Tumor targeting peptide modified gold-copper bimetallic cluster nano-enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano biological material preparation, and discloses a tumor targeting peptide modified gold-copper bimetallic cluster nano enzyme and a preparation method and application thereof, and the preparation method comprises the following steps: preparing the gold-copper bimetallic cluster nano enzyme by using natural protein; the tumor targeting peptide is modified on the surface of the gold-copper bimetallic cluster nano-enzyme through amidation reaction to obtain the tumor targeting peptide modified gold-copper bimetallic cluster nano-enzyme. The gold-copper bimetallic cluster nano-enzyme obtained by the invention has excellent biological safety, can specifically aggregate in tumor cells and induce copper death, further realizes a remarkable tumor cell growth inhibition effect on the basis of a synergistic effect of enzyme-like catalysis, and has a wide application prospect in the field of tumor treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomaterials and relates to a gold-copper bimetallic cluster nanozyme modified with a tumor targeting peptide and a preparation method and application thereof. Background Art

[0002] Cancer is one of the three major killers that endanger the life and health of all mankind. At present, the main treatments for cancer include surgery, radiotherapy, chemotherapy, immunotherapy and photodynamic therapy. However, these treatments are difficult to meet clinical needs due to their inherent limitations. For example, surgical treatment has limited therapeutic effects on small-sized and metastatic tumors, radiotherapy and chemotherapy can have strong toxic side effects on the human body and easily cause drug resistance, and immunotherapy is limited in its wide application due to problems such as immune escape and immune system diseases. New therapies such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) based on nanomaterials have limited light source penetration and potential toxicity of photosensitizers or sonosensitizers. Therefore, it is urgent to develop new and efficient tumor treatment strategies to further improve the efficacy of tumor treatment.

[0003] Recently, cuprotosis, as a programmed cell death mechanism different from any known form of cell death (such as ferroptosis, apoptosis, pyroptosis and necrosis), has attracted widespread attention in the field of tumor treatment. At present, a variety of copper-based nanomaterials that induce cuprotosis in tumor cells have been developed, such as copper ion chelates, copper oxide nanoparticles, and copper-based metal organic frameworks. However, due to insufficient delivery efficiency, the limited copper ion concentration in tumor cells is often difficult to reach the conditions for inducing cuprotosis. At the same time, glutathione, which is highly expressed in tumor cells as an endogenous chelator of copper, may reduce the tumor cell killing effect mediated by cuprotosis. Therefore, it is crucial to design and construct efficient targeted transport carriers for copper ions while regulating the high glutathione content in tumor cells to improve the efficacy of cuprotosis therapy. Summary of the invention

[0004] In view of the problem of limited efficiency of copper death-inducing tumor cell death in the prior art, the purpose of the present invention is to provide a tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme and a preparation method and application. The gold-copper bimetallic cluster nanozyme has tumor cell targeting ability, copper death-inducing efficacy and enzyme catalysis to regulate the intracellular microenvironment to synergistically enhance tumor cell killing performance.

[0005] To achieve the above object, the technical solution adopted by the present invention is: A method for preparing a tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme comprises the following steps: Preparation of gold-copper bimetallic cluster nanozymes using natural proteins; The tumor targeting peptide was modified on the surface of the gold-copper bimetallic cluster nanozyme through an amidation reaction to obtain the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme.

[0006] A further improvement of the present invention is that the natural protein is bovine serum albumin; the gold-copper bimetallic cluster nanozyme is prepared by the following process: the pH value of a mixed solution containing chloroauric acid, copper nitrate and bovine serum albumin is adjusted to 10-12, and a reaction is carried out under heating conditions to obtain the gold-copper bimetallic cluster nanozyme.

[0007] A further improvement of the present invention is that the concentration of bovine serum albumin in the mixed solution containing chloroauric acid, copper nitrate and bovine serum albumin is 25 mg / mL, the molar ratio of chloroauric acid to copper nitrate is 1-8:1, and the dosage ratio of bovine serum albumin to copper nitrate is 100 mg:0.0022-0.01 mmol.

[0008] A further improvement of the present invention is that the reaction temperature is 35-45° C., the reaction time is 10-14 hours, and the reaction is carried out under stirring at a speed of 1200 rpm.

[0009] A further improvement of the present invention is that the gold-copper bimetallic cluster nanozyme modified with a tumor-targeting peptide is prepared by the following process: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to the gold-copper bimetallic cluster nanozyme, stirred evenly and reacted in the dark for 30 minutes, then the tumor-targeting peptide is added and reacted for 6-12 hours to obtain the gold-copper bimetallic cluster nanozyme modified with a tumor-targeting peptide.

[0010] A further improvement of the present invention is that the sequence of the tumor targeting peptide is shown as SEQ ID NO.1.

[0011] A further improvement of the present invention is that the mass ratio of the gold-copper bimetallic cluster nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 25:8-16.

[0012] A further improvement of the present invention is that the mass ratio of the gold-copper bimetallic cluster nanozyme to the tumor targeting peptide is 100:1-4.

[0013] A tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme.

[0014] A tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme is used in the preparation of an anti-tumor chemodynamic therapy preparation and / or a catalytic therapy preparation.

[0015] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, bovine serum albumin (BSA), a natural protein rich in multiple functional groups and having high biocompatibility, is used as a ligand to synthesize a gold-copper bimetallic cluster nanozyme (BAC) with high biocompatibility, high stability and high catalytic activity. Then, the tumor targeting peptide is modified on the surface of the gold-copper bimetallic cluster nanozyme (RBAC) by an amidation reaction. The nanozyme is rich in copper and has an ultra-small size. Under the action of the tumor targeting peptide, it can achieve the specific high-concentration accumulation of copper in tumor cells and induce the occurrence of copper death. At the same time, the nanozyme constructs a wealth of catalytic active sites on the surface of the nanocluster by means of the synergistic effect of gold and copper elements, showing multi-enzyme activities of glutathione oxidase, peroxidase, and oxidase, and can synergistically enhance the copper death effect through the redox microenvironment regulation mechanism such as catalytic depletion of glutathione and generation of hydroxyl free radicals. The preparation method of the gold-copper bimetallic cluster nanozyme modified with tumor targeting peptides in the present invention is simple, the reaction conditions are mild, and the repeatability is good. The novel gold-copper bimetallic nanocluster nanozyme synthesized by the present invention has synergistic therapeutic multi-biological properties compared to existing copper-death nanopreparations, and its intrinsic fluorescence properties help to visualize the cellular uptake efficiency of the nanozyme and the accumulation in tumor tissues, and has broad application prospects in the efficient treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 are transmission electron microscope (TEM) images of RBAC and BAC prepared in Example 1; wherein (a) is BAC, and (b) is RBAC; Figure 2 is the characterization of the peroxidase-like activity of the RBAC and BAC prepared in Example 1; Figure 3 is the characterization of the glutathione oxidase-like activity of the RBAC and BAC prepared in Example 1; Figure 4 is the characterization of the oxidase-like activity of the RBAC and BAC prepared in Example 1; Figure 5 The killing ability of RBAC and BAC prepared in Example 1 on melanoma cells (B16-F10) is characterized; wherein (a) is 24 h, and (b) is 72 h; Figure 6 It is a characterization of the intracellular oxidative free radical (ROS) level of B16-F10 cells after being acted upon by RBAC and BAC prepared in Example 1; wherein (a) is a blank control group, (b) is BAC, and (c) is RBAC; Figure 7 It is a characterization of the expression of proteins related to the intracellular copper death mechanism after B16-F10 cells were acted upon by RBAC and BAC prepared in Example 1. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood.

[0018] The preparation method of the tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme of the present invention uses natural protein (bovine serum albumin, BSA) as a ligand, synthesizes the gold-copper bimetallic cluster nanozyme by a one-step method in an aqueous phase, and modifies the tumor-targeting peptide on the surface of the gold-copper bimetallic cluster nanozyme by an amidation reaction, thereby preparing a tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme, which can specifically target tumor cells and achieve enzyme catalysis and synergistic enhancement of copper death therapy. High biocompatibility and BSA ligands rich in multiple functional groups ensure the biosafety, stability and flexibility of surface functionalization of the gold-copper bimetallic cluster nanozyme. Further modification of the tumor-targeting peptide gives the nanozyme the characteristics of precise targeting of tumor tissues and efficient uptake of tumor cells. The gold-copper bimetallic active site enables the nanozyme to have multiple enzyme activities and multiple physical and chemical properties, thereby achieving amplified and enhanced tumor cell killing efficacy.

[0019] Specifically, the preparation method of a tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme of the present invention comprises the following steps: 1) Preparation of gold-copper bimetallic cluster nanozymes: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozymes were prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, the chloroauric acid solution and the copper nitrate solution were stirred evenly and added to the BSA solution, and then sodium hydroxide solution was added to adjust the pH of the reaction system to 10-12, and the gold-copper bimetallic cluster nanozymes were prepared under heating conditions.

[0020] Among them, the concentration of BSA in the reaction system is 25 mg / mL, the molar ratio of chloroauric acid to copper nitrate is 1-8:1, and the dosage ratio of BSA to copper nitrate is 100 mg:0.0022-0.01 mmol; the reaction heating temperature is 35-45 ° C, the reaction time is 10-14 hours, and the reaction is carried out under stirring at 1200 rpm.

[0021] 2) Preparation of tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme: The tumor-targeting peptide was modified on the surface of the gold-copper bimetallic cluster nanozyme by amidation reaction. Specifically, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to the gold-copper bimetallic cluster nanozyme, stirred evenly and reacted in the dark for 30 minutes, and then the tumor-targeting peptide RGDSP (the sequence of the tumor-targeting peptide is arginine-glycine-aspartic acid-serine-proline, Arg-Gly-Asp-Ser-Pro, RGDSP, and the amino acid sequence is shown in SEQ ID NO.1) was added to the reaction system to react and obtain the tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme.

[0022] The mass ratio of the gold-copper bimetallic cluster nanozyme to EDC is 25:8-16. The mass ratio of the gold-copper bimetallic cluster nanozyme to RGDSP is 100:1-4. The reaction time is 6-12 hours, and the reaction is carried out under stirring at 800 rpm.

[0023] 3) Biomedical applications: RBAC nanozymes were co-cultured with melanoma cells (B16-F10) to study the killing effect of nanozymes on tumor cells.

[0024] The tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme prepared by the present invention can be used in the preparation of anti-tumor chemodynamic therapy preparations and / or in catalytic therapy preparations.

[0025] The following are specific embodiments.

[0026] Example 1 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1.33 mL, 10 mmol / L) and copper nitrate solution (0.67 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0027] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (16 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (100 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 6 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0028] Example 2 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1 mL, 10 mmol / L) and copper nitrate solution (1 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0029] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (16 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (100 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 6 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0030] Example 3 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1.6 mL, 10 mmol / L) and copper nitrate solution (0.4 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0031] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (16 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (100 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 6 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0032] Example 4 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1.78 mL, 10 mmol / L) and copper nitrate solution (0.22 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0033] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (16 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (100 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 6 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0034] Example 5 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1.33 mL, 10 mmol / L) and copper nitrate solution (0.67 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0035] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (24 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (200 μL, 10 mg / mL) was added to the reaction system and stirred at 800 rpm at room temperature for 8 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0036] Example 6 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1.33 mL, 10 mmol / L) and copper nitrate solution (0.67 mL, 10 mmol / L) were stirred evenly and added to BSA solution (2 mL, 50 mg / mL), and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10-12. The reaction was carried out at 37 °C for 12 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0037] 2) Preparation of gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozymes by amidation reaction to obtain gold-copper bimetallic cluster nanozymes that can specifically target tumor cells. Specifically, EDC (32 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (200 μL, 10 mg / mL) was added to the reaction system and stirred at 800 rpm at room temperature for 12 hours to obtain gold-copper bimetallic cluster nanozymes modified with tumor-targeting peptides. The prepared gold-copper bimetallic cluster nanozymes were purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0038] Example 7 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1 mL, 10 mmol / L) and copper nitrate solution (1 mL, 10 mmol / L) were stirred evenly and then added to BSA solution (2 mL, 50 mg / mL) to obtain a mixed solution, in which the BSA concentration in the mixed solution was 25 mg / mL, and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 12. The reaction was carried out at 45 °C for 10 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0039] 2) Preparation of tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozyme by amidation reaction to obtain gold-copper bimetallic cluster nanozyme that can specifically target tumor cells. Specifically, EDC (20 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (120 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 6 hours to obtain tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0040] Example 8 1) Preparation of gold-copper bimetallic cluster nanozyme: Using BSA as the surface ligand and reducing agent, gold-copper bimetallic cluster nanozyme was prepared by reducing chloroauric acid and copper nitrate in the aqueous phase in a one-step method. Specifically, chloroauric acid solution (1 mL, 10 mmol / L) and copper nitrate solution (1 mL, 10 mmol / L) were stirred evenly and then added to BSA solution (2 mL, 50 mg / mL) to obtain a mixed solution. The BSA concentration in the mixed solution was 25 mg / mL, and the mixed solution was stirred at 1200 rpm for 2-5 minutes at room temperature. Subsequently, sodium hydroxide (0.2 mL, 1 mol / L) was added to adjust the pH of the reaction system to 10. The reaction was carried out at 35 °C for 14 hours to obtain a gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was ultrafiltered and centrifuged using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0041] 2) Preparation of tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme: RGDSP was modified on the surface of gold-copper bimetallic cluster nanozyme by amidation reaction to obtain gold-copper bimetallic cluster nanozyme that can specifically target tumor cells. Specifically, EDC (28 mg) was added to the gold-copper bimetallic cluster nanozyme solution (2 mL, 25 mg / mL) and reacted at room temperature in the dark for 30 minutes. Subsequently, RGDSP solution (150 μL, 10 mg / mL) was added to the reaction system and stirred at room temperature at 800 rpm for 10 hours to obtain the tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme. The prepared gold-copper bimetallic cluster nanozyme was purified by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa, and the obtained product was stored at 4 °C in the dark for later use.

[0042] Figure 1(a) and (b) are TEM images of RBAC and BAC prepared in Example 1 of the present invention. The results show that RBAC and BAC have good dispersibility and uniform distribution; the average particle size of BAC is about 2.2 nm. After modification with RGDSP, the particle size of RBAC increases slightly to 3.1 nm.

[0043] Figure 2 It is the characterization of the peroxidase-like activity of RBAC and BAC prepared in Example 1 of the present invention. The peroxidase-like activity of RBAC and BAC was detected using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The specific steps are: 50 uL of TMB (final concentration 1 mM), 50 uL of hydrogen peroxide (final concentration 20 mM), and 30 uL of nanozyme material (final concentration 1 mg / mL) were added to 870 μL of phosphate buffer solution (1 mM, pH = 7.4), reacted at 37 ° C for 20 min, and centrifuged to take the supernatant to detect the UV-visible absorption spectrum. In the presence of hydrogen peroxide, the peroxidase-like activity of the nanozyme catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radicals (·OH), thereby oxidizing TMB to an oxidized state oxTMB having a characteristic absorption peak at 652 nm. Figure 2 It can be seen that both the RBAC-treated group and the BAC-treated group showed obvious characteristic absorption peaks at 652 nm, indicating that both RBAC and BAC have excellent peroxidase-like activity.

[0044] Figure 3 It is the characterization of the glutathione oxidase activity of RBAC and BAC prepared in Example 1 of the present invention. The glutathione oxidase activity of RBAC and BAC was detected using 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) as a substrate. Glutathione reacts with the thiol group of DTNB to generate 2-nitro-5-thiobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. By analyzing the absorbance value of the reaction system at 412 nm, the change in glutathione content can be reflected, and the glutathione oxidase activity of RBAC and BAC can be further reflected. The specific steps are: add 50 uL of nanozyme material (final concentration of 4 mg / mL) to 150 uL of phosphate buffer solution (1mM, pH=7.4), and then add 50 uL of glutathione (final concentration of 1 mM). After reacting at 37 °C for a certain period of time, 200 uL of the reaction solution was added with 50 uL of DTNB (final concentration was 0.1 mM) and the absorbance of the mixed solution was detected at 412 nm. Figure 3 The results showed that under the action of glutathione oxidase-like enzymes of RBAC and BAC, the glutathione content was significantly reduced, indicating the excellent glutathione oxidase-like activity of RBAC and BAC.

[0045] Figure 4 This is the characterization of the oxidase-like activity of RBAC and BAC prepared in Example 1 of the present invention. The oxidase-like activity of RBAC and BAC was detected using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The specific steps are: 50 uL of TMB (final concentration 1 mM) and 50 uL of nanozyme material (final concentration 2 mg / mL) were added to 900 μL of phosphate buffer solution (1 mM, pH=7.4), reacted at 37 °C for 20 min, and the supernatant was centrifuged to detect the UV-visible absorption spectrum. The oxidase-like activity of the nanozyme will directly catalyze the reaction of oxygen to generate ·OH and superoxide anions (·O2 - ), thereby oxidizing TMB to the oxidized state oxTMB with a characteristic absorption peak at 652 nm. Figure 4 It can be seen that both the RBAC-treated group and the BAC-treated group showed obvious characteristic absorption peaks at 652 nm, indicating that RBAC and BAC have excellent oxidase-like activity. Figure 5 (a) and (b) are characterizations of the killing ability of RBAC and BAC prepared in Example 1 of the present invention on B16-F10 cells. The specific steps are: 100 μL of 4×10 5 / mL B16-F10 cell suspension was inoculated in a 96-well plate. After the cells were pre-cultured in the incubator for 24 hours until the cells adhered to the wall, the culture medium was aspirated, washed twice with DPBS, and the culture medium containing RBAC and BAC was added. The final concentration of the nanozyme material and the cells was 100, 200, 400, and 800 μg / mL. After the nanozyme material and the cells were co-cultured for 24 h and 72 h, the culture medium was aspirated, washed twice with DPBS, and CCK-8 (10%) working solution was added. After a period of culture, the absorbance value of the well plate at 450 nm was detected using an ELISA reader. Figure 5 (a) The results showed that after incubation of cells with RBAC at a concentration of 400 μg / mL for 24 hours, the cell survival rate was about 60%, while the cell survival rate of the group treated with BAC at the same concentration was about 90%; Figure 5 As shown in (b), after 72 hours of co-incubation, the cell survival rate of the RBAC-treated group was only 40%, while the cell survival rate of the BAC-treated group was 85% at the same concentration, indicating that RGDSP-mediated cell targeting significantly enhanced the cell uptake efficiency of RBAC, enabling it to exhibit a stronger cell killing effect.

[0046] Figure 6It is a characterization of intracellular ROS levels after the RBAC and BAC prepared in Example 1 of the present invention are co-incubated with B16-F10 cells. DCFH-DA (2′, 7′-dichlorofluorescein diacetate) is a ROS indicator probe that has no fluorescence itself. When it enters the cell, it can be hydrolyzed by the esterase in the cell to generate non-fluorescent DCFH (2′, 7′-dichlorodihydrofluorescein), and DCFH cannot pass through the cell membrane and easily accumulates in the cell. The ROS in the cell can oxidize the non-fluorescent DCFH to generate green fluorescent DCF (2′, 7′-dichlorofluorescein), and the intensity of the green fluorescence is proportional to the ROS level. Therefore, the DCFH-DA probe can reflect the intracellular ROS level. The specific steps are: according to the amount of 1 mL per dish, a concentration of 1×10 5 / mL B16-F10 cell suspension was inoculated in a confocal culture dish. After the cells were pre-cultured in the incubator for 24 hours until the cells attached to the wall, the culture medium was aspirated, washed twice with DPBS, and 1 mL of culture medium containing nanozyme material (400 μg / mL) was added. After incubation for 6 hours, the culture medium was aspirated, washed twice with DPBS, and phenol red-free culture medium containing DCFH-DA probe (stock solution diluted 1000 times) was added and cell fluorescence imaging was taken using a laser scanning confocal microscope. Figure 6 As shown in (a), (b) and (c), the cells of the RBAC-treated group and the BAC-treated group showed significantly enhanced green fluorescence compared with the control group, indicating that the peroxidase-like and oxidase-like activities of the gold-copper bimetallic cluster nanozymes induced the production of a large amount of ROS in the cells, and at the same time, under the auxiliary effect of its glutathione oxidase-like catalytic consumption of intracellular GSH, it was conducive to the accumulation of a large amount of ROS in the cells. In addition, the intracellular green fluorescence intensity of the RBAC-treated group was significantly stronger than that of the BAC-treated group, indicating that the tumor-targeting peptide RGDSP enhanced the cell's uptake of the gold-copper bimetallic cluster nanozymes, thereby inducing the production of more ROS.

[0047] Figure 7 The expression of proteins related to the intracellular copper death mechanism after the RBAC and BAC prepared in Example 1 of the present invention were co-incubated with B16-F10 cells was characterized. Lipoic acid amide synthase (LIAS) and dihydrolipoamide transacetylase (DLAT) are key regulators of copper death in cells. Copper ions directly bind to the acylated proteins of the tricarboxylic acid cycle, resulting in abnormal oligomerization of acylated DLAT and loss of Fe-S cluster proteins (such as LIAS). Therefore, LIAS and DLAT are significantly downregulated when cells undergo copper death. The expression levels of intracellular LIAS and DLAT were analyzed by Western blot to study the effect and mechanism of RBAC nanozymes inducing copper death in tumor cells. Figure 7The results showed that the expression levels of LIAS and DLAT in the cells of the RBAC-treated group and the BAC-treated group were downregulated, and the expression levels of related proteins in the RBAC-treated group were lower than those in the BAC-treated group, indicating that the gold-copper bimetallic cluster nanozyme can induce copper death in B16-F10 cells, and under the action of the tumor-targeting peptide RGDSP, it exhibits a stronger effect of inducing copper death.

[0048] The present invention constructs a gold-copper bimetallic cluster nanozyme and endows the nanomaterial with unique glutathione oxidase, peroxidase and oxidase activities based on the synergistic effect of gold-copper bimetallic elements. By consuming high concentrations of glutathione in tumor cells and generating highly cytotoxic hydroxyl radicals, the synergistic therapeutic effect of enhancing copper death is achieved. At the same time, the surface functionalization of the gold-copper bimetallic cluster nanozyme by tumor-targeting peptides can significantly improve the efficient accumulation of copper in tumor cells, further enhancing the therapeutic effect.

[0049] The present invention uses bovine serum albumin as a surface ligand and adopts a one-step method to obtain a gold-copper bimetallic nanocluster with multi-enzyme activities such as peroxidase, oxidase and glutathione oxidase. The synthesis method is simple, efficient and highly repeatable. The tumor targeting peptide is further modified on the surface of the gold-copper bimetallic nanocluster by an amidation reaction to obtain a gold-copper bimetallic cluster nanozyme with tumor targeting function and multi-enzyme activities. The gold-copper bimetallic cluster nanozyme obtained by the present invention has excellent biosafety, can specifically aggregate in tumor cells and induce copper death, and then achieve a significant tumor cell growth inhibition effect based on the synergistic effect of enzyme-like catalysis, and has broad application prospects in the field of tumor treatment.

[0050] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. However, all changes made within the protection scope of the independent claims of the present invention are within the protection scope of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

Claims

1. A method for preparing a tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme, characterized in that: The following steps are involved: Preparation of gold-copper bimetallic cluster nanozymes using natural proteins; The tumor targeting peptide was modified on the surface of the gold-copper bimetallic cluster nanozyme through an amidation reaction to obtain the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme.

2. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 1, characterized in that: The natural protein is bovine serum albumin; the gold-copper bimetallic cluster nanozyme is prepared by the following process: the pH value of a mixed solution containing chloroauric acid, copper nitrate and bovine serum albumin is adjusted to 10-12, and a reaction is carried out under heating conditions to obtain the gold-copper bimetallic cluster nanozyme.

3. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 2, characterized in that: In the mixed solution containing chloroauric acid, copper nitrate and bovine serum albumin, the concentration of bovine serum albumin is 25 mg / mL, the molar ratio of chloroauric acid to copper nitrate is 1-8:1, and the dosage ratio of bovine serum albumin to copper nitrate is 100 mg: 0.0022-0.01 mmol.

4. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 2, characterized in that: The reaction temperature is 35-45°C, the reaction time is 10-14 hours, and the reaction is carried out under stirring at 1200 rpm.

5. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 1, characterized in that: The gold-copper bimetallic cluster nanozyme modified with a tumor-targeting peptide is prepared by the following process: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to the gold-copper bimetallic cluster nanozyme, stirred evenly and reacted in the dark for 30 minutes, then the tumor-targeting peptide is added and reacted for 6-12 hours to obtain the gold-copper bimetallic cluster nanozyme modified with a tumor-targeting peptide.

6. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 5, characterized in that: The sequence of the tumor targeting peptide is shown in SEQ ID NO.

1.

7. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 5, characterized in that: The mass ratio of gold-copper bimetallic cluster nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 25:8-16.

8. The method for preparing the tumor targeting peptide-modified gold-copper bimetallic cluster nanozyme according to claim 5, characterized in that: The mass ratio of gold-copper bimetallic cluster nanozymes to tumor-targeting peptides is 100:1-4.

9. A tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme prepared according to the method according to any one of claims 1-8.

10. Use of a tumor-targeting peptide-modified gold-copper bimetallic cluster nanozyme prepared according to the method of claims 1-8 in the preparation of an anti-tumor chemodynamic therapy preparation and / or a catalytic therapy preparation.

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