X-ray activated tumor immunometabolic regulation and pyroptosis synergistic bacteria outer membrane vesicle and preparation method and application thereof

Abstract

The application discloses an X-ray activated tumor immune metabolism regulation and pyroptosis synergistic bacteria outer membrane vesicle and a preparation method and application thereof, and belongs to the technical field of biotechnology.The application introduces an azido group on the surface of the bacteria outer membrane vesicle through metabolic sugar engineering technology, modifies PEG, a singlet oxygen sensitive linkage and an immune metabolism regulator on the surface of the bacteria outer membrane vesicle in sequence through a click chemistry reaction, and further modifies an X-ray sensitive drug on the bacteria outer membrane vesicle, generates singlet oxygen by irradiating the tumor site with X-rays, causes the singlet oxygen sensitive linkage to break, responsively releases an IDO inhibitor, effectively blocks T cell inhibition induced by high expression of IDO after radiotherapy and restores strong T cell response, reverses the tumor immunosuppressive microenvironment caused by radiotherapy, and simultaneously induces pyroptosis of tumor cells by lipopolysaccharide on the bacteria outer membrane vesicle, further activates the immune response of the body and enhances the radiotherapy effect.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and biomedicine, specifically relating to a bacterial outer membrane vesicle with X-ray-activated tumor immune metabolic regulation and synergistic pyroptosis effects, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Radiotherapy, or radiation therapy, is a treatment method that primarily uses high-energy rays to kill tumor cells. Although radiotherapy has been successfully applied clinically to treat various tumors, several challenges remain. For example, radiotherapy increases the expression of immune checkpoint-related proteins, such as programmed death receptor ligand 1 (PD-L1) and indoleamine-2,3-dioxygenase (IDO), allowing tumor cells to evade the immune system. It also increases the expression of regulatory T cells (Tregs), limiting T cell infiltration and function, and may polarize macrophages from anti-tumor M1 to pro-tumor M2, thereby negatively regulating anti-tumor immunity and suppressing the tumor immune microenvironment, thus limiting the effectiveness of radiotherapy. Therefore, there is an urgent need to develop a strategy that can reverse the radiotherapy-induced tumor immunosuppressive microenvironment and activate the anti-tumor immune response to enhance the efficacy of radiotherapy.

[0004] Immunomodulatory therapy is a novel cancer treatment method that enhances the immune system's ability to recognize and kill tumor cells by regulating their metabolic pathways, thereby producing therapeutic effects. Currently, small molecule inhibitors, such as the IDO enzyme inhibitor (NLG-919), are commonly used immunometabolic therapy drugs. Because tumor cells overexpress IDO enzymes, which catalyze the degradation of tryptophan (Trp) into kynurenine (Kyn), they inhibit the function of effector T cells and promote the proliferation of Treg cells, resulting in immunosuppression. NLG-919 interferes with amino acid metabolism by inhibiting IDO enzyme activity in tumor cells, thereby reversing the immunosuppressive microenvironment. However, these small molecule inhibitors often suffer from insufficient targeting and are prone to toxic side effects on normal cells, limiting their further application. Therefore, there is a need to develop a responsive immunomodulatory nanomedicine that can release immunomodulatory drugs at specific sites on the tumor through X-ray irradiation, avoiding damage to normal tissues and effectively reversing the radiotherapy-induced immunosuppressive microenvironment.

[0005] Pyroptosis is a newly discovered form of programmed cell death. The lipopolysaccharide (LPS)-mediated non-classical pyroptosis pathway involves LPS binding to and activating caspase-11 or caspase-4 / 5, which further cleaves GSDM family proteins. The N-terminal fragments of these cleavage products can create pores in the cell membrane, increasing intracellular osmotic pressure, leading to cell swelling and rupture, releasing cell contents, and ultimately, cell death.

[0006] Gram-negative bacterial outer membrane vesicles (OMVs) are spherical vesicles ranging in size from 20 to 250 nm secreted by bacteria. They contain a large amount of components derived from the outer membrane and pericyte of the parent bacteria, including LPS, proteins, lipids, and nucleic acids. Therefore, OMVs can act as natural carriers to deliver LPS into tumor cells, inducing pyroptosis in tumor cells via an LPS-mediated non-classical pathway. The large amount of tumor cell contents released can act as antigens, activating the immune system. Combining the pyroptosis induced by OMVs with X-ray-responsive immunometabolic regulation holds promise for synergistically regulating the immune microenvironment, activating anti-tumor immune responses, and enhancing the efficacy of radiotherapy. However, how this pathway is implemented and its effects remain to be explored. Summary of the Invention

[0007] To address the aforementioned limitations, this invention provides a bacterial outer membrane vesicle with X-ray-activated tumor immune metabolic regulation and synergistic pyroptosis effects, and its preparation method.

[0008] As one aspect of the present invention, a bacterial outer membrane vesicle with X-ray-activated tumor immune metabolism regulation and pyroptosis synergistic effect to enhance radiotherapy is provided, comprising azide-modified bacterial outer membrane vesicles, diphenylcyclooctyne-polyethylene glycol-amino, singlet oxygen-sensitive 2,2-[propane-2,2-dimethylbis(thio)]diacetic acid, an immune metabolism regulator, and an X-ray sensitive drug;

[0009] The immunomodulator is linked to polyethylene glycol (PEG) via a singlet oxygen-sensitive linker. The PEG modified with a diphenylcyclooctynyl (DBCO) group is modified with azide-modified bacterial outer membrane vesicles via a click chemical reaction.

[0010] The X-ray-sensitive drug chemically modifies the bacterial outer membrane vesicles.

[0011] The immunometabolism modulators are selected from metabolic enzyme inhibitors, PD-1 / PD-L1 interaction inhibitors, and protein synthesis inhibitors;

[0012] Furthermore, the metabolic enzyme inhibitor is an IDO enzyme inhibitor;

[0013] The PD-1 / PD-L1 interaction inhibitor is BMS-1166;

[0014] The protein synthesis inhibitor is puromycin.

[0015] The preferred metabolic enzyme inhibitor is NLG-919.

[0016] The X-ray sensitive drug is a porphyrin-based X-ray sensitive drug, preferably Verteporfin or Ce6.

[0017] The outer membrane vesicles are modified with drugs, X-ray sensitive drugs, and small molecule inhibitors, resulting in a particle size of 100-150 nm.

[0018] The bacterial outer membrane vesicles described are BL21(DE3) Escherichia coli outer membrane vesicles cultured in low-sugar M9 medium containing 1,3,4,6-tetra-oxoacetyl-2-[(azidoacetyl)amino]-2-deoxy-β-D-galactopyranose.

[0019] The X-ray-sensitive drug Verteporfin loaded with X-rays can generate singlet oxygen under X-ray excitation, inducing... 1 O2Linker (2,2-[propane-2,2-dimethylbis(thio)diacetic acid) is cleaved, releasing the loaded small molecule inhibitor. NLG-919 is an IDO inhibitor that effectively regulates IDO overexpression induced by radiotherapy and restores a strong T-cell response.

[0020] As another aspect of the present invention, a method for preparing bacterial outer membrane vesicles with X-ray-activated tumor immune metabolic regulation and synergistic pyroptosis to enhance radiotherapy efficacy is provided, comprising the following steps:

[0021] Step 1: Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3:

[0022] Step 2: The small molecule inhibitor is linked to diphenylcyclooctyne-polyethylene glycol-amino via a singlet oxygen-sensitive linker 2,2-[propane-2,2-dimethylbis(thio)]diacetic acid. The diphenylcyclooctyne-polyethylene glycol-amino is then linked to azide-modified bacterial outer membrane vesicles via click chemistry. The surface of the bacterial outer membrane vesicles is chemically modified with X-ray-sensitive drugs to prepare the responsive tumor immune microenvironment modulating nanomedicine PLG@OMVs-VP.

[0023] This invention introduces azide groups onto the surface of bacterial outer membrane vesicles using metabolic glycoengineering technology. Through a click chemical reaction, PEG, singlet oxygen-sensitive linkages, and a small molecule immunomodulator (IDO inhibitor) are sequentially modified onto the surface of the bacterial outer membrane vesicles. Furthermore, an X-ray-sensitive drug (Verteporfin) is modified onto the bacterial outer membrane vesicles. When the tumor site is irradiated with X-rays, Verteporfin on the bacterial outer membrane vesicles absorbs the X-rays and generates singlet oxygen, leading to the breakage of the singlet oxygen-sensitive linkages and the responsive release of the IDO inhibitor. This effectively blocks the T-cell suppression induced by high IDO expression after radiotherapy and restores a strong T-cell response, reversing the tumor immunosuppressive microenvironment caused by radiotherapy. Simultaneously, lipopolysaccharide (LPS) on the bacterial outer membrane vesicles induces pyroptosis of tumor cells, further activating the body's immune response and enhancing the radiotherapy effect.

[0024] As a third aspect of the invention, it provides the application of the bacterial outer membrane vesicles, which enhance the radiotherapy effect through X-ray-activated tumor immune metabolic regulation and pyroptosis synergistic effect, in the preparation of tumor therapeutic drugs.

[0025] In this application, X-ray irradiation of the tumor site generates singlet oxygen, which causes the singlet oxygen sensitive bond to break, releasing an immunomodulator in response.

[0026] This invention utilizes the EPR response of nanomaterials to enrich tumors. The principle stems from the unique structure of tumor blood vessels, which leads to the selective accumulation and prolonged retention of macromolecules in tumor tissue. The high permeability, abnormal structure, poor lymphatic drainage, and increased interstitial fluid pressure of tumor blood vessels enable macromolecular drugs such as nanoparticles and liposomes to reach the tumor site and accumulate effectively through blood circulation.

[0027] The tumor treatment drug is a tumor immune microenvironment modulator. Further, the drug is an injectable or oral formulation; wherein the injectable formulation is an intravenous or intraperitoneal injection.

[0028] The oral preparations are powders, pills, tablets, granules, capsules, solutions, emulsions, or suspensions.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] 1. This invention provides a responsive nanomedicine for regulating the tumor immune microenvironment, specifically relating to a bacterial outer membrane vesicle that enhances the radiotherapy effect through X-ray-activated tumor immune metabolic regulation and pyroptosis synergistic effect.

[0031] 2. Bacterial outer membrane vesicles with synergistic effects on X-ray-sensitive tumor immunometabolism regulation and pyroptosis effectively deliver lipopolysaccharide (LPS) and the small molecule immunometabolism inhibitor NLG-919 into cells, inducing pyroptosis and immune regulation, improving the tumor microenvironment, and enhancing the effect of radiotherapy.

[0032] 3. Bacterial outer membrane vesicles that enhance the radiotherapy effect through synergistic effects of X-ray-sensitive tumor immune metabolism regulation and pyroptosis, loaded with PEG, effectively prolong the circulation time of OMVs in vivo. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 The diagram below shows the preparation method of the responsively modulating tumor immune microenvironment nanomedicine in Example 1. In this diagram, a is a schematic diagram of the preparation of PLG@OMVs-VP, b is a schematic diagram of the mechanism by which PLG@OMVs-VP releases the small molecule immunometabolism inhibitor NLG-919, and c is a schematic diagram of the effect of PLG@OMVs-VP in vivo.

[0035] Figure 2 The image shows transmission electron microscopy (TEM) images of OMVs and PLG@OMVs-VP from the experiment in Example 1.

[0036] Figure 3 The images show the DLS diagram and Zeta potential diagram of PLG@OMVs-VP in Example 1.

[0037] Figure 4 This is the UV spectrum of PLG@OMVs-VP in Example 1.

[0038] Figure 5 The diagrams shown are from the CCK8 cell killing experiment in Example 2. a is a cell activity diagram, and b is a flow cytometry diagram of tumor cell apoptosis after treatment with PLG@OMVs-VP+X-rays.

[0039] Figure 6 To verify the inhibition of solid tumor growth by PLG@OMVs-VP+X-ray in Example 3, a is a diagram of the tumor model construction, b is a curve of tumor volume change, and c is an H&E staining image of the tumor after treatment.

[0040] Figure 7The images show the enrichment of PLG@OMVs-VP within the tumor over time and the organ distribution images in Example 4. Among them, a is the in vivo fluorescence imaging at different time points after 4T1 tumor-bearing mice were injected into the tail vein with Cy5.5-OMVs, Cy5.5-OMVs-VP and Cy5.5-PLG@OMVs-VP, and b is the quantitative analysis of Cy5.5 fluorescence in the tumor site at different time points.

[0041] Figure 8 In Example 5, the experiment verified that PLG@OMVs-VP releases NLG-919 under X-ray activation to regulate amino acid metabolism.

[0042] Figure 9 The structure diagram of BMS-1166, the small molecule inhibitor in Example 6.

[0043] Figure 10 This is a structural diagram of puromycin in Example 6. Detailed Implementation

[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] Table 1 Main experimental reagents

[0046]

[0047] Example 1: Preparation of Nanomedicines that Responsively Modulate the Tumor Immune Microenvironment

[0048] like Figure 1 As shown, it includes the following steps:

[0049] Step 1: Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3:

[0050] (1) Prepare M9 medium containing Ac4GalNAz (500 nM). M9 medium is prepared by adding MgSO4 (5 mM), D-glucose (0.2%), vitamin B1 (0.5%), Casamino Acids (0.2%) and CaCl2 (1 mM) to 1×M9 stock solution containing Na2HPO4, KH2PO4 and NH4Cl.

[0051] (2) After Escherichia coli is cultured in the culture medium prepared in (1), it is centrifuged at 10000×g and 4℃ for 10min to remove whole Escherichia coli and large bacterial fragments.

[0052] (3) Filter the supernatant obtained in (2) through a 0.45 μm aqueous filter membrane to remove cell debris and larger bacterial secretions;

[0053] (4) The supernatant obtained in (3) was concentrated by ultrafiltration using a 15 mL, 100 kDa ultrafiltration tube, and the upper retentate solution was preserved.

[0054] (5) Centrifuge the solution obtained in (4) at 150000×g and 4℃ for 2h in an ultra-high speed centrifuge to remove small bacterial fragments, large vesicles, etc., leaving small bacterial vesicles, which is OMVs-N3.

[0055] (6) The OMVs-N3 obtained in (5) was resuspended in PBS and quantified by BCA protein concentration assay.

[0056] Step 2: The small molecule immunosuppressant NLG-919 is linked to diphenylcyclooctyne-polyethylene glycol-amino via a singlet oxygen-sensitive linker 2,2-[propane-2,2-dimethylbis(thio)]diacetic acid. The diphenylcyclooctyne-polyethylene glycol-amino is then linked to azide-modified bacterial outer membrane vesicles via click chemistry. The surface of the bacterial outer membrane vesicles is chemically modified with X-ray-sensitive drugs. This completes the preparation of the responsive tumor immune microenvironment modulating nanomedicine PLG@OMVs-VP.

[0057] (1) Take 500 μg of OMVs-N3 obtained in step one, add 75 μL of DBCO-PEG5k-NH2 (1 mM stock solution) and 25 μL of DMSO, and then add PBS to make up to 500 μL. Through the click chemical reaction between N3 and DBCO groups (37℃, 2 h), NH2-PEG@OMV is formed. Excess DBCO-PEG 5k -NH2 was removed by passing it through a 100kDa ultrafiltration tube at 5000×g for 3 minutes.

[0058] (2) 1 O2 Linker (1 mg, 5 mM) and EDC (3.1 mg, 20 mM) were added to PBS (1 mL) and stirred vigorously at room temperature in the dark for 1 h. Then, NH2-PEG@OMVs and NHS (1.2 mg, 10 mM) obtained in (1) were added and stirred at room temperature in the dark for 24 h. The product was collected by centrifugation at 120,000 × g for 90 min. The precipitate was collected and the residual precipitate was removed. 1 O2 Linker.

[0059] (3) Collect the product obtained in (2) and redisperse it together with EDC (3.1 mg, 20 mM) in PBS (1 mL), stir at room temperature in the dark for 1 h, then add NLG-919 (1 mg, 3.5 mM) and NHS (1.2 mg, 10 mM), stir at room temperature in the dark for 24 h, collect the product by centrifugation at 120000×g for 90 min, and obtain PLG@OMVs;

[0060] (4) Take 10 μL Verteporfin (7.1 μg / μL), 40 μL DMSO, 250 μg NHS and 375 μg EDC·HCl, and stir vigorously for 2 h in the dark. Gradually add the above solution to 200 μL of the product PLG@OMVs (2.5 μg / μL) obtained in (3), and stir vigorously for 1 h. Centrifuge at 120000×g for 90 min, wash 3 times with PBS, and resuspend in PBS to obtain the final product PLG@OMVs-VP.

[0061] The microstructure of OMVs and PLG@OMVs-VP was characterized by transmission electron microscopy (TEM). First, equal volumes of 4% paraformaldehyde were added to 20 μg of OMVs or PLG@OMVs-VP solution and fixed at room temperature for 15 min. Then, 100 μL of PBS was dropped onto a Parafilm sealing film. The copper mesh (film side down) was transferred to the PBS drop for washing using clean tweezers. Next, TEM samples were prepared using phosphotungstic acid negative staining, and the copper mesh was transferred to 50 μL of 2% phosphotungstic acid drop for 2 min. The above steps were repeated, washing twice with PBS. Morphology analysis of the completely dried OMVs and PLG@OMVs-VP was performed using TEM at an accelerating voltage of 100 kV. The results are shown below. Figure 2 As shown, transmission electron microscopy (TEM) images indicate that PLG@OMVs-VP retains the spherical structure of OMVs, with a particle size of approximately 120 nm. 10 μg of OMVs and PLG@OMVs-VP were diluted with PBS, and the particle size and Zeta potential of OMVs and PLG@OMVs-VP were measured using dynamic light scattering. The results are shown below. Figure 3 As shown, DLS results indicate that the hydrated particle size of PLG@OMVs-VP is 150 nm. Zeta potential results show that surface modification with VP makes the potential of OMVs more negative. Further modification with PEG, singlet oxygen-sensitive linkages, and NLG-919 results in a more negative Zeta potential for PLG@OMVs-VP compared to OMVs-VP. UV-Vis spectroscopy was performed on PLG@OMVs-VP, NLG-919 standard solution, Verteporfin standard solution, and OMVs to verify the characteristic peaks. The results are shown below. Figure 4As shown, the UV-Vis absorption spectrum of PLG@OMVs-VP shows characteristic peaks of VP and NLG-919 at 700 nm and 274 nm, respectively, indicating that OMVs were successfully modified with VP and NLG-919.

[0062] Example 2: Tumor cell killing experiment using the prepared vesicles.

[0063] 4T1 cancer cells were treated at a rate of 1×10 4 Cells were seeded at a density of 96% in 96 cell culture plates and cultured overnight for attachment. Cells were then treated with OMVs, OMVs-VP, and PLG@OMVs-VP (50 μg / mL, based on the equivalent dose of OMVs protein) for 2 hours; untreated cells served as a control. Afterward, cells were irradiated with X-ray (6 Gy) and cultured for another 24 hours. Then, each well was replaced with 100 μL of DMEM medium containing 10 μL of CCK-8 enhancement solution and incubated for 1 hour. The absorbance of each well at 450 nm (A) was measured using a microplate reader. Results are shown below. Figure 5 As shown in a. 4T1 cells (1×10⁴) 5 Cells were seeded per well in 12-well cell culture plates and incubated for 24 h. Then, 4T1 cancer cells were treated with PBS, OMVs, OMVs-VP, or PLG@OMVs-VP (50 μg / mL, equivalent dose based on OMV protein) for 2 h, followed by X-ray irradiation (6 Gy) and continued culture for 24 h. Cells were collected, stained with Annexin V and PI, and apoptosis was analyzed by flow cytometry. Results are shown below. Figure 5 As shown in b, the results of CCK-8 assay, Annexin V and PI double staining jointly indicate that PLG@OMVs-VP group induced higher tumor cell death under X-ray irradiation.

[0064] Example 3: Solid tumor suppression experiment using the prepared bacterial outer membrane vesicles.

[0065] 4T1 cells (1×10) 6 A tumor model was constructed by subcutaneous inoculation of cells (per mouse) into the right thigh of 6-week-old female BALB / c mice. Figure 6 As shown in a, when the tumor volume reaches 50 mm 3Mice were randomly assigned to 8 groups (n=5) receiving PBS (100 μL), OMVs (2.5 mg / kg), OMVs-VP (2.5 mg / kg), PLG@OMVs-VP (5 mg / kg), X-ray (6 Gy), OMVs+X-ray (2.5 mg / kg, 6 Gy), OMVs-VP+X-ray (2.5 mg / kg, 6 Gy), and PLG@OMVs-VP+X-ray (5 mg / kg, 6 Gy). Equivalent doses were based on OMV protein. Each group received three systemic administrations on days 0, 3, and 6. X-ray irradiation was administered 16 hours after administration of PBS, OMVs, OMVs-VP, or PLG@OMVs-VP. Mouse body weight and tumor volume were monitored daily with calipers over 25 days. Tumor volume was calculated using the following formula:

[0066] Tumor volume (V, mm) 3 = length × width × width / 2.

[0067] The results are as follows Figure 6 As shown in b, the PLG@OMVs-VP+X-ray group exhibited a more significant anti-tumor effect. After treatment, mouse tumors were harvested and photographed. Tumor sections were subjected to H&E staining and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining, and observed under a fluorescence microscope. Figure 6 As shown in Figure c, the results indicate that the tumor growth inhibition rate in the PLG@OMVs-VP+X-ray group was higher than that in the other control groups, suggesting that the regulation of the IDO pathway and the induction of pyroptosis can enhance the radiotherapy effect. Hematoxylin and eosin (H&E) staining of tumor tissue further confirmed that the tumor tissue in the PLG@OMVs-VP+X-ray group showed severe nuclear division and necrosis.

[0068] Example 4: Verification / proof experiment on the synergistic effect of X-ray-sensitive tumor immune metabolism regulation and pyroptosis to enhance radiotherapy efficacy of bacterial outer membrane vesicles loaded with PEG to effectively prolong the circulation time of OMVs in vivo.

[0069] To investigate the biodistribution of PLG@OMVs-VP in vivo, we labeled Cy5.5 on the bacterial outer membrane vesicles. The obtained Cy5.5-OMVs, Cy5.5-OMVs-VP, and Cy5.5-PLG@OMVs-VP were intravenously injected into 4T1 tumor-bearing BALB / c mice. Small animal fluorescence imaging was performed at 0, 2, 4, 6, 8, 16, 24, and 36 hours. The imaging results are shown below. Figure 7As shown, the Cy5.5-OMVs and Cy5.5-OMVs-VP groups exhibited the strongest fluorescence at the tumor site at 4 hours, while the Cy5.5-PLG@OMVs-VP group showed the maximum Cy5.5 fluorescence signal at the tumor site at 16 hours. This indicates that PEG modification on the surface of OMVs can effectively prolong their circulation time in vivo.

[0070] Example 5,

[0071] To verify the inhibitory effect of PLG@OMVs-VP on IDO activity, 4T1 cancer cells were first stimulated with interferon to overexpress IDO. Intracellular metabolism was then detected by measuring the Trp / Kyn content in the cell culture medium after different treatments using HPLC. Compared to the control group, to verify the inhibitory effect of the material on IDO activity, 4T1 cancer cells were first stimulated with interferon to overexpress IDO. Intracellular metabolism was then detected by measuring the Trp / Kyn content in the cell culture medium after different treatments using a colorimetric method. Figure 8 As shown, compared with the control group, the Trp / Kyn content in the PLG@OMVs-VP group decreased significantly after X-ray irradiation due to the inhibition of IDO by the released NLG-919.

[0072] In summary, the bacterial outer membrane vesicles of the present invention, which enhance radiotherapy efficacy through X-ray-activated tumor immune metabolic regulation and pyroptosis synergistic effect, have at least the following advantages:

[0073] (1) This invention relates to X-ray activation of Verteporfin to generate singlet oxygen, sensitive linkage bond breakage, release of small molecule inhibitor while LPS exposure, to achieve responsive release;

[0074] (2) The present invention delivers immune small molecule inhibitors and pyroptosis-inducing endotoxin LPS simultaneously, combined with X-ray radiotherapy for tumors, and NLG-919 can improve the side effects of radiotherapy, such as regulating the imbalance of Kyn / Trp amino acid ratio caused by radiotherapy, to achieve a therapeutic effect of 1+1+1>3.

[0075] Example 5: Other X-ray sensitive drugs used to prepare X-ray activated tumor immune microenvironment modulating nanomedicines

[0076] Besides Verteporfin, other porphyrin-based X-ray sensitive drugs, such as Ce6, can also react with membrane proteins and can replace Verteporfin in the preparation of X-ray activated tumor immune microenvironment modulating nanomedicines.

[0077] Example 6: Preparation of X-ray-activated tumor immune microenvironment modulating nanomedicines using other small molecule inhibitors

[0078] In one embodiment, the small molecule inhibitor is BMS-1166, with the structure as follows: Figure 9 As shown.

[0079] BMS-1166, another immunomodulator, can effectively block the binding of programmed death-ligand 1 (PD-L1) / programmed death 1 (PD1), thereby promoting the activity and function of T cells (Angew. Chem. Int. Ed. 2023, 62, e202305200).

[0080] In one embodiment, the small molecule inhibitor is puromycin, with the structure as follows: Figure 10 As shown.

[0081] Puromycin is an aminonucleoside that can terminate protein synthesis during translation to induce cell death (Adv. Mater. 2019, 31, 1905091).

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An X-ray activated bacterial outer membrane vesicle for synergistic immunometabolic modulation and pyroptosis of tumors, characterized in that, The azido-modified bacterial outer membrane vesicles, diphenylcyclooctyne-polyethylene glycol-amino, singlet oxygen-sensitive linker 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, immunometabolic modulators, and X-ray sensitive drugs; The immunometabolic modulators are connected to the diphenylcyclooctyne-polyethylene glycol-amino through a singlet oxygen-sensitive linker, and the diphenylcyclooctyne-polyethylene glycol-amino is connected to the azido-modified bacterial outer membrane vesicles through a click chemistry reaction; The X-ray sensitive drugs are chemically modified on the surface of the bacterial outer membrane vesicles; The immunometabolic modulators are NLG-919; The X-ray sensitive drugs are porphyrin X-ray sensitive drugs.

2. The X-ray activated tumour immunometabolic modulation and pyroptosis synergy bacterial outer membrane vesicles according to claim 1, characterized in that, The X-ray sensitive drugs are selected from Verteporfin or Ce6.

3. The X-ray activated immuno-metabolic modulation and pyroptosis synergy bacterial outer membrane vesicles of claim 1, wherein, The outer membrane vesicles have a particle size of 100-150 nm after modification of the X-ray sensitive drugs and the immunometabolic modulators.

4. The X-ray activated immuno-metabolic modulation and pyroptosis synergy bacterial outer membrane vesicles of claim 1, wherein, The bacterial outer membrane vesicles are BL21(DE3) Escherichia coli outer membrane vesicles cultured in an Ac4GalNAz-containing low-sugar M9 medium.

5. The method of claim 1, wherein the X-ray activated tumor immunometabolic modulation and pyroptosis synergistic bacterial outer membrane vesicles are prepared by, The method comprises the following steps: Step one, preparation of azido-modified bacterial outer membrane vesicles OMVs-N3: Step two, connection of the immunometabolic modulators to the diphenylcyclooctyne-polyethylene glycol-amino through the singlet oxygen-sensitive linker 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, connection of the diphenylcyclooctyne-polyethylene glycol-amino to the azido-modified bacterial outer membrane vesicles through a click chemistry reaction, chemical modification of the X-ray sensitive drugs on the surface of the bacterial outer membrane vesicles, and preparation of the responsive tumor immune microenvironment regulating nanodrug PLG@OMVs-VP.

6. Use of the X-ray activated tumor immunometabolic regulation and pyroptosis synergistic bacterial outer membrane vesicles according to claim 1 in the preparation of a tumor treatment drug.

7. Use according to claim 6, characterized in that, Irradiation of the tumor site with X-rays generates singlet oxygen, which causes the singlet oxygen-sensitive bond to break, and the immunometabolic modulators are released in response.

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