Pyroptosis inducer, preparation method and application thereof

Abstract

The scheme discloses a self-starting pyroptosis inducer and a preparation method and application thereof, the self-starting pyroptosis inducer is based on caspase-4 / 5 / 11 to realize pyroptosis of tumor cells, a combination drug of manganese dioxide nanozyme and outer membrane vesicle is provided, the self-starting pyroptosis inducer is prepared by coating the manganese dioxide nanozyme with the outer membrane vesicle, it is ensured that the self-starting pyroptosis inducer is only decomposed and releases manganese ions and lipopolysaccharide of the outer membrane vesicle in tumor cells with high glutathione level, a series of reactions between the elevated GSH level of tumor cells and manganese ions are utilized to produce reactive oxygen species to up-regulate the precursor expression of caspase-4 / 5 / 11, the lipopolysaccharide of the outer membrane vesicle OMVs is used to activate caspase-4 / 5 / 11, and a synergistic effect is produced with the reactive oxygen species produced by the MnNZ, so that the tumor cells are efficiently induced to pyroptosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a pyroptosis inducer with self-starting function and a preparation method and application thereof. BACKGROUND

[0002] Pyroptosis is a mode of programmed cell death, belonging to the category of inflammatory cell death. Its main feature is dependent on the activation of inflammasome and the activation of inflammatory caspases (such as caspase-1), ultimately leading to cell membrane rupture and release of cell contents, thereby triggering an inflammatory response. During pyroptosis, activated caspase cleaves Gasdermin, and the released N-terminal fragment is transferred to the cell membrane to form pores, leading to cell swelling and lysis. As a form of immunogenic cell death, pyroptosis of tumor cells promotes the release of tumor antigens and pro-inflammatory damage-associated molecular patterns (DAMPs) such as HMGB1 and ATP, increasing the opportunity for antigen presentation and helping to reverse the immunosuppressive microenvironment of tumors. Therefore, pyroptosis shows great potential in the research of stimulating anti-tumor immune response.

[0003] Radiotherapy and chemotherapy can induce pyroptosis through the GSDME pathway, however, in this case, caspase-3 and caspase-8 associated with apoptosis are also activated when cells undergo pyroptosis, and the accompanying apoptosis process does not have the specificity of pyroptosis and is difficult to trigger a strong immune response, so this mode of pyroptosis induction is not ideal. In addition, although the classic pathway mediated by caspase-1 and GSDMD is a common pyroptosis induction mechanism, this pathway depends on the activation of pattern recognition receptors (PRRs) such as TLR and NLRP3, and these receptors are mainly expressed in immune cells, with limited expression in tumor cells, thus leading to low efficiency of pyroptosis induction in tumor cells by this classic pathway. Therefore, how to effectively induce pyroptosis in tumor cells to strongly stimulate anti-tumor immune response remains a major challenge.

[0004] In addition to the classic cleavage of caspase-1, GSDMD can also be cleaved by caspase-4 / 5 (corresponding to caspase-11 in mice) through a non-classical pathway, however, the research on the mechanism of caspase-4 / 5 / / 11 in tumor cell pyroptosis and its potential application is limited, and no significant breakthrough has been made. SUMMARY

[0005] The purpose of the present application is to solve the above problems, and to provide a self-starting pyroptosis inducer.

[0006] The present application provides a preparation method of the self-starting pyroptosis inducer.

[0007] And propose the application of the self-starting pyroptosis inducer in the preparation of an antitumor immunotherapy drug.

[0008] A self-starting pyroptosis inducer, comprising a manganese dioxide nanomachine (MnNZ) and an outer membrane vesicle (OMV) coated with the manganese dioxide nanomachine.

[0009] In the self-starting pyroptosis inducer described above, the manganese dioxide nanomachine is in the shape of a spike, and the outer membrane vesicle is coated on the surface of the spike-shaped manganese dioxide nanomachine.

[0010] In the self-starting pyroptosis inducer described above, the outer membrane vesicle is extracted from Escherichia coli, and the membrane contains lipopolysaccharide (LPS);

[0011] The spike-shaped manganese dioxide nanomachine is obtained by reducing potassium permanganate using oleic acid.

[0012] In the self-starting pyroptosis inducer described above, the average size of the outer membrane vesicle is (28±2) nm, and the maximum is not more than 35 nm and the minimum is not less than 20 nm;

[0013] The average size of the manganese dioxide nanomachine is (157±5) nm, and the maximum is not more than 165 nm and the minimum is not less than 150 nm.

[0014] In the self-starting pyroptosis inducer described above, the size of the self-starting pyroptosis inducer is (200±5) nm, and the maximum is not more than 210 nm and the minimum is not less than 190 nm.

[0015] The application of the self-starting pyroptosis inducer in the preparation of an antitumor immunotherapy drug.

[0016] In the application of the self-starting pyroptosis inducer in the antitumor immunotherapy drug described above, the tumor cells against which the antitumor immunotherapy is directed have a high level of glutathione, higher than 1 millimole per liter;

[0017] The tumor cells against which the antitumor immunotherapy is directed have a high level of hydrogen peroxide, higher than 100 micromoles per liter.

[0018] In the application of the self-starting pyroptosis inducer in the antitumor immunotherapy drug described above, the mechanism by which the self-starting pyroptosis inducer starts the antitumor immunity is as follows:

[0019] The self-starting pyroptosis inducer is administered to the tumor tissue and endocytosed by the tumor cells, and is degraded in response to the high level of glutathione in the tumor cells;

[0020] Under the action of glutathione, the self-starting pyroptosis inducer releases manganese ions and outer membrane vesicle components;

[0021] Manganese ion catalyzes the generation of reactive oxygen species, thereby triggering the translocation of NF-κB into the nucleus of tumor cells;

[0022] The translocation of NF-κB into the nucleus of tumor cells induces the gene expression of caspase-4 / 5 / 11;

[0023] Meanwhile, the expressed caspase-4 / 5 / 11 is activated by the previously released outer membrane vesicle components;

[0024] The activated caspase-4 / 5 / 11 cleaves the GSDMD protein, triggering pyroptosis of tumor cells.

[0025] A preparation method of a self-starting pyroptosis inducer, the method comprising:

[0026] Extracting outer membrane vesicles from Escherichia coli;

[0027] Coating the outer membrane vesicles on the surface of the sharp manganese dioxide nanoscale enzyme to obtain the self-starting pyroptosis inducer.

[0028] In the above preparation method of the self-starting pyroptosis inducer, specifically comprising:

[0029] Using oleic acid to reduce potassium permanganate to obtain the sharp manganese dioxide nanoscale enzyme with an average size of (157±5) nm;

[0030] Extracting the outer membrane vesicles with an average size of (28±2) nm from Escherichia coli;

[0031] Using an ultrasonic method to coat the outer membrane vesicles on the sharp manganese dioxide nanoscale enzyme to form the self-starting pyroptosis inducer with a size of (200±5) nm.

[0032] The advantages of the present application are:

[0033] The present scheme focuses on caspase-4 / 5 (corresponding to caspase-11 in mice) to induce pyroptosis of tumor cells, and the results show that it has high efficacy and specificity, providing a new idea and direction for tumor immunotherapy;

[0034] The present scheme proposes the combination of MnNZ and OMV, and the OMV-coated MnNZ self-starting pyroptosis inducer can protect MnNZ from being cleared before being taken up by tumor cells, thereby ensuring that the self-starting pyroptosis inducer is only decomposed and releases manganese ions and OMV in tumor cells with high glutathione levels, ensuring the specificity and high efficacy of the drug;

[0035] In the self-starting pyroptosis inducer provided in the scheme, the OMV itself contains components such as LPS, which can directly activate caspase-4 / 5 / 11, and has a synergistic effect with the active oxygen ROS produced by the catalysis of MnNZ on the generation of hydrogen peroxide in tumor cells, thereby efficiently inducing tumor cell pyroptosis.

[0036] The self-starting pyroptosis inducer provided in the scheme does not depend on pattern recognition receptors PRRs in the process of tumor cell pyroptosis, and the OMV released thereby activates caspase-4 / 5 / 11, thereby ensuring the activation efficiency, and this process utilizes the high glutathione level of tumor cells to up-regulate the expression of precursors of caspase-4 / 5 / 11 through a series of reactions, thereby providing a material basis for subsequent activation, thereby ensuring the specificity and high efficiency of pyroptosis. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a preparation and treatment mechanism diagram of the self-starting pyroptosis inducer of the application;

[0038] Figure 2 is an X-ray photoelectron spectroscopy analysis diagram of MnNZ prepared for the preparation of the self-starting pyroptosis inducer;

[0039] Figure 3 is an X-ray diffraction spectroscopy analysis diagram of MnNZ prepared for the preparation of the self-starting pyroptosis inducer;

[0040] Figure 4 is the characteristic research result of the self-starting pyroptosis inducer prepared in the embodiment of the application;

[0041] Figure 5 is the energy dispersive X-ray spectroscopy analysis and element mapping of MnNZ and MnNZ@OMV, with manganese Mn being purple and phosphorus P being yellow;

[0042] Figure 6 is the influence of MnNZ@OMV on tumor cells by in-vitro induction of the application;

[0043] Figure 7 is the confocal laser scanning microscope (CLSM) diagram of B16 cells and Di I-labeled (red) OMV or MnNZ@OMV co-incubated for a specified time interval in the in-vitro experiment of the application;

[0044] Figure 8 is the hydrodynamic diameter and zeta potential of MnNZ@Lip and PLGA@OMV in the in-vitro experiment of the application, M=MnNZ, ML=MnNZ@Lip, P=PLGA, PV=PLGA@OMV;

[0045] Figure 9is the experimental result of verifying that MnNZ@OMV inhibits tumor development in vivo;

[0046] Figure 10 is the experimental result of verifying that MnNZ@OMV activates anti-tumor immunity and inhibits distant metastasis;

[0047] Figure 11 is Figure 9 The change in tumor volume of each mouse after the specified treatment in the experiment;

[0048] Figure 12 is Figure 9 TUNEL staining image of tumor tissue after the specified treatment in the experiment. DETAILED DESCRIPTION

[0049] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0050] The present embodiment provides a self-starting pyroptosis inducer, and gives a preparation method of the self-starting pyroptosis inducer, and performs effectiveness test and mechanism explanation on the application of the pyroptosis inducer in anti-tumor immunotherapy.

[0051] The present embodiment provides a manganese dioxide nanoscale enzyme (MnNZ) wrapped by an outer membrane vesicle (OMV) as a self-starting pyroptosis inducer (MnNZ@OMV) for tumor treatment. First, oleic acid Ole i c Ac i d is used to reduce potassium permanganate KMnO4 to synthesize a sharp MnNZ. Then, OMV with an average size of about 28 nm is extracted from Escherichia coli, and finally, an ultrasonic method is used to coat the OMV on the MnNZ obtained in the previous step to form the required MnNZ@OMV, and the average size of the inducer is about 200 nm. The mechanism of the above-mentioned MnNZ@OMV as a self-starting pyroptosis inducer to play a therapeutic role in tumor immunotherapy is as shown in Figure 1

[0052] ​After MnNZ@OMV is injected into tumor tissue and rapidly endocytosed by tumor cells, the MnNZ in MnNZ@OMV is reduced to release manganese ions under the action of high levels of glutathione GSH (concentration range about 2-10 millimoles / liter) in tumor cells. This process also causes the destruction of the structure of MnNZ@OMV, and the OMV coated on the surface of MnNZ is also released. In addition, the concentration of hydrogen peroxide H2O2 in tumor cells is also much higher than that in normal cells, and the released manganese ions further catalyze the high levels of H2O2 in tumor cells to convert it into active oxygen-hydroxyl radicals, increasing the concentration of hydroxyl radicals. The increased hydroxyl radicals increase the oxidative stress in the cell, triggering the transfer of NF-κB to the nucleus of the tumor cell, and then activating the gene expression of caspase-4 / 5 (corresponding to caspase-11 in mice). Subsequently, the expressed caspase-4 / 5 / 11 is activated by the previously released OMV, and cuts the GasderminD (GSDMD) protein to release its N-terminal fragment, which forms pores on the cell membrane, leading to leakage of cell contents, and ultimately triggering pyroptosis and DAMPs release of tumor cells.

[0053] This process, MnNZ@OMV first uses the responsiveness of MnNZ to GSH to achieve the simultaneous release of manganese ions and OMV, and activates the expression of caspase-4 / 5 / 11 through a series of reactions, and then uses the released OMV to activate caspase-4 / 5 / 11, the two components promote each other, and the two steps synergistically induce pyroptosis of tumor cells, thereby realizing the anti-tumor immune response. This process does not depend on pattern recognition receptors (PRRs), and the OMV released by MnNZ@OMV activates caspase-4 / 5 / 11, so it can ensure the activation efficiency, and this process uses the elevated GSH level in tumor cells to up-regulate the expression of the precursor of caspase-4 / 5 / 11 through a series of reactions, providing a basis for subsequent activation, thereby ensuring the effectiveness and specificity of pyroptosis. In addition, the pyroptosis induced by MnNZ@OMV promotes DC maturation and subsequent activation of effector and memory T cells, which can effectively inhibit the growth of primary tumors and distant metastasis.

[0054] In order to explore the principle of the self-starting pyroptosis inducer MnNZ@OMV having a therapeutic effect in anti-tumor treatment, the present embodiment prepared MnNZ@OMV by the following means:

[0055] 0.4 milligrams of KMnO4 was dissolved in 200 milliliters of water, and continuously stirred at room temperature, 4 milliliters of oleic acid was added dropwise, stirred for 5 hours, then the product was collected by centrifugation at 12000 revolutions / minute, and washed with ethanol and deionized water alternately three times to finally obtain sharp MnNZ. As Figure 2 andFigure 3 The characteristic peaks observed according to X-ray photoelectron spectroscopy and X-ray diffraction spectrum can determine that MnNZ is mainly composed of δ-MnO2.

[0056] The monoclonal colonies of E. coli were cultured in LB medium at 37°C, 200 rpm for 16 hours. The supernatant was collected by centrifugation at 3000g for 30 minutes, and filtered through a 0.45 micron filter to remove bacteria. Then the OMV was separated by using an ultracentrifuge at 150,000g for 2 hours, finally the OMV was resuspended in deionized water and stored at -80°C for standby.

[0057] The OMV solution (0.1 mg protein / ml) was mixed with the MnNZ solution (0.2 mg manganese / ml) and ultrasonicated in ice water for 1 hour. Then the mixture was centrifuged at 12000 rpm for 15 minutes, and the collected precipitate was dispersed in deionized water to finally obtain MnNZ@OMV.

[0058] Subsequently, the self-starting pyroptosis inducer MnNZ@OMV prepared in the foregoing was subjected to physicochemical property research, such as Figure 4 As shown in the figure:

[0059] A is a transmission electron microscope (TEM) image of OMV, MnNZ and MnNZ@OMV, V represents OMV, M represents MnNZ, and MV represents MnNZ@OMV;

[0060] B is the hydrodynamic diameter and zeta potential of OMV, MnNZ and MnNZ@OMV;

[0061] C is the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result of OMV, MnNZ and MnNZ@OMV;

[0062] D is the element mapping display of MnNZ@OMV by energy dispersive X-ray spectroscopy, manganese Mn is purple and phosphorus P is yellow;

[0063] E is the percentage of residual MnNZ@OMV after incubation in simulated body fluid (SBF) containing different GSH concentrations for 8 minutes, the upper right corner of the figure is a photo of MnNZ@OMV suspension treated with different GSH concentrations;

[0064] F is a graph of the change of absorbance of MnNZ@OMV with time at a GSH concentration of 1 mM;

[0065] G is the percentage of residual methylene blue (MB) at 8 minutes after treatment with MnNZ@OMV at 1 mM H2O2 concentration and different concentrations of GSH, MB is used to represent the amount of methylene blue remaining after a certain time in the presence of MnNZ@OMV, H2O2 and GSH, thereby evaluating the ability of MnNZ@OMV to generate hydroxyl radicals;

[0066] H is the plot of MB absorbance over time after treatment with MnNZ@OMV in the presence of 1 mM GSH concentration and 1 mM H2O2 concentration.

[0067] From Figure 4 The transmission electron microscopy (TEM) image in A can see that the surface of the sharp spike MnNZ is visible OMV membrane; Figure 4 B can see that after being coated with OMV, the size of the sharp spike MnNZ increases from 157.1 nm to 199.5 nm, and the zeta potential of MnNZ@OMV decreases slightly compared with MnNZ, close to the lower potential of OMV. In addition, as Figure 4 C, by SDS-PAGE analysis of OMV, MnNZ and MnNZ@OMV can see the matching of protein bands between OMV and MnNZ@OMV, which can confirm that MnNZ is successfully coated with OMV membrane. As Figure 4 D and Figure 5 As shown, the overlap of manganese and phosphorus on the particles indicates that OMV and MnNZ have been successfully integrated. Therefore, from Figure 4 A, B, C, D and Figure 5 It can be confirmed that the aforementioned preparation process obtains the required self-starting pyroptosis inducer MnNZ@OMV, which includes sharp spike manganese nanoscale enzyme, and outer membrane vesicles are coated on the surface of the sharp spike manganese nanoscale enzyme.

[0068] Further, as shown in Figure 4 E, MnNZ@OMV has a GSH concentration-dependent degradation property, and the external environment is almost free of GSH, and only a small amount of GSH exists in normal cells, which indicates that the decomposition of MnNZ@OMV will only occur after being endocytosed by tumor cells. As shown in Figure 4 F, in a high GSH environment (1 mM and above), the absorbance of MnNZ@OMV sequentially decreases within 8 minutes, which further indicates that MnNZ@OMV has a rapid response to GSH. In addition, as shown in Figure 4 G and 4H, GSH and time-dependent methylene blue (MB) degradation is detected in the presence of MnNZ@OMV and H2O2, which can indicate that MnNZ@OMV has the ability to generate hydroxyl radicals when exposed to GSH and H2O2.

[0069] Subsequently, the effect of MnNZ@OMV on tumor cells was studied to verify its induction of pyroptosis in tumor cells in vitro through the non-canonical caspase pathway, and the experimental results are shown in FIG. 1, wherein: Figure 6

[0070] A is the cytotoxicity of PLGA@OMV, MnNZ@Lip and MnNZ@OMV on B16 cells of mouse melanoma cells after 24 hours of incubation. MnNZ@LiP and PLGA@OMV are control groups of MnNZ@OMV, the former replaces OMV in MnNZ@OMV with liposome LiP, and the latter replaces MnNZ in MnNZ@OMV with polylactic acid polyglycolic acid copolymer PLGA.

[0071] B is the relative GSH level in B16 cells after 24 hours of treatment with PBS, PLGA@OMV, MnNZ@Lip and MnNZ@OMV (n=3).

[0072] C is the ROS level in B16 cells after 24 hours of treatment with different formulations.

[0073] D is the immunoblotting analysis of NF-κb, caspase-11 and GSDMD.

[0074] E is the ATP release assay of B16 cells treated with different formulations (n=3).

[0075] F is the CLSM image of HMGB1 (red) and cell nucleus (blue) of B16 cells treated with different formulations.

[0076] G is the flow cytometry analysis of CRT+B16 cells treated with different formulations.

[0077] H is the quantitative analysis of CRT+B16 cells (n=3).

[0078] I is the flow cytometry analysis of CD80+CD86+BMDC co-cultured with B16 cells pretreated with different formulations.

[0079] J is the quantitative analysis of CD80+CD86+BMDC (n=3).

[0080] All numerical values are expressed as mean ± standard deviation (SD). The asterisk indicates significant difference (*p<0.05).

[0081] The experimental process is as follows:

[0082] ​Firstly, confocal laser scanning microscopy (CLSM) was used to detect the cellular uptake. OMV and MnNZ@OMV were both labeled with fluorescent dye Dil, and co-incubated with mouse melanoma cells (B16). The nuclei were counterstained with DAPI (blue), and the B16 cell membrane was counterstained with DiO (yellow). As shown in Figure 7 , MnNZ@OMV was more easily taken up by B16 cells compared with native OMV.

[0083] Subsequently, the cytotoxicity of MnNZ@OMV was evaluated in B16 cells. PLGA-encapsulated OMV (PLGA@OMV) and MnNZ-encapsulated liposomes (MnNZ@Lip) were prepared as control groups of MnNZ@OMV, and their hydrodynamic diameters and zeta potentials were characterized as shown in Figure 8 . From Figure 6 A, it can be seen that MnNZ@OMV showed a stronger concentration-dependent killing effect than PLGA@OMV and MnNZ@Lip at equivalent OMV or Mn concentrations. From Figure 6 B, it can be seen that the GSH levels in cells treated with MnNZ@Lip and MnNZ@OMV were significantly reduced, indicating that GSH was consumed due to the reduction of MnNZ. In addition, as shown in Figure 6 C, the Fenton-like reaction mediated by released Mn2+ions led to an increase in hydroxyl radical ROS production in cells treated with MnNZ@Lip and MnNZ@OMV.

[0084] To verify the proposed pyroptosis pathway, the experiment further carried out Western blot analysis to evaluate the levels of downstream signals, as shown in Figure 6 D. B16 cells treated with MnNZ@Lip and MnNZ@OMV showed increased NF-κb phosphorylation and increased expression of caspase-11 precursor. In addition, the expression of activated caspase-11 was significantly increased in the MnNZ@OMV group, confirming the activation of caspase-11 precursor by LPS in OMV. At the same time, the expression of the N-terminal fragment of GSDMD was also significantly increased in the MnNZ@OMV group, confirming the occurrence of pyroptosis. Since the translocation of calreticulin (CRT) and the release of cellular contents such as ATP and HMGB1 accompany the process of cell pyroptosis, the levels of these DAMPs related to pyroptosis were also evaluated accordingly. Figure 6 E. Cells treated with MnNZ@OMV showed the highest amount of ATP release; and in Figure 6 F, CLSM images showed the least residual HMGB1 in these nuclei. In addition, Figure 6 G, H, it was detected by flow cytometry that the proportion of cells with CRT membrane translocation was the highest in the MnNZ@OMV group. These results collectively indicate that MnNZ and OMV have a synergistic effect in inducing pyroptosis of tumor cells.

[0085] In addition, as Figure 6 I,J, the present experiment also studied the role of pyroptosis cells induced by MnNZ@OMV in promoting DC maturation, and the bone marrow-derived DCs were co-cultured with tumor cells treated in different ways. The increase of CD80+CD86+DCs detected by flow cytometry can prove that tumor cells pretreated with MnNZ@OMV show the highest efficacy in inducing DC maturation.

[0086] Overall, these results show that MnNZ@OMV can be rapidly endocytosed by tumor cells and respond to high levels of GSH in cells to release manganese ions and OMVs, which leads to an increase in intracellular ROS levels and nuclear translocation of NF-κb, which in turn initiates caspase-11 expression, and activates caspase-11 through LPS in OMVs, ultimately inducing non-canonical GSDMD-mediated pyroptosis of tumor cells.

[0087] Finally, the therapeutic efficacy of MnNZ@OMV was evaluated using B16 melanoma model mice, as shown in Figure 9 and Figure 10 Among them, Figure 9

[0088] A is the treatment scheme of B16 tumor-bearing mice.

[0089] B is the change of tumor volume after treatment with different drug formulations (n=6).

[0090] C is the picture of tumor tissue after treatment with different drug formulations.

[0091] D is the tumor weight after treatment with different drug formulations (n=6).

[0092] E is the immunoblot analysis of NF-κb, caspase-11 and GSDMD in tumor tissue after treatment with different drug formulations.

[0093] F is the H&E pathological image of tumor tissue after treatment with different drug formulations.

[0094] All values are expressed as mean ± SD, and the asterisk indicates significant difference (*p<0.05).

[0095] Figure 10 Among them,

[0096] A is the flow cytometry analysis of CD86+DC cells in tumor tissue after treatment with different drug formulations.

[0097] B is the quantitative analysis of CD86+DC cells (n=3).

[0098] ​C is flow cytometry analysis of CD8+ T cells in tumor tissues after treatment with different drug formulations.

[0099] D is quantitative analysis of CD8+ T cells (n=3).

[0100] E is flow cytometry analysis of IFN-γ+ T cells in tumor tissues after treatment with different drug formulations.

[0101] F is quantitative analysis of IFN-γ+ T cells (n=3).

[0102] G is flow cytometry analysis of CD44+CD62L+ memory T cells in spleen tissues after treatment with different drug formulations.

[0103] H is quantitative analysis of CD44+CD62L+ memory T cells (n=3).

[0104] I is a schematic diagram of the treatment regimen to monitor tumor distal recurrence metastasis.

[0105] J is a curve of tumor-free incidence rate of mice in the designated group (n=5).

[0106] All values are expressed as mean ± SD. Asterisks indicate significant differences (*p<0.05).

[0107] As shown in Figure 9 B, C, D, and Figure 11 MnNZ@OMV treatment group showed the highest tumor inhibition rate, demonstrating that the combination of MnNZ and OMV achieved an enhanced therapeutic effect. Figure 9 E, immunoblot analysis was performed on the treated tumor tissue samples to verify that MnNZ@OMV induced pyroptosis of tumor cells in vivo through the caspase11 / GSDMD signaling pathway. Figure 9 F, Figure 12 In the middle, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin-eosin (H&E) staining confirmed the strong pyroptosis induction and tumor destruction after MnNZ@OMV treatment.

[0108] In addition, tumor tissue samples from mice treated with MnNZ@OMV contained more mature DC cells (see Figure 10 A and B), leading to enhanced CD8+ T cell infiltration (see Figure 10 C and D) and IFN-γ+ T cell activation (see Figure 10 E and F), due to the antigen presentation and pro-inflammatory factor release effects of mature DC cells. In addition, as shown in Figure 10G and H can be seen that the proportion of CD44+CD62L+ memory T cells in the spleen samples of mice treated with MnNZ@OMV is the highest, indicating that MnNZ@OMV can promote the activation of memory T cells.

[0109] As shown in Figure 10 I, the experiment also evaluates the ability of MnNZ@OMV to inhibit the growth of distant tumor metastasis. The B16 tumor-bearing mice were inoculated with a second tumor on the contralateral side as a distant metastasis model on the first day of treatment with different drug formulations. During the 15-day monitoring period after the second tumor inoculation, only the mice treated with MnNZ@OMV did not have the growth of distant tumors (see Figure 10 J), indicating that MnNZ@OMV can induce a distant effect in tumor treatment. Overall, MnNZ@OMV induces strong pyroptosis of tumor cells, promotes DC maturation, and subsequently activates effector and memory T cells, effectively inhibiting primary and distant tumor growth.

[0110] In summary, the present scheme highlights the potential strategy of enhancing anti-tumor immunity through pyroptosis induction. The provided MnNZ@OMV as a self-starting pyroptosis inducer can be rapidly endocytosed by tumor cells and quickly respond to intracellular high GSH levels, leading to the decomposition of MnNZ@OMV and the release of manganese ions and OMV. The released manganese ions further mediate Fenton-like reactions to generate ROS, which in turn induce NF-κb phosphorylation and activate caspase-11. Caspase-11 is then activated by LPS in OMV and cleaves GSDMD to induce cell pyroptosis. The synergistic effect of MnNZ and OMV in inducing tumor cell pyroptosis and activating anti-tumor immunity leads to significant inhibition of primary and distant tumor growth.

[0111] The specific embodiments described in this example are merely illustrative of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace them without deviating from the spirit of the present application or exceeding the scope defined by the appended claims.

[0112] Although the terms self-starting pyroptosis inducer, manganese dioxide nanozyme, outer membrane vesicle, glutathione, reactive oxygen species, etc. are used more frequently herein, the possibility of using other terms is not excluded. The use of these terms is merely to facilitate the description and explanation of the essence of the present application; any interpretation of them as any kind of additional limitation is contrary to the spirit of the present application.

Claims

1. Use of a self-starting pyroptosis inducer in the preparation of an antitumor immunotherapy drug, the self-starting pyroptosis inducer comprising a manganese dioxide nanomachine and an outer membrane vesicle encapsulating the manganese dioxide nanomachine; The tumor cells against which the antitumor immunotherapy acts have a high level of glutathione; The tumor cells against which the antitumor immunotherapy acts have a high level of hydrogen peroxide; The mechanism by which the self-starting pyroptosis inducer starts the antitumor immunity is as follows: The self-starting pyroptosis inducer is administered to the tumor tissue and endocytosed by the tumor cells, and is degraded in response to the high level of glutathione in the tumor cells; Under the action of glutathione, the self-starting pyroptosis inducer releases manganese ions and outer membrane vesicle components; The manganese ions catalyze the generation of reactive oxygen species, thereby triggering the translocation of NF-κB into the nucleus of the tumor cells; The translocation of NF-κB into the nucleus of the tumor cells induces the gene expression of caspase 4 / 5 / 11; At the same time, the expressed caspase 4 / 5 / 11 is activated by the previously released outer membrane vesicle components; The activated caspase 4 / 5 / 11 cleaves the GSDMD protein, triggering pyroptosis of the tumor cells; The manganese dioxide nanomachine is a spiky manganese dioxide nanomachine, and the outer membrane vesicle is encapsulated on the surface of the spiky manganese dioxide nanomachine; The outer membrane vesicle is extracted from Escherichia coli, and the membrane contains lipopolysaccharide.

2. Use of the self-starting pyroptosis inducer according to claim 1 in the preparation of an antitumor immunotherapy drug, characterized in that, The spiky manganese dioxide nanomachine is obtained by reducing potassium permanganate using oleic acid.

3. Use of the self-starting pyroptosis inducer according to claim 2 in the manufacture of an antitumor immunotherapy drug, characterized in that, The average size of the outer membrane vesicle is 28±2 nm, and the maximum is not more than 35 nm and the minimum is not less than 20 nm; the average size of the manganese dioxide nanomachine is 157±5 nm, and the maximum is not more than 165 nm and the minimum is not less than 150 nm.

4. The use of the self-starting pyroptosis inducer according to claim 3 in the preparation of an antitumor immunotherapy drug, characterized in that, The size of the self-starting pyroptosis inducer is 200±5 nm, and the maximum is not more than 210 nm and the minimum is not less than 190 nm.

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