Embolism microsphere capable of slowly releasing medicine and controlling degradation speed and preparation method of embolism microsphere

Abstract

The invention provides an embolism microsphere capable of slowly releasing drugs and controlling degradation speed and a preparation method thereof, the method comprises the following steps: dissolving a dispersant in an oil phase liquid, and stirring at a first preset temperature to obtain a dispersion liquid; dissolving gelatin in water at a second preset temperature to obtain a gelatin solution; the preparation method comprises the following steps: dissolving a compound with negative electricity groups in water, and adding alkali liquor and an initiator in an ice bath to obtain a modified solution; adding the modified solution into a gelatin solution, stirring to obtain a water-phase pre-polymerized solution, dropwise adding the water-phase pre-polymerized solution into the dispersion solution, premixing, adding a cross-linking agent and a catalyst, and directly grafting negative electricity groups onto gelatin molecules in a low-temperature free radical polymerization manner to obtain an intermediate product; and performing post-treatment to obtain the embolism microspheres. According to the preparation method disclosed by the invention, a non-degradable material is not needed, negative electricity groups can be directly grafted on the surface of gelatin, so that the embolism microspheres have efficient drug loading performance and good long-period drug sustained release, and the degradation speed of the embolism microspheres is controlled.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a microsphere-type embolic agent, and in particular to an embolic microsphere capable of sustained-release of drugs and controlled degradation rate, and a preparation method thereof. Background Art

[0002] At present, commonly used solid embolic agents include microsphere embolic agents and granular embolic agents. Among them, embolic microspheres are used to treat tumor diseases and have become a common clinical treatment method. The principle is to embolize the blood vessels in the affected area through microspheres, cut off the nutrient supply of tumor cells, and make tumor cells wither and die due to lack of nutrition, thereby achieving the therapeutic effect. According to different materials, embolic microspheres can be divided into two types: biodegradable and non-biodegradable. Non-degradable microspheres will aggregate in the body after embolism and produce toxicity, while biodegradable microspheres can automatically degrade into products that are harmless to the human body. Ideal biodegradable embolic microspheres can artificially control their degradation time, and blood vessels can be reopened after degradation. Repeated embolization treatments can be performed multiple times. They can gradually degrade under the action of relevant enzymes in the body and slowly release drugs.

[0003] At present, the main materials of embolic microspheres on the market are poly(methyl methacrylate), PMMA, polyvinyl alcohol (PVA), polyethylene glycol (PEG) or one or more of the above three materials, all of which are non-degradable materials. Although gelatin is also used in some products, the carriers that realize their drug-carrying function are all non-degradable materials. For example, drug-carrying embolic microspheres are made of gelatin, polyethylene glycol derivatives and drug-carrying functional monomers as aqueous phase materials and an oil phase containing an emulsifier to form a microemulsion, which is then cross-linked with an aldehyde cross-linking agent and a photoinitiator. Among them, the polyethylene glycol derivative is grafted on the gelatin, and the drug-carrying monomer is grafted on the polyethylene glycol derivative. The drug-carrying and drug-releasing capacity is limited, and the polyethylene glycol derivative is non-degradable. In this method, it is necessary to graft intermediate carriers for drug delivery, such as polyethylene glycol derivatives, onto gelatin. The process is complicated, and these intermediate carriers cannot be degraded, and repeated treatment is not possible, and there is a risk of inflammation or ectopic embolism. In addition, this method requires the use of specific photoinitiators, which are easily affected by light, have poor stability, and cause environmental pollution. The synthesis process is difficult and the production efficiency is low.

[0004] In addition, there is a method of dissolving a mixture of gelatin and drugs in water, adding a cross-linking agent during stirring, and then obtaining embolic microspheres of different particle sizes by spray drying. However, such embolic microspheres only use physical methods to achieve drug encapsulation, and the particle size is uneven, and in particular, the drug cannot be sustained-released, thereby failing to achieve long-term treatment and being difficult to meet the needs of practical applications. Summary of the invention

[0005] The present invention proposes an embolic microsphere capable of sustained-release drugs and controlled degradation rate and a preparation method thereof. Through the embolic microsphere capable of sustained-release drugs and controlled degradation rate and the preparation method thereof provided by the present invention, negatively charged groups can be directly grafted onto the surface of gelatin molecules by low-temperature free radical polymerization without the aid of an intermediate carrier, thereby achieving drug loading, so that the embolic microsphere has efficient drug loading performance, can slowly release drugs, and achieve the effect of targeted drug delivery. In addition, there is no need to add non-degradable materials, and complete degradation can be achieved, thereby achieving repeated treatment and avoiding inflammation and ectopic embolism. The obtained embolic microsphere has a good long-term drug sustained-release effect and can control the degradation rate of the embolic microsphere.

[0006] In order to solve the above technical problems, an embodiment of the present invention provides a method for preparing embolic microspheres capable of sustained-release of drugs and controlled degradation rate, which comprises at least the following steps:

[0007] Dissolving a dispersant in an oil phase liquid, heating the mixture to a first preset temperature, and stirring the mixture to obtain a dispersion;

[0008] dissolving gelatin in water at a second preset temperature to obtain a gelatin solution;

[0009] Dissolve the compound with negatively charged groups in water, and add alkali solution and initiator in an ice bath to obtain a modified solution;

[0010] Adding the modified liquid into the gelatin solution and stirring to obtain an aqueous prepolymer solution;

[0011] The aqueous phase prepolymer solution is added dropwise to the dispersion solution for premixing, and then a crosslinking agent and a catalyst are added for crosslinking and catalysis, and negatively charged groups are directly grafted onto gelatin molecules by low-temperature free radical polymerization to obtain an intermediate product;

[0012] The intermediate product is post-processed to obtain embolic microspheres.

[0013] In one embodiment of the present invention, the low-temperature free radical polymerization is a polymerization reaction occurring at a temperature of 30° C. to 40° C.

[0014] In one embodiment of the present invention, the compound with a negatively charged group includes at least one of a compound with a carboxylic acid group or a sulfonic acid group, wherein the carboxylic acid group is derived from acrylic acid, methacrylic acid or other carboxylic acid compounds or carboxylate compounds with a carboxylate group and a double bond, and the sulfonic acid group is derived from one or more of 2-acrylamido-2-methylpropanesulfonic acid or other sulfonic acid compounds or sulfonate compounds with a sulfonate group and a double bond.

[0015] In one embodiment of the present invention, the cross-linking agent includes at least one of an acetal cross-linking agent, an amide cross-linking agent or an cyclopentane cross-linking agent; the acetal cross-linking agent includes one or a combination of formaldehyde, glutaraldehyde or n-butyraldehyde; the amide cross-linking agent includes one or a combination of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate or diethylenetriamine; the cyclopentane cross-linking agent includes one or a combination of genipin, oleuropein or aucubin.

[0016] In one embodiment of the present invention, the catalyst includes one or a combination of N,N-dimethylaniline, sodium bisulfite, tetraethyleneimine, tetramethylethylenediamine, mercaptan, ferrous chloride or silver nitrate.

[0017] In one embodiment of the present invention, the dispersant includes one or a combination of Span 20, Span 80, Tween 20, Tween 80, PEG-40 or PEG-20, and the oil phase liquid includes one or a combination of liquid paraffin, glycerol, dimethyl silicone oil, white oil, vegetable oil or petroleum ether.

[0018] In one embodiment of the present invention, the alkali solution includes at least one of sodium hydroxide, ammonia water or potassium hydroxide, and the initiator includes one or a combination of ammonium persulfate, potassium persulfate, hydrogen peroxide, diacyl peroxide, benzoyl peroxide or azobisisobutyronitrile.

[0019] In one embodiment of the present invention, the mass ratio of the compound with negatively charged groups to the gelatin is 1:(2-4); and / or

[0020] The mass ratio of the catalyst to the gelatin is 1:(4-20); and / or

[0021] The mass ratio of the cross-linking agent to the gelatin is 1:(4-20).

[0022] In one embodiment of the present invention, the first preset temperature is 30°C to 40°C;

[0023] The second preset temperature is 38°C~45°C.

[0024] In one embodiment of the present invention, the manufacturing method further includes:

[0025] Add the aqueous prepolymer solution dropwise to the dispersion and shear at 250 rpm to 350 rpm for 10 to 60 minutes;

[0026] Add the catalyst dropwise, and continue the reaction at 250 rpm to 350 rpm for 150 to 600 minutes; and

[0027] The cross-linking agent is then added, and the mixture is stirred at 250 rpm to 350 rpm for reaction for 2 minutes to 60 minutes to obtain an intermediate product.

[0028] In one embodiment of the present invention, the post-processing includes:

[0029] After obtaining the intermediate product, filtering the intermediate product, adding a solvent containing a surfactant to wash for a first preset time, rinsing with pure water and filtering, and then rinsing with injection water to collect the embolic microsphere intermediate phase;

[0030] The embolic microsphere intermediate phase is pre-frozen and then freeze-dried for a second preset time to obtain the embolic microsphere.

[0031] In one embodiment of the present invention, the first preset time is 10 minutes to 120 minutes; the second preset time is 60 hours to 260 hours.

[0032] In one embodiment of the present invention, the surfactant comprises Tween 80.

[0033] An embodiment of the present invention also provides an embolic microsphere that can sustainably release drugs and control degradation rate, which is obtained by the above-mentioned preparation method. The embolic microsphere is a microsphere obtained by polymerizing gelatin molecules and compounds containing negatively charged groups and cross-linking with a cross-linking agent; wherein: the negatively charged groups are directly grafted onto the surface of the gelatin molecules.

[0034] In one embodiment of the present invention, the negatively charged group includes at least one of a sulfonic acid group or a carboxylic acid group; and the cross-linking agent includes at least one of an acetal cross-linking agent, an amide cross-linking agent or an cyclopentane cross-linking agent.

[0035] In summary, according to the embolic microspheres and preparation methods thereof that can release drugs slowly and control degradation rate proposed by the present invention, negatively charged groups can be directly grafted on the surface of gelatin molecules by low-temperature free radical polymerization, and no drug-carrying intermediates, especially non-degradable drug-carrying intermediates, are required as in the past, so that completely degradable embolic microspheres can be obtained, which have good biocompatibility, blood vessels can be reopened after degradation, and repeated embolization treatments can be performed without causing inflammatory reactions and ectopic embolism. In addition, the embolic microspheres obtained by the method of the present invention have efficient drug loading performance, and can play a role in slowly releasing drugs, achieve the effect of targeted drug delivery, and realize complete degradation of embolic microspheres. By controlling the type of cross-linking agent, the embolic microspheres have a good effect of long-term drug sustained release, and the degradation rate of the embolic microspheres is controlled, and its degradation time can be controlled as desired, and functional monomers with different cross-linking strengths can be designed and synthesized as needed to meet the needs of practical applications in different lesion scenarios, providing more options for patient treatment. In addition, according to the technical solution of the present invention, the number of grafted negatively charged groups formed on the embolic microspheres can be controlled, so that the drug loading can be controlled according to the needs of the disease, and the preparation process is simple, the manufacturing process is safe and easy to control, and the cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0037] Figure 1 FIG. 4 is a diagram showing the drug loading mechanism of embolic microspheres in one embodiment of the present invention.

[0038] Figure 2 This is a diagram of the drug loading mechanism of the embolic microspheres in Example 1.

[0039] Figure 3 This is a diagram of the drug loading mechanism of the embolic microspheres in Example 2.

[0040] Figure 4 Schematic diagram of the drug loading mechanism of the embolic microspheres in Examples 3-5.

[0041] Figure 5 FIG. 5 is a microscope photograph of embolic microspheres prepared by the method of the present invention in one embodiment of the present invention.

[0042] Figure 6 This is a microscope photo of the embolic microspheres after drug loading in one embodiment of the present invention. DETAILED DESCRIPTION

[0043] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0044] It should be understood that the present invention can be implemented in different forms and should not be interpreted as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make the disclosure thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0045] The technical scheme of the present invention is further described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] The present invention proposes an embolic microsphere capable of sustained drug release and controlled degradation rate. The embolic microsphere is a microsphere obtained by polymerizing gelatin molecules and compounds containing negatively charged groups and cross-linking with a cross-linking agent, wherein negatively charged groups are grafted onto the surface of the gelatin molecules, and the negatively charged groups include at least one of sulfonic acid groups or carboxylic acid groups, and the cross-linking agent includes at least one of acetal cross-linking agents, amide cross-linking agents, or cyclopentane cross-linking agents. By selecting gelatin as the main material, completely degradable embolic microspheres can be obtained, and they are harmless to the human body after degradation. By grafting negatively charged groups onto the surface of gelatin molecules, the embolic microspheres can have efficient drug loading performance. By controlling the type of cross-linking agent, the embolic microspheres have a good long-term drug sustained release effect, and the degradation rate of the embolic microspheres can be controlled. Embolic microspheres with different needs can be customized to meet different usage requirements.

[0047] In one embodiment of the present invention, the acetal crosslinking agent includes one or several combinations of formaldehyde, glutaraldehyde or n-butyraldehyde, etc., the amide crosslinking agent includes one or several combinations of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate or diethylenetriamine, etc., and the cyclopentane crosslinking agent includes one or several combinations of genipin, oleuropein or aucubin, etc. In another embodiment of the present invention, gelatin can also be replaced by natural polymer materials such as polylactic acid, chitosan, alginate or soluble starch materials to ensure that the obtained embolic microspheres can be degraded.

[0048] The present invention provides a method for preparing embolic microspheres capable of sustained-release drugs and controlled degradation rate, which is used for preparing the above-mentioned embolic microspheres. The preparation method at least comprises: dissolving a dispersant in an oil phase liquid, heating the oil phase liquid to a first preset temperature, and stirring the mixture to obtain a dispersion; dissolving gelatin in water at a second preset temperature to obtain a gelatin solution; dissolving a compound with a negatively charged group in water, and adding an alkali solution and an initiator under an ice bath to obtain a modified solution; adding the modified solution to the gelatin solution, stirring the mixture, and obtaining an aqueous prepolymer solution; dropping the aqueous prepolymer solution into the dispersion, premixing the mixture, and then adding a crosslinking agent and a catalyst to perform crosslinking and catalysis, and directly grafting the negatively charged group onto the gelatin molecule in a low-temperature free radical polymerization manner to obtain an intermediate product; and post-treating the intermediate product to obtain the embolic microspheres.

[0049] In one embodiment of the present invention, the dispersant includes, for example, one or a combination of dispersants such as Span 20, Span 80, Tween 20, Tween 80, PEG-40 or PEG-20, and the oil phase liquid includes, for example, one or a combination of materials such as liquid paraffin, glycerol, dimethyl silicone oil, white oil, vegetable oil or petroleum ether. In one embodiment of the present invention, for example, 0.1g~10g of the dispersant is dispersed in 1000ml of the oil phase liquid, and for example, it is carried out in a reactor or a stirring kettle, and the first preset temperature is, for example, 30°C~40°C, and stirring is continued at, for example, 200rpm / min (revolutions / minute)~250rpm / min to improve the uniformity of the obtained dispersion.

[0050] In one embodiment of the present invention, the second preset temperature is, for example, 38° C. to 45° C. The solvent water is, for example, purified water or water for injection, and the mass ratio of gelatin to water is, for example, (1-5):10.

[0051] In one embodiment of the present invention, the compound with negatively charged groups is, for example, at least one of compounds including carboxylic acid groups or sulfonic acid groups, the carboxylic acid group is derived from carboxylic acid compounds or carboxylate compounds with carboxylate groups and double bonds such as acrylic acid or methacrylic acid, and the sulfonic acid group is derived from one or a combination of sulfonic acid compounds or sulfonate compounds with sulfonate groups and double bonds such as 2-acrylamide-2-methylpropanesulfonic acid. The solvent water is, for example, purified water or water for injection, and the mass ratio of the compound with negatively charged groups to water is, for example, (0.1-30):20. After the compound with negatively charged groups is dissolved, an alkali solution and an initiator are added under ice bath conditions to obtain a modified solution, wherein the alkali solution includes, for example, at least one of sodium hydroxide, ammonia water or potassium hydroxide, and the initiator includes one or a combination of ammonium persulfate, potassium persulfate, hydrogen peroxide, diacyl peroxide, benzoyl peroxide or azobisisobutyronitrile. In one embodiment of the present invention, for example, 0.1 ml to 10 ml of 7.5 mol / L sodium hydroxide solution is added under ice bath, and then 0.01 g to 5 g of potassium persulfate is added. The ice bath can release the heat generated by the reaction, improve the safety of the synthesis, and ensure the performance of the modified solution.

[0052] In one embodiment of the present invention, the modified liquid is added to the gelatin solution, stirred evenly, and the bubbles are removed, for example, by ultrasound, to obtain an aqueous prepolymer solution. The mass ratio of the compound with negatively charged groups to gelatin is, for example, 1: (2~4). After obtaining the aqueous prepolymer solution, the aqueous prepolymer solution is added dropwise to the dispersion solution for premixing, and the premixing is, for example, shearing at 250rpm / min~350rpm / min for 10min~60min to make the aqueous prepolymer solution and the dispersion solution evenly mixed, and a crosslinking agent and a catalyst are added for crosslinking and catalysis, and the negatively charged groups are grafted onto the gelatin molecules by low-temperature free radical polymerization to obtain an intermediate product. The volume ratio of the aqueous prepolymer solution to the dispersion solution is, for example, (10~15):100. When adding the crosslinking agent and the catalyst, they can be added simultaneously or at intervals. For example, after adding the crosslinking agent and the catalyst at the same time, the reaction is continued for 150min~600min to obtain an intermediate product. In another embodiment of the present invention, for example, after the catalyst is added dropwise, the mixture is stirred at 250 rpm / min to 350 rpm / min and the reaction is continued for 150 min to 600 min, and then a cross-linking agent is added and the mixture is stirred at 250 rpm / min to 350 rpm / min for 2 min to 60 min to obtain an intermediate product. The particle size of the drug-loaded microspheres can be controlled by controlling the stirring speed.

[0053] In one embodiment of the present invention, the mass ratio of the crosslinking agent to gelatin is, for example, 1: (4-20), and the mass ratio of the catalyst to gelatin is, for example, 1: (4-20). Low-temperature free radical polymerization is a polymerization reaction that occurs at a temperature of 30°C to 40°C. In this embodiment, the temperature during low-temperature free radical polymerization is controlled by controlling the first temperature, the second temperature, and ice bath treatment. In other embodiments, the temperature during low-temperature free radical polymerization is controlled, for example, by a water bath or temperature control equipment. In the present invention, the initiator can reduce the reaction activation energy of the compound with negatively charged groups and gelatin, stimulate free radical polymerization between the two, and react at a lower temperature, that is, the low-temperature free radical polymerization system can directly graft the negatively charged groups onto the gelatin molecules, crosslink them into microspheres through the crosslinking agent, and catalyze the reaction through the catalyst to obtain an intermediate product. By controlling the polymerization temperature, the problem that gelatin has poor stability at high temperatures and is easy to form gel at low temperatures is solved, and the modification of gelatin is achieved. The present invention can directly graft negatively charged groups onto gelatin molecules, thereby improving the drug loading and drug release capabilities of the embolic microspheres, while avoiding the use of non-degradable materials such as polyethylene glycol, and achieving complete degradation of the embolic microspheres.

[0054] In one embodiment of the present invention, the catalyst includes one or several combinations of N,N-dimethylaniline, sodium bisulfite, tetraethyleneimine, tetramethylethylenediamine, mercaptan, ferrous chloride or silver nitrate, etc., the crosslinking agent includes at least one of acetal crosslinking agent, amide crosslinking agent or iridoid crosslinking agent, etc., the acetal crosslinking agent includes one or several combinations of formaldehyde, glutaraldehyde or n-butyraldehyde, etc., the amide crosslinking agent includes one or several combinations of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate or diethylenetriamine, etc., and the iridoid crosslinking agent includes one or several combinations of genipin, oleuropein or aucubin, etc. When the crosslinking agent is an amide crosslinking agent, the amide crosslinking agent and gelatin can undergo a crosslinking reaction under the action of the catalyst. By controlling the mass ratio of the compound with negatively charged groups and gelatin, the number of negatively charged groups grafted on the formed embolic microspheres can be controlled, so that the drug loading amount can be controlled according to the needs of the disease. By controlling the type and proportion of the cross-linking agent, the subsequent drug release rate and the degradation rate of the embolic microspheres can be controlled, thereby realizing the preparation of customizable embolic microspheres to meet different usage needs.

[0055] In one embodiment of the present invention, after obtaining the intermediate product, the post-processing includes: filtering the intermediate product, adding a solvent containing a surfactant to wash for a first preset time, rinsing with pure water and filtering, then rinsing with water for injection, collecting the embolic microsphere intermediate phase; pre-freezing the embolic microsphere intermediate phase, and then freeze-drying for a second preset time to obtain the embolic microsphere. Specifically, for example, the solvent and oil phase liquid are removed by sieve filtration, and the filtered solid is placed in a container such as a beaker, and a solvent containing a surfactant is added to wash for a first preset time, and then an appropriate amount of purified water is taken to rinse, and the solvent is removed by sieve filtration again, and then rinsed with water for injection to collect the embolic microsphere intermediate phase. Among them, the solvent containing a surfactant includes, for example, Tween 80 solution, etc., and is stirred during the washing process to improve the washing efficiency and effectively remove the oil phase liquid and residual catalyst that may remain on the surface of the intermediate product. The first preset time is, for example, 10min~120min.

[0056] In one embodiment of the present invention, after collecting the embolic microsphere intermediate phase, the embolic microsphere intermediate phase is transferred to a freeze-drying box for pre-freezing, and then freeze-dried for a second preset time after pre-freezing to obtain the embolic microsphere. The pre-freezing temperature is, for example, -20°C, the freeze-drying temperature is, for example, -40°C, and the second preset time is, for example, 60h to 260h. By freeze-drying, the stability of the embolic microspheres can be ensured, the activity of the embolic microspheres can be protected, and high-quality embolic microspheres can be obtained.

[0057] Hereinafter, the present invention will be explained in more detail by citing examples, which should not be construed as limiting. Appropriate modifications may be made within the scope consistent with the gist of the present invention, all of which fall within the technical scope of the present invention.

[0058] Example 1

[0059] Take 1 g of Span 80 and dissolve it in 1000 ml of liquid paraffin. Add it into a 2 L reactor, preheat it at 30°C, and stir it at 230 r / min to obtain a dispersion.

[0060] 40 g of gelatin was added to 100 ml of purified water and stirred to dissolve at 40°C to obtain a gelatin solution.

[0061] Dissolve 20 g of 2-acrylamido-2-methylpropanesulfonic acid in 20 ml of purified water, add 10 ml of 7.5 mol / L sodium hydroxide solution under ice water bath, and then add 5 g of potassium persulfate and stir to dissolve to obtain a modified solution.

[0062] The modified liquid is added into the gelatin solution, stirred evenly, and the bubbles are removed by ultrasonication to obtain a water-phase prepolymer solution.

[0063] The aqueous prepolymer was added dropwise to the 30°C dispersion using a peristaltic pump, and after shearing at 300 r / min for 50 min, 5 g of N,N-methylenebisacrylamide was added, and then 10 g of tetramethylethylenediamine was added dropwise, and the reaction was continued for 300 min to obtain an intermediate product.

[0064] The intermediate product was filtered through a mesh to remove the oil phase liquid and solvent, the solid was put into a beaker, an appropriate amount of Tween 80 solution was added and stirred for 60 minutes, an appropriate amount of purified water was taken to rinse and filter through a mesh, an appropriate amount of injection water was used to rinse and sieve, and the microspheres in the mesh were collected to obtain the embolic microsphere intermediate phase. The embolic microsphere intermediate phase was transferred to a freeze-drying box, pre-frozen at -20°C, and after the pre-freezing was completed, freeze-dried at -40°C for 200 hours to obtain the embolic microspheres.

[0065] Example 2

[0066] Take 1 g of Span 80 and dissolve it in 1000 ml of liquid paraffin. Add it into a 2 L reactor, preheat it at 35°C, and stir it at 230 r / min to obtain a dispersion.

[0067] 40 g of gelatin was added to 100 ml of purified water and stirred to dissolve at 40°C to obtain a gelatin solution.

[0068] Dissolve 20 g of acrylic acid in 20 ml of purified water, add 10 ml of 7.5 mol / L sodium hydroxide solution under ice water bath, and then add 5 g of potassium persulfate and stir to dissolve to obtain a modified solution.

[0069] The modified liquid is added into the gelatin solution, stirred evenly, and the bubbles are removed by ultrasonication to obtain a water-phase prepolymer solution.

[0070] The aqueous prepolymer was added dropwise to the dispersion at 35° C. using a peristaltic pump, and after shearing at 300 r / min for 50 min, 5 g of N,N-methylenebisacrylamide was added, and then 10 g of tetramethylethylenediamine was added dropwise, and the reaction was continued for 300 min to obtain an intermediate product.

[0071] The intermediate product was filtered through a mesh to remove the oil phase and solvent, and the solid was added to a beaker, and an appropriate amount of Tween 80 solution was added and stirred for 60 minutes. An appropriate amount of purified water was taken to rinse and filter through a mesh, and an appropriate amount of injection water was used to rinse and sieve, and the microspheres in the mesh were collected to obtain the embolic microsphere intermediate phase. The embolic microsphere intermediate phase was transferred to a freeze-drying box, pre-frozen at -20°C, and after the pre-freezing was completed, freeze-dried at -40°C for 200 hours to obtain the embolic microspheres.

[0072] Example 3

[0073] Take 1 g of Span 80 and dissolve it in 1000 ml of liquid paraffin. Add it into a 2 L reactor, preheat it at 40°C, and stir it at 230 r / min to obtain a dispersion.

[0074] 40 g of gelatin was added to 100 ml of purified water and stirred to dissolve at 40°C to obtain a gelatin solution.

[0075] Dissolve 10 g of 2-acrylamido-2-methylpropanesulfonic acid and 10 g of acrylic acid in 20 ml of purified water, add 10 ml of 7.5 mol / L sodium hydroxide solution under ice water bath, and then add 5 g of potassium persulfate and stir to dissolve to obtain a modified solution.

[0076] The modified liquid is added into the gelatin solution, stirred evenly, and the bubbles are removed by ultrasonication to obtain a water-phase prepolymer solution.

[0077] The aqueous prepolymer was added dropwise to the 40°C dispersion using a peristaltic pump, and after shearing at 300 r / min for 50 min, 5 g of N,N-methylenebisacrylamide was added, and then 10 g of tetramethylethylenediamine was added dropwise, and the reaction was continued for 300 min to obtain an intermediate product.

[0078] The intermediate product was filtered through a sieve to remove the oil phase liquid and solvent, and the solid was added to a beaker, and an appropriate amount of Tween 80 solution was added and stirred for 60 minutes, and an appropriate amount of purified water was taken to rinse and filter through a sieve, and an appropriate amount of injection water was used to rinse and sieve, and the microspheres in the sieve were collected to obtain the embolic microsphere intermediate phase. The embolic microsphere intermediate phase was transferred to a freeze-drying box, pre-frozen at -20°C, and after the pre-freezing was completed, freeze-dried at -40°C for 200 hours to obtain the embolic microspheres.

[0079] Example 4

[0080] Take 1 g of Span 80 and dissolve it in 1000 ml of liquid paraffin. Add it into a 2 L reactor, preheat it at 40°C, and stir it at 230 r / min to obtain a dispersion.

[0081] 40 g of gelatin was added to 100 ml of purified water and stirred to dissolve at 40°C to obtain a gelatin solution.

[0082] Dissolve 10 g of 2-acrylamido-2-methylpropanesulfonic acid and 10 g of acrylic acid in 10 ml of purified water, add 10 ml of 7.5 mol / L sodium hydroxide solution under ice water bath, and then add 5 g of potassium persulfate and stir to dissolve to obtain a modified solution.

[0083] The modified liquid is added into the gelatin solution, stirred evenly, and the bubbles are removed by ultrasonication to obtain a water-phase prepolymer solution.

[0084] Use a peristaltic pump to add the above-mentioned aqueous prepolymer droplets into the above-mentioned dispersion at 40°C, shear at 300 r / min for 50 minutes, add 10g of tetramethylethylenediamine, continue to react for 300 minutes, then add formaldehyde aqueous solution containing 5g of formaldehyde, and stir to react for 30 minutes.

[0085] The intermediate product was filtered through a sieve to remove the oil phase liquid and solvent, and the solid was added to a beaker, and an appropriate amount of Tween 80 solution was added and stirred for 60 minutes, and an appropriate amount of purified water was taken to rinse and filter through a sieve, and an appropriate amount of injection water was used to rinse and sieve, and the microspheres in the sieve were collected to obtain the embolic microsphere intermediate phase. The embolic microsphere intermediate phase was transferred to a freeze-drying box, pre-frozen at -20°C, and after the pre-freezing was completed, freeze-dried at -40°C for 200 hours to obtain the embolic microspheres.

[0086] Example 5

[0087] Take 1 g of Span 80 and dissolve it in 1000 ml of liquid paraffin. Add it into a 2 L reactor, preheat it at 40°C, and stir it at 230 r / min to obtain a dispersion.

[0088] 40 g of gelatin was added to 100 ml of purified water and stirred to dissolve at 40°C to obtain a gelatin solution.

[0089] Dissolve 10 g of 2-acrylamido-2-methylpropanesulfonic acid and 10 g of acrylic acid in 20 ml of purified water, add 10 ml of 7.5 mol / L sodium hydroxide solution under ice water bath, and then add 5 g of potassium persulfate and stir to dissolve to obtain a modified solution.

[0090] The modified liquid is added into the gelatin solution, stirred evenly, and the bubbles are removed by ultrasonication to obtain a water-phase prepolymer solution.

[0091] The above-mentioned aqueous prepolymer droplets were added into the above-mentioned dispersion at 40° C. using a peristaltic pump, and after shearing at 300 r / min for 50 min, 10 g of tetramethylethylenediamine was added, and the reaction was continued for 300 min. Then, a genipin acetic acid aqueous solution containing 5 g of genipin acetic acid was added, and the reaction was stirred for 30 min.

[0092] The intermediate product was filtered through a sieve to remove the oil phase liquid and solvent, and the solid was added to a beaker, and an appropriate amount of Tween 80 solution was added and stirred for 60 minutes, and an appropriate amount of purified water was taken to rinse and filter through a sieve, and an appropriate amount of injection water was used to rinse and sieve, and the microspheres in the sieve were collected to obtain the embolic microsphere intermediate phase. The embolic microsphere intermediate phase was transferred to a freeze-drying box, pre-frozen at -20°C, and after the pre-freezing was completed, freeze-dried at -40°C for 200 hours to obtain the embolic microspheres.

[0093] Comparative Example 1

[0094] In comparative example 1, embolic microspheres produced by Baidikang UK Ltd. were selected, and the model number was DC2V103 and the specification was 100-300 μm.

[0095] In the present invention, in Examples 1-5 and Comparative Example 1, the drug loading and release properties and degradation properties of the embolic microspheres were tested. The test results are shown in Tables 1 and 2.

[0096] In one embodiment of the present invention, the drug loading and release test is conducted by taking the cationic drug doxorubicin as an example, and the drug loading steps include: using a 2ml disposable syringe to measure 100mg of embolic microspheres respectively and placing them in a 10ml brown vial; using a pipette to transfer 3ml of 15mg / ml Doxorubicin solution to three brown vials, covering them with rubber stoppers, shaking and flipping, and starting timing; shaking and flipping the vials every 5 minutes for the first 30 minutes, and shaking and flipping the vials at irregular intervals thereafter; taking 20μl of liquid from the supernatant of each vial at 0.5h, placing it in a clean brown vial, and adding 4.98ml of purified water to mix, and testing its ultraviolet absorbance. During the drug loading process, a small amount of hydrochloric acid (HCl) can also be added, such as adding 20μl of 37% hydrochloric acid, to adjust the Ph value during the drug loading process to improve the solubility, stability and pharmacological activity of the drug.

[0097] In one embodiment of the present invention, the drug release step includes: assembling an overhead stirrer, a polytetrafluoroethylene stirring paddle, a 1000 ml three-necked flask, and a heat-collecting constant temperature heating magnetic stirrer and adding an appropriate amount of purified water; at a water bath temperature of 37° C., the exposed part of the three-necked flask is wrapped with tin foil and covered, and the prepared 0.15M saline solution is placed in a 37° C. water bath for preheating; the embolic microspheres after drug loading are filtered out by filter paper to remove the unloaded Doxorubicin solution, and all the embolic microspheres are transferred to the three-necked flask and 900 ml of preheated 0.15M saline solution is added, and the timing is started; 2.5 ml of dissolution medium is taken out from the three-necked flask at 0 min, 5 min, 10 min, 0.5 h, 1.5 h, 3.0 h, 24 h, and 48 h, respectively, and 2.5 ml of 0.15M saline solution is added to the flask. The absorbance of Doxorubicin solutions with contents of 100.0 μg / ml, 10.00 μg / ml and 1.000 μg / ml at a wavelength of 480 nm was measured respectively, and a standard curve of solution concentration and absorbance was prepared.

[0098] In one embodiment of the present invention, the degradation test includes: weighing 100 mg of embolic microspheres, putting them into a constant weight bottle to weigh the weight, putting them into a 105°C oven to dry for 2 hours, and weighing the total weight after constant weight; taking 100 mg of the dried sample and putting it into a test tube, adding 10 ml of buffer solution, the buffer solution is a phosphoric acid solution with a Ph value of 7.4. Seal the mouth of the test tube with tin foil, mark it with the corresponding sample batch number. Two groups are made in parallel for each sampling point. Put the sample into a water bath at 37±1°C, take out the sample at 3, 7, 14, 21, 30, and 60 days, perform a constant weight test, and record the weight change rate of the embolic microspheres corresponding to the degradation time.

[0099] Table 1. Drug loading and release rate of embolic microspheres in Examples 1-5 and Comparative Example 1

[0100]

[0101] Table 2. Degradation rate of embolic microspheres in Examples 1-5 and Comparative Example 1

[0102]

[0103] See also Figure 1 As shown, in one embodiment of the present invention, multiple negatively charged groups 20 are grafted onto the surface of the embolic microspheres 10. The negatively charged groups 20 can be the same type of negatively charged groups or a combination of multiple negatively charged groups. The number of negatively charged groups 20 on the surface of the embolic microspheres 10 can be controlled according to the ratio of raw materials in the manufacturing process. When loading drugs, the drugs 30 are carried on the embolic microspheres 10 through the negatively charged groups 20. During use, after reaching a specific position, the drugs 30 are gradually degraded under the action of relevant enzymes in the body, and the drugs 30 are slowly released, and the embolic microspheres 10 are degraded. Please refer to Figures 2 to 4 The above are the drug loading mechanism diagrams of the embolic microspheres of Examples 1-5, respectively. Figure 4 The diagram is a drug loading mechanism diagram of Examples 3-5. It can be seen from the figure that when the surface of the embolic microspheres is grafted with the same type of negatively charged groups, or a combination of multiple negatively charged groups, the obtained embolic microspheres can all be loaded with drugs. The embolic microspheres obtained by the present invention have controllable degradability and efficient drug loading capacity, good biocompatibility, can reduce the side effects of patients after interventional treatment, and provide more options for interventional treatment.

[0104] See also Figures 5 and 6 As shown, in one embodiment of the present invention, the obtained embolic microspheres have good roundness, smooth surface and uniform particle size. After drug loading, the drug can be evenly distributed on the embolic microspheres, indicating that the negatively charged groups are evenly distributed on the embolic microspheres, and uniform drug loading can be achieved.

[0105] Please refer to Table 1. Comparative Examples 1-5 and Comparative Example 1 show that the embolic microspheres obtained by the technical solution of the present application can increase the drug loading of the embolic microspheres and achieve sustained release of the drug. Comparative Examples 1-3 show that when the negatively charged group is a sulfonic acid group, the drug loading increases relative to the carboxylic acid group, and the drug release rate is slower. When the embolic microspheres are grafted with sulfonic acid groups and carboxylic acid groups at the same time, the drug loading further increases, and the drug release rate further decreases. Comparative Examples 3 and 5 show that when different cross-linking agents are used to obtain embolic microspheres, and when the gelatin in the embolic microspheres is grafted with the same amount of sulfonic acid groups and carboxylic acid groups at the same time, the drug loading and drug release rate are similar, indicating that the type of cross-linking agent has a weak effect on the drug loading, and the drug loading is mainly controlled by the type and proportion of the negatively charged groups. Therefore, controlling the type and proportion of negatively charged groups on the embolic microspheres can control the drug loading and release rate, thereby achieving performance with different drug loadings, and at the same time can play a role in slow drug release, achieve the effect of targeted drug delivery, and realize the preparation of customizable embolic microspheres to meet the needs of different drug loadings and release rates. The obtained embolic microspheres have good biocompatibility, and the blood vessels can be reopened after degradation. Repeated embolization treatments will not cause inflammatory reactions, providing more options for patient treatment.

[0106] As shown in Table 2, by comparing Examples 1-5 and Comparative Example 1, it can be seen that when the same cross-linking agent is selected, the degradation rates of the embolic microspheres are similar, and when different cross-linking agents are selected, the degradation rates of the embolic microspheres are different. Therefore, the degradation rate of the embolic microspheres obtained by selecting different cross-linking agents can be controlled, so that the degradation time can be controlled as desired, and functional monomers with different cross-linking strengths can be designed and synthesized according to actual needs to meet the needs of practical applications in different lesion scenarios.

[0107] In summary, the present invention proposes an embolic microsphere that can release drugs slowly and control the degradation rate and a preparation method thereof. By selecting gelatin as the drug-carrying body and adopting a low-temperature free radical polymerization system, negatively charged groups can be directly grafted on the surface of the gelatin molecule, and no drug-carrying intermediates, especially non-degradable drug-carrying intermediates, are required as in the past, so that completely degradable embolic microspheres can be obtained, which have good biocompatibility, and the blood vessels can be reopened after degradation, and repeated embolization treatments can be performed without causing inflammatory reactions and ectopic embolism. In addition, the embolic microspheres obtained by the method of the present invention have efficient drug-carrying performance, and can play a role in slowly releasing drugs, achieve the effect of targeted drug delivery, and realize complete degradation of the embolic microspheres. By controlling the type of cross-linking agent, the embolic microspheres have a good long-term drug sustained release effect, and the degradation rate of the embolic microspheres is controlled, and its degradation time can be controlled as desired, and functional monomers with different cross-linking strengths can be designed and synthesized as needed to meet the needs of practical applications in different lesion scenarios, providing more options for patient treatment. In addition, according to the technical solution of the present invention, the number of grafted negatively charged groups formed on the embolic microspheres can be controlled, so that the drug loading can be controlled according to the needs of the disease, and the preparation process is simple, the manufacturing process is safe and easy to control, and the cost is reduced.

[0108] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the inventive concept, such as a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in this application.

[0109] Except for the technical features described in the specification, the remaining technical features are known technologies to those skilled in the art. In order to highlight the innovative features of the present invention, the remaining technical features will not be described here in detail.

Claims

1. A method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate, characterized in that: At least the following steps are included: Dissolving a dispersant in an oil phase liquid, heating the mixture to a first preset temperature, and stirring the mixture to obtain a dispersion; dissolving gelatin in water at a second preset temperature to obtain a gelatin solution; Dissolve the compound with negatively charged groups in water, and add alkali solution and initiator in an ice bath to obtain a modified solution; Adding the modified liquid into the gelatin solution and stirring to obtain an aqueous prepolymer solution; The aqueous phase prepolymer solution is added dropwise to the dispersion solution for premixing, and then a crosslinking agent and a catalyst are added for crosslinking and catalysis, and negatively charged groups are directly grafted onto gelatin molecules by low-temperature free radical polymerization to obtain an intermediate product; The intermediate product is post-processed to obtain embolic microspheres.

2. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The low temperature free radical polymerization is a polymerization reaction occurring at a temperature of 30°C to 40°C.

3. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The compound with negatively charged groups includes at least one of compounds with carboxylic acid groups or sulfonic acid groups, wherein the carboxylic acid group is derived from acrylic acid, methacrylic acid or other carboxylic acid compounds or carboxylate compounds with carboxylate groups and double bonds, and the sulfonic acid group is derived from one or more of 2-acrylamido-2-methylpropanesulfonic acid or other sulfonic acid compounds or sulfonate compounds with sulfonate groups and double bonds.

4. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The cross-linking agent includes at least one of an acetal cross-linking agent, an amide cross-linking agent or an iridoid cross-linking agent; the acetal cross-linking agent includes one or a combination of formaldehyde, glutaraldehyde or n-butyraldehyde; the amide cross-linking agent includes one or a combination of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate or diethylenetriamine; the iridoid cross-linking agent includes one or a combination of genipin, oleuropein or aucubin.

5. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The catalyst includes one or a combination of N,N-dimethylaniline, sodium bisulfite, tetraethyleneimine, tetramethylethylenediamine, mercaptan, ferrous chloride or silver nitrate.

6. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The dispersant includes one or a combination of Span 20, Span 80, Tween 20, Tween 80, PEG-40 or PEG-20, and the oil phase liquid includes one or a combination of liquid paraffin, glycerol, dimethyl silicone oil, white oil, vegetable oil or petroleum ether.

7. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The alkali solution includes at least one of sodium hydroxide, ammonia water or potassium hydroxide, and the initiator includes one or a combination of ammonium persulfate, potassium persulfate, hydrogen peroxide, diacyl peroxide, benzoyl peroxide or azobisisobutyronitrile.

8. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The mass ratio of the compound with negatively charged groups to the gelatin is 1:(2-4); and / or The mass ratio of the catalyst to the gelatin is 1:(4-20); and / or The mass ratio of the cross-linking agent to the gelatin is 1:(4-20).

9. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The first preset temperature is 30°C to 40°C; The second preset temperature is 38°C~45°C.

10. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The production method further comprises: Add the aqueous prepolymer solution dropwise to the dispersion and shear at 250 rpm to 350 rpm for 10 to 60 minutes; Add the catalyst dropwise, and continue the reaction at 250 rpm to 350 rpm for 150 to 600 minutes; and The cross-linking agent is then added, and the mixture is stirred at 250 rpm to 350 rpm for reaction for 2 minutes to 60 minutes to obtain an intermediate product.

11. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 1, characterized in that: The post-processing includes: After obtaining the intermediate product, filtering the intermediate product, adding a solvent containing a surfactant to wash for a first preset time, rinsing with pure water and filtering, and then rinsing with injection water to collect the embolic microsphere intermediate phase; The embolic microsphere intermediate phase is pre-frozen and then freeze-dried for a second preset time to obtain the embolic microsphere.

12. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 11, characterized in that: The first preset time is 10 minutes to 120 minutes; the second preset time is 60 hours to 260 hours.

13. The method for preparing embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 11, characterized in that: The surfactant includes Tween 80.

14. An embolic microsphere capable of sustained drug release and controlled degradation rate, characterized in that: The embolic microspheres are obtained by the preparation method according to any one of claims 1 to 13, wherein the microspheres are obtained by polymerizing gelatin molecules and compounds containing negatively charged groups and cross-linking with a cross-linking agent; wherein: the negatively charged groups are directly grafted onto the surface of the gelatin molecules.

15. The embolic microspheres capable of sustained drug release and controlled degradation rate according to claim 14, characterized in that: The negatively charged group includes at least one of a sulfonic acid group or a carboxylic acid group; the cross-linking agent includes at least one of an acetal cross-linking agent, an amide cross-linking agent or an cyclopentane cross-linking agent.

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