Application of a cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation

Abstract

The present invention belongs to the technical field of biocatalyst preparation, specifically the application of a cell membrane-coated oxygen vacancy Cu2(OH)3Cl loaded Os single atom biomimetic enzyme preparation. v The molecular formula of @CCM is Os-Cu2(OH)3Cl@CCM. This biomimetic catalyst can be used in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the progression of tumors. The tumor is malignant melanoma. The catalyst has a single atomic osmium catalytic center and an oxygen vacancy (O v ) for the treatment of malignant melanoma. Benefiting from the excellent electronic properties and structural diversity of osmium (Os) active sites, the synthesized biocatalysts exhibit excellent ROS catalytic activity, including peroxidase (POD), haloperoxidase (HPO), and catalase (CAT).

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalyst preparation, and specifically relates to the application of a cell membrane-coated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation. Background Art

[0002] Malignant melanoma (MM) is a highly aggressive tumor that poses a significant risk to human health. Although MM only accounts for 5% of skin malignancies, it is the most aggressive form of skin cancer and accounts 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 major obstacles to treatment. A lot of effort has been invested in developing new and effective therapies for MM. ROS-mediated therapeutic approaches have achieved great success in the exploration of cancer treatment. Upregulation of intracellular ROS can lead to oxidative damage to lipids, proteins, and DNA, thereby causing cell apoptosis. ROS generation strategies have also been explored as promising alternative tumor treatments in the tumor hypoxic 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 O2 and H2O2, reported enzyme-mimicking biocatalysts still have problems with 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 hypoxic microenvironment and penetrate tumor cells to combat tumor progression in nanomaterials and biomedical sciences.

[0004] Nowadays, metal-coordinated single-atom biocatalysts have attracted great attention due to their maximization of metal active sites, especially in cancer treatment. However, compared with natural enzymes, single-atom biocatalysts still have the problem of low ROS catalytic efficiency and specificity. It is reported that fast electron transfer ability and sufficient catalytic substrate binding sites will help to reduce activation energy and improve catalytic efficiency. As one of the emerging tools in detection engineering, oxygen vacancies (O v ) can endow catalysts with rich electronic orbitals to interact with oxygen-containing small molecules to assist energy transfer and further optimize 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, the synergistic effect between single-atom sites and O v The impact of the synergistic effect between them on their tumor therapeutic properties remains a mystery. Summary of the Invention

[0005] One of the purposes of the present invention is to address the problems existing in the prior art and provide a cell membrane-coated oxygen vacancy Cu2(OH)3Cl loaded Os single atom biomimetic enzyme preparation. v @CCM), which has an Os center and O v , can be used in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting tumor progression; it can be used for targeted treatment of MM. The catalytic active sites and chemical structures of ROS generation benefit 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 mimetic activity (K m :18.5mM,V max :1.41μM s -1 and TON:259.9×10 -3 s -1 ), exhibiting excellent activity. Cancer cell membrane (CCM) coating has emerged as an ideal surface modification strategy to endow OsCu-Ov with excellent biointerface properties, including homologous targeting and efficient OsCu-Ov delivery. CCM-encapsulated OsCu-Ov nanoparticles (OsCu-Ov@CCM) actively penetrate into tumor cells, improve the hypoxic microenvironment, and disrupt mitochondria.

[0006] Furthermore, we characterized a gene signature strongly associated with apoptosis pathways. This study provides a general strategy to effectively promote ROS and nanomedicine penetration into cancer cells and opens new avenues for cancer therapy.

[0007] In order to achieve the above object of the invention, the specific technical solution of the present invention is:

[0008] One of the purposes of the present invention is to provide a cell membrane-coated oxygen vacancy Cu2(OH)3Cl loaded Os single atom biomimetic enzyme preparation OsCu-O v @CCM.

[0009] Furthermore, the molecular formula of the cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation is Os-Cu2(OH)3Cl (OsCu-Ov).

[0010] Furthermore, the cell membrane in the cell membrane-coated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation is a B16F10 tumor cell membrane.

[0011] The second purpose of the present invention is to protect the above-mentioned cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl loaded Os single-atom biomimetic enzyme preparation in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the progression of tumors.

[0012] Preferably, in the aforementioned application, the tumor is malignant melanoma.

[0013] A third object of the present invention is to prepare a method for protecting the aforementioned cell membrane-encapsulated oxygen vacancies Cu2(OH)3Cl-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 CuCl2 and hexamethylenetetramine (HMT) solution, and then Os-Ov catalytic centers were prepared by wet impregnation to obtain Os-Cu2(OH)3Cl(OsCu-Ov) biocatalyst.

[0015] Furthermore, in the preparation method of the cell membrane-coated oxygen vacancy Cu2(OH)3Cl loaded Os single-atom biomimetic enzyme preparation,

[0016] The molar ratio of CuCl2·xH2O 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 to 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 is 4 to 6 hours (specifically, it can be 4 hours, 5 hours, 6 hours, etc.); Cu-O v The mass ratio of iodine to osmium is 50:1 to 10:1 (specifically, 50:1, 40:1, 30:1, 20:1, 10:1, etc.).

[0017] Furthermore, the cell membrane-coated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation prepared by the aforementioned method or combination of method steps has a spherical morphology and an ultra-small nanoscale.

[0018] Preferably, the nanoscale of the cell membrane-coated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation prepared by the aforementioned method is about 30 nm.

[0019] Furthermore, the cell membrane-coated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation prepared by the method has excellent catalase (CAT), halogenase (HPO) and peroxidase (POD) simulation activities and superior ROS generation activity.

[0020] Furthermore, the simulated activity K of the above-mentioned biomimetic enzyme preparation is m :18.5mM,V max :1.41μM 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-encapsulated enzyme mimetic OsCu-Ov@CCM in the present invention, with a single-atom osmium center and oxygen vacancy structure, is a perfect and efficient nanoplatform that mimics the ROS-generating properties of natural enzymes to achieve efficient and targeted malignant melanoma (MM) treatment.

[0023] (2) Due to the structural advantages and ROS catalytic activity of OsCu-Ov@CCM, it can be used in targeted MM treatment. In vitro experiments verified that the synthesized biocatalyst exhibited excellent CAT, HPO, and POD mimetic activities due to the synergistic effect of the single-atom osmium active center and oxygen vacancies, thereby 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.

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

[0025] Furthermore, OsCu-Ov@CCM actively inhibited MM progression, as evidenced by significant changes in gene expression and increased activity of multiple regulatory pathways associated with apoptosis after treatment. This presents a promising avenue for developing high-performance ROS-generating biocatalysts for tumor therapy and other ROS-mediated diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 OsCu-O is a cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl loaded Os single atom biomimetic enzyme preparation v Schematic diagram of the preparation process of @CCM;

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

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

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

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

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

[0032] Figure 7 to mimic the ROS-generating activity of the enzyme of the OsCu-Ov artificial biocatalyst;

[0033] Figure 8 To verify the anti-tumor activity of OsCu-Ov@CCM by in vitro cell experiments;

[0034] Figure 9 For in vivo anti-tumor therapy;

[0035] Figure 10 To explore the mechanism of tumor killing induced by OsCu-Ov@CCM;

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

[0037] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples. It should be understood that the specific examples described herein are merely used to explain the present invention and are not intended to limit 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 the application.

[0038] Example 1:

[0039] A cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation (also known as a cell membrane-encapsulated osmium single-atom biomimetic catalyst with oxygen vacancies and multi-enzyme mimetic activity)

[0040] OsCu-O v The preparation method of @CCM is as follows: Figure 1 As shown, Figure 1In the image, the blue, orange, fluorescent green, pink, and light blue balls represent copper, chlorine, oxygen, hydrogen, and osmium atoms, respectively.

[0041] The preparation method comprises the following steps:

[0042] First, a Cu2(OH)3Cl (Cu-Ov) precursor containing oxygen vacancies was prepared by a typical hydrothermal method using CuCl2 and HMT (hexamethylenetetramine) solution. The molar ratio of CuCl2·xH2O to hexamethylenetetramine was 100:1 to 100:5. The hydrothermal reaction temperature was 95°C and the reaction time was 5 hours.

[0043] Then, Os-Ov catalytic centers were prepared by wet impregnation to obtain Os-Cu2(OH)3Cl (OsCu-Ov) biocatalyst. v The mass ratio of iodine to osmium is 50:1 to 10:1.

[0044] Specific steps: weigh 340.9 mg CuCl2·xH2O (Aladdin) and 3.154 g HMT (hexamethylenetetramine C6H 12 N4, urotropine) in 100mL hydrothermal axe containing 35mL deionized water, mix well, hydrothermally react at 95℃ for 5h, wash with alcohol and deionized water in sequence, and dry to prepare a Cu-Ov precursor; then, weigh 50mg of Cu-Ov precursor into a 20mL glass bottle, add 0.5mL OsCl3 (10mg / ml), stir at room temperature for 24h, filter, wash and dry to obtain.

[0045] The obtained catalyst was subjected to performance test. Figures 2 to 5 ; Figure 2 High-resolution transmission electron microscopy (HR-TEM) image of OsCu-Ov. Figure 3 (c) Selected area electron diffraction (SAED) pattern of OsCu-Ov and the intensity profile along the cyan box in (c). Figure 4 Atomic resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of OsCu-Ov and corresponding elemental analysis. Figure 5 Energy dispersive spectroscopy (EDS) mapping image of OsCu-Ov.

[0046] Figure 2Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM) revealed its spherical morphology and ultrasmall nanoscale (approximately 30 nm). Subsequently, B16F10 CCMs were extracted from B16F10 cells and coated on OsCu-Ov by ultrasound to form OsCu-Ov@CCMs. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to ensure that the CCMs were loaded on OsCu-Ov. X-ray diffraction (XRD) patterns and electron paramagnetic resonance (EPR) confirmed the successful synthesis of the Cu-Ov precursor, and no peaks of osmium particles were observed in the patterns of OsCu-Ov. Osmium doping did not significantly change the spherical morphology and crystal structure of Cu-Ov. The HR-TEM image of OsCu-Ov shows clear lattice fringes of 0.2614, 0.2763 and 0.3165 nm, corresponding to the (022), (121) and (021) planes of Cu-Ov, respectively. Figure 2 The intensity profile obtained by line scanning shows that the lattice fringes of 2.1 / 8 are very consistent with the (121) crystal plane. The selected area electron diffraction (SAED) pattern is consistent with the characteristic diffraction rings of Cu-Ov, confirming the results ( Figure 3 ). Further, atomically dispersed osmium (bright spots) and Cu-Ov crystal lattices ( Figure 4 At the same time, energy dispersive spectroscopy (EDS) mapping clearly shows that the signals of osmium atoms are evenly distributed, further confirming the formation of 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. Figure 6 ; Figure 6 Figures 1 and 2 show the electronic structure of the biocatalysts analyzed by XPS and XAS. (a) High-resolution XPS O 1s spectrum of OsCu-Ov, (b) Cu 2p spectrum, and (c) Os 4f spectrum. (d) Normalized XANES spectrum of the osmium L-edge. (e) The relationship between the osmium L-edge absorption energy (E0) and valence state for OsCu-Ov and reference materials. (f) Osmium k2-weighted Fourier transforms of osmium foil, OsCu-Ov, and OsO2, along with the corresponding fitting results for OsCu-Ov. (g) Osmium foil, (h) OsCu-Ov, and (i) WT image of OsO2 at the osmium L-edge.

[0049] Depend on Figure 6It can be seen that the XPS spectrum of OsCu-Ov shows the simultaneous presence of copper, oxygen, carbon, chlorine and osmium, which is consistent with the EDS results. The O1s and Cl 1s spectra of the OsCu-Ov biocatalyst show obvious peak shifts compared with those of OsCu-Ov, indicating the coordination of osmium and oxygen ( Figure 6 a). Figure 6 As shown in Figure b, taking the 2p3 / 2 region of the copper 2p spectrum in the biocatalyst as an example, the peaks at 933.14 and 935.28 eV are attributed to copper (I) and copper (II) oxidation states, respectively. For the osmium 4f spectrum of OsCu-Ov, the two split peaks at 51.26 and 53.23 eV are attributed to the 4f7 / 2 of osmium 4+ and osmium 3+ ( Figure 6 c). No obvious metallic peaks were observed in the osmium 4f spectrum of OsCu-Ov, further confirming the atomic-level dispersion of osmium ions. Subsequently, X-ray absorption spectroscopy (XAS) was applied to gain a deeper understanding of 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 showed that the white line intensity (red line) of OsCu-Ov was closer to the oxidation state of OsO2 ( Figure 6 d). Valence analysis of osmium in the biocatalyst showed that the average valence of OsCu-Ov was 4.2, which was higher than that of osmium foil and OsO2 (4.0) ( Figure 6 e). The Fourier transformed k2-weighted extended X-ray absorption fine structure (EXAFS) spectrum shows an approx. The main peak of , corresponding to the first coordination shell of osmium-oxygen ( Figure 6 f). The metallic osmium-osmium peak of OsCu-Ov is negligible, which indicates that osmium exists in the form of a single osmium atom. The fitting results indicate that the coordination structure of osmium-oxygen 2-chlorine is very consistent with the first shell of the osmium L-edge EXAFS of OsCu-Ov ( Figure 6 f). Wavelet transform (WT) is further summarized in Figure 6 g–i. Obviously, OsCu-Ov The main peak at is shown, corresponding to the first coordination shell of osmium-oxygen, which confirms the formation of single osmium atoms and is consistent with the HAADF-STEM and XPS results.

[0050] Example 3:

[0051] After verifying the single atom and electronic structure of osmium in OsCu-Ov (based on Example 2), we systematically evaluated and compared the ROS catalytic activities of the biocatalysts, such as POD mimetic activity and HPO mimetic activity. The POD mimetic activity of the biocatalysts was verified using a classic colorimetric test using 3,3′,5,5′-tetramethylbenzidine (TMB, which can be oxidized by ROS to form colored ox-TMB); the specific results are shown in Figure 7 .

[0052] Figure 7 Figure 2 shows the enzyme-mimicking ROS generation activity of the OsCu-Ov artificial biocatalyst; a) shows the POD-mimicking activity. b) shows the O2-generation activity. c) shows the reaction kinetics of the POD-mimicking activity of OsCu-Ov. d) shows the comparison of Vmax and TON of recently reported artificial biocatalysts using H2O2 as a substrate. e) shows the temperature and pH dependence of the POD-mimicking activity of OsCu-Ov. f) shows the free radical quenching results for the quencher of ·OH (TBA), the quencher of ·O2- (BQ), and the quencher of 1O2 (NaN3). g) shows the EPR spectra of the ·OH, ·O2-, and 1O2 signals. h) shows the HPO-mimicking activity.

[0053] like Figure 7 As shown in a, OsCu-Ov and OsCu-Ov@CCM showed excellent ROS generation activity at 652 nm by catalyzing TMB oxidation. Meanwhile, OsCu-Ov was able to effectively utilize H2O2 substrate to generate O2 in the O2 generation test ( Figure 7 b). Then, the catalytic constant (Km = 18.5 mM), maximum reaction rate (Vmax = 1.41 m s-1) and turnover number (TON = 259.9 × 10 -3 s -1 , the maximum number of substrates converted per catalytic atom)( Figure 7 c). On this basis, we systematically compared OsCu-Ov with several advanced POD mimetic biocatalysts, such as metal oxides (Fe3O4, CeO2, Co3O4, CuO, Mn3O4, etc.), metal nanoparticles (Ru NPs, Pt NCs, PtFe@Fe3O4, etc.), single-atom biocatalysts (Fe-N-C SAzymes, Co-NC SAzymes, Zn-NC SAzymes, etc.), and metal-organic frameworks (Fe-MOF, etc.). The results showed that OsCu-Ov biocatalyst showed excellent ROS catalytic activity among current nanomaterials ( Figure 7d). OsCu-Ov also showed high ROS generation activity under various reaction conditions, such as a wide range of temperature (20-50 °C) and pH value (3-6) ( Figure 7 e). In order to verify the ROS catalytic activity of OsCu-Ov after long-term storage, a cycling test was performed. Interestingly, the catalytic activity of OsCu-Ov did not decrease significantly after immersion for 30 days, indicating its good stability. In addition, the free radical quenching experiment ( Figure 7 f) Fluorescence probe and electron paramagnetic resonance (EPR) further revealed that the ROS species of OsCu-Ov during POD simulation were ·OH, ·O2- and 1O2( Figure 7 g). In addition to the POD mimetic activity, the HPO mimetic activity of OsCu-Ov was also evaluated by the lapis lazuli blue (CB) test. Figure 7 As shown in Figure 3h, OsCu-Ov exhibits a very high intensity ratio, indicating its strong activity in catalyzing the oxidation of chlorine to HOCl / OCl-.

[0054] Example 4:

[0055] Inspired by the excellent catalytic ROS generation properties of OsCu-Ov@CCM, we first evaluated its potential for treating tumors through artificial biocatalysts through cell experiments. Human venous endothelial cells (HUVECs) were cultured in 96-well plates for 24 hours, with 1.0×10^4 cells seeded per well. The culture medium in the 96-well plates was discarded and washed with phosphate buffered saline (PBS). Subsequently, OsCu-Ov@CCM was added to the 96-well plates. v @CCM complete medium (for HUVECs), OsCu-O v The concentrations of @CCM were 0, 10, 20, 40, 80, and 160 μg / mL, and the cells were cultured for another 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 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 assess cell viability.

[0056] Figure 8In vitro cell experiments verify the antitumor activity of OsCu-Ov@CCM. (a) Bio-TEM image of B16F10 cells engulfing OsCu-Ov@CCM. (b) Colocalization of OsCu-Ov and OsCu-Ov@CCM, cytoskeleton, and nucleus. B16F10 cells were stained to identify the cytoskeleton (green), nucleus (blue), and DiI-stained nanozymes (red) and observed by confocal laser scanning microscopy (CLSM). Scale bar = 50 μm. (c) Quantitative data of Calcein AM / PI staining of B16F10 cells after different treatments. (d) Quantitative analysis of Annexin V-FITC and PI co-staining of B16F10 cells. (e) DCF fluorescence distribution of B16F10 cells after different treatments and (f) quantitative analysis measured by flow cytometry. (g) Schematic diagram of the antitumor activity of OsCu-Ov@CCM. h) Bio-TEM image of B16F10 cells treated with OsCu-Ov@CCM. White triangles mark the disappearance of mitochondrial cristae and mitochondrial swelling. i) Quantitative data of JC-1 staining of B16F10 cells.

[0057] First, we confirmed that OsCu-Ov@CCM was internalized by cells. Figure 8 Figure a shows that OsCu-Ov@CCM was efficiently internalized into B16F10 cells after 6 hours of co-incubation, as observed by biological transmission electron microscopy (Bio-TEM). Furthermore, the intracellular localization behavior of OsCu-Ov and OsCu-Ov@CCM was investigated using confocal laser scanning microscopy (CLSM), revealing a distinct DiI signal. As expected, the DiI signal fluorescence intensity in the OsCu-Ov@CCM group significantly exceeded 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 capability provided by active cancer cell membrane proteins, such as TF antigen and E-cadherin.

[0058] Subsequently, we evaluated the cytocompatibility of OsCu-Ov@CCM using a cell counting kit-8 (CCK-8) assay with human umbilical vein endothelial cells (HUVECs). The results showed that OsCu-Ov@CCM had a mild cytotoxic effect on HUVECs. Furthermore, even at a concentration of 200 μg mL⁻¹, the hemolysis rate was less than 5%, demonstrating its excellent hemocompatibility. Subsequently, the antitumor activity of OsCu-Ov@CCM against B16F10 cells was confirmed using a CCK-8 assay in an acidic complete medium at pH 6.5 to simulate the tumor microenvironment (TME). The results showed that the survival rate of B16F10 cells decreased significantly with increasing OsCu-Ov@CCM concentration, so a concentration of 40 μg mL⁻¹ was used in subsequent experiments.

[0059] To compare the antitumor activity of OsCu-Ov@CCM and another component on B16F10 cells, we used Calcein AM (green represents live cells) and propidium iodide (red represents dead cells) co-staining. Both OsCu-Ov and OsCu-Ov@CCM groups showed more red fluorescence than the corresponding 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 may be due to the same targeting ability of cancer cell membranes. Subsequently, we performed detailed apoptosis examination by flow cytometry (FCM). We used membrane-bound 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 with other groups.

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

[0061] Mitochondria are the site of ROS regulation, and apoptosis is associated with mitochondrial dysfunction, so we examined mitochondrial damage to discuss the anti-tumor mechanism. Figure 8As shown in h, biological transmission electron microscopy (Bio-TEM) observed that the mitochondria of B16F10 cells treated with OsCu-Ov@CCM were damaged, including the disappearance of mitochondrial cristae and mitochondrial swelling. At the same time, we used 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidazolium carbocyanine iodide salt (JC-1) as a probe of mitochondrial membrane potential. In healthy polarized mitochondrial membranes, it appears as red fluorescent aggregates, while in damaged and depolarized mitochondrial membranes, it appears as green fluorescent monomers. A slight green fluorescence signal was detected in the control group, Cu-Ov and Cu-Ov@CCM-treated groups, while in the OsCu-Ov@CCM group, the JC-1 dye changed from red aggregates to green monomers, indicating the presence 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 B16F10 tumors was constructed. B16F10 cells (1×10 6 The tumors were resuspended in 100 μL of 1640 medium and injected subcutaneously into the right axilla of each mouse. During the experiment, the tumor volume was allowed to reach 1500 mm 3 When the tumor volume reaches about 100mm 3 The tumor-bearing BALB / c mice were randomly divided into five groups and injected intratumorally (n=3 per group): 1. control group; 2. Cu-O v (10 mg / kg, 100 μL); 3. Cu-O v @CCM(10mg / kg, 100μL); 4.OsCu-O v (10 mg / kg, 100 μL); 5. OsCu-O v @CCM (10 mg / kg, 100 μL). Measure the weight and tumor size of mice every other day. The formula for calculating tumor volume is: tumor volume = 1 / 2 × length × width 2. After two weeks of treatment, the mice were killed and the tumors were collected. Finally, tumors and major organs (heart, liver, spleen, lungs, and kidneys) from different groups were collected and sectioned for H&E staining, Ki-67, and TUNEL detection. Blood samples from each group of mice were centrifuged at 3000 rpm for 10 minutes to collect plasma, which was further used for 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. (a) Schematic diagram of the timeline of in vivo anti-cancer treatment. (b) Weight changes of tumor-bearing mice during treatment, with a 14-day treatment cycle. (c) Heat map of tumor volume during treatment, n = 3. (d) Tumor weights for each group. (e) Quantitative analysis of TUNEL and Ki67+ staining. (g) Representative images of H&E, TUNEL, and Ki-67 immunofluorescence staining of tumor sections from each group (scale bar = 100 μm).

[0065] Depend on Figure 9 It can be seen that during the treatment period, all mice had a slight increase in body weight ( Figure 9 b). During the treatment period, the increasing trend of tumor volume and tumor weight in OsCu-Ov and OsCu-Ov@CCM groups showed significant inhibition of tumor growth compared with the control, Cu-Ov and Cu-Ov@CCM groups ( Figure 9 c and 9d). In addition, the OsCu-Ov@CCM group showed the most significant tumor inhibitory effect. After the treatment, the mice were sacrificed, the tumors were removed, and the main organs (heart, liver, spleen, lungs and kidneys) were collected for further analysis. Hematoxylin-eosin (H&E) staining of the main organs showed no significant pathological damage, and there was no statistically significant difference in the serum biochemical analysis of mice with different treatments, indicating that the treatment had minimal side effects and was extremely safe. Tumor sections were subjected to H&E staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and antigen Ki-67 staining to further evaluate the therapeutic effect ( Figure 9 g). According to the results of H&E staining, the tumor cell nuclei in the OsCu-Ov and OsCu-Ov@CCM groups were fewer than those in the other groups. In addition, the OsCu-Ov@CCM-treated group showed the most significant apoptosis of tumor cells in the TUNEL assay ( Figure 9 e). In addition, immunohistochemical staining of Ki-67, a key marker 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, n=3 in each group) were sacrificed after different treatments, and total mRNA was extracted from their tumor tissues. Detailed results are shown in Figure 10 .

[0068] Figure 10 Figure 1: Schematic diagram of the tumor-killing mechanism induced by OsCu-Ov@CCM. (a) Schematic diagram of the RNAseq experimental workflow. (b) Volcano plot of all differentially expressed genes (DEGs) between the control and OsCu-Ov@CCM groups (expressed proteins were defined as having a |fold-change| > 1.5 and p < 0.05). Dots represent DEGs (FDR ≤ 0.001, at least a 2-fold difference); red indicates upregulation, and green indicates downregulation. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated genes between the OsCu-Ov@CCM and control groups. (d) KEGG pathway enrichment analysis of downregulated genes between the OsCu-Ov@CCM and Cu-Ov@CCM groups. (e) Heat map of differentially expressed DEGs enriched in the OsCu-Ov@CCM and control groups. f) Protein expressions of Cleaved-caspase 3, Bcl-2 and Bax in B16F10 cells of different groups and their statistical results. gh)

[0069] Depend on Figure 10 These mRNAs were subsequently analyzed by high-throughput mRNA sequencing ( Figure 10 a). Gene expression profiles of each group changed significantly after different treatments. RNA sequencing results showed that 1,296 genes were upregulated and 340 genes were downregulated in the OsCu-Ov@CCM-treated group compared with the control group ( Figure 10 b). Based on the above studies, OsCu-Ov@CCM has been shown to have strong anti-tumor efficacy both in vitro and in vivo. We used bioinformatics techniques to comprehensively elucidate the biological functions of the regulatory genes altered by OsCu-Ov@CCM. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis ( Figure 10 c) showed that the genes upregulated by OsCu-Ov@CCM were concentrated in several regulatory pathways related to apoptosis, such as IL-17, PI3K-Akt, and TNF signaling pathways. This suggests that tumor cytotoxicity may be related to apoptosis. Figure 10e showed that OsCu-Ov@CCM administration affected genes involved in cancer pathways, IL-17 signaling pathway, PI3K-Akt signaling pathway, and 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 downregulation of apoptosis-related genes, which was consistent with the results of previous tests ( Figure 10 d). Furthermore, gene set enrichment analysis (GSEA) indicated that OsCu-Ov@CCM could disrupt the inner mitochondrial membrane protein complex and the mitochondrial protein complex, and that OsCu-Ov@CCM treatment corresponded to increased activity of several apoptosis-related regulatory pathways, 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, potentially leading to improved MM treatment outcomes.

[0070] To confirm the changes in mRNA expression, we used Western blot analysis to measure protein levels of three key apoptotic markers: cleaved caspase-3, Bcl-2, and Bax. Compared to the control group, the OsCu-Ov@CCM group displayed significantly higher levels of cleaved caspase-3 and Bax, indicating enhanced apoptosis. Conversely, Bcl-2 protein levels decreased significantly in the OsCu-Ov@CCM group, further confirming increased apoptotic activity. Consistent with previous results, the OsCu-Ov@CCM group showed decreased Bcl-2 expression and increased levels of cleaved caspase-3 and Bax relative to the Cu-Ov@CCM group.

[0071] In summary, the novel cell-membrane-encapsulated enzyme-mimicking material (OsCu-Ov@CCM) prepared in this paper, featuring a single-atom osmium center and oxygen vacancies, represents a perfect and highly effective nanoplatform for mimicking the ROS-generating properties of natural enzymes, enabling efficient and targeted treatment of malignant melanoma (MM). It can also be used in the preparation of drugs or formulations for treating tumors or inhibiting tumor progression. The structural advantages and ROS-catalytic activity of OsCu-Ov@CCM were systematically investigated for their application in targeted MM therapy. In vitro experiments demonstrated that the synthesized biocatalyst exhibited excellent CAT, HPO, and POD-mimicking activities due to the synergistic effect of the single-atom osmium center and oxygen vacancies, resulting in superior ROS-generating activity compared to natural micromolecules and most reported ROS-catalyzing biocatalysts. In vivo experiments demonstrated minimal side effects and an extremely high safety profile. Furthermore, OsCu-Ov@CCM actively inhibited MM progression, as evidenced by significant changes in gene expression and increased activity of multiple apoptosis-related regulatory pathways following treatment. We believe that this study will open up a promising direction for developing high-performance ROS-generating biocatalysts in tumor therapy and other ROS-mediated diseases.

[0072] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

[0073] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of the description in this section that did not constitute prior art at the time of filing are neither explicitly nor implicitly 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 @CCM is used in the preparation of drugs or preparations for treating tumors, or drugs or preparations for inhibiting the progression of tumors; the biomimetic enzyme preparation OsCu-O v The molecular formula of @CCM is Os-Cu2(OH)3Cl@CCM.

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

3. The use according to claim 1, wherein: The cell membrane is a B16F10 tumor cell membrane.

4. The use according to any one of claims 1 to 3, characterized in that Preparation method of cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl loaded Os single atom biomimetic enzyme preparation, The following steps are involved: Firstly, Cu2(OH)3Cl precursor Cu-O containing oxygen vacancies was prepared by a typical hydrothermal method using CuCl2•xH2O and hexamethylenetetramine solution. v Then, the osmium-oxygen vacancy catalytic center Os-O was prepared by wet impregnation. v , thereby obtaining the Os-Cu2(OH)3Cl biomimetic enzyme preparation OsCu-O v .

5. The use according to claim 4, 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 iodine to osmium is 50:1 to 10:

1.

6. The use according to claim 4, characterized in that: The cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation is spherical in shape and has a nanoscale; the nanoscale is 30 nm.

7. The use according to claim 6, wherein: OsCu-Ov and OsCu-Ov@CCM showed excellent ROS generation activity at 652nm by catalyzing 3,3',5,5'-tetramethylbenzidine reagent. At the same time, OsCu-Ov was able to effectively utilize H2O2 substrate to produce O2 in the O2 generation test. The catalytic constant Km, maximum reaction rate Vmax, and turnover number TON of H2O2 were calculated using the Michaelis-Menten equation diagram. The simulated activity of the cell membrane-encapsulated oxygen vacancy Cu2(OH)3Cl-loaded Os single-atom biomimetic enzyme preparation was also investigated. K m : 18.5 mM, V max : 1.41 μM s -1 and TON: 259.9×10 -3 s -1 .

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