A porous copper-manganese bimetallic nanomaterial applied to tumor immunotherapy and a preparation method thereof

Abstract

The application provides a porous copper-manganese bimetallic nanomaterial applied to tumor immunotherapy and a preparation method thereof. The application obtains the hollow mesoporous copper-manganese bimetallic nanomaterial with uniform particle size through a cuprous oxide self-template etching method, then the nanomaterial is used to load a photosensitizer IR820 due to excellent drug loading capacity, and finally the nanomaterial is modified by mercapto polyethylene glycol to increase the dispersibility of the nanoparticles. The preparation method is stable and reliable, low in cost and wide in source, and has the advantages of simple process, practicality, strong controllability and the like. The nanomaterial has multifunctionality, can realize synergistic treatment of photothermal treatment / photodynamic treatment / chemical dynamic treatment, improve cell hypoxia, consume glutathione, remodel a tumor microenvironment, finally induce immunogenic death of tumor cells, enhance the immune response of tumor cells, efficiently inhibit tumor growth and metastasis, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine technology, specifically relating to a porous copper-manganese bimetallic nanovaccine for the immunotherapy of tumors, especially breast cancer, and its preparation method. Background Technology

[0002] Triple-negative breast cancer (TNBC) is one of the most refractory subtypes of breast cancer with poor prognosis and short survival due to its aggressiveness, high recurrence rate, and tendency to metastasize. Immunotherapy is a promising strategy for treating TNBC. However, the clinical application of immunotherapy still faces the challenge of low immune response activation. Weak immunogenicity during tumor development is a major reason for this low immune response activation. Therefore, enhancing the immunogenicity of tumor cells and specifically stimulating the immune response at the tumor site is of great significance. Immunogenic cell death (ICD) can lead to the release of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) to increase immunogenicity, and has been proven to be an effective way to enhance the anti-tumor immune response.

[0003] Non-invasive tumor treatments such as photothermal therapy (PTT), photodynamic therapy (PDT), and chemodynamic therapy (CDT) can not only effectively kill tumor cells and prevent them from developing drug resistance, but also induce immunogenicity (ICD) in cancer cells, showing broad application prospects in ICD immunogenicity research. Therefore, nanoreagents with synergistic therapeutic effects of CDT / PDT / PTT would be ideal therapeutic agents and immune response activators.

[0004] Manganese (Mn)-based materials have attracted significant attention in the biomedical field due to their low cost and low biotoxicity. Studies have shown that the tumor microenvironment exhibits a series of abnormal characteristics, including hypoxia, low pH, excessive H₂O₂, and high concentrations of glutathione (GSH), offering potential avenues for tumor diagnosis and novel treatment strategies. High-valence manganese can be reduced by GSH, producing large amounts of Mn. 2+ Used in T1-weighted magnetic resonance imaging, Mn-based materials can generate O2 and hydroxyl radicals (•OH) in the presence of H2O2. O2 can effectively alleviate tumor hypoxia, but the accumulation of ROS and depletion of GSH cause oxidative stress damage to tumor cells, inducing tumor cell apoptosis. Therefore, Mn-based materials can be used for both cancer treatment and disease diagnosis; at the same time, hollow mesoporous manganese-based materials can also efficiently load various drugs, and are expected to serve as high-performance anti-tumor drug carriers, exerting a synergistic therapeutic effect.

[0005] Current methods for preparing Mn-based materials involve complex synthesis steps, high costs, and are unsuitable for mass production. Furthermore, the synthesized products are prone to agglomeration, irregular morphology and size, and poor stability. For example, using silica as a hard template to etch hollow mesoporous MnO2 has successfully produced hollow mesoporous MnO2 with controllable morphology, but the preparation process is cumbersome, costly, and requires stringent experimental conditions, greatly limiting its application. Therefore, there is a need to find a more economical and efficient method for preparing hollow mesoporous Mn-based nanoparticles. Based on the above problems, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a porous copper-manganese bimetallic nanomaterial with multiple therapeutic functions including photothermal therapy, photodynamic therapy, and chemodynamic therapy, as well as its preparation method and its use in inducing ICD activation of tumor cells for tumor immunotherapy.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy includes the following steps:

[0009] (1) Preparation of cuprous oxide template:

[0010] (2) Preparation of copper-manganese bimetallic nanoparticles (CuMnO) x ):

[0011] Under stirring conditions at 25-30℃, a cuprous oxide aqueous dispersion was mixed with a potassium permanganate aqueous solution and dispersed evenly. Dopamine was then added, and the reaction was continued with stirring for 4-6 hours (final solution volume 15-25 mL). After washing and vacuum drying, brownish-red copper-manganese bimetallic nanoparticles (CuMnO) were obtained. x );

[0012] (3) Preparation of CMI nanoparticles: The copper-manganese bimetallic nanoparticles obtained in step (2) were uniformly dispersed in water, and new indocyanine green (IR820) was added. The mixture was stirred at 25-30℃ in the dark for 10-14 h. After the reaction was completed, the precipitate was collected, washed with water until the supernatant was colorless, and dried under vacuum to obtain CMI nanoparticles (CuMnO). x -IR820);

[0013] (4) Preparation of CMI-PEG: The CMI nanoparticles obtained in step (3) are uniformly dispersed in water, and mercapto polyethylene glycol is added. The mixture is stirred at 25-30℃ in the dark for 6-12 h. After the reaction is complete, the precipitate is collected, washed, and vacuum dried to obtain porous copper-manganese bimetallic nanomaterial CuMnO. x-IR820-PEG (CMI-PEG).

[0014] Specifically, in step (2), the mass ratio of cuprous oxide, potassium permanganate, and dopamine is 1:2~4:0.25~0.5.

[0015] Specifically, in step (3), the mass ratio of the copper-manganese bimetallic nanoparticles to IR820 is 1:0.5~3.

[0016] Furthermore, in step (4), the mass ratio of the CMI nanoparticles to mercapto polyethylene glycol is 1:0.5~2.

[0017] Furthermore, step (1), the preparation of the cuprous oxide template, specifically involves:

[0018] At 25℃-30℃, CuCl2 solution was added to polyvinylpyrrolidone (PVP) solution and stirred for 5-10 min. Then, sodium hydroxide solution was slowly added and stirred for 5-10 min. Ascorbic acid solution was then slowly added and reacted for 1-1.5 h. After the reaction was completed, the precipitate was collected, washed, and vacuum dried to obtain yellow cubic cuprous oxide powder (Cu2O).

[0019] More preferably, the concentration of the polyvinylpyrrolidone solution is 1.5-2 mol / L. -1 The final volume of the reaction system is 50-60 mL. The concentration of the CuCl2 solution is 0.016-0.018 mol / L. -1 The amount added is 1-2 mL; the concentration of the sodium hydroxide solution is 1-2 mol / L. - 1 The amount added is 2-3 mL; the concentration of the ascorbic acid solution is 0.5-0.7 mol / L. -1 The amount added is 2-3 mL.

[0020] This invention provides porous copper-manganese bimetallic nanomaterials prepared using the above-described method for use in tumor immunotherapy. The porous copper-manganese bimetallic nanomaterials comprise hollow mesoporous copper-manganese bimetallic nanoparticles, a photosensitizer IR820, and a mercapto-polyethylene glycol (SH-mPEG) surface modifier, wherein the loading of IR820 is 19%–38%. The maximum radial size of the porous copper-manganese bimetallic nanomaterial particles is 130.5–183.7 nm.

[0021] This invention also provides the application of the above-mentioned porous copper-manganese bimetallic nanomaterials in the preparation of tumor immunotherapy drugs. More preferably, the porous copper-manganese bimetallic nanomaterials are used in the preparation of breast cancer immunotherapy drugs. The copper-manganese bimetallic nanomaterials prepared by the above method are also used in photothermal-photodynamic-chemodynamic therapy combined with immunotherapy.

[0022] This invention obtains hollow mesoporous copper-manganese bimetallic nanocarriers (CuMnO) using the cuprous oxide self-sacrificial template method. x The nanomaterial is loaded with the photosensitizer IR820 and then modified with mercapto-polyethylene glycol to increase its in vivo circulation capacity. This nanomaterial can not only kill tumor cells through a combination of photothermal therapy, chemodynamic therapy, and photodynamic therapy, but also enhance the immunogenicity of tumor cells, significantly activate the body's anti-tumor immunity, and effectively inhibit tumor growth, thus achieving the goal of treating and preventing cancer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) This invention utilizes the cuprous oxide self-sacrificial template method to obtain hollow mesoporous copper-manganese bimetallic nanomaterials. The method is stable and reliable, the process is simple and practical, the raw materials are inexpensive and widely available, and the controllability is strong.

[0025] (2) The hollow mesoporous copper-manganese bimetallic nanomaterials prepared in this invention have a high specific surface area and large pore volume, uniform particle size, and good dispersibility, which is conducive to efficient drug loading and is very suitable as a drug carrier. At the same time, it can passively target the tumor site through the high permeability and retention effect (EPR effect) of solid tumors or actively target the tumor site with surface-modified targeting agents, thereby increasing the accumulation at the tumor site and reducing the toxic side effects on normal tissues. The material has high biocompatibility, is suitable for in vivo application, and has great potential for translational applications.

[0026] (3) The hollow mesoporous copper-manganese bimetallic nanomaterial prepared by the present invention has multiple therapeutic functions of photothermal therapy / photodynamic therapy / chemodynamic therapy, which is conducive to achieving synergistic treatment and achieving the effect of "1+1>2". At the same time, the nanomaterial can relieve tumor hypoxia, consume GSH, and cause oxidative stress damage to tumor cells, which is conducive to further enhancing the therapeutic effect.

[0027] (4) The hollow mesoporous copper-manganese bimetallic nanomaterials prepared in this invention can not only kill tumor cells quickly, but also induce immunogenic cell death, reverse the immunosuppressive state of the tumor microenvironment, activate the immune response, and significantly improve the level of cellular immunity and humoral immunity. It has a long-term regulatory effect on the elimination of residual tumor cells and the inhibition of tumor recurrence and metastasis. It has unique advantages for treating tumors that are located deep in the body and are prone to recurrence and metastasis. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation route of the hollow mesoporous copper-manganese bimetallic nanomaterial of the present invention;

[0029] Figure 2Cu₂O(A) and CuMnO in Example 1 of this invention x (C) Scanning electron microscope image of Cu2O (B) and CuMnO x Transmission electron microscopy image of (D), E is CuMnO x Energy spectral analysis mapping diagram;

[0030] Figure 3 Cu₂O and CuMnO in Example 1 of this invention x XRD pattern;

[0031] Figure 4 The IR820 and CuMnO used in Embodiment 1 of this invention x UV-Vis absorption spectra of CMI-PEG;

[0032] Figure 5 CuMnO in Example 1 of this invention x Analysis results of catalytic H2O2 production of •OH;

[0033] Figure 6 CuMnO in Example 1 of this invention x Analysis results of catalytic H2O2 oxygen production;

[0034] Figure 7 CuMnO in Example 1 of this invention x Results of in vitro GSH clearance analysis;

[0035] Figure 8 The image shows the photothermal properties of CMI-PEG in Example 1 of this invention; where A represents the photothermal properties of CMI-PEG solutions of different concentrations at a power density of 1.0 W / cm². -2 Temperature change curve under 808 nm laser irradiation, B is CMI-PEG solution (200 μg mL) -1 Temperature change curves at different laser power densities, where C represents the temperature change curve of the CMI-PEG solution after five cycles of laser on / off irradiation;

[0036] Figure 9 A statistical graph showing the relative survival rate of 4T1 cells after different treatments;

[0037] Figure 10 The expression of CRT in 4T1 cells after different treatments;

[0038] Figure 11 HMGB1 expression in 4T1 cells after different treatments;

[0039] Figure 12 The graph shows the changes in ATP content in 4T1 cells after different treatments.

[0040] Figure 13 This is a graph showing the change in relative tumor volume in 4T1 tumor-bearing mice during the end of treatment.

[0041] Figure 14 Images of tumors dissected after treatment in 4T1 tumor-bearing mice;

[0042] Figure 15 HE staining images of heart, liver, spleen, lung, and kidney tissue sections after dissection of 4T1 tumor-bearing mice after treatment completion;

[0043] Figure 16 The results of routine blood tests and blood biochemistry tests of 4T1 tumor-bearing mice were obtained after the treatment was completed. Detailed Implementation

[0044] To further understand the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention and are not intended to limit the scope of protection of the present invention.

[0045] In the following examples, all raw materials used are common commercially available products that can be purchased directly or can be prepared using conventional methods in the art.

[0046] Example 1

[0047] This embodiment provides a porous copper-manganese bimetallic nanomaterial for breast cancer immunotherapy, and its preparation method is as follows:

[0048] (1) Preparation of cubic cuprous oxide (Cu2O):

[0049] At 25℃, 50 mL of a 2 mol L solution was added. -1 Add 1 mL of 0.017 mol / L PVP solution -1 After stirring the CuCl2 solution for 8 minutes, slowly add 2.5 mL of 2 mol / L CuCl2 solution dropwise. -1 Add 2.5 mL of a 0.6 mol / L sodium hydroxide solution, stir for 8 minutes, and then slowly add dropwise. -1 The ascorbic acid solution was reacted for 1 h. After the reaction was completed, the precipitate was collected by centrifugation, washed three times each with water and ethanol, and dried under vacuum at 40 °C to obtain a yellow powder of cuprous oxide (Cu2O).

[0050] (2) Preparation of copper-manganese bimetallic nanoparticles (CuMnO) x ):

[0051] 10 mg of potassium permanganate was dissolved in 19 mL of water to obtain an aqueous solution of potassium permanganate; 4 mg of the above-mentioned cuprous oxide was uniformly dispersed in 1 mL of water to obtain an aqueous dispersion of cuprous oxide. The two solutions were mixed under stirring at 25 °C and ultrasonically dispersed for 10 min. Then, 1.5 mg of dopamine was added, and the mixture was ultrasonically dispersed for 10 min. The reaction was continued under stirring at 25 °C for 5 h. After the reaction was complete, the product was centrifuged at 16000 rpm, the precipitate was washed three times with ultrapure water, and dried under vacuum at 40 °C to obtain brownish-brown copper-manganese bimetallic nanoparticles (CuMnO₂). x ).

[0052] (3) CMI preparation:

[0053] 2 mg of copper-manganese bimetallic nanoparticles were ultrasonically dispersed in 2 mL of water, and 4 mg of IR820 was added. The mixture was ultrasonically mixed and stirred at 25 °C in the dark for 12 h. After the reaction was complete, the product was centrifuged at 16,000 rpm, and the precipitate was washed with water until the supernatant was colorless (approximately 4 washes were required). The product was then vacuum dried at 40 °C to obtain the CMI nanoparticles.

[0054] (4) Preparation of CMI-PEG:

[0055] 2 mg of CMI nanoparticles were ultrasonically dispersed in 2 mL of water, and 2 mg of mercapto-polyethylene glycol was added. The mixture was ultrasonically mixed and stirred at 25 °C in the dark for 6 h. After the reaction was complete, the product was centrifuged at 16000 rpm, the precipitate was washed three times with water, and dried under vacuum at 40 °C to obtain the porous copper-manganese bimetallic nanomaterial CuMnO. x -IR820-PEG (CMI-PEG).

[0056] Detection and characterization

[0057] The copper-manganese bimetallic nanoparticles (CuMnO) prepared in Example 1 above x The basic properties of CMI-PEG were characterized.

[0058] (1) Performance test experiment 1: Copper-manganese bimetallic nanoparticles (CuMnO) x Morphological analysis

[0059] The Cu₂O and CuMnO prepared in Example 1 were analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). x Characterization was performed, and the scanning electron microscope (SEM) and transmission electron microscope (TEM) images of Cu₂O are shown below. Figure 2 As shown in Figures A and 2B, the synthesized Cu₂O nanoparticles exhibit a cubic morphology with an average size of 133.3 nm. CuMnO x The scanning electron microscope and transmission electron microscope images are as follows: Figure 2 As shown in C and 2D, it can be seen that CuMnO x It exhibits a distinct hollow nanostructure, is spherical in shape, and has an average size of 163.6 nm. The CuMnO4 prepared was characterized by energy dispersive spectroscopy (EDS) mapping. x The elemental composition of nanoparticles, the results are as follows: Figure 2 E (scale bar is 100 nm) shows the co-location of C, N, O, Cu, and Mn elements. This indicates that CuMnO x It was successfully prepared.

[0060] (2) Performance test experiment 2: Copper-manganese bimetallic nanoparticles (CuMnO) x XRD analysis of )

[0061] X-ray diffraction was used to analyze Cu₂O and CuMnO prepared in Example 1. x Characterization was performed, and the XRD pattern of Cu2O is shown below. Figure 3 As shown, the diffraction pattern of the synthesized Cu₂O is at 2θ = 29.6. o 36.4 o 42.3 o 61.3 o The presence of distinct characteristic peaks at various locations, consistent with the peak shapes of the standard spectrum of cubic Cu₂O (PDF#05-0667), indicates the successful synthesis of Cu₂O. CuMnO x XRD pattern as shown Figure 3 As shown, at 2θ = 18.1 o At the position, there is a distinct diffraction peak, which corresponds to the diffraction peak of the tetragonal MnO2 (200) plane and is consistent with the standard spectrum of MnO2 (PDF#44-0141), indicating that Cu2O is converted into copper manganese oxide.

[0062] (3) Performance test experiment 3: IR820, CuMnO x Absorption spectroscopy test of CMI-PEG

[0063] The nanomedicine delivery system was scanned across the entire wavelength range using a UV-Vis spectrophotometer. Figure 4 As can be seen in: CuMnO x The absorption in the near-infrared region is very weak. After loading IR820, CMI-PEG showed the same peak at 808 nm as IR820, which proved the successful loading of IR820 and provided the basic conditions for subsequent photothermal therapy.

[0064] (4) Performance test experiment 4: Copper-manganese bimetallic nanoparticles (CuMnO) x The ability to produce •OH

[0065] The chemokinetic efficiency, i.e., Fenton-like catalytic activity, of CMI-PEG was evaluated using methylene blue (MB). The catalytic activity was divided into five groups: MB (10 μg / mL). -1 3 mL), MB+CuMnO x (MB: 10 μg mL) -1 CuMnO x 100 μg mL -1 ; 3 mL), MB+CuMnO x +H2O2 (MB: 10 μg mL) -1 CuMnO x 100 μg mL -1 ; H2O2: 5 mM; 3 mL), MB+CuMnO x +GSH+H2O2 (MB: 10 μg mL) -1 CuMnO x 100 μg mL -1 ; GSH: 3 mM; H2O2: 5 mM; 3 mL), MB+CuMnO x +GSH+H2O2+HCO3 - (MB: 10 μg mL) -1 CuMnO x 100 μg mL -1 (GSH: 3 Mm; H2O2: 5 mM; NaHCO3: 20 mM; 3 mL) were incubated at room temperature for 15 min, and the absorbance of each solution at λ=664 nm was measured using a spectrophotometer. The results are shown in the figure. Figure 5 .

[0066] like Figure 5 As shown, in CuMnO x When present alone, the absorbance of MB at 664 nm remains almost unchanged (i.e., the absorbance of the MB group and the MB+CuMnOx group almost overlaps), while in CuMnO... x The absorbance of MB decreased slightly under the combined action of MB and H2O2. However, with the addition of GSH, the absorbance of MB decreased further. Especially in the case of MB + CuMnO2... x +GSH+H2O2+HCO3 - In the group, the absorbance of MB was observed to be lower than that without HCO3. - The group is low. This is because CuMnO is in the presence of GSH. x It reacts with GSH to release Mn 2+ and Cu + In H2O2 and HCO3- Under certain conditions, Cu can be stimulated simultaneously. + and Mn 2+ The Fenton-like reaction is mediated. Therefore, due to the bimetallic chemodynamic interaction of manganese and copper ions, CuMnO... x It can efficiently catalyze the production of •OH from H2O2.

[0067] (5) Performance test experiment 5: Copper-manganese bimetallic nanoparticles (CuMnO) x oxygen production capacity

[0068] CuMnO of different concentrations x Mixed with H2O2 solution (CuMnO) x 0 μg mL -1 25 μg mL -1 50 μg mL -1 100 μg mL -1 (H2O2: 5 mM; 3 mL) The dissolved oxygen concentration was measured in real time every 30 seconds using a portable dissolved oxygen meter. The results are shown in […]. Figure 6 .

[0069] like Figure 6 As shown, CuMnO x It can efficiently catalyze the production of oxygen from hydrogen peroxide; the higher the concentration, the faster the oxygen production rate. Therefore, this CuMnO... x It is expected to respond to oxygen production in the tumor microenvironment and improve the hypoxic environment of the tumor.

[0070] (6) Performance test experiment 6: Copper-manganese bimetallic nanoparticles (CuMnO) x GSH clearance capacity in vitro

[0071] The content of glutathione (GSH) in solution was determined using 5,5'-dithio-(2-nitrobenzoic acid) (DTNB). Different concentrations of CuMnO4 were used... x Mixed with GSH solution (CuMnO) x 0 μg mL -1 10 μg mL -1 50 μg mL -1 100 μg mL -1 GSH: 3 mM; 2 mL), centrifuge for 30 min, collect the supernatant, add DTNB solution (5,5'-dithiobis(2-nitrobenzoic acid), 1 mg / mL) -1 (40 μL), its absorption at 412 nm was observed using a UV-Vis spectrophotometer. The results are shown in [Figure number missing]. Figure 7 .

[0072] The results are as follows Figure 7 As shown, CuMnO at different concentrations x After 30 minutes of addition, the absorbance at 412 nm decreased significantly. The decrease in DTNB absorbance indicates the consumption of GSH. These results demonstrate that CuMnO x It is effective in scavenging GSH; even low concentrations can efficiently remove GSH. Therefore, CuMnO x It is expected that consuming GSH can increase the production of intracellular ROS.

[0073] (7) Performance test experiment 7: Photothermal properties of porous copper-manganese bimetallic nanomaterials (CMI-PEG)

[0074] To demonstrate its excellent photothermal performance, CMI-PEG solutions of different concentrations were irradiated with an 808 nm laser to determine its photothermal conversion capability. The temperature change of CMI-PEG under different power densities was also detected.

[0075] The results are as follows Figure 8 As shown, different concentrations of CMI-PEG aqueous dispersions (0, 25, 50, 100, 200 μg mL) were observed. -1 At a power density of 1.0 W / cm² -2 Temperature change curve after 10 min of 808 nm laser irradiation ( Figure 8 A) and the same concentration of CMI-PEG aqueous dispersion (100 μg mL) -1 ) at different laser power densities (0, 0.75, 1, 1.5, 2 W cm⁻¹) -2 Temperature change curve after 10 minutes of irradiation () Figure 8 B).

[0076] Figure 8 The results showed that CMI-PEG exhibited excellent photothermal conversion capabilities at a material concentration of 100 μg / mL. -1 At that time, in an 808 nm laser (1 W cm⁻¹) -2 Under irradiation for 10 minutes, the temperature of the CMI-PEG aqueous dispersion increased by approximately 38.1℃, and this increase showed a certain concentration dependence. Figure 8 A). Additionally, with increasing laser power density, CMI-PEG (100 μg mL) -1 The increase in temperature rise of the aqueous dispersion indicates its dependence on laser power density. Figure 8 B).

[0077] To demonstrate its photothermal stability, the photothermal stability of the synthesized CMI-PEG was evaluated through a five-cycle laser on / off irradiation experiment (10 min laser irradiation followed by natural cooling). The results are shown in [Figure number missing]. Figure 8 C. Figure 8 C shows that the photothermal stability of IR820 is weaker than that of CMI-PEG. This indicates that loading IR820 onto CuMnO... x The photothermal stability of IR820 has been improved to a certain extent.

[0078] Therefore, the excellent photothermal conversion properties of CMI-PEG nanomaterials prove that they can be used for photothermal therapy of tumors.

[0079] Application Experiment 1

[0080] This embodiment verifies the in vitro cytotoxicity and in vitro antitumor effects of the porous copper-manganese bimetallic nanomaterial (CMI-PEG) prepared in Example 1.

[0081] Mouse breast cancer cell line 4T1 was used as the test cell line. 4T1 cells were cultured in a solution containing 10% fetal bovine serum (FBS) and penicillin (100 U / mL). -1 ) and streptomycin (100 μg mL) -1 Cells were incubated in RPMI-1640 medium. All cells were cultured at 37°C and 5% CO2. The same medium was used in the following application experiments.

[0082] To evaluate in vitro cytotoxicity, breast cancer cells 4T1 (5 × 10⁻⁶) were used. 3 Cells per well were seeded into 96-well plates and cultured for 24 h. The cells were then divided into 6 groups: ① Control group; ② NIR (1.0 W / cm²) control group; ③ [Unclear text - possibly related to cell count / well measurement]. -2 ) group; ③ IR820 + 1.0 Wcm -2 Group 4; CMI-PEG group; ⑤ CMI-PEG + 0.3 W cm -2 Group ⑥ CMI-PEG +1 W cm -2 Groups were formed. 100 μL of fresh culture medium containing different materials was added to each group, and the plates were cultured for another 24 h. Groups ②, ③, ⑤, and ⑥ were irradiated with an 808 nm laser for 5 min after 6 h of culture. After incubation, the culture medium containing the materials in the 96-well plates was discarded, and the plates were washed three times with PBS buffer (pH 7.4). Then, 50 μL of a 1 mg / mL solution was added to each well of the 96-well plate. -1MTT solution was added. After incubation in an incubator for 4 h, the liquid in the plate was discarded, and 100 μL of dimethyl sulfoxide was added. Finally, the plate was placed in a 37°C oven for 10 min, and then the absorbance at 490 nm was measured using a microplate reader to calculate the cell viability. The results are shown in the figure. Figure 9 .

[0083] The results are as follows Figure 9 As shown, the CMI-PEG group (100 μg mL) -1 The concentration of IR820 was 20 μg mL. -1 1.0 W cm -2 The group exhibited the most significant inhibitory effect, with a cell inhibition rate of over 90%, confirming that multifunctional synergistic therapy can effectively kill tumor cells.

[0084] Application Experiment 2

[0085] This embodiment verifies the ICD effect of the porous copper-manganese bimetallic nanomaterial (CMI-PEG) prepared in Example 1.

[0086] The ICD efficacy of CMI-PEG was detected using immunofluorescence staining and a detection kit.

[0087] (1) Seed 4T1 cells in a light-focusing dish (1 × 10⁻⁶ cells / mL). 4 Cells per well were divided into 6 groups: ① Control group; ② NIR (1.0 W cm⁻¹) -2 Group ); ③ IR820 + 1.0 W cm -2 Group (IR820: 20 μg mL) -1 ); ④ CMI-PEG group (CMI-PEG: 100 μg mL -1 IR820: 20 μg mL -1 );⑤ CMI-PEG + 0.3 W cm -2 Group (CMI-PEG: 100 μg mL) -1 IR820: 20 μg mL -1 );⑥ CMI-PEG + 1 W cm -2 Group (CMI-PEG: 100 μg mL) -1 IR820: 20 μg mL -1After 24 h of incubation, the culture medium was removed, and the cells were cultured for another 6 h in fresh culture medium containing different materials according to the pre-defined groups. Groups ②, ③, ⑤, and ⑥ were irradiated with an 808 nm laser for 5 min at 4 h and cultured for another 2 h. The culture medium was then removed, and the cells were washed with PBS, fixed with cell fixation solution (4% paraformaldehyde), and incubated with PBS containing 0.1% Triton X-100 for 20 min. After washing twice with PBS, the cells were blocked with PBS solution containing 5% BSA for 45 min. Primary antibodies against cell surface calreticulin (CRT) and nuclear high-mobility group box 1 (HMGB1) were diluted 1:100 with PBA solution containing 0.5% BSA and incubated overnight at 4°C. The liquid was discarded, and secondary antibody against goat anti-rabbit IgG (H+L) (green) (Elabscience) was added and incubated for 1 h. The cell nuclei were stained with DAPI. The expression of CRT and HMGB1 in 4T1 cells was observed using a laser confocal microscope after treatment. The results are shown in [Figure 1]. Figure 10 and Figure 11 .

[0088] CRT test results are as follows Figure 10 As shown, compared with the control group, no significant changes were observed in group ② cells, while groups ③, ④, ⑤, and ⑥ cells showed obvious green fluorescence, with group ⑥ showing the strongest fluorescence. This indicates that under the synergistic effect of CMI-PEG-induced PTT / PDT / CDT, CRT will translocate and be exposed on the cell membrane surface, resulting in a significant enhancement of CRT expression on the cell membrane.

[0089] HMGB1 test results are as follows Figure 11 As shown, contrary to the trend of CRT fluorescence changes, the HMGB1 fluorescence signal in groups ③④⑤⑥ was significantly weakened compared with the control group, with group ⑥ showing the weakest fluorescence. This indicates that under the synergistic effect of CMI-PEG-induced PTT / PDT / CDT, HMGB1 can be effectively triggered to be released from the cell nucleus.

[0090] (2) To detect ATP release, cells from different treatment groups were collected, and the intracellular ATP content was measured according to the operating procedures of the ATP assay kit. The ATP assay kit was used to detect the intracellular ATP content, and the results are shown in [Figure 1]. Figure 12 .

[0091] The results are as follows Figure 12 As shown, compared with the control group, the intracellular ATP levels in groups ③④⑤⑥ were significantly decreased, with group ⑥ (CMI-PEG +1 W cm⁻¹) showing the highest decrease. -2 The group treated with this method had the lowest intracellular ATP content. This indicates that CMI-PEG-induced PTT / PDT / CDT synergistic therapy can effectively induce apoptosis, thereby inducing the release of intracellular ATP into the extracellular space.

[0092] In summary, the multifunctional synergistic therapy of CMI-PEG can effectively induce tumor cells to release damage-related molecular patterns, leading to immunogenic cell death of tumor cells and thereby activating the body's immune response.

[0093] Application Experiment 3

[0094] This embodiment verifies the in vivo antitumor effect of the porous copper-manganese bimetallic nanomaterial (CMI-PEG) prepared in Example 1.

[0095] This invention uses 4T1 cells to construct a BALB / c mouse orthotopic tumor model. The specific operation is as follows: BALB / c mice weighing 16-18 g are selected, and the cultured 4T1 cells are prepared with physiological saline to a concentration of 1×10⁻⁶. 6 0.1 mL of each milliliter of cell suspension was inoculated into the fat pad of the second pair of mammary glands on the left side of BALB / c mice. After about 7 days of growth, the tumor volume could reach 100 mm. 3 Around 10:00 AM, the mouse tumor model was successfully constructed.

[0096] When the tumor volume in tumor-bearing mice reaches 100 mm 3 At approximately 10:00 PM, mice were randomly divided into 6 groups (n=4): ① Control group, intravenously injected with 200 μL PBS; ② NIR (1.0 W cm⁻¹) -2 Group 1 received an intravenous injection of 200 μL PBS, followed by an 808 nm laser (1.0 W cm⁻¹) 12 h later. -2 ) irradiation; ③ IR820 + 1.0 W cm -2 Group 1 received 200 μL of IR820 (100 μg / mL) intravenously. -1 ), laser irradiation 12 h after injection; ④ CMI-PEG group, 200 μL of CMI-PEG solution (CMI-PEG: 1 mg / mL) was injected intravenously. -1 ,); ⑤CMI-PEG + 0.3 W cm -2 Group A received an intravenous injection of 200 μL of CMI-PEG solution (CMI-PEG: 1 mg / mL). -1 ), laser irradiation 12 hours after injection; ⑥ CMI-PEG + 1 W cm -2 Group 1 received 200 μL of CMI-PEG (1 mg / mL) intravenously. -1 The mice were injected with laser and then irradiated with laser light 12 hours later. The laser irradiation time was 10 minutes. Tumor volume and body weight were monitored every other day. All mice were euthanized 14 days after treatment. The formula for calculating mouse tumor volume is: V = W. 2 ×L / 2 where: V represents tumor volume, W represents tumor short diameter, and L represents tumor long diameter. Results are shown in […]. Figure 13and Figure 14 .

[0097] like Figure 13 and Figure 14 The results showed that in the CMI-PEG group, CMI-PEG + 0.3 W cm -2 All groups showed some inhibitory effect on tumor growth, CMI-PEG + 1 W cm -2 The group exhibited the strongest tumor-inhibiting effect in mice. This indicates that CMI-PEG nanomaterials, under the combined action of photothermal-photodynamic-chemodynamic therapy and immunotherapy, can significantly inhibit the growth of cancer cells.

[0098] To evaluate the in vivo toxicity of CMI-PEG, after the in vivo tumor suppression experiment, the histological changes of the major organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice were observed using H&E staining. The results are shown in the table below. Figure 15 .

[0099] like Figure 15 As shown, compared with the control group, no significant pathological changes were observed in any organ in the other treatment groups, indicating that the nanomaterials have good in vivo safety.

[0100] Simultaneously, complete blood count and blood biochemistry tests were performed. Complete blood count indicators included white blood cell (WBC), red blood cell (RBC), platelet (PLT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hematocrit (HCT), and hemoglobin (HGB). Liver and kidney function indicators included alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and uric acid (UA). Results are shown below. Figure 16 .

[0101] like Figure 16 As shown, compared with the control group, the blood routine and liver and kidney function indicators of other treatment groups were all within the normal range, indicating that the nanomaterials have good in vivo safety.

[0102] The porous copper-manganese bimetallic nanomaterials for tumor immunotherapy described in this invention are prepared by first obtaining uniformly sized hollow mesoporous copper-manganese bimetallic nanomaterials through a cuprous oxide self-template etching method, then loading the photosensitizer IR820 onto these nanomaterials using their excellent drug-loading capacity, and finally modifying them with mercapto-polyethylene glycol to increase the dispersibility of the nanoparticles. This nanomaterial exhibits multifunctionality, enabling synergistic treatment with photothermal therapy, photodynamic therapy, and chemodynamic therapy. It simultaneously improves cellular hypoxia, depletes glutathione, remodels the tumor microenvironment, ultimately inducing immunogenic death of tumor cells, enhancing the immune response of tumor cells, and effectively inhibiting tumor growth and metastasis, demonstrating broad application prospects.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy, characterized in that, Includes the following steps: (1) Preparation of cuprous oxide; (2) Preparation of copper-manganese bimetallic nanoparticles: Under stirring conditions at 25-30℃, the aqueous dispersion of cuprous oxide was mixed with an aqueous solution of potassium permanganate and dispersed evenly. Then, dopamine was added and the reaction was continued to be stirred for 4-6 hours. After washing and vacuum drying, copper-manganese bimetallic nanoparticles were obtained. (3) Preparation of CMI nanoparticles: The copper-manganese bimetallic nanoparticles obtained in step (2) are uniformly dispersed in water, IR820 is added, and the mixture is stirred at 25-30℃ in the dark for 10-14 h. After the reaction is completed, the precipitate is collected, washed with water until the supernatant is colorless, and dried under vacuum to obtain CMI nanoparticles. (4) Preparation of CMI-PEG: The CMI nanoparticles obtained in step (3) are uniformly dispersed in water, mercapto polyethylene glycol is added, and the mixture is stirred at 25-30℃ in the dark for 6-12 h. After the reaction is completed, the precipitate is collected, washed, and vacuum dried to obtain the final product.

2. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 1, characterized in that, In step (2), the mass ratio of cuprous oxide, potassium permanganate, and dopamine is 1:2~4:0.25~0.

5.

3. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 1, characterized in that, In step (3), the mass ratio of the copper-manganese bimetallic nanoparticles to IR820 is 1:0.5~3.

4. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 1, characterized in that, In step (4), the mass ratio of CMI nanoparticles to mercapto polyethylene glycol is 1:0.5~2.

5. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 1, characterized in that, Step (1) is as follows: At 25℃-30℃, CuCl2 solution was added to polyvinylpyrrolidone solution and stirred. Sodium hydroxide solution was then added and stirred again. Ascorbic acid solution was then added and reacted for 1-1.5 h. After the reaction was completed, the precipitate was collected, washed, and vacuum dried to obtain the final product.

6. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 5, characterized in that, The concentration of the polyvinylpyrrolidone solution is 1.5-2 mol / L. -1 The final volume of the reaction system is 50-60 mL.

7. The method for preparing porous copper-manganese bimetallic nanomaterials for tumor immunotherapy as described in claim 6, characterized in that, The concentration of the CuCl2 solution was 0.016-0.018 mol / L. -1 The amount added is 1-2 mL; the concentration of the sodium hydroxide solution is 1-2 mol / L. - 1 The amount added is 2-3 mL; the concentration of the ascorbic acid solution is 0.5-0.7 mol / L. -1 The amount added is 2-3 mL.

8. Porous copper-manganese bimetallic nanomaterials for tumor immunotherapy prepared by any of the preparation methods described in claims 1 to 7.

9. The use of the porous copper-manganese bimetallic nanomaterial of claim 8 in the preparation of tumor immunotherapy drugs.

10. The use of the porous copper-manganese bimetallic nanomaterial of claim 8 in the preparation of immunotherapy drugs for breast cancer.

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