Preparation method and application of M1C nano-enzyme applied to tumor immunotherapy preparation

Abstract

The invention relates to a preparation method and application of M1C nano-enzyme applied to tumor immunotherapy, and the preparation method comprises the following steps: dissolving 1% lauryl sodium sulfate in ultrapure water, and stirring at room temperature for 30 minutes to obtain a solution a; respectively dissolving Na2MoO4. 2H2O and CuCl2 in 5ml of ultrapure water, and violently stirring at room temperature for 2 minutes to obtain a solution b; adding the solution b into the stirred solution a, transferring the mixed solution into a 50mL hydrothermal reaction kettle, and reacting at 180 DEG C for 12 hours; centrifuging the mixed solution to remove larger particles, dialyzing the upper solution for 12 hours to remove excessive SDS, putting the upper solution into a freeze dryer for 24 hours to obtain black blue solid powder CuMoO4, and storing the black blue solid powder CuMoO4 in a refrigerator at-20 DEG C; the method comprises the following steps: dissolving a powdery sample with PBS to prepare CuMoO4 (1 mg mL <-1 > and 0.5 mL), mixing the CuMoO4 with M1-Exos (1 mg mL <-1 > and 1 mL), oscillating at 4 DEG C for 24 hours, centrifuging at 10000 rpm for 10 minutes, and removing redundant M1-Exos to obtain the M1C nano-enzyme. The preparation method of the M1C nano-enzyme is stable, reliable, good in repeatability and good in biocompatibility and biological safety; the damage to other normal tissues is reduced. The tumor microenvironment is improved, and favorable conditions are provided for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of an M1C nanozyme for use in tumor immunotherapy preparations. Background Art

[0002] Cancer immunotherapy has become one of the most promising therapies, and many advanced malignancies have been successfully treated with immunotherapy. Among the many immunotherapies, immune checkpoint blockade (ICB) therapy has made important clinical progress, among which anti-PD-L1 / anti-PD-1 antibodies have become the most widely used immune checkpoint inhibitors. Colorectal cancer (CRC) has a high incidence and mortality rate worldwide. Since 2017, three immune checkpoint inhibitor drugs (pembrolizumab, nivolumab, and ipilimumab) have been clinically used to treat microsatellite unstable colorectal cancer subtypes. However, this type only accounts for about 15% of all colorectal cancer patients, with a narrow range of indications. 85% of microsatellite stable patients lack sensitive response to immune checkpoint inhibitors and cannot benefit from ICB treatment.

[0003] The tumor immune microenvironment plays a more important role than immune checkpoints in tumor immune surveillance and immune escape, which may lead to the ineffectiveness of ICB therapy and other new immunotherapies. The tumor immune microenvironment is characterized by lack of tumor antigens, poor antigen presentation efficiency, and low immunogenic T cell infiltration. Reversing the tumor immune microenvironment by increasing exposure to tumor antigens, promoting antigen presentation, and increasing T cell infiltration to re-stimulate the immune response is a promising strategy.

[0004] Nanozymes are widely used in enhancing anti-tumor immune responses due to their excellent catalytic activity. They can catalyze hydrogen peroxide to produce cytotoxic reactive oxygen species (ROS), trigger immunogenic cell death of tumor cells, and activate tumor immune responses by enhancing the recruitment of dendritic cells and T cells. However, nanozyme-based immunotherapy is easily hindered by the low immunogenicity of "cold" tumors and insufficient T cell infiltration. Photothermal therapy (PTT) is an effective method to convert "cold" tumors into "hot" tumors. PTT uses photothermal agents to convert light energy into heat energy for ablation of tumor cells, and the generated tumor antigens activate immune signals to reverse immunogenic "cold" tumors and turn them into "hot" tumors. At the same time, the photothermal effect of this process can enhance the catalytic activity of nanozymes and promote the generation of ROS in the tumor site. Therefore, it is necessary to develop photothermal nanozymes with intrinsic photothermal properties and catalytic anti-tumor activity to reverse immunogenic "cold" tumors, convert "cold" tumors into "hot" tumors, reverse the tumor immune microenvironment, and activate tumor immune responses. However, there are relatively few photothermal nanozymes developed so far, and related research is still in its infancy. Although nanozymes can kill tumor cells, most nanozymes exhibit nonspecific biodistribution and cannot actively target tumor sites, inevitably causing damage to normal tissues around the tumor.

[0005] Therefore, it is necessary to develop a photothermal nanozyme that actively targets the tumor site, exerts catalytic and photothermal properties at the tumor site, promotes the production of ROS at the tumor site, triggers immunogenic cell death of tumor cells, and activates tumor immune response by enhancing the recruitment of dendritic cells and T cells to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing M1C nanozymes for use in tumor immunotherapy preparations, which can accurately target tumor tissues, reduce damage to other normal tissues, and effectively convert "cold" tumors into "hot" tumors. At the same time, it can also repolarize M2 macrophages into M1 macrophages, improve the tumor microenvironment, and provide favorable conditions for tumor immunotherapy.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing M1C nanozyme, the preparation steps are as follows: S1. Dissolve 1% sodium dodecyl sulfate (SDS) in ultrapure water (20 ml), stir with a magnetic stirrer at room temperature for 30 min to obtain solution a for later use; S2, dissolve Na2MoO4·2H2O (0.605 g) and CuCl2 (0.336 g) in 5 ml ultrapure water respectively, and stir vigorously for 2 min at room temperature (speed 1400 rpm) to obtain solution b; S3, stir liquid a, then add liquid b into the stirred liquid a, stir vigorously for 30 min, transfer the mixed solution to a 50 ml hydrothermal reactor, and react at 180 °C for 12 h; S4. The mixed solution after the reaction was centrifuged (5000 rpm, 5 min) to remove larger particles, and then the upper solution was dialyzed for 12 h (dialysis membrane mw: 8000-14000) to remove excess SDS to obtain a sample solution; S5. Place the sample solution in a freeze dryer overnight to obtain dark blue solid powder CuMoO4, which is stored in a -20°C refrigerator for later use; S6. Dissolve the powdered sample in PBS to prepare CuMoO4 (1 mg mL -1 , 0.5 mL) and then with M1-Exos (1 mg mL -1 , 1 mL) were mixed and shaken at 4°C for 24 h, and then centrifuged at 10000 rpm for 10 min to remove excess M1-Exos and obtain M1C nanozyme.

[0008] Specifically, the molecular weight cut-off of the dialysis membrane used for dialysis in step S4 is 8000-14000mw.

[0009] Specifically, the application of M1C nanozyme prepared by the preparation method of M1C nanozyme in tumor immunotherapy preparations.

[0010] The beneficial effects of the present invention are: Through biocompatibility analysis, it can be seen that the preparation method of M1C nanozyme shown in this application is stable and reliable, with good repeatability, good biocompatibility and biosafety; M1C nanozyme can accurately target tumor tissue and reduce damage to other normal tissues. Through photothermal performance detection, it can be seen that M1C nanozyme can effectively convert "cold" tumors into "hot" tumors, and can also repolarize M2 macrophages into M1 macrophages, improve the tumor microenvironment, and provide favorable conditions for tumor immunotherapy.

[0011] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A preparation flow chart of an M1C nanozyme proposed in the present invention; Figure 2 This is a transmission electron microscope (TEM) image of the CuMoO4 nanodots shown in the present invention (Scale bar: 200 nm); Figure 3 This is a transmission electron microscope (TEM) image of M1-Exos shown in the present invention; Figure 4 This is the M1-Exos PKH26 staining imaging diagram shown in the present invention; Figure 5 This is an atomic force microscope image of M1-Exos shown in the present invention; Figure 6 This is an image of the immunoblotting method for identifying exosomes shown in the present invention; Figure 7 This is an analysis diagram of the immunoblotting method for identifying exosomes shown in the present invention; Figure 8 The size distribution of CuMoO4 and MIC nanoparticles is detected by dynamic light scattering (DLS) as shown in the present invention; Fig. 9 The surface charge diagram of the nanoparticles detected by the Zeta potential instrument shown in the present invention; Fig.10 This is a size distribution diagram of M1-Exos and MIC nanozymes shown in the present invention; Fig.11 It is the UV-visible absorption spectrum distribution diagram of TMB (oxTMB) oxidized by M1C nanozyme with different concentrations shown in the present invention; Fig.12 This is the UV-visible absorption spectrum distribution diagram of MB degradation by M1C nanozymes of different concentrations shown in the present invention; Fig.13 It is the UV-visible absorption spectrum distribution diagram of OPD (oxOPD) oxidized by M1C nanozyme with different concentrations shown in the present invention; Fig.14 The fluorescence spectrum distribution diagram of phthalic acid (TA) oxidized by M1C nanozymes of different concentrations shown in the present invention; Figure 15-16 This is a graph showing the consumption of GSH by the M1C nanozyme of the present invention; Fig.17 Infrared thermal images of M1C nanozymes of different concentrations under laser irradiation shown in the present invention; Fig.18 It is a temperature curve diagram of different concentrations of M1C nanozymes under laser irradiation at different times shown in the present invention; Fig.19 It is a fitting curve diagram of the cooling time and -lnɵ of the M1C nanozyme shown in the present invention; Fig. 20 This is a characterization diagram of the targeting of the M1C nanozyme prepared by the present invention to tumors: Fig. 20-a is a cell uptake diagram of the M1C nanozyme prepared by the present invention for tumor targeting characterization after the M1C nanozyme and tumor cells were co-incubated for 12 hours; Fig. 20 -b is a cell uptake diagram of CuMoO4 and tumor cells after co-incubation for 12 hours in the tumor targeting characterization diagram of the M1C nanozyme prepared by the present invention; Fig.21 This is a diagram showing the killing of tumor cells by the M1C nanozyme prepared in the present invention. DETAILED DESCRIPTION

[0013] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0014] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0015] See also Figure 1 The present invention provides an M1C nanozyme, and the preparation steps are as follows: S1. Dissolve 1% sodium dodecyl sulfate (SDS) in ultrapure water (20 mL) and stir with a magnetic stirrer at room temperature for 30 min to obtain solution a for later use. S2, dissolve Na2MoO4·2H2O (0.605 g) and CuCl2 (0.336 g) in 5 ml ultrapure water respectively, and stir vigorously for 2 min at room temperature (speed 1400 rpm) to obtain solution b; S3, stir liquid a, then add liquid b into the stirred liquid a, stir vigorously for 30 min, transfer the mixed solution to a 50 ml hydrothermal reactor, and react at 180 °C for 12 h; S4. The mixed solution after the reaction was centrifuged (5000 rpm, 5 min) to remove larger particles, and then the upper solution was dialyzed for 12 h (dialysis membrane mw: 8000-14000) to remove excess SDS to obtain a sample solution; S5. Place the sample solution in a freeze dryer for 24 hours to obtain dark blue solid powder CuMoO4, which is stored in a -20°C refrigerator for later use; S6. Dissolve the powdered sample in PBS to prepare CuMoO4 (1 mg mL -1 , 0.5 mL) and then with M1-Exos (1 mg mL -1 , 1 mL) were mixed and shaken at 4°C for 24 h, and then centrifuged at 10000 rpm for 10 min to remove excess M1-Exos and obtain M1C nanozyme.

[0016] See also Figure 2~Figure 10 , transmission electron microscopy analysis and dynamic light scattering of CuMoO4 nanoparticles showed that their size was about 10-20 nm, they were evenly dispersed in aqueous solution, and the Zeta potential showed that they were negatively charged. Then, we obtained M1 macrophage exosomes by ultra-high speed centrifugation, observed the double-layer capsule morphology of exosomes by transmission electron microscopy, stained exosomes with PKH26, characterized the relative size of exosomes under physiological conditions by atomic force microscopy, and analyzed the concentration and particle size of exosomes by nanoparticle tracking, indicating that exosomes were successfully obtained. In addition, we polarized M0 macrophages to obtain M1 macrophages, and cultured and extracted exosomes. Immunoblotting analysis of M1 macrophage exosomes (M1-Exos) and M0 macrophage exosomes (M0-Exos) showed that both expressed the exosome common protein CD63 protein, while M1-Exos also expressed the M1 characteristic protein iNOS, indicating the successful preparation of M1-Exos. Finally, we fused M1-Exos with CuMoO4 to construct MIC photothermal nanozymes, and the successful preparation of MIC was demonstrated by the changes in particle size and Zeta potential before and after fusion.

[0017] See also Figure 11~Figure 19 The catalytic performance and photothermal performance of the M1C nanozyme prepared by the present invention are detected, and the detection steps are as follows: S1. Add different concentrations of M1C nanozyme (final concentration: 10, 25, 50, 100, 200 μg / mL) into PBS (pH 5.5), then add 3,3',5,5'-tetramethylbenzidine (TMB, final concentration: 20 mg / mL) and H2O2 (final concentration: 150 mmol / L), mix well, react at 37°C in the dark for 30 min, and monitor the UV absorption peaks at 370 nm and 652 nm.

[0018] S2, add methyl blue (MB, final concentration: 500 μg mL –1 ), H2O2 (final concentration: 10 mmol / L), and different concentrations of M1C nanozymes (final concentrations: 25, 50, 100, 200 μg mL -1 ) were incubated in PBS buffer (pH=5.5) at 37°C for 30 min. The degradation of MB caused by ·OH was observed by UV absorption at 665 nm.

[0019] S3. Incubate the following groups with o-phenylenediamine (OPD, final concentration: 0.13 mmol / L) at room temperature for 30 min, and record the characteristic absorption peak at 450 nm. Group 1: H2O2 (final concentration 150 mmol / L); Group 2: M1C nanozyme (final concentration: 100ug / mL); Group 3: H2O2 (final concentration: 150 mmol / L) + M1C nanozyme (final concentration: 25 ug / mL); Group 4: H2O2 (final concentration: 150 mmol / L) + M1C nanozyme (final concentration: 50 ug / ml); Group 5: H2O2 (final concentration: 150 mmol / L) + M1C nanozyme (final concentration: 100 ug / mL); Group 6: H2O2 (final concentration: 150 mmol / L) + M1C nanozyme (final concentration: 200 ug / mL).

[0020] S4, terephthalic acid (TA, final concentration: 0.15 mmol / L) and H2O2 (10 mmol / L) were reacted with different concentrations of M1C nanozymes (final concentrations: 25, 50, 100, 200 μg / mL) in PBS buffer (pH = 4) at 25°C for 2 h. The fluorescence emission peak at 425 nm was observed using 315 nm laser excitation.

[0021] S5. Mix different concentrations of M1C nanozymes (final concentration: 25, 50, 100, 200 μg / mL) with glutathione (GSH, final concentration: 200 μg / mL) in a 1.5 mL centrifuge tube. After 1 h of reaction, add 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, final concentration: 50 μg / mL). After 10 min of reaction, centrifuge at 10000 rpm for 10 min to remove M1C nanozymes. The absorbance of the supernatant at 412 nm was measured by UV-visible spectrophotometer, which was the residual GSH. At the same time, H2O2 (50 mM) was also evaluated as a GSH depletion inducer (positive control).

[0022] S6. Photothermal performance detection: Different concentrations of M1C nanozymes (final concentrations: 0, 50, 100, 200, 400 μg / mL) were irradiated with 1064 nm laser for 10 min (1.5 W / cm 2 ). The infrared thermal imager accurately recorded the temperature curve and image. To further study the photothermal conversion efficiency, CuMoO4 nanodots (final concentration: 100 μg / mL) were exposed to 1064 nm laser (1.5 W / cm 2 ) reaches a steady-state temperature and cools naturally to room temperature.

[0023] See also Fig. 20The targeting of the M1C nanozyme prepared by the present invention to tumors is carried out in the following specific steps: S1. CuMoO4 rhodamine staining: 1 mg CuMoO4 was dissolved in 1 mL PBS (pH 7.4), 200 uL of 1 mg / mL rhodamine was added, stirred at room temperature for 24 h, ultrafiltered for 4 h, and stored at 4 °C in the dark. Exosome PKH26 staining: 20 uL PKH26 (1 mg / mL, dissolved in DMSO) was added to 600 uL exosomes (protein concentration was 4 mg / mL), incubated at room temperature for 20 min, and then PBS was added to 1 mL, and stored at 4 °C in the dark. CuMoO4 (1 mg / mL, 0.5 mL) was mixed with the stained exosomes (1 mg / mL, 1 mL) and shaken overnight at 4 °C, then centrifuged at 10000 rpm for 10 min to remove excess M1-Exos.

[0024] After S2 and HCT116 tumor cells grew to 50% on the confocal dish, the stained CuMoO4 and M1C photothermal nanozymes were co-incubated with the tumor cells for 12 hours. Then 4% paraformaldehyde was added for fixation for 15 minutes, washed 3 times with PBS, 1 mL of 0.2% Triton X-100-PBS was added for incubation for 5 minutes, washed 3 times with PBS, 400uL of prepared phalloidin (6T / mL) was added for incubation for 20 minutes, washed 3 times with PBS, 500uL of Hoechst dye (10ug / mL) was added for staining for 18 minutes, then washed 3 times with PBS, PBS containing anti-fluorescence quencher was added, and observed under a confocal microscope.

[0025] See also Fig.21 The M1C nanozyme prepared by the present invention kills tumor cells, and the specific steps are as follows: S1. Cell culture: LoVo tumor cells (cell density 50%) were plated in 6-well plates and cultured for 12 h.

[0026] S2. Grouping: Group 1: PBS control group; Group 2: CuMoO4 (100ug / mL); Group 3: CuMoO4 (100ug / mL) + laser; Group 4: M1C nanozyme; Group 5: M1C nanozyme (100ug / mL) + laser. Each group was incubated for 24 hours after adding CuMoO4 or M1C nanozyme. The laser group was treated with 1064 nm laser (1.5 W cm) 12 hours after adding CuMoO4. -2 ) Irradiate for 10 minutes.

[0027] S3. Live and dead cell staining: Prepare the staining solution according to the following ratio: 1uL PI (1000x) + 0.75uL AM (1000x) + 1mL staining buffer; add 1mL staining solution to each 6-well plate, react at room temperature in the dark for 30 minutes, and then take pictures under a fluorescence microscope.

[0028] See also Figure 11~Figure 19 Based on the fact that M1C nanozymes have POD-like enzyme activity, they can induce H2O2 to produce ·OH, and ·OH can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) to generate the corresponding oxidation products oxTMB and oxOPD. In addition, ·OH can degrade methylene blue (MB) and react with phthalic acid (TA) to generate fluorescent products. We conducted the following experiments to verify the POD-like enzyme activity of M1C nanozymes. After different concentrations of M1C nanozymes reacted with TMB (with H2O2 as substrate), characteristic absorption peaks of oxTMB were presented at 370nm and 650nm, and the intensity of the absorption peak was proportional to the concentration of M1C nanozymes; similarly, the characteristic absorption peak of oxOPD was presented at 417nm, and the intensity of the absorption peak was proportional to the concentration of CuMoO4. In addition, with the increase of M1C nanozyme concentration, the absorption peak of MB at 665nm decreased, indicating that MB was degraded. As the concentration of M1C nanozyme increases, the fluorescent product generated by the reaction with TA increases. The above results show that M1C nanozyme has POD-like enzyme activity and catalyzes H2O2 to produce more ·OH. Based on the principle that glutathione peroxidase (GPx)-like enzyme activity can eliminate glutathione (GSH), the GPx-like enzyme activity of M1C nanozyme is verified. GSH can react with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to produce yellow 2-nitro-5-mercaptobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. After adding M1C nanozyme, it can consume GSH, reduce TNB generation, and reduce the absorption peak at 412 nm. The above results show that M1C nanozyme has GPx-like enzyme activity.

[0029] In addition, we studied the photothermal conversion performance of M1C nanozymes of different concentrations. After irradiating M1C nanozymes of different concentrations with laser, the thermal imager took pictures every 2 minutes and recorded the temperature changes within 10 minutes. The results showed that 100ug / mL CuMoO4 achieved a maximum temperature of 45°C within 10 minutes. After removing the laser irradiation, the cooling time and temperature of M1C nanozymes were recorded, and the photothermal conversion efficiency of M1C nanozymes was calculated to be 38.2% through the fitting curve of time and -lnɵ.

[0030] See also Fig. 20a. In order to study the tumor targeting of M1C photothermal nanozyme, rhodamine-stained CuMoO4 and PKH26-stained M1C photothermal nanozyme were co-incubated with tumor cells to observe the uptake of tumor cells. The rhodamine-stained CuMoO4 and PKH26-stained M1C photothermal nanozyme were both red, the nucleus was blue, and the cytoskeleton was green. After incubation for 12 hours, the PKH26-stained M1C photothermal nanozyme entered almost all tumor cells, and the rhodamine-stained CuMoO4 entered a few tumor cells, indicating that the M1C photothermal nanozyme has tumor targeting.

[0031] See also Fig.21 Tumor cells were treated as follows: Control: PBS; CuMoO4 (100ug / mL); CuMoO4 (100ug / mL) + laser; M1C nanozyme; M1C nanozyme (100ug / mL) + laser. Each group was incubated for 24 hours after adding CuMoO4 or M1C nanozyme. The laser group was treated with 1064 nm laser (1.5 W cm) 12 hours after adding CuMoO4. -2 ) irradiated for 10 minutes. The treated cells were stained with the AM-PI live and dead cell kit and photographed under a fluorescence microscope. AM can stain live cells and show green, while PI can stain dead cells and show red. The results showed that compared with the control group, the CuMoO4 group killed a small number of cells, and after adding laser treatment, the number of dead cells increased; the M1C nanozyme group and the M1C nanozyme + laser group had excellent catalytic activity and photothermal conversion ability because they actively targeted tumor cells, and their ability to kill tumor cells was greater than 90%.

[0032] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0033] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing an M1C nanozyme, characterized in that: The preparation steps are as follows: S1. Dissolve 1% sodium dodecyl sulfate (SDS) in ultrapure water (20 ml), stir with a magnetic stirrer at room temperature for 30 min to obtain solution a for later use; S2, dissolve Na2MoO4·2H2O (0.605 g) and CuCl2 (0.336 g) in 5 ml ultrapure water respectively, and stir vigorously for 2 min at room temperature (speed 1400 rpm) to obtain solution b; S3, stir liquid a, then add liquid b into the stirred liquid a, stir vigorously for 30 min, transfer the mixed solution to a 50 ml hydrothermal reactor, and react at 180 °C for 12 h; S4. The mixed solution after the reaction was centrifuged (5000 rpm, 5 min) to remove larger particles, and then the upper solution was dialyzed for 12 h (dialysis membrane mw: 8000-14000) to remove excess SDS to obtain a sample solution; S5. Place the sample solution in a freeze dryer for 24 hours to obtain dark blue solid powder CuMoO4, which is stored in a -20°C refrigerator for later use; S6. Dissolve the powdered sample in PBS to prepare CuMoO4 (1 mg / mL -1 , 0.5 mL) and then with M1-Exos (1 mg mL -1 , 1 mL) were mixed and shaken at 4°C for 24 h, and then centrifuged at 10000 rpm for 10 min to remove excess M1-Exos and obtain M1C nanozyme.

2. The method for preparing an M1C nanozyme according to claim 1, characterized in that: The molecular weight cut-off of the dialysis membrane used for dialysis in step S4 is 8000-14000mw.

3. An application of the M1C nanozyme prepared by the preparation method of the M1C nanozyme as claimed in claim 1 in a tumor immunotherapy preparation.

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