Nanometer delivery system based on copper death induction and application of nanometer delivery system in preparation of tumor immunotherapy drugs

Abstract

The invention provides a nano delivery system based on copper death induction and application of the nano delivery system in preparation of tumor immunotherapy drugs, according to the novel copper death induction nano drug Cu / ZIF-90 (at) DSF, 30 mol% of Cu (acac) 2 and disulfiram are loaded through a high-porosity structure of ZIF-90, and pH / ROS dual-responsiveness copper ion release is achieved. Animal experiments show that the Cu < 2 + > accumulation concentration of the nano-drug at a tumor site reaches 23.7 mu M, and efficient copper death is induced (the apoptosis rate is 41.3% vs and the control group is 9.2%). More importantly, by releasing HMGB1 and ATP, the CD8 < + > T cell infiltration in a tumor microenvironment is increased by 5.8 times, and the CD8 < + > T cell infiltration and the alphaPD-1 antibody generate a synergistic effect, so that the complete remission rate is increased from 18% to 67%. According to the technical breakthrough, a brand new solution is provided for solving the cold tumor immunotherapy drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical fields of biology and pharmacy, and particularly relates to a nano-delivery system based on copper death induction and its application in the preparation of tumor immunotherapy drugs. Background Art

[0002] Existing tumor immunotherapy methods (such as αPD-L1 antibody therapy) face many challenges in clinical applications, mainly including the following aspects: (1) Immune checkpoint inhibitor resistance: Most tumors are "cold tumors" with poor responses to immune checkpoint inhibitors, resulting in poor treatment effects. Low expression of PD-L1 in the tumor microenvironment (<1% positive rate on the surface of tumor cells) leads to limited efficacy of αPD-L1 antibody (objective response rate ORR < 20%); Tumor stem cells induce T cell rejection through the Wnt / β-catenin pathway. (2) Insufficient immunogenicity of traditional nano-drugs: Existing nano-drugs have low immunogenicity in tumor treatment and are difficult to effectively activate the immune system, limiting their treatment effects. For example, existing nano-carriers (such as PLGA, liposomes) only deliver chemotherapy drugs and cannot effectively induce ICD (HMGB1 release < 50 ng / mL); Copper-based nano-drugs (such as CuO NPs) have defects such as low bioavailability (Cu 2+ plasma half-life < 1 h) and high toxicity; (3) In addition, the infiltration of the immune microenvironment around the tumor is insufficient, further weakening the effect of immunotherapy. Tumor antigens are the key for the immune system to recognize and attack tumor cells. However, tumor cells often resist immune attacks by downregulating antigen expression or escaping immune surveillance, which greatly reduces the effect of immunotherapy. (4) Blank in copper death regulation technology: Reported Cu 2+ prodrugs (such as GSX-325) are limited by cell uptake efficiency (<15%) and lack a tumor microenvironment-responsive copper ion controlled release system (the existing system has a pH response range of 5.0 - 6.5, while the tumor stroma pH is 6.0 - 6.5).

[0003] In view of these problems, it is necessary to provide a nano-drug that can significantly improve the antigen expression and immunogenicity of tumor cells, and at the same time improve the infiltration of the immune microenvironment around the tumor. Summary of the Invention

[0004] The object of the present invention is to provide a nano-delivery system based on copper death induction and its application in the preparation of tumor immunotherapy drugs. This nano-drug significantly improves the antigen expression and immunogenicity of tumor cells, and at the same time improves the infiltration of the immune microenvironment around the tumor, providing a new strategy for tumor immunotherapy.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a nano-delivery system based on cuproptosis induction is provided, and the nano-delivery system comprises the following components:

[0007] Metal-organic framework ZIF-90 modified with polyvinylpyrrolidone (PVP) on the surface;

[0008] And copper acetylacetonate (Cu(acac) 2 ) and disulfiram (DSF) loaded in the pores of the metal-organic framework ZIF-90;

[0009] Wherein, the total copper ion loading accounts for 15-45 mol% of the mass of the metal-organic framework ZIF-90.

[0010] Preferably, the total copper ion loading is 15 mol%. The nano-particles prepared with a total copper ion loading of 15 mol% have a smaller particle size, and the spherical shape has a higher specific surface area compared with other morphologies, and can load more active substances.

[0011] Furthermore, the mass percentage of the polyvinylpyrrolidone (PVP) to the metal-organic framework ZIF-90 is 5-15 wt.%.

[0012] Furthermore, the molar ratio of the disulfiram (DSF) to copper acetylacetonate (Cu(acac) 2 ) is 1:2-1:4.

[0013] Furthermore, the particle size of the nano-delivery system based on cuproptosis induction is controlled within 15-25 nm, and the Zeta potential is -25 to -35 mV; the copper ion release rate reaches more than 75% in 24 hours under the pH 5.5 environment.

[0014] In the second aspect of the present invention, a preparation method of the nano-delivery system is provided, and the method comprises:

[0015] Adopting a microwave-assisted one-pot synthesis process to prepare with copper acetylacetonate (Cu(acac) 2 ), disulfiram (DSF), PVP, and the synthesis raw materials 2-methylimidazolecarboxaldehyde (2-ICA) and zinc source of the metal-organic framework ZIF-90.

[0016] As a specific implementation manner, the zinc source is Zn(CH 3 COOH) 2 ·2H 2 O.

[0017] As a specific implementation manner, the ZIF-90 substrate is synthesized by a microwave-assisted one-pot method, and the specific steps are as follows:

[0018] a. Prepare 2 mL of 2-ICA solution (DMF solvent) with a concentration of 0.2 M and sonicate for 10 minutes;

[0019] b. Prepare 2 mL of Zn(CH 3 COOH) 2 ·2H 2 O solution (DMF solvent) with a concentration of 0.1 M and sonicate with stirring for 30 minutes;

[0020] c. Dropwise add the solution from step a to the solution from step b within 1 minute and vigorously stir at room temperature for 10 minutes to form a white precipitate;

[0021] d. Centrifuge (10000 rpm / min / 10 min) × 6 times, wash with ethanol, and vacuum dry for 12 hours;

[0022] As a specific implementation manner, the Cu / ZIF-90 loading is completed through the following steps:

[0023] i. Dissolve 76.8 mg of 2-ICA, 44 mg of Zn(CH 3 COOH)□·2H 2 O, 26 - 78 mg of Cu(acac) 2 and 100 mg of PVP 4W solution in 4 mL of DMF;

[0024] ii. Dropwise add the 2-ICA solution from step a to the mixture, stir at room temperature for 5 minutes, add 6 mL of DMF, and then centrifuge for purification;

[0025] Furthermore, the microwave reaction power is 800 W and the duration is ≤ 3 minutes.

[0026] Furthermore, in the synthesis raw materials of the metal-organic framework ZIF-90, the molar ratio of 2-methylimidazole aldehyde group (2-ICA) to the zinc source is 2:1.

[0027] In the third aspect of the present invention, there is provided the application of the described nano-delivery system in the preparation of tumor immunotherapy drugs.

[0028] The nano-drug is synthesized by a one-pot method and has excellent stability and biocompatibility. Its core mechanism is to efficiently deliver copper ions into tumor cells through a nano-carrier, resulting in intracellular copper overload, and then inducing cuproptosis. Cuproptosis is a new type of programmed cell death. During this process, immunogenic molecules such as high-mobility group protein B1 (HMGB1), calreticulin (CAT), and adenosine triphosphate (ATP) are released, triggering immunogenic cell death (ICD). As a damage-associated molecular pattern (DAMP), HMGB1 can activate dendritic cells (DCs) and promote antigen presentation, while CAT further promotes antigen presentation and T cell activation by enhancing the phagocytosis of tumor cells. In addition, as a chemoattractant, ATP can attract immune cells (such as T cells and natural killer cells) to infiltrate into the tumor microenvironment and enhance the killing activity of immune cells.

[0029] In the third aspect of the present invention, there is provided the use of the nano-delivery system and the combined drug composition composed of an αPD-1 antibody in the preparation of a tumor immunotherapy drug.

[0030] In the said combined application, the infiltration of CD8+ T cells in the tumor microenvironment increases by ≥3 times. The administration interval between the nano-drug and the αPD-1 antibody is 72 hours; the complete remission (CR) rate ≥40% is achieved in the tumor model.

[0031] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0032] By delivering copper ions into tumor cells, inducing cuproptosis and triggering immunogenic cell death (ICD), immunogenic molecules such as high-mobility group protein B1 (HMGB1) and calreticulin (CAT) are released. As a damage-associated molecular pattern (DAMP), HMGB1 can activate dendritic cells (DCs) and promote antigen presentation, while CAT further promotes antigen presentation and T cell activation by enhancing the phagocytosis of tumor cells. In addition, other immunogenic molecules (such as ATP) released during cuproptosis can also enhance the infiltration and activity of immune cells. Through these mechanisms, the present invention significantly improves the antigen expression and immunogenicity of tumor cells, and at the same time improves the infiltration of the immune microenvironment around the tumor, providing a new strategy for tumor immunotherapy. Specifically, it is reflected in the following aspects:

[0033] (1) The present invention uses a ZIF-90 metal-organic framework to load Cu(acac) 2 / DSF complex to achieve pH / ROS dual-responsive release, and the copper ion release rate is increased to 82% (vs 35% of the traditional system).

[0034] (2) In terms of the mechanism of action, the cuproptosis-ICD cascade reaction: Cu 2+ → ROS burst → mitochondrial membrane rupture → release of HMGB1 / ATP → activation of DCs (↑300% infiltration of CD8+ T cells), achieving for the first time the multi-level amplification of the cuproptosis and ICD signaling pathways.

[0035] (3) DSF-mediated inhibition of Sirt1 enhances tumor antigen presentation (a 5-fold increase in the surface expression of MHC I), solving the problem of immune checkpoint inhibitor resistance.

[0036] (4) In terms of the process, microwave-assisted one-pot synthesis (reaction time < 3 min, yield > 92%), while the traditional solvothermal method requires 24 h with a yield of only 65%. Brief Description of the Drawings

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a flowchart for the preparation of nano-drugs.

[0039] Figure 2 It is a schematic diagram of the mechanisms of cuproptosis and immunogenic cell death (ICD).

[0040] Figure 3 It is a morphology diagram of Cu / ZIF-90@DSF prepared in Examples 1 - 5.

[0041] Figure 4 It is a particle size distribution diagram of Cu / ZIF-90@DSF prepared in Examples 1 - 5.

[0042] Figure 5 It is an XRD pattern of the solid samples of ZIF-90, Cu / ZIF-90, and Cu / ZIF-90@DSF prepared in Example 1.

[0043] Figure 6 It is a BET surface property characterization.

[0044] Figure 7 It is the ultraviolet absorption spectra of ZIF-90, Cu / ZIF-90, and Cu / ZIF-90@DSF.

[0045] Figure 8 It is the result of detecting the release of CRT and HMGB1 by immunofluorescence staining technology.

[0046] Figure 9 The established A549 tumor-bearing nude mouse model, the administration protocol with one-day interval, and the results.

[0047] Figure 10 The results of the Lewis tumor-bearing C57BL / 6J mouse model, where Figure A is a schematic diagram of the treatment process, B is the body weight change curve, C is the tumor volume growth curve, D is the final tumor volume, and E is the tumor pictures after different treatment procedures.

[0048] Figure 11 The proportion of cDC cells in the tumor microenvironment after treatment with Cu / ZIF-90@DSF. Among them, A is the schematic diagram of cDC cell gating, B is the flow cytometry diagram of the proportion of cDC cells in different treatment groups, C is the flow cytometry diagram of the proportion of CD4+ T cells in different treatment groups, and D is the flow cytometry diagram of the proportion of CD8+ T cells in different treatment groups.

[0049] Figure 12 The particle size distribution diagram of ZIF-90 without loaded copper.

[0050] Figure 13 The morphological comparison diagrams of copper-doped Cu / ZIF-90 and ZIF-90.

[0051] Figure 14 The results of cell proliferation and toxicity analysis.

[0052] Figure 15 The results of observing the live and dead states of A549 cells after treatment with different drugs by a laser confocal fluorescence microscope.

[0053] Figure 16 The detection of intracellular ATP residues using an ATP detection kit.

[0054] Figure 17 The experimental results of the combination treatment group (Cu / ZIF-90@DSF-30 + αPD-1). Among them, A is the treatment protocol, B is the mouse body weight change curve, C is the tumor volume growth curve, D is the tumor pictures after different treatments, and E is the immunohistochemistry of PD-L1. Detailed implementation manners

[0055] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0056] Throughout the specification, unless otherwise specifically stated, the terms used herein shall be understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0057] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or by existing methods.

[0058] The present application will be described in detail below in conjunction with examples and experimental data.

[0059] Example 1: A nanodelivery system (Cu / ZIF-90@DSF-15) based on copper death induction and its preparation method

[0060] In this example, the amount of Cu(acac) 2 is 26 mg (corresponding to 15 mol%), and the specific preparation process is as follows:

[0061] (1) Preparation of ZIF-90: First, use N,N-dimethylformamide (DMF) as a solvent to prepare 2 mL of a 0.2 M 2-ICA solution, and perform sufficient ultrasonic treatment to ensure dissolution; then, use DMF to prepare 2 mL of a 0.1 M Zn(CH 3 COOH) 2 ·2H 2 O solution, and ultrasonically stir for 30 minutes; subsequently, drop the 2-ICA solution into the Zn(CH 3 COOH) 2 ·2H 2 O solution within 1 minute, and vigorously stir at room temperature for 10 minutes. At this time, it can be observed that the solution becomes white and turbid, indicating the formation of the ZIF-90 nanodelivery system. Next, centrifuge the mixture at 10000 rpm / min for 10 minutes, then wash it with ethanol, repeat this process 6 times, and finally dry it in vacuo for 12 hours for later use.

[0062] (2) Preparation of Cu / ZIF-90: First, use N,N-dimethylformamide (DMF) as a solvent, add 76.8 mg of 2-ICA, and perform sufficient ultrasonic treatment to ensure its complete dissolution. Subsequently, add 44 mg of Zn(CH 3 COOH) 2 ·2H 2 O, 26 mg of Cu(acac) 2And 100 mg of PVP 4W solution, and continue ultrasonic stirring until all components are completely dissolved. Then, quickly add the prepared 2-ICA solution dropwise to the mixed solution, and vigorously stir at room temperature for 5 min. At this time, the solution will turn green and turbid, indicating the formation of the Cu / ZIF-90 nanodelivery system. Then, add 6 mL of DMF and stir for 5 min. Subsequently, centrifuge the mixed solution at 10000 rpm for 10 minutes, and wash with ethanol. Repeat this process 6 times. Finally, dry the product in vacuo for 12 hours for later use.

[0063] (3) Preparation of Cu / ZIF-90@DSF complex: Weigh the Cu / ZIF-90 prepared in step (2) and DSF, mix them in a ratio of 1:2, and fully ultrasonic to ensure complete dissolution. Transfer the mixed solution to a vial and stir at room temperature for 12 h. Subsequently, centrifuge the obtained solution at 10000 rpm for 10 minutes, and wash with ethanol until the supernatant after washing is visibly clear to the naked eye and no ultraviolet absorption peak of DSF can be detected under UV-vis spectroscopy. Finally, place the product in a vacuum drying oven and dry it in vacuo for 12 hours to obtain a solid product, namely Cu / ZIF-90@DSF, and then store it at 4 °C for later use.

[0064] Example 2

[0065] In this example, the dosage of Cu(acac) 2 is 39 mg (Cu loading 22.5 mol%), and other steps are the same as in Example 1.

[0066] Example 3

[0067] In this example, the dosage of Cu(acac) 2 is 52 mg (Cu loading 30 mol%), and other steps are the same as in Example 1.

[0068] Example 4

[0069] In this example, the dosage of Cu(acac) 2 is 65 mg (Cu loading 37.5 mol%), and other steps are the same as in Example 1.

[0070] Example 5

[0071] In this example, the dosage of Cu(acac) 2 is 78 mg (Cu loading 45 mol%), and other steps are the same as in Example 1.

[0072] Experimental Example 1. Characterization of Cu / ZIF-90@DSF

[0073] 1. The morphology diagrams of Cu / ZIF-90@DSF prepared in Examples 1-5 are as follows Figure 3 shown. With the gradual progression from Example 1 to Example 5, the particle size of the samples shows a gradually decreasing trend, the morphology gradually changes from irregular to spherical, and the surface pore size increases significantly. The preferred example of the present invention is Example 1. The nanoparticles prepared in Example 1 have a smaller size, and the spherical shape has a higher specific surface area compared to other morphologies, which can load more active substances.

[0074] 2. The particle size distribution diagrams of Cu / ZIF-90@DSF prepared in Examples 1-5 are as follows Figure 4 shown. The particle size of the samples shows a single-peak distribution, and the peak interval is 16-22 nm (PDI < 0.3), indicating that the nanoparticles have high uniformity. The uniform particle size is beneficial to enhancing the EPR effect and improving the tumor penetration rate.

[0075] The particle size distribution diagram of ZIF-90 without copper loading is as follows Figure 12 shown, indicating that: compared with the particle size of ZIF-90 without copper loading, the particle size of Cu / ZIF-90@DSF is significantly reduced (18 ± 2 nm). This is because PVP is added during the synthesis of Cu / ZIF-90@DSF, and high-concentration PVP can more effectively adsorb on the surface of nanoparticles and inhibit their growth. The small size (<20 nm) of the particle size can effectively avoid being rapidly cleared by liver macrophages.

[0076] 3. XRD characterization: The test materials are freshly prepared solid samples of ZIF-90, Cu / ZIF-90, and Cu / ZIF-90@DSF. First, use a mortar to grind the samples into fine powders, and then spread them evenly on the stage according to the operation instructions to ensure the uniformity and consistency of the samples. Next, perform X-ray diffraction (XRD) tests with the parameter settings of the 2θ range from 0 to 40°. During the test, ensure that the equipment is correctly calibrated to obtain an accurate diffraction pattern for subsequent phase analysis and crystal structure research.

[0077] The XRD patterns of the solid samples of ZIF-90, Cu / ZIF-90, and Cu / ZIF-90@DSF prepared in Example 1 are as follows Figure 5 shown. It can be seen from Figure 5 that

[0078] The main peak is located at **2θ = 7.1°**, and a new peak appears at **2θ = 25.6°** for Cu / ZIF-90, which can be attributed to [Cu(acac)] of Cu(acac) 2 of[Cu(acac)] 2- Coordination unit (PDF 41 - 1482); After the combination of DSF (Cu / ZIF - 90@DSF), a broad peak appears at **2θ = 38.5°**, indicating that DSF forms a complex with Cu through S - S bonds. 2+ A complex is formed.

[0079] The diffraction intensity of the crystal plane of Cu / ZIF - 90 is 18% lower than that of ZIF - 90, suggesting that Cu 2+ doping leads to crystal distortion. The grain size is calculated by the Scherrer formula: Cu / ZIF - 90 is 19.2 nm (32% smaller than 28.5 nm of ZIF - 90).

[0080] 4. Specific surface area and pore structure

[0081] From Figure 6 it can be seen that Cu / ZIF - 90@DSF exhibits a high specific surface area of 112 m 2 / g (the original value of ZIF - 90 is 85 m 2 / g). The adsorption isotherm shows the characteristics of a type - I curve, indicating the presence of a large number of micropores (pore diameter < 2 nm, accounting for 68%).

[0082] An inflection point appears at P / Po = 0.4 in the desorption branch, corresponding to the mesopore diameter (2 - 50 nm).

[0083] After DSF modification, the nitrogen adsorption capacity increases by 22% (from 3.15 mmol / g to 3.85 mmol / g).

[0084] CO 2 adsorption test shows that the capacity reaches 6.7 wt.% at 25 °C / 1 bar, confirming that the amino groups introduced by DSF enhance the polar adsorption sites.

[0085] 5. Ultraviolet absorption spectrum

[0086] From Figure 7 it can be seen that:

[0087] Cu / ZIF - 90 shows a characteristic absorption peak at 320 nm, corresponding to the π→π* transition of Cu(acac) 2 . After the combination of DSF, the absorption peak redshifts to 345 nm (Δλ = 25 nm), indicating that the mercapto group (-SH) of DSF forms a coordination bond with Cu 22 (the Cu - S bond length can be calculated by the ligand - field theory ).

[0088] The absorbance intensity shows a linear relationship with the Cu 2+ concentration (R 2 = 0.98). The Cu 2+The loading amount reaches 30.7 mol%.

[0089] The encapsulation efficiency measured by UV-visible spectroscopy (92.3%) is in good agreement with the ICP-MS result (91.8%).

[0090] In summary, under normal temperature and pressure, we successfully synthesized ZIF-90. The synthesized ZIF-90 shows a smooth surface and uniform morphology. After copper doping, Cu / ZIF-90 is slightly rounder in morphology than ZIF-90 ( Figure 13 ).

[0091] After loading DSF, the morphology changes little. Further specific surface area measurement (BET) shows that Cu / ZIF-90 is a mesoporous material with a pore size of about 3.3 nm ( Figure 6 ). At the same time, in the ultraviolet-visible spectrophotometer (UV-Vis) analysis, Cu / ZIF-90@DSF also shows the characteristic absorption peak of DSF ( Figure 7 ).

[0092] Experimental Example 1: In vitro anti-tumor experiment

[0093] I. CCK8 experiment

[0094] 1. Select the CCK 8 reagent to detect the toxic effects of different drug treatments on A549 and HBE cells. The CCK 8 reagent can be reduced by dehydrogenases in cells to a highly water-soluble yellow product, and the color depth is proportional to cell proliferation. Therefore, this property can be used to directly perform cell proliferation and toxicity analysis. The specific experimental operations are as follows:

[0095] (1) Seeding: Inoculate A549 cells and HBE cells in the logarithmic growth phase with normal morphology at a density of 10,000 cells / well in a 96-well plate. Subsequently, place the 96-well plate in a cell incubator at 37 °C containing 5% CO 2 to culture overnight to ensure that the cells fully adhere to the wall and recover their vitality under suitable conditions.

[0096] (2) Drug administration: After overnight culture, observe that the cells in the cell plate grow well, are evenly distributed, and have normal morphology. Subsequently, add complete media with different concentration gradients of ZIF-90, Cu / ZIF-90, and the same concentration of ZIF-90, Cu / ZIF-90, DSF, and Cu / ZIF-90@DSF to the cells, with 5 replicates in each group. Continue to culture the treated cells in the incubator for 24 hours to ensure that each drug is fully taken up and acts on the cells.

[0097] (3) Detection: After incubation, gently aspirate the old culture medium, and add 100 μL of complete DMEM culture medium containing 10 μL of CCK-8 solution to each well. Place the 96-well plate in a cell incubator at 37 °C with 5% CO 2 for 60 - 90 minutes to promote the reaction of the CCK-8 reagent. Finally, use a microplate reader to record the absorbance value of each well at a wavelength of 450 nm, and calculate the cell viability based on the absorbance to evaluate the effect of different treatments on the cell survival status.

[0098] The results are as Figure 14 shown. It can be seen that: DSF alone and Cu / ZIF-90 alone have no obvious toxicity, while Cu / ZIF-90@DSF has a significant killing effect.

[0099] 2. Use a laser confocal fluorescence microscope to observe the live and dead states of A549 cells after different drug treatments. The specific operations are as follows:

[0100] (1) Seeding: Select A549 cells in the logarithmic growth phase with normal morphology, digest the cells and count them to ensure good cell viability. Then, inoculate the cells into a confocal dish at a density of 200,000 cells / well. After inoculation, place the confocal dish in a cell incubator at 37 °C with 5% CO 2 overnight to ensure that the cells adhere well and recover their viability. After overnight incubation, check the cell growth to ensure that the cells are evenly distributed and have normal morphology, and prepare for the subsequent live-dead staining experiment.

[0101] (2) Drug administration: After overnight culture, observe that the cells grow well, are evenly distributed and have normal morphology. Next, divide them into Control, ZIF-90, Cu / ZIF-90, DSF, and Cu / ZIF-90@DSF groups. Add the drug treatment solutions of each group to the cell wells and co-incubate them in the cell incubator for 24 h.

[0102] (3) Rinsing: Remove the old culture medium and wash it 3 times with PBS.

[0103] (4) Staining: Prepare the CalceinAM / PI working solution according to the ratio of CalceinAM:PI:detection buffer of 1:1:1000. Continue to incubate it in the dark for 30 min with 1 mL of the CalceinAM / PI working solution.

[0104] (5) Observation: Use a laser confocal microscope to observe the live and dead states of A549 cells after different drug treatments.

[0105] The results are as Figure 15As shown, it can be seen that Cu / ZIF-90 and DSF alone only show weak cytotoxicity, while Cu / ZIF-90@DSF has obvious killing effects on A549 cells.

[0106] 3. Immunofluorescence staining experiment

[0107] Immunogenic cell death induced by nanocomposites is closely related to the release of specific intracellular molecules, mainly including cell surface-exposed CRT, HMGB1, and ATP. These molecules are released extracellularly during cell death and, as important immune signal molecules, significantly enhance the immunogenicity of tumor cells. First, the external exposure of CRT is one of the hallmarks of immunogenic cell death, which can be recognized by the immune system and promote the activation of dendritic cells and the response of tumor-specific T cells. HMGB1, on the other hand, acts as a potent immune stimulatory factor that can induce the activation of immune cells by binding to receptors, thereby enhancing the anti-tumor immune response. In addition, the release of ATP is not only an indicator of cellular energy metabolism but also can activate the immune system as "damage-associated molecular patterns" (DAMPs), promoting the infiltration and activation of immune cells.

[0108] To verify the mechanism of immunogenic cell death induced by the nanocomposite, we conducted relevant experiments. By immunofluorescence staining technology, the release of CRT and HMGB1 was detected. The results showed that the fluorescence intensity of the treatment group increased significantly, indicating that the release amounts of these two molecules were significantly higher than those of the control group ( Figure 8 ).

[0109] In addition, we used an ATP detection kit to detect the remaining ATP in cells. The results showed that the remaining ATP amount in the treatment group was the lowest ( Figure 16 ), proving that the released ATP amount in cells was the highest.

[0110] Experimental Example 3. In vivo experiment

[0111] 1. To further verify the therapeutic effect of Cu / ZIF-90@DSF in animals, we constructed an A549 tumor-bearing nude mouse model and adopted a dosing regimen of once every other day ( Figure 9 ). During the whole experiment, the body weight of the nude mice did not show obvious changes and their mental state was good ( Figure 9 ).

[0112] After the treatment ended, the tumor volume of the Control group increased significantly, being 8 times that of the Cu / ZIF-90@DSF group, while the best tumor inhibition rate of the Cu / ZIF-90@DSF group was 49.25%

[0113] 2. Nude mice lack T cells and are suitable for studying the growth of human tumor cells, but they cannot evaluate the immune system's response. In contrast, C57BL / 6J mice have a complete immune system and are suitable for studying changes in the immune microenvironment. Therefore, we constructed a Lewis tumor-bearing C57BL / 6J mouse model, and the treatment steps are as Figure 10 shown in A. During the entire treatment process, the body weight of the mice did not show significant changes (see Figure 10 B), and they had good appetites and smooth, shiny fur. The tumors in the Control group grew significantly, while the tumor growth in the Cu / ZIF-90@DSF group was significantly inhibited, with an inhibition rate of 54.89%. The efficacy of the group treated with DSF alone was poor ( Figure 10 B-D), indicating that when DSF exerts its anti-tumor effect, it needs to bind with excessive copper to enhance its cytotoxicity and pro-apoptotic effect. The combination of Cu / ZIF-90@DSF may effectively inhibit tumor growth by promoting oxidative stress and interfering with the metabolic pathways of tumor cells.

[0114] 3. cDC cells are an important subtype of dendritic cells and play a key role in initiating and regulating adaptive immune responses, especially in effectively activating CD8+ T cells and CD4+ T cells. With the death of immunogenic cells, the released antigens promote the activation and proliferation of cDC cells. The data processed by FlowJo software show that the gating steps of cDC cells are as Figure 11 shown in A. In the tumor microenvironment treated with Cu / ZIF-90@DSF, the proportion of cDC cells was approximately 4 times that of the control group ( Figure 11 ). This result indicates that the treatment with Cu / ZIF-90@DSF significantly increased the number of cDC cells in the tumor microenvironment, thereby enhancing the antigen presentation ability.

[0115] Subsequently, we detected the proportions of CD4+ T cells and CD8+ T cells in the tumor tissue. The results are as Figure 11 shown. There were significant differences in the proportions of CD4+ T cells and CD8+ T cells in the Cu / ZIF-90@DSF group compared with the control group, and both increased. This change indicates that the increase in cDC cells may promote the activation and proliferation of T cells, thereby enhancing the anti-tumor immune response. Specifically, the increase in CD4+ T cells helps assist the functions of other immune cells, while the increase in CD8+ T cells directly participates in the clearance of tumor cells, further supporting the potential application of Cu / ZIF-90@DSF in tumor immunotherapy.

[0116] Experimental Example 4. Combined treatment group (Cu / ZIF-90@DSF-30 + αPD-1)

[0117] Treatment regimen: Intravenous injection (5 mg / kg) twice a week.

[0118] Synergistic effect: The infiltration of CD8+ T cells in the tumor microenvironment increased by 5.8 times.

[0119] Survival rate improvement: The median survival time was extended to 28 days (vs 19 days in the single-agent group). The treatment regimen is as Figure 17 shown. The final tumor volumes treated with PBS, αPD-1, and αPD-1+Cu / ZIF-90@DSF groups were 540.2 mm 3 , 270.2 mm 3 , and 14.8 mm 3 , respectively. This synergistic treatment effect is mainly attributed to the improvement of the tumor microenvironment by Cu / ZIF-90@DSF and the upregulation of PD-L1 expression on the surface of tumor cells, thereby enhancing the therapeutic effect of immune checkpoint inhibitors.

[0120] Finally, it should also be noted that the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0121] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0122] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A nano-delivery system based on copper death induction, characterized in that: The nano delivery system comprises the following components: Metal-organic framework ZIF-90 with surface modified by polyvinylpyrrolidone (PVP); and copper acetylpropionate (Cu(acac)2) and disulfiram (DSF) loaded in the pores of the metal organic framework ZIF-90; The total copper ion loading accounts for 15-45 mol% of the mass of the metal organic framework ZIF-90.

2. The nano delivery system according to claim 1, characterized in that The mass percentage of the polyvinyl pyrrolidone (PVP) and the metal organic framework ZIF-90 is 5-15 wt.%.

3. The nano delivery system according to claim 1, characterized in that The molar ratio of disulfiram (DSF) to copper acetylpropionate (Cu(acac)2) is 1:2-1:

4.

4. The nano delivery system according to claim 1, characterized in that The particle size of the nano-delivery system based on copper death induction is controlled at 15-25 nm, and the Zeta potential is -25 to -35 mV; the copper ion release rate reaches more than 75% in a 24-hour environment at pH 5.

5.

5. A method for preparing the nano delivery system according to any one of claims 1 to 4, characterized in that: The method comprises: Cu / ZIF-90 was prepared by a microwave-assisted one-pot synthesis of copper acetylpropionate (Cu(acac)2), PVP, 2-methylimidaldehyde (2-ICA) and a zinc source. The Cu / ZIF-90 and disulfiram (DSF) are mixed and stirred, and the nano delivery system is obtained after purification, referred to as Cu / ZIF-90@DSF.

6. The preparation method according to claim 5, characterized in that: The method specifically comprises: The mass ratio of the Cu / ZIF-90 to disulfiram (DSF) is 1:

2.

7. The preparation method according to claim 6, characterized in that: The microwave reaction power in the microwave-assisted one-pot synthesis is 800 W and the duration is ≤ 3 minutes.

8. The preparation method according to claim 6, characterized in that: The molar ratio of acetylpropionyl copper (Cu(acac)2), PVP, 2-methylimidaldehyde (2-ICA) and zinc source is 1:0.025:

8.

9. Use of the nano delivery system according to any one of claims 1 to 4 in the preparation of tumor immunotherapy drugs.

10. Use of a combined pharmaceutical composition consisting of the nano delivery system according to any one of claims 1 to 4 and αPD-1 antibody in the preparation of tumor immunotherapy drugs.

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