A nanomaterial for starvation therapy combined with copper death, and its preparation method and application

Abstract

The present invention belongs to the technical field of nanomaterials and anti-tumor therapeutic drugs, and specifically relates to a nanomaterial for starvation therapy combined with copper death, as well as a preparation method and application thereof. The nanomaterial comprises a metal organic framework (MOF-199) wrapped around manganese dioxide (MnO2) nanoparticles, and glucose oxidase (GOx) is modified outside the MOF-199 shell. After the nanomaterial is internalized by cancer cells, on the one hand, glucose is consumed to achieve the purpose of starvation therapy. On the other hand, the MOF-199 shell is degraded by glutathione in the cancer cells, and the exposed MnO2 exerts catalase-like activity to catalyze the production of oxygen. At the same time, a surge in Cu(I) in the cell and oligomerization of lipoylated protein S-acetyltransferase (DLAT) lead to loss of mitochondrial function, resulting in copper death. The present invention not only achieves cancer treatment but also has important scientific significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and anti-tumor therapeutic drugs, and particularly relates to a nanomaterial for starvation therapy combined with copper death, a preparation method thereof, and an application thereof. Background Art

[0002] Starvation therapy, which consumes glucose in tumors, represents an important anti-tumor treatment strategy. Glucose oxidase (GOx) can effectively consume glucose in tumor cells, but it consumes oxygen during the reaction, causing further hypoxia in the tumor microenvironment. Hypoxia is a common feature of most solid tumors, which is mainly due to the imbalance between oxygen consumption and oxygen uptake caused by the rapid proliferation of tumor cells and the inefficiency of the tumor microvascular system. Hypoxia not only reduces the efficacy of anti-cancer treatment, but also enhances the invasiveness and metastasis of tumors, and is the main cause of death in most cancer patients. Therefore, alleviating the hypoxic environment of the tumor is very important for starvation therapy. At the same time, the effect of tumor treatment relying on a single mode is often poor, so it is also necessary to develop a combined treatment method.

[0003] Metal-organic frameworks (MOFs) and their corresponding nanostructures have been widely used in catalysis, optical probes, and biomedicine. The copper-based MOF-199 nanomaterial also plays an important role in cancer treatment, such as depleting high glutathione concentrations in tumor cells and providing exogenous copper to tumor cells to disrupt intracellular copper homeostasis.

[0004] Copper, an essential mineral nutrient, is widely involved in copper overload-induced cell proliferation and death pathways. A recent study identified a unique copper-dependent death pathway, termed "copper death," which is induced by intracellular copper accumulation and triggers the aggregation of mitochondrial lipidated proteins. However, intracellular copper is maintained at extremely low levels by active homeostatic mechanisms. Therefore, disrupting copper homeostasis in tumor cells to achieve copper death is crucial for cancer treatment.

[0005] Based on the above, it is necessary to develop a new nanomaterial that can alleviate the hypoxia and starvation therapy combined with copper death in the tumor microenvironment. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a nanomaterial for starvation therapy combined with copper death, as well as its preparation method and application. By growing a MOF-199 shell on the outer layer of MnO2 nanoparticles and modifying MOF-199 with glucose oxidase, a multimodal treatment system for alleviating hypoxia, starvation therapy and copper death can be constructed.

[0007] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a nanomaterial for starvation therapy combined with copper death, wherein the nanomaterial is MnO2@MOF-199@GOx, comprising MnO2, MOF-199, and GOx, wherein the outside of the MnO2 is wrapped with MOF-199, and GOx is modified on the outer layer of MOF-199.

[0009] In a second aspect, the present invention provides a method for preparing a nanomaterial for starvation therapy combined with copper death, comprising the following steps:

[0010] Step S1: Preparation of MnO2 nanoparticles

[0011] KMnO4 was dispersed in water and bovine serum albumin dispersed in phosphate buffer was added dropwise to obtain a mixture; the mixture was stirred at 37°C for 2 hours to obtain MnO2 nanoparticles;

[0012] Step S2: Preparation of MnO2@MOF-199 nanoparticles

[0013] Step S2-1: dissolving trimesic acid in a triethylamine aqueous solution, stirring vigorously during the dissolution process, and then drying in a vacuum drying oven at 60° C. to synthesize benzene-1,3,5-tricarboxylic acid triethylamine salt;

[0014] Step S2-2: The MnO2 nanoparticles prepared in step S1 are dispersed in water, and then a mixture of ethanol and water is added and mixed. An aqueous solution of copper nitrate is added to the mixture, heated and stirred for a period of time, and then an aqueous solution of benzene-1,3,5-tricarboxylic acid triethylammonium salt is added dropwise. The precipitate is collected by centrifugation and then washed with ethanol to obtain MnO2@MOF-199 nanoparticles;

[0015] Step S3: Preparation of MnO2@MOF-199@GOx nanoparticles

[0016] The MnO2@MOF-199 nanoparticles prepared in step S2 were added to the GOx aqueous solution, stirred for a period of time, collected by centrifugation, and washed with water to obtain MnO2@MOF-199@GOx nanoparticles.

[0017] Preferably, in step S2-1, the mass ratio of trimesic acid to triethylamine is 2.1:3, and the stirring speed during the dissolution process is 1600 rpm.

[0018] Preferably, in step S2-2, the amount of MnO2 nanoparticles used is 5-20 mg, the ratio of ethanol to water is 1:1, the heating temperature is 40-60°C, the time is 30 min, the molar ratio of copper nitrate to benzene-1,3,5-tricarboxylic acid triethylamine salt is 1:1, and the dropwise addition rate of the benzene-1,3,5-tricarboxylic acid triethylamine salt aqueous solution is 50 μL / min.

[0019] Preferably, the amounts of ethanol and water used in step S2-2 are 10-20 mL respectively.

[0020] Preferably, in step S3, the mass ratio of MnO2@MOF-199 nanoparticles to GOx is 2:1-2, the stirring time is 1-2 h, and the stirring speed is 600-1000 rpm.

[0021] In a third aspect, the present invention provides an application of a nanomaterial combining starvation therapy with copper death in the preparation of an anti-tumor drug.

[0022] The present invention has the following beneficial effects: the present invention realizes wrapping a MOF-199 shell layer on the outer layer of MnO2 particles, and uses GOx to modify the surface of MOF-199. After being taken up by cancer cells, the prepared nanomaterial MnO2@MOF-199@GOx consumes glucose through the catalytic action of GOx to achieve starvation therapy. On the other hand, MOF-199 reacts with the high concentration of glutathione in cancer cells to expose the inner layer of MnO2 to exert catalase-like activity, generate oxygen and alleviate hypoxia in the tumor. At the same time, during the degradation of the MOF-199 shell, Cu(I) and lipoylated protein S-acetyltransferase (DLAT) oligomerization are generated, resulting in loss of mitochondrial function and copper death, which is of great scientific significance for achieving cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a transmission electron microscopy (TEM) image of the MnO2@MOF-199@GOx nanoparticles of the present invention;

[0024] Figure 2 is the element distribution diagram of MnO2@MOF-199@GOx nanoparticles in the present invention;

[0025] Figure 3 is the GOx gel electrophoresis diagram of the MnO2@MOF-199@GOx nanoparticles in the present invention;

[0026] Figure 4 This is the case where oxygen is generated after the reaction of MnO2@MOF-199@GOx with glutathione and hydrogen peroxide in the present invention;

[0027] Figure 5The consumption of GSH after incubation of PBS, MOF-199, and MnO2@MOF-199@GOx with cancer cells in the present invention;

[0028] Figure 6 The consumption of glucose after incubation of PBS, MOF-199, and MnO2@MOF-199@GOx with cancer cells in the present invention;

[0029] Figure 7 The aggregation of DLAT was observed using a laser confocal microscope after incubation of PBS, MOF-199, MnO2@MOF-199@GOx with cancer cells in the present invention;

[0030] Figure 8 The mitochondrial damage was observed using a laser confocal microscope after incubation of PBS, MOF-199, and MnO2@MOF-199@GOx with cancer cells in the present invention;

[0031] Figure 9 This is the survival status of cancer cells after MOF-199, MnO2@MOF-199@GOx in the present invention are incubated with cancer cells. Implementation Method

[0032] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention. Example 1

[0033] Step S1: Preparation of MnO2 nanoparticles

[0034] 6.32 mg of KMnO4 was dispersed in 0.6 mL of water, and a solution of 50 mg of bovine serum albumin dispersed in 1.4 mL of phosphate buffer was added dropwise to obtain a mixture; the mixture was stirred at 37°C for 2 hours to obtain MnO2 nanoparticles;

[0035] Step S2: Preparation of MnO2@MOF-199 nanoparticles

[0036] Step S2-1: Dissolve trimesic acid (2.1 g, 0.01 mol) in a 30 wt% aqueous solution of triethylamine (10 ml) with vigorous stirring at 1600 rpm. The solution was then dried in a vacuum drying oven at 60°C to synthesize benzene-1,3,5-tricarboxylic acid triethylamine salt.

[0037] Step S2-2: 10 mg of the MnO2 nanoparticles prepared in step S1 was dispersed in 1 mL of water, and then a mixture of 15 mL of ethanol and 15 mL of water was added and mixed. A 0.01 M aqueous solution of copper nitrate was added to the mixture, and the mixture was heated and stirred at 50°C for 30 min. Then, a 0.01 M aqueous solution of triethylamine salt of benzene-1,3,5-tricarboxylic acid (3 mL) was added dropwise at a dropping rate of 50 μL / min for 1 h. The precipitate was collected by centrifugation and then washed three times with ethanol to obtain MnO2@MOF-199 nanoparticles.

[0038] Step S3: Preparation of MnO2@MOF-199@GOx nanoparticles

[0039] Take 1 mg of MnO2@MOF-199 nanoparticles prepared in step S2 and add it to a GOx aqueous solution (1 mg / mL, 2 mL). Stir for 2 h and then collect by centrifugation at a stirring speed of 600-1000 rpm. Wash with water three times to obtain MnO2@MOF-199@GOx nanoparticles.

[0040] Characterization of MnO2@MOF-199@GOx

[0041] Figure 1 is a transmission electron microscope (TEM) image of the MnO2@MOF-199@GOx nanoparticles in the present invention, as shown in FIG. Figure 1 As shown in the figure, MnO2@MOF-199@GOx nanoparticles were successfully synthesized, and the MOF-199 shell was wrapped on the outer layer of the MnO2 nanoparticles.

[0042] Figure 2 is the element distribution diagram of the MnO2@MOF-199@GOx nanoparticles in the present invention, such as Figure 2 The Mn element is shown distributed on MOF-199.

[0043] Figure 3 This is the gel electrophoresis diagram of the MnO2@MOF-199@GOx nanoparticles in the present invention. By performing gel electrophoresis tests on GOx and MnO2@MOF-199@GOx nanoparticles, it was found that a band appeared at 76 kD for MnO2@MOF-199@GOx nanoparticles, proving that GOx was successfully modified onto MnO2@MOF-199. Example 2

[0044] Figure 4To investigate the generation of oxygen after the reaction of MnO2@MOF-199@GOx with glutathione and hydrogen peroxide in the present invention, 10 mg of the MnO2@MOF-199@GOx nanomaterial prepared in Example 1 was weighed and placed in a glass bottle containing a 10 mM hydrogen peroxide solution. After the solution was fully dissolved, glutathione solution was added dropwise to a concentration of 10 mM, and the generation of oxygen was measured using a dissolved oxygen meter. Figure 4 As shown in Table 1, compared with the control group, Example 1 produced a large amount of oxygen, about 8 mg / L, within 400 s, proving that MnO2 in the MnO2@MOF-199@GOx nanomaterial can generate oxygen, suggesting that it can alleviate the hypoxic environment of tumor cells.

[0045] . Example 3

[0046] Intracellular glutathione depletion study

[0047] Figure 5 The consumption of glutathione after incubation of PBS, MOF-199, MnO2@MOF-199@GOx with cancer cells in the present invention was investigated. 100,000 cells were seeded in each well of a six-well plate. After 12 h of incubation, serum-free culture medium containing PBS, MOF-199, and MnO2@MOF-199@GOx was added to each well. After further incubation for 12 h, the cells were blown off and lysed with lysis buffer for 5 min. The supernatant was then centrifuged and the glutathione detection kit of Beyuntian was used to determine the consumption of glutathione in cancer cells by MOF-199 nanoparticles and MnO2@MOF-199@GOx nanoparticles. Figure 5 As shown in Table 2, the consumption of GSH by MnO2@MOF-199@GOx nanoparticles is the most significant. The multiple relationship between the experimental group and the PBS group is shown in Table 2.

[0048] Example 4

[0049] Figure 6 To investigate the glucose consumption of cancer cells after incubation with PBS, MOF-199, and MnO2@MOF-199@GOx, 100,000 cells were seeded per well in a six-well plate. After 12 h of incubation, serum-free culture medium containing PBS, MOF-199, and MnO2@MOF-199@GOx was added to each well. After incubation for 8 h, the cells were blown off and lysed with lysis buffer for 5 min. The supernatant was then centrifuged and the glucose consumption of each group was determined using a Beyotime glucose kit. Figure 6As shown in Table 3, MnO2@MOF-199@GOx nanoparticles have a significant consumption of glucose compared to MOF-199, proving that GOx in MnO2@MOF-199@GOx nanomaterials can play a role in consuming glucose to achieve the purpose of starvation therapy. The data are shown in Table 3.

[0050] Example 5

[0051] Figure 7 In the present invention, PBS, MOF-199, MnO2@MOF-199@GOx were incubated with cancer cells and the aggregation of DLAT was observed using a laser confocal microscope. 50,000 cells were planted in a confocal dish and incubated for 12 hours. Serum-free culture medium containing PBS, MOF-199, MnO2@MOF-199@GOx was added to each well, and then incubated for 6 hours. Paraformaldehyde was added for fixation for 30 minutes, and 0.1% triton was used for permeabilization for 5 minutes. 5% BSA solution was used for blocking for 1 hour. DLAT primary antibody was added for overnight incubation, and then secondary antibody was added for observation using a laser confocal microscope. Figure 7 As shown in Figure 2, MOF-199 nanoparticles and MnO2@MOF-199@GOx nanoparticles prepared in Example 1 were incubated with cancer cells and then laser confocal microscopy was performed. The figure shows that after the treatment with MnO2@MOF-199@GOx nanoparticles, the fluorescence aggregation is more obvious, indicating that it induces cell copper death. Example 6

[0052] Figure 8 In the present invention, PBS, MOF-199, MnO2@MOF-199@GOx were incubated with cancer cells to observe the damage of mitochondria using a laser confocal microscope. 50,000 cells were planted in a confocal dish. After incubation for 12 hours, serum-free culture medium containing PBS, MOF-199, and MnO2@MOF-199@GOx was added to each well, and then incubated for another 6 hours. Mitochondrial fluorescent dye was added for staining and then observed using a confocal microscope. Figure 8 As shown in Figure 2, MOF-199 nanoparticles and MnO2@MOF-199@GOx nanoparticles prepared in Example 1 were incubated with cancer cells and then subjected to laser confocal microscopy. The figure shows that after treatment with MOF-199 nanoparticles and MnO2@MOF-199@GOx nanoparticles, mitochondrial membrane potential changed significantly, and the fluorescence of the MnO2@MOF-199@GOx nanoparticle group was dimmer, indicating greater damage to the mitochondria. Example 7

[0053] Figure 9 To investigate the survival of cancer cells after incubation of MOF-199 and MnO2@MOF-199@GOx with cancer cells, 10,000 cells were seeded per well in a 96-well plate. After 12 h of incubation, serum-free culture medium containing MOF-199 and MnO2@MOF-199@GOx at different concentrations was added to each well. After further incubation for 12 h, the CCK-8 method was used to determine the killing effect of MOF-199 nanoparticles and MnO2@MOF-199@GOx nanoparticles on cancer cells. The experimental results are shown in Figure 2. Figure 9 As shown in Table 4, the toxicity of MOF-199 and MnO2@MOF-199@GOx to cancer cells is shown.

[0054]

[0055] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any person skilled in the art without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. A nanomaterial for starvation therapy combined with copper death, characterized in that, The nanomaterial is MnO2@MOF-199@GOx, which includes MnO2, MOF-199, and GOx. The MnO2 is coated with MOF-199, and GOx is modified on the outer layer of MOF-199. After the MnO2@MOF-199@GOx is taken up by cancer cells, MOF-199 reacts with glutathione in the cancer cells to expose the inner layer of MnO2, which then exhibits catalase-like activity. The preparation steps of the nanomaterial are as follows: Step S1: Preparation of MnO2 nanoparticles KMnO4 was dispersed in water and bovine serum albumin dispersed in phosphate buffer was added dropwise to obtain a mixture; the mixture was stirred at 37°C for 2 hours to obtain MnO2 nanoparticles; Step S2: Preparation of MnO2@MOF-199 nanoparticles Step S2-1: dissolving trimesic acid in an aqueous triethylamine solution with vigorous stirring during the dissolution process, and then drying the solution in a vacuum drying oven at 60°C to synthesize benzene-1,3,5-tricarboxylic acid triethylamine salt, wherein the mass ratio of trimesic acid to triethylamine is 2.1:3, and the stirring speed during the dissolution process is 1600 rpm; Step S2-2: The MnO2 nanoparticles prepared in step S1 are dispersed in water, and then a mixed system of ethanol and water is added and mixed. An aqueous copper nitrate solution is added to the mixed system, heated and stirred for a period of time, and then an aqueous solution of benzene-1,3,5-tricarboxylic acid triethylammonium salt is added dropwise. The precipitate is collected by centrifugation and then washed with ethanol to obtain MnO2@MOF-199 nanoparticles, wherein the amount of MnO2 nanoparticles is 5-20 mg, the ratio of ethanol to water is 1:1, the heating temperature is 40-60°C, the time is 30 min, the molar ratio of copper nitrate to benzene-1,3,5-tricarboxylic acid triethylammonium salt is 1:1, and the dropwise addition rate of the aqueous solution of benzene-1,3,5-tricarboxylic acid triethylammonium salt is 50 μL / min; Step S3: Preparation of MnO2@MOF-199@GOx nanoparticles The MnO2@MOF-199 nanoparticles prepared in step S2 were added to the GOx aqueous solution, stirred for a period of time, collected by centrifugation, and washed with water to obtain MnO2@MOF-199@GOx nanoparticles; wherein the mass ratio of MnO2@MOF-199 nanoparticles to GOx was 2:1-2, the stirring time was 1-2 h, and the stirring speed was 600-1000 rpm.

2. The nanomaterial for starvation therapy combined with copper death according to claim 1, characterized in that: The amounts of ethanol and water used in step S2-2 are 10-20 mL, respectively.

3. Use of the nanomaterial of starvation therapy combined with copper death according to claim 1 in the preparation of anti-tumor therapeutic drugs.

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