Preparation method of in-situ growth type gold nano assembly based on micelle template and cell antioxidation application of in-situ growth type gold nano assembly

Abstract

The invention belongs to the field of biomedical nano materials, and discloses an ultra-small-size gold nano assembly (AuNAs) based on a micelle confinement in-situ growth strategy, a one-step preparation method and application. According to the assembly, an amphiphilic block copolymer pluronic F127 and n-dodecanethiol serve as a composite micelle template, reduction, nucleation and ordered assembly of a gold precursor are synchronously completed in a confinement space, the hydration particle size is 27.37 + / -4.95 nm, and the assembly is uniform in structure and excellent in stability. In-vitro experiments show that the assembly has efficient scavenging capacity on ABTS < + >, DPPH and OH (the scavenging rates are respectively 98.37%, 51.9% and 93.9% under the concentration of 10.0 nM), has no obvious toxicity on HK-2 cells within the concentration range of 0-20 nM, and can effectively remove excessive reactive oxygen species (ROS) in the cells. The preparation process is simple and convenient, the cost is controllable, the prepared AuNAs can be used for preparing ROS removal type cell protective agents or candidate drugs for treating oxidative stress related diseases, and the AuNAs have good industrial application prospects and clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials, specifically relating to an ultrasmall gold nanoassembly with highly efficient reactive oxygen species (ROS) scavenging ability, its one-step preparation method, and its applications. In particular, this invention provides a small-sized, structurally uniform gold nanoassembly synthesized based on a micelle-confined in-situ growth strategy. In in vitro experiments, it exhibits highly efficient scavenging activity against 2,2′-adiazono-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt (ABTS), 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radicals, and hydroxyl radicals (•OH). It also demonstrates significant intracellular ROS scavenging ability in cell models, and can be used to prepare ROS-scavenging cytoprotective agents or candidate drugs for treating oxidative stress-related diseases. Background Technology

[0002] The abnormal accumulation of reactive oxygen species (ROS) in cells is a major factor in triggering oxidative stress and related diseases. Developing efficient and biocompatible ROS scavenging materials has become a key research direction in the field of nanomedicine. Among numerous candidate materials, ultrasmall gold nanoparticles (AuNPs, d<3nm) exhibit excellent biocompatibility, easy surface functionalization, and high oxidative activity, making them uniquely advantageous in the field of antioxidants.

[0003] Self-assembly strategies based on precise surface engineering regulation are key to effectively improving the ROS scavenging performance of inorganic nanomaterials. On the one hand, constructing ordered small-sized assemblies can effectively increase the specific surface area of ​​the material, fully exposing catalytic active sites; on the other hand, surface ligand modification can regulate the surface charge, hydrophilicity / hydrophobicity, and specific recognition ability of the material, thereby enhancing its stability in physiological environments and its interaction efficiency with ROS molecules. This dual-regulation "structure-surface" self-assembly strategy provides an important direction for developing next-generation high-efficiency ROS scavengers. However, traditional synthetic methods (such as multi-step assembly methods) face significant challenges in constructing ultra-small AuNP assemblies (AuNAs): the assembly process is difficult to control precisely, resulting in uneven product size distribution, disordered structure, and generally excessively large size. These structural defects directly limit the scavenging performance of the material—excessively large size leads to the embedding of active sites, significantly reducing the specific surface area; while structural inhomogeneity causes batch-to-batch performance differences, seriously affecting the reproducibility and practical application potential of the material.

[0004] Amphiphilic block copolymers (ABCs) possess both hydrophilic and lipophilic structures, and their in-situ self-assembly strategy has been used to achieve the ordered self-assembly of metal nanoparticles, thereby enhancing the stability, optical properties, and biological interactions of individual nanomaterials. However, whether this in-situ self-assembly strategy can effectively enhance the oxidizing properties of gold nanomaterials and achieve efficient ROS scavenging remains to be seen. Summary of the Invention

[0005] To address the aforementioned issues, this study utilizes an in-situ self-assembly strategy to achieve the controllable preparation of small-sized, monodisperse gold nanoparticle assemblies (AuNAs) confined by micellar templates. This strategy employs a hydrophobic thiol ligand and a triblock copolymer to construct a uniformly sized composite micellar template above the critical micellar concentration (CMC), simultaneously completing the reduction, nucleation, and ordered assembly of the gold precursor within the confined space. The thiol ligand, n-dodecyl mercaptan, simultaneously functions as a reducing agent and stabilizer in this process. Its thiol group (-SH) exhibits weak reducing properties, forming Au(I)-SR covalent bonds to stabilize the nanoparticles, further facilitating the synthesis and assembly of nanoparticles. This fundamentally avoids the size loss and structural disorder problems caused by stepwise processing in traditional multi-step methods. This strategy opens a new avenue for constructing high-performance ROS scavenging materials with high specific surface area, fully exposed active sites, and excellent stability.

[0006] Specifically, the present invention addresses the bottlenecks of the prior art through the following means:

[0007] 1. Based on the micelle template method, small and uniform in-situ grown ultra-small gold nanoassemblies were successfully prepared. This method uses n-dodecathiol and Pluronic F127 (F127, a type of ABCs), commonly used in the n-ABC system, as templates. A uniformly sized composite micelle template was constructed above the critical micelle concentration, allowing for the simultaneous reduction, nucleation, and ordered assembly of the gold precursor within a confined space. Notably, the thiol ligand functions as both a reducing agent and a stabilizer in this process, further facilitating the synthesis and assembly of nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) results show (see reference...) Figure 1 , Figure 2 The in-situ grown ultrasmall gold nanoassemblies prepared by this method exhibited a uniform structure with a hydrated particle size (HD) of 27.37 ± 4.95 nm. In contrast, the control group ultrasmall gold nanoassemblies prepared by the two-step assembly method showed a larger size and a disordered assembly morphology (see reference). Figure 3The above results demonstrate that the in-situ self-assembly strategy based on micelle templates can effectively guide the ordered self-assembly of ultrasmall gold nanoparticles.

[0008] 2. The in-situ growth self-assembly strategy significantly improved the availability and stability of AuNAs active sites: the in-situ grown ultrasmall gold nanoassemblies exhibited excellent stability under physiological conditions of pH 7.4, with the HD remaining at 33.97 ± 9.12 nm after six days, showing no significant difference from the first day (refer to...). Figure 4 The scavenging capacity of 2,2′-adiazono-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS), 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radicals, and hydroxyl radicals (•OH) is a representative indicator for evaluating the antioxidant efficiency of drugs. ABTS... + • (refer to Figure 5 , Figure 6 ), DPPH• (refer to Figure 7 , Figure 8 ) solution or •OH (refer to Figure 9 , Figure 10 Incubation with in-situ grown ultrasmall gold nanoassemblies showed a significant decrease in absorbance at the response wavelength with increasing incubation dose, indicating that free radicals were scavenged. When the concentration of the in-situ grown ultrasmall gold nanoassemblies was 10.0 nM, ABTS... + The removal rates of •, DPPH• solution or •OH were as high as 98.3%, 51.9% and 93.9%, respectively. These results indicate that the in-situ self-assembly strategy based on micelle templates can effectively enhance the stability of AuNAs and improve their oxidation activity, thus enabling them to possess highly efficient ROS removal performance.

[0009] 3. This study further systematically evaluated the biosafety and intracellular ROS scavenging performance of this material. Cytotoxicity assays (CCK-8 assay) showed that after 24 hours of treatment with HK-2 cells at concentrations ranging from 0 to 20 nM, the in-situ grown ultrasmall gold nanoassemblies maintained a cell viability of over 80%, indicating that the material exhibited no significant cytotoxicity at the tested concentrations and possessed good biocompatibility (see reference). Figure 11 To verify its antioxidant activity at the cellular level, this study constructed an oxidative stress model of HK-2 cells using H2O2 stimulation and observed changes in intracellular ROS levels using laser confocal microscopy, with DCFH-DA as the ROS fluorescent probe. Figure 12As shown, compared with the H2O2 model group, the fluorescence intensity in cells pretreated with the in-situ grown ultrasmall gold nanoassemblies was significantly reduced, indicating that this material can effectively remove excess intracellular ROS induced by H2O2. These results demonstrate that the in-situ grown ultrasmall gold nanoassemblies not only possess good biosafety but also efficiently remove ROS at the cellular level, showing potential application value in the prevention or intervention of oxidative stress-related diseases.

[0010] This invention successfully constructed small-sized, uniformly morphological in-situ self-assembled ultrasmall gold nanoassemblies capable of efficiently removing intracellular ROS. This strategy provides an excellent design method for solving the challenges of "large size and high heterogeneity" of AuNAs, and offers important design ideas and technical support for promoting their clinical translation.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] I. Preparation methods of gold nanoassemblies

[0013] (1) Synthesis of in-situ self-assembled ultra-small gold nanoparticles: In this synthesis method, the concentration of ABCs is controlled to be higher than its critical micelle concentration (CMC, F127 is about 1%, 25℃). For example, 3% F127 is used as a functionalizing agent and stirred with the hydrophobic ligand n-dodecyl mercaptan at 25℃ for 45 minutes to form a composite micelle template. Then, chloroauric acid solution and reducing agent sodium borohydride are added sequentially to complete the reduction, nucleation and assembly of gold nanoparticles in situ in a micelle confinement environment. The reaction process is monitored by a UV-Vis spectrophotometer. After the reaction is completed, unreacted substances are removed by dialysis and the product is collected by high-speed centrifugation to obtain the in-situ grown ultra-small gold nanoparticles.

[0014] (2) Preparation of the control gold nanoparticle assembly using the traditional two-step method: First, in an ethanol system, n-dodecylthiol-functionalized hydrophobic gold nanoparticles were prepared by a one-step reduction method using n-dodecylthiol as a ligand and sodium borohydride as a reducing agent. Then, F127 was added to the system and used as an assembly template to assemble the hydrophobic gold nanoparticles and achieve hydrophilic functionalization. After the reaction was completed, impurities were removed by dialysis, and then the control gold nanoparticle assembly was obtained by ultrafiltration concentration.

[0015] In steps (1) and (2), the concentration of F127 is 3%, which is higher than its critical micelle concentration (~1%, 25°C); the reducing agent is sodium borohydride.

[0016] The purification methods in steps (1) and (2) are as follows: using a dialysis bag with a molecular weight cutoff of 10 kDa, dialyzing in ultrapure water for 24 hours, and changing the dialysis solution every 4 hours; ultrafiltration concentration is carried out using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa under centrifugation conditions of 4000g.

[0017] II. Structural and property characterization of gold nanoassemblies

[0018] The assembly prepared by the method is composed of gold nanoparticles with a single particle size of < 3 nm, exhibiting a uniform size with an HD of 27.37 ± 4.95 nm; while the control gold nanoparticle assembly exhibits a larger size and disordered assembly morphology.

[0019] The characterization methods for the AuNAs include: observing their morphology using transmission electron microscopy (TEM); dispersing the samples in a buffer solution at pH = 7.4 and determining their hydrated particle size using a nanoparticle size analyzer.

[0020] ABTS of the in-situ grown ultrasmall gold nanoparticle assembly + • The scavenging activity test method is as follows: different concentrations of in-situ grown ultra-small gold nano-assemblies solution are added to ABTS⁺ working solution and reacted at room temperature in the dark for 10 minutes. Then, the absorbance at 734 nm is measured using a UV-Vis spectrophotometer, and the scavenging efficiency is calculated based on the absorbance decrease rate.

[0021] The DPPH scavenging activity test method for the in-situ grown ultra-small gold nanoassemblies is as follows: different concentrations of AuNAs solution are added to the DPPH working solution, reacted in the dark at room temperature for 10 minutes, the absorbance at 517 nm is measured, and the scavenging efficiency is calculated.

[0022] The method for testing the •OH scavenging activity of the in-situ grown ultra-small gold nanoassemblies is as follows: different concentrations of AuNAs solution are added to the •OH test system, reacted in the dark at room temperature for 10 minutes, and the absorbance at 652 nm is measured, and the scavenging efficiency is calculated accordingly.

[0023] The application of the in-situ grown ultrasmall gold nanoassemblies in intracellular ROS scavenging includes the following steps: HK-2 cells were selected and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator; an oxidative stress model was constructed by adding exogenous H2O2; DCFH-DA (excitation wavelength 488 nm, emission wavelength 525 nm) was used as a ROS fluorescent probe, and changes in intracellular ROS levels were observed and analyzed using laser confocal microscopy.

[0024] III. Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] (1) The in-situ growth self-assembly synthesis process is simple to operate, has mild reaction conditions, readily available raw materials and controllable cost, and has good prospects for industrial application.

[0026] (2) The prepared in-situ grown ultra-small gold nano-assemblies have stable structures and uniform sizes, and exhibit efficient scavenging ability against various free radicals such as ABTS⁺•, DPPH• and •OH in the in vitro system;

[0027] (3) The material has good biocompatibility, low cytotoxicity, and excellent intracellular ROS scavenging performance, showing potential for application in the treatment of oxidative stress-related diseases at the cellular level. Attached Figure Description

[0028] Figure 1 The image is a transmission electron microscope image of the in-situ grown ultrasmall gold nanoassemblies prepared in Example 1.

[0029] Figure 2 This is a statistical diagram of the hydrated particle size distribution of the in-situ grown ultrasmall gold nanoassemblies prepared in Example 1.

[0030] Figure 3 The image shows a transmission electron microscope image of the conventional two-step gold nanoassembly prepared in Example 1.

[0031] Figure 4 This is a statistical chart of the 6-day hydration particle size distribution of the in-situ grown ultra-small gold nanoassemblies in Example 2.

[0032] Figure 5 This is an ABTS⁺•removal diagram of the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0033] Figure 6 The ABTS⁺• clearance statistics are shown for the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0034] Figure 7 This is a DPPH•removal diagram of the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0035] Figure 8 The DPPH•removal statistic is shown for the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0036] Figure 9 This is a diagram showing the removal of •OH from the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0037] Figure 10 This is a statistical chart of •OH scavenging in the in-situ grown ultrasmall gold nanoassemblies in Example 3.

[0038] Figure 11 This is a cytotoxicity diagram of the in-situ grown ultrasmall gold nanoassemblies in Example 4.

[0039] Figure 12The intracellular ROS generation under H2O2 induction: (-) No drug administration group; (+) Administered with in situ grown ultra-small gold nanoassemblies. Detailed Implementation

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection and implementation of the present invention are not limited thereto.

[0041] In the following examples, chloroauric acid (HAuCl4·3H2O) and sodium borohydride (NaBH4) were purchased from Shanghai Mairui Chemical Technology Co., Ltd.; n-dodecyl mercaptan (98%) was purchased from Shanghai Titan Technology Co., Ltd.; F127 was purchased from Sigma-Aldrich; cellulose dialysis bags with a molecular weight cutoff (MWCO) of 10 kDa were purchased from Sangon Biotech Co., Ltd.; dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology Co., Ltd.; 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH•) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. All the purchased chemical reagents and consumables were commercial products, and their purity was as defined at the time of purchase. No further processing was performed before use. The instruments used to characterize the properties of AuNAs materials and their efficient scavenging of intracellular ROS included: a Malvern Zetasizer ULTRA nanoparticle size analyzer (UK), a ThermoFisher Talos F200x field emission transmission electron microscope (USA), and a PerkinElmer Instruments Lambda 365 UV-Vis spectrophotometer (USA) for measuring the UV-Vis absorption spectrum of the dispersion.

[0042] Example 1: Synthesis of in-situ grown ultrasmall gold nanoassemblies

[0043] Before the experiment, all glassware was soaked in freshly prepared aqua regia (HCl: HNO3 = 3:1, v / v) for 30 minutes, then rinsed repeatedly with ultrapure water at least three times, and dried in a drying oven for later use. Using the hydrophobic molecule dodecyl mercaptan as a stabilizing ligand and F127 as a functionalizing reagent, micelles were formed through the interaction of dodecyl mercaptan and F127 in aqueous solution. HAuCl4 was then added, and NaBH4 was used as a reducing agent. This yielded an in-situ grown ultrasmall gold nanoparticle assembly. The specific steps are as follows:

[0044] First, F127 (450 mg) and n-dodecyl mercaptan (39.6 µL) were sequentially dispersed in 15 mL of deionized water. After vigorous stirring at room temperature for 20 minutes, the mixture was sonicated for 5 minutes, then stirred for another 20 minutes. Finally, HAuCl4 solution (100 mmol / L) was added. -1 (180 µL). Then immediately and rapidly add the reducing agent NaBH4 solution (8 mg / mL). -1 The reaction solution (850 µL) changed from yellow to yellowish-brown immediately, and the reaction time was determined by the change in the absorption spectrum of a UV-Vis spectrophotometer. The reaction was stopped after 3 hours at room temperature when the UV-Vis spectrophotometer absorption spectrum no longer changed, thus completing the synthesis of the in-situ grown ultrasmall gold nanoparticle assembly. The resulting solution was dialyzed at 37°C for 24 hours using a dialysis bag with a molecular weight of 10 kDa, and then centrifuged at 21,000 rpm for 15 minutes to remove large aggregates. The purified sample solution was stored at 37°C and used for subsequent experiments.

[0045] Example 2: Stability test of in-situ grown ultrasmall gold nanoassemblies

[0046] To investigate the stability of in-situ grown ultrasmall gold nanoparticle assemblies, their hydrated particle size was measured after being placed at pH 7.4 for 0, 1, 2, 3, 4, 5 and 6 days, and the results were compared and analyzed.

[0047] Example 3: Free radical scavenging activity test

[0048] (1) ABTS + • Scavenging activity test: ABTS was prepared by mixing ABTS with 2.45 mM K2S2O8 solution and incubating at room temperature in the dark for 12 hours. + • Working solution. Add gold nanoparticle assembly solutions of different concentrations (0–10.0 nM), react at room temperature in the dark for 10 minutes, measure absorbance at 734 nm, and calculate clearance rate.

[0049] (2) DPPH• scavenging activity test: The DPPH• scavenging activity test is based on the principle of characteristic absorption of DPPH• free radicals at 517 nm. During the test, a series of concentrations (0–10.0 nM) of gold nanoparticle assembly solutions were mixed with 0.1 mM DPPH• ethanol solution and reacted at room temperature in the dark for 10 minutes. The change in absorbance at 517 nm was then detected to evaluate the scavenging activity.

[0050] (3) •OH scavenging activity test: FeSO4·7H2O solution (1.5 mM) and TMB solution (1.5 mM) were added sequentially to 1.38 mL of sodium acetate buffer (0.1 M, pH 4.0). After mixing, H2O2 was added to initiate the reaction and form a •OH generation system. Then, solutions of gold nanoparticles at different concentrations (0–10.0 nM) were added and reacted at room temperature in the dark for 10 minutes. The absorbance at 652 nm was measured. The 0 nM group was used as a control. The decrease in absorbance indicated that the sample had a scavenging effect on •OH.

[0051] Example 4: Cytotoxicity and Intracellular ROS Scavenging Experiment

[0052] The cell viability of HK-2 cells treated with in-situ grown ultrasmall gold nanoparticle assemblies was assessed using the conventional CCK-8 assay. First, HK-2 cells were cultured at 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 1:1 in 96-well plates and incubated for 24 hours to allow cell adhesion. The culture medium in the plates was then replaced with medium containing different concentrations of in-situ grown ultrasmall gold nanoparticle assemblies (concentration range: 0 to 20 nM). After 30 hours of incubation, 10 μL CCK-8 was added, and incubation was continued for another 30 minutes. The absorbance at 450 nm was collected using a microplate reader. Cell viability was finally assessed by comparing the absorbance of cells treated with in-situ grown ultrasmall gold nanoparticle assemblies to that of blank cells.

[0053] Intracellular ROS scavenging assay

[0054] HK-2 cells were spaced at 10 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured at 37°C and 5% CO2 for 4 days. Once the cells had adhered and reached a suitable density, the original culture medium was removed and replaced with fresh medium containing 20 nM in-situ grown ultrasmall gold nanoassemblies, and cultured for another 24 hours. Subsequently, 500 μM H2O2 solution was added to each well, and incubation was performed for 30 minutes to induce oxidative stress. The culture medium was discarded, and DCFH-DA fluorescent probe working solution was added, followed by incubation in the dark for 10 minutes. After incubation, the cells were gently washed once with PBS to remove residual probes. Finally, fluorescence images of the cells were acquired using an OLYMPUS optical microscope for subsequent analysis.

[0055] The above embodiments are preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an ultrasmall gold nanoparticle assembly with highly efficient intracellular reactive oxygen species (ROS) scavenging capability, characterized in that: Includes the following steps: (1) 3% block copolymer Prönnick F127 was used as a functionalizing agent and mixed with hydrophobic ligand n-dodecyl mercaptan. The mixture was stirred at 25°C for 30 minutes to form a composite micelle template. (2) Chloroauric acid solution and sodium borohydride reducing agent are added sequentially to the composite micelle template obtained in step (1) to complete the reduction, nucleation and assembly reaction of the gold precursor in situ and simultaneously within the micelle confinement space; (3) The reaction solution obtained in step (2) is purified by dialysis, and then centrifuged and concentrated by ultrafiltration to obtain the ultra-small gold nano-assembly.

2. The preparation method according to claim 1, characterized in that: The concentration of the block copolymer F127 is 3%, which is higher than its critical micelle concentration (~1%, 25°C).

3. The preparation method according to claim 1, characterized in that: The hydrophobic ligand n-dodecyl mercaptan simultaneously functions as an auxiliary reducing agent and a stabilizer. Its thiol group forms an Au(I)-SR covalent bond with the gold nanoparticles to stabilize the nanoparticles.

4. The preparation method according to claim 1, characterized in that: The dialysis purification was performed using a dialysis bag with a molecular weight cutoff of 10 kDa, and the dialysis was carried out in ultrapure water at 37°C for 24 hours, with the dialysis solution being replaced every 4 hours.

5. The preparation method according to claim 1, characterized in that: The ultrafiltration concentration was completed using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa, centrifuged at 4000g and 4℃.

6. An ultrasmall gold nanoparticle assembly prepared by the method according to any one of claims 1-5, characterized in that: The assembly has a spherical structure with a hydrated particle size of 27.37±4.95 nm, and is assembled from gold nanoparticles with a single particle size of < 3 nm.

7. The ultrasmall gold nanoassembly according to claim 6, characterized in that: The assembly was stable when dispersed in phosphate buffer at pH 7.4 and stored at 37°C for 6 days. The hydrated particle size remained at 33.97±9.12 nm, with no significant difference from the initial particle size.

8. The application of the ultrasmall gold nanoassemblies according to claim 6 in scavenging free radicals, characterized in that: When the concentration of the gold nanoassemblies is 10.0 nM, for ABTS + The scavenging rate of · was 98.3%, the scavenging rate of hydroxyl radical (•OH) was not less than 93.9%, and the scavenging rate of DPPH• was 51.9%.

9. The application of the ultrasmall gold nanoassemblies according to claim 6 in the preparation of formulations for scavenging intracellular reactive oxygen species, characterized in that: The gold nanoassemblies showed no significant toxicity to HK-2 cells in the concentration range of 0-20 nM, and the cell survival rate was not less than 80%.

10. The application according to claim 9, characterized in that: In the H2O2-induced oxidative stress model of HK-2 cells, the gold nanoassemblies significantly reduced intracellular reactive oxygen species levels, resulting in a significant decrease in the fluorescence intensity of the intracellular DCFH-DA fluorescent probe.

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