A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight

Abstract

This invention discloses a method for preparing soluble regenerated silk fibroin lyophilized powder with controllable molecular weight, yielding a lyophilized powder that exists primarily in a SilkⅠ aggregated state and can be rapidly and completely dissolved. By optimizing the degumming, dissolution, dialysis purification, and lyophilization processes, combined with clarification filtration and low-temperature control, this invention overcomes the stability and quality problems of existing soluble regenerated silk fibroin preparation methods. By controlling key production process parameters, it is possible to prepare regenerated silk fibroin lyophilized powders with different molecular weight ranges for application in the preparation of biodegradable medical materials with varying properties.

Description

Technical Field

[0001] This invention relates to the field of biopolymer materials, specifically to a method for preparing a soluble regenerated silk fibroin freeze-dried powder with adjustable molecular weight. Background Technology

[0002] Silk has a long history of use in China. It is a continuous long fiber formed by the solidification of silk fluid secreted from the silk glands of mature silkworms when they spin their cocoons. With the advancement of science and technology, the application of silk has expanded from the traditional textile field to food, health products, cosmetics, and medicine. Silk is a natural high-molecular-weight protein fiber with unique structural characteristics. It is composed of fibroin (about 70%) and sericin (about 30%). Sericin is a water-soluble protein, which is usually removed by alkaline degumming to obtain the fibroin fibers encased within.

[0003] With the development of tissue engineering materials, natural biomaterials with good biocompatibility and in vivo absorption have enormous potential. Cell and tissue-inducing biomaterials have been included in the "Guidance Catalogue of Key Products and Services in Strategic Emerging Industries," and the natural biomaterial silk fibroin has been included in the "Diagnostic and Therapeutic Equipment and Biomedical Materials" key special project. Regenerative medicine is a cutting-edge direction that the country is focusing on and planning for, and silk fibroin, as an excellent regenerative biomaterial, has huge market prospects.

[0004] Silk fibroin contains 18 amino acids, including 8 essential amino acids for the human body. Glycine (Gly), alanine (Ala), and serine (Ser) account for more than 80% of its total composition. It exhibits good biocompatibility and has been the subject of considerable research in the biomedical field, such as regenerated silk fibroin membranes, porous regenerated silk fibroin scaffolds, regenerated silk fibroin hydrogels, and drug-loaded microspheres. However, current technologies lack methods for producing regenerated silk fibroin with controllable molecular weight. Furthermore, during industrialization, the self-assembly caused by the structural characteristics of regenerated silk fibroin leads to a transformation of its aggregated structure from Silk I to Silk II. The higher the molecular weight and concentration of the protein, the faster this transformation occurs. Even with cryopreservation, the protein can only be preserved for two weeks under stringent storage conditions. These existing technological limitations significantly hinder the development and application of regenerated silk fibroin. Summary of the Invention

[0005] The purpose of this invention is to address the lack of a production method for regenerated silk fibroin with controllable molecular weight in existing technologies, as well as the instability and storage difficulties of regenerated silk fibroin products. This invention provides a method for preparing soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight. The method can be used to obtain regenerated silk fibroin freeze-dried powders with different weight-average molecular weight ranges as needed. Regenerated silk fibroin raw materials with different molecular weight ranges can be used to prepare medical biodegradable materials with different properties. Furthermore, the method of this invention optimizes the degumming, dissolution, dialysis purification, and freeze-drying processes, combined with clarification filtration and low-temperature control. The resulting regenerated silk fibroin freeze-dried powder mainly exists in a Silk I aggregated state, which can be rapidly and completely dissolved, overcoming the stability and quality problems of existing soluble regenerated silk fibroin.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight includes the following steps:

[0008] Step 1: Add silk to a first sodium carbonate aqueous solution and boil for the first time. Remove and drain to obtain the first pre-degummed silk. Add the first pre-degummed silk to a fresh first sodium carbonate aqueous solution and boil for the second time. Remove and drain, wash and dry to obtain the first degummed silk.

[0009] Alternatively, the silk can be added to a second sodium carbonate aqueous solution, boiled for the first time, drained, and then the second pre-degummed silk can be obtained. The second pre-degummed silk can be added to a fresh second sodium carbonate aqueous solution, boiled for the second time, drained, washed, and dried to obtain the second degummed silk.

[0010] Alternatively, the silk can be added to a third sodium carbonate aqueous solution, boiled for the first time, drained, and then the third pre-degummed silk can be obtained. The third pre-degummed silk can be added to a fresh third sodium carbonate aqueous solution, boiled for the second time, drained, washed, and dried to obtain the third degummed silk.

[0011] Alternatively, add silk to a sodium carbonate aqueous solution and boil it for the first time. Then, remove the silk and drain it to obtain pre-degummed silk. Add the pre-degummed silk to a fresh sodium carbonate aqueous solution and boil it for the second time. Remove the silk, drain it, wash it, and dry it to obtain degummed silk.

[0012] Step 2: Soak the first, second, third or fourth degummed silk obtained in Step 1 in a lithium bromide aqueous solution to dissolve it, and obtain the first, second, third or fourth solution.

[0013] Step 3: Filter the first, second, third or fourth solution obtained in step 2 through a polyethersulfone membrane, collect the filtrate, adjust the protein concentration of the filtrate, and obtain the first, second, third or fourth filtrate.

[0014] Step 4: Dialyze the first, second, third, or fourth filtrate obtained in Step 3 to remove lithium bromide, collect the retentate, adjust the pH and protein concentration of the retentate to obtain the first, second, third, or fourth retentate, freeze-dry it to obtain the first, second, third, or fourth soluble regenerated silk fibroin freeze-dried powder.

[0015] The molecular weight distribution ranges of the first, second, third and fourth soluble regenerated silk fibroin freeze-dried powders are 200-300 kDa, 100-200 kDa, 50-100 kDa and 10-50 kDa, respectively.

[0016] In step 1, preferably, the silk is cut into lengths of 20-50cm and then added to the first, second, third, or fourth sodium carbonate aqueous solution; the silk is preferably 5A grade medical mulberry silk.

[0017] In step 1, the concentrations (c) of sodium carbonate in the first, second, third, and fourth sodium carbonate aqueous solutions range from 1 g / L < c ≤ 2 g / L, 2 g / L < c ≤ 5 g / L, 5 g / L < c ≤ 10 g / L, and 10 g / L < c ≤ 20 g / L, respectively. Other types of degumming agents that achieve equivalent degumming effects can also be used, such as sodium bicarbonate, potassium carbonate, sodium hydroxide, and urea.

[0018] In step 1, the solid-liquid ratio of the silk to the first, second, third, or fourth sodium carbonate aqueous solution is 1g:30-100mL; the solid-liquid ratio of the first, second, third, and fourth pre-degummed silk to their corresponding first, second, third, and fourth sodium carbonate aqueous solutions is 1g:30-100mL.

[0019] In step 1, the first boiling and the second boiling are both 20 to 60 minutes; the washing is done with pure water until the pH of the washing waste liquid is 6 to 8 and the conductivity is less than 20 μs / cm; the drying is done at a temperature of 60 to 80°C.

[0020] By degumming in step 1, not only can sericin in silk be completely removed, but regenerated silk fibroin with different molecular weight ranges can also be obtained by adjusting the concentration of sodium carbonate aqueous solution and the degumming time.

[0021] In step 2, the concentration of the lithium bromide aqueous solution is 8.5–9.5 M; the solid-liquid ratio of the first, second, third, or fourth silk fibroin to the lithium bromide aqueous solution is 1 g: 3–10 mL.

[0022] In step 2, the dissolution process is carried out at a temperature of 40–90°C for 0.5–5 hours, with the preferred temperature being 40–60°C.

[0023] In step 3, the pore size of the polyethersulfone membrane is 0.22–5 μm; the protein concentration of the filtrate is adjusted so that the protein concentration of the first, second, third, or fourth filtrate is 1–10 wt%. The protein concentration of the filtrate is adjusted by diluting it with pure water or buffer. The higher the protein concentration, the more difficult the subsequent dialysis becomes, and the more unstable the protein is.

[0024] In step 4, the dialysis is performed using a hollow fiber dialysis membrane or dialysis bag; the hollow fiber dialysis membranes or dialysis bags used for the first, second, third, and fourth filtrates have molecular weight cutoffs of any molecular weight in the range of 200-250 kDa, 100-150 kDa, 50-80 kDa, and 10-20 kDa, respectively; preferably, the hollow fiber dialysis membranes or dialysis bags used for the first, second, third, and fourth filtrates have molecular weight cutoffs of any molecular weight in the range of 200-220 kDa, 100-250 kDa, 100-80 kDa, and 10-20 kDa, respectively. The dialysis solution can be any molecular weight within 120 kDa, any molecular weight within 50–60 kDa, or any molecular weight within 10–15 kDa. The dialysate used is pure water. The volume of the dialysate used is 20–80 times the volume of the first, second, third, or fourth filtrate. The dialysis is cryogenic dialysis, which ends when the conductivity of the waste dialysate is less than 20 μS / cm. The waste dialysate refers to the dialysate that has undergone exchange and contains waste (lithium bromide) removed from the first, second, third, or fourth filtrate. Preferably, the cryogenic dialysis temperature is 3–10°C. At low temperatures, the self-assembly of silk fibroin in solution can be inhibited, thereby reducing precipitation and improving dialysis stability.

[0025] In step 4, the pH value and protein concentration of the retentate are adjusted so that the pH value of the first, second, third or fourth retentate is 6.0 to 7.0 and the protein concentration is 1 to 10 wt%.

[0026] Preferably, in step 4, the pH value and protein concentration of the retentate are adjusted so that the pH of the first, second, third, or fourth retentate is 6.0 to 6.5 and the protein concentration is 1 to 5 wt%.

[0027] Excessively high protein concentrations lead to decreased solubility of silk fibroin after freeze-drying. The first, second, third, or fourth retentate solutions are essentially aqueous solutions of silk fibroin. When the pH is adjusted to 6.0–7.0, the random coil structure content in the silk fibroin aqueous solution is highest, and the β-sheet structure content is lowest. Therefore, silk fibroin is relatively most stable at pH 6.0–7.0, which can maximally inhibit the self-assembly of high molecular weight silk fibroin into Silk II aggregates and subsequent precipitation during the preparation process. This also confirms that pH adjustment is a crucial method for controlling the state of silk fibroin in silkworm glands. Therefore, this invention adjusts the pH of the silk fibroin aqueous solution to 6.0–7.0, allowing silk fibroin to exist stably in an aqueous solution with a random coil structure.

[0028] In step 4, the pH of the retentate is adjusted using a phosphate buffer solution with a concentration of 0.1–0.2 M and a pH of 6–7, or a Tris buffer solution with a concentration of 0.05–0.2 M and a pH of 6–7; preferably, the pH of the retentate is adjusted using a sodium phosphate buffer solution with a concentration of 0.1 M and a pH of 6–6.5.

[0029] In step 4, the protein concentration of the retentate is adjusted by diluting it with pure water or a buffer solution.

[0030] Preferably, in step 4, before freeze-drying the first, second, third, or fourth retentate, a freeze-drying protectant can be added to the first, second, third, or fourth retentate and mixed thoroughly. The freeze-drying protectant is any one or more combinations of polysorbate 80, trehalose, and mannitol. The amount of freeze-drying protectant added is no more than 20 g / L. Polysorbate 80, trehalose, and mannitol are commonly used freeze-drying protectants and excipients, often used to protect protein samples, and also promote the redissolution of freeze-dried powder.

[0031] In step 4, the freeze-drying temperature is -60 to -25°C, and the time is 36 to 100 hours.

[0032] Preferably, all solutions in steps 2-4 are prepared using water for injection, and the purified water used is also water for injection. All reagents used are low-endotoxin reagents, thereby reducing the introduction of endotoxins into the regenerated silk fibroin lyophilized powder. The lyophilized powder from step 4 is tested for endotoxins; if the endotoxin content is less than 0.05 EU / mg, it can be directly used for the preparation of implantable materials.

[0033] Figure 1 This is a flow chart illustrating the preparation process of the molecular weight-controllable soluble regenerated silk fibroin freeze-dried powder of the present invention.

[0034] Beneficial effects:

[0035] (1) By controlling the key method parameters of the silk degumming step, the present invention can obtain regenerated silk fibroin with different molecular weight ranges. After processing by the subsequent steps of the present invention, regenerated silk fibroin freeze-dried powder with different molecular weights is finally obtained, realizing the controllable preparation of regenerated silk fibroin freeze-dried powder with the required molecular weight. The regenerated silk fibroin with different molecular weights can be directly used to prepare various implant-grade regenerated silk fibroin biodegradable materials with different properties.

[0036] (2) This invention inhibits the self-assembly of regenerated silk fibroin during its preparation by clarifying filtration, low-temperature dialysis, and adjusting the pH of the regenerated silk fibroin solution, thus preserving its Silk I structure. After freeze-drying, a rapidly soluble lyophilized powder is obtained, and the silk fibroin exhibits good stability after resolvation. The lyophilized silk fibroin powder prepared by this invention can be stored and transported at room temperature, greatly improving the convenience of raw material use and promoting the commercial application of silk fibroin biomaterials.

[0037] (3) The lyophilized silk fibroin powder prepared by the present invention has good biocompatibility. The results of the three basic biological tests (cytotoxicity, intradermal reaction and skin sensitization) show that the regenerated lyophilized silk fibroin powder prepared by the present invention has no cytotoxicity, no intradermal irritation reaction and no skin sensitization.

[0038] (4) The soluble regenerated silk fibroin prepared by this invention has good stability. After being sterilized by moist heat at 121°C for 15 minutes, its molecular weight does not change significantly. Attached Figure Description

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0040] Figure 1 This is a flow chart illustrating the preparation process of the molecular weight-controllable soluble regenerated silk fibroin freeze-dried powder of the present invention.

[0041] Figure 2 This is a statistical chart showing the yield of silk fibroin obtained from silk degumming using sodium carbonate aqueous solutions of different concentrations in Example 1.

[0042] Figure 3 The image shows SDS-PAGE gel electrophoresis images of the four regenerated silk fibroin proteins with different molecular weight ranges prepared in Example 1.

[0043] Figure 4 This is a photograph showing the test results of lithium bromide residue in the regenerated silk fibroin solution obtained after dialysis in Example 1.

[0044] Figure 5 This is a schematic diagram of the polyethersulfone (PES) membrane filtration process in Example 2.

[0045] Figure 6 This is a schematic diagram of the hollow fiber membrane filtration system in Example 2.

[0046] Figure 7 This is a photograph of the regenerated silk fibroin freeze-dried powder prepared in Example 2.

[0047] Figure 8 The image shows the SDS-PAGE gel electrophoresis results of the regenerated silk fibroin lyophilized powder prepared in Example 2 before and after moist heat sterilization.

[0048] Figure 9 This is a comparison chart of sample A (pH 6.2) and sample B (pH 7.5) after standing for 2 months in Example 2.

[0049] Figure 10 The image shows gels prepared from regenerated silk fibroin of different molecular weights with a protein concentration of 2 wt% in Example 3.

[0050] Figure 11 This is a photograph of the regenerated silk fibroin membrane prepared from freeze-dried powder 2 in Example 3.

[0051] Figure 12 The image shows a fluorescence inverted microscope image of the MTT cytotoxicity assay of the regenerated silk fibroin lyophilized powder (Figure b) and the negative control (Figure a) in Example 5. Detailed Implementation

[0052] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0053] The following examples illustrate the method for measuring the concentration of silk fibroin solution:

[0054] Using the evaporation-weighing method: Weigh the dried small petri dish (W0), add approximately 1 mL of silk fibroin solution (W1), and dry it in a 60℃ oven until it reaches equilibrium. Weigh the dried dish (W2). The concentration of the silk fibroin solution is calculated as follows: W% = (W2 - W0) / (W1 - W0) × 100%.

[0055] Example 1

[0056] A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight includes the following steps:

[0057] Step 1: Cut 100g of silk into 20cm lengths and add them to 10L of a 2g / L sodium carbonate aqueous solution. Boil for 30 minutes, stirring every 10 minutes to disperse the silk and prevent it from sticking together. After boiling for 30 minutes, remove and drain. Add the silk to another 10L of a 2g / L sodium carbonate aqueous solution for degumming again, boil for 30 minutes, remove and drain, wash thoroughly with water for injection several times, and dry in a 60℃ oven overnight to obtain dried silk fibroin. Weigh approximately 70g of degummed silk, labeled as Sample 1.

[0058] Step 1 was repeated four times, with the sodium carbonate solution concentration used in the two degumming processes (using the same sodium carbonate concentration in both degumming processes) replaced by 5 g / L, 10 g / L, and 15 g / L respectively. All other operations remained the same as in Step 1. Approximately 68 g, 62 g, and 57 g of degummed silk were obtained, labeled as Sample 2, Sample 3, and Sample 4, respectively. The yields of silk fibroin obtained from degumming at different sodium carbonate concentrations are shown below. Figure 2 As shown. Silk fibroin yield = (actual mass of silk fibroin / mass of silk) × 100%

[0059] Step 2: Take 20g of each of the four degummed silk obtained in Step 1, and add them to 100mL of 9.3M lithium bromide aqueous solution in three separate portions. Stir with a glass rod to fully soak the degummed silk, and place it in a 60℃ oven to dissolve for 4 hours to obtain a yellow solution.

[0060] Step 3: Filter the four solutions obtained in Step 2 through a polyethersulfone (PES) membrane with a pore size of 2 μm, collect the filtrates, and dilute the protein concentration to 1 wt% with water for injection.

[0061] Step 4: The four filtrates with a protein concentration of 1 wt% obtained in Step 3 were purified by dialysis using a hollow fiber dialysis membrane filtration system at 4–7°C.

[0062] PES filtrate of Sample 1: A hollow fiber dialysis membrane with a molecular weight cutoff of 200 kDa was used. Automatic water replenishment was employed. Dialysis was performed using 25 times the volume of water for injection as the filtrate. The pressure at the dialysis outlet was 0.15 MPa, and the pressure at the inlet was 0.3 MPa. Low-temperature cyclic dialysis was performed at 4°C for 6 hours to remove lithium bromide. The conductivity of the waste dialysis solution was measured to be 16 μS / cm. After dialysis, the retentate was collected to obtain regenerated silk fibroin solution 1.

[0063] PES filtrate of Sample 2: A hollow fiber dialysis membrane with a molecular weight cutoff of 100 kDa was used, with automatic water replenishment. Dialysis was performed using 30 times the volume of water for injection. The pressure at the dialysis outlet was 0.15 MPa, and the pressure at the inlet was preferably 0.2 MPa. Low-temperature cyclic dialysis was performed at 4°C for 7 hours to remove lithium bromide. The conductivity of the waste dialysis solution was measured to be 20 μS / cm. The retentate was collected to obtain regenerated silk fibroin solution 2.

[0064] PES filtrate of sample 3: A hollow fiber dialysis membrane with a molecular weight cutoff of 50 kDa was used. Automatic water replenishment was employed. Dialysis was performed using 50 times the volume of water for injection. The pressure at the dialysis outlet was 0.1 MPa, and the pressure at the inlet was 0.25 MPa. Low-temperature cyclic dialysis was performed at 4°C for 10 hours to remove lithium bromide. The conductivity of the waste dialysis solution was measured to be 15 μS / cm. After dialysis, the retentate was collected to obtain regenerated silk fibroin solution 3.

[0065] PES filtrate of sample 4: A hollow fiber dialysis membrane with a molecular weight cutoff of 10 kDa was used, with automatic water replenishment. Dialysis was performed using 40 times the volume of water for injection. The pressure at the dialysis outlet was 0.15 MPa, and the pressure at the inlet was 0.3 MPa. Low-temperature cyclic dialysis was performed at 4°C for 8 hours to remove lithium bromide. The conductivity of the waste dialysis solution was measured to be 13 μS / cm. After dialysis, the retentate was collected to obtain regenerated silk fibroin solution 4.

[0066] The pH of the above regenerated silk fibroin solutions 1-4 was adjusted to 6.3 using 0.2M phosphate buffer solution with a pH of 6.3. The protein concentration was diluted to 3wt% with water for injection, dispensed into vials, and completely frozen at -50°C in a freeze dryer. After freezing, the solutions were vacuum dried in a freeze dryer for 50 hours to obtain soluble regenerated silk fibroin freeze-dried powders 1-4 with different molecular weights.

[0067] The four different molecular weight lyophilized powders of regenerated silk fibroin prepared in the above steps were dissolved in water to form a protein solution of 2 mg / mL. A 5% w / v (5 g / 100 mL) Tris-Glycine stacking gel and a 12% w / v (12 g / 100 mL) Tris-Glycine separating gel were prepared. The gels were tested according to the electrophoresis method specified in General Chapter 0541, Method 5, SDS-polyacrylamide gel electrophoresis, as described in the Pharmacopoeia of the People's Republic of China. Figure 3 The SDS-PAGE gel electrophoresis image shown is illustrated in Table 1, where the molecular weight range of the proteins is listed in the "Molecular Weight Range" column. The weight-average molecular weight of the four regenerated silk fibroin lyophilized powders was then determined using rheological methods, and the results are shown in Table 1, "Weight-Average Molecular Weight".

[0068] Table 1. Molecular weights of soluble regenerated silk fibroin lyophilized powders 1-4

[0069] sample Concentration of sodium carbonate aqueous solution (g / L) Molecular weight distribution range (kDa) Weight-average molecular weight (kDa) freeze-dried powder 1 2 200～300 230±20 Freeze-dried powder 2 5 100～200 120±10 freeze-dried powder 3 10 50～100 60±10 freeze-dried powder 4 15 10～50 30±5

[0070] In this embodiment, four types of regenerated silk fibroin with different molecular weights were prepared by adjusting the concentration of the degumming solution and the molecular weight cut-off of the hollow fiber membrane. The molecular weight of the regenerated silk fibroin can be further adjusted by adjusting the degumming temperature and time in step one and the dissolution temperature and time in step two.

[0071] The protein concentration of the above-mentioned regenerated silk fibroin solution 2 is approximately 8.5 wt%. The YY / T 1950-2024 standard, "Silk Fiber Protein for Tissue Engineering Medical Devices," stipulates that the bromide ion content per unit mass of protein, on dry weight, should be ≤80 mg / g, and the lithium ion content should be ≤2 mg / g. Based on the silk fibroin concentration of 8.5 wt% after dialysis, a lithium bromide aqueous solution with the maximum content limit specified in the standard was prepared. 20 mL of the standard limit lithium bromide aqueous solution and regenerated silk fibroin solution 2 were taken separately, and 0.1 mol / L silver nitrate titrant was added dropwise to each, and the phenomena were observed. Figure 4 As shown in the figure, the beakers on the left and right contain lithium bromide aqueous solution (at the standard maximum content limit) and regenerated silk fibroin solution 2, respectively, after titration with silver nitrate. The figure shows that the lithium bromide aqueous solution at the standard maximum content limit has a distinct milky white precipitate, while the regenerated silk fibroin solution 2 is clear and transparent with no precipitate. This proves that when the conductivity of the dialysate is 20 μS / cm, the residual lithium bromide in the silk fibroin is within acceptable limits. The principle of this method is the reaction of lithium bromide and silver nitrate to produce silver bromide precipitate. Applied to the rapid detection of lithium bromide residue, it can serve as a key process control indicator in industrial production, significantly reducing process inspection costs.

[0072] Example 2

[0073] A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight includes the following steps:

[0074] Step 1: Cut 100g of silk into 20cm lengths and add it to 8L of 6g / L sodium carbonate aqueous solution. Boil for 30 minutes, stirring every 10 minutes to disperse the silk and prevent it from sticking together. After boiling for 30 minutes, remove and drain. Add it to 8L of 6g / L sodium carbonate aqueous solution again to degumme. Boil for 30 minutes, remove and drain. Wash it thoroughly with water for injection several times, and dry it in a 65℃ oven overnight. Weigh it to obtain approximately 68g of degummed silk.

[0075] Step 2: Take 25g of degummed silk obtained in Step 1 and add it to 100mL of 9.0M lithium bromide aqueous solution in 3 portions. Stir with a glass rod to fully soak the degummed silk and place it in an 80℃ oven to dissolve for 1.5 hours to obtain a yellow solution.

[0076] Step 3: Filter the solution obtained in Step 2 using a polyethersulfone (PES) membrane with a pore size of 5 μm (PES membrane filtration device such as...). Figure 5 (As shown), collect the filtrate and dilute its protein concentration to 1 wt% with water for injection.

[0077] Step 4: The filtrate obtained in Step 3 with a protein concentration of 1 wt% was purified by dialysis using a hollow fiber dialysis membrane filtration system at 4–7°C. A hollow fiber dialysis membrane with a molecular weight cutoff of 50 kDa was used, and dialysis was performed at low temperature for 7 hours with automatic water replenishment. Dialysis was performed using 35 times the volume of water for injection as the filtrate. The pressure at the dialysis outlet was 0.1 MPa, and the pressure at the inlet was 0.3 MPa (see schematic diagram of the hollow fiber membrane filtration system). Figure 6 (As shown). Dialysis was performed to remove lithium bromide. The conductivity of the waste dialysate was measured to be 8 μS / cm. After dialysis, the retentate was collected to obtain a regenerated silk fibroin solution.

[0078] The pH of the above regenerated silk fibroin solution was adjusted to 6.2 using 0.2M phosphate buffer (pH 6.2), and the resulting solution was named Sample A. The protein concentration was diluted to 1 wt% with water for injection (the measured protein concentration of the above regenerated silk fibroin solution was approximately 3 wt%). The solution was aliquoted into vials, completely frozen at -60°C in a freeze dryer, and then vacuum-dried for 55 hours to obtain a medium-molecular-weight soluble regenerated silk fibroin lyophilized powder with a weight-average molecular weight of 90 kDa. Figure 7 As shown.

[0079] The regenerated silk fibroin lyophilized powder prepared according to the above steps was sterilized by moist heat at 121℃ for 15 min. The sterilized regenerated silk fibroin lyophilized powder was then reconstituted with water for injection to prepare regenerated silk fibroin solutions with concentrations of 5 mg / mL and 2 mg / mL, respectively. Additionally, the unsterilized regenerated silk fibroin lyophilized powder was also reconstituted with water for injection to prepare regenerated silk fibroin solutions with concentrations of 5 mg / mL and 2 mg / mL, respectively. A 5% w / v Tris-Glycine stacking gel and a 15% w / v Tris-Glycine separating gel were prepared. The four silk fibroin solutions were subjected to SDS gel electrophoresis according to the electrophoresis method specified in General Chapter 0541, Method 5, SDS-polyacrylamide gel electrophoresis, of the Pharmacopoeia of the People's Republic of China. The electrophoresis results are as follows: Figure 8 As shown, the electrophoresis patterns before and after sterilization are basically the same, proving that the regenerated silk fibroin freeze-dried powder prepared by this invention has good stability under high temperature conditions.

[0080] In contrast, in step 4, when adjusting the pH of the regenerated silk fibroin solution (i.e., the retentate obtained in step 3), the pH is adjusted to 7.5. Other steps and related parameters are the same as in steps 1 to 4 above. The solution obtained after adjusting the pH of the regenerated silk fibroin solution is named sample B.

[0081] Dilute the protein concentration of samples A and B to 0.5 wt%, let stand for 2 months, and observe the protein precipitation. Figure 9 As shown in the figure, the left and right vials in the left image contain freshly prepared sample B with pH=7.5 and sample A with pH=6.2, respectively. The left and right vials in the right image contain sample B with pH=7.5 and sample A with pH=6.2, respectively, after standing for 2 months. It can be seen from the figure that after standing for 2 months, sample B with pH=7.5 showed relatively severe precipitation, while sample A with pH=6.2 showed slight precipitation, but the overall sample remained a transparent liquid.

[0082] Samples A and B, after standing for two months, were subjected to far-ultraviolet circular dichroism (FWD) scanning in the range of 190–250 nm. At pH 6.2, the FWD CD spectra of silk fibroin showed a strong negative Cotton effect at 197 nm (representing the relative abundance of random coil conformations). However, at pH 7.5, the Cotton effect almost disappeared, indicating that there was essentially no disordered silk fibroin present in the solution, consistent with the obvious precipitation observed by the naked eye. This demonstrates that the pH of the regenerated silk fibroin solution is a crucial factor in maintaining its stability. In this example, adjusting the pH to 6.2 significantly enhanced protein stability compared to adjusting it to pH 7.5.

[0083] Example 3

[0084] The regenerated silk fibroin lyophilized powders 1-4 with different molecular weights prepared in Example 1 were dissolved into regenerated silk fibroin solutions with a protein concentration of 2wt%. These solutions were sealed in vials and allowed to stand in a 40°C oven for 10 days to obtain the desired results. Figure 10 The regenerated silk fibroin gels 1-4 shown in the figure demonstrate that, for the same protein concentration, a higher weight-average molecular weight of the protein results in greater mechanical strength, while a lower molecular weight indicates better liquid affinity. In application, a regenerated silk fibroin gel with a specific molecular weight can be selected according to requirements. Table 2 below shows the applicable ranges for various molecular weight regenerated silk fibroin gels when used as dermal fillers.

[0085] Table 2. Applicable range of gels with different molecular weights

[0086]

[0087]

[0088] The regenerated silk fibroin lyophilized powders 1-4 with different molecular weights prepared in Example 1 were dissolved into regenerated silk fibroin solutions with a protein concentration of 2wt%. 5g of each protein solution was weighed and spread evenly in a glass petri dish, then dried in an 80℃ oven until equilibrium was reached. This yielded a transparent, dense, soft, smooth, and viscous protein film. Figure 11 The image shows the protein film formed by the lyophilized powder 2 in Example 1. The larger the molecular weight of the protein, the better the adhesion and the higher the tear resistance of the formed protein film; the smaller the molecular weight of the protein, the better the softness of the formed protein film, with good water vapor permeability and antibacterial properties, which can effectively shorten the wound healing time.

[0089] High molecular weight regenerated silk fibroin is suitable for further preparation into orthopedic materials (bone screws, cartilage scaffolds, etc.), exhibiting high mechanical strength, suitable degradation cycle, and good biocompatibility. Low molecular weight regenerated silk fibroin is suitable for further preparation into soft tissue fillers such as hydrogels and protein films, as well as wound healing promotion materials.

[0090] Example 4

[0091] The regenerated silk fibroin freeze-dried powder obtained in Example 2 was placed in a constant temperature and humidity test chamber, with the humidity set to 60% and the temperature set to 60°C, to conduct an accelerated aging test and examine the stability of the regenerated silk fibroin freeze-dried powder prepared by the present invention.

[0092] AAF (Accelerating Aging Factor) = Q 10 {(TAA-TRT) / 10} =2 {(60-25) / 10} =11.31;

[0093] RT: Expected storage time is 3 years;

[0094] TRT: Normal storage temperature is 25℃;

[0095] TAA: Accelerated aging temperature is 60℃;

[0096] Accelerated aging time in 1 year: AAT1 = RT1 / AAF = 365 / 11.31 ≈ 33 days;

[0097] Accelerated aging time over 2 years: AAT2 = RT2 / AAF = 730 / 11.31 ≈ 65 days;

[0098] Accelerated aging time over 3 years: AAT3 = RT3 / AAF = 1095 / 11.31 ≈ 97 days;

[0099] The regenerated silk fibroin lyophilized powder was removed from the constant temperature and humidity chamber on days 33, 65, and 97, and dissolved in water for injection to prepare a 1 wt% regenerated silk fibroin solution. The dissolution time, dissolution state, and the time to precipitation after standing at room temperature were recorded. The test results are shown in Table 3 below.

[0100] Table 3 Accelerated aging test of regenerated silk fibroin freeze-dried powder

[0101]

[0102]

[0103] Accelerated aging tests show that the regenerated silk fibroin freeze-dried powder prepared by this invention has good stability. After 3 years of storage, it can still dissolve rapidly and can be stably stored at room temperature for 5 to 6 weeks after dissolution, making it suitable for industrial production.

[0104] Example 5

[0105] The regenerated silk fibroin lyophilized powder sample obtained in Example 2 was subjected to three basic biological tests: cytotoxicity, intradermal reaction, and skin sensitization. Specific test methods and results are shown in Table 4. The test results indicate that the regenerated silk fibroin lyophilized powder obtained by the method of this invention has no cytotoxicity (e.g., ...). Figure 12 As shown in the figure, the regenerated silk fibroin freeze-dried powder prepared by this invention does not produce irritation or skin sensitization when injected into the skin, and has excellent biocompatibility.

[0106] Table 4. Cytotoxicity, Intradermal Reaction, and Skin Sensitization Tests

[0107]

[0108] Example 6

[0109] A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight includes the following steps:

[0110] Step 1: Cut 100g of silk into 20cm lengths and add them to 10L of a 5g / L sodium carbonate aqueous solution. Boil for 45 minutes, stirring every 10 minutes to disperse the silk and prevent it from sticking together. After boiling for 45 minutes, remove and drain. Add the silk to another 10L of a 5g / L sodium carbonate aqueous solution for degumming again and boil for 15 minutes. Remove and drain, wash thoroughly with water for injection several times, and dry in a 60℃ oven overnight to obtain dried silk fibroin. Weigh approximately 70g of degummed silk.

[0111] Step 2: Take 10g of degummed silk obtained in Step 1 and add it to 100mL of 9.3M lithium bromide aqueous solution in 3 portions. Stir with a glass rod to fully soak the degummed silk and place it in a 60℃ oven to dissolve for 2 hours to obtain a yellow solution.

[0112] Step 3: The solution obtained in Step 2 was purified by dialysis using a hollow fiber dialysis membrane filtration system. A hollow fiber dialysis membrane with a molecular weight cutoff of 10 kDa was used. Dialysis was performed at 5°C for 5 hours with automatic water replenishment. Dialysis was carried out using 20 times the volume of water for injection as the filtrate. The pressure at the dialysis outlet was 0.1 MPa, and the pressure at the inlet was 0.3 MPa. Dialysis was performed to remove lithium bromide. The conductivity of the waste dialysate was measured to be 10 μS / cm. After dialysis, the retentate was collected to obtain a regenerated silk fibroin solution.

[0113] In this embodiment, a polyethersulfone (PES) membrane with a pore size of 0.22–5 μm was not used to filter the solution and remove undissolved impurities. Silk fibroin is nucleation-dependent; if it is dialyzed directly without filtration, protein will precipitate during dialysis, adhering to the hollow fiber membrane, reducing membrane flux, increasing dialysis pressure, and thus affecting dialysis efficiency. Furthermore, after dialysis, some silk fibroin solution will precipitate, requiring further filtration or centrifugation, which will reduce the silk fibroin yield. This invention, by adding a 0.22–5 μm PES membrane filtration before dialysis desalination, can effectively improve the stability of regenerated silk fibroin during dialysis.

[0114] This invention provides a method and approach for preparing soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a soluble regenerated silk fibroin freeze-dried powder with controllable molecular weight, characterized in that, Includes the following steps: Step 1: Add silkworm silk to the first, second, third, or fourth sodium carbonate aqueous solution, boil, remove and drain to obtain the first, second, third, or fourth pre-degummed silkworm silk. Add the first, second, third, or fourth pre-degummed silkworm silk to the first, second, third, or fourth sodium carbonate aqueous solution respectively, boil, remove and drain, wash, and dry to obtain the first, second, third, or fourth degummed silkworm silk. The concentration c of sodium carbonate in the first, second, third, and fourth sodium carbonate aqueous solutions ranges from 1 g / L < c ≤ 2 g / L, 2 g / L < c ≤ 5 g / L, 5 g / L < c ≤ 10 g / L, and 10 g / L < c ≤ 20 g / L, respectively. Step 2: Soak the first, second, third, or fourth degummed silk obtained in Step 1 in a lithium bromide aqueous solution to dissolve it, thereby obtaining the first, second, third, or fourth solution respectively; wherein the concentration of the lithium bromide aqueous solution is 8.5 ~ 9.5 M; the dissolution is carried out at a temperature of 40 ~ 90℃ for 0.5 ~ 5 h; Step 3: Filter the first, second, third, or fourth solution obtained in Step 2 using a polyethersulfone membrane, collect the filtrate, and adjust the protein concentration of the filtrate to obtain the first, second, third, or fourth filtrate, respectively; wherein the protein concentration of the first, second, third, or fourth filtrate is 1~10 wt%. Step 4: Dialyze the first, second, third, or fourth filtrate obtained in Step 3, collect the retentate, adjust the pH and protein concentration of the retentate to obtain the first, second, third, or fourth retentate respectively, and freeze-dry them to obtain the first, second, third, or fourth soluble regenerated silk fibroin lyophilized powder respectively; wherein, the dialysis of the first, second, third, and fourth filtrate is performed using hollow fiber dialysis membranes or dialysis bags with molecular weight cutoffs of 200-250 kDa, 100-150 kDa, 50-80 kDa, and 10-20 kDa respectively; the pH of the first, second, third, or fourth retentate is 6.0-7.0, and the protein concentration is 1-10 wt%; the molecular weight distribution range of the first, second, third, and fourth soluble regenerated silk fibroin lyophilized powders is 200-300 kDa, 100-200 kDa, 50-100 kDa, and 10-50 kDa respectively.

2. The preparation method according to claim 1, characterized in that, In step 1, the solid-liquid ratio of the silk to the first, second, third, or fourth sodium carbonate aqueous solution is 1 g: 30 ~ 100 mL; the solid-liquid ratio of the first, second, third, and fourth pre-degummed silk to their corresponding first, second, third, and fourth sodium carbonate aqueous solutions is 1 g: 30 ~ 100 mL.

3. The preparation method according to claim 1, characterized in that, In step 1, the boiling time is 20 to 60 minutes; the washing is done with pure water; and the drying temperature is 60 to 80°C.

4. The preparation method according to claim 1, characterized in that, In step 2, the solid-liquid ratio of the first, second, third, or fourth degummed silk to the lithium bromide aqueous solution is 1 g : 3 ~ 10 mL.

5. The preparation method according to claim 1, characterized in that, In step 3, the pore size of the polyethersulfone membrane is 0.22~5 μm.

6. The preparation method according to claim 1, characterized in that, In step 4, the dialysate used in the dialysis is pure water; the amount of dialysate used is 20 to 80 times the volume of the first, second, third, or fourth filtrate; the dialysis is low-temperature dialysis, and the dialysis ends when the conductivity of the waste dialysate is less than 20 μs / cm.

7. The preparation method according to claim 1, characterized in that, In step 4, the pH of the retentate is adjusted using a phosphate buffer solution with a concentration of 0.1 to 0.2 M and a pH of 6 to 7, or a Tris buffer solution with a concentration of 0.05 to 0.2 M and a pH of 6 to 7.

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