Preparation method and application of dextran-modified DSF-loaded Cu-MOF nano-enzyme

Abstract

The invention provides a preparation method of dextran-modified DSF-loaded Cu-MOF nano-enzyme, which comprises the following steps: dropwise adding a methanol solution of copper chloride into a methanol solution of 3-amino-1, 2, 4-triazole, stirring, adding a methanol solution of disulfiram, continuously stirring, centrifuging, washing the precipitate, and carrying out vacuum drying to obtain DSF-loaded Cu-MOF nano-enzyme powder (D (at) MOF); and dissolving the D (at) MOF in deionized water, then dropwise adding a dextran (DEX) aqueous solution, stirring, centrifuging, washing the precipitate, and carrying out vacuum drying to obtain the dextran-modified DSF-loaded Cu-MOF nano enzyme (D (at) D (at) MOF). The invention also provides an application for preparing a medicine for treating triple negative breast cancer. The D (at) D (at) MOF prepared by the invention can target tumor tissues, simulate SOD, POD and GPx in TME, generate high-level ROS and reduce the GSH level at the same time so as to enhance chemical kinetic therapy (CDT), is combined with DSF-mediated chemical therapy to perform synergistic treatment on triple negative breast cancer (TNBC), and can be used for preparing drugs for triple negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of copper-based metal organic framework nanozymes, and specifically relates to a preparation method and application of a dextran-modified DSF-loaded Cu-MOF nanozyme. Background Art

[0002] Breast cancer (BC) is the most commonly diagnosed cancer in women and the second leading cause of cancer-related death worldwide. It can be divided into different subtypes, among which triple-negative breast cancer (TNBC) is one of the most aggressive and difficult to treat subtypes due to the lack of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2. The morbidity and mortality of TNBC account for approximately 15-20% of all breast cancer cases. In recent decades, great efforts have been made in the diagnosis and treatment of TNBC. Numerous strategies have been developed to prevent and manage this disease, including surgical intervention, chemotherapy, radiotherapy, and targeted therapy. However, TNBC is characterized by significant heterogeneity, high invasiveness, obvious dissemination, and resistance to chemotherapy, making it the subtype with the worst prognosis in breast cancer. Emerging nanoscience and nanotechnology are promoting innovative diagnostic and therapeutic approaches to deal with this cancer. To this end, numerous nanomaterials have been developed and implemented for photodynamic therapy, photothermal therapy, or magnetic therapy based on external stimuli such as light or field. However, external stimuli greatly limit the clinical translation and application of these nanomaterials. Therefore, non-invasive, advanced smart therapeutic nanomaterials that do not require drugs or external stimulation are attracting increasing attention, with representative materials such as nanozymes and enzyme-mimicking inorganic or organic nanomaterials.

[0003] Nanozymes are synthetic nanomaterials that mimic the catalytic functions of natural enzymes and can overcome the limitations of natural enzymes. They have the advantages of low cost, easy production, strong stability, and adjustable activity. As a promising approach in the field of healthcare, nanozymes have attracted widespread attention and have shown great potential as effective therapeutic agents for cancer treatment due to their ability to regulate intracellular reactive oxygen species (ROS) levels and their synergistic catalytic activity in the dynamic tumor microenvironment (TME). To date, various nanomaterials, including peroxidase mimics, metal nanoparticles that catalyze H2O2 to generate ·OH, metal oxide nanoparticles, carbon-based nanomaterials, and metal-organic frameworks (MOFs), have been designed and developed for tumor treatment via chemodynamic therapy (CDT). [8] Among them, MOFs are a new type of porous inorganic-organic hybrid materials composed of metal ions / clusters and organic ligands, which show great potential in the development of new nanozymes. [9]. The coordinated metals in MOFs serve as active centers for catalytic reactions and are used as Fenton reagents in CDT therapy. A variety of transition metals (including Fe, Cu, Mn, etc.) are involved in CDT therapy, among which Cu is the most effective Fenton reagent for generating toxic ·OH under weakly acidic conditions (pH 6.5-6.9) within the tumor microenvironment. Therefore, copper-based MOFs (Cu-MOFs) have been found to have higher peroxidase mimetic activity, which can catalyze overexpressed H2O2 in the tumor microenvironment and subsequently be used for CDT. In addition, MOFs nanosystems are also ideal carriers for delivering anti-tumor drugs to achieve synergistic therapeutic effects.

[0004] Disulfiram (DSF) is a drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of alcohol dependence and has demonstrated potent anticancer properties. It is easily reduced by endogenous glutathione (GSH) to diethylenedithiocarbamate (DTC), a highly efficient metal chelator that readily binds to Cu 2+ (CuET) forms a complex. Notably, CuET exhibits enhanced antitumor activity compared to DSF alone. However, the anticancer efficacy of DSF is always limited by trace amounts of endogenous copper or significant adverse reactions caused by direct application of CuET. Therefore, Cu-MOFs can be used as ion carriers to supplement copper; at the same time, they can also be designed to deliver DSF to achieve in situ triggered antitumor efficacy. This strategy can significantly improve the safety of DSF by regulating the transition from "non-toxic" to "toxic" under the stimulation of the tumor microenvironment (TME). In addition, CuET has been shown to induce copper death in many cancer cells, including 4T1, ABC1, and A549 cancer cells. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of a dextran-modified DSF-loaded Cu-MOF nanozyme in view of the deficiencies of the above-mentioned prior art. The dextran-modified DSF-loaded Cu-MOF nanozyme can be used to prepare drugs for triple-negative breast cancer.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme, the method comprising:

[0007] S1. Preparation of Cu-MOF nanozyme loaded with DSF:

[0008] A 0.02 mol / L methanol solution of copper chloride was added dropwise to a 0.02 mol / L methanol solution of 3-amino-1,2,4-triazole while stirring. After stirring for 60 min, a 0.015 mol / L methanol solution of disulfiram was added. Stirring was continued for 45 min. After centrifugation, the precipitate was washed with methanol 3 times and deionized water 3 times in sequence. After vacuum drying, a Cu-MOF nanozyme powder loaded with DSF was obtained, which was recorded as D@MOF.

[0009] S2. Preparation of dextran-modified DSF-loaded Cu-MOF nanozyme:

[0010] The D@MOF obtained in S1 was dissolved in deionized water, and then a 0.015 mol / L dextran (DEX) aqueous solution was dropped into it. After stirring for 24 h, the mixture was centrifuged and the precipitated material was washed three times with methanol and three times with deionized water. After vacuum drying, the dextran-modified DSF-loaded Cu-MOF nanozyme, D@D@MOF, was obtained.

[0011] Preferably, the stirring rate in S1 is 500 rpm.

[0012] Preferably, the molar ratio of the methanol solution of 3-amino-1,2,4-triazole with a concentration of 0.02 mol / L, the methanol solution of cupric chloride with a concentration of 0.02 mol / L, and the methanol solution of disulfiram with a concentration of 0.015 mol / L in S1 is 4:1:1.

[0013] Preferably, the centrifugal speed in S1 is 12000 rpm, and the centrifugal time is 5 min.

[0014] Preferably, the stirring rate in S2 is 500 rpm, the centrifugal rate is 12000 rpm, and the centrifugal time is 5 min.

[0015] Preferably, the particle size of the dextran-modified DSF-loaded Cu-MOF nanozyme described in S2 is 186.18±3.46 nm.

[0016] The present invention also provides an application of the dextran-modified DSF-loaded Cu-MOF nanozyme prepared by the above-mentioned preparation method, wherein the dextran-modified DSF-loaded Cu-MOF nanozyme is used to prepare a drug for triple-negative breast cancer.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The dextran-modified DSF-loaded Cu-MOF nanozyme prepared by the present invention can simulate the activity of superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GPx) in TME, produce high levels of ROS, and reduce GSH levels at the same time, thereby enhancing chemodynamic therapy (CDT), and combined with DSF-mediated chemotherapy, synergistically treat triple-negative breast cancer (TNBC). The present invention uses aminotriazole (3-AT) as an organic ligand for synthesizing Cu-MOF because it contains rich nitrogen-containing functional groups. In addition, 3-AT, as a catalase (CAT) inhibitor, can effectively prevent H2O2 hydrolysis, and synergistically enhance the intracellular H2O2 level with the SOD-like and GPx-like activities of Cu-MOF. The sharp increase in the intracellular H2O2 level accelerates the POD-like catalytic efficiency, thereby promoting the conversion of H2O2 into hydroxyl radicals and achieving efficient CDT. To promote precise catalytic therapy of tumors using nanozymes, we modified the nanozyme surface (D@D@MOF) with dextran (DEX) to improve its biocompatibility and tumor targeting ability. Nanozymes can be enriched in tumor tissues through specific binding interactions between dextran and glucose transporter 1 (GLUT1) expressed on the surface of tumor cells. Importantly, the cell death mechanism triggered by nanozymes was identified as copper death, which was mainly due to targeted intracellular copper delivery and GSH depletion, and cascade catalysis involving multiple enzyme activities further enhanced copper death. This work provides new insights into the treatment strategy of TNBC. The prepared dextran-modified DSF-loaded Cu-MOF nanozymes were used to prepare drugs for triple-negative breast cancer.

[0019] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the optimization results of the conditions for preparing Cu-MOF in Example 2 of the present invention.

[0021] Figure 2 This is a diagram showing the optimization results of the conditions for preparing D@MOF in Example 2 of the present invention.

[0022] Figure 3 It is a graph showing the potential and particle size results of Cu-MOF, D@MOF and D@D@MOF in Example 3 of the present invention.

[0023] Figure 4 It is the elemental analysis of D@D@MOF of Example 3 of the present invention and the XPS result diagram of Cu-MOF, D@MOF and D@D@MOF.

[0024] Figure 5It is the infrared result diagram of DSF, Cu-MOF+DSF, Cu-MOF, D@MOF and D@D@MOF of Example 3 of the present invention.

[0025] Figure 6 This is a diagram of the verification POD activity results of Example 3 of the present invention.

[0026] Figure 7 This is a graph showing the verification GPx activity results of Example 3 of the present invention.

[0027] Figure 8 This is a diagram showing the SOD activity results of Example 3 of the present invention.

[0028] Fig. 9 This is a quantitative graph of fluorescence distribution and fluorescence intensity of D@D@MOF in Example 3 of the present invention targeting tumors in mice.

[0029] Fig.10 This is a graph showing the anti-tumor effects of Cu-MOF, D@MOF and D@D@MOF of Example 3 of the present invention in tumor-bearing mice.

[0030] Fig.11 This is the preparation process of the multifunctional Cu-MOF nanozyme of Example 3 of the present invention and its promotion of copper death against triple-negative breast cancer by simulating the cascade catalysis of GPx, SOD and POD activities. DETAILED DESCRIPTION

[0031] Example 1

[0032] The preparation method of the dextran-modified DSF-loaded Cu-MOF nanozyme of this embodiment is as follows:

[0033] S1. Preparation of Cu-MOF nanozyme loaded with DSF:

[0034] Under the condition of stirring rate of 500 rpm, 1 mL of 0.02 mol / L copper chloride methanol solution was added dropwise to 4 mL of 0.02 mol / L 3-amino-1,2,4-triazole (3-AT) methanol solution while stirring, and then 0.015 mol / L disulfiram (DSF) methanol solution was added, and stirring was continued for 45 min. After centrifugation at a rate of 12000 rpm for 5 min, the precipitate was washed with methanol 3 times and deionized water 3 times in sequence, and vacuum dried to obtain DSF-loaded Cu-MOF nanozyme powder, recorded as D@MOF;

[0035] S2. Preparation of dextran-modified DSF-loaded Cu-MOF nanozyme:

[0036] The D@MOF obtained in S1 was dissolved in 1.5 mL of deionized water, and then 4 mL of a 0.015 mol / L dextran (DEX) aqueous solution was dropped into it. After stirring for 24 h at a stirring rate of 500 rpm, the mixture was centrifuged at a rate of 12000 rpm for 5 min. The precipitated material was washed three times with methanol and three times with deionized water. After vacuum drying, a dextran-modified DSF-loaded Cu-MOF nanozyme was obtained, named D@D@MOF.

[0037] This embodiment also provides the application of the dextran-modified DSF-loaded Cu-MOF nanozyme prepared by the above-mentioned preparation method, and the dextran-modified DSF-loaded Cu-MOF nanozyme is used to prepare a drug for triple-negative breast cancer.

[0038] Example 2

[0039] This example is the optimization of the conditions of the preparation method in Example 1:

[0040] (I) Preparation of Cu-MOF:

[0041] Cu-MOF nanoparticles were prepared by adsorption method. 4 mL of 3-amino-1,2,4-triazole (3-AT) solution (0.02 mol·L -1 ) was placed on a magnetic stirrer and stirred at a speed of 500 rpm. 1 mL of copper chloride solution (0.02 mol·L -1 ) was slowly added dropwise to the 3-AT solution and stirred for 60 min. The product was collected by centrifugation (12000 rpm, 5 min), washed three times with methanol and deionized water respectively, and dried in vacuum to obtain Cu-MOF powder.

[0042] The following is the condition optimization process:

[0043] 1) Investigation of reaction solvent:

[0044] The concentrations of the 3-AT solution and the copper chloride solution were fixed at 0.02 mol·L -1 , Cu 2+ The molar ratio with 3-AT was 1:4, the stirring speed was 500 rpm, the stirring time was 30 min, and the particle size and PDI were used as evaluation indicators to investigate the effects of different reaction solvents (methanol, water) on Cu-MOF.

[0045] like Figure 1 A. In methanol and water, the prepared nanoparticles have a smaller particle size when water is used as the solvent. However, considering that the drug DSF is insoluble in water, methanol was selected as the reaction solvent for the smooth conduct of subsequent research.

[0046] 2)Cu 2+Investigation of the molar ratio with 3-AT:

[0047] According to the screening results of the reaction solvent, set Cu 2+ The molar ratio of Cu to 3-AT was 1:1, 1:2, 1:3, and 1:4, and the particle size and PDI were used as evaluation indicators to investigate the 2+ Effect of the molar ratio of Cu to 3-AT on Cu-MOF.

[0048] like Figure 1 B, in the four conditions set, Cu 2+ When the molar ratio of Cu to 3AT is 1:4, the particle size is the smallest and the PDI is also small, so 1:4 is selected as Cu 2+ The optimal molar ratio with 3AT was used for subsequent studies.

[0049] 3)Cu 2+ Investigation of 3-AT concentration:

[0050] According to Cu 2+ The screening results of the molar ratio of Cu to 3-AT were set 2+ The concentrations of 3-AT were 0.2, 0.1, 0.05, 0.02 and 0.01 mol·L -1 , using particle size and PDI as evaluation indicators to investigate Cu 2+ Effect of 3-AT concentration on Cu-MOF.

[0051] like Figure 1 C, among the five conditions set, with Cu 2+ As the concentration of 3AT decreases, the particle size of the nanoparticles also decreases accordingly. 2+ With 3AT concentration of 0.01 mol·L -1 The particle size of the group with the smallest particle size was less but the product was less than 0.02 mol·L -1 The group was stable, and after comprehensive consideration, 0.02 mol·L was selected. -1 Cu 2+ The optimal reaction concentration with 3AT was used for subsequent studies.

[0052] 4) Investigation of stirring time:

[0053] According to Cu 2+ According to the screening results of 3-AT concentration, the stirring time was set to 5, 15, 30, 45, 60, and 90 min, and the particle size and PDI were used as evaluation indicators to investigate the effect of stirring time on Cu-MOF.

[0054] like Figure 1 D. When the stirring time is within 90 min, there is no significant effect on the particle size of the nanoparticles. Considering PDI comprehensively, the stirring time of 60 min was selected for subsequent research.

[0055] (II) Preparation of D@MOF:

[0056] Take 4 mL of 3-AT (3-amino-1,2,4-triazole) aqueous solution (0.02 mol·L -1 ) was placed on a magnetic stirrer and stirred at a speed of 500 rpm. 1 mL of copper chloride aqueous solution (0.02 mol·L -1 ) was slowly added dropwise to the 3-AT aqueous solution and stirred for 60 min. Then, 1 mL of DSF (disulfiram) methanol solution (0.015 mol·L -1 ), stirring was continued for 45 min, the product was collected by centrifugation (12000 rpm, 5 min), washed three times with methanol and deionized water respectively, and dried in vacuum to obtain D@MOF powder.

[0057] The following is the condition optimization process:

[0058] 1) 3-AT:Cu 2+ : Investigation of DSF molar ratio:

[0059] The concentrations of 3-AT aqueous solution, copper chloride aqueous solution and DSF aqueous solution were all fixed at 0.02 mol·L -1 , stirring speed was 500 rpm, stirring time was 45 min, and 3-AT:Cu was set 2+ The molar ratio of 3-AT:Cu:DSF was 1:1:1, 2:1:1, 3:1:1, 4:1:1, and 5:1:1. The particle size and PDI were used as evaluation indicators to investigate the 2+ :The effect of the molar ratio of DSF on D@MOF.

[0060] like Figure 2 A, among the five conditions set, 3AT:Cu 2+ When the molar ratio of 3AT:Cu:DSF is 4:1:1, the particle size of the nanoparticles is small and the most stable, so 4:1:1 is selected as the 3AT:Cu 2+ The optimal molar ratio of :DSF was used for subsequent experiments.

[0061] 2) Investigation of DSF concentration:

[0062] According to 3-AT:Cu 2+ :DSF molar ratio screening results, setting DSF concentration to 0.005, 0.01, 0.015, 0.02 mol·L -1 , the effect of DSF concentration on D@MOF was investigated with particle size and PDI as evaluation indicators.

[0063] like Figure 2 B. Among the four conditions set, the DSF concentration was 0.015 mol·L-1 When the nanoparticles have the smallest particle size and are relatively stable, 0.015 mol·L -1 Perform subsequent experiments for optimal DSF concentration.

[0064] 3) Investigation of stirring time:

[0065] According to the screening results of DSF concentration, the stirring time was set to 30, 45, 60, 120, and 150 min, and the particle size and PDI were used as evaluation indicators to investigate the effect of stirring time on D@MOF.

[0066] like Figure 2 C. When the stirring time is within 45-60 min, the particle size of the nanoparticles is the smallest. Considering the comprehensive PDI, the stirring time of 45 min was selected for subsequent research.

[0067] Design and synthesis of the nanozyme (D@MOF) in this example: aminotriazole (3-AT) is used as the organic ligand of Cu-MOF, because it is rich in nitrogen-containing functional groups, which is conducive to the coordination and stabilization of Cu ions. 3-AT also acts as a catalase (CAT) inhibitor, effectively preventing the hydrolysis of H2O2, and synergizing with the SOD-like and GPx-like activities of Cu-MOF to increase the intracellular H2O2 level. Loading DSF further enhances the chemotherapy effect of the nanozyme.

[0068] Cascade catalytic mechanism: Nanozymes (D@MOF) mimic the activities of SOD, POD, and GPx in TME (tumor microenvironment), promote ROS generation, and reduce GSH levels. The intracellular H2O2 level increases sharply, accelerating POD-like catalysis, converting H2O2 into highly toxic hydroxyl radicals, and achieving efficient CDT.

[0069] Improved biocompatibility and targeting: Dextran (DEX) is used to modify the surface of nanozymes (D@D@MOF) to improve biocompatibility and reduce side effects. DEX specifically binds to glucose transporter 1 (GLUT1) on the surface of 4T1 tumor cells (triple-negative breast cancer cells), enhancing the enrichment and targeting ability of nanozymes in tumor tissues.

[0070] Example 3

[0071] In this example, the performance of the dextran-modified DSF-loaded Cu-MOF nanozyme (D@D@MOF) prepared in Example 1 was tested:

[0072] The preparation method of the test substance designed in this embodiment is:

[0073] D@D@MOF: Preparation method of D@D@MOF in Example 1.

[0074] D@MOF: the preparation method of D@MOF in step S1 in Example 1.

[0075] Cu-MOF: Cu-MOF preparation method of Example 2.

[0076] Cu-MOF+DSF: a physical mixture of Cu-MOF powder obtained by the Cu-MOF preparation method in Example 2 and DSF (disulfiram) powder in a mass ratio of 1:1.

[0077] 1. The D@D@MOF prepared in Example 1 has uniform particle size and stable morphology and structure.

[0078] (1) The particle size and zeta potential of the prepared nanoparticles were measured using a laser particle size analyzer, and the morphology of the nanoparticles was observed using a transmission electron microscope (TEM).

[0079] The results are as follows Figure 3 As shown in A, the zeta potential of Cu-MOF is about 20.56mV. After the introduction of DSF, the zeta potential of D@MOF is reduced to 10.84mV. In addition, after modification with DEX, the zeta potential further decreased to -3.09mV. It is worth noting that after DEX modification, the zeta potential of the nanoparticles changed from positive to negative. In addition, the hierarchy of nanozyme particle size is as follows: D@D@MOF>D@MOF>MOF( Figure 3 BD), indicating that the introduction of DSF leads to an increase in the particle size of the nanozyme, while the modification of DEX further increases its particle size. Figure 3 As shown in the illustration of BD, the morphology of MOF is a cubic crystal structure. After the introduction of DSF, its shape changes to a spindle shape, and after DEX modification, a thin film is formed on the surface of D@MOF. The observed changes in the morphology of the nanozyme after the introduction of DSF may be attributed to the coordination between DSF and Cu(II). These results preliminarily indicate that D@D@MOF and its control formulation were successfully prepared.

[0080] (2) The elemental composition of D@D@MOF was then further confirmed by scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDS) (SEM-EDS) and X-ray photoelectron spectroscopy (XPS).

[0081] The elements S and C, N, and Cu from DSF are evenly distributed throughout the nanoparticles, indicating that DSF has been successfully and evenly integrated into the coordination structure of Cu-MOF. The dispersion of C and O elements on the particle surface confirms that DEX has been successfully coated on the nanoparticles ( Figure 4 A). The main oxidation state of copper atoms in Cu-MOF is divalent, and a small part is zero-valent or monovalent ( Figure 4 C) However, in D@MOF, the valence state of copper is mainly Cu0 / Cu + , only a small amount is in Cu 2+ state( Figure 4 D). This indicates that the introduced DSF has participated in the coordination structure of Cu-MOF, thereby changing the valence state of the copper element.

[0082] (3) In addition, Fourier transform infrared spectroscopy (FT-IR) analysis further demonstrated the successful preparation of D@D@MOF.

[0083] like Figure 5 As shown, in DSF and DSF+Cu-MOF samples, the - 1 and 914cm - There are characteristic peaks of C=S and CS at 1. However, these peaks are not observed in the D@MOF spectrum, indicating that DSF has been successfully encapsulated inside MOF rather than physically mixed or adsorbed on its surface. In addition, D@D@MOF has a - 1 shows the characteristic peak of hydroxyl at 1400-1100cm - The characteristic peaks of CO and CH are shown in the range of 1, which indicates the typical structure of DEX and further confirms that DEX has been successfully modified on the surface of D@D@MOF.

[0084] 2. The D@D@MOF prepared in Example 1 has multiple enzyme-like activities.

[0085] The D@D@MOF prepared in Example 1 has POD (peroxidase)-like activity, GPx (glutathione peroxidase)-like activity and SOD (superoxide dismutase)-like activity.

[0086] (1) The catalytic ability of D@D@MOF as a POD-like material was evaluated by MB fading experiment. First, a buffer solution (pH = 7.4 and 5.4) was prepared. The buffer solution was PBS (pH = 7.4 and 5.4) containing MB (methylene blue) (10 μg / mL), H2O2 (10 mM), and GSH (glutathione) (10 mM).

[0087] Weigh 1 mg Cu-MOF, 1 mg D@MOF, and 1 mg D@D@MOF, respectively, and dissolve them in 1 mL deionized water to obtain 1 mg / mL mother solution, which is diluted 100 times to obtain a liquid with a concentration of 10 μg / mL, that is, 10 μg / mL Cu-MOF aqueous solution, 10 μg / mL D@MOF solution, and 10 μg / mL D@D@MOF solution are obtained respectively;

[0088] At room temperature, 100 μL of Cu-MOF aqueous solution (10 μg / mL), 100 μL of D@MOF aqueous solution (10 μg / mL), and 100 μL of D@D@MOF aqueous solution (10 μg / mL) were added to 2 mL of the above buffer solution (pH = 5.5), and 100 μL of D@D@MOF aqueous solution (10 μg / mL) was added to 2 mL of the above buffer solution (pH = 7.4). After the liquids were fully mixed, the absorbance of each solution was measured using an ELISA reader.

[0089] Weigh 1 mg of D@D@MOF and dissolve it in 1 mL of deionized water to obtain a 1 mg / mL mother solution, which was diluted 400 times to obtain a D@D@MOF aqueous solution with a concentration of 2.5 μg / mL;

[0090] Then, 100 μL of D@D@MOF aqueous solution (2.5 μg / mL) was added to 2 mL of the above buffer solution (pH = 5.5), and the solution reaction was measured using an ELISA instrument at 0, 1, 3, 5, 7,

[0091] absorbance after 9, 11, 13, 15, and 20 min;

[0092] Weigh 1 mg of D@D@MOF and dissolve it in 1 mL of deionized water to obtain a 1 mg / mL stock solution. Dilute the solution to obtain different concentrations of 0, 0.5, 1.5, 2.5,

[0093] 3.5, 5, 10 μg / mL D@D@MOF aqueous solution;

[0094] Finally, 100 μL of D@D@MOF aqueous solutions with different concentrations (0, 0.5, 1.5, 2.5, 3.5, 5, 10 μg / mL) were added to 2 mL of the above buffer solution (pH = 5.5), and the absorbance of each solution was measured using an ELISA instrument after reacting for 5 min.

[0095] like Figure 6 As shown in A, under slightly acidic conditions (pH = 5.5), the three groups of nanoformulations have similar abilities to produce ·OH by reacting with H2O2. Figure 6 As the concentration of MB increases, the characteristic peak of MB at 665 nm decreases, indicating that the ·OH produced by the nanoformulation gradually increases. It also shows that the POD enzyme characteristics of D@D@MOF are concentration- and time-dependent.

[0096] The results showed that D@D@MOF has POD-like activity, which can increase the level of oxidative stress in tumor cells and reduce their reducing ability, leading to ROS burst and inducing tumor cell death.

[0097] (2) 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a GSH (glutathione) detection probe to explore the GSH consumption ability of nanoparticles (GPx-like activity). DTNB reacts with -SH groups to form 2-nitro-5-thiobenzoic acid anion (TNB 2- ), the reaction solution is bright yellow and has a characteristic absorption peak at 412nm.

[0098] First, a buffer solution was prepared, which was: PBS (pH = 5.5) containing GSH (10 mM) and DTNB (10 μg / mL);

[0099] At room temperature, 100 μL of Cu-MOF aqueous solution (10 μg / mL), D@MOF aqueous solution (10 μg / mL) and D@D@MOF aqueous solution (10 μg / mL) were respectively added to 2 mL of the above buffer solution. After the liquids were fully mixed, the absorbance of each solution was measured using an enzyme reader. Then, 100 μL of D@D@MOF aqueous solution of different concentrations (0, 0.5, 1.5, 2.5, 3.5, 5, 10 μg / mL) was added to 2 mL of the above buffer solution. After reacting for 5 minutes, the absorbance of each solution was measured using an enzyme reader.

[0100] Figure 7 The control in A is GSH+DTNB. Figure 7 As shown in A, after GSH was incubated with the three nanoformulations for 10 min, DTNB was added, and the absorbance at 412 nm was significantly reduced, indicating that the nanoformulations could effectively consume GSH, and D@D@MOF showed the strongest enzyme activity among the three preparations. Figure 7 As shown in (B), with the increase of D@D@MOF concentration, the ability of the nanoformulation to consume GSH also increases.

[0101] The results showed that D@D@MOF has GPx-like activity.

[0102] (3) Using nitro blue tetrazolium (NBT) as a detection probe, the NPs were evaluated for their ability to react with ·O2 - L-methionine and riboflavin will produce ·O2 under ultraviolet light. - , reducing NBT to formazan.

[0103] First, a buffer solution was prepared, which was PBS (pH = 5.5) containing L-methionine (0.033 mM), riboflavin (0.01 mol / L), and GSH (10 mM);

[0104] At room temperature, 100 μL of Cu-MOF aqueous solution (10 μg / mL), 100 μL of D@MOF aqueous solution (10 μg / mL), and 100 μL of D@D@MOF aqueous solution (10 μg / mL) were respectively added to 2 mL of the above buffer solution. The liquids were reacted under ultraviolet light for 5 min. After being fully mixed, 10 μg / mL of NBT solution was added to continue the reaction. The absorbance of different mixed solutions was measured using an enzyme reader. Figure 8 Control with UV refers to the control group irradiated with ultraviolet light, and Control without UV refers to the control group without ultraviolet light irradiation.

[0105] like Figure 8 As shown in Figure A, the fluorescence intensity of NBT at 510-700nm is significantly reduced after adding Cu-MOF, D@MOF and D@D@MO nanoformulations. As the concentration of D@D@MOF increases (Figure B), the intensity of the characteristic peak gradually decreases, indicating that the effect of D@D@MOF in inhibiting the reduction of NBT is gradually enhanced. This may be due to the fact that Cu + / Cu 2+ The presence of redox electron pairs, where O2 - Can be Cu + reduction to H2O2, indicating that D@D@MOF possesses properties as a SOD-like activity.

[0106] The results showed that D@D@MOF had SOD-like activity.

[0107] 3. The D@D@MOF prepared in Example 1 has a good anti-4T1 tumor effect in tumor-bearing mice.

[0108] (1) Tumor-bearing mouse model: First, 1×10 6 4T1 cells were subcutaneously injected into the right anterior axilla of BALB / c female mice to establish a tumor-bearing mouse model ( Fig. 9 A). The formula for calculating the tumor volume is as follows: Tumor volume = (tumor length) × (tumor width) 2 ×0.5.

[0109] (2) In vivo targeted distribution: ICG (indocyanine green) was used as a dye to evaluate the tissue distribution and tumor accumulation of the nanoformulation in mice. 1 mL of ICG (1 mg / kg) was mixed with 2 mL of D@D@MOF aqueous solution and ultrasonicated for 30 min to prepare D@D@MOF@ICG. Free ICG and D@D@MOF@ICG were injected into tumor-bearing mice through the tail vein, respectively. The mice were imaged using the IVIS in vivo imaging system at 0, 2, 4, 8, 12, and 24 h after administration ( Fig. 9A), and the fluorescence intensity in the mouse body was quantitatively analyzed to obtain the results as shown in Fig. 9 As shown in B.

[0110] like Fig. 9 As shown in A, during the entire observation period, the tumor site of mice injected with free ICG showed only a weak signal, and the fluorescence signal almost completely disappeared 24 hours after intravenous injection, indicating that free ICG can be completely metabolized in mice after 24 hours. In sharp contrast to this phenomenon, the gradual accumulation of D@D@MOF@ICG was observed in the tumor site, and it still showed significant fluorescence intensity 24 hours after administration. This shows that D@D@MOF can prolong the circulation time of ICG in mice. Fig. 9 As shown in B, the fluorescence intensity of the D@D@MOF@ICG group was approximately 25 times that of the free ICG group 24 h after administration.

[0111] The above results show that D@D@MOF has a good tumor targeting effect in tumor-bearing mice.

[0112] (3) In vivo antitumor study: Tumor-bearing mice were randomly divided into five groups (n=5): Control (normal saline), DSF, Cu-MOF, D@MOF, and D@D@MOF. Then, the mice were injected intravenously into the tail vein on days 0, 2, 4, 6, 8, 10, and 12, and the weight and tumor volume of the mice were recorded every two days. Fig.10 B) Weight ( Fig.10 C), and the tumor inhibition rate of each group was calculated by tumor weight ( Fig.10 D) Mice were killed 1 day after the last administration, and tumors and major organs were collected. Tumors in different treatment groups were weighed and photographed ( Fig.10 A). Fig.10 As shown in A, the tumors of mice in each group were collected and photographed, and it was clearly observed that the tumor volume of the D@D@MOF group was the smallest. Fig.10 The tumor growth curves of each group of mice in B also show that during the treatment period, the tumor volume of each group increased to 4.75 times, 3.95 times, and 2.77 times of the original, respectively, indicating that compared with the Control group and the DSF group, D@D@MOF played a therapeutic role, slowing down the growth of tumor volume and showing a significant inhibitory effect on tumors. Fig.10 The tumor weights of mice in each group C also show that the tumors in the D@D@MOF group are significantly lighter than those in other groups. Fig.10 In D, we can see that the tumor inhibition rate of the D@D@MOF group reached 71.15%, which is significantly higher than that of other drug-treated groups.

[0113] The above results all show that D@D@MOF has a good anti-tumor therapeutic effect on tumor-bearing mice.

[0114] In summary, dextran-modified DSF-loaded Cu-MOF nanozymes were used to prepare drugs for triple-negative breast cancer.

[0115] The dextran-modified DSF-loaded Cu-MOF nanozyme prepared by the present invention can simulate the activities of superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GPx) in TME, produce high levels of ROS, and reduce GSH levels, thereby enhancing chemodynamic therapy (CDT) and combining with DSF-mediated chemotherapy to synergistically treat triple-negative breast cancer (TNBC). Fig.11 As shown, we used aminotriazole (3-AT) as an organic ligand for the synthesis of Cu-MOF because it contains abundant nitrogen-containing functional groups. In addition, 3-AT, as a catalase (CAT) inhibitor, can effectively prevent the hydrolysis of H2O2 and synergistically enhance the intracellular H2O2 level with the SOD-like and GPx-like activities of Cu-MOF. The sharp increase in intracellular H2O2 levels accelerates the POD-like catalytic efficiency, thereby promoting the conversion of H2O2 into hydroxyl radicals and achieving efficient CDT. To promote precise catalytic therapy of tumors using nanozymes, we modified the surface of nanozymes (D@D@MOF) with dextran (DEX) to improve its biocompatibility and tumor targeting ability. Nanozymes can be enriched in tumor tissues through specific binding interactions between dextran and glucose transporter 1 (GLUT1) expressed on the surface of tumor cells. Importantly, the cell death mechanism triggered by nanozymes was determined to be copper death, which is mainly due to targeted intracellular copper delivery and GSH depletion, which is further enhanced by cascade catalysis involving multiple enzyme activities. This work provides new insights into treatment strategies for TNBC.

[0116] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme, characterized in that: The method is: S1. Preparation of Cu-MOF nanozyme loaded with DSF: A 0.02 mol / L methanol solution of copper chloride was added dropwise to a 0.02 mol / L methanol solution of 3-amino-1,2,4-triazole while stirring. After stirring for 60 min, a 0.015 mol / L methanol solution of disulfiram was added. Stirring was continued for 45 min. After centrifugation, the precipitate was washed with methanol 3 times and deionized water 3 times in sequence. After vacuum drying, a Cu-MOF nanozyme powder loaded with DSF was obtained, which was recorded as D@MOF. S2. Preparation of dextran-modified DSF-loaded Cu-MOF nanozyme: The D@MOF obtained in S1 was dissolved in deionized water, and then a 0.015 mol / L dextran aqueous solution was added dropwise. After stirring for 24 h, the mixture was centrifuged and the precipitated material was washed three times with methanol and three times with deionized water. After vacuum drying, the dextran-modified DSF-loaded Cu-MOF nanozyme was obtained, which was recorded as D@D@MOF.

2. The method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme according to claim 1, characterized in that: The stirring rate in S1 was 500 rpm.

3. The method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme according to claim 1, characterized in that: The molar ratio of the methanol solution of 3-amino-1,2,4-triazole with a concentration of 0.02 mol / L, the methanol solution of copper chloride with a concentration of 0.02 mol / L, and the methanol solution of disulfiram with a concentration of 0.015 mol / L in S1 is 4:1:

1.

4. The method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme according to claim 1, characterized in that: The centrifugal speed in S1 was 12000 rpm and the centrifugal time was 5 min.

5. The method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme according to claim 1, characterized in that: The stirring rate in S2 was 500 rpm, the centrifugal rate was 12000 rpm, and the centrifugal time was 5 min.

6. The method for preparing a dextran-modified DSF-loaded Cu-MOF nanozyme according to claim 1, characterized in that: The particle size of the dextran-modified DSF-loaded Cu-MOF nanozyme described in S2 was 186.18±3.46 nm.

7. An application of a dextran-modified DSF-loaded Cu-MOF nanozyme prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The dextran-modified DSF-loaded Cu-MOF nanozyme is used to prepare drugs for triple-negative breast cancer.

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