Self-setting injectable bi-component embolizing microspheres and method for preparing same

Abstract

The present application relates to the technical field of medical preparation, in particular to a self-solidifying injectable two-component embolization microsphere and a preparation method thereof, which is composed of hydrogel microspheres A containing positive charge groups and hydrogel microspheres B containing negative charge groups, wherein the hydrogel microspheres A containing positive charge groups and the hydrogel microspheres B containing negative charge groups can be injected in situ to solidify into a gel through a double syringe, or can be premixed to form a gel, and after the gel is formed, the gel can still be injected and solidified in situ through a syringe, a gel block is constructed by the microspheres, the self-assembly of the microspheres is induced to form a solidified state by the interaction between the particles, the injectability is maintained while the ectopic embolization of the material after embolization is effectively avoided, and the biocompatibility and degradability of the embolic agent are also beneficial.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to a self-coagulating injectable two-component embolic microsphere and its preparation method. Background Technology

[0002] Transcatheter arterial embolization (TAE) involves introducing an embolic agent into a blood vessel through a catheter to intentionally block vascular structures. These embolic agents cut off the tumor's supply of nutrients and oxygen to ensure its survival by blocking blood flow to the tumor, thereby achieving tumor devascularization and progressive shrinkage. Embolic agents include permanent and temporary types, such as repeatable TAEs (interventional tumor embolization therapy) specifically designed to control tumor growth, with biodegradable embolic materials being preferred. The shape of the embolic agent is also an important factor in its selection. Commercially available embolic agents come in various shapes, including coils, liquids, non-spherical, and microspheres. Non-spherical embolic agents cannot precisely control the embolization level, while microsphere embolic materials offer both precise control of the embolization level and smoother injection, but they are prone to deformation and displacement within the blood vessel. For example, Chinese patent application (publication number CN 116617445 A) discloses a biodegradable embolization microsphere, its preparation method and application. It uses a macromolecular crosslinking agent to crosslink the modified product of natural polymer, so that the provided embolization microsphere can be biodegraded by means of the macromolecular crosslinking agent, and can be loaded with drugs through electrostatic interaction for practical application. However, it is easy to deform and displace in blood vessels. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a self-coagulating injectable two-component embolizing microsphere. By constructing a gel block from the microspheres, the interaction between particles induces the microspheres to self-assemble into a solidified state. This maintains injectability while effectively preventing ectopic embolization of the material after embolization, and also improves the biocompatibility and biodegradability of the embolizing agent.

[0004] The present invention provides a self-curing injectable two-component embolizing microsphere, which is composed of hydrogel microsphere A containing positively charged groups and hydrogel microsphere B containing negatively charged groups. The hydrogel microsphere A containing positively charged groups and the hydrogel microsphere B containing negatively charged groups can be injected into the gel in situ by a dual-syringe syringe, or they can be premixed into a gel. After gelation, they can still be injected and cured in situ by a syringe.

[0005] As a preferred technical solution, the raw materials for preparing hydrogel microspheres A containing positively charged groups include at least type A gelatin and a cationic polymer containing multiple amino groups, wherein the isoelectric point of type A gelatin is 8-10, preferably 9.

[0006] Preferably, the cationic polymer containing multiple amino groups is selected from polylysine and polyglucosamine, with polylysine being the most preferred.

[0007] Preferably, the preparation process of the hydrogel microspheres A containing positively charged groups includes at least the following steps:

[0008] A1. Dissolve type A gelatin and a cationic polymer containing multiple amino groups in MES or PBS buffer to obtain an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups.

[0009] A2. Prepare an oil phase solution A containing a nonionic surfactant by dissolving the nonionic surfactant in an organic solvent;

[0010] A3. Dissolve the crosslinking agent in MES buffer to obtain crosslinking agent solution A containing the crosslinking agent;

[0011] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir, and obtain a mixed solution;

[0012] A5. Add the mixed solution dropwise to the oil phase solution A, and continue stirring to obtain an emulsion;

[0013] A6. Add an alkaline aqueous solution to the emulsion, stir, let stand, centrifuge, wash, and then sieve to collect hydrogel microspheres A containing positively charged groups.

[0014] Preferably, the preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0015] A1. Dissolve type A gelatin and a cationic polymer containing multiple amino groups in MES buffer solution at 50°C and pH=5 to obtain an aqueous phase solution A containing 10-20 wt% type A gelatin and 5 wt% cationic polymer containing multiple amino groups.

[0016] A2. Prepare an oil phase solution A containing 0.4-0.6% (v / v) Span-80 by dissolving Span-80 in liquid paraffin;

[0017] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10-20% (v / v) crosslinking agent;

[0018] A4. Pour the crosslinking agent solution A into the aqueous phase solution and stir for 5-20 seconds to obtain a mixed solution;

[0019] A5. Add the mixed solution dropwise to the oil phase solution A at a speed of 400-600 r / min, and continue stirring for 3-7 h to obtain an emulsion;

[0020] A6. Add sodium hydroxide aqueous solution to the emulsion, stir at 400-600 r / min for 0.5-3 h, let stand, centrifuge, wash and sieve to collect hydrogel microspheres A containing positively charged groups.

[0021] More preferably, the preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0022] A1. Dissolve type A gelatin and a cationic polymer containing multiple amino groups in MES buffer solution at 50°C and pH=5 to obtain an aqueous solution A containing 20 wt% type A gelatin and 5 wt% cationic polymer containing multiple amino groups.

[0023] A2. Prepare an oil phase solution A containing 0.5% (v / v) Span-80 by dissolving Span-80 in liquid paraffin;

[0024] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0025] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 5-20 seconds to obtain a mixed solution;

[0026] A5. The mixed solution is added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion is obtained after continuous stirring for 3-7 h.

[0027] A6. Add sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 0.5-3 h, let stand, centrifuge, wash and sieve to collect hydrogel microspheres A containing positively charged groups.

[0028] Preferably, the volume ratio of the crosslinking agent solution A to the aqueous phase solution A is (1-2):5, more preferably 2:5.

[0029] Preferably, the volume ratio of the aqueous solution A to the oil solution A is 1:1.

[0030] As a preferred technical solution, the raw materials for preparing the hydrogel microspheres B containing negatively charged groups include at least type B gelatin and negatively charged natural anionic polysaccharides, wherein the isoelectric point of type B gelatin is 4-6, preferably 5.

[0031] Preferably, the negatively charged natural anionic polysaccharide is selected from sodium alginate, sodium carboxymethyl cellulose, and sodium hyaluronate, with sodium alginate being the most preferred.

[0032] Preferably, the preparation process of the hydrogel microspheres B containing negatively charged groups includes at least the following steps:

[0033] B1. Dissolve type B gelatin and negatively charged natural anionic polysaccharides in PBS buffer to obtain an aqueous solution B containing type B gelatin and negatively charged natural anionic polysaccharides.

[0034] B2. Prepare an oil phase solution B containing a nonionic surfactant by dissolving the nonionic surfactant in an organic solvent;

[0035] B3. Add aqueous solution B dropwise to oil solution B, stir continuously to obtain an emulsion, and then carry out a cooling reaction;

[0036] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0037] B5. Add the cleaned microspheres to the crosslinking agent solution B, stir continuously to react, let stand, centrifuge, wash and sieve to collect hydrogel microspheres B containing negatively charged groups.

[0038] Preferably, the preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0039] B1. Dissolve type B gelatin and negatively charged natural anionic polysaccharides in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B containing 2-10 wt% type B gelatin and 2 wt% negatively charged natural anionic polysaccharides.

[0040] B2. Prepare an oil phase solution containing 0.4-0.6% (v / v) Span-80 by dissolving Span-80 in liquid paraffin;

[0041] B3. Add aqueous solution B dropwise to oil solution B at a speed of 400-600 r / min, stir continuously for 15-60 min to obtain an emulsion, and then carry out a cooling reaction at 2-8℃ for 1-2 h.

[0042] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0043] B5. Add the cleaned microspheres to a glutaraldehyde aqueous solution and stir continuously for 1-5 hours at 2-8℃ and 400-600r / min. After standing, centrifugation, washing, and sieving, collect the hydrogel microspheres B containing negatively charged groups.

[0044] More preferably, the preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0045] B1. Dissolve type B gelatin and negatively charged natural anionic polysaccharides in PBS buffer at 70°C and pH 7.2 ± 0.2 to obtain an aqueous solution B containing 2 wt% type B gelatin and 2 wt% negatively charged natural anionic polysaccharides.

[0046] B2. Prepare an oil phase solution B containing 0.5% (v / v) Span-80 by dissolving Span-80 in liquid paraffin;

[0047] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 15-60 min to obtain an emulsion, and then carry out a cooling reaction at 2-8℃ for 1-2 h.

[0048] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0049] B5. The cleaned microspheres were added to a 25wt% glutaraldehyde aqueous solution and stirred continuously at 2-8℃ and 500r / min for 1-5h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B were obtained.

[0050] Preferably, the volume ratio of the aqueous solution B to the oil solution B is 1:1.

[0051] Preferably, the volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of the aqueous phase solution B is (5-10):1.

[0052] The self-coagulating injectable bipartite embolic microspheres provided by this invention are based on gelatin material with excellent biocompatibility, controllable biodegradability and non-immunogenicity, combined with corresponding cationic polymers containing multiple amino groups and natural anionic polysaccharides. Through cross-linking, hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups are prepared. Based on the electrostatic interaction between hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups, a gel block is constructed, which effectively solves the problem of existing microspherical embolic agents being easily deformed and displaced in blood vessels.

[0053] In the process of constructing self-coagulating injectable bipartite embolizing microspheres, the inventors discovered that gel blocks could not be obtained using only type A gelatin and type B gelatin. Initially, the inventors used type A gelatin and polylysine, a cationic polymer containing multiple amino groups, as sources of positive charge. They found that even increasing the concentration of type B gelatin in hydrogel microspheres B (containing negatively charged groups) and the amount of crosslinking agent solution B did not result in agglomeration after mixing the two types of microspheres. The inventors then optimized the pH of the buffer solution in the aqueous phase solution A to 5, resulting in slight agglomeration after mixing the two types of microspheres. However, this agglomeration dispersed with gentle shaking, and the agglomerates were unstable. Further investigation revealed that by simultaneously optimizing type A gelatin and polylysine as sources of positive charge, and type B gelatin and sodium alginate (a negatively charged natural anionic polysaccharide) as sources of negative charge, strong electrostatic adsorption occurred between the two types of microspheres under the respective effects of crosslinking agent solutions A and B. This effectively solved the problem of agglomerate instability, and the agglomerates could be injected using a syringe.

[0054] As a preferred technical solution, the particle size of the hydrogel microspheres A containing positively charged groups and the hydrogel microspheres B containing negatively charged groups are both between 10 and 1000 μm, and the mass ratio of the two types of microspheres is (0.1-1):(0.1-1). Preferably, the particle size of the hydrogel microspheres A containing positively charged groups and the hydrogel microspheres B containing negatively charged groups are both 50-100 μm, and the mass ratio of the two types of microspheres is 1:1.

[0055] Another aspect of the present invention provides a method for preparing self-coagulating injectable two-component embolic microspheres. Hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups are delivered via a dual-syringe and a coaxial dual-catheter, and mixed and coagulated within the blood vessel to form an embolism. Alternatively, hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups are mixed to form a gel, which is then canned into a syringe barrel and delivered via injection through a syringe and catheter. After injection from the catheter tip, the gel returns to a coagulated state within the blood vessel, achieving embolism.

[0056] Beneficial effects

[0057] 1. This invention provides a self-coagulating injectable two-component embolization microsphere. By constructing a gel block through microspheres, the interaction between particles induces the microspheres to self-assemble into a solidified state. This maintains injectability while effectively avoiding ectopic embolization of the material after embolization, and also benefits the biocompatibility and degradability of the embolization agent.

[0058] 2. The self-coagulating injectable bipartite embolic microspheres provided by this invention are based on gelatin material with excellent biocompatibility, controllable biodegradability and non-immunogenicity, combined with corresponding cationic polymers containing multiple amino groups and natural anionic polysaccharides, to prepare hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups through cross-linking. Based on the electrostatic interaction between hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups, a gel block is constructed, which effectively solves the problem of existing microspherical embolic agents being easily deformed and lost in blood vessels.

[0059] 3. In this invention, by optimizing the pH of the buffer solution in the aqueous solution A to 5, a slight aggregation phenomenon occurs after mixing hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups.

[0060] 4. This invention optimizes the use of type A gelatin and polylysine, a cationic polymer containing multiple amino groups, as sources of positive charge, and type B gelatin and sodium alginate, a natural anionic polysaccharide, as sources of negative charge. Under the respective action of crosslinking agent solution A and crosslinking agent solution B, strong electrostatic adsorption can occur between the two types of microspheres, effectively solving the problem of unstable aggregates. Moreover, the aggregates can be injected using a syringe.

[0061] 5. The bicomponent embolization microspheres provided by the present invention can be delivered by combining hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups through a dual syringe and a coaxial dual catheter, and then mixed and coagulated in the blood vessel to form an embolism; or the hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups can be mixed to form a gel, then canned into a syringe syringe, and delivered by injection through a syringe and catheter. After being injected from the end of the catheter, the gel returns to a coagulated state in the blood vessel to achieve embolism. Attached Figure Description

[0062] Figure 1 This is a light micrograph of hydrogel microspheres A (AG) containing positively charged groups provided in this invention.

[0063] Figure 2 This is a light micrograph of hydrogel microspheres B(ai) containing negatively charged groups provided in this invention.

[0064] Figure 3 The figure shows the injection and coagulation performance test results of the two-component embolic microspheres provided in Example 1. In the figure, a represents the microspheres injected into physiological saline, and b represents the microspheres injected into serum protein aqueous solution.

[0065] Figure 4The figure shows the stability test results of the two-component embolic microspheres provided in Example 1. In the figure, a represents the microspheres injected into physiological saline, and b represents the microspheres injected into serum protein aqueous solution.

[0066] Figure 5 The figure shows the injection and coagulation performance test results of the two-component embolic microspheres provided in Example 2. In the figure, a represents the injection into physiological saline, and b represents the injection into serum protein aqueous solution.

[0067] The sources of the raw materials used in the embodiments and comparative examples of this invention are as follows:

[0068] Type A gelatin: Pigskin gelatin with a gel strength of 100 Bloom, sourced from Sigma.

[0069] Polylysine: This is Epsilon-type polylysine, sourced from Shanghai E-En Chemical Technology Co., Ltd.

[0070] Type B gelatin: Bovine gelatin with a gel strength of 250 blooms, sourced from Sigma.

[0071] Sodium alginate: viscosity 100 mPa·s, sourced from Shanghai E. En Chemical Technology Co., Ltd.

[0072] 4-(4,6-Dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride: CAS No.: 3945-69-5, source: Sigma. Detailed Implementation

[0073] Example 1

[0074] In one aspect, Embodiment 1 of the present invention provides a self-curing injectable two-component embolizing microsphere, which is composed of hydrogel microsphere A containing positively charged groups and hydrogel microsphere B containing negatively charged groups. The hydrogel microsphere A containing positively charged groups and the hydrogel microsphere B containing negatively charged groups can be injected into the gel in situ by a dual-syringe syringe, or they can be premixed into a gel. After gelation, they can still be injected and cured in situ by a syringe.

[0075] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0076] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0077] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in MES buffer solution at 50°C and pH=5 to obtain an aqueous phase solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0078] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0079] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0080] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0081] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0082] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named G) containing positively charged groups.

[0083] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 2:5.

[0084] The volume ratio of the aqueous solution A to the oil solution A is 1:1.

[0085] The raw materials for preparing the hydrogel microspheres B containing negatively charged groups include an aqueous solution B containing type B gelatin and a negatively charged natural anionic polysaccharide, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% glutaraldehyde aqueous solution, wherein the isoelectric point of type B gelatin is 5, and the negatively charged natural anionic polysaccharide is sodium alginate.

[0086] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0087] B1. Dissolve type B gelatin and negatively charged natural anionic polysaccharides in PBS buffer at 70℃ and pH=7.2±0.2 to obtain an aqueous solution B (50mL) containing 2wt% type B gelatin and 2wt% negatively charged natural anionic polysaccharides.

[0088] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0089] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0090] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0091] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named i) containing negatively charged groups were obtained.

[0092] The volume ratio of the aqueous solution B to the oil solution B is 1:1.

[0093] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 10:1.

[0094] The hydrogel microspheres A containing positively charged groups and B containing negatively charged groups both have a particle size of 50-100 μm, and the mass ratio of the two types of microspheres is 1:1.

[0095] Another aspect of the present invention provides a method for preparing self-coagulating injectable two-component embolic microspheres, wherein hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups are delivered through a dual syringe and a coaxial dual catheter, and mixed and coagulated in the blood vessel to form an embolism.

[0096] Example 2

[0097] Example 2 of the present invention provides a self-coagulating injectable two-component embolizing microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the preparation method of the self-coagulating injectable two-component embolizing microsphere is as follows: hydrogel microsphere A containing positively charged groups and hydrogel microsphere B containing negatively charged groups are mixed to form a gel and then filled into a syringe syringe. The gel is delivered by injection through a syringe and a catheter. After being injected from the end of the catheter, the microsphere returns to a coagulated state in the blood vessel, thereby achieving embolization.

[0098] Example 3

[0099] Example 3 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume ratio of the aqueous phase solution B is 5:1, and hydrogel microspheres B (named g) containing negatively charged groups are obtained.

[0100] Comparative Example 1

[0101] Comparative Example 1 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0102] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0103] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0104] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0105] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0106] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0107] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B (named a) were obtained.

[0108] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0109] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A of a cationic polymer containing type A gelatin, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide, wherein the isoelectric point of type A gelatin is 9, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0110] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0111] A1. Dissolve type A gelatin in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 10 wt% type A gelatin;

[0112] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0113] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0114] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0115] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0116] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A containing positively charged groups (named A).

[0117] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0118] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0119] Comparative Example 2

[0120] Comparative Example 2 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0121] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0122] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 20 wt% type B gelatin;

[0123] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0124] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0125] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0126] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named b) containing negatively charged groups were obtained.

[0127] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0128] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0129] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0130] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0131] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0132] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0133] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0134] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0135] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0136] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0137] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0138] Comparative Example 3

[0139] Comparative Example 3 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0140] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0141] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 30 wt% type B gelatin;

[0142] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0143] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0144] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0145] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named c) containing negatively charged groups were obtained.

[0146] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0147] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0148] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0149] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0150] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0151] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0152] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0153] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0154] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0155] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0156] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0157] Comparative Example 4

[0158] Comparative Example 4 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0159] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0160] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0161] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0162] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0163] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0164] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named d) containing negatively charged groups were obtained.

[0165] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 10:1.

[0166] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0167] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0168] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0169] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0170] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0171] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0172] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0173] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0174] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0175] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0176] Comparative Example 5

[0177] Comparative Example 5 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0178] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0179] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 20 wt% type B gelatin;

[0180] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0181] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0182] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0183] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B (named e) were obtained.

[0184] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 10:1.

[0185] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0186] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0187] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0188] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0189] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0190] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0191] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0192] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0193] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0194] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0195] Comparative Example 6

[0196] Comparative Example 6 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0197] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0198] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0199] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0200] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0201] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0202] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named f) containing negatively charged groups were obtained.

[0203] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 20:1.

[0204] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0205] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0206] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0207] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0208] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0209] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0210] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0211] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0212] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0213] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0214] Comparative Example 7

[0215] Comparative Example 7 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0216] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0217] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0218] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0219] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0220] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0221] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B (named a) were obtained.

[0222] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0223] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0224] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0225] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 10 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0226] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0227] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0228] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0229] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0230] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named B) containing positively charged groups.

[0231] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0232] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0233] Comparative Example 8

[0234] Comparative Example 8 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0235] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0236] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0237] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0238] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0239] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0240] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B (named a) were obtained.

[0241] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0242] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A of a cationic polymer containing type A gelatin, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide, wherein the isoelectric point of type A gelatin is 9, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0243] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0244] A1. Dissolve type A gelatin in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin;

[0245] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0246] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0247] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0248] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0249] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named C) containing positively charged groups.

[0250] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0251] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0252] Comparative Example 9

[0253] Comparative Example 9 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0254] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0255] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0256] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0257] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0258] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0259] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres containing negatively charged groups B (named a) were obtained.

[0260] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 5:1.

[0261] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0262] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0263] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in PBS buffer at 50°C and pH=7 to obtain an aqueous solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0264] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0265] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0266] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0267] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0268] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named D) containing positively charged groups.

[0269] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0270] No aggregation occurred when hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups were mixed.

[0271] Comparative Example 10

[0272] Comparative Example 10 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous phase solution B containing type B gelatin, an oil phase solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0273] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0274] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0275] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0276] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0277] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0278] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named f) containing negatively charged groups were obtained.

[0279] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 20:1.

[0280] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A of a cationic polymer containing type A gelatin, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide, wherein the isoelectric point of type A gelatin is 9, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0281] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0282] A1. Dissolve type A gelatin in PBS buffer at 50°C and pH=5 to obtain an aqueous solution A (50 mL) containing 10 wt% type A gelatin;

[0283] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0284] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0285] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0286] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0287] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named E) containing positively charged groups.

[0288] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 1:5.

[0289] When hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups are mixed, slight aggregation occurs, but they disperse after gentle shaking.

[0290] Comparative Example 11

[0291] Comparative Example 11 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0292] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0293] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0294] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0295] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0296] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0297] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named f) containing negatively charged groups were obtained.

[0298] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 20:1.

[0299] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A of a cationic polymer containing type A gelatin, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide, wherein the isoelectric point of type A gelatin is 9, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0300] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0301] A1. Dissolve type A gelatin in MES buffer solution at 50℃ and pH=5 to obtain an aqueous phase solution A (50mL) containing 20wt% type A gelatin;

[0302] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0303] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0304] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0305] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0306] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named E) containing positively charged groups.

[0307] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 2:5.

[0308] When hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups are mixed, slight aggregation occurs, but they disperse after gentle shaking.

[0309] Comparative Example 12

[0310] Comparative Example 12 of the present invention provides a self-coagulating injectable two-component embolic microsphere and its preparation method. The specific implementation method is the same as that of Example 1, except that the raw materials for preparing the hydrogel microsphere B containing negatively charged groups include an aqueous solution B containing type B gelatin, an oil solution B containing 0.5% (v / v) Span-80, and a 25wt% aqueous solution of glutaraldehyde, wherein the isoelectric point of type B gelatin is 5.

[0311] The preparation process of the hydrogel microspheres B containing negatively charged groups specifically includes the following steps:

[0312] B1. Dissolve type B gelatin in PBS buffer at 70℃ and pH 7.2 ± 0.2 to obtain an aqueous solution B (50 mL) containing 10 wt% type B gelatin;

[0313] B2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution B (50 mL) containing 0.5% (v / v) Span-80;

[0314] B3. Add aqueous solution B dropwise to oil solution B at a speed of 500 r / min, stir continuously for 30 min to obtain an emulsion, and then carry out a cooling reaction at 4℃ for 1.5 h.

[0315] B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away.

[0316] B5. The cleaned microspheres were added to a 25 wt% glutaraldehyde aqueous solution and stirred continuously at 4℃ and 500 r / min for 3 h. After standing, centrifugation, washing, and sieving, hydrogel microspheres B (named f) containing negatively charged groups were obtained.

[0317] The volume ratio of the 25wt% glutaraldehyde aqueous solution to the volume of aqueous solution B is 20:1.

[0318] The raw materials for preparing hydrogel microspheres A containing positively charged groups include an aqueous solution A containing type A gelatin and a cationic polymer containing multiple amino groups, an oil solution A containing 0.5% (v / v) Span-80, a crosslinking agent solution A containing 10% (v / v) crosslinking agent, and an aqueous solution of sodium hydroxide. The isoelectric point of type A gelatin is 9, the cationic polymer containing multiple amino groups is polylysine, and the concentration of the aqueous solution of sodium hydroxide is 1 mol / L.

[0319] The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps:

[0320] A1. Dissolve type A gelatin and a cationic polymer containing polyamino groups in MES buffer solution at 50°C and pH=5 to obtain an aqueous phase solution A (50 mL) containing 20 wt% type A gelatin and 5 wt% cationic polymer containing polyamino groups.

[0321] A2. Dissolve Span-80 in liquid paraffin to prepare an oil phase solution A (50 mL) containing 0.5% (v / v) Span-80;

[0322] A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10% (v / v) crosslinking agent;

[0323] A4. Pour the crosslinking agent solution A into the aqueous phase solution A, stir for 10 seconds to obtain a mixed solution;

[0324] A5. The mixed solution was added dropwise to the oil phase solution A at a speed of 500 r / min, and the emulsion was obtained after continuous stirring for 5 h.

[0325] A6. Add 10 mL of sodium hydroxide aqueous solution to the emulsion, stir at 500 r / min for 1.5 h, let stand, centrifuge, wash, and sieve to collect hydrogel microspheres A (named G) containing positively charged groups.

[0326] The volume ratio of the crosslinking agent solution A to the aqueous phase solution A is 2:5.

[0327] When hydrogel microspheres B containing negatively charged groups and hydrogel microspheres A containing positively charged groups are mixed, slight aggregation occurs, but they disperse after gentle shaking.

[0328] Performance testing methods

[0329] 1. Optical Microscopy Test: The hydrogel microspheres A (AG) containing positively charged groups and B (ai) containing negatively charged groups obtained in the embodiments and comparative examples of this invention were tested using an optical microscope. The test results are shown in [reference needed]. Figure 1 , Figure 2 .

[0330] 2. Injection and Coagulation Performance Tests: Hydrogel microspheres A(G) containing positively charged groups and B(i) containing negatively charged groups, as provided in Example 1, were respectively loaded into syringes and mixed using a dual-injection syringe. The mixture was then injected into physiological saline and serum protein, respectively. The results of the injection and coagulation performance tests are shown in [reference needed]. Figure 3 When injected with a dual-syringe syringe, thin gel strips can be seen appearing in the water, proving that the two can be injected and mixed to form a solid gel through electrostatic adsorption.

[0331] 3. Stability Test: After stirring injection and coagulation performance tests, the solid gel formed is observed to determine whether it disperses. See the results below. Figure 4 After stirring, it was found that all the substances clumped together, indicating that electrostatic attraction occurred between them, resulting in good stability.

[0332] 4. Injectability: The hydrogel microspheres A(G) containing positively charged groups and B(i) containing negatively charged groups provided in Example 2 were mixed and packaged into a syringe. Injection was performed to determine whether the microspheres were injectable and whether they could revert to their solidified state after injection. The results are shown in [reference needed]. Figure 5 The gel block can be injected with a syringe. It has good fluidity and can return to a solid state after injection.

Claims

1. A self-coagulating, injectable, two-component embolic microsphere, characterized in that, It is composed of hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups. The hydrogel microspheres A containing positively charged groups and the hydrogel microspheres B containing negatively charged groups are injected into the gel in situ by a dual-syringe or premixed into a gel. After gel formation, injection and in situ solidification are achieved by a syringe. The raw materials for preparing the hydrogel microspheres A containing positively charged groups include at least type A gelatin and a cationic polymer containing multiple amino groups, wherein the cationic polymer containing multiple amino groups is polylysine. The preparation process of the hydrogel microspheres A containing positively charged groups specifically includes the following steps: A1. Dissolve type A gelatin and a cationic polymer containing multiple amino groups in MES buffer at 50°C and pH=5 to obtain an aqueous solution A containing 10-20 wt% type A gelatin and 5 wt% cationic polymer containing multiple amino groups. A2. Prepare an oil phase solution A containing 0.4-0.6% (v / v) Span-80 by dissolving Span-80 in liquid paraffin; A3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in MES buffer to obtain crosslinking agent solution A containing 10-20% (v / v) crosslinking agent; A4. Pour the crosslinking agent solution A into the aqueous phase solution and stir for 5-20 seconds to obtain a mixed solution; A5. Add the mixed solution dropwise to the oil phase solution A at a speed of 400-600 r / min, and continue stirring for 3-7 h to obtain an emulsion; A6. Add sodium hydroxide aqueous solution to the emulsion, stir at 400-600 r / min for 0.5-3 h, let stand, centrifuge, wash and sieve to collect hydrogel microspheres A containing positively charged groups; The raw materials for preparing the hydrogel microspheres B containing negatively charged groups include at least type B gelatin and a negatively charged natural anionic polysaccharide, wherein the negatively charged natural anionic polysaccharide is sodium alginate.

2. The self-coagulating injectable two-component embolic microsphere according to claim 1, characterized in that, The isoelectric point of the type A gelatin is 8-10.

3. The self-coagulating injectable two-component embolic microsphere according to claim 1, characterized in that, The volume ratio of the crosslinking agent solution A to the aqueous solution A is (1-2):

5.

4. The self-coagulating injectable two-component embolic microsphere according to claim 1, characterized in that, The isoelectric point of the type B gelatin is 4-6.

5. The self-coagulating injectable two-component embolic microsphere according to claim 1, characterized in that, The preparation process of the hydrogel microspheres B containing negatively charged groups includes at least the following steps: B1. Dissolve type B gelatin and negatively charged natural anionic polysaccharides in PBS buffer to obtain an aqueous solution B containing type B gelatin and negatively charged natural anionic polysaccharides. B2. Prepare an oil phase solution B containing a nonionic surfactant by dissolving the nonionic surfactant in an organic solvent; B3. Add aqueous solution B dropwise to oil solution B, stir continuously to obtain an emulsion, and then carry out a cooling reaction; B4. After cooling reaction, the emulsion is subjected to solid-liquid separation to obtain microspheres, and the residual oil phase on the surface of the microspheres is washed away. B5. Add the cleaned microspheres to the crosslinking agent solution B, stir continuously to react, let stand, centrifuge, wash and sieve to collect hydrogel microspheres B containing negatively charged groups.

6. The self-coagulating injectable two-component embolic microsphere according to claim 1, characterized in that, The particle size of the hydrogel microspheres A containing positively charged groups and the hydrogel microspheres B containing negatively charged groups are both between 10 and 1000 μm, and the mass ratio of the two types of microspheres is (0.1-1):(0.1-1).

7. A method for preparing a self-coagulating injectable two-component embolic microsphere according to any one of claims 1-6, characterized in that, Hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups are prepared by injection using a dual syringe and a coaxial dual catheter, or by mixing hydrogel microspheres A containing positively charged groups and hydrogel microspheres B containing negatively charged groups to form a gel and then injecting it.

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