ROS (reactive oxygen species) response type persulfide biomacromolecule nano prodrug, nano preparation as well as preparation method and application of ROS response type persulfide biomacromolecule nano prodrug

Abstract

The invention relates to an ROS response type persulfide biomacromolecule nano prodrug, a nano preparation and a preparation method and application of the ROS response type persulfide biomacromolecule nano prodrug and the nano preparation, and belongs to the technical field of nano preparations. According to the invention, the hydrogen persulfide is protected by utilizing ROS to respond to a small molecule group, so that the defect that the existing hydrogen persulfide is unstable is overcome; meanwhile, biomacromolecules are used as a carrier of the hydrogen persulfide, so that the liver targeting property, the water solubility and the biological safety of the hydrogen persulfide are improved, and multi-site modification and adjustability of the drug loading capacity are realized; in addition, the nano preparation is simple in reaction process, mild in condition and beneficial to clinical transformation.

Description

Technical Field

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

[0002] Hepatic ischemia-reperfusion injury (IRI) is common after liver surgery or transplantation and may lead to liver failure. During hepatic ischemia-reperfusion, reactive oxygen species (ROS) are produced in large quantities, leading to a disorder of the balance between oxidative damage and antioxidant defense in the body, causing severe oxidative stress damage, and inducing the occurrence of downstream inflammatory cascades and apoptotic cascades, aggravating a large number of liver cell damage.

[0003] Many cellular redox signals rely on inorganic reactive sulfur species, and sulfur-containing compounds can play an antioxidant role by quenching free radical chain reactions. They are usually effective ROS scavengers, providing potential for the effective treatment of IRI. At present, the cysteine ​​prodrug N-acetylcysteine ​​(NAC), as the most widely used "antioxidant" in clinical research, is a typical therapeutic sulfur-containing compound (single thiol small molecule) that can quickly reduce ROS levels in the body and alleviate oxidative stress damage. It has been approved for the treatment of drug-induced acute liver injury. Compared with monosulfide compounds, each sulfur residue of hypersulfide has both nucleophilic and electrophilic properties, and compared with classical sulfur metabolites, it has a faster ROS scavenging rate and can self-catalyze regeneration cycles under physiological conditions in vivo. Studies have shown that low micromolar concentrations of polysulfide are sufficient to produce the same effective cell protection effect as millimolar concentrations of monosulfide. Despite this, hydropersulfide has poor stability and is easily oxidized. Currently, the in vivo delivery of hydropersulfide is mostly achieved through responsive hydropersulfide prodrugs, including esterase-responsive hydropersulfide donors, ROS-responsive hydropersulfide donors, pH-responsive hydropersulfide donors, light-responsive hydropersulfide donors, nitroreductase-responsive hydropersulfide donors, etc. However, the hydropersulfide prodrugs reported so far still have the characteristics of poor water solubility, short half-life, poor biological targeting, complex synthesis and high cost, which restricts the clinical application of hydropersulfide prodrugs. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and to provide a ROS-responsive persulfide biomacromolecule nanoprodrug, a nanoformulation, and a preparation method and application thereof.

[0005] The present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a ROS-responsive persulfide biomacromolecule nanoprodrug, the general structural formula of the nanoprodrug is shown in Formula I:

[0007]

[0008] Wherein, R1 is a biological macromolecule; n≥2;

[0009] R2 is shown in Formula II-III:

[0010]

[0011] The present invention synthesizes a ROS-responsive per(poly)sulfide biomacromolecule nanoprodrug by connecting a ROS-responsive group to a biomacromolecule carrier via a disulfide bond, and is used to treat IRI with ROS overload as an important pathological process; the prodrug utilizes the targeting of the biomacromolecule to the liver to deliver hydrogen per(poly)sulfide to the lesion site, reacts with the overloaded ROS at the lesion site, and the ROS-responsive group breaks off and leaves to expose hydrogen per(poly)sulfide (-SSH, -(S) n H), the exposed persulfide (poly) hydrogen sulfide acts as a good reducing agent to capture ROS free radicals and terminate free radical reactions, thereby protecting cells from oxidative damage and achieving the treatment of acute liver injury caused by IR. The present invention utilizes ROS responsive groups to protect persulfide (poly) sulfide precursors so that they can exist stably. In an environment where ROS is overloaded at the lesion site, the persulfide precursor reacts with reactive oxygen and exposes hydrogen persulfide (-SSH), thereby removing overloaded reactive oxygen to protect liver damage, and achieving stable delivery and controllable release of hydrogen persulfide.

[0012] Preferably, the biomacromolecule comprises one of bovine serum albumin (BSA), human serum albumin, porcine serum albumin, donkey serum albumin, mouse serum albumin, cystatin, and glutathione.

[0013] Preferably, in the formula I, 2≤n≤5.

[0014] In a second aspect, the present invention provides a method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug, comprising the following steps: mixing a thiol-containing biomacromolecule with a precursor compound, and performing a thiol-disulfide bond exchange reaction to obtain the ROS-responsive persulfide biomacromolecule nanoprodrug; the structure of the precursor compound is shown in Formula IV to Formula V:

[0015] In formula IV to formula V, the R3 are each independently One of them.

[0016] The present invention utilizes the targeting of biomacromolecules to the liver to efficiently deliver hydrogen persulfide (poly)sulfide to the liver; utilizes thiol-disulfide exchange reaction to synthesize asymmetric persulfide (poly)sulfide, with mild reaction conditions and simple synthesis process, which reduces the generation of reaction byproducts. At the same time, the prepared ROS-responsive persulfide (poly)sulfide biomacromolecule nanoformulation has good ROS scavenging effect in vitro and in vivo, and has good therapeutic effect in the liver ischemia-reperfusion injury disease model, and related indicators can be restored to normal levels.

[0017] Preferably, the ratio of the molar amount of thiol groups in the thiol-containing biomacromolecule to the molar amount of the precursor compound is 1:(1-25).

[0018] In the present invention, the molar ratio of the thiol group in the thiol-containing biomacromolecule to the molar ratio of the precursor compound is any one of or both of 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:16, 1:20, and 1:25.

[0019] Preferably, the solvent for the thiol-disulfide exchange reaction includes at least one of water, EDTA buffer solution, phosphate buffer, borate buffer, Tris buffer, HEPES buffer, dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, acetonitrile, acetone and tetrahydrofuran.

[0020] Preferably, the thiol-containing biomacromolecule is a biomacromolecule containing thiol itself or a biomacromolecule grafted with thiol.

[0021] Preferably, the biomacromolecule grafted with thiol groups is obtained by reducing the biomacromolecule; specifically, the preparation method of the biomacromolecule grafted with thiol groups comprises the following steps: reducing the disulfide bonds of the biomacromolecule with a reducing agent to obtain the biomacromolecule grafted with thiol groups; the reducing agent comprises at least one of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine, and a metal reducing agent.

[0022] By adjusting the ratio of the biomacromolecule to the reducing agent, the number of thiol groups on the biomacromolecule can be controlled, thereby adjusting the amount of the loaded precursor compound. Specifically, the ratio of the biomacromolecule to the reducing agent can be (10-100) mg: (0.001-0.03) mmol.

[0023] The present invention directly utilizes the sulfhydryl groups on the biomacromolecule as modification sites, or reduces the disulfide bonds on the biomacromolecule to convert the disulfide bonds into sulfhydryl groups as modification sites, and reacts the sulfhydryl groups with precursor compounds containing ROS responsive groups to connect the over (poly)sulfide containing ROS responsive groups to the biomacromolecule through disulfide bonds.

[0024] Preferably, when n=2, the preparation method of the precursor compound shown in formula IV comprises the following steps: ROS-responsive small molecule A and symmetrical persulfide are reacted by sulfhydryl-disulfide exchange reaction to obtain the precursor compound shown in formula IV; the structure of the ROS-responsive small molecule A is shown in formula A:

[0025]

[0026] Preferably, when n=2, the preparation method of the precursor compound shown in formula V comprises the following steps: a ROS-responsive small molecule B reacts with a symmetrical persulfide to obtain a precursor compound shown in formula V; the structure of the ROS-responsive small molecule B is shown in formula B:

[0027]

[0028] Preferably, when n=2, in the preparation method of the precursor compound shown in Formula IV or Formula V, the symmetrical persulfide includes one of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2,2'-dithiodipyridine (DTDP), and 2,2'-dithiobis(pyridine-1-oxide); the molar ratio of the ROS-responsive small molecule B to the symmetrical persulfide is 1:(1-2); the molar ratio of the ROS-responsive small molecule A to the symmetrical persulfide A is 1:(1-2); and the solvent for the thiol-disulfide exchange reaction includes at least one of anhydrous dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, dichloromethane, and chloroform.

[0029] The ROS-responsive small molecules A and B contain ROS-responsive groups, which can not only protect the hydrogen persulfide groups, but also expose hydrogen persulfide after responding and leaving the lesion site, thereby achieving controllable release at the lesion site.

[0030] Preferably, the ROS-responsive small molecule A is obtained by a substitution reaction between a phenylboronic acid pinacol ester small molecule and a sulfur-containing compound; the phenylboronic acid pinacol ester small molecule includes 4-(chloromethyl)phenylboronic acid pinacol ester, 4-(bromomethyl)phenylboronic acid pinacol ester, 4-(iodomethyl)phenylboronic acid pinacol ester, 4-(hydroxymethyl)phenylboronic acid pinacol ester, 4-(bromoethyl)phenylboronic acid pinacol ester, 4-(chloroethyl)phenylboronic acid pinacol ester, and 4-(iodoethyl)phenylboronic acid pinacol ester; the sulfur-containing compound includes one of thiourea, sodium sulfide, sodium hydrosulfide, ammonium hydrosulfide, and sulfur chloride; the molar ratio of the small molecule containing phenylboronic acid pinacol ester to the sulfur-containing compound is 1:(0.5-4); the solvent for the substitution reaction is at least one of ethanol, methanol, dichloromethane, chloroform, tetrahydrofuran, acetone, dimethyl sulfoxide, N,N-dimethylformamide, and formamide.

[0031] In the present invention, the molar ratio of the small molecule containing phenylboronic acid pinacol ester to the sulfur-containing compound is any one of 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4 or both of the range values.

[0032] Preferably, the ROS-responsive small molecule B is obtained by heating α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylpropionic acid with Lawesson's reagent.

[0033] Specifically, the preparation method of the ROS-responsive small molecule B comprises the following steps:

[0034] (1) Tert-butyl isobutyl ester and 2-bromobenzyl bromide are reacted by nucleophilic substitution to obtain 1,1-dimethylethyl 2-bromo-α,α-dimethylphenylpropionate; the synthetic route is as follows:

[0035]

[0036] (2) reacting the 1,1-dimethylethyl 2-bromo-α,α-dimethylphenylpropionate obtained in step (1) with bis(pinacol)diboron under alkaline conditions to obtain 1,1-dimethylethyl α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)phenylpropionate; the synthesis route is as follows:

[0037]

[0038] (3) hydrolyzing the 1,1-dimethylethyl α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylpropanoate obtained in step (2) in the presence of an acid to obtain α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylpropanoic acid; the synthesis route is as follows:

[0039]

[0040] (4) heating the α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylpropionic acid obtained in step (3) and Lawesson's reagent to obtain the ROS-responsive small molecule B; the synthesis route is as follows:

[0041]

[0042] Preferably, in step (3), the acid comprises at least one of trifluoroacetic acid, hydrochloric acid, phosphoric acid and sulfuric acid.

[0043] Polysulfides have stronger free radical capture ability than persulfides. When n>2, it can be expected that the nanoprodrug has equivalent or greater effect in treating liver damage.

[0044] Preferably, when n=3, the preparation method of the precursor compound shown in formula IV comprises the following steps: subjecting thioacetic acid to a thiol-disulfide exchange reaction with 2,2'-dithiodipyridine to obtain an asymmetric persulfide, subjecting the obtained asymmetric persulfide to a thiol-disulfide exchange reaction with 4-mercaptomethylphenylboronic acid pinacol ester to obtain an asymmetric phenylboronic acid pinacol ester-based persulfide precursor a, reducing the reaction to obtain a precursor b, and reacting the obtained precursor b with 2-pyridinethione to obtain the precursor compound shown in formula IV.

[0045] Preferably, the structure of the 4-mercaptomethylphenylboronic acid pinacol ester is shown in Formula C, the structure of the precursor a is shown in Formula D, and the structure of the precursor b is shown in Formula E:

[0046]

[0047] Specifically, in one embodiment of the present invention, when n=3, the preparation method of the precursor compound shown in Formula IV comprises the following steps: stirring thioacetic acid and 2,2'-dithiodipyridine in MeOH at room temperature under nitrogen atmosphere, monitoring the reaction progress by thin layer chromatography TLC (EtOAc), and obtaining an asymmetric persulfide (Py-SS-AC) containing an easy leaving group; dissolving Py-SS-AC in DCM, and then slowly adding 4-mercaptomethylphenylboronic acid dissolved in DCM; Pinacol ester, stirring to react, rotary evaporation to remove the solvent, column chromatography to purify the product, to obtain an asymmetric phenylboronic acid pinacol ester persulfide precursor a (PBAP-SS-AC); then PBAP-SS-AC, CuSO4.5H2O, 4.7-diphenyl-1.10-phenanthroline, Li2CO3, PhI(OPiv)2 and MeOH, stirring to react to obtain precursor b (PBAP-SSOMe); the obtained precursor b is reacted with 2-mercaptopyridine to obtain a precursor compound shown in formula IV.

[0048] Preferably, when n=4, the preparation method of the precursor compound shown in formula IV comprises the following steps: reacting benzyl alcohol and disulfur dichloride (S2Cl2) to obtain bis(benzyloxy)tetrasulfide; reacting the obtained bis(benzyloxy)tetrasulfide with α-mercaptopyridine to obtain 2,2'-tetrathiodipyridine; reacting the obtained 2,2'-tetrathiodipyridine with 4-mercaptomethylphenylboronic acid pinacol ester to obtain the precursor compound shown in formula IV.

[0049] Specifically, in one embodiment of the present invention, when n=4, the preparation method of the precursor compound shown in formula IV includes the following steps: stirring benzyl alcohol and triethylamine (NEt3) at -10°C to 5°C, adding S2Cl2, and continuing the reaction to obtain an intermediate product, bis(benzyloxy)tetrasulfide; reacting the intermediate product, bis(benzyloxy)tetrasulfide, with 2-mercaptopyridine to obtain 2,2'-tetrathiodipyridine; reacting the obtained 2,2'-tetrathiodipyridine with 4-mercaptomethylphenylboronic acid pinacol ester to obtain the precursor compound shown in formula IV.

[0050] Preferably, when n=5, the preparation method of the precursor compound shown in formula IV comprises the following steps: stirring the ROS-responsive small molecule A and pyridine at -70°C to -80°C, adding S2Cl2, and continuing the reaction to obtain an intermediate product, and reacting the intermediate product with 2,2'-dithiodipyridine at 150°C to 250°C to obtain the precursor compound shown in formula V.

[0051] In a third aspect, the present invention provides a nanoformulation, wherein the nanoformulation comprises the ROS-responsive persulfide biomacromolecule nanoprodrug.

[0052] The nano prodrug of the present invention can be used to prepare a nano preparation, which overcomes the shortcomings of short half-life, poor water solubility and lack of controlled release for pathological environments of hydropersulfide (poly)sulfide prodrugs. It can be used to treat IRI by injection. The method of preparing the nano prodrug into a nano preparation includes conventional methods such as desolvation method, coprecipitation method, salting-out method, etc. The nano preparation is prepared by self-assembly of the ROS-responsive persulfide biomacromolecule nano prodrug.

[0053] Specifically, the present invention provides a method for preparing the nano preparation, comprising the following steps: dissolving the ROS-responsive persulfide biomacromolecule nano prodrug in an organic solvent, injecting it into water, and centrifuging to obtain the nano preparation.

[0054] Preferably, the organic solvent comprises dimethyl sulfoxide.

[0055] The organic solution of the nanoprodrug is quickly injected into water, the hydrophobic end of the nanoprodrug forms a dense inner core, and the hydrophilic outer shell is arranged in the outer layer to assemble into nanoparticles, which are then collected by centrifug to obtain a nanoformulation.

[0056] In a fifth aspect, the present invention provides the use of the ROS-responsive persulfide biomacromolecule nanoprodrug and nanoformulation in the preparation of drugs for treating liver ischemia-reperfusion injury.

[0057] The present invention has the following beneficial effects:

[0058] (1) The present invention utilizes ROS-responsive groups to protect persulfide (poly)sulfide precursors so that they can exist stably. In the environment of ROS overload at the lesion site, persulfide precursors react with reactive oxygen and expose hydrogen persulfide (-SSH), thereby removing overloaded reactive oxygen to protect liver damage, and achieving stable delivery and controlled release of hydrogen persulfide. The synthesized nanoformulation has good stability (can be stably stored for 4 days at 4°C) and can be prepared into a freeze-dried preparation for long-term storage, which has clinical transformation prospects.

[0059] (2) The present invention uses biomacromolecules as carriers of hydropersulfide (polysulfide). Due to the steric hindrance of the macromolecules, the large amount of hydrogen sulfide byproducts produced by the reaction with glutathione is reduced, thereby avoiding damage to cells. At the same time, the use of biomacromolecules as carriers gives hydropersulfide (polysulfide) targeting, stability, and water solubility to the liver. In addition, as a natural component in the body, the biomacromolecule has good biological safety.

[0060] (3) The present invention prepares nanoprodrugs by utilizing thiol-disulfide exchange reaction. Compared with the traditional scheme for synthesizing persulfides, asymmetric persulfides connected by disulfide bonds are introduced and prepared under mild conditions. The synthesis conditions are mild and the synthesis steps are simple, which overcomes the shortcomings of the existing hydropersulfide prodrug synthesis process, which is complicated and costly, and has good reproducibility.

[0061] (4) The nanoformulation of the present invention has good controllability. By adjusting the number of thiol groups on the biomacromolecules during the preparation process and the reaction ratio of the persulfide small molecules containing ROS-responsive groups and the biomacromolecules, ROS-responsive persulfide biomacromolecules modified with different amounts of hydrogen persulfide groups can be obtained, thereby achieving a wide range of control over the drug loading amount. At the same time, the size of the nanoformulation can also be controlled.

[0062] (5) The present invention explores its in vivo pharmacodynamics in the classic IRI animal model and finds that the prepared ROS-responsive persulfide biomacromolecule nanoformulation has excellent therapeutic effects, and the relevant indicators can return to normal values, which has high clinical transformation value. At the same time, the optimal therapeutic dose is found. The drugs currently selected for liver injury require a large therapeutic dose. The therapeutic dose of this application is low (4-8 μmol / kg), the number of injections is small (single administration), and it has good clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a flow chart of the method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug of Example 1;

[0064] Figure 2 This is the NMR spectrum of PBAP-SH prepared in Example 1;

[0065] Figure 3 This is the NMR spectrum of PBAP-SS-Py prepared in Example 1;

[0066] Figure 4 The BSA-SS-PBAP nanoparticle size diagram obtained in Example 1 (I); the stability diagram of BSA-SS-PBAP placed in water at 4°C (II); the stability diagram placed in a 10mM, pH 7.4 PBS buffer solution at 4°C (III); the potential diagram (IV);

[0067] Figure 5 This is a graph showing the experimental data of in vitro ABTS free radical scavenging of the BSA-SS-PBAP nanoformulation obtained in Example 1;

[0068] Figure 6 This is a graph showing the experimental data of in vitro hydroxyl radical scavenging of the BSA-SS-PBAP nanoformulation obtained in Example 1;

[0069] Figure 7 In a mouse animal model of liver ischemia-reperfusion, different doses of the BSA-SS-PBAP nanoformulation (2 μmol·kg -1 , 4 μmol kg -1 , 8μmol·kg -1 ) Schematic diagram of the effect of ALT levels in mouse serum at 6h, 12h, and 24h after treatment;

[0070] Figure 8 In a mouse animal model of liver ischemia-reperfusion, different doses of the BSA-SS-PBAP nanoformulation (2 μmol·kg -1 , 4 μmol kg -1 , 8μmol·kg -1 ) Schematic diagram of the effect of AST content in mouse serum at 6h, 12h, and 24h after treatment;

[0071] Fig. 9 In a mouse animal model of liver ischemia-reperfusion, different doses of the BSA-SS-PBAP nanoformulation (2 μmol·kg -1 , 4 μmol kg -1 , 8μmol·kg -1 ) Schematic diagram of mouse liver 24 h after treatment;

[0072] Fig.10This is the NMR spectrum of PBAP-SSSS-Py prepared in Example 3;

[0073] Fig.11 This is the mass spectrum of PBAP-SSSS-Py prepared in Example 3;

[0074] Fig.12 In a mouse animal model of hepatic ischemia-reperfusion, different doses of the BSA-SS-PBAP nanoformulation obtained in Example 1 and the BSA-SSSS-PBAP nanoformulation obtained in Example 3 were used at 0 h and 3 h after reperfusion. BSA-SS-PBAP (8 μmol kg -1 )、BSA-SSSS-PBAP (8 μmol·kg -1 ) Schematic diagram of the effect of ALT levels in mouse serum at 6h, 12h, and 24h after treatment;

[0075] Fig.13 In a mouse animal model of hepatic ischemia-reperfusion, different doses of the BSA-SS-PBAP nanoformulation obtained in Example 1 and the BSA-SSSS-PBAP nanoformulation obtained in Example 3 were used at 0 h and 3 h after reperfusion. BSA-SS-PBAP (8 μmol kg -1 )、BSA-SSSS-PBAP (8 μmol·kg -1 ) Schematic diagram of the effect of AST content in mouse serum at 6h, 12h, and 24h after treatment. DETAILED DESCRIPTION

[0076] 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.

[0077] 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.

[0078] Example 1

[0079] The flow chart of the preparation method of the ROS-responsive persulfide biomacromolecule nanoprodrug provided in this embodiment is as follows Figure 1 As shown, the specific steps include:

[0080] (1) 4-bromomethylphenylboronic acid pinacol ester and thiourea were used as raw materials, the molar ratio of 4-bromomethylphenylboronic acid pinacol ester to thiourea was 1:1.1, methanol was used as solvent, the mixture was stirred in N2 atmosphere at room temperature, the solvent was evaporated by rotary evaporation, and then the obtained solid was dissolved in NaOH aqueous solution, refluxed for 1 h in N2 atmosphere, HCl was added dropwise at 0°C to adjust the pH of the obtained liquid to 2, the obtained reaction solution was extracted with ethyl acetate, the organic layer was separated and dried, and then purified by silica gel chromatography to obtain a ROS-responsive small molecule 4-mercaptomethylphenylboronic acid pinacol ester (PBAP-SH) containing a thiol group, and the NMR spectrum was shown in FIG. Figure 2 ; The synthesis path is as follows:

[0081]

[0082] (2) Using 4-mercaptomethylphenylboronic acid pinacol ester and DTDP as raw materials, the molar ratio of 4-mercaptomethylphenylboronic acid pinacol ester to DTDP is 1:2, and methanol is used as solvent. The reaction is stirred overnight at room temperature under N2 atmosphere. The resulting solution is concentrated by rotary evaporation and purified by silica gel chromatography to obtain a precursor compound (PBAP-SS-Py). The NMR spectrum is shown in Figure 3 ; The synthesis path is as follows:

[0083]

[0084] (3) A bovine serum albumin (BSA) aqueous solution and a tris(2-carboxyethyl)phosphine (TCEP) solution were mixed in a volume ratio of 1:1 (BSA concentration was 100 mg / mL; TCEP concentration was 15 mM), and the mixture was reacted on a shaker at a speed of 120 r / min for 10 minutes at a temperature ranging from room temperature to 50° C. The disulfide bonds on the BSA were reduced, and then the mixture was dialyzed and purified with ultrapure water to obtain a solution of BSA with polythiol groups (BSA-SH);

[0085] The BSA-SH solution was slowly added dropwise to the DMSO solution of PBAP-SS-Py at 200 μL / min (molar ratio of thiol: PBAP-SS-Py = 1:5), and a thiol-disulfide bond exchange reaction occurred at room temperature to obtain the nanoprodrug BSA-SS-PBAP, the structure of which is as follows:

[0086]

[0087] The obtained DMSO solution of nanoprodrug BSA-SS-PBAP is then quickly injected into water, and the hydrophobic end of BSA-SS-PBAP forms a dense inner core, and the hydrophilic outer shell is arranged in the outer layer to assemble into BSA-SS-PBAP nanoparticles, which are then collected by centrifugation to obtain BSA-SS-PBAP nanoparticles (NPs), i.e., nanoformulations.

[0088] Example 2

[0089] The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug provided in this embodiment comprises the following steps:

[0090] (1) Thioacetic acid (1 eq) and DTDP (2 eq) were stirred in MeOH under nitrogen atmosphere at room temperature, and the reaction progress was monitored by thin layer chromatography (TLC) (EtOAc) to obtain a small molecule (Py-SS-AC) containing an easy leaving group; the synthesis route is as follows:

[0091]

[0092] (2) Py-SS-AC (1.1 eq) was dissolved in DCM, and then 4-mercaptomethylphenylboronic acid pinacol ester (1 eq) prepared in Example 1 and dissolved in DCM was slowly added. After stirring at 25° C. for 12 h, the solvent was removed by rotary evaporation and purified by column chromatography to obtain precursor a (PBAP-SS-AC). The synthesis route is as follows:

[0093]

[0094] (3) PBAP-SS-AC (1 eq), CuSO4.5H2O, 4.7-diphenyl-1.10-phenanthroline, Li2CO3 (1 eq), PhI(OPiv)2 and MeOH were added to a Schlenk bottle, and the mixture was stirred at 20°C for 15 h. The mixture was quenched with saturated NaHCO3, extracted with dichloromethane, and then the organic phase was concentrated in vacuo. Precursor b (PBAP-SSOMe) was obtained by column chromatography; the synthetic route is as follows:

[0095]

[0096] (4) 2-Mercaptopyridine (1.1 eq), B(C6F5)3, PBAP-SSOMe (1 eq) and DCM / DMF were added to a Schlenk bottle, and the mixture was stirred at a constant temperature for 5-8 h under a N2 atmosphere, and then the mixture was concentrated in vacuo. The precursor compound was purified by column chromatography; the synthesis route is as follows:

[0097]

[0098] (5) The obtained precursor compound is prepared into a nano-prodrug according to step (3) in Example 1.

[0099] Example 3

[0100] The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug provided in this embodiment comprises the following steps:

[0101] (1) Add benzyl alcohol (1 eq) and disulfur dichloride (2 eq) into a reaction flask, use dichloromethane as solvent, and react at room temperature. After the reaction is terminated, remove the solvent and separate by column chromatography to obtain bis(benzyloxy)tetrasulfide: The synthesis route is as follows:

[0102]

[0103] (2) Add bis(benzyloxy)tetrasulfide (1 eq) and 2-mercaptopyridine (2 eq) into a reaction flask, use dichloromethane as solvent, react at room temperature, remove the solvent after the reaction is terminated, and separate by column chromatography to obtain 2,2'-tetrathiodipyridine; the synthesis route is as follows:

[0104]

[0105] (3) 2,2'-tetrathiodipyridine (2 eq) and 4-mercaptomethylphenylboronic acid pinacol ester (1 eq) were added to a reaction bottle, and anhydrous ethanol was used as a solvent. The reaction was carried out at room temperature under argon protection. After the reaction was terminated, the solvent was removed and column chromatography was performed to obtain the precursor compound PBA-SSSS-py; the NMR spectrum was as follows: Fig.10 As shown in the mass spectrometry Fig.11 As shown, the synthesis route is as follows:

[0106]

[0107] (4) The obtained precursor compound was prepared into nano-prodrug BSA-SSSS-PBAP according to step (3) in Example 1.

[0108] The obtained DMSO solution of nanoprodrug BSA-SSSS-PBAP is then quickly injected into water, and the hydrophobic end of BSA-SSSS-PBAP forms a dense inner core, and the hydrophilic outer shell is arranged in the outer layer to assemble into BSA-SSSS-PBAP nanoparticles, which are then collected by centrifugation to obtain BSA-SSSS-PBAP nanoparticles (NPs), i.e., nanoformulations.

[0109] Example 4

[0110] The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug provided in this embodiment comprises the following steps:

[0111] (1) PBAP-SH (1 eq) and pyridine (1 eq) prepared in Example 1 were added to 30 mL of anhydrous solvent in a thoroughly dried round-bottom flask, and reacted at -78°C for 1 h under a nitrogen atmosphere, and then S2Cl2 (0.6 eq) was added dropwise for 2 h. The temperature was then raised to room temperature, the reaction was quenched with deionized water, and the organic phase was separated and retained. The organic layer was washed with deionized water and a saturated sodium chloride solution, and the organic layer was dried with sodium sulfate, and the ROS-responsive small molecule containing a thiol group (PBAP-SSSS-PBAP) was obtained by rotary evaporation. The synthetic route is as follows:

[0112]

[0113] (2) In a flask, PBAP-SSSS-PBAP (1 eq) and DTDP (2 eq) were added, and the mixture was reacted at 180° C. for 1 h. The product was purified by silica gel chromatography to obtain a precursor compound (Py-SSSS-PBAP). The synthesis route is as follows:

[0114]

[0115] (3) The obtained precursor compound was prepared into nano prodrug BSA-SSSS-PBAP according to step (3) in Example 1. The synthesis route is as follows:

[0116]

[0117] Example 5

[0118] The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug provided in this embodiment comprises the following steps:

[0119] (1) A solution of n-butyl lithium in hexane (6.86 mL of a 1.6 M solution, 10.91 mmol) in diisopropylamine (1.53 mL, 10.91 mmol) was added to a solution of nitroformylhydrazine (HNF) (20 mL) at -78°C, and the resulting mixture was stirred under N2 for 15 min. Freshly distilled tert-butyl isobutyl ester (1.31 g, 9.09 mmol) dissolved in HNF (10 mL) was then added dropwise and stirred under N2 for 30 min. Next, 2-bromobenzyl bromide (2.50 g, 10.00 mmol) dissolved in HNF (10 mL) was added dropwise, and the mixture was allowed to warm to RT overnight. The reaction was quenched with H2O, extracted with CH2Cl2, and the organic phases were combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting product was purified by silica gel column to obtain 1,1-dimethylethyl 2-bromo-α,α-dimethylphenylpropionate. The synthetic route is as follows:

[0120]

[0121] (2) To a solution of 1,1-dimethylethyl 2-bromo-α,α-dimethylphenylpropionate (0.642 g, 2.05 mmol) in degassed 1,4-dioxane (20 mL) were added bis(pinacol)diboron (0.628 g, 2.47 mmol), Pd(dppf)Cl2 (154 mg, 0.21 mmol) and potassium acetate (0.607 g, 6.19 mmol), and the mixture was refluxed under N2 for 18 h, and then the resulting mixture was cooled to RT, concentrated under reduced pressure, and filtered by silica gel column chromatography. The solution volume was reduced, dissolved in CH2Cl2, washed with H2O, dried over Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain the product 1,1-dimethylethyl α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylpropionate; the synthesis route is as follows:

[0122]

[0123] (3) A solution of 1,1-dimethylethyl α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenepropanoate (0.219 g, 0.608 mmol), trifluoroacetic acid (698 μL, 9.12 mmol) and CH2Cl2 (6 mL) was stirred in N2 at RT until completely dissolved, and stirred in N2 at RT until the starting material disappeared, as shown by TLC, and then diluted with CH2Cl2, washed with 0.5 M HCl, dried over Na2SO4, and concentrated under reduced pressure to obtain the product α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenepropanoic acid; the synthetic route is as follows:

[0124]

[0125] (4) To a solution of α,α-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenepropanoic acid (0.150 g, 0.84 mmol) in toluene (10 mL) was added Lawesson's reagent (84 mg, 0.21 mmol). The mixture was heated to 120° C. in a thick-walled pressure vessel and stirred overnight at high temperature. The solution was then cooled, and the solvent was removed by rotary evaporation. The obtained mixture was dissolved in CH2Cl2, washed with 0.5M HCl and brine, and then concentrated under reduced pressure to obtain ROS responsive small molecule B (40 mg, 0.21 mmol). The obtained ROS responsive small molecule B (40 mg, 0.21 mmol), DTDP (85 mg, 0.21 mmol) and acetic acid (59 μL, 1.03 mmol) were then mixed in acetonitrile (100 mL, 1.03 mmol) and stirred overnight. The crude mixture was then diluted with CH2Cl2, washed with H2O, and then concentrated under reduced pressure. The obtained product was purified by column chromatography to obtain a precursor compound. The synthesis route is as follows:

[0126]

[0127] (5) The obtained precursor compound is prepared into a nano-prodrug according to step (3) in Example 1.

[0128] Comparative Example 1

[0129] This comparative example provides a method for preparing a nano prodrug without a ROS responsive group, which specifically comprises the following steps: (1) dissolving p-tert-butylbenzyl mercaptan (360 mg, 2 mmol, 1 eq) and DTDP (881.24 mg, 4 mmol, 2 eq) in 5 mL of EtOH. Stirring the mixture at RT for 24 h under N2, removing the solvent by rotary evaporation, and purifying the product by silica gel column chromatography; the synthesis route is as follows:

[0130]

[0131] (2) The obtained compound was prepared into nano-prodrug BSA-SS-TBBT according to step (3) in Example 1; the synthesis route is as follows:

[0132]

[0133] The obtained nano prodrug was then prepared into nanoparticles BSA-SS-TBBT NPs according to the method of Example 1, which is the nano preparation of this comparative example.

[0134] The particle size distribution and potential of the nanoformulation obtained in Example 1 were tested, and the nanoformulation was placed in 4°C water and 4°C 10mM PBS buffer solution, pH 7.4, to observe its stability. The results are as follows: Figure 4 As shown. Figure 4 As can be seen in the figure, the prepared BSA-SS-PBAP nanoformulation exhibited a small size (~18.2 nm), uniform size distribution (PDI: 0.22), negative surface charge (ζ potential: -17.5 mV) and excellent storage stability (stable for 4 days at 4 °C).

[0135] The BSA-SS-PBAP nanoformulation obtained in Example 1 was subjected to an in vitro ABTS free radical scavenging experiment and an in vitro hydroxyl free radical scavenging experiment. The experimental methods are as follows:

[0136] The in vitro ABTS free radical scavenging experimental steps are as follows:

[0137] Mix the ABTS stock solution (7.4mmol / L, 0.4mL) (take ABTS 0.0045g, distilled water 1.1025mL (MW=548.7)) and K2S2O8 stock solution (2.6mmol / L, 1.43mL): take K2S2O8 0.0025g, add distilled water 3.575mL (MW=270.32), mix the two, let stand at room temperature in the dark for 12 hours, and then dilute 50 times with anhydrous ethanol.

[0138] A0 value detection: Take 1.6mL of the ABTS solution and 0.6mL of anhydrous ethanol and mix them thoroughly, then measure the absorption value at 420nm. A value detection: Take 1.6mL of ABTS test solution and mix it thoroughly with 0.6mL of gradient concentration BSA-SS-PBAP solution, then measure the absorption value at 420nm. The experiment was repeated three times independently.

[0139] ROS clearance rate = (A0-A) / A0×100%.

[0140] The in vitro hydroxyl radical scavenging experimental steps are as follows:

[0141] 4 mM TMB solution (20 mg TMB was dissolved in 2 mL 95% ethanol and added to 18 mL 0.1 M pH 7.4 PBS solution), 139 μg / mL Fe2SO4 . 7H2O solution (11.12mgFe2SO4+20mLH2O), 100μM H2O2 solution and water were fully mixed at 1:1:1:1 and incubated at 37℃ for 120min, and the absorbance value A1 at 650nm was measured. The water was replaced with different nanoparticle solutions to measure the absorbance value A2;

[0142] ROS clearance rate = (A1-A2) / A1×100%.

[0143] The experimental results are shown in Figure 5 and Figure 6 In. Figure 5As shown in the in vitro ABTS free radical scavenging experiment, the nanoformulation BSA-SS-PBAP NPs has a good scavenging effect on total free radicals in vitro, and the scavenging ability is dose-dependent. When the concentration of the preparation reaches 42nM, the total free radical scavenging efficiency can reach 58%. Figure 6 As shown, the in vitro hydroxyl radical scavenging experiment showed that the nanoformulation BSA-SS-PBAP NPs had a good scavenging effect on hydroxyl radicals, and the scavenging ability was dose-dependent. When the concentration of the preparation reached 42nM / mL, the hydroxyl radical scavenging efficiency could reach more than 80%.

[0144] The BSA-SS-PBAP nanoformulation obtained in Example 1 was subjected to a mouse liver ischemia-reperfusion animal model experiment, and the experimental method was as follows:

[0145] Male mice were fasted for 12 hours before surgery and were allowed to drink water freely. Anesthesia was performed by intraperitoneal injection. After successful anesthesia, the mice were laid flat on the operating table with tape to fix their limbs. The mouse abdomen was dehaired and the surgical area was disinfected with iodine and 75% ethanol. A 1 cm midline incision was made in the abdomen, the abdominal cavity was opened, and the portal vein and hepatic artery were clamped with non-invasive vascular clamps. After 0.5 minutes, the blocked lobe turned grayish yellow, indicating that the blockage was successful. The skin incision was clamped with hemostatic forceps to temporarily close the abdominal cavity, and the mice were placed on a constant temperature heating blanket for insulation. After 30 minutes of continuous ischemia, the abdominal cavity was reopened, the vascular clamp was quickly removed, and the blood flow of the ischemic liver was restored. After about 0.5 minutes, the ischemic liver in the ischemic area gradually recovered from khaki to bright red, indicating successful reperfusion. The abdominal muscles and skin were sutured layer by layer to close the abdominal cavity, and the operation was completed. Drugs were injected through the tail vein, and the condition of the mice was observed. Blood was centrifuged and supernatant was obtained at 0, 6, 12, and 24 hours after the end of the experiment. All mice were uniformly killed and dissected after 24 hours.

[0146] The therapeutic effect of the BSA-SS-PBAP nanoformulation obtained in Example 1 on the mouse model of acute liver injury due to hepatic ischemia-reperfusion was further evaluated by evaluating the changes in serum AST and ALT levels 24 hours after a single intravenous injection of the preparation. Figure 7 , Figure 8 As shown. After ischemic injury, serum liver AST and ALT levels gradually increased, reaching peak values ​​at 6 and 12 hours, respectively. During the treatment with BSA-SS-PBAP nanoformulation, AST and ALT levels decreased in a dose-dependent manner. Immediate administration of a large dose (8 μmol / kg) reduced serum AST and ALT levels by 81.8% and 75.8%, respectively, at the beginning of treatment at 6 hours. After 24 hours of treatment, they even dropped to levels comparable to those of the normal group. Figure 7 and Figure 8It can also be seen that the BSA-SS-TBBT nanoformulation (8 μmol / kg) without ROS response groups in Comparative Example 1 has basically no therapeutic effect on the mouse model of acute liver injury caused by hepatic ischemia-reperfusion when administered at the same dose as in Example 1. During the entire treatment period, the AST and ALT levels were almost at the same level as the model group. Compared with the control group BSA-SS-TBBT nanoformulation administration group, the serum AST and ALT levels of the high-dose (8 μmol / kg) immediate administration were reduced by 77.8% and 78.2% at the beginning of the treatment, and by 87.8% and 89.9% at 24 hours.

[0147] Fig. 9 Schematic diagram of mouse liver after treatment. Fig. 9 As shown, injection of BSA-SS-PBAP NPs can restore the anatomical structure of mouse liver to a state almost identical to that of healthy liver, indicating that the preparation can significantly alleviate hepatocyte necrosis.

[0148] The BSA-SSSS-PBAP nanoformulation obtained in Example 3 was also subjected to the mouse liver ischemia-reperfusion animal model experiment according to the above method. The therapeutic effect of the BSA-SSSS-PBAP nanoformulation obtained in Example 3 on the mouse model of acute liver injury by liver ischemia-reperfusion was evaluated by evaluating the changes in serum AST and ALT levels 24 hours after a single intravenous injection of the preparation. The results are as follows: Fig.12 , Fig.13 shown. Fig.12 , Fig.13 The results showed that during the treatment period, the therapeutic effect of BSA-SSSS-PBAP (8 μmol / kg) was improved compared with BSA-SS-PBAP. Therapeutic administration of BSA-SSSS-PBAP (8 μmol / kg) reduced AST and ALT by 82.1% and 78.2% respectively in the initial 6 hours of treatment, by 85.9% and 84.9% in 12 hours, and even to the same level as the normal group in 24 hours. Prophylactic administration of BSA-SSSS-PBAP (8 μmol / kg) reduced AST and ALT by 89.5% and 87.2% respectively in the initial 6 hours of treatment, and almost reached the level of liver enzymes in normal mice in 12 hours.

[0149] 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-responsive persulfide biomacromolecule nanoprodrug, characterized in that: The general structural formula of the nano prodrug is shown in Formula I: In Formula I, R1 is a biomacromolecule; n≥2; R2 is shown in Formula II-III:

2. The ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 1, characterized in that: The biomacromolecule comprises one of bovine serum albumin, human serum albumin, porcine serum albumin, donkey serum albumin, mouse serum albumin, cystatin, and glutathione; and / or, in the formula I, 2≤n≤5.

3. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 1 or 2, characterized in that: The following steps are involved: The thiol-containing biomacromolecule and the precursor compound are subjected to a thiol-disulfide bond exchange reaction to obtain the ROS-responsive persulfide biomacromolecule nanoprodrug; the structure of the precursor compound is shown in Formula IV to Formula V: In formula IV to formula V, the R3 are each independently One of them.

4. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 3, characterized in that: The ratio of the molar amount of the thiol in the thiol-containing biomacromolecule to the molar amount of the precursor compound is 1:(1-25).

5. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 3, characterized in that: When n=2, the preparation method of the precursor compound shown in Formula IV or Formula V comprises the following steps: ROS-responsive small molecule A reacts with symmetrical persulfide through thiol-disulfide exchange reaction to obtain the precursor compound shown in Formula IV; ROS-responsive small molecule B reacts with symmetrical persulfide to obtain the precursor compound shown in Formula V; the structure of the ROS-responsive small molecule A is shown in Formula A, and the structure of the ROS-responsive small molecule B is shown in Formula B: The symmetrical persulfide includes one of 5,5'-dithiobis(2-nitrobenzoic acid), 2,2'-dithiodipyridine, and 2,2'-dithiobis(pyridine-1-oxide).

6. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 3, characterized in that: When n=3, the preparation method of the precursor compound shown in formula IV comprises the following steps: subjecting thioacetic acid to a thiol-disulfide bond exchange reaction with 2,2'-dithiodipyridine to obtain an asymmetric persulfide, subjecting the obtained asymmetric persulfide to a thiol-disulfide bond exchange reaction with 4-mercaptomethylphenylboronic acid pinacol ester to obtain an asymmetric phenylboronic acid pinacol ester-based persulfide precursor a, reducing the reaction to obtain a precursor b, and reacting the obtained precursor b with 2-pyridinethione to obtain the precursor compound shown in formula IV.

7. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 3, characterized in that: When n=4, the preparation method of the precursor compound shown in formula IV comprises the following steps: reacting benzyl alcohol and disulfur dichloride to obtain bis(benzyloxy)tetrasulfide; reacting the obtained bis(benzyloxy)tetrasulfide with α-mercaptopyridine to obtain 2,2'-tetrathiodipyridine; reacting the obtained 2,2'-tetrathiodipyridine with 4-mercaptomethylphenylboronic acid pinacol ester to obtain the precursor compound shown in formula IV.

8. The method for preparing the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 3, characterized in that: When n=5, the preparation method of the precursor compound shown in formula IV comprises the following steps: stirring the ROS-responsive small molecule A described in claim 5 and pyridine at -70°C to -80°C, adding S2Cl2, and continuing the reaction to obtain an intermediate product, and reacting the intermediate product with 2,2'-dithiodipyridine at 150°C to 250°C to obtain the precursor compound shown in formula IV.

9. A nano preparation, characterized in that: The nanoformulation comprises the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 1 or 2.

10. Use of the ROS-responsive persulfide biomacromolecule nanoprodrug according to claim 1 or 2 and the nanoformulation according to claim 9 in the preparation of a drug for treating liver ischemia-reperfusion injury.

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