Superoxide anion responsive nano-preparation, and preparation method and application thereof

Abstract

This invention belongs to the field of nanoparticle formulation and targeted drug delivery technology, specifically relating to a superoxide anion-responsive nanoparticle formulation, its preparation method, and its applications. This nanoparticle formulation is formed by random copolymerization of two monomers, wherein the hydrophobic portion contains a superoxide anion-responsive persulfide donor, and the hydrophilic portion is composed of glycyrrhetinic acid-polyethylene glycol, which also has hepatocyte targeting function. Using a nanoprecipitation method, the copolymer self-assembles into structurally stable nanoparticles through hydrophilic-hydrophobic interactions. This invention achieves the combination of a high-molecular-weight polymer and a persulfide donor, constructing a polymer prodrug system capable of actively targeting the liver and releasing therapeutically active ingredients at the lesion site. This formulation has advantages such as flexible design, specific response, and good biocompatibility, providing a new strategy and platform for targeted therapy of severe diseases such as acute liver injury.

Description

Technical Field

[0001] This invention belongs to the field of nano-formulation technology, specifically relating to a superoxide anion-responsive nano-formulation, its preparation method, and its application. Background Technology

[0002] Hepatic ischemia-reperfusion injury (HIRI) is progressive tissue damage caused by the restoration of perfusion (reperfusion) after a brief interruption of blood flow to the liver (ischemia). It is a major cause of acute liver injury (ALI) and organ failure, commonly seen in clinical settings such as liver transplantation, extensive hepatectomy, shock, and severe trauma. HIRI is a key factor limiting liver transplant success, potentially leading to early graft dysfunction and exacerbating rejection, severely impacting patient prognosis. Furthermore, there is currently a lack of effective and specific clinical interventions for HIRI. Therefore, effective prevention and treatment of HIRI is a significant challenge for improving the success rate of liver surgery and prolonging graft survival. The pathological mechanism of HIRI is a complex process involving multiple factors and pathways. At the cellular level, ischemia leads to impaired energy metabolism in hepatocytes, a sharp decrease in adenosine triphosphate (ATP) production, and the production of large amounts of lactic acid through anaerobic glycolysis, resulting in a decrease in intracellular pH. Following ischemia, ATP levels can drop sharply, causing the sodium / potassium and calcium pumps to malfunction, leading to cellular edema and calcium overload, severely impairing cell membrane stability and function. The core factor exacerbating HIRI damage is the reperfusion phase, during which blood flow and oxygenation are suddenly restored, triggering oxidative stress and an innate immune-mediated aseptic inflammatory response. Oxidative stress is one of the core components of the HIRI pathological mechanism. A large amount of reactive oxygen species (ROS) are explosively generated during reperfusion, including superoxide anions (O2). ·− Reperfusion oxidation (ROS) produces substances such as hydroxyl radicals (•OH). These ROS lead to cell membrane lipid peroxidation, DNA damage, and protein oxidative modification, ultimately driving various programmed cell death processes in hepatocytes, including apoptosis, necrotizing apoptosis, pyroptosis, and, in recent years, ferroptosis, which has received increasing attention. This results in severe liver dysfunction and tissue necrosis. Furthermore, reperfusion activates Kupffer cells in the liver and recruits neutrophils. Activated Kupffer cells release large amounts of pro-inflammatory cytokines (such as TNF-α and IL-β), exacerbating the inflammatory cascade. Neutrophil aggregation and the release of proteases further contribute to microcirculatory disturbances and tissue damage.

[0003] To overcome the limitations of traditional small molecule drugs (such as antioxidants and anti-inflammatory drugs) in HIRI therapy, such as short circulation time, poor bioavailability, and lack of targeting, nanoprodrug strategies have become a research hotspot in this field. Compared with traditional nanodrug delivery systems, nanoprodrugs have the advantage of being able to covalently bind active drugs or pharmacologically active functional groups to nanoframework materials, achieving high drug loading, excellent in vivo stability, intelligent response to the lesion environment, and on-demand release of active ingredients without introducing additional inactive carriers. This reduces ROS and inflammatory damage. However, existing nanoformulations still face the following challenges: (1) The oxidative / inflammatory inhibitory effect is not comprehensive enough, making it difficult to effectively intervene in multiple cell death pathways simultaneously; (2) The complexity or instability of nanocarrier preparation limits their clinical translation; (3) The targeting strategy still needs to be optimized to achieve more precise and faster targeted delivery to damaged hepatocytes or specific immune cells.

[0004] Therefore, developing a novel nanomedicine with multiple synergistic functions, precise targeting of damaged liver parenchymal cells, and feasible preparation process is crucial for the effective prevention and treatment of HIRI. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a superoxide anion-responsive nano-formulation, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a superoxide anion-responsive nanoformulation, which comprises a hydrophobic region and a hydrophilic region; the hydrophilic region includes polyethylene glycol methacrylate coupled with glycyrrhetinic acid and polyethylene glycol methacrylate; the hydrophobic region includes a hydrophobic monomer, the structure of which is shown in Formulas I-IV: Formula I, Formula II, Formula III, Formula IV.

[0007] This invention relates to a nanoformulation for treating acute liver injury. The formulation is based on a diphenylphosphonate bond (DPP) derivative, constructing an amphiphilic random polymer with superoxide anion responsiveness and hepatocyte targeting function. Its surface is modified with the targeting molecule glycyrrhetinic acid, enabling the nanoparticles to rapidly accumulate at the liver lesion site. In a pathological environment, this nanoformulation rapidly consumes superoxide anions through responsive cleavage, releasing the antioxidant persulfide (R-SSH). The released persulfide can efficiently capture free radicals, quench free radical chain reactions, thereby activating the Nrf2 pathway, inhibiting lipid peroxidation, and reducing inflammatory responses, thus achieving effective treatment for acute liver injury.

[0008] In the nano-formulation of the present invention, the hydrophobic region contains ROS-sensitive groups, free radical polymerized unsaturated alkanes, and hydrophobic drug molecules with therapeutic effects, and the hydrophilic region contains polyethylene glycol and zwitterionic segments.

[0009] Preferably, the molar ratio of the hydrophobic monomer, polyethylene glycol methacrylate coupled with glycyrrhetinic acid, and polyethylene glycol methacrylate is (10-40):(1-4):(2-8).

[0010] Preferably, the structure of the polyethylene glycol methacrylate coupled with glycyrrhetinic acid is as shown in Formula V: Formula V, where n = 4-500, specifically, n can be any one of 4, 9, 23, 46, 230, 460.

[0011] Preferably, the structure of the polyethylene glycol methacrylate is as shown in Formula VI: Equation VI, where n = 4-500, specifically, n can be any one of 4, 9, 23, 46, 230, 460.

[0012] Secondly, this invention provides a method for preparing a superoxide anion-responsive nanoparticle as described above. The method includes the following steps: free radical polymerization of a hydrophobic monomer, polyethylene glycol methacrylate coupled with glycyrrhetinic acid, and polyethylene glycol methacrylate to obtain a polymer; then, the obtained polymer is self-assembled to obtain the superoxide anion-responsive nanoparticle. This invention uses a DPP derivative hydrophobic monomer containing double bonds and a hydrophilic monomer to conduct a free radical polymerization reaction, constructing an amphiphilic random polymer with a diphenylphosphonate structure, which is then self-assembled to obtain the nanoparticle. In a ROS environment, the diphenylphosphonate bond in its structure can specifically respond to superoxide anions and break, releasing persulfide molecules, thereby efficiently clearing reactive oxygen species at the lesion site, reducing oxidative stress, inhibiting inflammation and lipid peroxidation, and thus achieving a therapeutic effect on acute liver injury. Furthermore, the glycyrrhetinic acid targeting molecules modified on the surface of the nanoparticle can significantly enhance its targeting recognition ability of hepatocytes.

[0013] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the free radical polymerization reaction includes any one of reversible addition-fragmentation chain transfer radical (RAFT) polymerization, ATRP (atomic transfer radical polymerization), NMP (nitrogen oxide stable radical polymerization), and SETRP (single electron transfer "living" radical polymerization).

[0014] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the polymer includes block copolymers and random copolymers, and the free radical polymerization reaction is a reversible addition-fragmentation chain transfer free radical polymerization reaction.

[0015] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the free radical polymerization reaction is carried out under the presence of a chain transfer agent and an initiator.

[0016] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the chain transfer agent includes at least one of dithiocarbonates, trithiocarbonates, xanthates, and dithiocarbamates; the initiator includes at least one of azo compounds (such as azobisisobutyronitrile, AIBN), organic peroxides (such as BPO or K2S2O8), and inorganic peroxides (such as potassium sulfate or ammonium persulfate).

[0017] Specifically, in the preparation method of the superoxide anion-responsive nano-formulation, the chain transfer agent includes 4-cyano-4-(thiobenzoylthio)valerate (CPADB); and the initiator includes azobisisobutyronitrile (AIBN).

[0018] Preferably, in the preparation method of the superoxide anion responsive nano-formulation, the molar ratio of the hydrophobic monomer, polyethylene glycol methacrylate coupled with glycyrrhetinic acid, and polyethylene glycol methacrylate is (10-40):(1-4):(2-8).

[0019] Preferably, in the preparation method of the superoxide anion responsive nano-formulation, the molar ratio of polyethylene glycol methacrylate, chain transfer agent and initiator is (5-20):(1-4):(0.1-0.4).

[0020] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the free radical polymerization reaction is carried out under light-protected conditions, the reaction temperature is 60℃-80℃, and the time is 16h-48h.

[0021] Preferably, in the preparation method of the superoxide anion-responsive nano-formulation, the self-assembly is carried out using a rapid nano-complexation method via an FNC nano-self-assembly platform; more preferably, in the rapid nano-complexation method, the oil phase solvent includes one of dimethyl sulfoxide, methanol, N,N-dimethylformamide, and 1,4-dioxane; the oil phase and aqueous phase are nano-complexed at a ratio of 1:(6-9) (e.g., 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 to form nano-complexes and nanoparticles; the solvent removal method is one or a combination of dialysis, rotary evaporation, vacuum drying, or lyophilization.

[0022] Preferably, the superoxide anion-responsive nano-formulation is in lyophilized form. Specifically, the aqueous solution of the nano-complexed nanoparticles is mixed with a lyophilization protectant and frozen at -80°C. The frozen product is then placed in a lyophilizer in the dark to produce lyophilized products. Salts are used as resuspension buffers in the lyophilized formulation to balance the pH value during the freeze-drying process. The lyophilization protectant is selected from alcohol-based and sugar-based lyophilization protectants. The alcohol-based lyophilization protectant is any one or a combination of xylitol, mannitol, sorbitol, or glycine. The sugar-based lyophilization protectant is any one or a combination of glucose, sucrose, lactose, trehalose, maltose, maltodextrin, fructan, or inulin. The concentration of the lyophilization protectant is selected from 0%, 5%, 10%, 15%, 20%, and 25%. The salt buffer is any one or a combination of sodium phosphate or potassium phosphate.

[0023] Preferably, the superoxide anion-responsive nanoparticles are spherical particles with a particle size of 30 nm to 100 nm, a polydispersity index (PDI) of 0.05 to 0.3, and a zeta potential of -15 mV to -40 mV.

[0024] Specifically, in the preparation method of the superoxide anion responsive nano-formulation, the polyethylene glycol methacrylate can be purchased commercially or prepared in-house. In a specific embodiment of the present invention, the preparation method of the polyethylene glycol methacrylate is as follows: polyethylene glycol and methacrylic anhydride are reacted under ice bath conditions in the presence of triethylamine and 4-dimethylaminopyridine to obtain the polyethylene glycol methacrylate.

[0025] Preferably, the preparation method of the polyethylene glycol methacrylate coupled with glycyrrhetinic acid includes the following steps: coupling polyethylene glycol methacrylate and glycyrrhetinic acid under the action of a condensing agent to obtain the polyethylene glycol methacrylate coupled with glycyrrhetinic acid.

[0026] Preferably, in the preparation method of polyethylene glycol methacrylate coupled with glycyrrhetinic acid, the molar ratio of polyethylene glycol methacrylate to glycyrrhetinic acid is (0.1-4):(0.1-4); in a specific embodiment of the present invention, the molar ratio of polyethylene glycol methacrylate to glycyrrhetinic acid is 1:1.

[0027] Preferably, in the preparation method of polyethylene glycol methacrylate coupled with glycyrrhetinic acid, the condensing agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU) hexafluorophosphate; the molar ratio of the condensing agent to glycyrrhetinic acid is (0.1-4):(0.1-4), and more preferably 1:1.

[0028] Preferably, the method for preparing the hydrophobic monomer shown in formula (I) includes the following steps: (1) P-cresol and diphenylphosphonic chloride were subjected to an acylation reaction under the action of a base and a catalyst to obtain 4-methylphenyl diphenylphosphonate; (2) The 4-methylphenyl diphenylphosphonate obtained in step (1) and N-bromosuccinimide were subjected to a substitution reaction under the action of an initiator to obtain 4-bromotolyl diphenylphosphonate; (3) The 4-bromotolyl diphenylphosphonate obtained in step (2) and thiourea were subjected to a substitution reaction under alkaline conditions to obtain 4-(mercaptomethyl)phenyl diphenylphosphonate; (4) The 4-(mercaptomethyl)phenyl diphenylphosphonate obtained in step (3) and 2-(2-pyridyldithio)methyl methacrylate ethyl ester are mixed and reacted to obtain the hydrophobic monomer shown in formula (I).

[0029] This invention utilizes diphenylphosphonic acid chloride as the synthetic source to synthesize toluene diphenylphosphonate, and constructs a disulfide bond structure that releases persulfate upon response. A double bond suitable for RAFT polymerization is introduced into the molecular structure as a hydrophobic monomer. This hydrophobic monomer is then polymerized with polyethylene glycol containing a double bond with a targeting group (glycyrrhetinic acid) to prepare an amphiphilic random copolymer. This copolymer, in response to superoxide anions, releases persulfate and consumes a large amount of reactive oxygen species, inhibiting lipid peroxidation and treating acute liver injury.

[0030] Preferably, in step (1), the molar ratio of p-cresol to diphenylphosphonic chloride is (0.1-4):(0.12-6); in a specific embodiment of the present invention, the molar ratio of p-cresol to diphenylphosphonic chloride is 1:1.2.

[0031] Preferably, in step (1), the base comprises triethylamine; and the catalyst comprises 4-dimethylaminopyridine.

[0032] Preferably, in step (2), the molar ratio of 4-methylphenyl diphenylphosphonate to N-bromosuccinimide is (0.1-4):(0.11-4.4); in a specific embodiment of the present invention, the molar ratio of 4-methylphenyl diphenylphosphonate to N-bromosuccinimide is 1:1.1.

[0033] Preferably, in step (2), the initiator includes azobisisobutyronitrile.

[0034] Preferably, in step (3), the molar ratio of 4-bromotolyl diphenylphosphonate to thiourea is (0.1-4):(0.11-4.4); in a specific embodiment of the present invention, the molar ratio of 4-bromotolyl diphenylphosphonate to thiourea is 1:1.1.

[0035] Preferably, in step (3), the alkaline conditions are obtained by adding an alkali, which includes n-hexylamine.

[0036] In step (4), the ethyl 2-(2-pyridyl dithio)methacrylate can be obtained commercially or prepared in-house. In a specific embodiment of the present invention, the ethyl 2-(2-pyridyl dithio)methacrylate is prepared by reacting 2,2′-dithiodipyridine and 2-mercaptoethanol to obtain 2-(2-pyridyl dithio)ethanol, and then reacting the obtained 2-(2-pyridyl dithio)ethanol and methacrylic anhydride under the action of triethylamine and 4-dimethylaminopyridine to obtain the ethyl 2-(2-pyridyl dithio)methacrylate.

[0037] Preferably, in step (4), the molar ratio of 4-(mercaptomethyl)phenyl diphenylphosphonate and 2-(2-pyridyldithio)methyl methacrylate is (0.1-4):(0.11-4.4); in a specific embodiment of the present invention, the molar ratio of 4-(mercaptomethyl)phenyl diphenylphosphonate and 2-(2-pyridyldithio)methyl methacrylate is 1:1.1.

[0038] Thirdly, the present invention provides the application of the superoxide anion-responsive nano-formulation in the preparation of drugs for treating acute liver injury.

[0039] The nano-formulation of this invention uses diphenylphosphonate bonds with a superoxide anion specific response, which has a superoxide anion responsive effect. The surface has a glycyrrhetinic acid structure that targets liver parenchymal cells. By targeting the lesion site on the surface, the nanoparticles are enriched at the lesion site. After the response, the polymer skeleton can release persulfides, which can eliminate reactive oxygen species, inhibit lipid peroxidation, and treat acute liver injury.

[0040] Preferably, the concentration range of the nano-formulation for treatment is 0.6 mg / mL - 10 mg / mL.

[0041] In a mouse liver ischemia-reperfusion model, the nano-formulation of this invention can precisely target the lesion site, prolong circulation time, and improve bioavailability. Upon response, the nano-formulation of this invention releases persulfide molecules to exert a therapeutic effect.

[0042] This invention offers the following advantages: The nanoformulation of this invention is prepared via rapid nanocomplexation technology from a random copolymer composed of a superoxide anion-responsive persulfate donor and a hepatocyte-targeting glycyrrhetinic acid-PEG monomer. The persulfate donor generated after the hydrophobic region responds eliminates reactive oxygen species, thereby inhibiting lipid peroxidation. The nanoformulation of this invention uses a modified persulfate precursor as the hydrophobic end of an amphiphilic random polymer. Upon reaching the inflammatory site, it releases persulfate molecules in response to superoxide anion stimulation, significantly improving the bioavailability of the persulfate. Secondly, compared to nanoformulations encapsulating therapeutic agents, the nanoformulation of this invention possesses an almost pure drug core, thus exhibiting higher drug loading and lower toxicity. Furthermore, this nanoformulation not only contains a single drug core but also boasts higher drug loading and fewer side effects. The nanoformulation itself is a drug, effectively addressing the challenges of early drug leakage and the difficulty in removing residues after drug release. This cutting-edge technology possesses unique characteristics and broad translational potential, providing a new approach for treating acute severe diseases such as acute liver injury. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the preparation method of the superoxide anion-responsive nano-formulation in Example 1; Figure 2 The 1H NMR spectrum of 4-methylphenyldiphenylphosphonate in Example 1; Figure 3 The 1H NMR spectrum of 4-bromotolyl diphenylphosphonate in Example 1; Figure 4 The 1H NMR spectrum of 4-(mercaptomethyl)phenyl diphenylphosphonate in Example 1; Figure 5 The 1H NMR spectrum of 2-(2-pyridinedithio)ethanol in Example 1; Figure 6 The 1H NMR spectrum of 2-(2-pyridinedithio)ethyl-methacrylate in Example 1; Figure 7 The 1H NMR spectrum of the hydrophobic monomer in Example 1; Figure 8 The 1H NMR spectrum of methacrylic acid-PEG-glycyrrhetinic acid in Example 1; Figure 9 The results of DLS testing and surface potential of the nano-formulation in Example 1 are shown. Figure 10 The graph shows the critical micelle concentration test results for the nano-formulation in Example 1. Figure 11 Hemolysis of the nano-formulation of Example 1 at different doses; Figure 12The serum ALT and AST levels are those obtained after treatment with the nano-formulation in Example 1. Detailed Implementation

[0044] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0045] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0046] Example 1 A flowchart of a method for preparing a superoxide anion-responsive nano-formulation is shown below. Figure 1 As shown, the specific steps include: (1) 5.00 g (46.27 mmol, 1.00 eq) of p-cresol, 6.78 g (55.53 mmol, 1.2 eq) of triethylamine, 0.47 g (4.63 mmol, 0.10 eq) of 4-dimethylaminopyridine, and 100 mL of anhydrous tetrahydrofuran were placed in a round-bottom flask. The resulting solution was cooled to 0 °C in an ice bath. Then, diphenylphosphonochloride (13.14 g, 55.53 mmol, 1.2 eq) was added dropwise using a constant-pressure dropping funnel. The reaction system was then restored to room temperature and the reaction continued. 16 After h, the generated salt was filtered off, tetrahydrofuran was removed by rotary evaporation, the product was redissolved in ethyl acetate and washed twice with saturated NaHCO3 solution and saturated brine solution, respectively. The organic layer was separated, dried with Na2SO4, concentrated by rotary evaporation, and purified by column chromatography to obtain a white solid 4-methylphenyl diphenylphosphonate, the 1H NMR spectrum of which is shown below. Figure 2 As shown, the synthesis path is as follows: ; In a round-bottom flask, 10.00 g of 4-methylphenyl diphenylphosphonate (32.43 mmol, 1.00 eq), 6.35 g of N-bromosuccinimide (35.68 mmol, 1.10 eq), and 80 mL of benzene were added. The mixture was stirred until the solid dissolved. After bubbling with nitrogen for 30 min, 0.53 g of azobisisobutyronitrile (0.10 eq) was added in a single injection using a syringe. The mixture was heated to reflux, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the reaction mixture was cooled to room temperature, washed with saturated brine, and the organic layer was separated. The organic layer was dried with Na2SO4 and purified by column chromatography to obtain a pale yellow solid, 4-bromotolyl diphenylphosphonate. Its 1H NMR spectrum is shown below. Figure 3As shown, the synthesis path is as follows: ; 10.00 g (25.83 mmol, 1.00 eq) of 4-bromotolyl diphenylphosphonate and 30 mL of methanol were placed in a round-bottom flask. The resulting suspension was bubbled with N2 for 15 minutes. Then, thiourea (2.16 g, 28.41 mmol, 1.10 eq) was added in a single dose under N2 protection. The reaction was stirred at room temperature, and the solid slowly dissolved to obtain a clear yellow solution. The methanol was then removed by rotary evaporation. The intermediate was then suspended in chloroform, and the suspension was bubbled with N2 for 15 minutes. Then, n-hexylamine (2.87 g, 28.41 mmol, 1.10 eq) was added in a single dose using a syringe. The suspension slowly dissolved to obtain a bright yellow solution. The organic phase was washed with 1M HCl, saturated NaHCO3, and saturated brine. The organic layer was separated and dried over anhydrous sodium sulfate. Column chromatography was used to purify the solution to obtain a white solid, 4-(mercaptomethyl)phenyl diphenylphosphonate. Its 1H NMR spectrum is shown below. Figure 4 As shown, the synthesis path is as follows: ; 2,2′-Dithiodipyridine (8.46 g, 38.40 mmol, 1.50 eq) was dissolved in dichloromethane, and 2-mercaptoethanol (2.00 g, 25.60 mmol, 1.00 eq) was added. After stirring at room temperature for 2 h, the solvent was removed by rotary evaporation. The solution was purified by column chromatography to obtain a pale yellow liquid, 2-(2-pyridinedithio)ethanol. Its 1H NMR spectrum is shown below. Figure 5 As shown, the synthesis path is as follows: ; 2-(2-pyridinedithio)ethanol (5.00 g, 26.70 mmol, 1.00 eq), triethylamine (8.11 g, 80.10 mmol, 3.00 eq), and 4-dimethylaminopyridine (0.33 g, 2.67 mmol, 0.10 eq) were dissolved in anhydrous dichloromethane. Then, methacrylic anhydride (12.35 g, 80.10 mmol, 3 eq) was added dropwise using a constant-pressure dropping funnel under ice bath conditions. After reacting in an ice bath for 30 min, the ice bath was removed, and the mixture was stirred overnight at room temperature. The reaction mixture was then washed multiple times with 1M dilute hydrochloric acid and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate. Column chromatography was used to purify the mixture into a pale yellow liquid, 2-(2-pyridinedithio)ethyl-methacrylate, whose 1H NMR spectrum is shown below. Figure 6 As shown, the synthesis path is as follows: ; 2-(2-pyridinedithio)ethyl-methacrylate (0.82 g, 3.23 mmol, 1.10 eq) was dissolved in dichloromethane, and 4-(mercaptomethyl)phenyl diphenylphosphonate (1.00 g, 2.94 mmol, 1.00 eq) was added. The mixture was stirred overnight at room temperature, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography to obtain a white solid, which was the hydrophobic monomer MA-SS-DPP. Its 1H NMR spectrum is shown below. Figure 7 As shown, the synthesis path is as follows: ; (2) PEG 1000 10.00 g (9.70 mmol, 1.00 eq), triethylamine (0.98 g, 9.70 mmol, 1.00 eq), and 4-dimethylaminopyridine (0.12 g, 0.97 mmol, 0.10 eq) were dissolved in anhydrous dichloromethane. Then, methacrylic anhydride (1.49 g, 9.70 mmol, 1.00 eq) was added dropwise using a constant-pressure dropping funnel under ice bath conditions. After reacting in the ice bath for 30 min, the ice bath was removed, and the mixture was stirred overnight at room temperature. The resulting solvent was concentrated using a rotary evaporator, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain a white powder of methacrylic acid-PEG (MA-PEG). 1000 The synthesis path is as follows: ; Glycyrrhetinic acid (GA) (17.12 g, 36.40 mmol, 1.00 eq) and EDCI (6.20 g, 40.04 mmol, 1.10 eq) were dissolved in anhydrous dichloromethane. MA-PEG was also dissolved in dichloromethane in another bottle. 1000 (40.00 g, 36.40 mmol, 1.00 eq) and DMAP were added dropwise to a polyethylene glycol solution under ice-water bath conditions. The mixture was stirred at room temperature for 6 h. The reaction solution was concentrated, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain methacrylic acid-PEG-glycyrrhetinic acid (MA-PEG). 1k -GA) white powder product, its 1H NMR spectrum is as follows Figure 8 As shown, the synthesis path is as follows: ; (3) Mix MA-DPP (1.60 g, 3287.36 μmol, 50 eq) and MA-PEG 1k (0.73 g, 657.48 μmol, 8 eq), MA-PEG 1k-GA (0.24 g, 164.36 μmol, 2 eq) was added to a Shrek flask, followed by 1148 μL of a 20 g / L solution of 4-cyano-4-(thiobenzoylthio)valerate (CPADB) (22.96 mg, 82.2 μmol, 1.00 eq) and 696 μL of a 2 g / L solution of azobisisobutyronitrile (AIBN) (1.36 mg, 8.24 μmol, 0.1 eq). After deoxygenation using a double-row tube freeze-thaw cycle at least three times, the reaction was carried out at 75 °C in the dark for 48 hours. After the reaction was completed, the orange-yellow oily product (DPP-PEG) was collected by sedimentation in ice-cold ether. 1K -GA). The synthesis path is as follows: ; (4) DPP-PEG dissolved in organic phase DMSO was injected into channel 1 of a three-channel confined impinging jet (CIJ) reactor using the FNC nano-self-assembly platform. 1k A solution of GA polymer (6 mg / mL) was prepared, and ultrapure water was injected into the other two channels. The solutions in the three channels were then thoroughly mixed at a constant preset flow rate using a CNC high-pressure injection pump to prepare uniform DTC-PEG-Man NPs nanoparticles. Then, the organic solvent DMSO was removed by dialyzing in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to obtain the superoxide anion responsive nano-formulation.

[0047] Example 2 A flowchart of a method for preparing a superoxide anion-responsive nano-formulation is shown below. Figure 1 As shown, the specific steps include: (1) 5.00 g (46.27 mmol, 1.00 eq) of p-cresol, 6.78 g (55.53 mmol, 1.2 eq) of triethylamine, 0.47 g (4.63 mmol, 0.10 eq) of 4-dimethylaminopyridine, and 100 mL of anhydrous tetrahydrofuran were placed in a round-bottom flask. The resulting solution was cooled to 0 °C in an ice bath. Then, diphenylphosphonochloride (13.14 g, 55.53 mmol, 1.2 eq) was added dropwise using a constant-pressure dropping funnel. The reaction system was then restored to room temperature and the reaction continued. After 16 h, the generated salt was filtered off, and the tetrahydrofuran was removed by concentration under reduced pressure. The solution was redissolved in ethyl acetate and washed twice with saturated NaHCO3 solution and saturated brine, respectively. The organic layer was separated, dried with Na2SO4, concentrated by rotary evaporation, and purified by column chromatography to obtain a white solid. The synthetic route is as follows: ; In a round-bottom flask, 10.00 g of 4-methylphenyl diphenylphosphonate (32.43 mmol, 1.00 eq), 6.35 g of N-bromosuccinimide (35.68 mmol, 1.10 eq), and 80 mL of benzene were added. The mixture was stirred until the solid dissolved. After bubbling with nitrogen for 30 min, 0.53 g of azobisisobutyronitrile (0.10 eq) was added in one go using a syringe. The mixture was heated to reflux, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the reaction mixture was cooled to room temperature, washed with saturated brine, and the organic layer was separated. The organic layer was dried with Na2SO4 and purified by column chromatography to obtain a pale yellow solid, 4-bromotolyl diphenylphosphonate. The synthetic route is as follows: ; 4-Bromotolyl diphenylphosphonate (10.00 g, 25.83 mmol, 1.00 eq) and 30 mL of methanol were placed in a round-bottom flask. The resulting suspension was bubbled with N2 for 15 minutes. Then, thiourea (2.16 g, 28.41 mmol, 1.10 eq) was added in a single dose under N2 protection. The reaction was stirred at room temperature, and the solid slowly dissolved to obtain a clear yellow solution. The methanol was then removed by rotary evaporation. Subsequently, the intermediate was suspended in chloroform, and the suspension was bubbled with N2 for 15 minutes. Then, n-hexylamine (2.87 g, 28.41 mmol, 1.10 eq) was added in a single dose using a syringe. The suspension slowly dissolved to obtain a bright yellow solution. The organic phase was washed with 1M HCl, saturated NaHCO3, and saturated brine. The organic layer was separated and dried over anhydrous sodium sulfate. Column chromatography was used to purify the solution to obtain a white solid, 4-(mercaptomethyl)phenyl diphenylphosphonate. The synthetic route is as follows: ; 2,2′-Dithiodipyridine (8.46 g, 38.40 mmol, 1.50 eq) was dissolved in dichloromethane, and 2-mercaptoethylamine (1.98 g, 25.60 mmol, 1.00 eq) was added. After stirring at room temperature for 2 h, the solvent was removed by rotary evaporation, and the solution was purified by column chromatography to obtain a pale yellow liquid. The synthetic route is as follows: ; 2-(2-pyridinedithio)mercaptoethylamine (4.77 g, 26.70 mmol, 1.00 eq), triethylamine (8.11 g, 80.10 mmol, 3.00 eq), and 4-dimethylaminopyridine (0.33 g, 2.67 mmol, 0.10 eq) were dissolved in anhydrous dichloromethane. Then, methacrylic anhydride (12.35 g, 80.10 mmol, 3 eq) was added dropwise using a constant-pressure dropping funnel under ice bath conditions. After reacting in an ice bath for 30 min, the ice bath was removed, and the mixture was stirred overnight at room temperature. The reaction mixture was then washed multiple times with 1M dilute hydrochloric acid and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate. Column chromatography was used to purify the mixture, yielding a pale yellow liquid. The synthetic route is as follows: ; N-(2-mercaptoethyl)methacrylamide (0.47 g, 3.23 mmol, 1.10 eq) was dissolved in dichloromethane, and 4-((pyridin-2-yldithioalkyl)methyl)phenyl diphenylphosphine ester (1.32 g, 2.94 mmol, 1.00 eq) was added. The mixture was stirred overnight at room temperature, and the solvent was removed by rotary evaporation. The product was purified by column chromatography to obtain a white solid, which is the hydrophobic monomer MA-SS-DPP. The synthetic route is as follows: ; (2) PEG 1000 10.00 g (9.70 mmol, 1.00 eq), triethylamine (0.98 g, 9.70 mmol, 1.00 eq), and 4-dimethylaminopyridine (0.12 g, 0.97 mmol, 0.10 eq) were dissolved in anhydrous dichloromethane. Then, methacrylic anhydride (1.49 g, 9.70 mmol, 1.00 eq) was added dropwise using a constant-pressure dropping funnel under ice bath conditions. After reacting in the ice bath for 30 min, the ice bath was removed, and the mixture was stirred overnight at room temperature. The resulting solvent was concentrated using a rotary evaporator, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain a white powder, PEG-methacrylic acid (MA-PEG). 1000 The synthesis path is as follows: ; GA (17.12 g, 36.40 mmol, 1.00 eq) and EDCI (6.20 g, 40.04 mmol, 1.10 eq) were dissolved in anhydrous dichloromethane. In another flask, MA-PEG1000 (40.00 g, 36.40 mmol, 1.00 eq) and DMAP were dissolved in dichloromethane. The activated carboxylic acid solution was added dropwise to the polyethylene glycol solution under an ice-water bath. The mixture was stirred at room temperature for 3–6 h. The reaction solution was concentrated, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain methacrylic acid-PEG-glycyrrhetinic acid (MA-PEG). 1k GA) is a white powder product. The synthetic route is as follows: ; (3) Mix MN-SS-DPP (1.60 g, 3287.36 μmol, 50 eq) and MA-PEG 1k (0.73 g, 657.48μmol, 8 eq), MA-PEG 1k -GA (0.24 g, 164.36 μmol, 2 eq) was added to a Shrek flask, followed by 1148 μL of a 20 g / L solution of 4-cyano-4-(thiobenzoylthio)valerate (CPADB) (22.96 mg, 82.2 μmol, 1.00 eq) and 696 μL of a 2 g / L solution of azobisisobutyronitrile (AIBN) (1.36 mg, 8.24 μmol, 0.1 eq). After deoxygenating using a double-row tube freeze-thaw cycle at least three times, the mixture was reacted at 75 °C in the dark for 48 hours. The product, an orange-yellow oil, was collected after sedimentation in ice-cold ether, which was the polymer DPP-PEG. 1K -GA. The synthesis path is as follows: ; (4) DPP-PEG dissolved in organic phase DMSO was injected into channel 1 of a three-channel confined impinging jet (CIJ) reactor using the FNC nano-self-assembly platform. 1k A 6 mg / ml solution of DPP-PEG polymer was prepared by injecting ultrapure water into the other two channels and then thoroughly mixing the solutions in all three channels at a constant preset flow rate using a CNC high-pressure injection pump to produce a homogeneous DPP-PEG. 1k -GA NPs nanoparticles were then dialyzed in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to remove the organic solvent DMSO, thus obtaining the superoxide anion-responsive nanoformulation.

[0048] Tests and characterization of the nano-formulation in Example 1: 1. Basic physical properties of nanoparticles Dynamic light scattering tests showed that the particle size of the nano-formulation in Example 1 was stable within the range of 40.00-45.00 nm with excellent repeatability, while the Zeta potential was approximately -25 mV. Figure 9 .

[0049] 2. Determination of the critical micelle concentration of polymers The critical micelle concentration (CMC) of the nanoformulation in Example 1 was determined using the classic pyrene fluorescent dye method. The test results are as follows: Figure 10 As shown, the measured value was 0.0389 mg / mL.

[0050] 3. Biocompatibility testing of nanoparticles like Figure 11 As shown, nanoparticles of different concentrations were dispersed in blood samples of a certain volume fraction and incubated at room temperature for 3 h. The supernatant was separated by centrifugation. Samples treated with surfactant (TX-100) and 1×PBS served as negative and positive controls, respectively. The hemolysis of different doses of nanoparticles was determined using an ELISA reader. The results are as follows. Figure 11 As shown, by Figure 11 It can be seen that even blood cells incubated with nanoparticles at a concentration of 1000 μg / mL still retain intact cell membranes, indicating that the nano-formulation of Example 1 has good blood compatibility and meets the safety standards for in vivo application.

[0051] 4. The therapeutic effect of nanoparticles on acute liver injury The specific procedures for establishing a mouse model of liver ischemia-reperfusion injury are as follows: Mice were fasted for 12 hours preoperatively but allowed free access to water. At the start of the experiment, mice were anesthetized and fixed in a supine position on the experimental board. The skin was prepared and disinfected with iodine. The abdominal skin and endothelium of the mouse were sequentially incised along the midline of the abdomen. After opening the abdomen, the portal vein and hepatic artery were clamped with non-invasive hemostatic clips. The color of the left middle lobe of the liver changed from red to yellowish-brown, indicating successful occlusion of hepatic blood flow. During the clamping period, the mouse was placed on a 37°C constant-temperature electric blanket, and the wound was covered with warm, moist gauze for protection. After 30 minutes, the hemostatic clips were removed, and the left middle lobe of the liver gradually returned to its rosy color, indicating restoration of blood supply. The abdominal skin was sutured layer by layer, and the mouse's condition was observed and recorded. After successful model establishment, medication was administered via tail vein injection. After successful model establishment, mice in different groups were injected with different doses of the drug. Blood samples were collected from the inner canthal vein of the eye at 0h, 6h, 12h, and 24h. The samples were incubated at room temperature for 3h, then centrifuged (3000 rpm, 8 min) to remove blood cells, and the supernatant serum was retained for later use. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two important liver function indicators. During liver ischemia-reperfusion injury, cell membrane damage leads to the release of ALT and AST into the blood, resulting in a significant increase in serum liver enzyme levels. Detecting serum ALT and AST levels can be used to assess liver health. The test results are shown below. Figure 12 .

[0052] Figure 12 The results of ALT and AST studies confirmed the restorative effect of the nano-formulation on liver function in HIRI mice.

[0053] 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A superoxide anion-responsive nano-formulation, characterized in that, The superoxide anion-responsive nanoformulation consists of two parts: a hydrophobic region and a hydrophilic region. The hydrophilic region comprises polyethylene glycol methacrylate coupled with glycyrrhetinic acid and polyethylene glycol methacrylate. The hydrophobic region comprises a hydrophobic monomer, the structure of which is shown in Formulas I-IV. Formula I, Formula II, Formula III, Formula IV.

2. The superoxide anion-responsive nano-formulation according to claim 1, characterized in that, The molar ratio of the hydrophobic monomer, polyethylene glycol methacrylate coupled with glycyrrhetinic acid, and polyethylene glycol methacrylate is (10-40):(1-4):(2-8).

3. The superoxide anion-responsive nano-formulation according to claim 1, characterized in that, The structure of the polyethylene glycol methacrylate coupled with glycyrrhetinic acid is shown in Formula V: Equation V, where n = 4-500.

4. A method for preparing a superoxide anion-responsive nano-formulation according to any one of claims 1-3, characterized in that, Includes the following steps: Hydrophobic monomers, polyethylene glycol methacrylate coupled with glycyrrhetinic acid and polyethylene glycol methacrylate are subjected to free radical polymerization to obtain a polymer. The obtained polymer is then self-assembled to obtain the superoxide anion responsive nano-formulation.

5. The method for preparing superoxide anion-responsive nano-formulation according to claim 4, characterized in that, The polymer includes block copolymers and random copolymers, and the free radical polymerization reaction is a reversible addition-fracture chain transfer free radical polymerization reaction.

6. The method for preparing superoxide anion-responsive nano-formulation according to claim 5, characterized in that, The free radical polymerization reaction is carried out in the presence of a chain transfer agent and an initiator. The chain transfer agent includes at least one of dithiocarbonates, trithiocarbonates, xanthates, and dithiocarbamates. The initiator includes at least one of azo compounds, organic peroxides, and inorganic peroxides. The molar ratio of polyethylene glycol methacrylate, chain transfer agent, and initiator is (5-20):(1-4):(0.1-0.4). The free radical polymerization reaction is carried out under light-protected conditions at a temperature of 60℃-80℃ for 16h-48h.

7. The method for preparing superoxide anion-responsive nano-formulation according to claim 4, characterized in that, The polyethylene glycol methacrylate coupled with glycyrrhetinic acid is prepared by the following steps: polyethylene glycol methacrylate and glycyrrhetinic acid are coupled together under the action of a condensing agent. The molar ratio of polyethylene glycol methacrylate to glycyrrhetinic acid is (0.1-4):(0.1-4). The condensing agent is selected from one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; The molar ratio of the condensing agent to glycyrrhetinic acid is (0.1-4):(0.1-4).

8. The method for preparing superoxide anion-responsive nano-formulation according to claim 4, characterized in that, The preparation method of the hydrophobic monomer shown in formula (Ⅰ) includes the following steps: (1) P-cresol and diphenylphosphonic chloride were subjected to an acylation reaction under the action of a base and a catalyst to obtain 4-methylphenyl diphenylphosphonate; (2) The 4-methylphenyl diphenylphosphonate obtained in step (1) and N-bromosuccinimide were subjected to a substitution reaction under the action of an initiator to obtain 4-bromotolyl diphenylphosphonate; (3) The 4-bromotolyl diphenylphosphonate obtained in step (2) and thiourea were subjected to a substitution reaction under alkaline conditions to obtain 4-(mercaptomethyl)phenyl diphenylphosphonate; (4) The 4-(mercaptomethyl)phenyl diphenylphosphonate obtained in step (3) and 2-(2-pyridyldithio)methyl methacrylate ethyl ester are mixed and reacted to obtain the hydrophobic monomer shown in formula (I).

9. The method for preparing superoxide anion-responsive nano-formulation according to claim 8, characterized in that, At least one of the following must be met: (1) In step (1), the molar ratio of p-cresol and diphenylphosphonic chloride is (0.1-4):(0.12-6). (2) In step (1), the base includes triethylamine; the catalyst includes 4-dimethylaminopyridine; (3) In step (2), the molar ratio of 4-methylphenyl diphenylphosphonate and N-bromosuccinimide is (0.1-4):(0.11-4.4). (4) In step (2), the initiator includes azobisisobutyronitrile; (5) In step (3), the molar ratio of 4-bromotolyl diphenylphosphonate and thiourea is (0.1-4):(0.11-4.4). (6) In step (3), the alkaline conditions are obtained by adding an alkali, which includes n-hexylamine; (7) In step (4), the molar ratio of 4-(mercaptomethyl)phenyl diphenylphosphonate and 2-(2-pyridyldithio)ethyl methacrylate is (0.1-4):(0.11-4.4).

10. The use of the superoxide anion-responsive nanoformulation according to any one of claims 1-3 or the superoxide anion-responsive nanoformulation according to any one of claims 4-9 in the preparation of drugs for treating acute liver injury.

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