Zn-penicillamine nano-chelating agent, and preparation method and application thereof

Abstract

The application belongs to the technical field of antitumor drugs, and relates to a Zn-penicillamine nano-chelating agent and a preparation method and application thereof. The nano-chelating agent is a non-water-soluble nanomaterial formed by chelating coordination of zinc ions and D-penicillamine. The preparation method is as follows: D-penicillamine and a divalent zinc salt are uniformly mixed in a solution, the pH is adjusted to neutral or alkaline, and continuous stirring reaction is carried out, and the Zn-penicillamine nano-chelating agent is obtained. The Zn-penicillamine nano-chelating agent provided by the application can interfere with cell ion homeostasis, realize dual inhibition of oxidative phosphorylation (OXPHOS) and glycolysis metabolism in breast cancer, and effectively activate an antitumor immune response.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, and relates to a Zn-penicillamine nanochelating agent, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Copper is a common metallic element and a transition element, exhibiting redox activity. Under normal chemical reaction and physiological conditions, reduced Cu... + It can be transformed into Cu oxide. 2+ Copper ions participate in a variety of biochemical reactions by donating or accepting electrons. They can bind to various proteins and enzymes, acting as cofactors or structural components, and participate in the regulation of multiple physiological processes such as energy metabolism, mitochondrial respiration, and antioxidant activity. The concentration of copper ions maintains a dynamic balance; imbalance can lead to oxidative stress and abnormal autophagy, thereby inducing various copper or copper-related diseases.

[0004] Copper death is a novel form of cell death distinct from other programmed cell death (such as apoptosis, pyroptosis, necrosis, and ferroptosis), and its regulation is closely related to mitochondrial metabolism. Excess copper binds directly to lipoylated proteins in the mitochondrial tricarboxylic acid (TCA) cycle, leading to abnormal aggregation of lipoylated proteins and loss of iron-sulfur cluster proteins in the respiratory chain complex, causing a protein toxicity stress response and ultimately resulting in cell death. However, this therapeutic approach of metal ion overload is often uncontrollable, potentially impacting normal tissues while killing tumor cells. Studies have shown that serum copper levels in breast cancer patients... 2+ The levels are higher than in normal individuals. Furthermore, mitochondrial copper chaperone proteins COX17 and SCO2 are upregulated in breast cancer patients, indicating that breast cancer cells have a higher demand for copper transport to mitochondria compared to normal cells. Depletion of mitochondrial copper would shift metabolism from respiration to glycolysis and reduce energy production, which could effectively combat oxidative phosphorylation-dependent (OXPHOS) cancer types with less impact on normal tissues. However, due to metabolic reprogramming in tumor cells, when OXPHOS is inhibited, tumor cells maintain the energy required for their vital activities by enhancing glycolytic metabolism. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a Zn-penicillamine nano-chelating agent, its preparation method, and its application. The Zn-penicillamine nano-chelating agent provided by the present invention can interfere with cellular ion homeostasis, and in breast cancer, it not only achieves dual inhibition of oxidative phosphorylation (OXPHOS) and glycolysis metabolism, but also effectively activates anti-tumor immune responses.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, there is a Zn-penicillamine nano-chelating agent, wherein the nano-chelating agent is a non-water-soluble nanomaterial formed by the chelation coordination of zinc ions and D-penicillamine.

[0008] The Zn-penicillamine nano-chelating agent provided by this invention can chelate copper in breast cancer cells while releasing Zn. 2+ Copper depletion inhibits oxidative phosphorylation (OXPHOS) metabolism by suppressing the activity of the mitochondrial copper enzyme cytochrome c oxidase complex IV (COXⅣ). Meanwhile, intracellular Zn... 2+ Elevated levels activate the cGAS-STING pathway, which interferes with breast cancer glycolytic metabolism by inhibiting hexokinase II (HK2) activity and triggers an immune response via IFN-β. Therefore, Zn-penicillamine nanochelates not only achieve dual inhibition of OXPHOS and glycolytic metabolism in the treatment of breast cancer, but also effectively activate anti-tumor immune responses.

[0009] Meanwhile, reagents that can chelate copper ions include not only D-penicillamine, but also other copper ion chelating agents such as trientine and tetrathiomolybdic acid. Compared with other copper ion chelating agents (such as trientine and tetrathiomolybdic acid), D-penicillamine has a simpler structure and possesses amino, carboxyl, and thiol groups. These groups make D-penicillamine readily chelate with metal ions (Zn). 2+ Chelating and coordinating with metal ions (Zn) forms insoluble nanomaterials, which are beneficial for long-term accumulation at tumor sites. Trientine and tetrathiomolybdic acid, on the other hand, bind with metal ions (Zn) to form water-insoluble nanomaterials. 2+ It easily forms water-soluble complexes and is quickly excreted by the body, failing to achieve a therapeutic effect. Furthermore, D-penicillamine is much cheaper, costing only 1 / 8 the price of trientine and 1 / 4 the price of tetrathiomolybdic acid, significantly reducing the cost of disease treatment. Therefore, compared to other copper ion chelating agents, Zn-penicillamine nano-chelating agents can exert a long-term effect at the tumor site, thus achieving better therapeutic results.

[0010] In addition, breast cancer inhibition experiments have shown that the combination of Zn and penicillamine has a synergistic promoting effect, enhancing the tumor-killing effect of the nano-chelating agent.

[0011] The nanomaterials described in this invention can be nanoparticles, nanofibers, etc. In some embodiments, the nanomaterials are nanofibers. Compared with nanomaterials of other morphologies, nanoparticles have smaller sizes (especially radial dimensions), resulting in deeper tumor penetration and thus better anti-tumor effects.

[0012] In some embodiments, the coordination ratio of zinc ions to D-penicillamine is 1:1.

[0013] On the other hand, a method for preparing a Zn-penicillamine nano-chelating agent involves mixing D-penicillamine and divalent zinc salt in a solution, adjusting the pH to neutral or alkaline, and continuously stirring the reaction to obtain the Zn-penicillamine nano-chelating agent.

[0014] The divalent zinc salts described in this invention are compounds whose cation is a divalent zinc ion, such as zinc chloride, zinc sulfate, and zinc nitrate. Studies using zinc chloride and zinc nitrate in this invention demonstrate that the anions of divalent zinc salts have little effect on the formation and morphology of nanomaterials.

[0015] The molar ratio of D-penicillamine to divalent zinc salt is 1:0.5–2.0. Studies have shown that different molar ratios have little effect on the morphology of Zn-penicillamine nano-chelating agents. In some embodiments, the molar ratio of D-penicillamine to divalent zinc salt is 1:0.9–1.1.

[0016] Adjusting the pH to 7–14, especially to 7–12, results in different complexation constants between D-penicillamine and zinc ions, leading to the formation of Zn-penicillamine nano-chelating agents with varying morphologies. In some embodiments, the pH is adjusted to 7.3–7.7. Studies have shown that these conditions can produce nanofiber-like Zn-penicillamine nano-chelating agents.

[0017] The reaction is continuously stirred for 2 to 14 hours. In some embodiments, the reaction is continuously stirred for 11 to 13 hours.

[0018] In some embodiments, solid-liquid separation is performed after continuous stirring of the reaction. Specifically, the solid-liquid separation method is centrifugation. More specifically, the centrifugation speed is 5000-10000 rpm and the centrifugation time is 5-15 min; preferably, the centrifugation speed is 9900-10000 rpm and the centrifugation time is 9-11 min.

[0019] Specifically, washing is performed after solid-liquid separation. Generally, the washing process is repeated 2 to 3 times.

[0020] Thirdly, a pharmaceutical composition comprising an active ingredient and pharmaceutical excipients, wherein the active ingredient is the Zn-penicillamine nano-chelating agent described in the first aspect of the present invention or the Zn-penicillamine nano-chelating agent obtained by the preparation method described in the second method of the present invention.

[0021] In some embodiments, the pharmaceutical excipients are water, physiological saline, buffer solutions, immune adjuvants, emulsifiers, suspending agents, fillers, disintegrants, or preservatives, etc.

[0022] In some embodiments, the pharmaceutical composition is administered via subcutaneous injection or intravenous injection, etc.

[0023] Fourthly, the use of the Zn-penicillamine nano-chelating agent described in the first aspect of the present invention, the Zn-penicillamine nano-chelating agent obtained by the preparation method described in the second method of the present invention, or the pharmaceutical composition described in the third aspect of the present invention in the preparation of an anti-breast cancer drug.

[0024] The positive surface charge of Zn-penicillamine promotes its internalization and accumulation in mitochondria of breast cancer cells (4T1). In the mitochondria, Zn-penicillamine binds to copper, leading to the deposition of Zn... 2+ The release of Zn interferes with mitochondrial function and induces apoptosis in 4T1 cells. Secondly, the released Zn... 2+ Stimulation of the cGAS-STING signaling pathway induces the release of interferon-β (IFN-β), further promoting DC maturation and enhancing the immune response. Additionally, copper deficiency and Zn... 2+ The surge hindered OXPHOS and glycolytic metabolism in 4T1 cells.

[0025] In some embodiments, the anti-breast cancer drug has the effect of inhibiting oxidative phosphorylation and glycolytic metabolism and / or activating anti-tumor immune responses.

[0026] The beneficial effects of this invention are as follows:

[0027] (1) Traditional chelation therapy involves intravenously injecting chelating agents to deplete excess metal elements in the host's body. Due to a lack of targeting, this method is prone to systemic toxicity. The Zn-penicillamine nano-chelating agent provided in this invention can accumulate at the tumor site for a long period, avoiding systemic toxicity. Furthermore, Zn-penicillamine can not only deplete copper, which is essential for the growth and development of breast cancer, but also release Zn with anti-tumor properties. 2+ .

[0028] (2) The Zn-penicillamine nano-chelating agent provided by this invention is Zn 2+ The chelation coordination with D-penicillamine resulted in an extremely high loading of penicillamine chelating agent compared to chelating agents loaded with nanomaterials.

[0029] (3) The Zn-penicillamine nano-chelating agent provided by the present invention not only achieves dual inhibition of OXPHOS and glycolysis metabolism in breast cancer, but also activates anti-tumor immune response and effectively hinders metastasis.

[0030] (4) The preparation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 This is a transmission electron microscope (TEM) image of the Zn-penicillamine nanochelating agent prepared in Example 1.

[0033] Figure 2 This is a TEM image of the Zn-penicillamine nanochelating agent prepared in Example 2.

[0034] Figure 3 This is a TEM image of the Zn-penicillamine nanochelating agent prepared in Example 3.

[0035] Figure 4 This is a TEM image of the Zn-penicillamine nanochelating agent prepared in Example 4.

[0036] Figure 5 The image shows the X-ray photoelectron spectroscopy (XPS) characterization of the Zn-penicillamine nanochelating agent prepared in Example 1.

[0037] Figure 6 XPS characterization of the nanomaterials prepared in Example 1 after Zn-penicillamine nanochelating agent was chelated with Cu.

[0038] Figure 7 The Zn released after the Zn-penicillamine nano-chelating agent prepared in Example 1 is chelated with Cu 2+ The representation of.

[0039] Figure 8 The flow cytometry diagram shows the chelation of Cu in 4T1 cells by the Zn-penicillamine nano-chelating agent prepared in Example 1.

[0040] Figure 9 The Zn-penicillamine nanochelating agent prepared in Example 1 releases Zn in 4T1 cells. 2+ flow cytometry instrumentation.

[0041] Figure 10The graph shows the toxicity test results of the Zn-penicillamine nanochelating agent prepared in Example 1 on tumor cells (4T1) and normal cells (HUVECs); a is 4T1 cells, and b is HUVECs cells.

[0042] Figure 11 This is a comparison of the toxicity test results of the Zn-penicillamine nano-chelating agent prepared in Example 1 with zinc ions and D-penicillamine on tumor cells (4T1).

[0043] Figure 12 The figure shows the test results of tumor cell (4T1) apoptosis induced by the Zn-penicillamine nanochelate prepared in Example 1.

[0044] Figure 13 The graph shows the COX IV activity test results of the Zn-penicillamine nanochelate prepared in Example 1 that inhibits tumor cells (4T1).

[0045] Figure 14 The graph shows the activity test results of HK2, prepared as a Zn-penicillamine nanochelate in Example 1, in inhibiting tumor cells (4T1).

[0046] Figure 15 The graph shows the metabolic test results of the Zn-penicillamine nano-chelator prepared in Example 1 inhibiting tumor cells (4T1); a is the glucose content, b is the lactic acid content, and c is the ATP content.

[0047] Figure 16 The image shows the test results of promoting DC maturation in tumor cells (4T1) treated with the Zn-penicillamine nanochelating agent prepared in Example 1. Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Example 1

[0050] I. Preparation method of Zn-penicillamine nano-chelating agent, the steps are as follows:

[0051] 1) Weigh 0.5 mmol of ZnCl2 and D-penicillamine into a 50 mL beaker.

[0052] 2) Add 20 mL of deionized water and dissolve by magnetic stirring.

[0053] 3) Adjust the pH of the above solution to 7.5 using NaOH solution.

[0054] 4) Stir at room temperature for 12 hours, then centrifuge at 10,000 rpm for 10 minutes to obtain a silvery-white solid Zn-penicillamine nano-chelating agent. Then wash with deionized water 3 times, centrifuge again, and finally obtain the Zn-penicillamine nano-chelating agent.

[0055] The Zn-penicillamine nanochelating agent prepared in Example 1 was characterized by transmission electron microscopy, such as... Figure 1 As shown in the figure, the prepared Zn-penicillamine nano-chelating agent is a fibrous nanomaterial.

[0056] II. XPS characterization of Zn-penicillamine nanochelating agents

[0057] 1) Dry the Zn-penicillamine nano-chelating agent prepared in the above steps at 70°C.

[0058] 2) Use an agate mortar to grind the solid Zn-penicillamine nano-chelating agent into powder.

[0059] 3) Finally, the XPS full spectrum of the Zn-penicillamine nano-chelating agent was tested.

[0060] like Figure 5 As shown, XPS characterization confirmed the presence of Zn, S, and N elements in the prepared Zn-penicillamine nanochelating agent.

[0061] III. XPS characterization of Zn-penicillamine nanochelating agents chelating Cu

[0062] 1) Disperse the prepared Zn-penicillamine nano-chelating agent in 20 mL of deionized water.

[0063] 2) Add 50mM CuCl2 solution and sonicate for 10min.

[0064] 3) Centrifuge the above solution at 10,000 rpm for 10 min to obtain Zn-penicillamine nano-chelating agent that chelates Cu.

[0065] 4) Subsequent testing steps are the same as those shown in step two.

[0066] like Figure 6 As shown, XPS full-spectrum characterization confirmed the presence of Cu, Zn, S, and N elements in the Zn-penicillamine nano-chelating agent that chelates Cu.

[0067] IV. Zn-Penicillamine nano-chelating agent chelates Cu 2+ Release Zn 2+ The representation

[0068] 1) Disperse the prepared Zn-penicillamine nano-chelating agent in 10 mL of deionized water, divide it into 5 equal portions, and each tube contains 2 mL of solution.

[0069] 2) Then add CuCl2 solution of different concentrations to each tube, namely 0, 6.25, 12.5, 25 and 50 mM, and continue sonication for 10 min.

[0070] 3) Centrifuge the above solution at 10,000 rpm for 10 min, and take the supernatant to analyze Zn using inductively coupled plasma mass spectrometry. 2+ The concentration.

[0071] like Figure 7 As shown, with Cu 2+ The increase in concentration of Zn in the supernatant solution 2+ The content gradually increased, which confirmed that the Zn-penicillamine nano-chelating agent chelated Cu. 2+ Zn can then be released. 2+ .

[0072] V. Characterization of Cu chelation by Zn-Penicillamine nanochelating agent in breast cancer

[0073] 1) 4T1 cells were seeded in 6-well plates (10 5 Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0074] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (10μg mL -1 (3) D-Penicillamine (23 μg mL) -1 (4) Zn-penicillamine (33 μg mL) -1 ).

[0075] 3) After 4 hours of different treatments, add the prepared copper ion fluorescent probe-RHB, incubate for 30 minutes, and wash 3 times with PBS.

[0076] 4) Use flow cytometry to detect changes in intracellular copper ions.

[0077] like Figure 8 As shown, the control group and Zn 2+ The similar intracellular copper content in breast cancer cells from the group indicates that Zn 2+ It does not affect the intracellular copper level in breast cancer cells. However, the intracellular copper content in cells treated with D-penicillamine and Zn-penicillamine was significantly reduced, confirming the ability of Zn-penicillamine to chelate copper in breast cancer cells.

[0078] VI. Zn-Penicillamine Nanochelating Agent Releases Zn in Breast Cancer 2+ The representation

[0079] 1) 4T1 cells were seeded in 6-well plates (10 5Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0080] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (10μg mL -1 (3) D-Penicillamine (23 μg mL) -1 (4) Zn-penicillamine (33 μg mL) -1 ).

[0081] 3) After 4 hours of different treatments, add the prepared zinc ion fluorescent probe-TSQ, incubate for 30 minutes, and wash 3 times with PBS.

[0082] 4) Use flow cytometry to detect changes in intracellular zinc ions.

[0083] like Figure 9 As shown, the intracellular zinc content in breast cancer cells of the D-penicillamine group was lower than that of the control group, which may be due to D-penicillamine also chelating intracellular Zn. Zn 2+ The intracellular zinc content in cells treated with Zn-penicillamine increased significantly, confirming that Zn-penicillamine releases Zn into breast cancer cells. 2+ The ability.

[0084] VII. Cytotoxicity of Zn-Penicillamine Nanochelating Agents to Breast Cancer Cells (4T1) and Normal Cells (HUVECs)

[0085] 1) HUVECs and 4T1 cells were seeded in 96-well plates (8×10⁶ cells / wells). 3 (cells / well).

[0086] 2) Use different concentrations (0, 20, 40, 60, 80, 100 μg / mL) -1 Cells in 96-well plates were treated with Zn-penicillamine nanochelating agent and cultured for 24 h.

[0087] 3) Cell viability was detected using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0088] like Figure 10 As shown, even at low concentrations (40 μg / mL) -1 At a concentration of 100 μg / mL, Zn-PEN inhibited the growth of 4T1 cells by 71.3%. -1 When the survival rate of 4T1 cells was less than 20%, the survival rate of HUVECs remained above 75%, which confirms that Zn-penicillamine nanochelates have high cytotoxicity to tumor cells and little effect on normal cells.

[0089] 8. Cytotoxicity of different treatments on breast cancer cells (4T1).

[0090] 1) 4T1 cells were seeded in 96-well plates (8 × 10⁻⁶ cells per well). 3 Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0091] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (30μg mL -1 (3) D-Penicillamine (70 μg mL) -1 (4) Zn-penicillamine (100 μg mL) -1 Cells in 96-well plates were treated according to the experimental groups and cultured for 24 hours.

[0092] 3) Cell viability was detected using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0093] like Figure 11 As shown, the survival rate of 4T1 cells treated with zinc ions alone was 46.2%, while the survival rate of 4T1 cells treated with D-penicillamine was 84.2%. The inhibition rate of Zn-PEN on 4T1 cells reached 79.5%, which was higher than the sum of zinc ions and D-penicillamine alone (70.6%). This indicates that the combination of Zn and penicillamine has a synergistic promoting effect, enhancing the tumor-killing effect of the nano-chelating agent.

[0094] IX. Detection of apoptosis in Zn-penicillamine nanochelates induced in breast cancer cells (4T1)

[0095] 1) 4T1 cells were seeded in 6-well plates (10 5 Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0096] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (30μg mL -1 (3) D-Penicillamine (70 μg mL) -1 (4) Zn-penicillamine (100 μg mL) -1 ).

[0097] 3) After 4 hours of different treatments, remove the old culture medium from the 6-well plate and wash 3 times with PBS.

[0098] 4) Digest the cells in the 6-well plate with trypsin, centrifuge at 1000 rpm for 5 min, and collect the pellet to obtain cells treated with different methods.

[0099] 5) Add the prepared apoptosis detection kit to the cells obtained above, gently pipette the cells to resuspend them, continue incubation for 30 min, and use flow cytometry to detect the apoptosis effect of the cells.

[0100] like Figure 12 As shown, the tumor cell apoptosis rate was highest in the Zn-penicillamine treatment group, at 62.3%.

[0101] 10. Detection of Zn-Penicillamine Nanochelating Agent Inhibiting 4T1 OXPHOS Metabolism

[0102] 1) 4T1 cells were seeded in culture dishes (4×10⁻⁶). 6 Cells / plate were incubated in an incubator at 37°C and 5% CO2 for 24 hours.

[0103] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (10μg mL -1 (3) D-Penicillamine (23 μg mL) -1 (4) Zn-penicillamine (33 μg mL) -1 ).

[0104] 3) After 4 hours of different treatments, remove the old culture medium from the culture dish and wash three times with PBS.

[0105] 4) Detect the COX-IV activity of 4T1 cells after different treatments according to the steps of the COX-IV detection kit.

[0106] like Figure 13 As shown, after treatment with the copper chelators D-penicillamine and Zn-penicillamine, the activity of COX IV in 4T1 cells decreased by 44.6% and 55.4% respectively compared with the control group, indicating that Zn-penicillamine inhibited the activity of COX IV through copper depletion, thereby inhibiting OXPHOS metabolism in breast cancer cells.

[0107] XI. Detection of the Inhibition of 4T1 Glycolytic Metabolism by Zn-Penicillamine Nanochelating Agent

[0108] 1) 4T1 cells were seeded in culture dishes (4×10⁻⁶). 6 Cells / plate were incubated in an incubator at 37°C and 5% CO2 for 24 hours.

[0109] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (10μg mL -1 (3) D-Penicillamine (23 μg mL) -1 (4) Zn-penicillamine (33 μg mL) -1 ).

[0110] 3) After 4 hours of different treatments, remove the old culture medium from the culture dish and wash three times with PBS.

[0111] 4) Detect the HK2 activity of 4T1 cells after different treatments according to the steps of the HK2 detection kit.

[0112] like Figure 14 As shown, compared with the control group, Zn 2+ Treatment with Zn-penicillamine and Zn-penicillamine resulted in a 77.1% and 79.2% decrease in HK2 enzyme activity in 4T1 cells, respectively. This indicates that Zn-penicillamine inhibits HK2 activity by activating STING, thereby suppressing glycolytic metabolism in breast cancer cells.

[0113] XII. Detection of Zn-Penicillamine Nanochelating Agent Inhibiting 4T1 Metabolism

[0114] 1) 4T1 cells were seeded in 6-well plates (10 5 Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0115] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (10μg mL -1 (3) D-Penicillamine (23 μg mL) -1 (4) Zn-penicillamine (33 μg mL) -1 ).

[0116] 3) After 4 hours of different treatments, remove the old culture medium from the 6-well plate and wash 3 times with PBS.

[0117] 4) Digest the cells in the 6-well plate with trypsin, centrifuge at 1000 rpm for 5 min, and collect the pellet to obtain cells treated with different methods.

[0118] 5) The changes in glucose, lactate and ATP levels in breast cancer cells after different treatments were detected using glucose assay kits, lactate assay kits and ATP assay kits, respectively.

[0119] like Figure 15 As shown, compared with the control group, Zn 2+ Treatment of 4T1 cells resulted in increased glucose levels and decreased lactate and ATP levels. This phenomenon is attributed to Zn. 2+ The elevated levels of glucose and lactate in D-penicillamine-treated 4T1 cells were due to the inhibition of glycolytic metabolism. ATP levels were slightly decreased. This phenomenon may be attributed to the inhibition of OXPHOS metabolism in breast cancer cells, leading to increased glycolytic metabolism. Finally, a significant decrease in ATP levels was found in Zn-penicillamine-treated 4T1 cells, indicating that copper depletion and Zn... 2+Increased inhibition of OXPHOS and glycolysis effectively disrupts the energy supply of breast cancer cells.

[0120] XIII. Detection of maturation of 4T1-induced DCs treated with Zn-penicillamine nanochelating agent

[0121] 1) 4T1 cells were seeded in 6-well plates (10 5 Cells / well), while DCs were seeded in the lower chamber (10 cells / well) of a Transwell 6-well plate. 5 Cells / well were incubated at 37°C and 5% CO2 for 24 hours.

[0122] 2) Four experimental groups were set up: (1) control group; (2) Zn 2+ (30μg mL -1 (3) D-Penicillamine (70 μg mL) -1 (4) Zn-penicillamine (100 μg mL) -1 ).

[0123] 3) After 4 hours of different treatments, the old culture medium in the 6-well plate was removed and the cells were washed 3 times with PBS.

[0124] 4) Digest 4T1 cells in a 6-well plate with trypsin, centrifuge at 1000 rpm for 5 min, and collect the precipitate to obtain 4T1 cells with different treatments.

[0125] 5) Seed 4T1 cells with different treatments into the upper chamber of the Transwell 6-well plate and continue to culture in an incubator at 37°C and 5% CO2 for 24 h.

[0126] 6) Digest DCs in the lower chamber of a Transwell 6-well plate with trypsin, centrifuge at 1000 rpm for 5 min, and collect the precipitate to obtain DCs.

[0127] 7) The DCs were stained with FITC anti-CD11c, PE anti-CD86 and APC anti-CD80 antibodies for 1 hour, and the maturation status of the DCs was analyzed by flow cytometry.

[0128] like Figure 16 As shown, the proportion of mature DCs in the control group was only 9.8%. However, Zn 2+ Treatment with Zn-penicillamine significantly enhanced the maturation of DCs, with proportions of 21.8% and 27.9%, respectively. These results confirm the immunoactivating effect of Zn-penicillamine.

[0129] Example 2

[0130] The preparation method of Zn-penicillamine nano-chelating agent is as follows:

[0131] 1) Weigh 0.5 mmol of ZnCl2 / Zn(NO3)2 and D-penicillamine into a 50 mL beaker.

[0132] 2) Add 20 mL of deionized water and dissolve by magnetic stirring.

[0133] 3) Adjust the pH of the above solution to 12 using NaOH solution.

[0134] 4) Stir at room temperature for 12 hours, then centrifuge at 10,000 rpm for 10 minutes to obtain a silvery-white solid Zn-penicillamine nano-chelating agent. Then wash with deionized water 3 times, centrifuge again, and finally obtain the Zn-penicillamine nano-chelating agent.

[0135] The Zn-penicillamine nanochelating agent prepared in Example 2 was characterized by transmission electron microscopy, such as... Figure 2 As shown in the figure, the prepared Zn-penicillamine nano-chelating agent at pH 12 is a large rhombic nanomaterial, and the different zinc salts have little effect on its morphology.

[0136] Example 3

[0137] The preparation method of Zn-penicillamine nano-chelating agent is as follows:

[0138] 1) Weigh ZnCl2 and D-penicillamine in 50mL beakers at different molar ratios of 0.5:1, 1:1, and 1:2.

[0139] 2) Add 20 mL of deionized water and dissolve by magnetic stirring.

[0140] 3) Adjust the pH of the above solution to 12 using NaOH solution.

[0141] 4) Stir at room temperature for 12 hours, then centrifuge at 10,000 rpm for 10 minutes to obtain a silvery-white solid Zn-penicillamine nano-chelating agent. Then wash with deionized water 3 times, centrifuge again, and finally obtain the Zn-penicillamine nano-chelating agent.

[0142] The Zn-penicillamine nano-chelating agent prepared in Example 3 was characterized by transmission electron microscopy, such as... Figure 3 As shown in the figure, at pH 12, the Zn-penicillamine nano-chelating agents prepared by reactants with different molar ratios are also large rhombic nanomaterials, and the different molar ratios of reactants have little effect on their morphology.

[0143] Example 4

[0144] The preparation method of Zn-penicillamine nano-chelating agent is as follows:

[0145] 1) Weigh 0.5 mmol of ZnCl2 and D-penicillamine into a 50 mL beaker.

[0146] 2) Add 20 mL of deionized water and dissolve by magnetic stirring.

[0147] 3) Adjust the pH of the above solutions to 7.5, 8, 9, 10, 11 and 12 respectively using NaOH solution.

[0148] 4) Stir at room temperature for 12 hours, then centrifuge at 10,000 rpm for 10 minutes to obtain a silvery-white solid Zn-penicillamine nano-chelating agent. Then wash with deionized water 2-3 times, centrifuge again, and finally obtain the Zn-penicillamine nano-chelating agent.

[0149] The Zn-penicillamine nano-chelating agent prepared in Example 4 was characterized by transmission electron microscopy, such as... Figure 4 As shown in the figure, the morphology of the prepared Zn-penicillamine nano-chelating agent varies significantly with different pH values. At pH 7.5, the prepared Zn-penicillamine nano-chelating agent is in the form of nanofibers. This difference in morphology may be due to the different reactions of D-penicillamine and Zn at different pH values. 2+ The difference in complexation constants is due to the different nanoparticle sizes. Studies have shown that the size of nanoparticles plays a crucial role in cellular uptake, with smaller nanomaterials exhibiting greater tumor penetration. Finally, smaller fibrous Zn-penicillamines generated under weakly alkaline conditions (pH = 7.5) were selected for further application research.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a Zn-penicillamine nanochelating agent or pharmaceutical composition in the preparation of an anti-breast cancer drug, characterized in that, The nano-chelating agent is a non-water-soluble nanomaterial formed by the chelation coordination of zinc ions and D-penicillamine. The pharmaceutical composition comprises an active ingredient and pharmaceutical excipients; the active ingredient is a Zn-penicillamine nano-chelating agent; the pharmaceutical excipients are water, physiological saline, buffer solution, immunoadjuvant, emulsifier, suspending agent, filler, disintegrant, or preservative. The anti-breast cancer drug is used to inhibit oxidative phosphorylation and glycolytic metabolism and / or activate anti-tumor immune responses; The Zn-penicillamine nano-chelating agent was prepared under conditions of pH 7.3-7.

7.

2. The application as described in claim 1, characterized in that, The nanomaterial is nanofiber.

3. The application as described in claim 1, characterized in that, The preparation method of Zn-penicillamine nano-chelating agent is as follows: D-penicillamine and divalent zinc salt are mixed evenly in solution, the pH is adjusted to neutral or alkaline, and the reaction is continuously stirred to obtain Zn-penicillamine nano-chelating agent; the pH value is adjusted to 7.3~7.

7.

4. The application as described in claim 3, characterized in that, The molar ratio of D-penicillamine to divalent zinc salt is 1:0.9~1.

1.

5. The application as described in claim 3, characterized in that, The reaction was continuously stirred for 11-13 hours.

6. The application as described in claim 1, characterized in that, The pharmaceutical composition is administered via subcutaneous injection or intravenous injection.

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