A silk fibroin gel and its preparation method

Abstract

This invention provides a silk fibroin gel and its preparation method, relating to the biomedical field. The preparation method includes: (1) dissolving degummed silk or regenerated silk fibroin in a lithium bromide solution to obtain a silk fibroin solution; (2) adding a crosslinking agent and sodium hyaluronate to the silk fibroin solution, mixing well, and then incubating to obtain a silk fibroin gel; wherein the amount of sodium hyaluronate is 2% to 14% of the amount of degummed silk or regenerated silk fibroin. The silk fibroin gel prepared by this invention has stable mechanical properties and can be stored stably for a long time, exhibiting excellent biocompatibility and filling properties.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a silk fibroin gel and its preparation method. Background Technology

[0002] As we age, the elasticity and smoothness of our skin gradually decrease, leading to wrinkles and a loss of radiance. To improve this, injectable fillers, which are minimally invasive, have a quick recovery time, and are relatively simple to administer, are often used to increase soft tissue volume and eliminate or improve wrinkles. Common injectable soft tissue fillers include collagen, sodium hyaluronate, polymethyl methacrylate (PMMA), calcium hydroxyapatite, and poly-L-lactic acid (PLLA). Collagen and PMMA, as biologically derived fillers, have good biocompatibility and few complications, but they are expensive, have high absorption rates, and short-lasting effects. Synthetic fillers such as PMMA, calcium hydroxyapatite, and PLA have longer-lasting effects, but due to poor biocompatibility, they may cause complications such as inflammation, infection, and foreign body granulomas.

[0003] Silk fibroin, derived from silkworm cocoons, is widely available and inexpensive. Existing chemical methods such as cross-linking, or physical methods such as ultrasound and shearing, can convert silk fibroin solutions into gels. Silk fibroin gels exhibit excellent biocompatibility, promoting cell migration, collagen deposition, and angiogenesis, making them ideal materials for injectable fillers. However, physically induced silk fibroin gels have poor mechanical strength, resulting in poor filling effects after subcutaneous injection. Chemically cross-linked silk fibroin gels possess excellent mechanical strength; processing them into gel particles followed by subcutaneous injection yields excellent filling effects, showing significant application potential in the medical aesthetics field. However, during storage, chemically cross-linked silk fibroin gels gradually become brittle and lose strength due to molecular conformational changes, causing the gel particles to rupture under injection shear forces and lose their filling effect. Therefore, there is an urgent need to provide a mechanically stable silk fibroin gel and its preparation method. Summary of the Invention

[0004] This invention provides a silk fibroin gel and its preparation method, which solves the problem that current silk fibroin gels cannot maintain stable mechanical properties over a long period of time.

[0005] In a first aspect, the present invention provides a method for preparing silk fibroin gel, the method comprising the following steps:

[0006] (1) Dissolve degummed silk or regenerated silk fibroin in lithium bromide solution to obtain silk fibroin solution;

[0007] (2) Add cross-linking agent and sodium hyaluronate to the silk fibroin solution, mix well and incubate to obtain silk fibroin gel; wherein the amount of sodium hyaluronate is 2% to 14% of the amount of degummed silk or regenerated silk fibroin.

[0008] Preferably, in step (1): the mass ratio of degummed silk or regenerated silk fibroin to the lithium bromide solution is 1:(5-20).

[0009] Preferably, in step (1), the concentration of the lithium bromide solution is 6-13 M.

[0010] More preferably, the concentration of the lithium bromide solution is 9-10 M.

[0011] Preferably, in step (1): the molecular weight of degummed silk or regenerated silk fibroin is 15-250 kDa.

[0012] Preferably, the molecular weight of degummed silk or regenerated silk fibroin is 50–150 kDa.

[0013] Preferably, the amount of sodium hyaluronate used is 2% to 10% of the amount of degummed silk or regenerated silk fibroin.

[0014] Preferably, in step (2): the crosslinking agent is butanediol diglycidyl ether and / or divinyl sulfone.

[0015] More preferably, the crosslinking agent is butanediol diglycidyl ether.

[0016] Preferably, in step (2): the molecular weight of the sodium hyaluronate is 200-3000 kDa.

[0017] More preferably, the molecular weight of the sodium hyaluronate is 1000-2000 kDa.

[0018] Preferably, in step (2): the amount of crosslinking agent used is 0.5% to 25% of the amount of degummed silk or regenerated silk fibroin.

[0019] Preferably, in step (2): the incubation temperature is 55-65°C and the incubation time is 2-4 hours.

[0020] Preferably, the process further includes the following after step (2):

[0021] The silk fibroin gel was soaked in a buffer solution, then cut through a sieve to obtain gel particles, which were then dispersed in sodium hyaluronate injection buffer and sterilized to obtain a silk fibroin gel dispersion.

[0022] Preferably, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 500-1500 kDa.

[0023] More preferably, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 1000-1500 kDa.

[0024] Preferably, the buffer salt used in the buffer solution and the sodium hyaluronate injection buffer solution is at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0025] More preferably, the buffer salts used in the buffer solution and the sodium hyaluronate injection buffer solution are both disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0026] More preferably, the pH of both the buffer solution and the sodium hyaluronate injection buffer solution is 6.0 to 7.5.

[0027] Preferably, the sodium hyaluronate injection buffer also includes an osmotic pressure regulator.

[0028] More preferably, the osmotic pressure regulator is at least one of sodium chloride and potassium chloride.

[0029] More preferably, the amount of the osmotic pressure regulator is 0% to 0.9% of the mass of the sodium hyaluronate injection buffer.

[0030] Preferably, the mass ratio of the gel particles to the sodium hyaluronate injection buffer is (2-8):1.

[0031] More preferably, the mass ratio of the gel particles to the sodium hyaluronate injection buffer is (3-5):1.

[0032] Secondly, the present invention provides a silk fibroin gel, which is prepared by any of the preparation methods described in the first aspect above.

[0033] Thirdly, the present invention provides an application of the silk fibroin gel of the second aspect described above, wherein the silk fibroin gel is used as a tissue filler material.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] This invention introduces sodium hyaluronate into a silk fibroin solution. The high hydrophilicity of sodium hyaluronate prevents conformational changes in silk fibroin molecules, enabling the silk fibroin gel particles to maintain stable mechanical properties over a long period. Thus, the silk fibroin gel product prepared using this method exhibits good stability and can be stored stably for at least 3 months, which is beneficial for product storage and has better prospects for industrialization. Simultaneously, this silk fibroin gel demonstrates excellent biocompatibility and can be used as an injectable tissue filler.

[0036] Instruction manual illustrations

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart of a method for preparing silk fibroin gel according to an embodiment of the present invention;

[0039] Figure 2 These are type I collagen immunohistochemical staining images of tissue sections from the injection site of the silk fibroin gel, control group, and HA group one month after injection, provided in this embodiment of the invention.

[0040] Figure 3 This is the type III collagen immunohistochemical staining of tissue sections from the injection site one month after the silk fibroin gel, control group, and HA group provided in the embodiments of the present invention;

[0041] Figure 4 This is an example of α-SMA immunohistochemical staining of tissue sections from the injection sites of the silk fibroin gel, control group, and HA group one month after injection, as provided in this embodiment of the invention.

[0042] Figure 5 The CD90 immunohistochemical staining of tissue sections from the injection sites one month after the injection of silk fibroin gel, control group, and HA group provided in this embodiment of the invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0044] This invention provides a method for preparing silk fibroin gel, such as... Figure 1 As shown, the preparation method includes the following steps:

[0045] (1) Dissolve degummed silk or regenerated silk fibroin in lithium bromide solution to obtain silk fibroin solution;

[0046] (2) Add cross-linking agent and sodium hyaluronate to silk fibroin solution, mix well and incubate to obtain silk fibroin gel; wherein, the amount of sodium hyaluronate is 2% to 14% of the amount of degummed silk or regenerated silk fibroin (for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%).

[0047] In this embodiment of the invention, sodium hyaluronate is introduced into the silk fibroin solution. The high hydrophilicity of sodium hyaluronate prevents conformational changes in the silk fibroin molecules, allowing the silk fibroin gel particles to maintain stable mechanical properties over a long period. Thus, the silk fibroin gel product prepared based on this method exhibits good stability, which is beneficial for product storage and offers better prospects for industrialization. Simultaneously, this silk fibroin gel demonstrates excellent biocompatibility and can be used as an injectable tissue filler.

[0048] 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) (for example, it can 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).

[0049] According to some preferred embodiments, in step (1): the concentration of the lithium bromide solution is 6 to 13 M (for example, it can be 6 M, 6.5 M, 7 M, 8 M, 9 M, 10 M, 11 M, 11.5 M, 12 M, 12.5 M or 13 M).

[0050] According to some more preferred embodiments, the concentration of the lithium bromide solution is 9 to 10 M (for example, it can be 9 M, 9.2 M, 9.5 M, 9.6 M, 9.8 M or 10 M).

[0051] In this invention, experiments have confirmed that if the amount of lithium bromide solution used is too large or the concentration is too high, too many lithium ions and bromide ions will remain. The remaining ions may react with the crosslinking agent, reducing the crosslinking efficiency and causing the formed gel network to be loose and its mechanical properties to decrease. However, if the amount of lithium bromide solution used is too small or the concentration is too low, some degummed silk or regenerated silk fibroin will not dissolve completely, and the subsequent silk fibroin gel will be easy to break and fracture.

[0052] According to some preferred embodiments, in step (1): the molecular weight of degummed silk or regenerated silk fibroin is 15 to 250 kDa (for example, it can be 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 100 kDa, 120 kDa, 150 kDa, 160 kDa, 180 kDa, 200 kDa, 220 kDa or 250 kDa).

[0053] In this embodiment of the invention, to ensure that the prepared silk fibroin gel has sufficiently long chains to form a stable network and to avoid aggregation due to excessive length, the molecular weight of degummed silk or regenerated silk fibroin is selected to be 15–250 kDa. This avoids the network defects, significant decrease in mechanical strength, and excessively rapid degradation rate that can occur with excessively low molecular weights, while also preventing the high viscosity of the silk fibroin solution from making it difficult to mix uniformly with the crosslinking agent / sodium hyaluronate, or even resulting in a brittle gel, due to excessively high molecular weights.

[0054] According to some preferred embodiments, the molecular weight of degummed silk or regenerated silk fibroin is 50-150 kDa (e.g., 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa or 150 kDa).

[0055] According to some preferred embodiments, in step (2): the crosslinking agent is butanediol diglycidyl ether and / or divinyl sulfone.

[0056] According to some preferred embodiments, in step (2): the amount of crosslinking agent used is 0.5% to 25% of the amount of degummed silk or regenerated silk fibroin (for example, it can be 0.5%, 1%, 1.5%, 3%, 5%, 10%, 15%, 20% or 25%).

[0057] According to some preferred embodiments, in step (2): the ratio of degummed silk or regenerated silk fibroin to crosslinking agent is 2g:0.44g.

[0058] According to some more preferred embodiments, the crosslinking agent is butanediol diglycidyl ether.

[0059] According to some more preferred embodiments, the amount of sodium hyaluronate used is 2% to 10% of the amount of degummed silk or regenerated silk fibroin used (e.g., it can 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%).

[0060] In this invention, experiments have confirmed that if the amount of sodium hyaluronate is less than 2%, it is difficult to form sufficient physical entanglement or chemical cross-linking sites with silk fibroin. Furthermore, the self-assembly of silk fibroin dominates, forming a brittle network dominated by β-sheets. Insufficient sodium hyaluronate also leads to increased water absorption in the silk fibroin gel, resulting in a loose structure that is prone to collapse. Consequently, the prepared silk fibroin gel is easily degraded and cannot maintain long-term stability. If the amount of sodium hyaluronate is higher than 14%, excessive sodium hyaluronate will disrupt the self-assembly of silk fibroin, reduce compatibility, and cause cracks in the gel. Moreover, the strong hydrophilicity and steric hindrance of sodium hyaluronate interfere with the arrangement of silk fibroin molecular chains, and the excessively soft hyaluronic acid chains dominating the network will lead to deterioration of its mechanical properties. In addition, excessive hyaluronic acid solution will consume the cross-linking agent, thereby reducing the degree of cross-linking, and high concentrations of hyaluronic acid will affect cell survival. Therefore, this invention limits the amount of sodium hyaluronate to 2%–14% of the amount of degummed silk or regenerated silk fibroin.

[0061] According to some preferred embodiments, in step (2): the molecular weight of sodium hyaluronate is 200 to 3000 kDa (for example, it can be 200 kDa, 300 kDa, 500 kDa, 600 kDa, 800 kDa, 1000 kDa, 1500 kDa, 2000 kDa, 2200 kDa, 2500 kDa, 2600 kDa, 2800 kDa or 3000 kDa).

[0062] According to some preferred embodiments, the molecular weight of sodium hyaluronate is 1000 to 2000 kDa (for example, it can be 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa or 2000 kDa).

[0063] In this embodiment of the invention, if the molecular weight of sodium hyaluronate is below 200 kDa, the prepared silk fibroin gel will have reduced mechanical strength due to its sparse network, and its rapid degradation will also reduce its bioactivity. If the molecular weight of sodium hyaluronate is above 3000 kDa, the prepared silk fibroin gel will increase the solution viscosity and inhibit silk fibroin assembly, thereby leading to reduced mechanical strength and difficulty in degradation, while also hindering nutrient transport. Therefore, this invention uses sodium hyaluronate with a molecular weight of 200–3000 kDa, which not only allows the interpenetrating network of silk fibroin and sodium hyaluronate to synergistically enhance mechanical properties, but also provides anti-inflammatory, cell proliferation and migration promotion, and tissue regeneration cycle matching functions.

[0064] According to some preferred embodiments, in step (2): the incubation temperature is 55-65°C (for example, it can be 55°C, 58°C, 60°C, 62°C or 65°C), and the time is 2-4h (for example, it can be 2h, 2.5h, 3h, 3.5h or 4h).

[0065] According to some preferred embodiments, after step (2), the method further includes:

[0066] The silk fibroin gel was soaked in a buffer solution, then cut through a sieve to obtain gel particles, which were then dispersed in sodium hyaluronate injection buffer and sterilized to obtain a silk fibroin gel dispersion.

[0067] According to some preferred embodiments, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 500-1500 kDa (e.g., it can be 500 kDa, 550 kDa, 600 kDa, 800 kDa, 900 kDa, 1000 kDa, 1200 kDa, 1300 kDa or 1500 kDa).

[0068] According to some preferred embodiments, the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer is 1000-1500 kDa (e.g., it can be 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa or 1500 kDa).

[0069] According to some preferred embodiments, the buffer salt used in the buffer solution and the sodium hyaluronate injection buffer solution is at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0070] It should be noted that "at least one" means any one or more of them mixed in any proportion.

[0071] According to some preferred embodiments, the buffer salts used in the buffer solution and the sodium hyaluronate injection buffer are both disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0072] According to some preferred embodiments, the pH of both the buffer solution and the sodium hyaluronate injection buffer solution is 6.0 to 7.5 (e.g., it can be 6.0, 6.2, 6.5, 6.6, 6.8, 7.0, 7.2 or 7.5).

[0073] In this invention, by storing silk fibroin gel particles in sodium hyaluronate injection buffer, the hydration state and structural integrity of the gel can be further maintained, preventing the gel from drying and cracking and deforming. It can also inhibit the embrittlement caused by secondary crystallization of silk fibroin in the air. At the same time, storing it in sodium hyaluronate injection buffer can also protect its biological activity and reduce the risk of inflammation by simulating the physiological environment.

[0074] According to some preferred embodiments, the sodium hyaluronate injection buffer also includes an osmotic pressure regulator.

[0075] According to some preferred embodiments, the osmotic pressure regulator is at least one of sodium chloride and potassium chloride.

[0076] According to some more preferred embodiments, the amount of osmotic pressure regulator is 0% to 0.9% of the mass of sodium hyaluronate injection buffer (e.g., it can be 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9%).

[0077] According to some preferred embodiments, the mass ratio of gel particles to sodium hyaluronate injection buffer is (2-8):1 (for example, it can 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).

[0078] According to some more preferred embodiments, the mass ratio of gel particles to sodium hyaluronate injection buffer is (3 to 5):1 (for example, it can be 3:1, 3.5:1, 4:1, 4.5:1 or 5:1).

[0079] In this embodiment of the invention, by limiting the mass ratio of gel particles to sodium hyaluronate injection buffer to (2-8):1, the sterilized silk fibroin gel dispersion can still be directly used as an injection filler, which can reduce the resistance of the gel through the needle, avoid blockage, and reduce the injection force to prevent the gel structure from being damaged by mechanical shearing.

[0080] The present invention also provides a silk fibroin gel, wherein the adsorbent resin microspheres are prepared by the preparation method provided in the present invention.

[0081] The present invention also provides an application of the silk fibroin gel prepared by any of the above methods, using the silk fibroin gel as a tissue filling material.

[0082] According to some preferred embodiments, silk fibroin gel dispersions are used as injectable soft tissue fillers.

[0083] To more clearly illustrate the technical solution and advantages of the present invention, the following detailed description of a silk fibroin gel and its preparation method is provided through several embodiments.

[0084] Example 1

[0085] Dissolve 2g of degummed silk or regenerated silk fibroin (molecular weight 75kDa) in 10mL of LiBr solution (concentration 9.3M) and incubate at 55-65℃ for 1-2h until the degummed silk or regenerated silk fibroin is completely dissolved to obtain a silk fibroin solution. Then add 0.44g of butylene glycol diglycidyl ether (BDDE) and 0.04g of sodium hyaluronate (HA) (molecular weight 1500kDa) and incubate at 55-65℃ for 2-4h to obtain silk fibroin gel (SF-HA (2%)). Cut the gel into small pieces, soak them in buffer solution, dialyze to remove residual reagents, and then cut them through a sieve to obtain gel particles. Finally, disperse the gel particles in sodium hyaluronate injection buffer (pH 7.0, molecular weight of sodium hyaluronate 1000kDa), sterilize at 121℃ for 15min, and store.

[0086] Example 2

[0087] Example 2 is basically the same as Example 1, except that the amount of sodium hyaluronate used is 0.1g, and silk fibroin gel (SF-HA (5%)) is obtained.

[0088] Example 3

[0089] Example 3 is basically the same as Example 1, except that the amount of sodium hyaluronate used is 0.2g, and silk fibroin gel (SF-HA (10%)) is obtained.

[0090] Example 4

[0091] Example 4 is basically the same as Example 3, except that the molecular weight of the sodium hyaluronate used for crosslinking is 200 kDa.

[0092] Example 5

[0093] Example 5 is basically the same as Example 3, except that the molecular weight of the sodium hyaluronate used for crosslinking is 1500 kDa.

[0094] Example 6

[0095] Example 6 is basically the same as Example 3, except that the molecular weight of the sodium hyaluronate used for crosslinking is 3000 kDa.

[0096] Example 7

[0097] Example 7 is basically the same as Example 3, except that the molecular weight of the sodium hyaluronate used for crosslinking is 200 kDa and the crosslinking agent is divinyl sulfone (DVS).

[0098] Example 8

[0099] Example 8 is basically 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).

[0100] Example 9

[0101] Example 9 is basically 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).

[0102] Example 10

[0103] Example 10 is basically the same as Example 3, except that it uses degummed silk or regenerated silk fibroin with a molecular weight of 15kDa.

[0104] Example 11

[0105] Example 11 is basically the same as Example 3, except that it uses degummed silk or regenerated silk fibroin with a molecular weight of 50kDa.

[0106] Example 12

[0107] Example 12 is basically the same as Example 3, except that it uses degummed silk or regenerated silk fibroin with a molecular weight of 150kDa.

[0108] Example 13

[0109] Example 13 is basically the same as Example 3, except that it uses degummed silk or regenerated silk fibroin with a molecular weight of 250kDa.

[0110] Comparative Example 1

[0111] Dissolve 2g of degummed silk or regenerated silk fibroin (molecular weight 75kDa) in 10mL of LiBr solution (concentration 9.3M) and incubate at 55-65℃ for 1-2h until the degummed silk or regenerated silk fibroin is completely dissolved to obtain a silk fibroin solution. Then add 0.44g of butylene glycol diglycidyl ether (BDDE) and incubate at 55-65℃ for 2-4h to obtain a silk fibroin gel (SF-HA(0%)). Cut the gel into small pieces, soak them in buffer solution, dialyze to remove residual reagents, and finally soak the sample in sodium hyaluronate injection buffer (pH 7.0, molecular weight of sodium hyaluronate 1000kDa) and sterilize at 121℃ for 15min for storage.

[0112] Comparative Example 2

[0113] Comparative Example 2 is basically the same as Example 1, except that the amount of sodium hyaluronate used is 0.3g, and silk fibroin gel (SF-HA (15%)) is obtained.

[0114] Comparative Example 3

[0115] Comparative Example 3 is basically the same as Example 1, except that the molecular weight of degummed silk or regenerated silk fibroin is 10 kDa.

[0116] Comparative Example 4

[0117] Comparative Example 4 is basically the same as Example 1, except that the molecular weight of sodium hyaluronate is 150 kDa.

[0118] The silk fibroin gels obtained in the above examples and comparative examples were cut using a sieve with a pore size of 150 micrometers to obtain silk fibroin gel particles. These particles were then dispersed in sodium hyaluronate injection buffer. The hyaluronic acid content and properties of the silk fibroin gel particles were detected. The long-term stability of the silk fibroin gel particles dispersed in the same concentration of sodium hyaluronate injection buffer was evaluated by maintaining them under different storage conditions and for different times. The results are shown in Table 1. Table 2 shows the effects of different molecular weights of sodium hyaluronate and different cross-linking agents on the physicochemical stability of the silk fibroin gel particles. The silk fibroin gel particles prepared in Examples 1 to 3 and Comparative Examples 1 and 2 were stored in the same concentration of sodium hyaluronate injection buffer for different times. The compressive stress-strain curves of the samples were measured using a universal testing machine (MTS-E44) at room temperature (25°C) (compression rate 5 mm / min). Young's modulus was calculated based on the experimental results. Specific data are shown in Table 3. The sodium hyaluronate content refers to the sodium hyaluronate content in the silk fibroin gel particles.

[0119] Table 1

[0120]

[0121] Table 2

[0122]

[0123] Table 3

[0124]

[0125] Table 1 shows that when the amount of sodium hyaluronate is 2% to 10% of the amount of degummed silk or regenerated silk fibroin, a cross-linked gel with stable physicochemical properties can be prepared, which is convenient for the development and use of subsequent products. Comparative Example 2 shows that when the amount of sodium hyaluronate is 15% of the amount of degummed silk or regenerated silk fibroin, the fibroin gel particles dispersed in sodium hyaluronate injection buffer are placed at 30°C for 3 months, and the gel properties change significantly, with very small white particles appearing in the colloid. This may be because the sodium hyaluronate concentration is too high, affecting the cross-linking stability of the silk fibroin, leading to precipitation of the silk fibroin during storage, which is detrimental to subsequent product development. In Comparative Example 3, degummed silk or regenerated silk fibroin with a molecular weight of 10 kDa cannot form a gel; however, in Comparative Example 4, a white gel is formed, and the elasticity of the white gel particles dispersed in sodium hyaluronate injection buffer is significantly reduced after being placed at 30°C for 3 months.

[0126] As shown in Table 2, when the amount of sodium hyaluronate is 10% of the amount of degummed silk, this proportion of sodium hyaluronate crosslinks with silk fibroin. Both BDDE and DVS crosslinking agents can produce crosslinked gels with stable physicochemical properties, and the crosslinking ability is not affected by the molecular weight of sodium hyaluronate. Similarly, the silk fibroin gels prepared in Examples 10 to 13 can also remain colorless and transparent gels after being stored at 30°C for 3 months.

[0127] Young's modulus reflects a material's ability to resist deformation under external forces. Table 3 shows that after one month of storage, the Young's modulus of silk fibroin gel SF-HA (0%) and silk fibroin gel SF-HA (2%) significantly decreased and then stabilized. This demonstrates that pure silk fibroin gel and silk fibroin gel with lower sodium hyaluronate content exhibit extremely poor mechanical stability after storage, making them highly susceptible to deformation under external forces. They also struggle to provide sufficient support after subcutaneous injection, resulting in poor product stability. After one month of storage, the Young's modulus of silk fibroin gel SF-HA (5%) decreased by approximately 30%, and after three months, it decreased by approximately 50%. This trend indicates that increasing the sodium hyaluronate content is beneficial for maintaining the stability of the mechanical properties of silk fibroin hydrogels. After one month of storage, the Young's modulus of silk fibroin gel SF-HA (10%) significantly increased from 28.5 kPa to 76.4 kPa. This may be because the high sodium hyaluronate concentration in the newly prepared silk fibroin gel SF-HA (10%) sample prevented its chains from fully stretching. After soaking, as the sample swelled, the sodium hyaluronate chains fully stretched, resulting in an increase in Young's modulus. Furthermore, after 3 months of storage, its Young's modulus remained around 70 kPa, without significant decrease, demonstrating that the mechanical properties of the silk fibroin gel SF-HA (10%) remained stable after prolonged storage, providing sufficient support after subcutaneous injection.

[0128] Furthermore, regarding 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 (the mass ratio of gel particles to sodium hyaluronate injection buffer was 4:1) was injected subcutaneously into rats using a 26G needle. One month after injection, the rats were sacrificed, and tissue from the injection site was collected, embedded, sectioned, and subjected to immunohistochemical staining. Figure 2 and Figure 3 These are immunohistochemical staining results of type I and type III collagen in tissue sections from the injection site one month after injection. Figure 2 and Figure 3 It was observed that, at the injection site subcutaneously, a large amount of type I and type III collagen was generated between the microspheres of each group of silk fibroin gel, demonstrating its excellent ability to induce collagen regeneration. In contrast, no type I and type III collagen were observed to be generated inside the commercially available sodium hyaluronate group (i.e., the HA group, which only received sodium hyaluronate injection buffer). Therefore, it can be concluded that silk fibroin gel has a better ability to induce collagen regeneration than sodium hyaluronate.

[0129] Figure 4 and Figure 5 These are immunohistochemical staining results for α-SMA and CD90 in tissue sections from the injection site one month after injection. α-SMA and CD90 are markers of fibroblasts. Figure 4 and Figure 5It was observed that, subcutaneously at the injection site, a large amount of α-SMA and CD90 were expressed between the microspheres of each silk fibroin gel group, indicating that a large number of fibroblasts migrated into the silk fibroin gel microspheres. In contrast, the commercial sodium hyaluronate group (i.e., the HA group, which only received sodium hyaluronate injection buffer) showed almost no α-SMA and CD90 expression at the injection site. Therefore, it can be concluded that silk fibroin gel can induce fibroblast migration to the injection area and induce them to produce type I and type III collagen, while commercial sodium hyaluronate injection filler has difficulty inducing fibroblast migration to the injection area.

[0130] It should be noted that, Figures 2 to 5 The Control group consisted of rats that received no treatment. Immunohistochemical staining was performed on tissue sections embedded at the injection sites. Figures 2 to 5 The scale bars in all of them are 200 micrometers.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. The parts of the present invention not described in detail are techniques known to those skilled in the art.

Claims

1. A method for preparing a silk fibroin gel dispersion, characterized in that, include: (1) Dissolve degummed silk or regenerated silk fibroin in lithium bromide solution to obtain silk fibroin solution; The molecular weight of degummed silk or regenerated silk fibroin is 15~250kDa; (2) After adding a cross-linking agent and sodium hyaluronate to the silk fibroin solution and mixing well, the mixture is incubated to obtain a silk fibroin gel; wherein, the amount of sodium hyaluronate is 2% to 14% of the amount of degummed silk or regenerated silk fibroin; the molecular weight of the sodium hyaluronate is 1000 to 2000 kDa; the incubation temperature is 55 to 65°C and the incubation time is 2 to 4 hours; the cross-linking agent is butanediol diglycidyl ether and / or divinyl sulfone; (3) The silk fibroin gel is soaked in a buffer solution, then cut through a sieve to obtain gel particles, and the gel particles are dispersed in a sodium hyaluronate injection buffer solution, and sterilized to obtain a silk fibroin gel dispersion; the molecular weight of sodium hyaluronate in the sodium hyaluronate injection buffer solution is 500~1500kDa.

2. The preparation method according to claim 1, characterized in that, In step (1): The mass ratio of degummed silk or regenerated silk fibroin to the lithium bromide solution is 1:(5~20).

3. The preparation method according to claim 1, characterized in that, In step (1): The concentration of the lithium bromide solution is 6~13M.

4. The preparation method according to claim 3, characterized in that, In step (1): The concentration of the lithium bromide solution is 9~10M.

5. The preparation method according to claim 1, characterized in that, In step (1): The molecular weight of degummed silk or regenerated silk fibroin is 50~150kDa.

6. The preparation method according to claim 1, characterized in that, In step (2): The amount of sodium hyaluronate used is 2% to 10% of the amount of degummed silk or regenerated silk fibroin.

7. The preparation method according to claim 1, characterized in that, In step (2): The crosslinking agent is butanediol diglycidyl ether.

8. The preparation method according to claim 1, characterized in that, In step (2): The amount of the crosslinking agent used is 0.5% to 25% of the amount of degummed silk or regenerated silk fibroin.

9. The preparation method according to claim 1, characterized in that, The hyaluronic acid sodium in the sodium injection buffer has a molecular weight of 1000~1500kDa.

10. The preparation method according to claim 1, characterized in that, The buffer solution and the sodium hyaluronate injection buffer solution use at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

11. The preparation method according to claim 10, characterized in that, The buffer solution and the sodium hyaluronate injection buffer solution both use disodium hydrogen phosphate and potassium dihydrogen phosphate as buffer salts.

12. The preparation method according to claim 10, characterized in that, The pH of both the buffer solution and the sodium hyaluronate injection buffer solution is 6.0~7.

5.

13. The preparation method according to claim 1, characterized in that, The sodium hyaluronate injection buffer also includes an osmotic pressure regulator.

14. The preparation method according to claim 13, characterized in that, The osmotic pressure regulator is at least one of sodium chloride and potassium chloride.

15. The preparation method according to claim 13, characterized in that, The amount of the osmotic pressure regulator is 0.1% to 0.9% of the mass of the sodium hyaluronate injection buffer.

16. The preparation method according to claim 1, characterized in that, The mass ratio of the gel particles to the sodium hyaluronate injection buffer is (2~8):

1.

17. The preparation method according to claim 16, characterized in that, The mass ratio of the gel particles to the sodium hyaluronate injection buffer is (3~5):

1.

18. A silk fibroin gel dispersion, characterized in that, It is prepared by any of the preparation methods described in claims 1 to 17.

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