X-ray activated bacterial outer membrane vesicle with synergistic effect of tumor immunometabolism regulation and pyroptosis as well as preparation method and application of bacterial outer membrane vesicle

Abstract

The invention discloses a bacterial outer membrane vesicle with a synergistic effect of X-ray activated tumor immunometabolism regulation and pyroptosis as well as a preparation method and application of the bacterial outer membrane vesicle, and belongs to the technical field of biology. An azide group is introduced to the surface of the bacterial outer membrane vesicle through a metabolic sugar engineering technology, PEG, a singlet oxygen sensitive connecting bond and an immunometabolism regulator are sequentially modified on the surface of the bacterial outer membrane vesicle through a click chemical reaction, and an X-ray sensitive drug is further modified on the bacterial outer membrane vesicle. The tumor site is irradiated with X rays to generate singlet oxygen, so that a singlet oxygen sensitive bond is broken, an IDO inhibitor is responsively released, T cell inhibition induced by IDO high expression after radiotherapy is effectively blocked, strong T cell response is recovered, a tumor immunosuppression microenvironment caused by radiotherapy is reversed, and the tumor immunosuppression effect is improved. Meanwhile, lipopolysaccharide on the outer membrane vesicles of the bacteria induces pyroptosis of tumor cells, further activates immune response of the organism and enhances the radiotherapy effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology and biomedicine, and specifically relates to a bacterial outer membrane vesicle with X-ray activated tumor immune metabolism regulation and pyroptosis synergistic effect, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Radiotherapy is a treatment method that mainly uses high-energy rays to kill tumor cells. Although radiotherapy has been successfully used in the clinical treatment of various tumors, there are still some challenges. For example, radiotherapy will increase the expression of proteins related to inhibitory immune checkpoints, such as programmed death receptor ligand 1 (PD-L1) and indoleamine-2,3-dioxygenase (IDO), prompting tumor cells to escape the attack of the immune system, highly express regulatory T cells (Tregs), limit the infiltration and function of T cells, and may polarize macrophages from anti-tumor M1 type to pro-tumor M2 type, thereby negatively regulating anti-tumor immunity, resulting in the suppression of the tumor immune microenvironment, and limiting the effect of tumor radiotherapy. Therefore, there is an urgent need to develop a strategy that can reverse the tumor immunosuppressive microenvironment caused by radiotherapy and activate the anti-tumor immune response to enhance the effect of radiotherapy.

[0004] Immunometabolism therapy is a new type of tumor treatment method. It regulates the metabolic pathways of tumor cells and enhances the immune system's ability to recognize and kill tumor cells, thereby producing a therapeutic effect. Currently, small molecule inhibitors, such as IDO enzyme inhibitors (NLG-919), are commonly used immunometabolism therapy drugs. Due to the overexpression of IDO enzyme in tumor cells, it can catalyze the degradation of tryptophan (Trp) to kynurenine (Kyn), 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 the activity of IDO enzyme in tumor cells, thereby reversing the immunosuppressive microenvironment. However, such small molecule inhibitors often have insufficient targeting and are prone to toxic side effects on normal cells, limiting their further application. Therefore, it is necessary to develop a responsive immune microenvironment regulating nanomedicine that can release immune metabolism regulating drugs at a fixed point at the tumor site through X-ray irradiation, avoid killing normal tissues, and effectively reverse the immunosuppressive microenvironment caused by radiotherapy.

[0005] Pyroptosis is a newly discovered programmed cell death. The non-classical pyroptosis pathway mediated by lipopolysaccharide (LPS) refers to the fact that LPS can bind to and activate Caspase-11 or Caspase-4 / 5, further cleave GSDM family proteins, and the N-terminal fragment of the cleavage product GSDM family protein can make holes in the cell membrane, increase the intracellular osmotic pressure, cause cell swelling and rupture, release cell contents, and trigger cell death.

[0006] Gram-negative bacterial outer membrane vesicles (OMVs) are spherical vesicles with a size of 20 to 250 nm secreted by bacteria. They contain a large number of components from the outer membrane and periplasm of the parent bacteria, including LPS, proteins, lipids, nucleic acids, etc. Therefore, OMVs can be used as a natural carrier to deliver LPS into tumor cells, triggering tumor cell pyroptosis through the LPS-mediated non-classical pathway. The large amount of released tumor cell contents can act as antigens to activate the immune system. Combining the pyroptosis caused by OMVs with X-ray responsive immune metabolic regulation is expected to synergistically regulate the immune microenvironment, activate anti-tumor immune responses, and enhance the effect of radiotherapy. However, how to implement this pathway and its effect have not yet been explored. Summary of the invention

[0007] In view of the above limitations, the present invention provides a bacterial outer membrane vesicle with X-ray activated tumor immune metabolism regulation and pyroptosis synergistic effect and a preparation method thereof.

[0008] As one aspect of the present invention, there is provided a bacterial outer membrane vesicle that can synergistically enhance the radiotherapy effect by regulating tumor immunometabolism with X-ray activation and pyroptosis, comprising a bacterial outer membrane vesicle modified with an azide group, diphenylcyclooctyne-polyethylene glycol-amino, a singlet oxygen-sensitive linker 2.2-[propane-2,2-diylbis(sulfide)]diacetic acid, an immunometabolism regulator, and an X-ray-sensitive drug;

[0009] The immunometabolism regulator is connected to polyethylene glycol (PEG) via a singlet oxygen-sensitive linker, and the polyethylene glycol modified with a diphenylcyclooctyne (DBCO) group is modified with an azide-modified bacterial outer membrane vesicle via a click chemistry reaction;

[0010] The X-ray sensitive drug is chemically modified in bacterial outer membrane vesicles.

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

[0012] Further, 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 metabolic enzyme inhibitor is further preferably NLG-919.

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

[0017] The outer membrane vesicle modified drug, X-ray sensitive drug and small molecule inhibitor have a particle size of 100-150 nm.

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

[0019] The loaded X-ray-sensitive drug Verteporfin can generate singlet oxygen under X-ray excitation, inducing 1 O2Linker (2.2-[propane-2,2-diylbis(sulfide)]diacetic acid) breaks and the loaded small molecule inhibitor is released. NLG-919 is an IDO inhibitor that effectively regulates IDO overexpression caused by radiotherapy and restores strong T cell responses.

[0020] As another aspect of the present invention, there is provided a method for preparing bacterial outer membrane vesicles that synergistically enhance the radiotherapy effect by X-ray-activated tumor immune metabolism regulation and pyroptosis, 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 connected to diphenylcyclooctyne-polyethylene glycol-amino through the singlet oxygen-sensitive linker 2.2-[propane-2,2-diylbis(sulfide)]diacetic acid, and the diphenylcyclooctyne-polyethylene glycol-amino is connected to the bacterial outer membrane vesicles modified with azide groups through click chemistry. The surface of the bacterial outer membrane vesicles is chemically modified with X-ray sensitive drugs to prepare the responsive nanodrug PLG@OMVs-VP that regulates the tumor immune microenvironment.

[0023] The present invention introduces an azide group on the surface of bacterial outer membrane vesicles through metabolic sugar engineering technology, and sequentially modifies the surface of bacterial outer membrane vesicles with PEG, singlet oxygen sensitive linker, and small molecule immunometabolism regulator (IDO inhibitor) through click chemistry reaction, and further modifies the bacterial outer membrane vesicles with X-ray sensitive drugs (Verteporfin). By irradiating the tumor site with X-rays, the Verteporfin on the bacterial outer membrane vesicles absorbs the X-rays to produce singlet oxygen, resulting in the breaking of the singlet oxygen sensitive bond, and responsively releasing the IDO inhibitor, effectively blocking the T cell inhibition induced by high expression of IDO after radiotherapy and restoring a strong T cell response, reversing the tumor immunosuppressive microenvironment caused by radiotherapy, and at the same time, the lipopolysaccharide (LPS) on the bacterial outer membrane vesicles induces tumor cell pyroptosis, further activating the body's immune response and enhancing the radiotherapy effect.

[0024] As a third aspect of the present invention, it provides the use of the bacterial outer membrane vesicles that synergistically enhance the radiotherapy effect by X-ray-activated tumor immune metabolism regulation and pyroptosis in the preparation of tumor therapeutic drugs.

[0025] In the application, irradiating the tumor site with X-rays generates singlet oxygen, which causes the singlet oxygen sensitive bonds to break, thereby responsively releasing the immunometabolism regulator.

[0026] The present invention utilizes the EPR response of nanomaterials to enrich tumors. The principle is derived from the unique structure of tumor blood vessels, which leads to the selective accumulation and long-term retention of macromolecular substances in tumor tissues. The high permeability, structural abnormalities, poor lymphatic return, and increased interstitial fluid pressure of tumor blood vessels enable macromolecular drugs such as nanoparticles and liposomes to reach the tumor site through blood circulation and accumulate effectively.

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

[0028] The oral preparation is a powder, a pill, a tablet, a granule, a capsule, a solution, an emulsion or a suspension.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention provides a nanomedicine that responsively regulates the tumor immune microenvironment, specifically a bacterial outer membrane vesicle that synergistically enhances the radiotherapy effect by X-ray-activated tumor immune metabolism regulation and pyroptosis.

[0031] 2. X-ray-sensitive tumor immunometabolism regulation and pyroptosis synergistic bacterial outer membrane vesicles can effectively deliver lipopolysaccharide (LPS) and small molecule immunometabolism inhibitor NLG-919 into cells, induce cell pyroptosis and immunoregulation, improve tumor microenvironment, and enhance the effect of radiotherapy.

[0032] 3. X-ray-sensitive tumor immune metabolism regulation and pyroptosis synergistically enhance the effect of radiotherapy in bacterial outer membrane vesicles. The loaded PEG effectively prolongs the circulation time of OMVs in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 This is a diagram of the method for preparing nanomedicines that responsively regulate the tumor immune microenvironment in Example 1, wherein a is a schematic diagram of the preparation of PLG@OMVs-VP, b is the mechanism of PLG@OMVs-VP releasing the small molecule immune metabolic inhibitor NLG-919, and c is a schematic diagram of the effect of PLG@OMVs-VP in vivo.

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

[0036] Figure 3 These are the DLS and Zeta potential diagrams of PLG@OMVs-VP in the experiment of Example 1.

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

[0038] Figure 5 These are the experimental diagrams of CCK8 cell killing in the experiment of Example 2, a is the cell activity diagram, and b is the apoptosis flow cytometer diagram of tumor cells after the action of PLG@OMVs-VP+X-rays.

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

[0040] Figure 7These are the enrichment images and organ distribution images of PLG@OMVs-VP in the tumor over time in the experiment of Example 4, where a is the in vivo fluorescence imaging at different time points after tail vein injection of Cy5.5-OMVs, Cy5.5-OMVs-VP and Cy5.5-PLG@OMVs-VP in 4T1 tumor-bearing mice, and b is the quantitative analysis of Cy5.5 fluorescence in the tumor site at different time points.

[0041] Figure 8 This is to verify in the experiment of Example 5 that PLG@OMVs-VP releases NLG-919 under X-ray activation to regulate amino acid metabolism.

[0042] Fig. 9 The structural diagram of the small molecule inhibitor BMS-1166 in Example 6.

[0043] Fig.10 This is the structural diagram of puromycin in Example 6. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0045] Table 1 Main experimental reagents

[0046]

[0047] Example 1: Preparation of nanomedicines that responsively regulate the tumor immune microenvironment

[0048] like Figure 1 As shown, the following steps are included:

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

[0050] (1) Prepare M9 medium containing Ac4GalNAz (500 nM), which 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 culturing E. coli with the medium prepared in (1), centrifugation was performed at 10,000×g and 4° C. for 10 min to remove whole E. coli bacteria and large bacterial fragments;

[0052] (3) Filter the supernatant obtained in (2) through a 0.45 μm water 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 saved;

[0054] (5) The solution obtained in (4) was centrifuged in an ultra-high speed centrifuge at 150,000 × g and 4°C for 2 h to remove small bacterial fragments, large vesicles, etc., leaving small bacterial vesicles to obtain OMVs-N3;

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

[0056] Step 2: The small molecule immunosuppressant NLG-919 is connected to diphenylcyclooctyne-polyethylene glycol-amino via a singlet oxygen sensitive linker 2.2-[propane-2,2-diylbis(sulfide)]diacetic acid, diphenylcyclooctyne-polyethylene glycol-amino is connected to azide-modified bacterial outer membrane vesicles via click chemistry, and the surface of bacterial outer membrane vesicles is chemically modified with X-ray sensitive drugs to prepare the responsive nanodrug PLG@OMVs-VP that regulates the tumor immune microenvironment:

[0057] (1) Take 500 μg of OMVs-N3 obtained in step 1, add 75 μL DBCO-PEG5k-NH2 (mother solution is 1 mM) and 25 μL DMSO, and then add PBS to make the volume to 500 μL. Through the click chemistry reaction between N3 and DBCO group (37 ° C, 2 h), NH2-PEG@OMV is formed. The excess DBCO-PEG 5k -NH2 was removed by passing through a 100 kDa ultrafiltration tube, 5000 × g, 3 min;

[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 obtained in (1) and NHS (1.2 mg, 10 mM) 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 1 O2 Linker.

[0059] (3) The product obtained in (2) was collected and redispersed with EDC (3.1 mg, 20 mM) in PBS (1 mL), stirred at room temperature in the dark for 1 h, and then NLG-919 (1 mg, 3.5 mM) and NHS (1.2 mg, 10 mM) were added, stirred at room temperature in the dark for 24 h, and the product was collected by centrifugation at 120,000 × g for 90 min to obtain PLG@OMVs;

[0060] (4) Take 10 μL of Verteporfin (7.1 μg / uL), 40 μL of DMSO, 250 μg of NHS and 375 μg of EDC·HCl, and stir vigorously for 2 h in the dark. The above solution was gradually added dropwise to 200 μL of the product PLG@OMVs (2.5 μg / μL) obtained in (3), and stirred vigorously for 1 h. Centrifuge at 120,000×g for 90 min, wash 3 times with PBS, and resuspend in PBS to obtain the final product PLG@OMVs-VP.

[0061] The microscopic morphology of OMVs and PLG@OMVs-VP was characterized by transmission electron microscopy (TEM). First, an equal amount of 4% paraformaldehyde was added to 20μg OMVs or PLG@OMVs-VP solution and fixed at room temperature for 15 minutes. Then 100μL of PBS was dropped on the Parafilm sealing film. Use clean tweezers to transfer the copper grid (membrane side down) to the PBS drop for cleaning. Then prepare the TEM sample with phosphotungstic acid negative staining, and transfer the copper grid to 50μL 2% phosphotungstic acid drop for 2 minutes. Repeat the above steps and wash with PBS twice. At an accelerating voltage of 100kV, the morphology of completely dried OMVs and PLG@OMVs-VP was analyzed by TEM, and the results are shown in the figure. Figure 2 As shown in Figure 1, transmission electron microscopy (TEM) images show that PLG@OMVs-VP retains the spherical structure of OMVs, with a particle size of about 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 by dynamic light scattering. The results are shown in Figure 1. Figure 3 As shown, the DLS results show that the hydrated particle size of PLG@OMVs-VP is 150nm. The Zeta potential results show that the surface modification of VP makes the potential of OMVs negative. After further modification of PEG, singlet oxygen sensitive linker, and NLG-919, the Zeta potential of PLG@OMVs-VP is more negative than that of OMVs-VP. PLG@OMVs-VP, NLG-919 standard solution, Verteporfin standard solution, and OMVs were tested by UV-visible spectroscopy to verify the characteristic peaks. The results are shown in Figure 4As shown, the UV-visible absorption spectrum of PLG@OMVs-VP showed 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 was carried out using the prepared vesicles.

[0063] 4T1 cancer cells were cultured at a rate of 1×10 4 The cells were seeded into a 96-well cell culture plate at a density of 1:1 and cultured overnight for attachment. The 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, and untreated cells were used as controls. After that, the cells were irradiated with X-ray (6 Gy) and cultured for 24 hours. Each well was replaced with 100 μL DMEM medium containing 10 μL CCK-8 enhancement solution and incubated for 1 hour. The absorbance of each well at 450 nm (A) was measured using a microplate reader, and the results are shown in Figure 2. Figure 5 a. 4T1 cells (1×10 5 Cells / well) were inoculated in 12-well cell culture plates and incubated for 24 hours. 4T1 cancer cells were then treated with PBS, OMVs, OMVs-VP or PLG@OMVs-VP (50 μg / mL, based on the equivalent dose of OMVs protein) for 2 hours, after which the cells were irradiated with X-rays (6 Gy) and cultured for another 24 hours. The cells were collected, stained with Annexin V and PI, and cell apoptosis was analyzed by flow cytometry. The results are shown in Figure 5 As shown in b, the results of CCK-8 experiment, Annexin V and PI double staining detection jointly showed that the PLG@OMVs-VP group caused higher tumor cell death under X-ray radiation.

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

[0065] 4T1 cells (1×10 6 The tumor model was established by inoculating 6-week-old female BALB / c mice subcutaneously on the right thigh. Figure 6 As shown in a, when the tumor volume reaches 50 mm 3At the time of the experiment, mice were randomly divided into 8 groups (n=5), including 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). The equivalent dose was based on OMVs protein. Each group received three systemic administrations on days 0, 3 and 6. X-ray irradiation was performed 16 hours after administration of PBS, OMVs, OMVs-VP or PLG@OMVs-VP. The weight and tumor volume of mice were monitored daily with calipers for 25 days. The tumor volume was calculated according to 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 showed a more significant anti-tumor effect. After the treatment, the mouse tumors were taken and photographed. The tumor sections were stained with H&E and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and observed under a fluorescence microscope. Figure 6 As shown in c, the results showed that the tumor growth inhibition rate in the PLG@OMVs-VP+X-ray group was higher than that in other control groups, indicating 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 tissues further confirmed that the tumor tissues in the PLG@OMVs-VP+X-ray group showed severe nuclear division and necrosis.

[0068] Example 4: X-ray-sensitive tumor immunometabolism regulation and pyroptosis synergistically enhance the effect of radiotherapy in bacterial outer membrane vesicles, and the verification / confirmation experiment that the loaded PEG effectively prolongs the circulation time of OMVs in vivo

[0069] To study the biodistribution of PLG@OMVs-VP in vivo, we labeled Cy5.5 on the bacterial outer membrane vesicle membrane, injected the obtained Cy5.5-OMVs, Cy5.5-OMVs-VP and Cy5.5-PLG@OMVs-VP intravenously into 4T1 tumor-bearing BALB / c mice, and observed the mice by small animal fluorescence imaging at 0, 2, 4, 6, 8, 16, 24, and 36 hours. The imaging results are shown in Figure 2. Figure 7As shown in the figure, the fluorescence of the tumor site was the strongest in the Cy5.5-OMVs and Cy5.5-OMVs-VP groups at 4 hours, and the Cy5.5 fluorescence signal of the tumor site reached the maximum at 16 hours in the Cy5.5-PLG@OMVs-VP group, indicating that PEG modification on the surface of OMVs can effectively prolong its circulation time in the body.

[0070] Embodiment 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, and then the intracellular metabolism was detected by measuring the Trp / Kyn content in the cell culture medium after different treatments using the HPLC method. Compared with 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, and then the intracellular metabolism was detected by measuring the Trp / Kyn content in the cell culture medium after different treatments using the 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 that synergistically enhance the radiotherapy effect by X-ray-activated tumor immune metabolism regulation and pyroptosis have at least the following advantages:

[0073] (1) The present invention involves X-ray activation of Verteporfin to generate singlet oxygen, breaking sensitive connecting bonds, releasing small molecule inhibitors and exposing LPS to achieve responsive release;

[0074] (2) The present invention simultaneously delivers immune small molecule inhibitors and endotoxin LPS that induces pyroptosis, combined with X-ray radiotherapy to treat 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 are used to prepare X-ray activated tumor immune microenvironment regulating nanomedicines

[0076] In addition to Verteporfin, other porphyrin X-ray sensitive drugs, such as Ce6, can also react with membrane proteins and can replace Verteporfin to prepare X-ray activated tumor immune microenvironment regulating nanomedicines.

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

[0078] In one embodiment, the small molecule inhibitor is BMS-1166, with a structure as shown in Fig. 9 shown.

[0079] BMS-1166, as 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 a structure such as Fig.10 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 only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bacterial outer membrane vesicle with X-ray-activated tumor immune metabolism regulation and pyroptosis synergistic effect, characterized in that: Including bacterial outer membrane vesicles modified with azide groups, diphenylcyclooctyne-polyethylene glycol-amino, singlet oxygen sensitive linker 2.2-[propane-2,2-diylbis(sulfide)]diacetic acid, immunometabolism regulators, and X-ray sensitive drugs; The immunometabolism regulator is connected to polyethylene glycol via a singlet oxygen-sensitive linker, and the polyethylene glycol modified with a diphenylcyclooctyne group is modified with an azide-modified bacterial outer membrane vesicle via a click chemistry reaction; The X-ray sensitive drug is chemically modified in bacterial outer membrane vesicles.

2. The X-ray activated bacterial outer membrane vesicles with synergistic effects of tumor immune metabolism regulation and pyroptosis according to claim 1, characterized in that: The immunometabolism regulator is selected from metabolic enzyme inhibitors, PD-1 / PD-L1 interaction inhibitors and protein synthesis inhibitors.

3. The X-ray activated tumor immune metabolism regulation and pyroptosis synergistic bacterial outer membrane vesicles according to claim 2, characterized in that: The metabolic enzyme inhibitor is an IDO enzyme inhibitor, preferably NLG-919; The PD-1 / PD-L1 interaction inhibitor is BMS-1166; The protein synthesis inhibitor is puromycin.

4. The X-ray activated bacterial outer membrane vesicles with synergistic effects of tumor immune metabolism regulation and pyroptosis according to claim 1, characterized in that: The X-ray sensitive drug is a porphyrin X-ray sensitive drug.

5. The X-ray activated bacterial outer membrane vesicles with synergistic effects of tumor immune metabolism regulation and pyroptosis according to claim 1, characterized in that: The X-ray sensitive drug is selected from Verteporfin or Ce6.

6. The X-ray activated bacterial outer membrane vesicles with synergistic effects of tumor immune metabolism regulation and pyroptosis according to claim 1, characterized in that: The outer membrane vesicle modified drug, X-ray sensitive drug and immune metabolism regulator have a particle size of 100-150nm.

7. The X-ray activated bacterial outer membrane vesicles with synergistic effects of tumor immune metabolism regulation and pyroptosis according to claim 1, characterized in that: The bacterial outer membrane vesicles are outer membrane vesicles of BL21 (DE3) Escherichia coli cultured in a low-sugar M9 medium containing 1,3,4,6-tetra-oxy-acetyl-2-[(azidoacetyl)amino]-2-deoxy-β-D-pyranose.

8. The method for preparing X-ray activated tumor immune metabolism regulation and pyroptosis synergistic bacterial outer membrane vesicles according to claim 1, characterized in that: The steps include: Step 1: Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3: Step 2. The immune metabolic regulator is connected to diphenylcyclooctyne-polyethylene glycol-amino through the singlet oxygen-sensitive linker 2.2-[propane-2,2-diylbis(sulfide)]diacetic acid, and the diphenylcyclooctyne-polyethylene glycol-amino is connected to the bacterial outer membrane vesicles modified with azide groups through 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 regulating nanodrug PLG@OMVs-VP.

9. Use of the bacterial outer membrane vesicles with X-ray-activated tumor immune metabolism regulation and pyroptosis synergistic effects according to claim 1 in the preparation of tumor therapeutic drugs.

10. The use according to claim 9, characterized in that: Irradiating the tumor site with X-rays produces singlet oxygen, which causes the singlet oxygen-sensitive bonds to break and responsively release immune metabolic regulators.

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