Polymer capable of assembling with protein-based drugs and hydrophobic small-molecule drugs, and preparation method therefor

Abstract

Disclosed are a block polymer capable of assembling with protein-based drugs and hydrophobic small-molecule drugs, and a preparation method therefor. The method comprises: selecting any one of a monomer containing an ammonium functional group, a monomer containing an amino functional group, and a monomer containing an epoxide functional group to serve as a matrix material, and subjecting the matrix material to a secondary ATRP reaction or a RAFT reaction to obtain a dual-block polymer having cavity functional groups; and if the matrix material is the monomer containing an epoxide functional group, subjecting the dual-block polymer having cavity functional groups to organic modification by grafting an organic molecule containing an amino functional group, followed by cationization of amine groups, so as to obtain a polymer capable of assembling with protein-based drugs and hydrophobic small-molecule drugs. The polymer capable of assembling with protein-based drugs and hydrophobic small-molecule drugs prepared according to the present invention is a degradable multi-block polymer, and can be combined with hydrophobic small-molecule targeted drugs, macromolecular immunotherapeutic drugs, and macromolecular targeted drugs through host-guest self-assembly, thereby enabling existing drug-loaded microspheres to effectively load small-molecule targeted drugs, macromolecular targeted drugs, and macromolecular immunotherapeutic drugs.

Description

Polymers that can be assembled with protein and hydrophobic small molecule drugs and their preparation methods Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a polymer that can be assembled with protein and hydrophobic small molecule drugs and its preparation method. Background Technology

[0002] According to the "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 Edition)," the currently recommended first-line drugs for the treatment of primary liver cancer include atezolizumab combined with bevacizumab, sintilimab combined with bevacizumab, donafenib, lenvatinib, sorafenib, and oxaliplatin. Currently, drug-eluting microsphere arterial chemoembolization (D-TACE) can load chemotherapy drugs to embolize tumor vessels, and simultaneously treat the tumor through the continuous release of chemotherapy drugs.

[0003] Currently, besides oxaliplatin, a chemotherapy drug, other first-line drugs for the clinical treatment of primary liver cancer include sorafenib, lenvatinib, donafenib, bevacizumab, atezolizumab, and sintilimab, which are all targeted and immune checkpoint inhibitors. First- and second-line targeted drugs, due to their poor water solubility, low bioavailability, and toxic side effects, cause considerable harm to patients during clinical treatment. First- and second-line immune checkpoint inhibitors can induce varying degrees of immune-related adverse reactions in patients' lungs, liver, kidneys, gastrointestinal system, endocrine system, and skin. Therefore, physically targeting these drugs to release them at the tumor site can effectively reduce excessive drug circulation in the bloodstream and decrease toxic side effects, while also effectively improving drug utilization at the tumor site. Currently, the clinically used drug-eluting microsphere arterial chemoembolization (D-TACE) combines chemotherapy drugs with embolizing microspheres, which are then filled into the tumor vessels through embolization, thereby continuously releasing the drug at the tumor site. However, the hydrophobic properties of targeted drugs and the macromolecular properties of immunosuppressant drugs mean that neither of these types of drugs can be loaded onto drug-loaded embolized microspheres currently available on the market. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention proposes a polymer that can be assembled with protein-based and hydrophobic small molecule drugs, and its preparation method. The aim is to enable the degradable multi-block polymer to combine with hydrophobic small molecule targeted drugs or macromolecular immunotherapeutic and targeted drugs through host-guest self-assembly, so that existing drug-loaded microspheres can effectively load small molecule targeted drugs, macromolecular targeted drugs, and macromolecular immunotherapeutic drugs.

[0005] A first aspect of the present invention provides a polymer that can be assembled with protein-based and hydrophobic small molecule drugs, characterized in that it has any one of the structural formulas shown in Formulas I-VI:

[0006] Wherein, R group is a functional group containing cyclodextrin or calixarene, n is the number of ammonium blocks, m is the number of hollow molecular blocks, and n and m are each independently selected from integers greater than or equal to 2.

[0007] Preferably, the ratio of n to m is in the range of (0.25 to 4):1.

[0008] In some embodiments of the present invention, the polymer is selected from the following structures:

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned block polymer that can be assembled with protein and hydrophobic small molecule drugs, comprising:

[0010] Select any one of the following monomers as the host material: an ammonium functional group monomer, an amino functional group monomer, or an epoxy alkyl functional group monomer. Obtain a polymer intermediate with grafting sites through a first ATRP reaction or a RAFT reaction. Perform a second ATRP reaction or a RAFT reaction on the polymer intermediate to graft a host polymer with a cavity structure or a polymer containing hydroxyl and ester groups. Modify the polymer containing hydroxyl and ester groups with a cavity-containing organic molecule to obtain a double block polymer with a cavity functional group. The host polymer with a cavity structure is any one of polypropyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, or polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin. The cavity-containing organic molecule is modified from sodium calix 4-azosulfonate[4]arene or sodium calix 4-azosulfonate[6]arene.

[0011] When the main material is a monomer containing ammonium functional groups, the cavity-functionalized diblock polymer is the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0012] When the main material is a monomer containing an amino functional group, the amyl group of the cavity functional group diblock polymer is cationicized to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0013] When the main material is a monomer containing an alkyl oxide functional group, the cavity functional group diblock polymer is modified by grafting an organic molecule containing an amino functional group to obtain an amination polymer, and then the amino group is cationized to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0014] In some embodiments of the present invention, the ammonium-containing functional group monomer includes any one of N-(3-aminopropyl)methacrylamide hydrochloride, 2-aminoethyl methacrylate hydrochloride, allylamine hydrochloride, 2-methylallylamine hydrochloride, O-allyl hydroxylamine hydrochloride, 3-butenamine hydrochloride, 3-methylbut-2-en-1-amine hydrochloride, 2-allyl aniline hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride.

[0015] In some embodiments of the present invention, the amino-functionalized monomer includes any one of 2-methylallylamine, acrylamide, N-(2-amino-2-oxoethyl)acrylamide, 2-butenamide, 4-aminostilbene, 3-aminostyrene, 4-aminostyrene, methacrylamide, 2-methylallylamine, and 4-vinylbenzylamine.

[0016] In some embodiments of the present invention, the monomer containing the alkylene oxide functional group includes any one of glycidyl methacrylate, allyl glycidyl ether, 3,4-epoxy-1-butene, methyl (3,4-epoxycyclohexyl) acrylate, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, 1,2-epoxy-4-ethylenecyclohexane, glycidyl acrylate, and glycidyl methacrylate.

[0017] In some embodiments of the present invention, the molar ratio of any one of the monomers containing ammonium functional groups, the monomers containing amine functional groups, and the monomers containing epoxy functional groups to the monomers containing cavity functional groups or the organic molecules containing cavities is (0.25-4):1.

[0018] In some embodiments of the present invention, the host molecular polymer with cavity structure includes any one of polypropyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin.

[0019] Optionally, the host molecular polymer with a cavity structure is polymerized from a monomer with a cavity functional group, wherein the monomer with a cavity functional group includes any one of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin.

[0020] Optionally, the method for preparing the monomer containing the cavity functional group includes:

[0021] Select any one of α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin as a monomer raw material with a cavity functional group, dissolve it in an alkaline solution, add toluenesulfonyl chloride as a modifier, react for several hours, then collect the filtrate and distill under reduced pressure and settle to obtain solid sample I;

[0022] Solid sample I was mixed and reacted with the modifier 1,3-propanediamine, and then distilled under reduced pressure and precipitated to obtain solid sample II.

[0023] Solid sample II and the modifier glycidyl methacrylate were dissolved in an organic solvent, nitrogen gas was introduced, the mixture was heated and reacted, and then the solid sample was obtained by sedimentation. This solid sample was the propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, or propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin corresponding to the raw materials.

[0024] In some embodiments of the present invention, the polymer containing hydroxyl and ester groups is obtained by polymerization of any one of the monomers selected from hydroxyethyl methacrylate, allyl 4-hydroxybenzoate, 6-(allyloxycarbonylamino)-1-hexanol, 3-hexenol lactate, hydroxyethyl acrylate, 6-hydroxyhexyl methacrylate, and hydroxypropyl acrylate.

[0025] The cavity-containing organic molecule is modified from 4-azosulfonate sodium calix[4] aromatic hydrocarbon or 4-azosulfonate sodium calix[6] aromatic hydrocarbon;

[0026] Optionally, the method for preparing the cavity-containing organic molecule includes: selecting sodium calix 4-azosulfonate[4]arene or sodium calix 4-azosulfonate[6]arene, dissolving it in an alkaline solution, adding the modifier epoxypropane, mixing, and then precipitating it with diethyl ether.

[0027] In some embodiments of the present invention, the organic molecules containing amino functional groups include diethylamine, 1,3-propanediamine, p-phenylenediamine, adipamide, N,N'-bis(2-aminoethyl)-1,3-propanediamine, 1,3-bis(aminomethyl)cyclohexane, N,N'-bis(3-aminopropyl)ethylenediamine, 6,6'-iminodihexylamine, 1,4-butanediol bis(3-aminopropyl) ether, 2-chloro-5-methyl-1,4-phenylenediamine, and 2-chloro-5-nitro-1,4-phenylenediamine. -phenylenediamine, 2-chloro-1,4-phenylenediamine, 1,3-cyclohexanediamine, 1,4-cyclohexanediamine, 2,6-diaminoanthraquinone, 6,6'-diamino-2,2'-bipyridine, 1,4-butanediamine, 3,6-diaminocarbazole, 1,10-diaminodecane, 3,3'-diaminodipropylamine, 1,12-diaminododecane, 2,7-diaminofluorene, 1,7-heptanediamine, 1,6-hexanediamine, 2,2'-diamino-N-methyldiethylamine, 3, 3'-Diamino-N-methyldipropylamine, 1,2-Diamino-2-methylpropane, 1,8-Diaminooctane, 1,3-Diaminopentane, 1,5-Diaminopentane, 1,2-Diaminopropane, 1,3-Propanediamine, 2-Hydroxy-1,3-Diaminopropane, 1,14-Diamino-3,6,9,12-Tetraoxatetradecane, 2,5-Diaminotoluene, 1,11-Diamino-3,6,9-Trioxaundecanane, 1,11-Diaminoundecane, Any one of 4,4'-diamino-3,3'-dimethoxybiphenyl, 2,6-dibromo-1,4-phenylenediamine, 2,5-dichloro-1,4-phenylenediamine, 2,2'-diaminodiethylamine, 3,3'-dihydroxybenzidine, 2,5-dimethyl-1,4-phenylenediamine, 2-methyl-1,5-diaminopentane, 2-methyl-1,3-propanediamine, naphthyl-2,6-diamine, 2-nitro-1,4-phenylenediamine, oxadiamide, and 1,4-phenylenediamine.

[0028] In some embodiments of the present invention, the specific steps of the preparation method are selected from any one of the following a to f:

[0029] a. Dissolve the ammonium-functionalized monomer, initiator, catalyst and catalyst ligand in a solvent, remove oxygen, heat and react for 10-18 hours, add a poor solvent to settle and centrifuge, repeat the process of adding a poor solvent to settle and centrifuge 1-5 times, and then vacuum dry the obtained solid sample to obtain a cationic polymer.

[0030] The cationic polymer, the host polymer with a cavity structure, the catalyst, and the catalyst ligand are dissolved in a solvent, deoxygenated, heated, and mixed for 10–18 h. A poor solvent is added for precipitation, followed by centrifugation. The resulting solid sample is repeated 2–5 times with the addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum dried to obtain a polymer that can be assembled with protein and hydrophobic small molecule drugs; or

[0031] The cationic polymer, the hydroxyl and ester polymer, and the initiator are dissolved in a solvent, deoxygenated, heated and reacted for 6-10 hours. After repeated addition of unsuitable solvents for precipitation and centrifugation, the obtained solid sample is vacuum dried to obtain a biblock polymer. The biblock polymer is mixed with cavity-containing organic molecules, reacted, and freeze-dried to obtain a polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0032] b. Dissolve the monomer containing amine functional groups, initiator, catalyst and catalyst ligand in solvent, remove oxygen, heat and react for 10-18 hours, add unsuitable solvent to settle and centrifuge, repeat the process of adding unsuitable solvent to settle and centrifuge 1-5 times, and then vacuum dry the obtained solid sample to obtain amine-containing polymer.

[0033] The amine-containing polymer, the host polymer with a cavity structure, the catalyst, and the catalyst ligand are dissolved in a solvent, deoxygenated, heated, and mixed for 10-18 hours. A poor solvent is added for precipitation, followed by centrifugation. The resulting solid sample is repeated 2-5 times with the addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer. Alternatively, the amine-containing polymer, the hydroxyl- and ester-containing polymer, and the initiator are dissolved in a solvent, deoxygenated, heated, and reacted for 6-10 hours. The poor solvent is added repeatedly for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer precursor. The diblock polymer precursor is mixed with a cavity-containing organic molecule, reacted, and freeze-dried to obtain the diblock polymer.

[0034] The diblock polymer was dissolved and acidified to a pH of 1-3. Then, it was subjected to sedimentation and centrifugation 2-5 times using a sedimentation agent. The obtained solid sample was vacuum dried to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0035] c. Dissolve the monomer containing alkyl oxide functional group, the catalyst and the catalyst ligand in a solvent, remove oxygen, heat and mix. Repeat the process of adding a poor solvent and centrifuging the resulting solution 2-5 times. After vacuum drying the obtained solid sample, the epoxy-containing polymer is obtained.

[0036] The epoxy-containing polymer, the cavity-structured host polymer, the catalyst, and the catalyst ligand are dissolved in a solvent, deoxygenated, heated, and mixed for 10–18 h. A poor solvent is added for sedimentation, followed by centrifugation. The resulting solid sample is then subjected to this process 2–5 times, with repeated sedimentation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer. Alternatively, the epoxy-containing polymer, the hydroxyl and ester-containing polymer, and the initiator are dissolved in a solvent, deoxygenated, heated, and reacted for 6–10 h. The process is repeated with repeated sedimentation and centrifugation, followed by vacuum drying of the obtained solid sample to obtain a diblock polymer precursor. The diblock polymer precursor is then mixed with a cavity-structured organic molecule, reacted, and freeze-dried to obtain the diblock polymer.

[0037] An organic molecule containing an amino functional group is placed in a solvent and the diblock polymer is added and stirred to react. The resulting solution is precipitated and centrifuged. The obtained solid sample is purified and dried to obtain the amino-modified diblock polymer.

[0038] The amino-modified diblock polymer was dissolved and acidified to a pH of 1-3. Then, it was subjected to sedimentation and centrifugation 2-5 times using a sedimentation agent. The obtained solid sample was vacuum dried to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0039] d. Add RAFT reagent, ammonium-functionalized monomer and initiator to solvent and heat to react for 6-10 h. Add unsuitable solvent to precipitate. Repeat the addition of unsuitable solvent to precipitate and centrifuge the obtained solid sample 1-5 times. Then, vacuum dry the obtained solid sample to obtain cationic polymer.

[0040] The cationic polymer, the bulk polymer with a cavity structure, and the catalyst are dissolved in a solvent, deoxygenated, heated, and mixed for 6-10 hours. A poor solvent is added for precipitation, followed by centrifugation. The resulting solid sample is repeated 2-5 times with the addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum dried to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs; or

[0041] The cationic polymer, the hydroxyl and ester polymer, and the initiator are dissolved in a solvent, deoxygenated, heated and reacted for 6-10 hours. After repeated addition of unsuitable solvents for precipitation and centrifugation, the obtained solid sample is vacuum dried to obtain a biblock polymer. The biblock polymer is mixed with cavity-containing organic molecules, reacted, and freeze-dried to obtain a polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0042] e. Add RAFT reagent, amine-functionalized monomer and initiator to solvent and heat for 6-10 hours. Add unsuitable solvent to precipitate. Repeat the process of adding unsuitable solvent to precipitate and centrifuging the obtained solid sample 1-5 times. Then, vacuum dry the obtained solid sample to obtain amine-functionalized polymer.

[0043] The amine-containing polymer, the bulk polymer with a cavity structure, and the RAFT reagent are dissolved in a solvent, deoxygenated, heated, and mixed for 6-10 hours. A poor solvent is added for precipitation, followed by centrifugation. The resulting solid sample is then subjected to this process 2-5 times, involving further addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer. Alternatively, the amine-containing polymer, the hydroxyl- and ester-containing polymer, and the initiator are dissolved in a solvent, deoxygenated, heated, and reacted for 6-10 hours. This process is repeated, involving further addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer precursor. This diblock polymer precursor is then mixed with a cavity-containing organic molecule, reacted, and freeze-dried to obtain the diblock polymer.

[0044] The diblock polymer was dissolved and acidified to a pH of 1-3. Then, it was subjected to sedimentation and centrifugation 2-5 times using a sedimentation agent. The obtained solid sample was vacuum dried to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0045] f. Add RAFT reagent, epoxy alkyl functional group monomer and initiator to solvent, heat and react for 6-10 h, add unsuitable solvent to precipitate, repeat the addition of unsuitable solvent to precipitate and centrifuge the obtained solid sample 1-5 times, and then vacuum dry the obtained solid sample to obtain epoxy-containing polymer.

[0046] The RAFT reagent, the epoxy-containing polymer, the host polymer with a cavity structure, the catalyst, and the catalyst ligand are dissolved in a solvent, deoxygenated, heated, and mixed for 10–18 h. A poor solvent is added for precipitation, followed by centrifugation. The resulting solid sample is repeated 2–5 times with the addition of a poor solvent for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer. Alternatively, the epoxy-containing polymer, the hydroxyl and ester-containing polymer, and the initiator are dissolved in a solvent, deoxygenated, heated, and reacted for 6–10 h. The poor solvent is added repeatedly for precipitation and centrifugation. The obtained solid sample is then vacuum-dried to obtain a diblock polymer precursor. The diblock polymer precursor is mixed with a cavity-containing organic molecule, reacted, and freeze-dried to obtain the diblock polymer.

[0047] An organic molecule containing an amino functional group is placed in a solvent and the diblock polymer is added and stirred to react. The resulting solution is precipitated and centrifuged. The obtained solid sample is purified and dried to obtain the amino-modified diblock polymer.

[0048] The amino-modified diblock polymer was dissolved and acidified to a pH of 1-3. Then, it was subjected to sedimentation and centrifugation 2-5 times using a sedimentation agent. The obtained solid sample was vacuum dried to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0049] In some embodiments of the present invention, the method for preparing the RAFT reagent includes: subjecting a nitrogen heterocyclic organic molecule to a negative charge under alkaline conditions; mixing the negatively charged nitrogen heterocyclic organic molecule with carbon disulfide; forming a thioester molecule with a negatively charged group by attacking the carbon-sulfur double bond in the carbon disulfide; mixing the thioester molecule with a benzyl halide-containing molecule; and having the negatively charged group of the thioester molecule attack the benzyl halide site of the benzyl halide-containing molecule to form the RAFT reagent.

[0050] A third aspect of the present invention provides a method for improving the drug-loading performance of drug-loaded microspheres, wherein the drug-loaded microspheres are added to a mixed solution of a polymer that can be assembled with protein and hydrophobic small molecule drugs and the drug to be loaded.

[0051] The biblock polymer prepared using the method of this invention can be assembled with hydrophobic small molecules and protein drugs. For hydrophobic small molecule drugs such as sorafenib and lenvatinib, both the biblock polymer and the hydrophobic small molecule drug are dissolved in DMSO solution. When water is added to the system, the insoluble drug rapidly assembles with the cavity-containing molecules in the biblock polymer, resulting in hydrophobic blocks in the assembled product. Through interaction with cationic blocks, vesicle structures are ultimately formed in the aqueous solution. The outer layer of the vesicle structure has a large number of hydrophilic amino cations, which interact with the negatively charged sulfonic acid groups on the microsphere polymer backbone, enabling the microspheres to load hydrophobic small molecule drugs such as sorafenib and lenvatinib.

[0052] For water-soluble protein drugs such as atezolizumab, biblock polymers attach to the protein surface in aqueous solution through multiple cavities in cyclodextrin-containing blocks, forming a layer of biblock polymer on the protein molecule surface. The cationic groups in the polymer interact with the negatively charged sulfonic acid groups on the microsphere polymer backbone, enabling the microspheres to load protein drugs such as atezolizumab.

[0053] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] Experimental methods not specified in the examples are generally performed under conventional conditions in the art or under conditions recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from the conventional market.

[0056] In the embodiments, the host molecular polymer with a cavity structure is polymerized from monomers containing cavity functional groups. In one specific embodiment, the preparation of polypropyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin includes the following steps:

[0057] Step 1: Add 15g (13.2mmol) of α-cyclodextrin, a monomer with a cavity functional group, to 125mL of purified water. Slowly add 5mL (41mmol) of NaOH solution and stir vigorously until the α-cyclodextrin is completely dissolved. Dissolve 2.5g (13.2mmol) of p-toluenesulfonyl chloride in 7.5mL of acetonitrile solution and slowly add it dropwise to the reaction system. After the addition is complete, react at 5℃ for 3h. After the reaction is complete, remove the solid by vacuum filtration and collect the filtrate. Distill the filtrate under reduced pressure and add acetone for precipitation. After precipitation is complete, collect the solid by vacuum filtration. Place the solid in a vacuum drying oven at 40℃ overnight.

[0058] Step 2: Mix 3g of the product from Step 1 with 62.5mL of 1,3-propanediamine and stir at 80℃ for 6 hours. Distill the reaction mixture under reduced pressure and add acetone for precipitation. After precipitation, collect the solid by vacuum filtration. Place the solid in a vacuum drying oven at 40℃ overnight.

[0059] Step 3: Dissolve 0.88 g of the product from Step 2 and 1.64 mL of glycidyl methacrylate in 10 mL of N,N-dimethylformamide. Purge with N2 for 30 min, then heat the system to 60 °C and react for 6 h. After the reaction is complete, add excess acetone dropwise to the reaction solution to allow precipitation. After precipitation, collect the solid by vacuum filtration. Place the solid in a vacuum drying oven at 40 °C overnight to obtain propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0060] The difference between the preparation methods of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin and polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin and the preparation method of polypropyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin is that the same molar amount of β-cyclodextrin or γ-cyclodextrin is used instead of α-cyclodextrin.

[0061] The molar ratio of any one of the monomers containing ammonium functional groups, amine functional groups, and epoxy functional groups to a monomer containing a cavity functional group or a cavity organic molecule includes (0.25-4):1.

[0062] Example 1: Preparation of polymer A1 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0063] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 400 mg N-(3-aminopropyl)methacrylamide hydrochloride, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain a cationic polymer.

[0064] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 4.5 mg of cationic polymer, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0065] Examples 2-9: Preparation of polymers A2-A9 by ATRP reaction that can be assembled with protein and hydrophobic small molecule drugs.

[0066] The difference between the preparation method of Example 2-Example 9 and Example 1 is only that the same molar amounts of 2-aminoethyl methacrylate hydrochloride, allylamine hydrochloride, 2-methylallylamine hydrochloride, O-allyl hydroxylamine hydrochloride, 3-butenamine hydrochloride, 3-methylbut-2-en-1-amine hydrochloride, 2-allyl aniline hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride are used in place of N-(3-aminopropyl)methacrylamide hydrochloride.

[0067] Example 10: Preparation of polymer A10 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0068] The difference between the preparation method and Example 1 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and the molar ratio of the monomer containing ammonium functional groups to the monomer with cavity functional groups is 0.25:1.

[0069] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 400 mg N-(3-aminopropyl)methacrylamide hydrochloride, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain a cationic polymer.

[0070] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 2.3 mg of cationic polymer, 64 mg of propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension, and centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times, and dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0071] Example 11: Preparation of polymer A11 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0072] The only difference between the preparation method and Example 1 is that the same amount of polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and the molar ratio of the monomer containing ammonium functional groups to the monomer with cavity functional groups is 1:0.25.

[0073] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 400 mg N-(3-aminopropyl)methacrylamide hydrochloride, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain a cationic polymer.

[0074] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 9 mg of cationic polymer, 18 mg of propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension, and centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times, and dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0075] Example 12: Preparation of polymer A12 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0076] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 400 mg N-(3-aminopropyl)methacrylamide hydrochloride, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain a cationic polymer.

[0077] Step 2: Take 4.5 mg of the above product, 5 mg of hydroxyethyl methacrylate, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above step 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer.

[0078] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain modified sodium calix[4] 4-azosulfonate. Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of modified sodium calix[4] 4-azosulfonate, and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain a polymer that can be assembled with protein and hydrophobic small molecule drugs by lyophilization.

[0079] Example 13: Preparation of polymer A13 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0080] The only difference between the preparation method of Example 13 and Example 12 is that the same amount of sodium 4-azosulfonate[6]arene is used instead of sodium 4-azosulfonate[4]arene in Example 12.

[0081] Example 14: Preparation of polymer A14 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0082] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 160 mg 2-methylallylamine, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an amine-containing polymer.

[0083] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 2 mg of an amine-containing polymer, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0084] Step 3: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0085] Examples 15–23: Preparation of polymers A14–A22 that can be assembled with protein and hydrophobic small molecule drugs by ATRP reaction

[0086] The only difference between the preparation methods of Examples 15 to 23 and Example 14 is that the same molar amounts of acrylamide, N-(2-amino-2-oxoethyl)acrylamide, 2-butenamide, 4-aminostilbene, 3-aminostyrene, 4-aminostyrene, methacrylamide, 2-methylallylamine, and 4-vinylbenzylamine are used in place of 2-methylallylamine.

[0087] Example 24: Preparation of polymer A24 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0088] The only difference between the preparation method and Example 14 is that the same amount of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and the molar ratio of the monomer containing amino functional groups to the monomer with cavity functional groups is 0.25:1.

[0089] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 160 mg 2-methylallylamine, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an amine-containing polymer.

[0090] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 1 mg of an amine-containing polymer, 64 mg of propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0091] Step 3: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0092] Example 25: Preparation of polymer A25 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0093] The preparation method differs from that in Example 14 only in that: propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used in place of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin in the same molar amount, and the ratio of the molar amount of the monomer containing ammonium functional groups to the molar amount of the monomer with cavity functional groups is 1:0.25.

[0094] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 160 mg 2-methylallylamine, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an amine-containing polymer.

[0095] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 4 mg of an amine-containing polymer, 18 mg of propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0096] Step 3: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0097] Example 26: Preparation of polymer A26 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0098] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 160 mg 2-methylallylamine, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an amine-containing polymer.

[0099] Step 2: Take 2 mg of the above solid product, 5 mg of allyl 4-hydroxybenzoate, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer precursor.

[0100] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain poly(sodium calix[4] 4-azosulfonate). Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of epibromopropane-modified poly(sodium calix[4] 4-azosulfonate), and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain the diblock polymer by lyophilization.

[0101] Step 4: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0102] Example 27: Preparation of polymer A27 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0103] The only difference between the preparation method of Example 27 and Example 26 is that the same amount of sodium 4-azosulfonate[6]arene is used instead of sodium 4-azosulfonate[4]arene in Example 12.

[0104] Example 28: Preparation of polymer A28 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0105] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 320 mg glycidyl methacrylate, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an epoxy-containing polymer.

[0106] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 3.5 mg of epoxy polymer, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0107] Step 3: Add 0.5 mL of propylenediamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Add 50 mg of the above product and stir the reaction at 60°C for 7 hours. Precipitate the resulting solution with diethyl ether, and centrifuge the precipitate to obtain the solid product. Purify the solid product by dialysis in purified water. Dry the purified solid product by lyophilization.

[0108] Step 4: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0109] Examples 29–37: Preparation of polymers A29–A37 that can be assembled with protein and hydrophobic small molecule drugs by ATRP reaction

[0110] The difference between the preparation methods of Examples 29 to 37 and Example 28 is that the same molar amounts of allyl glycidyl ether, 3,4-epoxy-1-butene, (3,4-epoxycyclohexyl) methyl acrylate, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, 1,2-epoxy-4-ethylenecyclohexane, glycidyl acrylate, glycidyl methacrylate, and 3,4-epoxy-1-butene allyl glycidyl ether are used in place of glycidyl methacrylate.

[0111] Examples 38–85: Preparation of polymers A38–A85 that can be assembled with protein and hydrophobic small molecule drugs by ATRP reaction.

[0112] The difference between the preparation methods of Examples 38 to 85 and Example 28 lies only in the fact that the same molar amounts of 1,3-propanediamine, p-phenylenediamine, adipamide, N,N'-bis(2-aminoethyl)-1,3-propanediamine, 1,3-bis(aminomethyl)cyclohexane, N,N'-bis(3-aminopropyl)ethylenediamine, 6,6'-iminodihexylamine, 1,4-butanediol bis(3-aminopropyl) ether, 2-chloro-5-methyl-1,4-phenylenediamine, and 2-chloro-5-nitrate were used sequentially. 1,4-phenylenediamine, 2-chloro-1,4-phenylenediamine, 1,3-cyclohexanediamine, 1,4-cyclohexanediamine, 2,6-diaminoanthraquinone, 6,6'-diamino-2,2'-bipyridine, 1,4-butanediamine, 3,6-diaminocarbazole, 1,10-diaminodecane, 3,3'-diaminodipropylamine, 1,12-diaminododecane, 2,7-diaminofluorene, 1,7-heptanediamine, 1,6-hexanediamine, 2,2'-diamino-N-methyldiethylamine Amines, 3,3'-diamino-N-methyldipropylamine, 1,2-diamino-2-methylpropane, 1,8-diaminooctane, 1,3-diaminopentane, 1,5-diaminopentane, 1,2-diaminopropane, 1,3-propanediamine, 2-hydroxy-1,3-diaminopropane, 1,14-diamino-3,6,9,12-tetraoxatetradecane, 2,5-diaminotoluene, 1,11-diamino-3,6,9-trioxaundecanane, 1,11-diaminodecadecane Monoalkyl, 4,4'-diamino-3,3'-dimethoxybiphenyl, 2,6-dibromo-1,4-phenylenediamine, 2,5-dichloro-1,4-phenylenediamine, 2,2'-diaminodiethylamine, 3,3'-dihydroxybenzidine, 2,5-dimethyl-1,4-phenylenediamine, 2-methyl-1,5-diaminopentane, 2-methyl-1,3-propanediamine, naphthyl-2,6-diamine, 2-nitro-1,4-phenylenediamine, oxalamide, and 1,4-phenylenediamine are used in place of diethylamine;

[0113] Example 86: Preparation of polymer A86 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0114] The preparation method differs from that of Example 28 only in that: propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin in the same molar amount, and the ratio of the molar amount of monomer containing epoxide functional groups to the molar amount of monomer with cavity functional groups is 0.25:1.

[0115] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 320 mg glycidyl methacrylate, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an epoxy-containing polymer.

[0116] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 1.8 mg of epoxy polymer, 64 mg of propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0117] Step 3: Add 0.5 mL of propylenediamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Add 50 mg of the above product and stir the reaction at 60°C for 7 hours. Precipitate the resulting solution with diethyl ether, and centrifuge the precipitate to obtain the solid product. Purify the solid product by dialysis in purified water. Dry the purified solid product by lyophilization.

[0118] Step 4: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0119] Example 87: Preparation of polymer A87 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0120] The preparation method differs from that of Example 28 in that: propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used in place of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin in the same molar amount, and the ratio of the molar amount of the monomer containing the epoxide functional group to the molar amount of the monomer with the cavity functional group is 1:0.25.

[0121] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 320 mg glycidyl methacrylate, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an epoxy-containing polymer.

[0122] Step 2: Add 0.34 mg of copper bromide, 6.5 μL of pentamethyldiethyltriamine, 7.2 mg of epoxy polymer, 18 mg of propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg of cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain the diblock polymer.

[0123] Step 3: Add 0.5 mL of propylenediamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Add 50 mg of the above product and stir the reaction at 60°C for 7 hours. Precipitate the resulting solution with diethyl ether, and centrifuge the precipitate to obtain the solid product. Purify the solid product by dialysis in purified water. Dry the purified solid product by lyophilization.

[0124] Step 4: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0125] Example 88: Preparation of polymer A88 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0126] Step 1: Add 40 μL benzyl chloride, 0.34 mg copper bromide, 6.5 μL pentamethyldiethyltriamine, 320 mg glycidyl methacrylate, and 1 mL N,N-dimethylformamide to a 10 mL reaction flask, and purge with high-purity nitrogen for 30 minutes. Add 4.28 mg cuprous bromide, and continue purging with high-purity nitrogen for another 30 minutes. Heat to 65 °C and stir for 14 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps three times. Dry the solid in a vacuum oven at 40 °C for 12 hours to obtain an epoxy-containing polymer.

[0127] Step 2: Take 3.5 mg of the above solid product, 5 mg of 6-(allyloxycarbonylamino)-1-hexanol, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide, and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer precursor.

[0128] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain modified sodium calix[4] 4-azosulfonate. Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of epibromopropane-modified sodium calix[4] 4-azosulfonate, and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain the diblock polymer by lyophilization.

[0129] Step 4: Add 0.5 mL of propylenediamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Continue by adding 50 mg of the above-mentioned diblock polymer and stirring the reaction mixture at 60°C for 7 hours. The resulting solution is precipitated with diethyl ether, and the precipitate is separated by centrifugation to obtain the solid product. The solid product is purified by dialysis in purified water. The purified solid product is then lyophilized.

[0130] Step 5: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0131] Example 89: Preparation of polymer A89 by ATRP reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0132] The difference between the preparation method of Example 89 and Example 88 is that the same amount of sodium calix[6] 4-azosulfonate is used to replace the sodium calix[4] 4-azosulfonate in Example 12.

[0133] Example 90: Preparation of RAFT reagent

[0134] Add 117.8 mg of potassium hydroxide to anhydrous dimethyl sulfoxide and stir vigorously until the potassium hydroxide dissolves. Then add 145 μL of pyrrole and stir for 2 hours. Slowly add 150 μL of carbon disulfide and continue stirring for 5 hours. Add 242 μL of benzyl chloride and stir the reaction at room temperature for 36 hours. Add excess acetone to the resulting red solution to obtain a suspension. Filter the suspension and dry the solid in a vacuum oven at 40 °C to obtain the final product.

[0135] Preferably, the nitrogen-containing heterocyclic organic molecule includes any one of pyrrole, carbazole, and indole;

[0136] Preferably, the benzyl halide molecule includes any one of benzyl chloride, benzyl bromide, and benzyl iodine.

[0137] Example 91: Preparation of polymer A91 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0138] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 0.7 g of N-(3-aminopropyl)methacrylamide hydrochloride, 2.5 mg of azobisisobutyronitrile, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add tetrahydrofuran for precipitation, and repeat the dissolution / precipitation process twice more using N,N-dimethylformamide / tetrahydrofuran. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven.

[0139] Step 2: Take 4.5 mg of the above solid product, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide, and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0140] Example 92-Example 99: Preparation of polymers A92-A99 by RAFT reaction that can be assembled with protein and hydrophobic small molecule drugs.

[0141] The difference between the preparation method of Example 92-Example 99 and Example 91 is only that: the same weights of 2-aminoethyl methacrylate hydrochloride, allylamine hydrochloride, 2-methylallylamine hydrochloride, O-allyl hydroxylamine hydrochloride, 3-butenamine hydrochloride, 3-methylbut-2-en-1-amine hydrochloride, 2-allyl aniline hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride are used in place of N-(3-aminopropyl)methacrylamide hydrochloride;

[0142] Example 100: Preparation of polymer A100 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0143] The only difference between the preparation method and Example 91 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0144] Example 101: Preparation of polymer A101 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0145] The only difference between the preparation method and Example 91 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0146] Example 102: Preparation of polymer A102 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0147] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 0.7 g of N-(3-aminopropyl)methacrylamide hydrochloride, 2.5 mg of azobisisobutyronitrile, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add tetrahydrofuran for precipitation, and repeat the dissolution / precipitation process twice more using N,N-dimethylformamide / tetrahydrofuran. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven.

[0148] Step 2: Take 4.5 mg of the above solid product, 5 mg of 3-hexenol lactate, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer.

[0149] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain modified sodium calix[4] 4-azosulfonate. Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of epibromopropane-modified sodium calix[4] 4-azosulfonate, and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain the product by lyophilization.

[0150] Example 103: Preparation of polymer A103 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0151] The difference between the preparation method of Example 103 and Example 102 is that the same amount of sodium calix 4-azosulfonate[6]arene is used instead of sodium calix 4-azosulfonate[4]arene in Example 12.

[0152] Example 104: Preparation of polymer A104 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0153] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 160 mg of 2-methylallylamine, 2.5 mg of azobisisobutyronitrile, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add tetrahydrofuran for precipitation, and repeat the dissolution / precipitation process twice more using N,N-dimethylformamide / tetrahydrofuran. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven to obtain the amine-containing polymer.

[0154] Step 2: Take 2 mg of the above solid product, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide, and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add tetrahydrofuran to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer.

[0155] Step 3: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0156] Examples 105–113: Preparation of polymers A101–A109 by RAFT reaction that can be assembled with protein and hydrophobic small molecule drugs.

[0157] The only difference between the preparation methods of Examples 105 to 113 and Example 104 is that the same molar amounts of acrylamide, N-(2-amino-2-oxoethyl)acrylamide, 2-butenamide, 4-aminostilbene, 3-aminostyrene, 4-aminostyrene, methacrylamide, 2-methylallylamine, and 4-vinylbenzylamine are used in place of 2-methylallylamine.

[0158] Example 114: Preparation of polymer A114 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0159] The only difference between the preparation method and Example 104 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0160] Example 115: Preparation of polymer A115 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0161] The only difference between the preparation method and Example 104 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0162] Example 116: Preparation of polymer A116 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0163] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 160 mg of 2-methylallylamine, 2.5 mg of azobisisobutyronitrile, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add tetrahydrofuran for precipitation, and repeat the dissolution / precipitation process twice more using N,N-dimethylformamide / tetrahydrofuran. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven to obtain the amine-containing polymer.

[0164] Step 2: Take 2 mg of the above solid product, 5 mg of hydroxyethyl acrylate, 2.5 mg of azobisisobutylene, and 1 mL of N,N-dimethylformamide and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer precursor.

[0165] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain modified sodium calix[4] 4-azosulfonate. Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of epibromopropane-modified sodium calix[4] 4-azosulfonate, and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain the diblock polymer by lyophilization.

[0166] Step 4: Weigh 0.5g of the diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, use anhydrous diethyl ether to precipitate the polymer and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0167] Example 117: Preparation of polymer A117 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0168] The difference between the preparation method of Example 117 and Example 116 is that the same amount of sodium calix[6] 4-azosulfonate is used to replace the sodium calix[4] 4-azosulfonate in Example 12.

[0169] Example 118: Preparation of polymer A118 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0170] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 320 mg of glycidyl methacrylate, 2.5 mg of azobisisobutyronitrile, and 1 mL of N,N-dimethylformamide to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add acetone for precipitation, and repeat the dissolution / precipitation process twice more using dimethyl sulfoxide / acetone. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven to obtain the epoxy-containing polymer.

[0171] Step 2: Take 3.5 mg of the above solid product, 56 mg of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, and 2.5 mg of azobisisobutylene and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add acetone to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven for 12 hours to obtain the diblock polymer.

[0172] Step 3: Add 0.1 mL of diethylamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Continue by adding 50 mg of the above product and stirring the reaction mixture at 60°C for 6 hours. Precipitate the resulting solution with acetone, and centrifuge the precipitate to obtain a solid product. Dry the solid product by lyophilization to obtain an aminolated diblock polymer.

[0173] Step 4: Weigh 0.5g of the amino-modified diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, precipitate the polymer using anhydrous diethyl ether and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0174] Examples 119–127: Preparation of polymers A119–A127 that can be assembled with protein and hydrophobic small molecule drugs by RAFT reaction

[0175] The difference between the preparation methods of Examples A119 to A127 and Example 118 is that the same molar amounts of allyl glycidyl ether, 3,4-epoxy-1-butene, (3,4-epoxycyclohexyl) methyl acrylate, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, 1,2-epoxy-4-ethylenecyclohexane, glycidyl acrylate, glycidyl methacrylate, and 3,4-epoxy-1-butene allyl glycidyl ether are used in place of glycidyl methacrylate.

[0176] Examples 128–175: Preparation of polymers A128–A175 that can be assembled with protein and hydrophobic small molecule drugs via RAFT reaction

[0177] The difference between the preparation methods of Examples A128 to A175 and Example 118 lies only in the fact that the same molar amounts of 1,3-propanediamine, p-phenylenediamine, adipamide, N,N'-bis(2-aminoethyl)-1,3-propanediamine, 1,3-bis(aminomethyl)cyclohexane, N,N'-bis(3-aminopropyl)ethylenediamine, 6,6'-iminodihexylamine, 1,4-butanediol bis(3-aminopropyl) ether, 2-chloro-5-methyl-1,4-phenylenediamine, and 2-chloro- 5-Nitro-1,4-phenylenediamine, 2-chloro-1,4-phenylenediamine, 1,3-cyclohexanediamine, 1,4-cyclohexanediamine, 2,6-diaminoanthraquinone, 6,6'-diamino-2,2'-bipyridine, 1,4-butanediamine, 3,6-diaminocarbazole, 1,10-diaminodecane, 3,3'-diaminodipropylamine, 1,12-diaminododecane, 2,7-diaminofluorene, 1,7-heptanediamine, 1,6-hexanediamine, 2,2'-diamino-N-methyl Diethylamine, 3,3'-diamino-N-methyldipropylamine, 1,2-diamino-2-methylpropane, 1,8-diaminooctane, 1,3-diaminopentane, 1,5-diaminopentane, 1,2-diaminopropane, 1,3-propanediamine, 2-hydroxy-1,3-diaminopropane, 1,14-diamino-3,6,9,12-tetraoxatetradecane, 2,5-diaminotoluene, 1,11-diamino-3,6,9-trioxaundecanane, 1,11-diamino Undecane, 4,4'-diamino-3,3'-dimethoxybiphenyl, 2,6-dibromo-1,4-phenylenediamine, 2,5-dichloro-1,4-phenylenediamine, 2,2'-diaminodiethylamine, 3,3'-dihydroxybenzidine, 2,5-dimethyl-1,4-phenylenediamine, 2-methyl-1,5-diaminopentane, 2-methyl-1,3-propanediamine, naphthyl-2,6-diamine, 2-nitro-1,4-phenylenediamine, oxalamide, and 1,4-phenylenediamine are used in place of diethylamine;

[0178] Example 176: Preparation of polymer A176 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0179] The only difference between the preparation method and Example 118 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0180] Example 177: Preparation of polymer A177 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0181] The only difference between the preparation method and Example 118 is that the same molar amount of polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin is used instead of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin.

[0182] Example 178: Preparation of polymer A178 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0183] Step 1: Add 3.5 mg of 2-phenyl-2-propylbenzodisulfide, 320 mg of glycidyl methacrylate, and 2.5 mg of azobisisobutyronitrile to a 10 mL reaction flask and react at 65 °C for 8 hours. After cooling the resulting solution, add tetrahydrofuran for precipitation, and repeat the dissolution / precipitation process twice more using N,N-dimethylformamide / tetrahydrofuran. Separate the resulting solid from the solution by centrifugation and dry it in a vacuum drying oven to obtain the epoxy-containing polymer.

[0184] Step 2: Take 3.5 mg of the above solid product, 5 mg of 6-hydroxyhexyl methacrylate or hydroxypropyl acrylate, 2.5 mg of azobisisobutyl acrylate, and 1 mL of N,N-dimethylformamide, and add them to a 10 mL reaction flask. Purge with high-purity nitrogen for 1 hour. React at 65°C for 8 hours. Add n-hexane to the resulting solution to obtain a suspension. Centrifuge the suspension at 5000 rpm to obtain a solid sample. Repeat the above steps 3 times. Dry the solid in a vacuum oven at 40°C for 12 hours to obtain the diblock polymer.

[0185] Step 3: Dissolve 60 mg of sodium calix[4] 4-azosulfonate in 10 mL of anhydrous DMF, and add 1 g of sodium carbonate (100 mg / mL) and 30 g of epibromopropane in sequence. Stir the solution at 25 °C for 24 h, and then precipitate it with diethyl ether to obtain modified sodium calix[4] 4-azosulfonate. Dissolve 8 mg of the product from Step 2 in 5 mL of sodium carbonate buffer (100 mM, pH = 8.5), add 30 mg of epibromopropane-modified sodium calix[4] 4-azosulfonate, and stir at 25 °C for 24 h. Remove unreacted calixarene by dialysis with deionized water, and obtain the product by lyophilization.

[0186] Step 4: Add 0.5 mL of diethylamine and 5 mL of N,N-dimethylformamide or N,N-dimethylacetamide to a 10 mL reaction flask and stir for 10 minutes. Add 50 mg of the above product and stir the reaction at 60°C for 7 hours. Precipitate the resulting solution with diethyl ether, and centrifuge the precipitate to obtain a solid product. Purify the solid product by dialysis in purified water. Dry the purified solid product by lyophilization to obtain the amination-modified diblock polymer.

[0187] Step 5: Weigh 0.5g of the amino-modified diblock polymer and dissolve it in 10mL of dimethyl sulfoxide (or a solvent capable of dissolving the compound). Stir with a magnetic stirrer (500rpm, 30min) until completely dissolved. Under ice bath conditions, slowly add hydrochloric acid (38%) or acetic acid, continuously monitoring the pH of the reaction environment and controlling the pH value between 1 and 3. After reaching the predetermined pH range, continue stirring for 1 hour. After the reaction is complete, precipitate the polymer using anhydrous diethyl ether and centrifuge. Repeat this process 2-5 times, then vacuum dry the obtained solid sample to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

[0188] Example 179: Preparation of polymer A179 by RAFT reaction, which can be assembled with protein and hydrophobic small molecule drugs.

[0189] The difference between the preparation method of Example 179 and Example 178 is that the same amount of sodium calix[6] 4-azosulfonate is used to replace the sodium calix[4] 4-azosulfonate in Example 12.

[0190] Comparative Example 1: This comparative example provides a commercially available embolization microsphere product, Callispheres, model number blue, with a size of 100-300μm.

[0191] Test Example 1: Testing the improvement of drug loading performance of commercially available embolization microspheres Callispheres using polymers A1-A89 and A91-A179;

[0192] The following tests were performed sequentially using polymer Ax (x values ​​range from 1 to 89 and 91 to 179):

[0193] Dissolve 68 mg of sorafenib and 100 mg of polymer Ax in 1 mL of DMSO to form a clear, transparent solution. Add 50 mL of purified water and shake vigorously for 5 min to form a milky white, semi-transparent solution. Add 1 mL of drug-loaded embolic microspheres (Callispheres), continue shaking for 5 min, let stand for 10 min, then shake for 1 min. Repeat the above steps for 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance measurement at a wavelength of 280 nm.

[0194] Table 1 shows the performance test data of Callispheres, a commercially available embolization microsphere product loaded with a targeted drug (sorafenib).

[0195] Test Example 2:

[0196] Testing on the improvement of drug loading performance of commercially available embolization microspheres Callispheres using polymers A1-A89 and A91-A179;

[0197] The following tests were performed sequentially using polymer Ax (x values ​​range from 1 to 89 and 91 to 179):

[0198] Dissolve 62 mg of lenvatinib and 100 mg of polymer Ax in 1 mL of DMSO to form a clear, transparent solution. Add 50 mL of purified water and shake vigorously for 5 min to form a milky white, semi-transparent solution. Add 1 mL of drug-loaded embolic microspheres (Callispheres), continue shaking for 5 min, let stand for 10 min, then shake for 1 min. Repeat the above steps for 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance measurement at a wavelength of 280 nm.

[0199] Table 2 shows the performance test data of the commercially available embolization microsphere product Callispheres loaded with a targeted drug (lenvatinib).

[0200] Test Example 3:

[0201] Testing on the improvement of drug loading performance of commercially available embolization microspheres Callispheres using polymers A1-A89 and A91-A179;

[0202] The following tests were performed sequentially using polymers A1-A89 and A91-A179:

[0203] Take 1 mL of atezolizumab injection (containing 60 mg of atezolizumab), add 50 mg of polymer Ax (x values ​​range from 1 to 89, 91 to 179) to dissolve, and shake vigorously for 5 min to form a clear and transparent solution. Add 1 mL of drug-loaded embolic microspheres (Callispheres), continue shaking for 5 min, let stand for 10 min, and then shake for 1 min. Repeat the above steps until 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance measurement at a wavelength of 280 nm.

[0204] Table 3 shows the performance test data of Callispheres, a commercially available embolization microsphere product loaded with an immunomodulatory drug (aterizolium).

[0205] Comparative Example 2: This comparative example provides a commercially available drug-loaded embolization microsphere DCBead, model number blue, with a size of 100-300μm.

[0206] Test Example 1: Testing the improvement of drug loading performance of commercially available embolization microsphere product DCBead using polymers A1-A89 and A91-A179;

[0207] The following tests were performed sequentially using polymers A1-A89 and A91-A179:

[0208] Dissolve 68 mg of sorafenib and 100 mg of polymer Ax (x values ​​range from 1 to 89, 91 to 179) in 1 mL of DMSO to form a clear, transparent solution. Add 50 mL of purified water and shake vigorously for 5 min to form a milky white, semi-transparent solution. Add 1 mL of commercially available drug-loaded embolization microspheres (DCBead), continue shaking for 5 min, let stand for 10 min, and then shake for 1 min. Repeat the above steps until 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance measurement at a wavelength of 280 nm.

[0209] Table 4 shows the performance test data of commercially available drug-loaded embolized microspheres (DCBead) carrying targeted drugs (sorafenib).

[0210] Test Example 2:

[0211] Testing on the improvement of drug loading performance of commercially available drug-loaded embolization microspheres (DCBead) using polymers A1-A89 and A91-A179;

[0212] The following tests were performed sequentially using polymer Ax (x values ​​range from 1 to 89 and 91 to 179):

[0213] Dissolve 62 mg of lenvatinib and 100 mg of polymer Ax (x values ​​range from 1 to 89, 91 to 179) in 1 mL of DMSO to form a clear, transparent solution. Add 50 mL of purified water and shake vigorously for 5 min to form a milky white, semi-transparent solution. Add 1 mL of drug-loaded embolic microspheres (DCBead), continue shaking for 5 min, let stand for 10 min, and then shake for 1 min. Repeat the above steps until 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance measurement at a wavelength of 280 nm.

[0214] Table 5 shows the performance test data of commercially available drug-loaded embolized microspheres (DCBead) carrying targeted drugs (lenvatinib).

[0215] Test Example 3:

[0216] Testing on the improvement of drug loading performance of commercially available drug-loaded embolization microspheres (DCBead) using polymers A1-A89 and A91-A179;

[0217] The following tests were performed sequentially using polymers A1-A89 and A91-A179:

[0218] Take 1 mL of atezolizumab injection (containing 60 mg of atezolizumab), add 50 mg of polymer Ax (x values ​​range from 1 to 89, 91 to 179) to dissolve, and shake vigorously for 5 min to form a clear, transparent solution. Add 1 mL of commercially available drug-loaded embolized microspheres (DCBead), continue shaking for 5 min, let stand for 10 min, and then shake for 1 min. Repeat the above steps until 1 h. After the microspheres have settled, take the supernatant solution for UV absorbance testing at a wavelength of 280 nm.

[0219] Table 6 shows the performance test data of commercially available drug-loaded embolized microspheres (DCBead) loaded with an immunomodulatory drug (aterizolium).

[0220] As can be seen from the above test examples, compared with the comparative examples, the biblock polymers A1-A86 and A88-A173 prepared using the examples, which can be assembled with hydrophobic small molecule and protein drugs, can effectively load small molecule targeted drugs, large molecule targeted drugs, and large molecule immunotherapies through host-guest self-assembly with hydrophobic small molecule targeted drugs and large molecule immunotherapies via existing drug-loaded microspheres such as Callispheres and DCBead. The reason for this is:

[0221] For hydrophobic small molecule drugs such as sorafenib and lenvatinib, both the biblock polymer and the hydrophobic small molecule drug prepared in this invention are in a dissolved state in DMSO solution. When water is added to the system, the insoluble drug rapidly assembles with the cavity-containing molecules in the biblock polymer, resulting in hydrophobic blocks in the assembled structure. Through interaction with cationic blocks, vesicle structures are ultimately formed in the aqueous solution. The outer layer of the vesicle structure has a large number of hydrophilic amino cations, which interact with the negatively charged sulfonic acid groups on the microsphere polymer backbone, enabling the microspheres to load hydrophobic small molecule drugs such as sorafenib and lenvatinib.

[0222] For water-soluble protein drugs such as atezolizumab, biblock polymers attach to the protein surface in aqueous solution through multiple cavities in cyclodextrin-containing blocks, forming a layer of biblock polymer on the protein molecule surface. The cationic groups in the polymer interact with the negatively charged sulfonic acid groups on the microsphere polymer backbone, enabling the microspheres to load protein drugs such as atezolizumab.

[0223] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0224] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A block polymer which can be assembled with protein and hydrophobic small molecule drugs, characterized in that, Any one of the following formulae I - VI: Wherein, R group is a functional group containing cyclodextrin or calixarene, n is the number of ammonium blocks, m is the number of hollow molecular blocks, and n and m are each independently selected from integers greater than or equal to 2.

2. [Corrected according to Rule 91, 08.04.2025] The block polymer according to claim 1, which can be assembled with protein and hydrophobic small molecule drugs, is characterized in that, The polymer is selected from the following structures:

3. A process for the preparation of a block polymer capable of assembling with proteinaceous and hydrophobic small molecule drugs as claimed in any one of claims 1 or 2, wherein, include: Select any one of the following monomers as the host material: an ammonium functional group monomer, an amino functional group monomer, or an epoxy alkyl functional group monomer. Obtain a polymer intermediate with grafting sites through a first ATRP reaction or a RAFT reaction. Perform a second ATRP reaction or a RAFT reaction on the polymer intermediate to graft a host polymer with a cavity structure or a polymer containing hydroxyl and ester groups. Modify the polymer containing hydroxyl and ester groups with a cavity-containing organic molecule to obtain a double block polymer with a cavity functional group. The host polymer with a cavity structure is any one of polypropyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, polypropyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, or polypropyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin. The cavity-containing organic molecule is modified from sodium calix 4-azosulfonate[4]arene or sodium calix 4-azosulfonate[6]arene. When the main material is a monomer containing ammonium functional groups, the cavity-functionalized diblock polymer is the polymer that can be assembled with protein and hydrophobic small molecule drugs. When the main material is a monomer containing an amino functional group, the amyl group of the cavity functional group diblock polymer is cationicized to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs. When the main material is a monomer containing an alkyl oxide functional group, the cavity functional group diblock polymer is modified by grafting an organic molecule containing an amino functional group to obtain an amination polymer, and then the amino group is cationized to obtain the polymer that can be assembled with protein and hydrophobic small molecule drugs.

4. The method for preparing block polymers that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The ammonium-containing functional group monomer includes any one of N-(3-aminopropyl)methacrylamide hydrochloride, 2-aminoethyl methacrylate hydrochloride, allylamine hydrochloride, 2-methylallylamine hydrochloride, O-allyl hydroxylamine hydrochloride, 3-butenamine hydrochloride, 3-methylbut-2-en-1-amine hydrochloride, 2-allyl aniline hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride.

5. The method for preparing block polymers that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The amino-functionalized monomer includes any one of 2-methylallylamine, acrylamide, N-(2-amino-2-oxoethyl)acrylamide, 2-butenamide, 4-aminostilbene, 3-aminostyrene, 4-aminostyrene, methacrylamide, 2-methylallylamine, and 4-vinylbenzylamine.

6. The method for preparing the block polymer that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The monomer containing the alkyl oxide functional group includes any one of glycidyl methacrylate, allyl glycidyl ether, 3,4-epoxy-1-butene, methyl (3,4-epoxycyclohexyl) acrylate, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, 1,2-epoxy-4-ethylenecyclohexane, glycidyl acrylate, and glycidyl methacrylate.

7. The method for preparing block polymers that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The bulk polymer with a cavity structure is polymerized from a monomer containing a cavity functional group, wherein the monomer containing a cavity functional group includes any one of propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, and propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin.

8. The method for preparing the block polymer that can be assembled with protein and hydrophobic small molecule drugs according to claim 7, characterized in that, The method for preparing the monomer containing the cavity functional group includes: Select any one of α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin as a monomer raw material containing a cavity functional group, dissolve it in an alkaline solution, add toluenesulfonyl chloride as a modifier, react for several hours, then collect the filtrate and distill under reduced pressure and allow it to settle to obtain solid sample I; Solid sample I was mixed and reacted with the modifier 1,3-propanediamine, and then distilled under reduced pressure and precipitated to obtain solid sample II. Solid sample II and the modifier glycidyl methacrylate were dissolved in an organic solvent, nitrogen gas was introduced, the mixture was heated and reacted, and then the solid sample was obtained by sedimentation. This solid sample was the propyl methacrylate-2-hydroxy-3-methylamino-α-cyclodextrin, propyl methacrylate-2-hydroxy-3-methylamino-β-cyclodextrin, or propyl methacrylate-2-hydroxy-3-methylamino-γ-cyclodextrin corresponding to the raw materials.

9. The method for preparing the block polymer that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The polymer containing hydroxyl and ester groups is obtained by polymerizing any one of the monomers selected from hydroxyethyl methacrylate, allyl 4-hydroxybenzoate, 6-(allyloxycarbonylamino)-1-hexanol, 3-hexenol lactate, hydroxyethyl acrylate, 6-hydroxyhexyl methacrylate, and hydroxypropyl acrylate.

10. The method for preparing the block polymer that can be assembled with protein and hydrophobic small molecule drugs according to claim 3, characterized in that, The method for preparing the cavity-containing organic molecule includes: selecting sodium calix 4-azosulfonate[4] aromatic hydrocarbon or sodium calix 4-azosulfonate[6] aromatic hydrocarbon, dissolving it in an alkaline solution, adding the modifier epoxypropane, mixing, and then precipitating it with diethyl ether.

11. The method for preparing a block polymer that can be assembled with protein and hydrophobic small molecule drugs according to claim 7 or 10, characterized in that, The molar ratio of any one of the monomers containing ammonium functional groups, amine functional groups, and epoxy functional groups to the monomer containing cavity functional groups or the organic molecule containing cavity is (0.25-4):

1.

12. A method of improving drug loading performance of drug-loaded microspheres, characterized by, Drug-loaded microspheres are added to a mixed solution of the drug to be loaded and the block polymer as described in any one of claims 1 or 2.

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