Application of cyano-bridged bimetallic nano-enzyme in preparation of tumor catalytic treatment medicine

Abstract

The invention discloses application of cyano-bridged bimetallic nano-enzyme in preparation of a tumor catalytic treatment medicine, and belongs to the technical field of medicines. The invention provides a design strategy of a bimetallic nano-enzyme taking coordination field engineering as a theoretical basis and cyano bridging as a structural carrier, and Cu2 [Fe (CN) 6] is adopted to prepare a tumor catalytic treatment drug. In the nano enzyme, the cyano group is used as a strong field bridging ligand and can form an electron delocalization network, and meanwhile, due to different electronegativity of copper and iron, the d orbital energy levels of copper and iron can generate synergistic distortion, so that electron transfer is synergistically driven, and the electron transfer efficiency is remarkably improved. In addition, by virtue of a cyano bridging effect, the stabilization effect of a strong coordination field can inhibit metal agglomeration, so that Cu is in monatomic distribution, and the catalytic treatment capability of the nano-enzyme is further enhanced. After being modified by an immunogenic tumor exosome, the nano-enzyme not only improves the targeting property to tumors, but also can exert high-efficiency peroxidase-like and glutathione-like oxidase activity in a tumor microenvironment, so that catalysis-immune synergistic treatment is realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of a cyano-bridged bimetallic nanozyme in the preparation of tumor catalytic therapeutic drugs. Background Technology

[0002] In recent years, nanozyme-based catalytic therapies have opened new avenues for cancer treatment due to their high stability, tunable catalytic activity, and cost-effectiveness. Nanozymes can mimic the catalytic function of natural enzymes, inducing cancer cell death by regulating biochemical reactions in the tumor microenvironment, making them a highly promising therapeutic strategy. However, both traditional and single-atom nanozymes still face the following challenges in multi-step catalytic reactions: the metal active center struggles to simultaneously optimize the adsorption and desorption barriers of key intermediates. The crux of this problem lies in the lack of a dynamic regulatory mechanism for the electronic structure of the active center, leading to an imbalance in the adsorption-desorption of reaction intermediates in single-metal nanozymes, severely limiting their catalytic efficiency and therapeutic efficacy.

[0003] To overcome this bottleneck, bimetallic catalysts have become a research hotspot. They can drive electron synergy through the redox potential difference of bimetallic sites to optimize the adsorption-desorption balance in enzyme-like activity. Existing diatomic nanozymes, such as MoCu diatomic nanozymes, enhance substrate adsorption and lower the energy barrier through the introduction of Cu atoms to form diatomic sites, exhibiting stronger catalytic activity than single-atom Mo enzymes. However, the electron transport mechanism of the diatomic sites in these nanozymes has inherent limitations. The metal sites (Mo-N4, Cu-N4) are indirectly coordinated through a nitrogen-doped carbon support. The π-conjugation effect of the carbon skeleton of the support can only provide limited electron transport capacity, resulting in the redox potential difference between the metal sites not being efficiently converted. The electronic structure of the active center is difficult to dynamically control, making it difficult to effectively regulate the intermediate adsorption-desorption imbalance in the process of nanozymes exerting POD-like enzyme activity to catalyze H2O2. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of a cyano-bridged bimetallic nanozyme in the preparation of tumor catalytic therapeutic drugs.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing cyano-bridged bimetallic nanozymes, comprising the following steps: dispersing citric acid and K4[FeⅡ(CN)6] in water to obtain solution A; dispersing CuSO4·5H2O in water to obtain solution B; adding solution B to solution A under stirring conditions to obtain the coordination field engineered cyano-bridged bimetallic nanozyme with the chemical formula Cu2[Fe(CN)6].

[0007] Furthermore, the molar ratio of citric acid, K4[FeⅡ(CN)6] and CuSO4·5H2O is (1~2):(0.1~2):(0.1~2).

[0008] Furthermore, the stirring speed is 200-1000 rpm, and the stirring time is more than 24 hours.

[0009] The present invention also provides a cyano-bridged bimetallic nanozyme prepared by the above preparation method, with the chemical formula Cu2[Fe(CN)6].

[0010] The present invention also provides the application of cyano-bridged bimetallic nanozymes in the preparation of tumor catalytic therapeutic drugs, wherein the cyano-bridged bimetallic nanozyme is Cu2[Fe(CN)6](SANE).

[0011] Technical Principle: This invention proposes a bimetallic nanozyme design strategy based on ligand field engineering and using cyano-bridged structures as the structural carrier. A cyano-bridged bimetallic nanozyme, Cu2[Fe(CN)6], is used to prepare tumor catalytic therapeutic drugs. Cu and Fe metals form a directional electron cloud shift (Fe→Cu) through an electronegativity gradient, precisely regulating the electronic structure of the active center to balance intermediate adsorption-desorption behavior. The cyano group (-C≡N) synergistically interacts with the electronegativity difference between Cu and Fe through its delocalized π bond, constructing an electron delocalized network that converts the bimetallic redox potential difference into a directional electron flow. Simultaneously, the single-atom dispersed coordination environment of copper hexacyanide inhibits metal aggregation, providing an atomic-level substrate for dynamically regulating the electronic structure. This fundamentally solves the problem of intermediate adsorption-desorption imbalance caused by insufficient electronic structure regulation in single-metal nanozymes, as well as the problem of traditional bimetallic systems being unable to achieve dynamic regulation due to low electron transfer medium efficiency.

[0012] The present invention also provides a tumor catalytic therapeutic drug, the raw materials of which include cyano-bridged bimetallic nanozyme (SANE) and tumor immunogenic exosome (iEV).

[0013] Furthermore, the particle mass ratio of the cyano-bridged bimetallic nanozyme to the tumor immunogenic exosome is (0.5~4):1.

[0014] Furthermore, the preparation method of the tumor catalytic therapeutic drug includes the following steps: mixing tumor immunogenic exosomes with cyano-bridged bimetallic nanozymes, and then performing ultrasonic treatment in an ice bath to obtain the tumor catalytic therapeutic drug.

[0015] Furthermore, the power of the ultrasound is 50~500W, and the duration of the ultrasound is more than 5 minutes.

[0016] Furthermore, the extraction method for the tumor immunogenic exosomes is differential ultracentrifugation.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] This invention employs a bimetallic nanozyme design strategy based on ligand field engineering theory and cyano-bridged structures as the structural carrier. It utilizes a cyano-bridged bimetallic nanozyme Cu2[Fe(CN)6] to prepare tumor-catalyzing therapeutic drugs. The cyano group (-C≡N) in Cu2[Fe(CN)6] acts as a strong-field bridging ligand, forming an electron delocalized network. This network, facilitated by Cu(d... 9 ) and Fe(d 6 The electronegativity gradient of the bimetallic active center induces synergistic distortion of the d-band energy level, synergistically driving the electron transfer process and significantly improving its efficiency. Furthermore, through cyano bridging, the stabilizing effect of its strong ligand field inhibits metal aggregation, resulting in a single-atom distribution of Cu, further enhancing the catalytic therapeutic capability of the nanozyme. After modification with immunogenic tumor exosomes (iEVs), this nanozyme not only improves its targeting of tumors but also exhibits highly efficient peroxidase-like (POD) and glutathione oxidase-like (GSHox) activities in the tumor microenvironment, achieving catalytic-immunotherapy synergistic therapy.

[0019] The tumor catalytic therapeutic drug iEV@SANE in this invention achieves a synergistic effect by efficiently generating reactive oxygen species (ROS) and depleting glutathione (GSH) in the tumor microenvironment, inducing ferroptosis in tumor cells and triggering an immune response. Through an innovative approach of "ligand field engineering—electronic structure regulation—catalytic efficiency optimization," it overcomes the challenges of adsorption-desorption imbalance and low electron transfer efficiency in traditional nanozymes, opening a new pathway for metal-ligand synergistic design in cancer catalytic therapy. Attached Figure Description

[0020] 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 undue limitation of the invention. In the drawings:

[0021] Figure 1 A schematic diagram illustrating the intracellular therapeutic mechanism of the ligand field-engineered cyano-bridged bimetallic nanozyme provided by the present invention in the preparation of tumor catalytic therapeutic drugs;

[0022] Figure 2Characteristic analysis of the SANE prepared in Example 1, where a is a TEM image, b is an XRD pattern, c is the XPS spectrum of Cu 2p, d is the XPS spectrum of Fe 2p, e is an atomic-level HAADF-STEM image, f is the Cu K-edge XANES spectrum of SANE and the reference sample, and g is the Fourier transform amplitude of the experimental Cu K-edge FT-EXAFS signal of SANE and the reference sample in R space.

[0023] Figure 3 The enzyme activity of SANE is characterized as follows: a represents the GSHox-like activity of PB and SANE; b and c represent the Michaelis saturation curves and double reciprocal fitting curves of PB and SANE at different concentrations of GSH; d represents the POD-like activity of PB and SANE; and ef represents the Michaelis saturation curves and double reciprocal fitting curves of PB and SANE at different concentrations of hydrogen peroxide.

[0024] Figure 4 Flow cytometry analysis of LLC cells stained with Annexin-VFITC and PI after treatment with SANE, CEV@SANE and iEV@SANE;

[0025] Figure 5 In the middle section, a is a transmission electron microscope image of LLC cells treated with iEV@SANE, and b is a Western blot image of GPX4 protein expression level in LLC cells treated with SANE, CEV@SANE and iEV@SANE.

[0026] Figure 6 In the middle section, a represents the flow cytometry analysis results of CRTs exposed on the surface of LLC cells after treatment with SANE, CEV@SANE, and iEV@SANE; b represents the intracellular ATP levels of LLC cells after treatment with SANE, CEV@SANE, and iEV@SANE.

[0027] Figure 7 Flow cytometry analysis results of DC2.4 cells after treatment with SANE, CEV@SANE and iEV@SANE;

[0028] Figure 8 The results of in vivo anti-tumor therapy evaluation are shown in Figure a, where a is the fluorescence image of mice after intravenous injection of different nanomedicines at different time points, and b is the fluorescence image of major organs and tumors in each group 24 hours after injection.

[0029] Figure 9 The results of in vivo anti-tumor treatment evaluation are shown in the following figures: a) is a photograph of the collected tumors; b) is the tumor growth curve of mice carrying LLC tumors after specified treatment (n=5); and c) is the average weight of tumors harvested from different groups (n=5). Detailed Implementation

[0030] 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Terminology Explanation:

[0033] Ligation field engineering: a molecular design technique that optimizes the catalytic, photoelectric, and other properties of materials by controlling the types, configurations, and electronic effects of ligands around a metal center.

[0034] Cyano bridging: The strong field effect (Δ0>P) of cyano ligands can induce specific electronic configurations (such as d... 6 d 7 Metal ions form low-spin configurations, while simultaneously utilizing configurations containing asymmetric electrons (such as d...). 9 With d 6 The bimetallic electronegativity gradient (combined) induces a coordinated distortion of the d-band energy level, which regulates the desorption kinetics of hydroxyl radicals through the Jahn-Teller effect.

[0035] The coordination field engineering in this invention refers to utilizing the synergistic effect between ligands and the metal center to induce energy splitting of the d orbitals of metal ions, thereby altering the electronic configuration and achieving dynamic control of the electronic structure of the active center. Prussian blue analogues (PBAs) possess a MI-N≡C-MII topology, combining a strong crystal field splitting effect with delocalized π-electron conjugation. Within their three-dimensional framework, the strong field effect of the cyano ligands leads to specific electronic configurations (such as d orbitals). 6 d 7 Metal ions exhibit a low-spin configuration. Simultaneously, they possess asymmetric electronic configurations (such as d...). 9 / d 6 The electronegativity gradient between the bimetals (in combination) triggers a cooperative twisting of the d-band energy levels, regulating the desorption kinetics of hydroxyl radicals (•OH) through the Jahn-Teller effect. This electron density rearrangement mechanism based on ligand field strength constructs a metal-ligand cooperative electron delocalization network through cyano bridging, converting the bimetallic redox potential difference into a directional electron flow. This fundamentally solves the problem of intermediate adsorption-desorption imbalance caused by insufficient electronic structure regulation in single-metal nanozymes, as well as the problem of the inability to achieve dynamic regulation in traditional bimetallic systems due to low electron transfer medium efficiency.

[0036] This invention provides a method for preparing cyano-bridged bimetallic nanozymes, comprising the following steps: dispersing citric acid and K4[FeⅡ(CN)6] in water to obtain solution A; dispersing CuSO4·5H2O in water to obtain solution B; adding solution B to solution A under stirring conditions to obtain the ligand field engineered cyano-bridged bimetallic nanozyme with the chemical formula Cu2[Fe(CN)6]. This invention successfully synthesizes the cyano-bridged bimetallic nanozyme Cu2[Fe(CN)6] via a one-step self-assembly method. Due to the strong coordination interaction between Cu ions and the N-terminus of the bridging ligand [FeⅡ(CN)6], in the presence of citric acid, the bridging ligand solution and the Cu ion solution immediately form a red wine-like suspension, thus successfully preparing Cu2[Fe(CN)6].

[0037] In a preferred embodiment, the molar ratio of citric acid, K4[FeⅡ(CN)6] and CuSO4·5H2O is (1~2):(0.1~2):(0.1~2), and more preferably 1.561:0.1:0.2.

[0038] In a preferred embodiment, with the amount of K4[FeⅡ(CN)6] being 0.1 mmol, the water content in solution A is 20 mL.

[0039] In a preferred embodiment, with CuSO4·5H2O used in an amount of 0.2 mmol, the water content in solution B is 10 mL.

[0040] In a preferred embodiment, the water in both solution A and solution B is ultrapure water.

[0041] In a preferred embodiment, the stirring speed is 200-1000 rpm, more preferably 600 rpm, and the stirring time is 24 hours or more, more preferably 48 hours or more.

[0042] In a preferred embodiment, the process further includes centrifugation and washing after stirring; the washing reagent is ultrapure water, and the washing is performed three times.

[0043] This invention also provides an application of cyano-bridged bimetallic nanozymes in the preparation of tumor-catalyzing therapeutic drugs, wherein the cyano-bridged bimetallic nanozyme is Cu2[Fe(CN)6]. This invention utilizes the iron-copper bimetallic nanozyme Cu2[Fe(CN)6] to prepare tumor-catalyzing therapeutic drugs, selecting Cu and Fe to construct an asymmetric Fe-C≡N-Cu active center, stemming from the unique electronic configuration complementarity between the two: Fe... 2+ d 6The configuration forms a low-spin t2g under the strong field of the cyano group. 6 e.g. 0 Configuration (Δ0>P) enhances H2O2 adsorption affinity by shifting the d-band center upward; while Cu 2+ d 9 The configuration optimizes the desorption pathway of hydroxyl radicals due to the Jahn-Teller effect. The two radicals form an electron cloud shift (Fe→Cu) through an electronegativity gradient, which precisely regulates the electronic structure of the active center to balance the adsorption-desorption behavior of the intermediate.

[0044] This invention also provides a tumor catalytic therapeutic drug, the raw materials of which include cyano-bridged bimetallic nanozymes (SANE) and tumor immunogenic exosomes (iEV).

[0045] In a preferred embodiment, the particle mass ratio of the cyano-bridged bimetallic nanozyme to the tumor immunogenic exosome is (0.5~4):1, more preferably 1:1.

[0046] In a preferred embodiment, the preparation method of the tumor catalytic therapeutic drug includes the following steps: mixing tumor immunogenic exosomes with cyano-bridged bimetallic nanozymes, and then performing ultrasonic treatment in an ice bath to obtain the tumor catalytic therapeutic drug.

[0047] In a preferred embodiment, the power of the ultrasound is 50~500W, more preferably 100W, and the duration of the ultrasound is 5 minutes or more, more preferably 6 minutes.

[0048] In a preferred embodiment, the method for extracting tumor immunogenic exosomes is differential ultracentrifugation.

[0049] In a preferred embodiment, the extraction method of the tumor immunogenic exosomes specifically includes the following steps: tumor cells are seeded into DMEM medium containing fetal bovine serum and penicillin-streptomycin and cultured in a humidified incubator at 37°C and a carbon dioxide volume concentration of 5%; when the cell density reaches 80%, the cells are washed three times with PBS buffer, and then the medium is replaced with DMEM medium containing penicillin-streptomycin, and 0.1-5 mg / L or more of doxorubicin is added. After culturing for another 24 hours, the supernatant is collected, the obtained supernatant is continuously centrifuged, the precipitate is collected, and then the precipitate is washed with PBS buffer to obtain the tumor immunogenic exosomes.

[0050] In a preferred embodiment, the tumor cells are selected from LLC cells; the seeding amount of the tumor cells is 2 × 10⁻⁶. 6 indivual.

[0051] In a preferred embodiment, the volume content of fetal bovine serum in the DMEM culture medium containing fetal bovine serum is 10%, and the volume content of penicillin-streptomycin is 1%.

[0052] In a preferred embodiment, the volume content of penicillin-streptomycin in the DMEM culture medium containing penicillin-streptomycin is 1%.

[0053] In a preferred embodiment, the continuous centrifugation process is as follows: centrifugation at 300 rpm for 10 minutes, centrifugation at 2000 rpm for 10 minutes, centrifugation at 10000 rpm for 30 minutes, and centrifugation at 100000 rpm for 70 minutes.

[0054] In a preferred embodiment, the precipitate is washed with PBS buffer at a speed of 100,000 rpm for 70 minutes.

[0055] In this embodiment of the invention, room temperature refers to "25±2℃".

[0056] In the following examples, citric acid (CA) and copper sulfate pentahydrate (CuSO4·5H2O) were purchased from Aladdin. 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate (TMB) was purchased from Solarbio. Potassium hexacyanoferrate (K4[FeⅡ(CN)6]) and hydrogen peroxide solution (hydrogen peroxide) were purchased from Sigma-Aldrich. Nano-Prussian blue (PB) and rhodamine B were purchased from Macklin.

[0057] Reactive oxygen species (ROS) assay kit, mitochondrial membrane potential assay kit, cell counting kit-8 (CCK-8), lipid peroxidation assay kit (containing BODIPY 581 / 591 C11), ATP assay kit, Annexin V-FITC apoptosis assay kit, GSH and GSSG assay kits, and Calreticulin (CRT) rabbit monoclonal antibody were purchased from Beyotime Biotechnology. Mouse CD3 (APC-labeled), anti-mouse CD8 (FITC-labeled), anti-mouse CD86 (PE-labeled), and anti-mouse CD80 (FITC-labeled) were purchased from Elabscience Biotechnology. Phosphate-buffered saline (PBS), DMEM high-glucose medium, penicillin-streptomycin (PS), and fetal bovine serum (FBS) were purchased from Gibco Biotechnology. High-mobility group box 1 (HMGB1) antibody was purchased from Boster Biological Technology.

[0058] Glutathione peroxidase 4 (GPX4), IFN-γ ELISA kit, TNF-α ELISA kit, IL-12 ELISA kit, and IL-6 ELISA kit were all purchased from Epizyme Biotech.

[0059] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0060] Example 1

[0061] A method for preparing tumor catalytic therapeutic drugs using cyano-bridged bimetallic nanozymes:

[0062] The raw materials for preparing tumor catalytic therapeutic drugs are cyano-bridged bimetallic nanozymes (SANE) and tumor immunogenic exosomes (iEV); the cyano-bridged bimetallic nanozyme (SANE) is Cu2[Fe(CN)6];

[0063] The preparation process of the tumor catalytic therapy drug is as follows: iEV and SANE are mixed at a particle mass ratio of 1:1 and then ultrasonically treated in an ice bath at a power of 100W for 6 minutes to obtain the tumor catalytic therapy drug iEV@SANE.

[0064] The preparation process of Cu2[Fe(CN)6] is as follows: 300 mg of CA and 0.1 mmol of K4[FeⅡ(CN)6] are dispersed in 20 mL of ultrapure water to obtain solution A. 0.2 mmol of CuSO4·5H2O is dispersed in 10 mL of ultrapure water to obtain solution B. Subsequently, solution B is added to solution A under vigorous stirring at 600 rpm, immediately producing a red wine-colored turbidity. Stirring is continued for 48 hours, and the precipitate is collected by centrifugation. The obtained precipitate is washed three times with ultrapure water to obtain Cu2[Fe(CN)6].

[0065] The extraction process for iEV is as follows: 2×10 6LLC cells were seeded into 150-well plates containing 10 wt.% FBS and 1 wt.% PS in DMEM medium and cultured in a humidified incubator at 37°C and 5% CO2. When the cell density reached 80%, the cells were washed three times with PBS buffer. The medium was then replaced with DMEM medium containing 1 wt.% PS and 1 mg / L doxorubicin was added. After culturing for another 24 hours, the supernatant was collected. The supernatant was then centrifuged continuously (300 rpm for 10 minutes, 2000 rpm for 10 minutes, 10000 rpm for 30 minutes, and 100000 rpm for 70 minutes) to collect the precipitate. The precipitate was then washed with PBS buffer (centrifuged at 100000 rpm for 70 minutes) to obtain tumor immunogenic exosomes (iEVs).

[0066] Comparative Example 1

[0067] The difference from Example 1 is that doxorubicin was not added during the extraction of tumor-derived exosomes, and the resulting tumor-derived exosomes were denoted as CEV, while the prepared tumor-catalyzing therapeutic drug was denoted as CEV@SANE.

[0068] Figure 1 A schematic diagram illustrating the intracellular therapeutic mechanism of the cyano-bridged bimetallic nanozyme provided by this invention in the preparation of tumor catalytic therapeutic drugs.

[0069] Characteristic analysis was performed on the SANE prepared in Example 1: Images of the SANE were acquired using a transmission electron microscope (TEM, Talos F200S) at an accelerating voltage of 200 kV. The results are shown in [Figure 1]. Figure 2 Part a of the diagram. Powder X-ray diffraction (XRD) patterns were obtained using a Persee XD-2 X-ray powder diffractometer, and the results are shown in [see section a]. Figure 2 Part b of the report. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Thermo Fisher Scientific ESCALAB 250 analyzer, and the results are shown in [Figure number missing]. Figure 2 The cd portion is shown in the figure (b is the XRD pattern, c is the XPS spectrum of Cu 2p, and d is the XPS spectrum of Fe 2p). Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC HAADF-STEM) images were acquired using FEI Themis Z aberration-corrected scanning transmission electron microscopy. The results are shown in the figure. Figure 2 The e-part of Cu. X-ray absorption fine structure (XAFS) spectra of the K-edge of Cu were collected at station BL14W1 of the Shanghai Synchrotron Radiation Facility (SSRF). XAFS data were processed in Athena (version 0.9.26) to calibrate the background, leading edge, and trailing edge; results are shown in […]. Figure 2The f-part in the equation. Subsequently, a Fourier transform fit was performed in Artemis (version 0.9.26). The fit for the Cu foil used k3 weighting, where k ranges from 3 to 12.5 Å. -1 The R-squared value ranged from 1 to 3A; the fitting of SANE was performed using k3 weighting, with k ranging from 3 to 11.5A. -1 R ranges from 1 to 2A. See the results below. Figure 2 The g part in the text.

[0070] from Figure 2 As can be seen from part a, the SANE nanoparticles exhibit a cubic structure with a size of 60 nm. From... Figure 2 As can be seen from part b, SANE exhibits high crystallinity, which closely matches the standard spectrum of copper hexacyanoferrate (JCPDS #02-0381). From... Figure 2 As can be seen from part c, the Cu 2p spectrum of SANE shows Cu 2+ (2p) 3 / 2 =933.2eV, 2p 1 / 2 =953.2eV) and Cu + (2p) 3 / 2 =931.4 eV, 2p 1 / 2 Cu exhibits mixed valence state characteristics (951.3 eV); compared to the standard spectrum of copper oxide, Cu... 2+ 2p 3 / 2 peak and 2p 1 / 2 The binding energies of the peaks decreased by 2.24 eV and 2.04 eV, respectively, indicating that in the cyano-bridged structure, the electron cloud density at the Cu site increased due to charge transfer from Fe to Cu—the more electronegative Cu (1.90 vs. 1.83 (Fe)) captured electrons through the Fe-C≡N conjugated π bond. Meanwhile, from Figure 2 As can be seen from the d-part, in the Fe 2p spectrum, Fe... 2+ 2p 3 / 2 The binding energy of the peak rises to 708.5 eV (compared to 707.8 eV in Prussian blue (PB)), 2p 1 / 2 The binding energy of the peak rises to 721.7 eV. From Figure 2 Part e clearly shows the precise dispersion of individual copper atoms within the carbon-nitrogen matrix (marked by white circles). Figure 2 The f section shows SANE and CuO. x Cu K-edge XANES spectra of nanozymes and Cu foil, where Cu-foil represents Cu foil, and CuO and Cu2O represent CuO. x Nanozymes. From Figure 2 As can be seen from part f, the absorption peak of SANE is located between Cu foil and copper oxide, indicating the presence of oxidized Cu in SANE.δ+ Species (0 < δ < 2). Figure 2 The g-part in the image shows the Fourier transform extended Cu K-edge EXAFS (FT-EXAFS) spectrum of SANE, and is represented by CuO. x Using nanozymes and Cu foil as references, a main peak appears at approximately 1.5 Å for the SANE single-atom enzyme, corresponding to the Cu-N bond. Notably, there is no significant peak at approximately 2.3 Å, indicating the absence of Cu-Cu coordination. This confirms the absence of metallic crystalline Cu species and the presence of only isolated single Cu atoms, consistent with HAADF-STEM measurements.

[0071] Performance testing:

[0072] 1. Catalytic activity (SANE's simulated activity against GSH)

[0073] Kinetic analysis of SANE and PB was performed using GSH as a substrate. Mixed solutions containing 50 μg / mL SANE or PB, 10 μg / mL DTNB, and different concentrations of GSH (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM) were prepared. The absorbance of the mixed solutions containing 50 μg / mL SANE or PB, 10 μg / mL DTNB, and 0.5 mM GSH was monitored. The results are shown in [Figure number missing]. Figure 3 Part a of the diagram; the absorbance of mixed solutions containing different concentrations of GSH at 412 nm was monitored, and the obtained absorbance data were fitted using GraphPadPrism 8 software to obtain Michaelis saturation curves and double reciprocal fitting curves. The results are shown in [Figure 1]. Figure 3 The bc part.

[0074] Kinetic analysis of SANE and PB was performed using H2O2 as a substrate. Mixed solutions containing 20 μg / mL SANE or PB, 50 μg / mL TMB, and different concentrations of H2O2 (0, 0.01, 0.02, 0.03, 0.04, and 0.05 M) were prepared. The absorbance of the mixed solutions containing 20 μg / mL SANE or PB, 50 μg / mL TMB, and 0.05 M H2O2 was monitored. The results are shown in [Figure number missing]. Figure 3 The d-part of the diagram; the absorbance of oxidized TMB at 650 nm in each reaction was monitored, and the obtained absorbance data were fitted using GraphPad Prism 8 software to obtain Michaelis saturation curves and double reciprocal fitting curves. The results are shown in [Figure 1]. Figure 3 The ef part in [the document / concept].

[0075] Figure 3The enzyme activity of SANE is characterized, where a represents the GSHox-like activity of PB and SANE, bc represents the Michaelis saturation curves and double reciprocal fitted curves of PB and SANE at different concentrations of GSH, d represents the POD-like activity of PB and SANE, and ef represents the Michaelis saturation curves and double reciprocal fitted curves of PB and SANE at different concentrations of hydrogen peroxide. Figure 3 As can be seen from the ac portion, SANE exhibits stronger GSH oxidase-like enzyme activity than PB. From... Figure 3 As can be seen from part d in the graph, SANE exhibits higher absorbance at 652 nm, while PB elicits a weaker color response. This may be because PB has only one active center and lower electron transfer efficiency. From... Figure 3 As can be seen from the ef portion, SANE exhibits stronger peroxidase activity than PB. These results demonstrate that the atomically dispersed active center and heteronuclear metal bridging structure successfully enhance enzyme activity similar to GSHox.

[0076] 2. In vitro antitumor evaluation

[0077] Cell viability was assessed using the CCK-8 cell counting kit after co-culturing 200 mg / L SANE, CEV@SANE, and iEV@SANE with LLC tumor cells and MEF-12 mouse embryonic fibroblasts for 24 hours, with water as a blank control.

[0078] The cells treated with the above methods were stained with Annexin V-FITC and PI. The results are shown in the figure. Figure 4 As can be seen, iEV@SANE treatment resulted in a higher apoptosis rate of tumor cells (23.1%) compared with the control group (5.62%) and the single-atom nanozyme SANE alone (11.9%).

[0079] JC-1 dye was used as a fluorescent indicator to track changes in membrane potential. When JC-1 dye accumulates in mitochondria with high membrane potential, it emits red fluorescence. If the mitochondrial membrane potential (MMP) decreases, JC-1 dye emits green fluorescence. Changes in mitochondrial morphology were observed using bio-transmission electron microscopy (bio-TEM), and the results are as follows: Figure 5 As shown in section a, compared with the control group, the iEV@SANE group exhibited mitochondrial atrophy and a reduced number of cristae. These results indicate that the single-atom nanozyme SANE can generate a large amount of reactive oxygen species (ROS) within cells, leading to mitochondrial damage and thus enhancing its in vitro antitumor effect.

[0080] Decreased glutathione (GSH) levels inactivate glutathione peroxidase 4 (GPX4), disrupting lipid repair mechanisms and triggering ferroptosis. SANE can deplete extracellular GSH. Therefore, a GSH / GSSG kit was used to detect intracellular GSH and GSSG levels in different treatment groups to verify whether SANE can effectively deplete intracellular GSH. The results are as follows: Figure 5 As shown in section b, both SANE and CEV@SANE can deplete intracellular GSH and increase GSSG levels. In particular, LLC cells treated with iEV@SANE showed a significant reduction in GPX4 expression, exhibiting the strongest ability to deplete intracellular GSH, indicating that SANE also possesses excellent GSH depletion capabilities within cells.

[0081] Ferroplasm is a form of immune cell death (ICD). This invention investigated typical damage-associated molecular patterns (DAMPs) of ICD, including high-mobility group box 1 (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP). The results are as follows: Figure 6 As shown in part ab.

[0082] CRT migration from the endoplasmic reticulum to the cell surface is another indicator for assessing ICD. LLC cells were spaced at 1 × 10⁻⁶ cells per well. 6 LLC cells were seeded at a density of [number] cells per well in six-well plates and treated with SANE, CEV@SANE, and iEV@SANE for 24 hours, respectively. After 24 hours of co-culture, cells were isolated from the culture plates using trypsin. Subsequently, LLC cells were stained with high-mobility group box 1 (HMGB1) antibody and CRT antibody, respectively, and analyzed by flow cytometry. The results are shown in [figure missing]. Figure 6 Part a in the text.

[0083] from Figure 6 As can be seen from part a in the figure, after iEV@SANE treatment, the expression of CRT on the cell surface increased significantly.

[0084] LLC cells were spaced at 1 × 10⁻⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in six-well plates and treated with SANE, CEV@SANE, and iEV@SANE for 24 hours, respectively. To detect extracellular ATP, the culture supernatant was used as the test sample. To detect intracellular ATP, the supernatant from lysed cells was centrifuged and used as the test sample. Subsequently, the ATP assay kit was used for detection, and the results are shown below. Figure 6 Part b in the text. From Figure 6As shown in section b, intracellular ATP was significantly reduced while extracellular ATP was significantly increased in the iEV@SANE group, indicating that iEV@SANE treatment can promote the secretion of ATP from the intracellular to the extracellular space. These results suggest that iEV@SANE treatment can effectively induce ICD in LLC cells. The DAMPs released during ICD can promote the maturation of dendritic cells (DCs), thereby activating CD8+ cells and achieving the effect of killing tumor cells.

[0085] DC 2.4 cells were planted at a density of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in six-well plates. After cell attachment, the culture medium was replaced with LLC cell supernatant that had been purified from SANE, CEV@SANE, and iEV@SANE, respectively, with water used as a blank control. After 24 hours of co-incubation, cells were separated from the wells using trypsin. Subsequently, DC 2.4 cells were stained with antibodies specifically targeting DC 2.4 cells (PE-CD86 and FITC-CD80), and analyzed by flow cytometry. The results are shown in [figure missing]. Figure 7 It can be seen that iEV@SANE has the strongest promoting effect on the maturation of DC2.4 cells.

[0086] 3. Antitumor effects of iEV@SANE in vivo

[0087] Four-week-old male C57BL / 6 mice were purchased from Jiangsu SLAC Laboratory Animal Co., Ltd., and were tested using 1×10⁻⁶ mice. 6 A 100 μL LLC cancer cell line was subcutaneously injected into the right back of mice to establish an LLC lung cancer model. When the tumor grew to approximately 100 mm... 3 Mice were randomly divided into four groups. Subsequently, mice were injected via tail vein with SANE, CEV@SANE, iEV@SANE, and PBS buffer containing rhodamine B. The experimental groups were: 1. Control group (PBS buffer, 1 mg / kg, 100 μL); 2. SANE group (1 mg / kg, 100 μL); 3. CEV@SANE group (1 mg / kg, 100 μL); 4. iEV@SANE group (1 mg / kg, 100 μL). Intravenous administration was performed on days 1, 3, and 5. Body weight and tumor size were measured every other day. The formula for calculating tumor volume was: Tumor volume = Length × Width. 2 / 2. Two weeks after treatment. Subsequently, the mice were euthanized, and tumor tissue, lymph nodes, and major organs were removed for further analysis.

[0088] The accumulation of drug at the tumor site at different time points after injection was observed using an in vivo imaging system (IVIS). The results are shown in [Figure number missing]. Figure 8Part a of the study shows that, due to the rich vascularity of the tumor site, iEV@SANE accumulated significantly at the tumor site after 2 hours. However, throughout the observation period, the fluorescence intensity at the tumor site in the iEV@SANE group was higher than that in the SANE group. Furthermore, 24 hours after injection, no fluorescence signal was observed at the tumor site in the SANE group, while a significant fluorescence signal was still observed at the tumor site in the iEV@SANE group. This indicates that tumor-derived exosomes possess in vivo "homing" homologous targeting properties, endowing iEV@SANE with the ability to target tumors.

[0089] Twenty-four hours later, the mice were euthanized, and their major organs and tumors were collected for fluorescence imaging. The results are shown in [Figure number missing]. Figure 8 Part b of the results shows that SANE mainly accumulates in the kidneys, while no significant fluorescent signal was observed in the tumor area. In contrast, iEV@SANE accumulation in the kidneys is reduced, which may explain the increased accumulation of iEV@SANE at the tumor site. These results are consistent with observations in in vivo experiments. Overall, when SANE is coated with iEV, it can effectively target the tumor site.

[0090] On day 14, the mice were euthanized, and the tumors were collected for further analysis. Tumor images, changes in tumor volume, and tumor weight after different treatments are shown in [see attached image]. Figure 9 As shown in the ac section, compared with the control group, all three materials inhibited tumor growth, mainly due to the excellent enzyme activity of the single-atom nanozyme SANE. Notably, the iEV@SANE group showed significantly better tumor growth inhibition than the SANE and CEV@SANE groups, primarily because iEV endows SANE with tumor-targeting capabilities while simultaneously activating the immune response.

[0091] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of a cyano-bridged bimetallic nanozyme in the preparation of tumor catalytic therapeutic drugs, wherein the cyano-bridged bimetallic nanozyme is a Cu2[Fe(CN)6] bimetallic nanozyme.

2. The application according to claim 1, characterized in that, The preparation method of the cyano-bridged bimetallic nanozyme includes the following steps: dispersing citric acid and K4[FeⅡ(CN)6] in water to obtain solution A; dispersing CuSO4·5H2O in water to obtain solution B; adding solution B to solution A under stirring conditions to obtain Cu2[Fe(CN)6], which is the cyano-bridged bimetallic nanozyme.

3. The application according to claim 2, characterized in that, The molar ratio of citric acid, K4[FeⅡ(CN)6] and CuSO4·5H2O is (1~2):(0.1~2):(0.1~2).

4. The application according to claim 2, characterized in that, The stirring speed is 200-1000 rpm, and the stirring time is more than 24 hours.

5. A tumor-catalyzing therapeutic drug, characterized in that, The raw materials include cyano-bridged bimetallic nanozymes and tumor immunogenic exosomes, wherein the cyano-bridged bimetallic nanozyme is a Cu2[Fe(CN)6] bimetallic nanozyme.

6. The tumor catalytic therapeutic agent according to claim 5, characterized in that, The particle mass ratio of the cyano-bridged bimetallic nanozyme to the tumor immunogenic exosome is (0.5~4):

1.

7. The tumor catalytic therapeutic agent according to claim 5, characterized in that, The preparation method of the tumor catalytic therapeutic drug includes the following steps: mixing tumor immunogenic exosomes with cyano-bridged bimetallic nanozymes, and then performing ultrasonic treatment in an ice bath to obtain the tumor catalytic therapeutic drug.

8. The tumor catalytic therapeutic agent according to claim 7, characterized in that, The power of the ultrasound is 50~500W, and the duration of the ultrasound is more than 5 minutes.

9. The tumor catalytic therapeutic agent according to claim 5, characterized in that, The extraction method for tumor immunogenic exosomes is differential ultracentrifugation.

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