Bacterial outer membrane vesicle with targeting function, GSH response type activated STING pathway and pyroptosis effect as well as preparation method and application of bacterial outer membrane vesicle

Abstract

The invention discloses a bacterial outer membrane vesicle with a targeting function and a GSH response type STING pathway activation synergistic pyroptosis effect as well as a preparation method and application of the bacterial outer membrane vesicle, and belongs to the technical field of biological medicines. A nucleic acid aptamer targeting nucleolin is modified on a bacterial outer membrane vesicle, a nano-drug is enriched at a tumor site through an active targeting effect, PEG, a GSH response connecting bond and an STING agonist are chemically modified on the surface of the bacterial outer membrane vesicle, in a high-concentration GSH environment in a tumor, a disulfide bond is broken, the STING agonist is responsively released, and the tumor activity is improved. According to the present invention, with the application of the lipopolysaccharide in the DC cell, the STING pathway in the DC cell can be effectively activated, the antigen presentation and the DC cell maturation can be promoted, the T cell immune response can be enhanced, the lipopolysaccharide naturally existing on the bacterial outer membrane vesicle can induce the caspase-4 / 5 / 11 mediated pyroptosis, the immune response can be further activated, and the tumor treatment effect can be enhanced;

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bacterial outer membrane vesicle with a GSH-responsive activation of the STING pathway and synergistic pyroptosis with targeting function, its preparation method and 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] Gram-negative bacteria produce outer membrane vesicles (OMVs) during their normal growth. OMVs possess a unique double-membrane structure, ranging in size from approximately 20 nm to 250 nm. These vesicles are rich in various components of the parent bacterium, including DNA, lipopolysaccharide (LPS), and proteins. Compared to the bacteria themselves, OMVs exhibit better biosafety, as they lack the ability to self-replicate or replicate. In recent years, bacterial OMVs have been successfully applied to drug delivery to construct highly efficient anti-tumor drug delivery systems.

[0004] Pyroptosis, a pro-inflammatory form of programmed cell death (PCD), is characterized by cell swelling and membrane rupture, leading to the release of cellular contents and triggering a strong inflammatory response. Lipopolysaccharide (LPS), a component of OMV membranes, is an effective pathogen-associated molecular model (PAMP) capable of activating atypical inflammasomes and Caspase-11 / 4 / 5. Activated Caspase-4 / 5 / 11 directly induces GSDMD (a key pyroptosis effector) to form membrane pores, promoting IL-1β release and ultimately initiating pyroptosis. Because pyroptosis is accompanied by the release of cellular contents such as IL-1β, ATP, and HMGB1, it is considered an immunogenic form of cell death. Pyroptosis holds promise for reshaping the immunosuppressive tumor microenvironment, transforming "cold" tumors into immunogenic "hot" tumors, and improving the efficacy of immunotherapy.

[0005] Nucleic acid aptamers are short, single-stranded DNA or RNA oligonucleotides whose unique three-dimensional conformation enables them to bind to target molecules with high affinity and specificity. AS1411, a synthetic 26-nucleotide phosphodiester oligodeoxynucleotide (i.e., unmodified DNA), is thermostable and non-immunogenic. Nucleolin is a target protein of AS1411. Nucleolin is highly expressed in malignant tumor cells but almost not expressed in normal cells, making it a highly promising tumor cell targeting site.

[0006] The cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulating factor (cGAS-STING) pathway, a key signaling axis in the innate immune system, plays a central role in the development and progression of inflammatory diseases. STING proteins can serve as targets in cancer therapy and are activated by various STING agonists, such as small molecule STING agonists, cyclic dinucleotide derivatives (CDNs), and specific metal ions. After activation, STING is transported from the endoplasmic reticulum to the perinuclear region, activating TANK-binding kinase 1 (TBK1) and leading to STING phosphorylation. Phosphorylated STING recruits interferon regulatory factor 3 (IRF3), which, after phosphorylation by TBK1, enters the nucleus and activates the transcription of type I IFNs. Type I IFNs promote cross-presentation of antigens by antigen-presenting cells.

[0007] A newly discovered oral small-molecule STING agonist, MSA-2, can bind to STING with nanomolar affinity, producing superior antitumor efficacy. However, since STING is expressed in both tumor and normal tissues, intraperitoneal or intravenous injection of STING agonists can trigger a systemic inflammatory storm, and local application of STING agonists also greatly limits their application. How to reduce the off-target toxicity of STING agonists and improve therapeutic efficacy is an urgent problem to be solved.

[0008] The prior application CN119733061A provides a bacterial outer membrane vesicle with X-ray-activated tumor immune metabolic regulation and pyroptosis synergistic effect, but the questions of whether it is applicable to STING agonists and how to reduce the off-target toxicity of STING agonists remain unresolved. Summary of the Invention

[0009] To address the aforementioned limitations, this invention provides a bacterial outer membrane vesicle with a GSH-responsive activation of the STING pathway and synergistic pyroptosis, along with its preparation method and application.

[0010] As one aspect of the present invention, a bacterial outer membrane vesicle with a targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis is provided, comprising azide-modified bacterial outer membrane vesicles, a targeted nucleic acid aptamer, bis(2-hydroxyethyl) disulfide, diphenylcyclooctylene-polyethylene glycol-carboxyl group and STING agonist. The STING agonist is linked to a GSH-responsive disulfide bond via an esterification reaction. The disulfide bond is linked to a polyethylene glycol-modified diphenylcyclooctynyl group and an azide-modified bacterial outer membrane vesicle via a click chemistry reaction. The targeted nucleic acid aptamer is modified onto bacterial outer membrane vesicles via hydrophobic interactions; The STING agonist is selected from small molecule STING agonists and cyclic dinucleotide derivatives.

[0011] This application combines the pyroptosis induced by OMVs with the GSH-responsive activation of the STING pathway, thereby reducing the occurrence of immune storms and actively targeting tumor cells to further reduce the toxicity of STING agonists to normal tissues.

[0012] In some embodiments, the STING agonist is selected from small molecule STING agonists di-ABZI, cGAMP, or MSA-2. Di-ABZI is a novel non-nucleotide STING agonist formed by two symmetrical benzimidazole (ABZI) molecules linked by chemical bonds to form a dimer structure, which significantly enhances its binding affinity to the STING receptor.

[0013] The STING agonist is further preferably the non-nucleotide STING agonist MSA-2.

[0014] In this invention, the agonist itself does not contain disulfide bonds; it releases the agonist upon cleavage of the disulfide bonds in response to GSH. The STING agonist binds to the STING protein, recruiting and activating downstream kinases such as TBK1 and IRF3, promoting IFN-β expression, and thereby enhancing the killing of tumor cells by immune cells.

[0015] The targeted nucleic acid aptamer is a nucleic acid aptamer targeting nucleolin, preferably AS1411, the nucleotide sequence of which is shown in SEQ ID No: 1.

[0016] The outer membrane vesicles are modified with drugs and nucleic acid aptamers, and their particle size distribution is 120-140 nm.

[0017] The bacterial outer membrane vesicles provided by this invention can be loaded with nucleic acid aptamers that can target tumor cells with high nucleolin expression. The loaded bis(2-hydroxyethyl) disulfide can cause disulfide bond breakage in the high concentration of GSH environment in the tumor, and the loaded STING agonist is released. MSA-2 is a STING agonist that effectively activates the STING pathway in dendritic cells (DC cells), promotes antigen presentation and DC maturation, thereby triggering a strong innate immune response.

[0018] As another aspect of the present invention, a method for preparing bacterial outer membrane vesicles with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis is provided, comprising the following steps: Step 1: Preparation of targeted azide-modified bacterial outer membrane vesicles Apt-OMVs-N3: Step 2: The STING agonist is linked to a bis(2-hydroxyethyl) disulfide via a GSH-responsive linkage, and the diphenylcyclooctyne-polyethylene glycol-carboxyl group is linked to azide-modified bacterial outer membrane vesicles via a click chemistry reaction to prepare the responsive STING pathway-activated nanomedicine Apt-OMVs@PSM.

[0019] Step one includes the following steps: S101, Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3; S102, the targeted nucleic acid aptamer is used to modify the azide-modified bacterial outer membrane vesicles prepared in step S101 to obtain Apt-OMVs-N3.

[0020] In step S102, the preparation method is as follows: OMV-N3 and AS1411-chol obtained in step S101 are incubated in PBS buffer. The nucleotide sequence of AS1411 is shown in SEQ ID No: 1.

[0021] Step two includes the following steps: S201, MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark, and then bis(2-hydroxyethyl) disulfide dissolved in N,N-dimethylformamide was added. The reaction was carried out at room temperature in the dark. After the reaction was completed, the product MSA-2-linker was obtained by post-treatment.

[0022] In step S202, diphenylcyclooctyne-polyethylene glycol-carboxyl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark. The MSA-2-linker obtained in step S201 was added and stirred in the dark at room temperature. After the reaction was completed, the product DBCO-PSM was obtained by rotary evaporation.

[0023] S203: Add DBCO-PSM, the product of step S202, dissolved in an organic solvent, to Apt-OMVs-N3 prepared in step one, and initiate a chemical reaction; after the reaction is complete, centrifuge and resuspend to obtain the final product Apt-OMVs@PSM.

[0024] Preferably, in step S201, the organic solvent is dichloromethane; in step S203, the organic solvent is dimethyl sulfoxide.

[0025] Preferably, in step S201, the molar ratio of MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine, and N,N-dimethylformamide bis(2-hydroxyethyl) disulfide is 2:2~4:2~4:7~9.

[0026] Preferably, in step S201, after the reaction is completed, the mixture of ice water and ethanol is added by rotary evaporation, the pH value is adjusted to 3±0.5 by hydrochloric acid solution, centrifuged, and the precipitate is dried under vacuum to obtain the product MSA-2-linker.

[0027] As a third aspect of the invention, it provides the application of the bacterial outer membrane vesicles with the GSH-responsive activation of the STING pathway and synergistic pyroptosis with targeting function in the preparation of tumor therapeutic drugs.

[0028] The tumor treatment drug is a tumor-targeting drug.

[0029] Furthermore, the drug is an injectable or oral preparation; wherein the injectable preparation is an intravenous or intraperitoneal injection. The oral preparations are powders, pills, tablets, granules, capsules, solutions, emulsions, or suspensions.

[0030] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a nanomedicine that responsively activates the STING pathway, specifically involving a GSH-responsive bacterial outer membrane vesicle with targeting function. This invention utilizes a nucleic acid aptamer capable of targeting nucleolin protein highly expressed in tumor cells, enabling the bacterial outer membrane vesicle to possess targeting function and enhancing the accumulation of nanomedicine at the tumor site. Simultaneously, since the GSH concentration within tumor cells is 2-10 mmol / L, which is 4 times higher than the concentration within normal cells, this invention utilizes GSH-responsive drug release, ensuring that the STING agonist is only released in the presence of high levels of GSH within the tumor. This dual effect ensures the controlled release of the STING agonist, preventing it from attacking normal cells and causing systemic toxicity.

[0031] 2. This invention provides bacterial outer membrane vesicles with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis. This invention utilizes bacterial outer membrane vesicles to effectively deliver LPS and the small molecule STING agonist MSA-2 into cells. LPS induces pyroptosis in tumor cells, releasing tumor antigens, and synergistically promotes DC cell maturation with MSA-2, effectively activating T cells and enhancing the therapeutic effect on tumors. Attached Figure Description

[0032] 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.

[0033] Figure 1The diagram shows the preparation route and mechanism of action of Apt-OMVs@PSM in Example 1; where a is a schematic diagram of the preparation of Apt-OMVs@PSM and the mechanism of GSH-responsive release of the STING agonist MSA-2, and b is a schematic diagram of the action of Apt-OMVs@PSM in vivo.

[0034] Figure 2 This is a transmission electron microscope (TEM) image of Apt-OMVs@PSM in Example 2.

[0035] Figure 3 The results show the particle size and zeta potential detection results of OMVs and Apt-OMVs@PSM in Example 2; where a is the DLS diagram of OMVs and Apt-OMVs@PSM, and b is the zeta potential diagram of OMVs and Apt-OMVs@PSM.

[0036] Figure 4 The results show the identification of Apt-OMVs@PSM in Example 2; where a is the UV absorption curve and b is the high performance liquid chromatogram.

[0037] Figure 5 The results of the tumor killing experiment of Apt-OMVs@PSM in Example 3 are shown; where a is the cell killing effect and b is the apoptosis flow cytometry.

[0038] Figure 6 The images show the immunoblotting experiment and related protein expression of STING pathway activation in Example 4; where a is an electrophoresis image of p-TBK1, p-IRF3, and p-STING expression levels, and b is a statistical analysis of p-STING, p-IRF3, and p-TBK1 expression levels.

[0039] Figure 7 The figures show the BMDC maturation status and cytokine secretion levels under different treatments in Example 4; where a is a flow cytometry plot of BMDC maturation; b is a quantitative statistical analysis of the BMDC maturation ratio; and c is the IFN-β content.

[0040] Figure 8 The images show the enrichment of Apt-OMVs@PSM within the tumor over time in Example 5; where a is the in vivo fluorescence imaging at different time points after tail vein injection of Cy5.5-OMVs, Cy5.5-OMVs@PSM, Cy5.5-Rnd-OMVs@PSM, and Cy5.5-Apt-OMVs@PSM 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 9Example 6 was used to verify the inhibition of solid tumor growth by Apt-OMVs@PSM; where a is a diagram of the tumor model construction, b is a curve of tumor volume change, and c is an image of tumor H&E staining and TUNEL staining after treatment. Detailed Implementation

[0042] 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.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0044] In some embodiments of the present invention, a bacterial outer membrane vesicle with a targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis is provided, comprising an azide-modified bacterial outer membrane vesicle, a targeted nucleic acid aptamer, a bis(2-hydroxyethyl) disulfide, a diphenylcyclooctyn-polyethylene glycol-carboxyl group, and a STING agonist; the STING agonist is linked to a GSH-responsive disulfide bond via an esterification reaction, and the disulfide bond connecting the polyethylene glycol-modified diphenylcyclooctyn group to the azide-modified bacterial outer membrane vesicle is modified via a click chemistry reaction; the targeted nucleic acid aptamer is modified on the bacterial outer membrane vesicle through hydrophobic interactions; in some embodiments, the STING agonist is a small molecule STING agonist di-ABZI, cGAMP, and MSA-2.

[0045] Different agonists bind to the STING protein in different ways. For example, di-ABZI binds to the cytoplasmic ligand-binding domain (CTD) of the STING protein, maintaining its "open" conformation and thus enabling it to exhibit immune activation. cGAMP and MSA-2 both bind to the cytoplasmic ligand-binding domain (LBD) of the STING protein, but cGAMP induces the STING protein to form tetramers or higher-order oligomers, completely encapsulating cGAMP and forming a "closed" conformation. MSA-2 binds to the STING protein in a non-covalent dimer form, forming a unique "cap" structure that provides a stable binding pocket, but does not completely encapsulate the ligand. This conformational change makes the binding process of MSA-2 to the STING protein reversible; it only binds to STING after dimerization, preventing continuous activation of STING and thus avoiding autoimmune responses.

[0046] like Figure 1As shown in Figures a and b, this invention introduces azide groups onto the surface of bacterial outer membrane vesicles through sugar metabolism and modifies these vesicles with a targeted nucleic acid aptamer (AS1411). Through click chemistry, PEG, GSH-responsive disulfide bonds, and a STING agonist are sequentially modified onto the surface of the bacterial outer membrane vesicles. AS1411 can actively target tumor cells. After delivering the nanomedicine to the tumor site, the abnormally elevated GSH levels within the tumor trigger the disulfide bonds on the bacterial outer membrane vesicles to react with GSH, releasing the STING agonist MSA-2. This activates the STING pathway in dendritic cells (DCs), promoting antigen presentation and DC maturation. Simultaneously, lipopolysaccharide (LPS) on the bacterial outer membrane vesicles induces pyroptosis in tumor cells, activating the body's adaptive immune response and enhancing the therapeutic effect.

[0047] In some embodiments, the targeted nucleic acid aptamer is a nucleic acid aptamer targeting nucleolin, preferably AS1411, the nucleotide sequence of which is shown in SEQ ID No: 1.

[0048] In some embodiments, the outer membrane vesicles modified with drugs and nucleic acid aptamers have a particle size distribution of 120-140 nm.

[0049] In other embodiments of the present invention, a method for preparing bacterial outer membrane vesicles with GSH-responsive activation of the STING pathway and synergistic pyroptosis with targeting function is provided, comprising the following steps: Step 1: Preparation of targeted azide-modified bacterial outer membrane vesicles Apt-OMVs-N3: Step one includes the following steps: S101, Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3; S102, the targeted nucleic acid aptamer is used to modify the azide-modified bacterial outer membrane vesicles prepared in step S101 to obtain Apt-OMVs-N3.

[0050] In step S102, the preparation method is as follows: OMV-N3 and AS1411-chol obtained in step S101 are incubated in PBS buffer. The nucleotide sequence of AS1411 is shown in SEQ ID No: 1.

[0051] Step 2: The STING agonist is linked to a bis(2-hydroxyethyl) disulfide via a GSH-responsive linkage, and the diphenylcyclooctyne-polyethylene glycol-carboxyl group is linked to azide-modified bacterial outer membrane vesicles via a click chemistry reaction to prepare the responsive STING pathway-activated nanomedicine Apt-OMVs@PSM.

[0052] Step two includes the following steps: S201, MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark, and then bis(2-hydroxyethyl) disulfide dissolved in N,N-dimethylformamide was added. The reaction was carried out at room temperature in the dark. After the reaction was completed, the product MSA-2-linker was obtained by post-treatment.

[0053] S202, take DBCO-PEG 2K -COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark; MSA-2-linker obtained in step S201 was added, and the mixture was stirred in the dark at room temperature; after the reaction was completed, the product DBCO-PSM was obtained by rotary evaporation.

[0054] S203: Add DBCO-PSM, the product of step S202, dissolved in DMSO, to Apt-OMVs-N3 prepared in step one, and initiate a chemical reaction; after the reaction is complete, centrifuge and resuspend to obtain the final product Apt-OMVs@PSM.

[0055] In step S201, the organic solvent is dichloromethane.

[0056] In step S201, the molar ratio of MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine, and N,N-dimethylformamide bis(2-hydroxyethyl) disulfide is 2:2~4:2~4:7~9.

[0057] In other embodiments, exceeding this material ratio can lead to incomplete material reaction.

[0058] In step S201, after the reaction is complete, the mixture of ice water and ethanol is added by rotary evaporation. The pH value is adjusted to 3±0.5 with hydrochloric acid solution. After centrifugation, the precipitate is dried under vacuum to obtain the product MSA-2-linker.

[0059] In other embodiments, after the reaction was complete, the pH was adjusted to 3 to precipitate a solid. After centrifugation, the precipitate was washed with ice water. Mass spectrometry results showed that the MSA-2 reaction was relatively complete, but some unreacted MSA-2 was not removed. This demonstrates that the post-processing method affects the yield of the product.

[0060] In some embodiments of the present invention, the application of the bacterial outer membrane vesicles with the GSH-responsive activation of the STING pathway and synergistic pyroptosis with targeting function is also provided in the preparation of tumor therapeutic drugs; the tumor therapeutic drugs are tumor-targeting drugs.

[0061] In some embodiments, the drug is an injection or an oral preparation; wherein the injection is an intravenous injection or an intraperitoneal injection; and the oral preparation is a powder, pill, tablet, granule, capsule, solution, emulsion, or suspension.

[0062] The technical solution of the present invention will be described in detail with reference to specific embodiments.

[0063] Table 1 Main experimental reagents

[0064] Table 2. Oligonucleotide sequences used in this experiment.

[0065] Example 1: Preparation of Nanomedicines with Targeting Function: Response-Activated STING Pathway Synergistic Pyroptosis like Figure 1 As shown, it includes the following steps: Step 1: Preparation of azide-modified bacterial outer membrane vesicles Apt-OMVs-N3: (1) In this example, M9 medium was used, which was prepared by adding Ac4GalNAz (500 nM), 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.

[0066] (2) After Escherichia coli is cultured in the M9 medium described in (1), it is centrifuged at 10000 ×g and 4 ℃ for 10 min to remove whole Escherichia coli and large bacterial fragments. (3) Filter the supernatant obtained in (2) through a 0.45 μm aqueous filter membrane to remove cell debris and larger bacterial secretions; (4) The filtrate obtained in (3) was concentrated by ultrafiltration using a 15 mL, 100 kDa ultrafiltration tube, and the upper retentate solution was preserved. (5) Centrifuge the upper layer of the retentate solution obtained in (4) at 120000 × g and 4 °C for 1.5 h in an ultra-high speed centrifuge to remove small bacterial fragments, large vesicles, etc., leaving small bacterial vesicles, which is OMVs-N3. (6) The OMVs-N3 obtained in (5) was resuspended in PBS and quantified by BCA protein concentration assay.

[0067] (7) Take 50 μg of OMV-N3 containing protein and 6 μL of AS1411-chol in 200 μL of PBS buffer (5mM MgSO4).2 + 100 mM Na + Apt-OMVs-N3 was obtained by incubating at 37 °C for 2 h in a solution of pH = 7.4.

[0068] Step 2: The STING agonist MSA-2 is linked to a diphenylcyclooctyn-polyethylene glycol-carboxyl group via a GSH-responsive linker bis(2-hydroxyethyl) disulfide. The diphenylcyclooctyn-polyethylene glycol is then linked to azide-modified bacterial outer membrane vesicles via click chemistry, thus preparing the responsive STING pathway-activating nanomedicine Apt-OMVs@PSM. This includes the following steps: (1) MSA-2 (5 mg, 0.02 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (5.75 mg, 0.03 mmol), and 4-dimethylaminopyridine (DMAP) (3.67 mg, 0.03 mmol) were stirred in 3 mL of anhydrous dichloromethane in the dark for 3 h. Then, bis(2-hydroxyethyl) disulfide (12.34 mg, 0.08 mmol) dissolved in 500 μL of anhydrous N,N-dimethylformamide was added, and the mixture was stirred in the dark at room temperature for 24 h. After the reaction was completed, the mixture was rotary evaporated, and 20 mL of the mixture (V) was added. 冰水 :V 乙醇 =3:1), adjust the pH to 3 with 2 mol / L dilute hydrochloric acid, centrifuge at 30,000 rpm for 10 min, and vacuum dry the precipitate to obtain the product MSA-2-linker.

[0069] (2) Take DBCO-PEG 2K -COOH (1 mg, 0.43 μmol), EDCI (1 mg, 5.2 μmol), and DMAP (1 mg, 8.2 μmol) were stirred in 2 mL of anhydrous dichloromethane in the dark for 3 h. MSA-2-linker (1 mg, 2.3 μmol) obtained in step (1) was added, and the mixture was stirred in the dark at room temperature for 24 h. After the reaction was complete, the product DBCO-PSM was obtained by rotary evaporation.

[0070] (3) Add 1 mg DBCO-PSM dissolved in 100 μL DMSO to 200 μL of reaction solution obtained in step (7) and perform a click chemical reaction at 37 °C for 2 h. After the reaction is completed, centrifuge at 120000 ×g and 4 °C for 90 min, and resuspend in PBS to obtain the final product Apt-OMVs@PSM.

[0071] Example 2: Physicochemical characterization of the prepared bacterial outer membrane vesicles Apt-OMVs@PSM (1) Morphological characterization: The microstructure of Apt-OMVs@PSM was characterized by transmission electron microscopy (TEM). First, an equal volume of 4% paraformaldehyde was added to 20 μg of Apt-OMVs@PSM solution and fixed at room temperature for 30 min. After washing with PBS, TEM samples were prepared by negative staining with 10 μL of 2% phosphotungstic acid and allowed to air dry. The morphology of the dried Apt-OMVs@PSM was then analyzed using TEM.

[0072] The results are as follows Figure 2 As shown in the TEM images, Apt-OMVs@PSM has a spherical structure, and the loaded nucleic acid aptamers and STING agonist do not change its shape, with a particle size of 70-120 nm.

[0073] (2) Particle size and zeta potential detection: 10 μg of OMVs and Apt-OMVs@PSM were used to test their particle size and zeta potential respectively.

[0074] The results are as follows Figure 3 As shown in Figures a and b, the hydrodynamic diameter of OMVs, measured by dynamic light scattering (DLS), is approximately 78.8 nm, while that of Apt-OMVs@PSM is approximately 122 nm. Compared to OMVs, the Zeta potential of Apt-OMVs decreased from -8.45 mV to -13.26 mV, indicating successful modification of the OMVs surface with negatively charged AS1411. Compared to Apt-OMVs, the Zeta potential of Apt-OMVs@PSM increased from -13.26 mV to -10.59 mV, indicating successful modification of the Apt-OMVs surface with positively charged MSA-2.

[0075] (3) Ultraviolet characteristic peak test: Take Apt-OMVs@PSM, OMVs, and MSA-2 standard solutions and use ultraviolet-visible spectrophotometry to test the ultraviolet absorption of the three.

[0076] The results are as follows Figure 4 As shown in Figure a, compared with the absorption spectrum of the MSA-2 standard solution, Apt-OMVs@PSM also shows an absorption peak at 328 nm. This indicates that MSA-2 was successfully attached to OMVs.

[0077] (4) Verification of GSH-responsive MSA-2 release: HPLC was used to verify whether Apt-OMVs@PSM could release MSA-2 in response to GSH. Apt-OMVs@PSM was incubated with 10 mM GSH and esterase, and the supernatant was analyzed.

[0078] The results are as follows Figure 4As shown in Figure b, the retention time of the supernatant of Apt-OMVs@PSM was the same as that of the MSA-2 standard, both being 1.5 min. This demonstrates that under high concentration GSH conditions, the disulfide bonds in Apt-OMVs@PSM break, thereby releasing MSA-2.

[0079] Example 3: Tumor cell killing experiment using the prepared vesicles Apt-OMVs@PSM Comparative Example 1: OMVs group. *E. coli* was cultured in LB medium and centrifuged at 10000 ×g, 4 ℃ for 10 min. The supernatant was filtered through a 0.45 μm aqueous filter membrane. The filtrate was concentrated by ultrafiltration using a 15 mL, 100 kDa ultrafiltration tube, and the upper retentate was preserved. The upper retentate was then centrifuged at 120000 ×g, 4 ℃ for 90 min to obtain OMVs. Comparative Example 2: OMVs@PSM group. The OMVs-N3 obtained in step (6) of Example 1 was added to 1 mg DBCO-PSM dissolved in 100 μL DMSO, and the reaction was carried out at 37 °C for 2 h. After the reaction was completed, the mixture was centrifuged at 120000 ×g at 4 °C for 90 min, and resuspended in PBS to obtain OMVs@PSM.

[0080] (1) CCK8 assay for cancer cell viability: mouse breast cancer cells (4T1 cells) were subjected to a 1×10⁻⁶ saturation solution. 4 Cells were seeded at a density of 10 μL per well in 96-well cell culture plates. The next day, the cells were treated with different concentrations of OMVs, OMVs@PSM, and Apt-OMVs@PSM. After culturing for 24 h, each well was replaced with 100 μL of DMEM medium containing 10 μL of CCK-8 working solution. After incubation for 30 min, the absorbance of each well at 450 nm was measured.

[0081] The results are as follows Figure 5 As shown in Figure a, OMVs showed negligible cytotoxicity against 4T1 cells, while OMVs@PSM and Apt-OMVs@PSM exhibited good cell-killing effects at a concentration of 100 μg / mL, with Apt-OMVs@PSM showing better killing effects than OMVs@PSM.

[0082] Comparative Example 1: PBS group. That is, no drug treatment was given to the cells.

[0083] Comparative Example 2: OMVs group. The preparation method is the same as that of Comparative Example 1 in Example 3 (1).

[0084] Comparative Example 3: MSA-2 group. MSA-2 powder was added to PBS solution to prepare MSA-2 solution.

[0085] Comparative Example 4: OMVs@PSM group. The preparation method is the same as that of Comparative Example 2 in Example 3 (1).

[0086] (2) Flow cytometry verification of the killing effect of Apt-OMVs@PSM on cancer cells: 4T1 cells were loaded with 2×10 5 Cells were seeded in 6-well cell culture plates. On the second day, the cells were treated with PBS, OMVs, MSA-2, OMVs@PSM, and Apt-OMVs@PSM (OMVs concentration of 60 μg / mL and MSA-2 concentration of 2.2 μg / mL). After 24 hours, the cells were collected, stained with Annexin V-FITC and PI, and the apoptosis status was analyzed by flow cytometry.

[0087] The results are as follows Figure 5 As shown in Figure b, the Apt-OMVs@PSM group induced higher tumor cell death compared to the PBS group.

[0088] Example 4: Activation of the STING pathway and in vitro induction of BMDC maturation using prepared bacterial outer membrane vesicles. Comparative Example 1: PBS group. The preparation method is the same as that of Comparative Example 1 in Example 3 (2).

[0089] Comparative Example 2: OMVs group. The preparation method is the same as that of Comparative Example 1 in Example 3 (1).

[0090] Comparative Example 3: MSA-2 group. The preparation method is the same as that of Comparative Example 3 in Example 3 (2).

[0091] Comparative Example 4: OMVs@PSM group. The preparation method is the same as that of Comparative Example 2 in Example 3 (1).

[0092] (1) After euthanizing C57BL / 6 mice, femurs and tibias were harvested, and bone marrow cells were washed with PBS. Cells were collected by centrifugation and cultured in RPMI-1640 medium containing 10% fetal bovine serum, with the addition of inducing stimulants GM-CSF and IL-4. On days 3 and 5, half of the culture medium was collected, centrifuged, and the cell pellet was redispersed in fresh medium and added back to the original medium. On day 7, non-adherent cells and loosely adherent cells were collected as bone marrow-derived dendritic cells (BMDCs).

[0093] (2) 4T1 cells were fed with 1×10 6 Cells were seeded in 6-well cell culture plates. On the second day, the cells were treated with PBS, OMVs, MSA-2, OMVs@PSM, and Apt-OMVs@PSM (OMVs concentration was 60 μg / mL and MSA-2 concentration was 2.2 μg / mL). After culturing for 24 hours, the cells were collected and co-cultured with the BMDC cells obtained in (1) above.

[0094] (3) Collect BMDC cells from each group after the above treatment, lyse the cells using RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors to extract proteins, and detect protein content using BCA reagent. After SDS-PAGE electrophoresis, transfer the separated proteins to a polyvinylidene fluoride (PVDF) membrane. Block with 5% BSA for 1.5 hours after transfer. After blocking, add diluted anti-p-STING antibody, anti-p-IRF3 antibody, and anti-p-TBK1 antibody, and incubate overnight at 4°C. After washing, add secondary antibody and incubate for 1.5 hours, then use ECL luminescence reagent and ChemiDoc™ Touch imaging system to capture images.

[0095] Experimental results are as follows Figure 6 As shown in Figures a and b, compared with the PBS and OMVs groups, the expression levels of p-STING, p-IRF3, and p-TBK1 were all increased in the MSA-2, OMVs@PSM, and Apt-OMVs@PSM groups, indicating that the STING pathway was activated.

[0096] (4) Collect the cells after co-culture (2), stain BMDC cells with anti-CD11c, anti-CD80 and anti-CD86 antibodies, analyze the BMDC maturity ratio using flow cytometry, and detect the IFN-β content in the culture supernatant using an ELISA kit.

[0097] Experimental results are as follows Figure 7 As shown, the BMDC cell maturation rate in the Apt-OMVs@PSM group was as high as 60%, which was 1-2 times higher than that in the groups using OMVs or MSA-2 alone. This indicates that bacterial outer membrane vesicles that activate the STING pathway in a GSH-responsive manner and synergistically induce pyroptosis can effectively promote DC cell maturation. The IFN-β content in the Apt-OMVs@PSM group was also higher than that in other groups, further demonstrating that the STING pathway was activated.

[0098] Example 5: Verification that modified AS1411 on the surface of bacterial outer membrane vesicles can target tumors. Comparative Example 1: Cy5.5-OMVs group. 4 μL of Cy5.5-se (5 μg / μL) was added to OMVs containing 50 μg of protein. The mixture was incubated at 37°C for 2 hours. After incubation, Cy5.5-OMVs were obtained. Unbound Cy5.5-se was removed by centrifugation using a 100 kDa ultrafiltration tube.

[0099] Comparative Example 2: Cy5.5-OMVs@PSM group. The preparation method is the same as Comparative Example 1 of Example 5, except that OMVs is replaced with OMVs@PSM.

[0100] Comparative Example 3: Cy5.5-Rnd-OMVs@PSM group. As in Example 1, Rnd-OMVs@PSM was prepared by replacing AS1411-chol with Random-DNA. The prepared Rnd-OMVs@PSM was co-incubated with Cy5.5-se to obtain Cy5.5-Rnd-OMVs@PSM, and the specific preparation method is the same as Comparative Example 1 in Example 5.

[0101] To investigate the biodistribution of Apt-OMVs@PSM in vivo, Cy5.5 was labeled on the bacterial outer membrane vesicles. The obtained Cy5.5-OMVs, Cy5.5-OMVs@PSM, Cy5.5-Rnd-OMVs@PSM and Cy5.5-Apt-OMVs@PSM were intravenously injected into 4T1 tumor-bearing BALB / c mice, and in vivo imaging was performed at five time points: 0.5, 1, 4, 8 and 12 h.

[0102] The results are as follows Figure 8 As shown in Figures a and b, the Cy5.5 fluorescence signal at the tumor site in the Cy5.5-Apt-OMVs@PSM group reached its maximum at 12 h, significantly higher than the fluorescence intensity of the other three groups. The fluorescence intensity of the major organs and ex vivo tumors in the Cy5.5-Apt-OMVs@PSM group was also significantly stronger than the other three groups at 12 h. These results indicate that the modified AS1411 can effectively target nanomedicines to tumors.

[0103] AS1411 is the first nucleolin-targeting aptamer to enter clinical trials, indicating that its targeting ability has been fully validated and it is currently the most promising candidate molecule in cancer treatment. Figure 8 The targeting specificity is evident: Apt-OMVs@PSM is modified with AS1411 and exhibits targeting activity, while Rnd-OMVs@PSM is unmodified with AS1411 and has no targeting activity. The higher fluorescence enrichment intensity of the Apt-OMVs@PSM group at the tumor site compared to the Rnd-OMVs@PSM group demonstrates the targeting ability of AS1411.

[0104] Example 6: Solid tumor suppression test using the prepared bacterial outer membrane vesicles.

[0105] Comparative Example 1: PBS group. Mice were not treated in any way.

[0106] Comparative Example 2: OMVs group. The preparation method is the same as that of Comparative Example 1 in Example 3 (1).

[0107] Comparative Example 3: MSA-2 group. The preparation method is the same as that of Comparative Example 3 in Example 3 (2).

[0108] Comparative Example 4: OMVs@PSM group. The preparation method is the same as that of Comparative Example 2 in Example 3 (1).

[0109] Construct a bilateral tumor mouse model. 4T1 cells (1×10⁻⁶) were used. 6 4 T1 cells (1 × 10⁻⁶ cells / mouse) were subcutaneously injected into the right thigh of 6-week-old BALB / c mice as the primary tumor. Seven days later, 4 T1 cells (1 × 10⁻⁶ cells / mouse) were subcutaneously injected into the left thigh of the mice. 6 (cells / each) as a distant tumor.

[0110] like Figure 9 As shown in Figure a, when the volume of the primary tumor reaches 200 mm... 3 Mice were randomly assigned to 5 groups (n = 5) receiving PBS, OMVs (10 mg / kg), MSA-2 (3.5 mg / kg), OMVs@PSM (10 mg / kg), or Apt-OMVs@PSM (10 mg / kg). Each group received the systemic drug three times on days 1, 4, and 7. Mouse body weight and tumor volume were monitored daily over 20 days. Tumor volume was calculated using the following formula: Tumor volume (V, mm) 3 = length × width × width / 2.

[0111] The results are as follows Figure 9 As shown in Figure b, the tumor growth inhibition rate of the Apt-OMVs@PSM group was higher than that of other groups, indicating that the activation of the STING pathway and the induction of pyroptosis can effectively inhibit the malignant growth of tumors.

[0112] After treatment, mouse tumor sections were taken for H&E staining and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining, and observed under a fluorescence microscope.

[0113] like Figure 9 As shown in Figure c, hematoxylin and eosin (H&E) staining further confirmed that the tumor tissues in the Apt-OMVs@PSM group exhibited severe mitotic figures and necrosis. Terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) also further verified that a large number of cells in the tumor tissues of the Apt-OMVs@PSM group underwent apoptosis.

[0114] In summary, the bacterial outer membrane vesicles Apt-OMVs@PSM of this invention, which target GSH-responsive activation of the STING pathway and synergistic pyroptosis, enable precise drug delivery. This invention combines the targeting nucleic acid aptamer AS1411 with bacterial outer membrane vesicles, loading the STING agonist MSA-2 after binding to form Apt-OMVs@PSM. Apt-OMVs@PSM accumulates in the tumor region by leveraging the function of AS1411 in actively targeting nucleolin on the surface of cancer cells. In the presence of GSH, it selectively releases MSA-2 to activate the STING pathway, simultaneously exposing LPS carried on the surface of OMVs, inducing pyroptosis in tumor cells, further stimulating DC cell maturation, and generating a strong immune response. Mouse models have demonstrated that Apt-OMVs@PSM effectively inhibits the growth of both primary and distant tumors. These results demonstrate the therapeutic potential of the bacterial outer membrane vesicles loaded with the STING agonist in synergistic pyroptosis in breast cancer.

[0115] 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. A bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis, characterized in that, These include azide-modified bacterial outer membrane vesicles, diphenylcyclooctyn-polyethylene glycol-carboxyl groups, GSH-responsive bis(2-hydroxyethyl) disulfide, STING agonists, and targeted functional nucleic acid aptamers; The STING agonist is linked to a GSH-responsive linker via an esterification reaction. The GSH-responsive linker connects a diphenylcyclooctynyl group-modified polyethylene glycol to an azide group-modified bacterial outer membrane vesicle via a click chemistry reaction. The targeted functional nucleic acid aptamer is modified on the surface of bacterial outer membrane vesicles.

2. The bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis as described in claim 1, characterized in that, The STING agonist is selected from small molecule STING agonists and cyclic dinucleotides.

3. The bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis as described in claim 2, characterized in that, The STING agonist is selected from small molecule STING agonists di-ABZI, cGAMP, or MSA-2, with MSA-2 being preferred.

4. The bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis as described in claim 1, characterized in that, The targeted functional nucleic acid aptamer is a nucleic acid aptamer that targets nucleolin.

5. The bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis as described in claim 1, characterized in that, The targeted functional nucleic acid aptamer is AS1411, and the nucleotide sequence of AS1411 is shown in SEQ ID No:

1.

6. The bacterial outer membrane vesicle with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis as described in claim 1, characterized in that, The bacterial outer membrane vesicles, after being modified with STING agonists and targeted functional nucleic acid aptamers, have a particle size distribution of 120-140 nm.

7. The method for preparing bacterial outer membrane vesicles with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis according to claim 1, characterized in that, Includes the following steps: Step 1: Preparation of targeted azide-modified bacterial outer membrane vesicles Apt-OMVs-N3: Step 2: The STING agonist is linked to a GSH-responsive linker via an esterification reaction. The GSH-responsive linker connects polyethylene glycol modified with a diphenylcyclooctynyl group to bacterial outer membrane vesicles modified with an azide group via a click chemistry reaction to prepare the responsive STING pathway-activated nanomedicine Apt-OMVs@PSM.

8. The method for preparing bacterial outer membrane vesicles with targeted GSH-responsive activation of the STING pathway and synergistic pyroptosis according to claim 7, characterized in that, Step two includes the following steps: S201, MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark, and then bis(2-hydroxyethyl) disulfide dissolved in N,N-dimethylformamide was added. The reaction was carried out at room temperature in the dark. After the reaction was completed, the product MSA-2-linker was obtained by post-treatment. The molar ratio of MSA-2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine, and N,N-dimethylformamide bis(2-hydroxyethyl) disulfide is 2:2~4:2~4:7~9; S202: Diphenylcyclooctylene-polyethylene glycol-carboxyl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine were stirred in an organic solvent in the dark; MSA-2-linker obtained in step S201 was added and stirred in the dark at room temperature; after the reaction was completed, the product DBCO-PSM was obtained by rotary evaporation. S203: Add DBCO-PSM, the product of step S202, dissolved in an organic solvent, to Apt-OMVs-N3 prepared in step one, and initiate a chemical reaction; after the reaction is complete, centrifuge and resuspend to obtain the final product Apt-OMVs@PSM.

9. The application of the GSH-responsive bacterial outer membrane vesicles with targeted function that activate the STING pathway and synergistically induce pyroptosis, as described in claim 1, in the preparation of tumor therapeutic drugs.

10. The application according to claim 9, characterized in that, The tumor treatment drug is a tumor-targeting drug.

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