Glutathione-responsive gold nanoparticle probe and preparation method and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a glutathione-responsive gold nanoparticle probe and a preparation method and application thereof. The preparation method comprises the following steps: stirring and mixing GSH aqueous solution and HAuCl4 aqueous solution, then adding TBAB solution, water-bathing, cooling the reaction system to room temperature, adding NaCl solution and anhydrous ethanol, centrifuging, collecting Au 25 SG 18 precipitate; dissolving the obtained Au 25 SG 18 and 800CW NHS Ester in PBS, vortexing, centrifuging, collecting the supernatant, and preparing the 800CW4-GS 18 -Au 25 gold nanoparticle probe. The preparation method of the gold nanoparticle probe is simple, has good response capacity to glutathione, has an ultrasmall (<6nm) core size, and can realize non-invasive detection of early acute liver injury and / or acute kidney injury through urine detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a glutathione-responsive gold nanoparticle probe, its preparation method, and its application. Background Technology

[0002] Glutathione (GSH) in the liver is crucial for maintaining the liver's redox environment and is a major antioxidant. Decreased liver GSH levels can easily lead to hepatic oxidative stress, accompanied by inflammatory responses, and are a root cause of many liver diseases, such as drug-induced liver injury, alcoholic / non-alcoholic fatty liver disease, steatohepatitis, liver fibrosis, and cirrhosis. However, currently, there is a lack of non-invasive in vivo methods for detecting GSH in clinical practice. Measurements of liver GSH concentration usually require surgery or liver biopsy, which introduces a greater risk burden to the organism. Furthermore, liver GSH depletion often occurs before hepatocyte death and elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST), meaning that clinically measured liver function biomarkers cannot effectively reflect the early stages of acute liver injury.

[0003] Acute kidney injury (AKI) is characterized by high morbidity (sepsis, surgical ischemia-reperfusion, and drug-induced AKI) and high mortality, making it a significant global health problem that causes 1.7 million deaths annually. Therefore, early detection of kidney injury facilitates timely kidney protection and prevents AKI from progressing to more serious diseases. Currently, serum creatinine and blood urea nitrogen (BUN) are used in clinical diagnosis to evaluate kidney function. However, as indicators of kidney function, serum creatinine and BUN only show significant increases when the glomerular filtration rate (GFR) decreases by more than 50%. Furthermore, using serum creatinine and BUN as standards for kidney function testing lacks representativeness, as cardiovascular diseases, intestinal diseases, and even high-protein diets can cause elevated levels. Therefore, it is difficult to detect AKI in its early stages when the GFR is declining. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a glutathione-responsive gold nanoparticle probe that can be cleared by the kidneys, its preparation method and application, so as to realize the early detection and diagnosis of acute liver injury and / or acute kidney injury in organisms, facilitate early intervention and treatment, and avoid the risks of inflammation, disease and tissue damage.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a method for preparing a glutathione-responsive gold nanoparticle probe, comprising the following steps: (1) Au 25 SG 18Preparation: Glutathione aqueous solution and HAuCl4 aqueous solution were stirred and mixed, then TBAB solution was added. The mixture was placed in a water bath, and after the reaction system cooled to room temperature, NaCl solution and anhydrous ethanol were added. After centrifugation, Au was collected. 25 SG 18 precipitation; (2) 800CW4-GS 18 -Au 25 Preparation: Au obtained in step (1) 25 SG 18 The 800CW NHS Ester was mixed with PBS buffer, vortexed, centrifuged, and the supernatant was collected to obtain the 800CW4-GS. 18 -Au 25 Gold nanoparticle probe.

[0006] GSH, acting as a ligand and reducing agent, reacts with HAuCl4, leading to the reduction of gold salt to gold atoms, which then form Au-S complexes with glutathione, i.e., gold nanoclusters. TBAB, as a surfactant, can form a charged ionic layer on the surface of the gold nanoclusters, which helps to stabilize and regulate the size and shape of the gold nanoclusters. In addition, tetrabutylammonium ions can interact with the negative charge on the surface of the gold nanoclusters, neutralizing the charge on the surface of the gold nanoclusters and improving their stability.

[0007] Furthermore, this invention utilizes gold nanoclusters Au 25 SG 18 A glutathione-responsive ultrasmall gold nanoparticle probe, 800CW4-GS, was prepared by linking it with the near-infrared fluorescent dye 800CW NHS Ester via Au-S bonds. 18 -Au 25 When the probe comes into contact with external GSH, a reaction occurs, causing the external GSH to replace the ligands of the 800CW-GSH on the probe. This results in the newly generated 800CW-GSH detaching from the nanoparticle surface, causing optical changes in the nanoprobe. Detection can be achieved using fluorescence spectrophotometry, ultraviolet spectrophotometry, and high-performance liquid chromatography. Furthermore, the 800CW NHS Ester exhibits strong resistance to serum proteins. 25 SG 18 When combined with 800CW NHS Ester, a gold nanoparticle probe 800CW4-GS was prepared. 18 -Au 25 It exhibits significant protein resistance, thereby regulating the biotransformation rate of gold nanoparticles in vivo by modulating their protein binding capacity, thus affecting their in vivo transport behavior.

[0008] Preferably, in step (1), the molar ratio of GSH to HAuCl4 is GSH:HAuCl4 = 4.96:1; and the molar ratio of TBAB to HAuCl4 is TBAB:HAuCl4 = 16:1.

[0009] The molar ratio of GSH to HAuCl4 can lead to different surface energies and solvation environments, thus affecting the morphology and size distribution of gold nanoclusters. Furthermore, GSH acts as both a reducing agent and a surface modifier in gold nanoclusters, and different molar ratios with HAuCl4 can affect the reducing power of GSH, thereby influencing the stability of the gold nanoclusters. Through experimental investigation, the inventors found that when the molar ratio of GSH to HAuCl4 is GSH:HAuCl4 = 4.96:1 and the molar ratio of TBAB to HAuCl4 is TBAB:HAuCl4 = 16:1, it is more effective than other fluorescent probes used for imaging (such as ICG-AuCl4). 25 Compared to other methods that strongly bind to serum proteins and have a hydrated particle size of approximately 8 nm, the nanoprobe described in this invention has an ultra-small (<6 nm) hydrated particle size. Dynamic light scattering tests show its hydrated particle size to be 2.59 ± 0.63 nm, and transmission electron microscopy shows its core size to be 1.28 ± 0.76 nm. It exhibits high purity and binds very little to serum proteins. After intravenous injection, it is easily transported to the kidneys and cleared in the urine, and is not easily captured by the mononuclear phagocytic system. This lays the foundation for subsequent urine detection methods, allowing for convenient and non-invasive detection of liver glutathione levels through simple urine analysis.

[0010] Preferably, in step (1), the water bath time is 15-17 hours and the water bath temperature is 35-40°C.

[0011] Preferably, the Au 25 SG 18 The mass ratio of Au to 800CW NHS Ester is: 25 SG 18 :800CW NHS Ester=1: (1-100).

[0012] 800CW NHS Ester is a near-infrared fluorescent dye. By attaching it to the surface of gold nanoclusters, fluorescent properties can be imparted to the gold nanoclusters. However, the amount of 800CW NHS Ester added needs to be carefully controlled to ensure excellent optical performance while minimizing adverse effects on the structure and properties of the gold nanoclusters. Experimental investigations have shown that when Au... 25 SG 18 The mass ratio of Au to 800CW NHSEster is: 25 SG 18When the NHS Ester ratio is 1: (1-100) = 800CW, a better optical effect can be achieved without significantly affecting the structure and properties of the gold nanoclusters.

[0013] Preferably, in step (2), the vortexing time is 2.5-3.5h; centrifugation is carried out using an ultracentrifuge tube with a molecular weight cutoff of 30kDa.

[0014] Secondly, the present invention provides a glutathione-responsive gold nanoparticle probe prepared by the above preparation method.

[0015] Thirdly, the present invention also provides the application of the above-mentioned glutathione-responsive gold nanoparticle probe in a formulation for detecting acute liver injury.

[0016] The gold nanoparticle probe 800CW4-GS prepared in this invention 18 -Au 25 The nanoparticles exhibit excellent responsiveness to glutathione, allowing for the analysis of their biotransformed metabolic components in urine using high-performance liquid chromatography (HPLC). This analysis determines the degree of biotransformation of the nanoparticles, reflecting the liver's biotransformation capacity and estimating glutathione levels in the liver. This enables early detection of liver diseases. When liver glutathione levels decrease, blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels may not yet show a significant increase, but liver function is already impaired. HPLC analysis of 800CW4-GS in urine... 18 -Au 25 The biotransformation index can detect the occurrence of liver damage at an early stage.

[0017] Preferably, the method for detecting acute liver injury using the glutathione-responsive gold nanoparticle probe includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 18 -Au 25 The 800CW4-GS was administered intravenously to an organism, and urine was collected within 30 minutes. Metabolic components in the urine were analyzed by HPLC, and the quantification was based on changes in the peak area (AUC) of each metabolic component in the HPLC results. 18 -Au 25 The biotransformation rate of liver injury in organisms under normal conditions is used to differentiate between acute liver injury and early detection; the 800CW4-GS 18 -Au 25 The biotransformation rate is lower in organisms with liver damage than in organisms with normal liver function.

[0018] Fourthly, the present invention also provides the application of the above-mentioned glutathione-responsive gold nanoparticle probe in a formulation for detecting acute kidney injury.

[0019] As the site of urine metabolism, the kidneys, through renal clearance of renal probes, can provide feedback on glomerular filtration rate. The fluorescence and metal element signals of the nanoprobes prepared in this invention can quantitatively reflect the renal metabolic capacity in vivo. Furthermore, in the early stages of kidney injury, serum creatinine and blood urea nitrogen do not show significant increases, but at this time, the clearance rate of the renal probes has already decreased, blood circulation time is prolonged, and the degree of biotransformation in the liver is more complete. Therefore, high-performance liquid chromatography (HPLC) can be used to analyze 800CW4-GS in urine. 18 -Au 25 The biotransformation rate can be used to detect the early occurrence of kidney damage.

[0020] Preferably, the method for detecting acute kidney injury using the glutathione-responsive gold nanoparticle probe includes the following steps: 800CW4-GS 18 -Au 25 The 800CW4-GS was administered intravenously to an organism, and urine was collected within 30 minutes. Metabolic components in the urine were analyzed by HPLC, and the quantification was based on changes in the peak area (AUC) of each metabolic component in the HPLC results. 18 -Au 25 The biotransformation rate under kidney injury and normal conditions in organisms is used to differentiate between acute liver injury and early detection.

[0021] Fifthly, the present invention also provides a kit for detecting acute liver injury and / or acute kidney injury, comprising the above-mentioned glutathione-responsive gold nanoparticle probe.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for preparing glutathione-responsive gold nanoparticle probes described in this invention is simple, mild, and has good biosafety. It also has good responsiveness to glutathione and has an ultra-small (<6nm) core size. It does not bind to serum proteins. After intravenous injection, it is easily transported to the kidneys and cleared with urine. It is not easily captured by the mononuclear phagocytic system, which lays the foundation for subsequent urine detection methods.

[0023] (2) The gold nanoparticle probe 800CW4-GS prepared in this invention 18 -Au 25 After intravenous injection, the GSH content in the liver can be non-invasively detected through urine testing, enabling timely monitoring of the body's antioxidant capacity, facilitating early intervention and treatment, and avoiding the risks of inflammation, disease, and tissue damage.

[0024] (3) The present invention uses a urine sample to detect gold nanoparticle probe 800CW4-GS in living organisms. 18 -Au 25Biotransformation rate can enable early detection of kidney damage in organisms, and this detection method precedes changes in clinical blood test indicators such as Scr and BUN. Attached Figure Description

[0025] Figure 1 For 800CW4-GS 18 -Au 25 Au 25 SG 18 The UV spectrum of 800CW (Figure A) and the retention time results in high performance liquid chromatography (Figure B). Figure 2 For 800CW4-GS 18 -Au 25 UV spectrum after reaction with glutathione (Figure A) and fluorescence intensity dependence results after reaction with different concentrations of glutathione (Figure B). Figure 3 They are respectively 800CW4-GS 18 -Au 25 The results of the core size (Figure A), hydrated particle size (Figure B), and ultraviolet spectrum (Figure C); Figure 4 800CW4-GS prepared for comparative examples 1-6 18 -Au 25 The ultraviolet spectra are shown, where the AF diagrams correspond to scales 1-6 respectively; Figure 5 ICG4-GS prepared for Comparative Example 7 18 -Au 25 Hydrated particle size distribution; Figure 6 Au in the blood after biotransformation 25 The graph shows the amount of residue (Figure A) and the tumor targeting efficiency (Figure B). Figure 7 Figure 1 shows the results of the following: glutathione content in normal mouse liver and liver treated with diethyl maleate (Figure A); determination of nanoparticle composition in mouse urine under various conditions by high performance liquid chromatography (Figure B); and changes in biotransformation rate in mice after diethyl maleate treatment (Figure C). Figure 8 The figure shows the changes in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice after induction with acetaminophen at doses of 200 mg / kg, 300 mg / kg, and 400 mg / kg. Figure 9Figure A shows the changes in liver glutathione in mice after induction with different doses of acetaminophen, the content of each component in urine at each dose according to high performance liquid chromatography (Figure B), and the linear relationship between bioconversion rate and liver glutathione at each induction dose (Figure C). Figure 10 Figure A shows the changes in serum creatinine and blood urea nitrogen in mice after induction with 10 mg / kg cisplatin, and the changes in glomerular filtration rate (Figure B). Figure 11 Figure A shows the retention time of each component in the urine of mice with kidney injury after induction with 10 mg / kg cisplatin, and Figure B shows the biotransformation rate of the mice with kidney injury. Detailed Implementation

[0026] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] All raw materials used in this invention were purchased from companies with legal production qualifications, including 800CW4-GS. 18 -Au 25 The 800CW NHS Ester used in the preparation was purchased from Beijing Yusheng Chemical Co., Ltd., and GSH and HAuCl4 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. In the in vivo experimental study, diethyl maleate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and acetaminophen and cisplatin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The total glutathione kit for testing the GSH content in animal tissues was purchased from Beyotime Biotechnology Co., Ltd., and the kits for testing serum alanine aminotransferase, aspartate aminotransferase, serum creatinine, and blood urea nitrogen were purchased from Nanjing Jiancheng Bioengineering Institute.

[0028] Example 1 This embodiment provides a method for preparing a glutathione-responsive gold nanoparticle probe. The specific steps of this method are as follows: (1) Au 25 SG 18 Preparation: 4 mL of GSH (31 mM) aqueous solution and 25 μL of HAuCl4 (1 M) aqueous solution were mixed and stirred for 5 min, then 2 mL of TBAB (0.2 M) aqueous solution was added, and the mixture was reacted in a water bath at 37 °C for 16 h. After the reaction system cooled to room temperature, 500 μL of saturated NaCl solution and 5 mL of anhydrous ethanol were added, and the precipitate was collected after centrifugation at 4200 rpm. (2) 800CW4-GS 18 -Au25 Preparation: 1 eq of Au 25 SG 18 The glutathione-responsive gold nanoparticle probe 800CW4-GS was mixed with 1 eq of 800CW NHS Ester and dissolved in 2 ml of pH 7.8 PBS. The mixture was vortexed for 3 h, and the reaction solution was centrifuged three times using a 30 kDa ultracentrifuge tube. The supernatant was collected to obtain the glutathione-responsive gold nanoparticle probe 800CW4-GS. 18 -Au 25 .

[0029] The ultrasmall gold nanoparticle probe 800CW4-GS synthesized using this method 18 -Au 25 The ultraviolet absorption spectrum is as follows Figure 1 As shown, 800CW successfully modified the surface of gold nanoparticles, promoting their H-type aggregation and resulting in a 74 nm blue shift. The retention time of the nanoparticles was determined by high-performance liquid chromatography (dextran gel chromatography column), indicating that the increase in relative molecular mass due to the successful modification of the gold nanoparticles with 800CW shortened the retention time from 9.53 min to 9.28 min.

[0030] 800CW4-GS synthesized using this method 18 -Au 25 After reacting with exogenous glutathione, such as Figure 2 As shown, the ultraviolet absorption spectrum shows that the 800CW ligand detaches from the nanoparticle surface due to the reaction between exogenous glutathione and the Au-S bond, resulting in the depolymerization of H-type aggregation and a 74nm red shift in the ultraviolet absorption of the nanoparticles. Simultaneously, the quenched fluorescence increases in a concentration-dependent manner with respect to GSH, which is also a characteristic of H-type aggregation. This concentration dependence provides a basis for subsequent urine analysis.

[0031] The ultrasmall gold nanoparticle probe 800CW4-GS synthesized using this method 18 -Au 25 Particle size such as Figure 3 As shown in (A, B), its core size was measured to be 1.28±0.76 nm by transmission electron microscopy and its hydrated particle size was measured to be 2.59±0.63 nm by dynamic light scattering, proving that it has an ultra-small (<6 nm) core size that can be effectively transported to the kidneys.

[0032] Ultrasmall gold nanoclusters Au synthesized using this method 25 SG 18 The ultraviolet absorption spectrum is as follows Figure 3 As shown in (C), Au 25 SG 18 There is a characteristic absorption peak at 675 nm. The presence or absence of this absorption peak can prove Au 25SG 18 Whether the synthesis was successful is generally considered to be determined when the ratio of its absorbance at 800 nm to its absorbance at 600 nm is close to 1.0, indicating the availability of Au. 25 SG 18 The purity is optimal, such as Figure 3 As shown in (C), the ultrasmall gold nanoclusters Au prepared using the above-mentioned raw material ratios are shown. 25 SG 18 It has the best purity.

[0033] Example 2 This embodiment provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this embodiment and Embodiment 1 is that: In step (2), Au of 1eq is... 25 SG 18 Mixed with 100eq of 800CW NHS Ester.

[0034] 800CW4-GS synthesized using this method 18 -Au 25 The UV absorption spectrum of the 800CW4-GS in Example 1 is similar to that of the 800CW4-GS in Example 1. 18 -Au 25 Essentially the same, 800CW was successfully modified on the surface of gold nanoparticles, promoting their H-type aggregation and causing a blue shift of 74 nm.

[0035] Comparative Example 1 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of GSH (31mM) aqueous solution used is 3ml, and the amount of TBAB (0.2M) aqueous solution used is 1.5mL.

[0036] Au was prepared by reacting with characteristic ultraviolet absorption peaks. 25 SG 18 The purity, the results are as follows Figure 4 As shown in (A), the reaction at this raw material ratio did not yield Au. 25 SG 18 The characteristic absorption peak at 675 nm is considered to indicate the absence of target product formation.

[0037] Comparative Example 2 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of GSH (31mM) aqueous solution used is 4.5ml and the amount of TBAB (0.2M) aqueous solution used is 2.5mL.

[0038] Au was prepared by reacting with characteristic ultraviolet absorption peaks. 25 SG 18 The purity, the results are as follows Figure 4 As shown in (B), the Au obtained 25 SG 18 Its UV absorbance at 800 nm to that at 600 nm is 0.4, reflecting that Au at this raw material dosage ratio... 25 SG 18 The purity is far lower than that of the Au prepared in Example 1. 25 SG 18 .

[0039] Comparative Example 3 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of GSH (31mM) aqueous solution used is 2.5ml.

[0040] The purity of the prepared sample is reflected by the characteristic peaks of ultraviolet absorption, and the results are as follows: Figure 4 As shown in (C), no Au was obtained from the reaction at this feed ratio. 25 SG 18 The characteristic absorption peak at 675 nm is considered to indicate the absence of target product formation.

[0041] Comparative Example 4 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of GSH (31mM) aqueous solution used is 5ml.

[0042] The purity of the prepared sample is reflected by the characteristic peaks of ultraviolet absorption, and the results are as follows: Figure 4 As shown in (D), the prepared Au 25 SG 18 Its UV absorbance ratio at 800 nm to that at 600 nm is 0.4, reflecting that Au at this ratio... 25 SG 18 The purity is far lower than that of the Au prepared in Example 1. 25 SG 18 .

[0043] Comparative Example 5 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of TBAB (0.2M) aqueous solution used is 1 ml.

[0044] The purity of the prepared sample is reflected by the characteristic peaks of ultraviolet absorption, and the results are as follows: Figure 4 As shown in (E), no Au was obtained from the reaction at this feed ratio. 25 SG 18 The characteristic absorption peak at 675 nm is considered to indicate the absence of target product formation.

[0045] Comparative Example 6 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (1), the amount of TBAB (0.2M) aqueous solution used is 3 ml.

[0046] The purity of the prepared sample is reflected by the characteristic peaks of ultraviolet absorption, and the results are as follows: Figure 4 As shown in (F), the Au obtained 25 SG 18 Its UV absorbance ratio at 800 nm to that at 600 nm is 0.6, reflecting that Au at this raw material dosage ratio... 25 SG 18 The purity is far lower than that of the Au prepared in Example 1. 25 SG 18 .

[0047] Comparative Example 7 This comparative example provides a method for preparing a glutathione-responsive gold nanoparticle probe. The difference between this comparative example and Example 1 is that: In step (2), indocyanine green (ICG) was used to replace 800CW NHS Ester to prepare ICG4-GS. 18 -Au 25 .

[0048] Because ICG4-GS 18 -Au 25 It has a strong binding capacity to serum proteins. In a physiological environment, it rapidly binds to proteins in the blood of organisms. Therefore, this comparative example simulates the physiological environment in vitro, namely ICG4-GS. 18 -Au 25 Bovine serum albumin (BSA) at a molar ratio of 1:1 was incubated at 37°C for 30 min, and the hydrated particle size was measured to be 7.89 ± 2.22 nm. Figure 5 As shown, this size is greater than the glomerular filtration threshold (6nm).

[0049] Experimental Example 1 This experimental example uses the 800CW4-GS prepared in Example 1. 18 -Au 25And ICG4-GS prepared in Comparative Example 7 18 -Au 25 Using samples, we investigated the levels of Au in the blood after the two substances underwent liver biotransformation. 25 The retention rate of Au in the blood is analyzed to determine its biotransformation rate within the organism. Specifically, pharmacokinetic parameters are used to define the concentration of Au in the blood. 25 The retention rate was determined through the following experimental procedure: Three groups of 6-week-old female mice were selected and injected intravenously with 800 CW4-GS. 18 -Au 25 ICG4-GS 18 -Au 25 and Au 25 SG 18 Three types of nanoparticles were used. Fresh blood was collected from the orbital venous plexus of mice at 2 min, 5 min, 10 min, 0.5 h, 1 h, 3 h, 5 h, 8 h, 12 h, and 24 h after nanoparticle injection and weighed. The blood at each time point was fully digested with freshly prepared aqua regia, and the gold content in the blood was determined using ICP-MS. Finally, Oringin 2021 software was used to plot the correlation curve between blood Au content and time, and the area under each curve was calculated using the same software. This value is the AUC of the drug, reflecting the speed and extent to which the drug enters the bloodstream from the administration site, thus quantifying the Au content in the blood. 25 The amount of residue.

[0050] Due to ICG4-GS 18 -Au 25 It can bind tightly to serum proteins, and after initiating specific liver targeting, it will undergo rapid hepatic glutathione-mediated biotransformation, while 800CW4-GS 18 -Au 25 Due to its strong protein-binding resistance, it exhibits a gradual biotransformation characteristic; therefore, the results are as follows: Figure 6 As shown in (A), due to the 800CW4-GS 18 -Au 25 The rate of biotransformation in the liver slows down, according to the levels of Au in the blood. 25 The retention situation was found to be related to ICG4-GS 18 -Au 25 Compared to the 800CW4-GS 18 -Au 25 Au nanoparticles after biotransformation 25 The retention rate increased by 2.4 times (229.10±23.77h*%ID / g vs 96.70±7.6h*%ID / g), while the retention rate was lower than that of free Au. 25 SG 18Compared to the previous year, it increased by nearly 5 times (49.30±3.56h*%ID / g); similarly, Figure 6 (B) Results showed that significantly enhanced retention in the blood also led to 800 CW4-GS 18 -Au 25 The tumor targeting efficiency was significantly improved (9.7±0.7% ID / g), which is approximately the same as that of ICG4-GS. 18 -Au 25 2 times (5.4±0.9%ID / g) and free Au 25 SG 18 The concentration was 3 times higher (3.4 ± 0.3% ID / g), indicating that the present invention achieved this by adjusting the prepared 800CW4-GS. 18 -Au 25 The protein-binding affinity of nanoparticles modulates their biotransformation rate in vivo, ultimately affecting their transport behavior. Furthermore, this influences their clearance from ICG4-GS via the hepatobiliary pathway. 18 -Au 25 The situation is different for the ICG-GS separated from it; the 800CW4-GS described in this invention is different. 18 -Au 25 The 800CW-GS isolated through biotransformation can be directly eliminated through urine, thus enabling non-invasive detection of GSH levels in the liver via urine testing.

[0051] Experimental Example 2 This experimental example investigated the 800CW4-GS prepared in Example 1. 18 -Au 25 The bioconversion rate in normal mice and mice with depleted liver glutathione was investigated using the following methods.

[0052] Diethyl maleate (DEM) was used to deplete the glutathione content in the liver of experimental mice, and then 800 CW4-GS was injected into the tail vein of the mice. 18 -Au 25 Urine samples were collected from mice within 30 minutes, and the metabolic components in the urine were analyzed by high-performance liquid chromatography (HPLC). The results were compared with urine samples from normal mice injected via the tail vein with 800CW4-GS18-Au25. The 800CW4-GS18-Au25 was quantified based on changes in the peak area (AUC) of each metabolic component in the HPLC analysis. 18 -Au 25 Bioconversion rate in normal mice and under conditions of liver glutathione depletion.

[0053] Figure 7 (A) shows the glutathione content in the livers of normal mice and mice treated with diethyl maleate. Figure 7(B) This describes the use of high-performance liquid chromatography (HPLC) to detect changes in the composition of nanoparticles in mouse urine under various conditions. Figure 7 (C) is 800CW4-GS 18 -Au 25 Bioconversion rate in normal mice and mice with depleted liver glutathione; due to the significant difference in liver glutathione content between normal mice and mice with depleted liver glutathione, 800CW4-GS 18 -Au 25 The components produced after metabolism in vivo differ, causing variations in the peak areas of each metabolic component in HPLC, thus allowing for the estimation and determination of 800CW4-GS. 18 -Au 25 Bioconversion rate in vivo; results showed that, compared with mice with normal livers, mice with depleted liver glutathione had significantly reduced levels of GSH in the liver, resulting in 800 CW4-GS 18 -Au 25 Its biotransformation rate within the body is significantly reduced.

[0054] Experimental Example 3 This experiment investigated the glutathione content and biotransformation rate in the livers of mice with different degrees of acute liver injury, as detailed below.

[0055] Acute liver injury of varying degrees was induced in experimental mice (ICR mice) using acetaminophen at concentrations of 200 mg / kg, 300 mg / kg, and 400 mg / kg, respectively. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma of the mice after drug induction were then measured, and the content of glutathione in the liver of the mice was investigated. Additionally, the mice were injected via tail vein with 800 CW4-GS prepared in Example 1. 18 -Au 25 Urine samples were collected from mice within 30 minutes. The linear relationship between the content of each component in the urine at each dose and the biotransformation rate at each induction dose and liver glutathione was investigated using high performance liquid chromatography.

[0056] Figure 8 This study showed the changes in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice after induction with acetaminophen at doses of 200 mg / kg, 300 mg / kg, and 400 mg / kg. The results indicated that liver function indicators in the mouse model did not show significant changes after drug induction compared to before induction, suggesting that clinically measured liver function biomarkers (ALT and AST) cannot effectively reflect the early stage of acute liver injury. Meanwhile, the glutathione level in the mouse liver had already decreased at this time, and this level gradually decreased with increasing drug induction dose. Figure 9As shown in (A). Furthermore, this invention, through high-performance liquid chromatography (HPLC), can distinguish organisms with acute liver injury caused by decreased glutathione levels in the liver, such as... Figure 9 As shown in (B), this method can visually present the differences in biotransformation rates in organisms in the form of "fingerprint maps" and quantify the biotransformation rates of mice with different degrees of damage.

[0057] In addition, such as Figure 9 As shown in (C), when the glutathione content in the liver of an organism is in the range of 0.9-6.0 μmol / g, the 800CW4-GS nanoparticles... 18 -Au 25 The conversion efficiency in vivo is linearly correlated with the liver glutathione content; that is, the lower the glutathione content, the lower the conversion efficiency of the nanoparticles. Therefore, the conversion efficiency can be determined by measuring the amount of 800 CW4-GS cleared from the body. 18 -Au 25 The conversion rate is used to assess the glutathione content in the liver. If the bioconversion efficiency of an organism is found to be 60%, the glutathione content in the liver can be calculated to have decreased from the normal value to about 2 μmol / g. This can be used to assess the antioxidant capacity of the organism and thus achieve early diagnosis of related diseases.

[0058] Test Example 4 This experiment investigated the biotransformation rate in mice with acute kidney injury. The specific methods are as follows.

[0059] Acute kidney injury was induced in experimental mice (ICR mice) using cisplatin at a dose of 10 mg / kg. Serum creatinine and blood urea nitrogen concentrations in the mice were then investigated. Additionally, the mice were injected via tail vein with 800CW4-GS prepared in Example 1. 18 -Au 25 Urine was collected from mice within 30 minutes, and the retention time and biotransformation rate of each component in the urine of mice with kidney injury were determined by high performance liquid chromatography.

[0060] like Figure 10 As shown, after cisplatin induction, serum creatinine in mice with kidney injury remained almost unchanged, while blood urea nitrogen increased significantly. However, the glomerular filtration rate (GFR) of these mice had already decreased by 44.7%. Since clinically used serum creatinine measurements often only show a significant increase after a 50% decrease in GFR, it is difficult to assess kidney function in the early stages of kidney injury. When the glomerular filtration rate decreases, the clearance rate of exogenous drugs injected into the body decreases, and the drug circulation time in the body increases, thus reducing the effectiveness of 800 CW4-GS. 18 -Au 25After injection, the increased blood circulation time and the greater likelihood of biotransformation in the liver lead to a higher biotransformation rate of nanoparticles cleared into the urine. Figure 11 As shown, when the glomerular filtration rate decreased by less than 50%, the high-performance liquid chromatography (HPLC) fingerprinting method was able to clearly distinguish between mice with acute kidney injury and normal mice. Meanwhile, the 800CW4-GS nanoparticles... 18 -Au 25 The bioconversion rate increased from 77.4% to 94.8%, enabling early diagnosis of acute kidney injury before the glomerular filtration rate decreased by less than 50%.

[0061] When mice were injected via tail vein with 800CW4-GS prepared from comparative examples 1-6, 18 -Au 25 When testing its bioconversion rate in vivo, the low purity and relatively large size of its nanoparticles make them easily detectable by the immune system, triggering an immune response and causing them to be captured by the mononuclear phagocytic system. This makes them difficult to transport to the kidneys for clearance in urine. In addition, large nanoparticles may deposit in liver tissue after injection, causing mechanical damage and triggering an inflammatory response, affecting the normal structure and function of the liver, resulting in a large error in the final test results.

[0062] 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a glutathione-responsive gold nanoparticle probe, characterized in that, Includes the following steps: (1) Au 25 SG 18 Preparation: Glutathione aqueous solution and HAuCl4 aqueous solution were stirred and mixed, then TBAB solution was added. The mixture was placed in a water bath, and after the reaction system cooled to room temperature, NaCl solution and anhydrous ethanol were added. After centrifugation, Au was collected. 25 SG 18 precipitation; (2) 800CW4-GS 18 -Au 25 Preparation: Au obtained in step (1) 25 SG 18 The 800CW NHS Ester was mixed with PBS buffer, vortexed, centrifuged, and the supernatant was collected to obtain the 800CW4-GS. 18 -Au 25 Gold nanoparticle probe; In step (1), the molar ratio of glutathione to HAuCl4 is glutathione:HAuCl4 = 4.96:1; the molar ratio of TBAB to HAuCl4 is TBAB:HAuCl4 = 16:

1. In step (2), Au 25 SG 18 The mass ratio of Au to 800CW NHS Ester is: 25 SG 18 :800CW NHS Ester =1: (1-100).

2. The method for preparing the gold nanoparticle probe according to claim 1, characterized in that, In step (1), the water bath time is 15-17 hours and the water bath temperature is 35-40℃.

3. The method for preparing the gold nanoparticle probe according to claim 1, characterized in that, In step (2), the vortexing time is 2.5-3.5h; centrifugation is carried out using ultracentrifuge tubes with a molecular weight cutoff of 30kDa.

4. A glutathione-responsive gold nanoparticle probe, characterized in that, It is prepared by the preparation method described in any one of claims 1-3.

5. The use of the glutathione-responsive gold nanoparticle probe as described in claim 4 in the preparation of a formulation for detecting acute kidney injury.

6. A kit for detecting acute kidney injury, characterized in that, It includes the glutathione-responsive gold nanoparticle probe of claim 4.

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