Nano prodrug as well as preparation method and application thereof

Abstract

The invention relates to a nano prodrug as well as a preparation method and application thereof, and belongs to the technical field of nano preparations. Two nano prodrugs based on a hyperbranched polymer and an amphiphilic random copolymer are constructed, the hyperbranched polymer takes a mercaptosuccinic acid small molecule as a metal ion chelating agent, and a novel hyperbranched polymer which is rich in a thioacetal branched structure inside and is modified by mannose on the surface is constructed through sulfydryl-alkyne click chemistry and esterification. The amphiphilic random copolymer is prepared by carrying out reversible addition-fragmentation chain transfer polymerization on a functional chelating monomer (DSF) with ROS responsiveness and PEG (Polyethylene Glycol) with targeted group-mannose. The two nano prodrugs have the characteristics of inert circulation, macrophage precise targeting, pathological microenvironment dynamic response exposed copper chelation sites and the like, intracellular overloaded copper ions can be efficiently removed, the limitation of traditional symptomatic treatment is broken through, and a brand new strategy is provided for acute liver injury prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano preparations, and in particular relates to a nano prodrug and a preparation method and application thereof. Background Art

[0002] Acute liver injury (ALI) is an acute liver dysfunction caused by drugs, toxins, infection, or metabolic abnormalities. Its pathological features include massive hepatocyte necrosis, an inflammatory cytokine storm, and an imbalance of oxidative stress. In severe cases, it can progress to liver failure or even death. Acetaminophen (APAP) overdose is the primary cause of drug-induced liver injury, accounting for 46%-50% of cases worldwide.

[0003] Currently, clinical treatment for APAP-induced acute liver injury (AILI) primarily relies on drugs such as N-acetylcysteine ​​(NAC), glucocorticoids, and acetaminophen antidotes to treat acute liver injury, but their mechanisms of action and efficacy have significant flaws. Although NAC can alleviate oxidative stress by supplementing glutathione, its therapeutic window is extremely narrow (only effective within 8-10 hours after poisoning), its oral bioavailability is less than 10%, and intravenous injection can easily cause severe allergic reactions. Although glucocorticoids are suitable for immune liver injury, they inhibit hepatocyte regeneration and significantly increase the risk of secondary infection. Acetaminophen antidotes only target specific drug-induced liver injury, cannot reverse the core pathological mechanism of mitochondrial oxidative damage, and are completely ineffective against non-drug liver injury such as ischemia-reperfusion. In addition, existing drugs are unable to specifically target liver injury lesions, resulting in high systemic exposure and insufficient local effective concentrations, making precise treatment difficult to achieve.

[0004] Therefore, developing a nanoformulation that effectively targets hepatocytes and has good therapeutic effects has important research significance and clinical value. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and provide a nano prodrug and its preparation method and application.

[0006] The present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a nanoprodrug comprising at least one of a hyperbranched polymer and an amphiphilic random copolymer; the hyperbranched polymer comprises a polymer backbone and a targeting structure A; the targeting structure A comprises polyethylene glycol and polyethylene glycol-mannose; the polymer backbone is obtained by click chemistry polymerization of a thiol compound and an alkynyl compound; the structure of the thiol compound is shown in Formula I, and the structure of the alkynyl compound is shown in Formula II:

[0008]

[0009] The amphiphilic random copolymer includes a functional monomer and a targeting structure B, wherein the targeting structure B includes poly(ethylene glycol) methacrylate and poly(ethylene glycol) methacrylate-mannose. The structure of the functional monomer is shown in Formula III:

[0010]

[0011] The present invention constructs two nanoprodrugs based on hyperbranched polymers and amphiphilic random copolymers. The hyperbranched polymers involve using the small molecule mercaptosuccinic acid (DMSA) as a metal ion chelator. Through thiol-alkyne click chemistry and esterification, a novel hyperbranched polymer with an internal thioacetal-rich branched structure and a mannose-modified surface is constructed. The amphiphilic random copolymers are prepared by reversible addition-fragmentation chain transfer polymerization of a ROS-responsive chelating monomer (DSF) and a PEG-containing targeting group (mannose). The nanoprodrugs of the present invention utilize the long circulation properties of polyethylene glycol-mannose and its high affinity for hepatocytes to achieve enrichment in damaged hepatocytes, thereby improving bioavailability. The functional monomers and polymer backbone dynamically respond to ROS, triggering the site-specific release of dimercaptosuccinic acid and diethyldithiocarbamate (DTC), while simultaneously chelating copper ions to block inflammatory responses. The responsively released chelated small molecules improve the inflammatory cascade caused by copper overload by reducing the concentration of copper ions, avoiding its continued effect on hepatic parenchymal cells, creating conditions for the renewal of hepatic parenchymal cells, and promoting the repair of damaged hepatic parenchymal cells to improve its bioavailability and therapeutic effect.

[0012] Preferably, the molecular weight of the polyethylene glycol is 100Da-20000Da, and the structure of the polyethylene glycol-mannose is shown in Formula A1: Wherein, n=4-500. Specifically, n can be any one of 4, 9, 23, 46, 230, and 460.

[0013] Preferably, the structure of the poly(ethylene glycol) methacrylate is shown in Formula B1, and the structure of the poly(ethylene glycol) methacrylate-mannose is shown in Formula B2:

[0014] In Formula B1 and Formula B2, n is independently 4-500. Specifically, n can be any one of 4, 9, 23, 46, 230, and 460.

[0015] Preferably, the molar ratio of the polymer backbone to the polyethylene glycol and polyethylene glycol-mannose is 1:(0.1-1):(0.01-0.1).

[0016] Preferably, the molar ratio of the functional monomer to the poly(ethylene glycol) methacrylate and poly(ethylene glycol) methacrylate-mannose is (30-60):(1-15):(1-4).

[0017] Preferably, the molar ratio of the thiol compound to the alkynyl compound is (1-10):(1-10).

[0018] Preferably, the method for preparing the hyperbranched polymer comprises the following steps: coupling the polymer backbone and the targeting structure A under the action of a condensing agent to obtain the hyperbranched polymer.

[0019] The hyperbranched polymer of the present invention uses dithiothiobutanedioic acid (DMSA) as a monomer, constructs a hyperbranched polymer skeleton through a thiol-alkyne click chemistry reaction, and is surface-modified with a macrophage-targeting ligand. The polymer skeleton is rich in ROS-sensitive bonds. The nanoformulation prepared from the nanoprodrug can be enriched in liver lesions, triggering the cleavage of sensitive bonds in a local high ROS environment, releasing active molecules, and exerting an AILI therapeutic effect by chelating copper ions and inhibiting the inflammatory cascade reaction.

[0020] Preferably, the condensing agent includes dicyclohexyldiaminomethane (DCC) and 4-dimethylaminopyridine (DMAP), and the coupling reaction time is 12h-72h.

[0021] Preferably, the method for preparing the polymer skeleton comprises the following steps: dissolving the alkynyl compound and the thiol compound in an organic solvent, and performing a click chemistry polymerization reaction under the irradiation of a photoinitiator and ultraviolet light to obtain the polymer skeleton.

[0022] Preferably, in the preparation method of the polymer skeleton, the organic solvent includes one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile, methanol, and tetrahydrofuran; the photoinitiator includes dimethyl benzoate (DMPA); the wavelength of the ultraviolet light irradiation is 365 nm; and the click chemistry polymerization reaction time is 2 h-8 h.

[0023] Preferably, the preparation method of the alkynyl compound comprises the following steps: subjecting 1,4-phenyldimethylthiol and an alkynylating agent 1-[(trimethylsilyl)ethynyl]-1,2-benzidoxyl-3(1H)-one (TMS-EBX) to an alkynylation reaction in the presence of a catalyst, followed by hydrolysis and deprotection to obtain the alkynyl compound.

[0024] The alkynylation reagent 1-[(trimethylsilyl)ethynyl]-1,2-benzidoyl-3(1H)-one (TMS-EBX) can be prepared in-house or commercially available. Specifically, the present invention provides a method for preparing the 1-[(trimethylsilyl)ethynyl]-1,2-benzidoyl-3(1H)-one, comprising the following steps: subjecting sodium periodate and 2-iodobenzoic acid to a reflux reaction in an acetic acid solution to obtain an intermediate product, o-iodobenzoic acid lactone; adding trimethylsilyl trifluoromethanesulfonate to the reaction until the mixture becomes colorless and transparent; then adding bis(trimethylsilyl)acetylene and pyridine dropwise; and continuing the reaction to obtain the 1-[(trimethylsilyl)ethynyl]-1,2-benzidoyl-3(1H)-one.

[0025] Preferably, the hydrolysis can be carried out under alkaline conditions, such as potassium carbonate, to achieve the removal of the trimethylsilyl group to produce the alkynyl compound.

[0026] Preferably, the preparation method of the amphiphilic random copolymer comprises the following steps: subjecting the functional monomer and the targeting structure B to a reversible addition-fragmentation chain transfer free radical polymerization reaction under the action of a macromolecular chain transfer agent and an initiator to obtain the amphiphilic random copolymer.

[0027] The amphiphilic random copolymer of the present invention uses a functional monomer with a ROS-responsive group as a hydrophobic monomer and polyethylene glycol-coupled mannose as a hydrophilic monomer. The amphiphilic random copolymer is constructed through reversible addition-fragmentation chain transfer polymerization. The nanoformulation made of this nanoprodrug can be enriched in liver lesions, triggering the cleavage of sensitive bonds in a local high ROS environment, releasing active molecules, and exerting a therapeutic effect on AILI by chelating copper ions and inhibiting the inflammatory cascade reaction.

[0028] Preferably, in the preparation method of the amphiphilic random copolymer, the macromolecular chain transfer agent includes at least one of dithiocarbonates, trithiocarbonates, xanthates, and dithiocarbamates; the initiator includes at least one of an azo compound (such as azobisisobutyronitrile, AIBN) and an organic peroxide (such as BPO or K2S2O8).

[0029] Specifically, in the preparation method of the amphiphilic random copolymer, the macromolecular chain transfer agent includes 4-cyano-4-(thiobenzoylthio)valeric acid (CPADB); and the initiator includes azobisisobutyronitrile (AIBN).

[0030] Preferably, the molar ratio of the functional monomer to the macromolecular chain transfer agent and the initiator is (30-60): (1-5): (0.1-5).

[0031] Preferably, the reversible addition-fragmentation chain transfer free radical polymerization reaction is carried out in the dark, at a reaction temperature of 60° C.-90° C., and for 12 h-24 h.

[0032] The poly(ethylene glycol) methacrylate in the targeting structure B of the present invention can be prepared in-house or purchased commercially. Specifically, the present invention provides a method for preparing poly(ethylene glycol) methacrylate, comprising the following steps: the method for preparing the poly(ethylene glycol) methacrylate comprises reacting polyethylene glycol and methacrylic anhydride under the action of triethylamine and 4-dimethylaminopyridine in an ice bath to obtain the poly(ethylene glycol) methacrylate; specifically, the molar ratio of the polyethylene glycol to methacrylic anhydride is (0.5-2):(0.5-1).

[0033] Preferably, the preparation method of the poly(ethylene glycol) methacrylate-mannose comprises the following steps: reacting polyethylene glycol-mannose and methacrylic anhydride under the action of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) in an ice bath to obtain the poly(ethylene glycol) methacrylate.

[0034] In the preparation method of poly(ethylene glycol) methacrylate-mannose, the molar ratio of the polyethylene glycol-mannose to methacrylic anhydride, triethylamine (TEA), and 4-dimethylaminopyridine is (1-5):(1-5):(1-5):(1-5).

[0035] Preferably, the preparation method of the functional monomer comprises the following steps: reacting 4-formylphenyl methacrylate and diethylammonium diethyldithiocarbamate in the presence of a catalyst to obtain the functional monomer; the structure of the 4-formylphenyl methacrylate is shown in Formula IV:

[0036]

[0037] Preferably, in the preparation method of the functional monomer, the molar ratio of the 4-formylphenyl methacrylate to the diethylammonium diethyldithiocarbamate is (1-5): (1-5).

[0038] Preferably, in the method for preparing the functional monomer, the catalyst includes boron trifluoride etherate (BF3·OEt2), the reaction temperature is 30°C-50°C, and the reaction time is 12h-24h.

[0039] Preferably, in the preparation method of the functional monomer, the preparation method of the 4-formylphenyl methacrylate comprises the following steps: subjecting p-hydroxymethylbenzaldehyde and methacrylic anhydride to an esterification reaction in the presence of a catalyst to obtain the 4-formylphenyl methacrylate.

[0040] Preferably, in the method for preparing 4-formylphenyl methacrylate, the catalyst comprises 4-dimethylaminopyridine (DMAP) and triethylamine (TEA).

[0041] Preferably, in the preparation method of 4-formylphenyl methacrylate, the esterification reaction is carried out in dichloromethane (DCM); and the molar ratio of p-hydroxymethylbenzaldehyde to methacrylic anhydride is (1-5): (1-5).

[0042] In a second aspect, the present invention provides a nanoformulation, wherein the nanoformulation is obtained by self-assembly of the nanoprodrug.

[0043] Preferably, the self-assembly adopts a rapid nanocomplexation method and is carried out through an FNC nano self-assembly platform; more preferably, in the rapid nanocomplexation method, the oil phase solvent includes one of dimethyl sulfoxide, methanol, N,N-dimethylformamide, and tetrahydrofuran; the oil phase and the aqueous phase are nanocomplexed in a ratio of 1:(6-9) (for example, 1:6, 1:7, 1:8, 1:9); the flow rate ratio of oil phase: aqueous phase: aqueous phase is (2-10):(9-45):(9-45) (2:9:9, 4:18:18, 8:36:36, 10:45:45); the polymer dissolved in the oil phase is mixed with the aqueous phase for nanocomplexation to form nanoparticles; the method for removing the solvent adopts one or a combination of dialysis, rotary evaporation, vacuum drying or freeze drying.

[0044] Preferably, the nanoformulation is in the form of spherical particles, the particle size of the nanoformulation is 20 nm-90 nm, the polydispersity index PDI of the nanoformulation is 0.03-0.33, and the zeta potential of the nanoformulation is -8 mV to -50 mV.

[0045] Preferably, the nanoformulation is in the form of a freeze-dried preparation; specifically, the nanocomplexed nanoparticle aqueous solution is mixed with a freeze-dried protective agent and frozen at -80°C, and the frozen product is placed in a freeze dryer in the dark for freeze-drying; the freeze-dried preparation uses salts as preparation resuspension buffers to balance the pH value during freeze-drying and storage; the freeze-dried preparation uses alcohol freeze-dried protective agents and sugar freeze-dried protective agents, and the alcohol freeze-dried protective agent is a combination of any one or more of xylitol, mannitol, sorbitol or glycine; the sugar freeze-dried protective agent is a combination of any one or more of glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan or inulin; the concentration of the freeze-dried protective agent is selected from one of 0%, 5%, 10%, 15%, 20% and 25%; the salt buffer is a salt selected from any one or a combination of sodium phosphate or potassium phosphate.

[0046] In a third aspect, the present invention provides the use of the nano-prodrug and nano-preparation in the preparation of drugs for treating acute liver injury.

[0047] Preferably, the concentration range of the nano-prodrug or nano-preparation for treatment is 5 mg / mL-30 mg / mL.

[0048] The present invention has the following beneficial effects:

[0049] (1) The present invention cleverly utilizes the characteristics of the high-concentration ROS microenvironment at the site of APAP-induced acute liver injury lesions, combined with the efficient chelation properties of DMSA and DTC for copper ions, to construct two ROS-stimulated responsive nanoprodrugs. By designing a ROS-responsive polymer carrier and introducing a thioacetal structure, controlled release of the drug under dynamic changes in ROS is achieved. At the same time, mannose modification gives the nanoprodrug the ability to target macrophages, significantly improving the enrichment efficiency of the drug at the lesion site and providing a guarantee for precise treatment.

[0050] (2) The nanoprodrug of the present invention not only achieves targeted enrichment of liver macrophages and responsive release in response to the inflammatory microenvironment, but also regulates copper ion homeostasis by selectively chelating intracellular copper ions, effectively inhibiting the inflammatory activation of Kupffer cells, blocking leukocyte recruitment, and creating favorable conditions for liver cell repair and regeneration. The nanoprodrug of the present invention integrates multiple functions such as efficient chelation, precise targeting, anti-inflammatory and antioxidant, providing a new treatment strategy for diseases related to excessive copper ions. In addition, the nanoprodrug of the present invention can achieve more efficient targeted delivery and sustained release effects, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Flowcharts of the preparation methods of the nanoprodrugs of Examples 1 and 2, wherein (A) is the synthesis route of the hyperbranched polymer of Example 1, and (B) is the synthesis route of the amphiphilic random copolymer of Example 2;

[0052] Figure 2 is the NMR spectrum of the alkynylating agent prepared in Example 1;

[0053] Figure 3 is the NMR spectrum of the alkynyl compound prepared in Example 1;

[0054] Figure 4 Detailed preparation process flow chart of the nano prodrug of Example 2;

[0055] Figure 5 This is the NMR spectrum of 4-formylphenyl methacrylate prepared in Example 2;

[0056] Figure 6 This is the NMR spectrum of the functionalized monomer prepared in Example 2;

[0057] Figure 7 This is a diagram showing the hemolysis of the nanoformulation prepared from the nanoprodrug of Example 1;

[0058] Figure 8 This is a diagram showing the cytotoxicity test results of the nanoformulation prepared from the nanoprodrug of Example 1;

[0059] Figure 9 Figure 1 shows the liver of mice before and after treatment with the nanoformulation prepared from the nanoprodrug of Example 1;

[0060] Figure 10 This is a graph showing the liver serum AST test results of the nanoformulation prepared from the nanoprodrug of Example 1;

[0061] Figure 11 This is a graph showing the liver serum ALT test results of the nanoformulation prepared from the nanoprodrug of Example 1. DETAILED DESCRIPTION

[0062] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0063] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0064] Example 1

[0065] A flow chart of the preparation method of nano prodrug is shown in FIG. Figure 1(A) specifically includes the following steps:

[0066] (1) Sodium periodate (15.44 g, 72.18 mmol) and 2-iodobenzoic acid (17.90 g, 72.18 mmol) were placed in a three-necked flask, 30% acetic acid solution was added, the temperature was raised to 115°C, refluxed for 4 h, and then cooled in the dark. The mixture was stirred at room temperature for 1 h, diluted with a large amount of water, and filtered through a Buchner funnel. The suspension was rinsed three times with ice water and then three times with ice acetone. The solid was vacuum dried to collect the product o-iodobenzoic acid lactone. Trimethylsilyl trifluoromethanesulfonate (5.6 mL, 80 mmol) was added dropwise to a solution of o-iodobenzoic acid lactone (6.0 g, 80 mmol) in acetonitrile. After the reaction became colorless and transparent, bis(trimethylsilyl)acetylene (5.6 mL, 80 mmol) was added dropwise. After reacting for 20 min, pyridine (4.8 mL) was added all at once and the reaction was continued for 30 min. The solvent was removed under reduced pressure and the obtained liquid was dissolved in dichloromethane. The organic phase was washed with water and collected, dried over anhydrous MgSO4 overnight, filtered to remove the solid, concentrated and recrystallized, and the product was collected to obtain the alkynylating agent TMS-EBX. Its nuclear magnetic spectrum is shown in FIG. Figure 2 ; The synthesis path is as follows:

[0067]

[0068] 1,4-Phenyldimethylthiol (500 mg, 3.52 mmol) and tetramethylguanidine (TMG, 971.75 mg, 8.44 mmol) were dissolved in 60 mL of tetrahydrofuran (THF) and stirred at room temperature for 5 minutes. Subsequently, the obtained alkynylating agent (2.66 g, 7.73 mmol) was quickly added, and the reaction mixture was continued to be stirred at room temperature for 5 minutes while maintaining contact with the atmosphere. After that, the reaction mixture was diluted with 10 mL of water and extracted with ethyl acetate (extraction three times, 10 mL each time). The collected organic layer was dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure. Finally, the crude product was separated by flash chromatography to obtain intermediate E; the synthesis route is as follows:

[0069]

[0070] The obtained intermediate product E (826 mg, 2.28 mmol, 1.00 eq) and potassium carbonate (K2CO3, 629 mg, 4.56 mmol, 2.00 eq) were dissolved in methanol (MeOH, 38 mL) and stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and then dichloromethane (CH2Cl2) was added for redispersion. The organic phase and the aqueous phase were washed three times, and then the solution was concentrated to finally obtain a white solid product, which is an alkynyl compound, recorded as compound F, and its nuclear magnetic spectrum is shown in FIG. Figure 3 ;

[0071] (2) Dimercaptopropionic acid (DMSA, 191 mg, 1.058 mmol, 4.00 equivalents) and compound F (50 mg, 0.264 mmol, 1.00 equivalents) were dissolved in DMF (0.3 mL) containing dimethyl phenylethanol (DMPA, 34.98 mg, 0.132 mmol, 0.50 equivalents). After three freeze-thaw cycles to remove oxygen, the reaction vessel was exposed to a 365 nm UV lamp and stirred at room temperature for 4 hours. After the reaction, the resulting solution was dialyzed against DMSO for 1 day and then transferred to water for 3 days. Finally, a white solid was obtained by freeze-drying, which was the polymer backbone and was designated as compound H. The synthesis route is as follows:

[0072]

[0073] (3) Compound H (200 mg), dicyclohexyldiaminomethane (DCC, 128 mg) and 4-dimethylaminopyridine (DMAP, 30 mg) were dissolved in 250 mL of DMF and cooled in an ice bath. Polyethylene glycol (PEG, 600 mg) and polyethylene glycol-mannose (PEG-Man, 50 mg) were added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the reaction mixture was dialyzed against DMF for 24 hours and then dialyzed against water for 3 days. Finally, a brown solid hyperbranched polymer was obtained by lyophilization, which was the nanoprodrug. The synthesis route is as follows:

[0074]

[0075] Example 2

[0076] A process flow chart of a nano-prodrug preparation method is shown in Figure 1(B), and the detailed synthesis process is shown in Figure 1(B). Figure 4 As shown, the specific steps include:

[0077] (1) p-Hydroxymethylbenzaldehyde (100 mg, 136.15, 1.00 eq), DMAP (13.46 mg, 122.17, 0.15 eq), and TEA (148.65 mg, 101.19, 2.00 eq) were dissolved in DCM (3 ml) and stirred at room temperature for 30 min. Then, methacrylic anhydride (135.88 mg, 154.17, 1.2 eq) dissolved in DCM was slowly added dropwise. The reaction was continued for 4 hours. After TLC monitoring, the solvent was removed by rotary evaporation. The mixture was then extracted three times with DCM and water and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography (petroleum ether: ethyl acetate 8:1) to obtain 4-formylphenyl methacrylate as a white solid. Its NMR spectrum is shown in FIG. Figure 5 ; The synthesis path is as follows:

[0078]

[0079] The obtained 4-formylphenyl methacrylate (141 mg, 204.23, 1 eq) was dissolved in 5 mL of chloroform and preheated at 40°C for 15 min. BF3·OEt2 (980 mg, 141.93, 10 eq) was added, and then diethylammonium diethyldithiocarbamate (461 mg, 222.41, 3 eq) dissolved in chloroform was slowly added dropwise. The reaction was carried out at 40°C for 16 hours and monitored by TLC. After the reaction was completed, water was added to quench the reaction. The crude product was extracted with ethyl acetate and water three times, dried over anhydrous Na2SO4, and purified by column chromatography (petroleum ether: ethyl acetate 2:1) to obtain a white solid functional monomer (MA-DTC). Its NMR spectrum is shown in FIG. Figure 6 ; The synthesis path is as follows:

[0080]

[0081] (2) Polyethylene glycol PEG 1K (1g, 987, 1.00eq), DMAP (18.56mg, 122.17, 0.15eq), TEA (205.01mg, 101.19, 2.00eq) were dissolved in DCM (10mL) and stirred at room temperature for 30min. Then, methacrylic anhydride (78.08mg, 154.17, 0.5eq) dissolved in DCM was slowly added dropwise. The reaction was continued for 4 hours. After TLC monitoring, the solvent was concentrated by rotary evaporation. Then, it was precipitated with ether and purified by column chromatography (dichloromethane: methanol 10:1) to obtain white solid poly (ethylene glycol) methacrylate (MA-PEG 1K ); the synthesis path is as follows:

[0082]

[0083] Mannose (Man-PEG 2K) (300 mg, 2162, 1.00 eq), DMAP (2.54 mg, 122.17, 0.15 eq), TEA (28.09 mg, 101.19, 2.00 eq) were dissolved in DCM (5 mL) and stirred at room temperature for 30 min. Then, methacrylic anhydride (23.54 mg, 154.17, 0.5 eq) dissolved in DCM was slowly added dropwise. The reaction was continued for 4 hours. After TLC monitoring, the solvent was concentrated by rotary evaporation and then precipitated with ether 3 times to obtain a white solid poly (ethylene glycol) methacrylate-mannose (MA-PEG 2K -Man); the synthetic route is as follows:

[0084]

[0085] (3) Combine CPADB (3mg, 279.37, 1.00eq), AIBN (0.18mg, 164.21, 0.1eq), MA-DTC (312.33mg, 484.75, 60eq), MA-PEG 1K (124.65mg, 1055, 15eq) and MA-PEG 2K -Man (95.77 mg, 2230 g, 4 eq) was added to a Shrek flask, and the mixture was freeze-thawed and deoxygenated three times using a double-row tube, and then reacted in the dark at 78°C for 16 hours. The mixture was precipitated in icy ether three times to obtain a pink solid DTC-PEG-Man polymer, which is the nanoprodrug. The synthesis route is as follows:

[0086]

[0087] The nanoprodrugs obtained in Example 1 and Example 2 were prepared into a nanoformulation by the following method: the nanoprodrug solutions obtained in Example 1 and Example 2 dissolved in the organic phase DMSO were injected into channel 1 of a three-channel confined impinging jet (CIJ) reactor using an FNC nano self-assembly platform, and ultrapure water was injected into the other two channels. The solutions in the three channels were then fully mixed at a constant preset flow rate using a digitally controlled high-pressure injection pump to prepare uniform nanoparticles. The organic solvent DMSO was then removed by dialyzing in ultrapure water using a dialysis bag to obtain the nanoformulation.

[0088] Characterization and testing of the nanoformulation (DSPM NPs) of Example 1:

[0089] 1. The nanoparticle preparation of Example 1 at different concentrations was dispersed in a certain volume fraction of blood samples and incubated at room temperature for 3 hours. The samples treated with surfactant (TX-100) and 1×PBS were used as negative and positive controls, respectively. The supernatant was separated and the hemolysis of the nanoparticles at different doses was determined by microplate reader. The results are shown in FIG. Figure 7 As shown. Figure 7 It can be seen that even when the blood cells are incubated with the nanoformulation of Example 1 at a concentration of 1000 μg / mL, the cell membrane is still intact, indicating that the nanoformulation obtained by the present invention has good blood compatibility and meets the safety standards for in vivo application.

[0090] 2. Macrophages were inoculated and cultured for 6 hours, and different concentrations of the nanoparticle preparation aqueous solution of Example 1 were added and cultured for 12 hours and 24 hours (the concentrations written here represent the concentrations of nanoparticles in all nanoparticle preparation aqueous solutions). Cytotoxicity results are shown in Figure 2. Figure 8 As shown, no cytotoxicity was observed. Even at 100 μg mL -1 At high equivalent drug concentrations, the cell viability still remained good and the survival rate exceeded 95%, indicating that the nanoformulation obtained by the present invention has good cell compatibility.

[0091] The acute liver injury (ALI) mouse model was established by intraperitoneal injection of APAP (300 mg / kg). ALI mice were killed and dissected 24 hours after administration of different methods and doses, and their livers were taken for photography. Figure 9 As shown. Furthermore, in a mouse model of acute liver injury induced by APAP overdose, the therapeutic efficacy of the nanoformulation of Example 1 was evaluated through preventive administration (dosing at 0 hours) and therapeutic administration (3 hours). NAC, a commonly used clinical APAP antidote, was used as a control. Blood was collected from mice at 6, 12, and 24 hours after treatment, and the supernatant was centrifuged to obtain serum. Serum ALT and AST levels were measured using commercially available kits. Figure 10 and Figure 11 Liver serum liver enzyme indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), n=8-16.

[0092] Figure 9 The results showed that the livers of mice in the drug-induced acute liver injury model established with APAP had large areas of hemorrhage and necrotic lesions. After treatment with NAC and the nanoformulation of Example 1, the hemorrhage and necrotic areas were significantly reduced, even approaching the normal liver morphology. Figure 9 Representative images of livers after dissection of mice in each group; scale bar: 1 cm.

[0093] pass Figure 10 and Figure 11 As can be seen, serum ALT and AST gradually increased after injury, indicating severe liver damage. Active treatment effectively reduced liver ALT and AST levels in the first 6 hours after injury and continued to reduce serum transaminase levels throughout the treatment period. The 0-hour preventive administration group even returned to normal levels 24 hours after injury.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nano prodrug, characterized in that: The invention comprises at least one of a hyperbranched polymer and an amphiphilic random copolymer; the hyperbranched polymer comprises a polymer backbone and a targeting structure A; the targeting structure A comprises polyethylene glycol and polyethylene glycol-mannose; the polymer backbone is obtained by click chemistry polymerization of a thiol compound and an alkynyl compound; the structure of the thiol compound is shown in Formula I, and the structure of the alkynyl compound is shown in Formula II: The amphiphilic random copolymer includes a functional monomer and a targeting structure B, wherein the targeting structure B includes poly(ethylene glycol) methacrylate and poly(ethylene glycol) methacrylate-mannose. The structure of the functional monomer is shown in Formula III:

2. The nano prodrug according to claim 1, characterized in that The molecular weight of the polyethylene glycol is 100 Da to 20,000 Da, the structure of the polyethylene glycol-mannose is shown in Formula A1, the structure of the poly(ethylene glycol) methacrylate is shown in Formula B1, and the structure of the poly(ethylene glycol) methacrylate-mannose is shown in Formula B2: In the formulas A1, B1 and B2, n is independently 4-500.

3. The nano prodrug according to claim 1, characterized in that The molar ratio of the polymer backbone to the polyethylene glycol and polyethylene glycol-mannose is 1:(0.1-1):(0.01-0.1); the molar ratio of the functional monomer to the poly(ethylene glycol) methacrylate and poly(ethylene glycol) methacrylate-mannose is (30-60):(1-15):(1-4); and the molar ratio of the thiol compound to the alkynyl compound is (1-10):(1-10).

4. The nano prodrug according to claim 1, characterized in that The preparation method of the hyperbranched polymer comprises the following steps: coupling the polymer skeleton and the targeting structure A under the action of a condensing agent to obtain the hyperbranched polymer; The preparation method of the amphiphilic random copolymer comprises the following steps: subjecting the functional monomer and the targeting structure B to a reversible addition-fragmentation chain transfer free radical polymerization reaction under the action of a macromolecular chain transfer agent and an initiator to obtain the amphiphilic random copolymer.

5. The nano prodrug according to claim 4, characterized in that The condensing agent includes dicyclohexyldiaminomethane and 4-dimethylaminopyridine, and the coupling reaction time is 12h-72h; the macromolecular chain transfer agent includes at least one of dithiocarbonates, trithiocarbonates, xanthates, and dithiocarbamates; the initiator includes at least one of an azo compound and an organic peroxide; the molar ratio of the functional monomer to the macromolecular chain transfer agent and the initiator is (30-60):(1-5):(0.1-5); the temperature of the reversible addition-fragmentation chain transfer free radical polymerization reaction is 60°C-90°C, and the time is 12h-24h.

6. The nano prodrug according to claim 4, characterized in that The preparation method of the polymer skeleton comprises the following steps: dissolving the alkynyl compound and the thiol compound in an organic solvent, and performing a click chemistry polymerization reaction under the irradiation of a photoinitiator and ultraviolet light to obtain the polymer skeleton; The preparation method of the functional monomer comprises the following steps: reacting 4-formylphenyl methacrylate and diethylammonium diethyldithiocarbamate in the presence of a catalyst to obtain the functional monomer; the structure of the 4-formylphenyl methacrylate is shown in Formula IV:

7. The nano prodrug according to claim 6, characterized in that In the preparation method of the polymer skeleton, the preparation method of the alkynyl compound comprises the following steps: 1,4-phenyldimethylthiol and the alkynylating agent 1-[(trimethylsilyl)ethynyl]-1,2-benzidoyl-3(1H)-one are subjected to an alkynylation reaction in the presence of a catalyst, followed by hydrolysis and deprotection to obtain the alkynyl compound.

8. The nano prodrug according to claim 6, characterized in that In the preparation method of the functional monomer, the molar ratio of the 4-formylphenyl methacrylate to the diethylammonium salt of diethyldithiocarbamate is (1-5):(1-5); the catalyst includes boron trifluoride etherate (BF3·OEt2); the reaction temperature is 30°C-50°C, and the reaction time is 12 hours-24 hours. The preparation method of the 4-formylphenyl methacrylate includes the following steps: esterifying p-hydroxymethylbenzaldehyde and methacrylic anhydride in the presence of the catalyst to obtain the 4-formylphenyl methacrylate.

9. A nano preparation, characterized in that: The nanoformulation is obtained by self-assembly of the nanoprodrug according to any one of claims 1 to 8.

10. Use of the nanoprodrug according to any one of claims 1 to 8 or the nanoformulation according to claim 9 in the preparation of a drug for treating acute liver injury.

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