Nanoscale coordination polymer np@doxcuc based on copper ions and doxorubicin and preparation method and application thereof

Abstract

The application discloses a nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin as well as a preparation method and application thereof, and relates to the technical field of ovarian cancer treatment. 2+ The nanoscale coordination polymer NP@DoxCu uses a ROS-sensitive polymer PSSP, doxorubicin Dox and Cu 2+ The application constructs a ROS-sensitive copper apoptosis nanoplatform NP@DoxCu. 2+ The platform is rapidly decomposed under the action of intracellular reductase and in a low-pH environment after entering cells; Cu 2+ cooperates with doxorubicin to not only directly kill cancer cells but also induce immune cell death; in addition, the NP@DoxCu remodels the tumor immune microenvironment, reorients tumor-associated macrophages (TAMs) and regulates T cells, thereby realizing effective immunotherapy in vivo; the combination of the copper apoptosis and the immunotherapy provides a new strategy for the treatment of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of ovarian cancer treatment technology, specifically to the nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is the deadliest gynecological cancer, severely impacting the health and quality of life of women worldwide. The standard treatment for this cancer is surgery and platinum-paclitaxel chemotherapy, but recurrence rates after these treatments remain high. Immunotherapy is the fourth pillar of cancer treatment. However, response rates to immunotherapy for ovarian cancer are not as high as expected. Ovarian cancer often exhibits characteristics of an "immunocold tumor," characterized by low immunogenicity and an immunosuppressive tumor microenvironment.

[0003] Copper death is a recently discovered non-apoptotic programmed cell death induced by excessive intracellular copper. It induces cell death through intracellular metabolic disturbances and oxidative stress, while simultaneously triggering immunogenic cell death (ICD) by activating the immune system's response to dying cells. However, simply increasing intracellular copper... 2+ The accumulated concentration is insufficient to induce copper poisoning. (Cu) 2+ Dissociation with ion support and Cu 2+ Binding to the DLAT protein constitutes two fundamental steps in copper adsorption. Therefore, finding a method that can effectively adsorb Cu... 2+ Developing excellent delivery vehicles to tumor cells is a challenge. The emergence of metal-based nanoparticles offers new opportunities to address this problem. Metal-based nanoparticles, linked by coordination bonds between transition metal ions and chemotherapeutic drugs, have become a hot topic in nanomedicine.

[0004] Doxorubicin (Dox) is a first-line chemotherapy drug for cancer and a topoisomerase IIα inhibitor, making it an excellent inducer of type ICD effects. Dox induces immunogenic cell death and promotes cytotoxic T-cell immune responses through damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), adenosine triphosphate (ATP), and high-mobility group protein B1 (HMGB1). However, when used as a monotherapy, the clinical application of Dox is always limited by low bioavailability and cardiotoxic adverse reactions.

[0005] Therefore, we have developed a nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, along with its preparation method and applications, to address the problems mentioned above.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, its preparation method and application, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, wherein the nanoscale coordination polymer NP@DoxCu uses the ROS-sensitive polymer PSSP, doxorubicin Dox, and Cu. 2+ Build.

[0009] This invention also provides a method for preparing the nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, comprising the following steps:

[0010] S1: Dissolve CuCl2 (1 mg) and Dox (5.43 mg) in DMSO (500 μL) and add it dropwise to deionized water;

[0011] S2: Then quickly add Tris solution (20 mmol, 6.6 mL) and mix rapidly;

[0012] S3: Next, add a PSSP (10 mg) solution dissolved in deionized water (200 μL);

[0013] S4: The final solution was purified by dialysis and collected to obtain NP@DoxCu.

[0014] Preferably, the dialysis molecular weight cutoff is 3500 Da.

[0015] This invention also provides the application of the above-mentioned copper ion and doxorubicin-based nanoscale coordination polymer NP@DoxCu in the preparation of ovarian cancer therapeutic drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention utilizes ROS-sensitive polymers PSSP, Dox, and Cu. 2+A ROS-sensitive copper apoptosis nanoplatform, NP@DoxCu, was constructed. After entering cells, this platform rapidly decomposes under the action of intracellular reductases and in a low pH environment; Cu 2+ In synergy with doxorubicin, it not only directly kills cancer cells but also induces immune cell death. In addition, NP@DoxCu reshapes the tumor immune microenvironment, redirects tumor-associated macrophages (TAMs), and regulates T cells, thereby achieving effective immunotherapy in vivo. This combination of copper apoptosis and immunotherapy provides a new strategy for the treatment of ovarian cancer.

[0018] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0019] Figure 1 (A) Schematic diagram of the preparation method of NP@DoxCu; (B) Schematic diagram of NP@DoxCu in chemotherapy and immunotherapy; (C) Analysis of the relationship between DLAT expression and overall survival in ovarian cancer patients using Kaplan-Meier plot; (D) Analysis of the correlation between DLAT expression and LIAS mRNA expression in ovarian cancer tumor tissue; (E) Analysis of the correlation between DLAT expression and FDX1 mRNA expression in ovarian cancer tumor tissue.

[0020] Figure 2 (A) UV-vis spectra of Cu2+, Dox, and NP@DoxCu; (B) Size distribution and TEM image of DoxCu; (C) Size distribution and TEM image of NP@DoxCu, scale bar: 100 nm; (D) Stability of NP@DoxCu in PBS over 7 days; (E) Stability of NP@DoxCu in 10% fetal bovine serum over 7 days; (F) Cu released from different pH values. 2+ (G) Dox released from different pH values; (H) Representative TEM image of NP@DoxCu at pH 5.5; (I) Different groups assessed by ⋅OH levels; *P<0.05, **P<0.01, ***P<0.001; ns indicates P>0.05;

[0021] Figure 3(A) CLSM image of SKOV3 cells after treatment with Rh B-labeled nanoparticles for 6 hours; scale bar: 25 μm; (B) Quantitative analysis of mean Rho B intensity of intracellular NPs; (C) Quantitative analysis of intracellular NPs by flow cytometry; *P<0.05, **P<0.01, ***P<0.001; ns indicates P>0.05;

[0022] Figure 4 (A) Relative survival rates of SKOV3 and ID8 cells after 72 hours of treatment with PBS, copper chloride, Dox, NP@Dox, and NP@DoxCu; (B) Live cells stained with fluorescein diacetate (green), and dead cell nuclei stained with PI (red); Scale bar: 100 micrometers; (C) Apoptotic images of SKOV3 cells after 24 hours of different treatments;

[0023] Figure 5 (A) DLAT aggregation in SKOV3 cells after different treatments, scale bar 10 μm; (B) Western blot and quantitative analysis of copper protein apoptosis-related proteins (FDX1 and LIAS) expression after different treatments; (C) GSH level in SKOV3 cells after treatment with different concentrations of NP@DoxCu; (D) ROS level in SKOV3 cells after treatment with different formulations; (E) ATP level in SKOV3 cells 24 hours after different treatments; (F) Representative confocal microscopy images of CRT, scale bar 100 μm; (G) Flow cytometry analysis of the maturation status of DCs collected from mouse lymph nodes after different treatments; *P<0.05, **P<0.01, ***P<0.001; ns indicates P>0.05;

[0024] Figure 6 (A) In vivo fluorescence distribution imaging of Cy5.5-labeled NP@DoxCu; (B) In vitro fluorescence distribution imaging of Cy5.5-labeled NP@DoxCu; (C) Schematic diagram of the establishment of an ovarian cancer PDX model and the in vivo treatment process; Tumor growth curves (D), mean tumor weight (E), and photographs (G) of mice after intravenous injection of different formulations; (H) H&E staining and Tunel staining results of tumor tissue after treatment, scale bar: 200 micrometers;

[0025] Figure 7(A) Schematic diagram of treatment regimens for C57BL / 6 mice; tumor images (B), tumor growth curves (C), and tumor weight (D) of mice in different treatment groups; representative FCM atlases (E) and proportions (F) of M1 macrophages (F40 / 8+ CD86+) in tumors of mice in different treatment groups; representative FCM atlases (G) and proportions (H) of M2 macrophages (F40 / 8+ CD206+) in tumors of mice in different treatment groups; (I) Immunohistochemical analysis of tumor-associated macrophage (TAMs) polarization in tumor tissues of mice after different treatments, scale bar: 100 micrometers; representative FCM atlases (J) and proportions (K) of CD8+ T cells in tumors of mice in different treatment groups; CD86+ T cells in tumors of mice in different treatment groups. Representative FCM map (L) and proportion (M) of T cells; the levels of inflammatory factors IL-6 (N), TNF-α (O), and IFN-γ (P) in the serum of mice after different treatments were measured by ELISA; data are presented as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001; ns indicates P>0.05.

[0026] Figure 8 PSSP synthesis roadmap;

[0027] Figure 9 : 1H NMR spectrum of PSSP in DMSO-d6;

[0028] Figure 10 A schematic diagram of the NP@DoxCu preparation process;

[0029] Figure 11 Zeta potentials of NP@Dox and NP@DoxCu;

[0030] Figure 12 STEM image of NP@DoxCu and corresponding elemental distribution map, scale bar: 100 nm;

[0031] Figure 13 Results of hemolysis tests for CuCl2, Dox, and NP@DoxCu;

[0032] Figure 14 Body weight changes in ovarian cancer PDX model mice during treatment;

[0033] Figure 15 : Levels of major serum biochemical indicators in different groups after treatment, n=3;

[0034] Figure 16Representative H&E stained histological images of tissues, including heart, liver, spleen, lung and kidney, in ovarian cancer PDX model mice after different treatments; scale bar: 100 μm; Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention provides a nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, using the ROS-sensitive polymer PSSP, doxorubicin Dox, and Cu. 2+ Build.

[0037] This invention also provides the application of the aforementioned copper ion and doxorubicin-based nanoscale coordination polymer NP@DoxCu in the preparation of ovarian cancer therapeutics. NP@DoxCu nanoparticles exhibit strong glutathione-consuming capacity, controllable drug release characteristics, and good in vitro and in vivo stability. When these nanoparticles are absorbed by tumor cells, the PSSP coating responds to high concentrations of glutathione and an acidic pH tumor microenvironment through biodegradation, thereby releasing doxorubicin and copper ions. On one hand, the released copper ions (Cu²⁺) trigger a Fenton-like reaction, enhancing the chemotherapeutic effect of doxorubicin and inducing tumor cell apoptosis. On the other hand, copper ion-induced copper protein apoptosis combines with doxorubicin-mediated immune effects, remodeling the immune microenvironment in ovarian cancer tissue.

[0038] 1. Characteristic analysis of NP@DoxCu

[0039] Copper-dependent apoptosis is a form of cell death driven by the lipoylation of over-esterified dihydroacetamide S-acetyltransferase (DLAT). The Kaplan-Meier graph database was used for the first time to investigate the relationship between DLAT expression levels and overall survival. Figure 1 As shown in Figure C, ovarian cancer patients with high DLAT expression had significantly longer overall survival than those with low expression. Furthermore, the study investigated two key copper-dependent apoptosis proteins in ovarian cancer—LIAS and FDX1—and the results showed that DLAT expression was correlated with LIAS (…). Figure 1 D) and FDX1 ( Figure 1 The expression of DLAT was positively correlated with that of E. In conclusion, increased DLAT expression is associated with poor prognosis in ovarian cancer patients. FDX1 and LIAS, as key players in copper-dependent apoptosis, are also closely related to DLAT expression in ovarian cancer.

[0040] The synthetic route of PSSP (glutathione-responsive polymer) is as follows: Figure 8 As shown, its structure is achieved through... 1 H NMR analysis confirmed this, see Figure 9 The preparation method of NP@DoxCu is as follows: Figure 1 A and Figure 10 As shown. Cu 2+ The coordination interaction with Dox was verified by significant changes in the UV-vis spectrum. Figure 2 A). Cu 2+ The optimal nanoparticles with a diameter of 87.8 ± 0.94 nm and a PDI of 0.35 ± 0.23 were formed with Dox. Figure 2 B). After PSSP coating, the particle size of NP@DoxCu increased to 104.06±0.98 nm, and the PDI was 0.24±0.01 (B). Figure 2 C). During the synthesis of NP@DoxCu, changes in the Zeta potential can be observed ( Figure 11 Transmission electron microscopy (TEM) revealed that the nanoparticles were spherical with a relatively uniform size distribution. Elemental mapping showed that C, N, O, and Cu were uniformly distributed in NP@DoxCu. Figure 12 The results showed that the particle size or PDI of NP@DoxCu did not change significantly over 7 days in PBS buffer and 10% FBS. Figure 2 D, Figure 2 E), which proves the stability of nanoparticles.

[0041] Since metal coordination bonds are generally stronger than hydrogen bonds, the responsiveness of nanoparticles was studied by testing the release of NP@DoxCu under different pH conditions. Figure 2 F and Figure 2 As shown in G, Dox and Cu 2+ The release trends were similar. At pH 7.4, the nanoparticles were relatively stable, with only about 10% Cu released after 72 hours of incubation. 2+ Dox was released. In contrast, under acidic conditions (pH=5.5), the release rate increased significantly, with over 80% of Dox released within the same experimental time. This increase was primarily due to the breaking of coordination bonds, which is highly sensitive to changes in external pH. Therefore, it is speculated that NP@DoxCu generally remains stable in blood and tissue fluid, avoiding premature drug release and ensuring a long half-life in blood circulation. Upon reaching the tumor site, the small molecule is rapidly released in the acidic tumor microenvironment.

[0042] At the tumor site, the components within the nanoparticles were released after the pH-sensitive connections were disrupted. Transmission electron microscopy (TEM) results further confirmed the pH responsiveness of NP@DoxCu. Figure 2 H). In addition, the released Cu 2+ By reacting with the generated hydroxyl radicals through the Fenton reaction, the intracellular redox balance is disrupted. Figure 2 I).

[0043] 2. Cellular uptake of NP@DoxCu

[0044] Cellular uptake of nanomaterial drugs is crucial for controlling their bioavailability in vivo. To assess the cellular uptake of NP@DoxCu, rhodamine B was encapsulated in nanoparticles as a probe, and its uptake was observed using confocal laser scanning microscopy (CLSM).

[0045] The uptake of nanoparticles was analyzed by confocal laser scanning microscopy (CLSM) and flow cytometry after incubation for 2 to 8 hours. A gradual increase in red fluorescence intensity was observed from 2 to 6 hours, indicating that NP@DoxCu was gradually absorbed intracellularly. Figure 3 A). The internalization process was quantified using flow cytometry. Figure 3 B), the results consistently showed that SKOV3 cells uptake increased almost sixfold after 6 hours of incubation. Figure 3 C), which indicates that NP@DoxCu can effectively enter the cell interior.

[0046] 3. Cytotoxicity of NP@DoxCu

[0047] After confirming the excellent uptake capacity of NP@DoxCu, its antitumor effects in SKOV3 and ID8 cells were further evaluated. Figure 4 As shown in Figure A, copper chloride was non-toxic in both cell lines, while the half-maximal inhibitory concentration (WMC) of Dox was 4.73 ± 1.27 μM in SKOV3 cells and 4.08 ± 0.56 μM in ID8 cells. Notably, NP@DoxCu exhibited the strongest antitumor effect, with a WMC of 1.72 ± 0.29 μM in SKOV3 cells and 2.22 ± 1.20 μM in ID8 cells. Subsequent experiments showed that after 48 hours of treatment, the apoptotic cell rate was only 4.96% in the PBS group, 5.28% in the copper chloride group, 25.02% in the Dox group, and as high as 31.00% in the NP@Dox group. Figure 4 C). In contrast, NP@DoxCu treatment significantly increased the proportion of apoptotic cells, reaching 72.00% ( Figure 4C). Further live / dead cell assays confirmed these results; NP@DoxCu resulted in a significant increase in red fluorescence, indicating a significant increase in the number of dead cells, consistent with the results of MTT and apoptosis assays. Figure 4 B).

[0048] 4. NP@DoxCu-induced cytotoxicity mechanism

[0049] Copper cell apoptosis is a newly discovered form of programmed cell death mediated by excess copper ions. To investigate the mechanism of NP@DoxCu-induced copper cell apoptosis, the oligomerization process of dihydrolipidamide S-acetyltransferase (DLAT) was observed using immunofluorescence imaging. Figure 5 As shown in Figure C, DLAT focal points were almost undetectable in cells treated with PBS, copper chloride, and Dox, while cells treated with NP@DoxCu showed significant DLAT aggregation. The FDX1 gene encodes a reductase that converts Cu2+ to the more toxic Cu+, which can downregulate Fe-S cluster proteins, such as LIAS, an important marker of copper cell apoptosis. Therefore, the expression of LIAS and FDX1 in SKOV3 cells treated with each group was detected by Western blotting. The results showed that the expression levels of LIAS and FDX1 decreased significantly after treatment, indicating that NP@DoxCu can effectively induce copper cell apoptosis. Figure 5 D).

[0050] High levels of intracellular reactive oxygen species (ROS) can induce immune cell death (ICD). ROS levels, measured by flow cytometry and fluorescence microscopy, show that... Figure 5 As shown in C and 5D, the ROS generation in the NP@DoxCu group was significantly higher than that in other groups. 2+ Doxorubicin (Dox) is a known potent ICD inducer. ICD promotes an immune antitumor response by releasing damage-associated molecular patterns (DAMPs) in dying tumor cells, among which HMGB1 and ATP are key biomarkers of ICD in cancer cells. Immunofluorescence staining showed that the HMGB1 green fluorescence was significantly reduced in the NP@DoxCu group compared to other groups ( Figure 5 F), which is consistent with the decrease in ATP levels ( Figure 5E). These data confirm that NP@DoxCu can effectively induce tumor cell death via the ICD pathway. To further investigate the effects of NP@DoxCu on the tumor microenvironment (TME), the maturation of dendritic cells (DCs) in SKOV3 cells was studied by flow cytometry. The results showed that the proportion of mature DCs (CD80+CD86+) was the highest in the NP@DoxCu group (21.2%), while the proportions in the PBS group (7.67%), copper chloride group (8.80%), Dox group (15.2%), and NP@Dox group (17.6%) were lower, indicating that NP@DoxCu can effectively promote DC maturation.

[0051] 5. In vitro processing and accumulation of NP@DoxCu

[0052] To investigate the biodistribution and tumor targeting efficiency of NP@DoxCu in vivo, the near-infrared cyan dye Cy5.5 was encapsulated within the hydrophobic core of NP@DoxCu nanoparticles. For example... Figure 6 As shown in Figure A, free Cy5.5 mainly accumulated in the kidneys and liver, and exhibited weak fluorescence at the tumor site 24 hours later, indicating that the free dye was primarily cleared by the liver and kidneys. In contrast, the NP@DoxCu group showed a relatively stable fluorescence signal at the tumor site. In vitro fluorescence imaging 24 hours after injection showed that the fluorescence intensity of NP@DoxCu-injected mice was significantly higher than that of free Cy5.5-injected mice. Figure 6 B) This indicates that NP@DoxCu has stronger tumor targeting and longer retention time.

[0053] To evaluate the in vivo therapeutic effect of NP@DoxCu, an ovarian cancer PDX mouse model was established ( Figure 6 C). Tumor volume and mouse weight were monitored daily, and treatment was initiated when the tumor volume reached 70 mm³. Tumor growth curves for different treatment groups are shown below. Figure 6 D. After 12 days of treatment, the tumor volume in the NP@DoxCu group was 210.09 mm³, while the mean tumor volume in the PBS-treated group was 1419.04 mm³. Figure 6 D). The tumor weight in the PBS group was 18.22 times that in the NP@DoxCu group (1.64±0.36 g vs. 0.09±0.08 g). Figure 6 E, 6G). After treatment, the mice were euthanized, and tumor tissue was collected for hematoxylin-eosin (H&E) staining. Significant nuclear shrinkage and fragmentation were observed in the NP@DoxCu group ( Figure 6 H). Similarly, stronger apoptosis markers were detected in tumor sections treated with NP@DoxCu. Figure 6H). To further elucidate the molecular mechanism of NP@DoxCu at the tumor site, the expression of LAIS and DLAT was examined. These results collectively validated the NP@DoxCu-induced apoptosis effect of copper proteins.

[0054] 6. NP@DoxCu reprograms the tumor microenvironment to activate anti-tumor immune responses in vivo.

[0055] To investigate the immune mechanism of NP@DoxCu against tumor progression, tumor samples from the ID8 model were further collected to analyze their immune parameters. Figure 7 A). In the experiment, C57BL / 6 mice received three intravenous injections of PBS, PD-L1, NP@DoxCu, and NP@ESCu + PD-L1 over three consecutive days. The results showed that tumor growth was significantly inhibited in the NP@DoxCu + PD-L1 group compared to the PBS, PD-L1, and NP@ESCu groups. Figure 7 B). Specifically, the tumor volumes of mice treated with PBS, PD-L1, NP@DoxCu, and NP@DoxCu+PD L1 were 694.70±71.12, 434.75±109.31, 377.14±60.57, 207.675, and 207.68±119.44 mm³, respectively. Figure 7 C). Furthermore, measurements of tumor weight also confirmed the above findings. Figure 7 D). In summary, the above results indicate that NP@DoxCu + PD-L1 has a significant anti-tumor effect on the ID8 tumor model.

[0056] Multiple studies have confirmed that effective immune checkpoint inhibitors (ICDs) can promote the maturation of dendritic cells (DCs) and the infiltration of cytotoxic T lymphocytes (CTLs), thereby reversing the tumor-induced immunosuppressive microenvironment. The maturation process of DCs and T cells was investigated by measuring cell surface marker levels using flow cytometry. Figure 7 As shown in E-7H, after treatment, the number of M2 macrophages in the NP@DoxCu treatment group was significantly reduced, while the number of M1 macrophages increased accordingly. Furthermore, the protein expression of iNOS (a marker for M1 tumor-associated macrophages) and CD206 (a marker for M2 tumor-associated macrophages) was analyzed by immunohistochemistry. Figure 7As shown in Figure I, iNOS was significantly increased and CD206 decreased in the NP@DoxCu group. These results suggest that NP@DoxCu may influence the immunosuppressive tumor microenvironment by modulating tumor-associated macrophages. Cytotoxic T lymphocytes (CTLs) play a crucial role in eliminating tumor cells by effectively inducing immune-associated cell death (ICD) and increasing T cell infiltration. Treatment effectively induced immune-associated cell death (ICD) and increased T cell infiltration.

[0057] Further analysis of tumor-infiltrating T cells was conducted. Results showed that the proportion of CD3+CD8+ T cells in tumors of mice treated with NP@DoxCu was significantly higher than that in mice treated with other drugs (30.6% in the NP@DoxCu group and 4.53% in the PBS group). Figure 7 J, Figure 7 K). Furthermore, the proportion of mature dendritic cells (DCs) in tumor tissue was investigated. Results showed that the proportion of mature DCs in the NP@DoxCu +PD-L1 group was 12.7%, which was 5 times higher than that in mice treated with PBS (2.53%). Figure 7 L, Figure 7 M).

[0058] Cytokines are key indicators for assessing the state of the immune response. This study measured the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). The results showed that in mice treated with ES@DoxCu+PD-1, the concentrations of TNF-α, IL-6, and IFN-γ were significantly increased. Figure 7 N- Figure 7 This further confirms the feasibility of NP@DoxCu + αPD-L1 in the treatment of ovarian cancer.

[0059] In summary, these data demonstrate that NP@DoxCu + PD-L1 can reprogram the tumor microenvironment and trigger an effective anti-tumor immune response in ovarian cancer.

[0060] Hemolysis tests showed that NP@DoxCu did not induce a significant hemolytic reaction, indicating that the nanoparticles do not interact with blood components and are therefore unlikely to have harmful effects after entering the bloodstream. Figure 13 To further evaluate the in vivo toxicity of NP@DoxCu, we assessed it using serum biochemistry, H&E staining, and body weight measurement. Results showed that mice treated with NP@DoxCu did not experience significant weight loss. Figure 14 No abnormalities in serum biochemistry were observed. Figure 15 ) or pathological changes ( Figure 16This indicates that NP@DoxCu has good biocompatibility. However, the safety of NP@DoxCu for clinical use still needs further validation.

[0061] We constructed NP@DoxCu nanoparticles via coordination bonds between copper ions and doxorubicin, and encapsulated them in a glutathione-responsive polymer. These nanoparticles are stable and reliable under physiological conditions and sensitive to the pH of the tumor site. After internalization, NP@DoxCu exerts its cytotoxicity through a synergistic effect of copper-induced cell death and chemotherapy. Furthermore, the combination of copper ion-induced cell death and doxorubicin-mediated immune effects remodels the immune microenvironment of ovarian cancer. Overall, NP@DoxCu demonstrates excellent antitumor efficacy through the synergistic effects of copper-induced apoptosis, chemotherapy, and immunotherapy, providing a novel strategy for the treatment of ovarian cancer.

[0062] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanoscale coordination polymer NP@DoxCu based on copper ions and doxorubicin, characterized in that: The nanoscale coordination polymer NP@DoxCu uses a polymer PSSP, doxorubicin Dox and Cu 2+ construction; The structural formula of the polymer PSSP is as follows: ； The preparation method of the nanoscale coordination polymer NP@DoxCu comprises the following steps: S1: 1 mg of CuCl2 and 5.43 mg of Dox are dissolved in 500 μL of DMSO, and are added dropwise into deionized water; S2: Then, 6.6 mL of a Tris solution with a concentration of 20 mmol is quickly added and is quickly mixed; S3: Next, 10 mg of a PSSP solution dissolved in 200 μL of deionized water is added; S4: The final solution is purified by dialysis with a molecular weight cut-off of 3500 Da, and NP@DoxCu is collected.

2. Use of the nanoscale coordination polymer NP@DoxCu according to claim 1 in the preparation of a drug for treating ovarian cancer.

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