Compound for preparing embolization bead, hydrogel bead, preparation method therefor and use thereof

Abstract

The present application provides a compound for preparing an embolization bead, a hydrogel bead, a preparation method therefor and a use thereof, the compound having a structure as shown in formula I. A hydrogel bead capable of carrying a chemical drug has a core-shell structure, comprising a shell formed from the compound, and a core formed from an ionic monomer polymeric segment. A hydrogel bead capable of carrying a protein has a core-shell structure, comprising a shell formed from the compound, and a core formed from a zwitterionic polymeric segment. The compound of the present application has good stability, which makes it possible to improve the stability of a hydrogel bead when the compound is used to prepare the hydrogel bead. The hydrogel beads of the present application have a super-hydrophilic core-shell structure, and the dense shell layer and high hydrophilicity cause the beads to have strong toughness, and not break when swelling. Dry beads of the hydrogel beads can quickly adsorb a drug solution, achieving high-efficiency, rapid and high-load drug loading, with significant sustained release effects for drugs or proteins, which can effectively regulate drug release.

Description

A compound for preparing embolic microspheres, hydrogel microspheres, and preparation method and application thereof Technical Field

[0001] The present application belongs to the technical field of hydrogel microspheres, and relates to a compound for preparing embolic microspheres, hydrogel microspheres, and a preparation method and application thereof. Background Art

[0002] Interventional therapy involves selectively directing a microcatheter into the blood vessels supplying the tumor site with the aid of medical imaging equipment. Embolic agents are then injected to cut off the tumor's blood supply, depriving it of nutrients and causing it to "starve to death." Therefore, the choice of embolic agent is crucial for interventional tumor treatment.

[0003] Drug-loaded hydrogel microspheres, with their excellent biocompatibility, drug loading, and release properties, have gradually developed into an important class of biomedical materials. Existing microspheres can be categorized by their primary matrix, including gelatin, starch, polylactic acid, chitosan, sodium alginate, polyvinyl alcohol, and ethyl cellulose. However, these microspheres have limitations that limit their clinical use.

[0004] Drug-eluting beads (DEB), as a new type of embolic agent, can not only effectively embolize the blood supply artery of the tumor, but also have the ability to load chemotherapy drugs. The drug loading time is long and stable, and the drug can be released slowly, which can increase the drug concentration around the tumor tissue and have a sustained killing effect on the tumor.

[0005] At present, the drug-loaded hydrogel microspheres commonly used in the clinical practice of TACE (transcatheter arterial chemoembolization) surgery in China include Callispheres, DCBead, etc., which are all microspheres with polyvinyl alcohol (PVA) as the main material. Although PVA-type microspheres can meet general clinical applications, some problems have also arisen during use. For example, the hydrophilicity and biocompatibility of the microspheres need to be further improved to avoid rejection in clinical practice. In particular, the existing PVA-type microspheres are in the form of wet spheres and need to swell in a specific buffer solution. When loading drugs, the drugs are adsorbed through ion exchange and hydrogen bonding between the microspheres and the drug solution, but they can only efficiently load cationic drugs.

[0006] However, for large molecule drugs, such as peptides, monoclonal antibodies, bispecific antibodies and other protein drugs, which have strong tumor-killing ability, existing drug-loaded microspheres often have problems such as low loading efficiency, slow loading speed, and difficult release for such drugs. Therefore, there are no embolic microspheres loaded with protein drugs that meet the needs of clinical use on the market.

[0007] Polyethylene glycol is the best known synthetic polymer material with good biocompatibility and good water solubility. Polyethylene glycol (PEG) is a general term for a class of polymers containing α,ω-dihydroxyl groups of ethylene glycol (HO-CH2CH2-OH). Its chemical formula is HO(CH2CH2O) n H, n represents a natural number greater than or equal to 2. These compounds exhibit good water solubility, as well as excellent lubricity, moisturizing properties, dispersibility, and adhesion. They can be used as antistatic agents and softeners, finding widespread application in industries such as cosmetics, pharmaceuticals, chemical fibers, rubber, plastics, papermaking, paints, electroplating, pesticides, metalworking, and food processing. Due to its excellent biocompatibility and hydrophilic properties, various polyethylene glycol-based hydrogel materials have garnered significant attention and research.

[0008] Polyethylene glycol molecules have hydroxyl groups at both ends, which, while somewhat reactive, make them difficult to directly use as a cross-linking raw material for synthesizing composite materials. Existing methods for modifying polyethylene glycol often involve complex, multi-step reactions to convert the hydroxyl groups of ethylene glycol into more reactive compounds such as azide, amino groups, or double bonds. Currently, the most widely used method for modifying polyethylene glycol is to convert the terminal hydroxyl groups into acrylates. The modified double bonds facilitate free radical cross-linking polymerization with a variety of acrylic acid and acrylamide monomers, making it easy to incorporate the polyethylene glycol structure into complex materials.

[0009] However, the ester bonds in acrylate-modified polyethylene glycol, namely polyethylene glycol diacrylate compounds, are easily broken under weak acid and alkaline conditions and are easily degraded in the human body environment, which seriously limits their reaction conditions and application scope.

[0010] Summary of the Invention

[0011] The present application provides a compound for preparing embolic microspheres, hydrogel microspheres, and a preparation method and application thereof.

[0012] In a first aspect, the present application provides a compound for preparing embolic microspheres, wherein the compound has a structure shown in the following formula I:

[0013] Wherein R1 and R2 are each independently selected from H, C1-C4 alkyl or -(CH2) q -OH; n = an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); R3 is selected from H or -CH3; m = 1 or 2; and q is 1 or 2.

[0014] In the present application, the compound is used as a material for preparing microspheres, which can ensure that the microspheres have higher stability.

[0015] In the present application, the C1-C4 alkyl group may be a C1, C2, C3 or C4 alkyl group, specifically a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group or the like.

[0016] In a second aspect, the present application provides a method for preparing the compound according to the first aspect, the preparation method comprising the following steps:

[0017] The glycidyl ether compound represented by formula II reacts with the N-hydroxyalkyl acrylamide compound represented by formula III to obtain the compound represented by formula I. The reaction formula is as follows:

[0018] Preferably, the glycidyl ether compound represented by formula II is selected from any one or a combination of at least two of ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, PEG200 diglycidyl ether or PEG500 diglycidyl ether; the structures of the glycidyl ether compounds listed here are as follows:

[0019] The general structural formula of PEG200 diglycidyl ether or PEG500 diglycidyl ether is For PEG200 diglycidyl ether n≈5, and for PEG500 diglycidyl ether n≈12.

[0020] Preferably, the N-hydroxyalkyl acrylamide compound is selected from N-hydroxymethyl acrylamide, N-hydroxyethyl acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, N-(2-hydroxypropyl)acrylamide, N-(hydroxymethyl)methacrylamide, N-(hydroxyethyl)methacrylamide, N-[tris(hydroxymethyl)methyl]methacrylamide or N-(2-hydroxypropyl)methacrylamide, and their structures are shown below:

[0021] Preferably, the reaction is carried out in the presence of a basic substance.

[0022] Preferably, the alkaline substance is sodium hydroxide and / or potassium hydroxide; and the alkaline substance is added to the reaction system in the form of an aqueous solution.

[0023] Preferably, the reaction is carried out at room temperature.

[0024] Preferably, the reaction time is 1-6 h, such as 1 h, 2 h, 3 h, 4 h, 5 h or 6 h.

[0025] In a third aspect, the present application provides a hydrogel microsphere capable of loading chemical drugs, wherein the hydrogel microsphere capable of loading chemical drugs has a core-shell structure, comprising a shell formed by the compound represented by formula I as described in the first aspect, and a core formed by ionic monomer polymer segments.

[0026] The drug-carrying hydrogel microspheres of the present application are of a super-hydrophilic core-shell structure, wherein the outer layer is a dense structure formed by polymerization of a large number of polyethylene glycol monomer derivatives, and the core layer is a loose structure formed by polymerization of ionic monomers.

[0027] Preferably, the ionic monomer is selected from sodium acrylate, sodium methacrylate, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), sodium p-styrenesulfonate, sodium vinylsulfonate, (3-acrylamidopropyl)trimethylammonium chloride, (3-methacrylamidopropyl)trimethylammonium chloride. The structures of the ionic monomers listed above are as follows:

[0028] Preferably, the particle size of the hydrogel microspheres capable of carrying chemical drugs is 30-1400 μm, for example, 30 μm, 50 μm, 80 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, 1200 μm or 1400 μm.

[0029] The hydrogel microspheres of the present application maintain their original morphology and particle size after being dried and swelled again; the drug solution is swelled and adsorbed on the dry spheres to load the drug.

[0030] In a fourth aspect, the present application provides a method for preparing the hydrogel microspheres capable of carrying chemical drugs as described above, wherein the preparation method comprises polymerizing the compound represented by formula I with an ionic monomer to obtain the hydrogel microspheres.

[0031] In this application, drug-carrying hydrogel microspheres are obtained by polymerizing a compound represented by Formula I with an ionic monomer. These microspheres possess a super-hydrophilic core-shell structure, with a dense outer layer formed by polymerization of a large number of polyethylene glycol derivative monomers, and a looser core layer formed by polymerization of ionic monomers. The dense and highly hydrophilic polyethylene glycol derivative structure of the outer layer imparts to the microspheres strong toughness, preventing them from bursting during swelling. The microspheres are then dehydrated and dried to obtain dry microspheres, which rapidly swell in pure water or aqueous solutions while retaining the original wet microsphere morphology and particle size.

[0032] Preferably, the molar amount of the ionic monomer is 1-8 times (eg, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times or 8 times) the molar amount of the compound represented by Formula I.

[0033] Preferably, the polymerization of the compound represented by formula I and the ionic monomer is carried out under the initiation of a free radical initiator.

[0034] Preferably, the free radical initiator is persulfate, azobisisobutylamidine hydrochloride (AIBA, V-50 initiator), azobisisobutylimidazoline hydrochloride (abbreviated as AIBI, VA-044 initiator), azobiscyanovaleric acid (abbreviated as ACVA, V-501), azobisisopropylimidazoline (abbreviated as AIP, VA-061 initiator), and the structures of some compounds are as follows:

[0035] Preferably, the persulfate is selected from any one of sodium persulfate, potassium persulfate or ammonium persulfate, or a combination of at least two thereof.

[0036] Preferably, the polymerization of the compound represented by formula I and the ionic monomer is carried out by reverse suspension polymerization.

[0037] Preferably, the reverse phase suspension polymerization is specifically as follows: the compound represented by formula I, the ionic monomer and the free radical initiator are configured into an aqueous solution; the aqueous solution is added to the oil phase to carry out reverse phase suspension polymerization to obtain the hydrogel microspheres capable of carrying chemical drugs.

[0038] The reaction scheme can be expressed as follows:

[0039] Preferably, the oil phase is butyl acetate, petroleum ether, n-hexane, cyclohexane or paraffin oil.

[0040] Preferably, 3-5% (3%, 3.5%, 4%, 4.5% or 5%) by mass of a surfactant is added to the oil phase.

[0041] Preferably, the surfactant comprises one or a mixture of two of cellulose acetate butyrate, Tween or Span.

[0042] Preferably, when the free radical initiator is a persulfate, a catalyst needs to be additionally added to the oil phase.

[0043] Preferably, the catalyst comprises tetramethylethylenediamine and / or triethylamine.

[0044] Preferably, the temperature of the reverse phase suspension polymerization is 50-90°C, for example 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

[0045] Preferably, the reverse phase suspension polymerization time is 2-12 h, for example, 2 h, 4 h, 6 h, 8 h, 10 h, 11 h or 12 h.

[0046] In the present application, after the reverse suspension polymerization is completed, the hydrogel microspheres capable of carrying chemical drugs are obtained through dehydration and drying.

[0047] In a fifth aspect, the present application provides a protein-loaded hydrogel microsphere having a core-shell structure, comprising a shell formed by the compound represented by Formula I, and a core formed by a zwitterionic monomer polymer segment;

[0048] The zwitterionic monomer has both cationic and anionic groups in its molecular structure, and specifically can be any one of 3-[(3-acrylamidopropyl)dimethylammonium]propane-1-sulfonate, 3-[(3-methacrylamidopropyl)dimethylammonium]propane-1-sulfonate, 4-[(3-acrylamidopropyl)dimethylammonium]butane-1-sulfonate, 4-[(3-methacrylamidopropyl)dimethylammonium]butane-1-sulfonate, 2-[(3-acrylamidopropyl)dimethylammonium]acetate, 2-[(3-methacrylamidopropyl)dimethylammonium]acetate, 3-[(3-acrylamidopropyl)dimethylammonium]propionate or 3-[(3-methacrylamidopropyl)dimethylammonium]propionate, or a combination of at least two thereof.

[0049] The protein-loaded hydrogel microspheres of the present application are super-hydrophilic core-shell structures, wherein the outer layer is a relatively dense structure formed by the polymerization of a large number of polyethylene glycol derivative monomers, and the core layer is a relatively loose structure formed by the polymerization of zwitterionic monomers.

[0050] In the present application, the structure of the zwitterionic monomers listed above is as follows:

[0051] Preferably, the particle size of the protein-loaded hydrogel microspheres is 30-1400 μm, for example, 30 μm, 50 μm, 80 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, 1200 μm or 1400 μm.

[0052] The protein-loaded hydrogel microspheres of the present application maintain their original morphology and particle size after being dried and swelled again; the drug solution is swelled and adsorbed on the dry microspheres to load the drug.

[0053] In a sixth aspect, the present application provides a method for preparing the protein-loaded hydrogel microspheres as described in the fifth aspect, the preparation method comprising: polymerizing the compound represented by formula I with a zwitterionic monomer to obtain the protein-loaded hydrogel microspheres.

[0054] In this application, protein-loaded hydrogel microspheres are obtained by polymerizing a compound represented by Formula I with a zwitterionic monomer. These microspheres have a superhydrophilic core-shell structure: a dense outer layer formed by polymerization of a large number of polyethylene glycol derivative monomers, and a looser core layer formed by polymerization of zwitterionic monomers. The dense and highly hydrophilic outer layer of the polyethylene glycol derivative structure imparts strong toughness to the microspheres, preventing them from bursting during swelling. The microspheres are then dehydrated and dried to obtain dry microspheres, which rapidly swell in pure water or aqueous solutions while retaining the original wet microsphere morphology and particle size.

[0055] Preferably, in the method for preparing protein-loaded hydrogel microspheres, the amount of the zwitterionic monomer used is 1-8 times (e.g., 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times or 8 times) the molar amount of the compound represented by Formula I.

[0056] Preferably, in the method for preparing protein-loaded hydrogel microspheres, the polymerization of the compound represented by Formula I and the zwitterionic monomer is carried out under the initiation of a free radical initiator.

[0057] Preferably, in the preparation method of protein-loaded hydrogel microspheres, the free radical initiator is persulfate, azobisisobutylamidine hydrochloride (AIBA, V-50 initiator), azobisisobutylimidazoline hydrochloride (abbreviated as AIBI, VA-044 initiator), azobiscyanovaleric acid (abbreviated as ACVA, V-501), azobisisopropylimidazoline (abbreviated as AIP, VA-061 initiator), and the structures of some compounds are as follows:

[0058] Preferably, the persulfate is selected from any one of sodium persulfate, potassium persulfate or ammonium persulfate, or a combination of at least two thereof.

[0059] Preferably, in the method for preparing protein-loaded hydrogel microspheres, the polymerization of the compound represented by Formula I and the zwitterionic monomer is carried out by reverse suspension polymerization.

[0060] Preferably, the reverse phase suspension polymerization is specifically as follows: the compound represented by formula I, the zwitterionic monomer and the free radical initiator are configured into an aqueous solution; the aqueous solution is added to the oil phase to carry out reverse phase suspension polymerization to obtain the hydrogel microspheres.

[0061] Preferably, the oil phase is butyl acetate, petroleum ether, n-hexane, cyclohexane or paraffin oil.

[0062] Preferably, 3-5% (3%, 3.5%, 4%, 4.5% or 5%) by mass of a surfactant is added to the oil phase.

[0063] Preferably, the surfactant comprises one or a mixture of two of cellulose acetate butyrate, Tween or Span.

[0064] Preferably, when the free radical initiator is a persulfate, a catalyst needs to be additionally added to the oil phase.

[0065] Preferably, the catalyst comprises tetramethylethylenediamine and / or triethylamine.

[0066] Preferably, the temperature of the reverse phase suspension polymerization is 50-90°C, for example 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

[0067] Preferably, the reverse phase suspension polymerization time is 2-12 h, for example, 2 h, 4 h, 6 h, 8 h, 10 h, 11 h or 12 h.

[0068] In the present application, after the reverse suspension polymerization is completed, the protein-loaded hydrogel microspheres are obtained by dehydration and drying.

[0069] In a seventh aspect, the present application provides a drug-loaded microsphere, which includes the hydrogel microsphere as described above and the drug loaded thereon.

[0070] Preferably, the drug includes any one of doxorubicin hydrochloride, irinotecan, sunitinib, oxaliplatin, VC sodium, gemcitabine, 5-Fu (5-fluorouracil), bovine serum albumin, catalase, glucose oxidase, atezolizumab or bevacizumab, or a combination of at least two thereof.

[0071] The operation of drug loading of the hydrogel microspheres of the present application is as follows: adding the hydrogel microspheres into an aqueous solution containing the drug to obtain the drug-loaded microspheres.

[0072] The hydrogel microspheres of the present application can quickly adsorb drug solutions through swelling, achieving efficient, rapid, and high-load loading of drugs; and the hydrogel microspheres of the present application have a relatively dense shell and ionic polymer chain structure, which has a significant sustained-release effect on drug molecules and can effectively regulate drug release.

[0073] Compared with the existing drug-loaded embolic microspheres, the hydrogel microspheres of the present application can be loaded with drugs in a dry ball state. The loaded drugs are not limited to cationic drugs, and the drug loading capacity of the microspheres is greatly increased and the drug release time is prolonged.

[0074] The protein-loaded hydrogel microspheres of the present application are composed of polyethylene glycol and zwitterionic polymers, do not non-specifically adsorb polypeptides and proteins, and can promote the full release of adsorbed protein drugs.

[0075] Compared with the prior art, this application has the following beneficial effects:

[0076] The compound of the present application does not contain an ester bond that is easily hydrolyzed, and the compound has good stability, so that the stability of the hydrogel microspheres can be improved when used to prepare the hydrogel microspheres.

[0077] The hydrogel microspheres of the present application have a super-hydrophilic core-shell structure, the shell layer is a relatively dense structure formed by the polymerization of a large number of polyethylene glycol derivative monomers, and the core layer is a relatively loose structure formed by the polymerization of ionic monomers. The dense shell layer and the highly hydrophilic polyethylene glycol derivative structure make the microspheres have strong toughness and will not burst when swelled. The microspheres are dehydrated and dried to obtain dry balls. The dry balls of the hydrogel microspheres can quickly adsorb the drug solution through swelling, thereby achieving efficient, rapid, and high-load loading of the drug. The hydrogel microspheres of the present application have a relatively dense shell and ionic polymer chain structure, which has a significant sustained-release effect on drug molecules and can effectively regulate drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is the H NMR spectrum of compound 2.

[0079] Figure 2 is the H NMR spectrum of compound 5.

[0080] FIG3 is a schematic diagram of the structure of the hydrogel microspheres of the present application.

[0081] FIG4 is a schematic diagram of the internal cross-linked network structure of the hydrogel microspheres of the present application.

[0082] FIG5 is an optical microscope image of the hydrogel microspheres prepared in Example 1.

[0083] FIG6 is an optical microscope image of the hydrogel microspheres prepared in Example 2.

[0084] FIG7 is an optical microscope image of the hydrogel microspheres prepared in Example 3.

[0085] FIG8 is an optical microscope image of the hydrogel microspheres prepared in Example 4.

[0086] FIG9 is an optical microscope image of the hydrogel microspheres prepared in Example 5.

[0087] FIG10 is an optical microscope image of the hydrogel microspheres prepared in Example 6.

[0088] FIG11 is an optical microscope image of the hydrogel microspheres prepared in Example 7.

[0089] FIG12 is an optical microscope image of the dry spheres of the hydrogel microspheres of Example 2.

[0090] FIG13 is an optical microscope image of the dry spheres of the hydrogel microspheres of Example 2 after absorbing the doxorubicin hydrochloride solution.

[0091] FIG14 is a drug release curve of doxorubicin hydrochloride from the hydrogel microspheres of Example 2.

[0092] FIG15 is a drug release curve of sodium salicylate from the hydrogel microspheres of Example 7.

[0093] FIG16 is an optical microscope image of the protein-loaded hydrogel microspheres of Example 9.

[0094] FIG17 is an optical microscope image of dry spheres obtained from the hydrogel microspheres of Example 9.

[0095] FIG18 is a cumulative protein release curve in pure water after the dry balls prepared from the hydrogel microspheres of Example 9 were loaded with protein solution.

[0096] FIG19 is a cumulative protein release curve in pure water after the dry balls prepared from the hydrogel microspheres of Example 14 were loaded with protein solution. DETAILED DESCRIPTION

[0097] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0098] Preparation Example 1

[0099] Accurately weigh 3g of ethylene glycol diglycidyl ether into a glass bottle, add 6mL of a 15% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9mL of a 50% (mass fraction) aqueous N-hydroxymethyl acrylamide solution to the bottle and continue stirring for 2h. Neutralize the solution with concentrated hydrochloric acid until neutral (this solution can be used directly in the subsequent preparation of hydrogel microspheres).

[0100] The reaction solution was dried under reduced pressure to obtain a crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 1:1) afforded 4.5 g (yield 70%) of pure compound 1 as a colorless oily liquid. H NMR spectrum data: 1 HNMR (400 MHz, D2O) δ 5.80-5.64 (m, 4H), 5.23-5.19 (m, 2H), 4.55 (s, 4H), 3.40-2.74 (m, 14H); mass spectrum data: MS (ESI): m / z 377 (M + H + ), 399(M + Na + ).

[0101] Preparation Example 2

[0102] Accurately weigh 3.2 g of diethylene glycol diglycidyl ether into a glass bottle, add 6 mL of a 12% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 9.5 mL of a 50% (mass fraction) aqueous solution of N-hydroxyethyl acrylamide to the bottle and continue stirring for 2 h. Neutralize the solution with concentrated hydrochloric acid until neutral (this solution can be used directly in the subsequent preparation of hydrogel microspheres).

[0103] The reaction solution was dried under reduced pressure to give a crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 1:1) yielded 4.3 g (65% yield) of pure compound 2 as a nearly colorless oily liquid. The H NMR spectrum is shown in Figure 1. The H NMR spectrum data are: 1 HNMR (400 MHz, D2O) δ 5.82-5.63 (m, 4H), 5.22-5.18 (m, 2H), 3.42-2.73 (m, 26H); mass spectrum data: MS (ESI): m / z 449 (M + H + ), 471(M + Na+).

[0104] Preparation Example 3

[0105] Accurately weigh 3.5 g of triethylene glycol diglycidyl ether into a glass bottle, add 6.4 mL of 18% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 9.5 mL of a 50% (mass fraction) aqueous solution of N-hydroxyethyl acrylamide and continue stirring for 1.5 h. Neutralize the solution with concentrated hydrochloric acid until neutral (this solution can be used directly in the subsequent preparation of hydrogel microspheres).

[0106] The reaction solution was dried under reduced pressure to obtain a crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 1:1) afforded 4.0 g (yield 61%) of pure compound 3 as a light yellow oily liquid. H NMR spectrum data: 1 HNMR (400 MHz, D2O) δ 5.81-5.62 (m, 4H), 5.24-5.18 (m, 2H), 3.44-2.73 (m, 30H); mass spectrum data: MS (ESI): m / z 493 (M+H+), 515 (M+Na+).

[0107] Preparation Example 4

[0108] Accurately weigh 2.8 g of tetraethylene glycol diglycidyl ether into a glass bottle, add 6 mL of a 20% aqueous solution of KOH, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 8.5 mL of a 50% (mass fraction) aqueous solution of N-(2-hydroxypropyl)acrylamide to the bottle and continue stirring for 2 h. Neutralize the solution with concentrated hydrochloric acid until neutral (this solution can be used directly in the subsequent preparation of hydrogel microspheres).

[0109] The reaction solution was evaporated to dryness under reduced pressure to yield the crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 1:1) afforded 2.9 g (57% yield) of pure compound 4 as a pale yellow oil. H NMR data: 1H NMR (400 MHz, D2O) δ 5.84-5.63 (m, 4H), 5.23-5.17 (m, 2H), 3.44-2.75 (m, 32H), 0.83 (s, 6H); MS (MALDI-TOF): m / z 587 (M+Na+).

[0110] Preparation Example 5

[0111] Accurately weigh 2.8 g of PEG200 diglycidyl ether into a glass bottle, add 8 mL of 25% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 10 mL of a 50% (mass fraction) aqueous solution of N-[tris(hydroxymethyl)methyl]acrylamide to the bottle and continue stirring for 2.5 h. Neutralize the solution with concentrated hydrochloric acid until neutral (the solution can be used directly for subsequent hydrogel microsphere preparation).

[0112] The reaction solution was dried under reduced pressure to obtain a crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 2:3) yielded 3.1 g (yield 41%) of pure compound 5 as a light yellow oily liquid. The H NMR spectrum is shown in Figure 2. The H NMR spectrum data are as follows: 1 HNMR (400 MHz, D2O) δ 5.82-5.64 (m, 4H), 5.21-5.16 (m, 2H), 3.44-2.75 (m, 42H); Mass spectral data: MS (MALDI-TOF): m / z 723 (M+Na + ).

[0113] Preparation Example 6

[0114] Accurately weigh 3.2 g of PEG500 diglycidyl ether into a glass bottle, add 10 mL of a 25% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 10 mL of a 50% (by weight) aqueous solution of N-hydroxyethyl methacrylamide and continue stirring for 2.5 h. Neutralize the solution with concentrated hydrochloric acid until neutral (this solution can be used directly for subsequent hydrogel microsphere preparation).

[0115] The reaction solution was dried under reduced pressure to obtain a crude compound. Further purification by column chromatography (C18 column, acetonitrile:water = 1:2) afforded 2.2 g (yield 46%) of pure compound 6 as a light yellow oily liquid. H NMR spectrum data: 1 HNMR (400 MHz, D2O) δ 5.86-5.60 (m, 2H), 5.24-5.18 (m, 2H), 3.44-2.75 (m, 66H), 2.43 (s, 6H); mass spectrum data: MS (MALDI-TOF): m / z 939,940.

[0116] Example 1

[0117] In this embodiment, hydrogel microspheres were prepared by the following method:

[0118] Aqueous phase preparation: To the reaction solution obtained by neutralizing the system solution with concentrated hydrochloric acid in Preparation Example 1 to neutrality, an aqueous solution containing 8.7 g of sodium methacrylate was added, and after stirring for 0.5 h, 160 mg of potassium persulfate was added, and the mixture was uniformly stirred for 0.5 h to prepare the aqueous phase.

[0119] Preparation of oil phase: Add 3 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 50°C to form a uniform oil phase system.

[0120] Reverse-Phase Suspension Polymerization: Slowly add the aqueous solution prepared above to the oil phase at 50°C with stirring at 300 rpm to form a water-in-oil reverse suspension polymerization system. Maintain the reaction at 50°C for 30 minutes. Then, add 1.0 mL of triethylamine (Et3N) to the oil phase and raise the temperature to 80°C for 4 hours.

[0121] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0122] The resulting hydrogel microspheres are schematically shown in Figure 3. They possess a core-shell structure, with the shell layer being a denser structure formed by the polymerization of a large number of polyethylene glycol monomer derivatives, and the core layer being a looser structure formed by the polymerization of ionic monomers. A schematic diagram of the internal cross-linked network structure of the hydrogel microspheres is shown in Figure 4.

[0123] Example 2

[0124] In this embodiment, hydrogel microspheres were prepared by the following method:

[0125] Aqueous phase preparation: neutralize the system solution in Preparation Example 2 with concentrated hydrochloric acid to a neutral reaction solution, add an aqueous solution containing 6.5 g of sodium acrylate, stir for 0.5 h, then add 180 mg of ammonium persulfate, stir evenly for 0.5 h, and prepare the aqueous phase.

[0126] Preparation of oil phase: Add 3 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 50°C to form a uniform oil phase system.

[0127] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oil phase at 50°C with stirring at 300 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction temperature at 50°C for 30 minutes. Then, add 1.2 mL of tetramethylethylenediamine (TMEDA) to the oil phase and heat to 80°C for 4 hours.

[0128] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0129] Example 3

[0130] In this embodiment, hydrogel microspheres were prepared by the following method:

[0131] Aqueous phase preparation: To the neutralized reaction solution in Preparation Example 3 using concentrated hydrochloric acid, add an aqueous solution containing 18 g of 2-acrylamido-2-methylpropanesulfonic acid (AMPS). After stirring for 0.5 h, add 180 mg of ammonium persulfate and stir evenly for 0.5 h to prepare the aqueous phase.

[0132] Preparation of oil phase: Add 3 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 50°C to form a uniform oil phase system.

[0133] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oil phase at 55°C with stirring at 300 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 55°C for 30 minutes. Then, add 1.2 mL of TMEDA to the oil phase and raise the temperature to 80°C for 5 hours.

[0134] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0135] Example 4

[0136] In this embodiment, hydrogel microspheres were prepared by the following method:

[0137] Aqueous phase preparation: neutralize the system solution in Preparation Example 4 with concentrated hydrochloric acid to a neutral reaction solution, add an aqueous solution containing 18 g of sodium methacrylate, stir for 0.5 h, then add 120 mg of ammonium persulfate, stir evenly for 0.5 h, and prepare the aqueous phase.

[0138] Preparation of oil phase: Add 3 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 50°C to form a uniform oil phase system.

[0139] Reverse-Phase Suspension Polymerization: Slowly add the aqueous solution prepared above to the oil phase at 50°C with stirring at 300 rpm to form a water-in-oil reverse suspension polymerization system. Maintain the reaction at 50°C for 30 minutes. Then, add 1.0 mL of triethylamine (Et3N) to the oil phase and raise the temperature to 85°C for 3.5 hours.

[0140] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0141] Example 5

[0142] In this embodiment, hydrogel microspheres were prepared by the following method:

[0143] Aqueous phase preparation: To the neutralized reaction solution in Preparation Example 5 with concentrated hydrochloric acid, add an aqueous solution containing 16 g of sodium p-styrenesulfonate, stir for 0.5 h, then add 120 mg of azobisisobutylamidine hydrochloride (V-50), stir evenly for 0.5 h, and prepare the aqueous phase.

[0144] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL n-hexane and stir to dissolve at 50°C to form a uniform oil phase system.

[0145] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at 70°C with stirring at 300 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 70°C for 30 minutes, then raise the temperature to 90°C for 6 hours.

[0146] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0147] Example 6

[0148] In this embodiment, hydrogel microspheres were prepared by the following method:

[0149] Aqueous phase preparation: neutralize the system solution in Preparation Example 6 with concentrated hydrochloric acid to a neutral reaction solution, add an aqueous solution containing 7.9 g of sodium vinyl sulfonate, stir for 0.5 h, then add 150 mg of azobisisobutylimidazoline hydrochloride (VA-044), stir evenly for 0.5 h, and then prepare the aqueous phase.

[0150] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL cyclohexane and stir to dissolve at 50°C to form a uniform oil phase system.

[0151] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at 70°C with stirring at 300 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 70°C for 30 minutes, then raise the temperature to 90°C for 6 hours.

[0152] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0153] Example 7

[0154] In this embodiment, hydrogel microspheres were prepared by the following method:

[0155] Aqueous Phase Preparation: Accurately weigh 3.0g of tetraethylene glycol diglycidyl ether into a glass bottle, add 6mL of 10% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9mL of a 50% (mass fraction) aqueous N-hydroxymethyl acrylamide solution to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 14g of (3-acrylamidopropyl)trimethylammonium chloride, stir for 0.5h, then add 100mg of azobiscyanovaleric acid (V-501), stir evenly for 0.5h, and prepare the aqueous phase.

[0156] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL paraffin oil and stir to dissolve at 50°C to form a uniform oil phase system.

[0157] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at 50°C with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 50°C for 30 minutes, then raise the temperature to 75°C for 5 hours.

[0158] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly 3-5 times with petroleum ether (30-60), butyl acetate, purified water and 5% sodium bicarbonate aqueous solution in sequence to obtain hydrogel microspheres.

[0159] Example 8

[0160] In this embodiment, hydrogel microspheres were prepared by the following method:

[0161] Aqueous Phase Preparation: Accurately weigh 3.0 g of tetraethylene glycol diglycidyl ether into a glass bottle, add 6 mL of 10% NaOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 9.2 mL of a 50% (mass fraction) aqueous solution of N-hydroxymethyl methacrylamide to the bottle and continue stirring for 2 h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 15 g of (3-methacrylamidopropyl)trimethylammonium chloride, stir for 0.5 h, then add 130 mg of azobisisopropylimidazoline (VA-061), stir evenly for 0.5 h, and prepare the aqueous phase.

[0162] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL paraffin oil and stir to dissolve at 50°C to form a uniform oil phase system.

[0163] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at 50°C with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 50°C for 30 minutes, then raise the temperature to 75°C for 5 hours.

[0164] Purification: After the reaction is completed, the obtained microspheres are washed repeatedly with petroleum ether (30-60), butyl acetate, purified water and 5% sodium bicarbonate aqueous solution for 3-5 times to obtain hydrogel microspheres.

[0165] The hydrogel microspheres prepared in Examples 1-8 were characterized using a scanning electron microscope. It can be seen that the hydrogel microspheres prepared using the scheme of the present application have a complete and smooth morphology, and a single microsphere can be identified under a Keyence (VHX-950F video display system) microscope as having a clear core-shell double-layer structure.

[0166] Performance testing:

[0167] Microsphere stability test: The hydrogel microsphere wet balls prepared in Example 2 were finely sieved through a sieve to obtain three specifications of 40-100μm, 100-200μm and 200-300μm (exceeding the limit ratio <5%), and acid and alkali tolerance tests were carried out cumulatively. Each batch of microspheres was soaked in 5% sodium hydroxide solution and 5% hydrochloric acid solution at room temperature for 24 hours, and then boiled in 5% sodium bicarbonate solution for 30 minutes. The morphology of the microspheres after each operation was observed under an optical microscope, and the particle size and average particle size changes in pure water were measured. If the chemical bonds in the microsphere structure are destroyed, the microsphere morphology is damaged or the particle size increases significantly; if the microsphere morphology remains good and the particle size changes little or does not change, it reflects that the microsphere microstructure is firmly linked and has excellent stability and acid and alkali resistance.

[0168] Sampling and swelling criteria: initial wet bulb volume is 10 mL; volume of each immersion water solution is 100 mL.

[0169] Average particle size test method: Using the above-mentioned optical microscope, take all microspheres within two fields of view and include them in the statistics. The total number of microspheres must be greater than 150. If the total number of microspheres is less than 150, take another field of view of microspheres. Use the instrument operating software to manually test the particle size of all microspheres included in the statistics, record the values ​​and calculate the average particle size. The results are shown in Table 1 below.

[0170] Table 1

[0171] As shown in Table 1 above, the hydrogel microspheres prepared by the PEG compound of the present application did not show significant changes in morphology and particle size after being treated in strong acid and alkali solutions for a long time, indicating that the hydrogel microspheres have excellent acid and alkali stability.

[0172] Preparation method of dry microspheres: The hydrogel microspheres prepared in Examples 1-8 were shrunk by adding acetone multiple times, and then dried at 80° C. for 3 h to constant weight to obtain dry granular microspheres.

[0173] The optical microscope image of the dried hydrogel microspheres obtained from Example 2 is shown in FIG12 . It can be seen that the microspheres still maintain an intact structure after drying without being broken.

[0174] Dry ball swelling rate test: 100 mg of the hydrogel microspheres prepared in Examples 1-8 were placed in 20 mL vials, and 10 mL of pure water, 0.9% NaCl aqueous solution, and 5% glucose aqueous solution were added. The time required for complete swelling was tested. The results are shown in Table 2.

[0175] Table 2

[0176] Dry ball drug loading test: A nearly saturated aqueous solution of each drug was prepared. 2 mL of the drug solution was taken each time into a 10 mL vial. 50 mg of the dry ball of each example was added. The dry ball rapidly swelled and absorbed the drug solution. The scanning electron micrograph of the dry ball obtained from the hydrogel microspheres of Example 2 after absorbing the doxorubicin hydrochloride solution is shown in Figure 13. It can be seen that doxorubicin hydrochloride can quickly penetrate the shell of the hydrogel microspheres and enter the microsphere core, facilitating the slow release of the drug.

[0177] The drug loading capacity of the hydrogel microspheres was calculated based on the absorption volume and drug concentration. The drug loading results are shown in Table 3.

[0178] Table 3

[0179] It can be seen from Table 3 that the hydrogel microspheres prepared according to the present application have excellent broad-spectrum drug loading function, which expands the scope of use of the microspheres compared to microspheres that can only load a single type of drug or a certain type of drug.

[0180] Drug release testing:

[0181] The drug-loaded hydrogel microspheres were transferred to 20 mL of normal saline at 37 °C for drug release testing. The extract was replaced at 30 minutes, 1 hour, 2 hours, 3 hours, 24 hours, 48 ​​hours, 72 hours, 90 hours, and 102 hours. The concentration of the drug in the extract was measured, and the cumulative drug release was calculated.

[0182] The drug release curve of the hydrogel microspheres of Example 2 for doxorubicin hydrochloride is shown in FIG14 . It can be seen that the hydrogel microspheres with carboxylate anions have an excellent sustained release effect for the cationic drug doxorubicin hydrochloride, with a release rate of 41.4% within 29 hours, achieving sustained slow release.

[0183] The drug release curve of the hydrogel microspheres of Example 7 for sodium salicylate is shown in FIG15 . It can be seen that the hydrogel microspheres with quaternary ammonium cations have an excellent sustained release effect for the anionic drug sodium saline saline, with a release rate of less than 50% within 120 hours, achieving sustained slow release.

[0184] The hydrogel microspheres prepared in Example 2 were finely sieved through a sieve to obtain three specifications of 40-100 μm, 100-200 μm, and 200-300 μm (overlimit ratio <5%). Three drying-swelling operations were performed cumulatively on each batch. The particle size changes of each batch of dry microspheres after full swelling in pure water, 0.9% NaCl solution, and 5 mg / mL doxorubicin hydrochloride solution were compared. The statistical data are shown in Table 4.

[0185] Sampling and swelling criteria: initial wet bulb volume is 1 mL; dry bulb mass is 100 mg; immersion water solution volume is 15 mL.

[0186] Average particle size test method: Using the above-mentioned optical microscope, take all microspheres within two fields of view and include them in the statistics. The total number of microspheres must be greater than 150. If less than 150, take another field of view of microspheres; using the instrument operating software, manually test the particle size of all microspheres included in the statistics, record the values ​​and calculate the average particle size.

[0187] Table 4

[0188] As can be seen from Table 4 above, the wet ball particle size of the microspheres of the present application does not change substantially before and after drying, and after three drying-swelling cycles, the microsphere morphology remains intact, with no damaged or ruptured microspheres, and the particle size remains unchanged compared to before drying, demonstrating that the hydrogel microspheres of the present application have excellent mechanical properties and toughness.

[0189] Example 9

[0190] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0191] Aqueous Phase Preparation: Accurately weigh 3.0g of triethylene glycol diglycidyl ether into a glass bottle, add 6mL of a 15% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9.2mL of a 50% by weight aqueous solution of N-hydroxyethyl acrylamide to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 18g of 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate, stir for 0.5h, then add 180mg of ammonium persulfate. Stir evenly for 0.5h to prepare the aqueous phase.

[0192] Preparation of oil phase: Add 3 g of cellulose acetate butyrate to 100 mL of butyl acetate and stir to dissolve at 50°C to form a uniform oil phase system.

[0193] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oil phase at 45°C with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 45°C for 30 minutes. Add 1.2 mL of TMEDA catalyst to the oil phase and heat to 80°C for 4 hours.

[0194] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0195] Example 10

[0196] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0197] Aqueous Phase Preparation: Accurately weigh 3.0g of triethylene glycol diglycidyl ether into a glass bottle, add 6mL of 15% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9.5mL of a 50% (mass fraction) aqueous solution of N-hydroxyethyl acrylamide to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 20g of 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonate, stir for 0.5h, then add 100mg of VA-044. Stir evenly for 0.5h to prepare the aqueous phase.

[0198] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL petroleum ether (90-120), stir and dissolve at room temperature to form a uniform oil phase system.

[0199] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 80°C for 8 hours.

[0200] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0201] Example 11

[0202] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0203] Aqueous phase preparation: Accurately weigh 3.5g of tetraethylene glycol diglycidyl ether into a glass bottle, add 8mL of 12% NaOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9.0mL of a 50% by weight aqueous solution of N-hydroxymethyl acrylamide to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid to neutrality, add an aqueous solution containing 22g of 4-[(3-acrylamidopropyl)dimethylammonio]butane-1-sulfonate, stir for 0.5h, then add 110mg of V-50. Stir evenly for 0.5h to prepare the aqueous phase.

[0204] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL petroleum ether (90-120), stir and dissolve at room temperature to form a uniform oil phase system.

[0205] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 70°C for 8 hours.

[0206] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0207] Example 12

[0208] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0209] Aqueous Phase Preparation: Accurately weigh 3.3g of tetraethylene glycol diglycidyl ether into a glass bottle, add 10mL of 12% NaOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9.0mL of a 50% by weight aqueous solution of N-hydroxymethyl acrylamide to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 24g of 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonate, stir for 0.5h, then add 120mg of VA-044. Stir evenly for 0.5h to prepare the aqueous phase.

[0210] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL petroleum ether (boiling point 90-120° C.), stir and dissolve at room temperature to form a uniform oil phase system.

[0211] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 80°C for 8 hours.

[0212] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0213] Example 13

[0214] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0215] Aqueous Phase Preparation: Accurately weigh 2.7g of diethylene glycol diglycidyl ether into a glass bottle, add 8mL of 18% NaOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 10.0mL of a 50% (mass fraction) aqueous solution of N-hydroxymethyl methacrylamide to the bottle and continue stirring for 2h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 15g of 2-[(3-acrylamidopropyl)dimethylammonium] acetate, stir for 0.5h, then add 90mg of V-501. Stir evenly for 0.5h to prepare the aqueous phase.

[0216] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL cyclohexane and stir to dissolve at room temperature to form a uniform oil phase system.

[0217] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 90°C for 4 hours.

[0218] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0219] Example 14

[0220] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0221] Aqueous Phase Preparation: Accurately weigh 3.2 g of triethylene glycol diglycidyl ether into a glass bottle, add 9 mL of 18% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 10.0 mL of a 50% (mass fraction) aqueous solution of N-hydroxymethyl methacrylamide to the bottle and continue stirring for 2 h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 16 g of 2-[(3-methacrylamidopropyl)dimethylammonium] acetate, stir for 0.5 h, then add 105 mg of VA-061. Stir evenly for 0.5 h to prepare the aqueous phase.

[0222] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL n-hexane and stir to dissolve at room temperature to form a uniform oil phase system.

[0223] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 75°C for 8 hours.

[0224] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0225] Example 15

[0226] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0227] Aqueous phase preparation: Accurately weigh 3.0g of diethylene glycol diglycidyl ether into a glass bottle, add 6mL of 25% NaOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 10.5mL of a 50% mass fraction N-[tris(hydroxymethyl)methyl]acrylamide aqueous solution to the bottle and continue stirring for 2.5h. Neutralize the system solution with concentrated hydrochloric acid to neutrality, add an aqueous solution containing 19g of 3-[(3-acrylamidopropyl)dimethylammonium]propionate, stir for 0.5h, then add 160mg of potassium persulfate and stir evenly for 0.5h to prepare the aqueous phase.

[0228] Preparation of oil phase: Add 3 g Span80 and 1 g Tween20 to 100 mL paraffin oil and stir to dissolve at 50°C to form a uniform oil phase system.

[0229] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oil phase at 50°C with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 50°C for 30 minutes. Add 1.3 mL of triethylamine as a catalyst to the oil phase and heat to 80°C for 8 hours.

[0230] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with petroleum ether, butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0231] Example 16

[0232] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0233] Aqueous Phase Preparation: Accurately weigh 2.5g of ethylene glycol diglycidyl ether into a glass bottle, add 7mL of 20% KOH aqueous solution, and stir on a magnetic stirrer at room temperature for 0.5h. Add 9mL of a 50% (mass fraction) aqueous solution of N-[tris(hydroxymethyl)methyl]acrylamide to the bottle and continue stirring for 2.5h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 20g of 3-[(3-methacrylamidopropyl)dimethylammonium]propionate, stir for 0.5h, then add 150mg of VA-061. Stir evenly for 0.5h to prepare the aqueous phase.

[0234] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL petroleum ether (90-120), stir and dissolve at room temperature to form a uniform oil phase system.

[0235] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at room temperature with stirring at 400 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at room temperature for 30 minutes, then raise the temperature to 75°C for 8 hours.

[0236] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0237] Example 17

[0238] In this example, protein-loaded hydrogel microspheres were prepared by the following method:

[0239] Aqueous Phase Preparation: Accurately weigh 3.8 g of polyethylene glycol 500 diglycidyl ether into a glass bottle, add 12 mL of 25% aqueous NaOH solution, and stir on a magnetic stirrer at room temperature for 0.5 h. Add 9.5 mL of a 50% (by mass) aqueous solution of N-hydroxyethyl methacrylamide to the bottle and continue stirring for 2.5 h. Neutralize the system solution with concentrated hydrochloric acid until neutral, add an aqueous solution containing 18 g of 2-[(3-acrylamidopropyl)dimethylammonium] acetate, stir for 0.5 h, then add 150 mg of VA-044. Stir evenly for 0.5 h to prepare the aqueous phase.

[0240] Preparation of oil phase: Add 3 g Span 80 and 1 g Tween 20 to 100 mL cyclohexane and stir to dissolve at room temperature to form a uniform oil phase system.

[0241] Inverse suspension polymerization: Slowly add the aqueous solution prepared above to the oily solution at 70°C with stirring at 300 rpm to form a water-in-oil inverse suspension polymerization system. Maintain the reaction at 70°C for 30 minutes, then raise the temperature to 90°C for 6 hours.

[0242] Purification: After the reaction is completed, the obtained microspheres are repeatedly washed 3-5 times with butyl acetate, purified water and 5% sodium bicarbonate aqueous solution to obtain protein-loaded hydrogel microspheres.

[0243] The protein-loaded hydrogel microspheres prepared in Examples 9-17 were characterized using a high-magnification optical microscope (Keyence, VHX-950F video display system). The optical microscope image of the protein-loaded hydrogel microspheres of Example 9 is shown in Figure 16. It can be seen that the protein-loaded hydrogel microspheres of this example have a complete and smooth morphology, and under the Keyence (VHX-950F video display system) microscope, it can be identified that a single microsphere has a clear core-shell double-layer structure.

[0244] Performance testing:

[0245] Preparation method of dry microspheres: After the protein-loaded hydrogel microspheres prepared in Examples 9-17 were shrunk by adding acetone multiple times, they were dried at 80° C. for 3 h to constant weight to obtain dry granular microspheres.

[0246] The optical microscope image of the dry spheres obtained from the protein-loaded hydrogel microspheres of Example 9 is shown in FIG17 . It can be seen that the protein-loaded hydrogel microspheres of this example have a complete and smooth morphology, and a single microsphere can be identified as having a distinct core-shell double-layer structure under a KEYENCE (VHX-950F video display system) microscope.

[0247] Dry ball swelling rate test: 100 mg of the protein-loaded hydrogel microspheres prepared in Examples 9-17 were placed in 20 mL vials and added with 10 mL of pure water, 0.9% NaCl aqueous solution, and 5% glucose aqueous solution. All of the dry balls were able to swell completely within 30 min. The detailed test results are shown in Table 5.

[0248] Table 5

[0249] Dry-sphere drug loading test: A near-saturated aqueous solution of each drug was prepared. 2 mL of each drug solution was placed in a 10 mL vial. 50 mg of dry-spheres from Examples 9-16 were added. The dry-spheres rapidly swelled and absorbed the drug solution. The drug loading capacity of the protein-loaded hydrogel microspheres was calculated based on the absorbed volume and drug concentration. The drug loading results are shown in Table 6.

[0250] Table 6

[0251] After the above-mentioned microspheres adsorbed the protein solution, a release experiment was carried out in pure water, which showed that sustained release could be achieved, and the cumulative release rate of each protein over 96 hours was greater than 80%, demonstrating that the zwitterionic protein-loaded hydrogel microspheres of the present application have the property of resisting protein-specific adsorption and can achieve sufficient release of protein drugs.

[0252] The cumulative protein release rate of the dry spheres prepared from the protein-loaded hydrogel microspheres of Example 9 after loading the protein solution in pure water is shown in Table 7, and the release curve is shown in FIG18 .

[0253] Table 7

[0254] As can be seen from Table 7 and Figure 18, the zwitterionic protein-loaded hydrogel microspheres of the present application have excellent sustained-release effects on a variety of protein compounds, and the release rate within 96 hours exceeds 85%, achieving sufficient release without obvious specific adsorption.

[0255] The cumulative protein release rate of the dry spheres prepared from the protein-loaded hydrogel microspheres of Example 14 after loading the protein solution in pure water is shown in Table 8, and the release curve is shown in FIG19 .

[0256] Table 8

[0257] As can be seen from Table 8 and Figure 19, the zwitterionic protein-loaded hydrogel microspheres of the present application have excellent sustained-release effects on a variety of protein compounds, and the release rate within 96 hours exceeds 91%, achieving sufficient release without obvious specific adsorption.

[0258] The applicant declares that while the above-mentioned embodiments are used to illustrate the process of the present application, the present application is not limited to the above-mentioned process steps, which does not mean that the present application must rely on the above-mentioned process steps in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials used in the present application, addition of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A compound for preparing embolic microspheres, having a structure as shown in Formula I below: in, R1 and R2 are each independently selected from H, C1-C4 alkyl or -(CH2) q -OH; n=an integer from 1 to 12; R3 is selected from H or -CH3; m=1 or 2; and q is 1 or 2.

2. The compound according to claim 1, wherein The C1-C4 alkyl group is methyl, ethyl, n-propyl, isopropyl or n-butyl.

3. A method for preparing the compound as claimed in claim 1 or 2, comprising the following steps: The glycidyl ether compound represented by formula II reacts with the N-hydroxyalkyl acrylamide compound represented by formula III to obtain the compound represented by formula I. The reaction formula is as follows:

4. The preparation method according to claim 3, wherein The glycidyl ether compound represented by formula II is selected from any one of ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, PEG200 diglycidyl ether or PEG500 diglycidyl ether, or a combination of at least two thereof; Preferably, the N-hydroxyalkyl acrylamide compound is selected from N-hydroxymethyl acrylamide, N-hydroxyethyl acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, N-(2-hydroxypropyl)acrylamide, N-(hydroxymethyl)methacrylamide, N-(hydroxyethyl)methacrylamide, N-[tris(hydroxymethyl)methyl]methacrylamide or N-(2-hydroxypropyl)methacrylamide; Preferably, the reaction is carried out in the presence of a basic substance; Preferably, the alkaline substance is sodium hydroxide and / or potassium hydroxide; and the alkaline substance is added to the reaction system in the form of an aqueous solution; Preferably, the reaction is carried out at room temperature; Preferably, the reaction time is 1-6 hours.

5. A hydrogel microsphere capable of carrying chemical drugs, which has a core-shell structure, wherein the core-shell structure comprises a shell formed by the compound as claimed in claim 1 or 2, and a core formed by an ionic monomer polymer segment.

6. The hydrogel microspheres capable of carrying chemical drugs according to claim 5, wherein: The ionic monomer is selected from sodium acrylate, sodium methacrylate, 2-acrylamide-2-methylpropane sulfonic acid, sodium p-styrene sulfonate, sodium vinyl sulfonate, (3-acrylamidepropyl)trimethylammonium chloride or (3-methacrylamidepropyl)trimethylammonium chloride; Preferably, the particle size of the hydrogel microspheres capable of carrying chemical drugs is 30-1400 μm.

7. A method for preparing hydrogel microspheres capable of carrying chemical drugs as claimed in claim 5 or 6, comprising: The compound represented by formula I is polymerized with an ionic monomer to obtain the hydrogel microspheres capable of carrying chemical drugs; Preferably, the amount of the ionic monomer is 1-8 times the molar amount of the compound represented by formula I; Preferably, the polymerization of the compound represented by formula I and the ionic monomer is carried out under the initiation of a free radical initiator; Preferably, the free radical initiator is persulfate, azobisisobutylamidine hydrochloride, azobisisobutylimidazoline hydrochloride, azobiscyanovaleric acid, azobisisopropylimidazoline; Preferably, the persulfate is selected from any one or a combination of at least two of sodium persulfate, potassium persulfate or ammonium persulfate; Preferably, the polymerization of the compound represented by formula I and the ionic monomer is carried out by reverse suspension polymerization; Preferably, the reverse phase suspension polymerization is specifically as follows: the compound represented by formula I, the ionic monomer and the free radical initiator are configured into an aqueous solution; the aqueous solution is added into the oil phase for reverse phase suspension polymerization to obtain the hydrogel microspheres; Preferably, the oil phase is butyl acetate, petroleum ether, n-hexane, cyclohexane or paraffin oil; Preferably, 3-5% by mass of a surfactant is added to the oil phase; Preferably, the surfactant comprises one or a mixture of two of cellulose acetate butyrate, Tween or Span; Preferably, when the free radical initiator is a persulfate, a catalyst is additionally added to the oil phase; Preferably, the catalyst comprises tetramethylethylenediamine and / or triethylamine; Preferably, the temperature of the inverse suspension polymerization is 50-90°C; Preferably, the time of the reverse suspension polymerization is 2-12 hours.

8. A protein-loaded hydrogel microsphere having a core-shell structure, wherein the core-shell structure comprises a shell formed by the compound according to claim 1 or 2, and a core formed by a zwitterionic monomer polymer segment; Preferably, the zwitterionic monomer is any one of 3-[(3-acrylamidopropyl)dimethylammonium]propane-1-sulfonate, 3-[(3-methacrylamidopropyl)dimethylammonium]propane-1-sulfonate, 4-[(3-acrylamidopropyl)dimethylammonium]butane-1-sulfonate, 4-[(3-methacrylamidopropyl)dimethylammonium]butane-1-sulfonate, 2-[(3-acrylamidopropyl)dimethylammonium]acetate, 2-[(3-methacrylamidopropyl)dimethylammonium]acetate, 3-[(3-acrylamidopropyl)dimethylammonium]propionate or 3-[(3-methacrylamidopropyl)dimethylammonium]propionate, or a combination of at least two thereof; Preferably, the particle size of the protein-loaded hydrogel microspheres is 30-1400 μm.

9. A method for preparing protein-loaded hydrogel microspheres as claimed in claim 8, comprising: The compound represented by formula I is polymerized with zwitterionic monomers to obtain the protein-loaded hydrogel microspheres; Preferably, the amount of the zwitterionic monomer is 1-8 times the molar amount of the compound represented by formula I; Preferably, the polymerization of the compound represented by formula I and the zwitterionic monomer is carried out under the initiation of a free radical initiator; Preferably, the free radical initiator is persulfate, azobisisobutylamidine hydrochloride, azobisisobutylimidazoline hydrochloride, azobiscyanovaleric acid, azobisisopropylimidazoline; Preferably, the persulfate is selected from any one or a combination of at least two of sodium persulfate, potassium persulfate or ammonium persulfate; Preferably, the polymerization of the compound represented by formula I and the zwitterionic monomer is carried out by reverse suspension polymerization; The reverse phase suspension polymerization specifically comprises: preparing a water phase solution with the compound represented by formula I, a zwitterionic monomer and a free radical initiator; adding the water phase solution to the oil phase for reverse phase suspension polymerization to obtain the protein-loaded hydrogel microspheres; Preferably, the oil phase is butyl acetate, petroleum ether, n-hexane, cyclohexane or paraffin oil; Preferably, 3-5% by mass of a surfactant is added to the oil phase; Preferably, the surfactant comprises one or a mixture of two of cellulose acetate butyrate, Tween or Span; Preferably, when the free radical initiator is a persulfate, a catalyst is additionally added to the oil phase; Preferably, the catalyst comprises tetramethylethylenediamine and / or triethylamine; Preferably, the temperature of the inverse suspension polymerization is 50-90°C; Preferably, the time of the reverse suspension polymerization is 2-12 hours.

10. A drug-loaded microsphere, comprising the hydrogel microsphere capable of loading chemical drugs as claimed in claim 5 or 6 or the hydrogel microsphere capable of loading protein as claimed in claim 8 and the drug loaded thereon; Preferably, the drug comprises any one of doxorubicin hydrochloride, irinotecan, sunitinib, oxaliplatin, VC sodium, gemcitabine, 5-fluorouracil, bovine serum albumin, catalase, glucose oxidase, atezolizumab or bevacizumab, or a combination of at least two thereof.

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