A GDY / CuO / Cy@PEG nanocomposite, a preparation method and application thereof

Abstract

The present application relates to a kind of GDY / CuO / Cy@PEG nano-composite material and its preparation method and application, belong to the field of biomedicine, solve the problem that copper atom in the prior art in the enrichment amount of tumor cell is limited, cannot play effective anti-tumor effect.The present application discloses a kind of preparation method of GDY / CuO / Cy@PEG nano-composite material, comprising the following steps: step 1: the dispersion liquid of graphdiyne, the dispersion liquid of copper oxide is ultrasonically mixed, obtain GDY / CuO dispersion liquid solution;Step 2: phthalocyanine dye, PEG-PLGA and the GDY / CuO dispersion liquid solution described in are stirred, obtain mixed solution;Step 3: water and the mixed solution described in step 2 are mixed, stirring, dialysis, obtain the GDY / CuO / Cy@PEG nano-composite material.GDY / CuO / Cy@PEG nano-composite material shows good anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a GDY / CuO / Cy@PEG nano-composite material and application thereof in preparation of an anti-tumor drug. BACKGROUND

[0002] Prostate cancer, an epithelial malignant tumor occurring in the prostate, is a common malignant tumor in the urinary system and is one of the main causes of death in male cancer, seriously threatening human life and health. Neuroendocrine prostate cancer (NEPC) is a rare but highly malignant subtype of prostate cancer. This disease type has characteristics such as rapid progression and easy resistance to conventional treatment.

[0003] At present, there are certain challenges in the treatment of NEPC. Traditional chemotherapeutic drugs, such as platinum compounds, are a first-line treatment, but patients often develop rapid drug resistance and are accompanied by severe and persistent side effects. The copper-dependent cell death mechanism, cuproptosis, regulates the content of copper ions in cells, binds copper to the acylated components of the tricarboxylic acid cycle (TCA), causes acylated protein aggregation and iron-sulfur cluster protein loss, thereby triggering protein toxicity stress and ultimately leading to cell death. Utilizing the cuproptosis mechanism can inhibit the proliferation and migration of tumor cells and exert an anti-tumor effect.

[0004] When using the cuproptosis mechanism to prepare an anti-tumor drug, the free state of copper ions in the body will bind to amino acid residues in the normal tissue cell membrane protein through coordination, causing oxidative stress and damage to the normal cell membrane, and the enrichment of metal copper ions in tumor cells is limited, which seriously limits the efficacy of the drug. On the other hand, the specific copper ion transporter protein (CTR) on the surface of the tumor cell membrane only allows divalent copper ions to be transported into cells, and monovalent copper ions cannot effectively complete transmembrane protein transport and are difficult to enter tumor cells. A large amount of reducing substances such as glutathione and reductase exist in the human body, which can convert copper atoms from high coordination (divalent copper atoms) to low coordination (monovalent copper atoms), which also limits the enrichment of copper ions in tumor cells to some extent. SUMMARY

[0005] In view of the above analysis, the present application aims to provide a GDY / CuO / Cy@PEG nano-composite material to at least solve one of the following problems:

[0006] 1. The enrichment of copper atoms in tumor cells is limited, which cannot effectively exert an anti-tumor effect;

[0007] 2. A large number of reducing substances such as glutathione, reductase, etc. in vivo can convert copper atoms from high coordination (divalent copper atoms) to low coordination (monovalent copper atoms), and monovalent copper ions cannot effectively complete transmembrane protein transport and are difficult to enter tumor cells.

[0008] The purpose of the present application is mainly realized by the following technical solutions:

[0009] In a first aspect, the present application discloses a preparation method of GDY / CuO / Cy@PEG nanocomposite, comprising the following steps:

[0010] Step 1: ultrasonic mixing of the dispersion solution of graphdiyne and the dispersion solution of copper oxide to obtain a GDY / CuO dispersion solution;

[0011] Step 2: stirring of the cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution to obtain a mixed solution;

[0012] Step 3: mixing of water and the mixed solution of step 2, stirring, dialysis to obtain the GDY / CuO / Cy@PEG nanocomposite.

[0013] Further, in step 1, the concentration of the dispersion solution of graphdiyne is 25-50 mg / mL, the mass concentration of the dispersion solution of copper oxide is 25-50 mg / mL, and the volume ratio of the dispersion solution of graphdiyne to the dispersion solution of copper oxide is 20-40:20-40, preferably 40:40.

[0014] Further, in step 1, the ultrasonic mixing time is 4-6 hours, preferably 6 hours, and the ultrasonic power is 500-720 W, preferably 720 W.

[0015] Further, in step 2, the stirring time is 1-2 hours, preferably 2 hours, and the stirring rate is 600-1000 rpm, preferably 1000 rpm.

[0016] Further, in step 2, the mass-volume ratio of the cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution is 1-2 mg:50-100 mg:40-80 μL.

[0017] Further, in step 3, the volume ratio of water to GDY / CuO / Cy@PEG mixed solution is 4-5:1, preferably 5:1; and the water used is preferably ultrapure water.

[0018] Further, in step 3, during the dialysis process, the molecular weight cut-off of the dialysis device used is 3000-5000 D, preferably 5000 D.

[0019] Further, in step 3, the dialysis device is placed in a container containing water, and the water in the container is replaced every 4-5 hours, preferably 5 hours; the water is replaced a total of 4-5 times, preferably 5 times.

[0020] In a second aspect, the present application discloses a GDY / CuO / Cy@PEG nano-composite material, which is in a spherical structure and has an average particle size of 40-160 nm.

[0021] In a third aspect, the present application discloses a pharmaceutical composition comprising the GDY / CuO / Cy@PEG nano-composite material and a pharmaceutically acceptable excipient; the pharmaceutical composition can be used alone or in combination with immunotherapy; the immunotherapy is an immune checkpoint inhibitor or cell therapy.

[0022] In a fourth aspect, the present application discloses an application of the GDY / CuO / Cy@PEG nano-composite material in preparation of an antitumor drug or a developing agent, wherein the tumor is a tumor of the urinary system.

[0023] In a fifth aspect, the present application discloses an application of the above-mentioned GDY / CuO / Cy@PEG nano-composite material in preparation of a tumor cell proliferation inhibitor, wherein the tumor cell is a tumor cell of the urinary system.

[0024] Compared with the prior art, the present application can achieve at least one of the following beneficial effects:

[0025] 1. The GDY / CuO / Cy@PEG nano-composite material prepared by the method of the present application has a large specific surface area, so that it can provide abundant sites for CuO loading. In addition, GDY can promote the conversion of copper atoms from low coordination (monovalent copper ions) to high coordination (divalent copper ions), and the divalent copper ions can smoothly enter tumor cells. Therefore, the use of GDY to load CuO is conducive to promoting the smooth entry of drugs into tumor cells under the action of transmembrane protein CTR, thereby improving the enrichment of drugs in tumor cells.

[0026] In the composite material prepared by the method of the present application, PEG-PLGA prolongs the residence time of the nano-material in the blood, reduces the clearance of the immune system, and improves the stability and biocompatibility of the nano-material.

[0027] The composite material in the present application realizes imaging-guided copper-induced cell death by introducing cyanine dye. The GDY / CuO / Cy@PEG nano-composite material of the present application can be imaged in vivo in mice, so that the tumor site can be visualized, and the effect of tracking the tumor can be achieved.

[0028] In summary, the present application promotes the conversion of copper atoms from low coordination (monovalent copper ions) to high coordination (divalent copper ions) by using GDY loaded with CuO, so that the drug can smoothly enter the tumor cells, solving the problem that monovalent copper ions cannot effectively complete the transmembrane protein transport and are difficult to enter the tumor cells, resulting in low drug efficiency. Further, by prolonging the residence time of the nanomaterial in the blood through PEG-PLGA, the immune system is reduced, and the drug efficiency is further improved. Finally, by introducing cyanine dye, the tumor site visualization is successfully realized.

[0029] 2、The GDY / CuO / Cy@PEG nanocomposite material obtained by the method of the present application presents a uniform dispersed spherical structure, and the particle size is consistent. The uniform dispersion of the nanomaterial is crucial for its stable transmission in the blood stream, so that it can more effectively reach the tumor microcirculation and improve the treatment effect.

[0030] The average particle size of the GDY / CuO / Cy@PEG nanocomposite material is 40-160 nm. This enables the nanomaterial to stay in the tumor microenvironment for a longer time, and through the enhanced permeability and retention (EPR) effect, the drug concentration in the tumor tissue is increased.

[0031] 3、The GDY / CuO / Cy@PEG nanocomposite material provided by the present application not only can promote the expression of copper death related proteins FDX1 and LIAS, but also can promote the expression of PI3K. After adding the PI3K inhibitor, the expression of PI3K protein is reduced, the expression amount of copper death related protein LIAS is also reversed, and the number of apoptotic cells is also reduced. In the copper death mechanism induced by the GDY / CuO / Cy@PEG nanocomposite material, PI3K promotes the uptake of copper ions by tumor cells.

[0032] 4、The GDY / CuO / Cy@PEG nanocomposite material provided by the present application has an immune activation effect, and when the GDY / CuO / Cy@PEG nanocomposite material is used in combination with an immune checkpoint inhibitor, it can effectively activate immune cells, and thus improve the effect of tumor immunotherapy.

[0033] 5. This invention increases the stability of nanoparticles by controlling the proportion of raw materials, ensuring the stable presence of copper ions within the nanoparticles. This allows more nanoparticles to utilize the EPR effect of tumors to ultimately accumulate and enrich within the tumor, thereby improving drug efficacy. Since drugs become unstable after entering the bloodstream, nanoparticles can experience two problems: firstly, unstable nanoparticles can cause copper ions to diffuse into other organs and tissues, leading to toxic side effects; secondly, the instability of nanoparticles can also affect drug accumulation in tumor tissues, reducing drug efficacy. For example, in step 3, the volume ratio of water to the mixed solution in step 2 is 4-5:1. This ratio range can increase the stability of the nanoparticles. Therefore, by setting the volume ratio of water to the mixed solution in step 2 to 4-5:1, the stability of the nanoparticles can be improved.

[0034] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0035] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals have the same meaning.

[0036] Figure 1 A schematic diagram illustrating the synthesis process and mechanism of action of GDY / CuO / Cy@PEG nanocomposites in animals;

[0037] Figure 2 Characterization diagram of GDY / CuO / Cy@PEG nanocomposite material;

[0038] Figure 3A Electron micrographs showing the inhibitory effects of high and low concentrations of GDY / CuO / Cy@PEG nanocomposite materials on the proliferation of human prostate cancer cells (PC-3) and human neuroendocrine prostate cancer cells (NCI-H660).

[0039] Figure 3B Three-dimensional columnar comparison of the inhibitory effects of GDY / CuO / Cy@PEG nanocomposite materials at different concentrations on the proliferation of human prostate cancer cells (PC-3);

[0040] Figure 3C Three-dimensional columnar comparison of the inhibitory effects of GDY / CuO / Cy@PEG nanocomposite materials at different concentrations on the proliferation of human neuroendocrine prostate cancer cells (NCI-H660);

[0041] Figure 3D Figure 11 is a graph showing the mitochondrial damage of cells treated with GDY / CuO / Cy@PEG nanocomposites observed using a transmission electron microscope (TEM);

[0042] Figure 4A Figure 15 is a graph showing the protein changes in FDX1 and PI3K expression after treatment with GDY / CuO / Cy@PEG nanocomposites in a Western blot experiment;

[0043] Figure 4B Figure 16 is a bar graph showing the protein expression of copper death-related proteins FDX1 and PI3K after treatment with GDY / CuO / Cy@PEG nanocomposites in a Western blot experiment;

[0044] Figure 5 Figure 17 is a graph showing the changes in copper death-related protein LIAS expression, PI3K protein expression, and apoptotic cells under the intervention of a PI3K inhibitor;

[0045] Figure 6 Figure 18 is a graph showing the immune activation effect of GDY / CuO / Cy@PEG nanocomposites in a flow cytometry analysis;

[0046] Figure 7a Figure 19 is a photograph showing the exfoliated tumor in a prostate cancer mouse model after treatment with GDY / CuO / Cy@PEG nanocomposites;

[0047] Figure 7b Figure 20 is a line graph showing the change in tumor volume over time in a prostate cancer mouse model after treatment with GDY / CuO / Cy@PEG nanocomposites;

[0048] Figure 8 Figure 21 is a graph showing the effect of GDY / CuO / Cy@PEG nanocomposites on the survival period of prostate cancer mice;

[0049] Figure 9 Figure 22 is a fluorescence imaging graph of GDY / CuO / Cy@PEG nanocomposites in a mouse in vivo. DETAILED DESCRIPTION

[0050] The preferred embodiments of the present application will be described in detail below with reference to the attached drawings, which form a part of this application. The present application is described in connection with the embodiments, but it is not intended to limit the scope of the present application to the preferred embodiments, and there can be various modifications and several specific examples.

[0051] For urinary system tumors, especially prostate cancer, which is highly invasive and prone to drug resistance, this invention provides a novel copper-based nanocomposite material that can precisely deliver copper ions into the mitochondria of tumor cells. The accumulation of copper ions in tumor cells can activate the cell copper death mechanism by binding to lipophilized proteins in mitochondria, thereby killing tumor cells.

[0052] A specific embodiment of the present invention discloses a method for preparing GDY / CuO / Cy@PEG nanocomposite materials. The preparation method first uses ultrasound to obtain a uniformly dispersed GDY / CuO dispersion solution; then, cyanine dye (Cy) and polylactic acid-glycolic acid polyethylene glycol copolymer (PEG-PLGA) are weighed in proportion and stirred with the GDY / CuO mixed dispersion to obtain a mixed solution; finally, water and the mixed solution are mixed, and the GDY / CuO / Cy@PEG nanocomposite material is obtained by self-assembly through dialysis.

[0053] Specifically, such as Figure 1 As shown, the preparation method of the GDY / CuO / Cy@PEG nanocomposite material includes the following steps:

[0054] Step 1: The dispersions of graphyne and copper oxide are ultrasonically mixed to obtain a GDY / CuO dispersion solution.

[0055] Step 2: Stir the cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution to obtain a mixed solution with a mass-to-volume ratio of 1-2 mg: 50-100 mg: 40-80 μL;

[0056] Step 3: Mix water and the mixed solution described in Step 2, stir, and dialyze to obtain the GDY / CuO / Cy@PEG nanocomposite material.

[0057] Compared with existing technologies, the GDY / CuO / Cy@PEG nanocomposites obtained by the above preparation method exhibit a uniformly dispersed spherical structure with consistent particle size. The average particle size of the GDY / CuO / Cy@PEG nanocomposites is 40-160 nm.

[0058] Specifically, in step 1, the mass concentration of the graphyne (GDY) dispersion is 25-50 mg / mL, such as 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, and 50 mg / mL; the mass concentration of the copper oxide (CuO) dispersion is 25-50 mg / mL, such as 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, and 50 mg / mL.

[0059] The mass concentration of the dispersion of graphdiyne and copper oxide is set to 25-50 mg / mL, which can achieve uniform dispersion of graphdiyne and copper oxide nanoparticles, and exceeding the range will result in dispersion of the dispersion or aggregation, affecting the preparation of the GDY / CuO / Cy@PEG nanocomposite.

[0060] The volume ratio of the dispersion of graphdiyne and the dispersion of copper oxide is 20-40:20-40, such as 40:20, 40:40, 25:15, 20:40, 20:20, and preferably 40:40.

[0061] Under the condition that the volume ratio of the dispersion of graphdiyne and the dispersion of copper oxide is 20-40:20-40, the effective loading and stability of graphdiyne on copper oxide can be achieved, and exceeding the range will affect the stability of the GDY / CuO / Cy@PEG nanocomposite.

[0062] The dispersion of copper oxide is a dimethyl sulfoxide dispersion of copper oxide.

[0063] The dispersion of graphdiyne is a dimethyl sulfoxide dispersion of graphdiyne, which is obtained by placing GDY nanosheets in dimethyl sulfoxide and treating for 24-48 hours by cell crushing.

[0064] In the preparation process of the GDY nanosheet, first, hexa (trimethylsilyl ethynyl) benzene (HEB-TMS) is deprotected to obtain hexaethynyl benzene (HEB) monomer, then the acetone solution of HEB is slowly added to a mixed solution of acetone:pyridine:N,N,N',N'-tetramethyl ethylenediamine (TMEDA) (volume ratio 100:5:1), copper foil is used as the substrate, after the reaction is completed, ultrasonic treatment is performed, and the GDY nanosheet is obtained by peeling in DMSO. The average size of the GDY nanosheet observed by transmission electron microscopy is 30±5 nm.

[0065] In step 1, the ultrasonic mixing time is 4-6 hours, such as 4 hours, 5 hours, or 6 hours, and the ultrasonic mixing time is preferably 6 hours; the ultrasonic power is 500-720 W, such as 500 W, 600 W, or 720 W, and the ultrasonic power is preferably 720 W. The above ranges of ultrasonic mixing time and ultrasonic power can achieve uniform dispersion and mixing of the material, and exceeding the range will result in incomplete or unstable mixing of the material.

[0066] Further, in step 1, after mixing the dispersion solution of graphdiyne and the dispersion solution of copper oxide, dimethyl sulfoxide is added to the above mixed solution, and ultrasonic mixing is performed to obtain a GDY / CuO dispersion solution; the volume ratio of the dimethyl sulfoxide to the mixed solution is 20-60:1, such as 20:1, 30:1, 40:1, 50:1, or 60:1.

[0067] The CuO nanoparticles prepared by step 1 are uniformly distributed on the surface of the GDY nanosheet, and GDY has a large specific surface area, so that the CuO loading can provide abundant sites. With the introduction of GDY, the Cu + The ratio of 2p3 / 2 to 2p1 / 2 decreases from 20.6% to 11.5%, which proves that there is an intermolecular interaction between GDY and CuO, and GDY promotes the conversion of copper atoms from low coordination (monovalent copper ions) to high coordination (divalent copper ions). Since only divalent copper ions can enter tumor cells smoothly, the use of GDY to load CuO is conducive to promoting the smooth entry of drugs into tumor cells under the action of transmembrane protein CTR, thereby improving the enrichment of drugs in tumor cells.

[0068] In step 2, the mass-volume ratio of the cyanine dye (Cy), PEG-PLGA, and the GDY / CuO dispersion solution is 1-2 mg (such as 1 mg, 1.5 mg, or 2 mg) to 50-100 mg (such as 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg) to 40-80 μL (such as 40 μL, 50 μL, 60 μL, 70 μL, or 80 μL); preferably, the mass-volume ratio of the cyanine dye, PEG-PLGA, and the GDY / CuO dispersion solution is 2 mg to 100 mg to 60 μL.

[0069] Under the condition that the mass-volume ratio of the cyanine dye, PEG-PLGA, and the GDY / CuO dispersion solution is 1-2 mg to 50-100 mg to 40-80 μL, the average particle size of the prepared GDY / CuO / Cy@PEG nanocomposite material can be 85-125 nm, which realizes high loading of the cyanine dye, GDY, and CuO while ensuring the stability of the solution; exceeding the range will result in insufficient loading or aggregation.

[0070] The PEG-PLGA is mPEG2k-PLGA2k(50:50).

[0071] In step 2, the stirring time is 1-2 hours, for example, 1 hour, 1.5 hours, or 2 hours, and the stirring time is preferably 2 hours; the stirring rate is 600-1000 revolutions per minute, such as 600 revolutions per minute, 800 revolutions per minute, or 1000 revolutions per minute, and the stirring rate is preferably 1000 revolutions per minute.

[0072] In step 3, a certain amount of water is weighed and quickly added to the mixed solution obtained in step 2, and electromagnetic stirring is performed to obtain a diluted solution; the electromagnetic stirring time is 1-2 hours, for example 1 hour, 1.5 hours, 2 hours, preferably 2 hours; the stirring power is 600-1000 revolutions per minute, such as 600 revolutions per minute, 800 revolutions per minute, 1000 revolutions per minute, preferably 1000 revolutions per minute; the volume ratio of water to mixed solution is 4-5:1, for example 4:1, 4.5:1, 4.8:1, 5:1, preferably 5:1; the water used is ultrapure water, tap water, distilled water, preferably ultrapure water. The above-mentioned volume ratio of water to mixed solution in the range of 4-5:1 can form a stable nanocomposite solution, and exceeding the range will cause the nanoparticles to be unstable.

[0073] After the drug enters the blood circulation, the nanoparticles will become unstable. On the one hand, nanoparticles with poor stability will cause copper ions to diffuse to other organs and tissues of the body, causing toxic side effects; on the other hand, the instability of the nanoparticles will also affect the enrichment of the drug in the tumor tissue, reducing the efficacy of the drug. Therefore, improving the stability of the nanoparticles and making the copper ions stably exist inside the nanoparticles will enable more nanoparticles to utilize the EPR effect of the tumor to achieve final accumulation and enrichment in the tumor.

[0074] In step 3, the above-mentioned diluted solution is transferred to a dialysis device, and then the dialysis device is placed in a water-containing container; the molecular weight cut-off of the dialysis device is 3000-5000 D, such as 3000 D, 4000 D, 5000 D, preferably 5000 D; the molecular weight cut-off of the dialysis device is selected in the range of 3000-5000 D, which is consistent with the molecular weight range of PEG-PLGA, and exceeding the range will result in incomplete dialysis.

[0075] In the above-mentioned water-containing container, water is added to dialyze the diluted solution, and the volume ratio of water to diluted solution is 10-30:1, such as 10:1, 15:1, 20:1, 25:1, 30:1.

[0076] In the above-mentioned water-containing container, a magnetic stirrer is added, and electromagnetic stirring is performed at a stirring rate of 300-500 revolutions per minute, such as 300 revolutions per minute, 400 revolutions per minute, 500 revolutions per minute, and the water in the container is replaced every 4-5 hours to ensure complete dialysis; the replacement time is preferably 5 hours; a total of 4-5 times of water replacement can ensure the effective circulation and penetration of the nanomaterials; exceeding the range will result in a shortened circulation process or the inability of the nanomaterials to penetrate into tumor cells; the number of replacements is preferably 5 times.

[0077] The PEG-PLGA is introduced in the application, which can prolong the residence time of the nanomaterial in the blood, reduce the clearance of the immune system, and improve the stability and biocompatibility of the nanomaterial.

[0078] The cyanine dye is introduced, which can realize imaging-guided copper-induced cell death, so that the GDY / CuO / Cy@PEG nanocomposite can be imaged in the mouse body, so that the tumor site can be visualized, and the effect of tracking the tumor can be realized.

[0079] Another specific embodiment of the application discloses a GDY / CuO / Cy@PEG nanocomposite prepared by the above method.

[0080] Specifically, the GDY / CuO / Cy@PEG nanocomposite has a uniform dispersed spherical structure and consistent particle size. The uniform dispersion of the nanomaterial is crucial for its stable transmission in the blood stream, so that it can more effectively reach the tumor microcirculation and improve the treatment effect.

[0081] Further, the average particle size of the GDY / CuO / Cy@PEG nanocomposite is 40-160 nm, such as 40 nm, 60 nm, 85 nm, 105 nm, 125 nm, 145 nm, and 160 nm, which enables the nanomaterial to stay in the tumor microenvironment for a longer time, increases the drug concentration in the tumor tissue through the enhanced permeability and retention (EPR) effect.

[0082] Another specific embodiment of the application discloses a pharmaceutical composition comprising the GDY / CuO / Cy@PEG nanocomposite and a pharmaceutically acceptable adjuvant.

[0083] Specifically, the pharmaceutically acceptable adjuvant is any one or a combination of at least two of a carrier, a diluent, an emulsifying agent, a cosolvent, a solubilizer, a catalyst, a surfactant, an antioxidant, a bacteriostatic agent, a binder, a coating material, a buffer.

[0084] Specifically, the dosage form of the pharmaceutical composition is one of a solution, a suspension, a tablet, a capsule, an enema, a cavity perfusion, an emulsion, and a gel preparation.

[0085] Another specific embodiment of the application discloses the use of the GDY / CuO / Cy@PEG nanocomposite in the preparation of an antitumor drug or a developing agent.

[0086] Specifically, the tumor is a tumor of the urinary system, which includes prostate cancer, kidney cancer, bladder cancer, preferably prostate cancer; the prostate cancer includes neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma or other types of prostate tumors; the NEPC includes primary NEPC and treatment-induced NEPC (t-NEPC).

[0087] In another specific embodiment of the present application, a method for administering the GDY / CuO / Cy@PEG nanocomposite alone or in combination with an immune checkpoint inhibitor, preferably a PD-1 inhibitor, is disclosed. The GDY / CuO / Cy@PEG nanocomposite has an immune activation effect, and thus, when used in combination with an immune checkpoint, can synergistically enhance the anti-tumor effect.

[0088] In another specific embodiment of the present application, the GDY / CuO / Cy@PEG nanocomposite is used to prepare a tumor cell proliferation inhibitor, wherein the tumor cells are urinary system tumor cells, such as human prostate cancer cells (PC-3) and human neuroendocrine prostate cancer cells (NCI-H660).

[0089] The GDY / CuO / Cy@PEG nanocomposite provided by the present application not only promotes the expression of copper death-related proteins FDX1 and LIAS, but also promotes the expression of PI3K in the mechanism of inducing copper death. The GDY / CuO / Cy@PEG nanocomposite shows a significant anti-tumor effect at the cellular level and in vivo, and can effectively inhibit tumor growth; further, when the GDY / CuO / Cy@PEG nanocomposite is used in combination with an immune checkpoint inhibitor, it can effectively activate immune cells, thereby improving the effect of tumor immunotherapy.

[0090] Example 1

[0091] Synthesis and characterization of GDY / CuO / Cy@PEG nanocomposite

[0092] Step 1: ultrasonic mixing of the dispersion solution of graphdiyne and the dispersion solution of copper oxide to obtain a GDY / CuO dispersion solution;

[0093] First, a dispersion solution of copper oxide and a dispersion solution of graphdiyne are prepared. The preparation method of the dispersion solution of copper oxide and the dispersion solution of graphdiyne is as follows:

[0094] Preparation of the dispersion solution of copper oxide: 100 mg of copper oxide is placed in 4 mL of dimethyl sulfoxide to obtain a dispersion solution of copper oxide (i.e., a CuO dispersion solution), and the mass concentration of the CuO dispersion solution is 25 mg / mL.

[0095] Preparation of Graphdiyne dispersion: 50 mg of GDY nanosheets were placed in 2 mL of dimethyl sulfoxide and subjected to cell disruption treatment for 24 hours to obtain a Graphdiyne dispersion (i.e., GDY dispersion). The mass concentration of the GDY dispersion was 25 mg / mL.

[0096] Preparation of GDY nanosheets: First, hexa(trimethylsilylethynyl)benzene (HEB-TMS) was deprotected with tetrabutylammonium fluoride (TBAF) to obtain hexaethynylbenzene (HEB). Then, HEB (10 mg) was dissolved in acetone (50 mL) and added dropwise over 3 hours to a mixed solution of acetone:pyridine:N,N,N',N'-tetramethylethylenediamine (TMEDA) (volume ratio 100:5:1). The treated copper foil was used as the growth substrate for GDY, and the reaction was carried out at 60°C in darkness under an argon atmosphere for 24 hours. After the reaction, the copper foil was washed with acetone and N,N-dimethylformamide (DMF), and then exfoliated in DMSO by ultrasonic treatment to obtain GDY nanosheets. The GDY nanosheets were observed by transmission electron microscopy (TEM) as follows. Figure 2 As shown in A, the average size of the GDY nanosheets observed by transmission electron microscopy is 30 ± 5 nm.

[0097] After preparing the dispersions of copper oxide and graphylene, 40 μL of GDY dispersion and 20 μL of CuO dispersion were transferred into sample vials using a pipette to obtain a mixture of GDY and CuO dispersions. Then, according to the volume ratio of dimethyl sulfoxide (DMSO) to the above mixture of 40:1, DMSO was added to the above sample vial using a pipette. The mixture was ultrasonically mixed for 6 hours at an ultrasonic power of 720 W to obtain a uniformly dispersed GDY / CuO dispersion solution.

[0098] The CuO nanoparticles obtained in step 1 are uniformly distributed on the surface of the GDY nanosheets, specifically as follows: Figure 2 As shown in C.

[0099] The interaction between GDY and CuO was verified by X-ray photoelectron spectroscopy (XPS). Figure 2 The upper and lower figures in D show the X-ray photoelectron spectra of CuO particles and GDY / CuO particles, respectively.

[0100] from Figure 2 As can be seen from D, the Cu 2p 3 / 2 XPS spectrum shows that Cu + 2p3 / 2 (933.0 eV) and Cu 2+ The typical peak of 2p³ / 2 (934.1 eV) and its satellite peaks. With the introduction of GDY, Cu +The ratio of 2p3 / 2 decreased from 20.6% to 11.5%, which proved the existence of intermolecular interaction between GDY and CuO. The incomplete charge transfer between CuO and GDY promoted the higher coordination number of Cu atom, indicating that GDY played an effective role in stabilizing CuO nanoparticles.

[0101] Step 2: Stir the cyanine dye, PEG-PLGA and GDY / CuO dispersion solution to obtain a mixed solution;

[0102] Weigh 2 mg of indigo five (Cy5) solid powder and 100 mg of PEG-PLGA solid, and pour the weighed Cy5 and PEG-PLGA solid into the sample bottle of the GDY / CuO dispersion solution prepared in step 1, then add a magnetic stirrer to the sample bottle, tighten the threaded bottle cap, and stir at a speed of 1000 revolutions per minute for 2 hours to obtain a mixed solution.

[0103] Step 3: Mix water with the mixed solution prepared in step 2, stir, dialyze, and obtain GDY / CuO / Cy@PEG nanocomposites.

[0104] According to the volume ratio of ultrapure water to mixed solution of 5:1, ultrapure water is removed and quickly added to the mixed solution prepared in step 2, and stirred at a speed of 1000 revolutions per minute for 2 hours to obtain a diluted solution.

[0105] The above diluted solution is transferred to a dialysis bag with a molecular weight cutoff of 5000 D using a plastic dropper, and then ultrapure water is added to the beaker, with a volume ratio of ultrapure water to the above diluted solution of 10:1; the dialysis bag is placed in the beaker.

[0106] A magnetic stirrer is added to the above beaker, and stirred at a speed of 500 revolutions per minute, and the ultrapure water in the beaker is replaced every 5 hours to remove DMSO in the original solution. After replacing the ultrapure water for 5 times, GDY / CuO / Cy@PEG nanocomposites are obtained.

[0107] As shown in E of Figure 2 The prepared GDY / CuO / Cy@PEG nanocomposites exhibit uniform spherical structure and uniform particle size. The uniform dispersion of nanomaterials is crucial for their stable transmission in blood flow, enabling them to more effectively reach tumor microcirculation and improve treatment effect.

[0108] As shown in E of Figure 2As shown in FIG. F, the average particle size of the GDY / CuO / Cy@PEG nanocomposite is 85 nm, and the particle size range is 50-150 nm. Nanoparticles of this size enable the nanomaterial to stay in the tumor microenvironment for a longer time, increasing the drug concentration in the tumor tissue through the enhanced permeability and retention (EPR) effect.

[0109] The optical absorption properties of the nanomaterial were analyzed by ultraviolet-visible spectroscopy (UV-Vis), and the structural composition of the nanocomposite was verified. The UV-Vis absorption spectrum shows the characteristic absorption signals of CuO, GDY, Cy, and GDY / CuO / Cy@PEG nanomaterials. In addition, the photoluminescence spectrum of the GDY / CuO / Cy@PEG nanocomposite shows a characteristic peak of Cy at 667.5 nm. All these results together indicate that GDY, CuO, and Cy have been successfully integrated.

[0110] Example 2

[0111] Synthesis and characterization of GDY / CuO / Cy@PEG nanocomposite

[0112] Step 1: ultrasonically mix the dispersion solution of graphdiyne and the dispersion solution of copper oxide to obtain a GDY / CuO dispersion solution;

[0113] First, prepare a dispersion solution of graphdiyne and a dispersion solution of copper oxide.

[0114] Preparation of the dispersion solution of copper oxide: 100 mg of copper oxide was placed in 2 mL of dimethyl sulfoxide to obtain a dispersion solution of copper oxide (i.e., CuO dispersion solution), and the mass concentration of the CuO dispersion solution was 50 mg / mL.

[0115] Preparation of the dispersion solution of graphdiyne: 100 mg of GDY nanosheets was placed in 2 mL of dimethyl sulfoxide, and after 48 hours of cell pulverization treatment, a dispersion solution of graphdiyne (i.e., GDY dispersion solution) was obtained, and the mass concentration of the GDY dispersion solution was 50 mg / mL.

[0116] Preparation of GDY nanosheets: GDY nanosheets were prepared according to the method for preparing GDY nanosheets in Example 1.

[0117] After the dispersion solution of graphdiyne and the dispersion solution of copper oxide were prepared, 25 μL of the GDY dispersion solution and 15 μL of the CuO dispersion solution were removed with a pipette and placed in a sample bottle to obtain a mixture of the GDY dispersion solution and the GDY dispersion solution, and then dimethyl sulfoxide (DMSO) was removed with a pipette according to a volume ratio of DMSO to the above mixture of 20:1 and added to the sample bottle, and ultrasonic mixing was performed for 4 h at an ultrasonic power of 500 W to obtain a uniformly dispersed GDY / CuO dispersion solution.

[0118] Step 2: stirring cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution to obtain a mixed solution;

[0119] 1 mg of Cy5 solid powder and 80 mg of PEG-PLGA solid were weighed, and the weighed Cy5 and PEG-PLGA were poured into the sample bottle of the above-mentioned GDY / CuO mixed dispersion solution, then a magnetic stirrer was added to the sample bottle, the threaded bottle cap was tightened, and electromagnetic stirring was performed for 1 hour at a stirring rate of 600 revolutions per minute to obtain a mixed solution.

[0120] Step 3: mixing water and the mixed solution of Step 2, stirring, dialysis, to obtain a GDY / CuO / Cy@PEG nanocomposite.

[0121] According to a volume ratio of ultrapure water to the mixed solution of 4:1, ultrapure water was removed and quickly added to the mixed solution of Step 2, electromagnetic stirring was performed for 1 hour at a stirring rate of 600 revolutions per minute to obtain a diluted solution.

[0122] The above-mentioned diluted solution was transferred to a dialysis bag with a molecular weight cut-off of 3000 D using a plastic dropper, then ultrapure water was added to the beaker, and the volume ratio of ultrapure water to the above-mentioned diluted solution was 20:1; the dialysis bag was placed in the beaker.

[0123] A magnetic stirrer was added to the above-mentioned beaker, electromagnetic stirring was performed at a stirring rate of 300 revolutions per minute, the ultrapure water in the beaker was replaced every 4 hours to remove DMSO in the original solution, and after replacing the ultrapure water for 4 times, a GDY / CuO / Cy@PEG nanocomposite was obtained.

[0124] The prepared GDY / CuO / Cy@PEG nanomaterials exhibited uniform dispersed spherical structures and uniform particle sizes. The average particle size of the GDY / CuO / Cy@PEG nanocomposite was 105 nm.

[0125] Example 3

[0126] Synthesis and characterization of GDY / CuO / Cy@PEG nanocomposite

[0127] Step 1: ultrasonic mixing of the dispersion solution of graphdiyne and the dispersion solution of copper oxide to obtain a GDY / CuO dispersion solution;

[0128] First, the dispersion solution of graphdiyne and the dispersion solution of copper oxide were prepared;

[0129] Preparation of the dispersion solution of copper oxide: 100 mg of copper oxide was placed in 2.5 mL of dimethyl sulfoxide to obtain a dispersion solution of copper oxide (i.e., CuO dispersion solution) with a concentration of 40 mg / mL.

[0130] Preparation of the dispersion of graphdiyne: 100 mg of GDY nanosheets was placed in 2.5 mL of dimethyl sulfoxide, and after 30 hours of cell pulverization treatment, a dispersion of graphdiyne (i.e., GDY dispersion) was obtained, and the mass concentration of the GDY dispersion was 40 mg / mL.

[0131] Preparation of GDY nanosheets: GDY nanosheets were prepared according to the method for preparing GDY nanosheets in Example 1.

[0132] After the dispersion of graphdiyne and the dispersion of copper oxide were prepared, 40 μL of the GDY dispersion and 20 μL of the CuO dispersion were first taken with a pipette and placed in a sample bottle to obtain a mixed solution of the GDY dispersion and the GDY dispersion, and then dimethyl sulfoxide (DMSO) was added to the sample bottle according to a volume ratio of DMSO to the mixed solution of 60:1, and the sample bottle was ultrasonically mixed for 5 h at an ultrasonic power of 600 W to obtain a uniformly dispersed GDY / CuO mixed dispersion solution.

[0133] Step 2: The cyanine dye, PEG-PLGA, and the GDY / CuO dispersion solution were stirred to obtain a mixed solution.

[0134] 1.5 mg of Cy3 solid powder and 90 mg of PEG-PLGA solid were weighed, and the weighed Cy3 and PEG-PLGA were poured into the sample bottle containing the GDY / CuO mixed dispersion solution, and then a magnet was added to the sample bottle, the threaded bottle cap was tightened, and electromagnetic stirring was performed at a stirring rate of 800 revolutions per minute for 1.5 hours to obtain a mixed solution.

[0135] Step 3: Water and the mixed solution of Step 2 were mixed, stirred, and dialyzed to obtain a GDY / CuO / Cy@PEG nanocomposite material.

[0136] According to a volume ratio of ultrapure water to the mixed solution of 4.8:1, ultrapure water was taken and quickly added to the mixed solution of Step 2, and electromagnetic stirring was performed at a stirring rate of 800 revolutions per minute for 1.5 hours to obtain a diluted solution.

[0137] The diluted solution was transferred to a dialysis bag with a molecular weight cut-off of 4000 D using a plastic dropper, and then ultrapure water was added to the beaker at a volume ratio of ultrapure water to the diluted solution of 30:1; the dialysis bag was placed in the beaker.

[0138] A magnet was added to the beaker, electromagnetic stirring was performed at a stirring rate of 400 revolutions per minute, the ultrapure water in the beaker was replaced every 4.5 h to remove the DMSO in the original solution, and after the ultrapure water was replaced for 5 times, a GDY / CuO / Cy@PEG nanocomposite material was obtained.

[0139] The prepared GDY / CuO / Cy@PEG nanocomposite presents a uniform dispersed spherical structure, and the particle size is uniform. The average particle size of the GDY / CuO / Cy@PEG nanocomposite is 125 nm.

[0140] Comparative Example 1

[0141] Preparation of the control drug CuO / Cy@PEG

[0142] The preparation method is the same as that in Example 1, except that no graphdiyne is added in step 1.

[0143] Comparative Example 2

[0144] Preparation of the control drug GDY / Cy@PEG

[0145] The preparation method is the same as that in Example 1, except that no copper oxide is added in step 1.

[0146] Explanation of the cell lines used in the following experimental examples: The prostate cancer cell lines (RM-1, PC-3, DU145, NCI-H660) were purchased from ATCC and cultured in RPMI-1640 medium containing 10% FBS. After resuscitation, the cells were inoculated in T25 culture bottles, and the state was observed regularly. Before the experiment, the density was adjusted to the appropriate density.

[0147] Experimental Example 1

[0148] CCK-8 cell proliferation activity experiment

[0149] The GDY / CuO / Cy@PEG nanocomposite prepared in Example 1 was used as the experimental group, and the control group included CuO / Cy@PEG prepared in Comparative Example 1 and GDY / Cy@PEG prepared in Comparative Example 2.

[0150] The concentration gradient of GDY / CuO / Cy@PEG used in the experiment was 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 μg / mL, respectively. The preparation method was as follows: the GDY / CuO / Cy@PEG stock solution (25 mg / mL) was diluted with phosphate buffer to the target dilution concentration.

[0151] The concentration gradient of CuO / Cy@PEG prepared by Comparative Example 1 and GDY / Cy@PEG prepared by Comparative Example 2 in the control group is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg / mL, respectively; and the preparation method is as follows: the CuO / Cy@PEG (25 mg / mL) and the GDY / Cy@PEG stock solution (25 mg / mL) are respectively diluted with a phosphate buffer to the target dilution concentration.

[0152] The cell proliferation ability and apoptosis change are evaluated by a Cell Counting Kit-8 (CCK-8, Solarbio, item number: CA1210) method.

[0153] In the experiment, PC-3 (human prostate cancer cells) and NCI-H660 (human neuroendocrine prostate cancer cells) cells are inoculated in a 96-well plate (the inoculation density is 5000 cells / well) and cultured overnight in an incubator. After the cells enter the rapid proliferation stage, the culture medium is replaced with a serum-free culture medium, and the GDY / CuO / Cy@PEG nanocomposite and the control group drugs CuO / Cy@PEG and GDY / Cy@PEG are added, respectively, and the culture is continued for 24 hours. After the incubation is completed, 10 μL of CCK-8 reagent is added to each well, and the incubation is carried out at 37°C for 1 hour. The absorbance (OD) is measured at a wavelength of 450 nm by using an enzyme label instrument. All tests are independently repeated three times.

[0154] The CCK-8 cell proliferation activity experiment proves that the GDY / CuO / Cy@PEG nanocomposite has a significant inhibitory effect on PC-3 (human prostate cancer cells) and NCI-H660 (human neuroendocrine prostate cancer cells), as shown in Figures 3A-3C The inhibitory effect is significantly improved with the increase of the concentration.

[0155] Specifically, as shown in Figure 3A The nanomaterial has a good inhibitory effect on PC-3 and NCI-H660 with the increase of the concentration. Specific analysis: with the continuous increase of the concentration, the PC-3 cells and NCI-H660 cells are significantly inhibited, the number starts to decrease, the cell membrane structure appears fuzzy and is accompanied by cell death debris. All cell lines show serious cell damage, the cell membrane structure is basically lost and a large number of dead cell clumps appear, further indicating that the killing effect of the nanocomposite on the cells is enhanced. As shown in Figure 3BAs shown in the GDY / CuO / Cy@PEG nanocomposite test group (blue column chart), with the increase of the concentration, the cell viability decreased significantly, indicating that the GDY / CuO / Cy@PEG nanocomposite had a significant inhibitory effect on PC-3 (human prostate cancer cells), and the inhibitory effect was significantly improved with the increase of the concentration. In the CuO@PEG control group (pink column chart), because it did not contain GDY nanosheets, its tumor inhibition effect was weaker than that of GDY / CuO / Cy@PEG nanocomposite. It is proved that the GDY carrier has a significant effect on improving the tumor inhibition effect of GDY / CuO / Cy@PEG nanocomposite. In the GDY / Cy@PEG control group (purple column chart), because it did not contain CuO, with the increase of the drug concentration, the cell viability did not change significantly, indicating that GDY / Cy@PEG had almost no inhibitory effect on PC-3 (human prostate cancer cells), thus proving that CuO is an active substance.

[0156] Similarly as Figure 3C As shown in the GDY / CuO / Cy@PEG nanocomposite test group (green column chart), with the increase of the concentration, the cell viability decreased significantly, indicating that the GDY / CuO / Cy@PEG nanocomposite had a significant inhibitory effect on NCI-H660 (human neuroendocrine prostate cancer cells), and the inhibitory effect was significantly improved with the increase of the concentration. In the CuO / Cy@PEG control group (red column chart), because it did not contain GDY nanosheets, its tumor inhibition effect was significantly lower than that of GDY / CuO / Cy@PEG nanocomposite. It is proved that the GDY carrier has a significant effect on improving the tumor inhibition effect of GDY / CuO / Cy@PEG nanocomposite. In the GDY@PEG control group (blue column chart), because it did not contain CuO, with the increase of the drug concentration, the cell viability did not change significantly, indicating that GDY / Cy@PEG had almost no inhibitory effect on NCI-H660 (human neuroendocrine prostate cancer cells), thus proving that CuO is an active substance.

[0157] Experimental Example 2

[0158] Transmission electron microscope observation experiment of tissue specimens

[0159] Transmission electron microscope (TEM) was used to observe the detailed microstructure of tumor cells in the tissue, to verify the mechanism of GDY / CuO / Cy@PEG nanocomposite prepared in Example 1 inducing mitochondrial dysfunction and copper death.

[0160] Experimental grouping: GDY / CuO / Cy@PEG nanocomposites prepared in Example 1 were used as the test group, and the control group included: GDY / Cy@PEG prepared in Comparative Example 1, CuO / Cy@PEG prepared in Comparative Example 2, cisplatin (CDDP), and phosphate buffer (NC).

[0161] The concentration of the GDY / CuO / Cy@PEG test group was 5 mg / mL; the preparation method was as follows: GDY / CuO / Cy@PEG (25 mg / mL) was diluted with phosphate buffer at a volume ratio of 4:1.

[0162] The concentrations of GDY / Cy@PEG and CuO / Cy@PEG were both 5 mg / mL; the preparation method was as follows: GDY / Cy@PEG (25 mg / mL) and CuO / Cy@PEG (25 mg / mL) were diluted with phosphate buffer at a volume ratio of 4:1, respectively.

[0163] Cisplatin (CDDP) was purchased from Shanghai Aladdin Biochem Technology Co., Ltd., and the concentration was 1 mg / mL; the preparation method was as follows: 10 mg of cisplatin powder was weighed using an electronic balance and dissolved in 10 mL of phosphate buffer to prepare a working solution with a concentration of 1 mg / mL, which was prepared and used immediately.

[0164] Phosphate buffer (NC) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with the product number AC13317-500 mL and a concentration of 0.01 mmol / L; this reagent was a ready-to-use commercial reagent, and the specific preparation method was as follows: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 were weighed and dissolved in 800 mL of distilled water, the pH value of the solution was adjusted to 7.4 using HCl, and finally distilled water was added to make up the volume to 1 L.

[0165] The specific experimental method was as follows: first, the test group and the control group were administered drugs through the tail vein of the mice on days 0, 4, 8, 12, and 16, with a concentration of 25 mg / kg (equivalent to 5 mg / mL) and a tail vein administration volume of 100 μL. The mice were euthanized on day 30, and the tumor tissues were observed under an electron microscope. After the mice were quickly euthanized, the specimens were cut into 1 mm 3The small piece is placed in 2.5% glutaraldehyde electron microscope fixing solution in a 4°C refrigerator for 4 hours, and then rinsed with pre-cooled PBS for 3 times (15 minutes each time), and then the sample is transferred to 1% osmium tetroxide in a 4°C environment for 2 hours. Then the tissue is dehydrated by 50%, 70%, 80%, and 90% ethanol gradient (15 minutes each time), and then the residual water in the tissue is removed by mixing the mixture of anhydrous ethanol and acetone (1:1). Then the tissue is gradually infiltrated with the mixture of epoxy resin and acetone at a ratio of 1:1 and 3:1 (2 hours each time), and finally the tissue is infiltrated with resin overnight, and then polymerized at 37°C, 45°C, and 60°C in stages to form a tissue embedding block. Then, the sample is embedded in resin for curing treatment at 60°C, and then trimmed and cut into tissue slices of about 50 nm using an ultramicrotome, and then pasted on a copper mesh for staining and transmission electron microscope observation.

[0166] The present application verifies that GDY / CuO / Cy@PEG can induce mitochondrial dysfunction and copper death mechanism through transmission electron microscope observation of tissue samples, such as Figure 3D As shown in the figure, the mitochondria of the cells treated by the GDY / CuO / Cy@PEG test group show obvious swelling, matrix dissolution and disappearance of cristae, and other typical mitochondrial damage phenomena, which further verifies the mechanism of inducing copper death.

[0167] Experimental Example 3

[0168] Western Blot protein immunoblotting analysis experiment

[0169] The influence of the GDY / CuO / Cy@PEG nanocomposite prepared in Example 1 on the expression amount of copper death related proteins FDX1 and PI3K is studied by Western Blot protein immunoblotting analysis.

[0170] Experimental grouping: GDY / CuO / Cy@PEG nanocomposite as test group, control group includes: GDY / CY@PEG, CuO / Cy@PEG, cisplatin (CDDP), phosphate buffer (NC).

[0171] The drug concentration and the corresponding preparation method of the test group and the control group are the same as those in Experimental Example 2.

[0172] The specific experimental steps are as follows:

[0173] Step 2.1, sample processing

[0174] The test group and the control group drugs were administered to the mice via the tail vein on days 0, 4, 8, 12, and 16 for intervention, with a concentration of 25 mg / kg (equivalent to 5 mg / mL) and a tail vein administration volume of 100 μL. The mice were euthanized on the 30th day of the experiment, and tumor tissue was taken for Western Blot protein immunoblotting analysis. The tumor tissue was placed in RIPA lysis buffer (Solarbio, Catalog No. R0010) containing a protein phosphatase inhibitor (Solarbio, Catalog No. P1260) and lysed on ice for 30 minutes to ensure complete lysis. The lysis buffer was centrifuged at 12,000 x g for 10 minutes at 4°C, and the supernatant was collected.

[0175] Step 2.2, Protein Quantification

[0176] The BCA protein quantification kit (Beyotime, Catalog No. P0012S) was used to determine the protein concentration according to the manufacturer's instructions.

[0177] Step 2.3, Electrophoresis and Membrane Transfer

[0178] 20 μg of protein was loaded per well, and SDS-PAGE protein separation was performed at 80 V for 30 minutes, followed by adjustment to 120 V for 30 minutes. The protein was transferred to a PVDF membrane (Vazyme, Catalog No. E801-02) using a wet transfer system at a current of 300 mA for 90 minutes.

[0179] Step 2.4, Blocking and Antibody Incubation

[0180] After the membrane transfer was completed, 5% skimmed milk was prepared in TBS buffer containing 0.1% Tween-20 (Solarbio, Catalog No. T1081) and incubated at room temperature for 1 hour, followed by TBS-T buffer washing for 3 times. Subsequently, the following primary antibodies were incubated at 4°C overnight:

[0181] PI3K Rabbit Polyclonal Antibody (Bi Yun Tian, Catalog No. AF7749, 1:000 dilution)

[0182] FDX1 Rabbit Polyclonal Antibody (HUABIO, Catalog No. HA500468, 1:500 dilution)

[0183] mTOR Rabbit Polyclonal Antibody (HUABIO, Catalog No. HA500126, 1:1000 dilution)

[0184] β-Actin Rabbit Polyclonal Antibody (HUABIO, Catalog No. R1207-1, 1:5000 dilution)

[0185] The primary antibody was diluted with 5% bovine serum albumin (BSA).

[0186] Step 2.5, secondary antibody incubation and development

[0187] After incubation with the primary antibody, the cells were washed with TBS-T buffer for 3 times, 10 minutes each time. Then, the cells were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (Beyotime, item number A0208, diluted at 1:1000) at room temperature for 1 hour. After washing again, the cells were developed using enhanced chemiluminescence (ECL) substrate (APPLYGEN, item number P1050-100), and images were collected by a Tanon full-automatic chemiluminescence imaging system.

[0188] Step 2.6, data analysis

[0189] The gray value of the protein band was quantitatively analyzed using ImageJ software, and the relative expression amount of FDX1, PI3K, and other related protein markers in the cells was detected, with β-Actin as the internal reference for standardization. All experiments were independently repeated at least three times.

[0190] The present application proves, through Western Blot protein immunoblotting analysis, that the GDY / CuO / Cy@PEG nanocomposite not only increases the expression amount of copper death-related proteins FDX1 and LIAS, but also promotes the expression of PI3K, as described in detail in Figure 4A 、 Figure 4B .

[0191] Specifically, as shown in a of Figure 4B , the GDY / CuO / Cy@PEG nanocomposite test group (pink column chart) significantly increased the expression amount of FDX1 protein, where FDX1 is a copper death-related protein, and the increase in the expression amount of FDX1 indicates that the GDY / CuO / Cy@PEG nanocomposite induces the copper death mechanism. As shown in b of Figure 4B , the GDY / CuO / Cy@PEG nanocomposite test group (pink column chart) significantly increased the expression amount of PI3K protein, where PI3K is a survival pathway of tumor cells, and tumors will increase the uptake of surrounding substances through the PI3K pathway, therefore, the increase in the expression amount of PI3K indicates that, in the copper death mechanism, PI3K promotes the uptake of copper ions by tumor cells.

[0192] Experimental Example 4

[0193] Immunohistochemical experiment

[0194] An immunohistochemical experiment was performed to study the effect of the GDY / CuO / Cy@PEG nanocomposite prepared in Example 1 on the expression amount of PI3K and LIAS proteins in tissue cells.

[0195] Experimental grouping: GDY / CuO / Cy@PEG nanocomposites as the experimental group, the control group includes GDY / CY@PEG, CuO / Cy@PEG, cisplatin (CDDP), phosphate buffer (NC).

[0196] The drug concentration and the corresponding preparation method of the experimental group and the control group are the same as those in Experimental Example 2.

[0197] The detailed experimental steps are as follows:

[0198] Step 3.1, the drugs of the experimental group and the control group were administered to the mice via tail vein on days 0, 4, 8, 12, and 16, with a concentration of 25 mg / kg (equivalent to 5 mg / mL) and a tail vein administration volume of 100 μL. The mice were euthanized on the 30th day of the experiment, and the tumor tissues were fixed with 4% paraformaldehyde for 24 hours. The 4% paraformaldehyde was purchased from Beijing Eprui Sheng Technology Co., Ltd., with the product number M329-01 and a concentration of 4%. The reagent is a ready-to-use commercial reagent, and the specific preparation method is as follows: weigh 40 g of paraformaldehyde and add it to 800 mL of 1X PBS (phosphate buffered saline), stir while heating to 60°C, but do not boil. Slowly increase the pH by adding 1N NaOH dropwise until the solution becomes clear. After cooling the solution, adjust the volume to 1 L with 1X PBS and check the pH, adjusting it to 6.9.

[0199] Step 3.2, use a fully automatic tissue dehydration embedding machine (Leica, Germany) to dehydrate and embed the fixed tissue specimens, and then use a paraffin microtome (Leica, Germany) to prepare tissue sections with a thickness of 4 mm.

[0200] Step 3.3, deparaffinize the paraffin sections to water: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water.

[0201] Step 3.4, antigen retrieval: place the tissue sections in a repair box filled with EDTA antigen retrieval buffer (pH 9.0) in a microwave oven for antigen retrieval, with medium heat for 8 min to boiling, turn off the heat for 8 min to keep warm, and then turn to medium-low heat for 10 min. During this process, prevent the buffer from evaporating too much and do not dry the slides. After natural cooling, place the slides in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time.

[0202] Step 3.5, Blocking endogenous peroxidase: Put the slice into 3% hydrogen peroxide solution, incubate at room temperature for 30 min in the dark, and wash the slide in PBS (pH 7.4) on a decolorizing shaker for 3 times, 5 min each time.

[0203] Step 3.6, Serum blocking: Add 3% BSA evenly to the histological circle to cover the tissue, and block at room temperature for 30 min.

[0204] Step 3.7, Adding primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slice, and incubate the slice in a wet box at 4°C overnight. (A small amount of water is added to the wet box to prevent evaporation of the antibody)

[0205] Step 3.8, Adding secondary antibody: Put the slide in PBS (pH 7.4) on a decolorizing shaker and wash for 3 times, 5 min each time. After gently shaking off the slice, add the secondary antibody (HRP-labeled) corresponding to the species of the primary antibody to cover the tissue in the circle, and incubate at room temperature for 60 min.

[0206] Step 3.9, DAB color development: Put the slide in PBS (pH 7.4) on a decolorizing shaker and wash for 3 times, 5 min each time. After gently shaking off the slice, add freshly prepared DAB color development solution in the circle, control the color development time under a microscope, and the positive result is brownish yellow. Rinse the slice with tap water to stop the color development.

[0207] Step 3.10, Re-staining the nucleus: Re-stain with hematoxylin for about 3 min, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, and return to blue with hematoxylin return-to-blue solution, and rinse with running water.

[0208] Step 3.11, Dehydration and mounting: Put the slice in 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, and then dehydrate and clear the slice. Dry the slice slightly, and mount it with neutral balsam.

[0209] Step 3.12, Microscopic examination and image acquisition and analysis.

[0210] Figure 5 The results of the immunohistochemical experiment show that after using the PI3K inhibitor, the expression amount of PI3K protein is reduced, and the expression amount of copper death-related protein LIAS is also reversed, indicating that under the action of the PI3K inhibitor, the expression amounts of PI3K protein and copper death-related protein LIAS are reversed. The above results show that the activation of PI3K has a direct impact on the expression amount of copper death-related protein LIAS.

[0211] Experimental Example 5

[0212] TUNEL staining experiment

[0213] The TUNEL staining experiment was used to observe the apoptotic cells under a microscope to study the influence of the GDY / CuO / Cy@PEG nanocomposite prepared in Example 1 on the apoptosis rate of tumor cells.

[0214] Experimental grouping: GDY / CuO / Cy@PEG was used as the test group, and the control group included GDY / Cy@PEG, CuO / Cy@PEG, cisplatin (CDDP), and phosphate buffer (NC).

[0215] The drug concentration and the corresponding preparation method of the test group and the control group were the same as in Experimental Example 2.

[0216] The specific experimental steps were as follows:

[0217] Step 4.1, the drugs of the test group and the control group were administered to the mice via the tail vein on days 0, 4, 8, 12, and 16, with a drug concentration of 25 mg / kg (equivalent to 5 mg / mL) and a tail vein administration volume of 100 μL. The mice were euthanized on day 30 of the experiment, and the tumor tissues were fixed with 4% paraformaldehyde for 24 hours. The 4% paraformaldehyde was purchased from Beijing Eprui Sheng Technology Co., Ltd., with the product number M329-01 and a concentration of 4%. The reagent was a ready-to-use commercial reagent, and the specific preparation method was as follows: 40 g of paraformaldehyde was added to 800 mL of 1X PBS (phosphate buffer), heated to 60°C while stirring, and the pH value was slowly increased by adding 1N NaOH dropwise until the solution became clear. After cooling the solution, the volume was adjusted to 1 L using 1X PBS, and the pH value was checked and adjusted to 6.9.

[0218] Step 4.2, the fixed tissue specimens were dehydrated and embedded using a fully automatic tissue dehydration embedding machine (Leica, Germany), and then tissue sections were prepared using a paraffin sectioning machine (Leica, Germany) to obtain paraffin sections with a thickness of 4 mm.

[0219] Step 4.3, paraffin section deparaffinization to water: the sections were sequentially placed in xylene I for 10 min, xylene II for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and distilled water.

[0220] Step 4.4, protease K repair: after the sections were slightly dried, a circle was drawn around the tissue using a histological pen (to prevent liquid from flowing away), protease K working solution was added to cover the tissue, and the sections were incubated in a 37°C incubator for 22 min. The slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for 3 times, 5 min each time. (Protease K working solution preparation method: stock solution: PBS = 1:9).

[0221] Step 4.5, membrane breaking: After the slice is slightly dried, add the membrane breaking solution to cover the tissue, and incubate at room temperature for 20 min. Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, each for 5 min.

[0222] Step 4.6, room temperature equilibration: After the slice is slightly dried, add the buffer to cover the tissue, and incubate at room temperature for 10 min.

[0223] Step 4.7, add reaction solution: Mix the TDT enzyme, dUTP and buffer in the tunel kit according to the slice number and tissue size at a ratio of 1:5:50, and add to the circle to cover the tissue. Place the slice in a wet box, and incubate in a 37°C incubator for 2 hours. Add a small amount of water to the wet box to maintain humidity.

[0224] Step 4.8, DAPI re-stain the nucleus: Wash the slice with PBS (pH 7.4) for 3 times, each for 5 min. After removing the PBS, add DAPI staining solution in the circle, and incubate at room temperature for 10 min in the dark.

[0225] Step 4.9, mounting: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, each for 5 min. Slightly dry the slice, and mount it with anti-fluorescence quenching mounting medium.

[0226] Step 4.10, microscopic examination and photographing: Observe the slice under a fluorescence microscope and collect images.

[0227] Figure 5 The TUNEL staining results in FIG. 6 show that after adding the PI3K inhibitor, the number of apoptotic cells is significantly reduced, indicating that the PI3K inhibitor reverses the activation of PI3K protein and LIAS protein by GDY / CuO / Cy@PEG, and further reverses the mitochondrial dysfunction and copper death induced by PI3K protein and LIAS protein, thereby reducing the number of apoptotic cells.

[0228] Figure 4A 、 Figure 4B and Figure 5 The research results collectively show that GDY / CuO / Cy@PEG can activate PI3K protein, as well as copper death related proteins LIAS and FDX1, thereby inducing mitochondrial dysfunction and copper death.

[0229] Experimental Example 6

[0230] Immune activation experiment

[0231] Detection of CD8 +The proportion of T cells was used to verify the immune activation effect of the GDY / CuO / Cy@PEG nanocomposites prepared in Example 1.

[0232] Experimental grouping: The GDY / CuO / Cy@PEG nanocomposites were used as the experimental group, and the control group included GDY / Cy@PEG, CuO / Cy@PEG, cisplatin (CDDP), phosphate buffer (NC), and the like.

[0233] The drug concentration and the corresponding preparation method of the experimental group and the control group were the same as in Example 2.

[0234] The drugs of the experimental group and the control group were administered to the mice via the tail vein on days 0, 4, 8, 12, and 16 for intervention, and the administration concentration was 25 mg / kg (equivalent to 5 mg / mL), and the tail vein administration volume was 100 μL. The mice were euthanized and sacrificed on the 30th day of the experiment, and the spleens of the mice were removed. The immune cells were isolated from the mouse spleen by a combination of mechanical disruption and density gradient centrifugation to obtain a single cell suspension. After isolation, the red blood cells were lysed using a red blood cell lysis solution (Biolegend, Catalog No.: 420302) to ensure the purity of the immune cells. The cells were washed twice with phosphate buffer solution (PBS) containing 2% fetal bovine serum (FBS) to maintain cell activity. Subsequently, the immune cells were stained with a set of fluorescently labeled antibodies for flow cytometry analysis. The following antibodies were used for surface and intracellular staining:

[0235] Brilliant Violet 421™ Anti-Mouse FOXP3 antibody (Biolegend, Catalog No.: 126419) was used to detect Foxp 3+ Regulatory T cells (Tregs). Intracellular staining of FOXP3 used FOXP3 Fix / Perm Kit (ThermoFisher, Catalog No.: 00-5523-00).

[0236] PE / Dazzle594 Anti-Mouse CD279 (PD-1) antibody (Biolegend, Catalog No.: 109116) was used to detect PD-1 + T cells.

[0237] PE-CY7 Anti-Mouse CD152 (CTLA-4) antibody (MultiSciences, Catalog No.: F2115205) was used to detect CTLA-4 + T cells.

[0238] FITC Anti-Mouse CD3ε antibody (Elabscience, Catalog No.: E-AB-F1103C) was used to detect total T cells.

[0239] APC anti-mouse CD4 antibody (Elabscience, catalog number: E-AB-F1097E) is used for the detection of CD4. + Helper T cells.

[0240] PerCP / Cyanine 5.5 anti-mouse CD8a antibody (Elabscience, catalog number: E-AB-F1104J) is used to detect CD8. + Cytotoxic T cells.

[0241] During staining, take 1×10⁻⁶ samples from each sample. 6 Cells were incubated with antibodies at 4°C in the dark for 30 minutes. They were then washed twice with PBS containing 2% FBS and fixed with 2% paraformaldehyde (PFA) if necessary. Flow cytometry was performed on a Sony ID7000. TM The procedure was performed on a spectrophotometer, and the data were analyzed using FlowJo software. Quantitative analysis of CD4 in immune cells was conducted using flow cytometry. + T cells, CD8 + T cells, Foxp 3+ Regulatory T cells, CTLA-4 + T cells and PD-1 + The proportion of T cells.

[0242] like Figure 6 As shown, compared with the treatment group treated with GDY / CuO / Cy@PEG nanocomposite alone, the treatment group treated with the combination of GDY / CuO / Cy@PEG nanocomposite and immune checkpoint inhibitors showed that... Figure 6 CD8+ tumor killer cells + The proportion of T cells increased from 28.69% to 30.99%, while the content of PD-1-positive T cells decreased from 22.73% to 17.33%, and the content of CTLA-4-positive T cells decreased from 29.55% to 7.86%. This demonstrates that the GDY / CuO / Cy@PEG nanocomposite material can effectively activate immune cells, thereby enhancing the efficacy of tumor immunotherapy. This provides a theoretical basis for the combined use of GDY / CuO / Cy@PEG with immune checkpoint inhibitors (ICIs).

[0243] Experimental Example 7

[0244] Antitumor effect of GDY / CuO / Cy@PEG nanocomposite material prepared in Example 1 in prostate cancer animal model

[0245] This study was approved by the Ethics Committee of Peking University School of Medicine (Approval No.: DLASBD0357), and all experimental procedures were strictly in accordance with the guidelines of the National Institutes of Health (NIH).

[0246] The experimental groups include a test group and a control group. The test group includes a GDY / CuO / Cy@PEG test group and a GDY / CuO / Cy@PEG+ICIs test group. The control group includes an immune checkpoint inhibitor aPD-1 (ICIs) control group, a GDY / Cy@PEG control group, a CuO / Cy@PEG control group, and a cisplatin (CDDP) control group.

[0247] The drug concentrations and corresponding configuration methods of the test group and the control group are the same as those in Experimental Example 2.

[0248] ICIs information: RecombiMAb Anti-Mouse PD-1 (CD279) (Bioxcell, Catalog No. CP162)

[0249] Specific experimental methods: 8-week-old male C57BL / 6J mice were selected for the experiment, which were purchased from the Experimental Animal Science Department of Peking University Medical Department / Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animals were raised in a SPF-level environment with a constant temperature of 25°C, a humidity of 50%-60%, and a 12-hour day-night cycle. The animals were adaptively fed for 2 weeks before the experiment to ensure their health. RM-1 prostate cancer tumor cells (viability ≥95% confirmed by trypan blue staining) from ATCC were used to prepare a cell suspension (5×10 6 Before the model was constructed, all surgical instruments, including ophthalmic scissors, ophthalmic forceps, needle holders, etc., were subjected to high-pressure steam sterilization to ensure a sterile environment during the experiment. Logarithmic growth phase RM-1 prostate cancer cells were collected, digested with 0.25% trypsin, resuspended with serum-free RPMI 1640 medium, and prepared into a single-cell suspension. Then, the tumor cell suspension (concentration 1×10 7 / mL) was mixed with Matrigel at a ratio of 3:1, placed on ice, and used. At the same time, an avertin anesthetic solution was prepared by diluting avertin anesthetic with sterile saline at a certain ratio to obtain a 1.25% anesthetic solution, which was stored in the dark.

[0250] Subsequently, the required amount of anesthetic solution was calculated according to the weight of the mouse (20 μL / g), and anesthesia was performed by intraperitoneal injection. After the anesthesia took effect, the mouse was gently supine and fixed on a sterile operating table, the abdominal hair was removed with a hair clipper, and iodophor was used for disinfection three times. Then, a small incision was made 2 mm above the pubic symphysis of the mouse with an ophthalmic scissors, and the abdominal muscle was exposed by cutting about 1 cm along the head of the abdomen. The abdominal muscle was carefully lifted and cut along the white line of the abdomen, and the intra-abdominal organs were carefully exposed using an ophthalmic forceps. During the operation, the organs were damaged as little as possible, and sterile gauze was used for hemostasis. The position of the bladder was probed with an ophthalmic forceps, and when a light yellow, clear and transparent balloon-shaped bladder was probed, the microscope was moved to the sterile operating table for microscopic positioning of the mouse prostate. In order to facilitate the exposure of the mouse prostate, the bladder can be gently clamped and lifted with an ophthalmic forceps, and the prostate can be seen as a typical gland-like structure with a light pink color at the junction of the bladder and urethra. At this time, 10 μL of tumor cell suspension on ice was gently injected into the mouse prostate with a microsyringe, and the mouse prostate capsule was gently observed to be slightly swollen without additional liquid leakage, indicating successful injection. After the injection of tumor cells was completed, the needle tip was gently rotated and slowly pulled out, and the anesthetized mouse was gently placed back on the sterile operating table, the bladder was in situ, and the muscle and mouse abdominal skin were sutured layer by layer with 4-0 silk thread, and then the wound was disinfected with iodophor before being placed back into the mouse cage. The mouse was observed to recover from anesthesia. In order to verify whether the mouse prostate cancer model was successfully constructed, 3 mice were randomly selected for euthanasia on the 7th day after the operation, and the position of the prostate in the abdominal cavity was probed to determine whether there was tumor growth, and the abdominal cavity was checked for tumor metastasis. After the model was successfully constructed, subsequent experiments were performed.

[0251] The experimental drugs CDDP (5 mg / kg) and nanocomposites (25 mg / kg) were administered by tail vein injection on days 0, 4, 8, 12, and 16, and the immune checkpoint inhibitors ICIs (5 mg / kg) were administered by tail vein injection on days 0, 7, and 14, each time 100 μl. The activity, food intake, and hair luster of the animals were recorded daily.

[0252] As shown in FIGS. 1-3, the anti-tumor effect of the GDY / CuO / Cy@PEG nanocomposite alone treatment group and the GDY / CuO / Cy@PEG nanocomposite combined with immune checkpoint inhibitors treatment group was significantly improved compared with the control group, and the anti-tumor effect of the GDY / CuO / Cy@PEG nanocomposite combined with immune checkpoint inhibitors group was significantly better than that of the GDY / CuO / Cy@PEG nanocomposite alone group, indicating that the combination of GDY / CuO / Cy@PEG nanocomposite and immune checkpoint inhibitors can play a synergistic effect in enhancing anti-tumor immunity and effectively inhibiting tumor growth. Figure 7a , 7b and Figure 8 As shown in FIGS. 1-3, the anti-tumor effect of the GDY / CuO / Cy@PEG nanocomposite alone treatment group and the GDY / CuO / Cy@PEG nanocomposite combined with immune checkpoint inhibitors treatment group was significantly improved compared with the control group, and the anti-tumor effect of the GDY / CuO / Cy@PEG nanocomposite combined with immune checkpoint inhibitors group was significantly better than that of the GDY / CuO / Cy@PEG nanocomposite alone group, indicating that the combination of GDY / CuO / Cy@PEG nanocomposite and immune checkpoint inhibitors can play a synergistic effect in enhancing anti-tumor immunity and effectively inhibiting tumor growth.

[0253] Experimental Example 8

[0254] Imaging experiment of GDY / CuO / Cy@PEG nanocomposites prepared in Example 1 in mice

[0255] Experimental grouping: GDY / CuO / Cy@PEG nanocomposites as experimental group, control group including CuO / Cy@PEG, GDY / Cy@PEG, free Cy, cisplatin.

[0256] The concentration of GDY / CuO / Cy@PEG is 5 mg / mL; the preparation method is that GDY / CuO / Cy@PEG (25 mg / mL) is diluted according to the volume 4:1 with phosphate buffer.

[0257] The concentration of CuO / Cy@PEG and GDY / Cy@PEG is 5 mg / mL; the preparation method is that CuO / Cy@PEG and GDY / Cy@PEG (25 mg / mL) are diluted according to the volume 4:1 with phosphate buffer.

[0258] The concentration of free Cy5 is 5 mg / mL; the preparation method is that Cy5 (25 mg / mL) is diluted according to the volume 4:1 with phosphate buffer.

[0259] Cisplatin (CDDP, 1 mg / mL), the preparation method is that 10 mg of cisplatin powder is weighed by using an electronic balance and dissolved in 10 mL of phosphate buffer to prepare a working solution with a concentration of 1 mg / mL, which is prepared and used immediately.

[0260] The in situ cancer mice are injected with the following preparations via tail vein: GDY / CuO / Cy@PEG, CuO / Cy@PEG, GDY / Cy@PEG, free Cy5 and cisplatin (CDDP, 1 mg / mL). After 24 hours of injection, the mice in anesthetized state are observed by using a small animal live imaging system.

[0261] As shown in Figure 9 , the experimental groups of GDY / CuO / Cy@PEG, CuO / Cy@PEG, GDY / Cy@PEG, free Cy5 and the like can be imaged in mice due to the presence of fluorescent groups, and the effect of tracking tumors can be achieved.

[0262] Further, from Figure 9It can be seen that the GDY / CuO / Cy@PEG test group has higher brightness than the CuO / Cy@PEG, GDY / Cy@PEG, free Cy5 and other test groups, indicating that the drug accumulation concentration of GDY / CuO / Cy@PEG at the tumor site is significantly higher than that of CuO / Cy@PEG, GDY / Cy@PEG, free Cy5 and other test groups. It is further verified that the use of GDY loaded with CuO can improve the enrichment of drugs in tumor cells.

[0263] The applicant declares that the present application is illustrated by the above examples to explain the detailed method of the present application, but the present application is not limited to the above detailed method, that is, it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing a GDY / CuO / Cy@PEG nanocomposite material, characterized in that, Includes the following steps: Step 1: The dispersions of graphyne and copper oxide are ultrasonically mixed to obtain a GDY / CuO dispersion solution. The graphyne dispersion is a dimethyl sulfoxide (DMSO) dispersion of graphyne, obtained by placing GDY nanosheets in DMSO and then subjecting them to cell disruption for 24-48 hours; the graphyne dispersion has a mass concentration of 25-50 mg / mL, the copper oxide dispersion has a mass concentration of 25-50 mg / mL, and the volume ratio of the graphyne dispersion to the copper oxide dispersion is 20-40 : 20-40. Step 2: Stir the cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution from Step 1 to obtain a mixed solution, wherein the mass-volume ratio of the cyanine dye, PEG-PLGA and the GDY / CuO dispersion solution is 1-2 mg: 50-100 mg: 40-80 μL. Step 3: Mix water and the mixed solution described in Step 2, stir, and dialyze to obtain the GDY / CuO / Cy@PEG nanocomposite material; The average particle size of the GDY / CuO / Cy@PEG nanocomposite is 40-160 nm.

2. The preparation method according to claim 1, characterized in that, In step 1, the ultrasonic mixing time is 4-6 hours and the ultrasonic power is 500-720 W.

3. The preparation method according to claim 1, characterized in that, In step 2, the stirring time is 1-2 hours and the stirring speed is 600-1000 rpm.

4. The preparation method according to claim 1, characterized in that, In step 3, the volume ratio of water to the mixed solution is 4-5:1, and the water used is ultrapure water; during the dialysis process, the molecular weight cutoff of the dialysis device is 3000-5000 D.

5. A GDY / CuO / Cy@PEG nanocomposite material prepared by the method according to any one of claims 1-4, characterized in that, The GDY / CuO / Cy@PEG nanocomposite material has a spherical structure with an average particle size of 40-160 nm.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the GDY / CuO / Cy@PEG nanocomposite material as described in claim 5 and pharmaceutically acceptable excipients; the pharmaceutical composition may be used alone or in combination with immunotherapy.

7. The application of the GDY / CuO / Cy@PEG nanocomposite material according to claim 5 in the preparation of antitumor drugs or contrast agents, characterized in that, The tumor is a urinary system tumor.

8. The application of the GDY / CuO / Cy@PEG nanocomposite material according to claim 7 in the preparation of antitumor drugs or contrast agents, characterized in that, Inhibits tumor cell proliferation or promotes the expression of FDX1, LIAS and / or PI3K, wherein the tumor cells are urinary system tumor cells.

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