Coagulation-promoting embolism microsphere and preparation method thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a coagulation-promoting embolism microsphere and a preparation method thereof. The sericin-based embolism microspheres are creatively prepared, and the sericin membrane layer on the surface of the microspheres can prevent the risk that a catheter is solidified during an operation due to thrombin burst release during the operation. On the other hand, sericin in the microspheres has excellent water absorption swelling property and lubricity, and is beneficial to embolism and prevention of reflux; moreover, sericin molecules contain a large amount of hydroxyl and carboxyl, so that the chelation of blood coagulation factor calcium ions is greatly improved. In addition, thrombin factors are loaded into the microspheres, calcium ions are chelated on the surfaces of the microspheres, the microspheres are endowed with rapid embolism capacity through the superimposed effect, blood flow blocking of target blood vessels can be effectively accelerated, and reflux is prevented. The prepared procoagulant embolism microspheres are suitable for being applied to treatment of vascular-rich tumors of the brain and can effectively prevent mistaken closure of many complex functional blood vessels of the brain due to reflux.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a degradable, absorbable, embolization-accelerating, anti-reflux embolization microsphere and a preparation method thereof. BACKGROUND

[0002] Intravascular embolization, as a minimally invasive method, has become an effective strategy for treating tumors and vascular malformations. Embolization microspheres inhibit tumor growth by effectively blocking the blood supply of target blood vessels. Currently, most commercial embolization microspheres are non-degradable, such as Contour SE embolization microspheres based on polyvinyl alcohol, HepaSphere embolization microspheres and Embball embolization microspheres based on acrylic acid polymer, and HydroPearl embolization microspheres based on polyethylene glycol. The embolization microsphere material is not degradable or degrades very slowly in the body, and is usually used for malignant tumor treatment that does not require recanalization, to achieve long-term embolization or permanent occlusion of blood vessels. However, non-degradable materials have the risk of long-term accumulation causing inflammation or immune response. Biodegradable embolization microspheres can reduce post-embolization syndrome, tissue inflammation and fibrosis, and reduce side effects caused by non-target embolization, especially for tumor recurrence types, which can achieve multiple embolization, achieving sustained and effective and controllable treatment. For example, CN117398507A discloses a porous degradable embolization microsphere (PLGA polymer) and a preparation method thereof. However, the problems of embolization materials in clinical use are becoming increasingly prominent, especially the uncontrollable drug release and embolization agent reflux. Moreover, before the present application is disclosed, there has been no report on silk fibroin-based embolization microspheres and the preparation of coagulation-accelerating embolization microspheres.

[0003] Silk fibroin and fibroin protein from silkworm silk are high-quality natural proteins composed of 20 amino acids, have excellent biocompatibility, controllable degradability and low immunogenicity, and are biologically safe materials for in vivo implantation. In particular, silk fibroin contains a large number of active functional groups, which is conducive to multi-channel functional modification. However, there are reports on the use of fibroin protein for microsphere preparation, most of which are used as drug release carriers, such as the preparation and performance of rifampicin-loaded fibroin protein microspheres (Tissue Engineering Research, 2022, 26: 1515-1521) and the like. CN106668845A, CN109157672A and CN118105535A disclose silk fibroin / chitosan, silk fibroin / short peptide and silk fibroin / alginic acid salt microspheres, but do not disclose use as embolization microspheres. According to the present literature query, so far there has been no disclosure of the preparation of silk fibroin-based embolization microspheres and coagulation-accelerating embolization microspheres. The main technical difficulty is that the particle size of the microspheres is difficult to improve, because effective embolization microspheres have strict shape and size requirements, and existing research cannot solve this problem. In addition, the coagulation accelerators disclosed in the existing literature are loaded by covalent cross-linking, surface adsorption or co-assembly into balls, and most of them have the problems of decreased activity of the coagulation accelerator or low loading rate. Summary of the Invention

[0004] Due to the technical bottleneck that silk proteins with excellent biocompatibility cannot meet the requirements for effective embolization, the purpose of this invention is to provide a degradable and absorbable drug-loaded embolization microsphere loaded with coagulation factors. Thrombin factors are loaded inside the microspheres and calcium ions are chelated on the surface of the microspheres. The synergistic effect gives the microspheres rapid embolization ability, which can effectively accelerate the blockage of blood flow in the target blood vessel and prevent reflux.

[0005] A method for preparing embolic microspheres includes the following steps: (1) Preparation of protein aqueous solution: S1. Degumming of raw silkworm silk with sodium carbonate aqueous solution to obtain degummed silk fibroin fibers, and dissolving them in lithium bromide solution to obtain silk fibroin protein solution; then filling the silk fibroin protein solution into a dialysis bag, dialyzing with deionized water and filtering to obtain silk fibroin protein aqueous solution. S2. Raw silkworm silk is dissolved in boiling water to obtain a sericin protein aqueous solution; Preferably, in step (1) S1, the mass concentration of the sodium carbonate aqueous solution is 0.1%, and the ratio of raw silkworm silk to sodium carbonate aqueous solution is 1:50 g / mL; the concentration of the lithium bromide solution is 9.3 M, the ratio of degummed silk fibroin fiber to lithium bromide solution is 1:10 g / mL, and the dissolution temperature is 60~70℃; the dialysis bag is a semi-permeable membrane with a molecular weight cutoff of 14~50 kDa, and dialysis with deionized water is performed for 3 days.

[0006] Preferably, in step (1) S2, the bath ratio of the silkworm raw silk when dissolved in boiling water is 1:20 g / mL, and the dissolution time in boiling water is 1-6 hours.

[0007] (2) Mix the sericin aqueous solution prepared in step (1) with the silk fibroin aqueous solution to obtain a silk fibroin aqueous solution; then add an emulsifier to the oil phase and stir for the first time. After stirring evenly, add the silk fibroin aqueous solution dropwise to the oil phase to carry out the emulsification reaction to obtain a reaction solution; add isopropanol to the reaction solution and stir again to obtain a mixed solution; after centrifuging the mixed solution, collect the precipitate and wash it with isopropanol and deionized water. After washing, collect the precipitate and freeze-dry it to obtain S embolization microspheres.

[0008] Preferably, in step (2), the mass ratio of the sericin aqueous solution to the silk fibroin aqueous solution is 1:10 to 1:100; the concentration of the resulting silk fibroin aqueous solution is 50 to 200 mg / mL; the volume ratio of the emulsifier to the oil phase is 0.5% to 10%, and the volume ratio of the silk fibroin aqueous solution to the oil phase is 1:0 to 1:15; the oil phase includes liquid paraffin; isopropanol is added to the reaction solution, wherein the volume ratio of the silk fibroin aqueous solution to isopropanol is 1:0 to 1:10; The temperature for the first and second stirrings is 50-60℃, for 20-40 minutes, and the speed is 800-900 r / min; the temperature for the emulsification reaction is 50-60℃, and the time is 30-40 minutes; the centrifugation conditions are: 10000 r / min for 15-20 minutes. The freeze-drying process is as follows: the precipitate is frozen at -20°C for 4 hours and then placed on a freeze dryer for freeze-drying for 24 hours.

[0009] (3) First, thrombin is dissolved in physiological saline to obtain thrombin solution; then the S embolization microspheres obtained in step (2) are immersed in a container containing thrombin solution; the container is provided with an inlet and an outlet, and then the thrombin solution is circulated under a certain pressure to continuously penetrate the S embolization microspheres; finally, the S embolization microspheres are taken out and freeze-dried to obtain thrombin-modified ST microspheres. Preferably, in step (3), the concentration of the thrombin solution is 100~1000U / mL, and the ratio of thrombin activity (U) to the mass (mg) of the S-embolization microspheres is (1~15)U:1mg. Under a certain pressure condition of 50~500mmHg, the thrombin solution is circulated, and the continuous penetration time of the S-embolization microspheres is 0.5~5 hours.

[0010] (4) Polyethylene glycol diglycidyl ether (PEGDE) was added to the aqueous solution of sericin to modify it, and a modified sericin solution was obtained; The thrombin-modified ST microspheres were then resuspended in a polyvinyl alcohol aqueous solution, and a modified sericin solution was added and mixed to obtain a mixed solution. The mixed solution was then frozen, thawed, and centrifuged to collect the precipitate, or the mixed solution was dynamically incubated and then centrifuged to collect the precipitate. Finally, the precipitate was washed, centrifuged, and freeze-dried to obtain STSS microspheres. Preferably, in step (4), the concentration of the sericin aqueous solution is 5~30 mg / mL; the mass ratio of the sericin aqueous solution to polyethylene glycol diglycidyl ether is 1:(0.5~1); the mass fraction of the polyvinyl alcohol aqueous solution is 0.5~7%; and the volume ratio of the modified sericin solution to the polyvinyl alcohol aqueous solution is 1:1. The freezing and thawing conditions are as follows: freezing at -20℃ for 4-12 hours, followed by thawing at room temperature; the dynamic incubation conditions are oscillation at 200-800 r / min at room temperature for 2-8 hours; and the rinsing solvent is deionized water.

[0011] (5) The STSS microspheres obtained in step (4) are immersed in calcium chloride solution overnight, and then centrifuged, rinsed and freeze-dried to obtain STSS@Ca silk protein drug-loaded embolization microspheres with dual modification of thrombin and calcium ions, which are the final products of procoagulant embolization microspheres.

[0012] Preferably, in step (5), the concentration of calcium chloride solution is 5~150 mg / mL, and the ratio of STSS microspheres to calcium chloride solution is 1~6 mg:1 mL.

[0013] In addition, various tumor treatment-related drugs can be added to the thrombin solution simultaneously. That is, in addition to the thrombin loading step, tumor treatment drugs can be loaded; furthermore, tumor treatment-related drugs can be loaded simultaneously in the reaction system for preparing microspheres and in the calcium ion loading step. These drugs include doxorubicin, paclitaxel, camptothecin, 5-fluorouracil, cisplatin, etc. Beneficial effects

[0014] The technical solution and inventiveness of this invention lie in the fact that the developed sericin-based embolic microspheres not only possess highly efficient embolic capacity but also maintain continuous inhibition of blood flow recurrence as the microspheres degrade. Because thrombin has a strong coagulation effect, the sericin membrane layer on the surface of the microspheres can prevent the risk of catheter coagulation during surgery due to sudden thrombin release. After the embolic microspheres are delivered to the target vascular site, calcium ions on the surface of the microspheres rapidly activate the coagulation system. As the sericin layer swells and ruptures, the thrombin in the microspheres is rapidly released through diffusion, accelerating coagulation and preventing backflow of the embolic microspheres.

[0015] On the other hand, the sericin in the microspheres has excellent water absorption, swelling, and lubrication properties, which help to embolize and prevent reflux. The design of the sericin membrane on the surface of the microspheres is not only to prevent the adverse effects of thrombin on the surgical procedure, but also because the sericin molecules contain a large number of hydroxyl and carboxyl groups, which greatly enhances the chelation of coagulation factor calcium ions.

[0016] Furthermore, the closed-loop dialysis method employed in this invention can significantly improve the encapsulation of thrombin or drugs, allowing for dosage adjustments based on treatment needs. The blood vessels in the brain are not only fine but also possess a rich and complex vascular network closely linked to human behavior, function, and life. Microspheres with a diameter of 45-150 μm are the most effective size for embolization treatment of conditions such as meningiomas. The procoagulant embolization microspheres prepared in this invention have a particle size distribution of 40-145 μm and a hemolysis rate of less than 0.4%, making them suitable for the treatment of highly vascularized tumors in the brain. This method can effectively prevent the accidental closure of many complex functional blood vessels in the brain due to reflux. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] Example 1: 1. Raw silkworm silk was placed in a 0.1% sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL and heated three times at 98-100℃ for 30 minutes each time. After each heating treatment, the silk was thoroughly rinsed with deionized water. After heating treatment, the silk was dried in a 60℃ oven for 12 hours to obtain degummed fibroin fibers. The degummed fibroin fibers were weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a water bath at 65±5℃ to obtain a fibroin protein solution. The fibroin protein solution was then poured into a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed with deionized water for 3 days to obtain a purified fibroin protein aqueous solution.

[0020] 2. Silkworm raw silk was dissolved in boiling water at 100℃ for 4 hours at a bath ratio of 1:20 g / mL to obtain a sericin aqueous solution; Then, the aqueous solutions of sericin and silk fibroin were mixed at a mass ratio of 1:24 to obtain an aqueous solution of silk fibroin with a concentration of 100 mg / mL.

[0021] 3. Using liquid paraffin as the oil phase, add 2% Span-80 emulsifier according to the volume of the oil phase, and stir at 900 r / min at 55℃ for 30 minutes; then add the silk protein aqueous phase solution dropwise to the oil phase at a water-to-oil volume ratio of 1:8, and stir at 55℃ for 30 minutes to emulsify and obtain a reaction solution; finally, slowly add isopropanol to the reaction solution at a silk protein aqueous phase solution to isopropanol volume ratio of 1:4, and stir at 55℃ for 30 minutes to obtain a mixed solution; centrifuge the mixed solution at 10000 r / min for 20 minutes, discard the supernatant, collect the precipitate, wash the precipitate thoroughly with isopropanol and deionized water, centrifuge again, collect the precipitate, freeze at -20℃ for 4 hours, and then freeze-dry it in a freeze dryer for 24 hours to obtain S-embolization microspheres.

[0022] 4. First, dissolve thrombin in physiological saline to obtain a thrombin solution (concentration of 500 U / mL). Select a closed container with an inlet and an outlet, place S-embolization microspheres in the container, and add thrombin solution at a concentration of 5 U / mg relative to the S-embolization microspheres to the closed container containing the microspheres. Immerse the S-embolization microspheres in the thrombin solution (preferably submerging the S-embolization microspheres). Under a pressure of 50 mmHg, maintain the thrombin solution level above the microspheres and allow it to circulate continuously. The flowing solution continuously penetrates the S-embolization microspheres for 1 hour. Finally, remove the S-embolization microspheres, freeze-dry them, and obtain thrombin-modified ST microspheres.

[0023] 5. Adjust the concentration of the sericin aqueous solution to 10 mg / mL, and then add polyethylene glycol diglycidyl ether at a mass ratio of 1:0.5 to modify it, thus obtaining the modified sericin solution.

[0024] The thrombin-modified ST microspheres were resuspended in a 2% (w / w) polyvinyl alcohol aqueous solution. Then, a modified sericin solution was added in equal volume to the polyvinyl alcohol aqueous solution and mixed. The precipitated microspheres were collected by freezing, thawing, and centrifugation. After rinsing, centrifugation, and freeze-drying, STSS microspheres were obtained.

[0025] In the above-mentioned preparation process of embolic microspheres, various anti-tumor drugs can be simultaneously loaded into the reaction system for microsphere preparation and during the thrombin loading process, depending on the characteristics of the drug (such as hydrophilicity and hydrophobicity). Especially in the circulating drug delivery technology, the required dosage and release rate can be controlled according to the therapeutic effect. Testing showed that the prepared microspheres had a particle size distribution of 40–100 μm, a hemolysis rate of less than 0.4%, and exhibited excellent coagulation and embolization properties. Compared to Example 5, the whole blood clotting time was reduced by 55%.

[0026] Example 2: 1. Raw silkworm silk was placed in a 0.1% sodium carbonate aqueous solution at a ratio of 1:50 g / mL and heated three times at 98-100℃ for 30 minutes each time. After each heating treatment, the silk was thoroughly rinsed with deionized water. After heating treatment, the silk fibers were dried in a 60℃ oven for 12 hours to obtain degummed silk fibroin fibers. The degummed silk fibroin fibers were weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a water bath at 65±5℃ to obtain a silk fibroin protein solution. The silk fibroin protein solution was poured into a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed with deionized water for 3 days to obtain a purified silk fibroin protein aqueous solution.

[0027] 2. Silkworm raw silk was dissolved in boiling water at 100℃ for 4 hours at a bath ratio of 1:20 g / mL to obtain a sericin aqueous solution; Then, the aqueous solutions of sericin and silk fibroin were mixed at a mass ratio of 1:24 to obtain an aqueous solution of silk fibroin with a concentration of 100 mg / mL.

[0028] 3. Liquid paraffin was used as the oil phase. 2% Span-80 emulsifier was added based on the oil phase volume, and the mixture was stirred at 900 r / min at 55°C for 30 minutes. Then, the silk protein aqueous phase solution was added dropwise to the oil phase at a water-to-oil volume ratio of 1:8, and emulsified at 55°C for 30 minutes to obtain the reaction solution. Finally, isopropanol was slowly added to the reaction solution at a silk protein aqueous phase solution to isopropanol volume ratio of 1:4, and the mixture was stirred at 55°C for 30 minutes. The mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was thoroughly washed with isopropanol and deionized water. After centrifugation, the precipitate was frozen at -20°C for 4 hours and then freeze-dried for 24 hours to obtain S-embolization microspheres.

[0029] 4. Adjust the concentration of the sericin aqueous solution to 10 mg / mL, and then add polyethylene glycol diglycidyl ether at a mass ratio of 1:0.5 to modify it, thus obtaining the modified sericin solution.

[0030] S-embolization microspheres were resuspended in a 2% (w / w) polyvinyl alcohol aqueous solution, and then a modified sericin solution was added in equal volume to the polyvinyl alcohol aqueous solution and mixed. The precipitated microspheres were collected by freezing, thawing, and centrifugation, and then rinsed, centrifuged, and freeze-dried to obtain STSS microspheres.

[0031] 5. STSS microspheres were immersed in a calcium chloride solution with a concentration of 50 mg / mL at a bath ratio of 4:1 mg / mL and incubated overnight at 4°C. Then, after centrifugation, rinsing, and lyophilization, S-SS@Ca silk protein embolization microspheres with dual modification of thrombin and calcium ions were obtained.

[0032] In the above-mentioned preparation process of embolic microspheres, various anti-tumor drugs can be simultaneously loaded into the reaction system for microsphere preparation and during the calcium ion loading process, depending on the characteristics of the drug (such as hydrophilicity and hydrophobicity). Testing showed that the prepared microspheres had a particle size distribution of 40–100 μm, a hemolysis rate of less than 0.4%, and exhibited excellent coagulation and embolization properties. Compared to Example 5, the whole blood clotting time was reduced by 20%.

[0033] Example 3: 1. Raw silkworm silk was placed in a 0.1% sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL and heated three times at 98-100℃ for 30 minutes each time. After each heating treatment, the silk was thoroughly rinsed with deionized water. After heating treatment, the silk was dried in a 60℃ oven for 12 hours to obtain degummed fibroin fibers. The degummed fibroin fibers were weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a water bath at 65±5℃ to obtain a fibroin protein solution. The fibroin protein solution was poured into a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed with deionized water for 3 days to obtain a purified fibroin protein aqueous solution. 2. Silkworm raw silk was dissolved in boiling water at 100℃ for 4 hours at a bath ratio of 1:20 g / mL to obtain a sericin aqueous solution; Then, the aqueous solutions of sericin and silk fibroin were mixed to obtain an aqueous solution of silk fibroin with a concentration of 100 mg / mL.

[0034] 3. Liquid paraffin was used as the oil phase. 2% Span-80 emulsifier was added based on the oil phase volume, and the mixture was stirred at 900 r / min at 55°C for 30 minutes. Then, the silk protein aqueous phase solution was added dropwise to the oil phase at a water-to-oil volume ratio of 1:8, and emulsified at 55°C for 30 minutes to obtain the reaction solution. Finally, isopropanol was slowly added to the reaction solution at a silk protein aqueous phase solution to isopropanol volume ratio of 1:4, and the mixture was stirred at 55°C for 30 minutes. The mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was thoroughly washed with isopropanol and deionized water. After centrifugation, the precipitate was frozen at -20°C for 4 hours and then freeze-dried for 24 hours to obtain S-embolization microspheres.

[0035] 4. First, dissolve thrombin in physiological saline to obtain a thrombin solution (concentration of 500 U / mL). Select a closed container with an inlet and an outlet, place S-embolization microspheres in the container, and add thrombin solution at a concentration of 5 U / mg relative to the S-embolization microspheres to the closed container containing the microspheres. Immerse the S-embolization microspheres in the thrombin solution (preferably submerging the S-embolization microspheres). Under a pressure of 50 mmHg, maintain the thrombin solution level above the microspheres and allow it to circulate continuously. The flowing solution continuously penetrates the S-embolization microspheres for 1 hour. Finally, remove the S-embolization microspheres, freeze-dry them, and obtain thrombin-modified ST microspheres.

[0036] 5. Adjust the concentration of the sericin aqueous solution to 10 mg / mL, and then add polyethylene glycol diglycidyl ether at a mass ratio of 1:0.5 to modify it, thus obtaining a modified sericin solution.

[0037] The thrombin-modified ST microspheres were resuspended in a 2% (w / w) polyvinyl alcohol aqueous solution. Then, a modified sericin solution was added in equal volume to the polyvinyl alcohol aqueous solution and mixed. The precipitated microspheres were collected by freezing, thawing, and centrifugation. After rinsing, centrifugation, and freeze-drying, STSS microspheres were obtained.

[0038] 6. STSS microspheres were immersed in calcium chloride solutions of different concentrations (50 mg / mL) at a bath ratio of 4:1 mg / mL and incubated overnight at 4°C. Then, the microspheres were centrifuged, washed, and lyophilized to obtain STSS@Ca silk protein embolization microspheres with dual modification of thrombin and calcium ions.

[0039] In the above-mentioned preparation process of embolic microspheres, various anti-tumor drugs can be simultaneously loaded into the reaction system for microsphere preparation and during the thrombin loading process, depending on the characteristics of the drug (such as hydrophilicity and hydrophobicity). Especially in the circulating drug delivery technology, the required dosage and release rate can be controlled according to the therapeutic effect. The prepared microspheres were found to have a particle size distribution of 40–100 μm. The hemolysis rate of the microspheres was less than 0.4%, exhibiting excellent coagulation properties and embolic function. Compared to Example 5, the whole blood clotting time was reduced by 70%.

[0040] Example 4: 1. Raw silkworm silk was placed in a 0.1% sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL and heated three times at 98-100℃ for 30 minutes each time. After each heating treatment, the silk was thoroughly rinsed with deionized water. After heating treatment, the silk fibers were dried in a 60℃ oven for 12 hours to obtain degummed fibroin fibers. The degummed fibroin fibers were weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a water bath at 65±5℃ to obtain a fibroin protein solution. The fibroin protein solution was poured into a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed with deionized water for 3 days to obtain a purified fibroin protein aqueous solution.

[0041] 2. Silkworm raw silk was dissolved in boiling water at 100℃ for 4 hours at a bath ratio of 1:20 g / mL to obtain a sericin aqueous solution; Then, the aqueous solutions of sericin and silk fibroin were mixed at a mass ratio of 3:47 to obtain an aqueous solution of silk fibroin with a concentration of 120 mg / mL.

[0042] 3. Liquid paraffin was used as the oil phase. 2% Span-80 emulsifier was added based on the oil phase volume, and the mixture was stirred at 900 r / min at 55°C for 30 minutes. Then, the silk protein aqueous phase solution was added dropwise to the oil phase at a water-to-oil volume ratio of 1:10, and emulsified at 55°C for 30 minutes to obtain the reaction solution. Finally, isopropanol was slowly added to the reaction solution at a silk protein aqueous phase solution to isopropanol volume ratio of 1:4, and the mixture was stirred at 55°C for 30 minutes. The mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was thoroughly washed with isopropanol and deionized water. After centrifugation, the precipitate was frozen at -20°C for 4 hours and then freeze-dried for 24 hours to obtain S-embolization microspheres.

[0043] 4. First, dissolve thrombin in physiological saline to obtain a thrombin solution (concentration of 500 U / mL). Select a closed container with an inlet and an outlet, place S-embolization microspheres in the container, and add thrombin solution at a concentration of 8 U / mg relative to the S-embolization microspheres to the closed container containing the microspheres. Immerse the S-embolization microspheres in the thrombin solution (preferably submerging the S-embolization microspheres). Under a pressure of 100 mmHg, maintain the thrombin solution level above the microspheres and allow it to circulate continuously. The flowing solution continuously penetrates the S-embolization microspheres for 1 hour. Finally, remove the S-embolization microspheres, freeze-dry them, and obtain thrombin-modified ST microspheres.

[0044] 5. Adjust the concentration of the sericin aqueous solution to 20 mg / mL, and then add polyethylene glycol diglycidyl ether at a mass ratio of 1:0.5 to modify it, thus obtaining the modified sericin solution.

[0045] The thrombin-modified ST microspheres were resuspended in a 2% (w / w) polyvinyl alcohol aqueous solution. Then, a modified sericin solution was added in equal volume to the polyvinyl alcohol aqueous solution and mixed. The precipitated microspheres were collected by freezing, thawing, and centrifugation. After rinsing, centrifugation, and freeze-drying, STSS microspheres were obtained.

[0046] 6. STSS microspheres were immersed in a calcium chloride solution with a concentration of 75 mg / mL at a bath ratio of 4:1 mg / mL and incubated overnight at 4°C. Then, the microspheres were centrifuged, washed, and lyophilized to obtain STSS@Ca silk protein embolization microspheres with dual modification of thrombin and calcium ions.

[0047] In the above-mentioned preparation process of embolic microspheres, various anti-tumor drugs can be simultaneously loaded into the reaction system for microsphere preparation and during the thrombin loading process, depending on the characteristics of the drug (such as hydrophilicity and hydrophobicity). Especially in the circulating drug delivery technology, the required dosage and release rate can be controlled according to the therapeutic effect. The prepared microspheres were found to have a particle size distribution of 60–145 μm. The hemolysis rate of the microspheres was less than 0.4%, exhibiting excellent coagulation properties and embolic function. Compared to Example 5, the whole blood clotting time was reduced by 92%.

[0048] Example 5 (Comparative Example): 1. Raw silkworm silk was placed in a 0.1% sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL and heated three times at 98-100℃ for 30 minutes each time. After each heating treatment, the silk was thoroughly rinsed with deionized water. After heating treatment, the silk fibers were dried in a 60℃ oven for 12 hours to obtain degummed fibroin fibers. The degummed fibroin fibers were weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a water bath at 65±5℃ to obtain a fibroin protein solution. The fibroin protein solution was poured into a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed with deionized water for 3 days to obtain a purified fibroin protein aqueous solution.

[0049] 2. Silkworm raw silk was dissolved in boiling water at 100℃ for 4 hours at a bath ratio of 1:20 g / mL to obtain a sericin aqueous solution; Then, the aqueous solutions of sericin and silk fibroin were mixed at a mass ratio of 1:24 to obtain an aqueous solution of silk fibroin with a concentration of 100 mg / mL.

[0050] 3. Liquid paraffin was used as the oil phase. 2% Span-80 emulsifier was added based on the oil phase volume, and the mixture was stirred at 900 r / min at 55°C for 30 minutes. Then, the silk protein aqueous phase solution was added dropwise to the oil phase at a water-to-oil volume ratio of 1:8, and emulsified at 55°C for 30 minutes to obtain the reaction solution. Finally, isopropanol was slowly added to the reaction solution at a silk protein aqueous phase solution to isopropanol volume ratio of 1:4, and the mixture was stirred at 55°C for 30 minutes. The mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was thoroughly washed with isopropanol and deionized water. After centrifugation, the precipitate was frozen at -20°C for 4 hours and then freeze-dried for 24 hours to obtain S-embolization microspheres.

[0051] 4. Adjust the concentration of the sericin aqueous solution to 10 mg / mL, and then add polyethylene glycol diglycidyl ether at a mass ratio of 1:0.5 to modify it, thus obtaining the modified sericin solution.

[0052] S-embolization microspheres were resuspended in a 2% (w / w) polyvinyl alcohol aqueous solution, and then a modified sericin solution was added in equal volume to the polyvinyl alcohol aqueous solution and mixed. The precipitated microspheres were collected by freezing, thawing, and centrifugation, and then rinsed, centrifuged, and freeze-dried to obtain STSS microspheres.

[0053] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing embolic microspheres, characterized in that, Includes the following steps: (1) Preparation of protein aqueous solution: S1. Degumming of raw silkworm silk with sodium carbonate aqueous solution to obtain degummed silk fibroin fibers, and dissolving them in lithium bromide solution to obtain silk fibroin protein solution; then filling the silk fibroin protein solution into a dialysis bag, dialyzing with deionized water and filtering to obtain silk fibroin protein aqueous solution. S2. Raw silkworm silk is dissolved in boiling water to obtain a sericin protein aqueous solution; (2) Mix the sericin aqueous solution prepared in step (1) with the silk fibroin aqueous solution to obtain a silk fibroin aqueous solution; then add an emulsifier to the oil phase, stir for the first time, and after stirring evenly, add the silk fibroin aqueous solution dropwise to the oil phase to carry out the emulsification reaction to obtain a reaction solution; and add isopropanol to the reaction solution, stir again to obtain a mixed solution; After centrifuging the mixed solution, the precipitate was collected and washed with isopropanol and deionized water. After washing, the precipitate was collected and lyophilized to obtain S-embolization microspheres. (3) First, dissolve thrombin in physiological saline to obtain thrombin solution; Then, the S-embolization microspheres obtained in step (2) are immersed in a container containing thrombin solution. The container has an inlet and an outlet. The thrombin solution is then circulated under a certain pressure to continuously penetrate the S-embolization microspheres. Finally, the S-embolization microspheres are taken out and freeze-dried to obtain thrombin-modified ST microspheres. (4) Polyethylene glycol diglycidyl ether was added to the sericin aqueous solution to modify it, and a modified sericin solution was obtained. Then, the thrombin-modified ST microspheres were resuspended in polyvinyl alcohol aqueous solution, and the modified sericin solution was added and mixed to obtain a mixed solution. The precipitate is collected by freezing, thawing and centrifuging of the mixed solution, or by dynamically incubating the mixed solution and then centrifuging to collect the precipitate. Finally, the precipitate is washed, centrifuged and freeze-dried to obtain STSS microspheres. (5) The STSS microspheres obtained in step (4) are immersed in calcium chloride solution overnight, and then centrifuged, rinsed and freeze-dried to obtain STSS@Ca silk protein drug-loaded embolization microspheres with dual modification of thrombin and calcium ions, which are the final products of procoagulant embolization microspheres.

2. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (1) S1, the mass concentration of sodium carbonate aqueous solution is 0.1%, and the ratio of raw silkworm silk to sodium carbonate aqueous solution is 1:50 g / mL; the concentration of lithium bromide solution is 9.3 M, and the ratio of degummed silk fibroin fiber to lithium bromide solution is 1:10 g / mL, and the dissolution temperature is 60~70℃; the dialysis bag is a semi-permeable membrane with a molecular weight cutoff of 14~50 kDa, and dialysis with deionized water is performed for 3 days.

3. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (1) S2, the bath ratio for dissolving raw silkworm silk in boiling water is 1:20 g / mL, and the dissolution time in boiling water is 1-6 hours.

4. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (2), the mass ratio of the sericin aqueous solution to the silk fibroin aqueous solution is 1:10 to 1:100; the concentration of the resulting silk fibroin aqueous solution is 50 to 200 mg / mL; the emulsifier accounts for 0.5% to 10% of the oil phase volume, and the volume ratio of the silk fibroin aqueous solution to the oil phase is 1:0 to 1:15; the oil phase includes liquid paraffin; isopropanol is added to the reaction solution, wherein the volume ratio of the silk fibroin aqueous solution to isopropanol is 1:0 to 1:10; The temperature for the first and second stirrings is 50-60℃, for 20-40 minutes, and the speed is 800-900 r / min; the temperature for the emulsification reaction is 50-60℃, and the time is 30-40 minutes; the centrifugation conditions are: 10000 r / min for 15-20 minutes. The freeze-drying process is as follows: the precipitate is frozen at -20°C for 4 hours and then placed on a freeze dryer for freeze-drying for 24 hours.

5. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (3), the concentration of thrombin solution is 100~1000U / mL, the ratio of thrombin activity to the mass of S embolization microspheres is (1~15)U:1mg; a certain pressure condition is 50~500mmHg, so that the thrombin solution circulates and continuously penetrates the S embolization microspheres for 0.5~5 hours.

6. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (4), the concentration of the sericin aqueous solution is 5~30 mg / mL; the mass ratio of sericin to polyethylene glycol diglycidyl ether in the sericin aqueous solution is 1:(0.5~1); and the volume ratio of the modified sericin solution to the polyvinyl alcohol aqueous solution is 1:

1.

7. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (4), the freezing and thawing conditions are: freezing at -20℃ for 4-12 hours, and then thawing at room temperature; the dynamic incubation conditions are oscillation at room temperature at 200~800 r / min for 2~8 hours; the rinsing solvent is deionized water.

8. The method for preparing a coagulation-promoting embolization microsphere according to claim 1, characterized in that, In step (5), the concentration of calcium chloride solution is 5~150 mg / mL, and the ratio of STSS microspheres to calcium chloride solution is 1~6 mg:1 mL.

9. Procoagulant embolization microspheres prepared by any one of claims 1-8.

10. The embolic microspheres according to claim 9, characterized in that, The procoagulant embolization microspheres have a particle size distribution of 40~145μm and a hemolysis rate of less than 0.4%.