Copper-manganese alloy drug loading system as well as preparation method and application thereof

Abstract

The invention discloses a copper-manganese alloy drug loading system and a preparation method and application thereof.The preparation method of the copper-manganese alloy drug loading system at least comprises the following steps that S1, gas-atomized copper-manganese powder serves as a raw material, the 3D printing technology is used for printing the gas-atomized copper-manganese powder, and a copper-manganese alloy precursor is obtained; s2, the copper-manganese alloy precursor is placed in a dealloying solution for dealloying, and the copper-manganese alloy of the porous nanostructure is obtained; and S3, loading an anti-tumor drug on the copper-manganese alloy with the porous nanostructure to obtain the copper-manganese alloy drug loading system. The copper-manganese alloy drug delivery system selectively releases manganese ions, can widely inhibit expression of cancer cell glycolysis related genes, hinder cancer cell glycolysis, mediate cancer cell pyroptosis and activate anti-tumor immunity, KHK-IN-2 can inhibit cancer-promoting macrophage fructose metabolism, finally macrophage metabolism-immune reprogramming is achieved, and the anti-tumor curative effect is further enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a copper-manganese alloy drug delivery system, a preparation method and application thereof. BACKGROUND

[0002] Traditional anti-tumor therapies, such as chemotherapy and radiotherapy, usually have great systemic toxicity, and new anti-tumor strategies with high efficiency and low toxicity are urgently needed. Energy metabolism disorder is a basic hallmark of cancer. Cancer cells meet their rapid growth energy demand through extensive metabolic reprogramming and reduce the oxidative stress pressure generated by proliferation. One of the major features of metabolic reprogramming is the increase in glucose uptake, and cells are transformed into a "glycolysis-dominated" metabolic pattern. The ability of cancer cells to absorb glucose is more than 10 times that of normal cells, and they rapidly produce ATP energy molecules required for proliferation through glycolysis. This metabolic pattern provides cancer cells with a great survival advantage and promotes their invasion, metastasis and resistance to treatment such as chemotherapy or radiotherapy. Therefore, intervention in glycolysis may be a promising metabolic therapy specific to cancer cells. A series of targeting strategies have been developed for cancer cell glycolysis. For example, inhibitors targeting key enzymes of glycolysis (such as HK2 and LDHA) are used to selectively inhibit the activity of certain glycolytic enzymes; and the activity of transport proteins that target and inhibit glycolytic metabolites is inhibited. Although these strategies have shown some anti-tumor effect, most of them target a single enzyme or gene, and the target is relatively limited. Moreover, these methods only cause cancer cell death, lack a more extensive anticancer effect, which also limits their application in combination therapy. SUMMARY

[0003] The purpose of the present application is to overcome the defects of the existing targeting treatment methods for cancer cell glycolysis, which lack anticancer effect and have poor anti-tumor effect.

[0004] In order to achieve the above purpose, the present application provides a preparation method of a copper-manganese alloy drug delivery system, which comprises at least the following steps:

[0005] Step S1: using a copper-manganese powder as raw material, printing the copper-manganese powder by using a 3D printing technology to obtain a copper-manganese alloy precursor;

[0006] Step S2: placing the copper-manganese alloy precursor in a dealloying solution to perform dealloying to obtain a porous nano-structured copper-manganese alloy;

[0007] Step S3: loading an anti-tumor drug on the porous nano-structured copper-manganese alloy to obtain a copper-manganese alloy drug delivery system.

[0008] Optionally, in step S1, the particle size of the copper-manganese powder is 20-40 μm.

[0009] Optionally, in step S1, the copper-manganese alloy precursor is prepared using laser powder melting technology. The laser beam diameter is 55 μm, the power is 90 W, the scanning speed is 700 mm / s, the scanning spacing is 105 μm, and the powder layer thickness is 25 μm. After the gas-atomized copper-manganese powder is printed, the copper-manganese alloy precursor is obtained.

[0010] Optionally, in step S1, the copper:manganese ratio in the gas-atomized copper-manganese powder is (40-50):(60-40) by mass.

[0011] Optionally, in step S2, the dealloying is carried out in a 0.25 mol / L sulfuric acid solution at a reaction temperature of 70°C, with the dealloying solution being replaced every 3 hours.

[0012] Optionally, in step S3, the antitumor drug is the fructose-metabolizing enzyme inhibitor KHK-IN-2.

[0013] Optionally, the ligament diameter of the porous nanostructured copper-manganese alloy is less than 100 nm.

[0014] The present invention also provides a copper-manganese alloy drug-loaded system, which is prepared by the preparation method described above.

[0015] The present invention also provides an application of a copper-manganese alloy drug-carrying system, which is used to prepare antitumor drugs.

[0016] Optionally, the antitumor drug is an anti-breast cancer drug.

[0017] Compared to the prior art, the beneficial effects of the present invention include at least the following:

[0018] This invention uses atomized copper-manganese powder as raw material and employs 3D printing technology to print the atomized copper-manganese powder to obtain a copper-manganese alloy precursor. The precursor is then placed in a dealloying solution for dealloying to obtain a porous nanostructured copper-manganese alloy with porous properties and a large surface area. Antitumor drugs are loaded onto this porous nanostructured copper-manganese alloy to obtain a copper-manganese alloy drug-loaded system. This system selectively releases manganese ions, which can broadly inhibit the expression of glycolysis-related genes in cancer cells, hinder cancer cell glycolysis, mediate cancer cell pyroptosis, and activate antitumor immunity. KHK-IN-2 can inhibit fructose metabolism in cancer-promoting macrophages, ultimately achieving macrophage metabolic-immune reprogramming and further enhancing the antitumor therapeutic effect. Attached Figure Description

[0019] Figure 1 These are transmission electron microscope (TEM) images and elemental surface scan images of the porous nanostructured copper-manganese alloy of the present invention; wherein,

[0020] A is a transmission electron microscope image of a porous nanostructured copper-manganese alloy;

[0021] B is an elemental surface scan image of a porous nanostructured copper-manganese alloy;

[0022] C is a surface scan image of manganese in a porous nanostructured copper-manganese alloy;

[0023] D is a surface scan image of copper in a porous nanostructured copper-manganese alloy.

[0024] Figure 2 This is a schematic diagram illustrating the effect of the porous nanostructured copper-manganese alloy of the present invention in inhibiting tumor cell glycolysis and mediating pyroptosis; wherein,

[0025] A represents the expression results of mRNAs of glycolysis-related genes in mouse breast cancer cell lines;

[0026] B represents the result of intracellular lactose content detection in mouse breast cancer cell lines;

[0027] C shows the cell viability test results of mouse breast cancer cell lines and normal control cell lines;

[0028] D represents the cell morphology of the mouse breast cancer cell line (cell vesicles are marked with red arrows).

[0029] Figure 3 This is a schematic diagram illustrating the effects of the copper-manganese alloy drug delivery system of the present invention on the regulation of macrophage metabolism and immune phenotype; wherein,

[0030] A represents the expression results of mRNAs of glycolysis-related genes in each group of mouse macrophage cell lines;

[0031] B shows the polarization flow cytometry plots and statistical results of the M1 mouse macrophage cell line in each group.

[0032] C shows the polarization flow cytometry plots and statistical results of the M2 mouse macrophage cell line in each group.

[0033] Figure 4 The figure shows the in vivo experimental results of the copper-manganese alloy drug delivery system of the present invention regulating the tumor microenvironment.

[0034] in,

[0035] A is a schematic diagram of a bilateral tumorigenic animal model;

[0036] B represents the bilateral tumor growth curves for each group;

[0037] C shows representative images of HE, Ki67, and pyroptosis staining of bilateral proximal tumor sections in each group;

[0038] D shows representative images of HE, Ki67, and pyroptosis staining of bilateral distal tumor sections in each group;

[0039] E represents the mRNA expression of tumor metabolism-related genes in each group;

[0040] F is a flow cytometry diagram and statistical results of the immunophenotypes of tumor-infiltrating macrophages in each group;

[0041] G represents the CD4 infiltration level in each tumor group. + Flow cytometry diagram and statistical results of T cell immune phenotypes;

[0042] H represents the CD8 infiltration rate of tumors in each group. + Flow cytometry diagram and statistical results of T-cell immunophenotypes. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] All experimental materials not otherwise mentioned in this invention are commercially available, and all experimental methods not otherwise mentioned in this invention are conventional experimental methods in the field.

[0045] The metabolic behavior of non-cancerous cells (including stromal cells and immune cells) in the tumor microenvironment plays a crucial role in tumor progression, metastasis, and recurrence. Macrophages, comprising approximately 50% of immune cells, exhibit a close correlation between their immune phenotype and metabolic state: pro-cancer macrophages are characterized by active fructose metabolism. Therefore, targeting macrophage fructose metabolism can modulate the immune microenvironment, thereby inhibiting tumor growth.

[0046] This invention provides a copper-manganese alloy drug delivery system, comprising a porous nanostructured copper-manganese alloy and a fructose-metabolizing enzyme inhibitor KHK-IN-2 loaded on the porous nanostructured copper-manganese alloy. The porous nanostructured copper-manganese alloy has the characteristics of selectively releasing manganese ions and releasing almost no copper ions. The fructose-metabolizing enzyme inhibitor KHK-IN-2 is used to inhibit fructose metabolism and simultaneously inhibit the fructose metabolism of cancer-promoting macrophages, thereby achieving macrophage metabolic-immune reprogramming. The released manganese ions can broadly inhibit the expression of cancer cell glycolysis-related genes, hinder cancer cell glycolysis, mediate cancer cell pyroptosis, activate anti-tumor immunity, and further enhance the anti-tumor efficacy.

[0047] I. Preparation of Copper-Manganese Alloy Drug Loading System

[0048] The preparation method of the copper-manganese alloy drug-carrying system of the present invention includes the following steps:

[0049] Step S1: Using gas-atomized copper-manganese powder as raw material, the gas-atomized copper-manganese powder is printed using 3D printing technology to obtain a copper-manganese alloy precursor.

[0050] Laser bed fusion (LPBF) technology was used with a TruPrint 1000 system (Trumpf Laser Systems GmbH, Germany). Gas-atomized copper-manganese powder (Chengdu Huaying Powder Technology Co., Ltd.) was used as the raw material, with an initial composition of Cu:Mn = 44:56 (mass percentage). The powder particle size distribution ranged from 20μm to 40μm. Due to the volatilization of Mn during additive manufacturing, the composition of the printed sample slightly shifted to Cu:Mn = 46:54 (mass percentage). The LPBF process was performed on a stainless steel substrate preheated to 100°C, with a laser beam diameter of 55μm, a power of 90W, a scanning speed of 700mm / s, a scanning spacing of 105μm, and a powder bed thickness of 25μm. After printing, the formed part was slowly cooled to room temperature (25°C) within the equipment and then manually separated from the substrate by wire cutting. The advantages of using laser powder bed fusion (LPBF) technology are that it can print complex structural parts, which is beneficial for subsequent implant printing. By forming specific precursor microstructures through LPBF technology, and then controlling dealloying parameters, such as holding time and dealloying medium, the diameter of ligaments in the copper-manganese alloy skeleton can be refined to form a nanoporous structure, thereby increasing the surface area of ​​the material and improving drug loading. In addition, the nanopores formed within the unit cells of the copper-manganese alloy, and the formation of the porous structure helps to increase the specific surface area of ​​the material, thereby improving the release efficiency of manganese ions within the unit cells.

[0051] Step S2: The copper-manganese alloy precursor is placed in a dealloying solution for dealloying to obtain a porous nanostructured copper-manganese alloy.

[0052] To generate nanoporous metallic materials that provide a large surface area for drug loading, dealloying was performed in a 0.25 mol / L sulfuric acid solution at 70°C, with the dealloying solution replaced every 3 hours. After treatment, the samples were thoroughly rinsed to remove residual solution and then stored in ethanol to prevent oxidation or contamination. The porous nature and large surface area of ​​the porous nanostructured copper-manganese alloy facilitate faster release of manganese ions.

[0053] Step S3: Load the antitumor drug onto the porous nanostructured copper-manganese alloy to obtain a copper-manganese alloy drug-loaded system.

[0054] In some embodiments, the antitumor drug is the fructose-metabolizing enzyme inhibitor KHK-IN-2.

[0055] The prepared porous nanostructured copper-manganese alloy was physically characterized, such as... Figure 1As shown in the projection electron microscopy images, the prepared copper-manganese alloy possesses a porous structure and exhibits two morphologies: granular and grain boundary states. The proportions of copper and manganese differ between the two morphologies: the granular state contains approximately 1 wt% Mn, while the grain boundary state contains approximately 47 wt% Mn. This difference in Mn activation energies between the two morphologies leads to preferential release of Mn from the grains, while Mn from the grain boundaries is released sequentially.

[0056] It should be noted that the release of manganese ions in this invention is achieved by placing one alloy piece (1 mm in diameter and 1.5 mm in height) in a certain volume (10 mL) of sterile ultrapure aqueous solution. The solution is then sealed and placed in a shaker, shaken at 500 rpm at room temperature. At predetermined time points, 1 mL of the solution is pipetted for testing, while simultaneously adding 1 mL of fresh sterile ultrapure water. Samples are taken at these predetermined time points, and the collected solutions are filtered, diluted, and then analyzed using ICP-MS to determine the content of copper and manganese ions. The amount of manganese ions released at different time points is calculated using a formula. The results show that, within the same time period, the release content of manganese ions is approximately 1000 times that of copper ions.

[0057] The reasons for the preferential release of manganese ions in this invention are as follows:

[0058] (1) Compared with copper, manganese ions are more active metal ions (based on their standard electrode potentials: Mn 2+ The standard electrode potential of / Mn is -1.185V, Cu 2+ The standard electrode potential of Cu is +0.337V. In electrochemical corrosion, the metal with the more negative potential acts as the anode, while the metal with the more positive potential acts as the cathode. When a copper-manganese alloy is immersed in an electrolyte (such as water or body fluid), countless tiny "manganese-copper" galvanic cells form on the alloy surface. According to the laws of thermodynamics, manganese, with its more negative electrode potential, will spontaneously act as the anode and be oxidized and dissolved, while copper acts as the cathode and is protected. This process is ubiquitous on the alloy surface, thus manganese ions are preferentially released.

[0059] (2) Corrosion products of manganese, such as Mn(OH)2, have relatively high solubility. Once formed, they readily detach from the metal surface and enter the solution, exposing new manganese atoms to continue the reaction. In contrast, corrosion products of copper, such as Cu2O or CuO, CuCO3·Cu(OH)2, have very low solubility. They adhere tightly to the copper surface, forming a protective film that greatly slows down the further release of copper ions.

[0060] (3) After the manganese atoms on the surface are selectively dissolved, a porous, copper-rich sponge-like structure is left behind. Although this copper-rich layer looks like copper, its porous structure cannot completely prevent the electrolyte from penetrating. More importantly, this copper-rich layer constitutes a huge cathode area, while the small amount of manganese atoms remaining at the bottom or inside of the pores become very small anodes. It drives the small anode (manganese) to dissolve at an extremely high current density (i.e., an extremely fast rate).

[0061] II. Porous nanostructured copper-manganese alloys inhibit tumor cell glycolysis and mediate tumor cell pyroptosis by selectively releasing manganese ions.

[0062] A porous nanostructured copper-manganese alloy was immersed in a culture medium solution for 24 hours. The extract was then used to treat mouse breast cancer cell lines, resulting in a treatment group (i.e., the mouse breast cancer cell lines were directly immersed in the extract). Figure 2 As shown, the control group (Ctrl) served as the culture medium control. It was found that compared to the control group (Ctrl), key genes in glycolysis (such as SLC2A1 and HK1) were extensively downregulated at the mRNA level in the treatment group (CuMn). Figure 2 (A). Glycolysis products are lactic acid, and intracellular lactic acid content detection also showed that the intracellular lactic acid content was reduced in the copper-manganese alloy treatment group. Figure 2 (B). Furthermore, the cytotoxic effect of copper-manganese alloy on cells was detected using a CCK8 assay kit. It was found that treatment with porous nanostructured copper-manganese alloy significantly inhibited the cell viability of tumor cells, far exceeding its effect on normal cells. Figure 2 Pyroptosis is a form of cell death, characterized by typical morphological changes such as cell shrinkage and vacuole dissolution. These morphological features were observed in cells treated with porous nanostructured copper-manganese alloy. Figure 2 (D). In summary, the above results indicate that manganese ions released from porous nanostructured copper-manganese alloys inhibit glycolytic metabolism in tumor cells, thereby mediating pyroptosis in tumor cells.

[0063] III. Copper-manganese alloy drug delivery system regulates macrophage metabolism and immune phenotype

[0064] like Figure 3 As shown, compared with the control group (Ctrl), the fructose metabolism enzyme genes of macrophages treated with the copper-manganese alloy drug-loaded system (CuMn-K) were significantly inhibited, while glycolysis-related genes were significantly upregulated, indicating that the porous nanostructured copper-manganese alloy loaded with drugs can remodel the metabolic state of macrophages. Figure 3 (A). The metabolic state of macrophages further regulates their immune phenotype. Flow cytometry results showed that the copper-manganese alloy drug delivery system upregulated the M1 polarization ratio and downregulated the M2 polarization ratio of macrophages, indicating an immune activated state. Figure 3 B and Figure 3(C). The above results suggest that the porous copper-manganese alloy system of the present invention can regulate the metabolic state and immune phenotype of macrophages after drug loading.

[0065] IV. In vivo experiments on the anti-tumor effects of copper-manganese alloy drug delivery system by regulating the microenvironment

[0066] like Figure 4 As shown, the in vivo effects of copper-manganese alloy were evaluated using a bilateral tumorigenesis model of mouse breast cancer cell lines: tumor cells were subcutaneously injected into the skin on both sides of the dorsal side of the mice, and porous nanostructured copper-manganese alloy was implanted in situ into the right tumor for treatment. Figure 4 A). For example Figure 4 As shown in Figure B, compared to the control group (Ctrl) without implantation of porous nanostructured copper-manganese alloy, both porous nanostructured copper-manganese alloy (CuMn) and the copper-manganese alloy drug-loaded system (CuMn-K) showed significant inhibitory effects on bilateral tumors. Tumor tissue section staining results showed that the proportion of tumor proliferation decreased and the proportion of pyroptosis increased with both porous nanostructured copper-manganese alloy (CuMn) and the copper-manganese alloy drug-loaded system (CuMn-K). Figure 4 (C), and tumor glycolysis-related genes are suppressed ( Figure 4 E). Evaluation of intratumoral macrophages and CD4+ using flow cytometry. + T cells, CD8 + T-cell immunophenotype analysis revealed that, compared to the control group (Ctrl) and the porous nanostructured copper-manganese alloy (CuMn), the copper-manganese alloy drug delivery system (CuMn-K) exhibited M1 polarization in intratumoral macrophages. Figure 4 F), immunosuppressive CD4 + T and CD8 + The proportion of T cells decreased ( Figure 4 (GH). The above results verify that the copper-manganese alloy drug delivery system can regulate tumor metabolism and immune microenvironment, and has a significant inhibitory effect on tumor growth.

[0067] In summary, this invention provides a copper-manganese alloy drug delivery system comprising a porous nanostructured copper-manganese alloy and a fructose-metabolizing enzyme inhibitor KHK-IN-2 loaded on the porous nanostructured copper-manganese alloy. The fructose-metabolizing enzyme inhibitor KHK-IN-2 can inhibit fructose metabolism in cancer-promoting macrophages. The porous nanostructured copper-manganese alloy selectively releases manganese ions and releases almost no copper ions. Manganese ions can broadly inhibit the expression of glycolysis-related genes in cancer cells, hinder cancer cell glycolysis, mediate cancer cell pyroptosis, and activate anti-tumor immunity, ultimately achieving macrophage metabolic-immune reprogramming and further enhancing the anti-tumor therapeutic effect.

[0068] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing a copper-manganese alloy drug-loaded system, characterized in that, Include at least the following steps: Step S1: Using gas-atomized copper-manganese powder as raw material, the gas-atomized copper-manganese powder is printed using 3D printing technology to obtain a copper-manganese alloy precursor. Step S2: The copper-manganese alloy precursor is placed in a dealloying solution for dealloying to obtain a porous nanostructured copper-manganese alloy. Step S3: Load the antitumor drug onto the porous nanostructured copper-manganese alloy to obtain a copper-manganese alloy drug-loaded system.

2. The preparation method according to claim 1, characterized in that, In step S1, the particle size of the copper-manganese powder is 20μm to 40μm.

3. The preparation method according to claim 1, characterized in that, In step S1, the copper-manganese alloy precursor is prepared using laser powder melting technology. The laser beam diameter is 55 μm, the power is 90 W, the scanning speed is 700 mm / s, the scanning spacing is 105 μm, and the powder layer thickness is 25 μm. After the gas-atomized copper-manganese powder is printed, the copper-manganese alloy precursor is obtained.

4. The preparation method according to claim 1, characterized in that, In step S1, the copper:manganese ratio of the gas-atomized copper-manganese powder is (40-50):(60-40) by mass percentage.

5. The preparation method according to claim 1, characterized in that, In step S2, the dealloying is carried out in a 0.25 mol / L sulfuric acid solution at a reaction temperature of 70°C, with the dealloying solution being replaced every 3 hours.

6. The preparation method according to claim 1, characterized in that, In step S3, the antitumor drug is the fructose metabolism enzyme inhibitor KHK-IN-2.

7. The preparation method according to claim 1, characterized in that, The ligament diameter of the porous nanostructured copper-manganese alloy is less than 100 nm.

8. A copper-manganese alloy drug delivery system, characterized in that, The copper-manganese alloy drug-carrying system is prepared by the preparation method described in any one of claims 1 to 7.

9. An application of the copper-manganese alloy drug delivery system as described in claim 8, characterized in that, The copper-manganese alloy drug delivery system is used to prepare anti-tumor drugs.

10. The application as described in claim 9, characterized in that, The anti-tumor drug is an anti-breast cancer drug.

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