED DESCRIPTION

[0060] DEFINITIONS

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

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

[0063] As used herein, the term "porous three-dimensional body" includes porous microspheres, hollow conduits, porous microparticles, porous irregular blocks, and is a kind of functional polymer material with many small holes, which is usually prepared by using polymer materials or inorganic materials. It has a three-dimensional spatial pore structure inside, and the pore size and specific surface area are relatively large. The porous three-dimensional body has many unique characteristics. Because there are a large number of micro-pores and defects inside, these micro-pores can provide additional surface area, thereby increasing the loading capacity of active substances. Secondly, the porous three-dimensional body material also has good mesoporous properties. The pore structure of different sizes, shapes and connectivity makes it have the characteristics of adjustable pore size distribution.

[0064] As used herein, the term "crosslinking" refers to intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into more stable structures such as gels. 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-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), adipic hydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3- epoxypropyl)-2,3-epoxycyclohexane, carbodiimides, and the like.

[0065] As used herein, the term "gel" refers to a thick liquid or semisolid preparation of solution, suspension or emulsion type. Gel bases are single phase dispersion systems, which are either aqueous or oleaginous. Aqueous gel bases are generally composed of water, glycerin or propylene glycol with cellulose derivatives, carbomer and alginates, tragacanth, gelatin, starch, and the like; oleaginous gel bases are composed of liquid paraffin with polyethylene or a fatty oil with colloidal silicon or aluminum soaps, zinc soaps, and the like.

[0066] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts can include hydrochloride, phosphate, pyrophosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and alkanoate (e.g., acetate, HOOC-(CH2)n-COOH, where n is 0-4). Furthermore, if the compounds herein are obtained in the form of an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the compounds herein are in the form of 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 an acid, in accordance with the general procedures for preparing acid addition salts from base compounds. Those of skill in the art will appreciate various synthetic methodologies that can be used to prepare the nontoxic pharmaceutically acceptable addition salts. n -COOH, where n is 0-4). Furthermore, if the compounds herein are obtained in the form of an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the compounds herein are in the form of 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 an acid, in accordance with the general procedures for preparing acid addition salts from base compounds. Those of skill in the art will appreciate various synthetic methodologies that can be used to prepare the nontoxic pharmaceutically acceptable addition salts.

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

[0068] As used herein, the term "kit" can include two or more agents in single or multiple doses, each packaged or formulated separately; or in single or multiple doses packaged or formulated in combination. Thus, one or more agents can be present in a first container, and the kit can optionally include one or more agents in a second container. The container(s) is placed into a package and the package can optionally include instructions for administration or dosing. The kit can include additional components such as a syringe or other means for administering the agents and diluents or other means for formulation. DETAILED DESCRIPTION

[0069] Injection filling is to inject natural or synthetic biomaterials into the dermis or subcutaneous layer to improve skin relaxation, depression and delay skin aging by tissue filling or stimulating collagen regeneration in the body, and the filling effect depends on the performance of the filling material. According to the biological absorption performance and mechanism of action, the injection filling materials are mainly divided into three categories: (1) endogenous biomaterials, which are similar to human extracellular matrix, such as hyaluronic acid (HA) and collagen; (2) synthetic polymer, which has good biodegradability and can stimulate tissue to generate collagen and fiber structure, thereby limitedly increasing the tissue volume, also known as semi-permanent filler, such as poly-L-lactic acid (PLLA), polycaprolactone (PCL) and calcium hydroxyapatite (CaHA) containing filler; (3) non-absorbable and degradable polymer materials, which permanently and irreversibly provide tissue occupation and cause immune response of the body, such as polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA) and polyacrylic acid (PAA). The ideal injection filling material should integrate “filling, repairing and anti-aging” in one, first quickly fill, and then stimulate collagen regeneration to achieve the effect of “safer, more natural and longer”.

[0070] Hyaluronic acid is an endogenous component of the human body, which has good biocompatibility and degradability. An exemplary injection filling product of hyaluronic acid composite system can include cross-linked sodium hyaluronate gel containing L-lactic acid-ethylene glycol copolymer microspheres, which is composed of cross-linked sodium hyaluronate, L-lactic acid-ethylene glycol copolymer microspheres, lidocaine hydrochloride and phosphate buffer system, and has the dual functions of “filling and stimulating regeneration”.

[0071] Silk fibroin (SF) is a natural polymer fibroin composed of 18 kinds of amino acids extracted from silk, which has excellent biocompatibility, biodegradability, low immunogenicity, tissue cell adhesion ability and mechanical properties. In one technical solution of the present application, a gel for injection filling is provided by combining rigid porous silk fibroin microsphere skeleton and flexible continuous gel matrix.

[0072] Examples

[0073] The technical solutions of the present application will be described in detail below in combination with examples, but those skilled in the art should understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0074] The main materials in the following examples are as follows:

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

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

[0077] Comparative Example 1: commercially available product Reborn No. 2 (modified sodium hyaluronate gel for injection), manufacturer GALDERM Co., Ltd., batch number 21771;

[0078] Comparative Example 2: commercially available product Aivlan (cross-linked sodium hyaluronate gel containing microspheres of L-lactic acid-ethylene glycol copolymer), manufacturer Changchun Shengboma Co., Ltd., batch number T23043014;

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

[0080] (1) Preparation of silk fibroin microspheres: 40 g of mulberry silk was added to 2700 ml of a 0.2% mass concentration sodium carbonate aqueous solution, and water bathed at 96-100°C for 60 min, filtered, and the filter cake was repeatedly water bathed for 3 times, and the filter cake from the last filtration was dried at 60°C, then slowly dissolved in 200 ml of a 9 mol / L lithium bromide aqueous solution, water bathed at 60°C for 6 h, then centrifuged at 8000 rpm for 5 min, and the supernatant was taken, and the supernatant after centrifugation was loaded into a dialysis bag (MW 8,000-14,000), and purified water was used as the dialysis liquid, and magnetic stirring dialysis was performed for three days, and the cut-off liquid was taken, and the purified water was prepared into a silk fibroin solution with a mass concentration of 5%.

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

[0082] (2) 0.7 g of sodium hyaluronate was dissolved in 7 ml of a 1% mass concentration sodium hydroxide solution to prepare a 0.1 g / ml sodium hyaluronate solution, and 2.167 g (0.07 / 0.0323=2.167 g) of silk fibroin particles were added to the sodium hyaluronate solution, mixed thoroughly, and then 56 μl of BDDE was added, mixed uniformly, and then placed at 40°C for 5 hours for crosslinking to form a silk fibroin hyaluronic acid composite gel material.

[0083] The silk fibroin hyaluronic acid composite gel material is added into a dialysis bag with MW 8,000-14,000, and dialysis treatment is carried out under magnetic stirring with PBS buffer solution with pH value of 7.4 as dialysate. After the dialysis treatment, the composite gel material is homogenized by a homogenizer under the condition of 24,000 rpm / min for 10 min, and then is extruded to pass through a 60-mesh sieve. The composite gel particles are collected, and after high-temperature high-pressure steam sterilization at 120℃ for 15 min, sterile packaging is carried out, and the composite gel particles are loaded into disposable syringes. Thus, 35 g of the silk fibroin hyaluronic acid composite gel for injection is obtained.

[0084] Example 1: Preparation of hyaluronic acid-silk fibroin gel (HA-SF gel)

[0085] Preparation of HA-SF gel particles: 1wt% 1,4-butanediol diglycidyl ether (BDDE) solution is prepared by mixing 1wt% sodium hydroxide aqueous solution. 0.5 g of HA (molecular weight 800-2000 kDa) and 0.05 g of SF (molecular weight 50-100 kDa) are precisely weighed, 5 mL of the above-mentioned BDDE solution is added, and stirring is uniformly carried out. The mixture is placed at 4℃ for 48 h for preliminary crosslinking, and then is placed in a water bath at 40℃ for 3 h for deep crosslinking. Thus, HA-SF co-crosslinked gel is formed. 1 g of the HA-SF co-crosslinked gel is added into phosphate buffer solution, and dialysis swelling is carried out for 10 h. After dialysis, the gel particles are taken out and passed through a 60-mesh and an 80-mesh sieve. Thus, HA-SF gel particles (HA 10wt%, SF 1wt%) are obtained.

[0086] Preparation of 5% sodium hyaluronate solution: 0.5 g of HA (molecular weight 800-2000 kDa) is dissolved in 10 mL of PBS buffer solution, and stirring and standing are carried out until the solution is swelled. The solution is used as a 5% sodium hyaluronate solution.

[0087] Preparation of HA-SF gel: HA-SF gel particles are precisely weighed, and the same weight of 5% sodium hyaluronate solution is added. After mixing, the mixture is loaded into a sterile syringe, and moist heat sterilization is carried out. Thus, HA-SF gel is obtained. 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 silk fibroin porous microspheres

[0090] 0.3 g of silk fibroin is dissolved in 3 mL of pure water to obtain a silk fibroin concentrate with a concentration of 10wt%. The silk fibroin concentrate is freeze-dried, and then is fumigated with ethanol or methanol vapor overnight (37℃). Then, the silk fibroin porous microspheres are pulverized by a multifunctional pulverizer and passed through a 120-mesh sieve.

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

[0092] 0.5 g of hyaluronic acid (800-2000 kDa), 0.1 g of silk fibroin (50-100 kDa), and 0.01 g of porous silk fibroin microspheres were precisely weighed, mixed uniformly, 5 mL of a solution containing 1 w / v% sodium hydroxide and 1 wt% BDDE was added, stirred uniformly, and then crosslinked at 4°C for 48 h and then at 40°C for 3 h. 1 g of the gel was cut into pieces and swelled by dialysis in a phosphate buffer for 7 h. After dialysis, the particles were passed through 60-mesh and 80-mesh screens to obtain HA-SF gel particles containing porous silk fibroin microspheres (SF porous microspheres 0.2 wt%, SF 2 wt%, and HA 10 wt%).

[0093] An equal proportion of 5% sodium hyaluronate solution was added, mixed uniformly, and then loaded into a sterile syringe and subjected to moist heat sterilization to obtain HA-SF gel containing porous silk fibroin microspheres, with the mass ratio of SF, HA, and BDDE in the gel being 1:5:0.5.

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

[0095] (1) Preparation of hyaluronic acid-infiltrated crosslinked porous silk fibroin microspheres

[0096] 1 w / v% sodium hydroxide aqueous solution 20 mL and 1.5 wt% BDDE solution (0.3 mL) were mixed uniformly, 1 g of sodium hyaluronate (800-2000 kDa) and 0.5 g of the porous silk fibroin microspheres in Example 2 were added, stirred uniformly, vacuum degassed, then slowly dripped into soybean oil, stirred uniformly, and then placed at -20°C for 3 days. After being taken out, the ice crystals were completely melted at room temperature, the pH was adjusted to neutral with a hydrochloric acid solution, and then the gel was purified by dialysis in a PBS buffer to obtain hyaluronic acid-infiltrated crosslinked porous silk fibroin microspheres.

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

[0098] 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 crosslinked porous silk fibroin microspheres were precisely weighed, mixed uniformly, 5 mL of a solution containing 1 w / v% sodium hydroxide and 1 wt% BDDE was added, stirred uniformly, and then crosslinked at 4°C for 48 h and then at 40°C for 3 h. 1 g of the gel was cut into pieces and swelled by dialysis in a PBS buffer for 7 h. After dialysis, the particles were passed through 60-mesh and 80-mesh screens to obtain HA-SF gel particles containing hyaluronic acid-infiltrated crosslinked porous silk fibroin microspheres.

[0099] The HA-SF composite gel containing the hyaluronic acid infiltrated crosslinked silk fibroin porous microspheres of the present application is prepared by adding 5% hyaluronic acid sodium solution in equal proportions, mixing, and then loading into a sterile syringe and hot-sterilizing to obtain the HA-SF composite gel containing the hyaluronic acid infiltrated crosslinked silk fibroin porous microspheres (HA infiltrated crosslinked SF porous microspheres 0.2 wt%, SF 2 wt%, HA 10 wt%, the mass ratio of SF, HA and BDDE in the gel is 1:5:0.5).

[0100] As shown in Figure 1, the HA-SF composite gel containing the hyaluronic acid infiltrated crosslinked silk fibroin porous microspheres is prepared by two-step chemical crosslinking, specifically, first preparing hyaluronic acid infiltrated silk fibroin porous microspheres, and then adding the porous microspheres into a hyaluronic acid and silk fibroin co-crosslinked gel system to prepare the HA-SF composite gel containing the microspheres. After being injected and filled into the dermis of the skin, the composite gel undergoes a triple degradation process: first, the outermost HA-SF composite gel has an immediate effect, which can fill the skin soft tissue depression, and with the increase of time, the outer composite gel is gradually degraded by HA enzymes in the skin, which is the first layer of degradation; then part of the hyaluronic acid infiltrated microspheres is exposed, which also has a supporting effect, and the microsphere structure has a stimulating effect on promoting skin tissue regeneration, which is the second layer of degradation; with further increase of time, the HA enzymes degrade the HA in the microspheres, and the porous SF microspheres are exposed, the porous microspheres at this time exert the unique properties of silk fibroin material, promote the adhesion and cell infiltration of the surrounding tissue, and make the cells gradually grow into the porous microspheres, promote the regeneration of skin collagen, and exert the third layer of degradation.

[0101] Example 4: Performance determination

[0102] The HA-SF gels with different HA concentrations and different SF concentrations were prepared according to the method of Example 1, and the rheological properties and push force were determined, and the prescription composition and results are shown in Table 1.

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

[0104] The determination method is as follows:

[0105] 1. Elastic modulus and viscous modulus

[0106] The Kinexus Lab+ rotary rheometer was used to detect the elastic modulus and viscous modulus of the sample. The parameter settings were: rotor: P40 Ti L; Gap value: 1.00 mm; temperature: 25℃; determination mode: oscillation frequency scanning; stress 1%; frequency range: 0.05-10 Hz. The elastic modulus and viscous modulus of the sample at 1 Hz were recorded. Each sample was repeated three times, and the results are shown in Table 1.

[0107] 2. Push force

[0108] Pushing force was measured by using a universal material testing machine. The sample was filled in a 1.0 mL syringe, a 30G injection needle was installed, a small amount of air was discharged from the front end of the syringe, and then it was installed on the universal material testing machine (Jinan Zhongzheng ZDW-T100 type). The testing parameters of the universal material testing machine were set, it was placed in room temperature for 1 h in advance, the test was measured at room temperature, the pushing speed was set to 30 mm / min, the test was started, the full-scale average force was read, each sample was repeated 5 times, and the results are shown in Table 1.

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

[0110] It is considered that when the pushing force of the facial filling composition 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, the injectability of the composition is good. The results of Table 1 show that when the SF concentration is in the range of 1 wt% to 5 wt%, the mechanical properties of the sample are good. The elastic modulus and viscous modulus of samples 6, 7 and 4 with different SF molecular weights are in the appropriate range. The mechanical properties and pushing force (injectability) of samples 1-7 are good, and are suitable for facial filling.

[0111] Table 2 Elastic modulus, viscous modulus and pushing force of gel containing SF porous microspheres (mean ± SD)

[0112] The results of Table 2 show that the mechanical properties of the gels prepared in Example 2 and Example 3 are good, and the viscosity and pushing force are moderate, which can be used for facial filling.

[0113] Example 5: Structure characterization of silk fibroin porous microspheres

[0114] The silk fibroin porous microspheres prepared in Example 2 and the silk fibroin microspheres prepared in Comparative Example 3 were cut into small pieces of about 2-3 mm from the cross section after freeze-drying for 35 h, and were sprayed with gold for 30 S twice, and then were placed under a scanning electron microscope for imaging observation. The results are shown in Figure 2.

[0115] The results show that Comparative Example 3 is a solid microsphere structure without pores; the SF porous microspheres prepared in Example 2 have obvious pores and are porous sponges, which are more conducive to cell entry and adhesion and are conducive to tissue regeneration.

[0116] Example 6: Anti-enzymatic ability

[0117] Hyaluronic acid is directly used for facial filling and can be quickly degraded by enzymes into glucuronic acid. Glucuronic acid reacts with carbazole reagent to produce a red-violet color, and the depth of the color is proportional to the content of glucuronic acid. Therefore, the content of glucuronic acid is determined by absorbance value, and the anti-enzymatic ability of the hyaluronic acid-containing gel is indirectly reflected.

[0118] The anti-enzymatic ability determination was carried out for Example 2, Example 3, Comparative Examples 1-3, and the experimental method was as follows:

[0119] 1) Solution preparation

[0120] Carbazole ethanol solution with a volume fraction of 0.125%: 0.125 g of carbazole was weighed and dissolved in 100 ml of anhydrous ethanol, transferred to a dark brown bottle, and stored in the dark, with a validity period of 15 days.

[0121] Glucuronic acid standard solution: 0.100 g of D-glucuronic acid was accurately weighed, dissolved in water, and diluted to 100.0 g, and mixed to serve as a stock solution, which was stored at 4°C±2°C for 3 hours. 5.0 ml of the stock solution was accurately measured into a 100 ml volumetric flask, water was added to make a solution containing 50 μg per 1 g, and shaken well, and stored at 2-8°C.

[0122] 0.025 mol / L sodium tetraborate sulfuric acid solution: 9.54 g of sodium tetraborate (Na2B4O7·10H2O) was weighed and added to 1 L of concentrated sulfuric acid, covered, and shaken from time to time until the sodium tetraborate was completely dissolved. It was stored at room temperature for 12 months.

[0123] 0.5 mol / L sulfuric acid solution: 5 ml of 98% sulfuric acid was added to a beaker containing 179 ml of water and mixed well.

[0124] 1 mol / L sodium hydroxide solution: 10 g of sodium hydroxide was added to 250 ml of water and stirred to dissolve.

[0125] 2) Hyaluronic acid sodium content determination method

[0126] 2.1 1 ml of each sample was taken into a 50 ml volumetric flask, 2 mg of HA enzyme was added, the volumetric flask was placed in a 37°C water bath and shaken for 72 h, and the sample was taken for determination.

[0127] 2.2 Preparation of control solution: 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucuronic acid solution was accurately measured into 20 ml test tubes with stoppers, and water was added to 1.0 ml. Three replicates were prepared in parallel. One blank tube was prepared with 0.

[0128] 2.3 Sample preparation: about 0.1 g of sample was accurately weighed (to the nearest 0.1 mg), 10 ml of 0.5 mol / L sulfuric acid solution was added, and it was heated to completely dissolve in a 95±5°C constant temperature oven, 10 ml of 1 mol / L sodium hydroxide solution was added, and water was added to dilute to 50 ml, and it was shaken well and mixed, and 1 ml was taken from it and placed in a test tube.

[0129] 2.4 Method

[0130] The 2.1 control solution and blank solution and 2.2 sample solution tubes were placed in an ice water bath, and 5 ml of 0.025 nol / L sodium tetraborate sulfuric acid solution, which was previously cooled in an ice bath, was added to each test tube, and the test tubes were shaken. The test tubes were placed in a boiling water bath for 10 minutes, and then cooled to room temperature. 0.2 ml of 0.125% carbazole ethanol solution was added to each test tube, which was then sealed with a cap, shaken, and placed in a boiling water bath for 15 minutes, and then cooled to room temperature. The blank tube was used as a blank zero, and the absorbance of each tube was measured at 530 nm using a calibrated ultraviolet spectrophotometer. The average concentration of each control solution and its corresponding absorbance value was used as a standard curve. According to the standard curve, the average concentration of D-glucuronic acid in the sample solution was calculated based on the corresponding absorbance value. (n = 3)

[0131] 2.5 Calculation

[0132] The mass concentration value C (mg / ml) of sodium hyaluronate in the sample was calculated according to the following formula.

[0133] Content

[0134] In the formula, m1 is the mass of the sample, g;

[0135] m2 is the mass of the sample and purified water, mg;

[0136] d1 is the density of the sample, g / ml;

[0137] d2 is the density of the sample and purified water, g / ml;

[0138] p1 is the content of D-glucuronic acid in the sample test solution, pg / ml.

[0139] Table 3 Anti-enzymatic activity of HA-SF gels containing SF microspheres (mean ± SD)

[0140] The results in Table 3 show that Examples 2, 3, Comparative Example 2 and Comparative Example 3 all have certain anti-enzymatic activity compared with Comparative Example 1, and Example 3 has stronger anti-enzymatic activity, which is significantly better than Comparative Examples 1-3.

[0141] Example 7: Cell proliferation experiment

[0142] Cell proliferation experiments were performed on Examples 1-3 and Comparative Examples 1-2. Logarithmic growth phase mouse fibroblast L929 cells were digested and inoculated into 24-well plates. 0.3 mL of each sample was placed in a TRANSWELL chamber and co-cultured with the cells for 7 days, and then the A450 nm absorbance value was detected to observe the cell viability. The results are shown in Figure 3.

[0143] The results show that the gels of Examples 1-3 can significantly promote the proliferation of fibroblasts, indicating good safety and being significantly better than Comparative Examples 1 and 2.

[0144] Example 8: Cell adhesion

[0145] Take 1 mL of each of the samples of Example 2, Example 3 and Comparative Example 3 and add to DMEM culture medium, and the obtained sterile extract is co-cultured with mouse fibroblast L929 cells for 3 days, and then washed with PBS to remove non-adherent cells. The morphology, quantity and distribution of adherent cells are observed and recorded, and the number of cells is counted (n = 5). The results are shown in Table 4.

[0146] Table 4 Cell adhesion of the filler of the application

[0147] The results show that the number of adherent cells of the sample 4 of Example 1, and Examples 2 and 3 of the application is significantly increased compared with the blank group, and is better than Comparative Examples 1 and 2; indicating that the gels prepared in Examples 1-3 of the application have good cell adhesion.

[0148] Example 9: Promoting collagen regeneration

[0149] 1. HA-SF gels with different molecular weights of SF

[0150] Take 1 g of each of the sample 4 of Example 1, sample 6 and sample 7, dissolve in DMEM culture medium to prepare a sterile extract, digest L929 cells in logarithmic growth phase, inoculate in a 96-well plate, and culture with the above sterile extract. After 14 days, the collagen content in the cell supernatant is detected by ELISA kit method. The results are shown in Figure 4.

[0151] The results show that compared with the blank control group which is not added any sample and is only cultured with DMEM complete medium, the SF with three different molecular weights all significantly promote the collagen regeneration of fibroblasts.

[0152] 2. Gels containing silk fibroin porous microspheres

[0153] Digest L929 cells in logarithmic growth phase, inoculate in a 24-well plate, and take 0.3 mL of each of the sample 4 of Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2 and Comparative Example 3 and place in TRANSWELL chamber. After 10 days of co-culture, the collagen in the fibroblast supernatant is detected by ELISA kit. The results are shown in Figure 5.

[0154] The results show that compared with the blank group (CTRL), the gels of the application have the effect of promoting collagen regeneration, and Examples 2 and 3 are better than Comparative Examples 1, 2 and 3.

[0155] Example 10: Biocompatibility - ICR mouse injection test

[0156] Example 3, Comparative Example 1 and Comparative Example 2 0.3 mL were injected into the back dermis of ICR mice, respectively, and after 14 days, the injection sites were observed for the presence or absence of abnormal lesions in the subcutaneous and muscle tissues, and the tissue response (presence or absence of hemorrhagic swelling, edema, cysts and hyperplasia) was observed under a low-power magnifying glass. If the biocompatibility is good, the tissue inflammatory response is small; otherwise, if the biocompatibility is poor, the tissue inflammatory response is large. The health of the tissue was also observed by HE staining of the pathological sections. The results are shown in Figure 6.

[0157] The results show that the surface of the skin tissue on the back of the normal mouse is a basket-shaped keratinization, the granular layer exists, the arrangement is regular, the prickle layer is normal, the base is regular and the interface with the dermal papillary layer is clear, the dermal layer can see rich collagen fibers, the shape and distribution are normal, the subcutaneous fat layer is basically normal, there is no inflammatory infiltration in the dermal layer and subcutaneous fat layer, and the skin health level is high. After treatment with Comparative Example 2, more neutrophils were observed in the dermal layer and subcutaneous fat layer of the mouse. After treatment with Example 3 and Comparative Example 1, a small amount of neutrophils were observed in the dermal layer and subcutaneous fat layer of the back skin of the mouse. Compared with Comparative Example 2, the skin health level of the mouse treated with Example 3 was higher. Compared with Comparative Example 1, the skin health level of the mouse treated with Example 3 was higher.

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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