A method for preparing silk fibroin nanoparticles

Abstract

A preparation method of silk fibroin nanoparticles belongs to the field of biomedical technology. In order to obtain silk fibroin nanoparticles with uniform particle size and smaller size, the silk fibroin obtained after degumming silk is dissolved by LiBr in the present invention, and a regenerated silk fibroin solution with a certain concentration is obtained through dialysis. Then, regenerated silk fibroin RSF with different molecular weights is obtained through ultrafiltration separation and freeze-drying, and silk fibroin nanoparticles are prepared by the self-assembly method induced by absolute ethanol. The silk fibroin RSF with molecular weights of 30-50KDa and >50KDa obtained by ultrafiltration in the present invention (30～50KDa) and RSF (>50KDa) self-assembled to form silk fibroin nanoparticles SFNPs (30～50KDa) and SFNPs (>50KDa) have uniform particle size and smaller size, and are beneficial to cell uptake and drug controlled release as drug delivery carriers.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing silk fibroin nanoparticles. Background Art

[0002] Silk fibroin is a natural high molecular weight fibrous protein extracted from silk, accounting for about 70% to 80% of silk. It is mainly composed of glycine, alanine and serine. The main chain contains two segments, a light chain (~26KDa) and a heavy chain (~390KDa). The heavy chain contains multiple hydrophobic and hydrophilic regions to form an amphiphilic block copolymer. Silk is a biomaterial approved by the U.S. Food and Drug Administration (FDA) for certain medical purposes. As a natural protein polymer, silk fibroin has excellent characteristics such as good biocompatibility, biodegradability, and low immunogenicity, and has broad application prospects in biomedical materials.

[0003] Silk fibroin is widely available, inexpensive, and rich in functional groups such as amino, carboxyl, and hydroxyl groups on its surface. It is easy to modify and prepare multifunctional materials. The conformational transformation and crystallization degree of silk fibroin can be effectively controlled by changing external conditions, thereby regulating the structure, micromorphology, and mechanical properties of silk fibroin materials. In recent years, a variety of silk fibroin biomaterials have been studied, including nanoparticles, microspheres, nanofibers, and hydrogels for the preparation of drug carriers, skin wound dressings, and biological scaffolds. Silk fibroin encapsulates drug molecules without being affected by immunoglobulins and can be used to assemble small molecule drugs, enzymes, growth factors, antibodies, and genes. In addition, silk fibroin can also be used to encapsulate drugs with poor water solubility or hydrophobic surfaces to increase their solubility.

[0004] As a non-toxic and biocompatible drug carrier, silk fibroin is a research hotspot in the field of biomedical materials. Synthesizing nanoparticles with uniform particle size and smaller size is beneficial for cell uptake, drug transport and controlled release, further expanding its application in biomedical materials. The main methods for preparing silk fibroin nanoparticles and microspheres include spray drying, emulsification, phase separation, salting out, etc. Summary of the invention

[0005] In order to make the prepared silk fibroin nanoparticles uniform in particle size and smaller in size, the present invention provides a method for preparing silk fibroin nanoparticles, comprising three preparation processes: extraction of silk fibroin, ultrafiltration separation of regenerated silk fibroin with different molecular weights, and self-assembly of silk fibroin nanoparticles into spheres. Specifically, silk is extracted to obtain a regenerated silk fibroin solution, and then the regenerated silk fibroin solution is ultrafiltered using ultrafiltration tubes with molecular weight cutoffs of 50 KDa and 30 KDa in sequence to obtain regenerated silk fibroin solutions with molecular weights of 30 to 50 KDa and >50 KDa, and regenerated silk fibroin RSF is obtained by freeze drying.(30～50KDa) and RSF (>50KDa) , and then use anhydrous ethanol to induce the regenerated silk fibroin to self-assemble into spheres.

[0006] It is further defined that the extraction is carried out according to the following steps:

[0007] S1. Weigh 4-12g of silk and put it into 600-1000mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 15-45min, stirring it during the process; after cooling down, take out the silk and squeeze out the water, put it into 0.5% Na2CO3 solution again, boil it for 15-45min, and stir it; boil it three times in total;

[0008] S2. After cooling, take out the silk and squeeze out the water, wash it with deionized water for 4 to 8 times, and test it with pH test paper until it is neutral; then squeeze out the water from the silk after the sericin is removed, and dry it in an oven at 40 to 45°C overnight;

[0009] S3. Place the dried silk into a 9.3 mol / L LiBr solution to ensure complete immersion, and heat in a 60°C water bath for 1 to 4 hours until there is no obvious filament in the solution, indicating that the silk is completely dissolved and the solution is dark yellow in color;

[0010] S4. Centrifuge the above solution to remove insoluble matter, put it into 8-14K ​​dialysis bag, dialyze it with deionized water for three days, centrifuge the dialyzed silk fibroin again, and pass the supernatant through 0.45μm and 0.22μm filter membranes in turn to obtain regenerated silk fibroin solution.

[0011] Preferably, the centrifugal speed in step S4 is 11000-13000 rpm, and the time is 15-25 min.

[0012] Preferably, the mass concentration of silk fibroin in the obtained regenerated silk fibroin solution is 1% to 5%.

[0013] Preferably, the volume of the LiBr solution in S3 is 25 to 30 mL.

[0014] Preferably, the dialysis temperature in S4 is 4°C.

[0015] It is further defined that the ultrafiltration is carried out in the following steps:

[0016] (1) placing the regenerated silk fibroin solution into an ultrafiltration tube with a molecular weight cutoff of 50 KDa, and centrifuging to separate the regenerated silk fibroin solution with a molecular weight > 50 KDa;

[0017] (2) placing the regenerated silk fibroin solution of less than 50 KDa filtered through the ultrafiltration tube in step (1) into an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and centrifuging to obtain a regenerated silk fibroin solution with a molecular weight of 30 to 50 KDa;

[0018] (3) freeze-drying the ultrafiltered regenerated silk fibroin solution to obtain regenerated silk fibroin RSF with a molecular weight of 30 to 50 KDa and >50 KDa (30～50KDa) and RSF (>50KDa) .

[0019] Preferably, the centrifugal force of the centrifugation in step (1) and step (2) is 5000g and the temperature is 4°C.

[0020] Preferably, the freeze-drying temperature in step (3) is -60 to -80°C.

[0021] It is further defined that the method for self-assembly of silk fibroin into spheres is as follows: freeze-dried regenerated silk fibroin is dissolved in deionized water to prepare a 10-30 mg / mL regenerated silk fibroin solution, anhydrous ethanol is added dropwise under stirring, stirring is continued for 2-5 minutes, and then the solution is placed in a -20°C refrigerator and frozen for 18-30 hours, and then allowed to stand and thaw at room temperature. After centrifugation, the precipitate is washed three times with deionized water, and finally the precipitate is freeze-dried to obtain silk fibroin nanoparticles.

[0022] Preferably, the volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:1.

[0023] Preferably, the centrifugal speed is 12000-15000 rpm, and the time is 10-20 min.

[0024] Beneficial effects of the present invention:

[0025] The present invention utilizes ultrafiltration to separate silk fibroin with different molecular weights, and then adds anhydrous ethanol to the silk fibroin solution to induce the silk fibroin to transform from a hydrophilic random coil structure to a hydrophobic β-folded structure, and then the β-folded structure further self-assembles into a nucleus, and finally utilizes the shear force formed during the freezing process to cause the formed nucleus to further self-assemble into nanoparticles. The preparation method described in the present invention is simple and pollution-free, and the materials and equipment used are readily available, suitable for large-scale and stable production; the preparation process conditions are mild, and no toxic reagents are introduced, and the resulting silk fibroin nanoparticles have good biocompatibility. The silk fibroin RSF with a molecular weight of 30 to 50 KDa and >50 KDa obtained by ultrafiltration in the present invention (30～50KDa) and RSF (>50KDa) Self-assembled silk fibroin nanoparticles SFNPs (30～50KDa) and SFNPs (>50KDa) The particle size is uniform and smaller, which is beneficial to cell uptake and controlled drug release as a drug delivery carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 SFNPs obtained in Example 1(<30KDa) Scanning electron microscopy images;

[0027] Figure 2 SFNPs obtained in Example 1 (30～50KDa) Scanning electron microscopy images;

[0028] Figure 3 SFNPs obtained in Example 1 (>50KDa) Scanning electron microscopy images;

[0029] Figure 4 This is the scanning electron microscope image of SFNPs obtained in Comparative Example 1;

[0030] Figure 5 The regenerated silk fibroin RSF obtained in Example 1 (<30KDa) 、RSF (30～50KDa) 、RSF (>50KDa) And the high performance liquid chromatogram of the regenerated silk fibroin RSF obtained in Comparative Example 1. DETAILED DESCRIPTION

[0031] Embodiment 1:

[0032] The preparation method of a silk fibroin nanoparticle of the present embodiment includes three processes: extraction of silk fibroin, ultrafiltration separation of regenerated silk fibroin of different molecular weights, and self-assembly of silk fibroin nanoparticles into spheres. Specifically, the method is carried out in the following steps:

[0033] Step 1: Preparation of regenerated silk fibroin solution

[0034] (a) Weigh 8 g of silk and place it in 800 mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 30 min while stirring. After cooling down, take out the silk and squeeze out the water, then place it in a new 0.5% Na2CO3 solution and boil it for 30 min while stirring. Repeat the third boiling of the silk.

[0035] (b) After cooling, the silk was taken out and squeezed to dry, and then washed with deionized water for 6 times, and pH test paper was used to detect neutrality; then the silk from which the sericin was removed was squeezed to dry, and dried in an oven at 42° C. overnight;

[0036] (c) Place the dried silk into 27 mL of 9.3 mol / L LiBr solution to ensure complete infiltration, and heat in a water bath at 60 °C for 3 h until there is no obvious filament in the solution, indicating that the silk is completely dissolved and the solution is dark yellow;

[0037] (d) The above solution was centrifuged (12000 rpm, 20 min) to remove insoluble matter, and placed in a 8-14K ​​dialysis bag with deionized water. The dialyzed silk fibroin was centrifuged again (12000 rpm, 20 min), and the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes in turn to obtain a regenerated silk fibroin solution.

[0038] Step 2: Ultrafiltration to separate regenerated silk fibroin with different molecular weights

[0039] (1) The regenerated silk fibroin solution extracted by the Na2CO3 / LiBr system is placed in an ultrafiltration tube with a molecular weight cutoff of 50 KDa, and centrifuged at 4°C and 5000 g to separate the regenerated silk fibroin solution with a molecular weight > 50 KDa;

[0040] (2) placing the regenerated silk fibroin solution of less than 50 KDa filtered through the ultrafiltration tube in step (1) into an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and centrifuging at 4°C and 5000 g to obtain regenerated silk fibroin solutions of molecular weights of less than 30 KDa and 30 to 50 KDa, respectively;

[0041] (3) The regenerated silk fibroin solution obtained by ultrafiltration was freeze-dried at -70°C to obtain regenerated silk fibroin RSF with molecular weights <30 KDa, 30-50 KDa and >50 KDa (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) .

[0042] Step 3: Self-assembly of silk fibroin nanoparticles into spheres

[0043] S1. Weigh the freeze-dried regenerated silk fibroin RSF with molecular weight <30KDa, 30-50KDa and >50KDa respectively. (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) , add deionized water and vortex to dissolve it to prepare a 20 mg / mL regenerated silk fibroin solution;

[0044] S2. Add anhydrous ethanol dropwise to the regenerated silk fibroin solution under stirring (the volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:1), continue stirring for 3 minutes, and then freeze in a -20°C refrigerator for 24 hours;

[0045] S3, standing and thawing at room temperature, the silk protein solution turned milky white, centrifuged (13000rpm, 15min), the precipitate was washed three times with deionized water, and finally the precipitate was freeze-dried to obtain silk protein nanoparticles SFNPs prepared from regenerated silk fibroin with different molecular weights (<30KDa) SFNPs (>50KDa)and SFNPs (30～50KDa) .

[0046] Embodiment 2:

[0047] The preparation method of a silk fibroin nanoparticle of the present embodiment includes three processes: extraction of silk fibroin, ultrafiltration separation of regenerated silk fibroin of different molecular weights, and self-assembly of silk fibroin nanoparticles into spheres. Specifically, the method is carried out in the following steps:

[0048] Step 1: Preparation of regenerated silk fibroin solution

[0049] (a) Weigh 4 g of silk and place it in 1000 mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 15 min while stirring. After cooling down, take out the silk and squeeze out the water, then place it in a new 0.5% Na2CO3 solution and boil it for 15 min while stirring. Repeat the third boiling of the silk.

[0050] (b) After cooling, the silk was taken out and squeezed to dry, and then washed with deionized water for 4 times and pH test paper was used to detect neutrality; then the silk from which the sericin was removed was squeezed to dry, and dried in an oven at 40° C. overnight;

[0051] (c) Place the dried silk into 25 mL of 9.3 mol / L LiBr solution to ensure complete immersion, and heat in a water bath at 60 °C for 1 h until there is no obvious filament in the solution, indicating that the silk is completely dissolved and the solution is dark yellow;

[0052] (d) The above solution was centrifuged (12000 rpm, 20 min) to remove insoluble matter, and placed in a 8-14K ​​dialysis bag with deionized water. The dialyzed silk fibroin was centrifuged again (11000 rpm, 15 min), and the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes in turn to obtain a regenerated silk fibroin solution.

[0053] Step 2: Ultrafiltration to separate regenerated silk fibroin with different molecular weights

[0054] (1) The regenerated silk fibroin solution extracted by the Na2CO3 / LiBr system is placed in an ultrafiltration tube with a molecular weight cutoff of 50 KDa, and centrifuged at 4°C and 5000 g to separate the regenerated silk fibroin solution with a molecular weight > 50 KDa;

[0055] (2) placing the regenerated silk fibroin solution of less than 50 KDa filtered through the ultrafiltration tube in step (1) into an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and centrifuging at 4°C and 5000 g to obtain regenerated silk fibroin solutions of molecular weights of less than 30 KDa and 30 to 50 KDa, respectively;

[0056] (3) The regenerated silk fibroin solution obtained by ultrafiltration was freeze-dried at -60°C to obtain regenerated silk fibroin RSF with molecular weights <30 KDa, 30-50 KDa and >50 KDa (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) .

[0057] Step 3: Self-assembly of silk fibroin nanoparticles into spheres

[0058] S1. Weigh the freeze-dried regenerated silk fibroin RSF with molecular weight <30KDa, 30-50KDa and >50KDa respectively. (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) , add deionized water and vortex to dissolve it to prepare a 10 mg / mL regenerated silk fibroin solution;

[0059] S2. Add anhydrous ethanol dropwise to the regenerated silk fibroin solution under stirring (the volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:1), continue stirring for 2 minutes, and then freeze in a -20°C refrigerator for 18 hours;

[0060] S3, standing and thawing at room temperature, the silk fibroin solution turned milky white, centrifuged (12000rpm, 10min), the precipitate was washed three times with deionized water, and finally the precipitate was freeze-dried to obtain silk fibroin nanoparticles SFNPs prepared from regenerated silk fibroin with different molecular weights (<30KDa) SFNPs (>50KDa) and SFNPs (30～50KDa) .

[0061] Embodiment 3:

[0062] The preparation method of a silk fibroin nanoparticle of the present embodiment includes three processes: extraction of silk fibroin, ultrafiltration separation of regenerated silk fibroin of different molecular weights, and self-assembly of silk fibroin nanoparticles into spheres. Specifically, the method is carried out in the following steps:

[0063] Step 1: Preparation of regenerated silk fibroin solution

[0064] (a) Weigh 12 g of silk and place it in 600 mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 45 minutes while stirring; after cooling down, take out the silk and squeeze out the water, place it in a new 0.5% Na2CO3 solution, and boil it for 45 minutes while stirring; repeat the third boiling of the silk;

[0065] (b) After cooling, the silk was taken out and squeezed to dry, and then washed with deionized water for 8 times, and pH test paper was used to detect neutrality; then the silk from which the sericin was removed was squeezed to dry, and dried in an oven at 45° C. overnight;

[0066] (c) Place the dried silk into 30 mL of 9.3 mol / L LiBr solution to ensure complete infiltration, and heat in a water bath at 60 °C for 4 h until there is no obvious filament in the solution, indicating that the silk is completely dissolved and the solution is dark yellow;

[0067] (d) The above solution was centrifuged (12000 rpm, 20 min) to remove insoluble matter, and placed in a 8-14K ​​dialysis bag with deionized water. The dialyzed silk fibroin was centrifuged again (13000 rpm, 25 min), and the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes in turn to obtain a regenerated silk fibroin solution.

[0068] Step 2: Ultrafiltration to separate regenerated silk fibroin with different molecular weights

[0069] (1) The regenerated silk fibroin solution extracted by the Na2CO3 / LiBr system is placed in an ultrafiltration tube with a molecular weight cutoff of 50 KDa, and centrifuged at 4°C and 5000 g to separate the regenerated silk fibroin solution with a molecular weight > 50 KDa;

[0070] (2) placing the regenerated silk fibroin solution of less than 50 KDa filtered through the ultrafiltration tube in step (1) into an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and centrifuging at 4°C and 5000 g to obtain regenerated silk fibroin solutions of molecular weights of less than 30 KDa and 30 to 50 KDa, respectively;

[0071] (3) The regenerated silk fibroin solution obtained by ultrafiltration was freeze-dried at -80°C to obtain regenerated silk fibroin RSF with molecular weights of <30 KDa, 30-50 KDa and >50 KDa. (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) .

[0072] Step 3: Self-assembly of silk fibroin nanoparticles into spheres

[0073] S1. Weigh the freeze-dried regenerated silk fibroin RSF with molecular weight <30KDa, 30-50KDa and >50KDa respectively. (<30KDa) 、RSF (30～50KDa) and RSF (>50KDa) , add deionized water and vortex to dissolve it to prepare a 30 mg / mL regenerated silk fibroin solution;

[0074] S2. Add anhydrous ethanol dropwise to the regenerated silk fibroin solution under stirring (the volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:1), continue stirring for 5 minutes, and then freeze in a -20°C refrigerator for 30 hours;

[0075] S3, standing and thawing at room temperature, the silk fibroin solution turned milky white, centrifuged (15000rpm, 20min), the precipitate was washed three times with deionized water, and finally the precipitate was freeze-dried to obtain silk fibroin nanoparticles SFNPs prepared from regenerated silk fibroin with different molecular weights (<30KDa) SFNPs (>50KDa) and SFNPs (30～50KDa) .

[0076] Comparative Example 1:

[0077] The preparation process of the silk fibroin nanoparticles SFNPs of this embodiment is as follows:

[0078] (1) Weigh 8 g of silk and place it in 800 mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 30 min while stirring. After cooling down, take out the silk and squeeze out the water, place it in a new 0.5% Na2CO3 solution, and boil it for 30 min while stirring. Repeat the third boiling of the silk.

[0079] (2) After cooling, the silk was taken out and squeezed to dry, and then washed with deionized water for 6 times and pH test paper was used to detect neutrality; then the silk from which the sericin was removed was squeezed to dry, and dried in an oven at 42° C. overnight;

[0080] (3) Place the dried silk into 27 mL of 9.3 mol / L LiBr solution to ensure complete immersion, and heat in a water bath at 60 °C for 3 h. If there is no obvious filament in the solution, the silk is completely dissolved and the solution is dark yellow in color.

[0081] (4) The above solution was centrifuged (12000 rpm, 20 min) to remove insoluble matter, and placed in a 8-14K ​​dialysis bag with deionized water. It was dialyzed at 4°C for three days. The dialyzed silk fibroin was centrifuged again (12000 rpm, 20 min), and the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes in turn to obtain a regenerated silk fibroin solution. After freeze-drying at -70°C, the regenerated silk fibroin RSF was obtained.

[0082] (5) Weighing the freeze-dried regenerated silk fibroin, adding deionized water and vortexing to dissolve it, to prepare a 20 mg / mL regenerated silk fibroin solution;

[0083] (6) adding anhydrous ethanol dropwise to the regenerated silk fibroin solution under stirring (the volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:1), stirring for 3 min, and then freezing in a -20 °C refrigerator for 24 h;

[0084] (7) The solution was allowed to thaw at room temperature until the silk fibroin solution turned milky white. After centrifugation (12000 rpm, 10 min), the precipitate was washed three times with deionized water and finally freeze-dried to obtain silk fibroin nanoparticles SFNPs.

[0085] Detection test:

[0086] Experiment 1: Characterization of the microscopic morphology of silk fibroin:

[0087] The silk fibroin nanoparticles SFNPs obtained in Example 1 were (<30KDa) SFNPs (30～50KDa) and SFNPs (>50KDa) The silk fibroin nanoparticles SFNPs obtained in Example 1 were ultrasonically dispersed in deionized water and appropriately diluted, and then dropped onto an aluminum foil attached to a conductive sample stage. After being allowed to stand and dry naturally at room temperature, they were sprayed with gold for 45 seconds under vacuum. The microscopic morphology and particle size of the nanoparticles were observed using a scanning electron microscope. The results are as follows: Figure 1 , Figure 2 , Figure 3 and Figure 4 shown.

[0088] Depend on Figure 1 , Figure 2 , Figure 3 and Figure 4 It can be seen that the silk fibroin nanoparticles prepared from regenerated silk fibroin with molecular weight <30KDa, 30-50KDa and >50KDa and regenerated silk fibroin without ultrafiltration separation are all regular spherical morphology with rough surface and good dispersion of nanoparticles. The particle size of silk fibroin nanoparticles SFNPs prepared from regenerated silk fibroin without ultrafiltration separation is uneven and large in size. The nanoparticles SFNPs prepared from regenerated silk fibroin with molecular weight 30-50KDa and >50KDa after ultrafiltration separation are (>50KDa) and SFNPs (30～50KDa) The particle size is uniform and smaller. The larger the molecular weight of silk fibroin, the smaller the particle size of the prepared nanoparticles, that is, SFNPs (>50KDa) <SFNPs (30～50KDa) <SFNPs (<30KDa) This may be because the larger the molecular weight of the silk fibroin, the more hydrophobic / hydrophilic segments it retains, and it is easier to transform into a β-folded structure under the induction of anhydrous ethanol, thereby forming a dense core, so the particle size of the obtained nanoparticles is smaller. The present invention uses a scanning electron microscope to observe the silk fibroin nanoparticles obtained in Example 2 and Example 3, and obtains the same conclusion as Example 1, that is, the nanoparticles SFNPs prepared from regenerated silk fibroin with a molecular weight of 30 to 50 KDa and >50 KDa after ultrafiltration separation (>50KDa) and SFNPs (30～50KDa) The particle size is uniform and smaller.

[0089] Experiment 2: High performance liquid chromatography analysis of the molecular weight of silk fibroin:

[0090] Used on Agilent 1260 Infinity II HPLC Protein BEH SEC 3.5μm chromatographic column for regenerated silk fibroin RSF, ultrafiltration separation of molecular weight <30KDa, 30~50KDa and >50KDa silk fibroin RSF (<30KDa) 、RSF (30～50KDa) 、RSF (>50KDa) All samples were filtered through a 0.22 μm filter membrane before loading, with a loading volume of 100 μL, a flow rate of 0.6 mL / min, and a running time of 50 min. The mobile phase used was 100 mM phosphate, 150 mM NaCl buffer (pH 6.0). The results are shown in Figure 5 As shown:

[0091] Since SEC-HPLC has a size exclusion function, the larger the molecular weight of a protein, the earlier it will flow through SEC-HPLC. Therefore, the molecular weight of a protein can be determined based on this phenomenon. Figure 5 It can be seen that compared with the regenerated silk fibroin RSF without ultrafiltration separation, the silk fibroin RSF with a molecular weight > 50KDa obtained by ultrafiltration separation (>50KDa) Mainly concentrated in the leftmost peak, and the peaks on the left of the two are similar; Silk protein RSF with molecular weight <30KDa (<30KDa) The protein peak is mainly concentrated on the right side of the regenerated silk protein, and the right peaks of the two are similar; while the silk protein RSF with a molecular weight between 30 and 50 KDa (<30～50KDa) The protein peak is mainly concentrated in the middle peak position of the regenerated silk fibroin; the order of the peak time of silk fibroin with different molecular weights is: RSF (>50KDa) <RSF (<30～50KDa) <RSF (<30KDa) ; In summary, regenerated silk fibroin can be divided into three types of silk fibroin with molecular weight <30KDa, 30-50KDa and >50KDa through ultrafiltration treatment.

[0092] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing silk fibroin nanoparticles, characterized in that: Specifically, the silk was extracted to obtain a regenerated silk fibroin solution, and then the regenerated silk fibroin solution was ultrafiltered using ultrafiltration tubes with a molecular weight cutoff of 50 KDa and 30 KDa to obtain a regenerated silk fibroin solution with a molecular weight of 30-50 KDa and >50 KDa, and then freeze-dried to obtain regenerated silk fibroin RSF (30～50KDa) and RSF (>50KDa) , and then use anhydrous ethanol to induce the regenerated silk fibroin to self-assemble into spheres.

2. The preparation method according to claim 1, characterized in that: The extraction is carried out according to the following steps: S1. Weigh 4-12g of silk and put it into 600-1000mL of 0.5% Na2CO3 solution, heat it in a water bath, and boil it for 15-45min, stirring it during the process; after cooling down, take out the silk and squeeze out the water, put it into 0.5% Na2CO3 solution again, boil it for 15-45min, and stir it; boil it three times in total; S2. After cooling, take out the silk and squeeze out the water, wash it with deionized water for 4 to 8 times, and test it with pH test paper until it is neutral; then squeeze out the water from the silk after the sericin is removed, and dry it in an oven at 40 to 45°C overnight; S3, placing the dried silk into a 9.3 mol / L LiBr solution to ensure complete immersion, and heating in a 60°C water bath for 1 to 4 h until no obvious filaments remain in the solution; S4. Centrifuge the above solution to remove insoluble matter, put it into 8-14K ​​dialysis bag, dialyze it with deionized water for three days, centrifuge the dialyzed silk fibroin again, and pass the supernatant through 0.45μm and 0.22μm filter membranes in turn to obtain regenerated silk fibroin solution.

3. The preparation method according to claim 2, characterized in that: The centrifugal speed in S4 is 11000-13000 rpm, and the time is 15-25 min.

4. The preparation method according to claim 2, characterized in that: The mass concentration of silk fibroin in the obtained regenerated silk fibroin solution is 1% to 5%.

5. The preparation method according to claim 2, characterized in that: The ultrafiltration is carried out according to the following steps: (1) placing the regenerated silk fibroin solution into an ultrafiltration tube with a molecular weight cutoff of 50 KDa, and centrifuging to separate the regenerated silk fibroin solution with a molecular weight > 50 KDa; (2) placing the regenerated silk fibroin solution of less than 50 KDa filtered through the ultrafiltration tube in step (1) into an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and centrifuging to obtain a regenerated silk fibroin solution with a molecular weight of 30 to 50 KDa; (3) freeze-drying the ultrafiltered regenerated silk fibroin solution to obtain regenerated silk fibroin RSF with a molecular weight of 30 to 50 KDa and >50 KDa (30～50KDa) and RSF (>50KDa) .

6. The preparation method according to claim 5, characterized in that: The centrifugal force of the centrifugation in step (1) and step (2) is 5000g and the temperature is 4°C.

7. The preparation method according to claim 5, characterized in that: The freeze-drying temperature in step (3) is -60 to -80°C.

8. The preparation method according to claim 5, characterized in that: The method for self-assembling silk fibroin into spheres is as follows: freeze-dried regenerated silk fibroin is dissolved in deionized water to prepare a 10-30 mg / mL regenerated silk fibroin solution, anhydrous ethanol is added dropwise under stirring, stirring is continued for 2-5 minutes, and then the solution is placed in a -20°C refrigerator for 18-30 hours, and then allowed to stand and thaw at room temperature. After centrifugation, the precipitate is washed three times with deionized water, and finally the precipitate is freeze-dried to obtain silk fibroin nanoparticles.

9. The preparation method according to claim 8, characterized in that: The volume ratio of the regenerated silk fibroin solution to anhydrous ethanol is 10:

1.

10. The preparation method according to claim 8, characterized in that: The centrifugal speed is 12000-15000 rpm, and the time is 10-20 min.

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