Copolymer with tissue adhesion and ROS (reactive oxygen species) response performance, drug-loaded nanoparticles as well as preparation method and application of copolymer and drug-loaded nanoparticles

Abstract

The invention relates to a copolymer with tissue adhesion and ROS (reactive oxygen species) response performance, a drug-loaded nanoparticle as well as a preparation method and application of the copolymer and the drug-loaded nanoparticle. The copolymer is formed by copolymerizing a diacrylate compound and an amino-containing compound, the diacrylate compound is selected from one or more of compounds as shown in a formula (I); the amino-containing compound is selected from one or more of compounds as shown in a formula (II-1) and a formula (II-2) or salts or stereoisomers thereof. The copolymer can deliver a drug for treating gastrointestinal oxidative stress related diseases to a diseased region in a targeted manner, can release a therapeutic drug in a responsive manner at the diseased region, has good gastric mucosa adhesion capability, and can improve local drug concentration, so that the therapeutic targeting and curative effect persistence of the drug are enhanced, and the curative effect of the drug is improved. The treatment effect is enhanced. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, relates to drug carriers, and specifically relates to a copolymer with tissue adhesion and ROS-responsive properties, a drug-loaded nanoparticle, and a preparation method and application thereof. Background Art

[0002] In current nano-delivery systems for treating gastrointestinal oxidative stress-related diseases (such as gastritis, gastric ulcer, and inflammatory bowel disease), existing technologies usually use inert polymers such as PLGA, PEG-PCL, or chitosan as carriers, and achieve the responsive release of drugs in an oxidative environment by introducing ROS-sensitive chemical bonds (such as thioether bonds, selenoether bonds, or phenolic ether structures). To enhance the local retention ability, such carriers are often surface-modified with charged polymers such as chitosan to improve their tissue adhesion. Drug loading is mainly in the form of single drugs, and is often used to deliver anti-inflammatory drugs such as N-acetylcysteine (NAC), ibuprofen, and prednisone, or proton pump inhibitors (such as omeprazole), and it is difficult to achieve multi-drug synergistic delivery. Moreover, there are still certain limitations in the persistence of targeted adhesion and the precision of responsive release of such delivery systems. They are prone to desorption under gastrointestinal peristalsis or mucus shear force, and have insufficient targeting and cannot selectively adhere to the diseased site. This will thus affect the actual therapeutic efficacy of the drug. Summary of the Invention

[0003] Based on this, the purpose of the present invention is to provide a novel carrier that can target and deliver drugs for treating gastrointestinal oxidative stress-related diseases to the diseased site, can responsively release therapeutic drugs at the diseased site, and has good gastric mucosa adhesion ability, can increase the local drug concentration, thereby enhancing the therapeutic targeting and efficacy persistence of the drug and enhancing the therapeutic effect.

[0004] The technical solutions to achieve the above purpose include the following.

[0005] In the first aspect, the present invention provides a copolymer with tissue adhesion and ROS-responsive properties, which is copolymerized from a diacrylate compound and an amino-containing compound; the diacrylate compound is selected from one or more of the compounds shown in formula (I);

[0006] The amino-containing compound is selected from one or more of the compounds shown in formula (II-1) and formula (II-2), or salts or stereoisomers thereof;

[0007]

[0008] Among them, X is selected from: C1-C 16 alkylene, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1-16 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1-16 atoms;

[0009] R1 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0010] R2 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0011] M is selected from: C6-C aryl optionally substituted by R3, 5-10 membered heteroaryl optionally substituted by R3; 10

[0012] R3 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl, halogen, C1-C6 alkoxy.

[0013] Preferably, the diacrylate compound is selected from one or more of the following compounds:

[0014]

[0015] Preferably, the amino-containing compound is selected from one or more of the following compounds:

[0016]

[0017] For example, the copolymer may have a structure represented by the following formula (A), formula (B) or formula (C):

[0018]

[0019] Wherein, n≧1, preferably an integer between 1 and 10000, more preferably an integer between 1 and 10;

[0020] X is selected from: C1-C 16 alkylene, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1-16 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1-16 atoms;

[0021] R1 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0022] R2 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0023] M is selected from: C6-C aryl optionally substituted by R3, 5-10 membered heteroaryl optionally substituted by R3; 10

[0024] R3 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl, halogen, C1-C6 alkoxy.

[0025] In a second aspect, the present invention also provides a method for preparing the copolymer, comprising the following steps:

[0026] In an organic solvent, the copolymer is obtained by reacting the diacrylate compound and the amino-containing compound under heating in the presence of an acid-binding agent.

[0027] In a third aspect, the present invention also provides a drug-loaded nanoparticle, which is prepared by self-assembly of the copolymer, small molecule drug and prodrug of the present invention in an aqueous medium;

[0028] The small molecule drug is a small molecule drug with anti-inflammatory or repair functions;

[0029] The prodrug is selected from the following compounds:

[0030]

[0031] Among them, the small molecule drug is selected from: one or more of N-acetylcysteine, glutathione, D-cysteine, sodium dimercaptopropanesulfonate, metadoxine, perampanel, idebenone, vitamin C, lansoprazole, aluminum hydroxide and bismuth potassium citrate, etc., and preferably N-acetylcysteine.

[0032] In a fourth aspect, the present invention also provides a method for preparing the drug-loaded nanoparticle, which includes the following steps:

[0033] Dissolve the copolymer, small molecule drug and prodrug in an organic solvent as the organic phase; use a buffer solution or pure water as the aqueous phase; drop the organic phase into the aqueous phase to promote the self-assembly of the three components in the aqueous phase to form nanoparticles, thereby obtaining the drug-loaded nanoparticles.

[0034] In a fifth aspect, the present invention provides the application of the copolymer as a carrier in the preparation of drugs for preventing and / or treating gastrointestinal oxidative stress-related diseases.

[0035] In a sixth aspect, the present invention provides the application of the drug-loaded nanoparticle in the preparation of drugs for preventing and / or treating gastrointestinal oxidative stress-related diseases.

[0036] In a seventh aspect, the present invention provides a drug for preventing and / or treating gastrointestinal oxidative stress-related diseases, which is prepared from the drug-loaded nanoparticle of the present invention and other pharmaceutically acceptable excipients.

[0037] Among them, the gastrointestinal oxidative stress-related diseases can be gastritis, gastric ulcer, inflammatory bowel disease, etc.

[0038] The present invention has the following beneficial effects:

[0039] By introducing structural units such as dopamine and / or phenylalanine into the copolymer main chain of the diacrylate compound, the obtained copolymer has material self-adhesion and self-reduction ability, and has good gastric mucosa adhesion and ROS response performance.

[0040] Loading small molecule anti-inflammatory or repair drugs and controllable prodrugs with the copolymer of the present invention can achieve ROS concentration-dependent drug release, responsive release in an inflammatory environment rich in reactive oxygen species (ROS), effectively achieve "lesion-specific" drug delivery, reduce the side effects of drugs in non-target areas, improve drug utilization rate, and are particularly suitable for the treatment of gastric diseases related to oxidative stress. That is, the copolymer of the present invention can target and deliver drugs for treating gastrointestinal oxidative stress-related diseases to the lesion site, and can responsive release therapeutic drugs at the lesion site, which can increase the local drug concentration, thereby enhancing the therapeutic targeting of the drug and enhancing the therapeutic effect.

[0041] The copolymer of the present invention can form a stable bond with the tissue surface through covalent and non-covalent interactions, has good gastric mucosa adhesion ability, can effectively prolong the residence time of nanoparticles at the lesion site, reduce drug loss, increase the local drug concentration, thereby further enhancing the therapeutic targeting and efficacy persistence, and greatly improving the therapeutic effect on gastrointestinal oxidative stress-related diseases such as gastric ulcer.

[0042] Further preferably, the copolymer of the present invention containing both dopamine and phenylalanine or its similar structural units can achieve sequential release of antacid drugs and anti-inflammatory drugs, so that drugs that inhibit gastric acid secretion (such as vonoprazan) are preferentially released, rapidly reducing the gastric acidity, providing a favorable microenvironment for the stable release and exertion of subsequent anti-inflammatory drugs, thereby simulating the strategy of "first inhibiting acid and then repairing" in clinical treatment, and can significantly improve the synergistic therapeutic effect of antacid drugs and anti-inflammatory drugs. Description of the Drawings

[0043] Figure 1 is the particle size of the drug-loaded nanoparticles.

[0044] Figure 2 is the PDI of the drug-loaded nanoparticles.

[0045] Figure 3 is the potential of the drug-loaded nanoparticles.

[0046] Figure 4 is the in vitro release curve of N-acetylcysteine in the drug-loaded nanoparticles.

[0047] Figure 5 is the in vitro release curve of vonoprazan in the drug-loaded nanoparticles.

[0048] Figure 6 is the test result of the retention ability of the nanoparticles on the gastric mucosa surface.

[0049] Figure 7 is the in vivo therapeutic effect of the drug-loaded nanoparticles on rabbits with gastric mucosal ulcer model. Detailed Embodiments

[0050] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0051] For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturers. All common chemical reagents used in the embodiments are commercially available products.

[0052] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0053] In addition, as used in the present invention, the term "or" is the inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0054] To achieve targeted therapy for gastric mucosal oxidative stress injury, the present invention constructs an intelligent responsive nanodrug delivery system with multifunctional characteristics. By introducing substances such as dopamine to increase viscosity and anti-inflammatory activity, and / or introducing amino acids such as phenylalanine to increase structural polarity and reactivity, a dimethacrylate copolymer with a synergistic structure of hydrophobic and hydrophilic chains is designed and synthesized. This copolymer has excellent tissue adhesiveness, antioxidant property, reactive oxygen species (ROS) responsive characteristics, and biocompatibility. Further, borate bond or ester bond is used as a cleavable linker to construct a prodrug structure with ROS responsive characteristics. The anti-inflammatory drug is combined with the carrier through a prodrug strategy, further improving the ROS responsive characteristics and enhancing the selectivity of drug release in the lesion microenvironment. Subsequently, a drug-loaded nanosystem is constructed by self-assembling the functional copolymer with the prodrug and small molecule anti-inflammatory or repair drugs (such as acetylcysteine and vonoprazan) to form nanoparticles. The drug-loaded nanoparticles prepared by the present invention have good gastric mucosal adhesion ability and ROS responsive characteristics, can target and deliver drugs for treating gastrointestinal oxidative stress-related diseases to the lesion site, can release therapeutic drugs responsively at the lesion site, can effectively prolong the residence time of nanoparticles at the lesion site, reduce drug loss, increase local drug concentration, thereby further enhancing the treatment targeting and efficacy persistence, and greatly improving the treatment effect on gastrointestinal oxidative stress-related diseases such as gastric ulcer.

[0055] In some embodiments of the present invention, a copolymer is involved, which is copolymerized from a dimethacrylate compound and an amino-containing compound; the dimethacrylate compound is selected from one or more of the compounds shown in formula (I);

[0056] The amino-containing compound is selected from one or more of the compounds shown in formula (II-1) and formula (II-2), or its salt, or its stereoisomer;

[0057]

[0058] Wherein, X is selected from: C1-C 16 alkylene, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1-16 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1-16 atoms;

[0059] R1 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0060] R2 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl;

[0061] M is selected from: an aryl group of C6-C 10 substituted or unsubstituted by R3, a 5-10 membered heteroaryl group substituted or unsubstituted by R3;

[0062] R3 is selected from: hydrogen, C1-C6 alkyl, hydroxy, carboxy, halogen, C1-C6 alkoxy.

[0063] As used herein, the term "alkyl" means a branched or straight-chain saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight-chain or branched-chain configuration. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

[0064] As used herein, the term "saturated hydrocarbon group containing one or more oxygen atoms" refers to a group in which one or more carbon atoms in an alkyl chain are replaced by one or more oxygen atoms, such as: -CH2-O-CH2-, -CH2CH2-O-CH2CH2-O-CH2CH2-, -CH(CH3)CH2-O-CH2CH(CH3)-, -CH2CH2-O-CH2CH2CH2CH2CH2CH2-O-CH2CH2-, -CH(CH3)CH2-O-CH(CH3)CH2-, -CH(CH3)CH2-O-CH(CH3)CH2-O-CH(CH3)CH2-.

[0065] As used herein, the term "saturated hydrocarbon group containing one or more sulfur atoms" refers to a group in which one or more carbon atoms in an alkyl chain are replaced by one or more sulfur atoms, such as: -CH2-S-CH2-, -CH2CH2-S-CH2CH2-S-CH2CH2-, -CH(CH3)CH2-S-CH2CH(CH3)-, -CH2CH2-S-CH2CH2CH2CH2CH2CH2-S-CH2CH2-, -CH(CH3)CH2-S-CH(CH3)CH2-, -CH(CH3)CH2-S-CH(CH3)CH2-S-CH(CH3)CH2-.

[0066] As used herein, the term "alkoxy" refers to a group of the structure -O-alkyl, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0067] As used herein, the term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S, which can be monocyclic, bicyclic, or polycyclic, and includes, for example, but is not limited to: quinolinyl, pyrazolyl, pyrrolyl, thienyl, furyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, etc.; "heteroaryl" is also understood to include N-oxide derivatives of any heteroaryl containing nitrogen. The heteroaryl can be linked through a carbon atom or through a heteroatom.

[0068] In some preferred embodiments, X is selected from: C1-C 12 alkylene, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1-12 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1-12 atoms.

[0069] In some preferred embodiments, R1 is selected from: hydrogen, C1-C3 alkyl, hydroxyl, carboxyl.

[0070] In some preferred embodiments, R2 is selected from: hydrogen, C1-C3 alkyl, hydroxyl, carboxyl.

[0071] In some preferred embodiments, M is selected from: phenyl, phenyl substituted with hydroxyl, indolyl, imidazolyl.

[0072] In some preferred embodiments, the diacrylate compound is selected from one or more of the following compounds:

[0073]

[0074] In some preferred embodiments, the amino-containing compound is selected from one or more of L-dopamine, dopamine, norepinephrine, phenylalanine, tyrosine, tryptophan, histidine (i.e., the following compounds):

[0075]

[0076] In some preferred embodiments, the molar ratio of the diacrylate compound to the amino-containing compound is 1:0.5-2, more preferably 1:0.9-1.2.

[0077] In some preferred embodiments, the diacrylate compound is a combination of 1,6-hexanediol diacrylate and triethylene glycol diacrylate, and the molar ratio is preferably 1:0.8-1.2.

[0078] In some preferred embodiments, the amino-containing compound is selected from one or more of dopamine, dopamine hydrochloride, and D-phenylalanine, preferably a combination of dopamine hydrochloride and D-phenylalanine, and the molar ratio is preferably 1-1.5:1.

[0079] In some preferred embodiments, the copolymer is copolymerized from 1,6 - hexanediol diacrylate, triethylene glycol diacrylate, dopamine hydrochloride, and D - phenylalanine, and the molar ratio is preferably 1:0.8 - 1.2:1 - 1.5:0.8 - 1.2.

[0080] In some preferred embodiments, the copolymer is copolymerized from 1,6 - hexanediol diacrylate, triethylene glycol diacrylate, and dopamine hydrochloride, and the molar ratio is preferably 1:0.8 - 1.2:1.8 - 2.2.

[0081] In some preferred embodiments, the copolymer is copolymerized from 1,6 - hexanediol diacrylate, triethylene glycol diacrylate, and D - phenylalanine, and the molar ratio is preferably 1:0.8 - 1.2:1.8 - 2.2.

[0082] The copolymer of the present invention may have the structure shown in the following formula (A), formula (B), or formula (C):

[0083]

[0084] Wherein, X, R1, R2, and M are as described above, n ≧ 1, preferably an integer between 1 and 10000, more preferably an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0085] For example, the copolymer may have the following structure:

[0086]

[0087]

[0088] Wherein, n ≧ 1, preferably an integer between 1 and 10000, more preferably an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0089] The end of the copolymer of the present invention can be vinyl, or can be capped with the amino - containing compound described in the present invention. Only the repeating structural unit is drawn in the structural formula.

[0090] The number - average molecular weight of the copolymer of the present invention is 2000 Da - 5000 Da, preferably 2600 Da - 4200 Da, more preferably 3900 Da - 4100 Da or 2600 Da - 3000 Da.

[0091] Some embodiments of the present invention also relate to a method for preparing the copolymer, comprising the following steps: in an organic solvent, the diacrylate compound and the amino-containing compound are reacted under heating and in the presence of an acid-binding agent to obtain the copolymer.

[0092] In some preferred embodiments, the organic solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and 1,4-dioxane.

[0093] In some preferred embodiments, the acid-binding agent is selected from at least one of triethylamine, diisopropylethylamine, pyridine, tetramethylethylenediamine, and piperidine.

[0094] In some preferred embodiments, the molar ratio of the acid-binding agent to the diacrylate compound is 0.4 - 0.8:1, preferably 0.5 - 0.7:1.

[0095] In some preferred embodiments, the reaction temperature is 65°C - 100°C, more preferably 85°C - 98°C.

[0096] And / or, the reaction time is 12h - 36h, more preferably 20h - 28h.

[0097] The present invention also provides a drug-loaded nanoparticle, which is prepared by self-assembly of the copolymer, small molecule drug, and prodrug of the present invention in an aqueous medium;

[0098] The small molecule drug is a small molecule drug with anti-inflammatory or repair function;

[0099] The prodrug is selected from the following compounds:

[0100]

[0101] Among them, the prodrug is obtained by reacting vonoprazan or N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)-pyrimidin-2-amine drug with (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid (the molar ratio is preferably 1:0.9 - 1.2) under the action of an activator and a base; the activator can be N-hydroxysuccinimide, etc., and the base is selected from triethylamine, diisopropylethylamine, pyridine, etc.; the solvent for this reaction is a mixed solvent of an organic solvent and water (the volume ratio is preferably 1:0.5 - 1.5), and the organic solvent can be tetrahydrofuran, 1,4-dioxane, etc.; the reaction temperature is 15°C - 37°C; the reaction time is 8h - 24h.

[0102] Among them, (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid can be obtained by reacting 4-(hydroxymethyl)phenylboronic acid pinacol ester and p-nitrophenyl chloroformate (the molar ratio is preferably 1:1 - 1.5) under the action of a base; the base in this reaction can be triethylamine, diisopropylethylamine, pyridine, etc.; the reaction solvent can be tetrahydrofuran, 1,4-dioxane, acetonitrile, etc.; the reaction temperature is 15°C - 37°C; the reaction time is 12h - 30h.

[0103] In some preferred embodiments, the small molecule drug is selected from one or more of: N-acetylcysteine, glutathione, D-cysteine, sodium dimercaptopropanesulfonate, metadoxine, perampanel, idebenone, vitamin C, lansoprazole, aluminum hydroxide, and bismuth potassium citrate, preferably N-acetylcysteine.

[0104] In some preferred embodiments, the molar ratio of the copolymer, small molecule drug, and prodrug is 1:(1.4 - 13.3):(1.0 - 13.3), preferably 1:(2 - 10):(2 - 10), more preferably 1:(8 - 10):(2 - 4).

[0105] In some preferred embodiments, the particle size of the drug-loaded nanoparticles is 70nm - 300nm, preferably 100nm - 200nm.

[0106] Some embodiments of the present invention also relate to a method for preparing the drug-loaded nanoparticles, comprising the following steps:

[0107] Dissolve the copolymer, small molecule drug, and prodrug in an organic solvent as the organic phase; use a buffer solution or pure water as the aqueous phase; drop the organic phase into the aqueous phase to promote the self-assembly of the three components in the aqueous phase to form nanoparticles, thus obtaining the drug-loaded nanoparticles.

[0108] In some preferred embodiments, the organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

[0109] In some preferred embodiments, the buffer solution is a phosphate buffer solution with a pH of 7 - 8 or a sodium acetate buffer solution with a pH of 4.5 - 5.5.

[0110] In some preferred embodiments, the total concentration of the copolymer, small molecule drug, and prodrug in the organic phase is 8mg / mL - 12mg / mL.

[0111] In some preferred embodiments, the volume ratio of the organic phase to the aqueous phase is 1:8 - 12.

[0112] In some preferred embodiments, the dropping temperature is 15°C - 37°C.

[0113] In some of the preferred embodiments, the dropping rate is 0.1 mL / min - 1 mL / min, preferably 0.1 mL / min - 0.2 mL / min.

[0114] In some of the preferred embodiments, during the dropping process, a mild stirring at a rotation speed of 300 rpm - 800 rpm or an ultrasonic assistance with a power of 40 kHz - 60 kHz is maintained to promote the self-assembly of the three components in the aqueous phase to form nanoparticles.

[0115] In some of the preferred embodiments, during the dropping process, a mild stirring at a rotation speed of 350 rpm - 450 rpm is maintained to promote the self-assembly of the three components in the aqueous phase to form nanoparticles.

[0116] In some of the embodiments, the preparation method of the prodrug comprises the following steps:

[0117] (1) 4-(Hydroxymethyl)phenylboronic acid pinacol ester and 4-nitrophenyl chloroformate react under the action of a base to obtain (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid;

[0118] (2) The voriconazole or N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)pyrimidin-2-amine is reacted with (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid under the action of an activator and a base to obtain the prodrug;

[0119] The reaction formula is as follows

[0120]

[0121]

[0122] In some of the preferred embodiments, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to 4-nitrophenyl chloroformate in step (1) is 1:1 - 1.5.

[0123] In some of the preferred embodiments, the base in step (1) is selected from at least one of triethylamine, diisopropylethylamine, and pyridine.

[0124] In some of the preferred embodiments, the molar ratio of the base to 4-(hydroxymethyl)phenylboronic acid pinacol ester in step (1) is 1 - 1.5:1.

[0125] In some of the preferred embodiments, the reaction solvent in step (1) is selected from at least one of tetrahydrofuran, 1,4-dioxane, and acetonitrile.

[0126] In some of the preferred embodiments, the reaction temperature in step (1) is 15°C - 37°C.

[0127] In some of the preferred embodiments, the reaction time in step (1) is 12 h - 30 h, preferably 20 h - 28 h.

[0128] In some of the preferred embodiments, the molar ratio of vonoprazan or N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)-pyrimidine-2-amine to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 1:0.9 - 1.2.

[0129] In some of the preferred embodiments, the activator in step (2) is N-hydroxysuccinimide.

[0130] In some of the preferred embodiments, the molar ratio of the activator to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 0.3 - 0.7:1.

[0131] In some of the preferred embodiments, the base in step (2) is selected from at least one of triethylamine, diisopropylethylamine, and pyridine.

[0132] In some of the preferred embodiments, the molar ratio of the base to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 1 - 1.5:1.

[0133] In some of the preferred embodiments, the reaction solvent in step (2) is a mixed solvent of an organic solvent and water, and the organic solvent is tetrahydrofuran and / or 1,4-dioxane.

[0134] In some of the preferred embodiments, the reaction temperature in step (2) is 15°C - 37°C.

[0135] In some of the preferred embodiments, the reaction time in step (2) is 8 h - 24 h, preferably 10 h - 14 h.

[0136] Some embodiments of the present invention also relate to the application of the drug-loaded nanoparticles of the present invention in the treatment of gastrointestinal oxidative stress-related diseases (such as gastric ulcer).

[0137] The following further describes the present invention in detail with specific embodiments.

[0138] Example 1 Synthesis of Copolymer 1

[0139]

[0140] First, 2 g of 1,6 - hexanediol diacrylate (8.84 mmol), 2.28 g of triethylene glycol diacrylate (8.84 mmol), 2.01 g of dopamine hydrochloride (10.61 mmol), 1.46 g of D - phenylalanine (8.84 mmol) and 1.07 g of triethylamine (10.61 mmol) were dissolved in 5 mL of DMSO. Then, the mixture was stirred at 95 °C for 24 h. Subsequently, the resulting reaction mixture was dialyzed in pure water using a 2000 Da dialysis bag for 3 days, with water being changed every 6 h. After dialysis, the product was freeze - dried to obtain copolymer 1, with a yield of approximately 90.7%. The number - average molecular weight was measured to be around 4000 Da, and the number of repeating units (i.e., n in the structural formula) was approximately 2.

[0141] The method for measuring the molecular weight is as follows: The copolymer sample was prepared into a solution with a concentration of approximately 5 mg / mL using deuterated chloroform as the solvent. A 400 MHz nuclear magnetic resonance spectrometer was used for 1 1H NMR measurement. In the obtained 1 1H NMR spectrum, the characteristic peaks of the polymer end groups and the main chain were selected for integration. According to the number of hydrogen atoms in the end groups and the number of hydrogen atoms in the main - chain repeating units, the average number of repeating units (DPn) of the copolymer was calculated. Finally, the number - average molecular weight (Mn) was calculated according to the following formula:

[0142] Mn = DPn×Mrepeat + Mend groups. Where Mrepeat is the molar mass of the repeating unit and Mend groups is the total molar mass of the two end groups.

[0143] DPn=(Imain / Hrepeat) / (Iend / Hend), where Imain, Hrepeat, Iend, and Hend represent the main - chain peak area, the number of main - chain repeating units, the end - group peak area, and the number of hydrogen atoms in the end groups, respectively.

[0144] 1H NMR data of copolymer 1:

[0145] 11H NMR (400 MHz, CDCl3) δ 7.29 - 7.19 (m, 5H), 6.72 - 6.53 (m, 6H), 6.15 - 6.06 (m, 4H), 6.00 (ddd, J = 12.6, 9.1, 5.0 Hz, 2H), 4.61 - 3.64 (m, 18H), 3.64 - 3.45 (m, 20H), 3.10 (s, 3H), 2.97 (dt, J = 12.6, 6.9 Hz, 2H), 2.88 (dt, J = 12.6, 7.0 Hz, 2H), 2.83 (t, J = 6.7 Hz, 8H), 2.77 (s, 6H), 2.71 - 2.61 (m, 2H), 2.53 (dt, J = 19.6, 6.9 Hz, 14H), 2.39 - 2.31 (m, 8H), 1.97 - 1.79 (m, 2H), 1.70 - 1.58 (m, 12H), 1.32 - 1.23 (m, 8H).

[0146] Synthesis of Copolymer 2 in Example 2

[0147]

[0148] First, dissolve 2 g of 1,6 - hexanediol diacrylate (8.84 mmol), 2.28 g of triethylene glycol diacrylate (8.84 mmol), 3.35 g of dopamine hydrochloride (17.68 mmol), and 1.07 g of triethylamine (10.61 mmol) in 5 mL of DMSO. Then stir the mixture at 95 °C for 24 h. After that, dialyze the resulting reaction mixture against pure water using a 2000 Da dialysis bag for 3 days, changing the water every 6 h. After dialysis, lyophilize the product to obtain Copolymer 2 with a yield of 92.6%. The number - average molecular weight was measured to be about 3000 Da, and the number of repeating units (i.e., n in the structural formula) was about 2. The testing method was the same as in Example 1.

[0149] 1H NMR data of Copolymer 2:

[0150] 1 1H NMR (400 MHz, CDCl3) δ 6.72 - 6.53 (m, 6H), 6.49 - 6.18 (m, 4H), 5.93 (d, J = 10.3 Hz, 1H), 5.76 (s, 1H), 4.32 - 3.87 (m, 14H), 3.67 - 3.50 (m, 10H), 3.25 -

[0151] 2.94 (m, 8H), 2.94 - 2.78 (m, 8H), 2.75 (s, 8H), 2.55 (d, J = 1.8 Hz, 8H), 2.55 - 2.37 (m, 4H), 1.58 (d, J = 15.2 Hz, 8H), 1.47 - 1.15 (m, 12H).

[0152] Synthesis of Copolymer 3 in Example 3

[0153]

[0154] First, 2 g of 1,6 - hexanediol diacrylate (8.84 mmol), 2.28 g of triethylene glycol diacrylate (8.84 mmol), 2.92 g of D - phenylalanine (17.68 mmol), and 1.07 g of triethylamine (10.61 mmol) were dissolved in 5 mL of DMSO. Then, the mixture was stirred at 95 °C for 24 h. After that, the resulting reaction mixture was dialyzed against pure water using a 2000 Da dialysis bag for 3 days, with water changed every 6 h. After dialysis, the product was freeze - dried to obtain copolymer 3 with a yield of 89.6%. The number - average molecular weight was measured to be about 2800 Da, and the number of repeating units (i.e., n in the structural formula) was about 2. The testing method was the same as in Example 1.

[0155] 1H NMR data of copolymer 3:

[0156] 1 H NMR (400 MHz, CDCl3) δ 7.29 - 7.22 (m, 5H), 6.16 - 5.95 (m, 6H), 4.30 - 4.21 (m, 8H), 4.04 (dt, J = 19.4, 6.2 Hz, 4H), 3.73 - 3.60 (m, 18H), 3.03 - 2.94 (m, 3H), 2.94 - 2.84 (m, 3H), 2.55 (t, J = 6.9 Hz, 4H), 2.39 - 2.32 (m, 4H), 1.71 - 1.58 (m, 8H), 1.31 - 1.24 (m, 4H).

[0157] Synthesis of Prodrug 1 (Vorolanib Prodrug) in Example 4

[0158]

[0159] First, 5 g of 4 - (hydroxymethyl)phenylboronic acid pinacol ester (21.36 mmol), 5.17 g of p - nitrophenyl chloroformate (25.63 mmol), and 1.85 g of triethylamine (25.63 mmol) were dissolved in 7 mL of tetrahydrofuran. Then, the reaction was carried out at room temperature for 24 h. After that, the resulting reaction mixture was extracted with saturated sodium bicarbonate solution and washed with 1 M hydrochloric acid solution until no bubbles were produced. After drying, it was rotary - evaporated to obtain the intermediate product (4 - [(4 - nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid with a yield of 87%.

[0160]

[0161] 1 g of voruprazan (2.90 mmol) and 917.97 mg of (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid (2.90 mmol), 166.66 mg of N-hydroxysuccinimide (1.45 mmol), and 351.58 mg of triethylamine (3.47 mmol) were dissolved in 1 mL of a mixed solvent of tetrahydrofuran and water with a volume ratio of 1:1. After stirring at room temperature for 12 h, the resulting reaction mixture was washed with water three times and then purified by silica gel column using dichloromethane and methanol (50:1) as the eluent to obtain prodrug 1. The yield was 76.9%.

[0162] 1H NMR data of prodrug 1:

[0163] 1 H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 1.9, 1.0 Hz, 1H), 8.69 (ddd, J = 4.0, 1.8, 0.9 Hz, 1H), 8.13 (dt, J = 7.5, 1.9 Hz, 1H), 7.75 (ddd, J = 8.1, 4.9, 1.1 Hz, 1H), 7.72 - 7.67 (m, 2H), 7.50 - 7.40 (m, 2H), 7.33 - 7.22 (m, 6H), 6.55 (d, J = 1.6 Hz, 1H), 6.15 (t, J = 1.9 Hz, 1H), 5.15 (t, J = 1.0 Hz, 2H), 4.45 (s, 2H), 2.97 (s, 3H).

[0164] Synthesis of prodrug 2 in Example 5

[0165]

[0166] 1 g of N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)-pyrimidin-2-amine (2.76 mmol), 1.05 g of (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid (3.31 mmol), 158.76 mg of N-hydroxysuccinimide (1.38 mmol), and 335.03 mg of triethylamine (3.31 mmol) were dissolved in 1 mL of a mixed solvent of tetrahydrofuran and water with a volume ratio of 1:1. After stirring at room temperature for 12 h, the resulting reaction mixture was washed with water three times and then purified by silica gel column using dichloromethane and methanol (volume ratio 50:1) as the eluent to obtain prodrug 2. The yield was 74.5%.

[0167] 1H NMR data of prodrug 2:

[0168] 11H NMR (400 MHz, CDCl3) δ 7.73 - 7.67 (m, 2H), 7.64 - 7.57 (m, 2H), 7.28 - 7.24 (m, 3H), 7.24 - 7.19 (m, 1H), 7.19 - 7.12 (m, 3H), 7.12 - 7.04 (m, 2H), 5.24 (q, J = 1.1 Hz, 2H), 4.84 - 4.77 (m, 1H), 4.12 (ddd, J = 12.5, 6.7, 5.1 Hz, 1H), 3.94 -

[0169] 3.86 (m, 1H), 3.02 (dddd, J = 6.4, 5.1, 2.4, 1.1 Hz, 2H), 2.44 (s, 3H), 1.60 (d, J = 7.0 Hz, 3H).

[0170] Example 6 Preparation of Drug - loaded Nanoparticles

[0171] Organic phase: The copolymer, the small - molecule drug with anti - inflammatory and repair functions, and the prodrug were respectively prepared into the copolymer stock solution, small - molecule drug stock solution, and prodrug stock solution with a concentration of 10 mg / ml using DMF. Then, the copolymer stock solution, small - molecule drug stock solution, and prodrug stock solution were mixed together according to the molar ratio of copolymer, small - molecule drug, and prodrug of 1:9:3 as the organic phase.

[0172] Aqueous phase: Sodium acetate buffer (pH = 8.5).

[0173] The organic phase was slowly added dropwise (dropwise addition rate of 0.12 mL / min) at room temperature (15 °C - 37 °C) to 10 times the volume of the aqueous phase. During the dropwise addition process, the system was maintained under gentle stirring (400 rpm) to promote the self - assembly of the three components in the aqueous phase to form drug - loaded nanoparticles. Then, the nanoparticle solution was dialyzed against 1 L of pure water for 2 hours using a dialysis bag with a cut - off molecular weight of 2000 Da for later use.

[0174] The following drug - loaded nanoparticles were prepared according to the above steps: P1 - N - 1, P2 - N - 1, P3 - N - 1, P1 - N - 2, P2 - N - 2, P3 - N - 2, P1 - GSH - 1, P2 - GSH - 1, P3 - GSH - 1, P1 - GSH - 2, P2 - GSH - 2, P3 - GSH - 2; where P1 represents copolymer 1, P2 represents copolymer 2, P3 represents copolymer 3; N represents the small - molecule drug N - acetylcysteine; GSH represents the small - molecule drug glutathione; 1 and 2 respectively represent prodrug 1 and prodrug 2 in sequence; for example, P1 - N - 1 represents the drug - loaded nanoparticles self - assembled from copolymer 1, N - acetylcysteine, and prodrug 1.

[0175] In addition, PLGA 1500 -PEG 1000 -PLGA1500 - is an ABA - type triblock copolymer composed of polyethylene glycol (PEG 1000 ) and poly (lactic - co - glycolic acid) (PLGA 1000 ). PLGA is a copolymer of poly (lactic acid) (PLA) and poly (glycolic acid) (PGA), and the molar ratio of lactic acid (LA): glycolic acid (GA) = 75:25. This copolymer has been used as a standard material in many studies to construct drug delivery systems. In this invention, drug - loaded nanoparticles PLGA - PEG - PLGA - N - 1 were prepared as a control material (abbreviated as PEG - PLGA - N - 1) using the same method as in this example with this copolymer as the carrier.

[0176] Measure the particle size and zeta potential of the drug - loaded nanoparticles prepared in this example.

[0177] The particle size and surface potential of the drug - loaded nanoparticles were measured by dynamic light scattering (DLS). Before measurement, the sample was diluted with deionized water to 0.1 mg / mL. Gently shake and mix well to avoid generating bubbles. The particle size measurement was carried out at 25 °C, the detection angle was set to 173° backscattering, and the Z - average particle size and polydispersity index (PDI) were recorded. The surface potential measurement was to measure the electrophoretic mobility at 25 °C and convert it to zeta potential, with the unit of millivolt (mV). Each sample was independently measured three times, and the average value was taken and the standard deviation was calculated.

[0178] The results are as Figures 1 - 3 shown: Most of the drug - loaded nanoparticles of this invention are superior to the control material PEG - PLGA - N - 1 in physicochemical properties, showing smaller particle sizes and more favorable surface charge characteristics. PEG - PLGA - N - 1 is negatively charged, while the drug - loaded nanoparticles of this invention are close to neutral or positively charged. Cell membranes, mucus, and tissue surfaces are generally negatively charged, and positively charged nanoparticles can adhere more strongly to these surfaces through electrostatic attraction. Among them, P1 - N - 1 and P1 - N - 2 show smaller particle sizes and lower polydispersity indices, indicating that their nanoparticle structures are stable and uniform. The excellent particle size and distribution characteristics are expected to significantly improve tissue penetration and drug loading efficiency in vivo. In contrast, PEG - PLGA - N - 1 has a larger particle size and a negative surface potential, which is not conducive to its effective interaction with the negatively charged biofilm surface, limiting its application potential in local retention, tissue adhesion, and targeted release. The drug - loaded nanoparticles containing glutathione have strong positive charges, which are beneficial for cell membrane adsorption, but their potential is too high, which may cause cytotoxicity and biocompatibility problems. Therefore, considering all indicators, the physicochemical properties of P1 - N - 1 are the best.

[0179] Example 7 In vitro release experiment

[0180] The drug-loaded nanoparticles were loaded into a dialysis bag with a cut-off molecular weight of 2000 Da and placed in different drug release media for in vitro release experiments. Three groups were set up in the experiment: the control group used phosphate buffer solution (PBS) with pH 7.4 as the drug release medium; the acidic group used PBS with pH 1.2 as the drug release medium; the oxidative stress group (pH 1.2 + H2O2) used PBS with pH 1.2 added with 100 mM hydrogen peroxide (H2O2) as the drug release medium to simulate the inflammatory environment. The samples of each group were incubated in a constant temperature shaker at 37 °C, and the drug release liquid samples were taken at time points such as 0 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, and 48 h. The drug concentration was measured by high performance liquid chromatography (HPLC) to evaluate the drug release behavior under different conditions.

[0181] The results are as Figure 4 and Figure 5 shown: For the two drug-loaded nanoparticles P1-N-1 and P2-N-1, in the simulated gastric acid environment (pH 1.2) and the oxidative stress environment (pH 1.2 + H2O2), the release rates and total release amounts of the two drugs (N-acetylcysteine and vorapaxar) were significantly higher than those under neutral conditions (control, pH 7.4). Among them, the pH 1.2 + H2O2 group showed the fastest release rate and the highest cumulative release percentage for both drugs, indicating that the drug-loaded nanosystem of the present invention has good responsiveness to acidic and reactive oxygen species (ROS) environments, indicating its targeted response release ability to the co-existing environment of gastric acid and inflammation, which is beneficial to the precise treatment of inflammation-related gastric diseases.

[0182] Among them, in the P1-N-1 drug-loaded nanoparticles, vorapaxar was rapidly released and tended to reach a plateau in the first 6 hours, while the release of N-acetylcysteine was relatively slow but continuous, showing a certain delivery characteristic of first inhibiting acid and then anti-inflammatory. The drug that inhibits gastric acid secretion was preferentially released, which can rapidly reduce the gastric acidity, provide a favorable microenvironment for the stable release and exertion of subsequent anti-inflammatory drugs, and can improve the synergistic therapeutic effect of the drugs; and the release rate and total release amount of vorapaxar of P1-N-1 were significantly higher than those of P2-N-1. This shows that the P1-N-1 drug-loaded nanoparticles have more advantages than P2-N-1 in terms of drug release control ability.

[0183] Moreover, the drug release performances of the two nanocarriers P1-N-1 and P2-N-1 under acidic conditions (pH 1.2) were significantly better than those of the PEG-PLGA-N-1 group. Especially under oxidative conditions (pH 1.2 + H2O2), the release curves of the two further increased, indicating that the drug-loaded nanoparticles constructed in the present invention have a significant release enhancement effect when dealing with gastric acid and oxidative environments, and their release effect is significantly better than that of the commonly used PLGA-PEG-PLGA standard material.

[0184] Example 8 Adhesion Experiment

[0185] In this example, nanoparticles encapsulated with fluorescent dyes were constructed and an in vitro adhesion experiment was carried out to evaluate the retention ability of the nanoparticles on the gastric mucosa surface.

[0186] First, the target copolymer was dissolved in N,N-dimethylformamide (DMF) to prepare an organic phase with a concentration of 10 mg / mL. Using 25 mM aqueous sodium acetate solution as the aqueous phase, 50 μL of the organic phase was slowly added dropwise (the dropping rate was 0.12 mL / min) to 500 μL of the aqueous phase at room temperature, and nanoparticle self-assembly was carried out under mild magnetic stirring (400 rpm). Subsequently, 20 μL of 1 mg / mL lipophilic fluorescent probe DIR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) was added to the obtained nanoparticle dispersion, and it was thoroughly mixed to obtain a nanoparticle solution with a fluorescent signal.

[0187] The experiment used freshly dissected gastric tissues of Babl / c mice. After thoroughly washing the surface residues with phosphate buffer solution (PBS) at pH 7.4, they were co-incubated with the above-mentioned nanoparticle solution at 37 °C for 10 minutes. After the incubation, it was thoroughly rinsed with artificial gastric juice (pH 1.2) to remove non-specifically adsorbed nanoparticles, and it was left to stand in a dark environment with 20 μL of artificial gastric juice placed at the bottom to keep it moist, and it was rinsed 5 times with artificial gastric juice before each imaging. Imaging was carried out on a small animal in vivo imaging system at 30 minutes, 6 hours, 18 hours, and 24 hours respectively, and the changes in fluorescent signals were recorded to evaluate the retention degree of the nanoparticles on the gastric tissue surface. The experiment was divided into four groups: the PEG-PLGA-DIR control group and the P1-N-1-DIR experimental group, and the PEG-PLGA-DIR control group and the P2-N-1-DIR experimental group.

[0188] The results are as Figure 6 shown: The fluorescence intensities of P1-N-1-DIR and P2-N-1-DIR were always significantly higher than those of PEG-PLGA-DIR at each time point, and remained at a relatively high level within 6 to 24 hours, showing good tissue enrichment and continuous retention ability. In contrast, the retention performance of P1-N-1-DIR in vivo was more prominent, indicating that it has stronger in vivo stability and therapeutic potential in drug delivery.

[0189] Example 9 In Vivo Therapeutic Effect

[0190] In this example, rabbits were used as experimental animals to establish a visual gastric mucosal ulcer model to test the healing-promoting effect of the drug-loaded nanoparticles of the present invention. Before the experiment, the animals were fasted but allowed to drink water for 24 hours and then anesthetized (intramuscular injection of ketamine-xylazine anesthesia, ketamine concentration 40 mg / kg, xylazine concentration 5 mg / kg) to ensure no pain response during the experiment.

[0191] Under sterile conditions, a wound was created by mechanical traction on the anterior wall of the gastric body using a living forceps through endoscopic operation, resulting in a focal circular ulcer injury with a diameter of about 2-5 mm. Only one ulcer was modeled in each animal. After successful modeling, the animals were randomly divided into:

[0192] Control group: Equal volume of normal saline was administered by gavage daily.

[0193] Drug administration group (P1-N-1 group): The drug-loaded nanoparticles P1-N-1 prepared by the present invention were administered by gavage daily (2 mL, administered through a spraying tube and endoscope).

[0194] Drug administration started from Day 0 (the day when modeling was completed) and was administered on days 1, 3, and 7 respectively. The animals were given liquid food daily and raised in a constant temperature environment. Body temperature, feeding behavior, and general status were closely monitored to ensure a stable anesthesia and recovery process.

[0195] On Day 1, Day 3, and Day 7, after anesthetizing the animals, an animal endoscope was used to observe the ulcer site and calculate its area. The ulcer area at each time point was based on the area on the day of modeling on Day 0 (100%), and the percentage of the remaining area was calculated to reflect the degree of ulcer healing. Finally, a trend graph of the residual rate of ulcer area over time was plotted to evaluate the differences in ulcer repair effects among groups.

[0196] The results are as Figure 7 shown. Since Day 0 after modeling, the ulcer area in the control group remained at a high level, and no obvious remission was observed until Day 3. The ulcer area was observed to decrease until Day 7. In contrast, in the drug administration group, the ulcer area showed a trend of decreasing day by day starting from Day 1, with a significant difference starting from Day 3, and the remaining ulcer area decreased to nearly 5% at Day 7. The results indicate that the drug-loaded nanoparticles prepared by the present invention can significantly accelerate the healing process of gastric ulcers and exhibit a good effect of promoting mucosal repair.

[0197] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A copolymer, characterized in that, It is copolymerized from a diacrylate compound and an amino-containing compound; the diacrylate compound is selected from one or more of the compounds shown in formula (I); The amino-containing compound is selected from one or more of the compounds shown in formula (II-1) and formula (II-2), or its salt or its stereoisomer; Among them, X is selected from: C1-C 16 an alkylene group, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1 to 16 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1 to 16 atoms; R1 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl; R2 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl; M is selected from: C6-C aryl which is substituted or unsubstituted with R3, 5-10 membered heteroaryl which is substituted or unsubstituted with R3; 10 aryl, 5-10 membered heteroaryl which is substituted or unsubstituted with R3; R3 is selected from: hydrogen, C1-C6 alkyl, hydroxyl, carboxyl, halogen, C1-C6 alkoxy.

2. The copolymer according to claim 1, characterized in that, X is selected from: C1-C 12 an alkylene group, a saturated hydrocarbon group containing one or more oxygen atoms with a chain length of 1 to 12 atoms, a saturated hydrocarbon group containing one or more sulfur atoms with a chain length of 1 to 12 atoms; And / or, R1 is selected from: hydrogen, C1-C3 alkyl, hydroxyl, carboxyl; And / or, R2 is selected from: hydrogen, C1-C3 alkyl, hydroxyl, carboxyl; And / or, M is selected from: phenyl, hydroxyl-substituted phenyl, indolyl, imidazolyl.

3. The copolymer according to claim 1, characterized in that, The diacrylate compound is selected from one or more of the following compounds: And / or, the amino-containing compound is selected from one or more of the following compounds:

4. The copolymer according to claim 1, wherein The molar ratio of the diacrylate compound to the amino-containing compound is 1:0.5-2, preferably 1:0.9-1.

2.

5. The copolymer according to claim 1, characterized in that, The diacrylate compound is a combination of 1,6-hexanediol diacrylate and triethylene glycol diacrylate, and its molar ratio is preferably 1:0.8-1.2; And / or, the amino-containing compound is selected from one or more of dopamine, dopamine hydrochloride and D-phenylalanine, preferably a combination of dopamine hydrochloride and D-phenylalanine, and its molar ratio is preferably 1-1.5:

1.

6. The copolymer according to claim 1, wherein, It is copolymerized from 1,6-hexanediol diacrylate, triethylene glycol diacrylate, dopamine hydrochloride and D-phenylalanine, and its molar ratio is preferably 1:0.8-1.2:1-1.5:0.8-1.2; Or, it is copolymerized from 1,6-hexanediol diacrylate, triethylene glycol diacrylate and dopamine hydrochloride, and its molar ratio is preferably 1:0.8-1.2:1.8-2.2; Or, it is copolymerized from 1,6-hexanediol diacrylate, triethylene glycol diacrylate and D-phenylalanine, and its molar ratio is preferably 1:0.8-1.2:1.8-2.

2.

7. The copolymer according to claim 1, wherein The copolymer has a structure shown in the following formula (A), formula (B) or formula (C): Wherein, X, R1, R2 and M are as described in any one of claims 1-3, n≥1, preferably an integer between 1 and 10000, more preferably an integer between 1 and 10.

8. The copolymer according to claim 1, characterized in that, The copolymer has the following structure: Wherein, n≥1, preferably an integer between 1 and 10000, more preferably an integer between 1 and 10.

9. The copolymer according to any one of claims 1-8, characterized in that, Its number average molecular weight is 2000Da-5000Da, preferably 2600Da-4200Da, more preferably 3900Da-4100Da or 2600Da-3000Da.

10. A method for preparing the copolymer according to any one of claims 1-9, characterized in that, It includes the following steps: In an organic solvent, the diacrylate compound and the amino-containing compound react under the conditions of heating and the presence of an acid-binding agent to obtain the copolymer.

11. The preparation method according to claim 10, characterized in that, The organic solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran and 1,4-dioxane; And / or, the acid-binding agent is selected from at least one of triethylamine, diisopropylethylamine, pyridine, tetramethylethylenediamine, and piperidine; And / or, the molar ratio of the acid-binding agent to the diacrylate compound is 0.4 - 0.8:1, preferably 0.5 - 0.7:1; And / or, the temperature of the reaction is 65°C - 100°C, preferably 85°C - 98°C; And / or, the reaction time is 12h - 36h, preferably 20h - 28h.

12. A drug-loaded nanoparticle, characterized in that, It is prepared by self-assembly of the copolymer, small molecule drug, and prodrug according to any one of claims 1 - 9 in an aqueous medium; The small molecule drug is a small molecule drug with anti-inflammatory or repair functions; The prodrug is selected from the following compounds:

13. The drug-loaded nanoparticles according to claim 12, characterized in that, The small molecule drug is selected from one or more of N-acetylcysteine, glutathione, D-cysteine, sodium dimercaptopropanesulfonate, metadoxine, perampanel, idebenone, vitamin C, lansoprazole, aluminum hydroxide, and bismuth potassium citrate, preferably N-acetylcysteine; And / or, the molar ratio of the copolymer, small molecule drug, and prodrug is 1:(1.4 - 13.3):(1.0 - 13.3), preferably 1:(2 - 10):(2 - 10), more preferably 1:(8 - 10):(2 - 4).

14. The drug-loaded nanoparticles according to claim 12 or 13, characterized in that, Its particle size is 70nm - 300nm, preferably 100nm - 200nm.

15. A method for preparing the drug-loaded nanoparticles according to any one of claims 12-14, characterized in that, It includes the following steps: Dissolve the copolymer, small molecule drug, and prodrug in an organic solvent as the organic phase; use a buffer solution or pure water as the aqueous phase; drop the organic phase into the aqueous phase to promote the self-assembly of the three components in the aqueous phase to form nanoparticles, thus obtaining the drug-loaded nanoparticles.

16. The preparation method according to claim 15, characterized in that, The buffer solution is a phosphate buffer solution with a pH of 7 - 8 or a sodium acetate buffer solution with a pH of 6.5 - 8.5; And / or, the organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide; And / or, the total concentration of the copolymer, small molecule drug, and prodrug in the organic phase is 8mg / mL - 12mg / mL; And / or, the volume ratio of the organic phase to the aqueous phase is 1:8 - 12; And / or, the dropping temperature is 15 - 37°C; And / or, the dropping rate is 0.1mL / min - 1mL / min, preferably 0.1mL / min - 0.2mL / min; And / or, during the dropping process, maintain a gentle stirring at a rotation speed of 300rpm - 800rpm or an ultrasonic assistance with a power of 40kHz - 60kHz to promote the self-assembly of the three components in the aqueous phase to form nanoparticles.

17. The preparation method according to claim 15 or 16, characterized in that, The preparation method of the prodrug includes the following steps: (1) 4-(Hydroxymethyl)phenylboronic acid pinacol ester reacts with p-nitrophenyl chloroformate under the action of a base to obtain (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid; (2) React the vonoprazan or N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)pyrimidin-2-amine with (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid under the action of an activator and a base to obtain the prodrug; The reaction formula is as follows 18. The preparation method according to claim 17, wherein The molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to 4-nitrophenyl chloroformate in step (1) is 1:1 - 1.5; and / or, the base in step (1) is selected from at least one of triethylamine, diisopropylethylamine, and pyridine; and / or, the molar ratio of the base to 4-(hydroxymethyl)phenylboronic acid pinacol ester in step (1) is 1 - 1.5:1; and / or, the reaction solvent in step (1) is selected from at least one of tetrahydrofuran, 1,4-dioxane, and acetonitrile; and / or, the reaction temperature of step (1) is 15°C - 37°C; and / or, the reaction time of step (1) is 12 h - 30 h, preferably 20 h - 28 h; and / or, the molar ratio of vonoprazan or N-(4-fluorophenyl)-4,5-dimethyl-6-(1-methyl-3,4-dihydroisoquinolin-2-yl)-pyrimidin-2-amine to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 1:0.9 - 1.2; and / or, the activator in step (2) is N-hydroxysuccinimide; and / or, the molar ratio of the activator to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 0.3 - 0.7:1; and / or, the base in step (2) is selected from at least one of triethylamine, diisopropylethylamine, and pyridine; and / or, the molar ratio of the base to (4-[(4-nitrophenoxy)carbonyloxymethyl]phenyl)boronic acid in step (2) is 1 - 1.5:1; and / or, the reaction solvent in step (2) is a mixed solvent of an organic solvent and water, and the organic solvent is tetrahydrofuran and / or 1,4-dioxane; and / or, the reaction temperature of step (2) is 15°C - 37°C; and / or, the reaction time of step (2) is 8 h - 24 h, preferably 10 h - 14 h.

19. Use of the copolymer according to any one of claims 1 - 9 as a carrier in the preparation of a drug for preventing and / or treating gastrointestinal oxidative stress-related diseases.

20. Use of the drug-loaded nanoparticles according to any one of claims 12 - 14 in the preparation of a drug for preventing and / or treating gastrointestinal oxidative stress-related diseases.

21. The application according to claim 19 or 20, characterized in that The gastrointestinal oxidative stress-related diseases are gastritis, gastric ulcer, and inflammatory bowel disease.

22. A drug for preventing and / or treating gastrointestinal oxidative stress-related diseases, characterized in that, Prepared from the drug-loaded nanoparticles according to any one of claims 12 - 14 and other pharmaceutically acceptable excipients.

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