ROS (reactive oxygen species) and esterase double-response type nano preparation as well as preparation method and application thereof

Abstract

The invention relates to an ROS (reactive oxygen species) and esterase dual-response type nano preparation as well as a preparation method and application thereof, and belongs to the technical field of nano preparations. According to the nano preparation, itaconic acid is modified to serve as a hyperbranched polymer skeleton, after the itaconic acid reaches an inflammation part, the hyperbranched polymer responds to disintegrate under the stimulation of ROS, and then the itaconic acid medicine active ingredients are released under the further action of esterase, so that the curative effect of the itaconic acid is improved. The cell membrane permeability and bioavailability of the exogenous itaconic acid are obviously improved; secondly, compared with a nano preparation for encapsulating a therapeutic agent, the nano preparation disclosed by the invention almost has a pure drug core, so that the nano preparation has relatively high drug loading capacity and relatively low toxic and side effects; according to the nano preparation, the therapeutic agent is used as a nano carrier skeleton, so that the problems of premature leakage of the therapeutic agent and removal of foreign substances after parent drugs are released can be effectively avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of nano preparations, and in particular relates to a ROS and esterase dual-responsive nano preparation and a preparation method and application thereof. Background Art

[0002] Acute liver injury (ALI) is a clinical syndrome caused by multiple factors in a short period of time, which leads to increased oxidative stress in the liver, inflammatory response, and apoptosis or necrosis of liver cells. It is usually caused by viral infection, alcohol abuse, ischemia, drug poisoning or other external factors. The incidence of drug-induced liver injury (DILI), especially acetaminophen (APAP, a common antipyretic analgesic), has been increasing and has become the main cause of acute liver failure.

[0003] Usually, patients with mild symptoms will only have abnormal liver function, which will recover on their own after stopping taking the relevant drugs. However, due to clinical uncertainty, other causes may be missed and further intervention treatment may not be given, resulting in worsening of the condition. There are currently two main treatments approved for the treatment of patients with drug-induced acute liver injury. One is to limit the absorption of toxic drugs by quickly removing them to prevent further liver toxicity. For example, activated charcoal is widely used to treat drug overdose, such as acetaminophen, barbiturates, and carbamazepine. However, since its therapeutic effect is limited to within 3-4 hours of acute ingestion of toxic drugs, this treatment method is highly dependent on the patient's timely admission to the hospital for treatment, the doctor's recognition of potential DILI, and the availability of medical resources. Another widely recognized treatment method is to use drug-specific antidotes, such as N-acetyl-L-cysteine ​​(NAC), which can promote the synthesis of glutathione (GSH) in hepatocytes to consume toxic N-acetyl-p-benzoquinoneimine (NAPQI) and is currently the only approved DILI antidote. However, in clinical treatment, NAC can only have a therapeutic effect within 8 hours after APAP poisoning. Once the best treatment time is misjudged, the only remedy may be liver transplantation. Even if medical treatment is sought in time, intravenous NAC can be accompanied by severe nausea, vomiting, allergies and other common adverse reactions.

[0004] Therefore, it is of great significance to develop new nanoformulations for the treatment of acute liver injury. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and to provide a ROS and esterase dual-responsive nanoformulation and a preparation method and application thereof.

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

[0007] In a first aspect, the present invention provides a ROS and esterase dual-responsive nanoformulation, the nanoformulation comprising a hydrophobic core and a hydrophilic shell; the hydrophobic core is formed by polycondensation of a branch point and a hydrophobic monomer, the branch point comprising at least one of 1,3,5-trihydroxybenzene, 1,3,5-benzenetrimethanol, cyanuric chloride, and glycerol; the general structural formula of the hydrophobic monomer is shown in Formula I:

[0008]

[0009] Wherein, R1 is as shown in Formula II to Formula VI:

[0010]

[0011] The present invention utilizes the high concentration of ROS microenvironment in the lesion site of drug-induced liver injury and the anti-inflammatory effect of itaconic acid to construct a stimulus-responsive nanoparticle for treating acute liver injury. Through condensation polymerization, a hyperbranched polymer with dual response of ROS and esterase based on itaconic acid skeleton is synthesized. The hydrophobic core of the nanoformulation is synthesized by polycondensation reaction with ROS-responsive thioketal alcohol and itaconic acid with anti-inflammatory effect as the main raw materials, and finally the hydrophilic shell is modified by esterification reaction. The rigid structure of the branching point and the hydrophobic monomer act together as the hydrophobic inner cavity of the nanoparticle to ensure the internal stability of the structure. Due to the poor permeability of exogenous itaconic acid on the cell membrane, the present invention uses itaconic acid as a hyperbranched polymer skeleton by modification to ensure that the drug can effectively reach the inflammatory site. Under the stimulation of ROS, the polymer thioketal skeleton of the nanoformulation first responds to disintegration, and then releases the itaconic acid drug active ingredient under the further action of esterase, significantly improving the cell membrane permeability and bioavailability of exogenous itaconic acid; secondly, compared with the nanoformulation encapsulating the therapeutic agent, the nanoformulation of the present invention has a pure drug core and therefore has a higher drug loading capacity and lower toxic side effects. In addition, the nanoformulation of the present invention uses the therapeutic agent as a nanocarrier skeleton to effectively avoid the problem of premature leakage of the therapeutic agent and removal of foreign substances after the release of the parent drug.

[0012] Preferably, the hydrophilic shell comprises polyethylene glycol and polyethylene glycol-mannose.

[0013] In order to achieve better therapeutic effects, the nanoformulation is effectively targeted and delivered into macrophages. The hydrophilic shell of the present invention includes polyethylene glycol and polyethylene glycol-mannose, wherein the polyethylene glycol derivative modified with mannose is used as the target head, and the mannose at the end of the hydrophilic chain has the ability to improve the nanoformulation's ability to target macrophages, which can effectively avoid being taken up by other parts and achieve long circulation in the body.

[0014] Preferably, the molar ratio of the hydrophobic core to the hydrophilic shell is 1:(0.1-0.4).

[0015] More preferably, the molar ratio of the hydrophobic core to the hydrophilic shell is 1:0.25.

[0016] More preferably, the relative molecular weight of the polyethylene glycol and the polyethylene glycol in the polyethylene glycol mannose is independently 0.2-10KDa, and each independently includes PEG 200 PEG 400 PEG 1K PEG 2K At least one of .

[0017] Preferably, the nanoformulation is obtained by coupling a hydrophobic core and a hydrophilic shell.

[0018] More specifically, the hydrophobic core is first coupled with polyethylene glycol, and then coupled with polyethylene glycol-mannose to obtain the nanoformulation.

[0019] The hydrophobic core and the hydrophilic shell are connected by an esterification reaction.

[0020] Preferably, the polyethylene glycol (eg, PEG 1000 ) and polyethylene glycol-mannose (e.g. PEG 1000 -Man) is 1:0.25.

[0021] In a second aspect, the present invention provides a method for preparing the ROS and esterase dual-responsive nanoformulation, comprising the following steps:

[0022] (1) itaconic anhydride and a ROS-responsive compound undergo esterification to obtain a hydrophobic monomer;

[0023] (2) subjecting the hydrophobic monomer and branch points obtained in step (1) to polycondensation under the action of a condensation agent to obtain a hydrophobic core;

[0024] (3) coupling the hydrophobic core obtained in step (2) with the hydrophilic shell under the action of a condensing agent, and then self-assembling to obtain the nanoformulation;

[0025] The structure of the ROS-responsive compound is shown in Formula V:

[0026] wherein R1 is the formula II to formula VI described in claim 1.

[0027] Preferably, when R1 in the ROS responsive compound is Formula II, the method for preparing the ROS responsive compound comprises the following steps:

[0028] S1. reacting mercaptopropionic acid and acetone in the presence of a catalyst to obtain an intermediate product;

[0029] S2. subjecting the intermediate product obtained in step S1 to a reduction reaction under the action of a reducing agent to obtain the ROS response compound;

[0030] The structure of the intermediate product is shown in Formula VI:

[0031]

[0032] The nano preparation of the present invention couples itaconic acid to both ends of the responsive thioketal as a hydrophobic monomer, polycondenses with the branching point to form a hydrophobic core, and the peripherally modified hydrophilic core forms a hyperbranched polymer, and the polymer is further self-assembled to obtain hyperbranched polymer nanoparticles capable of treating acute liver injury, especially drug-induced liver injury. After the hydrophobic core responds to ROS and esterase, the released itaconic acid can play an anti-inflammatory, anti-oxidative and cell death signaling pathway regulating role. The present invention realizes the rapid preparation of ROS and esterase dual-responsive hyperbranched polymers based on itaconic acid skeleton to efficiently deliver exogenous itaconic acid, so that the hyperbranched polymer nano preparation has multiple functions of accurate delivery, inflammatory response reduction and reversal of acute liver injury. The proposal of this technology has potential clinical application value and future application prospects, and provides a new direction for the treatment of drug-induced liver injury.

[0033] Preferably, in step S1, the catalyst comprises trifluoroacetic acid (TFA).

[0034] Preferably, in step S1, the molar ratio of the catalyst, acetone and mercaptopropionic acid is 0.01:(0.8-2.0):(2.0-3.0), more preferably 0.01:1.0:2.2.

[0035] Preferably, in step S2, the reducing agent includes borane dimethyl sulfide (BH3·SMe2).

[0036] Preferably, in step S2, the molar ratio of the intermediate product to the reducing agent is 1:(1.5-4.5), more preferably 1:3.

[0037] Preferably, when R1 in the ROS responsive compound is Formula III, the preparation method of the ROS responsive compound comprises the following steps: reacting selenium powder and 2-bromoethanol under the action of sodium borohydride to obtain the ROS responsive compound.

[0038] Preferably, the molar ratio of the selenium powder to the 2-bromoethanol is 1:(0.5-1.5); more preferably 1:0.95.

[0039] Preferably, in step (1), the molar ratio of the ROS responsive compound to itaconic anhydride is 1:1:(1.8-6).

[0040] More preferably, in step (1), the molar ratio of the ROS responsive compound to itaconic anhydride is 1:2.1.

[0041] Preferably, in step (1), the temperature of the esterification reaction is 25°C-80°C, and the time is 24h-72h.

[0042] Preferably, in step (1), the solvent for the esterification reaction includes toluene and n-hexane.

[0043] Preferably, in step (2), the molar ratio of the branch point to the hydrophobic monomer is 1:(1-6).

[0044] More preferably, in the step (2), the molar ratio of the branching point to the hydrophobic monomer is 1:3.

[0045] In the present invention, the molar ratio of the branch point to the hydrophobic monomer may be any one of 1:1, 1:3, and 1:6.

[0046] Preferably, in the step (3), the hydrophobic core is first coupled with polyethylene glycol and then coupled with polyethylene glycol-mannose to obtain a hydrophilic shell, and the molar ratio of the hydrophobic core to the polyethylene glycol and polyethylene glycol-mannose is 1:(0.1-1):(0.01-0.1); more preferably, the molar ratio of the hydrophobic core to the polyethylene glycol and polyethylene glycol-mannose is 1:0.25:0.0625.

[0047] Preferably, in step (2) and step (3), the condensing agent includes any one of dicyclohexylcarbodiimide (DCC) / N,N-diisopropylethylamine (DIPEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC) / 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / triethylamine (TEA).

[0048] Preferably, in the step (2), the polycondensation time is 12h-72h.

[0049] Preferably, in step (3), the coupling time is 12h-72h.

[0050] Preferably, the structural formula of the hydrophobic core is as shown in Formula VIII:

[0051]

[0052] Among them, R is One of;

[0053] R1 is One of them.

[0054] The structure shown in Formula VIII is a representative unit of the hyperbranched polymer structure of the present invention.

[0055] Preferably, in the step (3), the self-assembly adopts the rapid nanoprecipitation (FNC) method; more preferably, in the rapid nanoprecipitation method, the oil phase solvent includes one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile, methanol, and tetrahydrofuran; the oil phase and the water phase are nano-complexed in a ratio of 1:(6-9) (for example, 1:6, 1:8, 1:9); the flow rate ratio of oil phase: water phase: water phase is (1-3): (4.5-13.5): (4.5-13.5) (for example, 1:4.5:4.5, 2:9:9, 3:13.5:13.5); the method for removing the oil phase solvent includes at least one of dialysis, rotary evaporation, and vacuum drying.

[0056] The present invention uses a flash nanocomplexation (FNC) platform to regulate the parameters of nanoparticles to obtain nanoparticles with uniform size, good stability under pathological and physiological conditions, and strong pharmacokinetics.

[0057] In a specific embodiment of the present invention, the self-assembly process is as follows: through the FNC nano self-assembly platform, a condensed polymer (1-16 mg / mL) solution dissolved in the organic phase DMSO is injected into channel 1 of a three-channels confined impinging jet reactor (CIJ), and ultrapure water is injected into the other two channels. Then, a CNC high-pressure injection pump is used to fully mix the solutions in the three channels at a constant preset flow rate to prepare uniform nanoparticles, and then the organic phase is removed by dialyzing in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to obtain the nanoformulation.

[0058] Preferably, the nanoparticles prepared in the present invention are in the shape of spherical particles, the particle size of the nanoparticles is 50nm-150nm, the polydispersity index PDI of the nanoparticles is 0.05-0.35, and the zeta potential of the nanoparticles is -10mV to -40mV.

[0059] Preferably, the nanoparticle preparation of the present invention is stored in the form of a freeze-dried preparation; the self-assembled nanoparticle aqueous solution is mixed with a freeze-drying protective agent and frozen at -80°C, and the frozen product is placed in a freeze dryer away from light to prepare freeze-drying; the freeze-dried preparation uses salt as a preparation resuspension buffer to balance the pH value during freeze-drying storage;

[0060] The freeze-drying protective agent is selected from alcohol freeze-drying protective agents and sugar freeze-drying protective agents, wherein the alcohol freeze-drying protective agent is any one or more combinations of xylitol, mannitol, sorbitol or glycine; the sugar freeze-drying protective agent is any one or more combinations of glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan or inulin;

[0061] The concentration of the freeze-drying protective agent is selected from one of 0%, 5%, 10%, 15%, 20%, and 25%; the salt buffer is selected from any one of sodium phosphate or potassium phosphate or a combination of the two.

[0062] In a third aspect, the present invention provides the use of the ROS and esterase dual-responsive nanoformulation in the preparation of a drug for treating acute liver injury.

[0063] Preferably, the present invention provides the use of the ROS and esterase dual-responsive nanoformulation in the preparation of a drug for treating drug-induced liver injury.

[0064] The nano preparation of the present invention can be used in the form of injection to treat acute liver injury, especially drug-induced liver injury, such as drug-induced liver injury caused by APAP.

[0065] Preferably, the nanoformulation has a therapeutic itaconic acid concentration in the liver ranging from 0.5 mg / kg to 50 mg / kg.

[0066] The present invention has the following beneficial effects:

[0067] (1) After the nanoformulation of the present invention reaches the inflammation site, the thioketal reaction breaks the bond after contacting the high concentration ROS microenvironment, consuming ROS and reducing oxidative stress. At the same time, under the further action of esterase in the cell, the itaconic acid drug component in the nanoformulation is released, producing anti-inflammatory and antioxidant effects, and blocking multiple cell death signal pathways.

[0068] (2) The present invention uses chemical means such as reduction and esterification to synthesize hydrophobic monomers, and further uses recrystallization, column chromatography and extraction to separate and purify the products. Among them, the hydrophobic monomers of the present invention have a wide range of applications; the polycondensation method is simple, low cost, and high reaction efficiency; the molecular design capability is strong, and dendritic and star-shaped polymers can be prepared.

[0069] (3) The technology of the present invention has good universality and can flexibly regulate the ratio of hydrophobic monomers and branching points to construct a series of drug delivery platforms with different drug loadings. It also has the advantages of continuity, large scale, narrow distribution and high reproducibility between batches, providing a new idea for the treatment of acute and severe diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Schematic diagram of the process for preparing the ROS and esterase dual-responsive nanoformulation of Example 1;

[0071] Figure 2 The NMR spectrum of the intermediate product COOH-TK-COOH prepared in Example 1;

[0072] Figure 3 The NMR spectrum of the ROS responsive compound OH-TK-OH prepared in Example 1;

[0073] Figure 4 The NMR spectrum of the hydrophobic monomer IA-TK-IA prepared in Example 1;

[0074] Figure 5 The cytotoxicity results of the nanoformulation prepared in Example 1;

[0075] Figure 6 The hemolysis test results of the nanoformulation prepared in Example 1;

[0076] Figure 7 The results of the ROS scavenging ability test of the nanoformulation prepared in Example 1 at the cellular level;

[0077] Figure 8 The anti-apoptosis ability test results of the nanoformulation prepared in Example 1 at the cellular level;

[0078] Fig. 9 This is an anatomical diagram of liver tissue of the nanoformulation prepared in Example 1;

[0079] Fig.10 The results of liver tissue staining using the nanoformulation or NAC prepared in Example 1;

[0080] Fig.11 The graph is a liver serum ALT test result graph using the nanoformulation or NAC prepared in Example 1;

[0081] Fig.12 This is a graph showing the results of liver serum AST testing using the nanoformulation or NAC prepared in Example 1;

[0082] Fig.13 The antioxidant capacity evaluation results of the nanoformulation or NAC prepared in Example 1 at the tissue level;

[0083] Fig.14 The results of evaluating the anti-inflammatory ability of the nanoformulation or NAC prepared in Example 1 at the tissue level;

[0084] Fig.15 This is a graph showing the biocompatibility results of the nanoformulation prepared in Example 1. DETAILED DESCRIPTION

[0085] 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. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0086] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0087] Example 1

[0088] The schematic diagram of the preparation method of the ROS and esterase dual-responsive nanoformulation provided in this embodiment is as follows Figure 1 As shown, the specific steps include:

[0089] (1) Mercaptopropionic acid (2.2 eq) and trifluoroacetic acid (TFA) (0.01 eq) were dissolved in acetone (11 mL, 1 eq). The mixture was stirred at room temperature overnight to generate a large amount of white crystals. A large amount of ultrapure water was added to completely dissolve the crystals at 65°C. The crystals were then placed in a refrigerator for recrystallization overnight. The insoluble matter was washed with ultrapure water by suction filtration and dried in a vacuum drying oven to obtain an intermediate product (COOH-TK-COOH). The NMR spectrum is shown in Figure 2 ; The synthesis path is as follows:

[0090]

[0091] (2) COOH-TK-COOH (1.0 eq) was dissolved in anhydrous THF (15 mL). Nitrogen was introduced and stirred for 30 min under ice bath conditions. BH3·SMe2 (3.0 eq) solution was slowly added dropwise under ice bath conditions. The reaction was continued with stirring for 30 min and then continued with stirring at room temperature for 3 h. After the reaction was completed, the insoluble matter was dissolved with a small amount of MeOH, and water was slowly added to quench the reaction until no bubbles appeared in the reaction solution. Ethyl acetate was added to dissolve the organic phase, and the organic phase was extracted with NaHCO3, H2O and NaCl respectively, dried with anhydrous sodium sulfate, and separated and purified by column chromatography to obtain a ROS-responsive compound (OH-TK-OH). The NMR spectrum is shown in Figure 3 ; The synthesis path is as follows:

[0092]

[0093] (3) Itaconic anhydride (2.1 eq) and OH-TK-OH (1.0 eq) were poured into a round-bottom flask, oxygen was extracted by a water pump, a nitrogen balloon was inserted to maintain a nitrogen atmosphere, a mixed solution of toluene / n-hexane = 1 / 1 (v / v) was added into the flask by a syringe, and the mixture was stirred at 60° C. for 36 h. After the reaction was completed, the solution was evaporated into a colorless oil, dissolved in ethyl acetate, and extracted three times with a saturated sodium bicarbonate solution. The mixed aqueous phase was extracted once with ether, and the aqueous phase was acidified to pH = 2 with 6 M concentrated hydrochloric acid. The solution was extracted once with ethyl acetate, and dried with anhydrous sodium sulfate. The mixture was separated and purified by column chromatography to obtain a hydrophobic monomer (IA-TK-IA). The NMR spectrum is shown in FIG. Figure 4 ; The synthesis path is as follows:

[0094]

[0095] (4) IA-TK-IA (3.0 eq), 1,3,5-trihydroxybenzene (1.0 eq), DCC (6.5 eq) and DIPEA (6.5 eq) were dissolved in 10 mL of anhydrous DMF and heated and stirred for 60 h under nitrogen atmosphere. After the reaction, PEG was directly added. 1000 (0.25eq), DCC (0.25eq) and DIPEA (0.25eq), and continued stirring for 24h. After the reaction was completed, it was dialyzed with DMF for 60h, and then dialyzed with ultrapure water for 72h, and freeze-dried to obtain a target head-free polymer (HBP (IA) -PEG);

[0096] HBP (IA) -PEG was dissolved in DMF and PEG was added 1000-mannose (0.0625eq), DCC (0.0625eq) and DIPEA (0.0625eq) were stirred for reaction for 24h. After the reaction, the mixture was dialyzed with DMF for 60h and then with ultrapure water for 72h. The polymer (HBP) containing a hydrophilic shell was obtained by freeze-drying. (IA) -PEG-Man);

[0097] (5) HBP dissolved in organic phase DMSO was injected into channel 1 of the three-channel confined impact jet reactor through the FNC nano-self-assembly platform. (IA) -PEG-Man polymer (8 mg / mL) solution was added, and ultrapure water was injected into the other two channels, and then the solutions in the three channels were fully mixed at a constant preset flow rate by a digitally controlled high-pressure injection pump to prepare uniform nanoparticles, and then, DMSO in the nanoparticles was removed by dialyzing in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa), thereby obtaining the nanoparticle preparation (HBP (IA) -PEG-Man NPs).

[0098] Example 2

[0099] The preparation method of the ROS and esterase dual-responsive nanoformulation provided in this embodiment specifically comprises the following steps:

[0100] (1) Sodium borohydride (1.05 eq) was dissolved in deionized water (150 mL) under an argon stream. Then, Se powder (1.0 eq) was carefully added to obtain a dark solution. Then, 2-bromoethanol (0.95 eq) in 150 mL THF was injected under an argon stream. The reaction was carried out at 50° C. for 6 hours and then extracted with DCM. The organic phase was dried over magnesium sulfate overnight. The crude product was separated and purified by column chromatography (DCM / ethyl acetate=1 / 1, v / v) to obtain a ROS-responsive compound (OH-Se-Se-OH). The synthetic route is as follows:

[0101]

[0102] (2) Itaconic anhydride (2.1 eq) and OH-Se-Se-OH (1.0 eq) were dissolved in a mixed solution of toluene / n-hexane = 1 / 1 (v / v), maintained under nitrogen atmosphere, and refluxed at 60° C. for 36 h. After the reaction, the solution was evaporated to a colorless oil, dissolved in ethyl acetate, and extracted three times with a saturated sodium bicarbonate solution. The mixed aqueous phase was extracted once with ether, and the aqueous phase was acidified to pH = 2 with 6 M concentrated hydrochloric acid. The solution was extracted once with ethyl acetate, dried with anhydrous sodium sulfate, and separated by column chromatography to obtain a hydrophobic monomer (IA-Se-Se-IA). The synthesis route is as follows:

[0103]

[0104] (3) Prepare a hyperbranched polymer and a nanoformulation according to steps (4) and (5) of Example 1.

[0105] Example 3

[0106] The preparation method of the ROS and esterase dual-responsive nanoformulation provided in this embodiment specifically comprises the following steps:

[0107] (1) IA-TK-IA (3.0 eq), 1,3,5-benzenetrimethylol (1.0 eq), DCC (6.5 eq) and DIPEA (6.5 eq) prepared in Example 1 were dissolved in 10 mL of anhydrous DMF and heated and stirred under nitrogen for 60 h. After the reaction was completed, PEG was directly added. 1000 (0.25eq), DCC (0.25eq) and DIPEA (0.25eq), and continued stirring for 24h. After the reaction was completed, it was dialyzed with DMF for 60h, then dialyzed with ultrapure water for 72h, and freeze-dried to obtain a target head-free polymer;

[0108] HBP (IA) -PEG was dissolved in DMF and PEG was added 1000 - mannose (0.0625eq), DCC (0.0625eq) and DIPEA (0.0625eq) were stirred for reaction for 24h. After the reaction, the mixture was dialyzed with DMF for 60h and then with ultrapure water for 72h. The polymer containing the hydrophilic shell was lyophilized;

[0109] (2) Prepare the nanoformulation according to step (5) of Example 1.

[0110] Example 4

[0111] The preparation method of the ROS and esterase dual-responsive nanoformulation provided in this embodiment is different from that in Example 3 in that 1,3,5-benzenetrimethylol is replaced with glycerol, and the rest is the same as in Example 3 to obtain the nanoformulation of this embodiment.

[0112] Performance Test:

[0113] 1. Characterization of the therapeutic effect of the nanoformulation prepared in Example 1 at the cellular level

[0114] 1.1 Cytotoxicity assay

[0115] The isolated AML-12 and Raw264.7 cells were inoculated and cultured for 6 h, and different concentrations of nanoparticle aqueous solutions were added and cultured for 12 h (the concentrations written here represent the concentrations of nanoparticles HBP in all nanoparticle aqueous solutions). (IA) -PEG-Man NPs concentration). Cytotoxicity results are shown in Figure 5As shown, HBP exposed to AML-12 and Raw264.7, respectively (IA) -The dose of PEG-Man NPs (from 0 to 100 μg mL -1 ), no cytotoxicity was observed. -1 At high equivalent drug concentrations, cell viability was still well maintained and the survival rate exceeded 95%, indicating that the preparation had good cell compatibility.

[0116] 1.2 Nanoparticle hemolysis detection

[0117] Nanoparticles of different concentrations were 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 nanoparticles of different doses was determined by means of an ELISA instrument. The results are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that the blood cells incubated with nanoparticles at a concentration of 2000 μg / mL still retain intact cell membranes, indicating that the nanoparticles obtained by the present invention have good blood compatibility and meet the safety standards for in vivo application.

[0118] 1.3 Detection of ROS scavenging ability at the cellular level

[0119] After LPS induces macrophage damage, a large amount of hydrogen peroxide is produced in the mitochondria, and superoxide anions / hydroxyl free radicals cause oxidative stress. Excessive ROS seriously damages the structure and function of the cell membrane, mitochondria and endoplasmic reticulum. Figure 7 The results of the ROS scavenging ability test of the nanoformulation prepared in Example 1 at the cellular level are as follows: Figure 7 As shown, after the nanoparticles respond to ROS, they release itaconic acid to exert anti-inflammatory and antioxidant effects in the cells. This also explains why the nanoparticles can significantly reduce the ROS level in macrophages after the cells are attacked by LPS, showing a significant ROS scavenging effect. In all the tested concentration ranges, the nanoparticles with an IA concentration of 2 μg / mL have the best ability to scavenge ROS, which greatly promotes the recovery of the cellular redox state.

[0120] 1.4 Detection of anti-apoptosis ability at the cell level

[0121] According to the pathogenic mechanism of APAP, macrophages secrete a series of inflammatory factors and chemokines after activation, leading to an inflammatory cascade that further aggravates liver cell damage. The alternating culture method was used to culture macrophages first. After the preparation was administered, as the function of macrophages recovered, the hepatocyte AML-12 was further incubated with the conditioned medium of macrophages. Finally, the apoptosis of AML-12 was detected by flow cytometry. Figure 8As shown, the apoptosis flow cytometry results showed that the apoptosis of hepatocytes can be effectively inhibited by regulating the crosstalk between macrophages and hepatocytes, thereby reducing the proportion of apoptotic cells to a level close to normal.

[0122] 2. Characterization of the therapeutic effect of the nanoformulation prepared in Example 1 on the animal model of acetaminophen-induced liver injury

[0123] 2.1 Liver tissue anatomy

[0124] 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. Fig. 9 As shown, the livers of mice in the drug-induced acute liver injury model constructed with APAP had large areas of bleeding and necrotic lesions. After treatment with NAC and nanoparticles, the bleeding and necrotic areas were significantly reduced, even approaching the normal liver morphology.

[0125] Fig. 9 Representative liver images after dissection of mice in each group; scale bar: 1 cm.

[0126] 2.2 Liver histopathological evaluation

[0127] Pathological staining:

[0128] The liver tissue was fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin and sliced. The paraffin sections were then stained with H&E. Histopathological evaluation found that the nanoparticles were able to effectively reduce the area of ​​liver necrotic lesions after administration, while the NAC administration group only partially reduced the area of ​​liver damage lesions. This shows that our preparation has excellent liver protection function, thereby effectively restoring the morphology and function of the liver.

[0129] Fig.10 This is hematoxylin-eosin (H&E) staining of liver tissue. The black dotted area is the necrotic area. Scale bar: 100 μm.

[0130] 2.3 Monitoring of serum liver enzyme levels

[0131] In the APAP overdose-induced acute liver injury mouse model, the therapeutic effect of the preparation was evaluated by preventive administration (0h administration) and therapeutic administration (3h), and the APAP antidote positive drug NAC commonly used in clinical practice was used as a control. Blood was collected from mice at 6h, 12h and 24h during the treatment process, and the supernatant was centrifuged to obtain serum, and the ALT and AST levels in the serum were detected using commercially purchased kits.

[0132] Fig.11 and Fig.12Liver serum liver enzyme indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), n=8-16.

[0133] Serum ALT and ALT gradually increase after injury, indicating severe liver damage. HBP (IA) -PEG-Man NPs (in Fig.11 and Fig.11 HBP (IA) NPs) 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.

[0134] 2.4 Assessment of antioxidant capacity at the tissue level

[0135] The marker of oxidative stress damage is the increase in ROS content. The pathophysiological process of ALI is accompanied by the generation of a large amount of ROS. As expected ( Fig.13 A), APAP induces cells to produce a large amount of superoxide, which leads to the collapse of the antioxidant system. The dihydroethidium (DHE) stained sections of the ALI model group have a strong red fluorescence signal. In contrast, the DHE level after nanoparticle administration was significantly reduced, indicating that oxidative stress damage was alleviated to a certain extent. At the same time, compared with the model group, the disorder of the oxidative system led to an increase in MDA levels, while GSH and SOD decreased. The nanoparticle administration group had a certain degree of increase, which means that nanoparticles can reduce the production of MDA in mouse liver tissue and restore the activity of SOD. Especially when treated with a dose of 2mg / kg at 0h, the superoxide in the mouse body was close to the normal level, restoring the antioxidant function of the liver.

[0136] 2.5 Evaluation of anti-inflammatory capacity at the tissue level

[0137] Immunofluorescence staining of the liver revealed that in the damaged CD31-marked liver microvascular endothelial periphery in APAP-induced ALI mouse liver tissue, intercellular adhesion molecule 1 (ICAM-1) was significantly downregulated after nanoparticle administration ( Fig.14 A), while the expression of ICAM-1 was barely inhibited in the NAC-administered group.

[0138] After administration of the preparation, the expression level of F4 / 80 positive mononuclear macrophages ( Fig.14B) is reduced to normal levels, which means that after administration of the preparation, due to the blocking of inflammatory factors in resident macrophages and the effective inhibition of oxidative stress, further damage to the liver microvascular endothelium can be effectively prevented, and at the same time, the recruitment of mononuclear macrophages is reduced, thereby effectively restoring the homeostasis of the liver inflammatory microenvironment, which will effectively prevent further damage to liver cells.

[0139] 2.6 Biocompatibility assessment

[0140] To evaluate the in vivo toxicity of the nanoparticles, H&E staining of relevant biomedical indicators and major organs was performed, e.g. Fig.15 As shown in C, the results showed that all treatment groups did not cause pathological tissue abnormalities or necrosis, indicating the in vivo safety of the nanoparticles. At the same time, the results showed that all biomedical indicators, including creatinine (Cre) and urea nitrogen (BUN), were within the normal range, indicating that no systemic toxicity occurred in any treatment, such as Fig.15 A, as shown in 15B.

[0141] The nanoformulations of Example 2 and Example 1 have similar ROS response groups, and the nanoformulations of Example 3 and Example 4 have similar skeleton structures, and are expected to have similar therapeutic effects to the nanoformulation of Example 1.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A ROS and esterase dual-responsive nanoformulation, characterized in that: The nanoformulation comprises a hydrophobic core and a hydrophilic shell; the hydrophobic core is formed by polycondensation of branch points and hydrophobic monomers, and the branch points comprise at least one of 1,3,5-trihydroxybenzene, 1,3,5-benzenetrimethanol, cyanuric chloride, and glycerol; the general structural formula of the hydrophobic monomer is shown in Formula I: Wherein, R1 is as shown in Formula II to Formula VI:

2. The ROS and esterase dual-responsive nanoformulation according to claim 1, characterized in that: The hydrophilic shell includes polyethylene glycol and polyethylene glycol-mannose.

3. The ROS and esterase dual-responsive nanoformulation according to claim 1, characterized in that: The nanoformulation is obtained by coupling a hydrophobic core and a hydrophilic shell.

4. A method for preparing the ROS and esterase dual-responsive nanoformulation according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) itaconic anhydride and a ROS-responsive compound undergo esterification to obtain a hydrophobic monomer; (2) subjecting the hydrophobic monomer and branch points obtained in step (1) to polycondensation under the action of a condensation agent to obtain a hydrophobic core; (3) coupling the hydrophobic core obtained in step (2) with the hydrophilic shell under the action of a condensing agent, and then self-assembling to obtain the nanoformulation; The structure of the ROS-responsive compound is shown in Formula V: wherein R1 is the formula II to formula VI described in claim 1.

5. The method for preparing the ROS and esterase dual-responsive nanoformulation according to claim 4, characterized in that: In the step (1), the molar ratio of the ROS responsive compound to itaconic anhydride is 1:(1.8-6).

6. The method for preparing the ROS and esterase dual-responsive nanoformulation according to claim 4, characterized in that: In the step (2), the molar ratio of the branch point to the hydrophobic monomer is 1:(1-6).

7. The method for preparing the ROS and esterase dual-responsive nanoformulation according to claim 4, characterized in that: In the step (3), the hydrophobic core is first coupled with polyethylene glycol and then coupled with polyethylene glycol-mannose to obtain a hydrophilic shell, and the molar ratio of the hydrophobic core to the polyethylene glycol and polyethylene glycol-mannose is 1:(0.1-1):(0.01-0.1).

8. The method for preparing the ROS and esterase dual-responsive nanoformulation according to claim 4, characterized in that: When R1 in the ROS responsive compound is Formula II, the preparation method of the ROS responsive compound comprises the following steps: S1. reacting mercaptopropionic acid and acetone in the presence of a catalyst to obtain an intermediate product; S2. subjecting the intermediate product obtained in step S1 to a reduction reaction under the action of a reducing agent to obtain the ROS response compound; The structure of the intermediate product is shown in Formula VI:

9. The method for preparing the ROS and esterase dual-responsive nanoformulation according to claim 4, characterized in that: Satisfy at least one of the following: (1) In the step (1), the temperature of the esterification reaction is 25°C-80°C, and the time is 24h-72h; (2) In the steps (2) and (3), the condensing agent comprises any one of dicyclohexylcarbodiimide / N,N-diisopropylethylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 4-dimethylaminopyridine, dicyclohexylcarbodiimide / 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / triethylamine; (3) In step (2), the polycondensation time is 12h-72h; (4) In step (3), the coupling time is 12h-72h; (5) In step (3), the self-assembly is performed by a rapid nanoprecipitation method.

10. Use of the ROS and esterase dual-responsive nanoformulation according to any one of claims 1 to 3 in the preparation of a drug for treating acute liver injury.

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