Application of cell membrane coated oxygen vacancy Cu2 (OH) 3Cl loaded Os monatomic bionic enzyme preparation

Abstract

The invention belongs to the technical field of biocatalyst preparation, and particularly relates to application of a cell membrane coated oxygen vacancy Cu2 (OH) 3Cl loaded Os monatomic bionic enzyme preparation. The molecular formula of the biomimetic enzyme preparation OsCu-Ov (at) CCM is Os-Cu2 (OH) 3Cl (at) CCM. The biomimetic catalyst can be used for preparing medicines or preparations for treating tumors or medicines or preparations for inhibiting the development process of the tumors. The tumor is malignant melanoma. The catalyst has a monatomic osmium catalytic center and an oxygen vacancy (Ov), and is used for treating malignant melanoma. Benefited from excellent electronic characteristics and structural diversity of osmium (Os) active sites, the synthesized biocatalyst shows excellent ROS catalytic activity, including peroxidase (POD), halogen peroxidase (HPO), catalase (CAT) and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalyst preparation, and specifically relates to the application of an oxygen vacancy Cu 2 (OH) 3 Cl-loaded Os single-atom biomimetic enzyme preparation. Background Art

[0002] Malignant melanoma (MM) is a highly invasive tumor that poses a significant risk to human health. Although MM accounts for only 5% of skin malignancies, it is the most aggressive form of skin cancer, accounting for approximately 80% of skin cancer-related deaths. Despite continuous improvements in surgical methods and other treatment modalities, the invasiveness of MM cells and the complexity of the tumor microenvironment remain the main obstacles to treatment. Considerable efforts have been devoted to developing new and effective methods for treating MM. ROS-mediated treatment methods have achieved great success in cancer treatment exploration. Upregulation of intracellular ROS leads to oxidative damage of lipids, proteins, and DNA, thereby causing cell apoptosis. ROS generation strategies have also been explored as promising alternative tumor treatment methods in the tumor hypoxia microenvironment (THEM). Various ROS-catalyzing biocatalysts, including metal oxides, metal hydroxides, metal nanoparticles, metal-organic frameworks, single-atom catalysts, etc., have been studied for tumor inhibition.

[0003] Although ROS can be generated by catalyzing O 2 and H 2 O 2 the reported enzyme-mimicking biocatalysts still suffer from insufficient tumor inhibitory activity, such as low ROS catalytic efficiency, biocompatibility, tumor cell specificity, and tumor microenvironment. Therefore, there is an urgent need to develop novel and effective ROS-generating biocatalysts with the high efficiency and specificity of natural enzymes, which can regulate the tumor hypoxia microenvironment and penetrate tumor cells to combat tumor progression in nanomaterials and biomedical sciences.

[0004] Nowadays, metal-coordinated single-atom biocatalysts have received great attention due to their maximized metal active sites, especially in cancer treatment. However, compared with natural enzymes, single-atom biocatalysts still have problems of low ROS catalytic efficiency and specificity. It has been reported that fast electron transfer ability and sufficient catalytic substrate binding sites will help reduce the activation energy and improve the catalytic efficiency. As one of the emerging tools in detection engineering, oxygen vacancy (O v ) can endow the catalyst with rich electron orbits, interact with small molecules of oxygen-containing compounds to assist energy transfer and further optimize the band structure. The synergistic effect between single-atom sites and catalytic substrate defects has been widely studied as a promising way to enhance catalytic activity in various catalytic processes. However, single-atom sites and O vThe impact of the synergistic effect on its tumor treatment performance remains a mystery. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded single-atom Os bionic enzyme preparation in view of the problems existing in the prior art. The bionic enzyme preparation (OsCu-O v @CCM) has an Os center and O v and can be applied in the preparation of drugs or preparations for treating tumors or inhibiting the tumor development process; it can be used for targeted treatment of MM. The catalytic active sites and chemical structure for ROS generation, benefiting from the electronic properties of the single-atom Os center and the defect structure of Ov, OsCu-Ov exhibits excellent ROS catalytic behavior, including CAT, HPO, and POD mimicking activities (K m : 18.5 mM, V max : 1.41 μM s -1 and TON: 259.9×10 -3 s -1 ), showing excellent activity. The coating of cancer cell membrane (CCM) has become an ideal surface modification strategy, endowing OsCu-Ov with excellent biological interface characteristics, including homologous targeting and efficient delivery of OsCu-Ov. The CCM-hidden OsCu-Ov nanoparticles (OsCu-Ov@CCM) actively penetrate into tumor cells, improve the hypoxic microenvironment, and disrupt mitochondria.

[0006] In addition, we characterized the gene signature strongly associated with the apoptosis pathway. This study provides a general strategy for effectively promoting ROS and nanoparticle penetration into cancer cells and opens up a new avenue for cancer treatment.

[0007] To achieve the above invention objectives, the specific technical solutions of the present invention are as follows:

[0008] One of the objectives of the present invention is to provide a cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded single-atom Os bionic enzyme preparation OsCu-O v @CCM.

[0009] Furthermore, the molecular formula of the cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded single-atom Os bionic enzyme preparation is Os-Cu 2 (OH) 3 Cl (OsCu-Ov).

[0010] Furthermore, the cell membrane-coated oxygen vacancy Cu 2 (OH) 3 The cell membrane in the Cl-loaded Os single-atom biomimetic enzyme preparation is the B16F10 tumor cell membrane.

[0011] The second object of the present invention is to protect the above-mentioned cell membrane-coated oxygen vacancy Cu 2 (OH) 3 The application of the Cl-loaded Os single-atom biomimetic enzyme preparation in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the tumor development process.

[0012] Preferably, in the above-mentioned application, the tumor is malignant melanoma.

[0013] The third object of the present invention is to protect the above-mentioned cell membrane-coated oxygen vacancy Cu 2 (OH) 3 The preparation method of the Cl-loaded Os single-atom biomimetic enzyme preparation, comprising the following steps:

[0014] First, a Cu2(OH)3Cl (Cu-Ov) precursor containing oxygen vacancies was prepared by a typical hydrothermal method using CuCl 2 and hexamethylenetetramine (HMT) solution, and then an Os-Ov catalytic center was prepared by wet impregnation to obtain an Os-Cu 2 (OH) 3 Cl (OsCu-Ov) biocatalyst.

[0015] Furthermore, in the preparation method of the cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded Os single-atom biomimetic enzyme preparation,

[0016] The molar ratio of CuCl 2 ·xH 2 O to HMT is 100:1 to 100:5 (specifically, it can be 100:1, 100:2, 100:3, 100:4, 100:5, etc.); the hydrothermal reaction temperature is 90-100 °C (specifically, it can be 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C, 100 °C, etc.); the reaction time: 4-6 h (specifically, it can be 4 h, 5 h, 6 h, etc.); the mass ratio of Cu-O v to osmium is 50:1 to 10:1 (specifically, it can be 50:1, 40:1, 30:1, 20:1, 10:1, etc.).

[0017] Furthermore, the cell membrane-coated oxygen vacancy Cu prepared by the above method or a combination of method steps 2 (OH)3 Cl-loaded Os single-atom biomimetic enzyme preparation, with a spherical morphology and ultra-small nanoscale.

[0018] Preferably, the oxygen vacancy Cu coated with cell membrane prepared by the foregoing method 2 (OH) 3 The nanoscale of the Cl-loaded Os single-atom biomimetic enzyme preparation is about 30 nm.

[0019] Furthermore, the oxygen vacancy Cu coated with cell membrane prepared by the method 2 (OH) 3 The Cl-loaded Os single-atom biomimetic enzyme preparation has excellent catalase (CAT), haloperoxidase (HPO) and peroxidase (POD) mimicking activities, and superior ROS generation activity.

[0020] Furthermore, the mimicking activity K of the above-mentioned biomimetic enzyme preparation m : 18.5 mM, V max : 1.41 μM s -1 and TON: 259.9×10 -3 s -1 .

[0021] Compared with the existing technology, the beneficial effects of the present invention are:

[0022] (1) The novel cell membrane-coated enzyme mimicking OsCu-Ov@CCM in the present invention has a single-atom osmium center and an oxygen vacancy structure, is a perfect and efficient nanoplatform, mimics the ROS generation characteristics of natural enzymes, and realizes efficient and targeted treatment of malignant melanoma (MM).

[0023] (2) Due to the structural advantages and ROS catalytic activity of OsCu-Ov@CCM, it can be applied in targeted MM treatment. In vitro experiments verified that the synthesized biocatalyst showed excellent CAT, HPO and POD mimicking activities due to the synergistic effect of the single-atom osmium active center and the oxygen vacancy, thus showing superior ROS generation activity compared with natural micromolecules and most reported ROS catalytic biocatalysts. OsCu-Ov@CCM showed minimal side effects and extremely high safety in in vivo experiments.

[0024] (3) The CCM coating has excellent biointerface properties, including homologous targeting and efficient delivery of OsCu-Ov. Therefore, in vivo experiments showed that OsCu-Ov@CCM provided efficient therapeutic effects for MM by generating ROS and alleviating the hypoxic tumor microenvironment, while increasing the activities of several regulatory pathways related to apoptosis.

[0025] In addition, OsCu-Ov@CCM actively inhibits the MM trend due to significant changes in gene expression after treatment and increased activities of multiple regulatory pathways related to apoptosis. The present invention opens up a promising direction for the development of high-performance ROS-generating biocatalysts in tumor treatment and other ROS-mediated diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the preparation process of a cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded Os single-atom bionic enzyme preparation OsCu-O v @CCM;

[0027] Figure 2 High-resolution transmission electron microscope (HR-TEM) image of OsCu-Ov;

[0028] Figure 3 Selected area electron diffraction (SAED) pattern of OsCu-Ov and intensity profile along the cyan box in c;

[0029] Figure 4 Atomic resolution high-angle annular dark field scanning transmission electron microscope (HAADF-STEM) image of OsCu-Ov and corresponding elemental analysis;

[0030] Figure 5 Energy dispersive spectroscopy (EDS) mapping image of OsCu-Ov;

[0031] Figure 6 Electron structure analysis diagram of the biocatalyst by XPS and XAS;

[0032] Figure 7 Enzyme-mimicking ROS generation activity of the OsCu-Ov artificial biocatalyst;

[0033] Figure 8 In vitro cell experiments to verify the anti-tumor ability of OsCu-Ov@CCM;

[0034] Figure 9 In vivo anti-tumor treatment;

[0035] Figure 10 Mechanism exploration of tumor killing caused by OsCu-Ov@CCM;

[0036] Figure 11 Schematic diagram of the treatment mechanism of OsCu-Ov@CCM against malignant melanoma. DETAILED DESCRIPTION OF THE INVENTION

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the following further details the present invention with reference to examples. It should be understood that the specific examples described herein are only for explaining the present invention and not for limiting the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes and modifications to the present invention, but these equivalent forms also fall within the scope defined by the appended claims of this application.

[0038] Example 1:

[0039] A cell membrane-coated oxygen vacancy Cu 2 (OH) 3 Cl-loaded Os single-atom biomimetic enzyme preparation (also known as cell membrane-coated osmium single-atom biomimetic catalyst with oxygen vacancy and multi-enzyme mimicking activity)

[0040] OsCu-O v @CCM preparation method, the preparation process is as Figure 1 shown, Figure 1 wherein, the blue, orange, fluorescent green, pink and light blue spheres represent copper, chlorine, oxygen, hydrogen and osmium atoms respectively.

[0041] The preparation method includes the following steps:

[0042] First, a Cu 2 (OH) 2 Cl (Cu-Ov) precursor containing oxygen vacancies was prepared by using a typical hydrothermal method with CuCl 3 and HMT (hexamethylenetetramine) solution; the molar ratio of CuCl 2 ·xH 2 O to hexamethylenetetramine is 100:1 to 100:5; the hydrothermal reaction temperature is 95 °C; the reaction time is 5 h;

[0043] Then, an Os-Ov catalytic center was prepared by wet impregnation to obtain an Os-Cu 2 (OH) 3 Cl (OsCu-Ov) biocatalyst. The mass ratio of Cu-O v to osmium is 50:1 to 10:1.

[0044] Specific steps: Weigh 340.9 mg of CuCl 2 ·xH 2 O (Aladdin) and 3.154 g of HMT (hexamethylenetetramine C 6 H 12 N 4, hexamethylenetetramine) in a 100 mL hydrothermal autoclave containing 35 mL of deionized water, mixed evenly, hydrothermally reacted at 95 °C for 5 h, washed successively with alcohol and deionized water, and dried to prepare a Cu-Ov precursor; then, 50 mg of the Cu-Ov precursor was weighed into a 20 mL glass bottle, and 0.5 mL of OsCl 3 (10 mg / ml) was added, stirred at room temperature for 24 h, filtered, washed, and dried to obtain the product.

[0045] The obtained catalyst was subjected to performance testing, as shown specifically in Figures 2 to 5 ; Figure 2 is the high-resolution transmission electron microscope (HR-TEM) image of OsCu-Ov. Figure 3 is the selected area electron diffraction (SAED) pattern of OsCu-Ov and the intensity profile along the cyan box in c. Figure 4 is the atomic resolution high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of OsCu-Ov and the corresponding elemental analysis. Figure 5 is the energy-dispersive spectroscopy (EDS) mapping image of OsCu-Ov.

[0046] Figure 2 Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM) showed its spherical morphology and ultra-small nanoscale (about 30 nm). Subsequently, B16F10 CCM was extracted from B16F10 cells and coated on OsCu-Ov by ultrasonic method to form OsCu-Ov@CCM. In addition, we applied sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to ensure the loading of CCM on OsCu-Ov. X-ray diffraction (XRD) patterns and electron paramagnetic resonance (EPR) confirmed the successful synthesis of the Cu-Ov precursor, and no peak of osmium particles was observed in the pattern of OsCu-Ov. The doping of osmium did not significantly change the spherical morphology and crystal structure of Cu-Ov. The HR-TEM image of OsCu-Ov showed clear lattice fringes of 0.2614, 0.2763, and 0.3165 nm, corresponding to the (022), (121), and (021) crystal planes of Cu-Ov, respectively. The intensity profile obtained from the Figure 2 line scan showed that 2.1 / 8 of the lattice fringes was in good agreement with the (121) crystal plane. The selected area electron diffraction (SAED) pattern was consistent with the characteristic diffraction ring of Cu-Ov, confirming the results ( Figure 3 ). Further, atomically dispersed osmium (bright spots) and the crystal lattice of Cu-Ov were observed in the high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image ( Figure 4)。Meanwhile, energy-dispersive spectroscopy (EDS) mapping clearly shows that the signal of single osmium atoms is evenly distributed, further confirming the formation of single osmium atoms. Figure 5 )。

[0047] Example 2:

[0048] After verifying the existence of single-atom catalytic sites in Example 1, we then applied X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XAS) to explore the surface composition and coordination structure of the OsCu-Ov biocatalyst. See specifically Figure 6 ; Figure 6 for the electron structure analysis diagrams of the biocatalyst by XPS and XAS. Among them, a) is the high-resolution XPS O 1s spectrum of OsCu-Ov, b) is the Cu 2p spectrum, c) is the Os 4f spectrum. d) is the normalized XANES spectrum of the osmium L-edge. e) is the relationship between the absorption energy (E0) and valence state of the osmium L-edge of OsCu-Ov and the reference. f) is the k2-weighted Fourier transform of osmium foil, OsCu-Ov and OsO 2 and the corresponding fitting results of OsCu-Ov. g) is osmium foil, h) OsCu-Ov, i) is OsO 2 WT images at the osmium L-edge.

[0049] As can be seen from Figure 6 , the XPS spectrum of OsCu-Ov shows the simultaneous presence of copper, oxygen, carbon, chlorine and osmium elements, which is consistent with the EDS results. The O 1s and Cl 1s spectra of the OsCu-Ov biocatalyst show obvious peak shifts compared with OsCu-Ov, thus indicating the osmium-oxygen coordination ( Figure 6 a). As Figure 6 shown in b, taking the 2p3 / 2 region of the copper 2p spectrum in the biocatalyst as an example, the peaks located at 933.14 and 935.28 eV are attributed to the copper(I) and copper(II) oxidation states respectively. For the osmium 4f spectrum of OsCu-Ov, the two split peaks located at 51.26 and 53.23 eV are attributed to the 4f7 / 2 of osmium 4+ and osmium 3+ ( Figure 6 c). No obvious metal peaks are observed in the osmium 4f spectrum of OsCu-Ov, further confirming the atomic dispersion of osmium ions. Subsequently, X-ray absorption spectroscopy (XAS) was applied to deeply understand the precise coordination structure of the atomically dispersed osmium-oxygen catalytic sites in the OsCu-Ov biocatalyst. The X-ray absorption near-edge structure (XANES) spectrum of the osmium L3-edge shows that the oxidation state of the white line intensity (red line) of OsCu-Ov is closer to OsO2 compared with osmium foil ( Figure 6 d). The valence state analysis of osmium in the biocatalyst shows that the average valence state of OsCu-Ov is 4.2, higher than that of osmium foil and OsO2 (4.0) ( Figure 6e). The Fourier transform k2-weighted extended X-ray absorption fine structure (EXAFS) spectrum shows a main peak of approximately , corresponding to the first coordination shell of osmium-oxygen ( Figure 6 f). In the range of , the metal osmium-osmium peak of OsCu-Ov is negligible, indicating that osmium exists in the form of single osmium atoms. The fitting results indicate that the coordination structure of osmium-oxygen 2-chloride is in good agreement with the first shell of the osmium L-edge EXAFS of OsCu-Ov ( Figure 6 f). The wavelet transform (WT) is further summarized in Figure 6 g–i. Obviously, OsCu-Ov shows a main peak at around , corresponding to the first coordination shell of osmium-oxygen. This confirms the formation of single osmium atoms, which is consistent with the HAADF-STEM and XPS results.

[0050] Example 3:

[0051] After verifying the single osmium atoms and electronic structure in OsCu-Ov (based on Example 2), we systematically evaluated and compared the ROS catalytic activities of the biocatalysts, such as POD-mimicking activity and HPO-mimicking activity. The classical colorimetric method was used to test and verify the POD-mimicking activity of the biocatalyst, using 3,3′,5,5′-tetramethylbenzidine reagent (TMB, which can form colored ox-TMB through ROS oxidation); the specific results are shown in Figure 7 .

[0052] Figure 7 For the enzyme-mimicking ROS generation activity of the OsCu-Ov artificial biocatalyst; among them, a) is the POD-mimicking activity. b) is the O2 generation activity. c) is the reaction kinetic analysis of the POD-mimicking activity of OsCu-Ov. d) is the comparison of Vmax and TON of recently reported artificial biocatalysts with H 2 O 2 as the substrate. e) is the temperature and pH dependence test of the POD-mimicking activity of OsCu-Ov. f) is the radical quenching results, the quencher of ·OH (TBA), the quencher of ·O2− (BQ), and the quencher of 1O2 (NaN 3 ). g) is the EPR spectra of ·OH, ·O 2 − and 1O2 signals. h) is the HPO-mimicking activity.

[0053] As shown in Figure 7 a, OsCu-Ov and OsCu-Ov@CCM show excellent ROS generation activity at 652 nm by catalyzing the oxidation of TMB. At the same time, OsCu-Ov can effectively utilize the H2O2 substrate to generate O 2 in the O2 ( Figure 7 b). Then, the catalytic constant (Km = 18.5 mM), the maximum reaction rate (Vmax = 1.41 m s-1), and the turnover number (TON = 259.9×10 2 O 2 of H (the maximum number of substrates converted per catalytic atom) were calculated through the Michaelis-Menten equation graph ( -3 s -1 ). Figure 7 c). On this basis, we systematically compared OsCu-Ov with several advanced POD-mimicking biocatalysts, such as metal oxides (Fe 3 O 4 , CeO 2 , Co 3 O 4 , CuO, Mn 3 O 4 , etc.), metal nanoparticles (Ru NPs, Pt NCs, PtFe@Fe 3 O 4 , etc.), single-atom biocatalysts (Fe-N-C SAzymes, Co-N-C SAzymes, Zn-N-C SAzymes, etc.), metal-organic frameworks (Fe-MOF, etc.). The results showed that the OsCu-Ov biocatalyst exhibited excellent ROS catalytic activity among current nanomaterials ( Figure 7 d). OsCu-Ov also showed high ROS generation activity under various reaction conditions, such as a wide temperature range (20 - 50 °C) and a wide pH range (3 - 6) ( Figure 7 e). To verify the ROS catalytic activity of OsCu-Ov after long-term storage, cyclic tests were conducted. Interestingly, the catalytic activity of OsCu-Ov did not significantly decrease after soaking for 30 days, indicating its good stability. In addition, free radical quenching experiments ( Figure 7 f) fluorescence probes and electron paramagnetic resonance (EPR) further revealed that the ROS species in the POD-mimicking process of OsCu-Ov were ·OH, ·O2-, and 1O2 ( Figure 7 g). In addition to the POD-mimicking activity, the HPO-mimicking activity of OsCu-Ov was also evaluated through the Celestine Blue (CB) test. As Figure 7 shown in h, OsCu-Ov presented a very high intensity ratio, indicating its strong activity in catalyzing the oxidation of chloride to HOCl / OCl-.

[0054] Example 4:

[0055] Inspired by the excellent catalytic ROS generation characteristics of OsCu-Ov@CCM, we first evaluated its potential for treating tumors through artificial biocatalysts via cell experiments. Human umbilical vein endothelial cells (HUVECs) were cultured in a 96-well plate for 24 hours, with 1.0×10^4 cells seeded in each well. The medium in the 96-well plate was discarded and washed with phosphate buffer solution (PBS). Subsequently, complete medium (for HUVECs) containing OsCu-O v @CCM was added to the 96-well plate, and the concentrations of OsCu-O v @CCM were 0, 10, 20, 40, 80, and 160 μg / mL, respectively, and the cells were cultured for an additional 8 hours. Next, the medium in each well was replaced with 100 μL of fresh medium containing 10 μL of Cell Counting Kit-8 (CCK-8, K1018, APExBIO, USA), and the mixture was incubated at 37 °C for 2 hours. Finally, the absorbance of the reaction product was measured at a wavelength of 450 nm using a microplate reader to evaluate cell viability.

[0056] Figure 8 To verify the anti-tumor ability of OsCu-Ov@CCM in in vitro cell experiments. Among them, a) is the Bio-TEM image of B16F10 cells phagocytosing OsCu-Ov@CCM. b) is the co-localization of OsCu-Ov and OsCu-Ov@CCM, the cytoskeleton and the nucleus. B16F10 cells were stained to identify the cytoskeleton (green), the nucleus (blue), and the DiI-stained nanozyme (red), and observed by confocal laser scanning microscopy (CLSM). Scale bar = 50 μm. c) is the quantitative data of CalceinAM / PI staining of B16F10 cells after different treatments. d) is the corresponding quantitative analysis of the co-staining of B16F10 cells with Annexin V-FITC and PI. e) is the DCF fluorescence distribution of B16F10 cells after different treatments and f) the corresponding quantitative analysis measured by flow cytometry. g) is a schematic diagram of the anti-tumor ability of OsCu-Ov@CCM. h) is the Bio-TEM image of B16F10 cells treated with OsCu-Ov@CCM. White triangles mark the disappearance of mitochondrial cristae and mitochondrial swelling. i) is the quantitative data of JC-1 staining of B16F10 cells.

[0057] First, it was confirmed that OsCu-Ov@CCM was endocytosed by cells. Figure 8a shows that it was observed by Bio - TEM that after 6 - hour co - incubation, OsCu - Ov@CCM was effectively internalized into B16F10 cells. In addition, the intracellular localization behavior of OsCu - Ov and OsCu - Ov@CCM was studied using confocal laser scanning microscopy (CLSM), and the DiI signal was obvious. As expected, the fluorescence intensity of the DiI signal in the OsCu - Ov@CCM group was significantly higher than that in the OsCu - Ov group, confirming that cancer cell membrane camouflage enhanced the cellular uptake of OsCu - Ov@CCM. This enhancement was attributed to the homotypic targeting ability provided by active cancer cell membrane proteins such as TF antigen and E - cadherin.

[0058] Subsequently, we evaluated the cytocompatibility of OsCu - Ov@CCM by Cell Counting Kit - 8 (CCK - 8) assay in human umbilical vein endothelial cells (HUVECs). The results showed that OsCu - Ov@CCM had a slight toxic effect on HUVECs. In addition, even when the concentration of OsCu - Ov@CCM reached 200 μg mL−1, the hemolysis rate was lower than 5%, indicating its excellent hemocompatibility. Subsequently, the tumor microenvironment (TME) was simulated in acidic complete medium at pH 6.5, and the anti - tumor ability of OsCu - Ov@CCM against B16F10 cells was confirmed by CCK - 8 assay. The results showed that as the concentration of OsCu - Ov@CCM increased, the survival rate of B16F10 cells decreased significantly. Therefore, 40 μg mL−1 was used as the concentration in subsequent experiments.

[0059] To compare the anti - tumor ability of OsCu - Ov@CCM and another component on B16F10, we used Calcein AM (green represents live cells) and propidium iodide (red represents dead cells) co - staining assay. Both the OsCu - Ov and OsCu - Ov@CCM groups showed more red fluorescence than their respective control groups. The percentage of dead cells in the OsCu - Ov@CCM group was higher than that in the OsCu - Ov - treated group ( Figure 8 c), which might be due to the homotypic targeting ability of the cancer cell membrane. Subsequently, we performed a detailed apoptosis examination by flow cytometry (FCM). We used membrane - binding protein Annexin V - FITC and propidium iodide (PI) staining, as well as their respective semi - quantitative analysis. Consistent with the live / dead staining results, Figure 8 d shows that the OsCu - Ov@CCM group exhibited the most significant highest apoptosis rate compared to other groups.

[0060] To further determine the source of tumor-killing ability, we used the DCFH-DA probe to evaluate the generation of intracellular ROS. Compared with other groups, the cells treated with OsCu-Ov@CCM showed the most significant green fluorescence intensity. This indicates the generation of a large amount of ROS. The results of flow cytometry (FCM) also showed the same result ( Figure 8 e and 8f). This indicates that among all groups, OsCu-Ov@CCM generated the highest amount of ROS.

[0061] Mitochondria are the sites for regulating ROS, and apoptosis is related to mitochondrial dysfunction. Therefore, we detected mitochondrial damage to discuss the anti-tumor mechanism. As Figure 8 shown in h, it was observed by biological transmission electron microscopy (Bio-TEM) that the mitochondria of B16F10 cells treated with OsCu-Ov@CCM were damaged, including the disappearance of mitochondrial cristae and mitochondrial swelling. Meanwhile, we used 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanine iodide (JC-1) as a probe for mitochondrial membrane potential. In healthy polarized mitochondrial membranes, it appears as red fluorescent aggregates, while in damaged depolarized mitochondrial membranes, it appears as green fluorescent monomers. Slight green fluorescence signals were detected in the control group, Cu-Ov, and Cu-Ov@CCM treatment groups, while in the OsCu-Ov@CCM group, the JC-1 dye changed from red aggregates to green monomers, indicating the appearance of a large number of dysfunctional mitochondria ( Figure 8 i).

[0062] Example 5:

[0063] Inspired by the significant anti-tumor properties of OsCu-Ov@CCM in vitro, we investigated the anti-cancer effect of OsCu-Ov@CCM in vivo. A BALB / c mouse model with subcutaneous inoculation of B16F10 tumors was constructed: B16F10 cells (1×10 6 cells) were resuspended in 100 μL of 1640 medium and subcutaneously injected into the right axilla of each mouse. During the experiment, the tumor volume was allowed to reach 1500 mm 3 . When the tumor volume reached approximately 100 mm 3 , the tumor-bearing BALB / c mice were randomly divided into five groups and intratumoral injections were performed (n = 3 for each group): 1. Control group; 2. Cu-O v (10 mg / kg, 100 μL); 3. Cu-O v @CCM (10 mg / kg, 100 μL); 4. OsCu-O v (10 mg / kg, 100 μL); 5. OsCu-O v@CCM (10 mg / kg, 100 μL). The body weight and tumor size of the mice were measured every other day. The formula for calculating the tumor volume was: Tumor volume = 1 / 2 × length × width 2 . After two weeks of treatment, the mice were sacrificed and the tumors were collected. Finally, the tumors and major organs (heart, liver, spleen, lung, kidney) of different groups were collected and sectioned for H&E staining, Ki-67, and TUNEL assays. The blood samples of each group of mice were centrifuged at 3000 rpm for 10 minutes to collect plasma, which was further used for the analysis of serum creatinine (Crea), aspartate aminotransferase (AST), urea (Urea), creatine kinase (CK), and alanine aminotransferase (ALT). (All experimental protocols have been approved by the Animal Ethics Committee of North Sichuan Medical College, with the approval number NSMC2023117)

[0064] Figure 9 For anti-tumor treatment. Among them, a) is a schematic diagram of the timeline of in vivo anti-cancer treatment. b) is the change in body weight of tumor-bearing mice during treatment, and the treatment cycle is 14 days. c) is a heat map of tumor volume during treatment, n = 3. d) is the tumor weight of each group. e-f) are the quantitative analyses of TUNEL and Ki67+ staining. g) are representative images of H&E staining, TUNEL staining, and Ki-67 immunofluorescence staining of tumor sections of each group (scale bar = 100 μm)

[0065] It can be seen that Figure 9 during the treatment period, the body weight of all mice increased slightly ( Figure 9 b). During the treatment period, compared with the control, Cu-Ov, and Cu-Ov@CCM groups, the growth trends of tumor volume and tumor weight in the OsCu-Ov and OsCu-Ov@CCM groups showed significant inhibition of tumor growth ( Figure 9 c and 9d). In addition, the OsCu-Ov@CCM group showed the most significant tumor suppression effect. After the treatment, the mice were sacrificed, the tumors were excised, and the major organs (heart, liver, spleen, lung, and kidney) were collected for further analysis. Hematoxylin-eosin (H&E) staining of the major organs showed no significant pathological damage, and there were no statistically significant differences in the serum biochemical analysis of mice treated with different treatments, indicating minimal treatment side effects and extremely high safety. H&E staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and antigen Ki-67 staining were performed on tumor sections to further evaluate the treatment effect ( Figure 9 g). According to the H&E staining results, compared with other groups, the OsCu-Ov and OsCu-Ov@CCM groups had fewer tumor cell nuclei. In addition, the OsCu-Ov@CCM treatment group showed the most significant apoptosis of tumor cells in the TUNEL assay ( Figure 9e). Additionally, immunohistochemical staining of the key marker Ki-67, which is closely related to the invasive proliferation of cancer cells, also showed an inhibitory trend after treatment ( Figure 9 f).

[0066] Example 6:

[0067] To further explore the potential mechanism of the therapeutic effect of OsCu-Ov@CCM on malignant melanoma (MM), RNA sequencing analysis was performed to evaluate the regulation of gene expression and analyze differentially expressed genes. Nine mice (randomly assigned to the control group, Cu-Ov@CCM group, and OsCu-Ov@CCM group, with n = 3 in each group) were sacrificed after different treatments, and total mRNA was extracted from their tumor tissues. The specific results are shown in Figure 10 .

[0068] Figure 10 For the exploration of the mechanism by which OsCu-Ov@CCM causes tumor killing. Among them, a) is a schematic diagram of the RNAseq experimental process. b) is a volcano plot of all differentially expressed genes (DEGs) between the control group and the OsCu-Ov@CCM group (the expressed proteins are defined as |fold-change| > 1.5 and p < 0.05). The dots represent DEGs (FDR ≤ 0.001, at least 2-fold difference); red indicates upregulation, and green indicates downregulation. c) is the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the upregulated genes between the OsCu-Ov@CCM group and the control group. d) is the KEGG pathway enrichment analysis of the downregulated genes between the OsCu-Ov@CCM group and the Cu-Ov@CCM group. e) is a heat map of the differentially expressed DEGs enriched in the OsCu-Ov@CCM group and the control group. f) is the protein expression of Cleaved-caspase 3, Bcl-2, and Bax in B16F10 cells of different groups and g-h) their statistical results.

[0069] As Figure 10 can be seen, these mRNAs were subsequently analyzed by high-throughput mRNA sequencing ( Figure 10 a). The gene expression profiles of each group changed significantly after different treatments. The RNA sequencing results showed that compared with the control group, 1,296 genes were upregulated and 340 genes were downregulated in the OsCu-Ov@CCM treatment group ( Figure 10 b). On the basis of the above research, it has been demonstrated that OsCu-Ov@CCM has strong anti-tumor efficacy both in vitro and in vivo. We used bioinformatics techniques to comprehensively clarify the biological functions of the regulatory genes altered by OsCu-Ov@CCM. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis ( Figure 10c) showed that the genes upregulated by OsCu-Ov@CCM were concentrated in several regulatory pathways related to apoptosis, such as the IL-17, PI3K-Akt, and TNF signaling pathways. This indicates that tumor cytotoxicity may be related to apoptosis. Figure 10 e showed that the administration of OsCu-Ov@CCM affected genes involved in cancer pathways, the IL-17 signaling pathway, the PI3K-Akt signaling pathway, and the TNF signaling pathway. Subsequently, we compared the differentially expressed genes (DEGs) between the Cu-Ov@CCM group and the OsCu-Ov@CCM group. The Cu-Ov@CCM group showed a downregulation of apoptosis-related genes, consistent with the previous test results ( Figure 10 d). In addition, gene set enrichment analysis (GSEA) indicated that OsCu-Ov@CCM might disrupt the inner mitochondrial membrane protein complex and mitochondrial protein complex, and the treatment with OsCu-Ov@CCM corresponded to an increase in the activities of several regulatory pathways related to apoptosis, such as the PI3K-Akt signaling pathway, the calcium signaling pathway, and the TGF-β signaling pathway. These findings highlight the ability of OsCu-Ov@CCM to manipulate key cellular pathways, which may lead to improved treatment outcomes for MM.

[0070] To confirm the mRNA changes, we used Western blot analysis to detect the protein levels of three key apoptosis markers: Cleaved Caspase-3, Bcl-2, and Bax. Compared with the control group, the OsCu-Ov@CCM group showed significantly higher levels of Cleaved Caspase-3 and Bax, indicating enhanced apoptosis. In contrast, the protein level of Bcl-2 in the OsCu-Ov@CCM group decreased significantly, further confirming the increase in apoptotic activity. Consistent with the previous results, the OsCu-Ov@CCM group showed a decrease in Bcl-2 expression and an increase in the levels of Cleaved Caspase-3 and Bax compared with the Cu-Ov@CCM group.

[0071] In summary, in the present invention, the novel cell membrane-coated enzyme mimic OsCu-Ov@CCM we prepared, which has a single-atom osmium center and oxygen vacancy structure, is a perfect and efficient nanoplatform that mimics the ROS generation characteristics of natural enzymes to achieve efficient and targeted treatment of malignant melanoma (MM); it can also be applied in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the tumor development process. The structural advantages and ROS catalytic activity of OsCu-Ov@CCM were systematically studied for their application in targeted MM treatment. In vitro experiments verified that the synthesized biocatalyst exhibited excellent CAT, HPO, and POD mimic activities due to the synergistic effect of the single-atom osmium active center and oxygen vacancy, thus showing superior ROS generation activity compared to natural micromolecules and most reported ROS catalytic biocatalysts. OsCu-Ov@CCM showed minimal side effects and extremely high safety in in vivo experiments. In addition, OsCu-Ov@CCM actively inhibited the MM trend, which was due to significant changes in gene expression after treatment and increased activities of multiple regulatory pathways related to apoptosis. We believe that this study will open up a promising direction for the development of high-performance ROS-generating biocatalysts in tumor treatment and other ROS-mediated diseases.

[0072] The above-described embodiments merely represent the specific embodiments of the present application, and their descriptions are relatively specific and detailed, but should not be construed as limiting the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

[0073] This background art section is provided to generally present the context of the present invention. The work of the currently named inventors, to the extent described in this background art section, and aspects of the work that are not prior art as of the filing date of this application are neither expressly nor impliedly admitted to be prior art to the present invention.

Claims

1. A cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation OsCu-O v @The application of CCM in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the progression of tumors.

2. The use according to claim 1, characterized in that: The tumor is malignant melanoma.

3. The use according to claim 1, characterized in that: The biomimetic enzyme preparation OsCu-O v The molecular formula of @CMM is Os-Cu2(OH)3Cl@CMM.

4. The use according to claim 1, characterized in that: The cell membrane is a B16F10 tumor cell membrane.

5. The use according to any one of claims 1 to 4, characterized in that: Preparation method of cell membrane-coated oxygen vacancy Cu2(OH)3Cl loaded Os single atom bionic enzyme preparation, The following steps are involved: Firstly, Cu2(OH)3Cl (Cu-O) containing oxygen vacancies was prepared by a typical hydrothermal method using CuCl2·xH2O and hexamethylenetetramine solution. v ) precursor; then, Os-O v catalytic center, thereby obtaining Os-Cu2(OH)3Cl(OsCu-O v )Bionic enzyme preparations.

6. The use according to claim 5, characterized in that: The molar ratio of CuCl2·xH2O to hexamethylenetetramine is 100:1 to 100:5; the hydrothermal reaction temperature is 90 to 100°C; the reaction time is 4 to 6 hours; Cu-O v The mass ratio of tantalum to osmium is 50:1 to 10:

1.

7. The use according to claim 5, characterized in that: The cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom bionic enzyme preparation is spherical in shape and has an ultra-small nanoscale.

8. The use according to claim 7, characterized in that: The nanoscale is 30nm.

9. The use according to claim 7, characterized in that: The biomimetic enzyme preparation has excellent CAT, HPO and POD mimicking activities and superior ROS generation activity.

10. The use according to claim 7, characterized in that: Simulated active K of Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparations with oxygen vacancies encapsulated by cell membranes m :18.5mM,V max :1.41μMs -1 and TON:259.9×10 -3 s -1 .

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