Silk fibroin composition for injection and preparation method thereof

Abstract

The invention provides a silk fibroin composition for injection and a preparation method thereof, and belongs to the technical field of medical cosmetology. The silk fibroin composition is prepared from the following components in percentage by mass: 0.1 to 0.7 weight percent of hyaluronic acid or salt thereof, 0.1 to 10 weight percent of silk fibroin and the balance of phosphate buffer solution. The silk fibroin composition is used for water-light injection, can promote collagen production and inhibit collagen fibrosis, does not have adverse reactions such as skin allergy, hardening and the like, and has the effects of quickly improving the dry state of skin, fading fine wrinkles or wrinkles, replenishing water, preserving moisture and tightening the skin.

Description

Technical Field

[0001] This invention relates to the field of medical aesthetics technology, specifically to a silk fibroin composition for injection and its preparation method. Background Technology

[0002] As we age, facial skin exhibits signs of aging, most notably the loss of collagen and moisture, clinically manifesting as wrinkles, sagging, and roughness. Dry skin is prone to fine lines, wrinkles, and other signs of aging, while adequate hydration helps maintain smoothness and firmness. Collagen fibrosis is one of the contributing factors to skin aging. Therefore, preventing collagen fibrosis and increasing collagen content and skin elasticity are crucial for improving skin aging.

[0003] Currently used preparations for facial hyaluronic acid injections can cause excessive collagen production, leading to excessive collagen fiber proliferation and adverse reactions such as skin hardening, small papules, or nodules. They also disrupt the balance of facial physiological osmotic pressure, thus affecting the normal proliferation of skin cells. Therefore, there is an urgent need in the market for safe, effective products that can quickly improve facial skin aging. Summary of the Invention

[0004] The purpose of this invention is to provide an injectable silk fibroin composition and its preparation method. The silk fibroin composition contains silk fibroin and hyaluronic acid. After injection, it can quickly replenish skin moisture, effectively promote skin collagen regeneration, and inhibit collagen fibrosis, avoiding adverse reactions such as skin allergies and stiffness, and has good safety.

[0005] The technical solution adopted in this invention is as follows:

[0006] On one hand, the present invention provides a silk fibroin composition for injection, comprising, by weight percentage: 0.01-10 wt% hyaluronic acid or its salt, and 0.01-20 wt% silk fibroin.

[0007] Preferably, based on the total mass of the composition, the content of the hyaluronic acid or its salt is 0.05-5 wt%, preferably 0.1-1 wt%, more preferably 0.1-0.7 wt%, more preferably 0.3-0.7 wt%, even more preferably 0.4-0.6 wt%, and most preferably 0.5 wt%.

[0008] Preferably, based on the total mass of the composition, the content of the silk fibroin is 0.05-15 wt%, more preferably 0.1-10 wt%, more preferably 0.1-1 wt%, more preferably 0.3-0.7 wt%, and most preferably 0.5 wt%.

[0009] Preferably, the composition is a solution, reconfigurable powder, emulsion, ointment, gel, paste, or suspension, preferably a solution or reconfigurable powder, more preferably an aqueous solution, and most preferably an aqueous solution for use in hyaluronic acid injections.

[0010] Preferably, the hyaluronic acid has a molecular weight of 500-2000 kDa, more preferably 1000-1800 kDa; the silk fibroin has a molecular weight of 10-150 kDa, more preferably 10-50 kDa.

[0011] Alternatively, the composition may further include a buffer solution selected from one or more of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, and HEPES buffer, preferably phosphate buffer.

[0012] Alternatively, the silk fibroin composition may further include a stabilizer selected from one or more of sorbitol, mannitol, inositol, xylitol, urea peptide, sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium bicarbonate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, and potassium chloride.

[0013] Alternatively, the silk fibroin composition may further include an antioxidant, which is at least one of nicotinamide, vitamin C, tea polyphenols, or vitamin E.

[0014] In a preferred embodiment, based on the total mass of the silk fibroin composition, the composition comprises:

[0015] -0.05-5wt% of hyaluronic acid or its salt, preferably 0.1-1wt%, more preferably 0.1-0.7wt%, more preferably 0.3-0.7wt%, even more preferably 0.4-0.6wt%, and most preferably 0.5wt%;

[0016] -0.05-15wt% of silk fibroin, preferably 0.1-10wt%, more preferably 0.1-1wt%, more preferably 0.3-0.7wt%, and most preferably 0.5wt%;

[0017] - The remainder is phosphate buffer.

[0018] Preferably, the osmotic molar concentration of the silk fibroin composition is 200-400 mOsmol / kg, more preferably 280-350 mOsmol / kg; the kinematic viscosity is 10-1000 mPa·s, more preferably 100-600 mPa·s, and even more preferably 150-300 mPa·s; and the extrusion force is 8-20 N, more preferably 10-15 N, and even more preferably 12-15 N.

[0019] Preferably, the silk fibroin composition is used for injection into the face or neck, and more preferably into the face.

[0020] In a second aspect, the present invention provides a method for preparing the silk fibroin composition for injection described in the first aspect, comprising the following steps:

[0021] 1) Dissolve hyaluronic acid or its salt in phosphate buffer to obtain a hyaluronic acid solution;

[0022] 2) Dissolve silk fibroin in phosphate buffer to obtain a silk fibroin solution; and

[0023] 3) Mix the above hyaluronic acid solution and silk fibroin solution.

[0024] In a preferred embodiment, after step 2), the method further includes a step of freeze-drying the silk fibroin solution to obtain a freeze-dried silk fibroin block. Preferably, the freeze-dried silk fibroin block is sterilized by irradiation and then stored at room temperature.

[0025] In a preferred embodiment, the method further includes a step of compounding the lyophilized silk fibroin block with the hyaluronic acid solution obtained in step 1). Preferably, after the compounding step, the resulting solution is left to stand at room temperature for 3-10 minutes.

[0026] Preferably, the freeze-drying conditions are as follows: pre-freezing temperature -50℃ to -45℃, ramp time 0-60 min, holding time 120 min; primary drying temperature rise including -10℃ to 0℃, ramp time 0-120 min, holding time 300-600 min, vacuum setting 0.3 mbar; first stage of desorption drying, temperature setting 0℃-15℃, ramp time 0-30 min, holding time 500-800 min, vacuum setting 0.1 mbar; second stage of desorption drying 30℃-37℃, ramp time 30 min, holding time 200-400 min, vacuum setting 0.1 mbar; pressure recovery test, qualified standard: pressure change ≤ 0.05 mbar in 5 min.

[0027] Thirdly, the present invention also provides a reagent kit comprising:

[0028] (a) hyaluronic acid or a salt thereof, preferably hyaluronic acid or sodium hyaluronate, more preferably hyaluronic acid; and

[0029] (b) Silk fibroin,

[0030] Prior to use, the hyaluronic acid or its salt is reconstituted with the silk fibroin to form an injectable composition. Based on the total mass of the composition, the content of the hyaluronic acid or its salt is 0.01-10 wt%, preferably 0.05-5 wt%, preferably 0.1-1 wt%, more preferably 0.1-0.7 wt%, more preferably 0.3-0.7 wt%, even more preferably 0.4-0.6 wt%, and most preferably 0.5 wt%; the content of the silk fibroin is 0.01-20 wt%, preferably 0.05-15 wt%, preferably 0.1-10 wt%, more preferably 0.1-1 wt%, more preferably 0.3-0.7 wt%, and most preferably 0.5 wt%. Preferably, the injectable composition is a composition for facial or neck injection, and more preferably a composition for facial injection.

[0031] In a preferred embodiment, the hyaluronic acid or its salt and the silk fibroin are reconstituted into a solution, emulsion, ointment, gel, paste or suspension before use, preferably a solution, more preferably an aqueous solution, and most preferably an aqueous solution for use in hyaluronic acid injections.

[0032] In a preferred embodiment, the hyaluronic acid or its salt has a molecular weight of 500-2000 kDa, preferably 1000-1800 kDa; the silk fibroin has a molecular weight of 10-150 kDa, preferably 10-50 kDa.

[0033] In a preferred embodiment, the kit further includes a buffer selected from one or more of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, and HEPES buffer, preferably phosphate buffer.

[0034] In a preferred embodiment, the kit further includes a stabilizer selected from one or more of sorbitol, mannitol, inositol, xylitol, urea peptide, sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium bicarbonate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, and potassium chloride.

[0035] In a preferred embodiment, the kit further includes an antioxidant, which is at least one of nicotinamide, vitamin C, tea polyphenols, or vitamin E.

[0036] Fourthly, the present invention also provides the use of the silk fibroin composition for injection described in the first aspect in the preparation of a product for improving the skin condition of a subject, said skin condition including dry skin, wrinkles, fine lines, sagging skin, and rough skin.

[0037] Preferably, the improvement in skin condition manifests in at least one of the following ways:

[0038] 1) Increase the level of collagen in the skin, preferably, the collagen is type III collagen (Collagen III);

[0039] 2) Reduce α-SMA levels;

[0040] 3) Promotes the expression of AQP3.

[0041] Beneficial effects

[0042] This invention relates to a silk fibroin composition for facial dermal injection, which rapidly improves dry skin, quickly replenishes skin moisture, reduces fine lines and wrinkles, and firms the skin. This invention can enhance fibroblast vitality, promote keratinocyte proliferation, repair the skin barrier, and promote collagen production, especially type III collagen.

[0043] The silk fibroin composition of the present invention can reduce the level of α-SMA in the skin, inhibit collagen fibrosis, and promote the expression of AQP3, thereby promoting the transport of moisture in the skin and achieving a moisturizing effect.

[0044] This invention has good safety and will not cause adverse reactions such as skin hardening or redness. Moreover, the osmotic pressure meets the requirements and is more in line with the physiological characteristics of the skin. It has a significant effect on improving the skin condition after injection. Brief description of the attached diagram

[0045] Figure 1 The effect of the silk fibroin composition on the proliferation of L929 cells in Example 2 (compared with the CTRL group, **P<0.01, ****P<0.0001).

[0046] Figure 2 The total collagen content of L929 cells treated with the silk fibroin composition in Example 2 was measured 14 days later (compared to the CTRL group, ***P<0.001).

[0047] Figure 3 The results of type III collagen content 14 days after treatment of L929 cells with the silk fibroin composition in Example 2 (compared with the CTRL group, **P<0.01, ***P<0.001).

[0048] Figure 4 The effect of the silk fibroin composition on α-SMA expression in Example 2 (compared to the CTRL group, **P<0.01).

[0049] Figure 5AMasson staining and PSR staining images of skin tissue sections from the back of mice in each group (compared with the CTRL group, *P<0.05). In the Masson staining image, the blue part represents collagen and the red part represents muscle fibers. In the PSR image, the red and yellow parts represent type I collagen and the green part represents type III collagen. Figure 5B for Figure 5A The PSR staining image shows a bar chart of the relative content of type I and type III collagen, where the gray part represents type I collagen and the pink part represents type III collagen.

[0050] Figure 6A Immunofluorescence (IF) staining images of pathological sections of skin tissue from the backs of mice in each group. Figure 6B To display Figure 6A A bar chart of AQP3 staining area. Invention Details

[0052] definition

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] As used herein, the term "stabilizer" refers to a chemical that increases the stability of solutions, colloids, solids, and mixtures. Stabilizers can slow down reactions, maintain chemical equilibrium, reduce surface tension, and prevent photo-, thermal, or oxidative decomposition.

[0058] As used in this article, the term "antioxidant" refers to a substance that can prevent the adverse effects of oxygen. Its reduction potential should be lower than that of the easily oxidized active ingredient in the formulation. Therefore, it is oxidized before the active ingredient and is consumed at the same time, thus maintaining the stability of the formulation.

[0059] As used in this article, the term "hyaluronic acid injection" refers to the precise injection of nutrients and medications into specific layers of the skin using hollow microneedles. This effectively replenishes various nutrients, stimulates collagen production, makes the skin hydrated and radiant, effectively delays skin aging, and improves skin texture.

[0060] 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

[0061] Collagen is the most abundant protein in the extracellular matrix. Collagen fibers play a crucial role in maintaining skin tension and elasticity. With age, the loss of collagen in the skin leads to a loss of its supporting structure, making facial skin more prone to sagging and drooping, as well as wrinkles and fine lines. Type I collagen constitutes the majority of the skin's composition (80-85%), participating in the structure and integrity of the dermis and maintaining the skin's reticular structure. Type III collagen accounts for a smaller proportion of the skin (10-15%). In addition to participating in the dermal structure, it also provides tension, flexibility, and softness to the skin by altering the diameter of collagen fibers. Type III collagen is most abundant in infancy and is known as the "golden collagen" of the skin. With age, the irreversible reduction of type III collagen leads to facial depressions, decreased skin elasticity, and facial wrinkles. There are numerous reports on collagen supplementation or collagen production promotion; however, excessive collagen supplementation can cause fibrosis, leading to a series of problems such as hardened skin and nodules.

[0062] Hyaluronic acid, also known as hyaluronic acid, is a major component of the intercellular matrix and extracellular matrix of skin cells. It exists in the epidermis and dermis, possessing high viscoelasticity and malleability, and helps maintain skin moisture, plumpness, and elasticity. Clinically, it can be treated non-surgically, such as with mesotherapy injections. Under computer control, disposable 5-, 9-, or 13-well needles are used to draw the skin up through a negative pressure system, simultaneously injecting medication into the target area. This stimulates and activates surface skin cells, improving rough skin and wrinkles, achieving a skin rejuvenation effect.

[0063] The hydrating component of the injectable sodium hyaluronate complex solution used in mesotherapy is mainly sodium hyaluronate. In addition, it also contains L-carnosine, glycine, alanine, proline, and vitamin B2. Among them, glycine, alanine, proline, and L-carnosine can promote the production of collagen by skin fibroblasts, while vitamin B2 promotes the conversion of collagen into collagen fibers.

[0064] Silk fibroin is a structural protein extracted from natural silkworm silk, composed of 18 amino acids. Compared with other natural biopolymers, silk fibroin has good biocompatibility and degradability, low immunogenicity, and excellent mechanical properties, making it a highly promising new natural medical material. Prior to the date of this invention application, there were no silk fibroin-related products on the market for facial injection.

[0065] In view of the problems of poor collagen regeneration effect and poor safety of the mesotherapy currently used in clinical and medical aesthetic fields, this invention proposes for the first time a silk fibroin composition for injection.

[0066] Example

[0067] 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.

[0068] The materials used in the following embodiments are sourced from the following sources:

[0069] Hyaluronic acid (HA): Bloomage Biotechnology Co., Ltd., molecular weight 1000-1800kDa, batch number: 22061041;

[0070] Silk fibroin (SF): Shenzhen Huasi Biotechnology Co., Ltd., molecular weight 10-50kDa, batch number: 23041101;

[0071] Comparative Example 1: Sodium hyaluronate complex solution for injection (trade name: Haiti), batch number 230426, manufacturer: Aimeike Technology Development Co., Ltd.

[0072] Example 1

[0073] A silk fibroin composition for facial injection comprises, by weight percentage: 0.5 wt% hyaluronic acid, 0.1 wt% silk fibroin, and the balance being phosphate buffer.

[0074] The preparation method is as follows:

[0075] 1) Weigh 0.05g of hyaluronic acid (molecular weight 1000-1800kDa), dissolve it in 9mL of PBS buffer, place it at 4℃ for about 12h to swell, and then use magnetic stirring for 2-3h at 100-200rpm until fully swollen.

[0076] 2) Weigh 0.01g of silk fibroin (molecular weight 10-50kDa), add 1mL of PBS, stir to dissolve, and then refrigerate at 4℃ for 2-24h.

[0077] 3) According to the 2.5mL specification, add 2.25mL of hyaluronic acid solution and 0.25mL of silk fibroin solution in a volume ratio of 9:1 to the same vial, seal with an aluminum cap, invert and let stand to mix, and then sterilize by moist heat to obtain the silk fibroin composition of the present invention.

[0078] Example 2

[0079] A silk fibroin composition for facial injection comprises, by weight percentage: 0.5 wt% hyaluronic acid, 0.1 wt% silk fibroin, and the balance being phosphate buffer.

[0080] The preparation method is as follows:

[0081] 1) Weigh 0.05g of hyaluronic acid (molecular weight 1000-1800kDa), dissolve it in 9mL of PBS buffer, place it at 4℃ for about 12h to swell, and then use magnetic stirring for 2-3h at 100-200rpm until fully swollen.

[0082] 2) According to the 2.5mL specification, take 2.5mL of the above hyaluronic acid solution and fill it into a vial. Seal the vial with an aluminum cap, invert it and let it stand to mix well, and then sterilize it with moist heat.

[0083] 3) Preparation of silk fibroin solution: Weigh 0.01g of silk fibroin (molecular weight 10-50kDa), add 1mL of PBS, stir to dissolve, and refrigerate at 4℃ for 2-24h. Freeze-dry the silk fibroin solution to obtain a white block, sterilize by 20kgy irradiation, and store at room temperature.

[0084] The freeze-drying conditions were as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the pass standard is pressure change ≤0.05mbar within 5min.

[0085] 4) Before use, mix 2.5 mL of hyaluronic acid solution with 2.5 mg of lyophilized silk fibroin block and let stand at room temperature for 1-3 minutes to obtain the silk fibroin composition of the present invention.

[0086] Example 3: Performance Measurement

[0087] Samples 1-12 are silk fibroin compositions with different HA and SF concentrations (HA molecular weight 1000-1800kDa, SF molecular weight 10-50kDa) prepared according to the method of Example 1.

[0088] Sample 13 is a silk fibroin composition prepared according to the method of Example 2.

[0089] The viscosity and extrusion force of samples 1-13 and Comparative Example 1 (Hi-body) were tested using the following methods:

[0090] 1. Kinematic viscosity

[0091] The viscosity parameters of the samples were determined using a Kinexus Lab+ rotational rheometer. Before measurement, the samples were allowed to equilibrate at room temperature for 1 hour. Each measurement volume was 1 mL–1.2 mL, ensuring the sample filled the conical lamina. The V001 program was selected, with a temperature of 25 ± 0.1 °C and a shear rate measurement range of 0.1–1 s⁻¹. Measurements were performed according to the Malvern rheometer operating procedure, and data were recorded at the corresponding frequencies of 0.1, 0.25, 0.5, and 1 s⁻¹. The results are shown in Table 1.

[0092] 2. Pushing force

[0093] The extrusion force was measured 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). Test parameters were set, and the machine was allowed to equilibrate to room temperature for 1 hour beforehand. The extrusion speed was set to 30 mm / min. The test was then started, and the average force (mean ± SD) at full scale was read. The results are shown in Table 1.

[0094] Table 1. Viscosity and extrusion force of silk fibroin compositions with different HA and SF contents.

[0095]

[0096] For facial injectable injections, excessive viscosity can lead to a thick, poorly fluid consistency, poor absorption after injection, and the appearance of small, dot-like papules under the skin. Insufficient viscosity can result in a short retention time under the skin, rapid absorption, and fast metabolism. Excessive force makes injection difficult and may not reach the dermis, while insufficient force makes it difficult to determine the injection site, leading to errors in the injection location.

[0097] Therefore, it is believed that silk fibroin compositions with a pushing force of less than 15N have good injectability, while those with a kinematic viscosity of more than 700mPa·s have poor flowability. When the pushing force is 10-15N and the kinematic viscosity is 100-600mPa·s, they are more suitable for facial injection.

[0098] 3. Osmotic pressure

[0099] An osmometry meter (Tianjin Tianda Tianfa) was used for calibration. The instrument was calibrated using standard osmolarity concentration (OSC) solutions of 100 mOsmol / kg and 400 mOsmol / kg provided by the manufacturer. Test solutions were prepared. Following the instrument's instruction manual, an appropriate amount of freshly boiled and cooled water was first used to adjust the instrument's zero point. Then, the instrument was calibrated using the two standard solutions. Finally, 100 μL samples (samples 4, 7, 8, 13, and Comparative Example 1) were taken and their osmolarity was measured. The results are shown in Table 2.

[0100] Table 2. Osmotic pressure (mean ± SD) of the present invention and Comparative Example 1

[0101]

[0102] As shown in Table 2, the osmotic pressure of samples 4, 7, 8 and 13 are close to the normal osmotic pressure of the skin (280-320 mOsm / kg), indicating good safety.

[0103] Example 4: Cell Experiment

[0104] 1. Cell proliferation experiment

[0105] Following the method in Example 2, 1 mL of sterile hyaluronic acid solution and 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg of sterile lyophilized silk fibroin powder were taken to prepare silk fibroin compositions with HA 0.5 wt% and SF contents of 0.1 wt%, 0.3 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, and 10 wt%, respectively. 1 mg, 3 mg, 5 mg, and 10 mg of sterile lyophilized silk fibroin powder were dissolved in 1 mL of PBS buffer to prepare silk fibroin solutions of 0.1 wt%, 0.3 wt%, 0.5 wt%, and 1 wt%, respectively.

[0106] Experimental methods: Logarithmic growth phase mouse fibroblasts L929 were digested and seeded in 24-well plates. The silk fibroin composition of the present invention and Comparative Example 1 were placed in TRANSWELL chambers and co-cultured for 6 days. The number and viability of fibroblasts were then detected using the classic CCK-8 assay. Results are shown below. Figure 1 .

[0107] The results showed that, compared with the normal group (CTRL group) cultured in DMEM complete medium without the addition of other additives, the silk fibroin composition promoted the growth of fibroblasts and was superior to Comparative Example 1 (HA-SF). Compared with SF solution, HA-SF water-light preparation had a better proliferation-promoting effect at the same concentration.

[0108] 2. Detection of total collagen and type III collagen

[0109] Following the method in Example 2, 1 mL of sterile hyaluronic acid solution and 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg of sterile lyophilized silk fibroin powder were taken respectively to prepare silk fibroin compositions with HA 0.5% and SF contents of 0.1 wt%, 0.3 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, and 10 wt%, respectively. 1 mg, 3 mg, 5 mg, and 10 mg of sterile lyophilized silk fibroin powder were dissolved in 1 mL of PBS buffer to prepare silk fibroin solutions of 0.1 wt%, 0.3 wt%, 0.5 wt%, and 1 wt%, respectively.

[0110] Experimental Methods: Logarithmic growth phase mouse fibroblasts (L929) were digested and seeded in 24-well plates. The silk fibroin composition of the present invention and Comparative Example 1 were placed in TRANSWELL chambers and co-cultured for 14 days. The total collagen and type III collagen in the fibroblast supernatant were then measured. The assays were performed using an ELISA kit. Results are shown below. Figure 2 and Figure 3 .

[0111] The results showed that, compared with the normal group (CTRL group), the silk fibroin composition promoted collagen regeneration (see...). Figure 2 The results showed that the HA 0.5% and SF 0.3%, 0.5%, and 1% silk fibroin compositions, respectively, promoted the production of type III collagen (see [link to original text]). Figure 3 Compared to SF solution, HA-SF hydrogel preparations are more effective at promoting collagen production at the same concentration.

[0112] 3. Collagen fibrosis detection

[0113] Experimental Methods: Logarithmic growth phase mouse fibroblasts (L929) were digested and seeded in 24-well plates. The silk fibroin composition (HA 0.5%, SF 0.3%) prepared according to Example 2 and Comparative Example 1 were placed in TRANSWELL chambers and co-cultured for 14 days. The degree of fibrosis in the fibroblast supernatant was then detected. The expression of the collagen fibrosis marker protein α-smooth muscle actin (α-SMA) was measured using an ELISA kit. Results are shown below. Figure 4 .

[0114] The results showed that, compared with the normal group (CTRL group), the silk fibroin composition significantly reduced the expression of α-SMA. The silk fibroin composition of this invention may have an inhibitory effect on collagen fibrosis, thereby reducing adverse reactions and improving skin aging.

[0115] Example 5: Animal Experiment

[0116] Female ICR mice aged 6-8 weeks were selected. The hair on the backs of each mouse was shaved, and the skin was wiped with physiological saline. The following day, nine points were taken within a 1×1 cm area of ​​exposed skin. Physiological saline (normal group), the silk fibroin composition of this invention (prepared according to Example 2, HA concentration 0.5wt%, SF concentration 0.5wt%), Comparative Example 1 (HA-body), and SF solution (concentration 0.5wt%) were injected into the dermis at each point, approximately 0.03 mL per point, for a total of 0.3 mL per mouse. One month later, the back skin of the mice was harvested for skin histopathological sections. Masson staining and PSR staining were used to detect collagen expression in the skin. The results are shown below. Figure 5A and Figure 5B The expression of the skin water transport-related protein AQP3 was detected by IF staining, and the results are shown in [Figure number missing]. Figure 6A and Figure 6B .

[0117] Figure 5A and Figure 5B The results showed that the arrows indicated the content and distribution density of collagen in the dermis. Compared with the normal group, the proportion of blue in Example 2 and Comparative Example 1 was significantly increased, indicating that both could improve collagen content. However, the collagen distribution in Comparative Example 1 was sparse, while the collagen distribution in Example 2 (silk fibroin composition HA 0.5wt%, SF 0.5wt%) was dense, indicating that the silk fibroin composition of the present invention can improve the content and density of skin collagen after injection.

[0118] Collagen types were identified using PSR staining. Under a polarizing microscope, type I collagen appeared red or yellow, while type III collagen appeared green. In Comparative Example 1, the proportion of yellow fibers was significantly higher, suggesting that they promoted the production of type I collagen to a greater extent. However, in Example 2, type III collagen production was significantly promoted, consistent with the quantitative results from the bar chart.

[0119] Figure 6A and Figure 6B The results showed that, compared with the normal group, the silk fibroin composition significantly promoted the expression of water transport-related protein AQP3 in mouse skin, indicating that the silk fibroin composition can promote skin moisture transport and has a better moisturizing effect.

[0120] Example 6: Human Skin Experiment

[0121] The human trial samples were prepared using the same method as in Example 2, consisting of a sodium hyaluronate solution and lyophilized silk fibroin blocks, with the solution containing 0.5 wt% sodium hyaluronate and 0.5 wt% silk fibroin. The control group received saline injection. Electrotherapy (hydrotherapy) was used, and strict aseptic techniques were followed during the injection process to avoid cross-infection. Sterile 32G nine-needle injection needles were prepared in advance. Within 28 days after the injection treatment, at follow-up time points, the subjects' facial skin was assessed using the Allergan Fine Lines Scale (ALLAMS) for fine lines, moisture, elasticity, and skin texture (dermoscopy). The improvement in skin condition was evaluated, and the Global Aesthetic Effect Index (GAIS) was used to assess effectiveness and calculate the improvement rate.

[0122] Evaluation 1: Global Aesthetic Effect Rating (GAIS)

[0123] The overall facial cosmetic effect is graded into 5 levels: worsening, ineffective, slight improvement, significant improvement, and complete improvement. See Table 3 for specific definitions.

[0124] Table 3 Global Aesthetic Effect Rating (GAIS) Reference Table

[0125]

[0126] Evaluation 2: Allergan fine line severity rating scale, see Table 4 for details.

[0127] Table 4. Allergan Fine Line Severity Grading Reference Table

[0128]

[0129] Conclusion: Within 28 days post-injection, the GAIS scores of most subjects in the experimental group were 1 or 2, indicating that the skin condition of most subjects improved, the skin became smoother, and moisture content and elasticity also improved significantly. The secondary efficacy endpoint, the Allergan fine line score, showed a significant improvement of 1-2 degrees within 28 days of injection, with a reduction in fine lines. In the control group, there were no significant changes in moisture or elasticity, and neither the GAIS nor Allergan fine line scores changed significantly before and after injection.

[0130] Example 7: Freeze-drying experiment

[0131] Silk fibroin freeze-dried sample 1, the freeze-drying conditions are as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0132] Silk fibroin freeze-dried sample 2, the freeze-drying conditions are as follows: pre-freezing temperature -45℃, ramp time 60min, holding time 120min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0133] Silk fibroin freeze-dried sample 3, the freeze-drying conditions are as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including 0℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0134] Silk fibroin freeze-dried sample 4, the freeze-drying conditions are as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 0℃, ramp time 30min, holding time 800min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 200min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0135] Silk fibroin freeze-dried sample 5, the freeze-drying conditions are as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 33℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0136] Silk fibroin freeze-dried sample 6. The freeze-drying conditions for the silk fibroin were as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 60min; first drying temperature rise including -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the pass standard is pressure change ≤0.05mbar within 5min.

[0137] Silk fibroin freeze-dried sample 7. The freeze-drying conditions for the silk fibroin were as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -20℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0138] 8 lyophilized silk fibroin samples were prepared under the following conditions: pre-freezing temperature -50℃, ramp time 60 min, holding time 120 min; first drying temperature rise including -20℃, ramp time 120 min, holding time 600 min, vacuum setting 0.3 mbar; first stage of desorption drying, temperature setting 15℃, ramp time 30 min, holding time 200 min, vacuum setting 0.1 mbar; second stage of desorption drying 37℃, ramp time 30 min, holding time 400 min, vacuum setting 0.1 mbar; pressure recovery test, the pass standard is pressure change ≤0.05 mbar within 5 min.

[0139] Silk fibroin freeze-dried sample 9. The freeze-drying conditions for the silk fibroin were as follows: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; first drying temperature rise including -20℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; first stage of desorption drying, temperature set at 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; second stage of desorption drying at 45℃, ramp time 30min, holding time 100min, vacuum setting 0.1mbar; pressure recovery test, the qualified standard is pressure change ≤0.05mbar within 5min.

[0140] Lyophilized silk fibroin samples 1-9 were redissolved in 5% HA solution until completely clear and free of debris, and the dissolution time was recorded. The initial height of the lyophilized sample was recorded as H1, and the height after 24 hours at room temperature was recorded as H2. The degree of collapse was calculated as H2 / H1*100%. The moisture content of lyophilized silk fibroin samples 1-9 was determined. Moisture content was determined according to the coulometric titration method as described in the pharmacopoeia. An appropriate amount of Fischer reagent was added to the titration vessel to pre-titrate and remove moisture from the reagent and system. Then, an appropriate amount of the test sample (containing approximately 0.5–5 mg of water) was accurately measured and quickly transferred to the titration vessel, or dissolved in a suitable anhydrous solvent and quickly injected into the titration vessel. The endpoint was indicated by the permanent stop titration method (Pharmacopoeia General Chapter 0701). The moisture content of the test sample was directly read from the instrument display. Each 1 mg of water is equivalent to 10.72 coulombs of electricity. The results are shown in Table 5.

[0141] Table 5. Reconstitution time and collapse degree of SF freeze-dried samples under different freeze-drying conditions

[0142]

[0143] Table 5 shows that the reconstitution time of SF freeze-dried samples 1-5 was short, the moisture content was <5%, and almost no collapse was observed after 24 hours at room temperature, indicating good stability.

Claims

1. A silk fibroin composition for injection, characterized by A composition comprising hyaluronic acid or a salt thereof (preferably hyaluronic acid or sodium hyaluronate, more preferably hyaluronic acid), and silk fibroin, wherein the content of the hyaluronic acid or a salt thereof is 0.01-10 wt%, and the content of the silk fibroin is 0.01-20 wt%, based on the total mass of the composition.

2. The silk fibroin composition according to claim 1, wherein The content of the hyaluronic acid or a salt thereof is 0.05-5 wt%, preferably 0.1-1 wt%, more preferably 0.1-0.7 wt%, more preferably 0.3-0.7 wt%, more preferably 0.4-0.6 wt%, most preferably 0.5 wt%, based on the total mass of the composition.

3. The silk fibroin composition according to claim 1 or 2, wherein The content of the silk fibroin is 0.05-15 wt%, preferably 0.1-10 wt%, more preferably 0.1-1 wt%, more preferably 0.3-0.7 wt%, most preferably 0.5 wt%, based on the total mass of the composition.

4. The silk fibroin composition according to any one of claims 1 to 3, wherein The composition is a solution, a reconstitutable powder, an emulsion, an ointment, a gel, a paste or a suspension, preferably a solution or a reconstitutable powder, more preferably an aqueous solution, most preferably an aqueous solution for water-light injection.

5. The silk fibroin composition according to any one of claims 1 to 4, wherein The molecular weight of the hyaluronic acid or a salt thereof is 500-2000 kDa, preferably 1000-1800 kDa; and the molecular weight of the silk fibroin is 10-150 kDa, preferably 10-50 kDa.

6. The silk fibroin composition according to any one of claims 1 to 5, wherein The composition further comprises a buffer selected from one or several of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, preferably phosphate buffer.

7. The silk fibroin composition according to any one of claims 1 to 6, wherein The composition further comprises a stabilizer selected from one or several of sorbitol, mannitol, inositol, xylitol, urea peptide, sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium bicarbonate, sodium phosphate monobasic, potassium chloride, sodium chloride, potassium chloride.

8. The silk fibroin composition according to any one of claims 1 to 7, wherein The composition further comprises an antioxidant which is at least one of nicotinamide, vitamin C, tea polyphenol or vitamin E.

9. The silk fibroin composition according to any one of claims 1 to 8, wherein The composition comprises, based on the total mass of the composition: - 0.05-5 wt% of hyaluronic acid or a salt thereof, preferably 0.1-1 wt%, more preferably 0.1-0.7 wt%, more preferably 0.3-0.7 wt%, more preferably 0.4-0.6 wt%, most preferably 0.5 wt%; - 0.05-15 wt% of silk fibroin, preferably 0.1-10 wt%, more preferably 0.1-1 wt%, more preferably 0.3-0.7 wt%, most preferably 0.5 wt%; - the balance being phosphate buffer.

10. The silk fibroin composition according to any one of claims 1 to 9, wherein The composition has an osmolarity of 200-400 mOsmol / kg, preferably 280-350 mOsmol / kg; a kinematic viscosity of 10-1000 mPa·s, preferably 100-600 mPa·s, more preferably 150-300 mPa·s; and a push force of 8-20 N, preferably 10-15 N, more preferably 12-15 N.

11. The silk fibroin composition of any one of claims 1-10, wherein the composition is for injection into the face or the neck, preferably into the face.

12. The method of claim 1-11, wherein the silk fibroin composition is prepared by the steps of: comprising the steps of: 1) dissolving hyaluronic acid or a salt thereof in a phosphate buffer to obtain a hyaluronic acid solution; 2) dissolving fibroin in a phosphate buffer to obtain a fibroin solution; and 3) mixing the above hyaluronic acid solution and fibroin solution.

13. The method for preparing a fibroin composition according to claim 12, wherein after step 2) further comprising a step of freeze-drying the fibroin solution to obtain a fibroin lyophilized cake, preferably the fibroin lyophilized cake is sterilized by irradiation and stored at room temperature.

14. The method for preparing a fibroin composition according to claim 13, further comprising a step of reconstituting the fibroin lyophilized cake with the hyaluronic acid solution obtained in step 1), preferably after the reconstitution step the obtained solution is left to stand at room temperature for 3-10 min.

15. The method for preparing the silk fibroin composition according to claim 13 or 14, characterized in that, The conditions for the freeze-drying are: Pre-freezing temperature -50°C - 45°C, ramp time 0-60 min, holding time 120 min; Primary drying temperature -10°C - 0°C, ramp time 0-120 min, holding time 300-600 min, vacuum setting 0.3 mbar; Primary drying temperature -10°C - 0°C, ramp time 0-120 min, holding time 300-600 min, vacuum setting 0.3 mbar; Primary drying temperature -10°C - 0°C, ramp time 0-120 min, holding time 300-600 min, vacuum setting 0.3 mbar; 16. A kit comprising: (a) hyaluronic acid or a salt thereof, preferably hyaluronic acid or sodium hyaluronate, more preferably hyaluronic acid; and (b) fibroin, wherein the hyaluronic acid or a salt thereof and the fibroin are reconstituted into a composition for injection before use, and the content of the hyaluronic acid or a salt thereof is 0.01-10 wt%, preferably 0.05-5 wt%, preferably 0.1-1 wt%, more preferably 0.1-0.7 wt%, more preferably 0.3-0.7 wt%, more preferably 0.4-0.6 wt%, most preferably 0.5 wt%, based on the total mass of the composition; the content of the fibroin is 0.01-20 wt%, preferably 0.05-15 wt%, preferably 0.1-10 wt%, more preferably 0.1-1 wt%, more preferably 0.3-0.7 wt%, most preferably 0.5 wt%; preferably the composition for injection is a composition for facial or neck injection, more preferably a composition for facial injection.

17. The kit of claim 16, wherein The hyaluronic acid or a salt thereof and the fibroin are reconstituted into a solution, emulsion, ointment, gel, paste or suspension before use, preferably a solution, more preferably an aqueous solution, most preferably an aqueous solution for water-light injection.

18. The kit of claim 16 or 17, wherein The molecular weight of the hyaluronic acid or a salt thereof is 500-2000 kDa, preferably 1000-1800 kDa; the molecular weight of the fibroin is 10-150 kDa, preferably 10-50 kDa.

19. The kit of any one of claims 16-18, wherein, The kit further comprises a buffer selected from one or several of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, preferably a phosphate buffer.

20. The kit of any one of claims 16-19, wherein, The kit further comprises a stabilizer selected from one or several of sorbitol, mannitol, inositol, xylitol, urea peptide, sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium bicarbonate, sodium phosphate, potassium chloride, sodium chloride, potassium chloride.

21. The kit of any one of claims 16-20, wherein, The kit further comprises an antioxidant which is at least one of nicotinamide, vitamin C, tea polyphenols or vitamin E.

22. Use of the silk fibroin composition according to any one of claims 1-11 or the composition prepared according to the method of any one of claims 12-15 or the kit according to any one of claims 16-21 in the manufacture of a product for improving the skin condition of a subject, preferably the skin condition comprises dry skin, skin wrinkles, fine lines, skin laxity, skin roughness.

23. The use of claim 22, wherein, The improvement of the skin condition is manifested by at least one of: 1) increasing the level of collagen in the skin, preferably the collagen is collagen III (Collagen III); 2) reducing the level of excessive fibrosis of skin collagen a-SMA; 3) promoting the expression of skin aquaporin AQP3.

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