OMVs (at) Se bioactive preparation as well as preparation method and application thereof

Abstract

The invention discloses an OMVs (at) Se bioactive preparation as well as a preparation method and application thereof. According to the preparation, a core-shell structure is formed by coating bacterial outer membrane vesicles with selenium nanoparticles, targeted delivery to lung macrophages is achieved, ferroptosis of the lung macrophages is effectively inhibited, inflammatory response is cooperatively regulated, the immune clearance ability is enhanced, and therefore the efficient immunotherapy effect on bacterial pneumonia is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to an OMVs@Se bioactive formulation, where OMVs@Se refers to selenium nanoparticles loaded on bacterial outer membrane vesicles, as well as a method for preparing the selenium nanoparticles loaded on bacterial outer membrane vesicles and their application in the preparation of drugs for treating bacterial pneumonia. Background Technology

[0002] Bacterial pneumonia is a type of lower respiratory tract infection caused by various pathogens. It is characterized by high incidence and rapid progression, and in severe cases, it can develop into respiratory failure or sepsis. It has become one of the leading causes of infection-related deaths worldwide. Currently, clinical treatment mainly relies on antibiotics, but due to long-term overuse, drug-resistant strains, especially multidrug-resistant strains, are constantly emerging, significantly weakening the efficacy of traditional antibacterial agents.

[0003] Besides the bacteria themselves, an excessive immune response in the host to pathogens is also a significant factor contributing to the worsening of the disease. Recent studies have found that lung macrophages play a crucial role in immune regulation during bacterial infection. However, under conditions of infection and inflammatory stress, macrophages are prone to programmed cell death, particularly ferroptosis, a novel form of cell death dependent on iron ions and lipid peroxidation. Ferroptosis not only weakens the host's immune defenses but also promotes the continuous release of inflammatory factors, causing irreversible damage to lung tissue and thus exacerbating pneumonia.

[0004] Currently, there are no effective treatments to intervene in macrophage ferroptosis. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide an OMVs@Se bioactive formulation that can target and deliver selenium nanoparticles to lung macrophages, effectively inhibit their ferroptosis, synergistically regulate the inflammatory response, and enhance immune clearance capacity, thereby achieving highly effective immunotherapy for bacterial pneumonia.

[0006] The technical solution of the present invention is: this OMVs@Se bioactive preparation has a core-shell structure, with selenium nanoparticles (Se nps) as the core and encapsulated by bacterial outer membrane vesicles (OMVs).

[0007] The OMVs@Se bioactive formulation of the present invention can target and deliver selenium nanoparticles to lung macrophages, effectively inhibit their ferroptosis, synergistically regulate the inflammatory response, and enhance immune clearance capacity, thereby achieving highly effective immunotherapy for bacterial pneumonia.

[0008] A method for preparing OMVs@Se bioactive formulations is also provided, which includes the following steps: (1) Extraction of OMVs: Take 800 mL of sterile LB liquid medium and inoculate with OMVs. E. coli Nissle 1917 strain was cultured in a 37°C constant temperature shaker at 200 rpm for 7 hours until the logarithmic growth phase. After the culture, the supernatant was collected by centrifugation at 3000×g and 4°C for 30 minutes. The supernatant was filtered through a 0.22 μm sterile filter to remove residual bacteria. The filtrate was further concentrated by ultrafiltration using a 100 kDa molecular weight cutoff membrane at 3000×g and 4°C. The concentrate was transferred to an ultracentrifuge tube and centrifuged at 170,000×g and 4°C for 2 hours to obtain a black precipitate, which was the collected OMVs. (2) Synthesis of Senps: Weigh 1.038 g of sodium selenite and add it to 150 mL of Milli-Q ultrapure water, shake to dissolve; separately take 0.75 g of chitosan with a concentration of 200–600 mPa·s, dissolve it in 150 mL of 1% w / v acetic acid solution to prepare a 0.5% w / v chitosan solution, add the chitosan solution dropwise to the sodium selenite solution, and stir thoroughly to dissolve; separately weigh 7.045 g of vitamin C and dissolve it in 400 mL of Milli-Q ultrapure water as a reducing agent, and slowly add it to the above mixture system while stirring; after the addition is complete, place the reaction system in a constant temperature shaker at 4 ℃ for 24 hours, and keep shaking to promote the complete reaction; after the reaction is complete, load the reaction solution into a container with a molecular weight cutoff of 8000. The dialysis bag of Da was dialyzed in Milli-Q ultrapure water for 24 hours, with the dialysate being changed every 6 hours to remove small molecule impurities. After the dialysis was completed, the dialysis product was collected as the synthesized Senps. (3) Preparation of biological agent loaded with SE nps by OMVs: 0.3 mL of 5 μg / mL SE nps was mixed with 0.7 mL of 5 μg / mL OMVs solution; the mixture was placed in an ultrasonic cell disruptor and treated for 3 minutes at a power of 50 W to temporarily disturb the outer membrane vesicle structure and promote the encapsulation of SE nps; after ultrasonic treatment, the mixture was centrifuged at 6000×g and 4 ℃ for 15 minutes to remove unloaded free SE nps; after centrifugation, the precipitate was collected and resuspended with sterile PBS to obtain the target product OMVs@Se.

[0009] It also provides applications for OMVs@Se bioactive formulations, which are used to inhibit macrophage ferroptosis.

[0010] It also provides applications for OMVs@Se bioactive formulations, which are used to inhibit the excessive release of inflammatory factors and promote the restoration of anti-inflammatory signals.

[0011] It also provides applications for OMVs@Se bioactive formulations, which are used to improve mitochondrial membrane potential damage and maintain mitochondrial functional stability.

[0012] It also provides applications for OMVs@Se bioactive formulations, which are used in the preparation of drugs for treating bacterial pneumonia. Attached Figure Description

[0013] Figure 1 Characterization of the bioactive formulation is shown. (A) Morphology of OMVs, Senps, and OMVs@Se observed by transmission electron microscopy. (B) Energy dispersive spectroscopy (EDS) spectra of the corresponding OMVs@Se. (CE) Particle size and surface potential of OMVs@Se, OMVs, and Senps determined by dynamic light scattering. (F) Surface elemental composition of OMVs@Se and Senps analyzed by X-ray photoelectron spectroscopy.

[0014] Figure 2 This study demonstrates endocytosis and biosafety. (A) RAW 264.7 macrophages were incubated with C6 dye (10 μg / mL, green) labeled Senps and OMVs@Se for 1, 3, and 6 hours, respectively. Cell uptake efficiency was assessed using laser confocal microscopy, with Hoechst 33342 labeling of cell nuclei (blue). (B, C) Cell uptake efficiency of different agents was determined by flow cytometry. (D) RAW 264.7 macrophages were co-incubated with different concentrations of OMVs, Senps, and OMVs@Se for 24 hours, and cell viability was determined by CCK-8 assay. Results are presented as mean ± standard deviation of n samples, n=3, **** represents... p <0.0001.

[0015] Figure 3 The in vitro anti-inflammatory activity assay was shown. The mRNA expression levels of (A) tumor necrosis factor α (TNF-α), (B) interleukin-1β (IL-1β), (C) interleukin-4 (IL-4), and (D) interleukin-10 (IL-10) in cells after drug treatment were quantitatively analyzed by RT-qPCR. Results are presented as mean ± standard deviation of n samples, n=3, * represents... p <0.05, ** represents p <0.01, *** represents p <0.001, **** represents p <0.0001.

[0016] Figure 4 The image shows the use of the JC-1 probe to label mitochondrial membrane potential (green: low membrane potential; red: high membrane potential), and the assessment of changes in mitochondrial membrane potential by laser confocal microscopy. Hoechst 33342 is used to label the cell nucleus (blue).

[0017] Figure 5 The following describes the assay for inhibiting macrophage ferroptosis in vitro. (A) Intracellular ROS were labeled using the DCFH-DA probe, and ROS levels were assessed by flow cytometry. (B) Intracellular LPO was labeled using the BODIPY C11 probe, and LPO levels were assessed by flow cytometry. (C) Intracellular Fe was labeled using the FerroOrange probe. 2+ Fe was assessed by flow cytometry. 2+ Levels. (DE) Corresponding fluorescence statistics measured by flow cytometry. (G) Intracellular GPX4 activity. (H) Intracellular GSH level. Results are the mean ± standard deviation of n samples, n=3, * indicates p <0.05, ** represents p <0.01, *** represents p <0.001, **** represents p <0.0001.

[0018] Figure 6 In vivo targeting validation is shown. (AB) Mice with bacterial pneumonia were nebulized with Cy7.5-labeled OMVs@Se, and tissues were collected 24 hours later for fluorescence imaging and quantitative analysis. (CD) Quantitative analysis of the deposition efficiency of Se nps and OMVs@Se in lung tissue. (E) Flow cytometry determination of the proportion of particles in macrophages in lung tissue. Results are the mean ± standard deviation of n samples, n=5, **** represents p <0.0001.

[0019] Figure 7 The in vivo therapeutic activity assay is shown. (A) Experimental flowchart. (B) Weight changes in mice with bacterial pneumonia after treatment in different groups. (C) CFU statistics in lung tissue of each group. (DG) ELISA results of different inflammatory factors in lung tissue of each group, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). Results are the mean ± standard deviation of n samples, n=5, ** represents p <0.01, *** represents p <0.001, **** represents p <0.0001.

[0020] Figure 8 The images show hematoxylin-eosin (H&E) staining and myeloperoxidase (MPO) immunohistochemical analysis of the lungs of mice in each treatment group. Detailed Implementation

[0021] This OMVs@Se bioactive formulation has a core-shell structure, with selenium nanoparticles (Se nps) serving as the core and being encapsulated by bacterial outer membrane vesicles (OMVs).

[0022] The OMVs@Se bioactive formulation of the present invention can target and deliver selenium nanoparticles to lung macrophages, effectively inhibit their ferroptosis, synergistically regulate the inflammatory response, and enhance immune clearance capacity, thereby achieving highly effective immunotherapy for bacterial pneumonia.

[0023] A method for preparing OMVs@Se bioactive formulations is also provided, which includes the following steps: (1) Extraction of OMVs: Take 800 mL of sterile LB liquid medium and inoculate with OMVs. E. coli Nissle 1917 strain was cultured in a 37°C constant temperature shaker at 200 rpm for 7 hours until the logarithmic growth phase. After the culture, the supernatant was collected by centrifugation at 3000×g and 4°C for 30 minutes. The supernatant was filtered through a 0.22 μm sterile filter to remove residual bacteria. The filtrate was further concentrated by ultrafiltration using a 100 kDa molecular weight cutoff membrane at 3000×g and 4°C. The concentrate was transferred to an ultracentrifuge tube and centrifuged at 170,000×g and 4°C for 2 hours to obtain a black precipitate, which was the collected OMVs. (2) Synthesis of Senps: Weigh 1.038 g of sodium selenite and add it to 150 mL of Milli-Q ultrapure water, shake to dissolve; separately take 0.75 g of chitosan with a concentration of 200–600 mPa·s, dissolve it in 150 mL of 1% w / v acetic acid solution to prepare a 0.5% w / v chitosan solution, add the chitosan solution dropwise to the sodium selenite solution, and stir thoroughly to dissolve; separately weigh 7.045 g of vitamin C and dissolve it in 400 mL of Milli-Q ultrapure water as a reducing agent, and slowly add it to the above mixture system while stirring; after the addition is complete, place the reaction system in a constant temperature shaker at 4 ℃ for 24 hours, and keep shaking to promote the complete reaction; after the reaction is complete, load the reaction solution into a container with a molecular weight cutoff of 8000. The dialysis bag of Da was dialyzed in Milli-Q ultrapure water for 24 hours, with the dialysate being changed every 6 hours to remove small molecule impurities. After the dialysis was completed, the dialysis product was collected as the synthesized Senps. (3) Preparation of biological agent loaded with SE nps by OMVs: 0.3 mL of 5 μg / mL SE nps was mixed with 0.7 mL of 5 μg / mL OMVs solution; the mixture was placed in an ultrasonic cell disruptor and treated for 3 minutes at a power of 50 W to temporarily disturb the outer membrane vesicle structure and promote the encapsulation of SE nps; after ultrasonic treatment, the mixture was centrifuged at 6000×g and 4 ℃ for 15 minutes to remove unloaded free SE nps; after centrifugation, the precipitate was collected and resuspended with sterile PBS to obtain the target product OMVs@Se.

[0024] Preferably, in step (1), 1 mL of sterile PBS is added to resuspend the collected OMVs, and the mixture is gently blown to obtain a homogeneous OMVs suspension; the obtained product is inoculated onto a TSA plate to assess sterility; its morphology, particle size distribution and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); and the protein concentration of OMVs is quantified by BCA protein cassette.

[0025] Preferably, in step (2), the morphology, particle size distribution and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the Se element is characterized by X-ray photoelectron spectroscopy (XPS); after acid digestion, the content of Se element in the nanoparticles is determined by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0026] Preferably, in step (3), the morphology, particle size distribution and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the Se element is characterized by X-ray photoelectron spectroscopy (XPS); after acid digestion, the content of Se element in the complex is determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and its loading efficiency is calculated.

[0027] It also provides applications for OMVs@Se bioactive formulations, which are used to inhibit macrophage ferroptosis.

[0028] It also provides applications for OMVs@Se bioactive formulations, which are used to inhibit the excessive release of inflammatory factors and promote the restoration of anti-inflammatory signals.

[0029] It also provides applications for OMVs@Se bioactive formulations, which are used to improve mitochondrial membrane potential damage and maintain mitochondrial functional stability.

[0030] It also provides applications for OMVs@Se bioactive formulations, which are used in the preparation of drugs for treating bacterial pneumonia.

[0031] The embodiments of the present invention will be described in detail below. Example 1: Extraction and characterization of bacterial outer membrane vesicles (OMVs)

[0032] Take 800 mL of sterile LB liquid culture medium and inoculate. E. coli Nissle 1917 (EcN) strain was cultured at 37 °C in a shaker at 200 rpm for 7 hours until the logarithmic growth phase. After culture, the supernatant was collected by centrifugation at 3000 × g and 4 °C for 30 minutes. The supernatant was filtered through a 0.22 μm sterile filter to remove residual bacteria. The filtrate was further concentrated by ultrafiltration using a 100 kDa molecular weight cutoff membrane at 3000 × g and 4 °C. The concentrate was transferred to an ultracentrifuge tube and centrifuged at 170,000 × g and 4 °C for 2 hours. After centrifugation, a black precipitate was visible at the bottom of the tube, which was the collected bacterial outer membrane vesicles (OMVs). The OMVs were resuspended in 1 mL of sterile PBS and gently mixed by pipetting to obtain a homogeneous OMVs suspension. The resulting product was inoculated onto TSA plates to assess sterility. Its morphology, particle size distribution, and surface potential were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The protein concentration of OMVs was quantified using a BCA protein box. Example 2: Synthesis and Characterization of Selenium Nanoparticles (Se nps)

[0033] Weigh 1.038 g of sodium selenite (6 mmol) and add it to 150 mL of Milli-Q ultrapure water, shaking to dissolve. Separately, dissolve 0.75 g of chitosan (200–600 mPa·s) in 150 mL of acetic acid solution (1% w / v) to prepare a 0.5% w / v chitosan solution. Add this chitosan solution dropwise to the sodium selenite solution, stirring thoroughly to dissolve. Separately, weigh 7.045 g of vitamin C (40 mmol) and dissolve it in 400 mL of Milli-Q ultrapure water as a reducing agent, slowly adding it to the above mixture while stirring. After the addition is complete, place the reaction system in a constant temperature shaker at 4 ℃ for 24 hours, maintaining continuous shaking to promote complete reaction. After the reaction is complete, transfer the reaction solution into a dialysis bag with a molecular weight cutoff of 8000 Da and dialyze it in Milli-Q ultrapure water for 24 hours, changing the dialysate every 6 hours to remove small molecule impurities. After dialysis, the dialysis product was collected, which was the prepared Se nps. Its morphology, particle size distribution, and surface potential were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Se was characterized by X-ray photoelectron spectroscopy (XPS). After acid digestion, the Se content in the nanoparticles was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Example 3: Preparation and characterization of OMVs-loaded Se nps (OMVs@Se) biopharmaceutical

[0034] This invention employs an ultrasound-assisted loading method to prepare the biological agent OMVs@Se. Specifically, 0.3 mL of Se nps (5 μg / mL) was mixed with 0.7 mL of OMVs solution (5 μg / mL). The mixture was placed in an ultrasonic cell disruptor and treated at 50 W for 3 minutes to temporarily disturb the outer membrane vesicle structure and promote the encapsulation of Se nps. After ultrasonic treatment, the mixture was centrifuged at 6000 × g and 4 °C for 15 minutes to remove unloaded free Se nps. The precipitate was collected after centrifugation and resuspended in sterile PBS to obtain the target product OMVs@Se. Its morphology, particle size distribution, and surface potential were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Se was characterized by X-ray photoelectron spectroscopy (XPS). After acid digestion, the Se content in the complex was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and its loading efficiency was calculated. Example 4: Cellular internalization level of OMVs@Se

[0035] A coumarin-6 (C6) dye-labeled biological agent was prepared and named C6@OMVs@Se. C6@Se nps were used as a positive control. RAW 264.7 macrophages were seeded in 6-well plates containing sterile cell spreaders (5 × 10⁻⁶ cells / well). 5 (Cells / well). After overnight culture, C6 (10 μg / mL) labeled biological agent was added, and the cells were co-cultured for 1 hour, 3 hours, and 6 hours, respectively. The cell culture medium was removed, and the cells were washed three times with PBS. After fixation with cell fixation solution and Hoechst 33342 staining, cell slides were removed and mounted. The distribution of green fluorescence signals within the cells was observed using a laser confocal microscope. Simultaneously, in parallel, another group of samples was washed 2-3 times with PBS after removing the culture medium, centrifuged at 400×g, 4 ℃ for 3-5 minutes, and the cell suspension was collected. Intracellular fluorescence intensity was detected and quantitatively analyzed by flow cytometry to evaluate the endocytosis efficiency of cells to different agents. Example 5: Cellular safety of OMVs@Se

[0036] RAW 264.7 macrophages were seeded in 96-well plates (1×10⁻⁶ cells / well). 4Cells / well), after overnight culture, OMVs, Senps, and OMVs@Se were added to the cells, respectively. The concentration of Senps ranged from 0.1 to 20 μg / mL, and the corresponding concentration of OMVs ranged from 0.1 to 100 μg / mL. After 24 hours of incubation, CCK-8 solution was added according to the kit instructions, and cell viability was measured and calculated to evaluate the cytotoxicity of the formulation. Example 6: In vitro anti-inflammatory activity of OMVs@Se

[0037] (1) Construction of inflammation model RAW 264.7 macrophages were seeded into 6-well plates (5 × 10⁶ cells / well). 5 Cells / well), after overnight culture, were incubated with LPS (100 ng / mL) and IFN-γ (20 ng / mL) to induce an inflammatory model.

[0038] (2) Experimental grouping and treatment Blank control group: uninduced RAW 264.7 cells; Model group: Inflammation model cells induced by LPS and FN-γ; Drug administration groups: After the aforementioned inflammatory model was successfully established, OMVs, Senp, OMVs+Se nps, and OMVs@Se were added respectively. The concentration of Senp was uniformly set to 0.1 μg / mL, and the concentration of OMVs was uniformly set to 0.3 μg / mL, and the treatment time was 12 hours.

[0039] (3) Detection method Twelve hours after drug administration, the cell culture medium was removed, cells were washed with PBS, centrifuged to collect cells, total RNA was extracted, cDNA was obtained by reverse transcription, and qPCR was performed. The expression levels of inflammatory factor genes in each group were measured, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4), and interleukin-10 (IL-10). Example 7: Inhibitory effect of OMVs@Se on macrophage ferroptosis

[0040] (1) Construction of a macrophage ferroptosis model RAW 264.7 macrophages were seeded in 12-well plates (2 × 10⁻⁶ cells / well). 5 Cells / well), after overnight culture, were incubated with LPS (100 ng / mL) and IFN-γ (20 ng / mL) to induce macrophage ferroptosis model.

[0041] (2) Experimental grouping and treatment Blank control group: uninduced RAW 264.7 cells; Model group: LPS and FN-γ induced ferroptosis model cells; Drug administration group: After the aforementioned ferroptosis model was successfully constructed, OMVs, Senp, OMVs+Se nps and OMVs@Se were added respectively, with the concentration of Senp uniformly set to 0.1 μg / mL and the concentration of OMVs uniformly set to 0.3 μg / mL, and the treatment time was 12 hours.

[0042] (3) Detection method Mitochondrial membrane potential detection: Cells were washed with PBS, stained with JC-1 staining working solution, and incubated at 37 ℃ in the dark for 20 minutes. Cells were then washed with JC-1 staining buffer. Hoechst 33342 was added to label the cell nuclei, cells were washed with PBS, cell culture medium was added, and changes in intracellular fluorescence intensity were observed using a laser confocal microscope.

[0043] ROS detection: Cells were washed with PBS and collected. The fluorescent probe DCFDA (5 μM, 1 mL) was added and stained in a 37 ℃ incubator in the dark for 20 minutes. After centrifugation and washing, the cells were resuspended in serum-free medium and the changes in intracellular fluorescence intensity of various cells were quantitatively analyzed using flow cytometry.

[0044] Fe 2+ Detection: Cells were washed with serum-free medium and collected. FerroOrange working solution (5 μM, 1 mL) was added, and the cells were stained in a 37 ℃ incubator in the dark for 20 minutes. The changes in intracellular fluorescence intensity of various cells were quantitatively analyzed using flow cytometry.

[0045] LPO detection: Cells were washed with serum-free medium and collected. BODIPY C11 581 / 591 fluorescent probe (5 μM, 1 mL) was added and stained in a 37 ℃ incubator in the dark for 30 minutes. Cells were washed by centrifugation and resuspended in serum-free medium. Flow cytometry was used to quantitatively analyze the changes in intracellular fluorescence intensity of various cells.

[0046] GPX4 enzyme activity assay: Wash cells with PBS, centrifuge at 400×g, 4 ℃ for 3-5 minutes, collect cells, and lyse cells using cell lysis buffer. Centrifuge at 12000×g, 4 ℃ for 10 minutes, collect the supernatant, and determine GPX4 activity using a GPX4 assay kit.

[0047] GSH content determination: Wash cells with PBS, centrifuge at 400×g, 4 ℃ for 3-5 minutes, collect and weigh the cell pellet, then add protein removal reagent solution and vortex thoroughly. Perform two rapid freeze-thaw cycles using liquid nitrogen and a 37 ℃ water bath. Incubate on ice for 5 min, centrifuge at 10000×g, 4 ℃ for 10 minutes, collect the supernatant, and determine the GSH content using a GSH detection kit. Example 8: In vivo targeted evaluation of OMVs@Se

[0048] (1) Establishment of an acute pneumonia mouse model: Female BALB / c mice aged 6-8 weeks were selected and acclimatized for 1 week in an animal biosafety level 2 laboratory (ABSL-2). 20 µL of Pseudomonas aeruginosa suspension (1×10⁻⁶) was administered via intranasal drip. 9 (CFU / mL) to construct a mouse model of acute bacterial pneumonia.

[0049] (2) Nebulized administration: Cy7.5 was used as a fluorescent tracer molecule and loaded into Se nps and OMVs@Se, respectively, and administered to pneumonia mice by nebulization.

[0050] (3) Evaluation of tissue distribution: After the administration was completed, the mice were sacrificed and lung tissue was taken. The tissue fluorescence intensity was detected by an in vitro imaging system, and the retention difference of Se nps and OMVs@Se in lung tissue was compared.

[0051] (4) Evaluation of cell-level distribution: Lung tissue was minced, added to 6 mL of digestion solution, and digested at 37 ℃ and 200 rpm for 2 h. The cells were filtered through a 100 μm filter and centrifuged at 400×g for 5 min to collect the cell pellet. Red blood cell lysis buffer was added for 2 min, the supernatant was discarded after centrifugation, and the cells were resuspended in HBSS to prepare a single-cell suspension. The obtained single-cell suspension was then subjected to LIVE / DEAD staining, Fc receptor blocking with anti-mouse CD16 / 32 antibody, and then stained with anti-mouse CD45-BV510, CD11b-PE, and F4 / 80-FITC antibodies. After washing with HBSS, the cells were detected by flow cytometry. The targeting ability of OMVs@Se was evaluated by comparing the proportion of fluorescence signals in lung macrophages of the two formulations. Example 9: In vivo therapeutic evaluation of OMVs@Se for bacterial pneumonia

[0052] (1) Construction of an acute pneumonia mouse model The method for constructing the mouse model is the same as described in Example 8.

[0053] (2) Nebulized drug delivery therapy Two hours after modeling, mice were randomly divided into 6 groups. Inhalation was performed using a HY-JSE01 small animal nebulizer (Beijing Yuansen Kaide Biotechnology Co., Ltd.) at a nebulization rate of 0.2 mL / min for 10 minutes. Mice were inhaled with an OMVs@Se formulation, containing 0.2 mg / kg of Senps and 0.75 mg / kg of OMVs, administered every 2 hours for a total of 2 doses. The control group received equal doses of OMVs, Senps, an OMVs+Seps mixture, and PBS.

[0054] (3) Methods for evaluating treatment efficacy During treatment, body weight changes in mice were recorded over 72 hours. Twenty-four hours after treatment, mice were euthanized, and lung tissue was harvested under aseptic conditions. Multilobar lung tissue was added to 1 mL of sterile PBS and homogenized with sterile grinding beads. 100 μL of the homogenate was serially diluted and inoculated onto TSA plates, and colony-forming units (CFU) were counted. The supernatant from the lung tissue homogenate was collected by centrifugation, and the levels of TNF-α, IL-β, IL-6, and IL-10 were quantitatively detected using an ELISA kit. Single lung tissue was fixed in 4% paraformaldehyde for 48 hours, then routinely dehydrated, embedded in paraffin, sectioned, and stained with H&E and MPO immunohistochemically.

[0055] Results and Discussion: 1. Characterization of OMVs@Se biological agents like Figure 1 As shown, this invention successfully prepared OMVs, Senps, and their composite system OMVs@Se. Transmission electron microscopy revealed that OMVs exhibited a typical spherical vesicle structure with a particle size of approximately 100 nm; Senps appeared as uniformly dispersed nanoparticles; and OMVs@Se possessed a core-shell structure, with Senps acting as the core encapsulated within the OMVs membrane, indicating that Senps were successfully loaded into the vesicle lumen. Energy dispersive spectroscopy (EDS) further confirmed the presence of Se in OMVs@Se, proving successful composite formation. Dynamic light scattering (DLS) results showed that the average particle size of OMVs@Se was slightly larger than that of pure OMVs. Zeta potential measurements showed that OMVs had a negative potential, while Senps had a positive potential; the surface potential of the composite OMVs@Se was significantly higher than that of pure OMVs, but still remained negatively charged, indicating that the two composited through electrostatic interactions. X-ray photoelectron spectroscopy (XPS) results showed the simultaneous presence of characteristic peaks for C, N, O, and Se in OMVs@Se, further confirming that Senps were successfully loaded into OMVs.

[0056] like Figure 2As shown, confocal microscopy results revealed that both Senps and OMVs@Se were taken up by RAW 264.7 cells with prolonged incubation time. However, the OMVs@Se group exhibited stronger green fluorescence signals at 1, 3, and 6 hours, indicating that OMVs coating significantly enhanced the intracellular delivery capacity of Senps. Flow cytometry quantitative analysis further confirmed this trend, showing that the cellular uptake efficiency of OMVs@Se was significantly higher than that of the Senps group. Furthermore, in vitro CCK-8 assays demonstrated that the introduction of OMVs significantly reduced the cytotoxicity of Senps, validating the potential of OMVs in improving the biocompatibility of Senps.

[0057] like Figure 3 As shown, OMVs@Se exhibits significant anti-inflammatory regulatory effects in vitro. RT-qPCR results showed that in LPS-stimulated RAW 264.7 cells, the mRNA levels of pro-inflammatory cytokines TNF-α and IL-1β were significantly increased, while those treated with OMVs@Se were significantly downregulated, demonstrating a superior inhibitory effect compared to Se nps or OMVs alone. Conversely, the expression of anti-inflammatory cytokines IL-4 and IL-10 was significantly upregulated in the OMVs@Se treatment group, indicating that OMVs@Se can effectively inhibit the excessive release of inflammatory cytokines and promote the recovery of anti-inflammatory signals, exhibiting good immunomodulatory capabilities.

[0058] like Figure 4 As shown in the results, JC-1 probe detection revealed a significant decrease in mitochondrial membrane potential after LPS stimulation, manifested as enhanced green fluorescence and weakened red fluorescence. After treatment with OMVs@Se, the red fluorescence signal was significantly restored, indicating that this formulation can improve mitochondrial membrane potential damage and maintain mitochondrial functional stability.

[0059] like Figure 5 As shown, after LPS stimulation, the levels of reactive oxygen species (ROS) and lipid peroxidation products (LPO) in RAW 264.7 cells were significantly increased, accompanied by an increase in intracellular Fe²⁺ content, a decrease in glutathione peroxidase 4 (GPX4) activity, and a decrease in reduced glutathione (GSH) levels, indicating that the cells exhibited obvious oxidative stress and ferroptosis characteristics. After OMVs@Se treatment, the levels of intracellular ROS and LPO were significantly reduced, Fe²⁺ accumulation was inhibited, and GPX4 activity and GSH levels returned to near-normal levels. These results indicate that the OMVs@Se formulation provided by this invention can effectively alleviate LPS-induced oxidative stress, maintain intracellular redox balance, and prevent excessive accumulation of iron ions, thereby inhibiting ferroptosis-related damage at the molecular level, demonstrating good cell protection and application potential.

[0060] like Figure 6As shown, to verify the in vivo targeted distribution characteristics of the formulation of the present invention, OMVs@Se and Senps labeled with Cy7.5 fluorescent dye were administered to a bacterial pneumonia model mouse via nebulization. Twenty-four hours after administration, in vitro fluorescence imaging and signal intensity analysis of major organs showed that the OMVs@Se group was mainly concentrated in the lung tissue, while fluorescence accumulation in other tissues was low, indicating that the formulation has good lung enrichment characteristics after nebulized administration. Further imaging and quantitative analysis of lung tissue showed that, compared with the Senps group, OMVs@Se had a higher signal intensity in the lung, demonstrating significant lung-targeted delivery capability. Flow cytometry analysis of single-cell suspensions of lung tissue showed that the proportion of OMVs@Se taken up by macrophages in the lung tissue was significantly higher than that in the Senps group, indicating that the OMVs coating structure of the present invention can promote the specific enrichment of particles in lung macrophages, achieving effective targeted delivery.

[0061] like Figure 7 As shown, a bacterial pneumonia mouse model was constructed to explore the in vivo therapeutic effect of the formulation of this invention. The changes in body weight after treatment in different groups of mice showed that the weight loss in the OMVs@Se group was significantly less than that in the model group, indicating that the formulation can alleviate disease-related weight loss. CFU statistics of lung tissue showed that the bacterial load in the lungs of the OMVs@Se group was significantly lower than that in the model group and the Se nps group alone, indicating that the formulation has a stronger antibacterial effect. ELISA results showed that OMVs@Se significantly reduced the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in lung tissue, while significantly increasing the expression of the anti-inflammatory factor IL-10, indicating that OMVs@Se can effectively regulate the inflammatory response and achieve an anti-inflammatory effect.

[0062] like Figure 8 As shown, H&E staining results revealed significant inflammatory infiltration, alveolar structural damage, and interstitial edema in the lung tissue of the model group mice, while the lung tissue of mice treated with OMVs@Se showed relatively intact structure and significantly reduced inflammatory cell infiltration. Further MPO immunohistochemical analysis revealed that neutrophil infiltration in the lung tissue of the OMVs@Se group was significantly lower than that in the model group, suggesting that the preparation can effectively reduce pulmonary inflammatory response and tissue damage, exhibiting significant anti-inflammatory effects.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. OMVs@Se bioactive formulation characterized in that: It is in the core-shell structure, and the selenium nanoparticles (Se nps) are coated by the bacterial outer membrane vesicles (OMVs) as the core.

2. The process for the preparation of OMVs@Se bioactive formulation as claimed in claim 1, wherein: It comprises the following steps: (1) Extraction of OMVs: Take 800 mL of sterile LB liquid medium and inoculate with OMVs. E. coli Nissle 1917 strain was cultured in a 37 ℃ constant temperature shaker at 200 rpm for 7 hours until the logarithmic growth phase. After the culture, the supernatant was collected by centrifugation at 3000×g and 4 ℃ for 30 minutes. The supernatant was filtered through a 0.22 μm sterile filter to remove residual bacteria. The filtrate was further concentrated by ultrafiltration using a 100 kDa molecular weight cutoff membrane at 3000×g and 4 ℃. The concentrate was transferred to an ultracentrifuge tube and centrifuged at 170,000×g and 4 ℃ for 2 hours to obtain a black precipitate, which was the collected OMVs. (2) Synthesis of Se nps: weigh 1.038 g of sodium selenite and add it to 150 mL of Milli-Q ultrapure water, shake to dissolve; take another 0.75 g of chitosan with a viscosity of 200-600 mPa·s, dissolve it in 150 mL of 1% w / v acetic acid solution to prepare a 0.5% w / v chitosan solution, and add the chitosan solution to the sodium selenite solution dropwise, stirring to dissolve; take another 7.045 g of vitamin C and dissolve it in 400 mL of Milli-Q ultrapure water as a reducing agent, and slowly add it to the above mixture, stirring while adding; after the addition is completed, the reaction system is placed in a constant-temperature shaker at 4°C for 24 hours, and continuous shaking is maintained to promote complete reaction; after the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8000 Da, and dialyzed in Milli-Q ultrapure water for 24 hours, with the dialysis solution being replaced every 6 hours to remove small molecular impurities; after dialysis, the dialysis product is collected as the synthesized Se nps; (3) Preparation of OMVs-loaded Se nps biological agent: mix 0.3 mL of Se nps at 5 μg / mL with 0.7 mL of OMVs solution at 5 μg / mL; place the mixture in an ultrasonic cell disruptor and treat it at a power of 50 W for 3 minutes to temporarily disturb the outer membrane vesicle structure and promote the encapsulation of Se nps; after ultrasonic treatment, the mixture is centrifuged at 6000×g and 4°C for 15 minutes to remove the unloaded free Se nps; After centrifugation, the precipitate is resuspended with sterile PBS to obtain the target product OMVs@Se.

3. The method of preparation of OMVs@Se bioactive formulation as claimed in claim 2, wherein: In step (1), 1 mL of sterile PBS is added to resuspend the collected OMVs, which are gently blown and mixed to obtain a uniform OMVs suspension; the product is inoculated on a TSA plate to evaluate sterility; its morphology, particle size distribution, and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); and the protein concentration of the OMVs is quantified by BCA protein box.

4. The process for the preparation of OMVs@Se bioactive formulation as claimed in claim 3, wherein: In step (2), its morphology, particle size distribution, and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the Se element is characterized by X-ray photoelectron spectroscopy (XPS); and after acid digestion, the Se element content in the nanoparticles is determined by inductively coupled plasma emission spectrometer (ICP-OES).

5. The method of preparation of OMVs@Se bioactive formulation as claimed in claim 4, wherein: In step (3), its morphology, particle size distribution, and surface potential are characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the Se element is characterized by X-ray photoelectron spectroscopy (XPS); and after acid digestion, the Se element content in the complex is determined by inductively coupled plasma emission spectrometer (ICP-OES) to calculate the loading efficiency.

6. Use of OMVs@Se bioactive formulation according to claim 1, characterized by the fact that it is used as a prophylactic and / or therapeutic agent for the treatment of diseases caused by bacteria of the genus Helicobacter. It is used for inhibiting macrophage ferroptosis.

7. Use of OMVs@Se bioactive formulation according to claim 1, characterized by the fact that it is used as a prophylactic and / or therapeutic agent for the treatment of diseases caused by bacteria of the genus Helicobacter. It is used for inhibiting excessive release of inflammatory factors and promoting recovery of anti-inflammatory signals.

8. Use of OMVs@Se bioactive formulation according to claim 1, characterized by the fact that it is used as a prophylactic and therapeutic agent for the treatment of diseases caused by bacteria of the genus Helicobacter. It is used for improving mitochondrial membrane potential damage and maintaining mitochondrial function stability.

9. Use of OMVs@Se bioactive formulation according to claim 1, characterized by the fact that it is used as a prophylactic and / or therapeutic agent for the treatment of diseases caused by bacteria of the genus Helicobacter. It is applied to the preparation of a drug for treating bacterial pneumonia.

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