Embolic microspheres and preparation method thereof and drug-loaded embolic microspheres

Abstract

The present application relates to an embolic microsphere and a preparation method thereof and a drug-loaded embolic microsphere, belonging to the technical field of embolic microspheres. The embolic microspheres include a three-dimensional network structure formed by cross-linking polymerization of a polyhydroxy water-soluble polymer modified mainly by a photodegradation agent and a photosensitizer, a water-soluble monomer with a polymerizable double bond, and a water-soluble cross-linking agent, wherein the photosensitizer is covalently coupled to the photodegradation agent, and the photodegradation agent is covalently coupled to the polyhydroxy water-soluble polymer. The embolic microspheres can have the therapeutic effects of embolization and photodynamic therapy, and embolization therapy and photodynamic therapy can be synergistically treated to improve the therapeutic efficiency of tumors.

Description

Technical Field

[0001] The present application relates to the technical field of embolic microspheres, and in particular to embolic microspheres, a preparation method thereof, and drug-loaded embolic microspheres. Background Art

[0002] As a medical interventional embolic material, embolic microspheres can be injected into the blood vessels supplying lesions or organs through a catheter to cause occlusion and interrupt blood supply, thereby achieving the purpose of controlling bleeding, occluding vascular lesions, treating tumors, and clearing the functions of diseased organs.

[0003] In addition to the above-mentioned embolic effect, embolic microspheres can also be used as drug carriers, and can adsorb drugs to form drug-loaded embolic microspheres. After reaching the lesion, the drug-loaded embolic microspheres slowly release the drug to increase the local drug concentration to achieve the therapeutic effects of embolization and chemotherapy. However, the above-mentioned drug-loaded embolic microspheres are prone to drug resistance in the lesion during long-term treatment, resulting in a decrease in treatment efficiency. Summary of the invention

[0004] The purpose of the present application is to provide an embolic microsphere and a preparation method thereof and a drug-loaded embolic microsphere, wherein the embolic microsphere can have both embolic and photodynamic therapeutic effects, and the embolic therapy and the photodynamic therapy can synergistically treat and improve the treatment efficiency of tumors.

[0005] In the first aspect, an embodiment of the present application provides an embolic microsphere, comprising a three-dimensional network structure formed by cross-linking polymerization of a polyhydroxy water-soluble polymer mainly modified by a photodegradant and a photosensitizer, a water-soluble monomer with polymerizable double bonds, and a water-soluble cross-linking agent, wherein the photosensitizer is covalently coupled to the photodegradant, and the photodegradant is covalently coupled to the polyhydroxy water-soluble polymer.

[0006] In the above technical solution, the three-dimensional network structure included in the embolic microspheres is mainly formed by cross-linking polymerization of polyhydroxy water-soluble polymers modified by photodegradation agents and photosensitizers, water-soluble monomers with polymerizable double bonds, and water-soluble crosslinking agents, so that the embolic microspheres can have embolic functions. In addition, the hydroxy water-soluble polymers are modified by photodegradation agents and photosensitizers, the photosensitizers are covalently coupled with the photodegradants, and the photodegradants are covalently coupled with the polyhydroxy water-soluble polymers. When the photodegradants absorb light of a specific wavelength, they will be sheared and release the photosensitizers covalently coupled with them. The photosensitizers can convert oxygen into cytotoxic singlet oxygen under the irradiation of light, thereby damaging tumor cells, thereby achieving the purpose of photodynamic therapy of the embolic microspheres. Moreover, the photodynamic therapy of the embolic microspheres can be synergistic with embolization therapy to improve the treatment efficiency of tumors.

[0007] In a second aspect, the present application provides a method for preparing the embolic microspheres according to the first aspect of the present application, comprising the following steps:

[0008] S100, protecting the photosensitizer with an amino group using a protecting agent to obtain a first intermediate product;

[0009] S200, coupling the first intermediate product to a photodegradation agent to obtain a second intermediate product,

[0010] S300, grafting the second intermediate product to a polyhydroxy water-soluble polymer to obtain a modified polymer;

[0011] S400, mixing the modified polymer, the water-soluble monomer, the water-soluble crosslinking agent and the initiator to obtain an aqueous phase;

[0012] S500, adding the water phase into the oil phase to carry out a reverse suspension polymerization reaction to obtain embolic microspheres.

[0013] In the above technical solution, the photosensitizer is amino protected by a protective agent to mask the chemical and biological activity of the amino group in the photosensitizer. Then, the photodegradation agent is coupled with the first intermediate product to obtain a second intermediate product, and the obtained second intermediate product is grafted to a polyhydroxy water-soluble polymer to form a modified polymer, that is, a polyhydroxy water-soluble polymer modified by a photodegradation agent and a photosensitizer, so that the three-dimensional network structure formed by cross-linking polymerization of the modified polymer, the water-soluble monomer and the water-soluble cross-linking agent under the action of an initiator can have embolization and photodynamic therapy effects.

[0014] In a third aspect, an embodiment of the present application provides a drug-loaded embolic microsphere, comprising embolic microspheres and drugs, wherein the embolic microspheres include the embolic microspheres of the first aspect of the present application and / or the embolic microspheres prepared by the preparation method of the second aspect of the present application.

[0015] In the above technical solution, the drug-loaded embolic microspheres have both embolic and photodynamic therapeutic effects, and embolic therapy and photodynamic therapy can synergistically treat and improve the therapeutic efficiency of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 A schematic diagram of the process for preparing embolic microspheres provided in some embodiments of the present application.

[0018] Figure 2 This is a curve diagram showing the ALA release amount and time of the embolic microspheres provided in Examples 1 to 6 of the present application under light irradiation.

[0019] Figure 3 This is a graph showing the drug release rate and time of the drug-loaded embolic microspheres provided in Examples 1 to 6 of the present application.

[0020] Figure 4 This is a graph of the cell activity test results provided in the examples of the present application. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0023] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range.

[0024] In this application, unless otherwise specified, directional words such as "upper" and "lower" used refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the specification of this application, the terms "include", "comprise", etc. mean "including but not limited to".

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0026] The following is a detailed description of an embolic microsphere, a preparation method, and a drug-loaded embolic microsphere according to an embodiment of the present application.

[0027] The embodiment of the present application provides an embolic microsphere, comprising a three-dimensional network structure formed by cross-linking polymerization of a polyhydroxy water-soluble polymer mainly modified by a photodegradant and a photosensitizer, a water-soluble monomer with a polymerizable double bond, and a water-soluble cross-linking agent, wherein the photosensitizer is covalently coupled to the photodegradant, and the photodegradant is covalently coupled to the polyhydroxy water-soluble polymer.

[0028] In the above technical solution, the three-dimensional network structure included in the embolic microspheres is mainly formed by cross-linking polymerization of polyhydroxy water-soluble polymers modified by photodegradation agents and photosensitizers, water-soluble monomers with polymerizable double bonds, and water-soluble crosslinking agents, so that the embolic microspheres can have embolic functions. In addition, the hydroxy water-soluble polymers are modified by photodegradation agents and photosensitizers, the photosensitizers are covalently coupled with the photodegradants, and the photodegradants are covalently coupled with the polyhydroxy water-soluble polymers. When the photodegradants absorb light of a specific wavelength, they will be sheared and release the photosensitizers covalently coupled with them. The photosensitizers can convert oxygen into cytotoxic singlet oxygen under the irradiation of light, thereby damaging tumor cells, thereby achieving the purpose of photodynamic therapy of the embolic microspheres. Moreover, the photodynamic therapy of the embolic microspheres can be synergistic with embolization therapy to improve the treatment efficiency of tumors.

[0029] In the examples of the present application, selection of appropriate raw materials can make the prepared embolic microspheres have a higher therapeutic efficiency for tumors.

[0030] In some embodiments of the present application, the light absorption wavelength of the photodegradation agent is 780 nm to 1100 nm.

[0031] In the above embodiment, the light absorption wavelength of the photodegradation agent is controlled within the above range, that is, the light absorption wavelength of the photodegradation agent is within the near-infrared short-wave range. Light within this wavelength range has strong penetrating ability, which can help the absorption of the photodegradation agent and control the release of the photosensitizer.

[0032] Furthermore, the photodegradation agent includes coumarin and its derivatives.

[0033] In the above embodiment, coumarin and its derivatives are a type of light-sensitive photoresponsive groups, which can make them have strong light absorption ability, thus promoting their shearing and controlling the release of photosensitizers.

[0034] As an example, the photodegradation agent includes 7-aminocoumarin-4-acetic acid.

[0035] In some embodiments of the present application, the photosensitizer includes at least one of 5-aminolaevulinic acid (ALA) and its derivatives and protoporphyrin and its derivatives.

[0036] In the above embodiment, the photosensitizer can generate a large amount of active oxygen molecules under the irradiation of light, thereby helping to improve the effect of photodynamic therapy.

[0037] In some embodiments of the present application, the polyhydroxy water-soluble polymer includes at least one of polyvinyl alcohol, sodium alginate, sodium hyaluronate, sodium carboxymethyl cellulose and chitosan.

[0038] In some embodiments of the present application, the water-soluble monomer also includes an ionic group, and the ionic group includes at least one of a carboxylic acid group, a carboxylic acid group, an amino group, an amine group, and an ammonium group; in this way, the embolic microspheres also have a drug-carrying function, thereby realizing the triple therapeutic functions of the embolic microspheres of embolization therapy, chemotherapy therapy, and photodynamic therapy.

[0039] In some embodiments of the present application, the water-soluble monomer includes at least one of sodium 2-acrylamide-2-methylpropane sulfonate, sodium allyl sulfonate, sodium methacrylic acid, sodium methacrylate, sodium acrylate, and acrylamide.

[0040] In some embodiments of the present application, the water-soluble crosslinking agent includes at least one of N-(2,2-dimethoxy)-2-methylacrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylamide, N-(2,2-dimethoxy)-2-acrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylamide, N-(2,2-dimethoxy)-2-methylacrylate, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylate, N-(2,2-dimethoxy)-2-acrylate and N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylate.

[0041] In some embodiments of the present application, the particle size of the embolic microspheres is 40 μm to 1375 μm, and the specific particle size can be selected according to actual needs.

[0042] Exemplary, the particle size of the embolic microspheres can be, but is not limited to, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, 150 μm, 200 μm, 300 μm, 350 μm, 400 μm, 450 μm, 480 μm, 580 μm, 650 μm, 750 μm, 825 μm, 975 μm. , 1025μm, 1175μm, 1225μm, 1375μm or a range consisting of any two of the above values, for example, 40μm-70μm, 70μm-100μm, 100μm-150μm, 150μm-200μm, 200μm-300μm, 350μm-450μm, 480μm-580μm, 650μm-750μm, 825μm-975μm, 1025μm-1175μm, 1225μm-1375μm.

[0043] Second, see Figure 1As shown, the present application embodiment provides a method for preparing the embolic microspheres in the above embodiment, comprising the following steps:

[0044] S100, protecting the photosensitizer with an amino group using a protecting agent to obtain a first intermediate product;

[0045] S200, coupling the first intermediate product to a photodegradation agent to obtain a second intermediate product,

[0046] S300, grafting the second intermediate product to a polyhydroxy water-soluble polymer to obtain a modified polymer;

[0047] S400, mixing the modified polymer, the water-soluble monomer, the water-soluble crosslinking agent and the initiator to obtain an aqueous phase;

[0048] S500, adding the water phase into the oil phase to carry out a reverse suspension polymerization reaction to obtain embolic microspheres.

[0049] In the above technical solution, the photosensitizer is amino protected by a protective agent to mask the chemical and biological activity of the amino group in the photosensitizer. Then, the photodegradation agent is coupled with the first intermediate product to obtain a second intermediate product, and the obtained second intermediate product is grafted to a polyhydroxy water-soluble polymer to form a modified polymer, that is, a polyhydroxy water-soluble polymer modified by a photodegradation agent and a photosensitizer, so that the three-dimensional network structure formed by cross-linking polymerization of the modified polymer, the water-soluble monomer and the water-soluble cross-linking agent under the action of an initiator can have embolization and photodynamic therapy effects.

[0050] In some embodiments of the present application, in step S100, the protective agent includes di-tert-butyl dicarbonate (BOC for short). Di-tert-butyl dicarbonate can form a stable amide intermediate with the amino group in the photosensitizer, which can effectively protect the amino group in the photosensitizer from being affected by other reactions.

[0051] In some embodiments of the present application, the mass percentage of the protective agent to the photosensitizer is 100%-160%, which can be understood as the mass ratio of the protective agent to the photosensitizer is (1-1.6): 1. Exemplarily, the mass percentage of the protective agent to the photosensitizer can be, but is not limited to, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160% or a range consisting of any two of the above values.

[0052] In some embodiments of the present application, step S100 may specifically include:

[0053] A photosensitizer is added to a solvent and stirred to dissolve, and a mixed solution is obtained after the photosensitizer is dissolved; an organic base catalyst is added to the mixed solution, and a protective agent is added at 0°C to 5°C, and a first coupling reaction is carried out under stirring conditions. After the reaction is completed, the obtained product solution is dropped into ethanol, and the first intermediate product is obtained by filtering.

[0054] In the above embodiment, the solvent includes at least one of dichloromethane and chloroform, and the mass percentage of the solvent to the photosensitizer is 800% to 1200%, which can be understood as the mass ratio of the solvent to the photosensitizer is (8 to 12): 1. Exemplarily, the mass percentage of the solvent to the photosensitizer can be, but is not limited to, 800%, 850%, 900%, 950%, 1000%, 1050%, 1100%, 1150%, 1200%, or a range consisting of any two of the above values.

[0055] The organic base catalyst may include at least one of triethylamine and tetramethylethylenediamine, and the mass percentage of the organic base catalyst to the photosensitizer is 50% to 100%, which can be understood as the mass ratio of the organic base catalyst to the photosensitizer is (0.5 to 1): 1. The first coupling reaction time may be 10 hours to 15 hours.

[0056] In some embodiments of the present application, in step S200, the first intermediate product is catalytically coupled to the photodegradation agent by the first activator and the second activator under light-shielding conditions.

[0057] In some embodiments of the present application, the first activating agent includes N-hydroxysuccinimide.

[0058] In some embodiments of the present application, the mass percentage of the first activator in the first intermediate product is 30% to 60%, which can be understood as the mass ratio of the first activator to the first intermediate product is (0.3 to 0.6): 1. Exemplarily, the mass percentage of the first activator in the first intermediate product can be, but is not limited to, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or a range consisting of any two of the above values.

[0059] In some embodiments of the present application, the second activating agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0060] In some embodiments of the present application, the mass percentage of the second activator to the first intermediate product is 70% to 150%, which can be understood as the mass ratio of the second activator to the first intermediate product is (0.7 to 1.5): 1. Exemplarily, the mass percentage of the second activator to the first intermediate product can be, but is not limited to, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or a range consisting of any two of the above values.

[0061] In some embodiments of the present application, the mass percentage of the photodegradation agent in the first intermediate product is 80% to 160%, which can be understood as the mass ratio of the photodegradation agent to the first intermediate product is (0.8 to 1.6): 1. Exemplarily, the mass percentage of the photodegradation agent in the first intermediate product can be, but is not limited to, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160% or a range consisting of any two of the above values.

[0062] In some embodiments of the present application, step S200 may specifically include:

[0063] Under light-proof conditions, the first intermediate product is dissolved in water, and then the first activator and the second activator are added and stirred at room temperature for 30 minutes to 60 minutes, and then a photodegradation agent is added to carry out a second coupling reaction. After the reaction is completed, the obtained solution is dripped into an ethanol solution for filtration to obtain a second intermediate product.

[0064] In the above embodiment, the second coupling reaction time is 12 h to 16 h.

[0065] In some embodiments of the present application, step S300 is performed under light-proof conditions and in the presence of a third activating agent and a fourth activating agent.

[0066] In some embodiments of the present application, the third activating agent includes N-hydroxysuccinimide.

[0067] In some embodiments of the present application, the mass percentage of the third activator in the second intermediate product is 20% to 30%, which can be understood as the mass ratio of the third activator to the second intermediate product is (0.2 to 0.3): 1. Exemplarily, the mass percentage of the third activator in the second intermediate product can be, but is not limited to, 20%, 22%, 24%, 26%, 28%, 30%, or a range consisting of any two of the above values.

[0068] In some embodiments of the present application, the fourth activating agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0069] In some embodiments of the present application, the mass percentage of the fourth activator and the second intermediate product is 40% to 80%, which can be understood as the mass ratio of the fourth activator to the second intermediate product is (0.4 to 0.8): 1. Exemplarily, the mass percentage of the fourth activator to the second intermediate product can be, but is not limited to, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or a range consisting of any two of the above values.

[0070] In the above embodiments, the above activating agents and the range settings of their mass percentages can help to improve the conversion rate of the product and reduce the reaction cost.

[0071] In some embodiments of the present application, the mass percentage of the second intermediate product to the polyhydroxy water-soluble polymer is 15% to 30%, which can be understood as the mass ratio of the second intermediate product to the polyhydroxy water-soluble polymer is (0.15 to 0.3): 1. Exemplarily, the mass percentage of the second intermediate product to the polyhydroxy water-soluble polymer can be, but is not limited to, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or a range consisting of any two of the above values.

[0072] In some embodiments of the present application, step S300 may specifically include:

[0073] The polyhydroxy water-soluble polymer is added into water, and after being stirred and dissolved to form a water-soluble polymer aqueous solution, the temperature is cooled to room temperature; under light-proof conditions, the second intermediate product is dissolved in water, and then the third activator and the fourth activator are added and stirred at room temperature for 30 minutes to 60 minutes, and then the water-soluble polymer aqueous solution is added to carry out a third coupling reaction to obtain a modified polymer.

[0074] In the above embodiment, the mass percentage of the polyhydroxy water-soluble polymer in water is 15% to 25%. The dissolution temperature of the polyhydroxy water-soluble polymer is 90° C. to 100° C. The third coupling reaction time is 12 h to 16 h.

[0075] In some embodiments of the present application, in step S400, a water-soluble cross-linking agent is coupled to a modified polymer under an acidic catalyst, and then a water-soluble monomer and an initiator are added and stirred uniformly to obtain an aqueous phase.

[0076] In the above embodiment, the acidic catalyst includes at least one of hydrochloric acid, sulfuric acid, nitric acid and p-toluenesulfonic acid, and hydrochloric acid can be selected. The mass percentage of the acidic catalyst to the water-soluble polymer is 30% to 60%, which can be understood as the mass ratio of the acidic catalyst to the water-soluble polymer is (0.3 to 0.6): 1. The coupling reaction temperature is 20°C to 30°C, and the reaction time is 10h to 15h.

[0077] The mass percentage of the water-soluble monomer in the water phase is 30% to 80%, which can be understood as the mass ratio of the water-soluble monomer to the water phase is (0.3 to 0.8):1.

[0078] The initiator may include at least one of ammonium persulfate, sodium persulfate and potassium persulfate, and the mass percentage of the initiator to water is 0.3% to 0.8%.

[0079] In some embodiments of the present application, in step S500, the oil phase includes an oil-soluble dispersant and an oily solvent.

[0080] In some embodiments of the present application, the mass percentage of the oil-soluble dispersant in the oil phase is 2% to 5%.

[0081] In some embodiments of the present application, the oil-soluble dispersant includes cellulose acetate butyrate.

[0082] In some embodiments of the present application, the oily solvent includes at least one of butyl acetate, ethyl acetate, methyl acetate and propyl acetate.

[0083] In some specific embodiments of the present application, the oil phase can be prepared by the following method:

[0084] The oil-soluble dispersant is added to the oily solvent, and dissolved by stirring at 30° C. to 40° C. to form a uniform solution to obtain an oil phase.

[0085] In some embodiments of the present application, the temperature of the reverse phase suspension polymerization reaction is 60°C to 70°C.

[0086] In some embodiments of the present application, step S500 may specifically include:

[0087] Under stirring conditions, slowly add water phase to the oil phase to form an oil-in-water reverse suspension polymerization reaction system. After the water phase is added, the reaction system is heated to 60°C-70°C, and a certain amount of catalyst is continued to be added to the reaction system, and the reverse suspension polymerization reaction begins. After the reaction is completed, stirring and heating are stopped, the reaction system is allowed to stand to separate layers, the oil phase is separated, the microspheres are collected and repeatedly washed to obtain embolic microspheres, which can be sieved into microspheres of different sizes using a sieve as needed.

[0088] In the above embodiment, the catalyst may include tetramethylethylenediamine.

[0089] In a third aspect, an embodiment of the present application provides a drug-loaded embolic microsphere, comprising embolic microspheres and drugs, wherein the embolic microspheres include the embolic microspheres of the first aspect of the present application and / or the embolic microspheres prepared by the preparation method of the second aspect of the present application.

[0090] In the above technical solution, the drug-loaded embolic microspheres have the therapeutic effects of embolization, chemotherapy and photodynamic therapy, and embolization therapy, chemotherapy therapy and photodynamic therapy can synergize to improve the treatment efficiency of tumors.

[0091] In some embodiments of the present application, the drug includes an anti-tumor drug. As an example, the anti-tumor drug includes an anthracycline drug. Further, the anthracycline drug includes at least one of doxorubicin, aclarubicin, epirubicin, idarubicin and valrubicin.

[0092] In some embodiments of the present application, the drug-loaded embolic microspheres can be prepared by the following method:

[0093] After the embolic microspheres were mixed with the anticancer drug solution, they were stirred and reacted at 25° C. for 5 min, and the drug-loaded embolic microspheres were obtained after filtration.

[0094] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0095] Example 1

[0096] This embodiment provides a method for preparing embolic microspheres, comprising:

[0097] Amino group protection reaction: 16.7 g of photosensitizer 5-aminolevulinic acid (ALA) was added to 200 g of dichloromethane solvent and stirred to dissolve. After ALA was dissolved, a mixed solution was obtained. 10.1 g of triethylamine catalyst was added to the mixed solution, and 24.0 g of di-tert-butyl dicarbonate (BOC) was added at 5° C., and a coupling reaction was carried out under stirring. The product solution obtained after the reaction was allowed to react overnight was added dropwise to ethanol, and a solid first intermediate product was obtained by filtration, which was recorded as ALA-BOC. The reaction formula of ALA-BOC is as follows:

[0098]

[0099] Coupling photodegradation agent: Under light-proof conditions, dissolve 26g of the first intermediate product (ALA-BOC) in 100g of water, add 11.5g of N-hydroxysuccinimide and 19.1g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and stir at room temperature for 60min, then add 21.9g of 7-aminocoumarin-4-acetic acid (represented by CO) for coupling reaction. After 12h of reaction, add the obtained product solution dropwise into ethanol and filter to obtain a solid second intermediate product, recorded as CO-ALA-BOC. The reaction formula of CO-ALA-BOC is as follows:

[0100]

[0101] Coupling polyvinyl alcohol: add 200g polyvinyl alcohol (PVA) to 1000g water, heat to 100°C, stir and dissolve to form a uniform polyvinyl alcohol aqueous solution, and then cool to room temperature; then dissolve 48g of the second intermediate product (CO-ALA-BOC) in 100g water under light-proof conditions, add 11.5g N-hydroxysuccinimide and 19.1g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, stir at room temperature for 60min, then add the polyvinyl alcohol aqueous solution for coupling reaction, and after reacting for 12h, obtain a modified polyvinyl alcohol aqueous solution, which is recorded as PVA-CO-ALA-BOC. The reaction formula of PVA-CO-ALA-BOC is as follows:

[0102]

[0103] Coupling water-soluble crosslinking agent: add 6g of water-soluble crosslinking agent N-(2,2-dimethoxy)-2-methylacrylamide to the modified polyvinyl alcohol aqueous solution, stir evenly, add 70g of hydrochloric acid catalyst, continue stirring and react at 25°C for 10h, the protective group CO is removed under acidic conditions, and a modified polymer aqueous solution is obtained at the same time. The specific reaction formula is as follows:

[0104]

[0105] Prepare the oil phase: add 20 g of cellulose acetate butyrate into 500 mL of butyl acetate, stir and dissolve at 40°C to obtain the oil phase.

[0106] Prepare the aqueous phase: stir and mix 10 g of 2-acrylamide-2-methylpropanesulfonic acid, 0.3 g of potassium persulfate and 100 g of the modified polymer aqueous solution to obtain an aqueous phase. The reaction formula of the aqueous phase is as follows.

[0107]

[0108] Reverse suspension polymerization: Under stirring conditions, slowly add water phase to the oil phase to form an oil-in-water reverse suspension polymerization reaction system. After the water phase is added, the reaction system is heated to 60°C, and a certain amount of tetramethylethylenediamine catalyst is continued to be added to the reaction system. The reverse suspension polymerization reaction begins. After the reaction is completed, stirring and heating are stopped, the reaction system is allowed to stand to separate layers, the oil phase is separated, the microspheres are collected and repeatedly washed to obtain embolic microspheres, which can be sieved into microspheres of different sizes using a sieve as needed.

[0109] This embodiment also provides a method for preparing drug-loaded embolic microspheres, the preparation method comprising:

[0110] 2 mL of the above embolic microspheres were mixed with 4 mL of 20 mg / mL doxorubicin solution, stirred for reaction at 25° C. for 5 min, and filtered to obtain drug-loaded embolic microspheres.

[0111] Example 2

[0112] The present embodiment provides a method for preparing embolic microspheres, which differs from that of Example 1 in the step of coupling polyvinyl alcohol: the mass percentage of the second intermediate product (CO-ALA-BOC) to the polyvinyl alcohol is 10%.

[0113] Example 3

[0114] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of embodiment 1 in the step of coupling polyvinyl alcohol: the mass percentage of the second intermediate product (CO-ALA-BOC) to the polyvinyl alcohol is 15%.

[0115] Example 4

[0116] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of embodiment 1 in the step of coupling polyvinyl alcohol: the mass percentage of the second intermediate product (CO-ALA-BOC) to the polyvinyl alcohol is 30%.

[0117] Example 5

[0118] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of embodiment 1 in the step of coupling polyvinyl alcohol: the mass percentage of the second intermediate product (CO-ALA-BOC) to the polyvinyl alcohol is 35%.

[0119] Example 6

[0120] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of embodiment 1 in that in the step of coupling polyvinyl alcohol: sodium alginate replaces polyvinyl alcohol.

[0121] Comparative Example 1

[0122] This comparative example uses commercially available embolic microspheres, which are called DC dead.

[0123] Comparative Example 2

[0124] This comparative example uses commercially available embolic microspheres, which are called CalliSphere.

[0125] Test example

[0126] (1) Mechanical properties test of embolic microspheres

[0127] Test method: The embolic microspheres (particle size range 100 μm to 300 μm) in the above example were spread on a glass slide and placed under the probe of a texture analyzer (TA-XT plusC). The Hold Compressor mode was selected for testing. The results are shown in Table 1.

[0128] Table 1 Mechanical properties test results of embolic microspheres

[0129] experiment The mass percentage of the second intermediate product in water-soluble polymer Strength / g Compression elasticity / % Example 1 24％ 63.5 61.3 Example 2 10％ 80.6 55.2 Example 3 15％ 75.7 58.6 Example 4 30％ 52.4 59.9 Example 5 35％ 39.2 56.3 Example 6 24％ 64.9 56.8

[0130] According to Table 1, by comparing the test results of Examples 1 to 6, it can be seen that as the mass percentage of the second intermediate product (CO-ALA-BOC) in the water-soluble polymer increases, the strength of the microsphere embolization will decrease accordingly, and the compression elasticity will first increase (10% to 24%) and then decrease (24% to 35%).

[0131] (2) Drug loading performance test of embolic microspheres

[0132] Test method: 2 mL of embolic microspheres (particle size range 100 μm to 150 μm) prepared in Examples 1 to 6, 2 mL of embolic microspheres (DC dead, particle size range 100 μm to 300 μm) of Comparative Example 1, and 2 mL of embolic microspheres (Equalsphere, particle size of approximately 100 μm) of Comparative Example 2 were respectively measured, and the measured embolic microspheres were mixed with 4 mL and 20 mg / mL of doxorubicin solution, respectively. After the doxorubicin solution was completely adsorbed, the residual amount of doxorubicin in the supernatant solution was tested by high performance liquid chromatography, and the drug loading rate was calculated. The test results are shown in Table 2.

[0133] Table 2 Drug loading performance test results of embolic microspheres

[0134]

[0135]

[0136] Note: “N / A” in Table 2 1 and N / A 2 "Indicates that the drug loading cannot reach 95%, "N / A 3 and N / A4 " indicates that the mass percentage of the second intermediate product in the water-soluble polymer is 0.

[0137] According to Table 2, by comparing the test results of Examples 1 to 6, it can be seen that as the mass percentage of the second intermediate product (CO-ALA-BOC) in the water-soluble polymer increases, the drug loading rate and the drug loading rate of the microsphere embolization show a downward trend, but the drug loading rate and the drug loading rate are higher than those of Comparative Examples 1 to 2.

[0138] (3) 5-aminolevulinic acid (ALA) release test

[0139] Test method: The embolic microspheres of Examples 1 to 6 were placed in purified water at 37°C and incubated at 0.8W / cm 2 The samples were irradiated with 808nm near-infrared laser, and the ALA release was tested by HPLC. The curve was drawn according to the sampling time and its corresponding release amount. Figure 2 In addition, a control group was set up, that is, the embolic microspheres prepared in Example 1 were measured and placed in purified water at 37° C. and incubated in the dark, and samples were taken at regular intervals to test the ALA release amount using high performance liquid chromatography.

[0140] according to Figure 2 Comparing the test results of Examples 1 to 6 with the control group, it can be seen that as the mass percentage of the second intermediate product (CO-ALA-BOC) in the water-soluble polymer increases, the grafting rate of ALA on the embolic microspheres increases accordingly. 2 Under 808nm near-infrared laser irradiation, the release of ALA also increased. However, under light-protected conditions, no ALA release was detected in the control group.

[0141] (4) Doxorubicin release rate test

[0142] Test method: 2 mL of embolic microspheres (particle size range 100 μm to 300 μm) of Examples 1 to 6 were mixed with 8 mL and 20 mg / mL doxorubicin solution, respectively, and then the loaded embolic microspheres were added to a dialysis bag, and then the dialysis bag was placed in a centrifuge tube, 20 mL of normal saline was added to the centrifuge tube, the centrifuge tube was sealed and placed on a 37.0°C constant temperature oscillator at a speed of 100 rpm, samples were taken at regular intervals, the release amount of doxorubicin was tested, and a curve was drawn according to the sampling time and its corresponding release rate Figure 3 .

[0143] according to Figure 3 The test results show that the embolic microspheres prepared in the examples of the present application have a sustained release effect on doxorubicin, and can release cumulatively for 360 hours.

[0144] (5) Cell activity test

[0145] Test method: MTT colorimetry was used to determine cell activity, and the cells were divided into a blank group (complete culture medium group), group 1 (DOX group: pure drug doxorubicin), group 2 (light-proof conditions + embolic microspheres of Example 1), group 3 (light-proof conditions + embolic microspheres loaded with drugs in Example 1, i.e., embolic microspheres @DOX), group 4 (light-proof conditions + embolic microspheres loaded with drugs in Example 1), group 5 (light-proof conditions + embolic microspheres loaded with drugs in Example 1, i.e., embolic microspheres @DOX), and three parallel wells were set in each group. Ultrasonic conditions were 0.6 W / cm 2 , 60s. HepG2 liver cancer cells were 8×10 3 The cells were inoculated in a 96-well plate. After the cells adhered to the wall, the blank group and the experimental group were treated for 24 hours. The culture medium was removed and the cells were cultured with 0.5 mg / mL MTT solution at 37°C for 4 hours. The formazan crystals were dissolved with 100 μL dimethyl sulfoxide, and the absorbance was measured at 490 nm using a cell imaging microplate reader (Bio-Tek Cytation5). The test results are shown in Figure 4 shown.

[0146] according to Figure 4 The embolic microspheres in group 3 loaded with doxorubicin and under light-protected conditions and the embolic microspheres in group 4 not loaded with doxorubicin and under light both showed tumor killing effects; when the embolic microspheres in group 5 were loaded with doxorubicin and exposed to light, the activity of their tumor cells was the lowest compared with other groups, which indicates that the embolic microspheres provided in the present application have the therapeutic effects of embolization, chemotherapy and photodynamic therapy, and that embolization therapy, chemotherapy therapy and photodynamic therapy can synergize to improve the treatment efficiency of tumors.

[0147] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. An embolic microsphere, characterized in that: It includes a three-dimensional network structure formed by cross-linking polymerization of a polyhydroxy water-soluble polymer modified mainly by a photodegradant and a photosensitizer, a water-soluble monomer with a polymerizable double bond, and a water-soluble cross-linking agent, wherein: The photosensitizer is covalently coupled to the photodegradant, and the photodegradant is covalently coupled to the polyhydroxy water-soluble polymer.

2. The embolic microsphere according to claim 1, characterized in that: The light absorption wavelength of the photodegradation agent is 780nm to 1100nm; Optionally, the photodegradation agent includes coumarin and its derivatives; Optionally, the photodegradation agent comprises 7-aminocoumarin-4-acetic acid.

3. The embolic microsphere according to claim 1, characterized in that: The photosensitizer includes at least one of 5-aminolevulinic acid and its derivatives and protoporphyrin and its derivatives.

4. The embolic microsphere according to claim 1, characterized in that: The polyhydroxy water-soluble polymer includes at least one of polyvinyl alcohol, sodium alginate, sodium hyaluronate, sodium carboxymethyl cellulose and chitosan; and / or, The water-soluble monomer includes an ionic group, and the ionic group includes at least one of a carboxylic acid group, a carboxylic acid group, an amino group, an amine group, and an ammonium group; optionally, the water-soluble monomer includes at least one of sodium 2-acrylamide-2-methylpropane sulfonate, sodium allyl sulfonate, sodium methacrylic acid sulfonate, sodium methacrylate, sodium acrylate, and acrylamide; and / or, The water-soluble crosslinking agent includes at least one of N-(2,2-dimethoxy)-2-methylacrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylamide, N-(2,2-dimethoxy)-2-acrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylamide, N-(2,2-dimethoxy)-2-methylacrylate, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylate, N-(2,2-dimethoxy)-2-acrylate and N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylate.

5. A method for preparing embolic microspheres according to any one of claims 1 to 4, characterized in that: The steps include: S100, protecting the photosensitizer with an amino group using a protecting agent to obtain a first intermediate product; S200, coupling the first intermediate product to the photodegradation agent to obtain a second intermediate product, S300, grafting the second intermediate product to the polyhydroxy water-soluble polymer to obtain a modified polymer; S400, mixing the modified polymer, the water-soluble monomer having a polymerizable double bond, the water-soluble crosslinking agent and an initiator to obtain an aqueous phase; S500, adding the water phase into the oil phase to carry out a reverse phase suspension polymerization reaction to obtain the embolic microspheres.

6. The preparation method according to claim 5, characterized in that: In step S100, the protective agent includes di-tert-butyl dicarbonate; and / or, The mass percentage of the protective agent to the photosensitizer is 100%-160%.

7. The preparation method according to claim 5, characterized in that: In step S200, the first intermediate product is catalytically coupled to the photodegradation agent by the first activator and the second activator under light-shielding conditions; Optionally, the first activator comprises N-hydroxysuccinimide; Optionally, the mass percentage of the first activator to the first intermediate product is 30% to 60%; Optionally, the second activator comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Optionally, the mass percentage of the second activator to the first intermediate product is 70% to 150%; Optionally, the mass percentage of the photodegradation agent in the first intermediate product is 80% to 160%.

8. The preparation method according to claim 5, characterized in that: Step S300 is performed under light-shielding conditions and in the presence of a third activating agent and a fourth activating agent; Optionally, the third activator comprises N-hydroxysuccinimide; Optionally, the mass percentage of the third activator to the second intermediate product is 20% to 30%; Optionally, the fourth activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Optionally, the mass percentage of the fourth activator in the second intermediate product is 40% to 80%; Optionally, the mass percentage of the second intermediate product to the polyhydroxy water-soluble polymer is 15% to 30%.

9. The preparation method according to claim 5, characterized in that: In step S400, the water-soluble cross-linking agent is coupled to the modified polymer under an acidic catalyst, and then the water-soluble monomer and the initiator are added and stirred to obtain the aqueous phase; and / or, In step S500, the oil phase includes an oil-soluble dispersant and an oily solvent; optionally, the mass percentage of the oil-soluble dispersant in the oil phase is 2% to 5%; optionally, the oil-soluble dispersant includes cellulose acetate butyrate; optionally, the oily solvent includes at least one of butyl acetate, ethyl acetate, methyl acetate and propyl acetate; optionally, the temperature of the reverse suspension polymerization reaction is 60°C to 70°C.

10. A drug-loaded embolic microsphere, characterized in that: include: Embolic microspheres and drugs, wherein the embolic microspheres include the embolic microspheres according to any one of claims 1 to 4 and / or the embolic microspheres prepared by the preparation method according to any one of claims 5 to 9; Optionally, the drug includes an anti-tumor drug; Optionally, the anti-tumor drug includes anthracycline drugs, Optionally, the anthracycline drug includes at least one of doxorubicin, aclarubicin, epirubicin, idarubicin and valrubicin.

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