CN120617619A

CN120617619A - Silk fibroin gel and preparation method thereof

Info

Publication number: CN120617619A
Application number: CN202510854096.6A
Authority: CN
Inventors: 王祖元; 李秀娟; 崔云; 霍彩霞; 任恒春
Original assignee: Beijing Xinliyan Biotechnology Co ltd
Current assignee: Beijing Xinliyan Biotechnology Co ltd; Fuzhou Guoruikang Biotechnology Co.,Ltd.
Priority date: 2025-06-24
Filing date: 2025-06-24
Publication date: 2025-09-12

Abstract

The invention provides a silk fibroin gel and a preparation method thereof, and relates to the field of biological medicines, wherein the preparation method comprises the steps of (1) dissolving degummed silk or regenerated silk fibroin in a lithium bromide solution to obtain a silk fibroin solution, (2) adding a cross-linking agent and sodium hyaluronate into the silk fibroin solution, uniformly mixing, and then incubating to obtain the silk fibroin gel, wherein the dosage of sodium hyaluronate is 2% -14% of that of degummed silk or regenerated silk fibroin. The silk fibroin gel prepared by the invention has stable mechanical property, can be stored stably for a long time, and has excellent biocompatibility and filling property.

Description

Silk fibroin gel and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a silk fibroin gel and a preparation method thereof.

Background

With the gradual increase of age, the elasticity and smoothness of the skin gradually decrease, wrinkles are formed, and the surface gloss also gradually disappears. To improve this condition, soft tissue volume is often increased, wrinkles are eliminated and ameliorated using an injection fill formulation that is less traumatic, recovers quickly, and is relatively simple to handle. Common injectable soft tissue fillers include collagen, sodium hyaluronate, polymethyl methacrylate, calcium hydroxyapatite, poly-l-lactic acid, and the like. Collagen and sodium hyaluronate are used as fillers of biological sources, and have good biocompatibility, less complications, but high price, high absorptivity and short maintenance time. The synthetic filler such as polymethyl methacrylate, calcium hydroxyapatite and poly-L-lactic acid has lasting effect, but has poor biocompatibility, and can cause complications such as inflammatory infection and granuloma of foreign matters.

The silk fibroin is derived from silkworm cocoons, and has wide raw materials and low cost. The silk fibroin solution can be converted into gel by the existing chemical methods such as crosslinking or physical methods such as ultrasonic and shearing. The silk fibroin gel has excellent biocompatibility, can promote cell migration, collagen deposition and angiogenesis, and is an ideal material for preparing injection filling materials. However, the silk fibroin gel induced by the physical method has poor mechanical strength and poor filling effect after subcutaneous injection. The chemically crosslinked silk fibroin gel has excellent mechanical strength, can obtain excellent filling effect after being processed into gel particles by subcutaneous injection, and has great application potential in the medical field, however, the chemically crosslinked silk fibroin gel can become fragile gradually and the strength is reduced due to the change of molecular conformation in the storage process, so that the gel particles cannot bear the injection shearing force to break, and the filling effect is lost. Therefore, there is a need to provide a silk fibroin gel with stable mechanical properties and a preparation method thereof.

Disclosure of Invention

The embodiment of the invention provides a silk fibroin gel and a preparation method thereof, which solve the problem that the existing silk fibroin gel cannot maintain stable mechanical properties for a long time.

In a first aspect, the present invention provides a method for preparing a silk fibroin gel, the method comprising the steps of:

(1) Dissolving degummed silk or regenerated silk fibroin in a lithium bromide solution to obtain a silk fibroin solution;

(2) Adding a cross-linking agent and sodium hyaluronate into the silk fibroin solution, uniformly mixing, and then incubating to obtain silk fibroin gel, wherein the dosage of sodium hyaluronate is 2% -14% of that of degummed silk or regenerated silk fibroin.

Preferably, in the step (1), the mass ratio of the degummed silk or regenerated silk fibroin to the lithium bromide solution is 1 (5-20).

Preferably, in the step (1), the concentration of the lithium bromide solution is 6-13M.

More preferably, the concentration of the lithium bromide solution is 9-10M.

Preferably, in the step (1), the molecular weight of the degummed silk or regenerated silk fibroin is 15-250 kDa.

Preferably, the molecular weight of the degummed silk or regenerated silk fibroin is 50-150 kDa.

Preferably, the dosage of the sodium hyaluronate is 2% -10% of that of degummed silk or regenerated silk fibroin.

Preferably, in step (2), the cross-linking agent is butanediol diglycidyl ether and/or divinyl sulfone.

More preferably, the crosslinker is butanediol diglycidyl ether.

Preferably, in the step (2), the molecular weight of the sodium hyaluronate is 200-3000 kDa.

More preferably, the molecular weight of the sodium hyaluronate is 1000-2000 kDa.

Preferably, in the step (2), the amount of the cross-linking agent is 0.5% -25% of the amount of degummed silk or regenerated silk fibroin.

Preferably, in the step (2), the incubation temperature is 55-65 ℃ and the incubation time is 2-4 hours.

Preferably, after step (2), further comprising:

Soaking the silk fibroin gel in a buffer solution, then cutting through a screen to obtain gel particles, dispersing the gel particles in a sodium hyaluronate injection buffer solution, and sterilizing to obtain a silk fibroin gel dispersion.

Preferably, the molecular weight of the sodium hyaluronate in the sodium hyaluronate injection buffer solution is 500-1500 kDa.

More preferably, the molecular weight of the sodium hyaluronate in the sodium hyaluronate injection buffer is 1000-1500 kDa.

Preferably, the buffer solution and the buffer salt used in the sodium hyaluronate injection buffer solution are at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

More preferably, the buffer and the buffer salt used in the sodium hyaluronate injection buffer are disodium hydrogen phosphate and potassium dihydrogen phosphate.

More preferably, the pH of the buffer solution and the sodium hyaluronate injection buffer solution is 6.0-7.5.

Preferably, the sodium hyaluronate injection buffer also comprises an osmotic pressure regulator.

More preferably, the osmotic pressure regulator is at least one of sodium chloride and potassium chloride.

More preferably, the dosage of the osmotic pressure regulator accounts for 0% -0.9% of the mass of the sodium hyaluronate injection buffer.

Preferably, the mass ratio of the gel particles to the sodium hyaluronate injection buffer is (2-8): 1.

More preferably, the mass ratio of the gel particles to the sodium hyaluronate injection buffer is (3-5): 1.

In a second aspect, the invention provides a silk fibroin gel prepared by the preparation method of any one of the first aspects.

In a third aspect, the present invention provides the use of a silk fibroin gel of the second aspect as described above as a tissue filling material.

Compared with the prior art, the invention has at least the following beneficial effects:

according to the invention, sodium hyaluronate is introduced into the silk fibroin solution, and the high hydrophilicity of the sodium hyaluronate prevents the conformational transition of silk fibroin molecules, so that the silk fibroin gel particles can maintain stable mechanical properties for a long time. Therefore, the silk fibroin gel product prepared based on the method has good stability, can be stably stored for at least 3 months, is favorable for storing the product, and has better industrialization prospect. Meanwhile, the silk fibroin gel has excellent biocompatibility and can be used as a tissue filling material for injection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for preparing a silk fibroin gel according to an embodiment of the present invention;

FIG. 2 is a type I collagen immunohistochemical staining chart of the silk fibroin gel, the control group and the HA group provided by the embodiment of the invention, wherein the injection site tissue sections are injected 1 month later;

FIG. 3 shows immunohistochemical staining of type III collagen in tissue sections at the injection site 1 month after injection of silk fibroin gel, control group, HA group provided by the present invention;

FIG. 4 shows the immunohistochemical staining of α -SMA of tissue sections at the injection site 1 month after injection of silk fibroin gel, control group, HA group provided by the present invention;

FIG. 5 shows CD90 immunohistochemical staining of tissue sections at the injection site 1 month after injection of silk fibroin gel, control group, HA group provided by the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments of the present invention are all within the scope of protection of the present invention.

The invention provides a preparation method of silk fibroin gel, as shown in figure 1, which comprises the following steps:

(2) And adding a cross-linking agent and sodium hyaluronate into the silk fibroin solution, uniformly mixing, and incubating to obtain silk fibroin gel, wherein the dosage of sodium hyaluronate is 2% -14% (for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%) of the dosage of degummed silk or regenerated silk fibroin.

In the embodiment of the invention, the high hydrophilicity of the sodium hyaluronate is used for preventing the conformational transition of the silk fibroin molecules by introducing the sodium hyaluronate into the silk fibroin solution, so that the silk fibroin gel particles can maintain stable mechanical properties for a long time. Therefore, the silk fibroin gel product prepared based on the method has good stability, is favorable for storing the product, and has better industrialization prospect. Meanwhile, the silk fibroin gel has excellent biocompatibility and can be used as a tissue filling material for injection.

According to some preferred embodiments, in step (1) the mass ratio of degummed silk or regenerated silk fibroin to lithium bromide solution is 1 (5-20) (e.g. may be 1:5, 1:5.5, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18 or 1:20).

According to some preferred embodiments, in step (1), the concentration of the lithium bromide solution is 6-13M (for example, 6M, 6.5M, 7M, 8M, 9M, 10M, 11M, 11.5M, 12M, 12.5M or 13M may be used).

According to some more preferred embodiments, the concentration of the lithium bromide solution is 9-10M (e.g., may be 9M, 9.2M, 9.5M, 9.6M, 9.8M, or 10M).

In the invention, experiments prove that excessive lithium ions and bromide ions can remain if the dosage of the lithium bromide solution is excessive or the concentration is too high, the residual ions can react with a cross-linking agent to reduce the cross-linking efficiency, so that a formed gel network is loose and the mechanical property is reduced, but if the dosage of the lithium bromide solution is too low or the concentration is too low, the dissolution of part of degummed silk or regenerated silk fibroin is incomplete, and the subsequent silk fibroin gel is easy to break and fracture.

According to some preferred embodiments, in step (1), the molecular weight of the degummed silk or regenerated silk fibroin is 15-250 kDa (e.g., 15kDa, 20kDa, 30kDa, 40kDa, 50kDa, 100kDa, 120kDa, 150kDa, 160kDa, 180kDa, 200kDa, 220kDa or 250 kDa).

In the embodiment of the invention, in order to ensure that the prepared silk fibroin gel has enough long chains to construct a stable network and avoid aggregation caused by overlong chains, degummed silk or regenerated silk fibroin is selected to have the molecular weight of 15-250 kDa. Therefore, the silk fibroin gel can be prevented from generating network defects, greatly reducing mechanical strength, and excessively fast degradation rate due to the excessively low molecular weight, and the brittle gel can be even obtained even if the viscosity of the silk fibroin solution is difficult to uniformly mix with the cross-linking agent/sodium hyaluronate due to the excessively high molecular weight.

According to some preferred embodiments, the molecular weight of the degummed silk or regenerated silk fibroin is 50-150 kDa (e.g., can be 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa or 150 kDa).

According to some preferred embodiments, in step (2), the cross-linking agent is butanediol diglycidyl ether and/or divinyl sulfone.

According to some preferred embodiments, in step (2) the amount of cross-linking agent is 0.5% -25% (e.g. may be 0.5%, 1%, 1.5%, 3%, 5%, 10%, 15%, 20% or 25%) of the amount of degummed silk or regenerated silk fibroin.

According to some more preferred embodiments, in step (2) degummed silk or regenerated silk fibroin is used in an amount of 2g to 0.44g of cross-linking agent.

According to some more preferred embodiments, the cross-linking agent is butanediol diglycidyl ether.

According to some more preferred embodiments, the amount of sodium hyaluronate is 2% -10% (e.g. may be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%) of the amount of degummed silk or regenerated silk fibroin.

In the embodiment of the invention, experiments prove that if the dosage of sodium hyaluronate is less than 2%, enough physical entanglement or chemical crosslinking sites are difficult to form with the silk fibroin, self-assembly of the silk fibroin is dominant, a brittle network mainly comprising beta-sheets is formed, and the dosage of sodium hyaluronate is too small, so that the water absorption capacity of the silk fibroin gel is increased, the structure is loose and easy to collapse, and therefore, the prepared silk fibroin gel is easy to degrade and cannot maintain long-term stability. If the amount of sodium hyaluronate is higher than 14%, the self-assembly of the silk fibroin is destroyed by excessive sodium hyaluronate, the compatibility is reduced, the gel is cracked, the strong hydrophilicity and steric hindrance of sodium hyaluronate interfere with molecular chain arrangement of the silk fibroin, excessive soft hyaluronic acid chains dominate the network, the mechanical property is deteriorated, in addition, the crosslinking agent is consumed by excessive hyaluronic acid solution, the crosslinking degree is reduced, and the cell survival is affected by high concentration of hyaluronic acid. Therefore, the usage amount of the sodium hyaluronate is 2% -14% of that of degummed silk or regenerated silk fibroin.

According to some preferred embodiments, in step (2), the molecular weight of the sodium hyaluronate is 200-3000 kDa (e.g., may be 200kDa, 300kDa, 500kDa, 600kDa, 800kDa, 1000kDa, 1500kDa, 2000kDa, 2200kDa, 2500kDa, 2600kDa, 2800kDa or 3000 kDa).

According to some more preferred embodiments, the molecular weight of sodium hyaluronate is 1000 to 2000kDa (e.g., may be 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa or 2000 kDa).

In the embodiment of the invention, if the molecular weight of the sodium hyaluronate is lower than 200kDa, the mechanical strength of the prepared silk fibroin gel is reduced due to sparse network, and the degradation is too fast, so that the biological activity is also reduced. If the molecular weight of the sodium hyaluronate is higher than 3000kDa, the prepared silk fibroin gel can increase the viscosity of the solution and inhibit the assembly of silk fibroin, so that the mechanical strength of the silk fibroin gel is reduced and is difficult to degrade, and simultaneously, the transmission of nutrient substances is also hindered. Therefore, the molecular weight of the sodium hyaluronate is 200-3000 kDa, so that the silk fibroin and sodium hyaluronate interpenetrating network can synergistically enhance the mechanical property, and the functions of resisting inflammation, promoting cell proliferation and migration and matching the tissue regeneration period can be realized.

According to some preferred embodiments, in step (2), the incubation is performed at a temperature of 55-65 ℃ (e.g. 55 ℃, 58 ℃, 60 ℃, 62 ℃ or 65 ℃) for a time of 2-4 hours (e.g. 2 hours, 2.5 hours, 3 hours, 3.5 hours or 4 hours).

According to some preferred embodiments, after step (2) further comprises:

Soaking the silk fibroin gel in a buffer solution, then cutting through a screen to obtain gel particles, dispersing the gel particles in a sodium hyaluronate injection buffer solution, and sterilizing to obtain a silk fibroin gel dispersion liquid.

According to some preferred embodiments, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 500-1500 kDa (e.g., can be 500kDa, 550kDa, 600kDa, 800kDa, 900kDa, 1000kDa, 1200kDa, 1300kDa or 1500 kDa).

According to some more preferred embodiments, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 1000 to 1500kDa (e.g., may be 1000kDa, 1050kDa, 1100kDa, 1150kDa, 1200kDa, 1250kDa, 1300kDa, 1350kDa, 1400kDa, 1450kDa or 1500 kDa).

According to some preferred embodiments, the buffer and the buffer salt used in the sodium hyaluronate injection buffer are each at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate.

At least one kind is a mixture of any one or any plurality of kinds mixed in any proportion.

According to some more preferred embodiments, the buffer salt used in the buffer and the sodium hyaluronate injection buffer are disodium hydrogen phosphate and potassium dihydrogen phosphate.

According to some more preferred embodiments, the pH of both the buffer and the sodium hyaluronate injection buffer is 6.0-7.5 (e.g., may be 6.0, 6.2, 6.5, 6.6, 6.8, 7.0, 7.2, or 7.5).

In the invention, the hydration state and the structural integrity of the gel can be further maintained by storing the silk fibroin gel particles in the sodium hyaluronate injection buffer solution, the gel is prevented from being cracked and deformed, the embrittlement caused by secondary crystallization of silk fibroin in air can be inhibited, and meanwhile, the biological activity can be protected and the inflammation risk can be reduced by simulating the physiological environment by storing the silk fibroin gel particles in the sodium hyaluronate injection buffer solution.

According to some preferred embodiments, an osmolality adjusting agent is also included in the sodium hyaluronate injection buffer.

According to some more preferred embodiments, the osmolality adjusting agent is at least one of sodium chloride, potassium chloride.

According to some more preferred embodiments, the osmolality adjusting agent is used in an amount of 0% -0.9% (e.g., may be 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%) of the mass of the sodium hyaluronate injection buffer.

According to some preferred embodiments, the mass ratio of gel particles to sodium hyaluronate injection buffer is (2-8): 1 (e.g., may be 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, or 8:1).

According to some more preferred embodiments, the mass ratio of gel particles to sodium hyaluronate injection buffer is (3-5): 1 (e.g., may be 3:1, 3.5:1, 4:1, 4.5:1, or 5:1).

In the embodiment of the invention, the mass ratio of the gel particles to the sodium hyaluronate injection buffer solution is (2-8): 1, so that the sterilized silk fibroin gel dispersion liquid can be directly used as an injection filling liquid, the resistance of gel passing through a needle head can be reduced, the blockage is avoided, the injection pushing force is reduced, and the gel structure is prevented from being damaged due to mechanical shearing.

The invention also provides a silk fibroin gel, and the adsorption resin microsphere prepared by the preparation method provided by the invention.

The invention also provides an application of the silk fibroin gel prepared by any method, and the silk fibroin gel is used as a tissue filling material.

According to some preferred embodiments, the silk fibroin gel dispersion is used as an injectable soft tissue filler.

In order to more clearly illustrate the technical scheme and advantages of the present invention, a silk fibroin gel and a preparation method thereof are described in detail below through several examples.

Example 1

Dissolving 2g of degummed silk or regenerated silk fibroin (with a molecular weight of 75 kDa) in 10mL of LiBr solution (with a concentration of 9.3M), incubating for 1-2 h at 55-65 ℃ to obtain a silk fibroin solution after the degummed silk or regenerated silk fibroin is completely dissolved, adding 0.44g of butanediol diglycidyl ether (BDDE) and 0.04g of sodium Hyaluronate (HA) (with a molecular weight of 1500 kDa), incubating for 2-4 h at 55-65 ℃ to obtain silk fibroin gel (SF-HA (2%), cutting the gel into small blocks, immersing in a buffer solution, dialyzing to remove residual reagent, cutting by a screen to obtain gel particles, finally dispersing the gel particles in a sodium hyaluronate injection buffer solution (with a pH value of 7.0 and a molecular weight of 1000 kDa), sterilizing at 121 ℃ for 15min, and preserving.

Example 2

Example 2 was substantially the same as example 1 except that sodium hyaluronate was used in an amount of 0.1g to obtain a silk fibroin gel (SF-HA (5%)).

Example 3

Example 3 was substantially the same as example 1 except that sodium hyaluronate was used in an amount of 0.2g to obtain a silk fibroin gel (SF-HA (10%)).

Example 4

Example 4 is essentially the same as example 3 except that the molecular weight of the sodium hyaluronate used for crosslinking is 200kDa.

Example 5

Example 5 is essentially the same as example 3, except that the molecular weight of the sodium hyaluronate used for crosslinking is 1500kDa.

Example 6

Example 6 is essentially the same as example 3 except that the molecular weight of the sodium hyaluronate used for crosslinking is 3000kDa.

Example 7

Example 7 is essentially the same as example 3 except that the molecular weight of the sodium hyaluronate used for crosslinking is 200kDa and the crosslinking agent is divinyl sulfone (DVS).

Example 8

Example 8 is essentially the same as example 3 except that the molecular weight of the sodium hyaluronate used for crosslinking is 1500kDa and the crosslinking agent is divinyl sulfone (DVS).

Example 9

Example 9 is essentially the same as example 3 except that the molecular weight of the sodium hyaluronate used for crosslinking is 3000kDa and the crosslinking agent is divinyl sulfone (DVS).

Example 10

Example 10 is essentially the same as example 3, except that degummed silk or regenerated silk fibroin having a molecular weight of 15kDa is used.

Example 11

Example 11 is essentially the same as example 3, except that degummed silk or regenerated silk fibroin having a molecular weight of 50kDa is used.

Example 12

Example 12 is essentially the same as example 3, except that degummed silk or regenerated silk fibroin having a molecular weight of 150kDa is used.

Example 13

Example 13 is essentially the same as example 3, except that degummed silk or regenerated silk fibroin having a molecular weight of 250kDa is used.

Comparative example 1

Dissolving 2g of degummed silk or regenerated silk fibroin (with the molecular weight of 75 kDa) in 10mL of LiBr solution (with the concentration of 9.3M), incubating for 1-2 h at 55-65 ℃, obtaining silk fibroin solution after the degummed silk or regenerated silk fibroin is completely dissolved, adding 0.44g of butanediol diglycidyl ether (BDDE), incubating for 2-4 h at 55-65 ℃, obtaining silk fibroin gel (SF-HA (0%), cutting the gel into small pieces, immersing in buffer solution, dialyzing to remove residual reagent, finally immersing a sample in sodium hyaluronate injection buffer solution (with the pH value of 7.0 and the molecular weight of 1000 kDa), sterilizing at 121 ℃ for 15min, and preserving.

Comparative example 2

Comparative example 2 was substantially the same as in example 1 except that sodium hyaluronate was used in an amount of 0.3g to obtain a silk fibroin gel (SF-HA (15%)).

Comparative example 3

Comparative example 3 is essentially the same as example 1, except that the molecular weight of the degummed silk or regenerated silk fibroin is 10kDa.

Comparative example 4

Comparative example 4 is substantially the same as example 1 except that the molecular weight of sodium hyaluronate is 150kDa.

The silk fibroin gels obtained in the above examples and comparative examples were cut with a screen having a pore size of 150 μm to obtain silk fibroin gel particles, and then the gel particles were dispersed in sodium hyaluronate injection buffer. The long-term stability of the above-mentioned silk fibroin gel particles dispersed in sodium hyaluronate injection buffer solution of the same concentration was evaluated by detecting the content and properties of hyaluronic acid in the silk fibroin gel particles and respectively maintaining the above-mentioned silk fibroin gel particles under different conditions for different periods of time, and the results are shown in Table 1, wherein Table 2 shows the effects of different molecular weights of sodium hyaluronate and different crosslinking agents on the physicochemical stability of the silk fibroin gel particles. After the silk fibroin gel particles prepared by examples 1 to 3 and comparative examples 1 and 2 were stored in sodium hyaluronate injection buffer at the same concentration for different times, the compressive stress strain curve (compression rate 5 mm/min) of the sample was measured at room temperature (25 ℃) using a universal tester (MTS-E44), and young's modulus was calculated from the experimental results, and specific data are shown in table 3. Wherein the content of sodium hyaluronate is the content of sodium hyaluronate in the silk fibroin gel particles.

TABLE 1

TABLE 2

TABLE 3 Table 3

As can be seen from Table 1, when the dosage of sodium hyaluronate is 2% -10% of that of degummed silk or regenerated silk fibroin, crosslinked gel with stable physicochemical properties can be prepared, and development and use of subsequent products are facilitated. As is clear from comparative example 2, when the amount of sodium hyaluronate is 15% of the amount of degummed silk or regenerated silk fibroin, the silk fibroin gel particles dispersed in the sodium hyaluronate injection buffer solution are placed for 3 months under the storage condition of 30 ℃, the properties of the gel are obviously changed, and the colloid has extremely small white particles, probably because the concentration of sodium hyaluronate is too high, the crosslinking stability of the silk fibroin is affected, and the precipitation of the silk fibroin occurs in the placing process, so that the subsequent product development is not facilitated. The degummed silk or regenerated silk fibroin having a molecular weight of 10kDa in comparative example 3 failed to form a gel, while comparative example 4 formed a white gel, and the elasticity of the gel particles was significantly reduced after the white gel particles dispersed in the sodium hyaluronate injection buffer were left to stand for 3 months under a storage condition of 30 ℃.

As can be seen from table 2, when the amount of sodium hyaluronate is 10% of the amount of degummed silk, the sodium hyaluronate and silk fibroin in the ratio are crosslinked, and both BDDE and DVS crosslinking agents can produce crosslinked gel with stable physicochemical properties, and the crosslinking ability is not affected by the molecular weight of sodium hyaluronate. Also, the silk fibroin gels prepared from examples 10-13 were also capable of remaining as colorless transparent gels after 3 months of storage at 30 ℃.

Young's modulus reflects the ability of a substance to deform under an external force. As can be seen from Table 3, after the silk fibroin gel SF-HA (0%) and silk fibroin gel SF-HA (2%) were stored for 1 month, young's modulus was significantly reduced and then remained stable, which proves that the mechanical stability was extremely poor after the pure silk fibroin gel and silk fibroin gel with lower sodium hyaluronate content were stored, deformation was extremely easy to occur under external force, and sufficient supporting force was difficult to be provided after subcutaneous injection, resulting in poor product stability. After the silk fibroin gel SF-HA (5%) is stored for 1 month, the Young modulus is reduced by about 30%, and after the silk fibroin gel SF-HA is stored for 3 months, the Young modulus is reduced by about 50%, which shows that the content of sodium hyaluronate is increased, and the stability of the mechanical property of the silk fibroin gel is maintained. After storage of silk fibroin gel SF-HA (10%) for 1 month, young's modulus increased significantly from 28.5kPa to 76.4kPa. This is probably because the higher concentration of sodium hyaluronate in the freshly prepared silk fibroin gel SF-HA (10%) samples resulted in incomplete stretching of the segments, and after soaking, as the samples swelled, the sodium hyaluronate segments stretched sufficiently to increase Young's modulus. In addition, after 3 months of storage, the Young's modulus of the silk fibroin gel is still kept at about 70kPa, no obvious reduction is seen, and the mechanical property of the silk fibroin gel SF-HA (10%) is stable after long-term storage, and the silk fibroin gel SF-HA can provide sufficient supporting force after subcutaneous injection.

Furthermore, for the silk fibroin gel SF-HA (10%) prepared in examples 1 to 3, a dispersion of the silk fibroin gel SF-HA (10%) in sodium hyaluronate injection buffer (mass ratio of gel particles to sodium hyaluronate injection buffer 4:1) was injected subcutaneously into rats with a 26G needle. After 1 month of injection, rats were sacrificed, tissues at the injection site were collected, and immunohistochemical staining was performed after embedding sections. Wherein, FIGS. 2 and 3 are respectively type I and type III collagen immunohistochemical staining of injection site tissue sections 1 month after injection. From FIGS. 2 and 3, it can be seen that there is a large amount of type I and type III collagen formation between the microspheres of each group of silk fibroin gel under the skin at the injection site, which proves that it has excellent ability to induce collagen regeneration. In contrast, no type I and type III collagen production was seen inside the commercial sodium hyaluronate group (i.e., HA group, injected with sodium hyaluronate injection buffer alone). From this, it is clear that silk fibroin gel has better collagen regeneration-inducing ability than sodium hyaluronate.

Figures 4 and 5 are a-SMA and CD90 immunohistochemical staining of tissue sections at the injection site 1 month after injection, respectively. alpha-SMA and CD90 are markers for fibroblasts. As can be seen from FIGS. 4 and 5, there is a large amount of alpha-SMA and CD90 expression between the microspheres of each group of silk fibroin gel subcutaneously at the site of injection, demonstrating that a large amount of fibroblasts migrate between the silk fibroin gel microspheres. In contrast, the injection sites of the commercial sodium hyaluronate group (i.e., HA group, injected with sodium hyaluronate injection buffer alone) were substantially free of α -SMA and CD90 expression. From this, it is clear that silk fibroin gel is capable of inducing migration of fibroblasts to the injection region and inducing the same to form type I and type III collagen, whereas commercial sodium hyaluronate injection of fillers is difficult to induce migration of fibroblasts to the injection region.

The Control group in fig. 2 to 5 is a Control group, i.e., a rat not subjected to any treatment, and the tissue at the injection site was subjected to embedding and slicing and then subjected to immunohistochemical staining. And the scales in fig. 2-5 are each 200 microns.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention. The invention is not described in detail in a manner known to those skilled in the art.

Claims

1. A method for preparing a silk fibroin gel, comprising:

2. The method of claim 1, wherein in step (1):

The mass ratio of the degummed silk or regenerated silk fibroin to the lithium bromide solution is 1 (5-20), and/or,

The concentration of the lithium bromide solution is 6-13M, preferably 9-10M.

3. The method of claim 1, wherein in step (1):

the molecular weight of the degummed silk or regenerated silk fibroin is 15-250 kDa, preferably 50-150 kDa, and/or,

The dosage of the sodium hyaluronate is 2% -10% of that of degummed silk or regenerated silk fibroin.

4. The method of claim 1, wherein in step (2):

the cross-linking agent is butanediol diglycidyl ether and/or divinyl sulfone, preferably butanediol diglycidyl ether, and/or,

The molecular weight of the sodium hyaluronate is 200-3000 kDa, preferably 1000-2000 kDa.

5. The method of claim 1, wherein in step (2):

The dosage of the cross-linking agent is 0.5% -25% of that of degummed silk or regenerated silk fibroin, and/or,

The temperature of the incubation is 55-65 ℃ and the time is 2-4 hours.

6. The production method according to any one of claims 1 to 5, characterized by further comprising:

7. The method according to claim 6, wherein,

The molecular weight of the sodium hyaluronate in the sodium hyaluronate injection buffer is 500-1500 kDa, preferably 1000-1500 kDa, and/or,

The buffer solution and the sodium hyaluronate injection buffer solution are both prepared from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, preferably disodium hydrogen phosphate and potassium dihydrogen phosphate, and more preferably the pH values of the buffer solution and the sodium hyaluronate injection buffer solution are 6.0-7.5.

8. The method according to claim 6, wherein,

The sodium hyaluronate injection buffer solution also comprises an osmotic pressure regulator;

preferably, the osmotic pressure regulator is at least one of sodium chloride and potassium chloride;

9. The method according to claim 6, wherein the mass ratio of the gel particles to the sodium hyaluronate injection buffer is (2-8): 1, preferably (3-5): 1.

10. A silk fibroin gel prepared by the method of any one of claims 1 to 9.