Bacterial plasma membrane vesicle rich in c-di-GMP and preparation method and application thereof

Abstract

The invention belongs to the field of medicine, and particularly relates to a c-di-GMP-rich bacterial plasma membrane vesicle and a preparation method and application thereof.The c-di-GMP-entrapped bacterial plasma membrane vesicle is integrally prepared by directly utilizing bacteria capable of generating c-di-GMP, and the vesicle can effectively deliver c-di-GMP to APC and activate STING. The method can realize synchronous preparation of the carrier and c-di-GMP, and is simpler and more convenient than a traditional method. Mouse experiments confirm that the vesicle provided by the invention has an anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a bacterial plasma membrane vesicle rich in c-di-GMP, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance 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 those skilled in the art.

[0003] As the field of immuno-oncology matures and clinical evaluations continue, our understanding of the complexities of tumor-immune cell interactions is growing. While immunotherapy has become the standard of care for many cancers, 55-87% of patients remain unresponsive to checkpoint inhibitors. To improve response rates, there is growing interest in developing innate immune agonists, such as stimulator of interferon genes (STING) agonists, to drive intratumoral activation of antigen-presenting cells (APCs) and effector cells such as NK / T cells. STING activation mediates a multifaceted type I interferon (IFN-I) response, which in turn promotes dendritic cell (DC) maturation and migration, and primes cytotoxic T lymphocytes and natural killer (NK) cells for spontaneous immune responses.

[0004] Cyclic dinucleotides (CDNs) are natural, high-affinity ligands for STING. These include bacterially derived cyclic diguanylate (c-di-GMP), cyclic diadenylate (c-di-AMP), and cyclic diguanylate (3,3-cGAMP), as well as human-derived cyclic diguanylate (2,3-cGAMP). Clinical application of these STING agonists faces numerous challenges, including off-target effects from systemic administration of CDNs, which can easily trigger systemic immunotoxicity; activation of STING in off-target T cells, which can lead to apoptosis and ultimately hinder the formation of immune memory. Therefore, researchers have attempted to administer CDNs intratumorally to mitigate these off-target systemic effects and improve the overall efficacy of such approaches. However, the inherent negative charge of CDNs significantly limits their efficiency in cytoplasmic delivery. Furthermore, rapid enzymatic degradation and clearance of CDNs in vivo further hinder their clinical application. Summary of the Invention

[0005] To address the challenges faced in the clinical application of CDNs, designing a delivery system that selectively targets APCs while protecting them from degradation is expected to reduce off-target toxicity, improve efficacy, and promote their clinical application. Current research focuses on developing novel CDN delivery vectors that can then encapsulate CDNs to create effective CDN delivery systems. Using c-di-GMP as an example, this invention directly utilizes c-di-GMP-producing bacteria to create integrated bacterial plasma membrane vesicles that effectively deliver c-di-GMP to APCs and activate STING. This method allows for the simultaneous preparation of the vector and c-di-GMP, making it simpler than traditional methods.

[0006] In one aspect, the present invention provides a bacterial plasma membrane vesicle, wherein the bacterial plasma membrane vesicle contains cyclic diguanylate (c-di-GMP).

[0007] Furthermore, the c-di-GMP content in the bacterial plasma membrane vesicles is greater than or equal to 39.9 pg / mg.

[0008] Furthermore, the particle size of bacterial plasma membrane vesicles is in the range of 50-300 nm.

[0009] Furthermore, bacterial plasma membrane vesicles were obtained from Gram-negative Escherichia coli and Salmonella.

[0010] Furthermore, bacterial plasma membrane vesicles were obtained after the strain was enriched with c-di-GMP by an inducer.

[0011] Furthermore, the inducer is selected from one or more of ursodeoxycholic acid, arginine, and spermidine.

[0012] In another aspect, the present invention provides a method for preparing bacterial plasma membrane vesicles, comprising the following steps: (1) Inducing the bacteria with an inducer to enrich the bacteria with c-di-GMP; (2) Using lysozyme to degrade the outer membrane and obtain bacterial protoplasts rich in c-di-GMP; (3) Extrusion through a microporous membrane to obtain bacterial plasma membrane vesicles rich in c-di-GMP.

[0013] Furthermore, the inducer used in step (1) is selected from one or more of ursodeoxycholic acid, arginine, and spermidine.

[0014] Furthermore, the particle size in step (3) is in the range of 50-300 nm.

[0015] Furthermore, a method for preparing bacterial plasma membrane vesicles is provided, and the detailed steps are as follows: (1) Inoculate 10 μL of a bacterial solution (E. coli or Salmonella typhimurium) with an OD value of 1 into 5 mL of LB liquid medium and add an inducer (one or more of L-arginine, ursodeoxycholic acid, and spermidine). Incubate the solution at 37°C and 200 rpm for 12 h. During this process, the inducer stimulates the bacteria to produce a large amount of c-di-GMP, which accumulates in the bacterial cytoplasm. After the incubation period, collect the bacteria by centrifugation at 6000 g for 5 min.

[0016] (2) Resuspend the bacteria collected in step (1) in 2 mL of Tris-HCl buffer (50 mM, pH = 8.0) containing 20% ​​sucrose, 50 mM EDTA, and 2 mg / mL lysozyme, and incubate at 37°C, 100 rpm for 30 min. During this process, lysozyme degrades the toxic bacterial outer membrane. After incubation, centrifuge at 6000 g for 5 min, discard the supernatant containing the bacterial outer membrane components, and collect the bacterial protoplast pellet.

[0017] (3) The bacterial protoplasts collected in step (2) were resuspended in 2 mL of physiological saline and subsequently passed through microporous filters with pore sizes of 1 μm, 400 nm, and 200 nm, ultimately obtaining bacterial plasma membrane vesicles rich in c-di-GMP with a particle size of 50-300 nm. During the membrane extrusion process, the bacterial plasma membrane was broken down and rearranged to form smaller vesicles, which encapsulated c-di-GMP and entered the vesicles during the rearrangement.

[0018] In another aspect, the present invention provides a use of bacterial plasma membrane vesicles in the preparation of drugs for activating the STING pathway.

[0019] Another aspect of the present invention provides a use of bacterial plasma membrane vesicles in the preparation of anti-tumor drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention induces bacteria to produce large amounts of c-di-GMP. Then, during the preparation of bacterial plasma membrane vesicles, c-di-GMP is encapsulated by the vesicles to obtain c-di-GMP-loaded bacterial plasma membrane vesicles. The present invention achieves the simultaneous preparation of vesicles and c-di-GMP, making the preparation process simpler.

[0021] (2) The vesicles themselves are rich in the STING agonist c-di-GMP and have the function of activating the STING pathway in target cells without the need for additional STING agonists.

[0022] (3) The present invention intentionally removes the toxic bacterial outer membrane during the preparation of vesicles, reducing the level of systemic inflammation induced by toxic outer membrane components and significantly improving the safety of the preparation.

[0023] (4) In addition to the ability to activate STING, the bacterial plasma membrane vesicles prepared by the present invention also have the ability to activate TLRs, and synergistically activate APCs with c-di-GMP, significantly improving the APC activation ability of c-di-GMP and the ultimate anti-tumor effect.

[0024] (5) The present invention utilizes bacterial-derived plasma membrane vesicles as carriers for c-di-GMP delivery, which can actively target APCs, reduce off-target toxicity, and improve safety.

[0025] (6) The present invention can obtain a large number of preparations with STING activation ability by adding a low-cost single component, which significantly reduces the cost compared to the traditional STING agonist. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, 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.

[0027] Figure 1 : Schematic diagram of bacterial plasma membrane vesicles rich in c-di-GMP; Figure 2 : Transmission electron micrograph of Escherichia coli plasma membrane vesicles enriched with c-di-GMP; Figure 3 : IFN-β secretion by BMDCs after treatment with different Escherichia coli plasma membrane vesicles; Figure 4 : Antitumor effects of different Escherichia coli plasma membrane vesicles. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with embodiment, but the content of the present invention is not limited to the following embodiment. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0029] Example 1: Preparation of bacterial plasma membrane vesicles induced by L-arginine 10 μL of Escherichia coli culture at an OD value of 1 was inoculated into 5 mL of LB liquid medium, and L-arginine was added to a final concentration of 20 mg / mL. The E. coli was cultured at 37°C and 200 rpm for 12 hours. After the incubation period, the bacteria were harvested by centrifugation at 6000 g for 5 minutes. The bacteria were resuspended in 2 mL of Tris-HCl buffer (50 mM, pH 8.0) containing 20% ​​sucrose, 50 mM EDTA, and 2 mg / mL lysozyme, and incubated at 37°C and 100 rpm for 30 minutes. After the incubation period, the bacterial protoplasts were harvested by centrifugation at 6000 g for 5 minutes. The protoplasts were resuspended in 2 mL of saline and filtered sequentially through microporous filters with pore sizes of 1 μm, 400 nm, and 200 nm. E. coli plasma membrane vesicles with a particle size of 176 nm were obtained. L-arginine incubation increased the c-di-GMP content of the bacteria by 4.1 times. The c-di-GMP content in the vesicles is 46.5 pg / mg. A schematic diagram of bacterial plasma membrane vesicles rich in c-di-GMP is shown in Figure 1 , transmission electron microscopy images of plasma membrane vesicles are shown in Figure 2 , its shape is round and has obvious vesicle structure.

[0030] Example 2: Preparation of bacterial plasma membrane vesicles induced by spermidine 10 μL of Escherichia coli culture at an OD value of 1 was inoculated into 5 mL of LB liquid medium, and spermidine was added to a final concentration of 1 mg / mL. The E. coli cells were cultured at 37°C and 200 rpm for 12 hours. After incubation, the cells were harvested by centrifugation at 6000 g for 5 minutes. The cells were resuspended in 2 mL of Tris-HCl buffer (50 mM, pH 8.0) containing 20% ​​sucrose, 50 mM EDTA, and 2 mg / mL lysozyme, and incubated at 37°C and 100 rpm for 30 minutes. After incubation, the cells were centrifuged at 6000 g for 5 minutes to collect bacterial protoplasts. The protoplasts were resuspended in 2 mL of saline and filtered sequentially through microporous filters with pore sizes of 1 μm, 400 nm, and 200 nm. E. coli plasma membrane vesicles with a particle size of 181 nm were obtained. Spermidine incubation increased the c-di-GMP content of the cells by 3.8-fold. The c-di-GMP content in the vesicles was 39.9 pg / mg.

[0031] Example 3: Preparation of bacterial plasma membrane vesicles induced by taurodeoxycholic acid 10 μL of Salmonella culture at an OD value of 1 was inoculated into 5 mL of LB liquid medium, and taurodeoxycholic acid was added to a final concentration of 1 mg / mL. The Salmonella were cultured at 37°C and 200 rpm for 12 hours. After incubation, the bacteria were harvested by centrifugation at 6000 g for 5 minutes. The bacteria were resuspended in 2 mL of Tris-HCl buffer (50 mM, pH 8.0) containing 20% ​​sucrose, 50 mM EDTA, and 2 mg / mL lysozyme, and incubated at 37°C and 100 rpm for 30 minutes. After incubation, bacterial protoplasts were harvested by centrifugation at 6000 g for 5 minutes. The protoplasts were resuspended in 2 mL of saline and filtered sequentially through microporous filters with pore sizes of 1 μm, 400 nm, and 200 nm. Salmonella plasma membrane vesicles with a particle size of 179 nm were obtained. Incubation with taurodeoxycholic acid increased the c-di-GMP content of the bacteria by 4.5-fold. The c-di-GMP content in the vesicles was 49.8 pg / mg.

[0032] Example 4: Verification of c-di-GMP-rich Escherichia coli plasma membrane vesicles activating the STING pathway Bone marrow-derived DCs (BMDCs) were cultured at a rate of 2*10 5 Cells were seeded into 12-well plates at a density of 100 μg / mL and divided into three groups. After the cells adhered, physiological saline, 100 μg / mL of conventional E. coli plasma membrane vesicles, and 100 μg / mL of vesicles prepared in Example 1 were added, and the cultures were continued for 24 hours. After the culture was completed, the cell culture supernatant was aspirated and the IFN-β content was determined. Results ( Figure 3 ) showed that conventional E. coli plasma membrane vesicles could not effectively activate the STING pathway in BMDC cells, while E. coli plasma membrane vesicles rich in c-di-GMP significantly activated the STING pathway in BMDC cells and increased the secretion of IFN-β. This indicates that E. coli plasma membrane vesicles rich in c-di-GMP effectively activated the STING pathway in BMDC cells.

[0033] Example 5: Anti-tumor Effects of Escherichia coli Plasma Membrane Vesicles Rich in c-di-GMP Fifteen 6-week-old C57 mice were subcutaneously inoculated with 5*10 5 When the tumors grew to about 100 cubic millimeters, the mice were randomly divided into three groups and intravenously injected with normal saline, conventional E. coli plasma membrane vesicles, and vesicles prepared in Example 1. The intravenous injections were performed every two days. After 12 days, the tumors were dissected, the weights were recorded, and the tumor inhibition rate was calculated. The results are shown in Figure 4Because conventional vesicles cannot effectively activate the STING pathway in target cells (as demonstrated by the results of Example 4), their 15% tumor inhibition efficiency may be due to the activation of pathways other than STING by the vesicles, such as the TLR pathway. Compared with conventional vesicles, the vesicles prepared in Example 1 effectively activated the STING pathway, increasing the tumor inhibition efficiency from 15% to 31%, significantly improving the anti-tumor effect.

[0034] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A bacterial plasma membrane vesicle, characterized in that: The bacterial plasma membrane vesicles contain cyclic diguanylate.

2. The bacterial plasma membrane vesicle according to claim 1, characterized in that The content of cyclic diguanylate in the bacterial plasma membrane vesicles is greater than or equal to 39.9 pg / mg.

3. The bacterial plasma membrane vesicle according to claim 1, characterized in that The particle size of the bacterial plasma membrane vesicles is in the range of 50-300 nm.

4. The bacterial plasma membrane vesicle according to claim 1, characterized in that The bacterial plasma membrane vesicles are obtained from Gram-negative Escherichia coli and Salmonella.

5. The bacterial plasma membrane vesicle according to claim 1, characterized in that The bacterial plasma membrane vesicles are obtained after the strain is enriched with cyclic diguanylate by an inducer.

6. The method for preparing bacterial plasma membrane vesicles according to claim 1, characterized in that the steps as follows: (1) Inducing the bacteria with an inducer to make them rich in cyclic diguanylate; (2) Using lysozyme to degrade the outer membrane and obtain bacterial protoplasts rich in cyclic diguanylate; (3) Microporous membrane extrusion to obtain bacterial plasma membrane vesicles rich in cyclic diguanylate.

7. The method for preparing bacterial plasma membrane vesicles according to claim 6, characterized in that: The induction agent used in step (1) is selected from one or more of ursodeoxycholic acid, arginine and spermidine.

8. The method for preparing bacterial plasma membrane vesicles according to claim 6, characterized in that: The particle size of step (3) is in the range of 50-300 nm.

9. Use of the bacterial plasma membrane vesicles as claimed in claim 1 in the preparation of drugs for activating the STING pathway.

10. Use of the bacterial plasma membrane vesicles according to claim 1 in the preparation of anti-tumor drugs.

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