Nano-drug of albumin for treating acute liver injury, preparation method and application thereof

Abstract

The present invention belongs to the field of biotechnology, and discloses a nano drug, preparation method and application of albumin for treating acute liver injury. The drug reduces the disulfide bonds between BSA molecules into multiple thiol groups, produces a self-thiolated albumin structure BSA-SH with more functional thiol antioxidant groups under mild conditions, obtains BSA-SNO NCs by nitrosylating multiple thiol groups, and further obtains BSA-SNO nanoparticles BSA-SNO NPs by nanoprecipitation of BSA-SNO NCs. The present invention realizes the rapid preparation of multi-thiol functional molecules and the preparation of efficient delivery carriers of NO by nitrosation process, which has lower cost and good repeatability. Functionalized BSA molecules provide potential therapeutic strategies for the treatment of acute liver injury and inflammatory diseases, and have high application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a nano drug for treating acute liver injury using albumin, a preparation method and an application thereof. Background Art

[0002] The main physiological function of thiol in the body is to balance the oxidative stress state, so thiol modification can significantly improve the antioxidant capacity of nanomedicines. At present, the research field of related antioxidant polythiol polymers focuses on chemical modification preparation; in the prior art, there is a method of chemical modification to increase the number of thiol groups on HSA molecules to 7.52 mol / mol HSA by grafting thiol groups on nano-scale mannosylated human serum albumin (HSA) through the reaction of 2-iminothiolane with primary amino groups. However, this study only uses an external chemical grafting method to modify the amino group of HSA to achieve the preparation of polythiol molecules. The modifiable amino sites on the surface of HSA are limited, the thiol modification efficiency is low, and the obtained thiol molecules are few, which greatly limits the development prospects of thiol-type nanomedicines.

[0003] In addition, thiol groups have good antioxidant properties in vivo. Exogenous supplementation of thiol-containing drugs, such as small molecule drugs (such as L-cysteine, N-acetyl-L-cysteine, Bucillamine, etc.), thiol polymers or bioconjugates, can restore the redox state of the pathological microenvironment in diseases associated with oxidative stress, thereby quickly restoring tissue damage reversal and organ function recovery. However, the low oxidation efficiency of monothiol molecules, slow oxygen free radical scavenging reaction rate, poor pharmacokinetics, off-target effects, etc. lead to low bioavailability, narrow therapeutic window, and diverse allergic reactions. In addition, the chemical modification of polythiol groups into polymers is usually hindered by the problems of low thiol modification efficiency, easy oxidation inactivation and self-crosslinking of thiols, cumbersome and time-consuming reaction steps, and the use of large amounts of toxic organic solvents. In this case, these molecules may not have a wide and universal role as effective antioxidants in clinical practice.

[0004] In addition, thiols easily form disulfide compounds with cysteine ​​residues of endogenous proteins, which reduces the biosafety of intravenous macromolecular biothiol drugs. Biomolecules with multiple free thiols also face the problem of unnecessary disulfide bond formation during subsequent modification and storage.

[0005] Nitrosothiol (R-SNO) is the product of sulfhydryl after S-nitrosation reaction in vivo, and biological thiols in vivo regulate various biological processes through S-nitrosation reaction. This process provides inspiration for sulfhydryl protection and as an anti-inflammatory NO small molecule donor. It is worth noting that NO small molecules, one of the important second messengers of cell signaling in vivo, can play a cytoprotective role by inducing vasodilation, reducing vascular resistance, improving microcirculation, anti-apoptosis, inhibiting inflammatory cell infiltration, etc. In the prior art, there is currently a method for wound healing research by nitrosation modification on keratin molecules, but the preparation form prepared by this technology is an electrospinning composite pad form, which is limited to local topical treatment, and it is difficult to meet the requirements of the preparation form that needs to be injectable, and it cannot be extended to the application of diseases such as organ damage. In addition, the prior art is modified by nitrosation on the surface after the carrier is prepared, and it also has the problems of limited modifiable sulfhydryl sites, low modification efficiency, and cumbersome preparation process, which makes it difficult to achieve large-scale preparation of NO donor nanomedicines, limiting its application.

[0006] In addition, the native structure of BSA is stabilized by 17 pairs of intramolecular disulfide bonds (formed by 35 cysteine ​​residues), which provides a potential source of thiol groups. Furthermore, albumin itself has few thiol groups, such as only one free thiol group on the peptide chain of bovine serum albumin, and its antioxidant properties are poor. By using a reducing agent to reductively cleave disulfide bonds under native and mild conditions, this method is expected to provide a unique opportunity to effectively and strategically generate albumin structures with multi-thiol functional groups, whose backbones contain more exposed, strongly antioxidant multi-thiol moieties.

[0007] In the prior art, the reaction of increasing the thiol group of the protein is often carried out under strict conditions and complicated processes (such as the use of organic reagents such as 2-iminothiolane to modify and introduce additional thiols, and the introduction of cysteine ​​residues through protein recombination). The thiol group has strong reducing properties and is easily oxidized to reform disulfide bonds, resulting in the instability of BSA. Through the nitrosation reaction, the thiol group can be protected while delivering NO molecules, introducing new functions such as improving microcirculation, maintaining vasodilation response, and anti-inflammatory effects. The complex obtained before the nanoprecipitation method has an uneven size distribution. Injection as a preparation has a great impact on the in vivo pharmacokinetic behavior of the nano drug preparation. The uneven distribution in tissues and organs may further affect the in vivo pharmacodynamics of the preparation. Summary of the invention

[0008] In order to overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a nano-drug, preparation method and application of albumin for treating acute liver injury, and specifically relate to the preparation of a self-thiolated and nitrosated albumin nano-drug and its application in treating acute liver injury.

[0009] The purpose of the present invention is to obtain a BSA-SH molecule with multiple exposed thiol functional groups by reducing and destroying the disulfide bonds between bovine serum albumin molecules under natural, mild and non-destructive conditions using a reducing agent. The nitrosation process cleverly and flexibly prevents the oxidation and self-crosslinking process of the active thiol functional groups, and at the same time serves as a transport and exogenous reserve for NO gas small molecules, that is, as a potential donor of NO, achieving a dual purpose. Nanoprecipitation realizes the preparation of injectable nanoformulations, which is helpful for clinical transformation. The present invention innovatively proposes the dual effects of exposing the antioxidant thiol process and the nitrosation process on the BSA molecule, as well as the injectability of the nanoformulation. The colloid of the preparation has stability, storage stability, and storage stability of the freeze-dried preparation. The excellent therapeutic effect of the nanoformulation of the present invention in two classic animal models of liver injury, some indicators can be restored to normal values. Compared with other dosage forms of drugs, the nano-sized drug preparation of the present invention has the advantages of improving the stability of the drug, prolonging the circulation time in the body, increasing safety, and being able to achieve passive targeted delivery to the liver, and has advantages in improving the tissue distribution of the drug and thus improving the bioavailability.

[0010] In one aspect, the present application provides a nanomedicine for treating acute liver injury with albumin, which is a nanoparticle BSA-SNO NPs; the nanoparticles BSA-SNO NPs are prepared by reducing the disulfide bonds between BSA molecules into multiple thiol groups, generating a self-thiolated albumin structure BSA-SH with thiol antioxidant groups under suitable conditions, nitrosating the multiple thiol groups to obtain BSA-SNO NCs, and then obtaining the BSA-SNO NCs by a nanoprecipitation method.

[0011] In certain embodiments, the functional thiol groups with antioxidant properties are obtained by reducing the intermolecular disulfide bonds, and have one or more of the following characteristics: the albumin is selected from bovine serum albumin, human serum albumin, porcine serum albumin, donkey serum albumin, mouse serum albumin or any combination thereof; the disulfide bonds between the BSA molecules are reduced to multiple thiol groups, and the BSA and the reducing substance are dissolved using one or a combination of Tris buffer, TBST buffer, low concentration DMSO, and water;

[0012] The reducing substance includes one or a combination of dithiothreitol DTT, tris(2-carboxyethyl)phosphine TCEP, glutathione GSH and 2-mercaptoethanol beta-ME; the TCEP concentration is 5-45mM, and the pH is 7-10; the suitable conditions include one of natural, mild and non-destructive conditions; the number of antioxidant thiol functional groups is 1-18 per mol per mol of albumin molecules;

[0013] In certain embodiments, the present application provides a nitrosated functional group albumin nanoformulation that simultaneously serves as an NO donor and as a thiol protecting group;

[0014] During the nitrosation of the sulfhydryl groups, the molar ratio of the sulfhydryl groups to sodium nitrite is 1:0.4, 1:0.6, 1:0.8, 1:1.2 or 1:1.4;

[0015] In certain embodiments, the particles are precipitated by a nanoprecipitation method to form nanoparticles; in the process of forming BSA-SNO NCs particles by the nanoprecipitation method, the solvent is one of ethanol, acetone, propanol, and tetrahydrofuran; the nitrosation product and the solvent are mixed in a ratio of 1:0.1, 1:0.3, 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5 to perform BSA-SNO NCs nanoprecipitation; the method of removing the organic solvent includes one or a combination of dialysis, rotary evaporation, vacuum drying, and freeze drying. The nanoparticles have one or more of the following characteristics: the shape of the nanoparticles is spherical; the particle size of the nanoparticles is 20nm-100nm; preferably, the particle size of the nanoparticles is 20-40nm, 40-60nm, 60-80nm or 80-100nm; the polydispersity index (PDI) of the nanoparticles is 0.1-0.5; preferably, the polydispersity index (PDI) of the nanoparticles is 0.1-0.2, 0.2-0.3, 0.3-0.4 or 0.4-0.5; the zeta potential of the nanoparticles is -5mV to -30mV. Preferably, the zeta potential of the nanoparticles is -5mV to -10mV, -10mV to -20mV or -20mV to -30mV.

[0016] In certain embodiments, the nanoprecipitated BSA-SNO NCs are subjected to rotary evaporation to remove impurities, mixed with a lyoprotectant and frozen at -80°C, and the frozen product is placed in a lyophilizer away from light to prepare lyophilization; the lyophilized preparation uses salts as preparation resuspension buffers to balance the pH value during freeze-drying and storage; the lyoprotectants are selected from alcohol lyoprotectants and sugar lyoprotectants, and the alcohol lyoprotectants are one or a combination of xylitol, mannitol, sorbitol, and glycine; the sugar lyoprotectants are one or a combination of glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan and inulin; the concentration of the lyoprotectant is selected from one of 0%, 5%, 10%, 15%, 20%, and 25%; the salt buffer includes the salt selected from one or a combination of sodium phosphate and potassium phosphate.

[0017] In another method, the present application provides a preparation method comprising the following steps:

[0018] S1, BSA disulfide bond reduction modified BSA-SH;

[0019] S2, BSA-SH nitrosation reaction to form self-thiolated and nitrosylated serum albumin nanocomplexes BSA-SNONCs;

[0020] S3, nanoparticles BSA-SNO NPs were formed by nanoprecipitation of thiolated and nitrosylated serum albumin nanocomplexes BSA-SNO NCs.

[0021] In certain embodiments, in step S1, BSA disulfide bond reduction modifies BSA-SH to produce a self-thiolated BSA-SH solution having multiple antioxidant thiol functional groups, wherein the self-thiolated BSA-SH solution comprises the BSA-SH complex of any one of claims 1 to 2;

[0022] The preparation method is to open the disulfide bonds in BSA by reducing with DTT and TCEP to produce a BSA-SH complex with multiple thiol groups in the form of a solution, wherein the reaction formulas for opening the disulfide bonds in BSA by reducing with DTT and TCEP are as follows:

[0023]

[0024] In certain embodiments, in the BSA-SH nitrosation reaction of step S2, BSA-SH is nitrosated by NaNO2 under acidic conditions, and the preparation of the complex in BSA-SNO NCs specifically includes:

[0025] (2.1) Take the BSA-SH product and add concentrated HCl under vortexing to acidify the product;

[0026] (2.2) taking out all the products in the dialysis bag, centrifuging, collecting the supernatant, diluting, measuring the absorbance, calculating the protein content, and measuring the thiol concentration;

[0027] (2.3) Based on the measured -SH concentration, prepare NaNO2 of the same concentration dissolved in 0.1 M HCl and keep it away from light. Mix the BSA-SH solution with the NaNO2 solution and shake it at room temperature away from light.

[0028] In certain embodiments, the suspension of nanoparticles is subjected to precipitation polymerization by a nanoprecipitation method to form nanoparticles with a dense structure, and the step S3 uses the nanoparticles; specifically comprises:

[0029] (3.1) providing a suspension solution containing the nanocomposite and a solvent;

[0030] (3.2) selecting a suspension containing the nanocomposite and rapidly injecting an optional organic solvent in proportion under a vortex state;

[0031] (3.3) removing the organic solvent to obtain a solution containing the nanoformulation.

[0032] In another method, the present application provides an application of a functionalized albumin nanomedicine composition targeted to the liver for treating acute liver injury, which uses the thiol and further nitrosylation of the disulfide bonds existing in albumin itself to simultaneously deliver the functional antioxidant thiol groups and NO gas small molecules to the lesion site; the pharmaceutical composition is used for diseases that can be prevented or treated by the functionalized group molecules themselves;

[0033] In the disease to be prevented or treated, the therapeutic molecules are anti-inflammatory NO gas small molecules and antioxidant thiol groups, and the disease is acute liver injury in the subject; in the disease to be prevented or treated, the active functional group concentration range of BSA-SH, BSA-SNO NCs and BSA-SNONPs in liver cells for treatment is 250nM-1000nM; the acute liver injury includes acute liver injury induced by ischemia-reperfusion, acute liver injury induced by endotoxin, liver injury induced by acetaminophen, alcoholic hepatitis, and viral hepatitis; the subject is a mammal, such as rodents, primates, canines, and porcines; BSA-SNO NPs include BSA-18SNO NPs with 18 per mol of albumin molecules according to the number of antioxidant thiol functional groups, and the active functional group concentration of the BSA-18SNO NPs in animal treatment is 1μmol / kg to 100μmol / kg.

[0034] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0035] Combining all the above technical solutions, the advantages and positive effects of the present invention are as follows: The present invention provides a liver-targeted functional albumin nano drug delivery system: by utilizing the disulfide bonds existing in bovine serum albumin (BSA) to be thiolated and further nitrosylated, the functional antioxidant thiol groups and NO gas small molecules are efficiently delivered to the lesion site at the same time. BSA itself, as a macromolecular protein, has good biocompatibility and degradability. By reducing the disulfide bonds between BSA molecules to multiple thiol groups, a self-thiolated albumin structure (BSA-SH) with more functional thiol antioxidant groups is cleverly produced under mild conditions. By nitrosylating multiple thiol groups (BSA-SNO NCs), this process can improve the stability of the thiol groups, avoid intermolecular self-crosslinking that exposes multiple thiol groups, and give the BSA structure the ability to act as a NO transporter, thereby achieving efficient delivery of NO.

[0036] The present invention further obtains BSA-SNO nanoparticles (BSA-SNO NPs) with uniform structural components and good colloidal stability by a nanoprecipitation method. The prepared BSA-SNO NPs can be rapidly enriched in the damaged liver site in two clinically relevant ischemia-reperfusion-induced and endotoxin-induced acute liver injury mouse animal models, and are reduced to BSA-SH molecules and NO molecules with multiple functional antioxidant thiol groups in the lesions, achieving highly effective anti-inflammatory and antioxidant effects, activating antioxidant-related cell signaling pathways at the source, effectively inhibiting the inflammatory cascade signaling pathways in the damaged liver site, and blocking mitochondria-related cell apoptosis-related pathways, thereby effectively improving the redox state of the damaged cell microenvironment in the liver lesion site, saving dying liver cells, quickly reversing the process of liver damage, restoring liver function, and improving the survival rate.

[0037] The technical solution of the present invention is easy to prepare, low in toxicity and safety, and has a short preparation cycle. The rapid preparation of multi-thiol functional molecules and the preparation of efficient NO delivery carriers are achieved through a simple BSA intramolecular disulfide bond reduction process, and the nitrosation process is used to achieve lower costs and good repeatability. The functionalized BSA molecules provide a potential therapeutic strategy for the treatment of acute liver injury and inflammatory diseases, and have a high application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings herein are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the description, serve to explain the principles of the present disclosure;

[0039] Figure 1This is a flow chart of a method for preparing a nanomedicine for treating acute liver injury using albumin provided in an embodiment of the present invention;

[0040] Figure 2 This is a schematic diagram of the BSA disulfide bond reduction modification of BSA-SH provided in an embodiment of the present invention;

[0041] Figure 3 : is a schematic diagram of the formation of BSA-SNO by the nitrosation reaction of BSA-SH provided in an embodiment of the present invention;

[0042] Figure 4 is a reaction principle diagram of the Ellman detection method provided in an embodiment of the present invention;

[0043] Figure 5 1 is a test graph provided in an embodiment of the present invention; wherein, (1) is a graph showing the effect of different initial TCEP concentrations on the efficiency of BSA reducing disulfide bonds under the condition of a reaction buffer system pH 9.0; (2) is a graph showing the stability of BSA-SH at 4°C and 25°C; (3) is a graph showing the stability of BSA-SH at -20°C;

[0044] Figure 6 1 is a test diagram provided by an embodiment of the present invention; wherein, (1) is a UV-visible spectrum diagram of BSA-SNO NCs; (2) is a diagram showing the ratio of thiol groups remaining after nitrosation of BSA-SH;

[0045] Figure 7 is a particle size distribution diagram of BSA-SNO NCs provided in an embodiment of the present invention; and a test diagram provided in an embodiment of the present invention, wherein (1) is a freeze-drying process diagram of a BSA-SNO NPs preparation, (2) is a particle size distribution diagram of a preparation after reconstitution, (3) is a diagram showing changes in the particle size and dispersibility (PDI) of nanoparticles after reconstitution of a freeze-dried preparation with 10% trehalose added at 25°C for different storage times in order to verify the shelf stability of the freeze-dried preparation, and (4) is a diagram showing changes in the particle size and dispersibility (PDI) of nanoparticles after reconstitution of a freeze-dried preparation with 10% trehalose added at -20°C for different storage times in order to verify the shelf stability of the freeze-dried preparation.

[0046] Figure 8 It is a semi-quantitative fluorescence detection graph of NO release of the preparation provided in the embodiment of the present invention after incubation in the mouse liver cell line AML-12 for 1, 3, 6, and 12 hours;

[0047] Fig. 9 The following are (1) flow cytometric detection graphs and (2) semi-quantitative result graphs of ROS levels of different preparations in the H2O2-induced acute hepatocyte injury model (AML-12 cell line) after incubation for 12 hours in the characterization of intracellular reactive oxygen levels provided in the embodiments of the present invention.

[0048] Fig.10 The flow cytometry results show that different preparations were used for apoptosis in the acute AML-12 hepatocyte injury model induced by H2O2 for 12 hours.

[0049] Fig.11 The figure shows the recovery of cell viability after 12 hours of incubation with different preparations in the H2O2-induced acute AML-12 hepatocyte injury model;

[0050] Fig.12 The figure shows the biodistribution of the Cy5 fluorescently labeled preparation in important organs (heart, liver, spleen, lung, and kidney) and semi-quantitative results in the acute liver injury mouse model induced by ischemia-reperfusion after intravenous injection of the preparation.

[0051] Fig.13 This is the hematoxylin-eosin (H&E) staining picture of liver tissue;

[0052] Fig.14 Serum liver enzyme indicators include alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase.

[0053] Fig.15 The data statistical graphs of liver apoptotic cells provided by the embodiments of the present invention, wherein (A) is a statistical graph of the proportion of liver TUNEL-positive cells, i.e., the number of apoptotic cells, and (B) is a semi-quantitative statistical graph of liver superoxide DHE fluorescence intensity;

[0054] Fig.16 A test chart is provided for an embodiment of the present invention; wherein (A) is malondialdehyde in liver tissue homogenate, (B) is glutathione in liver tissue homogenate, (C) is a superoxide dismutase level chart in liver tissue homogenate, and (D) is a level chart of oxidized glutathione in liver tissue homogenate;

[0055] Fig.17 This is a picture of immunofluorescence co-staining of Kupffer cell CLEC4F and monocyte-derived macrophage F4 / 80 in frozen sections of liver tissue;

[0056] Fig.18 Western blot analysis of inflammatory factors in liver tissue homogenate, pro-inflammatory factor interleukin-6, pro-inflammatory factor tumor necrosis factor-α, and anti-inflammatory factor interleukin-10 levels;

[0057] Fig.19 Western blot analysis of liver tissue homogenate related protein expression, Nrf-2, HO-1, NQO1, p-IκBα, NF-κB, Cyto C, Bcl-2, Bax, Cleaved caspase-3 levels and semi-quantitative results;

[0058] Fig. 20 This is a diagram for the biosafety evaluation of functionalized albumin nanoformulations in an animal model of acute liver injury induced by ischemia-reperfusion;

[0059] Fig.21 This is the experimental process and survival rate analysis diagram of nanomedicine intervention for acute liver injury induced by lipopolysaccharide / D-galactosamine (LPS / D-gal);

[0060] Fig. 22 Serum liver enzyme indicators for the liver, including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase;

[0061] Fig.23 The fluorescence staining diagram provided in the embodiment of the present invention, wherein (1) is a quantitative diagram of the number of TUNEL fluorescence-positive cells of liver apoptotic cells, and (2) is a semi-quantitative diagram of the fluorescence intensity of DHE, an indicator of liver superoxide;

[0062] Fig.24 A test chart is provided for an embodiment of the present invention; wherein (A) is a graph of malondialdehyde in liver tissue homogenate, (B) is a graph of glutathione level in liver tissue homogenate, (C) is a graph of superoxide dismutase level in liver tissue homogenate, and (D) is a graph of oxidized glutathione level in liver tissue homogenate;

[0063] Fig.25 Serum creatinine, urea nitrogen levels and H&E staining of renal tissue;

[0064] Fig.26 Western blot analysis of key protein expression in liver tissue, including HMGB1, TLR4, NF-κB p-IκBα, pro-inflammatory factor tumor necrosis factor-α, pro-inflammatory factor interleukin-6, Cleaved caspase-1 and pro-inflammatory factor interleukin-1β levels; DETAILED DESCRIPTION

[0065] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0066] The innovative point of the albumin nanomedicine, preparation method and application for treating acute liver injury provided by the embodiments of the present invention is that the present invention utilizes a reducing agent to reduce and destroy the disulfide bonds between bovine serum albumin (BSA) molecules under natural, mild and non-destructive conditions, thereby generating BSA molecules (BSA-SH) that expose multiple antioxidant thiol groups. The BSA-SH molecules further undergo sulfhydryl s-nitrosylation and subsequent nanoprecipitation to form a self-thiolated, nitrosylated injectable albumin nanoformulation (BSA-SNO NP), which enables albumin to serve as a potential donor of NO and prevents the bottom surface oxidation and self-crosslinking of the thiol groups. This design, as a powerful carrier for delivering NO and polythiol groups, cleverly and flexibly combines the functions of the potent antioxidant thiol groups in maintaining the redox system with the functions of NO gas in regulating host defense, inhibiting inflammation, and restoring microcirculation, providing a powerful liver protective effect in typical ischemia-reperfusion injury and endotoxin-induced acute liver injury (ALI) animal models.

[0067] Through the analysis of the existing technology, it is found that the existing technology does not have the relevant technology for reconstructing disulfide bonds to obtain multi-antioxidant thiol structures and performing subsequent nitrosation modification, as well as nanoprecipitation to prepare nanoparticles. The existing technology method is limited to a certain link.

[0068] Example 1. The present invention provides a nanomedicine for treating acute liver injury with albumin, which is BSA-SNO nanoparticles BSA-SNO NPs for intravenous injection; the nanoparticles BSA-SNO NPs are prepared by reducing the disulfide bonds between BSA molecules into multiple thiol groups, generating self-thiolated albumin structures BSA-SH with thiol antioxidant groups under suitable conditions, nitrosating the multiple thiol groups to obtain BSA-SNO NCs, and then subjecting the BSA-SNO NCs to a nanoprecipitation method.

[0069] Exemplarily, it has one or more of the following characteristics: reducing the disulfide bonds between BSA molecules into multiple sulfhydryl groups, using one or a combination of Tris buffer, TBST buffer, low concentration DMSO, and water to dissolve BSA and reducing substances; the reducing substances include one or a combination of dithiothreitol DTT (Dithiothreitol), tris (2-carboxyethyl) phosphine TCEP (Tris (2-chloroethyl) phosphate), glutathione GSH (glutathione) and 2-mercaptoethanol beta-ME; the TCEP concentration is 5-45mM, and the pH is 7-10; the suitable conditions include one of natural, mild, and non-destructive conditions; the number of antioxidant sulfhydryl functional groups is 1-18 per mol per mol of albumin molecules;

[0070] Exemplarily, the present invention also provides a nitrosated functional group albumin nanoformulation that simultaneously serves as an NO donor and as a thiol protecting group; during the nitrosation of the thiol group, the molar ratio of the thiol group to sodium nitrite is 1:0.4, 1:0.6, 1:0.8, 1:1.2 or 1:1.4;

[0071] In certain embodiments, the particles are precipitated by a nanoprecipitation method to form nanoparticles; in the process of forming BSA-SNO NCs particles by the nanoprecipitation method, the solvent is one of ethanol, acetone, propanol, and tetrahydrofuran; the nitrosation product and the solvent are mixed in a ratio of 1:0.1, 1:0.3, 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5 to perform BSA-SNO NCs nanoprecipitation; the method of removing the organic solvent includes one or a combination of dialysis, rotary evaporation, vacuum drying, and freeze drying.

[0072] Exemplarily, the nanoparticles have one or more of the following characteristics: the shape of the nanoparticles is spherical particles; the particle size of the nanoparticles is 20nm-100nm; preferably, the particle size of the nanoparticles is 20-40nm, 40-60nm, 60-80nm or 80-100nm; the polydispersity index (PDI) of the nanoparticles is 0.1-0.5; preferably, the polydispersity index (PDI) of the nanoparticles is 0.1-0.2, 0.2-0.3, 0.3-0.4 or 0.4-0.5; the zeta potential of the nanoparticles is -5mV to -30mV. Preferably, the zeta potential of the nanoparticles is -5mV to -10mV, -10mV to -20mV or -20mV to -30mV.

[0073] In another exemplary embodiment, the BSA-SNO NCs after nanoprecipitation are subjected to rotary evaporation to remove impurities, mixed with a lyophilizing agent and frozen at -80°C, and the frozen material is placed in a lyophilizer in the dark to prepare lyophilization; the lyophilized preparation uses salts as preparation resuspension buffers to balance the pH value during freeze-drying and storage; the lyophilizing agent is selected from alcohol lyophilizing agents and sugar lyophilizing agents, and the alcohol lyophilizing agent is one or a combination of xylitol, mannitol, sorbitol, and glycine; the sugar lyophilizing agent is one or a combination of glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan and inulin; the concentration of the lyophilizing agent is selected from one of 0%, 5%, 10%, 15%, 20%, and 25%; the salt buffer includes the salt selected from one or a combination of sodium phosphate and potassium phosphate.

[0074] Embodiment 2, as Figure 1 As shown, the present invention provides a preparation method, comprising the following steps:

[0075] S1, BSA disulfide bond reduction modified BSA-SH;

[0076] S2, BSA-SH nitrosation reaction to form self-thiolated and nitrosylated serum albumin nanocomplexes BSA-SNONCs;

[0077] S3, nanoparticles BSA-SNO NPs were formed by nanoprecipitation of thiolated and nitrosylated serum albumin nanocomplexes BSA-SNO NCs.

[0078] In certain embodiments, in step S1, BSA disulfide bond reduction modifies BSA-SH to produce a self-thiolated BSA-SH solution having multiple antioxidant thiol functional groups, wherein the self-thiolated BSA-SH solution includes the BSA-SH complex;

[0079] The preparation method is to open the disulfide bonds in BSA by reducing with DTT and TCEP to produce a BSA-SH complex with multiple thiol groups in the form of a solution, wherein the reaction formulas for opening the disulfide bonds in BSA by reducing with DTT and TCEP are as follows:

[0080]

[0081] Exemplarily, in the BSA-SH nitrosation reaction of step S2, BSA-SH is nitrosated by NaNO2 under acidic conditions, and the preparation of the complex in BSA-SNO NCs specifically includes:

[0082] (2.1) Take the BSA-SH product and add concentrated HCl under vortexing to acidify the product;

[0083] (2.2) taking out all the products in the dialysis bag, centrifuging, collecting the supernatant, diluting, measuring the absorbance, calculating the protein content, and measuring the thiol concentration;

[0084] (2.3) Based on the measured -SH concentration, prepare NaNO2 of the same concentration dissolved in 0.1 M HCl and keep it away from light. Mix the BSA-SH solution with the NaNO2 solution and shake it at room temperature away from light.

[0085] In another exemplary embodiment, in some embodiments, the suspension of nanoparticles is subjected to precipitation polymerization by a nanoprecipitation method to form nanoparticles with a dense structure, and the step S3 uses nanoparticles; specifically includes:

[0086] (3.1) providing a suspension solution containing the nanocomposite and a solvent;

[0087] (3.2) Selecting a suspension containing the nanocomposite and rapidly injecting an optional organic solvent in a vortex state according to the ratio of claim 4;

[0088] (3.3) removing the organic solvent to obtain a solution containing the nanoformulation.

[0089] In the above-mentioned examples 1 and 2, the present invention further provides an application of a functionalized albumin nanomedicine composition targeted to the liver for treating acute liver injury, wherein the functionalized antioxidant thiol groups and NO gas small molecules are simultaneously delivered to the lesion site by thiolation and further nitrosylation of the disulfide bonds existing in the albumin itself; the pharmaceutical composition is used for diseases that can be prevented or treated by the functionalized group molecules themselves; in the diseases to be prevented or treated, the therapeutic molecules are anti-inflammatory NO gas small molecules and antioxidant thiol groups, and the disease is acute liver injury in the subject; in the diseases to be prevented or treated, BSA-SH, BSA-SNO NCs and BSA-SNO The concentration range of active functional groups of NPs in liver cells is 250nM-1000nM; the acute liver injury includes acute liver injury induced by ischemia-reperfusion, acute liver injury induced by endotoxin, liver injury induced by acetaminophen, alcoholic hepatitis, viral hepatitis; the subject is a mammal, such as rodents, primates, canines, and porcines; BSA-SNONPs include 18 per mol per mol of albumin molecules according to the number of antioxidant thiol functional groups, and the active functional group concentration of BSA-18SNO NPs in animal in vivo treatment is 1 μmol / kg to 100 μmol / kg. The pharmaceutical composition also contains pharmaceutically acceptable carriers and / or excipients.

[0090] It can be seen from the above Examples 1 and 2 that, compared with the prior art, the advantages of the present invention further include: (1) From the perspective of the selection of raw materials, the present invention aims at the problems of immunogenicity, low preparation efficiency, poor in vivo stability, and large side effects of the thiol preparations prepared from modifiable natural / synthetic polymer materials, which are difficult to meet the needs of clinical treatment. The present invention adopts biodegradable, biocompatible, safe, low immunogenicity, and a wide range of material sources and low prices as the main structure of the material.

[0091] (2) In the preparation of the preparation, the present invention obtains a therapeutic nano preparation with controllable particle size, uniform size, good colloidal stability and good storage stability through the reduction of disulfide bonds, thiol nitrosation and nanoprecipitation processes, and screens and obtains the best preparation in lyophilized form. Specifically, it can be mentioned that: the present invention prepares a protein molecular structure with multiple antioxidant thiol functional groups by reducing the disulfide bonds of BSA itself through a mild, simple and rapid one-step reduction method at room temperature. On the one hand, compared with traditional small molecule thiol antioxidants, the prepared molecules provide a potential source of multi-thiol molecules, which solves the poor antioxidant properties and slow reaction kinetics of current single-molecule thiol small molecule drugs, and solves the broad and general role of these small molecules as effective antioxidants that they do not currently have. In addition, the direct reductive preparation of BSA-SH avoids the generation of harsh highly reactive intermediates and complex catalytic conditions in traditional polythiol modification synthesis. The potential thiol source provided by the 17 pairs of disulfide bonds of the BSA molecule itself can cleverly and strategically introduce the polythiol structure, and the synthesis and purification steps of the BSA-thiol molecule can be completed efficiently and quickly within minutes. The preparation steps are simple and efficient. And through the precise regulation of factors such as the concentration of the reducing agent, pH, and the concentration of the reaction substrate, the proportion of exposed thiols on the obtained preparation can be flexibly controlled, and self-thiolated protein preparations with different molecular ratios can be obtained, meeting the requirements for the proportion of antioxidant thiol groups in different practices and therapeutic needs.

[0092] It is further understandable that albumin is a non-exclusive transport protein that can form a complex with poorly soluble or exogenous substances, becoming a "ferry" for such substances in the blood circulation, so that it is usually used as a carrier of other hydrophilic and hydrophobic drugs in the traditional sense to prepare pharmaceutical preparations. Compared with its traditional functions, the present invention uses the BSA molecular structure with multiple functional groups as a drug form by disulfide bond reduction and nitrosation modification, giving the carrier its own pharmacological function. Furthermore, albumin disulfide bond reduction is currently less studied, and most of them increase the load of other therapeutic hydrophobic drugs by reducing disulfide bonds to open hydrophobic cavities, or prepare corresponding preparations as gel forms by cross-linking with other molecules. Compared with other technical contents, the purpose of reducing disulfide bonds in the present invention is to expose more antioxidant thiol forms, so that it has certain pharmacological activity. And further, the present invention provides a nitrosation process, which not only solves the possible adverse oxidation process of the current polythiol compounds, but also avoids the self-crosslinking problem of thiol molecules in the preparation process, and provides further anti-inflammatory small molecule gas delivery as a donor of NO gas molecules. Also, the present invention adopts nano precipitation mode to obtain nanometer-scale, controllable size, uniformly distributed nano preparation form. Compared with the problems of poor stability, poor targeting, unstable pharmacokinetic distribution in traditional pharmaceutical preparations, the nanometerized form of the preparation further enhances its stability under physiological conditions, prolongs the internal circulation time, increases safety, and can achieve the advantages of passive targeted delivery of the liver, and provides greater advantages in improving the tissue distribution of the drug and thus improving bioavailability. In addition, the present invention adopts anhydrous ethanol as the organic solvent of nano precipitation, which is cheap and easy to remove, avoids the use of traditional toxic and difficult to remove organic solvents, and increases conversion accessibility. In a word, the preparation process of the present invention is fast, convenient, simple in preparation method, good reproducibility of preparation batches, high in output, adjustable in preparation process and parameters, low cost and easy to process, and does not involve many advantages such as a large amount of toxic organic solvents, so it has the potential for clinical transformation application.

[0093] (3) Based on the preparation process of the above-mentioned preparation, the present invention innovatively proposes a fusion mode of innovative preparation methods of the preparation, which is specifically embodied in the following aspects: for acute liver injury diseases with high morbidity and mortality, there is currently a lack of convincing evidence to support the efficacy of existing treatment options other than liver transplantation. However, the supportive liver protection and liver protection therapies currently used in clinical practice are difficult to reverse liver dysfunction caused by acute liver injury and are difficult to exert therapeutic effects. For this problem, the present invention provides solutions from the following aspects: First, for the rapid development of liver injury accompanied by multi-cascade pathological and physiological processes, including the simultaneous activation of multiple cell death signaling pathways such as mitochondrial apoptosis, endoplasmic reticulum stress, and cell pyroptosis, the preparation deeply explores its upstream common inducing factors and intervention targets and strategies. This preparation analyzes in detail the relationship and influence of the dosage, administration time, in vivo efficacy and in vivo safety of the nano-preparation. The optimal dosage and treatment time window for the preparation were screened out, and a prescription parameter information library that can exert the best drug effect at different stages of the progression of liver injury was screened out (screening the optimal proportion of nitrosated thiol functional groups, as well as the dosage, time and method of administration), thereby providing a multi-faceted, comprehensive, integrated and flexible treatment plan for liver injury diseases that are characterized by rapid progression, difficult treatment, and no specific targeted drugs. Secondly, the current development of drug preparations for the treatment of liver injury at home and abroad only targets a single pathophysiological mechanism. This solution combines the powerful antioxidant and anti-inflammatory effects of polythiols with NO gas molecules to achieve multi-target and multi-mechanism regulation in the complex and changeable inflammatory environment of liver injury, thereby maximizing the therapeutic effect.

[0094] (4) In order to verify the efficacy of the drug prepared in the preparation process, the present application selected an animal model and conducted experiments: its in vivo pharmacodynamics was explored in classic animal models of liver injury including ischemia-reperfusion injury and endotoxin-induced liver injury. The preparation has excellent therapeutic effects in a wide range of etiologically induced liver injury forms and has high clinical translation value.

[0095] (5) Based on the animal model experiments conducted, this application proposes the optimal drug dosage for subsequent treatment effects, which is specifically reflected in the following aspects: the drug preparations currently under study have large dosages, long treatment cycles (each course of treatment is greater than 14 days), and have problems such as allergies, hepatotoxicity, and hepatorenal toxicity. The adverse reactions are unclear, and there are many incompatibility taboos. The in-depth understanding of its related basic research, mechanism of action, and time and dosage of use is obviously insufficient. This preparation has a lower drug dosage (1000nM in vitro, 4μmol / L in vivo), a short treatment cycle (only a single intravenous injection is required after the liver injury model is established), good in vivo safety, and in-depth mechanism exploration. It solves the problems of the drug preparations currently under clinical research and has high application and transformation value.

[0096] (6) Based on the above analysis of the subsequent treatment effects, this application further explored the regulatory mechanism of the preparation, which is reflected in: In the treatment of acute liver injury, in addition to etiological treatment, supportive treatment and temporary liver protection treatment, there is currently no approved drug therapy that can comprehensively, synergistically and multi-facetedly improve the treatment of acute liver injury. In view of the defects of the current advanced acute liver injury treatment therapies at home and abroad, this program currently provides a drug-free, self-sulfurized, nitro-fixed injectable BSA nanoparticle preparation. This innovative formula combines the powerful antioxidant and anti-inflammatory effects of polythiol with NO gas molecules to achieve multi-target and multi-mechanism regulation in the complex and changeable inflammatory environment of liver injury, and quickly eliminates calcium overload in damaged cells, while inhibiting the initiation of multiple apoptosis, pyroptosis and inflammatory cascade-related signaling pathways, to achieve a full range of protective effects on damaged liver cells.

[0097] To further illustrate the effects of the embodiments of the present invention, the following experiment is conducted using bovine serum albumin as an example.

[0098] Experimental example.

[0099] 1.Material properties and functions.

[0100] (1) Bovine serum albumin (BSA). Definition: BSA is a globulin in bovine serum, containing 607 amino acid residues and a molecular weight of 66.446 KDa. Composition structure: Albumin is composed of 581 amino acid residues, of which 34 cysteine ​​residues form 17 intramolecular disulfide bonds, and there is a free thiol group at the 34th position of the peptide chain. The amino acid sequence SEQ ID NO: 1 is:

[0101] .

[0102] It has the following advantages: 1. Wide source and low cost; 2. Good biocompatibility in vivo and low cytotoxicity; 3. The presence of a cysteine ​​residue (-SH) at position 34 has good antioxidant properties; 4. The natural structure of BSA is stabilized by 17 pairs of intramolecular disulfide bonds (formed by 35 cysteine ​​residues), and the presence of multiple disulfide bonds provides a potential source of thiol groups; 5. It has a certain liver targeting effect; 6. There are two drug binding sites, which are suitable as a carrier for drug delivery; (The two binding sites are: Site I (or warfarin site) is located on domain 2 (upper right), which is more inclined to bind large heterocycles and negatively charged compounds, while Site II (or indole-benzodiazepine site) is located on domain 3 (below), which is the preferred site for small aromatic carboxylic acids.) 7. It can prolong the circulation half-life of other rapidly cleared drugs.

[0103] (2) NO Nitric oxide (NO): NO is an important messenger molecule in the body and one of the most effective vasodilator molecules. It can act as an antioxidant to reduce cell damage caused by superoxide anion free radicals. It can inhibit the expression of adhesion molecules in macrophages, thereby reducing inflammation. S-nitrosoalbumin is a reservoir of NO in endothelial cells in the human body.

[0104] 2. Experimental process.

[0105] a. BSA disulfide bond reduction modification BSA-SH. Figure 2 As shown. BSA disulfide bonds are thiolated to form BSA-SH: The disulfide bonds in BSA are opened by reduction with dithiothreitol (DTT) or tri(2-carboxyethyl)phosphine (TCEP), producing more thiol groups and improving the antioxidant function. TCEP has better selectivity than DTT, higher reduction efficiency, and lower working environment requirements. Through this reaction, BSA has more thiol sites, improving its antioxidant properties. The breaking of disulfide bonds usually requires a large number of free thiols and disulfide bond exchanges. Trivalent phosphine derivatives are favored by many researchers due to their strong reducing ability and selective reduction of disulfide bonds. The reduction of this type of reducing agent is a quantitative reaction process. The principle is that the lone electron pair carried by the central atom "P" can form a coordinated covalent bond with the oxygen atom and has reducing properties.

[0106] b. BSA-SH nitrosation reaction forms self-thiolated and nitrosylated bovine serum albumin nanocomplex BSA-SNO, such as Figure 3 As shown. BSA-SH nitrosation reaction to form BSA-SNO: Under certain conditions, BSA-SH reacts with sodium nitrite hydrochloric acid solution to form BSA-SNO, which is further made into nanoparticles. In the nitrosation process, the oxidation and self-crosslinking of the active thiol functional groups are prevented, and at the same time, it serves as a transporter and exogenous reserve for small molecules of NO gas, giving the albumin structure BSA-SH the ability to act as a NO transporter, thereby achieving NO delivery.

[0107] c. BSA-SNO nanoprecipitation to form nanoparticles. BSA-SNO is agglomerated and denatured to form nanoparticles BSA-SNO NPs by using an organic solvent such as tetrahydrofuran, anhydrous ethanol, and acetonitrile for nanoprecipitation. The prepared nanoparticles BSA-SNO NPs are rotary evaporated at 40°C to remove the organic solvent to obtain the final nanoformulation. Preferably, anhydrous ethanol is selected to perform nanoprecipitation to promote nanoparticle agglomeration.

[0108] d, preparation of BSA-SNO NPs lyophilized agent, adding an equal volume of trehalose solution and vacuum drying in a freeze vacuum dryer;

[0109] The freeze-drying protective agent in the BSA-SNO NPs freeze-dried agent is selected from alcohol and sugar freeze-drying protective agents, and the concentration is selected from 0%, 5%, 10%, 15%, 20%, and 25%. It can be understood that the present invention directly reduces the disulfide bonds in the middle of the protein molecules by exogenously adding a reducing agent to obtain a polythiol structure.

[0110] 3. Detailed material preparation process.

[0111] Step 1, reduction of disulfide bonds to obtain sulfhydryl groups: (1.1) Prepare 10 mM Tris buffer, adjust to pH 9.0 with 1 M hydrochloric acid, take 3 mL of buffer, add 300 mg of BSA, and sonicate to obtain a clear BSA solution. Similarly, take 3 ml of buffer, prepare a 15 mM tris (2-carboxyethyl) phosphine hydrochloride solution, and adjust to pH 9 with 5 M and 0.1 M NaOH.

[0112] (1.2) Add the above TCEP solution to the BSA solution under vortexing. After mixing evenly, a distinct rotten egg smell can be immediately detected. Shake on a shaker at room temperature for 10 min at 100-120 r / min.

[0113] (1.3) The reaction solution was placed in a dialysis bag (molecular weight cut-off: 14000 Mw) and dialyzed against 1 L of ultrapure water 5-6 times, 30 min each time, to remove small molecules.

[0114] Step 2, disulfide bond nitrosation. (2.1) Take the above product and quickly add concentrated HCl in proportion under vortexing (inject quickly within 3 seconds) to acidify the product so that its final concentration reaches 1.0-3.0.

[0115] (2.2) Take out all the products in the dialysis bag, centrifuge at 10000 r / min for 5 min, collect the supernatant, take out 200 μl, dilute 5 times, use Nanodrop protein assay BSA mode to measure the absorbance, calculate the protein content based on the standard curve, and determine the thiol concentration using Ellman's reagent method.

[0116] (2.3) According to the measured -SH concentration, prepare NaNO2 of the same concentration dissolved in 0.1M HCl, protect it from light with tin foil and use it immediately. Take BSA-SH solution and NaNO2 solution in a volume ratio of 1:1, vortex mix, and shake at room temperature in the dark for 1 hour at 100-120r / min. After the reaction is completed, the reaction solution is between yellow and orange.

[0117] Step 3, preparation of BSA-SNO nanoparticles. (3.1) Take out the above nitrosated product, place it in a 5 ml glass bottle, add different volumes of anhydrous ethanol with a pipette while vortexing, cover the bottle cap, vortex for 5 seconds, and protect from light with tin foil.

[0118] (3.2) Take out the product (about 2 ml of liquid), collect it in a small chicken heart bottle, and rotary evaporate (37°C, 80 r / min) until no bubbles are generated from the liquid.

[0119] (3.3) Accurately collect the product in the chicken heart bottle and dilute the BSA-SNO solution with 1× PBS (10 mM, pH 7.4) as needed.

[0120] Step 4, preparation of BSA-SNO NPs lyophilized agent. Take out the solution after rotary evaporation and add an equal volume of 20% trehalose solution (after mixing, the concentration is 10%), and vacuum dry it in a freeze vacuum dryer for 24 hours.

[0121] 4. Effect verification.

[0122] (A) Preparation of BSA-SNO NPs.

[0123] 1.1 Identification results of thiol products. The number of thiol groups produced by the reduction of BSA disulfide bonds by TCEP at different concentrations was identified by the Ellman method. -SH reacted with DTNB to produce a color-developing substance with ultraviolet absorption at 412nm. The reaction principle of the Ellman detection method is as follows: Figure 4 As shown. Figure 5 As shown in (1), the effect of different initial TCEP concentrations on the efficiency of BSA disulfide bond reduction under the condition of pH 9.0 in the reaction buffer system. The gray bar graph represents the relative molar number of thiol groups exposed after disulfide bond reduction, and the line graph represents the percentage of disulfide bonds remaining on the BSA molecule, n=3. Figure 5 (2) shows the stability of BSA-SH at 4°C and 25°C, n=3. Figure 5 (3) shows the stability of BSA-SH at -20°C, n=3. It can be seen that as the TCEP concentration increases, the number of exposed thiol groups increases from 5 to 30, and after being placed at -20°C for different time periods, the exposed thiol groups on each mole of BSA molecules remain stable and can be stored well.

[0124] 1.2 Identification of nitrosation products. Identification of -SNO groups produced by the reaction of different sulfhydryl and sodium nitrite ratios. Identification method: BSA-SH produces -SNO groups after nitrosation, which have ultraviolet absorption at 340nm. Figure 6(1) is the UV-visible spectrum of BSA-SNO NCs. The nitrosated thiol group (-SNO group) has a characteristic absorption at 340nm. This figure shows the absorption of different molar equivalents of NO2 - Effect of ions on the formation of -SNO groups n = 3; Figure 6 (2) is the ratio of remaining thiol groups after nitrosation of BSA-SH. Identification method: Ellman method, n=3.

[0125] 1.3 Nanoparticle identification results: The particle size of BSA-SNO NPs was 34.2 nm in the dispersion system of BSA-SNO NPs in PBS (0.01 M, pH 7.4). Figure 7 A is the particle size distribution diagram of BSA-SNO NCs, which shows the particle size distribution of nanoparticles obtained after nanoprecipitation of BSA-SNO NCs using ethanol of different concentrations.

[0126] 1.4 Freeze-drying parameter screening. Figure 7 (1) in B is the freeze-drying flow chart of BSA-SNO NPs preparation, where the trehalose concentration (wt%) is: 5% on the left and 10% on the right. Figure 7 (2) in B is the particle size distribution diagram of the reconstituted preparation; the range of the lyophilized preparation is 0, 5, 10, 15, 20.25, and the optimal is 5 and 10%; Figure 7 (3) in B is to verify the storage stability of the freeze-dried preparation, and the changes in the nanoparticle size and dispersibility (PDI) after the freeze-dried preparation was reconstituted after the preparation was stored at 25°C for different time periods, n = 3; Figure 7 (4) in B verifies the shelf stability of the freeze-dried preparation by adding 10% trehalose to the freeze-dried preparation and observing the changes in the nanoparticle size and dispersibility (PDI) after the preparation is reconstituted after being stored at -20°C for different periods of time, n=3.

[0127] 1.5 Structural analysis. When the disulfide bonds in BSA were reduced, the rigid structure was destroyed, the α-helix and β-fold content decreased, the random coil increased, and the protein structure gradually unfolded. After thiol nitrosation, the α-helix content in BSA-SNO NCs increased. When the organic solvent ethanol was added to nanoprecipitate BSA-SNO NCs to form BSA-SNO NPs, an increase in β-fold and β-turn was observed.

[0128] Table 1 Secondary structure content of different functionalized albumins

[0129]

[0130] (1) Reduction modification of BSA disulfide bonds: BSA and reducing substances are dissolved using one of Tris buffer, TBST buffer, low concentration DMSO, water and the like or a combination thereof. In some embodiments, the Tris buffer is adjusted to pH = 7.0-9.0 with hydrochloric acid, the buffer is taken, and BSA is added. A clear BSA solution with a concentration of 100 mg / ml is prepared by ultrasound, vortexing, shaking and the like. In some embodiments, the reducing substance is selected from dithiothreitol (DTT), glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP) and 2-mercaptoethanol (beta-ME). Taking TCEP as an example, the above Tris buffer is used to prepare a TCEP solution. In some embodiments, the concentration of TCEP is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, and the pH is adjusted to 7.0-9.0 with an alkaline substance (NaOH is selected in this embodiment). The TCEP solution was added to the BSA solution under vortex, oscillation, shaking, ultrasound, etc., and after being mixed evenly, the mixture was reacted on a shaker at a speed of 80r / min, 90r / min, 100r / min, 110r / min, and 120r / min for 5min-60min to ensure that the two were fully reacted. The reaction solution was placed in a dialysis bag (14000MW), and dialyzed with sufficient ultrapure water for multiple times until the small molecules were completely removed, and the dialyzate was analyzed by infrared and ultraviolet spectra to determine that the small molecules were completely removed.

[0131] (2) -SH nitrosation: Use hydrochloric acid, sulfuric acid and other substances to adjust the pH of the solution used for dialysis to 1.0-6.0, and inject the acid directly and quickly into the product after the previous dialysis step according to a certain ratio to fully acidify the protein. In this process, the product quickly appears as a white flocculent precipitate, and the white flocculent will dissolve and disappear within 2 seconds. At this time, the product state is a clear and transparent solution. Determine the content of BSA and the concentration of thiol groups, and the number of thiol groups formed by TCEP breaking disulfide bonds can be inferred from this. In some schemes, the protein determination BSA mode in Nanodrop is used to determine the absorption value and calculate the protein content using a standard curve. The thiol concentration is determined using the Ellman reagent method. According to the measured -SH concentration, prepare a NaNO2 solution and a GSNO solution of appropriate concentration to nitrosate the thiol group. A certain implementation scheme is to dissolve the same concentration of NaNO2 in 0.1M HCl, use tin foil to protect from light, and prepare it for use immediately. Take BSA-SH solution and NaNO2 solution, and mix them in volume ratios of 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, and 1:2, vortex mix, and shake at room temperature in the dark for 1 hour at 100-120r / min. This step converts the unstable -SH into -SNO. After the reaction, the reaction solution is between yellow and orange. The reaction solution after nitrosation is dialyzed to remove the remaining small molecules such as acid. The obtained BSA-SNO is relatively stable, but it is sensitive to strong light and reducing substances (especially Cu 2+ , Fe 2+ The product needs to be stored away from light, and a metal ion complexing agent can also be added to protect the stability of BSA-SNO.

[0132] (3) Formation of BSA-SNO nanoparticles. The above-mentioned nitrosated product is mixed with a solvent such as ethanol, acetone, propanol, tetrahydrofuran, etc. that is miscible with water but cannot dissolve BSA. Taking ethanol as an example, the nitrosated product and ethanol are mixed in a ratio of nitrosated product: ethanol = 1:0.1, 1:0.3, and 1:0.5. The mixed solution is fully mixed and reacted under conditions such as vortexing, oscillation, shaking, and ultrasound. At this time, BSA-SNO will precipitate and spontaneously agglomerate due to the reduced solubility in the mixed solution, and BSA-SNO nanoparticles are formed in the solution. The obtained nanoparticles are protected from light and the excess unreacted small molecules are removed by dialysis. The dialyzed product is accurately collected, and PBS (0.01M, pH 7.4) is added to disperse and store the nanoparticles. The present invention analyzes the obtained nanoparticles by particle size, particle size dispersion, electron microscopy, infrared, ultraviolet, etc. It is proved that the obtained nanoparticles are pure, small in size, uniform and controllable. Meets the requirements of use.

[0133] (4) Freeze-drying protection of the product. Take the product after rotary evaporation to remove impurities, mix it with a freeze-drying protective agent and freeze it at -80°C. Put the frozen product in a freeze dryer to make freeze-drying in the dark.

[0134] In certain embodiments, the freeze-drying protective agent is selected from alcohols and sugar freeze-drying protective agents, which will crystallize during slow freezing, thereby providing a support structure for the active component and will not react with the active component. Specifically, there are one of xylitol, mannitol, sorbitol, glycine, glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan and inulin, and their combinations. In addition, salts are used as buffers in certain experimental schemes to balance the pH value during freeze-drying storage to achieve a protective effect. In certain schemes, sodium phosphate, potassium phosphate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, and their combinations are selected and used.

[0135] In addition, the protection of sugars and alcohols during freeze-drying of products is directly related to their added concentration. In some schemes, the concentration of freeze-drying protectants is selected to be 0%, 5%, 10%, 15%, 20%, or 25%.

[0136] (B) Effect experiment

[0137] 2.1, the therapeutic effects of the preparations at the cellular level were characterized.

[0138] 2.2, Release of NO small molecules at the cellular level. Figure 8 The semi-quantitative graphs show the relative fluorescence intensity of NO produced by different preparations after different incubation times (1, 3, 6, 12 hours) in the normal mouse liver cell line AML-12; detection method: Diaminofluorescein-FM diacetate (DAF-FM DA) NO detection probe, probe concentration: 5 μM; incubation time after administration is 12 hours. n = 6.

[0139] 2.3, ROS scavenging effect at the cellular level. Fig. 9 ROS levels of different preparations after H2O2-induced acute hepatocyte injury for 12 hours

[0140] 2.4, Cell apoptosis analysis. Fig.10 The flow cytometric graphs of cell apoptosis after different incubations for 12 hours in the H2O2-induced acute hepatocyte injury model (AML-12 cell line) with different preparations reflect the cell apoptosis. The detection method is flow cytometric analysis (Beyotime, C1062M) after staining with Annexin V-FITC apoptosis detection kit. Annexin V positive indicates early apoptotic cells, and PI positive indicates necrotic cells. The incubation time after administration is 12 hours.

[0141] 2.5, cell viability restored. Fig.11The recovery of cell viability after different preparations were incubated for 12 hours in the H2O2-induced acute hepatocyte injury model (AML-12 cell line). Detection method: After incubation with the cck8 cell viability detection probe (Beyotime, C0037), the cell viability was detected by an ELISA instrument.

[0142] Experiments show that:

[0143] (1) NO release at the cellular level. Initially, it was observed that NO was gradually released within the first 3 hours after administration of the thiol nitrosated preparation and reached a high level at 6 hours. In the subsequent period (12 hours), the amount of NO released tended to decrease slowly. It is worth noting that during the administration period, the release of NO in the cells increased with the increase in the number of nitrosated thiols and the ratio of nitrosated thiols in the unit thiol nitrosated nanoformulation. At the same nitrosated thiol administration concentration, the intracellular NO content of BSA-18SNO NPs was higher than that of BSA-5SNO NPs within the unit incubation time (1, 3, and 6 hours), and this difference was more obvious at a higher nitrosated thiol administration concentration (nitrosated thiol concentration: 1000nM). The NO release efficiency is related to the ratio of BSA functional groups on a single molecule, that is, the higher the functional group ratio, the higher the NO release efficiency in the cells at the same administration concentration.

[0144] (2) ROS scavenging. It was investigated whether the supplementation of exogenous NO and antioxidant thiol groups to cells could serve as an effective antioxidant molecule reservoir to resist cellular damage caused by oxidative stress. Flow cytometry results showed that BSA-18SH could effectively scavenge the excessive ROS production in H2O2-stimulated damaged cells (reduced by 19.2% compared with the model group) due to the significant increase in the number of exposed antioxidant thiol groups in albumin molecules by cleaving intrachain disulfide bonds. Compared with BSA-SH, the relative ROS level in the BSA-SNO NCs-treated group decreased more significantly, decreasing by 66.1% compared with the model group, highlighting the increase in antioxidant activity mediated by NO released from the developed formulation. With the increase of thiol concentration and the ratio of thiol groups on BSA-SNO NPs, BSA-SNO NPs produced significant thiol dose-dependent inhibition of intracellular ROS, which emphasized that the strong antioxidant effect of the prepared formulation was related to the number of nitrosated thiol functional groups. In particular, at the same nitrosatable thiol concentration (250nM and 1000nM), the preparation with a higher ratio of nitrosatable thiol groups on the single molecule BSA (BSA-18SNO NPs) showed better antioxidant effect than the preparation with a low ratio of nitrosatable thiol groups (BSA-5SNO NPs), which was manifested as lower intracellular ROS intensity. Treatment with high-dose BSA-18SNO NPs (nitrosatable thiol concentration: 1000nM) resulted in a significant decrease in intracellular ROS levels, even by 82.3% compared with the model group. After treatment with high-dose BSA-18SNO NPs, intracellular ROS almost reached the same level as the normal group.

[0145] (3) Inhibition of cell apoptosis. Apoptosis analysis showed that BSA-SNO NCs and BSA-SNO NPs with different concentrations of nitrosated thiol functional groups significantly inhibited cell damage and apoptosis induced by exogenous H2O2 stimulation. BSA-18SNO NPs can significantly prevent H2O2-induced hepatocyte apoptosis and effectively reduce the proportion of apoptotic cells and necrotic cells.

[0146] (4) Restoration of cell viability. Self-thiolated and nitrosylated BSA preparations were able to restore cell viability to varying degrees, and the cytoprotective effects of different preparations increased with increasing functional group concentrations. BSA-SH restored cell viability to more than 70% at a thiol concentration of 250 μM, while BSA-SNO NCs also exerted similar cytoprotective properties (cell viability restored to more than 70% of normal cells) at a lower concentration of nitrosylated thiol administration (1 μM). This indicates that nitrosylated thiol groups deliver NO through the cytoplasm, promoting cytoprotection together with exposed thiol groups, and ultimately more effectively rescuing dying cells. After treatment with a high dose of BSA-SNO NPs (1.5 μM), cell viability was restored to 86.8%, indicating the effectiveness of functionally modified BSA nanoformulations in rescuing dying cells.

[0147] (III) Analysis of the therapeutic effect of the preparation in the animal model of acute liver injury induced by ischemia-reperfusion injury.

[0148] 3.1 Biodistribution in vivo. Fig.12 The biodistribution and semi-quantitative results of free BSA, BSA-18SNO NCs and BSA-18SNO NPs labeled with Cy5 in important organs (heart, liver, spleen, lung and kidney) in the ischemia-reperfusion-induced acute liver injury mouse model after intravenous injection of free BSA, BSA-18SNO NCs and BSA-18SNO NPs, n=3.

[0149] 3.2 Liver histopathological recovery. Fig.13 This is hematoxylin-eosin (H&E) staining of liver tissue (Figure C). The black solid line area is the severe necrotic area; the solid circle area is the hepatocyte vacuolar degeneration and balloon necrosis; the arrows represent hepatocyte lysis and necrosis, inflammatory cell infiltration and hepatocyte regeneration, respectively; scale bar: 100μm.

[0150] 3.3 Monitoring of serum liver enzyme levels. Fig.14 Serum liver enzyme indicators include alanine aminotransferase (ALT, Figure A), aspartate aminotransferase (AST, Figure B), and alkaline phosphatase (AKP, Figure C). n=8.

[0151] 3.4 Inhibit tissue cell apoptosis and relieve oxidative stress at the tissue level. Fig.15 (A) is a quantitative graph of the number of TUNEL-positive apoptotic cells in the liver; n = 6; (B) is a semi-quantitative graph of the fluorescence intensity of superoxide DHE fluorescence staining in the liver, n = 6

[0152] 3.5 Alleviate oxidative stress at the tissue level. Fig.21 (A) is the level of malondialdehyde (MDA, Figure A), (B) glutathione (GSH), and (C) superoxide dismutase (SOD) in liver tissue homogenate, n = 8; Fig.21 Middle (D) is the level of oxidized glutathione (GSSG) in liver tissue homogenate, n = 8;

[0153] 3.6 Inhibit the inflammatory cascade and restore the liver tissue microenvironment at the tissue level. Fig.17 Immunofluorescence staining of CLEC4F (red fluorescence: liver-resident macrophages, Kupffer cells) and F4 / 80 (green fluorescence: monocyte-derived macrophages) in frozen sections of liver tissue. Scale bar: 100 μm (Merge), 50 μm (Magnify); n=8;

[0154] Fig.18 Western blot analysis of inflammatory factors in liver tissue homogenate, pro-inflammatory factor interleukin-6 (IL-6, Figure A), pro-inflammatory factor tumor necrosis factor-α (TNF-α, Figure B), and anti-inflammatory factor interleukin-10 (IL-10, Figure C) levels. n = 3.

[0155] 3.7 Therapeutic mechanism at the molecular level. Fig.19 Western blot analysis of liver tissue homogenate related protein expression, Nrf-2 (nuclear factor erythroid-2-related factor 2, Figure A), HO-1 (heme oxygenase 1, Figure B), NQO1 (NAD (P) H: quinone oxidoreductase, Figure C), p-IκBα ((Phospho-InhibitorySubunit of NF Kappa B Alpha, Figure D), NF-κB (nuclear factor kappa-B, Figure E), Cyto C (cytochrome C, Figure F), Bcl-2 (B cell lymphoma / leukemia-2, Figure G), Bax (BCL2-AssociatedX, Figure H), Cleaved caspase-3 (Figure I) levels and semi-quantitative results. n = 3.

[0156] 3.8 Biocompatibility assessment. Fig.25The biosafety evaluation of functionalized albumin nanoformulation in an animal model of acute liver injury induced by ischemia-reperfusion. Serum creatinine level (Figure A), urea nitrogen level (Figure B), H&E tissue sections of mouse heart, spleen, lung and kidney, scale bar: 100 μm; n = 8.

[0157] Experiments show that:

[0158] (1) In vivo biodistribution. Near-infrared fluorescence imaging images showed that after intravenous injection of Cy5-labeled BSA-18SNO NCs / NPs, liver lesions showed obvious fluorescence signals at various time points compared with free Cy5-labeled BSA preparations. The Cy5-labeled BSA-18SNO NPs administration group showed more significant fluorescence signals in the liver than the BSA-18SNO NCs treatment group at each time point, indicating that the nanoformulated preparations accumulated rapidly and preferentially in the liver.

[0159] (2) Recovery of liver histopathology. In the acute liver injury mouse model induced by ischemia-reperfusion, a single dose of BSA-18SNO NCs (thiol concentration: 4 μmol / kg) significantly reduced liver congestion and focal necrosis area, and reduced inflammatory cell infiltration in liver tissue. An equal dose of BSA-18SNO NPs (thiol concentration: 4 μmol / kg) can effectively treat the liver tissue structure in acute liver injury model mice, and its morphology is even close to that of healthy liver. Further histological analysis showed that after treatment with BSA-18SNO NPs, some liver sections showed only slight histological damage, reduced inflammatory cell infiltration, no obvious necrosis of hepatocytes, and significant recovery of liver cord and sinusoid structures.

[0160] (3) Recovery of liver function. Serum ALT and AST gradually increased after ischemic injury and reached a peak at 12 h of reperfusion, indicating severe liver damage. BSA-18SNO NCs effectively reduced liver ALT and AST levels in the first 6 h of reperfusion, by 45.3% and 52.1%, respectively, compared with the model group, and continued to reduce serum transaminase levels throughout the treatment period. BSA-18SNO NPs caused a significant and rapid decrease in ALT and AST levels, by 74.0% and 76.3%, respectively, at 6 h compared with the model group, and even returned to normal levels after 24 h of reperfusion.

[0161] (4) Anti-apoptosis at the tissue level. After ischemia-reperfusion injury, TUNEL-positive cells in liver tissue increased significantly, reaching 19.9 times that of the normal group, while the proportion of apoptotic cells was significantly reduced after BSA-18SNO NCs treatment (reduced by 61.6%). BSA-18SNO NPs administration almost completely eliminated TUNEL-positive cells in mouse liver tissue (reduced by 85.6% compared with the model group), as manifested by almost invisible fluorescent signals in TUNEL immunofluorescence staining of liver sections, further supporting that single-dose treatment with BSA-18SNO NPs attenuated hepatocyte apoptosis and protected hepatocytes from the adverse effects of ischemia-reperfusion injury during acute liver injury.

[0162] (5) Alleviate oxidative stress at the tissue level. Compared with the model group, treatment with BSA-18SNO NCs inhibited oxidative stress damage, as shown by a significant decrease in superoxide levels in liver tissue homogenate (DHE fluorescence quantification decreased by up to 64.7% compared with the model group), recovery of antioxidant activity (SOD increased to 1.5 times that of the model group), and reduction in lipid peroxidation (MDA decreased by 42.0%). In particular, the level of ROS in the liver of mice treated with BSA-18SNO NPs was significantly reduced, as shown by a significant decrease in DHE fluorescence intensity (a decrease of 79.8% compared with the model group), a significant recovery of the activity of the antioxidant enzyme SOD (the level increased by up to 1.2 times), a decrease in the level of antioxidant GSH in liver tissue, and a decrease in the GSH / GSSG ratio.

[0163] (5) Inhibit the inflammatory cascade and restore the liver tissue microenvironment at the tissue level. The number of Kuppffer cells and mononuclear macrophages was greatly reduced in the group treated with a single dose of BSA-18SNO NCs (the dose of nitrosothiol was 4 μmol / kg). Compared with BSA-18SNO NCs, BSA-18SNO NPs treatment was able to more effectively inhibit the activation of inflammatory cells during ischemia-reperfusion injury, and there was almost no obvious expression of macrophage positive markers after 24 hours of treatment. In addition, mice treated with BSA-18SNO NPs (4 μmol / kg) showed high expression of the anti-inflammatory factor IL-10, and the levels of proinflammatory cytokines TNF-α and IL-6 in liver tissue were downregulated to normal levels. On the other hand, intravenous administration of BSA-18SNO NCs and BSA-18SNO NPs in an acute liver injury mouse model resulted in down-regulation of endothelial inflammatory marker ICAM-1 expression and a significant reduction in Ly6G-positive neutrophil recruitment, confirming a significant reduction in neutrophil activation, adhesion, and infiltration. This also means that the treatment of self-sulfurized, nitro-fixed BSA nanoformulations can not only alleviate the induction of local inflammation, but also reduce the possibility of neutrophil reverse migration into the circulation system and promote distal organ damage through neutrophil extracellular trap-associated thrombosis.

[0164] (6) Molecular mechanism exploration. Compared with the normal group, the expression of Nrf2, a key transcription factor and master regulator of the antioxidant defense pathway in the liver of mice with severe liver I / R, was significantly reduced by 73.6%. In mice treated with BSA-18SNO NCs and nano-BSA-18SNONPs, Nrf2 expression was significantly increased by 3.1 and 4.7 times compared with the model group, while the antioxidant HO-1 and NQO1 proteins in hepatocytes downstream of Nrf2 were significantly upregulated, increasing by about 2.3 and 1.5 times, respectively, and even returned to normal levels. Compared with the model group, treatment with BSA-18SNO NC preparations attenuated the phosphorylation level of IκBα, a liver inflammation-related protein in acute liver injury, by 27.1%, and downregulated the level of nuclear NF-κB p65 by 12.6%. In particular, the administration of BSA-18SNO NPs resulted in a significant reduction in the expression of NF-κB subunit p65 to normal levels. BSA-18SNP NCs and BSA-18SNO NPs inhibited oxidative stress, prevented the collapse of mitochondrial membrane potential, and reduced the release of Cyto C from the mitochondrial membrane, resulting in a 35.4% and 54.9% reduction in Cyto C, respectively. The expression of Cyto C was significantly downregulated after BSA-18SNO NPs treatment, and its level even reached a level comparable to that of the normal group. The preparation further significantly upregulated the anti-apoptotic protein Bcl-2 and downregulated the expression of apoptosis-related Bax protein after treatment, ultimately leading to an increase in the Bax / Bcl-2 ratio, thus indicating an effective inhibitory effect on the initiation of the mitochondrial apoptotic signaling pathway. The cleavage of caspase-3, a key downstream event of the mitochondrial apoptotic pathway, was further significantly inhibited, and its level was similar to that of the normal group, indicating an effective inhibition of the mitochondrial apoptotic pathway.

[0165] (7) Biocompatibility assessment. The in vivo biosafety of BSA-18SNO NPs was further evaluated by measuring serum biomarkers (such as urea nitrogen and creatinine) and assessing histological changes in major organs. In the ischemia-reperfusion-induced acute liver injury mouse model, injection of BSA-18SNO NCs and BSA-SNO NPs did not lead to changes in serum creatinine and BUN, nor did it cause structural damage to major organs (heart, spleen, kidney, lung, and kidney).

[0166] (IV) Analysis of the therapeutic effect of the preparation in the animal model of acute liver injury induced by lipopolysaccharide / D-galactosamine.

[0167] 4.1 Liver histopathological recovery. Fig.21Figure 2. Flow chart of the experiment of nanomedicine intervention on acute liver injury induced by lipopolysaccharide / D-galactosamine (LPS / D-gal), survival rate analysis (B), and H&E tissue staining results of liver of each group of mice. The dotted circle indicates vacuolar degeneration and balloon necrosis of hepatocytes, and the arrow area indicates hepatocyte lysis and necrosis as well as inflammatory cell infiltration. Scale bar: 100 μm.

[0168] 4.2 Liver function recovery. Fig. 22 Serum liver enzyme indexes included alanine aminotransferase (ALT, Figure A), aspartate aminotransferase (AST, Figure B), and alkaline phosphatase (AKP, Figure C). The sample size of each group at 6h, 12h, and 24h was: control group n=8, 8, 8; model group n=6, 4, 4; BSA-18SNO NCs group n=9, 8, 7; BSA-18SNO NPs (1μmol / kg) group: n=9, 7, 6; BSA-18SNO NPs (4μmol / kg) group: n=12, 11, 11.

[0169] 4.3 Anti-apoptotic effect at the tissue level. Fig.23 (1) shows the number of TUNEL-positive cells in the liver, n=6. (2) shows the quantitative results of DHE fluorescence staining in the liver, n=6

[0170] 4.4 Antioxidant effect at the tissue level. Fig.24 The levels of malondialdehyde (MDA, Figure A), glutathione (GSH, Figure B), superoxide dismutase (SOD, Figure C) and oxidized glutathione (GSSG, Figure D) in liver tissue homogenate were measured. The sample size of each group was: control group n = 8; model group n = 4; BSA-18SNO NCs group n = 7; BSA-18SNO NPs (1μmol / kg) group: n = 6; BSA-18SNO NPs (4μmol / kg) group: n = 11.

[0171] 4.5 Relief of renal damage. Fig.25 Serum creatinine (Figure A), urea nitrogen level (Figure B) and H&E staining of renal tissue (Figure C), scale bar: 200μm, 50μm (Magnify). Arrows represent glomerular atrophy, shedding of damaged cells, cast formation, tubular dilatation, and massive infiltration of inflammatory cells.

[0172] 4.6 Therapeutic mechanism in LPS / D-gal-induced liver injury animal model. Fig.26 Western blot analysis of key protein expression in liver tissue, including the levels of HMGB1 (High Mobility Group Protein 1, Figure A), TLR4 (Toll-like receptor 4, Figure B), NF-κB (nuclear factor kappa-B, Figure C) p-IκBα (Phospho-Inhibitory Subunit of NF Kappa B Alpha, Figure D), proinflammatory factor tumor necrosis factor-α (Tumor necrosis factor-α, TNF-α, Figure G), proinflammatory factor interleukin-6 (Interleukin-6, IL-6, Figure H), Cleaved caspase-1 (Figure I) and proinflammatory factor interleukin-1β (Interleukin-1β, IL-1β, Figure J), ​​and their semi-quantitative results. Liver tissue frozen sections showed the expression of NLRP3 (NOD-like receptor thermal protein domain associated protein 3, Figure E) and ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain, Figure F) Immunofluorescence staining results.

[0173] Experiments show that:

[0174] (1) Improve the survival rate of endotoxin-induced model animals and restore the pathological structure of liver tissue. The preparation was able to protect the liver, restore liver function, and significantly improve the survival rate in the clinically relevant LPS / D-gal-induced acute liver injury mouse model (endotoxin-related liver injury). The survival rate of the high-dose BSA-18SNO NPs (administered nitrosated thiol amount: 4μmol / kg) treatment group was significantly improved to 73.3%, indicating that it has a good therapeutic effect on improving endotoxin-induced acute liver injury. High-dose self-thiolated and nitro-fixed BSA-18SNO NPs preparations can significantly reverse ALI and protect liver damage, with the liver structure well preserved and sinusoidal congestion, edema, cavitation or necrosis significantly reduced.

[0175] (2) Reduction of tissue liver enzyme indicators. In contrast to the model group, which showed a continuous increase in serum liver enzyme levels, mice treated with BSA-18SNO NCs (administered nitrososulfhydryl amount: 4 μmol / kg) showed a certain degree of resistance to LPS / D-gal stimulation, as evidenced by a 53.5% and 56.4% decrease in serum ALT and AST levels, respectively, in the first 6 hours, which remained at low levels during the treatment period. BSA-18SNO NPs treatment showed a significant dose-dependent reduction in serum ALT and AST levels. In particular, serum ALT and AST levels were significantly reduced by 81.8% and 77.8% within 6 hours after treatment with a high-dose BSA-SNO NPs formulation (administered nitrososulfhydryl amount: 4 μmol / kg), and even dropped to levels similar to those of the normal group, while their levels remained within the normal range throughout the treatment period.

[0176] (3) Inhibition of cell apoptosis. LPS / D-gal administration led to a large number of liver cell apoptosis. The number of TUNEL-positive cells (apoptotic cells) in the liver tissue sections of mice treated with BSA-18SNO NPs was greatly reduced, approaching the level of normal tissue.

[0177] (4) Relieve oxidative stress. The prepared BSA-18SNO NPs significantly inhibited the secretion of superoxide in mouse liver (reduced DHE by about 66.6%), reduced the degree of lipid peroxidation (MDA decreased by about 64.6%), and restored the liver's antioxidant capacity (manifested by the significant recovery of SOD and GSH, even close to normal levels). In other words, the nanoscale self-sulfurized and nitro-fixed BSA preparation alleviated the oxidative stress of damaged tissues in the endotoxin-induced acute liver injury mouse model, thereby effectively restoring the cellular redox state.

[0178] (5) Alleviation of renal injury. BSA-18SNO NCs and BSA-SNO NPs also provided therapeutic benefits to the kidneys after treatment, showing that the administration of the formulation significantly restored renal histological damage, as manifested by a significant decrease in creatinine and urea nitrogen in the treatment group, as well as reduced glomerular atrophy, reduced tubular dilatation, and reduced epithelial cell shedding in the renal tissue.

[0179] (6) Therapeutic molecular mechanism. In this case, in the LPS / D-GalN-induced acute liver injury model, a single intravenous administration of BSA-18SNO NPs blocked the expression of key proteins in the downstream HMGB1-TLR4-IκBα-NF-κB axis in a dose-dependent manner through the synergistic effect of NO and antioxidant thiol groups to effectively inhibit the initiation of the inflammatory cascade, thereby blocking the expression of downstream inflammatory factors including IL-6 and TNF-α. BSA-18SNO NPs administration further inhibited the recruitment of NLRP3 inflammasomes and ACS, thereby blocking the upregulation of the proinflammatory cytokine IL-1β and the cleavage of the apoptotic protein caspase 1, thereby exerting an anti-pyroptotic effect in liver-damaged cells.

[0180] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of nanoparticles BSA-18SNO NPs in the preparation of albumin nanomedicine for treating acute liver injury, characterized in that: The number of antioxidant thiol functional groups of the nanoparticles BSA-18SNO NPs is 18 per mol of albumin molecules, the concentration of active functional groups of the nanoparticles BSA-18SNO NPs in animal in vivo treatment is 1 μmol / kg to 100 μmol / kg, the nanoparticles BSA-18SNO NPs are prepared by reducing the disulfide bonds between BSA molecules into multiple thiol groups, generating a self-thiolated albumin structure BSA-SH of thiol antioxidant groups under suitable conditions, nitrosating 18 thiol groups to obtain BSA-18SNO NCs, and then subjecting the BSA-18SNO NCs to a nanoprecipitation method; The functional thiol groups with antioxidant properties are obtained by reducing the intermolecular disulfide bonds, and have one or more of the following characteristics: The albumin is selected from bovine serum albumin, human serum albumin, porcine serum albumin, donkey serum albumin, mouse serum albumin or any combination thereof; Provide a nitrosated, self-thiol functional group albumin nanoformulation to deliver thiol and NO molecules; In the process of forming particles of BSA-18SNO NCs by nanoprecipitation, the solvent is one of ethanol, acetone, propanol and tetrahydrofuran; The solvent ratio is 1:0.1, 1:0.3, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, and the nitrosation product and the solvent are mixed to perform BSA-18SNO NCs nanoprecipitation; The method of removing the organic solvent includes one or a combination of dialysis, rotary evaporation, vacuum drying, and freeze drying; The method for preparing the nanoparticles BSA-18SNO NPs comprises the following steps: S1, BSA disulfide bond reduction modified BSA-SH, where the number of thiol functional groups is 1-18 per mol albumin molecules; S2, BSA-SH nitrosation reaction to form self-thiolated and nitrosylated serum albumin nanocomplexes BSA-18SNONCs; S3, nanoparticles BSA-18SNO NPs were formed by nanoprecipitation of self-thiolated and nitrosylated serum albumin nanocomplexes BSA-18SNO NCs.

2. The use according to claim 1, characterized in that The nanoparticles have one or more of the following characteristics: The nanoparticles are in the shape of spherical particles; The particle size of the nanoparticles is 20nm-100nm; The polydispersity index (PDI) of the nanoparticles is 0.1-0.5; The zeta potential of the nanoparticles is -5 mV to -30 mV.

3. The use according to claim 1, wherein the preparation comprises a freeze-dried preparation, characterized in that: The nanoprecipitated BSA-18SNO NPs are subjected to rotary evaporation to remove impurities, mixed with a freeze-drying protective agent and frozen at -80°C, and the frozen product is placed in a freeze dryer to be freeze-dried in the dark; the freeze-dried preparation uses salt as a preparation resuspension buffer to balance the pH value during freeze-drying storage; The freeze-drying protective agent is selected from alcohol freeze-drying protective agents and sugar freeze-drying protective agents, and the alcohol freeze-drying protective agent is one or a combination of xylitol, mannitol, sorbitol, and glycine; The carbohydrate freeze-drying protective agent is one or a combination of glucose, sucrose, lactose, trehalose, maltose, maltopolysaccharide, fructan and inulin; The concentration of the lyoprotectant is selected from one of 0%, 5%, 10%, 15%, 20%, and 25%; The buffer includes one or a combination of sodium phosphate and potassium phosphate.

4. The use according to claim 1, characterized in that The preparation method uses DTT and TCEP to reduce and open the disulfide bonds in BSA, thereby generating a BSA-SH complex having multiple sulfhydryl groups in the form of a solution; wherein the reaction formulas for DTT and TCEP to reduce and open the disulfide bonds in BSA are as follows:

5. The use according to claim 1, characterized in that: In the BSA-SH nitrosation reaction of step S2, BSA-SH is nitrosated by NaNO2 under acidic conditions, and the composite in the nanoparticle BSA-18SNO NCs is prepared by claim 1, specifically comprising: (2.1) Take the BSA-SH product and add concentrated HCl under vortexing to acidify the product; (2.2) taking out all the products in the dialysis bag, centrifuging, collecting the supernatant, diluting, measuring the absorbance, calculating the protein content, and measuring the thiol concentration; (2.3) Based on the measured -SH concentration, prepare NaNO2 of the same concentration dissolved in 0.1 M HCl and keep it away from light. Mix the BSA-SH solution with the NaNO2 solution and shake it at room temperature away from light.

6. The use according to claim 1, providing a nanoparticle preparation in an injectable form, characterized in that: The nanoparticles BSA-18SNO NCs used in any one of claims 1 to 3 are subjected to precipitation polymerization by a nanoprecipitation method to form nanoparticles with a dense structure, wherein the step S3 uses the nanoparticles BSA-18SNO NCs used in any one of claims 1 to 2; specifically comprising: (3.1) providing a nanocomposite suspension solution comprising any one of claims 1 to 2 and a solvent; (3.2) Selecting a nanocomposite suspension comprising any one of claims 1 to 2, and rapidly injecting an optional organic solvent in a vortex state according to the ratio of claim 1; (3.3) Removing the organic solvent according to the method of claim 1 to obtain a solution containing the nanoformulation.

7. A use of nanoparticles BSA-18SNO NPs in the preparation of liver-targeted functionalized albumin nanopharmaceutical compositions / preparations according to any one of claims 1 to 3, characterized in that: This application utilizes the thiol and further nitrosylation of the disulfide bonds in albumin itself to achieve the combination of functional antioxidant thiol groups with NO gas small molecules; In the drug preparation, the therapeutic molecule is a combination of an anti-inflammatory NO gas small molecule and an antioxidant thiol group.

Images