Hybrid membrane bionic nanoparticles and preparation method thereof and antibacterial and vaccine applications

Abstract

The present invention relates to a hybrid membrane bionic nanoparticle and a preparation method and application thereof, wherein the hybrid membrane bionic nanoparticle is a core-shell structure, wherein the core is a lipid nanoparticle, and the shell is a hybrid membrane; wherein the hybrid membrane is a hybrid membrane formed by the fusion of an immune cell membrane vesicle and a bacterial outer membrane vesicle; and the lipid nanoparticle is loaded with an antibiotic. The hybrid membrane bionic nanoparticle inherits the functions of targeting inflammatory sites and targeting homologous bacteria and being easily phagocytosed by bacteria, tends to the inflammatory sites caused by bacterial infection in the systemic circulation, and is then preferentially taken up by homologous bacteria, thereby achieving efficient delivery of antibiotics. The present invention shows good therapeutic and preventive effects in a mouse model of bacterial infection, and may become a promising strategy for combating Gram-negative bacterial infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a hybrid membrane bionic nanoparticle and a preparation method thereof and antibacterial and vaccine applications. Background Art

[0002] Multidrug-resistant bacteria refer to bacteria that are resistant to three or more drugs. They are highly contagious and lethal, and are a thorny issue in today's medical field. In the list of global priority antibiotic-resistant bacteria published by the World Health Organization, the highest-level resistant bacteria are all Gram-negative bacteria. Therefore, there is an urgent need to develop safe and effective therapies to prevent and treat bacterial infections, especially Gram-negative infections.

[0003] Currently, antibiotics are the main strategy to combat bacterial infections. They exert their antibacterial effects by interfering with or disrupting essential processes for bacterial growth, such as the synthesis of cell walls and cell membranes, and the production of DNA, RNA, or important proteins. Antibiotics have shortcomings in systemic circulation, such as high blood clearance and low levels at sites of bacterial infection. Therefore, it is necessary to increase the dosage of antibiotics to improve the antibacterial effect, especially for drug-resistant bacteria. However, this will also bring about greater biological toxicity. Therefore, increasing the accumulation of antibiotics at sites of bacterial infection, thereby enhancing bacterial phagocytosis of antibiotics, thereby achieving efficient antibacterial and good biosafety, is an important solution.

[0004] Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria retain the characteristic proteins of the original bacteria, display various pathogen-associated molecular patterns (PAMPs), and have functions such as specific localization to specific receptors and activation of the host's immune response. In the field of antibacterial, OMVs are used to encapsulate antibiotics, deliver drug nanoparticles, or as antibacterial vaccines, etc. Studies have found that OMVs of Gram-negative bacteria Escherichia coli can be used to encapsulate nanoparticles loaded with the antibiotic rifampicin, and achieve efficient intrabacterial delivery of antibiotics through bacterial homologous targeting and good membrane fusion properties. However, OMVs lack the ability to actively target bacterial infection sites in the blood circulation, and their surface antigens are easily recognized by the immune system, thereby accelerating the blood clearance of the antibacterial system coated by them, limiting their antibacterial function in vivo.

[0005] Neutrophils are one of the white blood cells in the blood, accounting for 50-70% of the total white blood cells, and are the first line of host defense against malignant tumors and invasive pathogens. In inflammatory diseases, neutrophils are activated and continuously recruited to the inflamed site under the stimulation of specific adhesion molecules (such as ICAM-1, VCAM-1, P-selectin and E-selectin). With the rise of cell membrane biomimetic nanocarriers, researchers have found that neutrophil membrane vesicles (NMVs) also have similar chemotactic functions and can continuously target inflammatory sites. For example, the NMVs developed by Dong et al. can specifically deliver the therapeutic agent ResolvinD2 (RvD2) to the inflamed brain endothelium, significantly reducing the inflammatory response of ischemic stroke and improving the neurological function of mice. Wang et al. used NMVs to co-deliver the antibiotic ceftazidime and the anti-inflammatory drug RvD1. The targeted aggregation of NMVs to the infected inflammatory part enhanced the inflammation resolution and bacterial killing effects of these two drugs in the Pseudomonas aeruginosa peritoneal infection model. However, the technical effects of existing technologies are not ideal, and it is difficult to achieve the purpose of step-by-step targeted antibacterial and efficient prevention of bacterial infection.

[0006] Therefore, it is urgent to provide new technical solutions based on existing reports and develop a nano-drug delivery system that can efficiently accumulate at the site of bacterial infection and be engulfed by bacteria, so as to achieve efficient delivery of antibiotics. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the present invention provides a hybrid membrane bionic nanoparticle and its preparation method and application. First, lipid nanoparticles (LNPs) loaded with quinolone antibiotics-norfloxacin (Nor) are synthesized by aqueous solvent diffusion method; then, NMVs and OMVs secreted by bacteria are extracted respectively, and the two membranes are fused by ultrasound and extrusion to form hybrid membrane vesicles (HMVs); finally, HMVs are coated on the surface of LNPs to obtain hybrid membrane bionic nanoparticles (LNP@HMVs). LNP@HMVs inherit the functions of NMVs targeting inflammatory sites and OMVs targeting homologous bacteria and being easily phagocytosed by bacteria. LNP@HMVs will first tend to the inflammatory sites caused by bacterial infection in the systemic circulation, and then further be preferentially taken up by homologous bacteria at the inflammatory sites, thereby realizing the efficient delivery of norfloxacin. In addition, the antigens of OMVs are retained on LNP@HMVs, which can activate the production of specific immunity against bacteria in the body. LNP@HMVs showed good therapeutic and preventive effects in mouse models of bacterial infection and may become a promising strategy to combat Gram-negative bacterial infections.

[0008] One object of the present invention is to provide a hybrid membrane bionic nanoparticle, wherein the hybrid membrane bionic nanoparticle is a core-shell structure, wherein the core is a lipid nanoparticle and the shell is a hybrid membrane;

[0009] in,

[0010] The hybrid membrane is a hybrid membrane formed by the fusion of immune cell membrane vesicles and bacterial outer membrane vesicles;

[0011] The lipid nanoparticles are loaded with antibiotics.

[0012] Furthermore, the hybrid membrane bionic nanoparticles contain antigens of bacterial outer membrane vesicles, and the immune cells are neutrophils.

[0013] Furthermore, the antibiotic is a quinolone antibiotic.

[0014] Furthermore, the hybrid membrane bionic nanoparticles have a particle size of 180 to 210 nm and a zeta potential of -11 to -13 mV.

[0015] Another object of the present invention is to provide a method for preparing the hybrid membrane bionic nanoparticles, comprising the following steps:

[0016] S1. Extract immune cell membrane vesicles and bacterial outer membrane vesicles;

[0017] S2. mixing the immune cell membrane vesicles with the bacterial outer membrane vesicles, and fusing them by ultrasound and extrusion to form a hybrid membrane;

[0018] S3. Preparation of lipid nanoparticles containing antibiotics;

[0019] S4. After mixing the lipid nanoparticles and the hybrid membrane, the mixture is subjected to ultrasound and extrusion treatment to obtain a product.

[0020] Furthermore, in step S1, the method for extracting immune cell membrane vesicles comprises the following steps:

[0021] Human myeloid leukemia cells are cultured in a culture medium containing a mixture of fetal bovine serum and penicillin-streptomycin, and then dimethyl sulfoxide and lipopolysaccharide are added for culture. After the immune cells are collected by centrifugation, they are ground and centrifuged. The supernatant is collected and centrifuged again and washed to obtain immune cell membrane vesicles.

[0022] Furthermore, in step S1, the method for extracting bacterial outer membrane vesicles comprises the following steps:

[0023] After culturing the bacterial strain, the supernatant is collected by centrifugation, the supernatant is centrifuged again, and the precipitate is collected and washed to obtain the bacterial outer membrane vesicles.

[0024] Furthermore, in step S2, the protein mass ratio of the immune cell membrane vesicles and the bacterial outer membrane vesicles is (1-1.5):1.

[0025] Furthermore, in step S3, the method for preparing lipid nanoparticles comprises the following steps:

[0026] Glyceride is dissolved in a solvent, and then antibiotics are added, heated, stirred, and ultrasonically treated to obtain an oil phase; an emulsifier is added to water, heated, stirred, and an aqueous phase is obtained; the oil phase is added to the aqueous phase, heated, stirred, and cooled to solidify to obtain the lipid nanoparticles.

[0027] Furthermore, the bacteria is preferably Escherichia coli.

[0028] Furthermore, the specific preparation method of the hybrid membrane bionic nanoparticles comprises the following steps:

[0029] First, neutrophil membrane vesicles NMVs and Escherichia coli outer membrane vesicles OMVs were extracted. The method for isolating NMVs was as follows: human myeloid leukemia cells (HL-60) were cultured in RPMI1640 medium containing a mixture of 20% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin. 1.3% dimethyl sulfoxide (DMSO) was added for stimulation and culture for 3 days, and then lipopolysaccharide (LPS, 1 μg mL -1 ) were co-incubated for 4 hours to induce cell differentiation, and then low-speed centrifugation (1000rpm, 3min) was used to collect neutrophil-like cells. The cells were dispersed in a hypotonic lysis buffer (containing 30mM Tris-HCl (pH 7.5), 225mM D-mannitol, 75mM sucrose, 0.5mM EDTA, 0.5% bovine serum albumin and 1% enzyme inhibitor). In an ice bath environment, the cells were ground using a Dounce homogenizer with a tight pestle. The ground solution was centrifuged at 4°C (3000g, 5min) to remove the cell precipitate and collect the supernatant. The supernatant was centrifuged again (20000g, 1h) to collect the NMVs precipitate, washed with phosphate buffered saline (PBS), and finally dispersed in 1mL PBS and stored at -80°C. The method for isolating and obtaining OMVs is as follows: the Escherichia coli (E.coli) strain was added to the MHB liquid culture medium and shaken for 24h (37°C, 220rpm). The bacterial precipitate was removed by low-speed centrifugation (4500 g, 20 min), the supernatant was centrifuged again (100000 g, 1 h) to collect the precipitate, which was washed with PBS and finally dispersed in 1 mL of PBS and stored at -80°C.

[0030] Then, NMVs and OMVs were mixed at a 1:1 protein mass ratio and fused by sonication and extrusion to form HMVs.

[0031] Subsequently, LNPs containing norfloxacin were prepared by solvent diffusion method, and then HMVs were added and encapsulated by ultrasound and extrusion. The specific method is as follows: 15 mg of glyceryl monostearate (GMS) was dissolved in 1.5 mL of ethanol, 2.1 mg of norfloxacin was added, and stirring (400 rpm) and water bath ultrasound were alternated at 85 ° C until norfloxacin was completely dissolved to obtain an oil phase. A 0.1% poloxamer 188 aqueous solution (15 mg poloxamer 188 was dissolved in 15 mL of water) was prepared and stirred at 75 ° C (400 rpm) to obtain an aqueous phase. The oil phase was quickly injected into the aqueous phase, maintained at 75 ° C, and continued to stir at 400 rpm for 10 min. The prepared emulsion was left to stand in an ice bath environment for 1 h, cooled and solidified to form drug-loaded LNPs. NMVs and OMVs were mixed at a protein weight ratio of 1:1, and ultrasound was performed for 10 min in an ice bath (15%, 2s on / 3s off). Then, LNPs were added at a ratio of 1:1 between total protein mass and nanoparticle mass, and water bath sonication was performed for 5 min. LNP@HMVs were obtained by extruding through polycarbonate films with pore sizes of 800 nm and 400 nm using a liposome extruder. According to demand, 6% (m / v) trehalose can be added to LNP@HMVs, and the mixture was freeze-dried at low temperature for 24 h to obtain LNP@HMVs freeze-dried powder, which was then dispersed in PBS when used.

[0032] Another object of the present invention is to provide the use of the hybrid membrane bionic nanoparticles in antibacterial and anti-biofilm applications.

[0033] The present invention has the following beneficial effects:

[0034] The present invention innovatively designs a hybrid membrane bionic nanoparticle to deliver antibiotics. The hybrid membrane bionic nanoparticles show powerful in vivo treatment and in vivo prevention effects in terms of bacterial infection. They can target bacterial infection sites in the systemic blood circulation and then be engulfed by bacteria, achieving efficient delivery of antibiotics and thus improving the bactericidal effect of antibiotics, which is significantly better than simple antibiotic treatment or lipid nanoparticle formulations that encapsulate antibiotics. In addition, the present invention has the function of activating specific immunity and playing a role in preventing bacterial infection. The present invention hybridizes the cell membrane of immune cells with bacterial outer membrane vesicles, overcoming the problem that drug-loaded nanoparticles coated only by bacterial outer membrane vesicles are easily cleared in the blood circulation and difficult to reach the bacterial infection site, making the hybrid membrane bionic nanoparticles have good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of the preparation process of hybrid membrane bionic nanoparticles is shown.

[0036] Figure 2 The figure shows the characterization results of hybrid membrane biomimetic nanoparticles;

[0037] in,

[0038] Figure 2 A shows the transmission electron microscopy images of NMVs and OMVs;

[0039] Figure 2 B shows the fluorescence colocalization image of a mixture of HMVs and NMVs with OMVs;

[0040] Figure 2 C shows The fusion of NMVs and OMVs was studied by using resonance energy transfer (FRET);

[0041] Figure 2 D shows the transmission electron microscopy images of LNPs, HMVs, and LNP@HMVs;

[0042] Figure 2 E shows the hydrated particle size and zeta potential of LNPs, OMVs, HMVs, and LNP@HMVs;

[0043] Figure 2 F shows the protein bands of NMVs, OMVs and LNP@HMVs analyzed by SDS-PAGE;

[0044] Figure 2 G shows the drug release profiles of LNPs and LNP@HMVs.

[0045] Figure 3 Inflammatory cell uptake performance test results are shown;

[0046] in,

[0047] Figure 3 A is a confocal microscopy image of untreated and TNF-a treated human umbilical vein endothelial cells co-incubated with LNP@HMVs;

[0048] Figure 3 B is a graph showing the results of flow cytometry analysis;

[0049] Figure 3 C is the CLSM test result of the inflammatory endothelial cell targeting ability of LNPs coated with different membranes;

[0050] Figure 3 D is the result of flow cytometry detection of the inflammatory endothelial cell targeting ability of LNPs coated with different membranes;

[0051] Figure 3 E is the cytotoxicity graph of LNPs and LNP@HMVs on HUVECs.

[0052] Figure 4 The results of homologous bacterial uptake performance testing are shown;

[0053] in,

[0054] Figure 4 A is a confocal microscopy image of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Staphylococcus aureus (S. aureus) co-incubated with LNP@HMVs;

[0055] Figure 4 B is the confocal microscopy image of LNPs and LNP@HMVs co-incubated with E. coli;

[0056] Figure 4 C is the confocal microscopy image of LNPs and LNP@HMVs co-incubated with K. pneumoniae;

[0057] Figure 4 D is the flow cytometry analysis of E. coli, K. pneumoniae co-incubated with S. aureus and LNP@HMVs;

[0058] Figure 4 E is the flow cytometry detection result of LNPs and LNP@HMVs co-incubated with E. coli or K. pneumoniae, respectively.

[0059] Figure 5 The results of in vitro antibacterial and anti-biofilm performance tests are shown;

[0060] in,

[0061] Figure 5 A is the bacterial plating image of norfloxacin (Nor) and two drug-loaded nanoparticles (LNPs and LNP@HMVs) after incubation with E. coli for 8 h at different drug concentrations;

[0062] Figure 5 B is the colony count results of norfloxacin (Nor) and two drug-loaded nanoparticles (LNPs and LNP@HMVs) after incubation with E. coli for 8 h at different drug concentrations;

[0063] Figure 5 C is the results of characterizing the biofilm amount using crystal violet staining after Nor, LNPs and LNP@HMVs were incubated with E. coli biofilm at different drug concentrations for 24 hours.

[0064] Figure 6 In vitro immunological performance test results are shown;

[0065] in,

[0066] Figure 6A is bone marrow-derived dendritic cells (CD11c + Flow cytometric analysis of surface maturation markers (CD80 and CD86) of BMDCs;

[0067] Figure 6 B is bone marrow-derived dendritic cells (CD11c + Histogram of the percentage of surface maturation markers (CD80 and CD86) in BMDCs;

[0068] Figure 6 C is the result of enzyme-linked immunosorbent assay (ELISA) to measure the levels of immune cytokines IL-6 and TNF-a secreted by BMDCs.

[0069] Figure 7 In vivo therapeutic performance testing results are shown;

[0070] in,

[0071] Figure 7 A is a diagram of the in vivo treatment plan;

[0072] Figure 7 B is the peritoneal fluid test result;

[0073] Figure 7 C is the spleen test result;

[0074] Figure 7 D is the lung test result;

[0075] Figure 7 E is the kidney test results.

[0076] Figure 8 In vivo immune performance test results are shown;

[0077] in,

[0078] Figure 8 A is the in vivo immunization study protocol;

[0079] Figure 8 B is the result of the anti-E.coli IgG antibody titer determination in mouse serum;

[0080] Figure 8 C is mouse lymph node CD11C + Cell percentage;

[0081] Figure 8 D is CD11C + CD80 in cells + percentage;

[0082] Figure 8 E is CD11C + CD86 in cells+ Cell percentage;

[0083] Figure 8 F is mouse spleen CD3 + Cell percentage;

[0084] Figure 8 G is CD3 + CD4 in cells + percentage;

[0085] Figure 8 H is CD3 + CD8 in cells + Cell percentage.

[0086] Fig. 9 In vivo preventive performance test results are shown;

[0087] in,

[0088] Fig. 9 A is a research plan for the prevention of bacterial infection in vivo;

[0089] Fig. 9 B is the survival rate of mice for 7 consecutive days;

[0090] Fig. 9 C is the result of colony count in peritoneal fluid of mice killed 1 day after infection;

[0091] Fig. 9 D is the result of spleen colony counts of mice killed 1 day after infection;

[0092] Fig. 9 E is the result of lung colony counts when mice were killed 1 day after infection;

[0093] Fig. 9 F shows the results of kidney colony counts of mice killed one day after infection. DETAILED DESCRIPTION

[0094] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials in the same field at home and abroad, and technical methods well known to those skilled in the art.

[0095] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or in other circumstances. In addition, the statement of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention. It should be understood that, except in any operating examples, or where otherwise indicated, all numbers representing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary according to the desired performance to be obtained by the present invention.

[0096] Norfloxacin in the examples of the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0097] The glyceryl monostearate in the examples of the present invention was purchased from Guangzhou Muniu Biotechnology Co., Ltd.

[0098] Poloxamer-188 in the examples of the present invention was purchased from Guangzhou Pubo Instrument Co., Ltd.

[0099] The Escherichia coli strain used in the examples of the present invention is numbered ATCC 25922, and its source is American Type Culture Collection (ATCC, USA).

[0100] The HL-60 cell line used in the examples of the present invention was purchased from Wuhan Pronocell Life Science Co., Ltd.

[0101] The experimental animals in the embodiments of the present invention use C57BL / 6 mice (6-8 weeks, male, weighing 20-25 grams, SPF grade) as animal experimental subjects, purchased from Guangzhou Ruige Biotechnology Co., Ltd., animal production license number: SCXK (Guangdong) 2021-0059, animal certificate number: 44827200002797. During the experiment, the mice had free access to water and were kept in a clean area.

[0102] Example 1

[0103] A hybrid membrane bionic nanoparticle, wherein the hybrid membrane bionic nanoparticle is a core-shell structure, the core is a lipid nanoparticle, and the shell is a hybrid membrane;

[0104] in,

[0105] The hybrid membrane is a hybrid membrane formed by the fusion of neutrophil membrane vesicles and Escherichia coli outer membrane vesicles;

[0106] The lipid nanoparticles are loaded with norfloxacin.

[0107] The method for preparing the hybrid membrane bionic nanoparticles comprises the following steps:

[0108] S1. Isolation and acquisition of NMVs: Human myeloid leukemia cells (HL-60) were cultured in RPMI1640 medium containing a mixture of 20% FBS and 1% penicillin-streptomycin. 1.3% DMSO was added for stimulation for 3 days, and LPS (1 μg ml -1 ) for 4 h to induce cell differentiation, and then collect the cells by low-speed centrifugation (1000 rpm, 3 min); disperse the cells in a hypotonic lysis buffer (containing 30 mM Tris-HCl (pH 7.5), 225 mM D-mannitol, 75 mM sucrose, 0.5 mM EDTA, 0.5% bovine serum albumin and 1% enzyme inhibitor); then grind the cells using a Dounce homogenizer with a tight pestle in an ice bath, centrifuge the grinded solution at 4°C (3000 g, 5 min) to remove the cell precipitate, and collect the supernatant; centrifuge the supernatant again (20000 g, 1 h) to collect the precipitate, wash with PBS, and finally disperse in 1 mL PBS and store at -80°C;

[0109] Isolation and acquisition of OMVs: E. coli strains were added to MHB liquid medium and cultured with shaking for 24 h (37°C, 220 rpm), and the bacterial precipitate was removed by low-speed centrifugation (4500 g, 20 min), and the supernatant was centrifuged again (100000 g, 1 h) to collect the precipitate, which was washed with PBS and finally dispersed in 1 mL PBS and stored at -80°C;

[0110] S2. NMVs and OMVs were mixed at a protein weight ratio of 1:1, and ultrasonicated for 10 min (15%, 2s on / 3s off) in an ice bath to obtain a hybrid membrane;

[0111] S3. Dissolve 15 mg of glyceryl monostearate (GMS) in 1.5 mL of ethanol, add 2.1 mg of norfloxacin, stir at 85° C. (400 rpm) and alternate with water bath ultrasound until norfloxacin is completely dissolved to obtain an oil phase; prepare a 0.1% poloxamer 188 aqueous solution, stir at 75° C. (400 rpm) to obtain an aqueous phase; quickly inject the oil phase into the aqueous phase, maintain 75° C., continue stirring at 400 rpm for 10 min, let the prepared emulsion stand in an ice bath environment for 1 h, and cool and solidify to obtain lipid nanoparticles;

[0112] S4. Add lipid nanoparticles at a ratio of 1:1 between total protein and lipid nanoparticles, perform water bath sonication for 5 min, and use a liposome extruder to extrude through polycarbonate films with pore sizes of 800 nm and 400 nm to obtain LNP@HMVs. If needed, add 6% trehalose to LNP@HMVs, freeze-dry at low temperature for 24 h, and obtain LNP@HMVs freeze-dried powder. Re-disperse in PBS when used.

[0113] Figure 1 A schematic diagram of the preparation process of hybrid membrane bionic nanoparticles is shown.

[0114] Figure 2 The figure shows the characterization results of hybrid membrane biomimetic nanoparticles;

[0115] in,

[0116] Figure 2 A shows the transmission electron microscopy images of NMVs and OMVs;

[0117] Figure 2 B shows the fluorescence colocalization image of a mixture of HMVs and NMVs with OMVs;

[0118] Figure 2 C shows The fusion of NMVs and OMVs was studied by using resonance energy transfer (FRET);

[0119] Figure 2 D shows the transmission electron microscopy images of LNPs, HMVs, and LNP@HMVs (the arrows indicate that LNP@HMVs has a double-layer structure);

[0120] Figure 2 E shows the hydrated particle size and zeta potential of LNPs, OMVs, HMVs and LNP@HMVs (n=3), where ns, no significant difference; **p<0.01;

[0121] Figure 2 F shows the protein bands of NMVs, OMVs and LNP@HMVs analyzed by SDS-PAGE;

[0122] Figure 2 G shows the drug release curves of NPs and LNP@HMVs (n=3).

[0123] according to Figure 2A It can be concluded that the transmission electron microscopy images show that both NMVs and OMVs have exosome-like morphology with a diameter of about 200 nm. In order to verify whether the two membranes can fuse, 1,1'-dioctadecyl-3,3,3',3'-tetramethylcarbocyanine perchlorate (DiI, red) was used to stain NMVs, while 3,3'-dioctadecylcarbocyanine perchlorate (DiO, green) was used to stain OMVs, as shown in Figure 2. Figure 2 As shown in B, the red and green fluorescence of NMVs and OMVs remain unchanged after mixing, while the red and green fluorescence of HMVs formed by ultrasound and extrusion overlap to form a yellow area, indicating successful membrane fusion. OMVs were labeled with dye pairs that have resonance energy transfer (FRET) properties and then fused with unlabeled NMVs at different protein mass ratios ( Figure 2 C). As the number of NMVs increases, the fluorescence emission of DiO increases and that of DiI decreases. This is because the insertion of NMVs weakens the FRET characteristics on OMVs, which also indicates that NMVs and OMVs can be successfully fused. Transmission electron microscopy images show that LNP@HMVs have a double-layer structure and display a spherical core-shell morphology, indicating that the LNPs surface is successfully coated with HMVs ( Figure 2 D). In the dynamic light scattering experiment, the hydrated particle sizes of LNPs, HMVs and LNP@HMVs were 134 nm, 197 nm and 194 nm, respectively ( Figure 2 E). The zeta potentials of LNPs, HMVs and LNP@HMVs were -18 mV, -12 mV and -12 mV, respectively. Figure 2 E). There was no significant difference in the hydrated particle size and zeta potential between HMVs and LNP@HMVs, indicating that the HMVs coating was successful. In the polyacrylamide gel electrophoresis test, most of the protein bands of NMVs and OMVs were observed on LNP@HMVs, indicating that the membrane proteins inherited from NMVs and OMVs were effectively retained on the surface of LNP@HMVs ( Figure 2 F). The norfloxacin loading efficiency of LNP@HMVs was 2.4%. LNP@HMVs and LNPs had similar norfloxacin release curves, both of which were relatively gentle. About 25% of the drug was released after 1 hour, and about 50% of the drug was released after 36 hours ( Figure 2 G). The above characterization results prove that hybrid membrane-coated drug-loaded nanoparticles LNP@HMVs have been successfully prepared and can be used for sustained drug release.

[0124] Test Example 1

[0125] Inflammatory cell uptake performance test

[0126] Test method: NMVs inherit the ability of neutrophils to bind to inflammatory endothelial cells. In order to verify whether LNP@HMVs retains this property of NMVs, LNP@HMVs (LNPs labeled with rhodamine B, HMVs labeled with DiO) were co-incubated with human umbilical vein endothelial cells (HUVECs), and then detected by confocal laser scanning microscopy (CLSM). In order to quantify the nanoparticles bound to or taken up by cells, flow cytometry was used to quantify the fluorescence intensity of LNP@HMVs in cells. The inflammatory endothelial cell targeting ability of LNPs coated with different membranes was then compared by CLSM and flow cytometry. The biocompatibility of LNP@HMVs to HUVEC cells was determined by thiazole blue colorimetry (MTT method).

[0127] Test results such as Figure 3 shown.

[0128] Figure 3 Inflammatory cell uptake performance test results are shown;

[0129] in,

[0130] Figure 3 A is a confocal microscopy image of untreated and TNF-a treated human umbilical vein endothelial cells co-incubated with LNP@HMVs;

[0131] Figure 3 B is a graph showing the results of flow cytometry analysis;

[0132] Figure 3 C is the CLSM test result of the inflammatory endothelial cell targeting ability of LNPs coated with different membranes;

[0133] Figure 3 D is the result of flow cytometry detection of the inflammatory endothelial cell targeting ability of LNPs coated with different membranes;

[0134] Figure 3 E is the cytotoxicity graph of LNPs and LNP@HMVs on HUVECs.

[0135] Confocal laser scanning microscopy (CLSM) images showed that in TNF-a-stimulated HUVECs, strong rhodamine B fluorescence and DiO fluorescence of LNP@HMVs were observed around the Hoechst-labeled nuclei. However, the fluorescence of LNP@HMVs in normal HUVECs (not treated with TNF-a) was weak, indicating that LNP@HMVs can better bind to TNF-a-induced inflammatory endothelial cells and have the ability to target inflammatory endothelial cells ( Figure 3A). Consistent with the CLSM results, the average fluorescence intensity of rhodamine B (labeled LNP@HMVs) in the TNF-a stimulated HUVECs group was significantly higher than that in the normal HUVECs group ( Figure 3 B), demonstrating the ability of LNP@HMVs to target inflammatory endothelial cells. Figure 3 C and D show that in inflammatory endothelial cells, the fluorescence of the LNP@NMVs group was the strongest, indicating that NMVs promoted the binding of inflammatory endothelial cells to LNPs. The fluorescence of the LNP@OMVs group was as negligible as that of the LNPs group, indicating that OMVs hardly interacted with inflammatory endothelial cells. As expected, the fluorescence of the LNP@HMVs group was stronger than that of the LNP@OMVs group, but weaker than that of the LNP@NMVs group. These results indicate that LNP@HMVs inherit the targeting ability of NMVs to inflammatory cells and have the potential to tend to and aggregate at the site of bacterial infection in vivo. Figure 3 The results showed that when the concentration of LNPs reached 160 μg·mL -1 When treated with LNP@HMVs, the cytotoxicity of LNP@HMVs was still low, and the cell survival rate was about 95%, indicating that LNP@HMVs had good cell compatibility.

[0136] Test Example 2

[0137] Homologous bacterial uptake performance test

[0138] Test method: Due to the similar membrane composition, nanoparticles encapsulated by OMVs derived from E. coli have been shown to be effectively internalized by E. coli. In order to verify whether LNP@HMVs retain this property of OMVs, LNP@HMVs (LNPs labeled with rhodamine B, HMVs labeled with DiO) were incubated with Gram-negative bacteria E. coli, K. pneumoniae and Gram-positive bacteria S. aureus, respectively, and CLSM detection was performed. The average fluorescence intensity of LNP@HMVs in different bacteria was then measured by flow cytometry. The homologous bacterial targeting ability of LNPs and LNP@HMVs was then compared by CLSM and flow cytometry.

[0139] The test results are as follows Figure 4 shown.

[0140] Figure 4 The results of homologous bacterial uptake performance testing are shown;

[0141] in,

[0142] Figure 4 A is a confocal microscopy image of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Staphylococcus aureus (S. aureus) co-incubated with LNP@HMVs;

[0143] Figure 4 B is the confocal microscopy image of LNPs and LNP@HMVs co-incubated with E. coli;

[0144] Figure 4 C is the confocal microscopy image of LNPs and LNP@HMVs co-incubated with K. pneumoniae;

[0145] Figure 4 D is the flow cytometry analysis of E. coli, K. pneumoniae co-incubated with S. aureus and LNP@HMVs;

[0146] Figure 4 E is the flow cytometry detection result of LNPs and LNP@HMVs co-incubated with E. coli or K. pneumoniae, respectively.

[0147] Figure 4 The CLSM image of A shows that the LNP@HMVs have strong rhodamine B fluorescence and DiO fluorescence inside E. coli, while the LNP@HMVs fluorescence inside S. aureus is weak, indicating that LNP@HMVs are more easily taken up by homologous bacteria and have a homologous bacterial targeting effect. At the same time, obvious LNP@HMVs fluorescence can also be observed inside the Gram-negative bacterium K. pneumoniae, indicating that K. pneumoniae can also effectively take up LNP@HMVs, which may be because Gram-negative bacteria have similar cell membrane components and structures. Figure 4 D The results show that E. coli has the strongest uptake of LNP@HMVs, which indicates that LNP@HMVs can selectively deliver antibiotics to specific bacteria. Figure 4 B and C show that unlike the strong fluorescence of the LNP@HMVs group, the fluorescence of LNPs in E. coli and K. pneumoniae is difficult to observe, indicating that the coating of HMVs gives LNPs the homotypic targeting function to Gram-negative bacteria. Flow cytometry also confirmed this conclusion ( Figure 4 E).

[0148] Combination Figure 3 and Figure 4 The results indicate that HMVs fused by NMVs and OMVs enable LNP@HMVs to effectively target inflammatory cells and bacteria, which will be beneficial for the delivery of antibiotics from the blood circulation to the site of bacterial infection in vivo and further into the interior of the bacteria, achieving efficient delivery of antibiotics and avoiding the biological toxicity caused by high-dose antibiotics.

[0149] Test Example 3

[0150] In vitro antibacterial and anti-biofilm performance testing

[0151] HMVs have been shown to promote the uptake of LNP@HMVs by homologous bacteria, which may facilitate the efficient delivery of norfloxacin (Nor) by LNP@HMVs into E. coli, thereby enhancing the in vitro antibacterial effect of LNP@HMVs. To verify this hypothesis, LNP@HMVs, LNPs and Nor containing different drug concentrations were co-incubated with E. coli and tested. Biofilms are composed of bacteria and their extracellular polymeric substances, which can reduce the bactericidal effect of the immune system and antibiotics on bacteria. It is usually more difficult to remove the formed biofilm than to remove planktonic bacteria, so the ability to destroy the biofilm has also become an important indicator for judging whether the antibacterial drug has clinical application potential. Therefore, the crystal violet staining method was further used to investigate its ability to remove E. coli biofilms.

[0152] Test method:

[0153] (1) Take 10 μL of E. coli frozen stock and add it to 10 mL of MHB medium for overnight culture. The next day, dilute the E. coli suspension with MHB to an OD600 of 0.07 (i.e., a bacterial density of about 10 8 CFU mL -1 ), and then diluted 1000 times with MHB to obtain a bacterial density of 10 5 CFU mL -1 100 μL of sample solution of different concentrations (diluted by MHB) and 100 μL of 10 5 CFU mL -1 After culturing on a shaker for 8 hours, take out the bacterial suspension and spread it on MHB agar plates. After culturing for 18-22 hours, take photos and count the colonies on the plates.

[0154] (2) Take 10 μL of E. coli frozen stock solution and add it to 10 mL of MHB medium for overnight culture. The next day, dilute the E. coli suspension with MHB to an OD600 of 0.07 (i.e., a bacterial density of about 10 8 CFU mL -1), add 100 μL / well to each well of a 96-well plate, and place in a constant temperature incubator for 48 hours. Take out the 96-well plate and remove the culture medium. Add different concentrations of sample solution diluted with MHB (100 μL / well), and place in a constant temperature incubator for stimulation for 24 hours. Take out the 96-well plate, remove the culture medium, add 0.05% crystal violet solution (diluted with deionized water, 200 μL / well), and place in a constant temperature incubator for incubation for 30 minutes. Remove the crystal violet solution and add deionized water to wash (300 μL / well). Remove the deionized water, add anhydrous ethanol (200 μL / well), and place in a shaker for incubation for at least 10 minutes. Use an enzyme-labeled absorbance meter (OD570).

[0155] The test results are as follows Figure 5 shown.

[0156] Figure 5 The results of in vitro antibacterial and anti-biofilm performance tests are shown;

[0157] in,

[0158] Figure 5 A is the bacterial plating image of norfloxacin (Nor) and two drug-loaded nanoparticles (LNPs and LNP@HMVs) after incubation with E. coli for 8 h at different drug concentrations;

[0159] Figure 5 B is the colony count results of norfloxacin (Nor) and two drug-loaded nanoparticles (LNPs and LNP@HMVs) after incubation with E. coli for 8 h at different drug concentrations;

[0160] Figure 5 C is the results of characterizing the biofilm amount using crystal violet staining after Nor, LNPs and LNP@HMVs were incubated with E. coli biofilm at different drug concentrations for 24 hours.

[0161] Figure 5 A and B show that when Nor is 0.06 μg mL -1 When the concentration of Nor was 0.12 μg mL -1 When the concentration of E. coli colonies was 0.0447 W / m, no E. coli colonies were observed in the LNP@HMVs group, while about 10 E. coli colonies were observed in the LNPs group and the Nor group. 4 CFU mL -1 These results indicate that the HMVs coating significantly enhances the in vitro antibacterial effect of Nor-loaded LNP@HMVs.

[0162] Figure 5 C shows that when Nor is 0.25 μg mL -1When the concentration of norfloxacin was 2 μg mL -1 When the concentration of LNP@HMVs was 0.0447 W / m, 97% of the biofilm in the LNP@HMVs group was removed, indicating that the coating of HMVs makes the low concentration LNP@HMVs have a strong antibacterial capsule ability.

[0163] Test Example 4

[0164] In vitro immune performance test

[0165] OMVs contain proteins that can serve as pathogen-associated molecular patterns (PAMPs), which can be recognized by dendritic cells and activate immune responses to eliminate bacteria. Previous studies have shown that most of the proteins of OMVs are retained on the surface of LNP@HMVs. Therefore, it may inherit the dendritic cell activation function of OMVs and is expected to be used in the treatment and prevention of E. coli infection in vivo. To verify this hypothesis, flow cytometry was used to analyze the expression of co-stimulatory molecules CD80 and CD86 in bone marrow-derived dendritic cells (CD11c + The expression level of TNF-α on BMDCs was used to characterize the maturation of BMDCs.

[0166] Then, the levels of immune cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secreted by BMDCs were detected by enzyme-linked immunosorbent assay (ELISA) to verify whether LNP@HMVs can activate dendritic cells and initiate subsequent immune responses.

[0167] Test method: The femur and humerus of one leg were extracted from healthy C57BL / 6 mice, and the cells were flushed out with PBS and cultured in a GM-CSF (20 ng mL -1 ) and IL-4 (10 ng mL -1 ) in 20 mL of RPMI 1640 complete medium (using a bacterial culture dish). On days 3 and 5, half of the medium was replaced. On day 6, immature BMDCs were plated at 10 6 The cells were seeded into 12-well plates at a density of 10 cells / mL. Then, the cells were stimulated with the samples for 24 hours, and PBS was used as a control. Subsequently, the cells were labeled with fluorescently labeled antibodies, including FITC Anti-CD11c, PEAnti-CD80, and PEAnti-CD86. Finally, all samples were analyzed by flow cytometry.

[0168] Test results such as Figure 6 shown.

[0169] Figure 6 In vitro immunological performance test results are shown;

[0170] in,

[0171] Figure 6 A is bone marrow-derived dendritic cells (CD11c + Flow cytometric analysis of surface maturation markers (CD80 and CD86) of BMDCs;

[0172] Figure 6 B is bone marrow-derived dendritic cells (CD11c + Histogram of the percentage of surface maturation markers (CD80 and CD86) in BMDCs;

[0173] Figure 6 C is the result of enzyme-linked immunosorbent assay (ELISA) to measure the levels of immune cytokines IL-6 and TNF-a secreted by BMDCs;

[0174] ns, no significant difference; *p<0.05; ***p<0.001.

[0175] Figure 6 A and B show that after incubation with LNP@OMVs and LNP@HMVs, the expression levels of CD80 on the surface of BMDCs were 54% and 57%, respectively, indicating that they have a strong function of activating and maturing BMDCs. In contrast, the CD80 expression levels of BMDCs after LNPs and LNP@NMVs treatment were similar to those of the PBS group, 12%, 14%, and 12%, respectively, indicating that they can hardly promote the maturation of BMDCs. The expression of CD86 in different groups has a similar trend to CD80, indicating that LNP@HMVs inherit the function of OMVs to activate and promote the maturation of dendritic cells.

[0176] Figure 6 C shows that, consistent with the expression trend of co-stimulatory molecules, the levels of IL-6 and TNF-a in the LNP@OMVs group and LNP@HMVs group were significantly higher than those in the PBS group, LNPs group, and LNP@NMVs group. In summary, LNP@HMVs can significantly promote the activation and maturation of BMDCs and enhance the immune response to kill bacteria.

[0177] Test Example 5

[0178] In vivo therapeutic performance testing

[0179] Test method: Healthy C57BL / 6 mice were randomly divided into 5 groups (Control group, Nor group, LNPs group, LNP@OMVs group and LNP@HMVs group), with 6 mice in each group. Mice were intraperitoneally injected with 1×10 7CFU / 20g of E.coli, different samples (25μg Nor / 20g) were injected into the tail vein 2h after infection, and mice were killed 24h after infection, and peritoneal fluid, spleen, lung and kidney were extracted. The peritoneal fluid was diluted with PBS for a certain multiple, spread on MHB agar plate, and counted after 18-22h of culture. The spleen, lung and kidney were weighed, soaked in 3mL PBS, and ground in an ice bath using a high-pressure homogenizer cell disruptor to obtain a suspension, which was diluted with PBS for a certain multiple and then spread on MHB agar plate. After 18-22h of culture, the colonies on the plate were counted.

[0180] The test results are as follows Figure 7 shown.

[0181] Figure 7 In vivo therapeutic performance testing results are shown;

[0182] in,

[0183] Figure 7 A is a diagram of the in vivo treatment plan;

[0184] Figure 7 B is the peritoneal fluid test result;

[0185] Figure 7 C is the spleen test result;

[0186] Figure 7 D is the lung test result;

[0187] Figure 7 E is the kidney test results.

[0188] Figure 7 The results showed that administration of LNP@HMVs improved the survival rate of infected mice ( Figure 7 BE). Comparing the colony counts in the peritoneal fluid of different treatment groups, although the Nor group, LNPs group, LNP@NMVs group and LNP@OMVs group were able to achieve a certain degree of bacterial clearance (the colony counts of the untreated group were reduced by 2.94-lg CFU, 3.10-lg CFU, 3.09-lg CFU and 3.07-lg CFU, respectively), the LNP@HMVs group showed stronger antibacterial ability (the colony count of the untreated group was reduced by 4.70-lg CFU), indicating that the coating of HMVs in vivo can also significantly improve the antibacterial function of drug-loaded nanoparticles. LNP@HMVs has stronger in vivo bactericidal ability than single membrane-coated drug-loaded nanoparticles LNP@NMVs and LNP@OMVs, which may be mainly due to the step-by-step targeting effect of the hybrid membrane ( Figure 7B). Similarly, in the results of colony counts in spleen, lung and kidney, LNP@HMVs showed higher bacterial clearance ability. The above results prove that the biomimetic nanoparticles formed by hybridization of NMVs and OMVs have excellent therapeutic effects on E. coli infection in vivo.

[0189] Test Example 6

[0190] In vivo immune performance testing

[0191] LNP@HMVs showed good ability to activate and promote BMDCs maturation in vitro, so its function of activating immunity in vivo was further investigated.

[0192] Test method: Healthy C57BL / 6 mice were randomly divided into 4 groups (Control group, LNPs group, LNP@OMVs group and LNP@HMVs group), with 6 mice in each group. 20μg / 20g sample was given for immunization for 3 days. The mice were bled from the eye sockets, then the mice were killed and the spleen and bilateral inguinal lymph nodes were collected.

[0193] E. coli lysate (100 ng / well, dissolved in PBS) was used as antigen to coat a 96-well plate and incubated overnight at 4°C. The plate was emptied and washed 3 times with wash buffer (1×PBST). Blocked with 1% BSA / PBS, i.e., incubated at 37°C for 1 h. The plate was emptied and washed 3 times with wash buffer. Diluted serum samples (100 μL / well) were used as primary antibodies, incubated at 37°C for 1 h, the plate was emptied and washed 3 times with wash buffer. HRP-conjugated goat anti-mouse IgG antibody (100 μL / well) was added, incubated at 37°C for 1 h, the plate was emptied and washed 3 times with wash buffer. TMB substrate (100 μL / well) was added and incubated at 37°C for 10 min. Finally, 1M HCl was added to stop the color development (100 μL / well), and UV-Vis absorbance was measured at 450 nm. The titer was defined as the lowest serum dilution at which the ELISA OD sample / OD negative control was ≥ 2.1.

[0194] The bilateral inguinal lymph nodes of the mice were mashed with a cell strainer and a sterile plunger, passed through a 70 μm cell sieve, and washed with PBS to obtain a single cell suspension. The cells were blocked with PBS / 0.1% BSA and stained with FITCAnti-CD11c, PEAnti-CD80, and PEAnti-CD86. Finally, flow cytometry analysis was performed.

[0195] The spleen of the mouse was crushed using a cell strainer and a sterile plunger, passed through a 70 μm cell sieve, and washed with PBS to obtain a single cell suspension. The cells were blocked with PBS / 0.1% BSA and stained with APC Anti-CD3, FITC Anti-CD4, and PE Anti-CD8a. Finally, flow cytometry analysis was performed.

[0196] Test results such as Figure 8 shown.

[0197] Figure 8 In vivo immunological performance test results are shown;

[0198] in,

[0199] Figure 8 A is the in vivo immunization study protocol;

[0200] Figure 8 B is the result of the anti-E.coli IgG antibody titer determination in mouse serum;

[0201] Figure 8 C is mouse lymph node CD11C + Cell percentage;

[0202] Figure 8 D is CD11C + CD80 in cells + Cell percentage;

[0203] Figure 8 E is CD11C + CD86 in cells + Cell percentage;

[0204] Figure 8 F is mouse spleen CD3 + Cell percentage;

[0205] Figure 8 G is CD3 + CD4 in cells + Cell percentage;

[0206] Figure 8 H is CD3 + CD8 in cells + Cell percentage;

[0207] Difference analysis compared with the untreated control group: ns, no significant difference; *p<0.05; **p<0.01; ***p<0.001.

[0208] Figure 8B shows that the ELISA results showed that the anti-E. coli IgG antibody titers of the Control group, LNP@OMVs group, and LNP@HMVs group were 1.4×10 4 , 1.9×10 5 With 1.9×10 5 The increase in the level of anti-E. coli antibodies in the blood indicates that bacterial outer membrane vesicles or hybrid membrane-coated nanoparticles can activate the specific humoral immunity of mice.

[0209] Figure 8 CE results showed that hybrid membrane biomimetic nanoparticles LNP@HMVs could significantly increase CD11C + CD80 + Cells and CD86 + At the cellular level, the effect is similar to that of bacterial outer membrane vesicle nanoparticles LNP@OMVs, indicating that LNP@HMVs can promote the maturation of dendritic cells in mouse lymph nodes, which is beneficial to enhance specific humoral and cellular immunity.

[0210] CD8 + The cells represent cytotoxic T cells, which recognize and destroy infected cells by releasing various cytotoxins, CD4 + The cells represent helper T cells, which can produce cytokines to assist cytotoxic T cell activation and support humoral immunity driven by B cells. Figure 8 FH results showed that CD3 + The cell proportions were 5.2%, 4.9%, 17.6% and 18.8%, respectively, indicating that LNP@HMVs significantly increased the level of T cells in the spleen. + CD4 in cells + The cell levels were 7.3%, 7.4%, 16.2% and 9.8% respectively; CD3 + CD8 in cells + The cell levels were 2.0%, 2.8%, 30.8% and 28.6% respectively. + With CD8 + The increase in the proportion of cells indicates that LNP@HMVs can activate specific cellular immunity in mice.

[0211] Test Example 7

[0212] In vivo preventive performance testing

[0213] Test method:

[0214] (1) Healthy C57BL / 6 mice were randomly divided into 4 groups (Control group, LNPs group, LNP@OMVs group, and LNP@HMVs group), with 6 mice in each group. Three days after immunization with 20 μg / 20 g sample, the mice were intraperitoneally injected with 5×10 7 CFU / 20g of E. coli, and record the number of surviving mice every 24 hours for 7 consecutive days to calculate the mouse survival rate.

[0215] (2) The antibacterial effect of hybrid membrane biomimetic nanoparticles in activating the immune system was verified by low-dose bacterial infection experiments: Healthy C57BL / 6 mice were randomly divided into 4 groups (Control group, LNPs group, LNP@OMVs group and LNP@HMVs group), with 6 mice in each group. After immunization with 20 μg / 20g sample for 3 days, the mice were intraperitoneally injected with 1×10 7 CFU / 20g of E.coli, mice were killed 1 day after infection, and peritoneal fluid, spleen, lung and kidney were extracted. The peritoneal fluid was diluted with PBS for a certain multiple, spread on MHB agar plate, and counted after 18-22h of incubation. The spleen, lung and kidney were weighed, soaked in 3mL PBS, and ground in an ice bath using a high-pressure homogenizer to obtain a suspension, which was diluted with PBS for a certain multiple and then spread on MHB agar plate. After 18-22h of incubation, the colonies on the plate were counted.

[0216] The test results are as follows Fig. 9 shown.

[0217] Fig. 9 In vivo preventive performance test results are shown;

[0218] in,

[0219] Fig. 9 A is a research plan for the prevention of bacterial infection in vivo;

[0220] Fig. 9 B is the survival rate of mice for 7 consecutive days;

[0221] Fig. 9 C is the result of colony count in peritoneal fluid of mice killed 1 day after infection;

[0222] Fig. 9 D is the result of spleen colony counts of mice killed 1 day after infection;

[0223] Fig. 9 E is the result of lung colony counts when mice were killed 1 day after infection;

[0224] Fig. 9 F is the result of kidney colony counts of mice killed 1 day after infection;

[0225] Difference analysis compared with the untreated control group: ns, no significant difference; **p<0.01.

[0226] Fig. 9 B Results showed that the LNPs group could not protect mice infected with a lethal dose of E. coli, and all mice died one day after infection; LNP@OMVs had an excellent protective effect, maintaining 100% survival of mice; the LNP@HMVs group had significantly improved protection compared to the LNPs group, and the survival rate of mice increased to 67%. This result shows that LNP@HMVs has inherited the immune function of bacterial OMVs well and has the effect of preventing E. coli infection in the mouse model. Fig. 9 CF shows the number of colonies in peritoneal fluid, spleen, lung and kidney. It can be seen that the LNPs group has almost no bacterial clearance function, while the number of bacteria in the LNP@OMVs group and LNP@HMVs group is significantly reduced, indicating that LNP@HMVs can enhance the in vivo antibacterial function of the immune system. The above results prove that the hybrid membrane biomimetic nanoparticles have inherited the immune activation effect of bacterial outer membrane vesicles well and have excellent in vivo preventive effect on E. coli infection.

[0227] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0228] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A hybrid membrane bionic nanoparticle, characterized in that: The hybrid membrane biomimetic nanoparticles are of a core-shell structure, wherein the core is a lipid nanoparticle and the shell is a hybrid membrane; in, The hybrid membrane is a hybrid membrane formed by the fusion of immune cell membrane vesicles and bacterial outer membrane vesicles; The lipid nanoparticles are loaded with antibiotics; The hybrid membrane bionic nanoparticles contain antigens of bacterial outer membrane vesicles, and the immune cells are neutrophils.

2. The hybrid membrane biomimetic nanoparticles according to claim 1, characterized in that: The antibiotic is a quinolone antibiotic.

3. The hybrid membrane biomimetic nanoparticles according to claim 1, characterized in that: The hybrid membrane bionic nanoparticles have a particle size of 180 to 210 nm and a zeta potential of -11 to -13 mV.

4. The method for preparing hybrid membrane bionic nanoparticles according to any one of claims 1 to 3, characterized in that: The steps include: S1. Extract immune cell membrane vesicles and bacterial outer membrane vesicles; S2. mixing the immune cell membrane vesicles with the bacterial outer membrane vesicles, and fusing them by ultrasound and extrusion to form a hybrid membrane; S3. Preparation of lipid nanoparticles containing antibiotics; S4. After mixing the lipid nanoparticles and the hybrid membrane, the mixture is subjected to ultrasound and extrusion treatment to obtain a product.

5. The method for preparing hybrid membrane bionic nanoparticles according to claim 4, characterized in that: In step S1, the method for extracting immune cell membrane vesicles comprises the following steps: Human myeloid leukemia cells are cultured in a culture medium containing a mixture of fetal bovine serum and penicillin-streptomycin, and then dimethyl sulfoxide and lipopolysaccharide are added for culture. After the immune cells are collected by centrifugation, they are ground and centrifuged. The supernatant is collected and centrifuged again and washed to obtain immune cell membrane vesicles.

6. The method for preparing hybrid membrane bionic nanoparticles according to claim 4, characterized in that: In step S1, the method for extracting bacterial outer membrane vesicles comprises the following steps: After culturing the bacterial strain, the supernatant is collected by centrifugation, the supernatant is centrifuged again, and the precipitate is collected and washed to obtain the bacterial outer membrane vesicles.

7. The method for preparing hybrid membrane bionic nanoparticles according to claim 4, characterized in that: In step S2, the protein mass ratio of the immune cell membrane vesicles to the bacterial outer membrane vesicles is (1-1.5):

1.

8. The method for preparing hybrid membrane bionic nanoparticles according to claim 4, characterized in that: In step S3, the method for preparing lipid nanoparticles comprises the following steps: Glyceride is dissolved in a solvent, and then antibiotics are added, heated, stirred, and ultrasonically treated to obtain an oil phase; an emulsifier is added to water, heated, stirred, and an aqueous phase is obtained; the oil phase is added to the aqueous phase, heated, stirred, and cooled to solidify to obtain the lipid nanoparticles.

9. Use of the hybrid membrane bionic nanoparticles according to any one of claims 1 to 3 in the preparation of antibacterial drugs and vaccines.

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