Copper-containing lentinan nano selenium and a preparation method and application thereof

Abstract

This invention provides a copper-containing lentinan nano-selenium, its application, and preparation method, comprising the following steps: (1) Preparation of stock solutions: Prepare ascorbic acid solution, sodium selenite solution, copper sulfate solution, and lentinan solution respectively; then, according to c(CuSO4):c(Na2SeO3) = 1%-100%, take a concentration value within this concentration range and dilute the copper sulfate solution with water to obtain a dilute copper sulfate solution; (2) Stir the sodium selenite solution, dilute copper sulfate solution, and lentinan solution in a reaction flask, then add ultrapure water and stir, then add ascorbic acid solution and stir, then place the reaction flask at low temperature for a certain time, and then stir the reaction at room temperature for a certain time; (3) After the reaction is completed, load the reaction solution into a dialysis belt and dialyze for a certain time; after dialysis, transfer the reaction solution, centrifuge, take the supernatant, and obtain the target product, copper-containing lentinan nano-selenium. This invention aims to explore the application of copper-containing nano-selenium drugs in the treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of synthetic medicinal chemistry technology, and specifically relates to a copper-containing lentinan nano-selenium, a method for preparing the copper-containing lentinan nano-selenium, and its application in drugs for treating malignant tumors. Background Technology

[0002] Cancer is one of the greatest threats to human health. The latest global cancer data for 2022 shows that nearly 20 million new cancer cases were diagnosed worldwide in 2022, with lung cancer and breast cancer ranking among the leading causes of infection and death. Currently, the main methods of cancer treatment include chemotherapy, radiotherapy, surgery, immunotherapy, targeted therapy, and combination therapy. Chemotherapy, as one of the commonly used methods for cancer treatment, despite the wide variety of chemotherapy drugs available, still faces significant obstacles due to its side effects, such as drug resistance and systemic toxicity.

[0003] Selenium (Se), as one of the essential trace elements for human health, plays a vital role in disease prevention. Through binding with selenoproteins, selenium exerts various effects, including antioxidant, anti-inflammatory, anti-tumor, and immunomodulatory functions. Selenium's chemical derivatives include inorganic compounds, such as selenites, and organic compounds, such as selenomethylselenocysteine ​​and selenomethionine. In recent years, increasing research has demonstrated the chemopreventive role of selenium in cancer risk and incidence; however, both inorganic and organic forms of selenium still possess significant toxicity, easily causing side effects. Nanoparticle-based selenium production can enhance its biocompatibility and bioavailability, and more importantly, reduce toxicity while maintaining high bioactivity.

[0004] Transition metals, due to their unique d or f orbitals, possess highly efficient catalytic activity. They induce the generation of reactive oxygen species (ROS) through the Fenton reaction, thereby increasing cellular oxidative stress, leading to DNA, protein, and lipid damage, and ultimately causing cancer cell death. Transition metals, represented by copper, have been extensively studied as components of metalloenzymes, signaling molecules, and important components of metallo-based anticancer drugs. Furthermore, copper-based drugs offer advantages such as relatively low cost, tunable structure, diverse composition, highly efficient photothermal / photodynamic therapy, unique imaging capabilities, and good biocompatibility. Based on this, designing and doping nano-selenium with copper to form copper-selenium compounds with catalytic activity and other effects, and studying their antitumor mechanisms, is of great significance for improving the antitumor activity of selenium nanoparticles. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the present invention aims to explore the application of copper-containing selenium nanoparticles obtained by doping copper sulfate with lentinan nanoparticles in different proportions in the treatment of tumors.

[0006] This application provides a method for preparing copper-containing lentinan nano-selenium, comprising the following steps:

[0007] (1) Preparation of stock solutions: Prepare ascorbic acid solution, sodium selenite solution, copper sulfate solution and lentinan solution respectively; then, according to c(CuSO4):c(Na2SeO3) = 1%-100%, take a concentration value within this concentration range and dilute the copper sulfate solution with water to obtain a dilute copper sulfate solution; it should be noted that diluting the copper sulfate solution with water according to the c(CuSO4):c(Na2SeO3) ratio refers to the concentration ratio of the diluted copper sulfate solution to the sodium selenite solution.

[0008] (2) Sodium selenite solution, dilute copper sulfate solution and lentinan solution were stirred in a reaction flask, then ultrapure water was added and stirred, then ascorbic acid solution was added and stirred, then the reaction flask was placed at low temperature for a certain period of time, and then stirred and reacted at room temperature for a certain period of time.

[0009] (3) After the reaction is completed, the reaction solution is loaded into a dialysis belt and dialyzed for a certain period of time. After the dialysis is completed, the reaction solution is transferred and centrifuged. The supernatant is collected to obtain the target product, copper-containing lentinan nano-selenium.

[0010] Specifically, the concentration ranges of the ascorbic acid solution, sodium selenite solution, copper sulfate solution, and lentinan solution are 180-220 mM, 40-60 mM, 40-60 mM, and 11.5-13.5 mg / mL, respectively.

[0011] Specifically, c(CuSO4):c(Na2SeO3) = any one of 1%, 10%, 30%, or 100%.

[0012] Specifically, step (2): stir sodium selenite solution, dilute copper sulfate solution and lentinan solution in a reaction flask for 4-6 minutes, then add ultrapure water and stir for 4-6 minutes, then add ascorbic acid solution and stir for 4-6 minutes, then place the reaction flask at 3-5℃ for 25-35 minutes, and then stir the reaction at room temperature for 11-13 hours.

[0013] Specifically, step (3): After the reaction is completed, the reaction solution is loaded into a dialysis belt with a molecular weight of 6000-8000 and dialyzed for 2.5-3.5 days; after the dialysis is completed, the reaction solution is transferred and centrifuged at 2500-3500 rpm for 9-11 min, and the supernatant is taken to obtain the target product containing copper-containing shiitake mushroom polysaccharide nano-selenium.

[0014] Specifically, it includes the following steps:

[0015] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 200mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 50mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 50mM copper sulfate solution; according to c(CuSO4):c(Na2SeO3)=10%, 30%, 100%, take any concentration value within this concentration range and dilute it with water to obtain a dilute copper sulfate solution; weigh 125mg of lentinan powder into a centrifuge tube and add ultrapure water to a total volume of 10mL to prepare a 12.5mg / mL lentinan solution;

[0016] (2) Take 1 mL of sodium selenite solution, 1 mL of dilute copper sulfate solution and 2 mL of lentinan solution into the reaction flask and stir for 5 min. Then add 5 mL of ultrapure water and stir for 5 min. Then add 1 mL of ascorbic acid solution and stir for 5 min. Then place the reaction flask at 4℃ for 30 min and then stir and react at room temperature for 12 h.

[0017] (3) After the reaction is completed, the reaction solution is loaded into a dialysis belt with a molecular weight of 6000-8000 and dialyzed for 3 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 3000 rpm for 10 min. The supernatant is taken to obtain the target product, copper-containing lentinan nano-selenium.

[0018] Copper-containing lentinan nano-selenium prepared according to any of the above preparation methods.

[0019] The above-mentioned application of copper-containing lentinan nano-selenium in drugs for the treatment of malignant tumors.

[0020] The improvements in this application bring the following advantages: Experiments have shown that the introduction of copper can significantly enhance the inhibitory effect of lentinan nano-selenium on tumor growth, making these copper-containing nano-selenium compounds promising for the treatment of tumors. Attached Figure Description

[0021] Figure 1 This is a hydrated particle size diagram of the copper-containing lentinan nano-selenium from an embodiment of this application.

[0022] Figure 2 This is an XRD pattern of copper-containing lentinan nano-selenium from an embodiment of this application;

[0023] Figure 3 This is a TEM image of copper-containing lentinan nano-selenium from an embodiment of this application. Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] Example 1: Preparation of copper-containing lentinan nano-selenium with different doping ratios

[0026] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 200mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 50mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 50mM copper sulfate solution, and dilute the copper sulfate solution according to a concentration of c(CuSO4):c(Na2SeO3) = 10% to obtain a dilute copper sulfate solution; weigh 125mg of lentinan powder into a centrifuge tube, add ultrapure water to a total volume of 10mL, and prepare a 12.5mg / mL lentinan solution;

[0027] (2) Take 1 mL of sodium selenite solution, 1 mL of dilute copper sulfate solution and 2 mL of lentinan solution into the reaction flask and stir for 5 min. Then add 5 mL of ultrapure water and stir for 5 min. Then add 1 mL of ascorbic acid solution and stir for 5 min. Then place the reaction flask at 4℃ for 30 min and then stir and react at room temperature for 12 h.

[0028] (3) After the reaction is completed, the reaction solution is loaded into a 7000 molecular weight dialysis belt and dialyzed for 3 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 3000 rpm for 10 min. The supernatant is taken to obtain the target product, copper-containing shiitake mushroom polysaccharide nano-selenium.

[0029] (4) Repeat steps (1)-(3) twice to prepare multiple copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios. The difference lies in the different dilution ratio of copper sulfate solution in step (1). Dilute the solution according to three different concentrations of c(CuSO4):c(Na2SeO3) = 1%, 30%, and 100% to obtain dilute copper sulfate solutions of different concentrations, so as to prepare copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios.

[0030] Example 2: Preparation of copper-containing lentinan nano-selenium with different doping ratios

[0031] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 180mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 40mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 40mM copper sulfate solution, and dilute the copper sulfate solution according to a concentration of c(CuSO4):c(Na2SeO3) = 10% to obtain a dilute copper sulfate solution; weigh 115mg of lentinan powder into a centrifuge tube, add ultrapure water to a total volume of 10mL, and prepare an 11.5mg / mL lentinan solution;

[0032] (2) Take 0.9 mL of sodium selenite solution, 0.9 mL of dilute copper sulfate solution and 1.8 mL of lentinan solution and stir in the reaction flask for 4 min. Then add 4 mL of ultrapure water and stir for 4 min. Then add 0.9 mL of ascorbic acid solution and stir for 4 min. Then place the reaction flask at 5 °C for 25 min. Then stir and react at room temperature for 11 h.

[0033] (3) After the reaction is completed, the reaction solution is loaded into a 6000 molecular weight dialysis tape and dialyzed for 2.5 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 2500 rpm for 9 min. The supernatant is taken to obtain the target product, copper-containing lentinan nano-selenium.

[0034] (4) Repeat steps (1)-(3) twice to prepare multiple copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios. The difference lies in the different dilution ratio of copper sulfate solution in step (1). Dilute the solution according to three different concentrations of c(CuSO4):c(Na2SeO3) = 1%, 30%, and 100% to obtain dilute copper sulfate solutions of different concentrations, so as to prepare copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios.

[0035] Example 3: Preparation of copper-containing lentinan nano-selenium with different doping ratios

[0036] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 220mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 60mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 60mM copper sulfate solution, and dilute the copper sulfate solution according to a concentration of c(CuSO4):c(Na2SeO3) = 10% to obtain a dilute copper sulfate solution; weigh 135mg of lentinan powder into a centrifuge tube, add ultrapure water to a total volume of 10mL, and prepare a 13.5mg / mL lentinan solution;

[0037] (2) Take 1.1 mL of sodium selenite solution, 1.1 mL of dilute copper sulfate solution and 2.2 mL of lentinan solution and stir in the reaction flask for 6 min. Then add 6 mL of ultrapure water and stir for 6 min. Then add 1.1 mL of ascorbic acid solution and stir for 6 min. Then place the reaction flask at 3℃ for 35 min. Then stir and react at room temperature for 13 h.

[0038] (3) After the reaction is completed, the reaction solution is loaded into an 8000 molecular weight dialysis belt and dialyzed for 3.5 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 3500 rpm for 11 min. The supernatant is taken to obtain the target product, copper-containing shiitake mushroom polysaccharide nano-selenium.

[0039] (4) Repeat steps (1)-(3) twice to prepare multiple copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios. The difference lies in the different dilution ratio of copper sulfate solution in step (1). Dilute the solution according to three different concentrations of c(CuSO4):c(Na2SeO3) = 1%, 30%, and 100% to obtain dilute copper sulfate solutions of different concentrations, so as to prepare copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios.

[0040] Example 4: Preparation of copper-containing lentinan nano-selenium with different doping ratios

[0041] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 190mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 45mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 45mM copper sulfate solution; dilute the copper sulfate solution according to a concentration of c(CuSO4):c(Na2SeO3) = 10% to obtain a dilute copper sulfate solution; weigh 120mg of lentinan powder into a centrifuge tube and add ultrapure water to a total volume of 10mL to prepare a 12mg / mL lentinan solution;

[0042] (2) Take 1 mL of sodium selenite solution, 1 mL of dilute copper sulfate solution and 2 mL of lentinan solution into the reaction flask and stir for 5 min. Then add 5 mL of ultrapure water and stir for 5 min. Then add 1 mL of ascorbic acid solution and stir for 5 min. Then place the reaction flask at 4℃ for 25 min. Then stir and react at room temperature for 11 h.

[0043] (3) After the reaction is completed, the reaction solution is loaded into a 6500 molecular weight dialysis belt and dialyzed for 3 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 2800 rpm for 10 min. The supernatant is taken to obtain the target product, copper-containing lentinan nano-selenium.

[0044] (4) Repeat steps (1)-(3) twice to prepare multiple copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios. The difference lies in the different dilution ratio of copper sulfate solution in step (1). Dilute the solution according to three different concentrations of c(CuSO4):c(Na2SeO3) = 1%, 30%, and 100% to obtain dilute copper sulfate solutions of different concentrations, so as to prepare copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios.

[0045] Example 5: Preparation of copper-containing lentinan nano-selenium with different doping ratios

[0046] (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 210mM ​​ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 55mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 55mM copper sulfate solution; dilute the copper sulfate solution according to a concentration of c(CuSO4):c(Na2SeO3) = 10% to obtain a dilute copper sulfate solution; weigh 125mg of lentinan powder into a centrifuge tube and add ultrapure water to a total volume of 10mL to prepare a 12.5mg / mL lentinan solution;

[0047] (2) Take 1 mL of sodium selenite solution, 1 mL of dilute copper sulfate solution and 2 mL of lentinan solution into the reaction flask and stir for 5 min. Then add 5 mL of ultrapure water and stir for 5 min. Then add 1 mL of ascorbic acid solution and stir for 5 min. Then place the reaction flask at 4℃ for 30 min and then stir and react at room temperature for 12 h.

[0048] (3) After the reaction is completed, the reaction solution is loaded into a 7500 molecular weight dialysis belt and dialyzed for 3 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 3300 rpm for 10 min. The supernatant is taken to obtain the target product, copper-containing shiitake mushroom polysaccharide nano-selenium.

[0049] (4) Repeat steps (1)-(3) twice to prepare multiple copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios. The difference lies in the different dilution ratio of copper sulfate solution in step (1). Dilute the solution according to three different concentrations of c(CuSO4):c(Na2SeO3) = 1%, 30%, and 100% to obtain dilute copper sulfate solutions of different concentrations, so as to prepare copper-containing shiitake mushroom polysaccharide nano-selenium with different doping ratios.

[0050] Experiment 6: Characterization of copper-containing lentinan nano-selenium

[0051] (1) ICP: Take 100 μL of sample (containing copper-containing lentinan nano-selenium) and nitrate it with aqua regia and then make up to 10 mL with ultrapure water. The sample was tested on an iCAP-RQ inductively coupled plasma mass spectrometer. As shown in Table 1, copper can be incorporated into lentinan nano-selenium at the theoretical doping ratio.

[0052] Table 1. Theoretical and actual doping of copper-containing lentinan nano-selenium drugs

[0053]

[0054] (2) Particle size: Take 200 μL of sample, dilute it to 2 mL with ultrapure water, and perform particle size testing using a Malvern potential particle size analyzer. Figure 1 As shown, the copper-containing lentinan nano-selenium has a suitable particle size.

[0055] (3) XRD: A small amount of sample powder was tested using an X-ray diffractometer, such as... Figure 2 As shown, the incorporation of Cu ions causes the XRD peak position to shift to the right.

[0056] (4) TEM: Take 200 μL of sample and drop it onto a copper grid, then perform the test using a transmission electron microscope, such as... Figure 3 As shown, the particle size decreases with increasing copper doping ratio.

[0057] Experiment 7: Investigation on the in vitro antitumor activity of copper-containing lentinan nano-selenium

[0058] The method for measuring the growth inhibition of tumor cells and normal cells (HeLa, HepG2, HK2 cells) by copper-containing lentinan nano-selenium in Experiment 1 using the MTT assay mainly includes the following steps:

[0059] (1) Place the copper-containing shiitake mushroom polysaccharide nano-selenium on an ultrasonic instrument and sonicate for 2 min. Take a certain amount of sample and dilute it to 64 μg / mL with DMEM medium, and perform serial dilution. The solution is ready for use.

[0060] (2) Different tumor cells in the logarithmic growth phase (US Model Culture Bank) were collected at a density of 2 × 10⁻⁶. 3Cells were seeded at 100 μL / well in 96-well plates and allowed to adhere for 24 hours. Then, 100 μL of different concentrations of the drug from step (1) were added to each well, and the plates were cultured for another 72 hours. After a certain degree of cell damage, 30 μL of MTT solution (5 mg / mL, PBS) was added to each well, and the plates were incubated for 4 hours. Next, the supernatant (DMEM high-glucose medium, Gibco) was aspirated from the 96-well plates, and 150 μL of dimethyl sulfoxide was added. The plates were gently shaken for 15 minutes to dissolve the purple crystals. The absorbance at 570 nm was measured using a multi-mode microplate reader, and cell viability was calculated. The half-maximal inhibitory concentration (IC50) was plotted. Cell viability (%) = (OD570 experimental group / OD570 control group) × 100%.

[0061] Test results: As shown in Table 2, the introduction of selenium can significantly enhance the inhibitory effect of copper-containing lentinan nano-selenium drugs on tumor growth. Among them, compared with undoped lentinan nano-selenium, copper-containing lentinan nano-selenium has a stronger inhibitory effect on tumor cells and a more significant inhibitory effect on HeLa cells.

[0062] Table 2. MTT toxicity test results of copper-containing lentinan nano-selenium drugs against tumor cells.

[0063]

[0064] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing copper-containing lentinan nano-selenium, characterized in that, Includes the following steps: (1) Preparation of stock solutions: Prepare ascorbic acid solution, sodium selenite solution, copper sulfate solution and lentinan solution respectively; then take a concentration value within the range of c(CuSO4):c(Na2SeO3)=1%-100% and dilute the copper sulfate solution with water to obtain a dilute copper sulfate solution. (2) Sodium selenite solution, dilute copper sulfate solution and lentinan solution were stirred in a reaction flask, then ultrapure water was added and stirred, then ascorbic acid solution was added and stirred, then the reaction flask was placed at low temperature for a certain period of time, and then stirred and reacted at room temperature for a certain period of time. (3) After the reaction is completed, the reaction solution is put into a dialysis bag and dialyzed for a certain period of time. After the dialysis is completed, the reaction solution is transferred and centrifuged. The supernatant is taken to obtain the target product, copper-containing lentinan nano-selenium.

2. The preparation method according to claim 1, characterized in that, The concentration ranges of the ascorbic acid solution, sodium selenite solution, copper sulfate solution, and lentinan solution are 180-220 mM, 40-60 mM, 40-60 mM, and 11.5-13.5 mg / mL, respectively.

3. The preparation method according to claim 1, characterized in that, c(CuSO4): c(Na2SeO3) = any one of 1%, 10%, 30%, or 100%.

4. The preparation method according to claim 1, characterized in that, Step (2): Stir the sodium selenite solution, dilute copper sulfate solution and lentinan solution in the reaction flask for 4-6 min, then add ultrapure water and stir for 4-6 min, then add ascorbic acid solution and stir for 4-6 min, then place the reaction flask at 3-5℃ for 25-35 min, and then stir the reaction at room temperature for 11-13 h.

5. The preparation method according to claim 1, characterized in that, Step (3): After the reaction is completed, the reaction solution is put into a dialysis bag with a molecular weight of 6000-8000 and dialyzed for 2.5-3.5 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 2500-3500 rpm for 9-11 min. The supernatant is collected to obtain the target product, copper-containing shiitake mushroom polysaccharide nano-selenium.

6. The preparation method according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of stock solution: Weigh a certain amount of ascorbic acid powder and dissolve it in ultrapure water to prepare a 200mM ascorbic acid solution; weigh a certain amount of sodium selenite powder and dissolve it in ultrapure water to prepare a 50mM sodium selenite solution; weigh a certain amount of copper sulfate pentahydrate and dissolve it in ultrapure water to prepare a 50mM copper sulfate solution. According to c(CuSO4):c(Na2SeO3)=10%, 30%, 100%, take any concentration value within this concentration range and dilute it with water to obtain a dilute copper sulfate solution; weigh 125mg of lentinan powder into a centrifuge tube and add ultrapure water to a total volume of 10 mL to prepare a 12.5 mg / mL lentinan solution. (2) Take 1 mL of sodium selenite solution, 1 mL of dilute copper sulfate solution and 2 mL of lentinan solution into the reaction flask and stir for 5 min. Then add 5 mL of ultrapure water and stir for 5 min. Then add 1 mL of ascorbic acid solution and stir for 5 min. Then place the reaction flask at 4℃ for 30 min and then stir the reaction at room temperature for 12 h. (3) After the reaction is completed, the reaction solution is put into a dialysis bag with a molecular weight of 6000-8000 and dialyzed for 3 days. After the dialysis is completed, the reaction solution is transferred and centrifuged at 3000 rpm for 10 min. The supernatant is taken to obtain the target product, copper-containing shiitake mushroom polysaccharide nano-selenium.

7. Copper-containing lentinan nano-selenium prepared by the preparation method according to any one of claims 1-6.

8. The application of the copper-containing lentinan nano-selenium according to claim 7 in the preparation of drugs for treating malignant tumors.

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