A kind of thermosensitive embolic microsphere and its preparation method and drug-loaded release application method

Abstract

The present application relates to a thermosensitive embolic microsphere and its preparation method and drug-loading release application method, belonging to the technical field of embolic microspheres. The critical transition temperature LCST of the thermosensitive embolic microsphere is ≥37°C, and it is polymerized by an ionic monomer containing a double bond, an acrylamide monomer containing a hydroxyl group, a thermosensitive monomer and a cross-linking agent; wherein the thermosensitive monomer includes N-isopropyl acrylamide, and the cross-linking agent includes a glycidyl ether compound with an end group of propylene oxide. The thermosensitive embolic microsphere has excellent thermosensitive responsiveness and drug-loading and release performance, and because the LCST of the thermosensitive embolic microsphere is not lower than the human body temperature of 37°C, when used in the human body, the controlled release of the drug in the microsphere can be achieved by adjusting the temperature, thereby effectively improving the therapeutic effect.

Description

Technical Field

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

[0002] Transcatheter Arterial chemoembolization (TACE) is an interventional radiology technique used to treat cancer. It involves selectively or super-selectively inserting a catheter into the artery that supplies the tumor, then injecting an appropriate amount of embolic agent and chemotherapy drugs to occlude the tumor's blood supply artery, causing ischemia and hypoxia in the tumor tissue, thereby inhibiting tumor growth and promoting tumor cell necrosis and apoptosis. TACE is currently one of the main treatments for mid- and late-stage liver cancer.

[0003] Drug-Eluting Beads TACE (DEB-TACE) can adsorb chemotherapy drugs and load drugs through ion exchange or physical adsorption to achieve smooth release of drugs, increase tumor ischemia intensity, prolong drug action time, and reduce systemic side effects. It combines drugs with embolization therapy to achieve a synergistic effect. The control of drug release in drug-loaded embolic microspheres can not only maintain effective drug concentrations for a long time, but also stabilize drug concentrations, avoid sudden release, and help reduce toxic side effects. However, the currently commercially available drug-loaded embolic microspheres do not have drug controlled release performance under stimulus response. It can be seen that the development of a responsive embolic microsphere with the ability to regulate drug release is an urgent need in clinical treatment. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present application provides a thermosensitive embolic microsphere and a preparation method and a drug-loading and releasing application method thereof. The thermosensitive embolic microsphere has good thermosensitive responsiveness and drug-loading and releasing properties, and can regulate drug release by regulating temperature, thereby effectively improving the therapeutic effect.

[0005] In a first aspect, the present application provides a thermosensitive embolic microsphere, wherein the critical transition temperature LCST of the thermosensitive embolic microsphere is ≥ 37° C. The thermosensitive embolic microsphere is polymerized by an ionic monomer containing a double bond, an acrylamide monomer containing a hydroxyl group, a thermosensitive monomer, and a crosslinking agent; wherein the thermosensitive monomer includes N-isopropyl acrylamide, and the crosslinking agent includes a glycidyl ether compound having an end group of propylene oxide.

[0006] In the above technical scheme, by using double-bond-containing ionic monomers, hydroxyl-containing acrylamide monomers, thermosensitive monomers and crosslinking agents for polymerization, and selecting the crosslinking agent as a glycidyl ether compound with an end group of propylene oxide, a large number of hydroxyl groups can be generated while bonding with the hydroxyl-containing acrylamide monomer by ring-opening reaction, thereby enhancing the hydrophilicity of the microsphere structure, thereby improving the LCST of the microsphere, so that the thermosensitive embolic microsphere has good thermosensitive responsiveness and drug loading and release performance, and the critical transition temperature LCST is not less than 37°C. In this way, when the external auxiliary temperature rises and is greater than 37°C to the preset temperature, the microspheres with different LCST will be activated to respond to the thermosensitive response during the process of heating from 37°C to the preset temperature, and the thermosensitive embolic microspheres will shrink due to the thermosensitive response, and the loaded drug will be squeezed, thereby accelerating the release. The higher the LCST temperature of the thermosensitive embolic microspheres, the later the temperature-controlled activation time of the microspheres, the shorter the time for the drug to be squeezed and released, and the lower the drug release rate, so that the release rate of the drug by the microspheres can be controlled by temperature. The critical transition temperature LCST of the thermosensitive embolic microspheres provided in the present application is not lower than the human body temperature of 37°C, so that they can have a good temperature-sensitive response in the human body, and can avoid structural changes before application to the human body or when implanted into the human body. When used, not only can the effective drug concentration be maintained for a long time, but the drug concentration can also be stabilized to avoid sudden release, which is beneficial to reducing toxic side effects, thereby effectively improving the therapeutic effect.

[0007] In a second aspect, the present invention provides a method for preparing thermosensitive embolic microspheres, comprising the following steps:

[0008] S1: reacting a hydroxyl-containing acrylamide monomer and a crosslinking agent in an alkaline solution to obtain a long-chain segment compound solution;

[0009] S2: adjusting the pH of the long-chain segment compound solution to neutral, adding a double-bond-containing ionic monomer, a thermosensitive monomer, and an initiator to form an aqueous phase;

[0010] S3: The water phase and the oil phase are mixed to form an oil-in-water reverse suspension polymerization system, and the thermosensitive embolic microspheres are obtained after the reaction.

[0011] In the above technical scheme, the hydroxyl-containing acrylamide monomer and the cross-linking agent are first cross-linked in an alkaline solution, and the glycidyl ether compound with the terminal group being an epoxy group undergoes epoxy ring opening under alkaline conditions, reacts with the hydroxyl group in the hydroxyl-containing acrylamide monomer, and obtains a terminal acrylamide-modified polyethylene glycol segment compound, which is then polymerized with the double-bond-containing ionic monomer and the thermosensitive monomer through double bond addition to form thermosensitive embolic microspheres. Among them, the glycidyl ether compound with the terminal group being an epoxy group and the hydroxyl-containing acrylamide monomer form a dense and super-hydrophilic network structure through the ring-opening reaction, and a large number of hydroxyl groups are simultaneously generated during the bonding process of the ring-opening reaction, which greatly improves the hydrophilicity of the microsphere structure, thereby improving the LCST of the microsphere. In addition, the preparation of thermosensitive embolic microspheres by the reverse suspension polymerization method has simple equipment and process requirements, mild reaction conditions, low system viscosity, easy discharge of reaction heat, and strong controllability of reaction parameters. The thermosensitive embolic microspheres prepared in the present application have good temperature responsiveness and a critical transition temperature LCST of not less than 37°C of the human body temperature, so that controllable drug release can be achieved when used in the human body, thereby effectively improving the therapeutic effect.

[0012] In a third aspect, an embodiment of the present application also provides a drug-loaded release application method of the above-mentioned thermosensitive embolic microspheres, comprising the steps of: setting the external auxiliary temperature to t1, the LCST temperature of the thermosensitive embolic microspheres to t2, and when it is necessary to accelerate the drug release rate of the thermosensitive embolic microspheres, setting t1>t2.

[0013] In the above technical scheme, the thermosensitive embolic microspheres provided in the present application have good thermosensitive responsiveness. When the temperature rises from below LCST to above LCST, the temperature of the thermosensitive embolic microspheres may show continuous contraction. Therefore, by simply setting the external auxiliary temperature, the structure of the thermosensitive embolic microspheres can be changed to achieve the regulation of drug release, thereby achieving the effect of improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] Figure 1 A process flow chart of a method for preparing temperature-sensitive embolic microspheres provided in an embodiment of the present application. DETAILED DESCRIPTION

[0016] 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.

[0017] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to the three situations of “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0018] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a ~ value b" includes the two end values ​​"a" and "b", and the "unit" in "value a ~ value b + unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0019] The drug-loaded embolic microspheres currently available on the market can achieve good drug release for some drugs, but none of them have the ability to control drug release under stimulus response.

[0020] Based on this, the first aspect of the present application provides a thermosensitive embolic microsphere, the critical transition temperature LCST of the thermosensitive embolic microsphere is ≥37° C. The thermosensitive embolic microsphere is polymerized by an ionic monomer containing a double bond, an acrylamide monomer containing a hydroxyl group, a thermosensitive monomer and a crosslinking agent; wherein the thermosensitive monomer includes N-isopropyl acrylamide, and the crosslinking agent includes a glycidyl ether compound with an end group of propylene oxide.

[0021] The present application adopts double-bond-containing ionic monomers, hydroxyl-containing acrylamide monomers, thermosensitive monomers and cross-linking agents for polymerization, and controls the types of thermosensitive monomers and cross-linking agents, so that the thermosensitive embolic microspheres have good temperature-sensitive response and drug-loading and release performance, and the critical transition temperature LCST is not lower than the human body temperature of 37°C. In this way, when the external auxiliary temperature rises and is greater than 37°C to the preset temperature, the microspheres with different LCSTs will be activated to respond to the temperature in the process of heating from 37°C to the preset temperature. The thermosensitive embolic microspheres will shrink due to the thermosensitive response, and the loaded drugs will be squeezed, thereby accelerating the release. The higher the LCST temperature of the thermosensitive embolic microspheres, the later the temperature-controlled activation time of the microspheres, the shorter the time for the drugs to be squeezed and released, and the lower the drug release rate, so that the release rate of the drugs by the microspheres can be controlled by temperature. When the thermosensitive embolic microspheres provided in the present application are used in the human body, they can not only maintain an effective drug concentration for a long time, but also stabilize the drug concentration, avoid sudden release, and help reduce toxic side effects, thereby effectively improving the therapeutic effect.

[0022] In the present application, the critical transition temperature LCST of the thermosensitive embolic microspheres can be 37-42°C, which is slightly higher than the human body temperature of 37°C, and can avoid structural changes before application to the human body or when implanted into the human body, which would prevent thermosensitive regulated drug release.

[0023] A second aspect of the embodiments of the present application provides a method for preparing the above-mentioned thermosensitive embolic microspheres. Figure 1 This is a process flow chart of the preparation method of the above-mentioned thermosensitive embolic microspheres provided in the embodiments of the present application. Figure 1 The preparation method of the thermosensitive embolic microspheres comprises the following steps:

[0024] S1: reacting a hydroxyl-containing acrylamide monomer and a crosslinking agent in an alkaline solution to obtain a long-chain segment compound solution.

[0025] Wherein, the cross-linking agent includes a glycidyl ether compound with a terminal group being an propylene oxide group. As an example, the cross-linking agent includes at least one of ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, PEG200 diglycidyl ether, PEG500 diglycidyl ether or glycerol diglycidyl ether.

[0026] The hydroxyl-containing acrylamide monomer includes at least one of N-hydroxymethyl acrylamide, N-hydroxyethyl acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, N-(2-hydroxypropyl)acrylamide, N-(hydroxymethyl)methacrylamide, N-(hydroxyethyl)methacrylamide or N-(2-hydroxypropyl)methacrylamide.

[0027] In the embodiment of the present application, the glycidyl ether compound with an end group of propylene oxide can form a dense and super-hydrophilic network structure through epoxy ring opening and cross-linking reaction with hydroxyl-containing acrylamide monomer. The present application selects a cross-linking agent to generate a large number of hydroxyl groups during the bonding process of the ring opening reaction, which greatly improves the hydrophilicity of the microsphere structure. The hydroxyl-containing acrylamide monomer contains hydroxyl groups and is an acrylamide compound, which is easy to further cross-link with thermosensitive monomers and ionic monomers containing double bonds. In addition, hydroxyl groups are hydrophilic groups. By regulating the types and addition ratios of the cross-linking agent and the hydroxyl-containing acrylamide monomer, the hydrophilicity and hydrophobicity of the thermosensitive embolic microspheres can be controlled accordingly, thereby achieving the regulation of LCST.

[0028] In some embodiments, step S1 comprises: adding a crosslinking agent to an alkaline solution and stirring for 0.5 to 1 hour; then adding a hydroxyl-containing acrylamide monomer to the alkaline solution and stirring at low temperature for 1 to 3 hours.

[0029] The alkaline solution may include a sodium hydroxide solution having a mass fraction of 10 to 30 wt %. For example, the alkaline solution is a sodium hydroxide solution having a mass fraction of 10 wt %, 20 wt %, or 30 wt %.

[0030] The temperature range of the low temperature may include 10-20° C. For example, the temperature of the low temperature is 10° C., 15° C., or 20° C.

[0031] S2: The pH of the long-chain segment compound solution is adjusted to neutral, and an ionic monomer containing a double bond, a thermosensitive monomer and an initiator are added to form an aqueous phase.

[0032] Among them, the temperature-sensitive monomer includes N-isopropylacrylamide. The phase transition temperature of N-isopropylacrylamide is about 37°C, which is close to the human body temperature, and it is easier to shrink the particle size quickly during clinical use.

[0033] The ionic monomer containing double bonds includes at least one of sodium itaconate, sodium acrylate, sodium methacrylate, sodium 2-acrylamido-2-methylpropanesulfonate, sodium p-styrenesulfonate, sodium vinylsulfonate, (3-acrylamidopropyl)trimethylammonium chloride or (3-methacrylamidopropyl)trimethylammonium chloride. The ionic monomer containing double bonds contains double bonds, so it can be copolymerized and cross-linked with the thermosensitive monomer, and also contains ionic functional groups, such as carboxylate, sulfonate, ammonium ions, etc., so as to improve the water solubility of the copolymer. At the same time, it can also be combined with drugs with opposite charges, so that the embolic microspheres also have drug-carrying functions.

[0034] In some embodiments, the molar ratio of the double bond-containing ionic monomer to the cross-linking agent is (1-8): 1. For example, the molar ratio of the double bond-containing ionic monomer to the cross-linking agent may be 1:1, 2:1, 5:1, 6:1, or 8:1.

[0035] In some embodiments, the molar ratio of the double-bond-containing ionic monomer to the thermosensitive monomer is (0.1-2): 1. As an example, the molar ratio of the double-bond-containing ionic monomer to the thermosensitive monomer can be 0.1: 1, 0.5: 1, 1: 1, 2: 1. By adjusting the amount of the added thermosensitive monomer, the particle size shrinkage degree of the embolic microspheres can be adjusted, thereby obtaining embolic microspheres of various specifications. The more thermosensitive monomers are added, the higher the particle size shrinkage rate and the narrower the particle size distribution.

[0036] By selecting ionic monomers with strong hydrophilicity and adjusting the dosage of the ionic monomers, the hydrophilicity of the microsphere structure can be further enhanced, thereby further improving the LCST.

[0037] In some embodiments, the initiator includes at least one of ammonium persulfate, sodium persulfate, or potassium persulfate.

[0038] It should be noted that the neutral pH in the present application refers to a pH of about 7, and the pH of the long-chain segment compound solution can be neutralized to neutrality by using concentrated hydrochloric acid with a mass fraction of 30 wt%.

[0039] S3: The water phase and the oil phase are mixed to form an oil-in-water reverse suspension polymerization system, and the thermosensitive embolic microspheres are obtained after the reaction.

[0040] In some embodiments, the method for preparing the oil phase includes: adding an oil-soluble dispersant to an oil-soluble solvent, and stirring at a certain temperature to obtain the oil phase.

[0041] Optionally, the oil-soluble dispersant includes at least one of cellulose acetate butyrate, a mixture of Tween series compounds and Span series compounds, OP series compounds or AEO series compounds; the oil phase solvent includes at least one of butyl acetate, petroleum ether, n-hexane, cyclohexane or paraffin oil.

[0042] Optionally, the mass fraction of the oil-soluble dispersant in the oil phase is 0.5-8wt%, and the stirring temperature is 40-50° C. As an example, the mass fraction of the oil-soluble dispersant in the oil phase can be 0.5wt%, 1.5wt%, 3wt%, 5wt%, 8wt%; the stirring temperature can be 40° C., 50° C.

[0043] In some embodiments, step S3 includes: under a first temperature condition, slowly adding the water phase to the oil phase to form an inverted suspension system during stirring; under a second temperature condition, maintaining stirring for a period of time; under a third temperature condition, adding a catalyst to the inverted suspension system for reaction.

[0044] In some embodiments, the first temperature is T1, the second temperature is T2, the third temperature is T3, and T1=T2<T3.

[0045] Optionally, the temperature ranges of T1 and T2 are both 35-55° C., and the temperature range of T3 is 45-70° C. As an example, T1 is 45° C., T2 is 45° C., and T3 is 70° C.

[0046] In some embodiments, the reaction time is 4 to 18 hours. For example, the reaction time can be 5 hours, 8 hours, 10 hours, 12 hours, or 18 hours.

[0047] Optionally, the stirring speed is 200-500 rpm. As an example, the stirring speed may be 300 rpm.

[0048] In some embodiments, after the reverse suspension reaction is completed, the process further includes: separation, washing, and sieving. As an example, after the reverse suspension reaction is completed, the lower gel phase is separated, the microspheres are collected, and they are washed with butyl acetate, sodium bicarbonate aqueous solution, and ultrapure water in sequence, and then sieved through sieves with different apertures to obtain thermosensitive embolic microspheres with different particle size distributions.

[0049] In the present application, a hydroxyl-containing acrylamide monomer and a cross-linking agent are first cross-linked in an alkaline solution, and a glycidyl ether compound with an end group of propylene oxide undergoes epoxy ring opening under alkaline conditions, reacts with the hydroxyl group in the hydroxyl-containing acrylamide monomer, and obtains a terminal acrylamide-modified long-chain segment compound, which is then polymerized by double bond addition with an ionic monomer and a thermosensitive monomer containing a double bond to form a thermosensitive embolic microsphere. Among them, the amide group present in the thermosensitive monomer N-isopropyl acrylamide can form a hydrogen bond with water, and the thermosensitive monomer also has a hydrophobic isopropyl group. When the temperature is lower than the LCST, N-isopropyl acrylamide has a strong hydrogen bonding force with water molecules, so that N-isopropyl acrylamide is in an extended state. When the temperature is higher than the LCST, the hydrogel is dominated by the hydrophobic isopropyl group, and the hydrogen bond formed by the amide group and water breaks with the increase of temperature, and the interaction between the hydrophobic isopropyl groups is enhanced, resulting in the molecular chain being more inclined to form a compact hydrophobic structure, that is, the gel shrinks. Moreover, the glycidyl ether compounds with propylene oxide terminal groups and hydroxyl-containing acrylamide monomers undergo a ring-opening reaction to form a dense and super-hydrophilic network structure, and a large number of hydroxyl groups are simultaneously generated during the bonding process of the ring-opening reaction, which greatly improves the hydrophilicity of the microsphere structure, thereby improving the LCST of the microspheres. In addition, the preparation of thermosensitive embolic microspheres by the reverse suspension polymerization method has simple equipment and process requirements, mild reaction conditions, low system viscosity, easy discharge of reaction heat, and strong controllability of reaction parameters. The thermosensitive embolic microspheres prepared in the present application have good temperature responsiveness and a critical transition temperature LCST not lower than the human body temperature of 37°C, so that controllable drug release can be achieved when used in the human body, thereby effectively improving the therapeutic effect.

[0050] In a third aspect, an embodiment of the present application also provides a drug-loaded release application method of the above-mentioned thermosensitive embolic microspheres, comprising the steps of: setting the external auxiliary temperature to t1, the LCST temperature of the thermosensitive embolic microspheres to t2, and when it is necessary to accelerate the drug release rate of the thermosensitive embolic microspheres, setting t1>t2.

[0051] Since the thermosensitive embolic microspheres provided in the present application have good thermosensitive responsiveness, when the temperature rises from below LCST to above LCST, the thermosensitive embolic temperature can show continuous contraction. Therefore, by simply setting the external auxiliary temperature, the structure of the thermosensitive embolic microspheres can be changed to achieve the regulation of drug release, thereby achieving the effect of improving the therapeutic effect.

[0052] The features and performance of the technical solution of the present application are further described in detail below in conjunction with the embodiments.

[0053] Example 1

[0054] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0055] (1) Preparation of aqueous phase

[0056] Take 4g of ethylene glycol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0057] Then, the pH value was adjusted to neutral, and 3.84 g of sodium acrylate aqueous solution, 9 g of N-isopropylacrylamide, and 0.24 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of ethylene glycol diglycidyl ether to sodium acrylate was 1:2; and the molar ratio of sodium acrylate to N-isopropylacrylamide was 0.5:1.

[0058] (2) Oil phase preparation

[0059] 2 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0060] (3) Inverse suspension polymerization

[0061] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0062] Example 2

[0063] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0064] (1) Preparation of aqueous phase

[0065] Take 4g of ethylene glycol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0066] Then, the pH value was adjusted to neutral, and 6.9 g of sodium itaconate aqueous solution, 9 g of N-isopropylacrylamide, and 0.31 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of ethylene glycol diglycidyl ether to sodium itaconate was 1:2; and the molar ratio of sodium itaconate to N-isopropylacrylamide was 0.5:1.

[0067] (2) Oil phase preparation

[0068] 4 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0069] (3) Inverse suspension polymerization

[0070] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 65°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 10h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, they were sieved through sieves of different pore sizes to obtain thermosensitive embolic microspheres with different particle size distributions.

[0071] Example 3

[0072] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0073] (1) Preparation of aqueous phase

[0074] Take 4g of ethylene glycol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0075] Then, the pH value was adjusted to neutral, and 9.84 g of 2-acrylamide-2-methyl-1-propane sodium sulfonate aqueous solution, 9 g of N-isopropylacrylamide, and 0.33 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase. Among them, the molar ratio of ethylene glycol diglycidyl ether to 2-acrylamide-2-methyl-1-propane sodium sulfonate was 1:2; the molar ratio of 2-acrylamide-2-methyl-1-propane sodium sulfonate to N-isopropylacrylamide was 0.5:1.

[0076] (2) Oil phase preparation

[0077] 3 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0078] (3) Inverse suspension polymerization

[0079] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 70°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 12h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, they were sieved through sieves of different pore sizes to obtain thermosensitive embolic microspheres with different particle size distributions.

[0080] Example 4

[0081] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0082] (1) Preparation of aqueous phase

[0083] Take 4g of ethylene glycol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0084] Then, the pH value was adjusted to neutral, and 1.92 g of sodium acrylate aqueous solution, 9 g of N-isopropylacrylamide, and 0.24 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of ethylene glycol diglycidyl ether to sodium acrylate was 1:1; and the molar ratio of sodium acrylate to N-isopropylacrylamide was 0.25:1.

[0085] (2) Oil phase preparation

[0086] 2 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0087] (3) Inverse suspension polymerization

[0088] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0089] Example 5

[0090] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0091] (1) Preparation of aqueous phase

[0092] Take 4g of ethylene glycol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0093] Then, the pH value was adjusted to neutral, and 5.76 g of sodium acrylate aqueous solution, 9 g of N-isopropylacrylamide, and 0.24 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of ethylene glycol diglycidyl ether to sodium acrylate was 1:3; and the molar ratio of sodium acrylate to N-isopropylacrylamide was 0.75:1.

[0094] (2) Oil phase preparation

[0095] 2 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0096] (3) Inverse suspension polymerization

[0097] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0098] Example 6

[0099] This embodiment provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0100] (1) Preparation of aqueous phase

[0101] Take 4g of glycerol diglycidyl ether in a flask, add 8mL of 20wt% sodium hydroxide aqueous solution, and stir for 0.5h. Add 12.5mL of 50wt% N-hydroxyethyl acrylamide aqueous solution into the flask, and continue stirring at 17℃ for 2h.

[0102] Then, the pH value was adjusted to neutral, and 3.84 g of sodium acrylate aqueous solution, 9 g of N-isopropylacrylamide, and 0.24 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of glycerol diglycidyl ether to sodium acrylate was 1:2; and the molar ratio of sodium acrylate to N-isopropylacrylamide was 0.5:1.

[0103] (2) Oil phase preparation

[0104] 2 g of cellulose acetate butyrate was added to 100 mL of butyl acetate and dissolved by stirring at 45°C to form a uniform oil phase.

[0105] (3) Inverse suspension polymerization

[0106] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0107] Comparative Example 1

[0108] This comparative example provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0109] (1) Preparation of aqueous phase

[0110] 9 g of N-isopropylacrylamide was dissolved in 27 mL of water, and then 9.6 mL of 12.5 wt % N,N′-methylenebisacrylamide aqueous solution and 0.24 g of ammonium persulfate were added and mixed repeatedly to obtain an aqueous phase.

[0111] (2) Oil phase preparation

[0112] Add 2 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 45°C to form a uniform oil phase system.

[0113] (3) Inverse suspension polymerization

[0114] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0115] Comparative Example 2

[0116] This comparative example provides a thermosensitive embolic microsphere, and the preparation method thereof comprises the following steps:

[0117] (1) Preparation of aqueous phase

[0118] 9 g of N-isopropylacrylamide was dissolved in 27 mL of water, and then 3.84 g of sodium acrylate aqueous solution, 9.6 mL of 12.5 wt% N,N′-methylenebisacrylamide aqueous solution, and 0.24 g of ammonium persulfate were added in sequence and mixed evenly to prepare an aqueous phase, wherein the molar ratio of sodium acrylate to N-isopropylacrylamide was 0.5:1.

[0119] (2) Oil phase preparation

[0120] Add 2 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 45°C to form a uniform oil phase system.

[0121] (3) Inverse suspension polymerization

[0122] Under stirring conditions of 45°C and 300rpm, the aqueous phase prepared above was slowly added to the oil phase to form an oil-in-water reverse suspension polymerization system. Stirring was maintained at 45°C for 1h, then the temperature was raised to 55°C, and 1.2mL of tetramethylethylenediamine was added to the suspension system and reacted for 8h. The gel phase below was separated, and the microspheres were collected. After washing with butyl acetate, sodium bicarbonate aqueous solution and ultrapure water in turn, the thermosensitive embolic microspheres with different particle size distributions were obtained by sieving through sieves of different pore sizes.

[0123] Performance testing and result analysis

[0124] 1. Critical transition temperature (LCST) test

[0125] When it is lower than LCST, there is a strong hydrogen bonding force between the N-isopropylacrylamide unit in the N-isopropylacrylamide molecular chain and the surrounding water molecules. This strong hydrogen bonding force makes the N-isopropylacrylamide molecular chain present an extended state, that is, the microsphere expands. When it is higher than LCST, the hydrophilic force between the water molecules and the amide group is weakened, and the interaction between the hydrophobic isopropyl groups is enhanced, resulting in the molecular chain being more inclined to form a compact hydrophobic structure, that is, the microsphere shrinks. According to this characteristic, a constant temperature-controlled ultraviolet-visible spectrophotometer is used to measure the transmittance changes of the thermosensitive embolic microsphere solutions in the embodiments and comparative examples at different temperatures, and the microsphere LCST is obtained according to the transmittance changes. The test results are shown in Table 1.

[0126] Table 1 LCST of thermosensitive embolic microspheres in Examples 1 to 6 and Comparative Examples 1 to 2

[0127] Group LCST Example 1 38℃ Example 2 40℃ Example 3 41℃ Example 4 37℃ Example 5 42℃ Example 6 39℃ Comparative Example 1 32℃ Comparative Example 2 35℃

[0128] As can be seen from the results in Table 1, the microspheres prepared in each group of embodiments and comparative examples are temperature responsive, wherein the LCST of the microspheres prepared in Examples 1 to 6 is not lower than the normal human body temperature of 37°C, while the LCST of Comparative Example 1 is 32°C, and the LCST of Comparative Example 2 is 35°C. The LCST of the thermosensitive embolic microspheres prepared in Examples 1 to 6 has certain differences, which is related to the hydrophilicity-hydrophobicity of the microspheres. Since the hydrophilic groups between different ionic monomers are different, the microspheres prepared by the more hydrophilic ionic monomers are more hydrophilic, which will further increase their LCST. In addition, it can be seen from Examples 1, 4, and 5 that by adjusting the addition ratio of the hydrophilic ionic monomer to the thermosensitive monomer, the LCST of the microspheres is also different, and the group with a higher content of ionic monomers has a higher LCST. It can be seen from Comparative Examples 1 and 2 that the LCST of Comparative Example 2 is higher, mainly because the introduction of ionic monomers enhances the hydrophilicity of the microspheres and thus makes the LCST higher. It can be seen from Examples 1, 6 and Comparative Example 2 that the crosslinking agents used in Examples 1 and 6 generate more hydroxyl groups during the reaction, making the thermosensitive embolic microspheres more hydrophilic, so the LCST is higher than that of Comparative Example 2. The microspheres prepared in Example 6 have a higher degree of crosslinking, so the hydrophilicity is better.

[0129] In the present embodiment, the LCST of the modified thermosensitive embolic microspheres is adjusted to be not lower than the human body temperature of 37° C., so as to avoid structural changes of the microspheres when implanted in the body, thereby preventing the thermosensitive regulated drug release from being achieved.

[0130] 2. Drug loading performance test

[0131] Commercially available DC Bead (Biocompatibles, UK) was used as a comparison, and 3 groups of 1 ml of the thermosensitive embolic microspheres prepared in Examples 1 to 6 were taken, the surface moisture of the microspheres was removed with filter paper, the microspheres were transferred into a vial, 5 mL of 10 mg / mL gemcitabine solution and 5 mg / mL lenvatinib solution were added, respectively, mixed and shaken, and the solution concentrations were tested at 1 min, 5 min, 15 min and 30 min, respectively, and the drug loading rate of the thermosensitive embolic microspheres was calculated. The test results are shown in Table 2.

[0132] Table 2 Drug loading rates of thermosensitive embolic microspheres and DC Beads in Examples 1 to 6

[0133]

[0134] As shown in Table 2, the thermosensitive embolic microspheres in Examples 1 to 6 of the present application can achieve almost complete loading of the two drugs within 30 minutes. Compared with the commercially available product DC Bead, the thermosensitive embolic microspheres in the present application have excellent drug loading performance, and the excellent drug loading performance provides a good foundation for long-term drug release and has greater application potential.

[0135] 3. Drug release performance test

[0136] The drug-loaded microspheres were added to a dialysis bag, and then the dialysis bag was placed in a beaker. Physiological saline (20 mL) was added to the beaker and the beaker was sealed. At 25°C and 42°C, samples were taken every 5 min, 30 min, and 60 min to determine the concentration of the drug in the solution and calculate the cumulative release of the drug-loaded embolic microspheres. The test results at 25°C are shown in Table 3, and the test results at 42°C are shown in Table 4.

[0137] Table 3 Drug release rates of thermosensitive embolic microspheres and DC Beads at 25°C in Examples 1 to 6

[0138]

[0139] Table 4 Drug release rates of thermosensitive embolic microspheres and DC Beads at 42°C in Examples 1 to 6

[0140]

[0141] As can be seen from Tables 3 and 4, there is no significant difference in drug release of the commercially available product DC Bead at different temperatures, while the thermosensitive embolic microspheres in Examples 1 to 6 of the present application show obvious temperature responsiveness in drug release, and release slowly at 25°C, with good sustained release performance; at 42°C, the drug release rate is significantly higher than the drug release rate at 25°C, which is mainly due to the change in temperature that causes the microsphere structure to change. When it is higher than the LCST, when the temperature rises from the human body temperature of 37°C to 42°C, when the external auxiliary temperature reaches the LCST of the microsphere, the thermosensitive embolic microsphere shrinks, so that the drug inside the microsphere is released faster. According to this characteristic, in practical applications, the appropriate temperature can be selected as needed to adjust the rate of microsphere release. In addition, the hydrophilicity of the microsphere can be affected by adjusting the monomer type, monomer content and cross-linking agent type, and its LCST will respond to changes. Combining the data in Tables 1, 3 and 4, it can be obtained that the smaller the LCST, that is, the group with relatively weak hydrophilicity, has a higher drug release rate at 42°C than at 25°C. The higher the LCST, the slower the triggering temperature-sensitive response, the smaller the shrinkage of the microspheres, and therefore the slower the drug release. In summary, the present application utilizes the temperature stimulus responsiveness of the microspheres, and through temperature regulation, the controlled release of the drug in the microspheres can be effectively achieved. In clinical applications, the release of the drug can be controlled according to the treatment needs, so as to achieve better therapeutic effects in clinical treatment.

[0142] 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. A thermosensitive embolic microsphere, characterized in that: The critical transition temperature LCST of the thermosensitive embolic microspheres is ≥37°C; The thermosensitive embolic microspheres are polymerized from ionic monomers containing double bonds, acrylamide monomers containing hydroxyl groups, thermosensitive monomers and crosslinking agents; Wherein, the temperature-sensitive monomer includes N-isopropyl acrylamide, and the cross-linking agent includes a glycidyl ether compound with a terminal group being an epoxypropene group.

2. The thermosensitive embolic microspheres according to claim 1, characterized in that: The double bond-containing ionic monomer includes at least one of sodium itaconate, sodium acrylate, sodium methacrylate, sodium 2-acrylamido-2-methylpropane sulfonate, sodium p-styrene sulfonate, sodium vinyl sulfonate, (3-acrylamidopropyl)trimethylammonium chloride or (3-methacrylamidopropyl)trimethylammonium chloride.

3. The thermosensitive embolic microspheres according to claim 1, characterized in that: The hydroxyl-containing acrylamide monomer includes at least one of N-hydroxymethyl acrylamide, N-hydroxyethyl acrylamide, N-[tris(hydroxymethyl)methyl] acrylamide, N-(2-hydroxypropyl) acrylamide, N-(hydroxymethyl)methacrylamide, N-(hydroxyethyl)methacrylamide or N-(2-hydroxypropyl)methacrylamide; And / or, the glycidyl ether compound having a terminal group of propylene oxide includes at least one of ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, PEG200 diglycidyl ether, PEG500 diglycidyl ether or glycerol diglycidyl ether.

4. A method for preparing the thermosensitive embolic microspheres according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: reacting a hydroxyl-containing acrylamide monomer and a crosslinking agent in an alkaline solution to obtain a long-chain segment compound solution; S2: adjusting the pH of the long-chain segment compound solution to neutral, adding a double-bond-containing ionic monomer, a temperature-sensitive monomer and an initiator to form an aqueous phase; S3: The water phase and the oil phase are mixed to form a water-in-oil reverse suspension polymerization system, and the temperature-sensitive embolic microspheres are obtained after the reaction.

5. The preparation method according to claim 4, characterized in that: In step S1, Adding the crosslinking agent to the alkaline solution and stirring for 0.5 to 1 hour; then adding the hydroxyl-containing acrylamide monomer to the alkaline solution and stirring for 1 to 3 hours at low temperature; And / or, the alkaline solution comprises a sodium hydroxide solution with a mass fraction of 10 to 30 wt%; And / or, the low temperature has a temperature range of 10-20°C.

6. The preparation method according to claim 4, characterized in that: In step S3, the method for preparing the oil phase includes: adding an oil-soluble dispersant to an oil-soluble solvent, stirring at a certain temperature, to obtain an oil phase; Optionally, the mass fraction of the oil-soluble dispersant in the oil phase is 0.5 to 8 wt %; Optionally, the temperature is 40-50°C.

7. The preparation method according to claim 4, characterized in that: In step S3, During stirring under the first temperature condition, slowly adding the water phase into the oil phase to form an inverse suspension system; Under the second temperature condition, stirring is maintained for a period of time; Under the third temperature condition, adding a catalyst into the inverted suspension system to carry out a reaction; Optionally, the catalyst comprises tetramethylethylenediamine.

8. The preparation method according to claim 7, characterized in that: The first temperature is T1, the second temperature is T2, and the third temperature is T3, wherein T1=T2<T3; Optionally, the temperature ranges of T1 and T2 are both 35-55°C, and the temperature range of T3 is 45-70°C.

9. The preparation method according to claim 4, characterized in that: The molar ratio of the double bond-containing ionic monomer to the temperature-sensitive monomer is (0.1-2):1; And / or, the molar ratio of the double bond-containing ionic monomer to the cross-linking agent is (1-8):

1.

10. A drug-loaded release application method of thermosensitive embolic microspheres, characterized in that: The thermosensitive embolic microspheres are the thermosensitive embolic microspheres according to any one of claims 1 to 3; Or, the thermosensitive embolic microspheres are thermosensitive embolic microspheres prepared by the preparation method according to any one of claims 4 to 9; The drug-loaded release application method comprises the steps of: setting the external auxiliary temperature to t1, the LCST temperature of the temperature-sensitive embolic microspheres to t2, and when it is necessary to accelerate the drug release rate of the temperature-sensitive embolic microspheres, setting t1>t2.

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