Silk fibroin / sodium hyaluronate composite microsphere gel, preparation method and application thereof

Abstract

The present application relates to the technical field of degradable high molecular biological material, and particularly relates to a silk fibroin / sodium hyaluronate composite microsphere gel, a preparation method and application thereof. The present application first prepares silk fibroin / sodium hyaluronate composite microspheres, then crosslinks the sodium hyaluronate gel, and further obtains the silk fibroin / sodium hyaluronate composite microsphere gel. The silk fibroin / sodium hyaluronate composite microsphere gel is regular spherical, and the particle size is controllable. The silk fibroin / sodium hyaluronate composite microsphere gel has stable structure, good mechanical properties, and is easy to be stored for a long time. The composite microspheres and the crosslinked gel have excellent structural stability, and the combination of the microspheres and the gel endows the material with good mechanical properties. The silk fibroin / sodium hyaluronate composite microsphere gel has slow degradation speed and excellent biocompatibility. The preparation method is simple, controllable and high in yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of degradable high-molecular biological materials, in particular to a silk fibroin / sodium hyaluronate composite microsphere gel and a preparation method and application thereof. BACKGROUND

[0002] Sodium hyaluronate is an inherent component in the dermis of human skin, has good biocompatibility and degradability, is an ideal natural moisturizing material, and has been widely used in the fields of biological medicine and tissue engineering. Silk fibroin also starts clinical application due to its excellent biocompatibility and low immunogenicity, and silk fibroin products are currently on the market, such as silk fibroin film dressing (national equipment registration 20203140593).

[0003] At present, many materials can be applied to skin fillers, such as sodium hyaluronate gel, polycaprolactone microspheres, poly-L-lactic acid microspheres, silk fibroin microspheres, etc., but various problems still need to be solved. For example, pure sodium hyaluronate gel is only suitable for skin tissue filling and moisturizing, and the material needs to be implanted again after rapid degradation. The microsphere product is either freeze-dried into powder and needs to be compounded for use, or the microsphere solution is easy to become unstable after sterilization. Silk fibroin microspheres as tissue filling materials can promote cell proliferation in the filling site and have the effect of nourishing tissues, but silk fibroin microspheres are easy to aggregate, precipitate and cannot be injected out of the injection needle after sterilization, and have poor heat resistance and stability, which affects product use. SUMMARY

[0004] Therefore, the present application aims to provide a preparation method and application of silk fibroin / sodium hyaluronate composite microsphere gel. The silk fibroin / sodium hyaluronate composite microsphere gel prepared by the present application has good heat resistance and structural stability, and a slow degradation rate, and can be applied to cosmetic, medical injection or gel dressing.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a preparation method of silk fibroin / sodium hyaluronate composite microsphere gel, comprising the following steps: mixing a sodium hyaluronate solution, a silk fibroin solution and an organic solvent, freezing and crystallizing the obtained mixed solution, and then thawing to obtain a silk fibroin / sodium hyaluronate composite emulsion;

[0007] Spray drying the silk fibroin / sodium hyaluronate composite emulsion to obtain silk fibroin / sodium hyaluronate composite microspheres;

[0008] Mixing the silk fibroin / sodium hyaluronate composite microspheres, sodium hyaluronate powder and alkali solution until the sodium hyaluronate powder is completely dissolved to obtain a dissolving solution; mixing the dissolving solution and a crosslinking agent to perform a crosslinking reaction to form a gel;

[0009] Placing the gel in water to swell, and then taking out to place in a buffer solution to perform shrinkage strengthening to obtain the silk fibroin / sodium hyaluronate composite microsphere gel.

[0010] Preferably, after obtaining the silk fibroin / sodium hyaluronate composite microsphere gel, the silk fibroin / sodium hyaluronate composite microsphere gel is sterilized to obtain a sterile silk fibroin / sodium hyaluronate composite microsphere gel.

[0011] Preferably, the mass ratio of silk fibroin and sodium hyaluronate in the mixed solution is (3-7):1.

[0012] Preferably, the temperature of the freeze crystallization is -80 to -15℃, and the freeze crystallization time is 12-48h.

[0013] Preferably, the spray drying conditions include: the inlet air temperature is 100-200℃, the feeding speed is 5-30mL / min, and the fan frequency is 10-25Hz.

[0014] Preferably, the mass ratio of the silk fibroin / sodium hyaluronate composite microspheres to the sodium hyaluronate powder is 1:(1.5-3).

[0015] The concentration of the silk fibroin / sodium hyaluronate composite microspheres in the dissolving solution is 20-50mg / mL.

[0016] The mass concentration of the alkali in the alkali solution is 0.01-2%.

[0017] The alkali in the alkali solution is sodium hydroxide and / or potassium hydroxide.

[0018] Preferably, the crosslinking agent includes 1,4-butanediol diglycidyl ether; and the mass of the crosslinking agent is 0.1-5% of the mass of the dissolving solution.

[0019] The temperature of the crosslinking reaction is 37-65℃, and the time is 6-24h.

[0020] Preferably, the buffer solution is a phosphate buffer solution or a sodium chloride solution, and the osmotic pressure of the buffer solution is 300-400mOsmol / kg.

[0021] The present application provides a silk fibroin / sodium hyaluronate composite microsphere gel prepared by the preparation method described in the above scheme, which includes a sodium hyaluronate gel and silk fibroin / sodium hyaluronate composite microspheres distributed in the sodium hyaluronate gel.

[0022] The application provides application of the silk fibroin / sodium hyaluronate composite microsphere gel in preparation of an injection or a gel dressing for cosmetic and medical purposes.

[0023] The application provides a preparation method of a silk fibroin / sodium hyaluronate composite microsphere gel, which comprises the following steps: mixing a sodium hyaluronate solution, a silk fibroin solution and an organic solvent, freezing and crystallizing the obtained mixed solution, and then thawing to obtain a silk fibroin / sodium hyaluronate composite emulsion; performing spray drying on the silk fibroin / sodium hyaluronate composite emulsion to obtain silk fibroin / sodium hyaluronate composite microspheres; mixing the silk fibroin / sodium hyaluronate composite microspheres, sodium hyaluronate powder and an alkali solution until the sodium hyaluronate powder is completely dissolved to obtain a dissolved solution; mixing the dissolved solution and a crosslinking agent to perform a crosslinking reaction to form a gel; and placing the gel in pure water to perform swelling, and then taking out and placing in a buffer solution to perform shrinkage strengthening to obtain the silk fibroin / sodium hyaluronate composite microsphere gel.

[0024] The application first prepares silk fibroin / sodium hyaluronate composite microspheres, and then crosslinks the sodium hyaluronate gel to obtain the silk fibroin / sodium hyaluronate composite microsphere gel; the silk fibroin / sodium hyaluronate composite microspheres have higher stability and heat resistance than single pure substance microspheres; the microspheres containing the sodium hyaluronate component are also more easily uniformly dispersed into the sodium hyaluronate gel; the composite microspheres and the sodium hyaluronate are crosslinked through stable crosslinking points, so that even large particle size microspheres will not appear to be precipitated and aggregated in the system; the ball gel crosslinking system is more stable and has good mechanical properties, and is easy to be stored for a long time.

[0025] The silk fibroin / sodium hyaluronate composite microsphere gel of the application can still maintain good stability after high-temperature sterilization, does not appear to be precipitated and accumulated, is uniformly dispersed, and is easy to be injected as an injection.

[0026] The preparation method of the silk fibroin / sodium hyaluronate composite microsphere gel provided by the application only uses two high molecular materials of sodium hyaluronate and silk fibroin; the organic solvent, the alkali solution and the crosslinking agent and other auxiliary materials added in the production process are also effectively removed in the process; the prepared silk fibroin / sodium hyaluronate composite microsphere gel has good biocompatibility; and the silk fibroin is subjected to beta folding through freezing and crystallization, so that the in-vivo degradation rate is delayed.

[0027] The preparation method of the silk fibroin / sodium hyaluronate composite microsphere gel provided by the application adopts the method of material compounding, freezing and crystallization, spray drying and ball gel combination to form the silk fibroin / sodium hyaluronate composite microsphere gel, and has the advantages of simple process, strong controllability, high yield, and controllable particle size of the prepared composite microspheres. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A scanning electron microscope image of the silk fibroin / sodium hyaluronate composite microspheres prepared in Example 1;

[0029] Figure 2 A scanning electron microscope image of the silk fibroin / sodium hyaluronate composite microspheres prepared in Example 1, magnified 12,000 times;

[0030] Figure 3 A particle size distribution graph of the silk fibroin / sodium hyaluronate composite microspheres prepared in Example 1;

[0031] Figure 4 An enzyme degradation graph of the silk fibroin / sodium hyaluronate composite microsphere gel prepared in Example 2;

[0032] Figure 5 A stability test result graph of Example 3 (B) and Comparative Example (A). DETAILED DESCRIPTION

[0033] The present application provides a method for preparing a silk fibroin / sodium hyaluronate composite microsphere gel, comprising the steps of: mixing a sodium hyaluronate solution, a silk fibroin solution, and an organic solvent, freezing and crystallizing the resulting mixture, and then thawing to obtain a silk fibroin / sodium hyaluronate composite emulsion;

[0034] Spray drying the silk fibroin / sodium hyaluronate composite emulsion to obtain silk fibroin / sodium hyaluronate composite microspheres;

[0035] Mixing the silk fibroin / sodium hyaluronate composite microspheres, sodium hyaluronate powder, and alkali solution until the sodium hyaluronate powder is completely dissolved to obtain a dissolved solution; mixing the dissolved solution and a crosslinking agent to perform a crosslinking reaction to form a gel;

[0036] Placing the gel in water to swell, and then taking it out to place in a buffer solution to perform shrinkage reinforcement to obtain the silk fibroin / sodium hyaluronate composite microsphere gel.

[0037] In the present application, unless otherwise specified, the raw materials used are commercially available products well known in the art.

[0038] The present application mixes a sodium hyaluronate solution, a silk fibroin solution, and an organic solvent, freezes and crystallizes the resulting mixture, and then thaws to obtain a silk fibroin / sodium hyaluronate composite emulsion.

[0039] In the present application, the concentration of the sodium hyaluronate solution is preferably 1-10 mg / mL, and in specific embodiments, the concentration of the sodium hyaluronate solution can be 2 mg / mL, 4 mg / mL, 5 mg / mL, 8 mg / mL or 10 mg / mL; the average molecular weight of the sodium hyaluronate is preferably 1200-1500 kDa, and in specific embodiments, the average molecular weight of the sodium hyaluronate can be 1200 kDa, 1300 kDa, 1400 kDa or 1500 kDa. In the present application, the solvent of the sodium hyaluronate solution is water. Sodium hyaluronate is a glycosaminoglycan and an inherent component in the dermis of human skin, has good biocompatibility and biodegradability, and is an ideal natural moisturizing material. Its super water-locking effect can improve the water storage capacity of the skin and prevent skin aging, and its application in the fields of biological medicine, biological materials, functional foods and cosmetics has been widely recognized and welcomed.

[0040] In the present application, the concentration of the silk fibroin solution is preferably 5-50 mg / mL, and in specific embodiments, the concentration of the silk fibroin solution can be 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL; the average molecular weight of the silk fibroin is 20-200 kDa, and in specific embodiments, the average molecular weight of the silk fibroin can be 20 kDa, 50 kDa, 100 kDa, 150 kDa or 200 kDa. Silk fibroin is derived from silk and is a natural high molecular weight protein material, has excellent biocompatibility, good low immunogenicity, and can be biodegraded in vivo. Silk fibroin can be prepared into various forms of materials, implanted into the body to serve as tissue repair materials, and slowly degraded into amino acids to provide nutrition for cell growth, promote cell proliferation, and finally degraded into CO2 and H2O and discharged outside the body.

[0041] In the present application, the organic solvent is preferably an alcohol solvent, and the alcohol solvent preferably includes one or more of methanol, ethanol, propanol and isopropanol; the volume ratio of the total volume of the silk fibroin solution and sodium hyaluronate mixed solution to the volume of the organic solvent is preferably (2-25):1, and in specific embodiments, it can be 2:1, 5:1, 10:1, 15:1 or 20:1.

[0042] In the present application, the mixing is preferably: first mixing the sodium hyaluronate solution with the silk fibroin solution, then adding the organic solvent to obtain a mixed solution.

[0043] In the present application, the mass ratio of silk fibroin to sodium hyaluronate in the mixed solution is preferably (3-7):1, and in specific embodiments, it can be 3:1, 4:1, 5:1, 6:1 or 7:1.

[0044] In the present application, the temperature of the freeze crystallization is preferably -80 to -15℃, and the time of the freeze crystallization is preferably 12 to 48 hours; in specific embodiments, the temperature of the freeze crystallization can be -80℃, -70℃, -60℃, -50℃, -40℃, -30℃, -20℃ or -15℃; and the time of the freeze crystallization can be 12 hours, 24 hours, 28 hours, 36 hours or 48 hours. The present application makes the silk fibroin change in the beta pleated sheet conformation through freeze crystallization, and wraps the sodium hyaluronate in the formed particles. By controlling the temperature and time of the freeze crystallization, the present application can control the beta pleated sheet degree of the silk fibroin, so as to achieve the purpose of regulating the degradation rate.

[0045] In addition, the present application can also adjust the particle size of the silk fibroin / sodium hyaluronate composite microspheres by regulating the volume ratio of the mixed solution of the silk fibroin and the sodium hyaluronate to the organic solvent and controlling the freezing temperature.

[0046] In the present application, the thawing preferably comprises: first placing at -4 to 25℃ for 6 to 24 hours, and if not completely thawed, continuing to place at room temperature until completely thawed.

[0047] After obtaining the silk fibroin / sodium hyaluronate composite emulsion, the present application sprays and dries the silk fibroin / sodium hyaluronate composite emulsion to obtain the silk fibroin / sodium hyaluronate composite microspheres.

[0048] Before the spray drying, the present application preferably dilutes the silk fibroin / sodium hyaluronate composite emulsion with pure water, and the dilution ratio is preferably 1 to 10 times, and in specific embodiments, the dilution ratio can be 2 times, 4 times, 5 times, 7 times or 10 times, which will not be listed here.

[0049] In the present application, the conditions of the spray drying comprise: the inlet air temperature is preferably 100 to 200℃, the feeding speed is preferably 5 to 30 mL / min, and the fan frequency is preferably 10 to 25 Hz; in specific embodiments, the inlet air temperature can be 100℃, 120℃, 140℃, 160℃, 180℃ or 200℃; the feeding speed can be 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min or 30 mL / min; and the fan frequency can be 10 Hz, 15 Hz, 20 Hz or 25 Hz.

[0050] In the present application, the particle size of the silk fibroin / sodium hyaluronate composite microspheres is preferably 100 nm to 10 μm.

[0051] After obtaining the silk fibroin / sodium hyaluronate composite microspheres, the silk fibroin / sodium hyaluronate composite microspheres, sodium hyaluronate powder and alkali liquor are mixed until the sodium hyaluronate powder is completely dissolved to obtain a dissolved liquor; the dissolved liquor and a crosslinking agent are mixed to perform crosslinking reaction to form a gel.

[0052] In the present application, the mass ratio of the silk fibroin / sodium hyaluronate composite microspheres to the sodium hyaluronate powder is preferably 1:(1.5-3), and in specific embodiments, the mass ratio of the silk fibroin / sodium hyaluronate composite microspheres to the sodium hyaluronate powder can be 1:1.5, 1:1.67, 1:2, 1:2.5, 1:2.67 or 1:3.

[0053] In the present application, the alkali in the alkali liquor is preferably sodium hydroxide and / or potassium hydroxide; and the mass concentration of the alkali in the alkali liquor is preferably 0.01-2%, and in specific embodiments, the mass concentration of the alkali in the alkali liquor can be 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 1.5% or 2%.

[0054] In the present application, the alkali liquor is used to dissolve the sodium hyaluronate powder, and the silk fibroin / sodium hyaluronate composite microspheres are not dissolved.

[0055] In the present application, the concentration of the silk fibroin / sodium hyaluronate composite microspheres in the dissolved liquor is preferably 20-50 mg / mL, and in specific embodiments, can be 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL or 50 mg / mL.

[0056] In the present application, the crosslinking agent preferably includes 1,4-butanediol diglycidyl ether; and the mass of the crosslinking agent is preferably 0.1-5% of the mass of the dissolved liquor, and in specific embodiments, the mass of the crosslinking agent can be 0.1%, 0.5%, 1.0%, 1.5%, 2%, 3%, 3.5%, 4.0%, 4.5% or 5% of the mass of the dissolved liquor.

[0057] In the present application, the temperature of the crosslinking reaction is preferably 37-65°C, and the time is preferably 6-24 h; and in specific embodiments, the temperature of the crosslinking reaction can be 37°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C; and the time of the crosslinking reaction can be 6 h, 10 h, 15 h, 20 h or 24 h.

[0058] In the present application, during the crosslinking reaction, the hydroxyl groups between the sodium hyaluronate in the silk fibroin / sodium hyaluronate composite microspheres and the sodium hyaluronate molecules added later are crosslinked to form network substances.

[0059] After obtaining the gel, the gel is placed in water for swelling, and then taken out to be placed in a buffer for shrinkage reinforcement to obtain the silk fibroin / sodium hyaluronate composite microsphere gel.

[0060] In the present application, the water is preferably pure water; the number of swelling is preferably 2-4, more preferably 3; the time of each swelling is preferably 3-15h, and in specific embodiments, the time of each swelling can be 3h, 5h, 8h, 10h, 12h or 15h; the mass of water used for each swelling is preferably 20-50 times the mass of the gel, and in specific embodiments, can be 20 times, 30 times, 40 times or 50 times. The present application can improve the performance of the gel by swelling, to obtain better elasticity and toughness, and the silk fibroin / sodium hyaluronate composite microsphere gel after swelling has a reduced swelling degree, which can delay the degradation rate when used.

[0061] In the present application, the buffer is preferably a phosphate buffer or a sodium chloride solution, and the osmotic pressure of the buffer is preferably 300-400mOsmol / kg, and in specific embodiments, the osmotic pressure of the buffer can be 300mOsmol / kg, 320mOsmol / kg, 350mOsmol / kg, 370mOsmol / kg or 400mOsmol / kg. In the present application, the number of shrinkage reinforcement is preferably 2-4, more preferably 3; the time of each shrinkage reinforcement is preferably 6-15h, and in specific embodiments, the time of each shrinkage reinforcement can be 6h, 8h, 10h, 12h or 15h. The mass of buffer used for each shrinkage reinforcement is preferably 15-30 times the mass of the gel, and in specific embodiments, can be 15 times, 20 times, 25 times or 30 times. The present application increases the osmotic pressure of the gel by shrinkage reinforcement, to prevent swelling when implanted in the human body.

[0062] The silk fibroin / sodium hyaluronate composite microsphere gel after shrinkage reinforcement is preferably sieved into small gel particles, and then filled into pre-filled syringes for use. In the present application, the mesh size of the sieve used for sieving is preferably 50-300μm, and in specific embodiments, can be 50μm, 100μm, 150μm, 200μm, 250μm, 250μm or 300μm.

[0063] In the present application, the silk fibroin / sodium hyaluronate composite microsphere gel obtained after the above steps is not subjected to sterilization treatment, and the present application preferably further comprises sterilizing the silk fibroin / sodium hyaluronate composite microsphere gel to obtain sterile silk fibroin / sodium hyaluronate composite microsphere gel.

[0064] In the present application, the sterilization is preferably moist heat sterilization, the sterilization temperature is preferably 110-135℃, and the sterilization time is preferably 8-35min.

[0065] The application provides a preparation method of silk fibroin / sodium hyaluronate composite microsphere gel.

[0066] The application provides the silk fibroin / sodium hyaluronate composite microsphere gel prepared by the preparation method, which comprises a sodium hyaluronate gel and silk fibroin / sodium hyaluronate composite microspheres distributed in the sodium hyaluronate gel.

[0067] The silk fibroin / sodium hyaluronate composite microspheres have higher stability and heat resistance than single pure material microspheres, and the microspheres containing the sodium hyaluronate component are more easily uniformly dispersed into the sodium hyaluronate gel.

[0068] The silk fibroin / sodium hyaluronate composite microsphere gel of the application can maintain good stability after high-temperature sterilization, does not appear to be precipitated and accumulated, is uniformly dispersed, and is easy to be injected as an injection.

[0069] The application provides application of the silk fibroin / sodium hyaluronate composite microsphere gel in preparation of an injection or a gel dressing for cosmetic and medical purposes.

[0070] The silk fibroin / sodium hyaluronate composite microsphere gel, the preparation method and the application thereof provided by the application are described in detail in combination with examples, but they cannot be understood as limitations to the protection scope of the application.

[0071] Example 1

[0072] The preparation of the silk fibroin / sodium hyaluronate composite microspheres is as follows:

[0073] (1) Preparation of silk fibroin / sodium hyaluronate composite emulsion:

[0074] A mixed solution of 500 mL of silk fibroin (average molecular weight of about 150 kDa) and sodium hyaluronate (average molecular weight of about 1200 kDa) (the mixed solution contains 15 g of silk fibroin and 2.5 g of sodium hyaluronate) was prepared, and then 50 mL of an ethanol solution was added. After mixing, the mixture was placed in a -23 °C environment for 24 h of freezing crystallization. After the freezing treatment, the mixture was treated at 0 °C for 24 h, and then thawed at room temperature to obtain a milky white silk fibroin / sodium hyaluronate composite emulsion.

[0075] (2) Preparation of silk fibroin / sodium hyaluronate composite microspheres:

[0076] The composite emulsion was diluted 4 times with pure water, and then composite microspheres were prepared by a spray dryer (the spray drying parameters were: inlet air temperature of 150 °C, feed liquid feeding speed of 10 mL / min, and fan frequency of 15 Hz). The high temperature can effectively remove the organic solvent ethanol after dilution, and pure and dry silk fibroin / sodium hyaluronate composite microspheres were collected.

[0077] Microscopic morphology characterization of silk fibroin / sodium hyaluronate composite microspheres:

[0078] The silk fibroin / sodium hyaluronate composite microspheres prepared in step (2) of Example 1 were observed by scanning electron microscopy. The overall situation of the microspheres could be clearly observed, the microspheres were round, the outline was regular and clear, the boundaries between the microspheres were obvious, and the microspheres were uniform (see Figure 1 ). The microspheres were observed at a magnification of about 10,000 times (see Figure 2 ), and the surface of the microspheres was regular without edges and corners.

[0079] Particle size analysis of silk fibroin / sodium hyaluronate composite microspheres:

[0080] The silk fibroin / sodium hyaluronate composite microspheres prepared in step (2) of Example 1 were dispersed with pure water, and then particle size analysis was performed by a laser particle size analyzer. The light intensity of the microspheres with a particle size of 1-6 μm accounted for about 83%, and details are shown in Figure 3 .

[0081] (3) Preparation of silk fibroin / sodium hyaluronate composite microsphere gel:

[0082] The silk fibroin / sodium hyaluronate composite microspheres and sodium hyaluronate powder were dissolved in a 0.5% sodium hydroxide solution (the concentration of the silk fibroin / sodium hyaluronate composite microspheres was 20 mg / mL, and the concentration of the sodium hyaluronate was 50 mg / mL), to obtain a dissolving solution, then 3 wt% of 1,4-butanediol diglycidyl ether was added to the dissolving solution, the solution was stirred uniformly and then placed in a constant temperature environment at 55°C for crosslinking reaction for 8 h, to finally form a gel. The gel was placed in pure water for swelling for 3 times, the amount of pure water used was 40 times the mass of the gel each time, and the swelling time was 6 h each time; then the swollen gel was placed in a sodium chloride solution with an osmotic pressure of 330 mOsmol / kg for shrinkage strengthening for 3 times, the amount of the sodium chloride solution used was 40 times the mass of the gel each time, and the strengthening time was 10 h each time.

[0083] (4) Preparation of sterile silk fibroin / sodium hyaluronate composite microsphere gel:

[0084] The silk fibroin / sodium hyaluronate composite microsphere gel after strengthening was sieved by using a sieve with a mesh size of 20 μm, then was filled into pre-filled syringes by using a filling device, then was subjected to moist heat sterilization at 121°C for 12 min, to obtain a sterile silk fibroin / sodium hyaluronate composite microsphere gel.

[0085] Example 2

[0086] The silk fibroin / sodium hyaluronate composite microsphere gel was prepared as follows:

[0087] (1) Preparation of silk fibroin / sodium hyaluronate composite emulsion:

[0088] A mixed solution of silk fibroin (average molecular weight about 20 kDa) and sodium hyaluronate (average molecular weight about 1400 kDa) was prepared, 500 mL (the mixed solution contained 10 g of silk fibroin and 2 g of sodium hyaluronate), then 100 mL of a mixed solution of ethanol and isopropyl alcohol (volume ratio of ethanol to isopropyl alcohol was 1:1) was added, the mixture was uniformly mixed and then was placed in a-20°C environment for freezing crystallization for 48 h. After the freezing treatment was completed, the mixture was treated at 25°C for 8 h, and at the same time was thawed to obtain a milky white silk fibroin / sodium hyaluronate composite emulsion.

[0089] (2) Preparation of silk fibroin / sodium hyaluronate composite microspheres:

[0090] The composite emulsion was diluted 7 times with pure water, then was subjected to high-temperature removal of organic solvents by spray drying (the spray drying parameters were: inlet air temperature 150°C, feed rate of the solution 10 mL / min, and frequency of the air blower 20 Hz), and the pure and dry silk fibroin / sodium hyaluronate composite microspheres were collected.

[0091] (3) Preparation of silk fibroin / sodium hyaluronate composite microsphere gel:

[0092] Silk fibroin / sodium hyaluronate composite microspheres and sodium hyaluronate powder were dissolved in a 0.5% sodium hydroxide solution (the concentration of silk fibroin / sodium hyaluronate composite microspheres was 35 mg / mL and the concentration of sodium hyaluronate was 70 mg / mL) to obtain a solution. Then, 2 wt% of 1,4-butanediol diglycidyl ether was added to the solution. After stirring the solution evenly, it was placed in a constant temperature environment of 40℃ for cross-linking reaction for 8 hours, and finally a gel was formed.

[0093] Swelling properties of silk fibroin / sodium hyaluronate composite microsphere gel:

[0094] The silk fibroin microspheres / sodium hyaluronate composite microsphere gel was pre-weighed and recorded as W1. The material was then completely immersed in pure water and kept in a water bath at 37°C for 24 hours. The material was then removed, rinsed three times with pure water, and the remaining moisture on the surface of the gel was removed with absorbent kitchen paper. The weight was then recorded as W2.

[0095] The swelling ratio was calculated using N = (W2 - W1) / W1 × 100%, and the calculated swelling ratio of the gel was 36.6 ± 5.1%, which is relatively low compared to pure sodium hyaluronate gel.

[0096] Degradation properties of silk fibroin / sodium hyaluronate composite microsphere gel:

[0097] Silk fibroin / sodium hyaluronate composite microsphere gels were swollen in PBS solution for 12 hours, and the wet weight W1 after removing surface moisture was measured. The swollen material was then sterilized by moist heat at 121℃ for 8 minutes, followed by the addition of 2 U / mL type I collagenase solution and PBS buffer (pH 7.4). The samples were then incubated at 37℃ for degradation. Samples were taken at 2h, 4h, 8h, 16h, 24h, 72h, 96h, 120h, and 144h, and the wet weight W2 after removing surface liquid was measured. The degradation solution was changed daily.

[0098] The remaining mass ratio was calculated using the formula W2 / W1×100%, and the results are shown below. Figure 4 ,from Figure 4 As can be seen, the degradation rate of the gel under the action of type I collagenase is higher than that in PBS, indicating that the silk fibroin microsphere / sodium hyaluronate composite microsphere gel material is biodegradable.

[0099] The gel was swollen twice in pure water, with the amount of pure water being 35 times the mass of the gel each time, and the swelling time being 5 hours each time. Then, the swollen gel was placed in phosphate buffer (PBS solution) with an osmotic pressure of 300 mOsmol / kg and shrunk three times, with the amount of PBS solution being 35 times the mass of the gel each time, and the shrunk time being 8 hours each time.

[0100] (4) Preparation of sterile silk fibroin / sodium hyaluronate composite microsphere gel:

[0101] After the strengthening is completed, the silk fibroin / sodium hyaluronate composite microsphere gel is sieved with a sieve with a pore size of 50 μm, then filled into pre-filled syringes through a filling device, then 121℃ moist heat sterilized for 8 min, to obtain sterile silk fibroin / sodium hyaluronate composite microsphere gel.

[0102] Example 3

[0103] (1) Preparation of silk fibroin / sodium hyaluronate composite emulsion:

[0104] A mixed solution of silk fibroin (average molecular weight 100 kDa) and sodium hyaluronate (average molecular weight about 1500 kDa) 500 mL (the mixed solution contains 20 g of silk fibroin and 4 g of sodium hyaluronate) is prepared, then 80 mL of a mixed solution of propanol and isopropanol is added, and after mixing uniformly, it is placed in a-30℃ environment for freezing crystallization for 48 h. After the freezing treatment is completed, it is annealed at-2℃ for 12 h, then thawed at room temperature to obtain a milky white silk fibroin / sodium hyaluronate composite emulsion.

[0105] (2) Preparation of silk fibroin / sodium hyaluronate composite microspheres:

[0106] The composite emulsion is diluted 6 times with pure water, then high-temperature removal of organic solvents is carried out by spray drying (spray drying parameters: inlet air temperature 180℃, feed liquid feeding speed 20 mL / min, fan frequency 20 Hz), and the pure and dry silk fibroin / sodium hyaluronate composite microspheres are collected.

[0107] (3) Preparation of silk fibroin / sodium hyaluronate composite microsphere gel:

[0108] The silk fibroin / sodium hyaluronate composite microspheres and sodium hyaluronate powder are dissolved in a 0.5% sodium hydroxide solution (wherein the concentration of silk fibroin / sodium hyaluronate composite microspheres is 20 mg / mL and the concentration of sodium hyaluronate is 50 mg / mL), to obtain a dissolved feed liquid, then 1 wt% of 1,4-butanediol diglycidyl ether is added to the dissolved feed liquid, and after the solution is stirred uniformly, it is placed in a constant temperature environment of 55℃ for crosslinking reaction for 10 h, to finally form a gel. The gel is placed in pure water for swelling 3 times, the amount of pure water used each time is 40 times the mass of the gel, and the swelling time each time is 6 h; then the swollen gel is placed in a 350 mOsmol / kg sodium chloride solution for shrinkage strengthening 3 times, the amount of sodium chloride solution used each time is 30 times the mass of the gel, and the strengthening time each time is 10 h.

[0109] (4) Preparation of sterile silk fibroin / sodium hyaluronate composite microsphere gel:

[0110] After the strengthening is completed, the silk fibroin / hyaluronic acid sodium composite microsphere gel is screened with a screen with a pore size of 20 μm, then filled into a pre-filled syringe through a filling device, then sterilized by wet heat at 121 °C for 8 min, to obtain sterile silk fibroin microsphere / hyaluronic acid sodium composite microsphere gel.

[0111] Stability test of sterile silk fibroin / hyaluronic acid sodium composite microsphere gel:

[0112] The sterilized product is subjected to centrifugal test (centrifugal force is 4500g, centrifugal time is 10 minutes), and no product layering phenomenon is observed (see Figure 5 B) in the middle.

[0113] Referring to the test method of the push force in YY / T 0962-2021 "Sodium Hyaluronate for Plastic Surgery", the push force of the product filled into the syringe is tested by a medical packaging physical performance tester to understand the actual use of the composite microsphere gel. Through the test, it is found that the maximum value of the push force when the push is stable is 20.35N, and the minimum value is 16.87N, the difference between the maximum value and the minimum value is very small, indicating that the push process is stable, and the average push force value is about 18N.

[0114] Comparative example

[0115] The silk fibroin / hyaluronic acid sodium composite microspheres prepared in step (2) of Example 3 are compounded with non-crosslinked hyaluronic acid sodium solution, the osmotic pressure of the non-crosslinked hyaluronic acid sodium is 350 mOsmol / kg, and the osmotic pressure regulator is sodium chloride. The composition of the product after compounding is that the concentration of silk fibroin / hyaluronic acid sodium composite microspheres is 10 mg / mL, and the concentration of hyaluronic acid sodium is 12 mg / mL. Screened with a screen with a pore size of 20 μm, then filled into a pre-filled syringe through a filling device, after sterilization by wet heat at 121 °C for 8 min, centrifugal test (centrifugal force is 4500g, centrifugal time is 10 minutes), the product can be seen. Obvious layering phenomenon (see Figure 5 A in the middle, the arrow points to). It is shown that crosslinking of silk fibroin / hyaluronic acid sodium composite microspheres with hyaluronic acid sodium gel can improve the stability of the system.

[0116] Example 4

[0117] (1) Preparation of silk fibroin / hyaluronic acid sodium composite emulsion:

[0118] A mixed solution of 500 mL of fibroin (average molecular weight 100 kDa) and sodium hyaluronate (average molecular weight about 1500 kDa) (the mixed solution contains 15 g of fibroin and 4 g of sodium hyaluronate) was prepared, and then 30 mL of a mixed solution of methanol and ethanol was added. After mixing, the solution was frozen and crystallized at -35°C for 36 h. After the freezing process, the solution was annealed at -4°C for 8 h, and then thawed at room temperature to obtain a milky white fibroin / sodium hyaluronate composite emulsion.

[0119] (2) Preparation of fibroin / sodium hyaluronate composite microspheres:

[0120] The composite emulsion was diluted four times with pure water, and then high-temperature methanol and ethanol were removed by spray drying (inlet temperature 180°C, feed rate 15 mL / min, and fan frequency 20 Hz) to obtain pure and dry fibroin / sodium hyaluronate composite microspheres.

[0121] (3) Preparation of fibroin / sodium hyaluronate composite microsphere gel:

[0122] The fibroin / sodium hyaluronate composite microspheres and sodium hyaluronate powder were dissolved in a 1% sodium hydroxide solution (the concentration of the fibroin / sodium hyaluronate composite microspheres was 30 mg / mL, and the concentration of the sodium hyaluronate was 50 mg / mL) to obtain a dissolved solution. Then, 0.5 wt% of 1,4-butanediol diglycidyl ether was added to the solution, which was stirred uniformly and then placed in a 37°C constant-temperature environment for crosslinking reaction for 24 h to form a gel. The gel was swelled in pure water three times, with the amount of pure water being 30 times the mass of the gel each time, and the swelling time being 6 h each time. Then, the swelled gel was placed in a 300 mOsmol / kg sodium chloride solution for shrinkage strengthening three times, with the amount of the sodium chloride solution being 30 times the mass of the gel each time, and the strengthening time being 10 h each time.

[0123] (4) Preparation of sterile fibroin / sodium hyaluronate composite microsphere gel:

[0124] The strengthened fibroin / sodium hyaluronate composite microsphere gel was sieved through a sieve with a pore size of 50 μm, and then filled into pre-filled syringes through a filling device. The syringes were subjected to moist heat sterilization at 115°C for 30 min to obtain sterile fibroin / sodium hyaluronate composite microsphere gel.

[0125] Example 5

[0126] (1) Preparation of fibroin / sodium hyaluronate composite emulsion:

[0127] A mixed solution of 500 mL of silk fibroin (average molecular weight 100 kDa) and sodium hyaluronate (average molecular weight about 1200 kDa) (the mixed solution contains 15 g of silk fibroin and 2.5 g of sodium hyaluronate) was prepared, and then 50 mL of ethanol was added. After mixing, the mixture was frozen and crystallized at -20 °C for 48 h. After the freezing treatment, the mixture was annealed at 0 °C for 18 h, and then thawed at room temperature to obtain a white silk fibroin / sodium hyaluronate composite emulsion.

[0128] (2) Preparation of silk fibroin / sodium hyaluronate composite microspheres:

[0129] The composite emulsion was diluted 5 times with pure water, and then high-temperature ethanol removal was performed by spray drying (inlet temperature 200 °C, feed rate 20 mL / min, and fan frequency 25 Hz). The pure and dry silk fibroin / sodium hyaluronate composite microspheres were collected.

[0130] (3) Preparation of silk fibroin / sodium hyaluronate composite microsphere gel:

[0131] The silk fibroin / sodium hyaluronate composite microspheres and sodium hyaluronate powder were dissolved in a 1% sodium hydroxide solution (the concentration of the silk fibroin / sodium hyaluronate composite microspheres was 30 mg / mL, and the concentration of the sodium hyaluronate was 80 mg / mL), and then 2% 1,4-butanediol diglycidyl ether was added. After stirring, the solution was placed in a constant-temperature environment at 50 °C for 24 h to form a gel. The gel was swelled in pure water for 3 times, with the amount of pure water being 50 times the mass of the gel each time, and the swelling time being 15 h each time. Then, the swelled gel was placed in a 320 mOsmol / kg phosphate buffer solution for shrinkage strengthening for 3 times, with the amount of sodium chloride solution being 30 times the mass of the gel each time, and the strengthening time being 15 h each time.

[0132] (4) Preparation of sterile silk fibroin / sodium hyaluronate composite microsphere gel:

[0133] The strengthened silk fibroin / sodium hyaluronate composite microsphere gel was sieved through a 50 μm mesh, and then filled into pre-filled syringes through a filling device. After 121 °C moist heat sterilization for 8 min, a sterile silk fibroin / sodium hyaluronate composite microsphere gel was obtained.

[0134] Cell toxicity test of silk fibroin / sodium hyaluronate composite microsphere gel:

[0135] The test was performed according to the MTT method in Appendix C of GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test".

[0136] Sample preparation:

[0137] MEM complete medium: MEM medium without phenol red, containing 10% fetal bovine serum, 4 mmol / L glutamine, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0138] Sample group: The sterile silk fibroin / sodium hyaluronate composite microsphere gel of Example 5 was mixed with 0.9% sodium chloride injection at a volume ratio of 2:1, and the mixed solution was extracted with a medium containing serum at a ratio of 0.2 g / mL at (37±1) °C for (24±2) h to prepare the extract.

[0139] 100% test solution: 1 mL of the extract stock solution

[0140] 75% test solution: 0.75 mL of the extract stock solution + 0.25 mL of MEM complete medium

[0141] 50% test solution: 0.50 mL of the extract stock solution + 0.50 mL of MEM complete medium

[0142] 25% test solution: 0.25 mL of the extract stock solution + 0.75 mL of MEM complete medium

[0143] Positive control: 1 mL of 20% DMSO: 0.2 mL of DMSO + 0.8 mL of MEM complete medium.

[0144] Negative control: 1 mL of MEM complete medium.

[0145] Blank control: 2 mL of MEM complete medium, treated under the same extraction conditions.

[0146] Each group was placed in a 96-well plate with mouse fibroblasts, and each group had 6 parallel samples. The plates were incubated in a (37±1) °C incubator with 5% CO2. After 24 h, the morphological changes of the cells in each group were observed under a microscope, and then the cell survival rate of each test group (sample group, negative control group, positive control group) relative to the blank control group was determined by the MTT method.

[0147] The average absorbance and survival rate of each test group are shown in Table 1:

[0148] Table 1 Results of cytotoxicity test

[0149]

[0150]

[0151] Blank control OD 570b The average value of the optical density of the test sample was 1.108, and the average values of the left and right columns of blanks differed from the average value of all blanks by 1.828%, which met the acceptance criteria.

[0152] The negative control and positive control meet the expected response, and the test is valid.

[0153] The survival rate of the 50% test sample extract is higher than that of the 100% extract, and the test is valid.

[0154] The survival rate of the 100% test sample extract is 78.77%, which is greater than 70%, and the silk fibroin / sodium hyaluronate composite microsphere gel has no cytotoxicity.

[0155] Intradermal reaction test of silk fibroin / sodium hyaluronate composite microsphere gel:

[0156] According to the requirements of intradermal reaction recommended in GB / T 16886.10-2017 "Biological evaluation of medical devices Part 10: Cytotoxicity tests", the test sample of silk fibroin / sodium hyaluronate composite microsphere gel was tested for biological compatibility.

[0157] The test sample was extracted in 0.9% sodium chloride injection (SC) and cottonseed oil (CSO) to prepare the extract of the test sample. The preparation process is as follows: take the sterile silk fibroin / sodium hyaluronate composite microsphere gel of Example 5, according to the proportion of 0.2g / mL, the extraction medium is 0.9% sodium chloride injection and cottonseed oil respectively; (37℃±1)℃, (72±2)h to prepare the extract, and the control solution is prepared in the same way without adding the test sample. Inject the test sample extract and the control solution into the intradermal of the spine of the rabbit (for each extract) on both sides, the injection amount is 0.2mL per point. After injection, observe the reaction of each injection site at 24h, 48h, 72h, and score the tissue reaction of erythema and edema of each injection site according to Table 2. Calculate the difference between the average scores of intradermal reaction of the test sample and the corresponding solvent control, the results are shown in Table 3 and Table 4.

[0158] Table 2 Intradermal reaction score standard

[0159]

[0160] Table 3 Intradermal reaction observation table (SC group)

[0161]

[0162] Table 4 Intradermal reaction observation table (CSO group)

[0163]

[0164]

[0165] From Table 3 and Table 4, it can be seen that the intradermal reaction observation shows that all the test animals are in normal state. After the last observation, the difference between the final scores of the rabbit intradermal reaction of the 0.9% sodium chloride injection (SC) extract of the product is 0.3, and the difference between the final scores of the rabbit intradermal reaction of the cottonseed oil (CSO) extract is 0, both of which are not greater than 1.0. The test results meet the requirements, and the silk fibroin / sodium hyaluronate composite microsphere gel has no intradermal reaction.

[0166] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a silk fibroin / sodium hyaluronate composite microsphere gel, characterized in that, Includes the following steps: A sodium hyaluronate solution, a silk fibroin solution, and an organic solvent were mixed. The resulting mixture was then frozen to crystallize and thawed to obtain a silk fibroin / sodium hyaluronate composite emulsion. The silk fibroin / sodium hyaluronate composite emulsion was spray-dried to obtain silk fibroin / sodium hyaluronate composite microspheres. The silk fibroin / sodium hyaluronate composite microspheres, sodium hyaluronate powder, and alkaline solution are mixed until the sodium hyaluronate powder is completely dissolved to obtain a solution; the solution is then mixed with a crosslinking agent to carry out a crosslinking reaction and form a gel. The gel was placed in water to swell, and then removed and placed in a buffer solution for shrinkage reinforcement to obtain the silk fibroin / sodium hyaluronate composite microsphere gel.

2. The preparation method according to claim 1, characterized in that, After obtaining the silk fibroin / sodium hyaluronate composite microsphere gel, the process further includes sterilizing the silk fibroin / sodium hyaluronate composite microsphere gel to obtain a sterile silk fibroin / sodium hyaluronate composite microsphere gel.

3. The preparation method according to claim 1, characterized in that, The mass ratio of silk fibroin to sodium hyaluronate in the mixture is (3-7):

1.

4. The preparation method according to claim 1 or 3, characterized in that, The freezing crystallization temperature is -80 to -15°C, and the freezing crystallization time is 12 to 48 hours.

5. The preparation method according to claim 1, characterized in that, The spray drying conditions include: an inlet air temperature of 100–200°C, a feed rate of 5–30 mL / min, and a fan frequency of 10–25 Hz.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the silk fibroin / sodium hyaluronate composite microspheres to sodium hyaluronate powder is 1:(1.5-3); The concentration of silk fibroin / sodium hyaluronate composite microspheres in the dissolving solution is 20-50 mg / mL; The mass concentration of alkali in the alkaline solution is 0.01-2%; The alkali in the alkaline solution is sodium hydroxide and / or potassium hydroxide.

7. The preparation method according to claim 1, characterized in that, The crosslinking agent includes 1,4-butanediol diglycidyl ether; the mass of the crosslinking agent is 0.1-5% of the mass of the dissolved solution; The cross-linking reaction is carried out at a temperature of 37–65°C for a time of 6–24 hours.

8. The preparation method according to claim 1, characterized in that, The buffer solution is a phosphate buffer or a sodium chloride solution, and the osmotic pressure of the buffer solution is 300-400 mOsmol / kg.

9. The silk fibroin / sodium hyaluronate composite microsphere gel prepared by the preparation method according to any one of claims 1 to 8, characterized in that, It includes sodium hyaluronate gel and silk fibroin / sodium hyaluronate composite microspheres cross-linked and distributed in the sodium hyaluronate gel.

10. The use of the silk fibroin / sodium hyaluronate composite microsphere gel according to claim 9 in the preparation of injectable or gel dressings for cosmetic and medical purposes.

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