Silk fibroin composition for injection and preparation method therefor

Abstract

A silk fibroin composition for injection, a preparation method therefor, and a use thereof. The silk fibroin composition comprises the following components in percentage by mass: 0.1-0.7 wt % of hyaluronic acid or a salt thereof, 0.1-10 wt % of silk fibroin, and a balance being a phosphate buffer solution. The composition promotes collagen production and improves skin condition.

Description

Silk fibroin composition for injection and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medical cosmetology, and particularly relates to a silk fibroin composition for injection and a preparation method thereof. BACKGROUND

[0002] With the increase of age, the facial skin will appear "aging" problems, among which the loss of collagen and moisture is the most obvious, which is clinically manifested as wrinkles, sagging, roughness and the like. Dry skin is prone to fine lines, wrinkles and other signs of aging, and sufficient moisture helps to keep the skin smooth and firm. Collagen fibrosis is one of the causes of skin aging. Therefore, preventing collagen fibrosis, increasing collagen content and skin elasticity are crucial to improve skin aging.

[0003] At present, the preparation for facial water-light injection can cause excessive collagen, resulting in excessive collagen fiber proliferation, and adverse reactions such as skin hardening, papules or nodules, and is also not conducive to maintaining the balance of physiological osmotic pressure of the face, which will further affect the normal proliferation state of skin cells. Therefore, there is an urgent need in the market for a safe and effective product that can quickly improve the "aging" of facial skin. SUMMARY

[0004] The purpose of the present application is to provide a silk fibroin composition for injection and a preparation method thereof, which contains silk fibroin and hyaluronic acid, can quickly replenish skin moisture after injection, effectively promote skin collagen regeneration, and inhibit collagen fibrosis, thereby avoiding adverse reactions such as skin allergy and stiffness, and has good safety.

[0005] The technical solution adopted by the present application is as follows:

[0006] In one aspect, the present application provides a silk fibroin composition for injection, which comprises the following components by mass percentage: 0.01-10wt% of hyaluronic acid or a salt thereof, and 0.01-20wt% of silk fibroin.

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

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

[0009] Preferably, 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 needle.

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

[0011] As an optional way, 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.

[0012] As an optional way, the silk fibroin 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 dihydrogen phosphate, potassium chloride, sodium chloride, potassium chloride.

[0013] As an optional way, the silk fibroin composition further comprises an antioxidant, which is at least one of nicotinamide, vitamin C, tea polyphenol or vitamin E.

[0014] In one preferred technical solution, based on the total mass of the silk fibroin composition, the composition comprises:

[0015] - 0.05-5 wt% of hyaluronic acid or its salt, 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%;

[0016] - 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%;

[0017] - the balance is phosphate buffer.

[0018] Preferably, the silk fibroin 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.

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

[0020] In a second aspect, the present application provides a method for preparing the silk fibroin composition for injection according to the first aspect, comprising the following steps:

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

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

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

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

[0025] In a preferred embodiment, the method further comprises a step of reconstituting the silk fibroin freeze-dried block with the hyaluronic acid solution obtained in step 1), preferably, the resulting solution is left to stand at room temperature for 3-10 min after the reconstitution step.

[0026] Preferably, the freeze-drying conditions are: pre-freezing temperature -50℃ to -45℃, ramp time 0-60 min, holding time 120 min; primary drying temperature -10℃ to 0℃, ramp time 0-120 min, holding time 300-600 min, vacuum setting 0.3 mbar; secondary drying phase 1, temperature setting 0℃ to 15℃, ramp time 0-30 min, holding time 500-800 min, vacuum setting 0.1 mbar; secondary drying phase 2, 30℃ to 37℃, ramp time 30 min, holding time 200-400 min, vacuum setting 0.1 mbar; pressure rise test, qualified standard: pressure change ≤0.05 mbar in 5 min.

[0027] In a third aspect, the present application further provides a 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] wherein the hyaluronic acid or salt thereof and the fibroin are reconstituted into a composition for injection before use, and the content of the hyaluronic acid or 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.

[0031] In a preferred embodiment, the hyaluronic acid or salt thereof and the fibroin are reconstituted into a solution, emulsion, ointment, gel, paste or suspension, preferably a solution, more preferably an aqueous solution, most preferably an aqueous solution for water-light needle, before use.

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

[0033] In a preferred embodiment, 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 phosphate buffer.

[0034] In a preferred embodiment, 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 dihydrogen phosphate potassium chloride, sodium chloride, potassium chloride.

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

[0036] In a fourth aspect, the present application also provides use of the fibroin composition for injection of the first aspect in the preparation of a product for improving the skin condition of a subject, which includes dry skin, skin wrinkles, fine lines, skin laxity, skin roughness.

[0037] Preferably, the improvement of the skin condition is manifested as at least one of the following:

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

[0039] 2) reducing the level of alpha-SMA;

[0040] 3) promoting the expression of AQP3. Beneficial effects

[0041] The silk fibroin composition of the present application is used for facial dermis injection, which has the effects of quickly improving the dry state of the skin, quickly supplementing the moisture of the skin, lightening fine lines or wrinkles, and tightening the skin. The present application can improve the activity of fibroblasts, promote the proliferation of keratinocytes, repair the skin barrier, and promote the generation of collagen, especially collagen III.

[0042] The silk fibroin composition of the present application can reduce the level of alpha-SMA in the skin, inhibit collagen fibrosis, promote the expression of AQP3, and promote the transport of skin moisture, thereby achieving the effect of moisturizing.

[0043] The present application has good safety and will not cause adverse reactions such as skin hardening and redness, and the osmotic pressure meets the requirements, which is more in line with the physiological characteristics of the skin, and the skin state is obviously improved after injection.

[0044] Brief description of the drawings

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

[0046] Figure 2 is the total collagen content result of L929 cells treated with the silk fibroin composition of Example 2 after 14 days (compared with the CTRL group, ***P<0.001).

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

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

[0049] Fig. 5A is a Masson staining and PSR staining chart of the back skin histopathological section of each group of mice (compared with the CTRL group, *P < 0.05), in which the blue part in the Masson staining chart represents collagen, and the red part represents muscle fibers, and the red and yellow parts in the PSR chart represent type I collagen, and the green part represents type III collagen. Fig. 5B is a column chart showing the relative contents of type I and type III collagens in the PSR staining chart of Fig. 5A, in which the gray part represents type I collagen, and the pink part represents type III collagen.

[0050] Fig. 6A is an immunofluorescence (IF) staining detection chart of the back skin histopathological section of each group of mice. Fig. 6B is a column chart showing the AQP3 staining area in Fig. 6A.

[0051] DETAILED DESCRIPTION

[0052] DEFINITIONS

[0053] 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.

[0054] As used herein, the term "hyaluronic acid" is a biodegradable polymer component composed 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 synovial fluid of the eye and joints. HA is commercially available in pure form. Small gel particles of HA fillings 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.

[0055] 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-hydroxyethylsulfonate, 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 a free base, an 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 conventional procedures for making acid addition salts from base compounds. Those skilled in the art will appreciate that a variety of synthetic procedures can be used to prepare the nontoxic pharmaceutically acceptable addition salts.

[0056] 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 useful in human therapy and / or veterinary applications. 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 properties of a solution, colloid, solid, mixture. Stabilizers can slow reactions, maintain chemical equilibrium, reduce surface tension, prevent photolytic or oxidative decomposition, etc.

[0058] As used herein, the term "antioxidant" refers to a substance that is capable of preventing the adverse effects of oxygen, which should have a reduction potential lower than that of the active ingredient susceptible to oxidation in the preparation, thus being oxidized before the active ingredient, while itself being consumed, thereby maintaining the stability of the preparation.

[0059] As used herein, the term "water-light injection" refers to the injection of nutrients and drugs into specific layers of the skin through hollow microneedles, effectively supplementing various nutrients and stimulating collagen production, making the skin moist and shiny, effectively delaying skin aging, and improving skin quality.

[0060] As used herein, the term "kit" can include single or multiple doses of two or more agents, each packaged or formulated separately; or single or multiple doses of two or more agents 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

[0061] Collagen is the most abundant protein in the extracellular matrix. Collagen fibers play a crucial role in maintaining skin tension and elasticity. The loss of skin collagen with age leads to the loss of support structure of the skin, making facial skin more prone to sagging and drooping, and also prone to wrinkles and fine lines. Type I collagen accounts for the majority of the skin (80-85%), participates in the structure and integrity of the dermis layer of the skin, and maintains the skin network structure. Type III collagen accounts for a small proportion of the skin (10-15%), in addition to participating in the dermal structure, it can also provide tension, flexibility and softness to the skin by changing the diameter of collagen fibers. Type III collagen has the highest content in infancy and is known as the "golden collagen" of the skin. With age, type III collagen decreases irreversibly, leading to facial depression, decreased skin elasticity, and facial wrinkles. There have been a large number of reports on skin collagen supplementation or promotion, but excessive collagen supplementation can cause fibrosis, leading to a series of problems such as skin hardening and nodules.

[0062] Hyaluronic acid, also known as hyaluronic acid, is the main component of the intercellular matrix and extracellular matrix of skin cells, exists in the epidermis and dermis of the skin, has high viscoelasticity and plastic deformation, and can maintain skin moisture, fullness and elasticity. Clinically, non-surgical methods such as water light needle injection technology can be used. Under the control of a microcomputer, a one-time 5-hole, 9-hole or 13-hole needle is used to adsorb the skin through a negative pressure system, and at the same time, drugs are injected into the corresponding parts to stimulate and activate the skin surface cells, improve the roughness and wrinkle state of the skin, and achieve the effect of skin rejuvenation.

[0063] The main water supplement component of the hyaluronic acid sodium complex solution for injection used in the water light needle is sodium hyaluronate, in addition, it also contains L-carnosine, glycine, alanine, proline and vitamin B2. Glycine, alanine, proline and L-carnosine can promote the generation of collagen by skin fibroblasts, and vitamin B2 can promote the transformation of collagen into collagen fibers.

[0064] Silk fibroin is a structural protein extracted from natural silk, which is composed of 18 amino acids. Compared with other natural biopolymers, silk fibroin has good biocompatibility and biodegradability, low immunogenicity and excellent mechanical properties, and is a potential new natural medical material. Before the application date of the present application, there is no silk fibroin related product for facial injection on the market.

[0065] In view of the problems of the water light needle used in the field of clinical and medical cosmetology, such as poor effect of promoting collagen regeneration and poor safety, the present application first proposes a silk fibroin composition for injection.

[0066] Embodiment

[0067] The technical solutions of the present application will be described in detail below in conjunction with the embodiments, but those skilled in the art should understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0068] The material sources of the following examples are as follows:

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

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

[0071] Comparative Example 1: Hyaluronic acid sodium injection complex solution (trade name: Hybody), batch number 230426, manufacturer: Aimeike Technology Development Co., Ltd.

[0072] Example 1

[0073] A silk fibroin composition for facial injection, comprising the following components by mass percentage: hyaluronic acid 0.5 wt%, silk fibroin 0.1 wt%, and phosphate buffer in the remainder.

[0074] The preparation method is as follows:

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

[0076] 2) Weigh 0.01 g of silk fibroin (molecular weight 10-50 kDa) and add 1 mL of PBS. After stirring and dissolving, store at 4°C for 2-24 h.

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

[0078] Example 2

[0079] A silk fibroin composition for facial injection, comprising the following components by mass percentage: hyaluronic acid 0.5 wt%, silk fibroin 0.1 wt%, and phosphate buffer in the remainder.

[0080] The preparation method is as follows:

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

[0082] 2) According to the 2.5 mL specification, take 2.5 mL of the above hyaluronic acid solution into a test tube, seal with an aluminum cap, invert and mix, then wet heat sterilization.

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

[0084] The freeze-drying conditions are as follows: pre-freezing temperature -50°C, ramp time 60 min, holding time 120 min; primary drying temperature -10°C, ramp time 120 min, holding time 600 min, vacuum setting 0.3 mbar; analytical drying phase 1, temperature setting 15°C, ramp time 30 min, holding time 600 min, vacuum setting 0.1 mbar; analytical drying phase 2, 37°C, ramp time 30 min, holding time 400 min, vacuum setting 0.1 mbar; pressure rise test, qualified standard 5 min pressure change ≤0.05 mbar.

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

[0086] Example 3: Performance determination

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

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

[0089] Viscosity and push force detection of samples 1-13 and Comparative Example 1 (Hyskin) were carried out, and the experimental method is as follows:

[0090] 1. Kinematic viscosity

[0091] Viscosity parameters of the samples were determined using a Kinexus Lab+ rotational rheometer. The sample was equilibrated at room temperature for 1 h before determination, and the amount of sample for each test was 1-1.2 mL, with the sample filling the cone. The V001 program was selected, the temperature was 25±0.1℃, the shear rate determination range was 0.1-1 s-1, and the determination was performed according to the operating procedures of the Malvern rheometer, and the data at the corresponding frequencies of 0.1, 0.25, 0.5 and 1 s-1 were read. The results are shown in Table 1.

[0092] 2. Pushing force

[0093] The pushing force was determined by a universal material testing machine. The sample was filled into 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 the syringe was installed on the universal material testing machine (Jinan Zhongzheng ZDW-T100). The test parameters were set, equilibrated at room temperature for 1 h, determined at room temperature, and the pushing speed was set to 30 mm / min. Start the test and read the full-scale average force (mean ± SD). The results are shown in Table 1.

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

[0095] For facial injection, too high viscosity can cause the injection to be sticky and have poor flowability, poor absorption after injection, and easy to form small nodules under the skin; too low viscosity can cause short storage time under the skin, too fast absorption, and fast metabolism; too large pushing force can make injection difficult and not easy to inject into the dermis; and too small pushing force can make it difficult to determine the injection site by force, and can cause injection site errors.

[0096] Therefore, it is considered that the silk fibroin composition has good injectability when the pushing force is less than 15 N, and the kinematic viscosity is more than 700 mPa·s. When the pushing force is 10-15 N and the kinematic viscosity is 100-600 mPa·s, it is more suitable for facial injection.

[0097] 3. Osmotic pressure

[0098] An osmotic pressure tester (Tianjin Tianda Tianfa) was used. The instrument was calibrated using the 100 mOsmol / kg and 400 mOsmol / kg standard solutions provided by the manufacturer. The test sample solution was prepared. According to the instrument manual, first adjust the instrument zero point with a suitable amount of freshly boiled and cooled water, then calibrate the instrument with the two standard solutions, and then measure the osmotic pressure of 100 μL of sample 4, sample 7, sample 8, sample 13 and comparative example 1, respectively. The results are shown in Table 2.

[0099] Table 2 Osmotic pressure of the present application and comparative example 1 (mean ± SD)

[0100] From Table 2, it can be seen that the osmotic pressure of sample 4, sample 7, sample 8 and sample 13 is close to the normal osmotic pressure of skin (280-320 mOsm / kg), and the safety is better.

[0101] Example 4: Cell experiment

[0102] 1. Cell proliferation experiment

[0103] According to the method of Example 2, 1 mL of sterile hyaluronic acid solution, 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg of sterile freeze-dried silk fibroin powder were taken to prepare a silk fibroin composition with HA 0.5wt%, and SF content of 0.1wt%, 0.3wt%, 0.5wt%, 1wt%, 2wt%, 5wt%, and 10wt%, respectively. 1 mg, 3 mg, 5 mg, and 10 mg of sterile freeze-dried silk fibroin powder were dissolved in 1 ml of PBS buffer to prepare 0.1wt%, 0.3wt%, 0.5wt%, and 1wt% silk fibroin solutions, respectively.

[0104] Experimental method: logarithmic growth period mouse fibroblast L929 was digested and inoculated in a 24-well plate. The above silk fibroin composition of the application and Comparative Example 1 were placed in TRANSWELL chamber, and co-cultured for 6 days. The number and activity of fibroblasts were detected, and the classic CCK-8 method was used for determination. The results are shown in Figure 1.

[0105] The results show that compared with the normal group (CTRL group) cultured with only DMEM complete medium without adding other additives, the silk fibroin composition can promote the growth of fibroblasts, and is better than Comparative Example 1 (Hyaluronic acid); compared with the SF solution, the HA-SF water-light preparation has better proliferation-promoting effect at the same concentration.

[0106] 2. Total collagen and type III collagen detection

[0107] According to the method of Example 2, 1 mL of sterile hyaluronic acid solution, 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg of sterile freeze-dried silk fibroin powder were taken to prepare a silk fibroin composition with HA 0.5wt%, and SF content of 0.1wt%, 0.3wt%, 0.5wt%, 1wt%, 2wt%, 5wt%, and 10wt%, respectively. 1 mg, 3 mg, 5 mg, and 10 mg of sterile freeze-dried silk fibroin powder were dissolved in 1 ml of PBS buffer to prepare 0.1wt%, 0.3wt%, 0.5wt%, and 1wt% silk fibroin solutions, respectively.

[0108] Experimental method: select logarithmic growth period of mouse fibroblast L929 digestion, inoculated in 24-well plates, the above silk fibroin composition and comparative example 1 were placed in TRANSWELL chamber, co-cultured for 14 days, the total amount of collagen and type III collagen in the fibroblast supernatant was detected respectively. The determination method was carried out according to the ELISA kit method. The results are shown in Figures 2 and 3.

[0109] The results show that, compared with the normal group (CTRL group), the silk fibroin composition can promote the regeneration of collagen (see Figure 2), and is better than comparative example 1 (hyaline body). The silk fibroin composition with HA 0.5%, SF content of 0.3%, 0.5%, 1% can promote the production of type III collagen (see Figure 3); compared with the SF solution, the HA-SF water-light preparation has better effect on promoting collagen under the same concentration.

[0110] 3. Collagen fibrosis detection

[0111] Experimental method: select logarithmic growth period of mouse fibroblast L929 digestion, inoculated in 24-well plates, the silk fibroin composition (HA 0.5%, SF 0.3%) prepared according to the method of example 2 and comparative example 1 were placed in TRANSWELL chamber, co-cultured for 14 days, and the fibrosis degree in the fibroblast supernatant was detected. The expression of collagen fibrosis marker protein α-smooth muscle actin (α-SMA) was determined according to the ELISA kit method. The results are shown in Figure 4.

[0112] The results show that, compared with the normal group (CTRL group), the silk fibroin composition can significantly reduce the expression of α-SMA. The silk fibroin composition of the application may have the effect of inhibiting collagen fibrosis, thereby reducing adverse reactions and improving skin aging.

[0113] Example 5: animal experiment

[0114] Select 6-8 week old female ICR mice, cut off the hair on the back of each mouse, wipe the mouse skin with normal saline, the next day take 9 points in the 1x1 cm area of the exposed skin of the mouse, inject normal saline (normal group), silk fibroin composition of the application (prepared according to the method of example 2, HA concentration 0.5wt%, SF concentration 0.5wt%), comparative example 1 (hyaline body) and SF solution (concentration 0.5wt%) into the dermis layer respectively, each point injection about 0.03mL, each mouse injection 0.3mL. After 1 month, take the skin of the mouse back, make skin histopathological sections, and detect the expression of skin collagen by Masson staining and PSR staining, the results are shown in Figures 5A and 5B. Detect the expression of skin water-transporting related protein AQP3 by IF staining, the results are shown in Figures 6A and 6B.

[0115] The results of FIGS. 5A and 5B show that the collagen content and distribution density of the dermis layer indicated by arrows are significantly increased in the Example 2 and Comparative Example 1 groups compared with the normal group, indicating that the collagen content can be increased, but the collagen distribution in the Comparative Example 1 group is sparse, and the collagen distribution in the Example 2 group (silk fibroin composition HA 0.5wt%, SF 0.5wt%) is tight, indicating that the silk fibroin composition of the application can increase the content and density of skin collagen after injection.

[0116] The collagen types were identified by PSR staining. Under a polarizing microscope, type I collagen appears red or yellow, and type III collagen appears green. There are significantly more yellow fibers in the Comparative Example 1 group, indicating that it promotes the production of type I collagen to a greater extent, but the Example 2 group significantly promotes the production of type III collagen, which is consistent with the quantitative results of the column chart.

[0117] The results of FIGS. 6A and 6B show that the silk fibroin composition significantly promotes the expression of water transport-related protein AQP3 in the skin of mice compared with the normal group, which indicates that the silk fibroin composition can promote the transport of skin moisture and has a better moisturizing effect.

[0118] Example 6: Human skin experiment

[0119] The human experiment sample was prepared according to the preparation method of Example 2, and after compounding the sodium hyaluronate solution and the silk fibroin freeze-dried block, the solution contained 0.5wt% sodium hyaluronate and 0.5wt% silk fibroin. The control group was injected with normal saline. Electronic injection (hydroderma therapy) was used, and the injection process strictly followed the aseptic operation principle to avoid cross infection. Sterile nine-needle 32G injection needles were prepared in advance. Within 28 days after injection treatment, the facial skin of the subjects was scored for fine lines (Allergan fine line change scale), moisture, elasticity and skin quality (dermatoscope) at the follow-up time points, and the improvement of skin condition was evaluated, and the effectiveness was evaluated by global aesthetic improvement scale (GAIS), and the improvement efficiency was calculated.

[0120] Evaluation 1: Global Aesthetic Improvement Scale (GAIS)

[0121] The global aesthetic improvement scale of the face was divided into five levels, namely, deterioration, no effect, slight improvement, significant improvement and complete improvement, and the specific definitions are shown in Table 3.

[0122] Table 3: Global Aesthetic Improvement Scale (GAIS) Reference Table

[0123] Evaluation 2: Allergan Fine Line Severity Grading Score, see Table 4 for details.

[0124] Table 4: Allergan Fine Line Severity Grading Score Reference Table

[0125] Conclusion: The GAIS score of the test sample group was mostly 1 or 2 within 28 days after injection, indicating that the skin condition of most subjects was improved, the skin was more delicate, and the moisture measurement results showed that the skin water content was also improved, and the skin elasticity was also greatly improved. The Allergan fine line score of the secondary efficacy index of the subjects before injection ranged from 2 to 3, and the score improved by 1-2 degrees within 28 days after injection of the sample, indicating that the skin fine lines were reduced. The moisture and elasticity of the control group had no significant change, and the GAIS score and Allergan fine line score had no significant change before and after injection.

[0126] Example 7: Freeze-drying experiment

[0127] Silk fibroin freeze-dried sample 1, the freeze-drying conditions are: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; primary drying temperature rise includes -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; analytical drying one stage, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; analytical drying two stages 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure rise test, qualified standard 5min pressure change ≤0.05mbar.

[0128] Silk fibroin freeze-dried sample 2, the freeze-drying conditions are: pre-freezing temperature -45℃, ramp time 60min, holding time 120min; primary drying temperature rise includes -10℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; analytical drying one stage, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; analytical drying two stages 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure rise test, qualified standard 5min pressure change ≤0.05mbar.

[0129] Silk fibroin lyophilized sample 3, the conditions of the freeze-drying were: pre-freezing temperature -50°C, ramp time 60 min, holding time 120 min; primary drying ramp to include 0°C, ramp time 120 min, holding time 600 min, vacuum set 0.3 mbar; primary drying phase 1, temperature set 15°C, ramp time 30 min, holding time 600 min, vacuum set 0.1 mbar; primary drying phase 2 37°C, ramp time 30 min, holding time 400 min, vacuum set 0.1 mbar; pressure rise test, pass criteria 5 min pressure change < 0.05 mbar.

[0130] Silk fibroin lyophilized sample 4, the conditions of the freeze-drying were: pre-freezing temperature -50°C, ramp time 60 min, holding time 120 min; primary drying ramp to include -10°C, ramp time 120 min, holding time 600 min, vacuum set 0.3 mbar; primary drying phase 1, temperature set 0°C, ramp time 30 min, holding time 800 min, vacuum set 0.1 mbar; primary drying phase 2 37°C, ramp time 30 min, holding time 200 min, vacuum set 0.1 mbar; pressure rise test, pass criteria 5 min pressure change < 0.05 mbar.

[0131] Silk fibroin lyophilized sample 5, the conditions of the freeze-drying were: pre-freezing temperature -50°C, ramp time 60 min, holding time 120 min; primary drying ramp to include -10°C, ramp time 120 min, holding time 600 min, vacuum set 0.3 mbar; primary drying phase 1, temperature set 15°C, ramp time 30 min, holding time 600 min, vacuum set 0.1 mbar; primary drying phase 2 33°C, ramp time 30 min, holding time 400 min, vacuum set 0.1 mbar; pressure rise test, pass criteria 5 min pressure change < 0.05 mbar.

[0132] Silk fibroin lyophilized sample 6, the conditions of the freeze-drying were: pre-freezing temperature -50°C, ramp time 60 min, holding time 60 min; primary drying ramp to include -10°C, ramp time 120 min, holding time 600 min, vacuum set 0.3 mbar; primary drying phase 1, temperature set 15°C, ramp time 30 min, holding time 600 min, vacuum set 0.1 mbar; primary drying phase 2 37°C, ramp time 30 min, holding time 400 min, vacuum set 0.1 mbar; pressure rise test, pass criteria 5 min pressure change < 0.05 mbar.

[0133] Silk fibroin lyophilized sample 7, the freeze-drying conditions of the silk fibroin are: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; primary drying temperature ramp -20℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; primary drying stage 1, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; primary drying stage 2 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure rise test, qualified standard 5min pressure change ≤0.05mbar.

[0134] Silk fibroin lyophilized sample 8, the freeze-drying conditions of the silk fibroin are: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; primary drying temperature ramp -20℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; primary drying stage 1, temperature setting 15℃, ramp time 30min, holding time 200min, vacuum setting 0.1mbar; primary drying stage 2 37℃, ramp time 30min, holding time 400min, vacuum setting 0.1mbar; pressure rise test, qualified standard 5min pressure change ≤0.05mbar.

[0135] Silk fibroin lyophilized sample 9, the freeze-drying conditions of the silk fibroin are: pre-freezing temperature -50℃, ramp time 60min, holding time 120min; primary drying temperature ramp -20℃, ramp time 120min, holding time 600min, vacuum setting 0.3mbar; primary drying stage 1, temperature setting 15℃, ramp time 30min, holding time 600min, vacuum setting 0.1mbar; primary drying stage 2 45℃, ramp time 30min, holding time 100min, vacuum setting 0.1mbar; pressure rise test, qualified standard 5min pressure change ≤0.05mbar.

[0136] The silk fibroin lyophilized samples 1-9 were respectively reconstituted with 5% HA solution until completely clear and no debris, and the complete dissolution time was recorded. The original height of the lyophilized sample was recorded as H1, and the height after being taken out and placed at room temperature for 24h was recorded as H2, and the collapse degree = H2 / H1*100%. The moisture of silk fibroin lyophilized samples 1-9 was detected. The moisture determination was according to the pharmacopoeia method coulometric titration method, and a suitable Fehling's solution was added to the titration cup, and the moisture in the solution and system was pre-titrated first, then a suitable amount of test sample (containing about 0.5-5mg of water) was accurately taken and quickly transferred to the titration cup, or dissolved in a suitable anhydrous solvent, then quickly injected into the titration cup, and the end point was indicated by the permanent stop titration method (general rules of pharmacopoeia 0701), and the content of moisture in the test sample was directly read from the instrument display screen, wherein 1mg of water was equivalent to 10.72 coulomb electric quantity. The results are shown in Table 5.

[0137] Table 5 Reconstitution time and collapse degree of SF lyophilized samples under different lyophilization conditions

[0138] Table 5 shows that the reconstitution time of SF lyophilized samples 1-5 is shorter, the moisture content is <5%, and almost no collapse is observed after being placed at room temperature for 24h, and the stability is better.

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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