Gold nanoparticles capable of loading aromatic drugs as well as preparation method and application of gold nanoparticles

Abstract

The invention relates to gold nanoparticles capable of loading aromatic drugs as well as a preparation method and application of the gold nanoparticles, and belongs to the technical field of biological medicines. The gold nanoparticles comprise gold nanospheres, 4-mercaptobenzoic acid ligands and methoxy-terminated polyethylene glycol-amino groups, wherein the gold nanospheres, the 4-mercaptobenzoic acid ligands and methoxy polyethylene glycol are sequentially connected through chemical bonds; wherein the core diameter of the gold nanoparticles is smaller than 3 nm, and the hydrodynamic diameter of the gold nanoparticles is smaller than 6 nm. The gold nanoparticles provided by the invention can improve the activity and bioavailability of aromatic drugs loaded on the gold nanoparticles. The gold nanoparticles are small in size and can be removed out of a body through the kidney, the gold nanoparticles are prevented from being captured by the liver, non-specific uptake of other organs is reduced, the bioavailability of the medicine is improved, and meanwhile side effects are reduced.

Description

Technical Field

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

[0002] Acute kidney injury (AKI) is a clinical syndrome caused by rapid decline of renal function due to various etiologies. "Early diagnosis and early treatment" of acute kidney injury is extremely important for the prognosis of patients. However, due to the serious lag of serum creatinine, a commonly used renal function detection index in clinical practice, the creatinine level usually does not show a significant increase until the glomerular filtration rate of the patient drops by more than 50%. At this time, the renal function has suffered irreversible damage. Therefore, it is urgent to achieve early diagnosis of AKI and timely treatment when the renal function is less severely damaged, which can effectively protect the renal function, reduce the mortality of AKI patients, and at the same time inhibit the transformation of patients into chronic kidney disease (CKD). During the progression of AKI, cell damage caused by intracellular oxidative stress is one of the main reasons for the progression of AKI. Therefore, developing antioxidant drugs targeting reactive oxygen species (ROS) can counteract the progression of AKI and effectively protect renal function. Aromatic natural antioxidants (such as curcumin, resveratrol, quercetin, etc.) have excellent antioxidant activity and economy. However, due to their strong hydrophobicity, their bioavailability is low, and it is difficult to reach or distribute into the kidneys.

[0003] Currently, with the continuous development of nanomaterials, many nanomaterials have been synthesized and applied to the treatment of AKI; for example, nanozymes with antioxidant properties themselves are used to treat acute kidney injury. In addition, loading substances with antioxidant activity onto nanomaterials to achieve the treatment of AKI for the oxidative stress pathological state of the kidneys in the case of AKI has also become a common strategy. Currently, the drug delivery methods of nanomaterials for the treatment of AKI are roughly divided into two types. One is to connect antioxidants or therapeutic drugs to nanomaterials through chemical bond modification. Although the drug delivery systems prepared by such chemical modifications can improve the bioavailability and targeting efficiency of drugs, there are also problems such as changes in drug structure caused by chemical modifications, which affect the activity of drugs. In addition, such delivery systems usually do not have universality, and the delivery of different drugs requires specific design and cannot perform multi-class drug delivery. The labor cost is relatively high.

[0004] In addition, due to size issues, most nanomaterial delivery systems are limited in their therapeutic applications for kidney diseases. Large nanoparticles are difficult to penetrate the glomerular membrane and renal extracellular matrix, making it difficult for drugs to effectively reach the target site. Due to size issues, most of them are cleared by the liver, and even if they enter the target site, they may remain in the body for a long time, which may trigger toxic reactions, especially in an organ that requires efficient filtration and excretion. Moreover, large-sized nanodelivery systems are less controllable in drug release kinetics than small particles, which may lead to drug release at inappropriate times and locations. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a gold nanoparticle capable of loading aromatic drugs, its preparation method and application. The gold nanoparticle has a small size, can be cleared from the body through the kidneys, avoids being captured by the liver, reduces non-specific uptake by other organs, and improves drug bioavailability while reducing side effects.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: In the first aspect, the present invention provides a gold nanoparticle, which comprises a gold nanosphere, a 4-mercaptobenzoic acid ligand, and a methoxy-capped polyethylene glycol-amine. The gold nanosphere, the 4-mercaptobenzoic acid ligand, and the methoxy-capped polyethylene glycol-amine are sequentially connected by chemical bonds; the core diameter of the gold nanoparticle is less than 3 nm; the hydrodynamic diameter of the gold nanoparticle is less than 6 nm.

[0007] Specifically, the gold nanosphere is connected to the methoxy-capped polyethylene glycol-amine through the 4-mercaptobenzoic acid ligand; more specifically, the gold nanosphere replaces the mercapto hydrogen on the 4-mercaptobenzoic acid ligand, and the amino group of the methoxy-capped polyethylene glycol-amine undergoes a condensation reaction with the carboxyl group on the 4-mercaptobenzoic acid ligand.

[0008] As a preferred embodiment of the first aspect of the present invention, the core diameter of the gold nanoparticle is 1.83 ± 0.9 nm.

[0009] The present invention uses 4-mercaptobenzoic acid (4-MBA) as a ligand to prepare a gold nanocarrier with drug delivery function. 4-MBA has a benzene ring structure, which can form π-π stacking interactions with various drugs containing aromatic rings and serve as a drug loading site for the gold nanodelivery system. After 4-MBA is modified with PEG, the circulation time of gold nanoparticles in vivo is prolonged, which can effectively change the pharmacokinetics of the loaded drugs and avoid the rapid clearance of drugs in the body. At the same time, the core diameter of the gold nanoparticles of the present invention is less than 3 nm, and the hydrodynamic diameter is less than 6 nm. It has a size smaller than the renal filtration threshold (6 nm), can be cleared to the outside of the body through the kidneys, avoid the side effects caused by liver capture, and reduce the non-specific uptake of other organs, improving the curative effect while reducing side effects. Especially when the core diameter of the gold nanoparticles of the present invention is 1.83 ± 0.9 nm, the size is smaller and it is more conducive to entering the kidneys and being cleared through the kidneys.

[0010] In the technical field, the core diameter of gold nanoparticles refers to the physical size (such as diameter or radius) of the core part composed of metallic gold (Au). Therefore, the core diameter of the gold nanoparticles described in the present invention is the diameter of the gold nanospheres. Usually, a transmission electron microscope (TEM) is used to directly observe and measure its core size.

[0011] In the technical field, the hydrodynamic diameter is an important parameter used to describe the behavior characteristics of particles in a fluid. It usually reflects the "apparent size" of nanoparticles in a solution, including the thickness of the surface modification layer or adsorbed molecules, and is usually larger than the core diameter. The hydrodynamic diameter is determined by analyzing the Brownian motion of particles in a solution. The smaller the particles, the faster the Brownian motion and the larger the diffusion coefficient. Therefore, the measurement range of the hydrodynamic diameter of the gold nanoparticles described in the present invention includes the diameter of the gold nanospheres and the thickness of their surface modification layer, and the modification layer includes 4-mercaptobenzoic acid ligand and methoxy-capped polyethylene glycol-amine. It is usually measured by dynamic light scattering (DLS) technology. By irradiating the nanoparticles suspended in a liquid with a laser and analyzing the change of the scattered light, the Brownian motion of the particles is calculated, and thus the hydrodynamic diameter is obtained.

[0012] 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.

[0013] The hydrodynamic diameter of the gold nanoparticles of the present invention is less than 6 nm, with a size smaller than the renal filtration threshold (6 nm), and can be cleared from the body through the kidneys, avoiding side effects caused by liver capture. The hydrodynamic diameter is determined by analyzing the Brownian motion of the particles in solution. The smaller the particles, the faster the Brownian motion and the larger the diffusion coefficient. In particular, when the hydrodynamic diameter of the gold nanoparticles of the present invention is 3.82 ± 1.49 nm, the size is smaller, which is more conducive to entering the kidneys. At the same time, the faster the Brownian motion, the larger the diffusion coefficient, and the higher the bioavailability.

[0014] As a preferred embodiment of the first aspect of the present invention, the gold nanoparticles further comprise a loaded hydrophobic drug; the hydrophobic drug is connected to the benzene ring on the 4-mercaptobenzoic acid ligand through physical adsorption (π-π stacking).

[0015] 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.

[0016] Specifically, the measured range of the hydrodynamic diameter of the gold nanoparticles loaded with hydrophobic drugs of the present invention includes the diameter of the gold nanospheres and the thickness of their surface modification layer and adsorbed drug molecules. The modification layer includes 4-mercaptobenzoic acid ligand and methoxy-capped polyethylene glycol-amine.

[0017] After loading the hydrophobic drug, the hydrodynamic diameter of the gold nanoparticles of the present invention is less than 6 nm, which is less than the renal filtration threshold of 6 nm, and is more conducive to entering the kidneys. The drug can enter the renal tubules through glomerular filtration, improving the utilization rate and therapeutic effect of the drug.

[0018] As a preferred embodiment of the first aspect of the present invention, the hydrophobic drug is an aromatic drug or an anti-tumor drug.

[0019] As a preferred embodiment of the first aspect of the present invention, the aromatic drug is any one of curcumin, resveratrol, and naringenin; the anti-tumor drug is a STING agonist.

[0020] 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: (1) Mix the gold nanoparticles with an ethanol solution of the drug, stir and react for 12 - 16 h, and then rotary evaporate to remove the ethanol in the reactants; (2) Redissolve the product after rotary evaporation, centrifuge to remove the precipitate, and the supernatant obtained is the gold nanoparticles loaded with aromatic drugs.

[0021] In the second aspect, the present invention provides the application of the gold nanoparticles as described above in any one of the following: (1) Use in the preparation of drugs for treating or preventing acute kidney injury.

[0022] (2) Use in the preparation of drugs for treating or preventing tumors.

[0023] Loading the aromatic drugs with the gold nanoparticles of the present invention can improve the therapeutic effect of aromatic drugs or anti-tumor drugs. Compared with single drugs, the bioavailability of the drugs is improved. It is proved by experiments that loading the aromatic drugs for treating or preventing acute kidney injury with the gold nanoparticles of the present invention can improve the therapeutic effect of the drugs, and loading the aromatic drugs for treating or preventing tumors with the gold nanoparticles of the present invention can improve the anti-tumor therapeutic effect of the drugs.

[0024] In the third aspect, the present invention provides a method for preparing gold nanoparticles, comprising the following steps: Step 1: Mix a chloroauric acid solution with a 4-mercaptobenzoic acid solution. After white flocculates appear in the mixed solution, add an NaOH solution and a sodium borohydride solution. After the reaction, obtain reaction system solution 1; Step 2: Centrifuge reaction system solution 1 to remove the precipitate in the reaction system solution, and purify the supernatant to obtain a gold nanoparticle system with 4-mercaptobenzoic acid ligands; Step 3: Activate the gold nanoparticle system with 4-mercaptobenzoic acid ligands obtained in Step 2, and add methoxy-terminated polyethylene glycol-amine to the activated gold nanoparticle system with 4-mercaptobenzoic acid ligands. After the reaction, obtain reaction system solution 2; Step 4: Centrifuge reaction system solution 2 to remove the precipitate in the reaction system, and purify the supernatant to obtain methoxy polyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles.

[0025] The preparation method of the present invention uses physical modification of gold nanoparticles. Compared with the delivery system of chemical modification methods, the present invention uses biocompatible materials, reducing the immune response and toxicity to the body. The drug loading method of the present invention is loading by physical adsorption (π-π stacking), without involving chemical modification, retaining the drug structure, and thus avoiding the influence of possible chemical reaction by-products on the drug activity. The physical modification system of the present invention can achieve controlled release of drugs by adjusting physical parameters (such as particle size, surface charge, etc.), thereby improving the therapeutic effect of the drugs.

[0026] As a preferred embodiment of the third aspect of the present invention, in Step 1, after adding the NaOH solution, the pH value is adjusted to 8-10. Preferably, the pH value is 9.

[0027] The research of the present invention finds that when the pH value is less than 8, the nanoparticles generated after adding a reducing agent are unstable and it is difficult to synthesize nanoparticles. When the pH value is greater than 10, the complex formed by 4-MBA and HAuCl4 undergoes sedimentation to form a black precipitate, and nanoparticles cannot be synthesized. Therefore, when the present invention controls the pH value within 8 - 10, ultra-small diameter nanoparticles can be obtained. Especially 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.

[0028] As a preferred embodiment of the third aspect of the present invention, in step 1, the molar ratio of chloroauric acid to 4-mercaptobenzoic acid is 1:(2 - 3).

[0029] As a preferred embodiment of the third aspect of the present invention, in step 1, the molar ratio of chloroauric acid to 4-mercaptobenzoic acid is 1:2.

[0030] The research of the present invention finds that when the molar ratio of HAuCl4 to 4-MBA < 1:2, small-sized gold nanoparticles cannot be formed; when the molar ratio of HAuCl4 to 4-MBA > 1:3, the formed gold nanoparticles are larger in size, unstable, and difficult to pass through the renal filtration threshold. Therefore, when the present invention controls the molar ratio of HAuCl4 to 4-MBA within 1:(2 - 3), ultra-small diameter nanoparticles can be obtained. Especially when the molar ratio of HAuCl4 to 4-MBA is 1:2, 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.

[0031] As a preferred embodiment of the third aspect of the present invention, in the chloroauric acid solution in step 1, the concentration of chloroauric acid is 10 - 30 mM; in the 4-mercaptobenzoic acid solution, the concentration of 4-mercaptobenzoic acid is 10 - 30 mM.

[0032] As a preferred embodiment of the third aspect of the present invention, in reaction system solution 2, the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to methoxy-capped polyethylene glycol-amine is 1:(1 - 5); and / or, the molecular weight of the methoxy-capped polyethylene glycol-amine (MPEG-NH2) is 750 - 1500.

[0033] As a preferred embodiment of the third aspect of the present invention, in reaction system solution 2, the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to methoxy-capped polyethylene glycol-amine is 1:2; and / or, the molecular weight of the methoxy-capped polyethylene glycol-amine (MPEG-NH2) is 1000.

[0034] The research of the present invention has found that the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to methoxy-terminated polyethylene glycol-amine, and the molecular weight of MPEG-NH2 are the key factors affecting the particle size of the gold nanoparticles of the present invention. If the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to methoxy-terminated polyethylene glycol-amine is too small or too large, PEG cannot be effectively connected to PEG. If the molecular weight of MPEG-NH2 is too small, PEG cannot be stably modified. If the molecular weight is too large, the particle size of the material will be too large, exceeding the renal filtration threshold and unable to achieve renal clearance, or it is prone to aggregation, affecting the drug-loading function. Therefore, in the present invention, when the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to methoxy-terminated polyethylene glycol-amine 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 ligand to methoxy-terminated polyethylene glycol-amine 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.

[0035] As a preferred embodiment of the third aspect of the present invention, the method for activation in step 3 is: using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the surface of gold nanoparticles with 4-mercaptobenzoic acid ligand.

[0036] 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 ligand. 1-ethyl-(3-dimethylaminopropyl)carbodiimide is a carbodiimide compound, which can form an active intermediate with the carbonyl oxygen atom of carboxylic acid or carboxylic acid ester to promote the activation of carboxyl groups. Using N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to jointly activate the carboxyl groups can improve the activation efficiency.

[0037] As a preferred embodiment of the third aspect of the present invention, the molar ratio of gold nanoparticles with 4-mercaptobenzoic acid ligand to 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(1 - 5):(1 - 5).

[0038] As a preferred embodiment of the third aspect of the present invention, the purification method in step 2 or step 3 is to use an ultrafiltration centrifugal tube with a cut-off value of 3000 Da for purification.

[0039] Compared with the prior art, the beneficial effects of the present invention are: The present invention uses 4-mercaptobenzoic acid (4-MBA) as a ligand to prepare a gold nanoparticle carrier with drug delivery ability. 4-MBA has a benzene ring structure, which can generate π-π stacking interactions with various drugs containing aromatic rings and serve as a drug loading site for the gold nanoparticle delivery system. After PEG-modifying 4-MBA, the circulation time of gold nanoparticles in vivo is prolonged, which can effectively change the pharmacokinetics of the loaded drugs and avoid the rapid clearance of drugs in the body. The activity and bioavailability of the loaded hydrophobic drugs are significantly improved. Compared with other drug delivery systems, gold nanoparticles have a small size, can be cleared out of the body through the kidneys, avoid the side effects caused by liver clearance, reduce the non-specific uptake of other organs, and improve the therapeutic effect while reducing side effects. Utilizing the property of ultra-small gold nanoparticles being cleared by the kidneys, a universal renal therapeutic drug delivery system has been invented.

[0040] The preparation method of the present invention uses physical action to load drugs. Compared with the delivery system of the chemical modification method, the present invention usually uses physical action to load drugs, does not change the structure of the drugs themselves, reduces the immune response and toxicity to the body. It does not involve chemical reactions, so it avoids the influence of possible chemical reaction by-products on the drug activity. The physical modification system can achieve the controlled release of drugs by adjusting physical parameters (such as particle size, surface charge, etc.), thereby improving the therapeutic effect. In addition, physical modification can also combine multiple functions, such as magnetism, photosensitivity, etc., to achieve multimodal therapy and diagnosis. Usually, it does not require complex chemical synthesis steps, the production process is relatively simple, and the cost is low. This method can not only deliver a single drug, but also has a universal delivery ability for drug molecules with the same properties.

[0041] The present invention uses a drug loading method based on physical action to invent a drug delivery system of ultra-small gold nanoparticles, and based on this, hydrophobic natural antioxidants are delivered to achieve the treatment of acute kidney injury. The hydrodynamic diameter of the ultra-small gold nanoparticles is less than 6 nm, the kidney filtration threshold, enabling them to enter the renal tubules through glomerular filtration, thereby achieving the delivery of antioxidant drugs. The prepared drug delivery system of ultra-small gold nanoparticles can be cleared by the kidneys, avoiding the liver toxicity caused by liver clearance. In addition, due to its drug loading principle being physical force, it has a good loading effect on drugs with an aromatic structure such as benzene rings, and this system has a certain universality. Depending on the different loaded drugs, the treatment of different diseases may be achieved. For example, the gold nanoparticles of the present invention loaded with aromatic drugs for the treatment or prevention of acute kidney injury can improve the therapeutic effect of the drug, making it have the efficacy of treating or preventing acute kidney injury; the gold nanoparticles of the present invention loaded with aromatic drugs for the treatment or prevention of tumors can improve the tumor treatment effect of the drug, making it have the efficacy of treating or preventing tumors. Description of the Drawings

[0042] Figure 1 Schematic diagram of the synthesis route of PEG-MBA-AuNPs; Figure 2 Schematic diagram of the core diameter and hydrodynamic diameter of PEG-MBA-AuNPs; Figure 3 Schematic diagram of the excitation / emission spectrum and ultraviolet-visible absorption spectrum of PEG-MBA-AuNPs; Figure 4 Schematic diagram of the preparation route of drug-loaded PEG-MBA-AuNPs; Figure 5 Schematic diagram of the ultraviolet-visible absorption spectrum of drug-loaded PEG-MBA-AuNPs with aromatic structure drugs (Figure A is curcumin; Figure B is naringenin; Figure C is resveratrol; Figure D is STING agonist DMXAA); Figure 6 Schematic diagram of the hydrodynamic diameter of Cur@PEG-AuNPs; Figure 7 Schematic diagram of the antioxidant activity of Cur@PEG-AuNPs: Figure 8 Schematic diagram of the serum creatinine and blood urea nitrogen levels of AKI mice after treatment with Cur@PEG-AuNPs (Normal represents the normal group; AKI+PBS represents acute kidney injury + administration of phosphate buffer solution; 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); Figure 9 Schematic diagram of the kidney tissue pathological sections and tubular injury scores of AKI mice after treatment with Cur@PEG-AuNPs (Figure A: Normal represents the normal group; Figure B: AKI+PBS represents acute kidney injury + administration of phosphate buffer solution; 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 the injury scores of the above several groups); Figure 10 Schematic diagram of the serum creatinine and blood urea nitrogen levels of AKI mice after prevention with Cur@PEG-AuNPs; Figure 11 Schematic diagram of the kidney tissue pathological sections and tubular injury scores of AKI mice after prevention with Cur@PEG-AuNPs (Figure A is the kidney tissue pathological section diagram; Figure B is the tubular injury score diagram); Figure 12It is the tumor volume and weight inhibition curve diagram of DMXAA@PEG-AuNPs (Figure A is the tumor photo diagram; Figure B is the tumor volume and weight inhibition curve diagram); Figure 13 It is that DMXAA@PEG-AuNPs activates the STING pathway to induce CD80 + CD86 + Percentage schematic diagram of DCs induced by DMXAA@PEG-AuNPs (Figure A is the activation schematic diagram of the STING pathway by DMXAA@PEG-AuNPs; Figure B is the induction schematic diagram of DCs); Figure 14 It is the schematic diagram of the number of metastatic lung nodules of DMXAA@PEG-AuNPs (Figure A is the photo diagram of the number of metastatic lung nodules; Figure B is the comparison schematic diagram of the number of metastatic lung nodules in different groups); Figure 15 It is the schematic diagram of the lung metastasis result analyzed by H&E staining in the DMXAA@PEG-AuNPs group; Figure 16 It is the cumulative statistical schematic diagram of the distribution of Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L) in the liver, spleen, and kidney under normal and AKI conditions respectively; Figure 17 It is the schematic diagram of the serum creatinine and urea nitrogen levels in AKI mice after being prophylactically treated with Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L) respectively; Figure 18 It is the schematic diagram of the kidney tissue pathological sections and renal tubular injury scores of AKI mice after being treated with Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L) respectively (Figure A is the kidney tissue pathological section diagram; Figure B is the renal tubular injury score schematic diagram); Figure 19 It is the pharmacokinetic curve diagram of MBA-AuNPs and PEG-MBA-AuNPs-1. Detailed implementation manners

[0043] 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.

[0044] Table 1: Chinese names involved in the present invention and their corresponding abbreviations Example 1 This example provides a preparation method of gold nanoparticles -1 to -15 ( Figure 1 ), including the following steps: Preparation method of gold nanoparticles -1: (1) Preparation of 4-mercaptobenzoic acid ligand-functionalized gold nanoparticles: a. Preparation of 20 mM chloroauric acid solution: Weigh accurately about 0.0394 g of chloroauric acid trihydrate, dissolve it in about 5 mL of ultrapure water, and stir evenly. The prepared 20 mM chloroauric acid solution is stored in a brown glass bottle and kept in the refrigerator at 4 °C in the dark to prevent decomposition and oxidation.

[0045] b. Preparation of 20 mM 4-mercaptobenzoic acid solution: Weigh accurately about 0.0304 g of 4-mercaptobenzoic acid, dissolve it in about 7 mL of absolute ethanol, and stir evenly. Volume fixation: Transfer the solution to a 10 mL volumetric flask, and make up the volume to the scale line with absolute ethanol, and shake well. Storage: The prepared 20 mM 4-mercaptobenzoic acid solution is stored in a brown glass bottle and kept in the refrigerator at 4 °C in the dark to prevent decomposition and oxidation.

[0046] c. Preparation of 4-mercaptobenzoic acid ligand-functionalized gold nanoparticle system c-1: Mix the above chloroauric acid solution and 4-mercaptobenzoic acid solution for reaction, stir well, and wait for white flocs to appear in the reaction solution; among them, the molar ratio of chloroauric acid to 4-mercaptobenzoic acid is HAuCl4:4-MBA = 1:2; c-2: Add 1M NaOH solution to the reaction system in step c-1, and adjust the pH to 9; c-3: Add 112 mM sodium borohydride solution to the reaction system in step c-2, stir and react for 10 h to obtain reaction system solution 1; d. After the reaction is completed, centrifuge at 15000 rpm for 10 min to remove the precipitate in reaction system solution 1, and the supernatant is purified more than 3 times using an ultrafiltration centrifugal tube with a cut-off molecular weight of 3000 Da. After purification, a 4-mercaptobenzoic acid ligand-functionalized gold nanoparticle (denoted as: MBA-AuNPs) system is obtained.

[0047] (2) Preparation of methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand-functionalized gold nanoparticles a. Activation of 4-mercaptobenzoic acid ligand-functionalized gold nanoparticles: Mix the MBA-AuNPs system with EDC and NHS, and the mixing molar ratio is MBA-AuNPs:EDC:NHS = 1:1.5:1.5. After stirring and reacting for 1 h, an activated MBA-AuNPs system is obtained. All MBA-AuNPs mentioned in the following steps are activated MBA-AuNPs; b. Add MPEG-NH2 to the reaction solution of MBA-AuNPs activated in step a. After stirring and reacting for 10 h, obtain reaction system solution 2; wherein, the molar ratio of MBA-AuNPs to MPEG-NH2 is MBA-AuNPs:MPEG-NH2 = 1:2; and the molecular weight of MPEG-NH2 is 1000.

[0048] c. After the reaction ends, centrifuge at 15000 rpm for 10 min to remove the precipitate in reaction system solution 2. The supernatant is purified more than 3 times using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000 Daltons. After purification, obtain methoxypolyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles (denoted in this invention as: PEG-MBA-AuNPs-1).

[0049] The preparation method of PEG-MBA-AuNPs-2 is different from that of PEG-MBA-AuNPs-1 as follows: (1) In step (1) c-1, HAuCl4:4-MBA = 1:3; (2) In step (1) c-2, the pH value is 8; (3) In step (1) c-3, the stirring reaction time is 12 h; (4) In step (2) a, the mixing molar ratio is MBA-AuNPs:EDC:NHS = 1:1:1; (5) In step (2) b, the molecular weight of MPEG-NH2 is 750; (6) In step (2) b, the stirring reaction time of MBA-AuNPs and MPEG-NH2 is 12 h; (7) In step (2) b, MBA-AuNPs:MPEG-NH2 = 1:1.

[0050] The preparation method of PEG-MBA-AuNPs-3 is different from that of PEG-MBA-AuNPs-1 as follows: (1) In step (1) c-1, HAuCl4:4-MBA = 1:2.5; (2) In step (1) c-2, the pH value is 10; (3) In step (1) c-3, the stirring reaction time is 8 h; (4) In step (2) a, the mixing molar ratio is MBA-AuNPs:EDC:NHS = 1:5:5; (5) In step (2) b, the molecular weight of MPEG-NH2 is 1500; (6) In step (2) b, the stirring reaction time of MBA-AuNPs and MPEG-NH2 is 8 h; (7) In step (2) b, the ratio of MBA-AuNPs to MPEG-NH2 is 1:5.

[0051] The preparation method of PEG-MBA-AuNPs-4 is different from that of PEG-MBA-AuNPs-1 in that: in step (1) c-1, the ratio of HAuCl4 to 4-MBA is 1:1.

[0052] The preparation method of PEG-MBA-AuNPs-5 is different from that of PEG-MBA-AuNPs-1 in that: in step (1) c-1, the ratio of HAuCl4 to 4-MBA is 1:4.

[0053] The preparation method of PEG-MBA-AuNPs-6 is different from that of PEG-MBA-AuNPs-1 in that: the pH value in step (1) c-2 is 7.

[0054] The preparation method of PEG-MBA-AuNPs-7 is different from that of PEG-MBA-AuNPs-1 in that: the pH value in step (1) c-2 is 11.

[0055] The preparation method of PEG-MBA-AuNPs-8 is different from that of PEG-MBA-AuNPs-1 in that: sodium citrate (CIT) is used to replace sodium borohydride, and the specific method is as follows; 1. Accurately weigh about 94.4 mg of chloroauric acid trihydrate with an analytical balance. Dissolve the weighed HAuCl4·3H2O with ultrapure water and quantitatively dilute it to 10 mL to obtain 24 mM HAuCl4.

[0056] 2. Prepare a 2.2 mM 50 mL CIT solution. Accurately weigh about 32.351 mg of sodium citrate dihydrate with an analytical balance. Add ultrapure water and gently stir to help dissolve and make up the volume to 50 mL.

[0057] 3. Prepare a 60 mM 50 mL CIT solution. Accurately weigh about 176.46 mg of sodium citrate dihydrate with an analytical balance. Dissolve the weighed sodium citrate with ultrapure water and quantitatively dilute it to 10 mL for standby.

[0058] 4. Prepare the above three reagents with different concentrations. Preheat the heating table to 100 °C in advance. Take 50 mL of 2.2 mM sodium citrate solution in a flask. Wait until the solution boils, then add 333 μL of 24 mM HAuCl4 and react for 10 min. The solution turns light pink. Let the solution cool to 90 °C, then add 333 μL of 60 mM sodium citrate solution, and then add 333 μL of 24 mM HAuCl4 and react for 30 min. The solution turns wine red. Stop the reaction after detecting an ultraviolet absorption peak in the range of 510 nm - 520 nm in the ultraviolet spectrum, and PEG-MBA-AuNPs-8 is thus prepared.

[0059] The preparation method of PEG-MBA-AuNPs-9 is different from that of PEG-MBA-AuNPs-1 in that: sodium cyanoborohydride is used to replace sodium borohydride, with a concentration of 112 mM, and other steps are the same.

[0060] The preparation method of PEG-MBA-AuNPs-10 is different from that of PEG-MBA-AuNPs-1 in that: in step (2) b, the molecular weight of MPEG-NH2 is 350.

[0061] The preparation method of PEG-MBA-AuNPs-11 is different from that of PEG-MBA-AuNPs-1 in that: in step (2) b, the molecular weight of MPEG-NH2 is 5000.

[0062] The preparation method of PEG-MBA-AuNPs-12 is different from that of PEG-MBA-AuNPs-1 in that: in step (2) b, the molar ratio of MBA-AuNPs:MPEG-NH2 = 1:7.

[0063] The preparation method of PEG-MBA-AuNPs-13 is different from that of PEG-MBA-AuNPs-1 in that: in step (2) b, the molar ratio of MBA-AuNPs:MPEG-NH2 = 1:0.5.

[0064] The preparation method of PEG-MBA-AuNPs-14 is different from that of PEG-MBA-AuNPs-1 in that: the order of steps is different. Swap steps (1) c-2 and c-3, and the rest of the steps are the same. The specific steps are as follows: c Prepare the gold nanoparticle system of 4-mercaptobenzoic acid ligand c-1: Mix the above chloroauric acid solution and 4-mercaptobenzoic acid solution for reaction, stir well, and wait until white flocculants appear in the reaction solution; among them, the molar ratio of chloroauric acid to 4-mercaptobenzoic acid is HAuCl4:4-MBA = 1:2; c-2: Add 112 mM sodium borohydride solution to the reaction system in step c-1 and stir the reaction for 10 h; c-3: Add 1M NaOH solution to the reaction system of step c-2 and adjust the pH to 9 to obtain reaction system solution 1.

[0065] 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 the 3-mercaptobenzoic acid ligand.

[0066] Example 2 Physical and Chemical Characterization of PEG-MBA-AuNPs Characterization method: Use transmission electron microscopy (TEM) to characterize the core size and morphology of nanoparticles; use dynamic light scattering (DLS) to characterize the hydrodynamic diameter of nanoparticles; use ultraviolet-visible spectrophotometer (UV-Vis) to characterize the characteristic absorption of nanoparticles; use fluorescence spectrometer (FL) to characterize the fluorescence properties of nanoparticles. The results are shown in the following table: Table 2 Particle size of gold nanoparticles prepared under different conditions like Figure 2 As shown in Figure 2, 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 has fluorescence properties, with the best excitation wavelength at 350 nm and the maximum emission wavelength at 810 nm. In the range of 400-500 nm, there is no obvious UV-Vis absorption of the nanoparticles, proving that there is no surface plasmon resonance and they are ultra-small size nanoparticles.

[0067] It can be seen from PEG-MBA-AuNPs-15 that although the 3-mercaptobenzoic acid ligand and the 4-mercaptobenzoic acid ligand of the present invention only differ in the position of the sulfhydryl group, they cannot stably connect to PEG. At the same time, the PEG-MBA-AuNPs prepared by the present invention is a drug loading system, which relies on the benzene ring structure of its MBA for drug loading, while the sulfhydryl group of 3-MBA is located in the meta position of the benzene ring, and the steric hindrance is large, and it is unstable when combined with the metal surface, and 3-MBA is not conducive to combining with the methoxy-terminated polyethylene glycol-amino (MPEG-NH2) from the steric hindrance, which is more unfavorable for the subsequent drug loading. The 4-MBA selected by the present invention has a steric hindrance less than that of the sulfhydryl group and the carboxyl group in the para position of the benzene ring, and is more directly combined with the metal surface and has higher stability. Therefore, 4-MBA is more conducive to the loading of aromatic drugs of the present invention relative to 3-MBA.

[0068] Example 3 This example takes curcumin as an example to illustrate the application of the PEG-MBA-AuNPs drug-loading system prepared by the present invention. The reaction route is as Figure 4 shown, and the specific method is as follows: (1) Dissolve equimolar curcumin in anhydrous ethanol solution to obtain curcumin ethanol solution.

[0069] (2) Take PEG-MBA-AuNPs-1 and add it to the curcumin ethanol solution to obtain a reaction system. Among them, the volume ratio of the curcumin ethanol solution to PEG-MBA-AuNPs in the reaction system is 3:1. After stirring and reacting for 12 - 24 h, rotate and evaporate to remove ethanol in the reactants, and redissolve the product after rotary evaporation with ultrapure water. Centrifuge at 15000 rpm for 10 min. The precipitate is unloaded curcumin (Curcumin), remove the precipitate, and the supernatant is the curcumin-loaded nanoparticles. Only retain the supernatant.

[0070] As Figure 5 shown, the characterization results by ultraviolet absorption spectrum prove that curcumin is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical interaction, denoted as Cur@PEG-AuNPs in the present invention.

[0071] As Figure 6 shown, by using dynamic light scattering (DLS) to characterize, the hydrodynamic diameter (hydrated particle size) of Cur@PEG-AuNPs is 4.84 ± 0.89 nm, which meets the size range of ultra-small gold nanoparticles.

[0072] Example 4 This example takes resveratrol as an example to illustrate the application of the PEG-MBA-AuNPs drug-loading prepared by the present invention. The specific method is as follows: (1) Dissolve equimolar resveratrol in anhydrous ethanol solution to obtain resveratrol solution.

[0073] (2) Take PEG-MBA-AuNPs-1 and add it to the resveratrol solution to obtain a reaction system. Among them, the volume ratio of ethanol to water in the reaction system is 3:1. After stirring and reacting for 16 h, rotate and evaporate to remove ethanol in the reactants, and redissolve the product after rotary evaporation with ultrapure water. Centrifuge at 15000 rpm for 10 min to remove the precipitate of unloaded resveratrol, and the supernatant is the resveratrol-loaded nanoparticles.

[0074] As Figure 5 shown, the characterization results by ultraviolet absorption spectrum prove that resveratrol is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical interaction, denoted as Res@PEG-AuNPs in the present invention.

[0075] Example 5 This example takes naringenin 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: (1) Dissolve naringenin with an equimolar ratio in an anhydrous ethanol solution to obtain a naringenin solution.

[0076] (2) Add PEG-MBA-AuNPs-1 to the naringenin solution to obtain a reaction system, wherein the volume ratio of ethanol to water in the reaction system is 3:1. After stirring and reacting for 14 h, rotary evaporate to remove the ethanol in the reactants, and redissolve the rotary-evaporated product with ultrapure water. Centrifuge at 15000 rpm for 10 min to remove the unloaded naringenin precipitate, and the supernatant is the naringenin-loaded nanoparticles.

[0077] As Figure 5 shown, the characterization results by ultraviolet absorption spectroscopy prove that naringenin is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, denoted as Nar@PEG-AuNPs in the present invention.

[0078] Example 6 This example takes 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: (1) Dissolve DMXAA with an equimolar ratio in an anhydrous ethanol solution to obtain a DMXAA solution.

[0079] (2) Add PEG-MBA-AuNPs-1 to the DMXAA solution to obtain a reaction system, wherein the volume ratio of ethanol to water in the reaction system is 3:1. After stirring and reacting for 15 h, rotary evaporate to remove the ethanol in the reactants, and redissolve the rotary-evaporated product with ultrapure water. Centrifuge at 15000 rpm for 10 min to remove the unloaded DMXAA precipitate, and the supernatant is the DMXAA-loaded nanoparticles.

[0080] As Figure 5 shown, the characterization results by ultraviolet absorption spectroscopy prove that DMXAA is successfully loaded on the surface of PEG-MBA-AuNPs-1 through physical action, denoted as DMXAA@PEG-AuNPs in the present invention.

[0081] Comparison of the antioxidant properties of Cur@PEG-AuNPs and Curcumin in Example 7 Experimental method: The antioxidant property of the nanoparticles in Example 3 was characterized using the ABTS method. ABTS is oxidized to green ABTS·+ under the action of an oxidant, and has characteristic absorption peaks at 734 nm or 405 nm. In the presence of antioxidants, the generation of ABTS·+ is inhibited, causing the reaction system to fade, and the absorbance at 405 nm decreases. Within a certain range, the change in its absorbance is proportional to the degree of free radical scavenging. The ability of the sample to scavenge ABTS free radicals can be reflected by the degree of decrease in absorbance.

[0082] (1) ABTS was dissolved in a sodium acetate solution with a pH value of 4.5 to prepare an ABTS solution with a concentration of 7.4 mmol / L. The ABTS solution was mixed with a 2.6 mmol / L potassium persulfate solution at a volume ratio of 1:1 and allowed to stand in the dark at room temperature for 12 h to obtain a reaction solution.

[0083] (2) Detection method: Phosphate buffer solution (PBS) of Cur@PEG-AuNPs with different concentrations (10, 20, 40, and 80 μg / mL) was mixed with the reaction solution in step (1) at a volume ratio of 1:9. After reacting in the dark for 30 min, the absorbance was measured at 734 nm, zeroed with distilled water, and the total antioxidant activity at different concentrations was calculated. A single curcumin solution with the same concentration was used as the control group.

[0084] The experimental results are as Figure 7 shown. 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 property, indicating that Cur@PEG-AuNPs has good antioxidant activity.

[0085] Therapeutic effect of Cur@PEG-AuNPs on AKI in Example 8 Experimental method: (1) Establishment of an acute kidney injury model: A model of acute kidney injury induced by rhabdomyolysis (induced by intramuscular injection of 8 mL / kg of 50% glycerol into both lower limbs) was used. Ultra-small gold nanoparticles loaded with curcumin (Cur@PEG-AuNPs) were injected 2 hours after glycerol injection, and a blank control, a model control group (injected with PBS), and a control group (injected with curcumin) were set up simultaneously.

[0086] (2) At 24 hours after the injection of gold nanoparticles, the biochemical indexes of blood and kidney function (creatinine, blood urea nitrogen) and kidney pathological sections of the mice were detected to determine the degree of AKI and the therapeutic effect. The specific operations are as follows: a. Detection of biochemical indexes: After the mice were anesthetized with isoflurane gas, blood was first drawn by puncture from the heart position. After the blood coagulated naturally, the serum was separated, and the levels of creatinine and blood urea nitrogen in the serum were detected using an automatic biochemical analyzer.

[0087] b. Pathological section examination of kidney: The abdomen was opened longitudinally along the midline of the abdomen, 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 tubular damage. Ten high-power fields were randomly selected from each kidney section and scored according to the following criteria: 1 point for obvious tubular dilation and flattened cells; 1 point for brush border damage and 2 points for shedding; 2 points for casts and 1 point for shedding and necrotic cells (not in casts or cell fragments) in the lumen of the renal tubules; the highest score for each field was 4 points and the lowest score was 0 points.

[0088] 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 and returned to normal levels compared with untreated mice, while the serum creatinine and urea nitrogen levels of the model control group (PBS injection) and the control group (curcumin injection) were still high.

[0089] 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 can be found that the degree of renal tubular damage was significantly reduced.

[0090] Example 9 Preventive effect of Cur@PEG-AuNPs on AKI 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.

[0091] ICR mice aged 6 to 8 weeks were selected. After anesthesia, bilateral kidneys were exposed through incision, and the renal pedicles (including renal arteries, renal veins, and ureters) were exposed. Non-damaging vascular clamps or mouse renal pedicle clamps were used to clamp bilateral renal pedicles to block the blood flow to the kidneys. During the clamping period, the kidneys were kept moist to avoid dryness and thermal damage. After the predetermined 30-min ischemia time, the vascular clamps were quickly released to restore the blood flow to the kidneys and start reperfusion. The color change of the kidneys was observed, and it gradually returned from pale to normal color, indicating successful reperfusion. The abdominal incision was sutured and disinfected to complete the operation. Blood samples were collected 24 h and 48 h after reperfusion to detect renal function indicators (serum creatinine, urea nitrogen). The experimental mice were euthanized 48 h later, and renal tissue samples were collected for H&E staining histopathological examination and evaluation of the degree of renal injury.

[0092] 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 after Cur@PEG-AuNPs prevention were significantly reduced and maintained at normal levels.

[0093] The experimental results are as follows Figure 11 shown. After paraffin sectioning and H&E staining of the kidneys of the above mice, it can be found that the degree of renal tubular injury is significantly reduced.

[0094] Antitumor immune effect of DMXAA@PEG-AuNPs in Example 10 Female BALB / c mice aged 6 - 8 weeks were selected. 3×10 6 4T1 cells (mouse breast cancer cells) were subcutaneously injected into the right back of the mice to establish a subcutaneous tumor-bearing mouse model. 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 drugs were administered once every two days, and Radiation (4 Gy) was given 24 h after injection. A total of 3 doses were administered. The in vivo therapeutic effect of the drugs 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, the tumors were collected, photographed, and immunohistochemistry experiments were performed. In addition, lymph nodes and tumor tissues of the tumor-bearing mice were collected to prepare single-cell suspensions for flow cytometry analysis of immune cells.

[0095] The experimental results are as follows Figure 12 shown. Compared with the control group, the DMXAA@PEG-AuNPs + Radiation treatment group showed the highest level of anti-cancer activity, the smallest tumor volume, and a tumor growth inhibition rate of 87.4%. This can be attributed to the fact that DMXAA@PEG-AuNPs can be effectively delivered to the tumor site and exert an anti-tumor effect under the action of radiation. During the entire treatment intervention period, there were no statistically significant changes in body weight, indicating good in vivo safety of DMXAA@PEG-AuNPs.

[0096] The experimental results are as follows Figure 13As shown, the activated STING further recruits and activates TBK1 (TANK-binding kinase 1), which then phosphorylates and activates IRF3 (interferon regulatory factor 3). The immunohistochemical staining results of phosphorylated IRF3 (pIRF3) showed that compared with free DMXAA, the positive area of DMXAA@PEG-AuNPs + Radiation was the largest, indicating the effective expression of pIRF3. This is mainly because PEG-AuNPs effectively delivered DMXAA to the tumor site, thereby activating the STING pathway, while free DMXAA was quickly metabolized in vivo. In addition, the flow analysis results showed that DMXAA@PEG-AuNPs + Radiation could effectively induce the maturation of dendritic cells (DCs), and the percentage of + CD80 + CD86

[0097] Example 11 Anti-lung tumor metastasis effect of DMXAA@PEG-AuNPs The 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, G7: DMXAA@PEG-AuNPs + Radiation group. On the 7th day, 3×10 5 4T1-luc cells in 100 μL of PBS were injected into the tail vein of the mice to form lung metastases. Then, the mice were intravenously injected with different preparations to treat the primary tumor. Then the mice were monitored for 2 weeks. On the 21st day, the mice were sacrificed, the lung tissues of each group of mice were collected for photography, the lung metastasis nodules were manually counted, and H&E staining was performed to analyze the lung metastasis situation.

[0098] The experimental results were as Figure 14 shown. A large number of metastases were observed in the PBS, Radiation, and free DMXA groups, while only a small number of metastases were observed in the DMXAA@PEG-AuNPs + Radiation treatment group. The lung tissue was clear and regular at the edge, indicating that DMXAA@PEG-AuNPs + Radiation had a significant effect on preventing lung metastasis. The quantitative analysis results also confirmed that the number of metastatic lung nodules in the DMXAA@PEG-AuNPs + Radiation group was significantly reduced by 93.3% compared with the control group.

[0099] The experimental results were as Figure 15The HE staining results showed that the lung tissue structure of the mice in the control group was abnormal, with obvious metastatic foci in the lungs, large areas of tissue necrosis, large atypia of cancer cell nuclei, and most of the lung tissue occupied by tumors, with uneven edges. However, there were no obvious visible lesions in the lungs of the DMXAA@PEG-AuNPs + Radiation treatment group. These results indicated that DMXAA@PEG-AuNPs + Radiation effectively inhibited the metastasis of tumor cells.

[0100] Preparation of Cur@PEG-AuNPs (L) in Comparative Example 1 Comparative Example 1 provided a large-sized gold nanoparticle AuNPs drug delivery system, and its preparation method was as follows: PEG-MBA-AuNPs-11 prepared in the above-mentioned 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.

[0101] The curcumin ethanol solution was mixed with the prepared PEG-MBA-AuNPs (L), and shaken and stirred for 12 - 24 h. A dialysis bag with a cut-off molecular weight of 5000 was used to dialyze to remove unbound curcumin and ethanol in the reaction system. The retained product was denoted as Cur@PEG-AuNPs (L). Characterization results: The hydrodynamic diameter of Cur@PEG-AuNPs (L) was 31.32 ± 10.42 nm.

[0102] Comparative Example 1 The distributions of Cur@PEG-AuNPs (denoted as: Cur@PEG-AuNPs (S)) prepared in Comparative Example 3 and Cur@PEG-AuNPs (L) prepared in Comparative Example 1 in the liver, spleen, and kidneys under normal conditions and AKI conditions were as follows: Experimental method: ICR experimental mice aged 6 - 8 weeks were prepared and injected with Cur@PEG-AuNPs (S) and Cur@PEG-AuNPs (L) (the curcumin content loaded in S / L was the same), with 3 mice in each group. The experimental mice were euthanized 24 h after injection, and the liver, kidneys, and spleen were taken and weighed.

[0103] Fresh aqua regia (nitric acid: hydrochloric acid = 1:3) was prepared, and the taken tissue organs were soaked in fresh aqua regia for 48 h. The content of gold element in the organs was measured by ICP-MS. The formula: the content of gold element measured in the tissue organ / the weight of the measured tissue organ * 100% = the content of gold element in the tissue (unit % ID / g) was used to calculate and compare the distributions in different tissue organs.

[0104] The experimental results were as Figure 16As shown, for the same drug-loading structure, compared with Cur@PEG-AuNPs (L), Cur@PEG-AuNPs (S) of the present invention exhibits excellent kidney accumulation. Under normal conditions and in cases of acute kidney injury, Cur@PEG-AuNPs (L) mainly accumulates in the liver and spleen, while the Cur@PEG-AuNPs (S) of the present invention has a much higher accumulation amount in the kidney than Cur@PEG-AuNPs (L), showing a significant difference.

[0105] Comparative Example 2 Compare the therapeutic effects of Cur@PEG-AuNPs prepared in Comparative Example 3 (denoted as: Cur@PEG-AuNPs (S)) and Cur@PEG-AuNPs (L) prepared in Comparative Example 1 on AKI, as follows: Experimental method: Use the experimental method of Example 8.

[0106] The experimental results are as Figure 17 shown. For the acute kidney injury model, after 24 hours of treatment, compared with the untreated mice (AKI + PBS), the serum creatinine and blood urea nitrogen levels of the mice treated with Cur@PEG-AuNPs (S) were significantly reduced and restored to normal levels. After treatment with Cur@PEG-AuNPs (L), the serum creatinine and blood urea nitrogen levels of the mice were partially restored, and the serum creatinine index showed no obvious improvement. Cur@PEG-AuNPs (S) demonstrated a better therapeutic effect.

[0107] The experimental results are as Figure 18 shown. After paraffin sectioning and H&E staining of the kidneys of the above mice, it was found that the degree of renal tubular injury in the mice treated with Cur@PEG-AuNPs (L) was significantly reduced. However, there were still obvious injuries in the kidneys of the mice treated with Cur@PEG-AuNPs (L), with dilated renal tubular lumens and missing brush borders. This indicates that the therapeutic effect of Cur@PEG-AuNPs (S) is significantly better than that of the large-sized nano-system.

[0108] Comparative Example 3 Compare the un-methoxy-capped polyethylene glycol-amino-modified gold nanoparticles capable of loading aromatic drugs prepared in Comparative Example 3 (denoted as: MBA-AuNPs) with PEG-MBA-AuNPs-1. The preparation method of MBA-AuNPs is the same as that of PEG-MBA-AuNPs-1, except that methoxypolyethylene glycol modification is not used.

[0109] The results are as Figure 19The pharmacokinetic curve showing MBA-AuNPs indicates that MBA-AuNPs are cleared faster in vivo. Compared with PEG-MBA-AuNPs-1, the AUC of MBA-AuNPs is significantly smaller than that of PEG-MBA-AuNPs-1. The larger the AUC, the more drug is absorbed. And it can be used to evaluate the proportion of the drug entering the blood circulation. Similarly, the higher the CL value, the faster the body clears the drug. MBA-AuNPs are rapidly cleared from the body. The modification of PEG significantly increases the blood circulation time of MBA-AuNPs.

[0110] In summary, the ultra-small gold nanoparticle delivery system prepared in 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. The ultra-small gold nanoparticle delivery system prepared in the present invention can not only increase the accumulation of drugs in the kidneys, reduce the accumulation in the liver and spleen, but also improve the water solubility and antioxidant properties of antioxidant drugs. The average hydrodynamic diameter of the ultra-small gold nanoparticle delivery system responsible for drugs is only 4.8 ± 0.89 nm, which is smaller than the renal filtration threshold of 6 nm, enabling it to enter the renal tubules through glomerular filtration and improve the therapeutic effect.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A gold nanoparticle, characterized in that, The gold nanoparticles include gold nanospheres, 4-mercaptobenzoic acid ligands, and methoxy-capped polyethylene glycol-amino groups; the gold nanospheres, 4-mercaptobenzoic acid ligands, and methoxy-capped polyethylene glycol-amino groups are sequentially connected by chemical bonds; 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. The gold nanoparticle according to claim 1, characterized in that, The gold nanoparticles further include a hydrophobic drug; the hydrophobic drug is connected to the benzene ring on the 4-mercaptobenzoic acid ligand by physical adsorption.

3. The gold nanoparticle according to claim 2, characterized in that, The hydrophobic drug is an aromatic drug or an anti-tumor drug.

4. The application of the gold nanoparticle according to any one of claims 1 - 3 in any of the following: (1) Application in the preparation of a drug for treating or preventing acute kidney injury; (2) Application in the preparation of a drug for treating or preventing tumors.

5. A preparation method of the gold nanoparticle according to claim 1, characterized in that, It includes the following steps: Step 1: Mix a chloroauric acid solution with a 4-mercaptobenzoic acid solution. After white flocculates appear in the mixed solution, add a NaOH solution and a sodium borohydride solution. After the reaction, obtain reaction system solution 1; Step 2: Centrifuge reaction system solution 1 to remove the precipitate, and purify the supernatant to obtain a gold nanoparticle system with 4-mercaptobenzoic acid ligands; Step 3: Activate the gold nanoparticle system with 4-mercaptobenzoic acid ligands obtained in Step 2, and add methoxy-capped polyethylene glycol-amino groups to the activated gold nanoparticle system with 4-mercaptobenzoic acid ligands. After the reaction, obtain reaction system solution 2; Step 4: Centrifuge reaction system solution 2 to remove the precipitate, and purify the supernatant to obtain methoxy polyethylene glycol-modified 4-mercaptobenzoic acid ligand gold nanoparticles, which are the gold nanoparticles described above.

6. The preparation method according to claim 5, characterized in that, The pH value of reaction system solution 1 in Step 1 is 8-10.

7. The preparation method according to claim 5, characterized in that, The molar ratio of chloroauric acid to 4-mercaptobenzoic acid in reaction system solution 1 in Step 1 is 1:(2-3).

8. The preparation method according to claim 5, characterized in that, The molar ratio of the gold nanoparticles with 4-mercaptobenzoic acid ligands to methoxy-capped polyethylene glycol-amino groups in reaction system solution 2 in Step 3 is 1:(1-5); or / and, the molecular weight of the methoxy-capped polyethylene glycol-amino groups is 750-1500.

9. The preparation method according to claim 5, characterized in that, The activation method in Step 3 is: use 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.

10. The preparation method according to claim 9, characterized in that, 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).

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