Antimony nanopowder, preparation method and application thereof

Abstract

The application relates to antimony nanopowder and a preparation method and application thereof, and belongs to the technical field of metal powder processing. The method comprises the following steps: dispersing antimony nanopowder in a solvent to obtain a dispersion liquid; mixing the dispersion liquid with 5-10 times the mass of nitrobenzene diazonium salt (p-NBD) of the antimony nanopowder, and then reacting under the condition of light irradiation at 0-4 DEG C for 30-60 h to obtain passivated antimony nanopowder. The diazonium chemical reaction forms a stable covalent bond on the surface of Sb NPs, and blocks the interaction with respiratory tract cells. Compared with the original Sb NPs group, the nitrobenzene diazonium salt modified Sb NPs can reverse the GPX4 activity inhibition effect, make the GPX4 protein expression up-regulated and restored to the level close to the control group, and lose the ability to induce cell ferroptosis, so that the lung toxicity problem of Sb NPs is solved from the ferroptosis mechanism level.

Description

Technical Field

[0001] This invention belongs to the field of metal powder processing technology, specifically relating to an antimony nanopowder, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Antimony nanopowder (Sb NPs) is widely used in industrial and medical fields due to its excellent properties, but occupational exposure can cause serious respiratory hazards. Nanoscale antimony dust is pulmonary toxic, easily penetrates the respiratory barrier, and induces ferroptosis in epithelial cells (BEAS-2B) and macrophages (THP-1) by inhibiting glutathione peroxidase 4 (GPX4), leading to lung inflammation, fibrosis, and even the risk of lung cancer. Its toxicity is 3 to 5 times greater than that of micron-sized particles, threatening the respiratory health of occupational workers.

[0004] Surface passivation of antimony nanopowder can reduce its hazards; in existing technologies, physical coating methods are cumbersome and the coating layer is prone to detachment; chemical passivation methods are based on surface modification with reagents such as thiols and silane coupling agents, but are inefficient and may introduce new toxicities; biological modification methods are limited by environmental constraints and cannot be applied on a large scale. In particular, existing passivation methods are not designed for the ferroptosis pathway and cannot solve the lung toxicity problem of Sb NPs at the mechanistic level. Summary of the Invention

[0005] In view of the current state of technology, the purpose of this invention is to provide an antimony nanopowder, its preparation method, and its application. Passivated antimony nanopowder is prepared using nitrobenzene diazonium salt (p-NBD) as a raw material. Stable covalent bonds are formed on the surface of Sb NPs through a diazonium chemical reaction, blocking their interaction with respiratory cells. This method has the advantages of simple process (low temperature and ambient pressure reaction), low cost, and high biocompatibility, and can solve the pulmonary toxicity problem of Sb NPs at the mechanistic level.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, a method for preparing antimony nanopowder includes: dispersing antimony nanopowder in a solvent to obtain a dispersion, mixing the dispersion with 5 to 10 times the mass of antimony nanopowder in a nitrobenzene diazonium salt, and reacting the mixture under light irradiation at 0 to 4°C for 30 to 60 hours to obtain passivated antimony nanopowder.

[0008] Secondly, the antimony nanopowder obtained by the above-mentioned method for preparing antimony nanopowder.

[0009] Thirdly, the above-mentioned method for preparing antimony nanopowder has applications in reducing the toxicity of antimony nanopowder.

[0010] The beneficial effects of this invention are as follows:

[0011] 1. This invention utilizes a diazonium chemical reaction to form stable covalent bonds on the surface of Sb NPs, blocking their interaction with respiratory cells. This addresses the pulmonary toxicity of Sb NPs at the level of ferroptosis mechanisms, reducing occupational health risks. Compared to the original Sb NPs group before treatment, modification with nitrobenzene diazonium salt reverses the inhibitory effect on GPX4 activity, upregulating GPX4 protein expression and restoring it to near-control group levels, thus eliminating its ability to induce ferroptosis. This method boasts advantages such as simple process (low temperature and ambient pressure reaction), low cost, and high biocompatibility, making it suitable for the batch processing of industrial-grade nanomaterials and providing a novel solution for occupational health protection of antimony nanomaterials.

[0012] 2. The antimony nanopowder prepared by this invention retains its potential to inhibit tumor proliferation after surface modification to significantly reduce lung toxicity. It still has excellent anti-tumor activity and can be normally applied to its original uses such as biomedicine, achieving a high degree of unity between toxicity reduction and efficacy enhancement. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 The graphs show the stability test results of Sb@PDA@M in different physiological solvents in Test Example 1; A is the UV absorption curve of Sb@PDA@M dispersed in water, B is the UV absorption curve of Sb@PDA@M dispersed in PBS solution, and C is the UV absorption curve of Sb@PDA@M dispersed in 1640 culture medium.

[0015] Figure 2 The graph shows the stability test results of Sb-p-NBD@PDA in different physiological solvents in Test Example 1; A is the UV absorption curve of Sb-p-NBD@PDA dispersed in water, B is the UV absorption curve of Sb-p-NBD@PDA dispersed in PBS solution, and C is the UV absorption curve of Sb-p-NBD@PDA dispersed in 1640 medium.

[0016] Figure 3The graph shows the stability test results of Sb-p-NBD@PDA@M in different physiological solvents in Test Example 1; A is the UV absorption curve of Sb-p-NBD@PDA@M dispersed in water, B is the UV absorption curve of Sb-p-NBD@PDA@M dispersed in PBS solution, and C is the UV absorption curve of Sb-p-NBD@PDA@M dispersed in 1640 medium.

[0017] Figure 4 The following are the X-ray photoelectron spectroscopy (XPS) analysis results of Sb-p-NBD in Test Example 2: A is the semi-quantitative analysis result of different oxidation states of Sb-p-NBD-2 obtained in Comparative Example 2, B is the semi-quantitative analysis result of different oxidation states of Sb-p-NBD obtained in Example 1, and C is the semi-quantitative analysis result of different oxidation states of Sb-p-NBD-1 obtained in Example 2.

[0018] Figure 5 The figures show the cell viability test results of BEAS-2B cells co-incubated with different concentrations of Sb@PDA@M and GSH for 24 hours in Test Example 3; A shows the test results of the "Sb@PDA@M" group and the "Sb@PDA@M+GSH" group; B shows different concentrations of GSH and a fixed concentration of Sb@PDA@M (75 ug / mL), where the concentrations of Sb@PDA@M and GSH in the Control group were both 0.

[0019] Figure 6 The figure shows the cell viability test results of BEAS-2B cells co-incubated with Sb@PDA@M (50 ug / mL) and different concentrations of Fer-1 for 24 hours in Test Example 3. In the Control group, the concentrations of Sb@PDA@M and Fer-1 were both 0.

[0020] Figure 7 The figures show the cell viability test results of THP-1 induced macrophages in Test Example 4 after 24 hours of co-incubation with different concentrations of Sb@PDA@M and GSH. Figure A shows the test results of the "Sb@PDA@M" group and the "Sb@PDA@M+GSH" group; Figure B shows the conditions with different concentrations of GSH and a fixed concentration of Sb@PDA@M (75 ug / mL), where the concentrations of Sb@PDA@M and GSH in the Control group were both 0.

[0021] Figure 8 The test result is the cell survival rate of macrophages differentiated by THP-1 in Test Example 4, which were co-incubated with Sb@PDA@M (50 ug / mL) and different concentrations of Fer-1 for 24 hours. The concentrations of Sb@PDA@M and Fer-1 in the Control group were both 0.

[0022] Figure 9 These are the electron paramagnetic resonance (EPR) spectra of Sb NPs, Sb-p-NBD, and Sb-MPA in Test Example 5; A is a comparison diagram of Sb NPs and Sb-p-NBD; B is a comparison diagram of Sb-MPA and Sb-p-NBD.

[0023] Figure 10 This is a graph showing the cell viability test results of BEAS-2B cells co-incubated with different concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M for 24 hours in Test Example 6; A represents the cell viability when the concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M are both 0-100 ug / mL; B represents the cell viability when the concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M are both 75 ug / mL, where the concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M in the Control group are both 0.

[0024] Figure 11 The images show the mitochondrial morphology of BEAS-2B cells under electron microscopy in Test Example 6. A represents Control; B represents Sb@PDA@M; and C represents Sb-p-NBD@PDA@M. The red arrows indicate characteristic lesions of ferroptosis in mitochondria (scale bar: 1 μm).

[0025] Figure 12 The images show the results of Western blot analysis of Sb@PDA@M and Sb-p-NBD@PDA@M co-incubated with BEAS-2B cells in Test Example 6. A is the Western blot of GPX4 protein; B is the quantitative analysis of GPX4.

[0026] Figure 13 The figures show the cell viability results of THP-1-induced differentiated macrophages in Test Example 7 after co-incubation for 24 hours with different concentrations of Sb@PDA@M, Sb-p-NBD@PDA@M, or Sb-p-NBD-1@PDA@M. Figure A shows the cell viability results of Sb@PDA@M and Sb-p-NBD@PDA@M at concentrations of 0-100 ug / mL; Figure B shows the cell viability when the concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M are both 50 ug / mL; Figure C shows the cell viability results when the concentrations of Sb@PDA@M and Sb-p-NBD-1@PDA@M are both 0-100 ug / mL; and the Control group had a concentration of 0 for both Sb@PDA@M and Sb-p-NBD@PDA@M.

[0027] Figure 14The images show the mitochondrial morphology of THP-1-induced differentiated macrophages under electron microscopy in Test Example 7. A represents Control; B represents Sb@PDA@M; and C represents Sb-p-NBD@PDA@M. The red arrows indicate characteristic lesions of ferroptosis in mitochondria (scale bar: 1 μm).

[0028] Figure 15 The images show the results of Western blot analysis of Sb@PDA@M and Sb-p-NBD@PDA@M co-incubated with THP-1-induced differentiated macrophages in Test Example 7. A is the Western blot of GPX4 protein; B is the quantitative analysis of GPX4.

[0029] Figure 16 This is a graph showing the results of the RM-1 cell proliferation test in Test Example 8.

[0030] Figure 17 This is a graph showing the results of Western blot analysis in test example 8. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions. All raw materials and reagents used in the following examples are commercially available unless otherwise indicated.

[0034] p-Nitrobenzene tetrafluoroborate diazonium salt, chemical formula C6H4BF4N3O2, CAS number 456-27-9.

[0035] 4-Nitrochlorodiazobenzene, chemical formula C6H4ClN3O2, CAS number 100-05-0.

[0036] 3-Mercaptopropionic acid, chemical formula C3H6O2S, CAS number 107-96-0.

[0037] N,N-Dimethylformamide, chemical formula C3H7NO, CAS number 68-12-2.

[0038] Acetonitrile, molecular formula CH3CN, CAS number 75-05-8.

[0039] Dimethyl sulfoxide, molecular formula C2H6SO, CAS number 67-68-5.

[0040] Reduced glutathione (reduced form) was purchased from Aladdin (product number G105427).

[0041] Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, was purchased from Targetmol (catalog number T6500).

[0042] One or more embodiments of the present invention provide a method for preparing antimony nanopowder, comprising: dispersing antimony nanopowder in a solvent to obtain a dispersion, mixing the dispersion with 5 to 10 times the mass of antimony nanopowder in nitrobenzene diazonium salt (p-NBD), and reacting under light irradiation at 0 to 4°C for 30 to 60 hours to obtain passivated antimony nanopowder.

[0043] In the above preparation process, a stable covalent bond is formed on the surface of Sb NPs using a diazonium chemical reaction in a solvent. This passivates the antimony nanopowder, causing it to lose its ability to induce ferroptosis and preventing the inhibitory effect of GPX4 activity in cells, thus addressing the pulmonary toxicity problem of Sb NPs at the mechanistic level. A slightly higher p-NBD content ensures that all vacancies on the surface of Sb NPs are occupied by p-NBD. During the preparation process, the reaction temperature is lowered, and a protective gas is used to prevent Sb NPs from being oxidized to the more toxic trivalent antimony during prolonged stirring.

[0044] Optionally, the antimony nanopowder has a particle size of 30–500 nm. Antimony nanopowder has applications in biomedicine, energy storage, and optoelectronic materials. However, antimony ions in this particle size range can cause harm to the respiratory tract of workers by inhibiting glutathione peroxidase 4 (GPX4) and inducing ferroptosis in epithelial cells (BEAS-2B) and macrophages (THP-1).

[0045] Optionally, the solvent is selected from acetonitrile and dimethyl sulfoxide, and the mass ratio of the nitrobenzene diazonium salt to the volume ratio of the solvent is 35~45 mg / mL.

[0046] Optionally, the nitrobenzene diazonium salt is p-nitrobenzene tetrafluoroborate diazonium salt or 4-nitrochlorodiazobenzene; it can be dissolved in solvents such as acetonitrile or dimethyl sulfoxide to passivate the surface of antimony nanopowder dispersed in the solvent.

[0047] Optionally, dispersion methods include: ultrasonic treatment for 15-20 minutes; or dispersing antimony nanopowder, which is insoluble in solvent, in a solvent and fully exposing the surface.

[0048] Optionally, after adding nitrobenzene diazonium salt, ultrasonic treatment is performed for 5-10 minutes to complete the dissolution of nitrobenzene diazonium salt and allow it to fully react with antimony nanopowder.

[0049] Optionally, the reaction is carried out in a protective gas, which is one or more of nitrogen and argon, to remove oxygen mixed in the reaction system and prevent antimony nanoparticles from being oxidized into more toxic trivalent antimony (trivalent antimony will further convert into pentavalent antimony) during long-term stirring, thereby reducing the toxicity of Sb NPs products; an oxygen removal device can also be used to remove oxygen mixed in the reaction system.

[0050] Optionally, the reaction temperature can be controlled by using an ice-water bath or circulating cold water during the reaction; this is also a method to prevent antimony nanoparticles from oxidizing into more toxic trivalent antimony during prolonged stirring, thereby enhancing the biocompatibility of the product.

[0051] Optionally, the reaction is carried out under illumination; illumination is used to activate p-NBD to generate free radicals. Illumination conditions include broadband irradiation with an intensity of 8000–15000 lx (lux); the light source is an incandescent lamp, which can achieve the reaction simply and at low cost; alternatively, the illumination wavelength is 320–415 nm. The N≡N bond in the diazonium salt compound is a photosensitive group, and its optimal wavelength for photolytic cleavage is usually in the ultraviolet region. Common and effective wavelengths for initiating the decomposition of such diazonium salts and subsequent reactions are in the ultraviolet band (320 nm–415 nm). Although shorter wavelengths of ultraviolet light (such as 254 nm) have a strong absorption peak in p-NBD and higher photon energy, and can also effectively cleave the N≡N bond, they are accompanied by more side reactions.

[0052] Optionally, the reaction can be stirred or carried out using a spiral reaction tube.

[0053] Optionally, the reaction time is 30-60 min; stable covalent bonds are formed on the surface of antimony nanopowder through a diazonium chemical reaction, thus completing the passivation of antimony nanopowder by nitrobenzene diazonium salt.

[0054] Optionally, the process may also include the following steps: after the reaction is complete, the product is washed multiple times with a solvent to remove unreacted nitrobenzene diazonium salt; after removing the unreacted nitrobenzene diazonium salt, purified passivated antimony nanopowder is obtained.

[0055] One or more embodiments of the present invention provide passivated antimony nanopowder obtained by the above-described method for preparing antimony nanopowder.

[0056] Passivated antimony nanopowder loses its ability to induce ferroptosis at the mechanistic level, providing a novel solution for occupational health protection with antimony nanopowder. Furthermore, the passivated antimony nanopowder still retains the potential to inhibit tumor cell proliferation, thus preserving its original purpose.

[0057] One or more embodiments of the present invention provide the application of the above-described antimony nanopowder surface passivation method in reducing the toxicity of antimony nanopowder.

[0058] The present invention will be further described below with reference to specific embodiments.

[0059] Example 1

[0060] An antimony nanopowder is prepared using acetonitrile as a solvent and p-nitrobenzene tetrafluoroborate diazonium salt as the nitrobenzene diazonium salt. A transparent reaction vessel is used, sealed with a rubber stopper. Two needles are inserted into the reaction vessel through the rubber stopper to deliver gas. The tip of one needle is above the liquid surface to expel the gas above the liquid surface from the vessel. The other needle is longer and its tip is inserted below the liquid surface to introduce argon gas into the solvent 30 minutes before the passivation reaction and throughout the passivation reaction, so that the reaction system reacts under conditions of no oxygen or extremely low dissolved oxygen content.

[0061] The specific steps include: using the above-mentioned reaction vessel, adding 20 mg of antimony nanopowder (Sb NPs) to 5 mL of acetonitrile, and sonicating for 15 min under ice-water bath (2±2℃) conditions to obtain a dispersion; adding 200 mg of p-nitrobenzene tetrafluoroborate diazonium salt (nitrobenzene diazonium salt, p-NBD) to the dispersion, and continuing to sonicate for 5 min under ice-water bath conditions to fully dissolve it, thereby obtaining a mixture.

[0062] During the passivation reaction, the reaction vessel was placed in an ice-water bath and the mixture was continuously stirred for 60 hours under low temperature (2±2℃) light irradiation (light intensity of 10000 lx) conditions to ensure full reaction. Incandescent lamps were used as the light source.

[0063] After the reaction, the reaction mixture was washed repeatedly with acetonitrile (centrifugation, 12,000 rpm, precipitate collected) to remove unreacted diazonium salts; then washed repeatedly with anhydrous ethanol (centrifugation, 12,000 rpm, precipitate collected) to remove toxic residual solvent acetonitrile and improve the biocompatibility of the reaction product. After drying, the reaction product became passivated antimony nanoparticles, denoted as Sb-p-NBD.

[0064] Example 2

[0065] An antimony nanopowder is prepared using acetonitrile as a solvent and p-nitrobenzene tetrafluoroborate diazonium salt as the nitrobenzene diazonium salt. A spiral reaction tube is used as the reaction vessel. The upstream of the reaction vessel is connected to a microfluidic mixer via a feed pipe. The upstream of the microfluidic mixer is connected to an Sb NPs dispersion tank and a diazonium salt / acetonitrile solution tank, respectively. An inert gas is continuously supplied to the tank below the liquid surface via a bubbling device. The Sb NPs dispersion tank and the diazonium salt / acetonitrile solution tank are respectively set to avoid light. An online deoxidizer is integrated into the feed pipe. The reaction tube is spiral-shaped and equipped with a narrow-band LED light source to irradiate a set length range of the reaction tube. The entire device is shielded from light in areas outside the irradiation range. The wavelength of the narrow-band LED light source is 320~415 nm, and the irradiation intensity at the reaction tube position is 10000 lx.

[0066] The specific steps include: connecting the outlets of the two material tanks to a microfluidic mixer; mixing antimony nanopowder with p-nitrobenzene tetrafluoroborate diazonium salt (nitrobenzene diazonium salt, p-NBD) in the microfluidic mixer at a mass ratio of 1:5; and then deoxygenating the mixture through an online deoxidizer to ensure extremely low dissolved oxygen content in the material entering the spiral reaction tube. Because the material is strictly deoxygenated and protected from light before entering the irradiation area of ​​the reaction tube, the reaction time can be precisely controlled to 30 minutes by controlling the flow rate of the liquid in the reaction tube and the irradiation range. This allows the diazonium salt to rapidly decompose in the irradiation section and graft onto the antimony nanoparticles for passivation, suppressing side reactions and limiting the oxidation rate of Sb NPs, thus minimizing the total irradiation dose. Similar to Example 1, the reaction temperature is controlled at 2±2℃. After the reaction, the reaction mixture was washed repeatedly with acetonitrile (centrifugation, 12,000 rpm, precipitate collected) to remove unreacted diazonium salts; then washed repeatedly with anhydrous ethanol (centrifugation, 12,000 rpm, precipitate collected) to remove toxic residual solvent acetonitrile and improve the biocompatibility of the reaction product. After drying, the reaction product became passivated antimony nanoparticles, denoted as Sb-p-NBD-1.

[0067] This embodiment uses a different apparatus than that in Embodiment 1 to address the problem of Sb NPs being easily oxidized in aerobic solutions, and strictly controls the irradiation time through a unidirectional flow reaction tube to reduce byproducts.

[0068] Comparative Example 1

[0069] An antimony nanopowder is prepared by passivating the surface of Sb NPs with 3-mercaptopropionic acid (MPA). The method includes: dispersing 20 mg of antimony nanopowder with a particle size of 50 nm in 10 mL of N,N-dimethylformamide (DMF), sonicating in a water bath for 40 min to obtain a dispersion, transferring the dispersion to a round-bottom flask, adding 5 mL of 3-mercaptopropionic acid (MPA) and stirring to dissolve, heating at 130 °C in a nitrogen atmosphere for 24 h to obtain MPA-treated antimony nanopowder, denoted as Sb-MPA; removing residual MPA by vacuum distillation, and resuspending the obtained product in DMF for later use.

[0070] Comparative Example 2

[0071] An antimony nanopowder, prepared according to the method of Example 1, uses acetonitrile as a solvent and p-nitrobenzene tetrafluoroborate diazonium salt as the nitrobenzene diazonium salt. The specific steps include:

[0072] Using the same reaction vessel as in Example 1, 20 mg of antimony nanopowder (Sb NPs) was added to 5 mL of acetonitrile and sonicated at room temperature for 15 min to obtain a dispersion. 100 mg of p-nitrobenzenetetrafluoroborate diazonium salt (nitrobenzene diazonium salt, p-NBD) was added to the dispersion and sonicated at room temperature for 5 min to dissolve completely, obtaining a mixture. The mixture was continuously stirred for 48 h under light irradiation (10000 lx) at room temperature (25°C) to allow for complete reaction. The reaction mixture was then washed multiple times with acetonitrile (centrifuged at 12000 rpm, and the precipitate was collected) to remove unreacted diazonium salt. The product, after drying, became passivated antimony nanopowder, denoted as Sb-p-NBD-2.

[0073] Example 3

[0074] Using the unpassivated antimony nanopowder raw material from Example 1 as a comparison, denoted as Sb NPs, the following operations and tests were performed using Sb NPs, Sb-p-NBD, Sb-MPA, and Sb-p-NBD-2 as raw materials to obtain the technical effect of reducing the toxicity of antimony nanopowder.

[0075] Preparation Example 1: Preparation of a cell membrane and polydopamine-coated antimony nanopowder composite.

[0076] Using a publicly available method, Sb NPs encapsulated in polydopamine (PDA) nanospheres were formed by dopamine self-assembly, denoted as Sb@PDA. Then, a cell membrane (M) was coated on the surface of Sb@PDA to obtain Sb@PDA encapsulated in cell membrane (M), denoted as Sb@PDA@M. Using the same method, Sb-p-NBD@PDA and Sb-p-NBD@PDA@M were prepared sequentially using Sb-p-NBD as raw material. Using the same method, Sb-MPA@PDA@M was prepared using Sb-MPA as raw material.

[0077] Test Example 1, characterization of the water solubility of Preparation Example 1.

[0078] Sb@PDA@M was dispersed in physiological solutions such as ultrapure water, phosphate-buffered saline (PBS), and 1640 medium, and its UV-Vis spectra were measured periodically. Time-dependent absorption curves were plotted, and the results are shown below. Figure 1 As shown, Figure 1 In the figure, A represents the curve of ultraviolet absorbance dispersed in water as a function of time. Figure 1 In the figure, B is the curve showing the change of UV absorbance of the PBS solution dispersed in the solution over time. Figure 1 C in the figure represents the curve of UV absorbance of Sb@PDA@M dispersed in 1640 medium over time. The water solubility and stability of Sb@PDA@M were determined based on the degree of change in the curve. It can be seen that the absorption curves of Sb@PDA@M in water, PBS, and 1640 medium all show a relatively slow decreasing trend. Correspondingly, the curve of UV absorbance of Sb-p-NBD@PDA dispersed in water over time is shown in the figure. Figure 2 As shown in Figure A, the curve of UV absorbance dispersed in PBS solution changing over time is as follows. Figure 2 As shown in Figure B, the curve of UV absorbance dispersed in 1640 medium as a function of time is as follows. Figure 2 As shown in C; the curve of UV absorbance of Sb-p-NBD@PDA@M dispersed in water as a function of time is shown in Figure C. Figure 3 As shown in Figure A, the curve of UV absorbance dispersed in PBS solution changing over time is as follows. Figure 3 As shown in Figure B, the curve of UV absorbance dispersed in 1640 medium as a function of time is as follows. Figure 3 As shown in C, Sb@PDA@M, Sb-p-NBD@PDA, and Sb-p-NBD@PDA@M exhibit good solubility and stability in physiological solutions, making them suitable for subsequent testing.

[0079] Test Example 2: Oxidation performance test of Sb NPs obtained by different passivation methods.

[0080] To compare the oxidation degree of the Sb NPs passivation method in Example 1 with that in Comparative Example 2, X-ray photoelectron spectroscopy (XPS) was used to obtain the energy spectra reflecting the surface chemical properties of the Sb NPs materials obtained by the two passivation methods. The contents of different valence state components in the Sb NPs composite material were obtained by analyzing, fitting, and calculating the energy spectra using Avantage software (version 5.979, Thermo Fisher Scientific). The results are as follows: Figure 4 As shown, Figure 4 In this context, A represents the analytical result of the passivation material in Comparative Example 2. Figure 4 In this example, B represents the analysis results of the passivation material in Example 1. By reducing the dissolved oxygen content in the reaction system and using low-temperature reaction, Example 1 significantly reduced the proportion of oxidized products in the product compared to Comparative Example 2 (antimony oxide content decreased from 89.43% to 23.67%, of which the content of more toxic trivalent antimony decreased from 30.45% to 21.13%), thereby significantly improving the biocompatibility of the passivation material (Sb-p-NBD). Furthermore, the passivation material Sb-p-NBD-1 obtained by the method of Example 2 inhibited the conversion of the nano-antimony surface layer from zero-valent antimony Sb(0) to more toxic trivalent antimony Sb(III), and oxidized more into pentavalent antimony Sb(V), which has much less cytotoxicity, thereby effectively improving the biocompatibility of the passivation material.

[0081] Test Example 3: BEAS-2B cell rescue experiment.

[0082] To verify the occurrence of intracellular ferroptosis, reduced glutathione (GSH) or the ferroptosis inhibitor ferrostatin-1 (Fer-1) were used as inhibitors to intervene in BEAS-2B cells. Cells were divided into three groups: "Control", "Sb@PDA@M", and "Sb@PDA@M+GSH" (when Fer-1 was used, the third group was Sb@PDA@M+Fer-1), n=3. Sb@PDA@M concentration gradients were set at 0, 12.5, 25, 50, 75, and 100 ug / mL. BEAS-2B or THP-1-induced macrophages were divided into 10 wells. 4 Cells were seeded in 96-well plates and incubated in a cell culture incubator for 24 hours. Then, according to pre-defined groups, the "Sb@PDA@M" group and the "Sb@PDA@M+GSH" group were added with a predetermined concentration of Sb@PDA@M, and the "Sb@PDA@M+GSH" group was added with GSH to a final concentration of 1 mM. After incubation for another 24 hours, 10 μL of CCK8 was added. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated by normalizing the absorbance values ​​to the Control group. The experimental results are as follows: Figure 5 As shown, where, Figure 5In the figure, A represents the test results for the "Sb@PDA@M" group and the "Sb@PDA@M+GSH" group. Figure 5 The comparison graph shows that group B represents the control group, the group with GSH concentration of 0 and Sb@PDA@M concentration of 75 ug / mL, and the group with GSH concentration of 1 mM and Sb@PDA@M concentration of 75 ug / mL. It can be seen that GSH can significantly rescue BEAS-2B cells from Sb@PDA@M toxicity, suggesting that this damage is caused by ferroptosis. When Fer-1 is used instead of GSH, the experimental results are as follows... Figure 6 As shown, Fer-1 can significantly rescue BEAS-2B cells from Sb@PDA@M toxic damage, suggesting that this damage is caused by ferroptosis.

[0083] Test Example 4: THP-1-induced macrophage rescue experiment.

[0084] To verify the occurrence of intracellular ferroptosis, multiple inhibitors, including reduced glutathione (GSH) and the ferroptosis inhibitor ferrostatin-1 (Fer-1), were used to intervene in THP-1-induced differentiated macrophages, following the treatment method for BEAS-2B cells in Test Example 3. The inhibition results of reduced glutathione (GSH) are as follows: Figure 7 As shown, where, Figure 7 In the figure, A represents the test results for the "Sb@PDA@M" group and the "Sb@PDA@M+GSH" group. Figure 7 The comparison results of group B (Control group), group with GSH concentration of 0 and Sb@PDA@M concentration of 75 ug / mL, and group with GSH concentration of 1 mM and Sb@PDA@M concentration of 75 ug / mL) are shown in the figure. GSH can significantly rescue THP-1-induced differentiated macrophages from Sb@PDA@M toxic damage, suggesting that this damage is caused by ferroptosis. When Fer-1 is used instead of GSH, the inhibition results of Fer-1 are as follows. Figure 8 As shown, Fer-1 can significantly rescue THP-1-induced differentiated macrophages from Sb@PDA@M toxic damage, suggesting that this damage is caused by ferroptosis.

[0085] Test Example 5: Characterization of surface vacancies in antimony nanopowder.

[0086] The presence of vacancies on the material surface can be directly reflected by detecting unpaired electrons using electron spin resonance (ESR). ESR was used to test the surface antimony (Sb) vacancies in Sb NPs, Sb-p-NBD, and Sb-MPA, respectively, with the magnetic field range set to no less than 3440–3560 GHz. The comparison results for Sb NPs and Sb-p-NBD are as follows: Figure 9As shown in Figure A, Sb NPs exhibit a strong ESR signal at g=2.0004, while the ESR signal of Sb-p-NBD is significantly attenuated, indicating that there is a high concentration of vacancies on the surface of the original Sb NPs, and the vacancy concentration on the surface of Sb-p-NBD is significantly reduced. This suggests that the original Sb NPs can induce ferroptosis at the cellular level, while the passivated Sb NPs lose their ability to induce ferroptosis, thus confirming the key role of surface vacancies in Sb NP-induced ferroptosis. Meanwhile, the comparison results between Sb-p-NBD and Sb-MPA are shown below. Figure 9 As shown in B, the passivation material Sb-MPA obtained by the passivation method of Comparative Example 1 (3-mercaptopropionic acid, MPA) has a significantly stronger ESR signal than Sb-p-NBD, indicating that its surface vacancy concentration after passivation is significantly weaker than that of Sb-p-NBD.

[0087] Test Example 6: Antimony Nanopowder Complex Co-incubation of Cells Test 1.

[0088] To test the cell viability of antimony nanopowder composites co-incubated, BEAS-2B cells were cultured at 10 cells per well. 4 Cells were seeded in 96-well plates and incubated for 24 hours at a density of 75 ± 5%. The cells were then divided into three groups: "Control", "Sb@PDA@M", and "Sb-p-NBD@PDA@M" (n=3). The "Control" group (control group) received no antimony nanopowder complex. The "Sb@PDA@M" group received Sb@PDA@M at concentrations of 0, 12.5, 25, 50, 75, and 100 ug / mL. The "Sb-p-NBD@PDA@M" group received Sb-p-NBD@PDA@M at concentrations of 0, 12.5, 25, 50, 75, and 100 ug / mL. After incubation for another 24 hours, 10 μL of CCK8 was added. Absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated by normalizing the absorbance values ​​to the Control group. Results are as follows. Figure 10 As shown, Figure 10 In the figure, A represents the cell viability results after co-incubation with different concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M. Figure 10B in the figure represents the cell viability results when the concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M are both 75 ug / mL. This indicates that when BEAS-2B cells are co-incubated with different concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M, Sb-p-NBD@PDA@M generally shows a significantly higher survival rate. At a concentration of 75 ug / mL, the survival rate of BEAS-2B cells co-incubated with Sb@PDA@M is less than 60%, while the survival rate of BEAS-2B cells co-incubated with Sb-p-NBD@PDA@M increases significantly, showing no difference from the "Control" group.

[0089] When ferroptosis occurs, the characteristic ultrastructural features of mitochondria include shrinkage, membrane rupture, increased membrane density, and a reduction or even disappearance of cristae. Therefore, transmission electron microscopy (TEM) is the most direct method for observing cell ultrastructure and ferroptosis. Cells from each group were fixed with 2.5% glutaraldehyde for at least 4 hours and then semi-in situ fixed. Cells were collected by scraping, washed with 0.1 M PBS, and fixed with 2% osmium tetroxide for 2 hours. After washing with deionized water, they were stained overnight with 3% uranium acetate solution. After gradient dehydration with ethanol and transition with acetone, the cells were embedded in Epon 812 resin and polymerized at 37℃, 45℃, and 60℃. Ultrathin sections were prepared using a Leica EM UC7 microtome. After double staining with uranium acetate / lead citrate, the distribution and morphological changes of mitochondria in each group were observed using an HT7700 TEM. The experimental results are as follows: Figure 11 show, Figure 11 The A in the data shows that the mitochondria in the "Control" group cells have normal mitochondrial morphology (uniform distribution of mitochondrial crests). Figure 11 In section B, the red arrow indicates that the "Sb@PDA@M" group shows obvious characteristic ferroptosis damage, while Figure 11 The C-value in the data shows that the mitochondrial morphology of the cells in the “Sb-p-NBD@PDA@M” group was restored to normal, and the characteristic damage of ferroptosis was recovered.

[0090] GPX4 is a key enzyme in the lipid repair system. Its inactivation inhibits the conversion of lipid peroxides to harmless lipid alcohols, leading to the accumulation of toxic lipid peroxides in cells and triggering ferroptosis. GPX4 is a negative regulator of ferroptosis. Therefore, lipid peroxides can be considered a key biomarker for ferroptosis. Cells from the "Control," "Sb@PDA@M," and "Sb-p-NBD@PDA@M" groups were lysed using RIPA (Radio Immunoprecipitation Assay) lysis buffer. After centrifugation at 4°C (12,000 rpm), the supernatant was collected to obtain total protein. Protein quantification was performed using the BCA (Bicinchoninic acid) method. GPX4 antibody was purchased from Abcam (catalog number ab125066). Western blot analysis was performed using standard methods. The experimental results are as follows: Figure 12 As shown in Figure A, the protein expression level was analyzed using ImageJ software, and the results are as follows: Figure 12 The results in B showed that GPX4 protein expression in BEAS-2B cells treated with Sb@PDA@M was significantly downregulated, while GPX4 protein expression was significantly upregulated after treatment with "Sb-p-NBD@PDA@M", returning to a level close to that of the "Control" group. This suggests that Sb@PDA@M damages the respiratory tract by inhibiting glutathione peroxidase 4 (GPX4)-induced ferroptosis in epithelial cells (BEAS-2B), while Sb-p-NBD@PDA@M can reverse the inhibitory effect of GPX4 activity.

[0091] This indicates that Sb@PDA@M has significant cytotoxicity to respiratory epithelial cells (BEAS-2B), while Sb-p-NBD@PDA@M modified by the method provided in this invention can significantly reduce the cytotoxicity to the above-mentioned cells, reverse the inhibitory effect of GPX4 activity, and reduce the damage caused by ferroptosis.

[0092] Test Example 7: Test II of co-incubating cells with antimony nanopowder complex.

[0093] To test the viability of cells co-incubated with Sb@PDA@M or Sb-p-NBD@PDA@M, THP-1-induced differentiated macrophages were treated according to the treatment method for BEAS-2B cells in Test Example 6. The results are as follows: Figure 13 As shown, Figure 13 In the figure, A represents the cell viability results after co-incubation with different concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M. Figure 13B in the table represents the cell survival rate results when both Sb@PDA@M and Sb-p-NBD@PDA@M concentrations were 50 ug / mL. This indicates that when THP-1-induced macrophages were co-incubated with different concentrations of Sb@PDA@M and Sb-p-NBD@PDA@M, Sb-p-NBD@PDA@M generally showed a significantly higher survival rate. At a concentration of 50 ug / mL, the cell survival rate of THP-1-induced macrophages co-incubated with Sb@PDA@M was approximately 60%, while the cell survival rate co-incubated with Sb-p-NBD@PDA@M significantly increased, showing no difference from the "Control" group. The passivation material obtained in Example 2 was tested using the same method, and the results are as follows... Figure 13 As shown in C: The passivation material Sb-p-NBD-1 obtained by the continuous flow reaction method, after being coated with PDA and cell membrane (denoted as Sb-p-NBD-1@PDA@M), achieved a cell survival rate similar to or higher than that before passivation (Sb@PDA@M) at different concentrations. This indicates that the passivation process effectively inhibited the generation of the more toxic trivalent oxidation product Sb(III), thereby effectively improving the biocompatibility of the passivation material.

[0094] Cells in this test case were stained with uranium acetate / lead citrate double staining as described in Test Case 5. The distribution and morphological changes of mitochondria in each group were observed using an HT7700 transmission electron microscope. The experimental results are as follows: Figure 14 show, Figure 14 The A in the data shows that the mitochondria in the "Control" group cells have normal mitochondrial morphology (uniform distribution of mitochondrial crests). Figure 14 B in the image shows that the "Sb@PDA@M" group exhibits obvious characteristic ferroptosis damage, while Figure 14 The C-values ​​in the "Sb-p-NBD@PDA@M" group showed that the mitochondrial morphology of the cells returned to normal, and the characteristic damage of ferroptosis was significantly reduced.

[0095] Following the method described in Test Example 6, Western blot analysis was performed on the cells in this test example. The experimental results are as follows: Figure 15 As shown in Figure A, the protein expression level was analyzed using ImageJ software, and the results are as follows: Figure 15 Figure B shows that macrophages differentiated by THP-1 after treatment with Sb@PDA@M showed significantly downregulated GPX4 protein expression, while macrophages treated with "Sb-p-NBD@PDA@M" showed significantly upregulated GPX4 protein expression, recovering to a level close to that of the "Control" group.

[0096] This indicates that Sb@PDA@M has significant cytotoxicity to macrophages (THP-1), while Sb-p-NBD@PDA@M modified by the method provided in this invention can significantly reduce the cytotoxicity to the above-mentioned cells, reverse the inhibitory effect of GPX4 activity, and reduce the damage caused by ferroptosis.

[0097] Test Example 8: Antimony Nanopowder Complex Co-incubation Cell Test III.

[0098] Antimony nanopowder has the ability to inhibit tumor cell proliferation. To test whether passivated antimony nanopowder has the ability to inhibit tumor cell proliferation, RM-1 cells (a murine prostate cancer cell line) were seeded into 96-well plates, with 500 cells per well and 3 replicates per group. After seeding, the cells were cultured for 24 h, and then different concentrations of Sb@PDA@M or passivated Sb-p-NBD@PDA@M were added. The cells were incubated for 120 h, and cell viability was detected every 24 h using a CCK-8 assay kit. The results are as follows: Figure 16 As shown, the passivated Sb material (Sb-p-NBD@PDA@M material) still has the potential to significantly inhibit tumor cell proliferation.

[0099] Following the analysis method in Test Example 6, RM-1 cells were then treated with Sb@PDA@M or Sb-MPA@PDA@M, respectively. Western blot analysis was then performed on the treated RM-1 cells, and the results are as follows: Figure 17 As shown, RM-1 cells treated with Sb@PDA@M showed a significant downregulation of GPX4 protein expression. However, after treatment with "Sb-MPA@PDA@M", the GPX4 protein expression failed to recover to a level close to that of the "Control" group. This indicates that Sb-MPA@PDA@M failed to effectively reduce the toxicity of Sb NPs powder, and its technical effect in reducing the toxicity of antimony nanopowder is significantly inferior to that of Sb-p-NBD@PDA@M.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., 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 antimony nanopowder, characterized in that, include: Antimony nanopowder is dispersed in a solvent to obtain a dispersion. The dispersion is then mixed with 5 to 10 times the mass of antimony nanopowder in nitrobenzene diazonium salt and reacted under light irradiation at 0 to 4°C for 30 to 60 hours to obtain passivated antimony nanopowder. The reaction is carried out in a protective gas, which is one or more of nitrogen and argon. The passivated antimony nanopowder can reverse the GPX4 activity inhibition effect and reduce damage caused by ferroptosis.

2. The method for preparing antimony nanopowder according to claim 1, characterized in that, The antimony nanopowder has a particle size of 30–500 nm.

3. The method for preparing antimony nanopowder according to claim 1, characterized in that, The solvent is selected from acetonitrile and dimethyl sulfoxide, and the mass ratio of the nitrobenzene diazonium salt to the volume ratio of the solvent is 35~45 mg / mL.

4. The method for preparing antimony nanopowder according to claim 1, characterized in that, Dispersion methods include: ultrasonic treatment for 15-20 minutes; ultrasonic treatment for 5-10 minutes after adding nitrobenzene diazonium salt.

5. The method for preparing antimony nanopowder according to claim 1, characterized in that, The reaction temperature is controlled by an ice-water bath or circulating cold water during the reaction.

6. The method for preparing antimony nanopowder according to claim 1, characterized in that, The light intensity is 8000~15000 lx; ​​the light source is an incandescent lamp, or the light wavelength is 320~415 nm.

7. The method for preparing antimony nanopowder according to claim 1, characterized in that, Stirring is used during the reaction, or a spiral reaction tube is used for the reaction.

8. The method for preparing antimony nanopowder according to claim 1, characterized in that, The nitrobenzene diazonium salt is p-nitrobenzene tetrafluoroborate diazonium salt or 4-nitrobenzene diazonium chloride.

9. Antimony nanopowder obtained by the method for preparing antimony nanopowder according to any one of claims 1-8.

10. The application of a method for preparing antimony nanopowder as described in any one of claims 1-8 in reducing the toxicity of antimony nanopowder.

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