Inflammation-targeting bionic extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses an inflammation-targeting bionic extracellular vesicle as well as a preparation method and application thereof, and particularly relates to the field of medicines. According to the invention, a new generation of aminoglycoside antibiotic teicoplanin (Teic), which is high in albumin binding rate and weak in intracellular antibacterial effect, is loaded on neutrophil-derived extracellular vesicles (NEV) with natural drug carrying capacity and inflammatory response chemotactic capacity to prepare the drug-loaded vesicles (Teic-NEV), wherein the new generation of aminoglycoside antibiotic teicoplanin (Teic) is used for loading the neutrophil-derived extracellular vesicles (NEV). The drug-loaded extracellular vesicle Teic-NEV can be used for targeted therapy of cells and tissues infected by MRSA (Methicillin-Resistant Staphylococcus Aureus). In vitro, the Teic (at) NEV can be used for destroying MRSA mature biological membranes and killing intracellular MRSA. According to the invention, the in-vivo and in-vitro biological safety of the TeicatNEV is evaluated. Therefore, the invention provides a certain theoretical basis for the application of NEV-based drug delivery in the field of intracellular bacteria treatment, and also provides a new treatment strategy for in-vivo targeted treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical materials, and more specifically, relates to an inflammation-targeted bionic extracellular vesicle and a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is an important and potentially fatal opportunistic pathogen that exists in about one-third of the population and is a zoonotic pathogen. S. aureus is one of the main pathogens causing hospital-acquired and community-acquired infections. It can cause a variety of bacterial infectious diseases including skin and wound infections, meningitis, pneumonia, endocarditis and osteomyelitis. In severe cases, it can cause sepsis and even threaten life. In recent decades, due to the widespread use of antibiotics, drug-resistant bacteria have increased, and the incidence of infectious diseases caused by drug-resistant bacteria has been on the rise worldwide. After the first report of methicillin-resistant Staphylococcus aureus (MRSA) in 1961, it spread rapidly, had a high infection rate, strong virulence, lacked effective treatment, had a high recurrence rate and a high mortality rate. It is reported that the number of deaths caused by MRSA infection in the United States has exceeded the number of deaths from AIDS.

[0003] In order to adapt to the living environment, bacteria can adhere to the surface of non-living or active tissues and grow, forming a dense biofilm by producing a sticky and uneven bacterial extracellular matrix, which improves the drug resistance and pathogenicity of bacteria and helps bacteria escape the attack of host immune cells. Once formed, the biofilm is resistant to various bactericidal treatments, including the use of antibiotics. The polymer matrix of the biofilm can hinder the diffusion of antibiotics, making it more difficult for antibiotics to penetrate the biofilm to eliminate pathogens. The antibiotic resistance of bacteria in the biofilm may be 1,000 times that of free bacteria.

[0004] Although scientific researchers are constantly exploring new anti-infective drugs, it is still difficult to completely eliminate pathogenic bacteria due to the difficulty of water-soluble substances entering cells or bacterial biofilms, and it may even promote the emergence of new drug resistance. Therefore, there is an urgent need for a carrier that can effectively deliver drugs to treat diseases caused by stubborn bacterial infections.

[0005] Delivery based on extracellular vesicles (EVs) has great potential in replacing exosomes as drug delivery carriers. All cells can secrete EVs wrapped in phospholipid bilayers, which can be divided into three types: exosomes, microvesicles, and apoptotic bodies according to their different sizes or functions. The membrane components of EVs secreted by cells are similar to cell membranes, and compared with exosomes, cell membranes have a larger yield and are easier to obtain. Therefore, researchers believe that cell membranes can be used to artificially prepare EVs as drug carriers, and even in the process of EV preparation, cell membranes can be engineered and modified in various ways such as physical, chemical or biological methods in order to achieve better drug delivery and therapeutic effects. The method currently used more in research is the liposome extrusion method, which is to prepare nanoscale EVs by continuously extruding the cell membrane purified by centrifugation through a polycarbonate membrane with gradient pores.

[0006] The chemotactic ability of inflammatory cells to the inflammatory or tumor microenvironment makes them a carrier for targeted delivery. For example, macrophage-mediated drug systems have been successfully used in the treatment of inflammatory diseases and cancer. However, macrophages are rare, accounting for only 5% of white blood cells, which is not enough for drug delivery. Their complex process preparation and invasiveness further limit the use of macrophage-mediated drug systems. Some researchers have proposed that neutrophils (Neu) may be used for cell-based drug delivery, because Neu is not only the most abundant white blood cell in mammalian blood circulation, accounting for 50-70% of the total white blood cells in the blood circulation, but also the main type of white blood cell recruited to the inflammatory site during infection and cancer development. During the inflammatory response, Neu is the first white blood cell recruited to the inflammatory site. Vascular endothelial cells are first activated when an inflammatory response occurs, and their adhesion molecule expression is upregulated. Neu cells adhere to activated endothelial cells and then roll and crawl along the blood vessels to activate. Activated Neu crosses the vascular barrier and migrates to the inflammatory site. Due to their ability to rapidly respond to inflammation and their natural ability to penetrate physiological barriers, Neu cells are used for drug delivery targeted to the inflammatory microenvironment, and Neu cell-based therapeutic strategies have great potential in the treatment of inflammation and cancer. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides an inflammation-targeted bionic extracellular vesicle and a preparation method and application thereof.

[0008] According to the first aspect of the present invention, the present invention provides an inflammation-targeted bionic extracellular vesicle, which is composed of a bionic extracellular vesicle derived from a neutrophil and a drug loaded therein. According to a preferred embodiment of the present invention, in the bionic extracellular vesicle, the drug loaded is preferably teicoplanin (Teic), which is a new generation of aminoglycoside antibiotics with high albumin binding rate and weak intracellular antibacterial effect.

[0009] According to a preferred embodiment of the present invention, in the bionic extracellular vesicles, the concentration of teicoplanin (Teic) is 1 μg / mL to 1 g / mL.

[0010] According to a preferred embodiment of the present invention, in the bionic extracellular vesicles, the mass ratio of neutrophil membrane to drug is 100:1 to 1:4000.

[0011] According to a preferred embodiment of the present invention, the drug loading amount of the drug is 1wt%-90wt%.

[0012] In a second aspect, the present invention provides a method for preparing the bionic extracellular vesicles, comprising:

[0013] 1) Extracting peripheral blood from mice with aseptic inflammation; separating neutrophils expressing chemotaxis-related proteins from the peripheral blood by density gradient separation; and further separating and obtaining the cell membrane of the neutrophils;

[0014] 2) Adding drugs to the product of step 1) and obtaining biomimetic extracellular vesicles derived from neutrophil membranes with complete structures by liposome extrusion.

[0015] Furthermore, the sterile inflammation mice refer to mice with inflammation without pathogen infection caused by extracts of pathogens such as lipopolysaccharide, such as mice with lipopolysaccharide-induced peritonitis.

[0016] The filter membrane used for liposome extrusion in step 2) is a hydrophilic polycarbonate membrane of 50 nm-5 μm; the temperature is 0-4° C. The liposome extrusion in step 2) includes but is not limited to manual, pneumatic and electric liposome extruders.

[0017] Specifically, according to an optional embodiment of the present invention, the step 1) includes the following steps:

[0018] 1.1) Peripheral blood was collected from ICR mice with lipopolysaccharide (LPS)-induced peritonitis using the retro-orbital venous plexus blood sampling method.

[0019] 1.2) Activated neutrophils were isolated from the peripheral blood of ICR mice using a peripheral blood neutrophil isolation kit.

[0020] 1.3) Use hypotonic solution and Dounce homogenizer to separate the plasma membrane, and collect the cell membrane by ultracentrifugation.

[0021] In a third aspect, the present invention provides an inflammation-targeting material, wherein the material comprises the aforementioned bionic extracellular vesicles.

[0022] In a fourth aspect, the present invention provides an inflammation-targeted drug, wherein the antibacterial drug comprises the aforementioned antibacterial bionic extracellular vesicles.

[0023] In a fifth aspect, the present invention provides an immunomodulator, wherein the immunomodulator comprises the aforementioned biomimetic extracellular vesicles.

[0024] In a sixth aspect, the present invention provides a use of the aforementioned bionic extracellular vesicles in the preparation of a drug for bacterial pneumonia.

[0025] Preferably, the bacterial pneumonia is caused by methicillin-resistant Staphylococcus aureus infection.

[0026] Preferably, the biomimetic extracellular vesicles are used as drugs / materials in vivo and / or in vitro in humans and / or animals.

[0027] Preferably, the biomimetic extracellular vesicles are used as MRSA bacterial biofilm inhibitors.

[0028] Preferably, the bionic extracellular vesicles are used as materials for regulating the immune microenvironment of the lungs of pneumonia mice.

[0029] Specifically: Neutrophils have a self-limiting "swarming" behavior. When local neutrophils gather to form neutrophil clusters, local attractants such as chemokine CXC ligand 2 (CXCL2) and leukotriene B4 (LTB4) activate G protein-coupled receptor kinase 2 (GRK2), inducing GPCR desensitization and the cessation of "swarming" behavior. This feedback mechanism allows neutrophils to self-limit their aggregation behavior when forming a group and ensure optimal elimination of bacteria. However, the specific action pathway of this feedback mechanism is still unknown. This may be related to the mutual communication between neutrophils themselves and the interaction of membrane proteins. Therefore, the use of bionic materials derived from neutrophil cell membranes has great potential for early restriction of "swarming" behavior.

[0030] Preferably, the biomimetic extracellular vesicles derived from neutrophil membranes separated in the present invention highly express neutrophil membrane proteins, especially proteins that escape macrophage phagocytosis and chemotaxis-related proteins.

[0031] Neutrophil membrane proteins mainly include: cytoskeleton β-actin (β-actin), cell membrane sodium-potassium pump (Na-K-ATPase), integrin-associated protein (IAP, CD47) related to Neu mature chemotactic ability, protein integrin-β2 (integrin-β2), L-selectin (L-selectin), chemokine receptor 4 (chemokine receptor 4, CXCR-4), adhesion factor integrin CD11c, also known as integrin αX (integrin AlphaX), vascular cell adhesion molecule 1 (vascular cell adhesion molecule 1, VCAM-1).

[0032] Preferably, the isolated neutrophil membrane-derived biomimetic extracellular vesicles hardly express cytoskeletal proteins such as β-actin.

[0033] Preferably, when the encapsulated drug is the antibiotic teicoplanin (Teic), the bionic extracellular vesicle is denoted as Teic@NEV, and the particle size range of the bionic extracellular vesicle (Teic@NEV) is 100 to 600 nm; preferably, the average particle size of the material is 179.9±232.4 nm.

[0034] Preferably, the Zeta potential range of the bionic extracellular vesicle (Teic@NEV) is -10 to -30 mV, and the Zeta potential value of the material is -19.07±0.6766 mV.

[0035] The bionic extracellular vesicles have sustained release ability. In a constant temperature shaker at 37°C and 80rpm, Teic@NEV can effectively achieve the sustained release of Teic. The release amount in 5 hours is about 25%, about 80% can be released in 20 hours, and the total release amount exceeds 90% in 40 hours.

[0036] The bionic extracellular vesicles of the present invention have the ability to treat MRSA pneumonia, and the bionic extracellular vesicles can increase the survival rate of mice with acute severe pneumonia caused by MRSA from 10%-20% to 60%-70%.

[0037] The beneficial effects of the present invention are as follows: the present invention designs a bionic extracellular vesicle with inflammation targeting. This targeted material can avoid tissue inflammation damage that may be caused by elastase in neutrophils while utilizing the chemotactic properties of neutrophils. In addition, the present invention uses bionic extracellular vesicles derived from neutrophils with high biocompatibility, strong targeting ability, and the ability to persist in the circulatory system as drug carriers to encapsulate antibiotics with weak cell penetration ability and unable to achieve effective intracellular antibacterial ability, and prepares a new bionic extracellular vesicle Teic@NEV with targeted delivery ability, which effectively improves the targeting, effectiveness and biosafety of antibiotics. The weight loss and body temperature drop caused by MRSA pneumonia in mice can be significantly improved by significantly reducing the dosage of antibiotics. At the same time, Teic@NEV treatment can significantly reduce the bacterial load in mouse lung tissue and bronchoalveolar lavage fluid, significantly reduce pulmonary edema and pulmonary congestion caused by MRSA pneumonia, reduce the lung wet / dry weight ratio, and reduce the intensity of inflammatory response in lung tissue and the degree of lung damage. Through the study of alveolar lavage fluid, it was found that Teic@NEV treatment can significantly reduce the concentration of white blood cells in alveolar lavage fluid, reduce the chemotaxis and aggregation of neutrophils in lung tissue, and alleviate the inflammatory response caused by infection. Therefore, the Teic@NEV of the present invention can effectively and accurately treat MRSA pneumonia in mice, and has great potential to become a clinical material for the treatment of acute bacterial pneumonia, and has strong clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The particle size, transmission electron microscopy and Zeta potential characterization results of Teic@NEV;

[0039] Figure 2 Characterization of NEV-specific proteins and determination of the sustained release capacity of Teic@NEV;

[0040] Figure 3 The results of the test of Teic@NEV's ability to kill free bacteria and intracellular bacteria in vitro;

[0041] Figure 4 To test the ability of Teic@NEV to destroy mature MRSA biofilm;

[0042] Figure 5 Evaluation results of Teic@NEV’s ability to target inflammatory sites;

[0043] Figure 6 To determine the ability of Teic@NEV to reduce MRSA load in lung tissue;

[0044] Figure 7 To evaluate the efficacy of Teic@NEV in treating MRSA pneumonia in mice;

[0045] Figure 8 Teic@NEV inhibits Neu aggregation and characterizes the level of negative regulation of immunity;

[0046] Fig. 9 Biosafety determination of Teic@NEV. DETAILED DESCRIPTION

[0047] The invention is further described below in conjunction with examples and drawings. In this example, the preparation of inflammation-targeted bionic extracellular vesicles and in vitro and in vivo antibacterial data and in vivo treatment of MRSA pneumonia data are used to illustrate the antibacterial and pneumonia treatment effects.

[0048] Embodiment 1:

[0049] The biomimetic extracellular vesicle Teic@NEV has excellent in vitro bactericidal and anti-biofilm capabilities.

[0050] (1) Preparation and physical characterization of Teic@NEV biomimetic extracellular vesicles

[0051] Neu cells were extracted from the peripheral blood of ICR mice with LPS-induced peritonitis by density gradient centrifugation. The cells were resuspended in pre-cooled hypotonic lysis buffer, and after repeated homogenization and ultracentrifugation, the final precipitate was collected and used to purify Neu membrane for subsequent experiments. The purified Neu membrane was gradient extruded using a jacketed temperature-controlled liposome extruder to prepare EVs. Teic solutions of different concentrations were prepared, mixed with Neu cell membranes at a certain mass concentration ratio, incubated and sonicated, and then extruded and centrifuged in the same manner as above to prepare NEV (Teic@NEV) solutions encapsulated with Teic. Figure 1 As shown in Figure A, the average particle size of Teic@NEV is 179.9±2.324nm, and the structure is intact with a complete lipid bilayer; its Zeta potential value is -19.07±0.6766mV ( Figure 1 B), indicating that their surfaces are all negatively charged. The above results are basically consistent with the exosome or extracellular vesicle data reported in the literature and are in line with research expectations.

[0052] (2) Physiological characterization and sustained release assay of Teic@NEV biomimetic extracellular vesicles

[0053] like Figure 2 As shown in A, the SDS-PAGE analysis showed that the NEV protein composition is basically membrane protein, and the expression of adhesion and chemotaxis-related proteins is higher than that of the total protein of whole cell lysis. At the same time, the cell membrane proteins that mediate cell migration and chemotaxis are almost not expressed or only expressed in small amounts in the cytoplasm. Based on the above protein concentration determination, it can be inferred that NEV concentrates more membrane proteins than the total protein of Neu cells, and contains almost no cytoskeletal proteins such as β-actin. Figure 2 The release experiment of B showed that Teic@NEV can be effectively released under simulated physiological conditions (PBS solution at 37°C).

[0054] (3) Determination of the antibacterial ability of Teic@NEV biomimetic extracellular vesicles in vitro

[0055] The MIC of Teic@NEV against MRSA bacteria was determined by the doubling dilution method, and the same concentration of Teic was used as a control to obtain the bactericidal curve. Figure 3 It can be clearly seen from A in that the MIC of Teic@NEV is about 2μg / mL, which is consistent with the MIC concentration of Teic, that is, at 24h, the bactericidal performance of Teic@NEV is not significantly different from that of free Teic. In order to more accurately determine the therapeutic effect of Teic@NEV on intracellular MRSA infection, we also used flow cytometry to characterize the number of cells infected with intracellular MRSA. The results showed that the positive rate of intracellular GFP-MRSA infection in macrophages treated with Teic@NEV decreased, while the positive rate of intracellular GFP-MRSA infection in macrophages treated with free Teic was still high ( Figure 3 B).

[0056] (4) Determination of the antibacterial ability of Teic@NEV biomimetic extracellular vesicles in vitro

[0057] To evaluate the effect of Teic@NEV on mature MRSA biofilm, PBS, NEV, Teic, and Teic@NEV were used to intervene in mature MRSA biofilm, with PBS as the negative control group. Figure 4 As can be seen from Figure A), both Teic and Teic@NEV can effectively reduce the damage to mature biofilms. Compared with free Teic, Teic@NEV loaded with NEV cell membrane has stronger ability to damage biofilms. The absorbance detection ( Figure 4 B), it was found that the absorbance of the biofilm after Teic treatment at OD 590nm was 0.42±0.022, which was significantly lower than that of the PBS group (0.72±0.059) and the NEV group (0.66±0.113). The Teic@NEV treatment group was better than the Teic group alone, with an absorbance of 0.20±0.013. In order to more intuitively characterize the effect of Teic@NEV on mature MRSA biofilm, CLSM was used to reconstruct the biofilm in 3D to characterize the thickness of the biofilm and the life and death of the bacteria inside. Figure 4As shown in Figure C, it can be found that the biofilm thickness of Teic and Teic@NEV treatments was significantly reduced compared with the control group. The biofilm thickness of the Teic@NEV treatment group was smaller than that of the free Teic group, and its internal red fluorescence signal (PI, dead bacteria) was more, indicating that Teic@NEV has a stronger ability to destroy MRSA biofilm than free Teic.

[0058] Embodiment 2:

[0059] The biomimetic extracellular vesicle Teic@NEV has excellent in vivo lung inflammation targeting and immunomodulatory capabilities and can effectively treat MRSA pneumonia in mice.

[0060] (1) Targeting of Teic@NEV biomimetic extracellular vesicles to lung inflammation in vivo

[0061] Cy7-Teic loaded with NEV accumulated more in the MRSA-infected lung tissue. At 8 h, the fluorescence signal in the lung tissue of mice in the Cy7-Teic@NEV-treated group was significantly stronger than that in the lung tissue of mice in the free Cy7-Teic-treated group. At the same time, Cy7-Teic@NEV allowed Cy7-Teic to stay longer in the infected lung tissue, and there was still a relatively obvious fluorescence signal at 24 h ( Figure 5 A in Figure 5 As shown in Figure B, fluorescence imaging of the main organs of mice was performed 24 hours after drug injection. The fluorescence signals of mice treated with Cy7-Teic@NEV were mainly located in the lungs, with a small amount of signals located in the kidneys and gallbladder, while the fluorescence signals of mice treated with free Cy7-Teic were mainly distributed in the liver and kidneys. HPLC detection of Teic concentration in lung tissue at different times showed that Teic@NEV could maintain high Teic concentration for a longer period of time than Teic ( Figure 5 C), indicating that NEV encapsulation can increase the accumulation of Teic in the MRSA-infected lungs while reducing drug concentrations in the liver and kidneys, reducing potential drug toxicity and side effects and improving treatment outcomes.

[0062] To further explore whether the ability of Teic@NEV to transport Teic to lung tissue after MRSA infection is related to inflammation caused by infection, we used normal mice (control group, denoted as Ctrl) and MRSA-infected pneumonia mice (experimental group, denoted as Pneumonia) as animal models, injected Teic@NEV into the tail vein, and continuously detected its blood drug concentration. Figure 5As shown in D, the blood drug concentration of uninfected mice was significantly higher than that of infected mice, but the Teic concentration in the lung tissue of pneumonia mice was significantly higher than that of uninjected mice 48 hours after injection, suggesting that the lung tissue targeting of Teic@NEV is related to MRSA infection. In order to explore the relationship between the targeting of Teic@NEV and its specific protein, we prepared Teic@RVC, a drug delivery biomimetic vesicle encapsulated with Teic by red blood cell membrane, in a similar way. For MRSA pneumonia mice ( Figure 5 E), the blood concentration of Teic@RVC was higher than that of the free form of Teic at 6h, 12h and 24h, indicating that cell membrane encapsulation can increase the blood concentration of Teic and increase its retention time in the systemic circulation. However, the blood concentration of Teic@NEV was lower than that of Teic@RVC at 6h, 12h and 24h, which suggests that the Teic carried by Teic@NEV may be mainly retained in the inflammatory site.

[0063] (2) Teic@NEV reduces bacterial load in the lungs of mice with MRSA pneumonia

[0064] After the fluorescently labeled MRSA pneumonia model was established, the mice were imaged in vivo before treatment and 2h, 4h, and 6h after treatment to investigate the bacterial load in the lungs. Figure 6 As shown in A, before treatment, the fluorescence signals in the lungs of mice were similar, and after treatment, the fluorescence signals in the lungs were reduced. The fluorescence intensity of bacteria in the lungs of the Teic@NEV group was more significantly reduced than that of the free Teic group, indicating that Teic@NEV is more effective than free Teic in treating MRSA infection in the lungs and can quickly reduce the bacterial load in the lungs. The bacterial load of lung tissue homogenates at 24h and 48h after treatment was measured, and Teic@NEV can significantly reduce the live bacterial load in the lung tissue ( Figure 6 B and C in ).

[0065] (3) Teic@NEV reduced the degree of lung tissue damage in mice with MRSA pneumonia and significantly improved the survival rate.

[0066] We analyzed the survival of MRSA-infected mice. Figure 7 As shown in A, compared with the PBS group and the NEV group, the survival of the Teic and Teic@NEV treatment groups was better, and the survival of Teic@NEV was the best, that is, Teic@NEV can significantly improve the survival rate of MRSA pneumonia mice.

[0067] In order to visually observe the changes in the lungs of mice with MRSA pneumonia after different treatments, we took pictures of the lungs of mice. Figure 7As shown in Figure B, the lung tissues of mice in each group showed different degrees and ranges of congestion, edema and other lung tissue consolidation manifestations. Among them, the lung tissue damage area of ​​mice in the Teic@NEV group was the most limited. The body temperature of mice in each group within 48 hours after treatment ( Figure 7 C) and weight ( Figure 7 Dynamic measurements of Teic@NEV in D) showed that the treatment with Teic@NEV could significantly improve the body temperature and weight loss of mice and maintain the stability of their vital signs. In order to evaluate the edema of the lung tissue of MRSA pneumonia mice after various treatments, the edema of the lung tissue of MRSA pneumonia mice was measured at 24h and 48h after treatment ( Figure 7 (E and F) The dry-to-wet weight ratio of lung tissue was measured, and it can be seen that Teic@NEV treatment can alleviate pulmonary edema.

[0068] In order to study the leukocyte infiltration in the alveoli and the severity of inflammatory response in mice with MRSA pneumonia after different treatments, we performed alveolar lavage on mice 24h and 48h after treatment and counted the total number of leukocytes in the alveoli ( Figure 5 G and H), Teic@NEV was able to reduce the concentration of leukocytes, suggesting that Teic@NEV effectively inhibited the severity of infection and may have immunomodulatory ability. Figure 5 H&E staining of lung tissue sections in Figures I and J also showed that Teic@NEV can reduce the degree of lung tissue damage.

[0069] (4) Teic@NEV has immunomodulatory capabilities, which can inhibit the “swarming” behavior of Neu in the lungs and reduce Neu accumulation in lung tissues

[0070] The proportion of Neu in leukocytes in bronchoalveolar lavage fluid was determined by flow cytometry, and it was found that after 24 hours of treatment, the proportion of Neu in BALF in the Teic@NEV-treated group decreased by nearly 10% ( Figure 8 Myeloperoxidase (MPO) immunohistochemical staining of lung tissue sections also confirmed that Teic@NEV can significantly reduce the accumulation of Neu in the lungs ( Figure 8 B).

[0071] The 10×Genomics Chromium Single-Cell 3'kit was used to perform single-cell analysis on mouse lung tissue suspension to characterize the changes in cell types and numbers in the lung tissue of mice with MRSA pneumonia after Teic@NEV treatment. The sequencing results were analyzed and interpreted using Illuminabcl2fastq, Cell Ranger, and Seurat software, and specific marker gene markers were used to focus on defining subpopulations of neutrophils in the lung tissue. Figure 8C in the figure suggests that Teic@NEV treatment can reduce Neu cell aggregation in the lung tissue of mice with MRSA pneumonia and inhibit the inflammatory response in the lung.

[0072] Embodiment 3:

[0073] The biomimetic extracellular vesicle Teic@NEV has good biosafety.

[0074] The body weight of mice was measured one week after Teic@NEV injection, and the blood routine and liver and kidney function of mice were tested 7 days after injection. The heart, liver, spleen, lung and kidney were examined by H&E section examination. Fig. 9 , indicating that Teic@NEV has no obvious damage to the functions of various organs of the body.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. An inflammation-targeted biomimetic extracellular vesicle, characterized in that: It consists of neutrophil-derived biomimetic extracellular vesicles and drugs loaded therein.

2. The biomimetic extracellular vesicle according to claim 1, characterized in that In the bionic extracellular vesicles, the drug contained may be the antibiotic teicoplanin (Teic), and the concentration thereof is 1 μg / mL to 1 g / mL.

3. The biomimetic extracellular vesicle according to claim 1, characterized in that In the bionic extracellular vesicles, the mass ratio of neutrophil membrane to drug is 100:1 to 1:4000.

4. The biomimetic extracellular vesicle according to claim 1, characterized in that The drug loading amount is 1wt%-90wt%.

5. A method for preparing the biomimetic extracellular vesicles according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) Extracting peripheral blood from mice with sterile inflammation; separating neutrophils expressing chemotaxis-related proteins from the peripheral blood by density gradient separation; and further separating and obtaining the cell membrane of the neutrophils; 2) Adding drugs to the product of step 1) and obtaining biomimetic extracellular vesicles derived from neutrophil membranes with complete structures by liposome extrusion.

6. The method according to claim 5, characterized in that The filter membrane used for liposome extrusion in step 2) is a hydrophilic polycarbonate membrane of 50 nm-5 μm; the temperature is 0-4°C.

7. A material, characterized in that: The material comprises the bionic extracellular vesicles according to any one of claims 1 to 4.

8. An immunomodulator, characterized in that: The immunomodulator comprises the biomimetic extracellular vesicles according to any one of claims 1 to 4.

9. An inflammation targeting material, characterized in that: The inflammation targeting material comprises the bionic extracellular vesicle according to any one of claims 1-4.

10. Use of the bionic extracellular vesicle according to any one of claims 1 to 4 or the bionic extracellular vesicle prepared by the method according to claim 5 or 6 in preparing a drug for treating bacterial pneumonia.

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