Self-starting pyroptosis inducer as well as preparation method and application thereof

Abstract

The invention discloses a self-starting pyroptosis inducer as well as a preparation method and application thereof, the self-starting pyroptosis inducer aims at realizing pyroptosis of tumor cells by caspase 4 / 5 / 11, a combined medicine of manganese dioxide nano-enzyme and outer membrane vesicles is provided, the manganese dioxide nano-enzyme is coated by the outer membrane vesicles to prepare the self-starting pyroptosis inducer, and the self-starting pyroptosis inducer has the advantages that the self-starting pyroptosis inducer is used for inducing the pyroptosis of tumor cells; it is guaranteed that the self-starting pyroptosis inducer can be decomposed in tumor cells with the high glutathione level to release manganese ions and lipopolysaccharide of outer membrane vesicles, and reactive oxygen is generated through a series of reactions between the increased GSH level of the tumor cells and the manganese ions to up-regulate precursor expression of caspase-4 / 5 / 11. Lipopolysaccharide of the outer membrane vesicles OMVs activates caspase-4 / 5 / 11, the lipopolysaccharide and active oxygen generated by MnNZ generate a synergistic effect, and pyroptosis of tumor cells is efficiently induced.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a self-starting pyroptosis inducer and a preparation method and application thereof. Background Art

[0002] Pyroptosis is a form of programmed cell death that falls into the category of inflammatory cell death. Its main feature is that it depends on the activation of inflammasomes and inflammatory caspases (such as caspase-1), which ultimately leads to cell membrane rupture and release of cell contents, thereby initiating an inflammatory response. During pyroptosis, activated caspases cleave Gasdermin, and the released N-terminal fragments are transferred to the cell membrane to form pores, causing cell swelling and lysis. As an immunogenic form of 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, which increases the opportunity for antigen presentation and helps reverse the immunosuppressive microenvironment of the tumor. Therefore, pyroptosis shows great potential in the study of stimulating anti-tumor immune responses.

[0003] Radiotherapy and chemotherapy can induce pyroptosis through the GSDME pathway. However, in this case, when cells undergo pyroptosis, caspase-3 and caspase-8, which are usually associated with apoptosis, will also be activated. The accompanying apoptosis process does not have the specificity of pyroptosis and is difficult to induce a strong immune response. Therefore, this pyroptosis induction method is not ideal. In addition, although the classical pathway mediated by caspase-1 and GSDMD is a common pyroptosis induction mechanism, this pathway relies on the activation of pattern recognition receptors (PRRs), such as TLR and NLRP3, which are mainly expressed in immune cells and have limited expression in tumor cells. Therefore, this classical pathway has low efficiency in inducing pyroptosis in tumor cells. Therefore, how to effectively induce pyroptosis in tumor cells to potently stimulate anti-tumor immune responses remains a major challenge.

[0004] In addition to the classical cleavage by caspase-1, GSDMD can also be cleaved by caspase-4 / 5 (corresponding to caspase-11 in mice) through a non-classical pathway. However, the current research on the mechanism of action of caspase-4 / 5 / / 11 in tumor cell pyroptosis and its potential application is relatively limited, and no significant breakthrough results have been achieved. Summary of the invention

[0005] The purpose of the present invention is to provide a self-starting pyroptosis inducer in view of the above problems;

[0006] Provide a method for preparing a self-starting pyroptosis inducer;

[0007] The application of self-initiated pyroptosis inducers in the preparation of anti-tumor immunotherapy drugs is also proposed.

[0008] A self-initiated pyroptosis inducer comprises a manganese dioxide nanozyme (MnNZ) and an outer membrane vesicle (OMV) encapsulating the manganese dioxide nanozyme.

[0009] In the above-mentioned self-initiated pyroptosis inducer, the manganese dioxide nanozyme is spike-shaped, and the outer membrane vesicles are coated on the surface of the spike-shaped manganese dioxide nanozyme.

[0010] In the above-mentioned self-initiated pyroptosis inducer, the outer membrane vesicles are extracted from Escherichia coli, and the membrane contains lipopolysaccharide (LPS);

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

[0012] In the above-mentioned self-initiated pyroptosis inducer, the average size of the outer membrane vesicles is (28±2) nm, and the maximum size does not exceed 35 nm, and the minimum size is not less than 20 nm;

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

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

[0015] The self-starting pyroptosis inducer is used in the preparation of anti-tumor immunotherapy drugs.

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

[0017] Tumor cells targeted by anti-tumor immunotherapy have high levels of hydrogen peroxide, greater than 100 micromoles per liter.

[0018] In the application of the above-mentioned self-initiated pyroptosis inducer in anti-tumor immunotherapy drugs, the mechanism by which the self-initiated pyroptosis inducer initiates anti-tumor immunity is as follows:

[0019] Self-priming pyroptosis inducers are administered to tumor tissues and internalized by tumor cells, where they are degraded in response to high levels of glutathione in tumor cells;

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

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

[0022] NF-κB translocates to the nucleus of tumor cells and induces gene expression of caspase-4 / 5 / 11;

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

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

[0025] A method for preparing a self-starting pyroptosis-inducing drug, the method comprising:

[0026] Extraction of outer membrane vesicles from Escherichia coli;

[0027] The outer membrane vesicles are coated on the surface of the spike-shaped manganese dioxide nanozyme to obtain a pyroptosis inducer that can self-initiate pyroptosis.

[0028] In the above-mentioned method for preparing the self-initiated pyroptosis-inducing drug, the method specifically comprises:

[0029] Using oleic acid to reduce potassium permanganate to obtain the spike-shaped manganese dioxide nanozyme 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] The outer membrane vesicles are coated on the spike-shaped manganese dioxide nanozyme using an ultrasonic method to form the self-initiated pyroptosis-inducing drug with a size of (200±5) nm.

[0032] The advantages of the present invention are:

[0033] This protocol focuses on caspase-4 / 5 (corresponding to caspase-11 in mice) to induce pyroptosis of tumor cells. The results show that it has high efficacy and specificity, providing new ideas and directions for tumor immunotherapy.

[0034] This scheme proposes the combination of MnNZ and OMV. By coating the self-initiated pyroptosis inducer with MnNZ by OMV, MnNZ can be protected from being cleared before being taken up by tumor cells, thereby ensuring that the self-initiated pyroptosis inducer will be decomposed to release manganese ions and OMV in tumor cells with high glutathione levels, thus ensuring the specificity and potency of the drug.

[0035] In the self-initiated pyroptosis inducer provided in this scheme, OMV itself contains LPS and other ingredients, which can directly activate caspase-4 / 5 / 11, and produce a synergistic effect with MnNZ to catalyze the reactive oxygen species ROS generated by hydrogen peroxide in tumor cells, thus efficiently inducing tumor cell pyroptosis;

[0036] The self-initiated pyroptosis inducer provided in this scheme does not rely on pattern recognition receptors PRRs during tumor cell pyroptosis. The OMVs released by them activate caspase-4 / 5 / 11, thereby ensuring the activation efficiency. In addition, this process utilizes the higher glutathione level of tumor cells and upregulates the precursor expression of caspase-4 / 5 / 11 through a series of reactions, providing a material basis for subsequent activation, thereby ensuring the specificity and potency of pyroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a diagram of the preparation and therapeutic mechanism of the self-starting pyroptosis inducer of the present invention;

[0038] Figure 2 This is an X-ray photoelectron spectroscopy analysis of MnNZ prepared for the preparation of self-initiated pyroptosis inducers;

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

[0040] Figure 4 It is the result of the study on the characteristics of the self-starting pyroptosis inducer prepared in the embodiment of the present invention;

[0041] Figure 5 Energy dispersive X-ray spectroscopy analysis and elemental mapping of MnNZ and MnNZ@OMV, with manganese Mn in purple and phosphorus P in yellow;

[0042] Figure 6 The present invention studies the effect of MnNZ@OMV on tumor cells through in vitro induction;

[0043] Figure 7 It is a confocal laser scanning microscopy (CLSM) image of B16 cells in the in vitro experiment of the present invention after co-incubation with Di I-labeled (red) OMV or MnNZ@OMV for a specified time interval;

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

[0045] Fig. 9It is the experimental result verifying that MnNZ@OMV inhibits tumor development in vivo;

[0046] Fig.10 It is the experimental result that verifies that MnNZ@OMV activates anti-tumor immunity and inhibits distant metastasis;

[0047] Fig.11 yes Fig. 9 Changes in tumor volume of each mouse after specified treatment under the experiment;

[0048] Fig.12 yes Fig. 9 TUNEL staining images of tumor tissues after designated treatments in the experiment. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0050] This embodiment provides a self-initiated pyroptosis inducer, gives a method for preparing the self-initiated pyroptosis inducer, and conducts effectiveness tests and mechanism descriptions on the application of the pyroptosis inducer in anti-tumor immunotherapy.

[0051] This embodiment provides a manganese dioxide nanozyme (MnNZ) encapsulated in an outer membrane vesicle (OMV) as a self-initiated pyroptosis inducer (MnNZ@OMV) for tumor treatment. First, oleic acid Oleic Acid is used to reduce potassium permanganate KMnO4 to synthesize spike-shaped MnNZ. Then, OMVs with an average size of about 28 nm are extracted from Escherichia coli, and finally, they are coated on the MnNZ obtained in the previous step by ultrasound to form the required MnNZ@OMV. The average size of the inducer is about 200 nm. The mechanism by which the above-mentioned MnNZ@OMV plays a therapeutic role as a self-initiated pyroptosis inducer in tumor immunotherapy is as follows Figure 1 As shown:

[0052] After MnNZ@OMV is injected into tumor tissue and rapidly internalized by tumor cells, MnNZ@OMV undergoes a reduction reaction to release manganese ions under the action of high levels of glutathione GSH (concentration range of about 2–10 mmol / L) in tumor cells. This process also causes the structural destruction 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 much higher than that in normal cells. The released manganese ions further catalyze the high level of H2O2 in tumor cells to convert it into reactive oxygen - hydroxyl radicals, which increases the concentration of hydroxyl radicals. The increased hydroxyl radicals increase the oxidative stress in the cells, triggering NF-κB to transfer to the nucleus of tumor cells, thereby activating the gene expression of caspase-4 / 5 (corresponding to caspase-11 in mice). Subsequently, the expressed caspase-4 / 5 / 11 will be activated by the previously released OMVs and cleave the GasderminD (GSDMD) protein, releasing its N-terminal fragment, which forms holes in the cell membrane, causing leakage of cellular contents, and ultimately triggering tumor cell pyroptosis and DAMPs release.

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

[0054] In order to explore the mechanism of the efficacy of the self-activated pyroptosis inducer MnNZ@OMV in anti-tumor treatment, this example prepared MnNZ@OMV by the following means:

[0055] 0.4 mg of KMnO4 was dissolved in 200 ml of water and stirred magnetically at room temperature. 4 ml of oleic acid was added dropwise and stirred for 5 hours. The product was then collected by centrifugation at 12,000 rpm and washed alternately with ethanol and deionized water three times to obtain the spiky MnNZ. Figure 2 and Figure 3 As shown, according to the characteristic peaks observed from X-ray photoelectron spectroscopy and X-ray diffraction spectrum, it can be determined that MnNZ is mainly composed of δ-MnO2.

[0056] A single colony of E. col i was cultured in LB medium at 37°C and 200 rpm for 16 hours. The supernatant was collected by centrifugation at 3000 g for 30 minutes and filtered through a 0.45 μm filter to remove bacteria. The OMVs were then isolated by ultracentrifugation at 150,000 g for 2 hours using an ultracentrifuge, and finally, the OMVs were resuspended in deionized water and stored at -80°C until use.

[0057] The OMV solution (0.1 mg protein / mL) was mixed with the MnNZ solution (0.2 mg Mn / mL) and sonicated in ice water for 1 h. The mixture was then centrifuged at 12,000 rpm for 15 min, and the collected precipitate was dispersed in deionized water to obtain MnNZ@OMV.

[0058] Subsequently, this example studied the physicochemical properties of the self-activated pyroptosis inducer MnNZ@OMV prepared above. Figure 4 As shown, where:

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

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

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

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

[0063] E is the percentage of residual MnNZ@OMV after incubation in simulated body fluid (SBF) containing different GSH concentrations for 8 min. The inset in the upper right corner is a photograph of the MnNZ@OMV suspension treated with different GSH concentrations as shown;

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

[0065] G is the percentage of residual methylene blue MB after 8 minutes of treatment with MnNZ@OMV at 1 mM H2O2 concentration and different concentrations of GSH. MB is used to indicate the amount of methylene blue remaining after a certain period of time in the presence of MnNZ@OMV, H2O2, and GSH, and is thus used to evaluate the ability of MnNZ@OMV to produce hydroxyl radicals;

[0066] H is the change 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 shows that the OMV film is visible on the surface of the spiky MnNZ; Figure 4 B shows that after OMV coating, the size of the spike-like MnNZ increased from 157.1 nm to 199.5 nm, while the zeta potential of MnNZ@OMV decreased slightly compared with that of MnNZ, approaching the lower potential of OMV. Figure 4 C, SDS-PAGE analysis of OMV, MnNZ and MnNZ@OMV showed that the protein bands between OMV and MnNZ@OMV matched, confirming that MnNZ was successfully coated by the OMV membrane. Figure 4 D and Figure 5 As shown in Figure 2, the overlap of Mn and P on the particles indicates the successful integration of OMV and MnNZ. Figure 4 A, B, C, D and Figure 5 It can be confirmed that the aforementioned preparation process obtains the desired self-initiated pyroptosis inducer MnNZ@OMV, which includes a spike-like manganese dioxide nanozyme, and the outer membrane vesicles are coated on the surface of the spike-like manganese dioxide nanozyme.

[0068] Furthermore, if Figure 4 As shown in Figure E, MnNZ@OMV has a degradation property that is dependent on the concentration of GSH, while there is almost no GSH in the external environment and only a small amount of GSH in normal cells. This result indicates that the decomposition of MnNZ@OMV will only occur after being internalized by tumor cells. Figure 4 As shown in Figure F, in a high GSH environment (1 mM and above), the absorbance of MnNZ@OMV decreased sequentially within 8 minutes, which further demonstrated the rapid response of MnNZ@OMV to GSH. Figure 4 As shown in Figures G and 4H, GSH and time-dependent methylene blue (MB) degradation were detected in the presence of MnNZ@OMV and H2O2, indicating 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 that it induced tumor cell pyroptosis in vitro through the non-classical caspase pathway. Figure 6 As shown, where:

[0070] A is the cytotoxicity of PLGA@OMV, MnNZ@Lip and MnNZ@OMV on mouse melanoma B16 cells after 24 hours of incubation. MnNZ@LiP and PLGA@OMV are the control groups of MnNZ@OMV, the former uses liposomal LiP to replace the OMV in MnNZ@OMV, and the latter uses poly(lactic acid-polyglycolic acid) copolymer PLGA to replace the MnNZ in MnNZ@OMV.

[0071] B is the relative GSH levels in B16 cells treated with PBS, PLGA@OMV, MnNZ@Lip and MnNZ@OMV for 24 h (n=3).

[0072] C ROS levels in B16 cells after treatment with different formulations for 24 h.

[0073] D is immunoblot 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 images of HMGB1 (red) and nucleus (blue) of B16 cells treated with different formulations.

[0076] G is flow cytometric 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 cytometric analysis of CD80+CD86+BMDCs co-cultured with BMDCs and B16 cells pretreated with different formulations.

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

[0080] All values ​​are expressed as mean ± standard deviation (SD). Asterisks indicate significant differences (*p<0.05).

[0081] The experimental process is as follows:

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

[0083] Subsequently, the cytotoxicity of MnNZ@OMV was evaluated in B16 cells. PLGA-encapsulated OMVs (PLGA@OMV) and MnNZ-encapsulated liposomes (MnNZ@Lip) were prepared as the control group of MnNZ@OMV, and their hydrodynamic diameter and zeta potential were characterized as shown in Figure 8 As shown. Figure 6 A shows that at equivalent OMV or Mn concentrations, MnNZ@OMV showed a stronger concentration-dependent killing effect than PLGA@OMV and MnNZ@Lip. Figure 6 B shows that the GSH level in cells treated with MnNZ@Lip and MnNZ@OMV was significantly reduced, indicating that GSH was consumed due to MnNZ reduction. Figure 6 C, The released manganese ions mediated a Fenton-like reaction leading to increased generation of hydroxyl radicals (ROS) in cells treated with MnNZ@Lip and MnNZ@OMV.

[0084] To validate the proposed pyroptotic pathway, immunoblotting analysis was further performed to assess the levels of downstream signals, such as Figure 6 As shown in 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 are accompanied by the process of cell pyroptosis, the levels of these DAMPs associated with pyroptosis were also evaluated accordingly. Figure 6 As shown in E, cells treated with MnNZ@OMV showed the highest ATP release; Figure 6 The CLSM images in F show that the residual HMGB1 in the nuclei of these cells is minimal. Figure 6 G, H show that the proportion of cells with CRT membrane translocation was the highest in the MnNZ@OMV group as detected by flow cytometry. These results collectively indicate that MnNZ and OMV have a synergistic effect in inducing tumor cell pyroptosis.

[0085] In addition, if Figure 6 As shown in I and J, this experiment also studied the effect of MnNZ@OMV-induced pyroptotic cells on promoting DC maturation, and bone marrow-derived DCs were co-cultured with tumor cells after different treatments. The increase in CD80+CD86+DCs detected by flow cytometry proved that tumor cells pretreated with MnNZ@OMV showed the highest efficacy in inducing DC maturation.

[0086] Collectively, these results suggest that MnNZ@OMVs can be rapidly internalized by tumor cells and respond to high levels of intracellular GSH to release manganese ions and OMVs, which leads to increased intracellular ROS levels and the translocation of NF-κb to the nucleus, thereby initiating caspase-11 expression and activating caspase-11 through LPS in OMVs, ultimately inducing non-classical GSDMD-mediated tumor cell pyroptosis.

[0087] Finally, the therapeutic efficacy of MnNZ@OMV was evaluated using B16 melanoma model mice. Fig. 9 and Fig.10 As shown. Among them, Fig. 9 middle:

[0088] A is a schematic diagram of the treatment plan for B16 tumor-bearing mice.

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

[0090] C is a 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 tissues 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 asterisks indicate significant differences (*p < 0.05).

[0095] Fig.10 middle:

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

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

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

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

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

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

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

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

[0104] I is a schematic diagram of the treatment plan for monitoring distant recurrence and metastasis of the tumor.

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

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

[0107] like Fig. 9 B, C, D and Fig.11 As shown, the MnNZ@OMV-treated group displayed the highest tumor inhibition rate, demonstrating that the combination of MnNZ and OMV achieved an enhanced therapeutic effect. Fig. 9 As shown in E, immunoblot analysis of treated tumor tissue samples verified that MnNZ@OMV induced tumor cell pyroptosis via the caspase11 / GSDMD signaling pathway in vivo. Fig. 9 F. Fig.12 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin-eosin (H&E) staining confirmed 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 DCs compared with those from other groups (see Fig.10 A and B), leading to enhanced CD8+ T cell infiltration (see Fig.10 C and D) and IFN-γ+ T cell activation (see Fig.10 E and F), which is due to the antigen presentation and pro-inflammatory cytokine release of mature DC cells. Fig.10As can be seen in G and H, the spleen samples of mice treated with MnNZ@OMV had the highest proportion of CD44+CD62L+ memory T cells, indicating that MnNZ@OMV may promote the activation of memory T cells.

[0109] like Fig.10 As shown in Figure 1, the ability of MnNZ@OMV to inhibit the growth of distant tumor metastases was also evaluated in this experiment. B16 tumor-bearing mice were inoculated with a second tumor on the contralateral side on the first day of treatment with different drug formulations as a distant metastasis model. During the 15-day monitoring period after the second tumor inoculation, only the mice treated with MnNZ@OMV did not show the growth of distant tumors (see Fig.10 J), indicating that MnNZ@OMV can induce distal effects in tumor therapy. Overall, MnNZ@OMV induced strong tumor cell pyroptosis, promoted DC maturation, and subsequently activated effector and memory T cells, effectively inhibiting primary tumor and distal growth.

[0110] In summary, this protocol highlights a potential strategy for enhancing anti-tumor immunity through pyroptosis induction. The provided MnNZ@OMV, as a self-initiated pyroptosis inducer, can be rapidly internalized by tumor cells and rapidly respond to high intracellular GSH levels, leading to the decomposition of MnNZ@OMV and the release of manganese ions and OMVs. The released manganese ions further mediate a Fenton-like reaction to produce ROS, which in turn induces NF-κb phosphorylation and activates caspase-11. Caspase-11 is subsequently activated by LPS in OMVs and cleaves GSDMD to induce cell pyroptosis. It can be seen that the synergistic effects of MnNZ and OMVs in inducing tumor cell pyroptosis and activating anti-tumor immunity led to significant inhibition of primary and distant tumor growth.

[0111] The specific embodiments described in this embodiment are merely examples of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0112] Although the terms such as self-initiated pyroptosis inducer, manganese dioxide nanozyme, outer membrane vesicle, glutathione, and reactive oxygen species are used more frequently in this article, the possibility of using other terms is not excluded. The use of these terms is only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A self-starting pyroptosis inducer, characterized in that It includes manganese dioxide nanozyme and outer membrane vesicles that encapsulate the manganese dioxide nanozyme.

2. The self-initiated pyroptosis inducer according to claim 1, characterized in that The manganese dioxide nanozyme is a spike-shaped manganese dioxide nanozyme, and the outer membrane vesicles are coated on the surface of the spike-shaped manganese dioxide nanozyme.

3. The self-initiated pyroptosis inducer according to claim 2, characterized in that The outer membrane vesicles are extracted from Escherichia coli, and the membrane contains lipopolysaccharide; The spike-shaped manganese dioxide nanozyme is obtained by reducing potassium permanganate with oleic acid.

4. The self-initiated pyroptosis inducer according to claim 3, characterized in that The average size of the outer membrane vesicles is (28±2) nm, with the maximum size not exceeding 35 nm and the minimum size not less than 20 nm; The average size of the manganese dioxide nanozyme is (157±5) nm, with a maximum size not exceeding 165 nm and a minimum size not less than 150 nm.

5. The self-initiated pyroptosis inducer according to claim 4, characterized in that The size of the self-initiated pyroptosis inducer is (200±5) nm, with the maximum not exceeding 210 nm and the minimum not less than 190 nm.

6. Use of the self-initiated pyroptosis inducer according to any one of claims 1 to 5 in the preparation of anti-tumor immunotherapy drugs.

7. The use of the self-activated pyroptosis inducer in anti-tumor immunotherapy drugs according to claim 6, characterized in that: Tumor cells targeted by anti-tumor immunotherapy have high levels of glutathione; Tumor cells targeted by anti-tumor immunotherapy have high levels of hydrogen peroxide.

8. The use of the self-activated pyroptosis inducer in anti-tumor immunotherapy drugs according to claim 7, characterized in that: The mechanism by which self-activated pyroptosis inducers initiate anti-tumor immunity is as follows: Self-priming pyroptosis inducers are administered to tumor tissues and internalized by tumor cells, where they are degraded in response to high levels of glutathione in tumor cells; Under the action of glutathione, the self-priming pyroptosis inducer releases manganese ions and outer membrane vesicle components; Manganese ions catalyze the generation of reactive oxygen species, thereby triggering the translocation of NF-κB into the nucleus of tumor cells; NF-κB translocates to the nucleus of tumor cells and induces 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; Activated caspase-4 / 5 / 11 cleaves GSDMD protein, triggering pyroptosis of tumor cells.

9. A method for preparing a self-activated pyroptosis-inducing drug, characterized in that: The method includes: Extraction of outer membrane vesicles from Escherichia coli; The outer membrane vesicles are coated on the surface of the spike-shaped manganese dioxide nanozyme to obtain a pyroptosis inducer that can self-initiate pyroptosis.

10. The method for preparing the self-activated pyroptosis inducing drug according to claim 9, characterized in that: Specifically include: Using oleic acid to reduce potassium permanganate to obtain the spike-shaped manganese dioxide nanozyme with an average size of (157±5) nm; Extracting the outer membrane vesicles with an average size of (28±2) nm from Escherichia coli; The outer membrane vesicles were coated on the spike-shaped manganese dioxide nanozymes using ultrasound to form a self-initiated pyroptosis-inducing drug with a size of (200±5) nm.

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