A temperature-sensitive liquid embolic agent and its preparation method and a temperature-sensitive composite embolic agent

Abstract

The present application relates to a thermosensitive liquid embolic agent and a preparation method thereof and a thermosensitive composite embolic agent, and belongs to the technical field of embolic agents. A thermosensitive liquid embolic agent, the thermosensitive liquid embolic agent comprises a copolymer and a solvent; wherein the copolymer comprises a network structure polymerized by a thermosensitive monomer, an acrylic monomer and a zwitterionic monomer, and the copolymer further comprises a thiol group. The coagulation time of the embolic agent can be adjusted, the drug release rate can be regulated, and a rich variety of drug types can be loaded.

Description

Technical Field

[0001] The present application relates to the technical field of embolic agents, and in particular to a temperature-sensitive liquid embolic agent and a preparation method thereof, and a temperature-sensitive composite embolic agent. Background Art

[0002] At present, the common embolic agents in the market are mainly solid embolic agents and liquid embolic agents. Among them, solid embolic agents include regular embolic microspheres or irregular embolic particles, which are usually designed with different particle sizes to match blood vessels of different sizes, and achieve vascular occlusion through physical embolization to achieve the purpose of ischemia and hypoxia. At present, the main solid embolic agents include blank microspheres Embosphere, 8sphere, Wizsphere, etc. and drug-loaded microspheres DC Bead, Callisphere, Diasphere, etc. However, embolic microspheres cannot evenly and completely fill irregularly shaped blood vessels such as arteriovenous malformations, cannot penetrate into the smaller vascular system at the distal end, are prone to ectopic embolism risks, and lack controlled release of loaded drugs. .

[0003] Liquid embolic agents have better fluidity and can flow to the ends of microvessels and their branches. Compared with solid embolic agents, they can achieve better embolic effects on the distal end. Traditional liquid embolic agents mainly include anhydrous ethanol, iodized oil, α-butyl cyanoacrylate (N-butyl2-cyanoacrylate, NBCA), etc. In recent years, some new liquid embolic agents have also been put into clinical use, such as Onyx and thermosensitive peptide Pushen. At present, these liquid embolic agents are usually only used for embolization, and there are few studies on combined drug loading, and there is a lack of controlled release of loaded drugs, which greatly limits the application scenarios of liquid embolic agents in treatment. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the embodiments of the present application includes providing a temperature-sensitive liquid embolic agent and a preparation method thereof and a temperature-sensitive composite embolic agent; by giving the liquid embolic agent functional regulation, the multi-scenario therapeutic application of the liquid embolic agent and the temperature-sensitive composite embolic agent containing the liquid embolic agent can be achieved.

[0005] In a first aspect, an embodiment of the present application provides a thermosensitive liquid embolic agent, which includes a copolymer and a solvent; wherein the copolymer includes a network structure polymerized from a thermosensitive monomer, an acrylic monomer and a zwitterionic monomer, and the copolymer also includes a thiol group.

[0006] The present application can regulate the hydrophilicity of the copolymer through the synergistic ratio of acrylic acid monomers and zwitterionic monomers to adjust the coagulation time of the embolic agent, and at the same time, the synergistic thiol group can regulate the degree of cross-linking of the embolic agent after coagulation to regulate the drug release rate; in addition, the addition of zwitterionic monomers can enrich the drug loading types.

[0007] In a second aspect, the present application provides a method for preparing the temperature-sensitive liquid embolic agent as provided in the first aspect, comprising:

[0008] S1, dispersing the temperature-sensitive monomer, acrylic acid monomer, zwitterionic monomer and initiator in a solvent according to mass percentage to carry out polymerization reaction to obtain a primary copolymer product;

[0009] S2, dissolving the initial copolymer product obtained in step S1 in a solvent, adding a thiol compound containing an amine group to carry out a bonding reaction, and obtaining a thiol-modified copolymer;

[0010] S3, dissolving the copolymer obtained in step S2 in a solvent to obtain a temperature-sensitive liquid embolic agent.

[0011] The thermosensitive liquid embolic agent prepared by the above-mentioned preparation method in the present application is easy to control its coagulation time, can effectively regulate the drug release rate, and can be loaded with a variety of drug types.

[0012] In a third aspect, an embodiment of the present application provides a temperature-sensitive composite embolic agent, comprising the temperature-sensitive liquid embolic agent provided in the first aspect, and further comprising embolic microspheres, wherein the embolic microspheres are dispersed in the temperature-sensitive liquid embolic agent.

[0013] The present application can obtain a thermosensitive composite embolic agent by dispersing embolic microspheres in the above-mentioned thermosensitive liquid embolic agent. The thermosensitive composite embolic agent combines the advantages of embolic microspheres and liquid embolic agents, and can not only effectively regulate the drug release rate, but also load a rich variety of drug types. DETAILED DESCRIPTION

[0014] 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.

[0015] In order to solve the problems that the current liquid embolic agents are limited in application and treatment due to the single function, on the one hand, an embodiment of the present application provides a thermosensitive liquid embolic agent, which includes a copolymer and a solvent; wherein the copolymer includes a network structure polymerized by a thermosensitive monomer, an acrylic monomer and a zwitterionic monomer, and the copolymer also includes a thiol group, and optionally, the solvent includes an aqueous solution.

[0016] The present application configures a cross-linked copolymer into a copolymer aqueous solution as a liquid embolic agent, wherein a thermosensitive monomer is introduced into the raw material of the synthetic copolymer, thereby giving the synthesized liquid embolic agent a thermosensitive property, so that it can be kept in a solution state at room temperature. After being injected into the human body, the liquid embolic agent solidifies to form a gel at a temperature of 37°C, thereby producing an embolic effect.

[0017] The raw materials for synthesizing the copolymer of the present application can regulate the hydrophilicity of the copolymer through the synergistic ratio of acrylic monomer and zwitterionic monomer, thereby regulating the solidification time of the liquid embolic agent and enhancing the practical application effect; at the same time, since the copolymer contains thiol groups, the self-crosslinking property of the thiol groups is utilized to make the copolymer have the property of self-crosslinking. When the liquid embolic agent is injected into the human body and solidifies, the thiol groups in the molecular chains of the copolymer approach each other due to the solidification effect, thereby self-crosslinking occurs, so that a certain degree of crosslinking occurs after the liquid embolic agent solidifies. When the liquid embolic agent is used as a drug load, the crosslinking structure of the copolymer can be used to regulate the drug release rate; in addition, the addition of zwitterionic monomers in the liquid embolic agent can be effectively combined with drugs of different electrical properties, enriching the drug loading types and application scenarios.

[0018] In an optional embodiment of the present application, the temperature-sensitive monomer includes at least one of N-isopropylacrylamide (NIPAM), N,N-diethylacrylamide, and N,N-dimethylaminoethyl methacrylate.

[0019] In an optional embodiment of the present application, the zwitterionic monomer includes at least one of sulfonated betaine (SBMA), carboxylated betaine (CBMA), and 2-methacryloyloxy-2'-trimethylamine ethyl phosphate (MPC). The above-mentioned zwitterionic monomers are used to coordinate with the acrylic acid monomer to efficiently adjust the coagulation time of the embolic agent. As an example, the zwitterionic monomer consists of three parts: a polymerizable skeleton, an anionic group, and a cationic group. The skeleton is a polyolefin skeleton, such as (meth) acrylamide, (meth) acrylate, etc., the cationic group is, for example, a quaternary ammonium salt cation, a quaternary phosphonium salt cation, a pyridinium ion, an imidazolium ion, etc., and the anionic group is, for example, a sulfonate anion, a carboxylate anion, a phosphate anion, etc.

[0020] In an optional embodiment of the present application, the thiol groups on the copolymer are obtained by chemically bonding a thiol compound containing an amino group to the copolymer; wherein the thiol compound containing an amino group includes cysteamine.

[0021] In the embodiment of the present application, the amino group on the thiol compound and the carboxyl group carried by the acrylic acid monomer in the copolymer are chemically bonded, so that the copolymer is modified with a thiol group, which can effectively control the cross-linking degree of the liquid embolic agent after solidification to control the drug release rate.

[0022] In an optional embodiment of the present application, the mass percentage of the acrylic acid monomer to the mass percentage of the thermosensitive monomer is 10%-30%. As an example, the mass percentage of the acrylic acid monomer to the mass percentage of the thermosensitive monomer can be, but is not limited to, 10%, 12%, 15%, 16%, 18%, 20%, 22%, 25%, 26%, 28%, 30%. The acrylic acid monomer with a suitable mass ratio can be efficiently coordinated with the zwitterionic monomer ratio to adjust the coagulation time of the embolic agent.

[0023] In some embodiments of the present application, the mass percentage of zwitterionic monomer to the mass percentage of thermosensitive monomer is 5%-20%. As an example, the mass percentage of zwitterionic monomer to the mass percentage of thermosensitive monomer is 5%, 6%, 8%, 10%, 12%, 15%, 16%, 18%, 20%. The zwitterionic monomer with appropriate mass can be efficiently coordinated with the acrylic acid monomer to adjust the coagulation time of the embolic agent.

[0024] In an optional embodiment of the present application, the acrylic acid monomer includes at least one of acrylic acid (AA) and methacrylic acid. The acrylic acid monomer is used to cooperate with the zwitterionic monomer to efficiently adjust the coagulation time of the embolic agent.

[0025] In a second aspect, the present application provides a method for preparing the above-mentioned temperature-sensitive liquid embolic agent, comprising:

[0026] S1, dispersing the temperature-sensitive monomer, acrylic acid monomer, zwitterionic monomer and initiator in a solvent according to mass percentage to carry out polymerization reaction to obtain a primary copolymer product;

[0027] S2, dissolving the initial copolymer product obtained in step S1 in a solvent, adding a thiol compound containing an amine group to carry out a bonding reaction, and obtaining a thiol-modified copolymer;

[0028] S3, dissolving the copolymer obtained in step S2 in a solvent to obtain a temperature-sensitive liquid embolic agent.

[0029] The thermosensitive liquid embolic agent prepared by the above-mentioned preparation method in the present application is easy to control its coagulation time, can effectively regulate the drug release rate, and can be loaded with a variety of drug types.

[0030] In an optional embodiment of the present application, in step S1, the reaction temperature of the polymerization reaction is 60-80°C; the reaction time is 6-12 hours. The use of a suitable temperature and a suitable time for the polymerization reaction is conducive to the polymerization reaction of the thermosensitive monomer, the acrylic acid monomer, and the zwitterionic monomer to form a copolymer primary product with a network structure.

[0031] In an optional embodiment of the present application, the initiator includes at least one of azobisisobutyronitrile, ammonium persulfate, and potassium persulfate. The above initiator is used to promote the polymerization reaction of the temperature-sensitive monomer, acrylic acid monomer, and zwitterionic monomer.

[0032] In an optional embodiment of the present application, the mass percentage of the initiator mass to the mass percentage of the temperature-sensitive monomer mass is 0.1%-2%. As an example, the mass percentage of the initiator mass to the mass percentage of the temperature-sensitive monomer mass can be, but is not limited to, 0.1%, 0.2%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8%, 2%.

[0033] In an optional embodiment of the present application, in step S2, the bonding reaction between the copolymer primary product and the thiol compound containing an amine group is carried out by a catalyst. The use of a catalyst is conducive to catalytically promoting the bonding reaction between the copolymer primary product and the thiol compound containing an amine group. As an example, the catalyst may include, but is not limited to, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide).

[0034] In an optional embodiment of the present application, the reaction temperature of the bonding reaction is 20-30°C, and the reaction time is 6-12 hours. The use of a suitable temperature and a suitable time for the bonding reaction is conducive to the bonding reaction of the initial copolymer product and the thiol compound containing an amine group, so that the carboxyl group carried by the acrylic acid monomer in the copolymer and the amino group on the thiol compound undergo amide bonding to obtain a thiol-modified copolymer.

[0035] In an optional embodiment of the present application, in step S2, the thiol group is obtained by amide bonding between the amine group on the thiol compound and the carboxyl group on the copolymer. The amine group on the thiol compound and the carboxyl group carried by the acrylic acid monomer in the copolymer undergo amide bonding, thereby modifying the thiol group on the copolymer.

[0036] In some embodiments of the present application, the molar amount of the thiol compound added is greater than the molar amount of the acrylic acid monomer added. Further, the molar ratio of the thiol compound to the acrylic acid monomer is (4-10):1. As an example, the molar ratio of the thiol compound to the acrylic acid monomer can be, but is not limited to, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. The molar amount of the added thiol compound is greater than the molar amount of the acrylic acid monomer, which is an appropriate ratio to ensure nearly 100% thiolization of the acrylic acid.

[0037] In some embodiments of the present application, step S3 further includes adding a therapeutic drug to the solvent to obtain a drug-loaded thermosensitive liquid embolic agent. The addition of the therapeutic drug and the copolymer can load the drug on the thermosensitive liquid embolic agent.

[0038] In some embodiments of the present application, the therapeutic drug includes at least one of a positively charged drug, a negatively charged drug, a zwitterionic compound, and a nucleic acid drug. Specifically, the positively charged drug may include, but is not limited to, at least one of anthracycline drugs and camptothecin drugs; wherein the anthracycline drug may include, but is not limited to, at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin; the camptothecin drug may include, but is not limited to, at least one of irinotecan and topotecan; the negatively charged drug may include, but is not limited to, at least one of methotrexate disodium and sodium dichlorophenate; the zwitterionic compound may include, but is not limited to, raltitrexed; the nucleic acid drug may include, but is not limited to, siRNA. The thermosensitive liquid embolic agent can load the above-mentioned rich drug types.

[0039] In the third aspect, the embodiment of the present application provides a thermosensitive composite embolic agent, including the above-mentioned thermosensitive liquid embolic agent, and also including embolic microspheres, wherein the embolic microspheres are dispersed in the thermosensitive liquid embolic agent. By dispersing the embolic microspheres in the above-mentioned thermosensitive liquid embolic agent, a thermosensitive composite embolic agent can be obtained, which combines the advantages of the embolic microspheres and the liquid embolic agent components, not only exerts a strong embolic effect, but also can utilize the functional groups contained in the embolic microsphere structure to load the corresponding drugs, and at the same time can also utilize the functional groups contained in the liquid embolic agent components to load the corresponding drugs, on the one hand, it can enrich the types of loaded drugs, and on the other hand, the cross-linked structure formed by the copolymer component in the liquid embolic agent can effectively regulate the drug release rate.

[0040] In some embodiments of the present application, the embolic microspheres include at least one of polyvinyl alcohol microspheres, acrylic microspheres, and polyethylene glycol microspheres. The above-mentioned embolic microspheres can form the above-mentioned temperature-sensitive composite embolic agent with the above-mentioned temperature-sensitive liquid embolic agent.

[0041] A thermosensitive composite embolic agent provided in the present application also includes a loaded drug. Specifically, the thermosensitive composite embolic agent includes a loaded first drug, and the embolic microspheres include the loaded first drug. Before the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the first drug is first loaded on the embolic microspheres to form drug-loaded microspheres, and then the drug-loaded microspheres are dispersed in the thermosensitive embolic agent solution to form the thermosensitive composite embolic agent; optionally, the first drug includes a cationic drug, so that the negatively charged groups carried by the above-mentioned embolic microspheres can attract the cationic groups of the first drug to form ionic bonding to achieve drug loading. In the embodiment of the present application, the embolic microspheres are loaded with cationic drugs, thereby achieving the loading of cationic drugs by the thermosensitive composite embolic agent.

[0042] Optionally, the cationic drug includes at least one of anthracycline drugs or camptothecin drugs. Before the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the cationic drug can be loaded on the embolic microspheres to form drug-loaded microspheres, so as to form a thermosensitive composite embolic agent loaded with the cationic drug.

[0043] In an optional embodiment of the present application, the anthracycline drugs include at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin, and the camptothecin drugs include at least one of irinotecan and topotecan.

[0044] In another optional thermosensitive composite embolic agent, the thermosensitive composite embolic agent further includes a loaded drug. Specifically, the thermosensitive composite embolic agent includes a loaded second drug, and the second drug is loaded in the liquid embolic agent component in the thermosensitive composite embolic agent, that is, the liquid embolic agent loads the second drug, and after the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the second drug is added to form the thermosensitive composite embolic agent. In the embodiment of the present application, the thermosensitive composite embolic agent utilizes the positively charged groups and negatively charged groups carried by the zwitterionic monomers in the liquid embolic agent component to achieve the loading of the second drug.

[0045] Optionally, the second drug includes at least one of a positively charged drug, a negatively charged drug, a zwitterionic compound, and a nucleic acid drug. Specifically, the positively charged drug may include, but is not limited to, at least one of anthracycline drugs and camptothecin drugs; wherein, anthracycline drugs may include, but are not limited to, at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin; camptothecin drugs may include, but are not limited to, at least one of irinotecan and topotecan; negatively charged drugs may include, but are not limited to, at least one of methotrexate disodium and sodium dichlorophenate; zwitterionic compounds may include, but are not limited to, raltitrexed; nucleic acid drugs may include, but are not limited to, siRNA. In the embodiment of the present application, after the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the second drug is added to form a thermosensitive composite embolic agent loaded with the second drug.

[0046] In another optional thermosensitive composite embolic agent, the thermosensitive composite embolic agent further includes loading drugs. Specifically, the thermosensitive composite embolic agent includes loading the above-mentioned first drug and the second drug. Before the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the first drug is first loaded on the embolic microspheres to form drug-loaded microspheres. After the drug-loaded microspheres are dispersed in the thermosensitive embolic agent solution, the second drug is added, thereby forming a thermosensitive composite embolic agent that loads the first drug and the second drug at the same time.

[0047] Optionally, the first drug includes a cationic drug, and the cationic drug includes at least one of anthracycline drugs or camptothecin drugs; specifically, anthracycline drugs may include but are not limited to at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin; camptothecin drugs may include but are not limited to at least one of irinotecan and topotecan.

[0048] Optionally, the second drug includes at least one of a positively charged drug, a negatively charged drug, a zwitterionic compound, and a nucleic acid drug. Specifically, the positively charged drug may include, but is not limited to, at least one of anthracycline drugs and camptothecin drugs; wherein, anthracycline drugs may include, but are not limited to, at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin; camptothecin drugs may include, but are not limited to, at least one of irinotecan and topotecan; negatively charged drugs may include, but are not limited to, at least one of methotrexate disodium and sodium dichlorophenate; zwitterionic compounds may include, but are not limited to, raltitrexed; nucleic acid drugs may include, but are not limited to, siRNA. The embodiment of the present application simultaneously loads the first drug and the second drug on the thermosensitive composite embolic agent, and the second drug can be selected from a variety of types, that is, the thermosensitive composite embolic agent can load a rich variety of drug types, which is beneficial to the therapeutic application of the thermosensitive composite embolic agent in multiple scenarios.

[0049] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0050] Example 1

[0051] This embodiment provides a temperature-sensitive liquid embolic agent, and the preparation method thereof comprises:

[0052] (1) 10 g of N-isopropylacrylamide, 1 g of acrylic acid, 0.5 g of sulfobetaine and 0.12 g of azobisisobutyronitrile were dissolved in 100 mL of aqueous solution, fully mixed and dispersed, and then heated to 70° C. for reaction for 10 h; the product obtained after the reaction was placed in a dialysis bag with a molecular weight cutoff of 3000, and the water was changed every 6 h for 3 days to obtain a dialysis product, and the obtained dialysis product was freeze-dried to obtain a copolymer;

[0053] (2) Weigh 10 g of the copolymer obtained in step (1) and dissolve it in 50 mL of aqueous solution, add 5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 5 g of cysteamine, and stir and react at room temperature for 8 h; the product obtained after the reaction is placed in a dialysis bag with a molecular weight cutoff of 3000, and the water is changed every 6 h. The dialysis bag is dialyzed for 3 days to obtain a dialysis product, and the dialysis product is freeze-dried to obtain a thiol-modified copolymer;

[0054] (3) dissolving the thiol-modified copolymer obtained in step (2) in an aqueous solution to prepare a solution with a mass percentage of 75% to obtain a temperature-sensitive liquid embolic agent.

[0055] Example 2

[0056] This embodiment provides a temperature-sensitive liquid embolic agent, which is different from the embodiment 1 in that the amount of sulfobetaine added is 1 g.

[0057] Example 3

[0058] This embodiment provides a temperature-sensitive liquid embolic agent, which is different from the embodiment 1 in that the amount of sulfobetaine added is 1.5 g.

[0059] Example 4

[0060] This embodiment provides a temperature-sensitive liquid embolic agent, which is different from the embodiment 1 in that the amount of sulfobetaine added is 2 g.

[0061] Example 5

[0062] This embodiment provides a temperature-sensitive liquid embolic agent, which is different from the embodiment 4 in that the amount of acrylic acid added is 2 g.

[0063] Example 6

[0064] This embodiment provides a temperature-sensitive liquid embolic agent, which is different from the embodiment 4 in that the amount of acrylic acid added is 3 g.

[0065] Example 7

[0066] This embodiment provides a temperature-sensitive liquid embolic agent, which differs from Embodiment 1 in that the amount of sulfobetaine added is 0.3 g.

[0067] Example 8

[0068] This embodiment provides a temperature-sensitive liquid embolic agent, which differs from Embodiment 1 in that the amount of sulfobetaine added is 2.5 g.

[0069] The composition ratios of the thermosensitive liquid embolic agents provided in Examples 1-8 are shown in Table 1.

[0070] Table 1 Ratio of components in thermosensitive liquid embolic agents

[0071] Group NIPAM AA SBMA Example 1 10g 1g 0.5g Example 2 10g 1g 1g Example 3 10g 1g 1.5g Example 4 10g 1g 2g Example 5 10g 2g 2g Example 6 10g 3g 2g Example 7 10g 1g 0.3g Example 8 10g 1g 2.5g

[0072] Performance testing and result analysis

[0073] 1. Solidification time test of thermosensitive liquid embolic agents

[0074] This test example tests the solidification of the thermosensitive liquid embolic agents provided in Examples 1-8. The specific method is: take each thermosensitive liquid embolic agent through a 5mL syringe, inject it into a PBS solution in a constant temperature water bath at 37°C, observe and record the time required for it to solidify into a gel; each group is paralleled twice and the average value is taken. The results are shown in Table 2.

[0075] Table 2 Solidification time of temperature-sensitive liquid embolic agents

[0076] Group Curing time Example 1 5.3s Example 2 7.2s Example 3 9.8s Example 4 11.8s Example 5 12.2s Example 6 13.8s Example 7 3.5s Example 8 15.6s

[0077] From the results in Table 2, it can be seen from the results of Examples 1-4 that when the amount of NIPAM and acrylic acid is fixed, as the content of zwitterionic SBMA increases, the time required for the thermosensitive liquid embolic agent to solidify into a gel becomes longer and longer; this is because zwitterions have better hydrophilicity, and as the content of zwitterionic molecules increases, the hydrophilic ratio in the thermosensitive liquid embolic agent increases, so the curing time required to change from hydrophilic to hydrophobic at 37°C is longer. This indicates that the addition of zwitterions to the thermosensitive liquid embolic agent provided in the present application can regulate the content of zwitterions to regulate the hydrophilic ratio in the thermosensitive liquid embolic agent, thereby regulating the coagulation time of the thermosensitive liquid embolic agent.

[0078] Comparing the results of Examples 5-6, it can be seen that, based on Example 4, the further increase in the amount of AA, the longer the time required for the thermosensitive liquid embolic agent to solidify into a gel; this is because AA will be subsequently modified with thiol groups, showing weak hydrophilicity, which will also increase the hydrophilic ratio in the thermosensitive liquid embolic agent, and also prolong the solidification time, but the impact is smaller than that of zwitterions. This shows that the addition of acrylic acid to the thermosensitive liquid embolic agent provided in the present application can further coordinate with zwitterions to regulate the hydrophilic ratio in the thermosensitive liquid embolic agent, thereby regulating the solidification time of the thermosensitive liquid embolic agent.

[0079] Comparing the results of Examples 7-8, it can be seen that the content of zwitterions in Example 7 is reduced, and the thermosensitive liquid embolic agent gels rapidly, which may cause the thermosensitive liquid embolic agent solution to solidify directly in the catheter during clinical operation, blocking the catheter and failing to reach the target position. The content of zwitterions in Example 8 is increased, and the gel curing time of the thermosensitive liquid embolic agent is further prolonged, which may cause the thermosensitive liquid embolic agent to be unable to solidify in time after being injected into the target blood vessel, and to penetrate into the blood vessel extremities, causing stronger adverse reactions. It is explained that by regulating the appropriate content range of zwitterions in the thermosensitive liquid embolic agent provided in the present application, the appropriate coagulation time of the thermosensitive liquid embolic agent can be controlled to avoid the thermosensitive liquid embolic agent solidifying too quickly and failing to reach the target position, or solidifying too slowly and penetrating into the blood vessel extremities, which is not conducive to therapeutic applications.

[0080] From the above results, it can be seen that the present application can regulate the hydrophilicity of the copolymer by synergizing the ratio of acrylic acid monomer and zwitterionic monomer, so as to efficiently adjust the coagulation time of the embolic agent and enhance the practical application effect. According to the simulated clinical experiment, the gel curing time of the thermosensitive liquid embolic agent is preferably between 5-15s, so the content of zwitterions should not be too high or too low. The thermosensitive liquid embolic agent provided in Examples 1-6 is more suitable for clinical use, among which the gel curing time of the thermosensitive liquid embolic agent provided in Examples 4-6 is better, leaving more operating space for the delivery of the embolic agent.

[0081] 2. Test on the release performance of the first drug by the thermosensitive composite embolic agent

[0082] (1) Loading of the first drug by the thermosensitive composite embolic agent

[0083] 2 mL of Callisphere microspheres (Hengrui Medical) were loaded with 80 mg of the first drug, such as doxorubicin solution (20 mg / mL). After loading, the supernatant was removed and then mixed with 2 mL of the temperature-sensitive liquid embolic agent provided in Examples 4, 5, and 6 to uniformly disperse the drug-loaded microspheres in the temperature-sensitive liquid embolic agent to form a temperature-sensitive composite embolic agent loaded with doxorubicin, which were marked as implementation groups 1, 2, and 3, respectively.

[0084] At the same time, 2 mL of Callisphere microspheres (Hengrui Medical) were loaded with 80 mg of doxorubicin solution (20 mg / mL). After loading, the supernatant was removed to form an embolic agent loaded with doxorubicin, which served as comparison group 1.

[0085] (2) Release test of the first drug by the thermosensitive composite embolic agent

[0086] In this test example, the thermosensitive composite embolic agents loaded with the first drug provided by the implementation groups 1-3 and the comparison group 1 were tested for the release of the first drug.

[0087] The specific method is: take each drug-loaded embolic agent through a 5mL syringe, inject it into a 50mL PBS solution in a constant temperature water bath at 37°C, observe the release of doxorubicin, take samples regularly to detect the doxorubicin concentration in the PBS solution, and obtain the release rate. The results are shown in Table 3.

[0088] Table 3 Drug release results of drug-loaded embolic agents provided by implementation groups 1-3 and comparison group 1

[0089]

[0090] As shown in Table 3, compared with the implementation group 1-3 and the comparison group 1, the implementation group 1-3 uses the thermosensitive liquid embolic agent provided in Example 4-6 to mix with the embolic microspheres to form a thermosensitive composite embolic agent, and loads doxorubicin, and its drug release rate is significantly reduced. And compared with the implementation group 1-3, with the increase of the acrylic acid content in the thermosensitive liquid embolic agent provided in Example 4-6, the rate of drug release is gradually reduced; this is because the higher the content of acrylic acid, the higher the content of thiol groups that can be modified, the higher the density of thiol groups, the higher the probability of self-crosslinking during gel curing, and the denser the cross-linked structure formed, thereby achieving the effect of reducing the drug release rate. It is explained that the thermosensitive liquid embolic agent provided in Example 4-6 of the present application uses an appropriate content of acrylic acid and an appropriate content of zwitterion ratio to coordinate, and can regulate the appropriate coagulation time and degree of self-crosslinking, mix it with the embolic microspheres to form a thermosensitive composite embolic agent, and load doxorubicin, which can effectively control the reduction of the drug release rate.

[0091] 3. Test of the loading and release performance of the second drug in the thermosensitive composite embolic agent

[0092] (1) Loading of a second drug on a thermosensitive composite embolic agent The present application provides a thermosensitive composite embolic agent loaded with a first drug and a second drug, and a preparation method thereof includes:

[0093] 2 mL of Callisphere microspheres (Hengrui Medical) were loaded with 80 mg of a first drug, such as a doxorubicin solution (20 mg / mL). After loading, 2 mL of a second drug, such as a methotrexate disodium solution (10 mg / mL), was added, and then mixed with 2 mL of the temperature-sensitive liquid embolic agent provided in Examples 1 to 6 of the present application to uniformly disperse the drug-loaded microspheres in the temperature-sensitive liquid embolic agent to form a temperature-sensitive composite embolic agent loaded with doxorubicin and methotrexate disodium, which were labeled as sample groups 1 to 6.

[0094] At the same time, 2 mL of Callisphere microspheres (Hengrui Medical) were loaded with 80 mg of doxorubicin solution (20 mg / mL). After loading, 2 mL of methotrexate disodium solution (10 mg / mL) was added and the supernatant was removed to form a thermosensitive composite embolic agent loaded with doxorubicin and methotrexate disodium, which was marked as comparison group 2.

[0095] The composite embolic agents provided by sample groups 1 to 6 and control group 2 were placed at 37° C. for solidification, and samples were taken at regular intervals to determine the content of methotrexate disodium in the remaining solution, thereby obtaining the amount of drug loaded on the composite embolic agent. The results are shown in Table 4.

[0096] Table 4 shows the drug loading of the second drug methotrexate disodium provided by sample groups 1 to 6 and comparison group 2

[0097] Group 5min drug loading Drug loading in 10 min 20min drug loading Sample set 1 6.5mg 8.5mg 12.3mg Sample set 2 10.8mg 12.9mg 15.3mg Sample set 3 15.8mg 17.2mg 18.9mg Sample set 4 19.2mg 20.0mg 20.0mg Sample set 5 14.6mg 19.2mg 20.0mg Sample set 6 11.2mg 15.3mg 19.9mg Comparison group 2 3.2mg 3.2mg 3.2mg

[0098] It can be seen from the results in Table 4 that, compared with sample groups 1-4, sample groups 1-4 use the thermosensitive liquid embolic agent provided in Example 1-4 to mix with embolic microspheres to form a thermosensitive composite embolic agent, and load doxorubicin. As the zwitterion content in the thermosensitive liquid embolic agent provided in Example 1-4 increases, the loading rate of the negatively charged drug methotrexate disodium is gradually accelerated.

[0099] Comparing sample group 5-6 with sample group 4, sample group 5-6 uses the thermosensitive liquid embolic agent provided in Example 5-6 to mix with embolic microspheres to form a thermosensitive composite embolic agent, and loads doxorubicin. As the AA content in the thermosensitive liquid embolic agent provided in Example 5-6 increases, its loading rate for the negatively charged drug methotrexate disodium gradually decreases; this is because the more AA content, the denser the relative structure, and the increased transmission resistance, so the loading rate of the drug decreases, but the total loading amount is close to 100%.

[0100] Compared with control group 2, control group 2 only includes embolic microspheres. Since the embolic microspheres only contain negative ions, the loading and adsorption effect on the negatively charged drug methotrexate disodium is very limited.

[0101] It is explained that the thermosensitive liquid embolic agent provided in Examples 1-6 of the present application uses acrylic acid and zwitterions in a coordinated ratio, which can adjust the degree of self-crosslinking. When it is mixed with embolic microspheres to form a thermosensitive composite embolic agent and loaded with two drugs with different electrical properties at the same time, the loading rate of the drug can be effectively adjusted, and the loading amount of the drug is close to 100%, which is conducive to therapeutic applications in multiple scenarios.

[0102] (2) Release detection of the second drug by the thermosensitive composite embolic agent

[0103] The drug-loaded composite embolic agents of sample groups 1 to 6 and control group 2 were taken out by 5 mL syringes, respectively, and injected into 50 mL PBS solution placed in a constant temperature water bath at 37°C for drug release. The concentration of methotrexate disodium in the PBS solution was measured by sampling at regular intervals to obtain the release rate. The results are shown in Table 5.

[0104] Table 5 Drug release results of drug-loaded embolic agents provided by sample groups 1 to 6 and comparison group 2

[0105] Group 1h release rate 5h release rate 15h release rate 30h release rate Release the time required Sample set 1 37.6％ 56.7％ 82.9％ 97.0％ 35h Sample set 2 29.0％ 46.7％ 60.9％ 89.0％ 42h Sample set 3 25.2％ 36.7％ 52.9％ 72.0％ 56h Sample set 4 19.0％ 26.7％ 40.9％ 59.0％ 92h Sample set 5 14.5％ 18.9％ 29.0％ 47.8％ 106h Sample set 6 10.3％ 15.6％ 23.1％ 35.8％ 122h Comparison group 2 100％ / / / Within 10 minutes

[0106] As shown in Table 5, compared with sample groups 1-4, sample group 1-4 uses the thermosensitive liquid embolic agent provided in Example 1-4 to mix with embolic microspheres to form a thermosensitive composite embolic agent, and loads doxorubicin and methotrexate disodium. As the zwitterion content in the thermosensitive liquid embolic agent provided in Example 1-4 increases, its drug release rate slows down significantly. In the thermosensitive liquid embolic agent provided in Example 1-3, due to the low zwitterion content and the limited total amount of loaded drugs, the complete release is completed in a relatively short time.

[0107] Comparing sample group 5-6 with sample group 4, sample group 5-6 uses the thermosensitive liquid embolic agent provided in Example 5-6 to mix with embolic microspheres to form a thermosensitive composite embolic agent, and loads doxorubicin and methotrexate disodium. As the AA content in the thermosensitive liquid embolic agent provided in Example 5-6 increases, the content of modified thiol groups thereon increases, and the degree of cross-linking increases, so the drug release becomes slower and slower. The thermosensitive composite embolic agent provided in sample group 6 has the most obvious sustained release effect on the drug.

[0108] Compared with control group 2, control group 2 only contained embolic microspheres, which had no specific adsorption to the anion drug methotrexate disodium and released it quickly within 10 minutes, and could not play a sustained-release effect on the drug.

[0109] It is explained that the thermosensitive liquid embolic agent provided in Examples 1-6 of the present application uses a synergistic ratio of acrylic acid and zwitterions to regulate the appropriate coagulation time and degree of self-crosslinking. When it is mixed with embolic microspheres to form a thermosensitive composite embolic agent and loaded with two drugs with different electrical properties at the same time, the release rate of the drugs can also be effectively controlled to be reduced, thereby facilitating therapeutic applications in multiple scenarios.

[0110] The examples of this application only list the research on drug loading and release of thermosensitive composite embolic agents, which illustrates the controlled release of drugs by the liquid embolic agent component in the thermosensitive composite embolic agent; however, the same is applicable to pure liquid embolic agents, which will not be elaborated here.

[0111] In the embodiment of the present application, when the thermosensitive composite embolic agent is loaded with the first drug and the second drug at the same time, the drug loading and release analysis of the present application is only performed on the second drug, but its related mechanism is also applicable to the first drug, which will not be elaborated here.

[0112] 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 temperature-sensitive liquid embolic agent, characterized in that: The temperature-sensitive liquid embolic agent comprises a copolymer and a solvent; wherein, The copolymer includes a network structure polymerized from a thermosensitive monomer, an acrylic monomer and a zwitterionic monomer, and the copolymer also includes a thiol group.

2. The temperature-sensitive liquid embolic agent according to claim 1, characterized in that: The thiol group is obtained by chemically bonding a thiol compound containing an amino group and the copolymer; wherein, The amino group-containing thiol compound includes cysteamine.

3. The temperature-sensitive liquid embolic agent according to claim 1, characterized in that: The mass percentage of the acrylic acid monomer to the mass of the temperature-sensitive monomer is 10%-30%; And / or, the mass percentage of the zwitterionic monomer to the thermosensitive monomer is 5%-20%.

4. The temperature-sensitive liquid embolic agent according to claim 1, characterized in that: The zwitterionic monomer includes at least one of sulfonate betaine, carboxylate betaine, and 2-methacryloyloxy-2'-trimethylamine ethyl phosphate; And / or, the temperature-sensitive monomer includes at least one of N-isopropylacrylamide, N,N-diethylacrylamide, and N,N-dimethylaminoethyl methacrylate; And / or, the acrylic acid monomer includes at least one of acrylic acid and methacrylic acid; And / or, the solvent comprises an aqueous solution.

5. A method for preparing the temperature-sensitive liquid embolic agent according to any one of claims 1 to 4, characterized in that: include: S1, dispersing the temperature-sensitive monomer, the acrylic acid monomer, the zwitterionic monomer and the initiator in the solvent according to mass percentage to carry out polymerization reaction to obtain a primary copolymer product; S2, dissolving the copolymer primary product obtained in step S1 in the solvent, adding the thiol compound containing an amine group to carry out a bonding reaction, and obtaining the thiol-modified copolymer; S3, dissolving the copolymer obtained in step S2 in the solvent to obtain the temperature-sensitive liquid embolic agent.

6. The preparation method according to claim 5, characterized in that: In step S1, the polymerization reaction temperature is 60-80°C; the reaction time is 6-12h; and / or, The initiator comprises at least one of azobisisobutyronitrile, ammonium persulfate and potassium persulfate; and / or, The mass percentage of the initiator to the mass of the temperature-sensitive monomer is 0.1%-2%; and / or; In step S2, the bonding reaction is carried out by a catalyst, and optionally, the catalyst includes (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide); and / or; The reaction temperature of the bonding reaction is 20-30° C., and the reaction time is 6-12 hours.

7. The preparation method according to claim 5, characterized in that: In step S2, the thiol group is obtained by amide bonding between the amine group on the thiol compound and the carboxyl group on the copolymer; and / or, The molar amount of the thiol compound added is greater than the molar amount of the acrylic acid monomer added; optionally, the molar ratio of the thiol compound to the acrylic acid monomer is (4-10):1; and / or; Step S3 also includes adding a therapeutic drug to the solvent to obtain a drug-loaded thermosensitive liquid embolic agent. Optionally, the therapeutic drug includes at least one of a positively charged drug, a negatively charged drug, a zwitterionic compound, and a nucleic acid drug.

8. A thermosensitive composite embolic agent, characterized in that: The thermosensitive liquid embolic agent according to any one of claims 1 to 4 further comprises embolic microspheres, wherein the embolic microspheres are dispersed in the thermosensitive liquid embolic agent; Optionally, the embolic microspheres include at least one of polyvinyl alcohol microspheres, acrylic microspheres, and polyethylene glycol microspheres.

9. The thermosensitive composite embolic agent according to claim 8, characterized in that: The embolic microspheres include a loaded first drug. Before the embolic microspheres are dispersed in the thermosensitive embolic agent solution, the first drug is first loaded on the embolic microspheres to form drug-loaded microspheres. Then, the drug-loaded microspheres are dispersed in the thermosensitive embolic agent solution to form the thermosensitive composite embolic agent. The first drug includes a cationic drug, and the cationic drug includes at least one of anthracycline drugs or camptothecin drugs; Optionally, the anthracycline drugs include at least one of epirubicin, doxorubicin, pirarubicin, and idarubicin, and the camptothecin drugs include at least one of irinotecan and topotecan.

10. The thermosensitive composite embolic agent according to claim 8 or 9, characterized in that: The liquid embolic agent further includes a loaded second drug, and after the embolic microspheres or the drug-loaded microspheres are dispersed in the thermosensitive embolic agent solution, the second drug is added to form the thermosensitive composite embolic agent; The second drug includes at least one of a positively charged drug, a negatively charged drug, a zwitterionic compound, and a nucleic acid drug; Optionally, the positively charged drug includes at least one of anthracycline drugs and camptothecin drugs, the negatively charged drug includes at least one of methotrexate disodium and dichlorophenazone sodium; the zwitterionic compound includes raltitrexed, and the nucleic acid drug includes siRNA.

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