Copper selenide nano-enzyme with effect of simultaneously inducing pyroptosis and copper death of tumor cells as well as preparation method and application of copper selenide nano-enzyme
By preparing copper selenide nanoenzymes with black tea extract as template, using its catalytic activity and copper ion accumulation, various cell death pathways are activated, and the recurrence and toxic side effects of traditional anti-tumor treatments are solved, and efficient tumor cell killing effect is achieved.
Patent Information
- Application Number
- CN202510461554.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-08-01
AI Technical Summary
Traditional anti-tumor treatment methods are prone to recurrence and metastasis, and have problems such as large toxic side effects and low treatment efficiency. Existing nanoenzymes have failed to effectively activate various cell death pathways in tumor treatment.
Using black tea extract as a template, copper selenide nanoenzyme was prepared by reactions of vitamin C, sodium selenite and copper sulfate. It was used to catalyze H2O2 to generate ROS and oxidize GSH, activate Caspase-1, combine with copper ion accumulation, and induce tumor cell pyrolysis and copper death.
It achieves efficient killing of tumor cells, and improves the therapeutic effect by inducing calcification and copper death at the same time, and the preparation process is simple and green.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical fields of nanomaterials and anti-tumor therapeutic drugs, and particularly relates to a copper selenide nanozyme with the effects of simultaneously inducing pyroptosis and cuproptosis of tumor cells, a preparation method thereof, and an application thereof. Background Art
[0002] Cancer, also known as malignant tumor, is a class of diseases caused by abnormal and excessive proliferation of body cells, and has the characteristics of fast proliferation rate, low differentiation degree, and easy metastasis. Traditional cancer treatment methods mainly include surgery, chemotherapy, and radiotherapy. However, these traditional anti-tumor means are prone to recurrence and metastasis, can damage the immune system, resulting in large toxic side effects and low treatment efficiency. Therefore, the research on cancer treatment methods has always been a research hotspot. With the development of nanobiotechnology, the application of nanomaterials has become more and more extensive. Applying nanobiotechnology to tumor treatment has attracted extensive attention from researchers.
[0003] A nanozyme is a nanomaterial that combines the physicochemical properties of nanomaterials and the catalytic activity of natural enzymes. Based on the unique advantages of nanozymes, the application of nanozymes in tumor treatment has always been a research hotspot. Since nanozymes have good biocompatibility and high catalytic activity, and the tumor microenvironment has the characteristics of excessive GSH and H2O2 and mild acidity, nanozymes can mimic peroxidase to produce harmful ROS under acidic conditions, and can also mimic the activity of glutathione peroxidase to catalytically oxidize GSH to reduce ROS consumption, thereby specifically killing tumors.
[0004] Pyroptosis, also known as cell inflammatory necrosis, is a programmed cell death mode mainly dependent on inflammasomes and Caspase-1. When pyroptosis occurs, cells will continuously swell and cause the cell membrane to rupture, and then cell contents and a large number of pro-inflammatory factors are released, activating the inflammatory response and Caspase-1, thereby causing cell death. Cuproptosis is a new type of cell death induced by excessive copper ions. Excessive copper directly binds to the lipoylated components of the tricarboxylic acid (TCA) cycle, causing oligomerization of lipoylated proteins, triggering proteotoxic stress leading to cell death, and loss of iron-sulfur cluster proteins.
[0005] Based on the multiple enzyme activities of nanozymes and the characteristics of the tumor microenvironment, different pathways of cell death can be activated to induce tumor cell death and play an anti-tumor role. Summary of the Invention
[0006] In order to overcome the disadvantages and deficiencies in the prior art, the primary object of the present invention is to provide a preparation method of copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells; this method uses black tea extract as a template, mixes vitamin C, sodium selenite, and copper sulfate for reaction to obtain copper selenide nanozyme.
[0007] Another object of the present invention is to provide a copper selenide nanozyme prepared by the above preparation method and having the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells; this copper selenide nanozyme can catalyze the overexpressed H2O2 in tumor cells to generate ROS, and at the same time catalyze the oxidation of GSH, reduce the consumption of ROS. A large amount of ROS can indirectly activate Caspase-1, cleave the GSDMD protein into GSDMD-N, cause cytoplasmic membrane perforation, and release inflammatory factor IL 1β; at the same time, the copper selenide nanozyme can cause the accumulation of intracellular copper ions, lead to the oligomerization of DLAT protein, increase pyruvate, trigger proteotoxic stress, and the loss of iron-sulfur cluster proteins, and at the same time induce pyroptosis and cuproptosis of tumor cells, providing a new strategy for tumor treatment.
[0008] Another object of the present invention is to provide an application of the above copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells.
[0009] The object of the present invention is achieved by the following technical solutions:
[0010] A preparation method of copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells, including the following operating steps:
[0011] (1) Preparation of nano-selenium solution;
[0012] (2) Preparation of copper selenide nanozyme.
[0013] The preparation of the nano-selenium solution in step (1) is specifically carried out according to the following steps: using black tea extract as a soft template, adding vitamin C solution and sodium selenite solution to the black tea extract solution, and standing in a water bath at 4 - 100 °C for 5 - 180 min, and obtaining a nano-selenium solution after the reaction;
[0014] The preparation of the copper selenide nanozyme in step (2) is specifically carried out according to the following steps:
[0015] Adding copper sulfate solution to the nano-selenium solution obtained in step (1), standing the obtained mixed solution in a water bath at 4 - 100 °C for 5 - 180 min, and dialyzing with a MWCO 8000Da dialysis bag for 24 h to obtain a copper selenide nanozyme solution;
[0016] The concentration of sodium selenite in the sodium selenite solution in the mixed solution is 10 mmol / L; the concentration of vitamin C in the vitamin C solution in the mixed solution is 20 - 200 mmol / L; the concentration of the dark tea extract in the dark tea extract solution in the mixed solution is 100 - 1000 mg / L; the concentration of copper sulfate in the copper sulfate solution in the mixed solution is 2.5 - 40 mmol / L.
[0017] The dark tea extract is the water extract, tea polysaccharide, tea brown pigment, etc. of dark tea processed from fresh tea leaves and tender shoots of tea trees by the processes of fixation, rolling, piling, and drying.
[0018] The concentration of the dark tea extract in the dark tea extract solution in the mixed solution is preferably 300 mg / L;
[0019] The concentration of vitamin C in the vitamin C solution in the mixed solution is preferably 60 mmol / L;
[0020] The concentration of copper sulfate in the copper sulfate solution in the mixed solution is preferably 10 mmol / L;
[0021] The temperature of the water bath for static settlement is 30 °C, and the time is 15 min.
[0022] A copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells prepared by the above preparation method.
[0023] The inhibitory effect of copper selenide nanozyme on the proliferation of different tumor cells was detected by the MTT method to evaluate the anti-cancer activity of the copper selenide nanozyme obtained in the present invention;
[0024] The above tumor cell models include: human colon cancer cell line HCT-116, human breast cancer cell line MCF-7, human liver cancer cell line HepG2, human cervical cancer cell line Hela, preferably the HCT-116 cell line.
[0025] The application of the above copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells in the preparation of products related to catalyzing hydrogen peroxide to produce ROS and consuming GSH.
[0026] The application of the above copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells in the preparation of cell pyroptosis and cuproptosis inducers.
[0027] The present invention has the following advantages and beneficial effects compared with the prior art:
[0028] (1) The present invention uses black tea extract as a template, and uses vitamin C, sodium selenite, and copper sulfate as raw materials to synthesize copper selenide nanozyme by the sacrificial template method. The preparation process is simple, rapid, and green. The copper selenide nanozyme has a small particle size, uniform particle distribution, good electrostatic stability, and good peroxidase-like activity and glutathione peroxidase-like activity.
[0029] (2) The copper selenide nanozyme prepared by the present invention has a concentration-dependent killing effect on tumor cells. The copper selenide nanozyme can catalyze H2O2 to produce ROS, and at the same time catalyze the oxidation of GSH to reduce the consumption of ROS. A large amount of ROS activates Caspase-1, cleaves the GSDMD protein into GSDMD-N, causes cytoplasmic membrane perforation, and releases the inflammatory factor IL 1β; at the same time, it causes the accumulation of intracellular copper ions, causes the oligomerization of DLAT, increases pyruvate, triggers proteotoxic stress, and the loss of iron-sulfur cluster proteins, thereby simultaneously triggering pyroptosis and cuproptosis of tumor cells. Description of the Drawings
[0030] Figure 1 It is a characterization diagram of the colloidal chemical properties of copper selenide nanozyme.
[0031] Figure 2 It is the peroxidase-like activity and glutathione peroxidase-like activity of copper selenide nanozyme.
[0032] Figure 3 It is a graph of the inhibitory effect of copper selenide nanozyme on the proliferation of different tumor cells (MTT).
[0033] Figure 4 It is a graph of the inhibitory effect of copper selenide nanozyme on the proliferation of HCT-116 cells (MTT).
[0034] Figure 5 It is a morphological diagram of pyroptosis of tumor cells after treatment with copper selenide nanozyme.
[0035] Figure 6 It is a fluorescence microscope image of the intracellular ROS level of tumor cells after treatment with copper selenide nanozyme.
[0036] Figure 7 It is the intracellular GSH level of tumor cells after treatment with copper selenide nanozyme.
[0037] Figure 8 It is the intracellular ATP level of tumor cells after treatment with copper selenide nanozyme.
[0038] Figure 9 It is the intracellular Cu 2+ content.
[0039] Figure 10The content of pyruvate in tumor cells after treatment with copper selenide nanozyme.
[0040] Figure 11 The relative protein levels (A) of pyroptosis and the corresponding quantitative results (B) in tumor cells after treatment with copper selenide nanozyme.
[0041] Figure 12 The relative protein levels (A) of cuproptosis and the corresponding quantitative results (B) in tumor cells after treatment with copper selenide nanozyme. Detailed implementation mode
[0042] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.
[0043] In order to make the purpose and technical solutions of the present invention clearer, the present invention will be further described in detail below by taking the aqueous extract of Pu-erh tea, crude polysaccharide of Pu-erh tea, and tea brown pigment of Pu-erh tea as examples. The equipment, instruments, and reagents used in the present invention are all commonly used in the art. It should be understood that the examples described herein are only used to explain the present invention and are not used to limit the present invention.
[0044] Example 1: Copper selenide nanozyme was prepared by the following method:
[0045] (1) The aqueous extract of Pu-erh tea, tea polysaccharide, and tea brown pigment were respectively dissolved in ultrapure water to obtain aqueous extract of Pu-erh tea, tea polysaccharide, and tea brown pigment solutions with a concentration of 2.5 g / L; 3 mL was respectively taken from the above solutions and added to 14 mL of ultrapure water, magnetically stirred evenly, 3 mL of vitamin C solution with a concentration of 500 mM was added, magnetically stirred for 5 min, left standing for 15 min, then 2.5 mL of sodium selenite solution with a concentration of 100 mM was added, magnetically stirred for 5 min, and left standing in a water bath at 30 °C for 15 min to undergo a reduction reaction to obtain Pu-erh tea-nano selenium, tea polysaccharide-nano selenium, and tea brown pigment-nano selenium;
[0046] (2) Then 2.5 mL of copper sulfate solution with a concentration of 100 mM was respectively added, magnetically stirred for 5 min, and left standing in a water bath at 30 °C for 15 min; in a dialysis bag with a MWCO of 8000 Da, it was placed in ultrapure water for dialysis to remove impurities for 24 h, and the ultrapure water was changed every 2.5 h. What was obtained after dialysis was copper selenide nanozyme, namely Pu-erh tea-selenium copper (PET-Cu 2-x Se), tea polysaccharide-selenium copper (TPs-Cu 2- x Se), tea brown pigment-selenium copper (TBs-Cu 2-x Se).
[0047] Example 2: Colloidal chemical properties of copper selenide nanozyme
[0048] Take the three copper selenide nanozyme solutions prepared in Example 1 above, and use DLS technology and LDV technology to measure the average hydrodynamic diameter (D H ) of the system, light scattering intensity, polydispersity index (PDI), and Zeta potential at 25 °C. The results are as Figure 1 shown.
[0049] As Figure 1 shown, the average hydrodynamic particle size of the copper selenide nanozymes prepared using different dark tea extracts as templates is 80 - 90 nm, the light intensity is 900 - 1245 kcps, the Zeta potential value is -32 to -35 mV, and the PDI value is less than 0.2, indicating that the copper selenide nanozymes prepared using dark tea extracts as templates have small particle sizes, uniform particle distributions, and good electrostatic stability.
[0050] Example 3: Study on the activities of peroxidase - like (POD) and glutathione peroxidase - like (GPX)
[0051] Use 3,3',5,5'-tetramethylbenzidine (TMB) as the detection indicator for POD activity. TMB can be oxidized by ·OH to blue oxTMB with a characteristic absorption peak at 652 nm. The peroxidase - like activity of the three copper selenide nanozymes prepared in Example 1 was detected by H2O2 and TMB. Use 2 - nitrobenzoic acid (DTNP) as the detection indicator for GPX activity. DTNP can react with GSH to form the yellow product 5 - thio - 2 - nitrobenzoic acid (DTNB). The glutathione peroxidase - like activity of the copper selenide nanozymes prepared in Example 1 was detected by H2O2 and DTNP. The results of the peroxidase - like and glutathione peroxidase - like activities of the copper selenide nanozymes obtained Figure 2 .
[0052] As Figure 2 shown, the copper selenide nanozymes prepared using dark tea extracts as templates all have good peroxidase - like activity and glutathione peroxidase - like activity.
[0053] Example 4: Proliferation inhibitory effect of copper selenide nanozymes on different tumor cells.
[0054] Inoculate different tumor cells into 96 - well plates at a concentration of 5×10 4 / mL. After culturing for 24 h, co - incubate with the theabrownin - copper selenide of 0, 25, 50, 100 μg / mL from Example 1 for 24 h. Use the MTT method to detect the proliferation inhibitory effect of theabrownin - copper selenide on different tumor cells, and use an enzyme - linked immunosorbent assay reader to read the absorbance at 490 nm. The results obtained are as Figure 3 shown.
[0055] As Figure 3As shown, with the increase in the concentration of theabrownin-copper selenide, the survival rates of different tumor cells gradually decreased, indicating that copper selenide nanozyme has a proliferation inhibitory effect on different tumor cells and shows concentration dependence.
[0056] Example 5: Proliferation inhibitory effect of copper selenide nanozyme on HCT-116 cells.
[0057] HCT-116 cells were inoculated into 96-well plates at a concentration of 5×10 4 / mL. After culturing for 24 h, they were co-incubated with the three copper selenide nanozymes of Example 1 at concentrations of 0, 25, 50, and 100 μg / mL for 24 h. The MTT method was used to detect the proliferation inhibitory effect of the three copper selenide nanozymes on HCT-116 cells, and the absorbance at 490 nm was read with an enzyme-labeling instrument. The results are as Figure 4 shown.
[0058] As Figure 4 shown, with the increase in the concentration of the three copper selenide nanozymes, the survival rate of HCT-116 cells gradually decreased, indicating that the copper selenide nanozymes prepared using dark tea extract as a template all have a proliferation inhibitory effect on tumor cells HCT-116 and have anti-tumor activity.
[0059] Example 6: Copper selenide nanozyme induces pyroptosis in tumor cells
[0060] HCT-116 cells were inoculated into 6-well plates at a concentration of 2.0×10 5 / mL. After incubating for 24 h, they were co-incubated with theabrownin-copper selenide nanozyme (TBs-Cu 2-X Se) prepared in Example 1 at concentrations of 0, 25, 50, and 100 μg / mL for 24 h. The morphology of HCT-116 cells was observed through an inverted microscope. The results are as Figure 5 shown.
[0061] As Figure 5 shown, after treatment with theabrownin-copper selenide nanozyme, obvious changes occurred in the morphology of HCT-116 cells. The cells swelled and bulged, small pores appeared on the cell surface, and obvious transparent bubbles were observed, showing typical morphological characteristics of pyroptotic cells. Moreover, with the increase in the concentration of theabrownin-copper selenide nanozyme, the proportion of cells showing such abnormal morphology increased, indicating that pyroptosis occurred in the cells after treatment with copper selenide nanozyme.
[0062] Example 7: Detection of the effect of theabrownin-copper selenide nanozyme on the ROS level in HCT-116 cells by DCFH-DA staining
[0063] HCT-116 cells were inoculated at a concentration of 2.0×10 5Inoculate in a 6-well plate at a concentration of / mL, and after culturing for 24 h, co-incubate with the theabrownin-seleniumized copper nanozyme (TBs-Cu 2-X Se) prepared in Example 1 at 0, 25, 50, 100 μg / mL for 24 h, then stain with DCFH-DA for 0.5 h, and observe the effect of the theabrownin-seleniumized copper nanozyme on the ROS level of HCT-116 cells under a fluorescence microscope. The results are as Figure 6 shown.
[0064] As Figure 6 shown, it was observed through a fluorescence microscope that after treatment with theabrownin-seleniumized copper nanozymes at different concentrations, the green fluorescence in HCT-116 cells was significantly enhanced, and the higher the concentration of the theabrownin-seleniumized copper nanozyme, the stronger the green fluorescence in the cells, indicating that the seleniumized copper nanozyme can induce a large amount of ROS production in HCT-116 cells through its peroxidase-like catalytic activity.
[0065] Example 8: Detection of the effect of theabrownin-seleniumized copper nanozyme on the GSH level of HCT-116 cells by GSH kit
[0066] Inoculate HCT-116 cells in a 6-cm culture dish at a concentration of 2.0×10 5 / mL, and after culturing for 24 h, co-incubate with the theabrownin-seleniumized copper nanozyme (TBs-Cu 2-X Se) prepared in Example 1 for 24 h. After collecting the cells, detect the GSH level in HCT-116 cells according to the method of the GSH kit. The results are as Figure 7 shown.
[0067] As Figure 7 shown, with the increase in the concentration of the theabrownin-seleniumized copper nanozyme, the GSH level in HCT-116 cells gradually decreased, indicating that after the theabrownin-seleniumized copper nanozyme enters HCT-116, it can catalyze the oxidation of GSH to form GSSG; on the other hand, the theabrownin-seleniumized copper nanozyme contains Cu 2+ , and Cu 2+ can undergo an oxidation-reduction reaction with GSH to consume overexpressed GSH. It shows that the seleniumized copper nanozyme can reduce the overexpressed GSH in tumor cells through its glutathione peroxidase-like activity, promote the conversion of the intracellular redox balance to oxidative stress, and further enhance tumor cell damage.
[0068] Example 9: Detection of the effect of theabrownin-seleniumized copper nanozyme on the ATP level of HCT-116 cells by ATP kit
[0069] Inoculate HCT-116 cells in a 6-cm culture dish at a concentration of 2.0×10 5Inoculate in a 6-cm culture dish at a concentration of / mL, and after culturing for 24 h, co-incubate with 0, 25, 50, 100 μg / mL of the theabrownin-seleniumized copper nanozyme (TBs-Cu 2-X Se) prepared in Example 1 for 24 h. After collecting the cells, detect the ATP level in HCT-116 cells according to the method of the ATP kit, and the results are as Figure 8 shown.
[0070] It can be seen from Figure 8 that as the concentration of the theabrownin-seleniumized copper nanozyme increases, the ATP content in HCT-116 cells gradually decreases. This indicates that the seleniumized copper nanozyme induces the cells to produce a large amount of ROS, causing mitochondrial damage and hindering ATP synthesis.
[0071] Example 10: Detection of the effect of the theabrownin-seleniumized copper nanozyme on the Cu 2+ level in HCT-116 cells by a kit 2+ of the theabrownin-seleniumized copper nanozyme on the Cu
[0072] Inoculate HCT-116 cells in a 6-cm culture dish at a concentration of 2.0×10 5 / mL, and after culturing for 24 h, co-incubate with 0, 25, 50, 100 μg / mL of the theabrownin-seleniumized copper nanozyme (TBs-Cu 2-X Se) prepared in Example 1 for 24 h. After collecting the cells, detect the Cu 2+ level in HCT-116 cells according to the method of the Cu 2+ kit, and the results are as Figure 9 shown.
[0073] It can be seen from Figure 9 that as the concentration of the theabrownin-seleniumized copper nanozyme increases, the Cu 2+ level in HCT-116 cells gradually increases. This result indicates that seleniumized copper can cause excessive accumulation of copper ions in HCT-116 cells.
[0074] Example 11: Detection of the effect of the theabrownin-seleniumized copper nanozyme on the pyruvate level in HCT-116 cells by a pyruvate kit
[0075] Inoculate HCT-116 cells in a 6-cm culture dish at a concentration of 2.0×10 5 / mL, and after culturing for 24 h, co-incubate with 0, 25, 50, 100 μg / mL of the theabrownin-seleniumized copper nanozyme (TBs-Cu 2-X Se) prepared in Example 1 for 24 h. After collecting the cells, detect the pyruvate level in HCT-116 cells according to the method of the pyruvate kit, and the results are as Figure 10 shown.
[0076] From Figure 10 It can be seen that as the concentration of theabrownine-seleniumized copper nanozyme increases, the pyruvate content in HCT-116 cells gradually increases. This indicates that after the seleniumized copper nanozyme enters tumor cells, it can cause the accumulation of copper ions in the cells, hinder the TCA cycle, damage mitochondrial function, prevent pyruvate from normally entering the TCA cycle, and thus lead to the accumulation of pyruvate in tumor cells.
[0077] Example 12: Western blot detection of the expression of pyroptosis-related proteins Caspase-1, GSDMD-N, and IL 1β in HCT-116 cells by theabrownine-seleniumized copper nanozyme
[0078] After treating HCT116 cells with 0, 25, 50, and 100 μg / mL of the theabrownine-seleniumized copper nanozyme (TBs-Cu 2-X Se) obtained in Example 1 for 24 h, the cell proteins of each group were extracted and prepared into loading working solutions. Using a 10% PAGE electrophoresis gel, electrophoresis was performed at 4 °C. After electrotransferring the proteins to the PVDF membrane on ice bath, the PVDF membrane strip was cut according to the position of the Marker. It was blocked with a 5% skim milk blocking solution for 1 h, and the strips were respectively placed in the primary antibody dilution solutions of Caspase-1, GSDMD-N, and IL 1β, and incubated overnight on a shaker at 4 °C. After thoroughly washing the strips with TBST, they were placed in the secondary antibody dilution solution and incubated at room temperature for 1 h. The proteins were developed using a hypersensitive ECL chemiluminescence reagent, and the gray values were analyzed using Fluorchem5500 software, and the results are as Figure 11 shown.
[0079] From Figure 11 It can be seen that as the concentration of theabrownine-seleniumized copper nanozyme increases, the expression of Caspase-1 protein in HCT-116 cells decreases, while the expressions of GSDMD-N and IL 1β proteins increase, demonstrating that the seleniumized copper nanozyme obtained in the present invention indeed has the efficacy of inducing cell pyroptosis.
[0080] Example 13: Western blot detection of the expression of cuproptosis-related proteins DLAT, FDX1, and HSP70 in HCT-116 cells by theabrownine-seleniumized copper nanozyme
[0081] After treating HCT116 cells with 0, 25, 50, and 100 μg / mL of the theabrownine-seleniumized copper nanozyme (TBs-Cu 2-XAfter treating HCT116 cells for 24 h, the cell proteins of each group were extracted and the sample loading working solution was prepared. Using a 10% PAGE electrophoresis gel, electrophoresis was carried out at 4 °C. After electrotransferring the proteins to the PVDF membrane on ice bath, the PVDF membrane strip was cut according to the position of the Marker. The strip was blocked with 5% skim milk blocking solution for 1 h, and the strip was placed into the primary antibody dilution solutions of DLAT, FDX1, and HSP70 respectively, and incubated overnight on a shaker at 4 °C. After washing the strip thoroughly with TBST, it was placed into the secondary antibody dilution solution and incubated at room temperature for 1 h. The hypersensitive ECL chemiluminescence reagent was used to develop the proteins, and the gray value was analyzed with Fluorchem5500 software, and the results were as follows Figure 12 shown
[0082] It can be seen from Figure 12 this that as the concentration of the theabrownin-seleniumized copper nanozyme increases, DLAT oligomerizes in HCT-116 cells, the expression of HSP70 protein increases, and the expression of FDX1 protein decreases, which proves that the seleniumized copper nanozyme obtained in the present invention indeed has the effect of inducing cuproptosis in cells.
[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. A preparation method of copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells, characterized in that It includes the following operation steps: (1) Preparation of nano-selenium solution; (2) Preparation of copper selenide nanozyme. The preparation of the nano-selenium solution described in step (1) is specifically carried out according to the following steps: Using black tea extract as a soft template, adding vitamin C solution and sodium selenite solution to the black tea extract solution, and standing still in a water bath at 4-100 °C for 5-180 min. After the reaction, a nano-selenium solution is obtained; The preparation of the copper selenide nanozyme described in step (2) is specifically carried out according to the following steps: Adding copper sulfate solution to the nano-selenium solution obtained in step (1), standing still the obtained mixed solution in a water bath at 4-100 °C for 5-180 min, and dialyzing through a MWCO 8000Da dialysis bag for 24 h to obtain a copper selenide nanozyme solution; The concentration of sodium selenite in the sodium selenite solution in the mixed solution is 10 mmol / L; the concentration of vitamin C in the vitamin C solution in the mixed solution is 20-200 mmol / L; the concentration of black tea extract in the black tea extract solution in the mixed solution is 100-1000 mg / L; the concentration of copper sulfate in the copper sulfate solution in the mixed solution is 2.5-40 mmol / L.
2. The preparation method of a copper selenide nanozyme with the functions of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 1, characterized in that: The black tea extract is an aqueous extract, tea polysaccharide, and tea brown pigment of black tea processed from fresh tea leaves and tender shoots of tea trees through the processes of fixation, rolling, piling, and drying.
3. The preparation method of a copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 1, characterized in that: The concentration of black tea extract in the black tea extract solution in the mixed solution is 300 mg / L.
4. The preparation method of a copper selenide nanoenzyme with the functions of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 1, characterized in that: The concentration of vitamin C in the vitamin C solution in the mixed solution is 60 mmol / L.
5. The preparation method of a copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 1, characterized in that: The concentration of copper sulfate in the copper sulfate solution in the mixed solution is 10 mmol / L.
6. The preparation method of a copper selenide nanozyme with the functions of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 1, characterized in that: The temperature of the standing still in the water bath is 30 °C, and the time is 15 min.
7. A copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells, prepared by the preparation method described in any one of claims 1-6.
8. Application of the copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 7 in the preparation of products that catalyze hydrogen peroxide to produce ROS and consume GSH.
9. Application of the copper selenide nanozyme with the efficacy of simultaneously inducing pyroptosis and cuproptosis of tumor cells according to claim 7 in the preparation of tumor cell pyroptosis and cuproptosis inducers.