Gold nanoparticles capable of loading aromatic drugs, preparation method and application thereof

Abstract

The present invention relates to gold nanoparticles capable of loading aromatic drugs, and their preparation and application, belonging to the field of biomedicine technology. The gold nanoparticles of the present invention comprise gold nanospheres, 4-mercaptobenzoic acid ligands, and methoxy-terminated polyethylene glycol-amino groups, wherein the gold nanospheres, 4-mercaptobenzoic acid ligands, and methoxypolyethylene glycol are sequentially connected by chemical bonds; wherein the core diameter of the gold nanoparticles is less than 3 nm and the hydrodynamic diameter is less than 6 nm. The gold nanoparticles of the present invention can improve the activity and bioavailability of the aromatic drugs loaded thereon. The gold nanoparticles are small in size and can be cleared from the body by the kidneys, avoiding capture by the liver and reducing nonspecific uptake by other organs, thereby improving drug bioavailability while reducing side effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to gold nanoparticles capable of loading aromatic drugs, and a preparation method and application thereof. Background Art

[0002] Acute kidney injury (AKI) refers to a clinical syndrome characterized by a rapid decline in renal function due to a variety of etiologies. Early diagnosis and treatment of AKI are crucial for patient prognosis. However, serum creatinine, a commonly used clinical renal function test, has a significant lag effect. A significant increase in creatinine levels often occurs only after the glomerular filtration rate (GFR) has decreased by more than 50%, at which point irreversible kidney damage has already occurred. Therefore, early diagnosis of AKI and prompt treatment, when renal impairment is relatively mild, are urgently needed. This can effectively protect renal function, reduce mortality in AKI patients, and inhibit the progression to chronic kidney disease (CKD). During the progression of AKI, cellular damage caused by intracellular oxidative stress is one of the main causes of AKI progression. Therefore, the development of antioxidant drugs targeting reactive oxygen species (ROS) could mitigate the progression of AKI and effectively protect renal function. Natural aromatic antioxidants (such as curcumin, resveratrol, and quercetin) possess excellent antioxidant activity and are economical. However, due to its strong hydrophobicity, it has low bioavailability and is difficult to reach or distribute in the kidneys.

[0003] At present, with the continuous development of nanomaterials, many nanomaterials have been synthesized and applied in the treatment of AKI; for example, nanozymes with their own antioxidant properties can achieve the treatment of acute kidney injury. In addition, loading substances with antioxidant activity into nanomaterials to treat AKI in response to the pathological state of oxidative stress in the kidneys under AKI conditions has become a common strategy. Currently, there are roughly two ways to deliver drugs for the treatment of AKI using nanomaterials. One is to connect antioxidants or therapeutic drugs to nanomaterials through chemical bond modification. Although drug delivery systems prepared by this type of chemical modification can improve the bioavailability and targeting efficiency of drugs, there are also changes in the drug structure caused by chemical modification, which affect the activity of the drug. In addition, such delivery systems are usually not universal, and specific design is required for the delivery of different drugs, making it impossible to deliver multiple types of drugs. The labor cost is high.

[0004] Furthermore, the size of most nanomaterial delivery systems limits their application in the treatment of renal diseases. Large nanoparticles have difficulty penetrating the glomerular membrane and renal extracellular matrix, making it difficult for drugs to effectively reach the target site. Due to their size, most are cleared through the liver, and even if they reach the target site, they may remain in the body for a long time, potentially triggering toxic reactions, especially in such organs that require efficient filtration and excretion. Furthermore, large-sized nanodelivery systems are less controllable in drug release kinetics than small particles, potentially leading to drug release at inappropriate times and locations. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of existing technologies by providing gold nanoparticles capable of loading aromatic drugs, as well as their preparation method and application. These gold nanoparticles are small in size and can be cleared from the body by the kidneys, avoiding capture by the liver and reducing nonspecific uptake by other organs, thereby increasing drug bioavailability and minimizing side effects.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a gold nanoparticle, comprising a gold nanosphere, a 4-mercaptobenzoic acid ligand, and a methoxy-terminated polyethylene glycol-amino group, wherein the gold nanosphere, the 4-mercaptobenzoic acid ligand, and the methoxy-terminated polyethylene glycol-amino group are sequentially connected by chemical bonds; the core diameter of the gold nanoparticle is less than 3 nm; and the hydrodynamic diameter of the gold nanoparticle is less than 6 nm.

[0008] Specifically, the gold nanospheres are connected to the methoxy-terminated polyethylene glycol-amino group through the 4-mercaptobenzoic acid ligand; more specifically, the gold nanospheres replace the thiol hydrogen on the 4-mercaptobenzoic acid ligand, and the amino group of the methoxy-terminated polyethylene glycol-amino group undergoes a condensation reaction with the carboxyl group on the 4-mercaptobenzoic acid ligand.

[0009] As a preferred embodiment of the first aspect of the present invention, the core diameter of the gold nanoparticles is 1.83±0.9 nm.

[0010] This invention utilizes 4-mercaptobenzoic acid (4-MBA) as a ligand to prepare a gold nanoparticle with drug delivery capabilities. 4-MBA, with its benzene ring structure, can form π-π stacking interactions with various aromatic drugs and serve as drug loading sites in the gold nanoparticle delivery system. PEGylation of 4-MBA prolongs the in vivo circulation time of the gold nanoparticles, effectively altering the pharmacokinetics of the loaded drug and preventing rapid drug clearance from the body. Furthermore, the gold nanoparticles of this invention have a core diameter of less than 3 nm and a hydrodynamic diameter of less than 6 nm, a size below the renal filtration threshold (6 nm). They can be cleared from the body via the kidneys, avoiding side effects caused by liver capture and reducing nonspecific uptake by other organs, thereby enhancing efficacy while minimizing side effects. In particular, when the core diameter of the gold nanoparticles of this invention is 1.83±0.9 nm, the smaller size facilitates renal entry and clearance.

[0011] In this technical field, the core diameter of a gold nanoparticle refers to the physical dimensions (e.g., diameter or radius) of the core portion of the gold metal (Au). Therefore, the core diameter of the gold nanoparticles described herein refers to the diameter of the gold nanospheres. Core size is typically measured by direct observation using a transmission electron microscope (TEM).

[0012] In this technical field, the hydrodynamic diameter (HD) is an important parameter used to describe the behavior of particles in fluids. It generally reflects the "apparent size" of a nanoparticle in solution, including the thickness of surface modifications or adsorbed molecules, and is typically larger than the core diameter. The hydrodynamic diameter is determined by analyzing the Brownian motion of the particles in solution. The smaller the particle, the faster the Brownian motion and the larger the diffusion coefficient. Therefore, the hydrodynamic diameter measurement range of the gold nanoparticles described in the present invention includes the diameter of the gold nanospheres and the thickness of their surface modification layer, which includes 4-mercaptobenzoic acid ligands and methoxy-terminated polyethylene glycol-amino groups. Dynamic light scattering (DLS) is typically used to measure the hydrodynamic diameter. Laser light is used to illuminate the nanoparticles suspended in a liquid, and the changes in the scattered light are analyzed to calculate the Brownian motion of the particles, thereby obtaining the hydrodynamic diameter.

[0013] As a preferred embodiment of the first aspect of the present invention, the hydrodynamic diameter of the gold nanoparticles is 3.82±1.49 nm.

[0014] The gold nanoparticles of the present invention have a hydrodynamic diameter of less than 6 nm, a size below the renal filtration threshold (6 nm). They can be cleared from the body by the kidneys, avoiding side effects caused by hepatic capture. The hydrodynamic diameter is determined by analyzing the Brownian motion of the particles in solution. Smaller particles exhibit faster Brownian motion and a greater diffusion coefficient. In particular, when the hydrodynamic diameter of the gold nanoparticles of the present invention is 3.82 ± 1.49 nm, their smaller size facilitates kidney entry. Furthermore, faster Brownian motion and a greater diffusion coefficient contribute to higher bioavailability.

[0015] As a preferred embodiment of the first aspect of the present invention, the gold nanoparticles further include a loaded hydrophobic drug; the hydrophobic drug is connected to the benzene ring on the 4-mercaptobenzoic acid ligand via physical adsorption (π-π stacking).

[0016] As a preferred embodiment of the first aspect of the present invention, the hydrodynamic diameter of the gold nanoparticles loaded with hydrophobic drugs is 4.84±0.89 nm.

[0017] Specifically, the range of the hydrodynamic diameter measurement of the gold nanoparticles loaded with hydrophobic drugs described in the present invention includes the diameter of the gold nanospheres and the thickness of their surface modification layer and adsorbed drug molecules, wherein the modification layer includes 4-mercaptobenzoic acid ligands and methoxy-terminated polyethylene glycol-amino groups.

[0018] The hydrodynamic diameter of the gold nanoparticles loaded with hydrophobic drugs is less than 6 nm, which is less than the kidney filtration threshold of 6 nm, making it easier for the gold nanoparticles to enter the kidneys. The drugs can enter the renal tubules through glomerular filtration, thereby improving the utilization rate and therapeutic effect of the drugs.

[0019] As a preferred embodiment of the first aspect of the present invention, the hydrophobic drug is an aromatic drug or an anti-tumor drug.

[0020] As a preferred embodiment of the first aspect of the present invention, the aromatic drug is any one of curcumin, resveratrol, and naringenin; and the anti-tumor drug is a STING agonist.

[0021] As a preferred embodiment of the first aspect of the present invention, the preparation method of the gold nanoparticles loaded with aromatic drugs is as follows:

[0022] (1) mixing the gold nanoparticles with an ethanol solution of the drug, stirring and reacting for 12-16 hours, and then removing the ethanol from the reaction by rotary evaporation;

[0023] (2) The product after rotary evaporation is redissolved and the precipitate is removed by centrifugation. The resulting supernatant is the nanoparticles loaded with aromatic drugs.

[0024] In a second aspect, the present invention provides the use of the gold nanoparticles in any of the following:

[0025] (1) Application in the preparation of drugs for treating or preventing acute kidney injury.

[0026] (2) Application in the preparation of drugs for treating or preventing tumors.

[0027] The gold nanoparticles of the present invention loaded with aromatic drugs can enhance the therapeutic efficacy of aromatic drugs or anti-tumor drugs, and increase the bioavailability of the drugs compared to single drugs. Experiments have shown that loading the gold nanoparticles of the present invention with aromatic drugs for treating or preventing acute kidney injury can enhance the therapeutic efficacy of the drugs, and loading aromatic drugs for treating or preventing tumors can enhance the tumor therapeutic efficacy of the drugs.

[0028] In a third aspect, the present invention provides a method for preparing gold nanoparticles, comprising the following steps:

[0029] Step 1, mixing a tetrachloroauric acid solution and a 4-mercaptobenzoic acid solution, and adding a NaOH solution and a sodium borohydride solution after white floccules appear in the mixed solution, and reacting to obtain a reaction system solution 1;

[0030] Step 2: centrifuging the reaction system solution 1 to remove the precipitate in the reaction system solution, purifying the supernatant to obtain a gold nanoparticle system with a 4-mercaptobenzoic acid ligand;

[0031] Step 3, activating the gold nanoparticle system of 4-mercaptobenzoic acid ligand obtained in step 2, and adding methoxy-terminated polyethylene glycol-amino to the activated gold nanoparticle system of 4-mercaptobenzoic acid ligand, and reacting to obtain a reaction system solution 2;

[0032] Step 4: centrifuge the reaction system solution 2 to remove the precipitate in the reaction system, purify the supernatant, and obtain methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles.

[0033] The preparation method of the present invention utilizes physically modified gold nanoparticles. Compared to delivery systems based on chemical modification, the present invention utilizes biocompatible materials, reducing immune responses and toxicity. The drug loading method of the present invention utilizes physical adsorption (π-π stacking), which does not involve chemical modification. This method preserves the drug structure and thus avoids the potential impact of chemical reaction byproducts on drug activity. The physical modification system of the present invention can achieve controlled drug release by adjusting physical parameters (such as particle size and surface charge), thereby enhancing the drug's therapeutic efficacy.

[0034] As a preferred embodiment of the third aspect of the present invention, after adding the NaOH solution in step 1, the pH value is adjusted to 8-10. Preferably, the pH value is 9.

[0035] The present invention has found that when the pH value is less than 8, the nanoparticles produced after adding a reducing agent are unstable and it is difficult to synthesize the nanoparticles. When the pH value is greater than 10, the complex formed by 4-MBA and HAuCl4 settles to form a black precipitate, and the nanoparticles cannot be synthesized. Therefore, the present invention controls the pH value at 8-10 to obtain nanoparticles with ultra-small diameters. In particular, when the pH value is 9, the core diameter of the nanoparticles of the present invention is 1.83±0.9 nm and the hydrodynamic diameter is 3.82±1.49 nm.

[0036] As a preferred embodiment of the third aspect of the present invention, in step 1, the molar ratio of tetrachloroauric acid to 4-mercaptobenzoic acid is 1:(2-3).

[0037] As a preferred embodiment of the third aspect of the present invention, the molar ratio of tetrachloroauric acid to 4-mercaptobenzoic acid in step 1 is 1:2.

[0038] The present invention has found that when the molar ratio of HAuCl4 to 4-MBA is less than 1:2, small gold nanoparticles cannot be formed. When the molar ratio of HAuCl4 to 4-MBA is greater than 1:3, the resulting gold nanoparticles are larger, unstable, and difficult to pass the renal filtration threshold. Therefore, the present invention controls the molar ratio of HAuCl4 to 4-MBA within 1:2-3 to obtain nanoparticles with ultra-small diameters. In particular, when the molar ratio of HAuCl4 to 4-MBA is 1:2, the nanoparticles of the present invention have a core diameter of 1.83±0.9 nm and a hydrodynamic diameter of 3.82±1.49 nm.

[0039] As a preferred embodiment of the third aspect of the present invention, the concentration of tetrachloroauric acid in the tetrachloroauric acid solution in step 1 is 10-30 mM; the concentration of 4-mercaptobenzoic acid in the 4-mercaptobenzoic acid solution is 10-30 mM.

[0040] As a preferred embodiment of the third aspect of the present invention, the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups in the reaction system solution 2 is 1:(1-5); or / and the molecular weight of the methoxy-terminated polyethylene glycol-amino group (MPEG-NH2) is 750-1500.

[0041] As a preferred embodiment of the third aspect of the present invention, the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups in the reaction system solution 2 is 1:2; or / and the molecular weight of the methoxy-terminated polyethylene glycol-amino group (MPEG-NH2) is 1000.

[0042] Research conducted by the present invention has found that the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups, as well as the molecular weight of MPEG-NH2, are key factors influencing the particle size of the gold nanoparticles of the present invention. Too low or too high a molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups results in ineffective PEG attachment. Too low a molecular weight of MPEG-NH2 prevents stable PEG modification, while too high a molecular weight results in excessively large particle size, exceeding the renal filtration threshold and preventing renal clearance, or prone to aggregation, impairing drug loading. Therefore, when the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups is 1:(1-5) and the molecular weight of MPEG-NH2 is 750-1500, the diameter of the PEG-MBA-AuNPs prepared by the present invention is less than 3 nm. In particular, when the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-terminated polyethylene glycol-amino groups is 1:2 and the molecular weight of MPEG-NH2 is 1000, the core diameter of the PEG-MBA-AuNPs prepared by the present invention is only 1.83±0.9 nm and the hydrodynamic diameter is 3.82±1.49 nm.

[0043] As a preferred embodiment of the third aspect of the present invention, the activation method in step 3 is: using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the surface of the gold nanoparticles of the 4-mercaptobenzoic acid ligand.

[0044] The present invention uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the surface of gold nanoparticles with 4-mercaptobenzoic acid ligands. 1-ethyl-(3-dimethylaminopropyl)carbodiimide is a carbodiimide compound that can form an active intermediate with the carbonyl oxygen atom of a carboxylic acid or a carboxylic ester, thereby promoting the activation of the carboxyl groups. Using N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide together to activate the carboxyl groups can improve the activation efficiency.

[0045] As a preferred embodiment of the third aspect of the present invention, the molar ratio of the gold nanoparticles with 4-mercaptobenzoic acid ligand to 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(1-5):(1-5).

[0046] As a preferred embodiment of the third aspect of the present invention, the purification method in step 2 or step 3 is to purify using an ultrafiltration centrifuge tube with a cut-off capacity of 3000 Daltons.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] This invention utilizes 4-mercaptobenzoic acid (4-MBA) as a ligand to prepare a gold nanoparticle with drug delivery capabilities. 4-MBA, with its benzene ring structure, can form π-π stacking interactions with various aromatic drugs and serve as drug loading sites within the gold nanoparticle delivery system. PEGylation of 4-MBA prolongs the in vivo circulation of gold nanoparticles, effectively altering the pharmacokinetics of the loaded drug and preventing rapid clearance from the body. This significantly improves the activity and bioavailability of the loaded hydrophobic drug. Compared to other drug delivery systems, gold nanoparticles are small in size and can be cleared from the body via the kidneys, avoiding the side effects of hepatic clearance and reducing nonspecific uptake by other organs, thereby enhancing efficacy while minimizing side effects. By leveraging this renal clearance property of ultrasmall gold nanoparticles, a universal drug delivery system for renal therapy has been developed.

[0049] The preparation method of the present invention uses physical action to load the drug. Compared with the delivery system of the chemical modification method, the physical action loading drug commonly used in the present invention does not change the structure of the drug itself, thereby reducing the immune response and toxicity to the body. No chemical reaction is involved, thus avoiding the influence of possible chemical reaction byproducts on the activity of the drug. The physical modification system can achieve controlled release of the drug by adjusting physical parameters (such as particle size, surface charge, etc.), thereby improving the therapeutic effect. In addition, physical modification can also be combined with multiple functions, such as magnetism, photosensitivity, etc., to achieve multimodal treatment and diagnosis. Usually, no complex chemical synthesis steps are required, the production process is relatively simple, and the cost is low. This method can not only be used for the delivery of a single drug, but also has universal delivery capabilities for drug molecules with the same properties.

[0050] This invention utilizes a physical drug-carrying mechanism to develop an ultrasmall gold nanoparticle drug delivery system. This system delivers hydrophobic natural antioxidants to treat acute kidney injury. The hydrodynamic diameter of the ultrasmall gold nanoparticles is less than 6 nm, the renal filtration threshold. This allows them to enter the renal tubules through glomerular filtration, thereby delivering antioxidant drugs. The resulting ultrasmall gold nanoparticle drug delivery system is cleared by the kidneys, avoiding the hepatotoxicity associated with hepatic clearance. Furthermore, because its drug-carrying mechanism relies on physical forces, it exhibits excellent loading for aromatic drugs containing benzene rings, making the system universally applicable. Depending on the drug loaded, different diseases can be treated. For example, the gold nanoparticles of the present invention loaded with aromatic drugs for treating or preventing acute kidney injury can enhance the drug's therapeutic efficacy, making it effective in treating or preventing acute kidney injury. Similarly, the gold nanoparticles of the present invention loaded with aromatic drugs for treating or preventing tumors can enhance the drug's therapeutic efficacy, making it effective in treating or preventing tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the synthesis route of PEG-MBA-AuNPs;

[0052] Figure 2 Schematic diagram of the core diameter and hydrodynamic diameter of PEG-MBA-AuNPs;

[0053] Figure 3 Schematic diagram of the excitation / emission spectrum and UV-visible absorption spectrum of PEG-MBA-AuNPs;

[0054] Figure 4 Schematic diagram of the preparation route of PEG-MBA-AuNPs loaded with drugs;

[0055] Figure 5 Schematic diagram of the UV-visible absorption spectra of aromatic drugs loaded on PEG-MBA-AuNPs (Figure A is curcumin; Figure B is naringenin; Figure C is resveratrol; Figure D is the STING agonist DMXAA);

[0056] Figure 6 Schematic diagram of the hydrodynamic diameter of Cur@PEG-AuNPs;

[0057] Figure 7 Schematic diagram of the antioxidant activity of Cur@PEG-AuNPs:

[0058] Figure 8Schematic diagram of serum creatinine and urea nitrogen levels in AKI mice after treatment with Cur@PEG-AuNPs (Normal represents the normal group; AKI+PBS represents acute kidney injury + administration of phosphate buffered saline; AKI+Curcumin represents acute kidney injury + administration of curcumin; AKI+Cur@PEG-AuNPs represents acute kidney injury + administration of curcumin-loaded PEG-AuNPs gold nanoparticles);

[0059] Figure 9 Schematic diagram of kidney tissue pathological sections and renal tubular injury scores of AKI mice after Cur@PEG-AuNPs treatment (Figure A: Normal represents the normal group; Figure B: AKI+PBS represents acute kidney injury + administration of phosphate buffered saline; Figure C: AKI+Cur@PEG-AuNPs represents acute kidney injury + administration of curcumin-loaded PEG-AuNPs gold nanoparticles; Figure D: AKI+Curcumin represents acute kidney injury + administration of curcumin; Figure E: Schematic diagram of injury scores for the above groups);

[0060] Figure 10 Schematic diagram of serum creatinine and urea nitrogen levels in AKI mice after prevention by Cur@PEG-AuNPs;

[0061] Figure 11 Schematic diagram of kidney tissue pathology sections and renal tubular injury scores in AKI mice after prevention by Cur@PEG-AuNPs (Figure A is a diagram of kidney tissue pathology sections; Figure B is a diagram of renal tubular injury scores);

[0062] Figure 12 Figure 2 is the inhibition curve of tumor volume and weight of DMXAA@PEG-AuNPs (Figure A is a tumor photo; Figure B is the inhibition curve of tumor volume and weight);

[0063] Figure 13 DMXAA@PEG-AuNPs activate the STING pathway to induce CD80 + CD86 + Schematic diagram of the percentage of DCs (Figure A is a schematic diagram of the activation of the STING pathway by DMXAA@PEG-AuNPs; Figure B is a schematic diagram of the induction of DCs);

[0064] Figure 14 Schematic diagram of the number of DMXAA@PEG-AuNPs metastatic lung nodules (Figure A is a photograph of the number of metastatic lung nodules; Figure B is a schematic diagram comparing the number of metastatic lung nodules in different groups);

[0065] Figure 15 Schematic diagram of H&E staining analysis of lung metastasis in the DMXAA@PEG-AuNPs group;

[0066] Figure 16 Schematic diagram of the cumulative statistical distribution of Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L) in the liver, spleen, and kidney under normal conditions and AKI conditions, respectively;

[0067] Figure 17 Schematic diagram of serum creatinine and urea nitrogen levels in AKI mice after prevention by Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L);

[0068] Figure 18 Schematic diagram of kidney tissue pathology sections and renal tubular injury scores of AKI mice treated with Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L), respectively (Figure A is a kidney tissue pathology section; Figure B is a schematic diagram of renal tubular injury scores);

[0069] Figure 19 Pharmacokinetic curves of MBA-AuNPs and PEG-MBA-AuNPs-1. DETAILED DESCRIPTION

[0070] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0071] Table 1: Chinese names of the present invention and their corresponding abbreviations

[0072]

[0073] Example 1

[0074] This embodiment provides a method for preparing gold nanoparticles-1 to -15 ( Figure 1 ), including the following steps:

[0075] Preparation method of gold nanoparticles-1:

[0076] (1) Preparation of gold nanoparticles with 4-mercaptobenzoic acid ligand:

[0077] a. Prepare a 20 mM tetrachloroauric acid solution: Accurately weigh approximately 0.0394 g of tetrachloroauric acid trihydrate, dissolve it in approximately 5 mL of ultrapure water, and stir thoroughly. Store the prepared 20 mM tetrachloroauric acid solution in a brown glass bottle at 4°C in a refrigerator away from light to prevent decomposition and oxidation.

[0078] b. Prepare a 20 mM 4-mercaptobenzoic acid solution: Accurately weigh approximately 0.0304 g of 4-mercaptobenzoic acid and dissolve it in approximately 7 mL of anhydrous ethanol. Stir thoroughly. To make up the volume: Transfer the solution to a 10 mL volumetric flask, dilute to the mark with anhydrous ethanol, and shake thoroughly. Storage: Store the prepared 20 mM 4-mercaptobenzoic acid solution in a brown glass bottle at 4°C in a refrigerator, away from light to prevent decomposition and oxidation.

[0079] c Preparation of gold nanoparticle system with 4-mercaptobenzoic acid ligand

[0080] c-1: Mix the above tetrachloroauric acid solution and 4-mercaptobenzoic acid solution and react with each other, stirring thoroughly until white flocs appear in the reaction solution; wherein the molar ratio of tetrachloroauric acid to 4-mercaptobenzoic acid is HAuCl4:4-MBA=1:2;

[0081] c-2: Add 1M NaOH solution to the reaction system in step c-1 and adjust the pH to 9;

[0082] c-3: Add 112 mM sodium borohydride solution to the reaction system in step c-2, and stir for 10 h to obtain reaction system solution 1;

[0083] d. After the reaction, the precipitate in the reaction system solution 1 was removed by centrifugation at 15,000 rpm for 10 min. The supernatant was purified more than three times using an ultrafiltration centrifuge tube with a cutoff of 3,000 Daltons to obtain a 4-mercaptobenzoic acid ligand gold nanoparticle system (referred to as MBA-AuNPs in this invention).

[0084] (2) Preparation of methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles

[0085] a. Activation of gold nanoparticles with 4-mercaptobenzoic acid ligands:

[0086] The MBA-AuNPs system was mixed with EDC and NHS at a molar ratio of MBA-AuNPs:EDC:NHS=1:1.5:1.5, and the reaction was stirred for 1 h to obtain an activated MBA-AuNPs system. The MBA-AuNPs described in the following steps were all activated MBA-AuNPs.

[0087] b. MPEG-NH2 was added to the MBA-AuNPs reaction solution activated in step a, and the reaction was stirred for 10 hours to obtain a reaction system solution 2; wherein the molar ratio of MBA-AuNPs to MPEG-NH2 was MBA-AuNPs:MPEG-NH2=1:2; and the molecular weight of MPEG-NH2 was 1000.

[0088] c. After the reaction, the precipitate in the reaction system solution 2 was removed by centrifugation at 15,000 rpm for 10 min. The supernatant was purified more than three times using an ultrafiltration centrifuge tube with a cutoff of 3,000 Daltons to obtain methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles (denoted as PEG-MBA-AuNPs-1 in the present invention).

[0089] The preparation method of PEG-MBA-AuNPs-2 differs from that of PEG-MBA-AuNPs-1 in the following ways:

[0090] (1) In step (1) c-1, HAuCl4:4-MBA=1:3;

[0091] (2) The pH value in step (1) c-2 is 8;

[0092] (3) The stirring reaction time in step (1) c-3 is 12 h;

[0093] (4) In step (2) a, the mixing molar ratio is MBA-AuNPs:EDC:NHS=1:1:1;

[0094] (5) The molecular weight of MPEG-NH2 in step (2)b is 750;

[0095] (6) In step (2) b, the stirring reaction time of MBA-AuNPs and MPEG-NH2 was 12 h;

[0096] (7) In step (2) b, MBA-AuNPs:MPEG-NH2=1:1.

[0097] The preparation method of PEG-MBA-AuNPs-3 differs from that of PEG-MBA-AuNPs-1 in the following ways:

[0098] (1) In step (1) c-1, HAuCl4:4-MBA=1:2.5;

[0099] (2) The pH value in step (1) c-2 is 10;

[0100] (3) The stirring reaction time in step (1) c-3 is 8 hours;

[0101] (4) In step (2) a, the mixing molar ratio is MBA-AuNPs:EDC:NHS=1:5:5;

[0102] (5) The molecular weight of MPEG-NH2 in step (2)b is 1500;

[0103] (6) In step (2) b, the stirring reaction time of MBA-AuNPs and MPEG-NH2 was 8 h;

[0104] (7) In step (2) b, the ratio of MBA-AuNPs to MPEG-NH2 is 1:5.

[0105] The preparation method of PEG-MBA-AuNPs-4 differs from that of PEG-MBA-AuNPs-1 in that in step (1) c-1, HAuCl4:4-MBA=1:1.

[0106] The preparation method of PEG-MBA-AuNPs-5 differs from that of PEG-MBA-AuNPs-1 in that in step (1) c-1, HAuCl4:4-MBA=1:4.

[0107] The preparation method of PEG-MBA-AuNPs-6 differs from that of PEG-MBA-AuNPs-1 in that the pH value in step (1) c-2 is 7.

[0108] The preparation method of PEG-MBA-AuNPs-7 differs from that of PEG-MBA-AuNPs-1 in that the pH value in step (1) c-2 is 11.

[0109] The preparation method of PEG-MBA-AuNPs-8 differs from that of PEG-MBA-AuNPs-1 in that sodium citrate (CIT) is used instead of sodium borohydride. The specific method is as follows;

[0110] 1. Accurately weigh approximately 94.4 mg of tetrachloroauric acid trihydrate using an analytical balance. Dissolve the weighed amount of HAuCl4·3H2O in ultrapure water and quantify to 10 mL to obtain 24 mM HAuCl4.

[0111] 2. To prepare 50 mL of a 2.2 mM CIT solution, accurately weigh approximately 32.351 mg of sodium citrate dihydrate using an analytical balance. Add ultrapure water and gently stir to dissolve and bring the volume to 50 mL.

[0112] 3. Prepare 50 mL of a 60 mM CIT solution. Accurately weigh approximately 176.46 mg of sodium citrate dihydrate using an analytical balance. Dissolve the weighed sodium citrate in ultrapure water to a volume of 10 mL.

[0113] 4. Prepare the three reagent concentrations listed above. Preheat the heating plate to 100°C. Add 50 mL of 2.2 mM sodium citrate solution to a flask. When the solution boils, add 333 μL of 24 mM HAuCl₄ and react for 10 minutes. The solution will turn light pink. Allow the solution to cool to 90°C, then add 333 μL of 60 mM sodium citrate solution and 333 μL of 24 mM HAuCl₄. React for 30 minutes. The solution will turn wine red. Stop the reaction when a UV absorption peak appears in the 510-520 nm range. This is the prepared PEG-MBA-AuNPs-8.

[0114] The preparation method of PEG-MBA-AuNPs-9 differs from that of PEG-MBA-AuNPs-1 in that sodium borohydride was replaced by sodium cyanoborohydride at a concentration of 112 mM, and the other steps were the same.

[0115] The preparation method of PEG-MBA-AuNPs-10 differs from that of PEG-MBA-AuNPs-1 in that the molecular weight of MPEG-NH2 in step (2)b is 350.

[0116] The preparation method of PEG-MBA-AuNPs-11 differs from that of PEG-MBA-AuNPs-1 in that the molecular weight of MPEG-NH2 in step (2)b is 5000.

[0117] The preparation method of PEG-MBA-AuNPs-12 differs from that of PEG-MBA-AuNPs-1 in that the molar ratio of MBA-AuNPs:MPEG-NH2 in step (2)b is MBA-AuNPs:MPEG-NH2=1:7.

[0118] The preparation method of PEG-MBA-AuNPs-13 differs from that of PEG-MBA-AuNPs-1 in that the molar ratio of MBA-AuNPs:MPEG-NH2 in step (2)b is MBA-AuNPs:MPEG-NH2=1:0.5.

[0119] The preparation method of PEG-MBA-AuNPs-14 differs from that of PEG-MBA-AuNPs-1 in that the order of the steps is different, and step (1) c-2 and c-3 are swapped. The remaining steps are the same, and the specific steps are as follows:

[0120] c Preparation of gold nanoparticle system with 4-mercaptobenzoic acid ligand

[0121] c-1: Mix the above tetrachloroauric acid solution and 4-mercaptobenzoic acid solution and react with each other, stirring thoroughly until white flocs appear in the reaction solution; wherein the molar ratio of tetrachloroauric acid to 4-mercaptobenzoic acid is HAuCl4:4-MBA=1:2;

[0122] c-2: Add 112 mM sodium borohydride solution to the reaction system in step c-1 and stir for 10 h;

[0123] c-3: Add 1 M NaOH solution to the reaction system of step c-2 and adjust the pH to 9 to obtain reaction system solution 1.

[0124] The preparation method of PEG-MBA-AuNPs-15 is different from that of PEG-MBA-AuNPs-1 in that the 4-mercaptobenzoic acid ligand in Example 1 is replaced by 3-mercaptobenzoic acid ligand.

[0125] Example 2 Physical and Chemical Characterization of PEG-MBA-AuNPs

[0126] Characterization methods: Transmission electron microscopy (TEM) was used to characterize the core size and morphology of the nanoparticles; dynamic light scattering (DLS) was used to characterize the hydrodynamic diameter of the nanoparticles; ultraviolet-visible spectrophotometry (UV-Vis) was used to characterize the characteristic absorption of the nanoparticles; and fluorescence spectrophotometry (FL) was used to characterize the fluorescence properties of the nanoparticles. The results are shown in the following table:

[0127] Table 2 Particle size of gold nanoparticles prepared under different conditions

[0128]

[0129] like Figure 2 As shown in Figure 3, the core diameter of PEG-MBA-AuNPs-1 is 1.83 ± 0.9 nm and is spherical, which is consistent with the size range of ultrasmall gold nanoparticles. Figure 3 As shown, PEG-MBA-AuNPs-1 exhibit fluorescence properties, with an optimal excitation wavelength of 350 nm and a maximum emission wavelength of 810 nm. Furthermore, within the 400-500 nm range, the nanoparticles exhibit no significant UV-Vis absorption, demonstrating the absence of surface plasmon resonance and the presence of ultra-small nanoparticles.

[0130] As shown in PEG-MBA-AuNPs-15, although the 3-mercaptobenzoic acid ligand differs from the 4-mercaptobenzoic acid ligand of the present invention only in the position of the sulfhydryl group, it is unable to stably attach to PEG. Furthermore, the PEG-MBA-AuNPs prepared in the present invention are a drug-loading system that relies on the phenyl ring structure of MBA for drug loading. However, the sulfhydryl group of 3-MBA is located in the meta position of the phenyl ring, which sterically hinders its instability when binding to metal surfaces. Furthermore, 3-MBA is sterically unfavorable for binding to the methoxy-terminated polyethylene glycol-amino group (MPEG-NH2), further hindering subsequent drug loading. The 4-MBA selected in the present invention, with its sulfhydryl and carboxyl groups located in the para position of the phenyl ring, offers less steric hindrance, more direct binding to metal surfaces, and greater stability. Therefore, 4-MBA is more advantageous than 3-MBA for loading aromatic drugs in the present invention.

[0131] Example 3

[0132] This example uses curcumin as an example to illustrate the application of the PEG-MBA-AuNPs drug loading system prepared by the present invention. The reaction scheme is as follows: Figure 4 The specific method is as follows:

[0133] (1) Curcumin in an equimolar ratio is dissolved in an anhydrous ethanol solution to prepare a curcumin ethanol solution.

[0134] (2) PEG-MBA-AuNPs-1 was added to the curcumin ethanol solution to obtain a reaction system, wherein the volume ratio of the curcumin ethanol solution to the PEG-MBA-AuNPs in the reaction system was 3:1. After stirring and reacting for 12-24 h, the ethanol in the reactant was removed by rotary evaporation, and the product after rotary evaporation was redissolved in ultrapure water. The product was centrifuged at 15,000 rpm for 10 min to precipitate the unloaded curcumin. The precipitate was removed, and the supernatant was the curcumin-loaded nanoparticles. Only the supernatant was retained.

[0135] like Figure 5 As shown, the UV absorption spectrum characterization results proved that curcumin was successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, which is recorded as Cur@PEG-AuNPs in the present invention.

[0136] like Figure 6 As shown, the hydrodynamic diameter (hydrated particle size) of Cur@PEG-AuNPs was characterized by dynamic light scattering (DLS) to be 4.84 ± 0.89 nm, which is consistent with the size range of ultrasmall gold nanoparticles.

[0137] Example 4

[0138] This example uses resveratrol as an example to illustrate the application of PEG-MBA-AuNPs loaded with drugs prepared by the present invention. The specific method is as follows:

[0139] (1) Dissolve resveratrol in anhydrous ethanol solution at an equal molar ratio to prepare a resveratrol solution.

[0140] (2) PEG-MBA-AuNPs-1 was added to the resveratrol solution to obtain a reaction system in which the volume ratio of ethanol to water was 3:1. After stirring for 16 h, the ethanol in the reaction was removed by rotary evaporation, and the product after rotary evaporation was redissolved in ultrapure water. The solution was centrifuged at 15,000 rpm for 10 min to remove the unloaded resveratrol precipitate. The supernatant was the resveratrol-loaded nanoparticles.

[0141] like Figure 5 As shown, the UV absorption spectrum characterization results proved that resveratrol was successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, which is recorded as Res@PEG-AuNPs in the present invention.

[0142] Example 5

[0143] This example uses naringenin as an example to illustrate the application of PEG-MBA-AuNPs loaded with drugs prepared by the present invention. The specific method is as follows:

[0144] (1) Naringenin solution was prepared by dissolving naringenin in anhydrous ethanol solution at an equal molar ratio.

[0145] (2) PEG-MBA-AuNPs-1 was added to the naringenin solution to obtain a reaction system in which the volume ratio of ethanol to water was 3:1. After stirring for 14 h, the ethanol in the reaction was removed by rotary evaporation, and the product after rotary evaporation was re-dissolved in ultrapure water. The solution was centrifuged at 15,000 rpm for 10 min to remove the unloaded naringenin precipitate. The supernatant was the naringenin-loaded nanoparticles.

[0146] like Figure 5 As shown in FIG, the UV absorption spectrum characterization results prove that naringenin is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, which is recorded as Nar@PEG-AuNPs in the present invention.

[0147] Example 6

[0148] This example uses the STING agonist (DMXAA) as an example to illustrate the application of the drug-loaded PEG-MBA-AuNPs prepared by the present invention. The specific method is as follows:

[0149] (1) DMXAA was dissolved in anhydrous ethanol solution at an equal molar ratio to prepare a DMXAA solution.

[0150] (2) PEG-MBA-AuNPs-1 was added to the DMXAA solution to obtain a reaction system in which the volume ratio of ethanol to water was 3:1. After stirring for 15 h, the ethanol in the reaction was removed by rotary evaporation, and the product after rotary evaporation was re-dissolved in ultrapure water. The product was centrifuged at 15,000 rpm for 10 min to remove the unloaded DMXAA precipitate. The supernatant was the DMXAA-loaded nanoparticles.

[0151] like Figure 5 As shown in FIG, the UV absorption spectrum characterization results prove that DMXAA is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, which is recorded as DMXAA@PEG-AuNPs in the present invention.

[0152] Example 7 Comparison of Antioxidant Properties of Cur@PEG-AuNPs and Curcumin

[0153] Experimental Method: The antioxidant properties of the nanoparticles in Example 3 were characterized using the ABTS assay. ABTS is oxidized to green ABTS·+ in the presence of an oxidant, exhibiting characteristic absorption peaks at 734 nm or 405 nm. The presence of antioxidants inhibits the production of ABTS·+, causing the reaction system to fade and the absorbance at 405 nm to decrease. Within a certain range, the change in absorbance is proportional to the degree of free radical scavenging. The degree of absorbance decrease reflects the sample's ability to scavenge ABTS free radicals.

[0154] (1) ABTS was dissolved in a sodium acetate solution with a pH of 4.5 to prepare a 7.4 mmol / L ABTS solution. The ABTS solution was then mixed with a 2.6 mmol / L potassium persulfate solution in a 1:1 volume ratio. The mixture was allowed to stand at room temperature in the dark for 12 h to obtain a reaction solution.

[0155] (2) Detection method: Cur@PEG-AuNPs phosphate buffer solution (PBS) at different concentrations (10, 20, 40, and 80 μg / mL) was mixed with the reaction solution from step (1) at a volume ratio of 1:9. After 30 min of reaction in the dark, the absorbance was measured at 734 nm and zeroed with distilled water. The total antioxidant activity at different concentrations was calculated. A single curcumin solution of equal concentration was used as the control group.

[0156] The experimental results are as follows Figure 7 As shown in the results, compared with single curcumin, the ultrasmall gold nanoparticle delivery system PEG-MBA-AuNPs prepared in the present invention improved the water solubility of the drug curcumin and enhanced its antioxidant activity, indicating that Cur@PEG-AuNPs has good antioxidant activity.

[0157] Example 8 The therapeutic effect of Cur@PEG-AuNPs on AKI

[0158] Experimental methods:

[0159] (1) Construction of acute kidney injury model: A rhabdomyolysis-induced acute kidney injury model was used (induced by 8 mL / kg 50% glycerol injected into the lower limb muscles). Ultrasmall gold nanoparticles loaded with curcumin (Cur@PEG-AuNPs) were injected 2 hours after glycerol injection. At the same time, a blank control, a model control group (injected with PBS), and a control group (injected with curcumin) were set up.

[0160] (2) 24 hours after the injection of gold nanoparticles, the blood renal function biochemical indicators (creatinine, urea nitrogen) and renal pathological sections of the mice were tested to determine the degree of AKI and the treatment effect. The specific procedures are as follows:

[0161] a. Biochemical Index Testing: After mice were anesthetized with isoflurane gas, blood was collected by cardiac puncture. After the blood was naturally coagulated, serum was separated and the levels of creatinine and urea nitrogen in the serum were measured using an automated biochemical analyzer.

[0162] b. Renal pathology section examination: The abdomen was opened longitudinally along the midline, and the kidneys were removed and fixed with 4% paraformaldehyde. After paraffin embedding, the sections were stained with hematoxylin and eosin (H&E) to analyze the degree of renal tubular damage. Ten high-power fields were randomly selected from each kidney section and scored according to the following criteria: 1 point for marked tubular dilation and flattened cells; 1 point for brush border damage and 2 points for detachment; 2 points for casts; and 1 point for the presence of detached or necrotic cells (not forming casts or cell fragments) in the renal tubular lumen. The maximum score for each field was 4, and the minimum score was 0.

[0163] The experimental results are as follows Figure 8 As shown in the figure, for the acute kidney injury model, after 24 hours of treatment, the serum creatinine and urea nitrogen levels of mice treated with Cur@PEG-AuNPs were significantly reduced compared with those of untreated mice and returned to normal levels, while the serum creatinine and urea nitrogen levels of the model control group (PBS injection) and the control group (curcumin injection) were still high.

[0164] The experimental results are as follows Figure 9 As shown, after paraffin sectioning and H&E staining of the kidneys of the above mice, it was found that the degree of renal tubular damage was significantly reduced.

[0165] Example 9 Preventive Effect of Cur@PEG-AuNPs on AKI

[0166] Experimental method: Cur@PEG-AuNPs were administered to the animal model ten minutes in advance, and then an acute kidney injury model induced by ischemia-reperfusion was immediately established.

[0167] Six- to eight-week-old ICR mice were selected for anesthesia. An incision was made to expose both kidneys and the renal pedicles (including the renal artery, renal vein, and ureter). A non-invasive vascular clamp or a mouse renal pedicle clamp was used to clamp both renal pedicles, blocking renal blood flow. During clamping, the kidneys were kept moist to prevent desiccation and thermal damage. After the predetermined 30-minute ischemia time, the clamps were quickly released to restore renal blood flow and initiate reperfusion. The kidneys were observed for color changes; a gradual return from pale to normal color indicated successful reperfusion. The abdominal incision was sutured and disinfected to complete the surgery. Blood samples were collected 24 and 48 hours after reperfusion to measure renal function markers (serum creatinine and urea nitrogen). Mice were euthanized 48 hours later, and renal tissue samples were collected for histopathological examination with H&E staining to assess the extent of renal injury.

[0168] The experimental results are as follows Figure 10 As shown, compared with mice that were not injected with Cur@PEG-AuNPs in advance, the serum creatinine and urea nitrogen levels of mice were significantly reduced after Cur@PEG-AuNPs prevention and maintained at normal levels.

[0169] The experimental results are as follows Figure 11 As shown, after paraffin sectioning and H&E staining of the kidneys of the above mice, it was found that the degree of renal tubular damage was significantly reduced.

[0170] Example 10 Anti-tumor immune effect of DMXAA@PEG-AuNPs

[0171] Female BALB / c mice aged 6 to 8 weeks were selected and injected subcutaneously on the right side of the back with 3 × 10 6 4T1 cells (mouse breast cancer cells) were used to establish a subcutaneous tumor model in mice. When the tumor volume reached 80 mm 3 The mice were randomly divided into 7 groups and given different treatments: G1: PBS group, G2: Radiation group, G3: Free DMXAA group, G4: PEG-AuNPs group, G5: PEG-AuNPs + Radiation group, G6: DMXAA@PEG-AuNPs group, G7: DMXAA@PEG-AuNPs + Radiation group. The drug was given once every two days, and Radiation (4 Gy) was given 24 hours after injection. A total of 3 doses were given. The in vivo therapeutic effect of the drug was observed by monitoring the tumor volume and body weight of the mice. Tumor volume = 1 / 2 × L × W 2(L refers to the long diameter of the tumor, and W refers to the short diameter of the tumor.) At the end of the experiment, tumors were harvested and photographed for immunohistochemistry. Additionally, lymph nodes and tumor tissues from tumor-bearing mice were harvested to prepare single-cell suspensions for flow cytometric analysis of immune cells.

[0172] The experimental results are as follows Figure 12 As shown, compared with the control group, the DMXAA@PEG-AuNPs + Radiation treatment group exhibited the highest level of anticancer activity, with the smallest tumor volume and an 87.4% tumor growth inhibition rate. This can be attributed to the effective delivery of DMXAA@PEG-AuNPs to the tumor site and their antitumor effect under the action of radiation. There were no statistically significant changes in body weight throughout the treatment intervention period, indicating that DMXAA@PEG-AuNPs have a good in vivo safety profile.

[0173] The experimental results are as follows Figure 13 As shown, activated STING further recruits and activates TBK1 (TANK binding kinase 1), which then phosphorylates and activates IRF3 (interferon regulatory factor 3). The results of immunohistochemical staining of phosphorylated IRF3 (pIRF3) showed that compared with free DMXAA, DMXAA@PEG-AuNPs + Radiation had the largest positive area, indicating that pIRF3 was effectively expressed. This is mainly because PEG-AuNPs effectively delivers DMXAA to the tumor site, thereby activating the STING pathway, while free DMXAA is quickly metabolized in the body. In addition, flow cytometry analysis results showed that DMXAA@PEG-AuNPs + Radiation can effectively induce the maturation of dendritic cells (DCs), CD80 + CD86 + The percentage of DCs reached about 27.0%, thereby recruiting T cells for anti-tumor immune response.

[0174] Example 11 Anti-lung tumor metastasis effect of DMXAA@PEG-AuNPs

[0175] 4T1 tumor-bearing mice were randomly divided into 7 groups: G1: PBS group, G2: Radiation group, G3: Free DMXAA group, G4: PEG-AuNPs group, G5: PEG-AuNPs + Radiation group, G6: DMXAA@PEG-AuNPs group, and G7: DMXAA@PEG-AuNPs + Radiation group. On the 7th day, 3×10 54T1-luc cells were injected into the tail vein of mice to establish lung metastases. Subsequently, the mice received routine intravenous infusions of various agents to treat the primary tumor. The mice were then monitored for two weeks. On day 21, the mice were sacrificed, and lung tissue was collected from each group for imaging. Lung metastases were manually counted and analyzed using H&E staining.

[0176] The experimental results are as follows Figure 14 As shown, a large number of metastatic lesions were observed in the PBS, Radiation, and free DMXA groups, while only a small number of metastatic lesions were observed in the DMXAA@PEG-AuNPs + Radiation group. The lung tissue margins were clear and regular, indicating that DMXAA@PEG-AuNPs + Radiation has a significant effect in preventing lung metastasis. Quantitative analysis also confirmed that the number of metastatic lung nodules in the DMXAA@PEG-AuNPs + Radiation group was significantly reduced by 93.3% compared to the control group.

[0177] The experimental results are as follows Figure 15 HE staining results show abnormal lung tissue structure in the control group, with visible metastatic lesions, extensive areas of tissue necrosis, and significant nuclear atypia. Tumors occupy most of the lung tissue, with uneven margins. In contrast, no obvious lesions were observed in the lungs of the DMXAA@PEG-AuNPs + Radiation treatment group. These results demonstrate that DMXAA@PEG-AuNPs + Radiation effectively inhibits tumor cell metastasis.

[0178] Comparative Example 1 Preparation of Cur@PEG-AuNPs (L)

[0179] Comparative Example 1 provides a large-sized gold nanoparticles AuNPs drug delivery system, and its preparation method is as follows:

[0180] The PEG-MBA-AuNPs-11 prepared in the above example was used as a representative of large nanoparticles, denoted as PEG-MBA-AuNPs (L). The subsequent experimental results were based on PEG-MBA-AuNPs-11 as an example.

[0181] The ethanolic curcumin solution was mixed with the prepared PEG-MBA-AuNPs(L) and stirred with shaking for 12-24 hours. Unbound curcumin and ethanol in the reaction system were removed by dialysis using a 5000 cutoff dialysis bag. The retained product was designated Cur@PEG-AuNPs(L). Characterization results showed that the hydrodynamic diameter of Cur@PEG-AuNPs(L) was 31.32±10.42nm.

[0182] Comparative Example 1

[0183] The distribution of Cur@PEG-AuNPs (denoted as Cur@PEG-AuNPs (S)) prepared in Example 3 and Cur@PEG-AuNPs (L) prepared in Comparative Example 1 in the liver, spleen, and kidney under normal conditions and AKI conditions is compared as follows:

[0184] Experimental methods:

[0185] Six- to eight-week-old ICR mice were injected with Cur@PEG-AuNPs(S) or Cur@PEG-AuNPs(L) (with the same curcumin content in the S / L) solutions, with three mice per group. Twenty-four hours after injection, the mice were euthanized, and their livers, kidneys, and spleens were harvested and weighed.

[0186] Prepare fresh aqua regia (nitric acid: hydrochloric acid = 1:3) and soak the collected tissues and organs in fresh aqua regia for 48 hours. Use ICP-MS to determine the gold content in the organs. Use the formula: measured gold content of tissues and organs / measured weight of tissues and organs*100%=gold content in tissues (unit: % ID / g) to calculate and compare the distribution of gold in different tissues and organs.

[0187] The experimental results are as follows Figure 16 As shown in the figure, for the same drug loading structure, the present invention's Cur@PEG-AuNPs (S) exhibits superior renal accumulation compared to Cur@PEG-AuNPs (L). Under normal conditions and in the case of acute kidney injury, Cur@PEG-AuNPs (L) mainly accumulate in the liver and spleen, while the present invention's Cur@PEG-AuNPs (S) accumulates much more in the kidney than Cur@PEG-AuNPs (L), showing a significant difference.

[0188] Comparative Example 2

[0189] The therapeutic effects of Cur@PEG-AuNPs (S) prepared in Example 3 and Cur@PEG-AuNPs (L) prepared in Comparative Example 1 on AKI were compared as follows:

[0190] Experimental method: The experimental method of Example 8 was adopted.

[0191] The experimental results are as follows Figure 17As shown in the figure, in the acute kidney injury model, after 24 hours of treatment, serum creatinine and urea nitrogen levels in mice treated with Cur@PEG-AuNPs (S) were significantly reduced and returned to normal levels compared to untreated mice (AKI + PBS). Serum creatinine and urea nitrogen levels in mice treated with Cur@PEG-AuNPs (L) were partially restored, but serum creatinine indicators did not significantly improve. Cur@PEG-AuNPs (S) demonstrated a superior therapeutic effect.

[0192] The experimental results are as follows Figure 18 As shown, paraffin sections and H&E staining of the kidneys of the mice above revealed significantly reduced renal tubular damage in mice treated with Cur@PEG-AuNPs (L). However, the kidneys of mice treated with Cur@PEG-AuNPs (L) still showed significant damage, with dilated renal tubular lumens and a loss of the brush border. This indicates that the therapeutic effect of Cur@PEG-AuNPs (S) is significantly superior to that of larger nanoparticles.

[0193] Comparative Example 3

[0194] The gold nanoparticles loaded with aromatic drugs (denoted as MBA-AuNPs) prepared in Comparative Example 3 and modified with polyethylene glycol-amino groups without methoxyl end-capping were compared with PEG-MBA-AuNPs-1. The preparation method of MBA-AuNPs was the same as that of PEG-MBA-AuNPs-1, except that methoxy polyethylene glycol modification was not used.

[0195] The results are as follows Figure 19 The pharmacokinetic curves of MBA-AuNPs demonstrate faster clearance from the body. Compared to PEG-MBA-AuNPs-1, the AUC of MBA-AuNPs is significantly lower. A higher AUC indicates greater drug absorption and can be used to assess the proportion of a drug entering the bloodstream. Similarly, a higher CL value indicates faster drug clearance from the body. MBA-AuNPs are rapidly cleared from the body. PEG modification significantly increases the blood circulation time of MBA-AuNPs.

[0196] In summary, the ultrasmall gold nanodelivery system prepared by the present invention (i.e., methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles, PEG-MBA-AuNPs) has a small particle size of only 1.83±0.9 nm. This ultrasmall gold nanodelivery system not only increases drug accumulation in the kidneys and reduces accumulation in the liver and spleen, but also enhances the water solubility and antioxidant properties of antioxidant drugs. The average hydrodynamic diameter of the drug-carrying ultrasmall gold nanodelivery system is only 4.8±0.89 nm, which is less than the renal filtration threshold of 6 nm. This allows the drug to pass through glomerular filtration and enter the renal tubules, enhancing therapeutic efficacy.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing gold nanoparticles, characterized in that: The steps include: Step 1, mixing a tetrachloroauric acid solution and a 4-mercaptobenzoic acid solution, adding a NaOH solution after white floccules appear in the mixed solution, adjusting the pH value to 8-10, and then adding a sodium borohydride solution. After reaction, a reaction system solution 1 is obtained; The molar ratio of tetrachloroauric acid to 4-mercaptobenzoic acid is 1:(2-3); Step 2: centrifuging the reaction system solution 1 to remove the precipitate, purifying the supernatant, and obtaining a gold nanoparticle system with a 4-mercaptobenzoic acid ligand; Step 3: activating the gold nanoparticle system of 4-mercaptobenzoic acid ligand obtained in step 2, and adding methoxy-terminated polyethylene glycol-amino to the activated gold nanoparticle system of 4-mercaptobenzoic acid ligand, and reacting to obtain a reaction system solution 2; wherein the molecular weight of the methoxy-terminated polyethylene glycol-amino is 750-1500; The molar ratio of the gold nanoparticles with 4-mercaptobenzoic acid ligand to the methoxy-terminated polyethylene glycol-amino group is 1:(1-5); The activation method in step 3 is as follows: using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the surface of the gold nanoparticles with 4-mercaptobenzoic acid ligands; the molar ratio of the gold nanoparticles with 4-mercaptobenzoic acid ligands to 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(1-5):(1-5); Step 4: centrifuging the reaction system solution 2 to remove the precipitate, purifying the supernatant, and obtaining methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles, namely the gold nanoparticles; The core diameter of the gold nanoparticles is less than 3 nm; or / and, The hydrodynamic diameter of the gold nanoparticles is less than 6 nm.

2. Gold nanoparticles prepared by the method for preparing gold nanoparticles according to claim 1, characterized in that: The gold nanoparticles include gold nanospheres, 4-mercaptobenzoic acid ligands and methoxy-terminated polyethylene glycol-amino groups; the gold nanospheres, 4-mercaptobenzoic acid ligands and methoxy-terminated polyethylene glycol-amino groups are sequentially connected through chemical bonds; The connection method is that the gold nanospheres replace the thiol hydrogen on the 4-mercaptobenzoic acid ligand, and the amino group of the methoxy-terminated polyethylene glycol-amino group undergoes a condensation reaction with the carboxyl group on the 4-mercaptobenzoic acid ligand; The core diameter of the gold nanoparticles is less than 3 nm; or / and, The hydrodynamic diameter of the gold nanoparticles is less than 6 nm.

3. The gold nanoparticles according to claim 2, wherein The gold nanoparticles also include a hydrophobic drug; the hydrophobic drug is connected to the benzene ring on the 4-mercaptobenzoic acid ligand through physical adsorption.

4. The gold nanoparticles according to claim 3, wherein The hydrophobic drug is an aromatic drug or an anti-tumor drug.

5. Use of the gold nanoparticles according to any one of claims 2 to 4 in any one of the following: (1) Use in the preparation of drugs for treating or preventing acute kidney injury; (2) Application in the preparation of drugs for treating or preventing tumors.

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