Preparation method of silk fibroin particles and preparation method of drug-loaded particles

Abstract

The invention belongs to the technical field of material extraction, and particularly relates to a preparation method of silk fibroin particles and a preparation method of drug-loaded particles. The invention provides a preparation method of silk fibroin particles, which comprises the following steps: degumming silkworm cocoons to obtain degummed silk; the degummed silk is dissolved, a dissolved solution is obtained, and reagents adopted for dissolution comprise CaCl2, H2O and CH2OH; purifying the dissolving solution to obtain a silk fibroin solution; mixing the silk fibroin solution with absolute ethyl alcohol, and carrying out self-polymerization to obtain a self-polymer; and dispersing the autopolymer to obtain the silk fibroin particles. According to the invention, absolute ethyl alcohol is utilized to induce a silk fibroin solution to form particles, the obtained particles are narrow in particle size range and uniform in size, and the particle size uniformity, the drug loading efficiency and the stability can be further balanced. Meanwhile, the preparation method provided by the invention is safe, environment-friendly, low in cost and high in particle yield.

Description

Technical Field

[0001] The invention belongs to the technical field of material extraction, and particularly relates to a method for preparing silk fibroin microparticles and a method for preparing drug-loaded microparticles. Background Art

[0002] Silk fibroin (SF) is a natural polymer extracted from silk. With its excellent biocompatibility, degradability, and tunable mechanical properties, it exhibits tremendous potential for application in biomedicine, tissue engineering, drug delivery, and other fields. Bombyx mori silk, the most common source of SF, primarily consists of SF (70-80% of dry weight) and sericin (20-30% of dry weight). The extraction process involves degumming, dissolution, purification, and regeneration to produce a highly pure SF solution. By further manipulating the regeneration conditions, silk fibroin materials in various forms, such as films, microparticles, and hydrogels, can be prepared.

[0003] The preparation of silk fibroin microparticles requires a balance between particle size uniformity, drug loading efficiency, and stability. Currently, these methods rely primarily on self-assembly, emulsification-solvent evaporation, and electrospray ionization. The self-assembly method modulates the pH, ionic strength, or temperature of the solution to induce aggregation of hydrophobic regions of silk fibroin to form microparticles, but the particle size distribution is relatively broad (50 to 500 nm). The emulsification method utilizes surfactants to form an oil / water interface, enabling the preparation of monodisperse microparticles. However, residual organic solvents can cause toxicity issues. While electrospray ionization allows for precise particle size control, it suffers from high equipment costs and low yields. Summary of the Invention

[0004] The present invention aims to provide a method for preparing silk fibroin microparticles and a method for preparing drug-loaded microparticles. The preparation method provided by the present invention is safe and environmentally friendly, and the obtained microparticles are uniform in size.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing silk fibroin microparticles, comprising the following steps:

[0007] Degumming the silk cocoons to obtain degummed silk;

[0008] Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH;

[0009] Purifying the dissolved solution to obtain a silk fibroin solution;

[0010] mixing the silk fibroin solution and anhydrous ethanol to perform self-polymerization to obtain a self-polymer;

[0011] The self-polymer is dispersed to obtain the silk fibroin microparticles.

[0012] Preferably, the degumming agent used in the degumming comprises at least one of Na2CO3 and NaHCO3;

[0013] The degumming agent is used in the form of a degumming agent solution, the mass concentration of the degumming agent solution is 0.5-2.0%; the mass ratio of the silk cocoon to the degumming agent solution is 1:10-20.

[0014] Preferably, the molar ratio of CaCl2, H2O and CH2OH in the reagent is 1:8:2.

[0015] Preferably, the usage ratio of the degummed silk and the reagent is 10 g: 100-200 mL.

[0016] Preferably, the purification method is dialysis;

[0017] The dialysis conditions include: the molecular weight cut-off of the dialysis bag used is 3500-14000Da; the dialysis temperature is 0-4°C, the dialysis time is 48-72h, and the dialysis water is replaced every 6-8h during the dialysis period.

[0018] Preferably, the mass concentration of the silk fibroin solution is 2-12%;

[0019] In the system obtained by mixing the silk fibroin solution and anhydrous ethanol, the volume proportion of anhydrous ethanol is 5-35%.

[0020] Preferably, the self-polymerization is carried out in an ice-water bath with stirring, and the stirring time is 10 to 30 minutes.

[0021] Preferably, after the self-polymerization, the process further comprises centrifuging the obtained system to obtain a self-polymer;

[0022] The rotation speed of the centrifugal separation is 10000-12000 rpm, and the time is 15-30 minutes.

[0023] Preferably, the dispersing process comprises: suspending the self-polymer in deionized water and performing vortex or ultrasonic dispersion;

[0024] The particle size of the silk fibroin microparticles is 100-160 nm.

[0025] The present invention also provides a method for preparing drug-loaded microparticles, comprising the following steps:

[0026] Degumming the silk cocoons to obtain degummed silk;

[0027] Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH;

[0028] Purifying the dissolved solution to obtain a silk fibroin solution;

[0029] mixing the silk fibroin solution, the drug and anhydrous ethanol, and performing self-polymerization to obtain a drug-loaded self-polymer;

[0030] The drug-loaded self-polymer is dispersed to obtain the drug-loaded microparticles.

[0031] The present invention provides a method for preparing silk fibroin microparticles, comprising the following steps: degumming silk cocoons to obtain degummed silk; dissolving the degummed silk to obtain a solution, wherein the dissolution reagents include CaCl2, H2O, and CH2OH; purifying the solution to obtain a silk fibroin solution; mixing the silk fibroin solution with anhydrous ethanol to induce self-polymerization to obtain a self-polymer; and dispersing the self-polymer to obtain the silk fibroin microparticles. The present invention utilizes anhydrous ethanol to induce the silk fibroin solution to form microparticles. The resulting microparticles have a narrow particle size range and are uniform in size, further balancing particle size uniformity, drug loading efficiency, and stability. The preparation method provided by the present invention is also safe, environmentally friendly, low-cost, and has a high yield of microparticles.

[0032] The present invention also provides a method for preparing drug-loaded microparticles. This method can further improve drug encapsulation efficiency and exhibits excellent biocompatibility and biosafety. The drug-loaded microparticles provided herein can be used in the preparation of anti-tumor drugs to effectively control their release and enhance their anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of the process for preparing silk fibroin provided by the present invention;

[0034] Figure 2 1 is the XRD pattern of the silk fibroin solution and silk fibroin particles obtained in Example 1;

[0035] Figure 3 1 is a DSC graph of the silk fibroin solution and silk fibroin microparticles obtained in Example 1;

[0036] Figure 4 IR spectra of the silk fibroin solution and silk fibroin particles obtained in Example 1;

[0037] Figure 5 The ultraviolet spectrum of the silk fibroin solution and silk fibroin particles obtained in Example 1;

[0038] Figure 61 is a Fourier self-deconvolution (FSD) peak fitting diagram of the silk fibroin solution and silk fibroin particles obtained in Example 1;

[0039] Figure 7 1 is a SEM image of the silk fibroin solution and silk fibroin particles obtained in Example 1;

[0040] Figure 8 This is the cumulative release process of the drug-loaded microparticles obtained in Example 4 at a pH of 6.8;

[0041] Figure 9 This is a graph showing the inhibitory effect of the drug-loaded microparticles obtained in Example 4 on the growth of 4T1 cells;

[0042] Figure 10 This figure shows the results of drug-loaded microparticles obtained in Example 4 inducing apoptosis of 4T1 cells within 12 hours;

[0043] Figure 11 This is a statistical analysis chart of the apoptosis level of drug-loaded microparticles obtained in Example 4. DETAILED DESCRIPTION

[0044] The present invention provides a method for preparing silk fibroin microparticles, comprising the following steps:

[0045] Degumming the silk cocoons to obtain degummed silk;

[0046] Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH;

[0047] Purifying the dissolved solution to obtain a silk fibroin solution;

[0048] mixing the silk fibroin solution and anhydrous ethanol to perform self-polymerization to obtain a self-polymer;

[0049] The self-polymer is dispersed to obtain the silk fibroin microparticles.

[0050] The present invention degummes silkworm cocoons to obtain degummed silk. The present invention does not specifically limit the source of the cocoons; any method known to those skilled in the art can be used. In the present invention, prior to degumming, the cocoons are preferably shredded to increase their specific surface area and accelerate degumming. In the present invention, the degumming agent used in the degumming preferably comprises at least one of Na2CO3 and NaHCO3; the degumming agent is preferably used in the form of a degumming agent solution, with a mass concentration of preferably 0.5-2.0%, specifically 0.5%, 1.0%, 1.5%, or 2.0%. In the present invention, the mass ratio (i.e., bath ratio) of the cocoons to the degumming agent solution is preferably 1:10-20, specifically 1:10, 1:15, or 1:20. In the present invention, the degumming is preferably carried out under boiling conditions, and the degumming time is preferably 30-60 minutes, specifically 30 minutes, 40 minutes, 50 minutes, or 60 minutes. In the present invention, after the glue coating, the obtained system is preferably washed with water and dried. The number of times of washing is preferably 3 times, and the drying temperature is preferably 60°C.

[0051] After obtaining the degummed silk, the present invention dissolves the degummed silk to obtain a dissolving solution. In the present invention, the reagents used for the dissolution include CaCl2, H2O and CH2OH; the molar ratio of CaCl2, H2O and CH2OH in the reagent is preferably 1:8:2. The present invention has no special limitation on the preparation method of the reagent, and the raw materials can be directly mixed evenly. In the present invention, the dosage ratio of the degummed silk and the reagent is preferably 10g:100~200mL. In the present invention, the temperature of the dissolution is preferably 70°C; the dissolution is preferably carried out under stirring. In the present invention, after the dissolution, it is also preferred to cool the obtained system and then filter it; the filtration is preferably carried out using a 200-mesh nylon mesh or a 200-mesh filter cloth to remove undissolved particles or impurities by filtration.

[0052] After obtaining the dissolved solution, the present invention purifies the dissolved solution to obtain a silk fibroin solution. In the present invention, the purification method is preferably dialysis; the dialysis conditions preferably include: the molecular weight cutoff of the dialysis bag used is 3500-14000Da; the dialysis temperature is 0-4°C, the time is 48-72h, and the dialysis water is replaced every 6-8h during the dialysis. In the present invention, the silk fibroin solution can be stored at 4°C for 2 weeks. In the present invention, after obtaining the silk fibroin solution, it is also preferably included to dry the silk fibroin solution, the drying method is preferably freeze-drying, and the freeze-drying time is preferably 24-48h.

[0053] After obtaining the silk fibroin, the present invention mixes the silk fibroin solution with anhydrous ethanol to perform self-polymerization to obtain a self-polymer. In the present invention, the volume proportion of anhydrous ethanol in the system obtained by mixing the silk fibroin solution and anhydrous ethanol is preferably 5-35%, specifically 5%, 10%, 20%, 30%, 35%. In the present invention, the self-polymerization is preferably carried out under the conditions of ice water bath and stirring, and the stirring time is preferably 10-30 minutes. In the present invention, after the self-polymerization, it is also preferred to centrifuge the obtained system to obtain a self-polymer; the rotation speed of the centrifugal separation is preferably 10000-12000 rpm, and the time is preferably 15-30 minutes.

[0054] After obtaining the self-polymer, the present invention disperses the self-polymer to obtain the silk fibroin microparticles. In the present invention, the dispersion process preferably includes: suspending the self-polymer in deionized water and performing vortex or ultrasonic dispersion. The present invention has no particular limitation on the vortex or ultrasonic dispersion process, as long as the self-polymer can be redispersed.

[0055] In the present invention, the particle size of the silk fibroin microparticles is preferably 100 to 160 nm.

[0056] The present invention also provides a method for preparing drug-loaded microparticles, comprising the following steps:

[0057] Degumming the silk cocoons to obtain degummed silk;

[0058] Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH;

[0059] Purifying the dissolved solution to obtain a silk fibroin solution;

[0060] mixing the silk fibroin solution, the drug and anhydrous ethanol, and performing self-polymerization to obtain a drug-loaded self-polymer;

[0061] The drug-loaded self-polymer is dispersed to obtain the drug-loaded microparticles.

[0062] In the present invention, the preparation method of the drug-loaded microparticles is preferably based on the preparation method of the silk fibroin microparticles described in the above technical solution, except that the drug to be coated is added during the self-polymerization process.

[0063] In the present invention, the mass ratio of silk fibroin to drug in the silk fibroin solution is preferably 8:1-4. The present invention does not specifically limit the type of drug, and any drug known to those skilled in the art can be used. In a specific embodiment of the present invention, the drug is a soluble biological protein TRAIL (sTRAIL).

[0064] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0065] Figure 1 Schematic diagram of the process for preparing silk fibroin in the present invention.

[0066] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0067] The instruments used in the following embodiments and test examples include: an ultrapure water meter (ULPHW, Zhengzhou ULP Instrument Equipment Co., Ltd.); a pH meter (a-AB33pH, Ohaus International Trade Co., Ltd.); an electronic balance (BT-25S, Sartorius Scientific Instrument Co., Ltd.); a heating magnetic stirrer (HS4, Aika Instrument Equipment Co., Ltd.); a freeze dryer (LGJ-10, Beijing Songyuan Huaxing Technology Development Co., Ltd.); a Fourier transform infrared spectrometer (TENSOR27, Bruker, Germany); an analytical balance (XPR analytical balance, Mettler-Toledo); and a water bath. (TW20, Ulabo Technology Co., Ltd.); ultrasonic cleaner (IDH20, AAM Technology Co., Ltd., Germany); vacuum concentrator (Concentratorplus, Eppendorf AG, Germany); electric constant temperature blast drying oven (BPG-9106B, Shanghai Yiheng Scientific Instrument Co., Ltd.); high-speed refrigerated centrifuge (5430R, Eppendorf AG, Germany); amino acid analyzer (S433D, Secham Scientific Instrument Co., Ltd., Germany); XRD powder diffractometer (smartlab-SE, Rigaku Corporation, Japan); differential scanning calorimeter (DSC 3500, Netzsch Instrument Manufacturing Co., Ltd., Germany); thermogravimetric analyzer (TGA4000, PerkinElmer, USA).

[0068] The materials and reagents used include: silkworm cocoons (purchased from the Northwest Silkworm Breeding Base); sodium chloride (Shanghai Shenggong Bioengineering Co., Ltd.); anhydrous sodium carbonate (Nantong Feiyu Biotechnology Co., Ltd.), anhydrous calcium chloride (Nanjing Weiwo Biotechnology Co., Ltd.), anhydrous ethanol (Nanjing Wanqing Chemical Glass Instrument Co., Ltd.), sodium chloride (Shanghai Titan Technology Co., Ltd.); dialysis bags (Nanjing Kemer Biotechnology Center).

[0069] Example 1

[0070] Weigh 10 g of anhydrous CaCl2, add 20 mL of anhydrous ethanol, and slowly add 80 mL of deionized water while stirring until completely mixed to obtain the reagent;

[0071] After the cocoons were cut into pieces, a 0.5 wt% Na2CO3 solution was added at a bath ratio of 1:20 for boiling and degumming for 30 minutes; then the cocoons were cleaned with deionized water, and the water washing was repeated 3 times, and then dried in an oven at 60°C for use to obtain degummed silk;

[0072] Weigh 10 g of dried degummed silk, add 200 mL of the reagent, and then place the mixture in a 70°C water bath to dissolve it while stirring occasionally until it is completely dissolved into a uniform viscous liquid. After cooling the liquid, filter it with a 200-mesh nylon mesh to remove undissolved particles or impurities to obtain a dissolved solution;

[0073] The dissolved solution was transferred to a dialysis bag (molecular weight cut-off of 3500-14000 Da), the dialysis bag was placed in deionized water, and dialyzed at 4° C. for 72 h, during which the dialysis water was replaced every 6-8 h to obtain a silk fibroin solution;

[0074] Adjust the mass concentration of silk fibroin solution to verify the effect of different concentrations of SF solution on the particle size;

[0075] Silk fibroin solutions (SF solutions) with mass concentrations of 2%, 4%, 6%, 8%, 10%, and 12% were mixed with anhydrous ethanol, where the volume proportion of anhydrous ethanol in the resulting system was 25%. The mixture was subjected to self-polymerization in an ice-water bath with stirring for 30 minutes. The resulting system was centrifuged at a speed of 12,000 rpm for 20 minutes to obtain a self-polymer.

[0076] suspending the self-polymer in deionized water and performing ultrasonic dispersion to obtain silk fibroin microparticles;

[0077] The particle size, polydispersity index and particle yield of the obtained silk fibroin microparticles were tested;

[0078] The test results are shown in Table 1;

[0079] Table 1 Characterization of silk fibroin microparticles

[0080]

[0081]

[0082] It can be seen from Table 1 that compared with other concentrations, when the silk fibroin concentration is 8%, the microparticle yield is the highest, reaching 87.0%, and the particle size distribution range is narrow and the dispersibility is good.

[0083] Example 2

[0084] Silk fibroin microparticles were prepared according to the method of Example 1, wherein the mass concentration of the silk fibroin solution was 8%, and the volume proportions of anhydrous ethanol in the obtained system were 5%, 15%, 25% and 35%;

[0085] The particle size, polydispersity index and particle yield of the obtained silk fibroin microparticles were tested;

[0086] The test results are shown in Table 2;

[0087] Table 2 Characterization of silk fibroin microparticles

[0088] Ethanol volume share Particle size / nm Polydispersity Index (PDI) Microparticle yield% 5 117.15±2.83 0.22±0.03 66.52±1.42 15 119.60±1.91 0.29±0.04 70.55±1.56 25 128.27±2.17 0.12±0.02 83.13±2.62 35 133.65±3.59 0.10±0.01 77.01±1.89

[0089] It can be seen from Table 2 that compared with other concentrations, when the silk fibroin concentration is 8% and the volume of anhydrous ethanol is 25%, the microparticle yield is the highest, reaching 83.1%, and the particle size distribution range is narrow and the dispersibility is good.

[0090] Example 3

[0091] Silk fibroin microparticles were prepared according to the method of Example 1, wherein the mass concentration of the silk fibroin solution was 8%, the volume proportion of anhydrous ethanol in the obtained system was 25%, and the mixing and stirring times for inducing the self-polymerization process were 10 minutes, 20 minutes, and 30 minutes, respectively;

[0092] The particle size, polydispersity index and particle yield of the obtained silk fibroin microparticles were tested;

[0093] The test results are shown in Table 3;

[0094] Table 3 Characterization of silk fibroin microparticles

[0095] Time / min Particle size / nm Polydispersity Index (PDI) Microparticle yield% 10 113.62±1.67 0.17±0.02 79.43±1.57 20 118.25±1.58 0.19±0.02 81.52±2.74 30 127.17±2.14 0.13±0.01 80.84±2.83

[0096] As can be seen from Table 3, when the silk fibroin concentration is 8% and the volume of anhydrous ethanol is 25%, continuous stirring for 30 min to induce protein self-aggregation can achieve the highest microparticle yield, which can reach 81.5%, and the particle size distribution range is narrow and the dispersibility is good.

[0097] Performance Testing

[0098] Test Example 1

[0099] The silk fibroin solution obtained in Example 1 was freeze-dried to obtain silk fibroin, and the yield was calculated according to the following formula:

[0100] Yield = mass of silk fibroin / mass of cocoon*100%.

[0101] According to calculation, the yield of silk fibroin in Example 1 is 85.21%.

[0102] Test Example 2

[0103] The silk fibroin solution and silk fibroin microparticles (SF-Spheres) obtained in Example 1 (taking 8% SF solution as an example) were freeze-dried, and the amino acid content distribution of the obtained silk fibroin and silk fibroin microparticles (SF-Spheres) was tested;

[0104] Weigh 50 mg of sample into a 200 mL hydrolysis tube. Hydrolyze with 100 mL of hydrochloric acid solution, freeze in liquid nitrogen, evacuate with a vacuum pump, fill with nitrogen for 1 minute, and tighten the cap. Place the hydrolysis tube in a 110°C constant temperature drying oven and hydrolyze for 24 hours. Mix thoroughly, open the tube, filter, and accurately pipette an appropriate amount of filtrate into a rotary evaporator. Evaporate at 60°C to dryness. If necessary, add a small amount of water and repeat the evaporation process 1–2 times. Redissolve the sample in 3–5 mL of sodium citrate buffer to a concentration of 50–250 nmol / mL. Shake thoroughly, filter, or centrifuge. Separate the supernatant on a sulfonic acid cation exchange column in an amino acid analyzer. The amino acid and ninhydrin were mixed and reacted at 135°C. The primary amine and ninhydrin generated a blue-purple compound, and the secondary amine and ninhydrin generated a yellow compound. The two derivative products were detected by visible light spectrophotometry at wavelengths of 570 nm and 440 nm, respectively. The retention time was used for qualitative analysis, and the external standard working curve method was used for quantitative analysis.

[0105] The specific test results are shown in Table 4:

[0106] Table 4 Amino acid content of silk fibroin and silk fibroin microparticles

[0107]

[0108]

[0109] Amino acid content analysis of SF and SF-Spheres revealed that glycine, alanine, serine, and tyrosine are the primary components of silk fibroin, accounting for over 87% of the total amino acid content. This ratio remained unchanged after ethanol-induced SF-Sphere formation. A hexapeptide sequence consisting of glycine, alanine, serine, and tyrosine, Gly-Ala-Gly-Ala-Gly-Ser, in a ratio of approximately 3:2:1, forms a stable antiparallel β-sheet structure within SF, endowing silk fibroin with excellent drug-carrying properties.

[0110] Test Example 3

[0111] X-ray diffraction (XRD) test;

[0112] The silk fibroin solution and silk fibroin microparticles (SF-Spheres) obtained in Example 1 (taking 8% SF solution as an example) were freeze-dried and then tested using an X-ray diffractometer. Crystals will diffract when irradiated with X-rays, and the spatial distribution orientation and intensity of the diffraction lines are closely related to the crystal structure. Silk is considered to be a semi-crystalline polymer. The ordered aggregation structure in the sample to be tested can be determined by X-ray street light method, that is, the crystal region composed of β-folds or regular helical structures. The freeze-dried sample was torn into small pieces, and a small amount of the small pieces were taken and filled into a sample tank cleaned with ethanol. It was gently flattened with a coverslip and placed on the test bench in the diffractometer to test the internal crystalline structure of the sample. The test conditions were set as follows: the diffraction angle range was 5-80°, the scanning speed was 2° / min, the current was 40mA, the tube voltage was 40kV, and CuKα rays.

[0113] The test results obtained are as follows Figure 2 As shown, from Figure 2 It can be seen that SF shows an obvious β-fold characteristic diffraction single peak at 22°, and the absorption peak is enhanced after ethanol induction (i.e., silk fibroin particles), indicating that the β-fold structure after ethanol-induced reorganization is still the main phase, which is beneficial to increase the drug loading capacity.

[0114] Test Example 4

[0115] Differential scanning calorimetry (DSC);

[0116] Differential scanning calorimetry (DSC) measures the temperature and heat changes of a substance to determine thermal changes in its structure and chemical reaction constants. It can be used to determine the glass transition temperature, melting temperature, and crystallization temperature of silk fibroin-related materials.

[0117] The silk fibroin solution and silk fibroin microparticles (SF-Spheres) obtained in Example 1 (taking 8% SF solution as an example) were freeze-dried and used as samples. The samples were heated from room temperature to 300°C at a heating rate of 10°C / min under nitrogen protection to analyze and measure the thermal stability of the samples. The test results are as follows: Figure 3 shown.

[0118] from Figure 3As can be seen, both SF and SF-Spheres have two distinct endothermic peaks, located near 70°C and 285°C, respectively. The peak around 78°C represents the evaporation of free water within the sample. At this stage, the mass loss of both samples is comparable, indicating similar water contents. As the temperature increases, a distinct endothermic peak appears near 285°C, indicating that the SF molecular chains begin to break from the amorphous to the crystalline region and that its β-pleated structure gradually decomposes under the heat. Furthermore, the β-pleated conformation degradation peak in the DSC curve of the SF raw material is at 282°C, while that of SF-Spheres shifts to 286°C. This is likely due to ethanol-induced transformation of the random coil structure into a β-pleated structure, forming more crystalline structures and enhancing its thermal stability.

[0119] Test Example 5

[0120] Infrared spectroscopy (FT-IR) test conditions: instrument model is Shimadzu IRAffinity-1S, test range is 400~4000cm -1 , resolution 0.5cm -1 ;

[0121] FT-IR spectroscopy can be used to qualitatively analyze and identify protein secondary structures through the presence of amide bonds in the molecular structure.

[0122] The test results obtained are as follows Figure 4 As shown, from Figure 4 It can be seen that both SF and SF-Spheres are at 3500 cm -1 There is a characteristic absorption peak near 1625cm, which is mainly due to the stretching vibration of -OH in the silk protein structure. -1 、1520cm -1 and 1230cm -1 There is a strong characteristic absorption peak on the left and right, with sharp peaks, corresponding to the absorption peaks of silk fibroin amide I, amide II and amide III, respectively, showing a typical β-sheet conformation. Among them, amide I corresponds to a mixed conformation of β-sheet and random coil, indicating that the β-sheet ratio in SF-Spheres increases after ethanol induction.

[0123] Test Example 6

[0124] Ultraviolet spectroscopy (UV);

[0125] Ultraviolet spectroscopy is a traditional analytical method for qualitative and structural analysis of substances. Freshly prepared silk fibroin or silk fibroin microparticles (using an 8% SF solution as an example) are prepared with ultrapure water to a specified concentration of stock solution. This is then diluted stepwise to create seven gradient sample solutions. Using ultrapure water as a control, the control and the sample to be tested are injected into separate quartz cuvettes and placed in corresponding positions on a full-wavelength scanning UV spectrometer for UV absorption analysis. The wavelength scan range is set to 200-400 nm.

[0126] The test results obtained are as follows Figure 5 As shown, from Figure 5 As can be seen, both SF and SF-Spheres exhibit a characteristic tyrosine absorption peak at 280 nm, consistent with the amino acid analysis results, demonstrating that the protein structure remains intact after ethanol induction. Furthermore, the absence of significant impurity peaks in the spectra indicates that the purification process effectively removed residual reagents.

[0127] Test Example 7

[0128] Fourier self-deconvolution (FSD) peak fitting;

[0129] Peak fit software was used to perform FSD peak fitting on the amide I band to further quantitatively characterize the changes in secondary structure components. The test results are shown in Table 5 and Figure 6 As shown;

[0130] Table 5 Ethanol-induced secondary structure changes

[0131] Secondary structure <![CDATA[Wavenumber / cm -1 > Silk fibroin Silk fibroin microparticles β-sheet 1611-1629 23.53 31.09 Random curl 1637-1648 21.96 28.32 α-helix 1646-1661 25.20 22.88 β turn 1665-1694 29.31 17.72

[0132] The results showed that the amide I band (1600-1700 cm -1 ) was fitted by Fourier self-deconvolution peak fitting, and the β-sheet in SF (1611~1629cm -1 , 23.53%), random curl (1637~1648cm -1 , 21.96%), α-helix (1646~1661cm -1 , 25.20%) and β angle (1665~1694cm -1 , 29.31%), SF-Spheres were formed after ethanol-induced β-folding (1611~1629cm -1 , 31.09%), random curl (1637~1648cm -1 , 28.32%), α-helix (1646~1661cm -1 , 22.88%) and β angle (1665~1694cm -1 , 17.72%), confirming that this process promotes the transformation of the random structure into a β-sheet network in SF.

[0133] Test Example 8

[0134] Microscopic morphology observation

[0135] The microscopic morphology of the silk fibroin and silk fibroin microparticles (SF-Spheres) (taking 8% SF solution as an example) in the silk fibroin solution obtained in Example 1 can be observed using a scanning electron microscope.

[0136] Figure 7 The left picture is the SEM image of silk fibroin (scale bar is 200 μm), and the right picture is the TEM image of silk fibroin microparticles;

[0137] SEM observation showed that SF had a porous sponge-like structure, which was beneficial for drug adsorption and sustained release. After ethanol induction, TEM observation showed that a uniform spherical structure was formed, which was beneficial for drug dispersion.

[0138] Example 4

[0139] A silk fibroin solution was prepared according to the method of Example 1;

[0140] An 8% silk fibroin solution, a soluble biological protein TRAIL (sTRAIL), and anhydrous ethanol were mixed, wherein the anhydrous ethanol content in the resulting system was 25%, and the mixture was subjected to self-polymerization in an ice-water bath with stirring for 20 minutes. The resulting system was centrifuged at a speed of 12,000 rpm for 20 minutes to obtain a drug-loaded self-polymer.

[0141] suspending the drug-loaded self-polymer in deionized water and performing ultrasonic dispersion to obtain drug-loaded microparticles;

[0142] The particle size and polydispersity coefficient were measured, the final SF concentration was determined by the BCA method, the sTRAIL content was detected by an ELISA kit, and the drug loading and encapsulation efficiency were calculated. The test results are shown in Table 6.

[0143] Table 6 Test results of drug-loaded microparticles

[0144] SF:sTRAIL Particle size (nm) PDI Drug loading (%) Encapsulation efficiency (%) 8:1 119.6±1.46 0.173±0.12 9.62±0.21 86.55±0.64 8:2 117.2±1.32 0.112±0.14 15.31±0.45 76.05±0.83 8:3 114.8±1.27 0.107±0.13 21.60±0.63 79.19±0.58 8:4 112.3±1.28 0.111±0.10 17.65±0.32 52.09±0.70 8:5 113.5±1.16 0.182±0.07 8.41±0.11 36.67±0.19 8:6 111.2±1.11 0.191±0.09 5.46±0.15 12.75±0.36

[0145] It can be seen that when the mass ratio of silk fibroin to sTRAIL is 8:3, microparticles with uniform particle size and stable dispersion can be obtained, the drug loading capacity can reach 21.6%, and the encapsulation efficiency can reach up to 79.19%.

[0146] The drug-loaded microparticles (SF) were obtained with a mass ratio of SF:sTRAIL of 8:3. sTRAIL ) as an example, in vitro release, cytotoxicity and apoptosis activity tests were performed;

[0147] (1) Using dialysis to simulate physiological environment, study SF sTRAIL In the cumulative release process at pH 6.8, the test results are as follows Figure 8 As described above, the results showed that the release time of sTRAIL can be extended to more than 7 days.

[0148] (2) The MTT method was used to compare the effects of saline, sTRAIL, and SF sTRAIL The toxic effect on 4T1 cells, the experimental results are as follows Figure 9 As shown in the figure, it can be seen that SF can effectively guarantee the anti-tumor activity of sTRAIL, and under the same sTRAIL concentration conditions, SF sTRAIL The anti-tumor activity is stronger.

[0149] (3) Quantitative determination of free sTRAIL and SF by FITCAnnexinV-PI flow cytometry dual-color assay sTRAIL Induced apoptosis ratio of 4T1 cells, the experimental results are as follows Figures 10-11 As shown. SF sTRAIL The ability to induce 4T1 cell apoptosis was slightly stronger than that of free sTRAIL, with apoptosis rates of 23.2% and 27.2%, respectively. This indicates that the SF delivery vector-based encapsulation strategy in this study can effectively ensure the tumor cell apoptosis-promoting activity of sTRAIL.

[0150] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing silk fibroin microparticles, characterized in that: The following steps are involved: Degumming the silk cocoons to obtain degummed silk; Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH; Purifying the dissolved solution to obtain a silk fibroin solution; mixing the silk fibroin solution and anhydrous ethanol to perform self-polymerization to obtain a self-polymer; The self-polymer is dispersed to obtain the silk fibroin microparticles.

2. The preparation method according to claim 1, characterized in that The degumming agent used in the degumming comprises at least one of Na2CO3 and NaHCO3; The degumming agent is used in the form of a degumming agent solution, the mass concentration of the degumming agent solution is 0.5-2.0%; the mass ratio of the silk cocoon to the degumming agent solution is 1:10-20.

3. The preparation method according to claim 1, characterized in that The molar ratio of CaCl2, H2O and CH2OH in the reagent is 1:8:

2.

4. The preparation method according to claim 3, characterized in that The usage ratio of the degummed silk and the reagent is 10g:100-200mL.

5. The preparation method according to claim 1, characterized in that The purification method is dialysis; The dialysis conditions include: the molecular weight cut-off of the dialysis bag used is 3500-14000Da; the dialysis temperature is 0-4°C, the dialysis time is 48-72h, and the dialysis water is replaced every 6-8h during the dialysis period.

6. The preparation method according to claim 1, characterized in that The mass concentration of the silk fibroin solution is 2-12%; In the system obtained by mixing the silk fibroin solution and anhydrous ethanol, the volume proportion of anhydrous ethanol is 5-35%.

7. The preparation method according to claim 1, characterized in that The self-polymerization is carried out in an ice-water bath with stirring, and the stirring time is 10 to 30 minutes.

8. The preparation method according to claim 1, characterized in that After the self-polymerization, the obtained system is centrifuged to obtain a self-polymer; The rotation speed of the centrifugal separation is 10000-12000 rpm, and the time is 15-30 minutes.

9. The preparation method according to claim 1, characterized in that The dispersion process includes: suspending the self-polymer in deionized water and performing vortex or ultrasonic dispersion; The particle size of the silk fibroin microparticles is 100-160 nm.

10. A method for preparing drug-loaded microparticles, characterized in that: The following steps are involved: Degumming the silk cocoons to obtain degummed silk; Dissolving the degummed silk to obtain a dissolving solution, wherein the dissolving reagents include CaCl2, H2O and CH2OH; Purifying the dissolved solution to obtain a silk fibroin solution; mixing the silk fibroin solution, the drug and anhydrous ethanol, and performing self-polymerization to obtain a drug-loaded self-polymer; The drug-loaded self-polymer is dispersed to obtain the drug-loaded microparticles.

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