Bionic non-iron-based nano enzyme and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and discloses a bionic non-iron-based nano-enzyme and application thereof, a preparation method of the bionic non-iron-based nano-enzyme comprises the following steps: S1, preparing meso-porous silicon nanoparticles MSNs; s2, loading platinum gold and palladium gold on the meso-porous silicon nanoparticles MSNs, so as to obtain MPP nano-enzyme; s3, carrying out functionalization on the MPP nano-enzyme by grafting cinnamyl aldehyde on the surface of the MPP nano-enzyme to obtain an MPPC nano-enzyme; and S4, coating the MPP nano enzyme or the MPPC nano enzyme with a 4T1 cell membrane to obtain an MPP (at) CM nano enzyme or an MPPC (at) CM nano enzyme, namely the bionic non-iron-based nano enzyme. The biomimetic non-iron-based nano-enzyme disclosed by the invention has the activity similar to that of a catalytic enzyme (CAT), peroxidase (POD) and oxidase (OXD), and can generate reactive oxygen species (ROS) in tumor cells to exhaust glutathione (GSH) to trigger ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a biomimetic non-ferrous-based nanozyme and its application. Background Art

[0002] Radiation therapy is one of the standard options for cancer treatment such as breast cancer. With the progress of radiation therapy technology, precise dose delivery to the tumor site has become possible, while reducing systemic toxicity. Radiation therapy achieves tumor cell death by directly damaging biomolecules (such as DNA and mitochondria) and indirectly by inducing oxidative stress and the generation of reactive oxygen species (ROS). However, certain characteristics of the tumor microenvironment (TME), such as hypoxia, low pH value, and high interstitial pressure, pose challenges to the sensitivity of radiation therapy, resulting in poor treatment effects.

[0003] Hypoxia in the tumor microenvironment is one of the main factors contributing to radiation therapy resistance. Hypoxia can reduce the direct DNA damage effect of radiation therapy and support tumor survival by increasing HIF-1α signaling and the regulation of cancer-associated fibroblasts (CAFs). In addition, radiation therapy can trigger immunogenic cell death (ICD), but the presence of radiation-resistant inhibitory cell types in the TME often leads to immunosuppression. This complex interaction within the TME not only affects the immune response to radiation therapy but also promotes radiation resistance, resulting in tumor recurrence.

[0004] Ferroptosis is a newly discovered non-apoptotic form of cell death, which is closely related to the efficacy of radiation therapy. The characteristics of ferroptosis are the accumulation of intracellular iron ions, the increase in lipid peroxide levels, and the imbalance of the antioxidant system. The depletion of glutathione (GSH) and the accumulation of iron ions are important markers of ferroptosis. Clinical studies have shown that increasing the level of ferroptosis can improve treatment outcomes and prolong the progression-free survival of patients receiving radiation therapy. During radiation therapy, externally generated ROS can effectively disrupt the redox balance, leading to the formation and accumulation of lipid peroxidation products (LPOs), or regulate genomic factors to promote ferroptosis. However, GSH can neutralize ROS, thereby hindering the reaction metabolism of LPO as a substrate for GPX4.

[0005] In order to improve the efficacy of radiation therapy, using nanozyme technology to regulate the redox state has become an important method. Nanozymes are composed of noble metals and have excellent catalytic activities, including functions similar to peroxidase (POD), oxidase (OXD), catalase (CAT), and superoxide dismutase (SOD), as well as the ability to reduce metal ions. These nanozymes have high enzyme activities due to their excellent electron conductivity, abundant active sites, and large specific surface area. In particular, noble metal-based nanozymes exhibit enhanced catalytic performance due to their synergistic effects, making them very effective in tumor treatment applications.

[0006] In summary, the development of novel nanozymes to enhance the efficacy of radiotherapy, especially in promoting ferroptosis and regulating the tumor microenvironment, is of great significance for improving cancer treatment outcomes. Summary of the Invention

[0007] The object of the present invention is to provide a biomimetic non-iron-based nanozyme and its application, using a nanozyme with oxidative promotion activity to trigger ferroptosis and enhance the efficacy of radiotherapy.

[0008] To achieve the above object, the present application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a biomimetic non-iron-based nanozyme, and its preparation method includes the following steps:

[0010] S1. Prepare mesoporous silica nanoparticles MSNs;

[0011] S2. Load platinum and palladium on the mesoporous silica nanoparticles MSNs to obtain MPP nanozyme;

[0012] S3. Functionalize the MPP nanozyme by surface grafting cinnamaldehyde to obtain MPPC nanozyme;

[0013] S4. Coating 4T1 cell membranes on the MPP nanozyme or the MPPC nanozyme to obtain MPP@CM nanozyme or MPPC@CM nanozyme, which is the biomimetic non-iron-based nanozyme.

[0014] In the above technical solution, in S1, the specific preparation method of the mesoporous silica nanoparticles MSNs is as follows: Dissolve CTAB in deionized water, adjust the pH to 10, add TEOS, stir under a nitrogen atmosphere, collect the precipitate by centrifugation, wash with ethanol or deionized water, then treat with 1% HCl ethanol solution, stir, and vacuum dry overnight to obtain the mesoporous silica nanoparticles MSNs.

[0015] In the above technical solution, in S2, the specific preparation method of the MPP nanozyme is as follows: Disperse the mesoporous silica nanoparticles MSNs in a methanol solution containing chloroplatinic acid and palladium chloride, stir, concentrate, wash with methanol, and freeze-dry to obtain the MPP nanozyme.

[0016] In the above technical solution, in S3, the specific preparation method of the MPPC nanozyme is as follows: Ultrasonically disperse the MPP nanozyme in methanol, add 3-aminopropyltriethoxysilane, wash with methanol after the reaction is completed, then disperse in methanol, add cinnamaldehyde, wash with methanol after the reaction is completed, and freeze-dry to obtain the MPPC nanozyme.

[0017] In the above technical solution, in S4, the specific preparation method of the MPP@CM nanozyme or the MPPC@CM nanozyme is as follows: A solution containing the 4T1 cell membrane and the MPP nanozyme or the MPPC nanozyme are co-extruded through a polycarbonate membrane with a pore size of 200 nm, and thus the MPP@CM nanozyme or the MPPC@CM nanozyme is obtained.

[0018] In a second aspect, the present invention provides the use of the above-mentioned biomimetic non-ferrous nanozyme in the preparation of radioimmunotherapy drugs.

[0019] In a third aspect, the present invention provides the use of the above-mentioned biomimetic non-ferrous nanozyme in the preparation of drugs for promoting ferroptosis.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned biomimetic non-ferrous nanozyme in the preparation of drugs for regulating the tumor microenvironment.

[0021] In a fifth aspect, the present invention provides a drug containing the above-mentioned biomimetic non-ferrous nanozyme.

[0022] In a sixth aspect, the present invention provides a reagent or a kit containing the above-mentioned biomimetic non-ferrous nanozyme.

[0023] The beneficial effects of the present invention are as follows:

[0024] (1) The biomimetic non-ferrous nanozyme of the present invention has activities similar to those of catalase (CAT), peroxidase (POD) and oxidase (OXD), generates reactive oxygen species (ROS) in tumor cells, depletes glutathione (GSH), and triggers ferroptosis.

[0025] (2) The biomimetic non-ferrous nanozyme of the present invention uses a cancer cell membrane coating with the ability to enhance tumor-specific targeting, which can improve the cancer treatment effect. Description of the Drawings

[0026] Figure 1 : Synthesis and characterization of nanozymes.

[0027] Figure 1 A shows the morphology and size of the MPP nanozyme observed by transmission electron microscopy (TEM), showing a uniform spherical structure and surface features.

[0028] Figure 1 B shows the infrared spectra of the MPP and MPPC nanozymes obtained by Fourier transform infrared spectroscopy (FTIR), revealing the characteristic peak changes after grafting cinnamaldehyde.

[0029] Figure 1 C shows the elemental distribution mapping of the MPPC BNs, showing the spatial distribution of elements.

[0030] Figure 1 D demonstrated the particle size distribution of nanozymes through dynamic light scattering (DLS) analysis, providing quantitative information on the size of nanozymes.

[0031] Figure 1 E to 1G demonstrated the chemical states on the surface of nanozymes through X-ray photoelectron spectroscopy (XPS) analysis, especially the electronic states and binding energies of Pt and Pd.

[0032] Figure 1 H demonstrated the atomic percentages of Pd and Pt in MPPC.

[0033] Figure 1 I demonstrated the morphology and size of cancer cell membrane-coated MPPC@CM BNs observed by TEM, showing a uniform spherical structure and surface characteristics.

[0034] Figure 1 J demonstrated the protein expression in MPPC, CM, and MPPC@MC through SDS-PAGE protein expression analysis.

[0035] Figure 1 K demonstrated the Zeta potential measurement results of MPPC, CM, and MPPC@CM, revealing the surface charge characteristics.

[0036] Figure 2 : Evaluation of the enzymatic activity of nanozymes.

[0037] Figure 2 A demonstrated the evaluation results of the similar catalase (CAT) activity of MPP@CM and MPPC@CM nanozymes by measuring the concentration of O2 generated after reacting with H2O2.

[0038] Figure 2 B and 2C demonstrated the GSH consumption evaluated through 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) experiments, showing the GSH consumption ability of MPPC@CM nanozymes.

[0039] Figure 2 D demonstrated the evaluation of the similar oxidase (OXD) activity of MPP@CM and MPPC@CM nanozymes by measuring the absorbance change of TMB oxidation.

[0040] Figure 2 E and 2F demonstrated the evaluation of the similar peroxidase (POD) activity of MPP@CM and MPPC@CM nanozymes by measuring the absorbance change of TMB under the action of nanozymes.

[0041] Figure 2 G demonstrated the enzymatic kinetic reactions of MPP@CM and MPPC@CM, and the reaction kinetics followed typical Michaelis-Menten characteristics.

[0042] Figure 2 H and 2I show the ESR spectra of ·OH radicals and ·O2- radicals captured by DMPO.

[0043] Figure 2 J and 2K show the evaluation of radical scavenging rate by ABTS and DPPH.

[0044] Figure 2 L and 2M show the POD-like enzyme activity of MPP@CM and the effect of ROS scavengers on the enzyme-like activity of MPPC@CM.

[0045] Figure 3 : Detection and evaluation of ferroptosis in cells.

[0046] Figure 3 A shows the cytotoxicity of IEC6 and 4T1 cells after co-culture with gradient-diluted MPPC@CM, and the cytotoxicity is greater in 4T1 cells than in IEC6 cells.

[0047] Figure 3 B shows the GSH detection results, indicating a significant decrease in the intracellular GSH level after treatment with MPPC@CM.

[0048] Figure 3 C and 3D show the relative expression levels of GPX4 under different treatments, indicating that the intracellular GPX4 protein level is the lowest after treatment with MPPC@CM combined with radiotherapy.

[0049] Figure 3 E shows the MDA detection results under different treatment conditions, indicating that the intracellular MDA level increases most significantly after treatment with MPPC@CM combined with radiotherapy.

[0050] Figure 3 F and 3G show the BODIPY 581 / 591 staining results, indicating a significant increase in the intracellular lipid peroxidation level after treatment with MPPC@CM combined with radiotherapy.

[0051] Figure 3 H and 3I show the relative expression levels of HIF-1α under different treatments, indicating that the intracellular HIF-1α protein level is the lowest after treatment with MPPC@CM combined with radiotherapy.

[0052] Figure 3 J shows the DCFH-DA staining results, revealing a significant increase in the intracellular ROS level after treatment with MPPC@CM combined with radiotherapy.

[0053] Figure 3K shows the results of live / dead cell staining, where live cells show green fluorescence (Calcein AM) and dead cells show red fluorescence (PI), indicating a decrease in cell viability and an increase in the number of dead cells after treatment with MPPC@CM combined with radiotherapy.

[0054] Figure 3 L and 3M show the apoptosis rates of cells in different treatment groups, indicating an increase in the apoptosis rate and a decrease in the viability of cells after treatment with MPPC@CM combined with radiotherapy.

[0055] Figure 3 N shows the fluorescence imaging of γ-H2AX foci formation in different treatment groups, indicating the most severe DNA damage after treatment with MPPC@CM combined with radiotherapy.

[0056] Figure 3 O and 3P show the results of colony formation assays at different doses and the cell survival curves fitted based on the single-hit multi-target model, indicating that MPPC@CM has a radiosensitizing effect.

[0057] Figure 4 : Evaluation of immunogenic cell death (ICD) of cells.

[0058] Figure 4 A shows a schematic diagram of the dendritic cell maturation assay.

[0059] Figure 4 B to 4E show the immunofluorescence images and quantitative analysis results, revealing the exposure of CRT on the cell surface and the release of HMGB1 after treatment with MPPC@CM, which are key markers of ICD.

[0060] Figure 4 F and 4G show the results of dendritic cell (DC) maturation assays, showing an increase in the DC maturation level after treatment with MPPC@CM, by analyzing the expression of the maturation markers CD80 and CD86 on the surface of DCs by flow cytometry. Detailed implementation manners

[0061] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will be defined only by the claims.

[0062] Example 1: Synthesis and characterization of materials

[0063] 1. Synthesis of mesoporous silica nanoparticles (MSNs):

[0064] Dissolve 1 g of CTAB (cetyltrimethylammonium bromide) in 50 mL of deionized water, adjust the pH to 10, add 1 mL of TEOS (tetraethyl orthosilicate), stir at 40 °C for 4 hours under a nitrogen atmosphere, collect the precipitate by centrifugation, wash it with ethanol or deionized water, then treat it with 20 mL of 1% HCl ethanol solution, stir at 60 °C for 6 hours, and finally dry it overnight under vacuum to obtain MSNs.

[0065] 2. Synthesis of MSNs loaded with platinum and palladium (MPP):

[0066] Disperse 133 mg of MSNs in 20 mL of methanol solution containing 0.1 mmol of chloroplatinic acid and 0.1 mmol of palladium chloride, stir at 70 °C for 3 hours, concentrate by rotary evaporation, wash with methanol, and freeze-dry to obtain MPP nanozyme.

[0067] 3. Synthesis of MPP grafted with cinnamaldehyde (MPPC):

[0068] Ultrasonically disperse 50 mg of MPP nanozyme in 10 mL of methanol, add 3-aminopropyltriethoxysilane in an equimolar amount to MPP, seal and react overnight in a metal bath at 65 °C, wash with methanol, then disperse it in methanol, add 10 mol of cinnamaldehyde, wash with methanol after the reaction is completed, and freeze-dry to obtain MPPC nanozyme.

[0069] 4. Synthesis of MPP@CM nanozyme or MPPC@CM nanozyme:

[0070] First, homogenize 4T1 cells, then perform low-speed centrifugation to remove unbroken cells, collect the supernatant and perform high-speed centrifugation, discard the precipitate, obtain the supernatant, and purify the cell membrane by repeated differential centrifugation. Subsequently, co-extrude 100 μg / mL cell membrane solution and 50 mg of MPP or MPPC solution through a polycarbonate membrane with a pore size of 200 nm to obtain encapsulated MPP@CM nanozyme or MPPC@CM nanozyme.

[0071] 5. Material characterization (corresponding to Figure 1 description):

[0072] Use transmission electron microscopy (TEM) to observe the morphology and size of the nanozyme. As Figure 1 shown in A, it shows the uniform spherical structure and surface characteristics of MPP nanozyme.

[0073] As Figure 1 shown in E, analyze the surface chemical state of the nanozyme by X-ray photoelectron spectroscopy (XPS); as Figure 1 shown in F and 1G, show the electronic state and binding energy of Pt and Pd.

[0074] Dynamic light scattering (DLS) was used to determine the particle size distribution of nanozymes, as Figure 1 shown in D, which shows the average particle size of the nanozymes.

[0075] Fourier transform infrared spectroscopy (FTIR) was used to analyze the infrared spectral characteristics of nanozymes, as Figure 1 shown in B, which shows the characteristic peaks after grafting cinnamaldehyde.

[0076] Ultraviolet-visible absorption spectroscopy (UV-vis) was used to determine the absorption characteristics of nanozymes, as Figure 1 shown in C, which shows the absorbance of nanozymes at different wavelengths.

[0077] As Figure 1 shown in H, which shows the atomic percentage of Pd and Pt in the PtPd deposit.

[0078] The morphology and size of cancer cell membrane-coated MPPC@CM BNs observed by TEM, as Figure 1 shown in I, which shows a uniform spherical structure and surface characteristics.

[0079] Protein expression analysis by SDS-PAGE showed the protein expression in MPPC, CM, and MPPC@MC, as Figure 1 shown in J, and the protein profile of MPPC@CM was exactly the same as that of CM.

[0080] The Zeta potential of MPPC, CM, and MPPC@CM was measured, as Figure 1 shown in K, and the potential decreased from -11.3 mV to -22.3 mV after camouflage, indicating successful coating.

[0081] Example 2: Evaluation of the enzyme activity of nanozymes

[0082] 1. Evaluation of catalase (CAT)-like activity:

[0083] The CAT activity was evaluated by measuring the concentration of O2 bubbles after mixing MPP@CM or MPPC@CM nanozymes with H2O2 using a dissolved oxygen meter.

[0084] 2. Evaluation of oxidase (OXD)-like activity:

[0085] TMB was added to the MPP@CM or MPPC@CM solution, and the change in absorbance at 652 nm was measured to evaluate the OXD activity.

[0086] 3. Evaluation of peroxidase (POD)-like activity:

[0087] MPP@CM or MPPC@CM was added to the H2O2 solution containing TMB, and the UV-visible absorption spectrum at 652 nm was measured to evaluate the POD activity.

[0088] 4. Results of enzyme activity evaluation (corresponding to Figure 2 )

[0089] The CAT-like activity was evaluated by measuring the production of O2, as shown in Figure 2 A, showing the increasing amount of O2 production by MPPC@CM nanozyme over time.

[0090] The OXD-like activity was evaluated by the TMB oxidation experiment, as shown in Figure 2 D, showing the increase in superoxide anions produced by MPPC@CM nanozyme.

[0091] The POD-like activity was evaluated by UV-vis absorption spectrum, as shown in Figure 2 E and 2F, showing the increase in the absorbance of TMB after treatment with MPPC@CM nanozyme, indicating the enhancement of POD activity.

[0092] The GSH consumption was evaluated by the DTNB experiment, as shown in Figure 2 B and 2C, showing the GSH consumption ability of MPPC@CM nanozyme.

[0093] The Michaelis constant and maximum reaction rate of MPPC@CM were calculated by kinetic analysis, as shown in Figure 2 G, showing the enzyme kinetic reactions of MPP@CM and MPPC@CM, and the reaction kinetics follows typical Michaelis-Menten characteristics.

[0094] The generation of ·OH radicals and ·O2- radicals was detected by electron paramagnetic resonance (ESR), as shown in Figure 2 H and 2I, showing the ESR spectra of ·OH radicals and ·O2- radicals captured by DMPO.

[0095] The ROS scavenging efficiency was evaluated by ABTS and DPPH methods. As shown in Figure 2 J and 2K, with the increase of concentration, the ABTS radical scavenging rate of MPP@CMBNs decreased, while the MPPC@CM group maintained a low-level constant.

[0096] Furthermore, the types of ROS, including singlet oxygen ( 1 O2), hydroxyl radical (·OH) and superoxide anion (·O2-), were explored by benzoquinone, tert-butanol and sodium azide. As shown in Figure 2 L and 2M, MPPC@CM still inhibited the absorption peak of oxTMB in the presence of benzoquinone or tert-butanol, while the absorption decreased significantly after treatment with sodium azide.

[0097] Example 3: Detection and Evaluation of Ferroptosis in Cells

[0098] 1. Cell Culture and Treatment:

[0099] Seed 4T1 breast cancer cells in 96-well plates. After the cells adhere to the plate, add MPPC@CM at different concentrations for treatment, and add an equal volume of PBS to the control group.

[0100] After culturing for a certain period of time, perform the following detections.

[0101] 2. Cell Viability Assessment (CCK8 method):

[0102] Use the Cell Counting Kit-8 (CCK8) to measure cell viability. Add the CCK8 reagent to each well, continue to culture for 1 - 2 hours, and then measure the optical density at 450 nm to determine cell viability.

[0103] 3. Cell Apoptosis Analysis (Annexin V-FITC / PI method):

[0104] Detect cell apoptosis by Annexin V-FITC / PI staining. Collect the treated cells, stain them with Annexin V-FITC and PI, and then analyze them using a flow cytometer.

[0105] 4. Detection of Intracellular Reactive Oxygen Species (ROS) Level (DCFH-DA method):

[0106] Use DCFH-DA as a fluorescent probe for ROS. Treat the treated cells with DCFH-DA staining solution, incubate in the dark for 30 minutes, and then use a confocal laser scanning microscope (CLSM) to detect the fluorescence intensity of intracellular ROS.

[0107] 5. Detection of Ferroptosis in Cells (BODIPY 581 / 591 method):

[0108] Use the BODIPY 581 / 591 fluorescent probe to detect the level of intracellular lipid peroxidation. Treat the treated cells with BODIPY 581 / 591 staining solution, incubate in the dark for 30 minutes, and then use CLSM to detect the fluorescence intensity of intracellular lipid peroxidation.

[0109] 6. Detection of Intracellular Glutathione (GSH) Level:

[0110] Use a GSH detection kit to measure the intracellular GSH level. Collect the treated cells, operate according to the kit instructions, and measure the GSH level.

[0111] 7. Live / Dead Cell Staining (Calcein AM and PI method):

[0112] Live and dead cell staining was performed using Calcein AM and PI. The treated cells were incubated with a staining solution containing Calcein AM and PI for 30 minutes in the dark, and then the cell fluorescence was detected using a fluorescence microscope or flow cytometry. Live cells showed green fluorescence, and dead cells showed red fluorescence.

[0113] In Figure 3 , we presented the evaluation results of ferroptosis in cells after MPPC@CM treatment. Figure 3 A shows the results of cell viability detection after co - culturing IEC6 and 4T1 cells with gradient - diluted MPPC@CM, indicating that MPPC@CM has greater cytotoxicity in 4T1 cells than in IEC6 cells.

[0114] Figure 3 B shows the results of GSH detection, indicating a significant decrease in the intracellular GSH level after MPPC@CM treatment.

[0115] Figure 3 C and 3D show the relative expression levels of GPX4 under different treatments, indicating that the intracellular GPX4 protein level is the lowest after the combined treatment of MPPC@CM and radiotherapy.

[0116] Figure 3 E shows the results of MDA detection under different treatment conditions, indicating that the intracellular MDA level increases most significantly after the combined treatment of MPPC@CM and radiotherapy.

[0117] Figure 3 F and 3G show the results of BODIPY 581 / 591 staining, indicating a significant increase in the intracellular lipid peroxidation level after the combined treatment of MPPC@CM and radiotherapy.

[0118] Figure 3 H and 3I show the relative expression levels of HIF - 1α under different treatments, indicating that the intracellular GPX4 protein level is the lowest after the combined treatment of MPPC@CM and radiotherapy.

[0119] Figure 3 J shows the results of DCFH - DA staining, revealing a significant increase in the intracellular ROS level after the combined treatment of MPPC@CM and radiotherapy.

[0120] Figure 3 K shows the results of live and dead cell staining, where live cells show green fluorescence (Calcein AM) and dead cells show red fluorescence (PI), indicating a decrease in cell viability and an increase in the number of dead cells after the combined treatment of MPPC@CM and radiotherapy.

[0121] Figure 3L and 3M showed the apoptosis rates of cells in different treatment groups, indicating that the apoptosis rate increased and the survival rate decreased after treatment with MPPC@CM combined with radiotherapy.

[0122] Figure 3 N showed the fluorescence imaging of γ-H2AX foci formation in different treatment groups, indicating that the DNA damage was the most severe after treatment with MPPC@CM combined with radiotherapy.

[0123] Figure 3 O and 3P showed the results of colony formation experiments at different doses and the cell survival curves fitted based on the single-hit multi-target model, indicating that MPPC@CM had a radiosensitizing effect.

[0124] Example 4: Evaluation of immunogenic cell death (ICD) of cells

[0125] 1. Evaluation of cell ICD:

[0126] 4T1 breast cancer cells were seeded in 24-well plates. After the cells adhered, different concentrations of MPPC@CM were added for treatment, and the control group was added with an equal volume of PBS.

[0127] After treatment, the cell culture supernatants were collected for subsequent dendritic cell (DC) maturation experiments.

[0128] 2. Dendritic cell (DC) maturation experiment:

[0129] Dendritic cells were extracted from the bone marrow of BALB / c mice, and the cell culture supernatant of 4T1 cells treated with MPPC@CM was added to the DC culture.

[0130] After incubation, flow cytometry was used to analyze the expression of mature markers (such as CD80 and CD86) on the surface of DCs to evaluate the DC maturation level.

[0131] 3. Detection of exposure of calreticulin (CRT) on the cell surface and release of HMGB1:

[0132] 4T1 cells were seeded in 24-well glass bottom plates. After the cells adhered, different concentrations of MPPC@CM were added for treatment, and the control group was added with an equal volume of PBS.

[0133] After treatment, immunofluorescence staining was performed with anti-CRT antibody and anti-HMGB1 antibody to detect the exposure of CRT on the cell surface and the release of HMGB1.

[0134] In Figure 4 we showed the evaluation results of cell ICD after treatment with MPPC@CM. Figure 4 A showed the schematic diagram of dendritic cell maturation experiment; Figure 4B to 4E show immunofluorescence images and quantitative analysis results, revealing the exposure of CRT on the cell surface and the release of HMGB1 after MPPC@CM treatment, which are key markers of ICD. Figure 4 F and 4G show the results of dendritic cell maturation experiments, in which the expression of the mature DC surface markers CD80 and CD86 increases, indicating an improvement in the level of DC maturation after MPPC@CM treatment.

[0135] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A bionic non-ferrous nanozyme, characterized in that: The preparation method thereof comprises the following steps: S1. Prepare mesoporous silica nanoparticles MSNs; S2. Load platinum and palladium on the mesoporous silica nanoparticles MSNs to obtain MPP nanozymes; S3. Functionalize the MPP nanozymes by surface grafting cinnamaldehyde to obtain MPPC nanozymes; S4. Coating the 4T1 cell membrane on the MPP nanozymes or the MPPC nanozymes to obtain MPP@CM nanozymes or MPPC@CM nanozymes, which are the bionic non-iron-based nanozymes.

2. The bionic non-ferrous nanozyme according to claim 1, wherein: In S1, the specific preparation method of the mesoporous silica nanoparticles MSNs is as follows: Dissolve CTAB in deionized water, adjust the pH to 10, add TEOS, stir under a nitrogen atmosphere, collect the precipitate by centrifugation, wash with ethanol or deionized water, then treat with 1% HCl ethanol solution, stir, and vacuum dry overnight to obtain the mesoporous silica nanoparticles MSNs.

3. The biomimetic non-ferrous nanozyme according to claim 1, wherein: In S2, the specific preparation method of the MPP nanozymes is as follows: Disperse the mesoporous silica nanoparticles MSNs in a methanol solution containing chloroplatinic acid and palladium chloride, stir, concentrate, wash with methanol, and freeze-dry to obtain the MPP nanozymes.

4. The biomimetic non-ferrous nanozyme according to claim 1, wherein: In S3, the specific preparation method of the MPPC nanozymes is as follows: Ultrasonically disperse the MPP nanozymes in methanol, add 3-aminopropyltriethoxysilane, wash with methanol after the reaction is completed, then disperse in methanol again, add cinnamaldehyde, wash with methanol after the reaction is completed, and freeze-dry to obtain the MPPC nanozymes.

5. The biomimetic non-ferrous nanozyme according to claim 1, wherein: In S4, the specific preparation method of the MPP@CM nanozymes or MPPC@CM nanozymes is as follows: Co-extrude the solution containing the 4T1 cell membrane and the MPP nanozymes or the MPPC nanozymes through a polycarbonate membrane with a pore size of 200 nm to obtain the MPP@CM nanozymes or MPPC@CM nanozymes.

6. Use of the bionic non-iron-based nanozyme according to any one of claims 1-5 in the preparation of radioimmunotherapy drugs.

7. Use of the bionic non-iron-based nanozyme according to any one of claims 1-5 in the preparation of drugs for promoting ferroptosis.

8. Use of the bionic non-iron-based nanozyme according to any one of claims 1-5 in the preparation of drugs for regulating the tumor microenvironment.

9. A drug, characterized in that: It contains the bionic non-iron-based nanozyme according to any one of claims 1-5.

10. A reagent or kit, characterized in that: It contains the bionic non-iron-based nanozyme according to any one of claims 1-5.

Images