Preparation method of gold (I) albumin nanoparticles and application of gold (I) albumin nanoparticles in antitumor activity

Abstract

The invention discloses a preparation method of gold (I) albumin nanoparticles and application of the gold (I) albumin nanoparticles in antitumor activity. Belongs to the field of medicine, and the preparation method comprises the following steps: dissolving BSA in ultrapure water, titrating with N sodium hydroxide until the pH value is 8.5, dropwise adding an ethanol solution of a gold (I) compound, dropwise adding absolute ethyl alcohol, adding 8% glutaraldehyde, stirring, removing the free gold (I) compound and ethanol through dialysis, centrifuging for three times to obtain purified nano-particles, and drying to obtain the gold (I) compound. And freeze-drying to obtain the nanoparticle freeze-dried powder. According to the invention, the input ratio of BSA to the gold (I) compound is determined through preparation of the nanoparticles, the characterization of TEP NPs shows that the particle size has good stability, and the release behavior is shown under the pH condition close to the tumor microenvironment. The result shows that the TEP NPs can retain most of the gold (I) compounds in the nanoparticles, and after the nanoparticles are accumulated at the tumor site through the ERP effect, a large amount of the gold (I) compounds are released to achieve the tumor cell killing effect of the nanoparticles.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and relates to a preparation method of gold (I) albumin nanoparticles and the application of the nanoparticles in anti-tumor activity. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary liver cancer. The development of HCC is a complex multi-step process involving persistent inflammatory damage, including hepatocyte necrosis and regeneration, accompanied by fibrosis deposition. The risk of HCC is associated with the development of cirrhosis and increases with the progression of liver function damage. In HCC, viral pathogenic factors, including ROS, nitrous oxide species, detoxification system inactivation, telomere shortening, DNA methylation, and replication stress in the TME, all contribute to genomic instability. There are five treatments that can extend the life expectancy of HCC patients: surgical resection, liver transplantation, radiofrequency ablation, chemoembolization, and multikinase inhibitor sorafenib. The disadvantages are that conventional chemotherapy has poor bioavailability, low therapeutic index, and many side effects. Although the molecular targeted drugs that have emerged in recent years, such as sorafenib and lenvatinib, have fewer side effects than chemotherapy drugs, the therapeutic effect of these drugs is poor due to the occurrence of tumor resistance or other reasons. Therefore, the development of a new drug has become a difficulty in the current field of tumor treatment. For metal drugs, although recent studies have shown that gold compounds have shown great potential in anti-tumor effects, how to prevent gold compounds from being destroyed by biological macromolecules and endogenous substances in the body has always been a difficult problem that needs to be overcome in the field of metal anti-cancer drugs. Summary of the invention

[0003] In view of the above problems, the present invention aims to provide a method for preparing gold (I) albumin nanoparticles and their application in anti-tumor activity.

[0004] The technical scheme of the present invention is: a method for preparing gold (I) albumin nanoparticles according to the present invention, wherein the gold (I) albumin nanoparticles include a gold (I) compound and blank albumin nanoparticles, and the gold (I) compound includes a nitrogen heterocyclic carbene gold (I) compound and a phosphine ligand gold (I) compound;

[0005] The synthetic route of the nitrogen heterocyclic carbene gold (I) compound:

[0006] Step (a): thiamine, sodium hydroxide, react at room temperature for 2-7 days;

[0007] Step (b): Formamide, heated to reflux for 3 hours;

[0008] Step (c): NaH, ethyl bromide, tetrahydrofuran, heating under reflux for 2 hours;

[0009] Step (d): acetonitrile, reflux for 3-5 days;

[0010] Step (e): silver oxide and acetonitrile, under nitrogen protection, react at room temperature in the dark overnight; gold (I) chloride and sodium bromide, under nitrogen protection, react at room temperature in the dark for 4 hours;

[0011] Step (f): sodium bromide, nitrogen protection, and light-proof at room temperature for 6 hours;

[0012] The specific synthesis route is as follows:

[0013]

[0014] The synthetic route of the phosphine ligand gold (I) compound:

[0015] Step (a): dichloromethane, nitrogen protection, room temperature and dark reaction for 2.5 hours;

[0016] Step (b): dichloromethane, nitrogen protection, room temperature and dark for 4 hours;

[0017] The specific synthesis route is as follows:

[0018]

[0019] The preparation method of Au4 is as follows: weigh 100 mg of gold (I) chloride (dimethyl sulfide) in a 50 mL flask, add dichloromethane to dissolve, then weigh 89.4 mg of triphenylphosphine, add 4 mL of DCM, and react at room temperature in the dark for 2.5 hours under nitrogen protection; after the reaction, spin dry the reaction solution, add dichloromethane to dissolve the white solid, and then add n-hexane until no solid precipitates; let stand for 1 hour and then filter, wash the filter cake with n-hexane and transfer it to a flask and dry it under vacuum to obtain a white solid;

[0020] The preparation method of Au5 is as follows: 50 mg of dimethyl sulfide gold (I) chloride is weighed into a 50 mL flask, dichloromethane is added to dissolve, 98.4 mg of 1,3,5-triaza-7-phosphaadamantane is weighed, 5 mL of DCM is added, and the mixture is reacted at room temperature in the dark for 4 hours under nitrogen protection; after the reaction is completed, the reaction solution is spin-dried and dried under vacuum conditions to obtain a white solid.

[0021] Furthermore, the preparation steps of the blank albumin nanoparticles are as follows: first, 40 mg of bovine serum albumin is dissolved in 1 mL of ultrapure water, and the solution is titrated to a pH value of 8.5 using 0.1 N sodium hydroxide, and then an ethanol solution is added dropwise after stirring, and then 10 μL of 8% glutaraldehyde is added, and after stirring for 16 hours, the ethanol is removed by dialysis, and then purified nanoparticles are obtained after 3 times of centrifugation, and finally freeze-dried to obtain nanoparticle freeze-dried powder, i.e., blank albumin nanoparticles;

[0022] Wherein, the stirring condition is: 600rpm;

[0023] The centrifugation conditions were: 5000 rpm, 10 minutes.

[0024] Furthermore, the preparation steps are as follows: first, 40 mg of BSA is dissolved in 1 mL of ultrapure water, and the solution is titrated to a pH value of 8.5 using 0.1 N sodium hydroxide, and then an ethanol solution of a gold (I) compound is added dropwise after stirring, and then anhydrous ethanol is added dropwise, and then 10 μL of 8% glutaraldehyde is added, and after stirring for 16 hours, the free gold (I) compound and ethanol are removed by dialysis, and then purified nanoparticles are obtained after 3 times of centrifugation, and finally freeze-dried to obtain nanoparticle freeze-dried powder; namely, gold (I) albumin nanoparticles;

[0025] Wherein, the stirring condition is: 600rpm;

[0026] The centrifugation conditions were: 5000 rpm, 10 minutes.

[0027] Furthermore, 1 mg of gold (I) albumin nanoparticles was weighed and dissolved in 1 mL of ultrapure water to prepare a 1 mg / mL nanoparticle aqueous solution. After ultrasonication in a water bath for 1 minute, the particle size distribution and Zeta potential of the nanoparticles were measured using a Malvern particle size analyzer.

[0028] Furthermore, the in vitro antiproliferative activity detection process of gold (I) albumin nanoparticles is:

[0029] (1) Cell plating: HepG2, Hepa1-6, Hep3B, and NCM460 cells in the logarithmic growth phase were digested with trypsin and centrifuged. The supernatant was discarded, and the cells were evenly pipetted with complete culture medium and counted. After adjusting the cell density, they were inoculated into 96-well plates with 100 μL of complete culture medium per well, and cultured in a 37°C incubator overnight until the cells adhered to the wall;

[0030] (2): Cell administration: Prepare different concentrations of compounds, nanoparticles and positive drug working solutions, and the final drug concentration in each well is 0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM. At the same time, 0.1% DMF or complete culture medium is added to the negative control group, and finally 200μL of complete culture medium is added to each well; the cells are cultured in a 37°C incubator for 72 hours;

[0031] (3): MTT method to detect antiproliferative activity: 20 μL MTT / well was added to a 96-well plate, and the plate was cultured in a 37°C incubator. After 4 hours, the supernatant was discarded, 200 μL DMSO was added, and the plate was shaken for 10 minutes. The absorbance at 570 nm was detected using an enzyme-labeled instrument. The cells without drug addition were used as the blank control group, and 4 replicate wells were set up to calculate the cell viability.

[0032] Cell viability (%)=(OD d-OD 0) / (OD c-OD 0)×100%

[0033] Wherein OD d: absorbance value of drug administration group, OD 0: absorbance value of zero adjustment well, OD c: absorbance value of negative control group.

[0034] Furthermore, the cell culture uses DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and an incubator at 37° C. and 5% CO 2 as the culture environment.

[0035] Further, the centrifugal conditions are: 1000 rpm, 5 minutes;

[0036] The 20 μL MTT / well has a concentration of 5 mg / mL and is dissolved in PBS.

[0037] Further, the gold (I) albumin nanoparticle size stability detection process is: using dynamic light scattering to analyze the size stability of the nanoparticles in PBS solution for 72 hours;

[0038] The nanoparticles can be obtained as solid powder after freeze-drying. The PBS solution prepared with the freeze-dried powder of the nanoparticles is stored at room temperature at different times, and the particle size of the nanoparticles is measured uniformly at last. According to the last measurement time, the aqueous solution of the freeze-dried powder of the nanoparticles is prepared 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, and 0 hours in advance, respectively, and the particle size is detected by a machine at the measurement time.

[0039] Furthermore, the volume of the aqueous solution is: 1 mg / mL.

[0040] Furthermore, the gold (I) albumin nanoparticles prepared by the preparation method are used in anti-tumor activity.

[0041] Principle: One of the important characteristics of cell carcinogenesis is redox imbalance. When reactive oxygen species (ROS) accumulate to a certain level in normal cells and exceed the load of the cell's antioxidant system, it will cause malignant transformation of normal cells. In addition, tumor cells require higher ROS levels to maintain their malignant levels, so increasing ROS generation or reducing antioxidant defense to make intracellular ROS levels reach the toxic threshold is an effective treatment strategy. Thioredoxin reductase (TrxR) is an important member of the redox system and is related to multiple signal transduction pathways of cellular redox balance. In addition, the expression of TrxR is generally increased in liver cancer cells, so targeting TrxR is an important strategy for liver cancer treatment. Inhibition of TrxR activity will cause intracellular redox imbalance, leading to increased ROS levels and triggering a series of downstream reactions. Increased ROS levels will cause endoplasmic reticulum stress (ERS), affecting the normal function of the endoplasmic reticulum, leading to misfolding of endoplasmic reticulum proteins, and producing a ROS cascade reaction. ER stress can stimulate damage-associated molecular patterns (DAMPs), thereby causing immunogenic cell death (ICD); in addition, redox homeostasis disorders play an important role in ferroptosis. When the lipid peroxide (LPO) produced by ferroptosis exceeds the buffering capacity provided by the defense system, it will lead to excessive accumulation of LPO on the cell membrane and cause cell death.

[0042] The invention uses bovine serum albumin as a carrier to construct a gold (I) albumin nano-delivery system, prepares nanoparticles of different gold compounds, determines the optimal feed ratio of the albumin carrier to the gold (I) compound by screening, then detects the anti-tumor activity of the gold (I) albumin nanoparticles on liver cancer cells by MTT method, selects human normal intestinal epithelial cells NCM460 as a selective control, and screens out triethylphosphine gold (I) albumin nanoparticles (TEP NPs) with significant anti-proliferation activity on different liver cancer cells (HepG2, Hepa1-6, Hep3B); transmission electron microscopy results show that the TEP NPs have a spherical structure, a uniform particle size distribution, and no adhesion phenomenon; the particle size stability of the TEP NPs indicates that the particle size remains stable within 72 hours; and the different release behaviors of the TEP NPs under pH 4.7 and pH 7.0 conditions indicate that the TEP NPs have a significant anti-proliferation activity in the tumor microenvironment (Tumor The results of matrix-assisted laser desorption / ionization-time of flight-mass spectrometry (MALDI-TOF-MS) showed that compared with BSA, the molecular weight of TEP NPs increased by the same molecular weight as that of two molecules of triethylphosphine gold, indicating that BSA and chloro(triethylphosphine)gold(I) were stably covalently bound.

[0043] The present invention evaluates the in vivo therapeutic effect of TEP NPs and explores its anti-liver cancer mechanism; according to the in vivo results, compared with the single TEP treatment group, TEP NPs can improve the therapeutic effect of the drug, and the inductively coupled plasma-mass spectrometry (ICP-MS) results of tumor tissue show that TEP NPs can increase the accumulation of gold compounds in tumor tissue; and then through in vitro exploration, it was found that the activity of TrxR decreased significantly after TEP NPs treatment, resulting in redox imbalance and increased intracellular ROS levels, which on the one hand caused the mitochondrial membrane potential to decrease and cause cell apoptosis, and on the other hand, induced the release of cellular DAMPs by inducing endoplasmic reticulum stress and activated the response of immune cells; in addition, TEP NPs can also inhibit the expression of glutathione peroxidase (Glutathione peroxidase4, GPX4), increase the accumulation of intracellular LPO and induce cell ferroptosis; according to the immunohistochemistry and immunofluorescence results of tumor tissue, TEP The expression of TrxR and GPX4 in the tumor tissues of mice in the NPs administration group was significantly inhibited, while the expression of apoptosis-related proteins and immune markers was significantly upregulated.

[0044] The beneficial effects of the present invention are as follows: the present invention determines the input ratio of BSA and gold (I) compound through the prescription study of nanoparticle preparation, and then prepares nanoparticles of different gold (I) compounds for subsequent anti-tumor activity study; MTT results show that TEP NPs have the best anti-tumor activity, so it is selected as the object of subsequent study; MALDI-TOF-MS results show that TEP and BSA are combined in a stable covalent binding manner, and the characterization experiment of TEP NPs shows that its particle size has good stability within 72 hours and shows good release behavior under pH conditions close to the tumor microenvironment; the results show that TEPNPs can retain most of the gold (I) compound inside the nanoparticles, avoid premature exposure during its in vivo transportation process and thus avoid reaction with endogenous macromolecules rich in thiol groups, and after the nanoparticles are accumulated in the tumor site through the ERP effect, a large amount of gold (I) compound is released to achieve its tumor cell killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a preparation flow chart of the present invention;

[0046] Figure 2The figures are characterization diagrams of TEP NPs in the embodiments of the present invention; wherein, (A) is an electron microscope image of TEP NPs; (B) is the particle size stability of TEP NPs in PBS solution for 72 hours; (C) is the release of gold compounds from TEP NPs under different pH conditions; (D, E) are MALDI-TOF-MS spectra of TEP NPs and BSA; Scale bar: 100 nm;

[0047] Figure 3 Schematic diagram of the in vivo anti-tumor effect of TEP NPs in the embodiment of the present invention; (A), schematic diagram of xenograft tumor model; (B), photo of mouse tumor 14 days after drug administration; (C), mouse tumor weight; (D), change of mouse tumor volume during drug treatment; (E), change of mouse weight during drug treatment; (F), Au content in mouse tumor tissue;

[0048] Figure 4 Schematic diagram of gene expression differences of samples in the embodiment of the present invention;

[0049] Figure 5 is a cluster heat map of differentially expressed genes in the embodiment of the present invention;

[0050] Figure 6 Schematic diagram of TEP NPs-induced mitochondrial damage in an embodiment of the present invention; (A) After 6 hours of TEP NPs treatment, the changes in mitochondrial membrane potential of HepG2 cells were detected by JC-1 probe; (B, C) After 6 hours of TEP NPs treatment, the mitochondrial membrane potential level was detected by flow cytometry;

[0051] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0052] Figure 7 Schematic diagram of TEP NPs inhibiting TrxR activity of HepG2 cells in an embodiment of the present invention; (A) Inhibitory effect of TEP NPs on intracellular TrxR enzyme activity after 24 hours of action; (B) Probe labeling method to detect TrxR expression after 24 hours of TEP NPs treatment; (C) DTNB method to detect pure enzyme inhibitory activity of TEP NPs; (D, E) Protein immunoblot analysis to detect TrxR expression after 24 hours of TEPNPs treatment;

[0053] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0054] Figure 8Schematic diagram of the effect of TEP NPs on intracellular ROS level in an embodiment of the present invention; (A), DCFH-DA probe detects the intracellular ROS level of HepG2 cells after incubation with TEP NPs for 6 hours; (B), DHE probe detects the intracellular ROS level of HepG2 cells after incubation with TEP NPs for 6 hours; (C, D), flow cytometry detects the intracellular ROS level of HepG2 cells;

[0055] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0056] Fig. 9 : It is the effect diagram of ICD effect induced by TEP NPs in the embodiment of the present invention; (A, B), immunofluorescence detection of intracellular localization of CRT and HMGB1 after 24 hours of TEP NPs and oxaliplatin treatment; (C, E), flow cytometry detection of intracellular migration of CRT after TEP NPs and oxaliplatin treatment; (D, F), flow cytometry detection of HMGB1 content; (G), extracellular ATP release after administration of TEP NPs and oxaliplatin; (H, I, J), protein immunoblot analysis detection of CRT and HMGB1 expression 24 hours after administration of TEP NPs and oxaliplatin;

[0057] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:10μm;

[0058] Fig.10 Schematic diagram of TEP NPs inducing DCs maturation in an embodiment of the present invention; (A, B), flow cytometry detection of DCs maturation;

[0059] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3;

[0060] Fig.11 Schematic diagram of TEP NPs causing cell ferroptosis in an embodiment of the present invention; (A) MTT method to detect the study of Ferrostatin-1 inhibiting ferroptosis; (B, C) Western blotting analysis to detect the expression of GPX4, a key protein in ferroptosis; (D) lipid peroxide probe to detect intracellular LPO peroxide levels;

[0061] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0062] Fig.12 Schematic diagram of TEP NPs-induced cell apoptosis in an embodiment of the present invention; (A, B, C), flow cytometry analysis of cell apoptosis 48 hours after administration;

[0063] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3;

[0064] Fig.13 Schematic diagram of immunofluorescence and immunohistochemical analysis of tumor tissue sections in the embodiment of the present invention; (A, C), HE and Tunel staining results of tumor tissue; (B), immunohistochemical analysis of AIF expression in tumor tissue;

[0065] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0066] Fig.14 Schematic diagram of immunofluorescence analysis of tumor tissue sections in an embodiment of the present invention; (A, C) immunofluorescence analysis of CRT and (B, D) TrxR in tumor tissue;

[0067] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0068] Fig.15 Schematic diagram of immunohistochemical analysis of tumor tissue sections in an embodiment of the present invention; (A), immunohistochemical analysis of CD4, CD8 and CD86 in tumor tissue; (B), immunohistochemical analysis of GPX4 in tumor tissue;

[0069] Significant difference*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=3,Scale bar:100μm;

[0070] Fig.16 Schematic diagram of the hypothesis of the mechanism of action of TEP NPs in an embodiment of the present invention. DETAILED DESCRIPTION

[0071] The specific technical solutions of the present invention are further described in detail below in conjunction with specific examples.

[0072] 1. The role of gold (I) compounds in the treatment of tumors. HSA-Au (III) nanoparticles prepared with HSA as a carrier have uniform particle size, good stability, and certain pH-responsive release behavior, which can improve treatment efficiency, bioavailability and targeting ability. Albumin appears as a powerful macromolecular carrier in medical treatment and diagnostic applications. Recently, albumin nanoparticles have been used to improve the targeting ability of anticancer drugs and reduce their side effects.

[0073] The present invention uses bovine serum albumin as a carrier to construct a nano delivery system of a gold (I) compound, evaluates the activity of the obtained gold (I) albumin nanoparticles through an in vitro anti-proliferation activity experiment, and characterizes the nanoparticles using a particle size analyzer, a transmission electron microscope, and MALDI-TOF-MS.

[0074] 2. Materials and Methods

[0075] (1): Instruments and reagents: nuclear magnetic resonance spectroscopy, low-resolution mass spectrometry, high performance liquid chromatography (HPLC), rotary evaporator, PCD-11-10 ultrapure water machine, particle size analyzer, inductively coupled plasma mass spectrometer (ICP-MS), glassware, ultrafiltration tube, dichloromethane, methanol, petroleum ether, ethyl acetate, anhydrous ethanol, sodium hydroxide, glutaraldehyde, thin layer chromatography plate, column chromatography silica gel, deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDCl 3), chloro(triethylphosphine)gold(I), bovine serum albumin (BSA), triphenylphosphine and 1,3,5-triaza-7-phosphaadamantane;

[0076] (2): Synthesis and characterization of gold (I) compounds: Au1 in the present invention is a commercial product; the rest are synthesized in this laboratory. The gold compounds and their synthesis routes involved are:

[0077]

[0078] Synthesis route of nitrogen heterocyclic carbene gold (I) compound: (a), thiamine, sodium hydroxide, reaction at room temperature for 2-7 days; (b), formamide, heating and reflux for 3 hours; (c), NaH, ethyl bromide, tetrahydrofuran, heating and reflux for 2 hours; (d), acetonitrile, reflux for 3-5 days; (e), silver oxide (0.6 equivalent), acetonitrile, nitrogen protection, reaction at room temperature in the dark overnight; (dimethyl sulfide) gold (I) chloride (1.0 equivalent), sodium bromide, nitrogen protection, reaction at room temperature in the dark for 4 hours; (f), sodium bromide, nitrogen protection, reaction at room temperature in the dark for 6 hours;

[0079]

[0080] Synthesis route of phosphine ligand gold (I) compound: (a), dichloromethane, nitrogen protection, room temperature and light-proof reaction for 2.5 hours; (b), dichloromethane, nitrogen protection, room temperature and light-proof reaction for 4 hours;

[0081] Synthesis and characterization: Au4: Weigh 100 mg of (dimethyl sulfide) gold (I) chloride in a 50 mL flask, add a small amount of dichloromethane to dissolve, then weigh 89.4 mg of triphenylphosphine, add 4 mL of DCM, and react at room temperature in the dark for 2.5 hours under nitrogen protection; after the reaction, spin dry the reaction solution, add an appropriate amount of dichloromethane to just dissolve the white solid, and then add n-hexane until no solid precipitates; let stand for 1 hour and then filter, wash the filter cake with n-hexane and transfer it to a flask and dry it under vacuum to obtain 130 mg of white solid with a yield of 77.4%. 1H NMR(500MHz,DMSO-d 6)δ7.68–7.51(m,15H,ArH).13CNMR(126MHz,DMSO-d 6).13C NMR(126MHz,DMSO-d 6) δ134.28(d,J=13.78Hz), 132.80(d,J=2.74Hz), 130.15(d,J=11.80Hz), 128.71(d,J=62.26Hz);

[0082] Au5: Weigh 50 mg of dimethyl sulfide gold (I) chloride in a 50 mL flask, add a small amount of dichloromethane to dissolve, then weigh 98.4 mg of 1,3,5-triaza-7-phosphaadamantane, add 5 mL of DCM, and react at room temperature in the dark under nitrogen protection for 4 hours; after the reaction, spin dry the reaction solution and dry it under vacuum to obtain 128 mg of white solid with a yield of 98.3%. 1H NMR (500MHz, DMSO-d 6) δ4.52 (d, J=12.68Hz, 3H, PCH 2N), 4.37 (d, J=12.92Hz, 3H, PCH 2N), 4.35 (d, J=1.92Hz, 6H, NCH 2N). 13C NMR (126MHz, DMSO-d 6) δ72.27 (d, J = 8.61Hz, 3C, PCH2N), 51.35 (d, J = 23.47Hz, 3C, NCH 2N).

[0083] (3): Preparation of blank albumin nanoparticles: First, 40 mg of bovine serum albumin (BSA) was dissolved in 1 mL of ultrapure water, and the solution was titrated to pH = 8.5 using 0.1 N sodium hydroxide (NaOH). An ethanol solution was slowly added dropwise under stirring (600 rpm), and then 10 μL of 8% glutaraldehyde was added. After stirring for 16 hours, the ethanol was removed by dialysis, and then the purified nanoparticles were obtained after 3 times of centrifugation (5000 rpm, 10 minutes), and finally the nanoparticle freeze-dried powder was obtained by freeze drying;

[0084] (4): Preparation of gold (I) albumin nanoparticles: Gold (I) albumin nanoparticles were prepared by desolvation technology; first, 40 mg BSA was dissolved in 1 mL ultrapure water, and the solution was titrated to pH = 8.5 using 0.1 N sodium hydroxide (NaOH), and then the ethanol solution of the gold (I) compound was slowly added dropwise under stirring (600 rpm) (some gold compounds have poor solubility in ethanol solution, and DMSO can be added to promote their dissolution), followed by slow addition of anhydrous ethanol, and then 10 μL of 8% glutaraldehyde was added. After stirring for 16 hours, the free gold (I) compound and ethanol were removed by dialysis, and then purified nanoparticles were obtained after 3 times of centrifugation (5000 rpm, 10 minutes), and finally freeze-dried to obtain nanoparticle freeze-dried powder;

[0085] (5): Determination of the particle size and Zeta potential of gold (I) albumin nanoparticles: 1 mg of gold (I) albumin nanoparticles was weighed using an electronic balance and dissolved in 1 mL of ultrapure water to prepare a 1 mg / mL nanoparticle aqueous solution. After ultrasonication in a water bath for 1 minute, the particle size distribution and Zeta potential of the nanoparticles were determined using a Malvern particle size analyzer;

[0086] (6): In vitro antiproliferative activity detection of gold (I) albumin nanoparticles: All cells used in the present invention were purchased from a cell bank; cells were cultured using DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, in an incubator at 37°C and 5% CO2 as the culture environment;

[0087] (1.1) Cell plating: HepG2, Hepa1-6, Hep3B, and NCM460 cells in the logarithmic growth phase were digested with trypsin and centrifuged (1000 rpm, 5 minutes). The supernatant was discarded, and the cells were evenly pipetted with complete medium and counted. After adjusting the cell density, they were inoculated into 96-well plates with 100 μL complete medium per well, and cultured in a 37°C incubator overnight until the cells adhered to the wall;

[0088] (1.2) Cell administration: Compounds, nanoparticles and positive drug (Auranofin) working solutions of different concentrations were prepared, and the final drug concentration in each well was 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. Meanwhile, 0.1% DMF or complete medium was added to the negative control group, and finally 200 μL of complete medium was added to each well; cells were cultured in a 37°C incubator for 72 hours;

[0089] (1.3) Antiproliferative activity was detected by MTT assay: 20 μL MTT / well (5 mg / mL, dissolved in PBS) was added to a 96-well plate and cultured in a 37°C incubator. After 4 hours, the supernatant was discarded and 200 μL DMSO was added. After slow shaking for 10 minutes, the absorbance at 570 nm was detected using an enzyme-labeled instrument.

[0090] The cells without drug addition were used as the blank control group, and 4 replicate wells were set up to calculate the cell viability (Cell viability, %), Cell viability (%) = (OD d-OD 0) / (OD c-OD 0) × 100%

[0091] OD d: absorbance value of the drug-treated group, OD 0: absorbance value of the zero-adjusted well, OD c: absorbance value of the negative control group;

[0092] (7): Particle size stability of gold (I) albumin nanoparticles: Dynamic light scattering was used to analyze the particle size stability of the nanoparticles in PBS solution for 72 hours; the above nanoparticles were freeze-dried to obtain solid powders, and the PBS solution prepared for the freeze-dried powders of the nanoparticles was stored at room temperature at different times, and the particle size of the nanoparticles was finally measured uniformly; according to the last measurement time, aqueous solutions (1 mg / mL) of the freeze-dried powders of the nanoparticles were prepared 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, and 0 hours in advance, and the particle size was detected by a machine at the measurement time;

[0093] (8): Release of gold (I) compounds from gold (I) albumin nanoparticles:

[0094] (1.1) Preparation of pH 4.7 and pH 7.0 buffer solutions: Preparation of 0.2 mol / L sodium dihydrogen phosphate solution: weigh 42.98 g sodium dihydrogen phosphate (Na2HPO4), add 600 mL ultrapure water, and stir until completely dissolved;

[0095] Preparation of 0.1 mol / L citric acid solution: weigh 21.0 g citric acid, add 600 mL ultrapure water, and stir until completely dissolved;

[0096] Preparation of pH 4.7 and pH 7.0 buffer:

[0097] While stirring with a stirrer, add the citric acid solution to the disodium hydrogen phosphate solution, and use a pH meter to detect the pH of the solution until the pH value shows pH 4.7 / pH 7.0;

[0098] (1.2) Release experiment of gold (I) compounds in albumin nanoparticles:

[0099] Nanoparticles containing 2.5 mg of gold (I) compound were dissolved in buffers of different pH values ​​and transferred to a dialysis bag, which was then placed in 100 ml of pH 4.7 or pH 7.4 buffer for dialysis. 1 ml of buffer was taken from a beaker at 3 hours, 6 hours, 12 hours, 2 hours, 48 ​​hours, and 72 hours, and 1 ml of the corresponding buffer was added to the beaker. The gold content released from the dialysis bag was determined by ICP-MS.

[0100] 3. Experimental Results and Discussion

[0101] (1): Formulation of gold (I) albumin nanoparticles: In the preparation process of gold (I) albumin nanoparticles, the addition ratio of BSA and gold (I) compound is the key factor affecting the drug loading of nanoparticles; in order to explore at what ratio of BSA and gold (I) compound to obtain nanoparticles with suitable particle size and optimal drug loading, verification and analysis were then carried out; BSA and Au1 (TEP) compound were added to the reaction at a molar ratio of 1:3, 1:4, 1:5 and 1:6, and the corresponding nanoparticles (TEP NPs) were obtained according to the preparation method of nanoparticles; the particle size and Zeta potential of blank nanoparticles and nanoparticles with different ratios were measured using a Malvern particle size analyzer, and the drug loading was detected by ICP-MS. The results are shown in Table 1;

[0102] As shown in the results in Table 1, when the input ratio of BSA to gold (I) compound is 1:3, 1:4, and 1:5, the particle size of gold (I) nanoparticles decreases with the increase of the ratio, and is smaller than the blank BSA nanoparticles without drug loading. This may be because the addition of gold compounds makes the internal structure of the nanoparticles more compact, so the particle size shows a trend of gradually decreasing; however, when the ratio increases to 1:6, the particle size of the nanoparticles increases to about 160nm, which has exceeded the particle size range of the nanoparticles. Excessive addition of compounds will cause protein aggregation, which increases the particle size of the nanoparticles; from the results of Zeta potential, the potential of drug-loaded TEP NPs is lower than that of blank BSA nanoparticles. The potential of NPs indicates that the addition of gold (I) compounds changes the surface potential of the nanoparticles. It is the combination of gold (I) compounds and amino acid residues on the surface of the protein that changes the surface potential of albumin. The drug loading results show that the drug loading increases with the increase of the input ratio, showing a positive correlation with the dosage. In summary, when the input ratio of BSA to gold (I) compounds is 1:5, nanoparticles with a compact structure, a qualified particle size range, a certain surface potential, and the largest drug loading can be obtained.

[0103] Table 1 Particle size, Zeta potential and drug loading of nanoparticles with different proportions

[0104]

[0105] (2): Preparation of gold (I) albumin nanoparticles with different gold compounds: Different gold (I) compounds have different anti-tumor properties due to their different structural characteristics. In order to explore the anti-tumor activity of nanoparticles of different compounds, nanoparticles of different gold compounds (Au1-Au5) were prepared, and their particle size, Zeta potential and drug loading were measured, as shown in Table 2. The results showed that the particle sizes of Au1NPs, Au2NPs and Au3NPs were all within 100nm, all belonging to the nanometer level. The particle sizes of Au4NPs and Au5NPs were 139.2nm and 161.4nm, respectively, which exceeded the nanometer level, which was due to the charge carried by the compound. In addition, the drug loading results showed that all five nanoparticles showed appropriate drug loading.

[0106] Table 2 Particle size, Zeta potential and drug loading of different gold (I) compound nanoparticles

[0107]

[0108] (3): Antitumor activity of gold (I) albumin nanoparticles: The in vitro cytotoxicity of five gold (I) compounds (Au1-Au5) was detected in HCC cell lines HepG2, Hepa1-6, Hep3B and normal cell line NCM 460 using the MTT method, with auranofin as the positive control. The results were analyzed using GraphPad Prism9 software and the corresponding IC50 values ​​were calculated. As shown in Table 3, it was found that the nanoparticles corresponding to Au1 (TEP NPs) had significant cytotoxicity in various liver cancer cells, and its activity in liver cancer cells (HepG2: 3.71 μM, Hepa1-6: 1.16 μM, Hep3B: 2.71 μM) was higher than that in normal cells (NCM460: 6.72 μM). The results showed that compared with the inhibitory activity of TEP NPs on normal cells NCM 460, TEP NPs had better selectivity for HepG2 cells.

[0109] Table 3 Antiproliferative activity of compounds and nanoparticles in different tumor cells (unit: μM)

[0110]

[0111] [a]nd means that it shows no significant antiproliferative activity against cells;

[0112] (4): Characterization of gold (I) albumin nanoparticles: Figure 2 As shown, to test the stability of gold (I) albumin nanoparticles, dynamic light scattering was used to analyze the particle size stability of the nanoparticles in PBS solution for 72 hours; the results are shown in Figure 2 As shown in (B), the particle size of the nanoparticles remained between 32-35 nm within 72 hours, indicating that the particle size of gold (I) albumin nanoparticles can maintain a stable particle size at room temperature and remain in a relatively stable state without particle agglomeration. In order to confirm the binding mode of gold (I) compounds and BSA, the mass spectra of gold (I) albumin nanoparticles TEP NPs and unloaded albumin nanoparticles were analyzed using MALDI-TOF-MS spectra. Figure 2 As can be seen in (D\E), compared with unloaded albumin nanoparticles, the molecular weight of TEP NPs increased by 628, which is equivalent to the molecular weight of two molecules of TEP minus two molecules of chlorine atoms, and no peaks of other molecular weights were observed in the mass spectrum of TEP NPs, indicating that TEP and BSA were covalently bound, resulting in a change in molecular weight;

[0113] (5): Release behavior of gold (I) albumin nanoparticles: To determine the possibility of targeted delivery of gold (I) albumin nanoparticles, the release behavior of gold (I) albumin nanoparticles was studied by simulating blood circulation and tumor microenvironment. The release amount of gold (I) albumin nanoparticles in pH 4.7 and pH 7.0 buffers was detected by ICP-MS. The results are shown in Figure 5. Figure 2 (C) shows that only 10% of the gold (I) compound was released within 72 hours in a buffer environment of pH 7.0; however, when the pH value was 4.7, about 82% of the gold compound was released from the nanoparticles; the results show that gold (I) albumin nanoparticles do not release a large amount of gold (I) compounds under physiological conditions, but in the weakly acidic tumor microenvironment, the gold (I) compound is released under this condition, increasing its concentration in the tumor site and improving its effect;

[0114] In summary, the input ratio of BSA and gold (I) compounds was determined through the preparation of nanoparticles, and then nanoparticles with different gold (I) compounds were prepared for subsequent antitumor activity; MTT results showed that TEP NPs had the best antitumor activity, and it was selected as the subsequent object; MALDI-TOF-MS results showed that TEP and BSA were combined in a stable covalent manner, and the characterization experiment of TEP NPs showed that its particle size had good stability within 72 hours, and showed good release behavior under pH conditions close to the tumor microenvironment; the results showed that TEPNPs could retain most of the gold (I) compounds inside the nanoparticles, avoiding premature exposure during its transport in the body and thus avoiding reaction with endogenous macromolecules rich in thiol groups. After the nanoparticles accumulated in the tumor site through the ERP effect, a large amount of gold (I) compounds were released to achieve its tumor cell killing effect; in summary, it was proved that TEP NPs had certain antitumor potential;

[0115] IV. Anti-liver cancer activity of gold (I) albumin nanoparticles in vivo and in vitro:

[0116] (1) Gold (I) albumin nanoparticles TEP NPs with good in vitro activity were obtained, and the feasibility of the gold (I) albumin nanodelivery system was confirmed by characterization; in order to evaluate the anti-tumor activity of the nanoparticles, C57BL / 6 mice were used as experimental subjects, and the in vivo anti-tumor ability of the nanoparticles was verified by establishing a mouse tumor-bearing model; in order to explore the mechanism of action of the nanoparticles, it is intended to reveal the effects of the nanoparticles in inhibiting TrxR activity, inducing ROS generation, affecting mitochondrial membrane potential, inducing apoptosis, ICD and ferroptosis at the in vitro level, and further support the above-mentioned effects of the nanoparticles by immunohistochemistry and immunofluorescence analysis of tumor tissues;

[0117] (2) Materials and methods

[0118] (2.1) Instruments and reagents: BS120S electronic balance, TGL-16C desktop high-speed centrifuge, IKA VortexGenius 3 vortex mixer, PCD-11-10 ultrapure water machine, Leica DMi8 fluorescence microscope, TU-100 heated metal bath, JY88-IIN ultrasonic cell disruptor, Tocan gel analysis system, electric constant temperature water bath, BCD-179D refrigerator, Redu PT-6100 microplate reader, DYY-6C electrophoresis instrument, BCD-186KB refrigerator, Olympus EM208S, Accuri C6 personal flow cytometer, TCS SP8 laser confocal microscope, clean bench, carbon dioxide incubator, manual single-channel adjustable pipette, manual multi-channel adjustable pipette, cell counting chamber, TRFS-green, TrxR activity detection kit, reduced coenzyme II tetrasodium salt, auranofin, concentrated nitric acid, concentrated hydrochloric acid, hydrogen peroxide, N,N-dimethylformamide (DMF), chemiluminescence ultrasensitive colorimetric kit, PVDF membrane, BCA protein quantitative assay kit, protein marker, rabbit, mouse secondary antibody and fluorescent secondary antibody, ferroptosis inhibitor (Ferrostatin-1), MTT, mitochondrial membrane potential detection probe (JC-1), Annexin V-FITC / PI apoptosis detection kit, ATP detection kit, reactive oxygen species detection kit, serum-free cell freezing solution, penicillin x streptomycin double antibody, trypsin cell digestion solution, fetal bovine serum (Fetal bovine serum, FBS), DMEM (Dulbecco's modified eagle medium), PBS (phosphate buffered solution), rmGM-CSF and IL-4, CRT primary antibody, HMGB1 primary antibody, CD80, CD86, CD11c, etc.;

[0119] (2.2) In vivo tumor bearing experiment: Mice were kept under constant temperature and humidity conditions, with no restriction on food and water. Hepa1-6 cells inoculated in a 10 cm cell culture dish were digested, resuspended with PBS and counted, and the cell density was adjusted to 1×10 6 The mice were inoculated with 100 μL of 1% iodine per mouse under the left armpit. The size of the tumor was monitored and the average tumor volume was greater than 100 mm. 3At 14:00 pm, the mice were randomly divided into 5 groups, each group was respectively: compound TEP group (5 mg / kg), nanoparticle TEP NPs group (5 mg / kg), oxaliplatin group (5 mg / kg), BSA group and model group; administration method: tail vein injection, administration frequency: once every two days, and the weight of mice and tumor size were recorded, and the volume calculation formula was: V (mm 3) = a 2 × (b / 2), (a: short diameter; b: long diameter; V: tumor volume); after two weeks, the mice were killed after blood was collected from the eyeballs, and the tumors and main organs were removed for subsequent experiments;

[0120] (2.3) ICP-MS detection: About 50 mg of mouse tumors from the compound TEP group and nanoparticle TEP NPs group were cut and chopped with scissors. 100 μL of concentrated nitric acid was added and heated at 95°C for about 1 hour. Then 50 μL of 30% hydrogen peroxide was added and heated at 95°C for about 1 hour. 75 μL of concentrated hydrochloric acid was added and heated at 95°C until about 50 μL of the solution remained. The volume was fixed to 200 μL and the gold content was detected by ICP-MS. Finally, the gold content in the tumor group was calculated.

[0121] (2.4) Intracellular TrxR activity detection: The cell treatment was the same as (2.6), and the drug was administered the next day. A control group and groups with different doses of TEP NPs (1.25, 2.5, and 5 μM) were set up. After 24 hours of drug administration, the cells were collected and operated according to the TrxR activity detection kit, and the protein concentration was detected;

[0122] (2.5) Detection of intracellular enzyme activity with TrxR probe: The cell treatment was the same as (2.6). After the cells attached to the wall, the drugs were administered. A control group, different doses of TEP NPs groups (2.5, 5, 10 μM) and a compound TEP group (10 μM) were set up. After 24 hours of administration, the whole culture was discarded, the cells were washed once with PBS, and TRFS-green staining working solution was added. The cells were stained at 37°C for 30 minutes, the supernatant was discarded, and the cells were washed twice with PBS and photographed with an inverted fluorescence microscope.

[0123] (2.6) Detection of pure TrxR enzyme activity: The pure TrxR enzyme activity was detected by the DTNB method; Dilution of pure TrxR enzyme: Take 95 μL of pure TrxR enzyme, add 690 μL of ddH2O to prepare a diluted enzyme solution, and keep it on ice for later use; Compound configuration: Use pure water to prepare the nanoparticles into a solution of appropriate concentration (so that the final concentration after adding 225 μL of reaction solution is 0.625, 1.25, 2.5, 5, 10 and 20 μM), add 25 μL of enzyme solution and 25 μL of nanoparticle solution to a 96-well plate, incubate at room temperature, and prepare the reaction solution (1 mL of reaction solution) according to Table 4 below;

[0124] Table 4 Preparation of reaction solution for determination of pure enzyme activity of TrxR by DTNB method

[0125]

[0126] The volume of the reaction solution is prepared according to the number of samples. The above reaction solution is 1 mL.

[0127] 225 μL of reaction solution was added to the 96-well plate, and after incubation, 25 μL of DTNB was added, followed by immediate detection of the OD value change at 405 nm;

[0128] (2.7) Western blotting analysis to detect TrxR protein expression level

[0129] (2.7.1) Sample pretreatment and protein content determination: Cell treatment was the same as (2.6) and inoculated in a 10 cm dish. After the cells attached to the wall, the drug was administered. Different groups were treated and incubated for corresponding time. The cells were washed once with PBS, and cell lysis buffer was added. The cells were ground on ice. The cell samples were ultrasonically broken in an ice bath, and then centrifuged at 14000 rpm for 20 minutes. Part of the supernatant was taken to determine the protein concentration according to the instructions. The remaining samples were adjusted to the same concentration with cell lysis buffer according to the protein concentration determined by BCA. Finally, 5× loading buffer was added, mixed thoroughly, and boiled in a metal bath at 100°C for 10 minutes. The protein samples were stored in a -80°C refrigerator for later use.

[0130] (2.7.2) Preparation before electrophoresis: prepare SDS-PAGE gel, electrophoresis buffer solution, transfer buffer solution, TBST buffer solution, etc. according to experimental requirements;

[0131] Loading: Add protein samples and pre-stained protein markers into the lanes;

[0132] Gel electrophoresis: 80V constant voltage electrophoresis until below the boundary between the concentrated gel and the separation gel, then adjust the voltage to 120V;

[0133] Transfer: After electrophoresis is completed, the PVDF membrane is activated with methanol, and the membrane transfer clip is placed in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge. Be careful not to generate bubbles between the PVDF membrane and the gel. After the sponge clip is clamped, constant current transfer is performed, with a current of 200mA for 120 minutes. If the protein molecular weight is small, the transfer time can be appropriately reduced;

[0134] Blocking: Transfer the PVDF membrane to 5% milk (dissolved in TBST buffer) and block for 2 hours at room temperature;

[0135] Wash the membrane: Wash the membrane 3 times with TBST buffer, 10 minutes each time;

[0136] Primary antibody incubation: Add corresponding primary antibody and incubate overnight at 4°C on a shaker;

[0137] Wash the membrane: Wash the membrane 3 times with TBST buffer, 10 minutes each time;

[0138] Secondary antibody incubation: Add the secondary antibody corresponding to the primary antibody and incubate on a shaker at room temperature for 2 hours;

[0139] Development: Prepare developer solution (A solution: B solution = 1:1), develop in a darkroom, and analyze the gray value of the bands using ImageJ software;

[0140] (2.8) ROS generation detection

[0141] (2.8.1) Intracellular ROS probe experiment: After 6 hours of incubation, the cells were washed once with PBS, stained with ROS probe (DCFH-DA or DHE) for 30 minutes, the supernatant was discarded, and the cells were washed twice with PBS and photographed with an inverted fluorescence microscope;

[0142] (2.8.2) Detection of ROS generation by flow cytometry After cell inoculation and drug administration, cells from each group were collected after 6 hours of culture, incubated with ROS probe (DHE) working solution, and detected by flow cytometer. The data were processed using FlowJo software.

[0143] (2.9) Mitochondrial membrane potential detection:

[0144] (2.9.1) Intracellular JC-1 probe experiment: After incubation for 6 hours, wash once with PBS, add JC-1 probe to stain for 30 minutes, discard the supernatant, wash twice with PBS, and take pictures with an inverted fluorescence microscope;

[0145] (2.9.2) Flow cytometry detection of MMP: After incubation for 6 hours, cells from each group were collected and stained with JC-1 probe for 30 minutes before detection by flow cytometer. The data were processed using FlowJo software.

[0146] (2.10) Immunofluorescence detection of HMGB1 and CRT in cells:

[0147] (2.10.1) Cell slides: Sterile cell slides were placed in 24-well plates. HepG2 cells in the logarithmic growth phase were digested with trypsin and counted, then inoculated in the 24-well plates and cultured overnight.

[0148] (2.10.2), drug incubation: control group, TEP NPs group (5 μM) and oxaliplatin group (5 μM)) were incubated for 24 h;

[0149] (2.10.3) Antibody incubation: Fixation: discard the culture medium, wash three times with PBS, add 4% paraformaldehyde to fix for 15 minutes (room temperature); Blocking / permeabilization: wash three times with PBS, 10 minutes each time; add PBS containing 1% BSA + 0.1% Triton, and block at 37°C for 1.5 hours;

[0150] Primary antibody incubation: Wash three times with PBS, 10 minutes each time; add diluted primary antibody working solution (HMGB1 and CRT prepared according to the instructions, 1% BSA as diluent), incubate at 4°C overnight;

[0151] Secondary antibody incubation: Wash three times with PBS the next day, 10 minutes each time, add fluorescent (FITC) labeled secondary antibody working solution, wrap with tin foil, and shake slowly on a shaker at room temperature for 2 hours. Keep away from light during the whole process;

[0152] Nuclear staining: Wash three times with PBS, 10 minutes each time, add DAPI staining solution to the slides for nuclear staining, and incubate at room temperature in the dark for 10 minutes;

[0153] Sealing: Wash three times with PBS, 5 minutes each time; remove the cell slides from the well plate, seal with anti-fluorescence quencher and place on a slide; take pictures using a TCS SP8 laser confocal microscope;

[0154] (2.11) Flow cytometry was used to detect the expression of CRT and HMGB1 in cells: cells (HepG2) in the logarithmic growth phase were taken; the cells were seeded into 6-well plates and administered with different concentrations for 24 hours;

[0155] Step 1: Wash twice with PBS, digest and collect cells by centrifugation;

[0156] Step 2: Wash once with PBS and add 500 μL of 4% paraformaldehyde and fix on a shaker (37°C, 30 min);

[0157] Step 3: Centrifuge and wash once with PBS, then add 1% BSA (prepared in PBS) and block on a shaker at 37°C for 1.5h (for detection of HMGB1 antibody, add 1% BSA + 0.1% Triton prepared in PBS);

[0158] Step 4: Centrifuge and wash once with PBS, then add CRT or HMGB1 primary antibody (prepared according to the antibody instructions) and incubate overnight on a shaker at 4°C;

[0159] Step 5: The next day, after centrifugation to recover the primary antibody, wash once with PBS, then add fluorescent secondary antibody (prepared in cell staining buffer) and incubate at 37°C in the dark for 1 hour;

[0160] Step 6: Centrifuge to discard the fluorescent secondary antibody, wash once with PBS, and analyze by flow cytometry;

[0161] (2.12) ATP release detection: cells (HepG2) in the logarithmic growth phase were taken; the cells were seeded in a 6-well plate and treated with different groups (control group, TEP NPs group (1.25, 2.5, 5 μM), oxaliplatin group (5 μM) and TEP group (5 μM)) for 24 hours, the cell culture fluid was collected, and the chemiluminescence value was detected using a multifunctional microplate reader;

[0162] (2.13) DCs maturation detection: C57BL / 6 mice (6-8 weeks old) were killed by cervical dislocation and immersed in 75% alcohol. The mice were cut open along the lower abdomen, and the intact thighs were taken out. The excess muscle tissue was removed to obtain the femoral head of the mice; the femurs were cut open at both ends, and the bone marrow cells in the femurs were flushed out with serum-free 1640 culture medium. The bone marrow suspension was filtered through a cell sieve and centrifuged (2000 rpm, 5 minutes), the supernatant was discarded, and red blood cell lysis buffer was added and lysed at room temperature for 2 minutes; 1640 complete culture medium was added to terminate the lysis. The complete culture medium contained mGM-CSF (20 ng / mL) and IL-4 (20 ng / mL); centrifuged (2000 rpm , 5 minutes), then add complete culture medium and blow evenly, inoculate in 6-well plates, and change half of the medium on the 2nd and 4th days respectively; on the 6th day, collect cells, wash once with PBS, and evenly inoculate in 6-well plates and culture overnight; on the second day, collect the cell culture medium of Hepa1-6 cells cultured for 24 hours in different drug administration groups, and co-culture with immature DCs; after 24 hours of co-culture, collect DCs, add CD11, CD86 and CD80 antibodies (antibodies are prepared according to the antibody instructions), stain at room temperature for 15 minutes, and then centrifuge (2000rpm, 5 minutes), discard the supernatant, resuspend with cell loading buffer and analyze by flow cytometry, and the data are processed using FlowJo software;

[0163] (2.14) Western blot analysis to detect the expression levels of HMGB1 and CRT: Western blot analysis to detect the expression levels of HMGB1 and CRT, the specific experimental procedures are the same as (2.8);

[0164] (2.15) Detection of the reversal of the antiproliferative activity of nanoparticles in vitro by ferroptosis inhibitors: Take cells (HepG2) in the logarithmic growth phase; discard the supernatant, pipette with complete culture medium and count, add 100 μL to each well, and culture in a 37°C incubator overnight until the cells adhere to the wall; set up pretreatment groups with different concentrations of ferroptosis inhibitor Ferrostatin-1 (0, 1, 2 and 4 μM); the drug administration operation is the same as (2.2);

[0165] (2.16) Detection of lipid peroxides: Take cells (HepG2) in the logarithmic growth phase; inoculate the cells in a 6-well plate, and after the cells adhere to the wall, incubate the cells in different groups (control group, TEP NPs group (5 μM), TEP NPs (5 μM) + Ferrostatin-1 (4 μM) group, and TEP group (5 μM)) for 6 hours, add lipid peroxide probe (C11 BODIPY) for staining (30 minutes), wash twice with PBS, and take pictures under an inverted fluorescence microscope;

[0166] (2.17) Western blotting analysis to detect the expression level of GPX4, a key protein in ferroptosis: Western blotting analysis to detect the expression level of GPX4, the specific experimental operation is the same as (2.8);

[0167] (2.18) Cell apoptosis detection:

[0168] (2.18.1) Cell culture and drug incubation: HepG2 cells in the logarithmic growth phase were seeded in a 6-well plate. After the cells adhered to the wall, different groups (control group, TEP NPs group (5, 10, 15 μM), and TEP group (5, 10, 15 μM)) were treated for 48 hours;

[0169] (2.18.2), cell staining, flow cytometry detection: discard the culture medium, wash the cells twice with PBS, use EDTA-free trypsin to digest the cells, collect the cells of each group, wash once with PBS, centrifuge (800g, 5 minutes), add 500μL cell staining buffer, then add 5μL Propidium Iodide and 5μL Annexin V-FITC, incubate at room temperature in the dark for 15 minutes, analyze by flow cytometry, and process the data using FlowJo software;

[0170] (2.19) Transcriptomics experiment: Take cells (HepG2) in the logarithmic growth phase; inoculate the cells in a 6 cm culture dish, add TEP NPs (10 μM), add PBS to the blank control group, incubate for 24 hours, wash once with PBS, add TRIzol and blow evenly to make the cells fall off, collect the cells, and freeze them quickly with liquid nitrogen; all data are analyzed using the Majorbio cloud platform (https: / / cloud.majorbio.com);

[0171] (2.20) Statistical analysis: The experimental data were expressed as Mean ± SD. GraphPad Prism9 software was used for data processing and statistical analysis. One-way analysis of variance was used for inter-group comparison. P < 0.05 indicated a statistically significant difference.

[0172] (3) Experimental results and discussion

[0173] (3.1) Evaluation of the antitumor efficacy of nanoparticles in vivo and their tumor targeting: TEP NPs have a good inhibitory effect on the proliferation of three types of liver cancer cells. To verify the antitumor effect of TEP NPs in vivo, a xenograft tumor model was established in immune-competent C57BL / 6 mice. Because the experimental mice were immune-competent C57BL / 6 mice, mouse liver cancer cells Hepa1-6 were used for the experiment. After 40 6-8 week-old C57BL / 6 female mice were adaptively raised for one week, the experiment began. Hepa1-6 cells were collected and the cell density was adjusted to 1×10 6 100 μL was inoculated into the left armpit of each mouse, and the size of the tumor was monitored. When the average tumor volume was greater than 100 cm 3 At the same time, the mice were randomly divided into 5 groups, each group was: compound TEP group (5 mg / kg), nanoparticle TEP NPs group (5 mg / kg), oxaliplatin group (5 kg / mg), BSA group and model group; the drug was administered according to the experimental scheme and the weight of the mice and the size of the tumor were recorded. The volume calculation formula was: V (cm 3 )=a 2×(b / 2), (a: short diameter; b: long diameter; V: tumor volume); blood was collected from the eyeballs two weeks later, and then the mice were killed, and the tumors and main organs were removed for subsequent experiments;

[0174] In vivo antitumor results Figure 3 As shown in the figure, it can be observed that the tumors in the model group and the BSA-treated group grew rapidly, while the tumor growth in the mice treated with TEP NPs was significantly inhibited, and the effect was better than that in the oxaliplatin-treated group and the TEP-treated group ( Figure 3 BC); and the body weight of mice remained stable during the treatment ( Figure 3 D); HE staining of tumor sections showed that the proportion of dead cells in the TEP NPs treatment group was significantly higher than that in the model group, BSA treatment group, TEP treatment group and oxaliplatin treatment group; at the end of treatment, no organ damage was observed ( Figure 3 F), indicating that the systemic toxicity of TEP NPs is low;

[0175] To detect the bioavailability of TEP / TEP NPs in vivo, the Au content in the tumor tissues of mice in the TEP treatment group and TEP NPs treatment group was detected by ICP MS, as shown in Figure 2 . Figure 3 As shown in F, the Au content in the tumors of mice in the TEP NPs treatment group was significantly higher than that in the TEP treatment group, indicating that compared with TEP, TEP NPs tend to accumulate in the tumor site.

[0176] (3.2) Transcriptomics study: To explore the anti-tumor mechanism of TEP NPs, mRNA sequencing was used to detect changes in cellular gene expression at the transcriptional level after TEP NPs administration. The analysis parameters of log2FC greater than 1 and log2FC less than 1, and p-adjust less than 0.05 were selected to analyze genes with different expression levels. The results showed that a total of 131 genes showed different expression levels between the TEP NPs treatment group and the control group, of which the expression levels of 60 genes were downregulated and the expression levels of 71 genes were upregulated ( Figure 4 ); In addition, by analyzing the RNA-seq clustering heat map of differential expression genes (DEGs), it was found that the expression patterns of each group of samples were very similar;

[0177] Enrichment analysis showed that ( Figure 5 ), TEP NPs mainly affected pathways related to sugar metabolism, such as fructose and mannose metabolism, galactose metabolism, glycolysis / gluconeogenesis, etc., and the occurrence of these processes requires a large amount of ATP provided by mitochondria, which is closely related to the normal function of mitochondria. Therefore, the mitochondrial function will be further tested in the next study;

[0178] (3.3) TEP NPs cause mitochondrial damage: Transcriptomics showed that TEP NPs affected fructose and mannose metabolism, galactose metabolism, glycolysis and gluconeogenesis and other related pathways. These energy metabolism pathways are closely related to the normal operation of mitochondrial function. Therefore, the effect of TEP NPs on mitochondrial function was explored. The JC-1 kit was used to detect the changes in mitochondrial membrane potential (MMP) of HepG2 after treatment with different concentrations of TEPNPs (2.5μM, 5μM, 10μM) and TEP (10μM) for 6 hours; the results are as follows: Figure 6 A, After TEP NPs treatment, green fluorescence (monomer) was enhanced, while red fluorescence (polymer), this result indicated that mitochondrial membrane potential decreased; Figure 6 The flow cytometry results of BC were also verified;

[0179] (3.4) Inhibitory effect of nanoparticles on thioredoxin reductase (TrxR): TrxR is usually overexpressed in various cancer cells and HCC cells, and TrxR is considered to be the target of gold compounds; therefore, the significant anti-tumor effect of TEP NPs may be attributed to its inhibition of TrxR in HepG2 cells; therefore, the TrxR inhibitory activity of TEP NPs was explored, and the inhibitory effect of compound 5a on TrxR was systematically studied from multiple aspects and angles using the intracellular TrxR activity level, intracellular TrxR probe, and TrxR pure enzyme; the results are shown in Figure 2. Figure 7As shown in Figure A, after HepG2 cells were treated with different concentrations of nanoparticles (1.25μM, 2.5μM, 5μM) for 24 hours, the changes in intracellular TrxR enzyme activity were detected using a TrxR enzyme activity kit, and it was found that TEP NPs had a dose-dependent inhibitory effect on the TrxR enzyme activity in HepG2 cells; to verify that TEP NPs can inhibit intracellular TrxR enzyme activity, the probe TRFS-green was used to detect the inhibitory effect of TEP NPs (2.5μM, 5μM, 10μM) on TrxR in HepG2 cells. The results are shown in Figure 2. Figure 7 As shown in B, after 24 h of treatment with TEP NPs, the TrxR activity in HepG2 cells was inhibited; in addition, Figure 7 The results in C show that after the purified TrxR pure enzyme was incubated with TEP NPs (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM) for 2 hours, the activity was detected by DTNB method, and it was found that the TrxR enzyme activity was significantly inhibited; the expression level of TrxR after treatment with different concentrations of TEP NPs (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM) was quantitatively analyzed by protein immunoblotting, and it was found that there was no significant difference in the expression level of TrxR compared with the control group; In summary, although nanoparticles TEP NPs did not affect the expression of TrxR in cells, they could inhibit the activity of TrxR in a dose-dependent manner. As a key enzyme for maintaining intracellular redox homeostasis, the inhibition of TrxR activity seriously disrupted the intracellular redox balance; Therefore, the above results show that TEP NPs can destroy the redox balance of HepG2 cells;

[0180] (3.5) Nanoparticles induce intracellular ROS generation: Continuous oxidative stress caused by ROS generated by environmental factors or mitochondrial dysfunction is believed to be related to the occurrence of liver cancer. Compared with normal cells, cancer cells are more dependent on the redox system and maintain their malignant phenotype by increasing ROS. ROS, as a secondary messenger in the cell signaling pathway, plays an important role in various biological processes of normal cells and cancer cells. Excessive ROS levels show anti-tumor activity by inducing cell cycle arrest and cell death.

[0181] To explore whether TEP NPs can affect intracellular ROS levels, DCFH-DA and DHE probes were used to detect intracellular ROS levels in HepG2 cells after treatment with different concentrations of TEP NPs (2.5 μM, 5 μM, 10 μM) and their contents TEP (10 μM); Figure 8As shown in Figures A and 8B, the fluorescence intensity in cells was significantly enhanced after 6 hours of TEP NPs treatment in a dose-dependent manner, indicating that TEP NPs caused the accumulation of ROS in cells. In addition, flow cytometry was used to quantify the level of ROS in cells. The results of flow cytometry analysis are shown in Figure 8A and 8B. Figure 8 As shown in C and 8D, TEP NPs can significantly increase the intracellular ROS level; therefore, the results indicate that inhibition of TrxR leads to increased intracellular ROS levels;

[0182] (3.6) Nanoparticle-mediated DAMPs release triggers ICD effect: Evidence shows that several conventional chemotherapeutic drugs can treat cancer by activating the immune system; in addition, the ability of anticancer drugs to induce ICD has been shown to be dependent on their ability to induce ROS production in the process of cell death; previous studies have shown that TEP NPs can induce intracellular ROS production, which suggests that TEP NPs may kill tumor cells by inducing immunogenic cell death; immunogenic cell death is characterized by the release of DAMPs, including HMGB1 efflux, ATP release, and CRT exposure on the cell surface;

[0183] Next, the indicators of ICD were characterized; by immunofluorescence and flow cytometry analysis, the exposure of CRT on the cell membrane surface was observed after treatment with TEP NPs (5 μM) and oxaliplatin (5 μM). Fig. 9 A and Fig. 9 C, E), and the results of Western blot analysis also showed that the expression of CRT increased significantly after drug treatment ( Fig. 9 H); at the same time, confocal microscopy was used to observe that HMGB1 in the nucleus of HepG2 cells was released into the cytoplasm after administration ( Fig. 9 B), flow cytometry analysis ( Fig. 9 D, F) and Western blot analysis ( Fig. 9 H) The results were consistent with the immunofluorescence results; the ATP detection kit was used to detect the extracellular ATP level of HepG2 cells after treatment, and it was found that compared with the control group, the extracellular ATP level of cells treated with TEP NPs and oxaliplatin increased significantly ( Fig. 9 J); In summary, TEP NPs significantly stimulated the release of DAMPs in HepG2 cells;

[0184] The ability of TEP NPs to induce DCs maturation was then evaluated. First, mouse bone marrow cells were extracted and induced to differentiate into immature DCs using stimulatory factors. The culture medium of Hepa1-6 cells treated with TEP NPs for 24 hours was co-cultured. Finally, DCs surface antigens were labeled with CD11c-APC, CD86-FITC, and CD80-PE, and the maturation ratio of DCs was detected by flow cytometry. The results are shown in Figure 2. Fig.10A and Fig.10 As shown in B, TEP NPs can significantly increase the proportion of mature DCs in DCs, which is comparable to the effect of oxaliplatin treatment;

[0185] (3.7) Nanoparticles cause cell ferroptosis: Changes in redox homeostasis are very important in the occurrence of ferroptosis. When the intracellular ROS level rises to a certain level and exceeds the buffering capacity provided by the antioxidant defense system, LPO will accumulate on the cell membrane or mitochondrial membrane, leading to ferroptosis. GPX4 is a key regulator of ferroptosis, which can work synergistically with DHODH to convert LPO into corresponding alcohol substances. Compared with normal tissues, GPX4 is usually overexpressed in liver cancer cells. Therefore, inhibiting the expression of GPX4 and increasing the accumulation of intracellular LPO to induce ferroptosis is a promising therapeutic strategy.

[0186] In addition, TEP NPs can also lead to ROS surge and decreased mitochondrial membrane potential, which may lead to cell ferroptosis. To explore the ability of TEP NPs to induce cell ferroptosis, Ferrostatin-1 was first used for pretreatment, and the cell survival rate was detected by MTT method. The results showed that Ferrostatin-1 pretreatment reversed the killing effect of TEP NPs on HepG2 cells to a certain extent ( Fig.11 A); Subsequently, LPO probe was used to detect the accumulation of LPO in cells; the results showed that ( Fig.11 D), TEP NPs treatment for 6 hours can increase the accumulation of intracellular LPO (green fluorescence), and Ferrostatin-1 pretreatment; Western blot analysis was used to detect the expression of GPX4, a key protein regulating ferroptosis, such as Fig.11 As shown in B and C, the expression of GPX4 was inhibited after TEPNPs treatment, and this inhibitory effect could be improved to a certain extent after pretreatment with Ferrostatin-1; in addition, the effect of TEP in inducing intracellular LPO accumulation and inhibiting GPX4 expression was not as good as that of TEP NPs, which may be related to the fact that TEPNPs promoted the accumulation of drugs in cells;

[0187] (3.8) Nanoparticles induce cell apoptosis: It was also found that TEP NPs can inhibit the activity of TrxR, induce increased ROS levels and decreased mitochondrial membrane potential. These phenomena indicate that TEP NPs are likely to kill tumor cells by inducing cell apoptosis. Therefore, flow cytometry was used to further detect TEP NPs and TEP-induced cell apoptosis. Fig.12 The results of AC showed that the apoptosis rate of HepG2 cells increased significantly after administration of different concentrations of TEP NPs (5μM, 10μM, 15μM) and TEP (5μM, 10μM, 15μM) for 48 hours;

[0188] (3.9) Nanoparticle integration of chemotherapy and immunotherapy: To determine the potential mechanism of TEP / TEP NPs in tumor suppression and induction of immune response in C57BL / 6 mice, the tumor tissues were subjected to pathological analysis, and the levels of AIF in tumor cells were analyzed by TUNEL assay, HE staining and immunohistochemical analysis to evaluate the apoptosis induced by TEP and TEP NPs in vivo. Fig.13 AC); compared with the model group, TEP and oxaliplatin administration groups, the tumor tissues administered with TEP NPs showed obvious apoptosis and increased AIF levels, indicating that after administration, the ability of TEP NPs to induce tumor cell apoptosis was stronger than that of TEP and oxaliplatin; the immunofluorescence results of tumor tissues showed that compared with the model group and BSA group, the expression level of TrxR was significantly reduced, and the expression of TrxR was significantly different from that of the TEP and oxaliplatin administration groups ( Fig.14 A, C), indicating that TEP NPs can inhibit the expression of TrxR in tumor cells in vivo; the effect of TEP / TEP NPs in inducing DAMPs release in vivo was also analyzed by immunofluorescence; compared with the model group and BSA group, the ICD-related marker CRT in the tumor tissue of the TEP NPs group showed obvious efflux phenomenon ( Fig.14 B, D), and the effect of TEP NPs in inducing CRT efflux was stronger than that of TEP and oxaliplatin groups; by analyzing the immune response in tumor tissues, it was found that compared with the model group and BSA group, the CD86 level in tumor tissues of the TEP NPs group was significantly increased ( Fig.15 A), while the expression level of CD86 in tumor tissues of TEP group and oxaliplatin group was lower than that of TEP NPs group; on the other hand, it was also found that TEP NPs could inhibit the expression of GPX4, a ferroptosis marker in tumor tissues ( Fig.15 B), and the inhibitory effect is stronger than that of TEP; the results show that TEP NPs are better than TEP in inducing DAMPs release, immune response and ferroptosis in vivo, which may be related to the ability of TEP NPs to target the tumor immune microenvironment and increase the accumulation of compounds in the tumor immune microenvironment;

[0189] Through in vitro and in vivo bioactivity studies, the potential mechanism of action of chloro(triethylphosphine)gold(I)albumin nanoparticles was further explored. The results showed that using albumin as a carrier of gold compounds for targeted therapy is an effective strategy. In vivo pharmacodynamic evaluation showed that TEP NPs can effectively target tumor tissues and exhibit better anti-tumor activity by increasing Au accumulation in tumor tissues. In vitro studies found that TEP NPs can effectively inhibit the activity of TrxR, induce ROS surge and decrease mitochondrial membrane potential, and induce cell apoptosis by destroying the redox balance in cells. At the same time, it can cause the release of DAMPs and activate the immune response in the tumor microenvironment, thereby causing ICD. In addition, TEP NPs can also inhibit the expression of GPX4, causing the accumulation of LPO in cells, thereby inducing ferroptosis. Immunohistochemical and immunofluorescence analysis of tumor tissues also showed that TEP NPs can integrate the dual effects of chemotherapy and immunotherapy, showing excellent chemoimmunotherapy effects. The possible mechanism of action of TEP NPs is as follows: Fig.16 shown.

[0190] The present invention constructs a class of albumin nano-drug delivery systems with good tumor targeting based on the natural biomacromolecule bovine serum albumin (BSA); in vitro anti-proliferation activity studies show that TEP NPs have a good inhibitory effect on liver cancer cells from different sources. Subsequently, it is characterized in vitro and a series of biological activity studies are carried out to explore its potential mechanism of action.

[0191] The results of MALDI-TOF-MS showed that chloro(triethylphosphine)gold(I) was stably covalently bound to BSA, and the particle size stability of TEP NPs illustrated the stability of this binding mode; TEP NPs released most of TEP at pH 4.7 and retained most of TEP at pH 7.0, which indicated that TEP could be retained inside the nanoparticles under normal physiological conditions to prevent it from reacting with endogenous substances in the body and losing its activity prematurely; in vivo antitumor experiments and higher gold content in tumor tissues proved that TEP NPs had better in vivo antitumor effects.

[0192] The results of transcriptomics showed that after TEP NPs treatment, signal pathways related to the sugar metabolism pathway were affected, and the normal maintenance of sugar metabolism is related to mitochondrial function; therefore, the mitochondrial membrane potential was detected, and the results showed that after the action of TEP NPs, the mitochondrial membrane potential was reduced and thus the mitochondrial function was affected, which was consistent with the transcriptomic results; further anti-tumor mechanism showed that, on the one hand, TEP NPs significantly inhibited TrxR activity, resulting in the accumulation of intracellular ROS, which induced cell apoptosis after reaching the toxic threshold; in addition, the destruction of redox homeostasis caused the accumulation of intracellular LPO and induced cell ferroptosis; on the other hand, TEP NPs also caused tumor cells to release DAMPs, which caused an immune response by inducing the maturation of DCs.

Claims

1. A method for preparing gold (I) albumin nanoparticles, characterized in that: The gold (I) albumin nanoparticles include a gold (I) compound and blank albumin nanoparticles, and the gold (I) compound includes a nitrogen heterocyclic carbene gold (I) compound and a phosphine ligand gold (I) compound; The synthetic route of the nitrogen heterocyclic carbene gold (I) compound: Step (a): thiamine, sodium hydroxide, react at room temperature for 2-7 days; Step (b): Formamide, heated to reflux for 3 hours; Step (c): NaH, ethyl bromide, tetrahydrofuran, heating under reflux for 2 hours; Step (d): acetonitrile, reflux for 3-5 days; Step (e): silver oxide, acetonitrile, nitrogen protection, reaction at room temperature in the dark overnight; gold (I) chloride, sodium bromide, nitrogen protection, reaction at room temperature in the dark for 4 hours; Step (f): sodium bromide, nitrogen protection, and light-proof at room temperature for 6 hours; The specific synthesis route is as follows: The synthetic route of the phosphine ligand gold (I) compound: Step (a): dichloromethane, nitrogen protection, room temperature and dark reaction for 2.5 hours; Step (b): dichloromethane, nitrogen protection, room temperature and dark for 4 hours; The specific synthesis route is as follows: The preparation method of Au4 is as follows: weigh 100 mg of gold (I) chloride (dimethyl sulfide) in a 50 mL flask, add dichloromethane to dissolve, then weigh 89.4 mg of triphenylphosphine, add 4 mL of DCM, and react at room temperature in the dark for 2.5 hours under nitrogen protection; after the reaction, spin dry the reaction solution, add dichloromethane to dissolve the white solid, and then add n-hexane until no solid precipitates; let stand for 1 hour and then filter, wash the filter cake with n-hexane and transfer it to a flask and dry it under vacuum to obtain a white solid; The preparation method of Au5 is as follows: 50 mg of dimethyl sulfide gold (I) chloride is weighed into a 50 mL flask, dichloromethane is added to dissolve, 98.4 mg of 1,3,5-triaza-7-phosphaadamantane is weighed, 5 mL of DCM is added, and the mixture is reacted at room temperature in the dark for 4 hours under nitrogen protection; after the reaction is completed, the reaction solution is spin-dried and dried under vacuum conditions to obtain a white solid.

2. The method for preparing gold (I) albumin nanoparticles according to claim 1, characterized in that: The preparation steps of the blank albumin nanoparticles are as follows: first, 40 mg of bovine serum albumin is dissolved in 1 mL of ultrapure water, the solution is titrated to a pH value of 8.5 using 0.1 N sodium hydroxide, an ethanol solution is added dropwise after stirring, and then 10 μL of 8% glutaraldehyde is added, and after stirring for 16 hours, the ethanol is removed by dialysis, and then purified nanoparticles are obtained after 3 times of centrifugation, and finally freeze-dried to obtain nanoparticle freeze-dried powder, namely blank albumin nanoparticles; Wherein, the stirring condition is: 600rpm; The centrifugation conditions were: 5000 rpm, 10 minutes.

3. The method for preparing gold (I) albumin nanoparticles according to claim 1, characterized in that: The preparation steps are as follows: first, 40 mg BSA is dissolved in 1 mL ultrapure water, and the solution is titrated to a pH value of 8.5 using 0.1 N sodium hydroxide, and then an ethanol solution of a gold (I) compound is added dropwise after stirring, and then anhydrous ethanol is added dropwise, and then 10 μL of 8% glutaraldehyde is added, and after stirring for 16 hours, the free gold (I) compound and ethanol are removed by dialysis, and then purified nanoparticles are obtained after three centrifugations, and finally freeze-dried to obtain nanoparticle freeze-dried powder; namely, gold (I) albumin nanoparticles; Wherein, the stirring condition is: 600rpm; The centrifugation conditions were: 5000 rpm, 10 minutes.

4. The method for preparing gold (I) albumin nanoparticles according to claim 3, characterized in that: 1 mg of gold (I) albumin nanoparticles was weighed and dissolved in 1 mL of ultrapure water to prepare a 1 mg / mL nanoparticle aqueous solution. After ultrasonication in a water bath for 1 minute, the particle size distribution and Zeta potential of the nanoparticles were measured using a Malvern particle size analyzer.

5. The method for preparing gold (I) albumin nanoparticles according to claim 3, characterized in that: The in vitro antiproliferative activity detection process of gold (I) albumin nanoparticles is: (1) Cell plating: HepG2, Hepa1-6, Hep3B, and NCM460 cells in the logarithmic growth phase were digested with trypsin and centrifuged. The supernatant was discarded, and the cells were evenly pipetted with complete culture medium and counted. After adjusting the cell density, they were inoculated into 96-well plates with 100 μL of complete culture medium per well, and cultured in a 37°C incubator overnight until the cells adhered to the wall; (2): Cell administration: Prepare different concentrations of compounds, nanoparticles and positive drug working solutions, and the final drug concentration in each well is 0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM. At the same time, 0.1% DMF or complete culture medium is added to the negative control group, and finally 200μL of complete culture medium is added to each well; the cells are cultured in a 37°C incubator for 72 hours; (3): MTT method to detect antiproliferative activity: 20 μL MTT / well was added to a 96-well plate, and the plate was cultured in a 37°C incubator. After 4 hours, the supernatant was discarded, 200 μL DMSO was added, and the plate was shaken for 10 minutes. The absorbance at 570 nm was detected using an enzyme-labeled instrument. The cells without drug addition were used as the blank control group, and 4 replicate wells were set up to calculate the cell viability. Cell viability (%)=(OD d-OD 0) / (OD c-OD 0)×100% Wherein OD d: absorbance value of drug administration group, OD 0: absorbance value of zero adjustment well, OD c: absorbance value of negative control group.

6. The method for preparing gold (I) albumin nanoparticles according to claim 5, characterized in that: The cells were cultured using DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator at 37° C. and 5% CO 2 as the culture environment.

7. The method for preparing gold (I) albumin nanoparticles according to claim 5, characterized in that: The centrifugal conditions are: 1000 rpm, 5 minutes; The 20 μL MTT / well has a concentration of 5 mg / mL and is dissolved in PBS.

8. The method for preparing gold (I) albumin nanoparticles according to claim 3, characterized in that: The gold (I) albumin nanoparticle size stability detection process is: using dynamic light scattering to analyze the size stability of the nanoparticles in a PBS solution for 72 hours; The nanoparticles can be obtained as solid powder after freeze-drying. The PBS solution prepared with the freeze-dried powder of the nanoparticles is stored at room temperature at different times, and the particle size of the nanoparticles is measured uniformly at last. According to the last measurement time, the aqueous solution of the freeze-dried powder of the nanoparticles is prepared 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, and 0 hours in advance, respectively, and the particle size is detected by a machine at the measurement time.

9. The method for preparing gold (I) albumin nanoparticles according to claim 7, characterized in that: The volume of the aqueous solution is: 1 mg / mL.

10. Use of gold (I) albumin nanoparticles prepared by the preparation method according to any one of claims 1 to 9 in anti-tumor activity.

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