DC targeting peptide modified human neutrophile granulocyte membrane coated PLGA rOmpW nano vaccine as well as preparation method and application thereof

Abstract

The invention relates to rOmpW, in particular to an rOmpW nano vaccine with human neutrophile granulocyte membrane coated PLGA modified by DC targeting peptide as well as a preparation method and application of the rOmpW nano vaccine. The core of the nano delivery system is composed of PLGA nano particles, a layer of human neutrophile cell membrane is wrapped outside the PLGA nano particles, and DCpep is modified on the outer surface of the human neutrophile cell membrane. According to the invention, the DCpep modified human neutrophile granulocyte membrane coated PLGA nano-carrier is used for loading the acinetobacter baumannii epitope vaccine rOmpW for the first time, so that the degradation of polypeptide is prevented, and meanwhile, the immune efficacy is improved.

Description

Technical Field

[0001] The present invention relates to rOmpW, and in particular to a DC targeting peptide modified human neutrophil membrane coated PLGA rOmpW nano vaccine and a preparation method and application thereof Background Art

[0002] According to CHINET data in 2023, among 445,199 clinical isolates, Acinetobacter baumannii (Ab) infection ranked fourth. The resistance rate of Ab to piperacillin-tazobactam reached 71%, and the resistance to meropenem reached 68.1%. Due to the pressure of antimicrobial selection, the multi-drug resistance and pan-drug resistance rates of Ab increased year by year. The conventional treatment strategy in clinical practice is the combined use of colistin, polymyxin and tigecycline. However, the commonly used combined treatment strategy is still ineffective for some pan-resistant strains; Ab infection not only threatens the health of patients, but also brings a heavy economic burden to society and families. Many scholars are committed to developing new antibacterial drugs to fight against bacillus infections, but few new antibiotics have been developed in the past 30 years. Therefore, in addition to new antimicrobial drugs, there is an urgent need to find new treatments. Vaccines have been proven to be safe and effective in preventing many bacterial infections, and the development of vaccines against Ab has become a hot topic for many inventors.

[0003] OmpW is a highly conservative small molecule hydrophobic outer membrane porin in Ab. An invention shows that OmpW has active and passive immune protection against Acinetobacter baumannii infection in mouse models, but has a significant inhibitory effect on the growth of 293FT and A549 cells. CN117603319A constructs and chemically synthesizes a recombinant multi-epitope polypeptide rOmpW, which not only retains the immunogenicity of the protein, but also avoids cytotoxicity.

[0004] As an epitope vaccine, rOmpW has significant advantages over traditional vaccines in terms of durability, safety, stability, production preparation and immune response, and has broad prospects for clinical application. However, subunit vaccines are easily degraded by proteases in the body, mainly due to their small fragments, so their immunogenicity is relatively weak.

[0005] In order to overcome these limitations and enhance the immune efficacy of subunit antigens, inventors often use adjuvants or delivery systems to protect antigens from degradation and enhance their immune efficacy. The present invention is the first to use DCpep-modified human neutrophil membrane-coated PLGA nanocarriers to load the Acinetobacter baumannii epitope vaccine rOmpW to prevent the polypeptide from being degraded and increase the immune efficacy. Summary of the invention

[0006] Purpose of the Invention

[0007] The present invention provides a DC targeting peptide modified human neutrophil membrane coated with PLGA rOmpW nano vaccine and its preparation method and application. The core of the nano delivery system is composed of PLGA nanoparticles, which are coated with a layer of human neutrophil membrane, and DCpep is modified on the outer surface of the human neutrophil membrane.

[0008] Technical Solution

[0009] A DC targeting peptide modified human neutrophil membrane coated PLGA rOmpW nano vaccine, characterized by containing rOmpW, PLGA, PVA, human neutrophil membrane and DCpep peptide.

[0010] The DC targeting peptide modified human neutrophil membrane coated PLGA rOmpW nano vaccine is characterized in that the DCpep peptide is DSPE-PEG2000-DCpep.

[0011] The method for preparing the DC targeting peptide-modified human neutrophil membrane-coated PLGA rOmpW nanovaccine is characterized in that it is prepared by the following steps:

[0012] A. Preparation of PLGA-rOmpW:

[0013] (1) Preparation of the internal aqueous phase: 2 mg of rOmpW was dissolved in a solution containing 0.003% sodium alginate;

[0014] (2) Preparation of organic phase: 80 mg of PLGA was dissolved in 2 ml of dichloromethane;

[0015] (3) Preparation of external aqueous phase: 10 ml of 2% PVA solution was mixed with 1 ml of 5% anhydrous calcium chloride and an appropriate amount of emulsifier Tween;

[0016] (4) Preparation of colostrum: adding the inner aqueous phase solution containing the polypeptide and sodium alginate to the organic phase, and treating it with an ultrasonic crusher for 2 minutes under ice bath conditions to form water-in-oil colostrum;

[0017] (5) Emulsion formation: Then, the colostrum was added to the external aqueous phase and mixed thoroughly, and then ultrasonic treatment was performed again in an ice bath for 2 min to form a water / oil / water emulsion structure to prepare PLGA-rOmpW;

[0018] B. Preparation of NM-PLGA-rOmpW

[0019] After mixing PLGA-rOmpW and human neutrophil membrane at a volume ratio of 5:1, NM-PLGA-rOmpW was obtained by extrusion;

[0020] C. Preparation of Nanovaccine DCpep-NM-PLGA-rOmpW

[0021] DSPE-PEG2000-DCpep is added to NM-PLGA-rOmpW solution, mixed at a volume ratio of 1:5, and stirred, and the DC targeting peptide modified human neutrophil membrane coated PLGA rOmpW nanovaccine is used in Acinetobacter baumannii vaccine.

[0022] Beneficial Effects

[0023] Poly lactic-co-glycolic acid (PLGA) is a high molecular organic compound, which is randomly polymerized from glycolic acid and lactic acid. Its degradation products are water and carbon dioxide, which are non-toxic to the human body. Due to its good biocompatibility and capsule and film-forming properties, it was approved by the US FDA in 1989 and can be used for clinical treatment. PLGA is an ideal carrier for epitope vaccines because of its good biological tissue distribution, significant controlled release characteristics, and ability to enhance antigen presentation and immune response. However, traditional nano-delivery systems are often recognized by the body as "non-self" substances and are cleared, making targeted delivery to the lesion difficult. Since the surface of nanoparticles is covered with cell membranes, it is a new type of biomimetic nanoparticle preparation method developed in recent years. Since neutrophils play a core role in inflammatory reactions, the present invention uses neutrophil cell membranes to reduce the body's immune clearance and enhance immune responses.

[0024] Dendritic cells (DCs) are professional antigen presenting cells that play an important role in innate and acquired immune responses. DCs targeting peptide (DCpep) is a targeting peptide composed of 12 amino acids (FYPSYHSTPQRP). DC targeting peptide specifically binds to receptors expressed on DCs, promotes DCs migration and activation, enhances the presentation of immunogens to T lymphocytes and B lymphocytes, and activates Th1, Th2 and Th17 immune responses. The present invention uses DCpep-modified NM-PLGA (PLGA nanocarrier) to prepare nanovaccines for the first time.

[0025] The rOmpW fragment is relatively small, and is easily degraded by proteases in vivo, and its immunogenicity is relatively weak. In order to overcome these limitations and enhance its immunogenicity, we use PLGA as an adjuvant and carrier, and to avoid being removed by the body as "non-self", the present invention wraps the PLGA carrier by neutrophil membrane to reduce the immune clearance of the body, and DCpep modifies the above-mentioned carrier simultaneously, and successfully obtains nano vaccine. The vaccine spherical structure, uniform morphology, good dispersibility, and the release characteristics in vitro (rOmpW presents the biphasic characteristics of early burst release and late sustained release), rOmpW has obvious burst release at 1h, and then gradually slowly releases, and can be released continuously for 72h, indicating that the nano vaccine protection time is longer, and the cellular immune response of stronger Th1 and Th17 types can be induced, and more B lymphocytes and T lymphocytes can be promoted to differentiate into lymph nodes, and more DCs and macrophages can be induced to gather in lymph nodes. The BALB / c mice were challenged with a lethal dose of ATCC17978 intratracheally to establish an acute lethal pneumonia model. In the nanovaccine group, only a small amount of inflammatory cells infiltrated, the alveolar cavity structure was intact, there was no edema and thickening of the pulmonary interstitium, and the inflammation area was significantly lower than that of the NS, rOmpW and DCpep-NM-PLGA-PBS groups (all P < 0.001); the body weight of the surviving mice in the nanovaccine group gradually increased and basically returned to the pre-challenge level in about 7 days, and the mouse symptom score basically returned to normal in about 7 days; the specific IgG level in the mouse serum ( Fig.11 ), compared with the NS, rOmpW and DCpep-NM-PLGA-PBS groups, the OD values ​​of serum specific IgG in the nanovaccine group increased significantly at five time points (P<0.001), and the nanovaccine group still had a high level at the 9th week, while the serum specific IgG level in the rOmpW group gradually decreased at the 5th week, and there was no significant difference with the NS group at the 9th week (P>0.05); the nanovaccine had no effect on liver and kidney function and the pathology of major organs (heart, liver, spleen, lung and kidney). It fully proves that the nanovaccine has good safety.

[0026] Specifically:

[0027] PLGA-rOmpW was successfully prepared by the double emulsion solvent evaporation method. After PLGA-rOmpW was mixed with human neutrophil membrane, NM-PLGA-rOmpW was obtained by extrusion. DCpep was surface modified by the insertion method to successfully prepare the nano vaccine DCpep-NM-PLGA-rOmpW, and its characterization was identified. The particle size was less than 200nm. It was observed under a transmission electron microscope to have a smooth spherical structure with uniform morphology and good dispersion. We observed the release characteristics of the nano vaccine in vitro, and the results showed that rOmpW showed a biphasic characteristic of early burst release and late sustained release. Early rapid release can induce a strong immune response, while late sustained slow release can continuously stimulate the body to produce antibodies and related cytokines, thereby enhancing the immune response. Safety is the key to whether the vaccine can be used in vivo. We evaluated the safety of the nano vaccine in vivo and in vitro, and found that it had no effect on the proliferation of A549 cells, and had no damage to liver and kidney function and important organs, indicating that the nano vaccine is safe and non-toxic and can be used for in vivo experimental inventions.

[0028] To detect the immune protection effect of nano vaccines, we detected the humoral immune response (serum rOmpW specific IgG, IgG1 and IgG2a) and cellular immune response (IFN-γ, IL-4 and IL-17A) levels of mice. Humoral immunity is one of the important mechanisms for the body to resist pathogens. The serum specific antibody level and its subtype are important bases for evaluating vaccine humoral immunity. The present invention found that nano vaccines can induce higher specific IgG, IgG1 and IgG2a antibody levels in mouse serum. CD4+T cells can differentiate into cell subsets such as Th1, Th2 and Th17. Th1 cells mainly secrete cytokines such as IL-2 and IFN-r to mediate cellular immune responses. Th17 cells secrete IL-17A. IL-17A can induce a large number of chemokines and promote neutrophil recruitment, playing a key role in the process of bacterial clearance. We found that nano vaccines can induce stronger Th1 (IFN-r) and Th17 (IL-17A) immune responses. In addition, the duration of antibody is also an important indicator for evaluating the effect of vaccines. We found that the level of rOmpW-specific IgG antibodies induced by nanovaccines lasted longer, indicating that the nanovaccines may have a longer immune protection effect. At the same time, we found by flow cytometry that the percentages of CD11c+DCs, F4 / 80+ macrophages, CD3+T lymphocytes, and CD19+B lymphocytes in lymph nodes increased significantly, indicating that part of the mechanism of nanovaccines inducing immune responses may be related to the aggregation of DCs and macrophages in lymph nodes and the induction of differentiation of more B lymphocytes and T lymphocytes.

[0029] The establishment of infection model is a key step in evaluating the protective effect of vaccines. We used intratracheal instillation of a lethal dose of Ab standard strain to construct Ab acute lethal pneumonia model, and found that nano vaccine can significantly reduce the bacterial load in lung tissue and blood in acute lethal Acinetobacter baumannii pneumonia model, improve mouse symptom scores, and reduce mortality. This shows that nano vaccine DCpep-NM-PLGA-rOmpW can play an effective immune protective role. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a photograph of human peripheral blood neutrophils, in which the CDiff-Ouik Stain kit is used to identify human peripheral blood neutrophils (with lobed nuclei).

[0031] Figure 2 The mass spectrum analysis diagram and liquid chromatography diagram of DCpep, wherein (A) the molecular weight of DCpep is 1479.63Da after mass spectrum analysis; (B) the purity is 98.23% after HPLC identification.

[0032] Figure 3 Particle size distribution and Zeta potential of PLGA-rOmpW and DCpep-NM-PLGA-rOmpW, where (A) particle size of PLGA-rOmpW; (B) particle size of DCpep-NM-PLGA-rOmpW; (C) Zeta potential of PLGA-rOmpW; (D) Zeta potential of DCpep-NM-PLGA-rOmpW.

[0033] Figure 4 The transmission electron micrographs are (A) PLGA, (B) NM, (C) NM-PLGA and (D) DCpep-NM-PLGA. DCpep-NM-PLGA is spherical, with a small particle size and good dispersibility.

[0034] Figure 5 SDS-PAGE was used to detect the neutrophil membrane surface proteins of DCpep-NM-PLGA, among which DCpep-NM-PLGA, NM-PLGA and NM showed similar electrophoretic bands.

[0035] Figure 6 UV-visible-near-infrared spectra of FITC-DCpep, NM-PLGA and FITC-DCpep-NM-PLGA, wherein FITC-DCpep has an absorption peak at 495 nm, and FITC-DCpep-NM-PLGA also has absorption at 495 nm.

[0036] Figure 7This is the co-localization of DCpep-NM-PLGA in macrophages. NM, DCpep and PLGA have significant co-localization in macrophages.

[0037] Figure 8 Figure 2 is the standard curve (A) and in vitro release curve (B) of rOmpW, in which the rOmpW concentration is linearly related to the peak area at the corresponding concentration. The release curve of rOmpW from DCpep-NM-PLGA-rOmpW in pH 7.4 saline from 1 to 72 h has the characteristics of early burst release and late slow release.

[0038] Fig. 9 The immune response of BALB / c mice one week after immunization. The expression levels of specific IgG (A), IgG1 (B), and IgG2a (C) in mouse serum. The expression levels of IFN-γ (D), IL-4 (E), and IL-17A (F) in spleen cell supernatant. The percentages of CD19+B cells, CD3+T cells (G), CD11c+ dendritic cells, and F4 / 80+ macrophages (H) in lymph nodes. The data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

[0039] Fig.10 This is the immune protective effect of DCpep-NM-PLGA-rOmpW on BALB / c mice.

[0040] Note: Bacterial load in mouse blood (A) and lung (B) 24 hours after challenge. Lung tissue sections were stained with hematoxylin-eosin and observed under a microscope (200×) for daily survival rate (D), weight change (E) and clinical score (F) after challenge with Acinetobacter baumannii. Data are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

[0041] Fig.11 is the duration of specific IgG in mouse serum. Data are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

[0042] Fig.12 Safety evaluation of DCpep-NM-PLGA-rOmpW, cytotoxicity of DCpep-NM-PLGA-rOmpW and DCpep-NM-PLGA-PBS on A549 cells (AB), liver and kidney function of different groups (CF), HE staining of major organs (G). DETAILED DESCRIPTION

[0043] Example 1

[0044] 1. Preparation of PLGA-rOmpW

[0045] PLGA-coated rOmpW was prepared by the double emulsion solvent evaporation method, and the specific steps are as follows:

[0046] (1) Preparation of the internal aqueous phase: 2 mg of rOmpW was dissolved in a solution containing 0.003% sodium alginate;

[0047] (2) Preparation of organic phase: 80 mg of PLGA (polylactic acid-polycaprolactone copolymer, with a molecular weight of 30,000-60,000 and a ratio of lactic acid to caprolactone of 50:50) was dissolved in 2 ml of dichloromethane;

[0048] (3) Preparation of external aqueous phase: 10 ml of 2% polyvinyl alcohol (PVA) solution was mixed with 1 ml of 5% anhydrous calcium chloride and an appropriate amount of emulsifier Tween;

[0049] (4) Preparation of colostrum: adding the inner aqueous phase solution containing polypeptides and sodium alginate to the organic phase, and treating with an ultrasonic crusher for 2 min in an ice bath to form water-in-oil (W / O) colostrum;

[0050] (5) Emulsion formation: colostrum was then added to the external aqueous phase and mixed thoroughly, followed by ultrasonic treatment for 2 min in an ice bath to form a water / oil / water (W / O / W) emulsion structure;

[0051] (6) Volatilization of organic solvent: In order to volatilize the dichloromethane, we subjected the emulsion to magnetic stirring overnight treatment to obtain a nanoemulsion with uniform drug distribution;

[0052] (7) Centrifugal washing: The prepared nanoemulsion was ultracentrifuged for 40 min at a speed of 14,000 rpm, and the supernatant was discarded to obtain the precipitate. Subsequently, ultrapure water was used for three washes to remove unbound rOmpW and excess PVA;

[0053] (8) Freeze-dried storage: After adding 5% trehalose to the precipitate, freeze-dried treatment was performed to finally obtain PLGA-rOmpW, which was stored in a -80°C refrigerator for subsequent use.

[0054] 2. Preparation of Human Neutrophil Membranes

[0055] Ten healthy adults were extracted with anticoagulation tubes, 15 ml / person, a total of 150 ml (approved by the ethics of the Second Affiliated Hospital of Nanjing Medical University, ethics number 2023-KY-165-01), and peripheral blood neutrophils from healthy adults were extracted with a peripheral blood neutrophil separation kit. Neutrophils were identified using a CDiff-Ouik Stain kit, and the washed neutrophils were resuspended in a hypotonic solution (PBS: ultrapure water = 1:10) and lysed at room temperature for 1 hour. Subsequently, these lysed cells were crushed in an ultrasonic cell crusher at 4°C for 5 minutes and treated with medium frequency. The first centrifugation was performed at 3500 rpm at 4°C for 5 minutes, followed by a second centrifugation at 4°C and 14500 rpm for 40 minutes. In order to obtain a neutrophil membrane with uniform particle size, we used a polycarbonate membrane with a pore size of 200 nm and extruded the neutrophil membrane using a mini extruder. Take 20 μL of the prepared human neutrophil membrane solution and drop it onto the copper grid, wait for it to dry, and then perform TEM characterization and identification.

[0056] 3. Preparation of DC targeting peptide (DCpep)

[0057] We commissioned Nanjing Putao Biotechnology Co., Ltd. to chemically synthesize DCpep, whose amino acid sequence is FYPSYHSTPQRP, see SEQ ID NO. 1. The molecular weight of DCpep was identified as 1479.63 Da by mass spectrometry, and the purity of the synthetic polypeptide was identified as 98.23% by HPLC. In order to modify and verify the nanovaccine by phospholipid insertion, we commissioned the company to prepare DSPE-PEG2000-DCpep and DSPE-PEG2000-DCpep-FITC.

[0058] 4. Preparation of NM-PLGA-rOmpW

[0059] After PLGA-rOmpW and human neutrophil membrane were mixed at a volume ratio of 5:1, the mixture was extruded through a polycarbonate membrane with a pore size of 200 nm using a mini extruder, and then centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and PBS buffer was used to resuspend, finally obtaining NM-PLGA-rOmpW, which was stored in a -20°C refrigerator for subsequent use.

[0060] 5. Preparation of DCpep-NM-PLGA-rOmpW

[0061] DSPE-PEG2000-DCpep or DSPE-PEG2000-DCpep-FITC was added to the NM-PLGA-rOmpW solution at a volume ratio of 1:5, and stirred for 2 h in the dark, centrifuged for 15 min at 10,000 rpm, the supernatant was discarded, the precipitate was taken, and resuspended with PBS buffer to obtain DCpep-NM-PLGA-rOmpW, which was stored at -20°C for later use.

[0062] 6. Particle size, PDI and Zeta potential detection

[0063] 3 ml of each PLGA-rOmpW and DCpep-NM-PLGA-rOmpW emulsion was diluted with an appropriate amount of ultrapure water and placed in a cuvette. The average particle size, dispersion coefficient (Polydiseperse Index, PDI) and Zeta potential were detected by a particle size analyzer. Each sample was repeated 5 times and the average value was taken to ensure the accuracy of the results.

[0064] 7.Transmission Electron Microscopy (TEM)

[0065] To observe the morphology of neutrophil membranes, PLGA, NM-PLGA, and DCpep-NM-PLGA, the four substances were first diluted 20 times with ultrapure water. Then, a small amount of each dilution was dropped on the copper mesh and left to stand for 2 minutes to allow the liquid to penetrate naturally. Next, the excess liquid on the surface of the copper mesh was gently absorbed with filter paper to ensure that it dried naturally at room temperature. After drying, 0.1% phosphotungstic acid was used for negative staining, and the staining time was 5 minutes. After staining, the sample was placed under a transmission electron microscope (TEM) to carefully observe its microscopic morphology.

[0066] 8. Membrane protein properties of DCpep-NM-PLGA

[0067] To verify the neutrophil membrane proteins on the surface of DCpep-NM-PLGA, we used polyacrylamide gel electrophoresis (SDS-PAGE) technology to verify it. In the four electrophoresis channels, PLGA, NM, NM-PLGA and DCpep-NM-PLGA solutions were added, with a total volume of 20μl. After the electrophoresis was completed, Coomassie Brilliant Blue solution was added for staining for 30 minutes, and then after 1 hour of washing, we took pictures of the gel.

[0068] 9. Intracellular Colocalization

[0069] China Pharmaceutical University was commissioned to coat the dye CY3, which is completely insoluble in water and soluble in chloroform, with PLGA; the neutrophil cell membrane was incubated with DIL in the dark for 20 minutes, centrifuged at 1500rpm for 5 minutes after the incubation, the precipitate was taken and resuspended with PBS; 0.5mg of PLGA was mixed with 0.1mg of neutrophil cell membrane, a PVDF membrane with a pore size of 200nm was selected, and a small mini extruder was used for extrusion to obtain NM-PLGA, and referring to the phospholipid insertion method, 0.1mgFITC-DSPE-PEG2000-DCpep and 0.5mg of NM-PLGA were stirred in the dark for 1h, and then centrifuged at 10000rpm for 5min, the precipitate was taken and resuspended with PBS to obtain DCpep-NM-PLGA; 50ul of PLGA, NM-PLGA and DCpep-NM-PLGA were incubated with RAW264.7 cells for 1h, and the intracellular co-localization was observed by laser scanning confocal microscopy.

[0070] 10. Encapsulation efficiency detection

[0071] To determine the encapsulation efficiency of rOmpW in the nanovaccine, a standard curve was prepared, and then the amount of rOmpW not encapsulated was indirectly calculated by HPLC, and finally the encapsulation efficiency (EE) was calculated. Encapsulation efficiency (%) = (total amount of rOmpW input - amount of rOmpW in the supernatant) / total amount of rOmpW input × 100.

[0072] 11. In vitro release test

[0073] The DCpep-NM-PLGA-rOmpW 5ml solution was placed in a shaker at 37°C and 100rpm to simulate the release process under physiological conditions. The release amount of rOmpW polypeptide was detected at 1h, 2h, 4h, 8h, 13h, 24h, 30h, 36h, 48h, 54h, 60h and 72h. After the sample was centrifuged at high speed, the supernatant was collected and the rOmpW content was accurately determined by HPLC.

[0074] 12. In vitro safety

[0075] A549 cells were incubated with PBS (as a control group) and 10 μL of different concentrations of DCpep-NM-PLGA-rOmpW (containing rOmpW 10, 20, 40 and 80 μg / ml) for 24 h and 48 h, respectively, and their effects on the proliferation level of A549 cells were detected by CCK-8 method.

[0076] 13. Mouse grouping, immunization and model establishment

[0077] Sixty BALB / c mice were randomly divided into four groups, namely, DCpep-NM-PLGA-rOmpW group, DCpep-NM-PLGA-PBS group, rOmpW group and normal saline (NS) group; mice were immunized with DCpep-NM-PLGA-rOmpW containing rOmpW 50μg / 200μl, an equal volume of DCpep-NM-PLGA-PBS, 50μg rOmpW dissolved in 100μl NS and an equal volume of Freund's adjuvant, and an equal volume of normal saline. Subcutaneous immunization was performed once every 2 weeks, for a total of three immunizations. One week after the last immunization, the level of rOmpW-specific antibodies in the peripheral blood of mice was detected by indirect ELISA, the expression levels of IgG1 and IgG2a in the serum of mice and IL-4, IL-17A and INF-r in the supernatant of spleen cells were detected by ELISA, and the CD19 in the inguinal lymph nodes was detected by flow cytometry. + 、CD3 + 、CD11c + and F4 / 80 + The percentage of cells.

[0078] Two weeks after the last immunization, mice were challenged intratracheally with ATCC17978 to establish an Ab-induced lethal pneumonia model. Peripheral blood and right lung colony counts were performed 24 hours later, and left lung tissue was stained with HE. The survival status, weight changes, and symptom scores of mice in each group were recorded every day for 1 week.

[0079] 14. rOmp-specific antibody detection

[0080] First, rOmpW was diluted to 10 μg / mL and covered in a 96-well plate. Then, 100 μL of the resulting solution was added to each well and incubated at 4°C for 12-18 hours. After washing five times with PBS+0.05% Tween20 (PBST), 200 μL of 2% bovine serum albumin (BSA) was added to each well and incubated at 37°C for 2 hours. Mouse serum was diluted with PBS at a ratio of 1:200 and added to the wells of the OmpW immune group and PBS group, 100 μl per well, incubated at 37°C for 2 hours, and the plate was washed five times; freshly diluted goat anti-mouse IgG was added, labeled with HRP and diluted to 1:10000, 100 μl per well, incubated at 37°C for 1 hour, and the plate was washed five times; TMB substrate was added, 100 μl per well and protected from light for 20 minutes; finally, 50 μl of stop solution was added to each well, and the data was read at OD450 nm.

[0081] 15. Cytokine Detection

[0082] One week after the last immunization, peripheral blood of mice was collected, and the expression levels of IgG1 and IgG2a in mouse serum were detected using IgG1 and IgG2a cytokine detection kits. Splenocytes were isolated one week after the last immunization and adjusted to 1×10 6 / mL, 200 μL of cell suspension was added to each well of a 96-well plate and stimulated with rOmpW (20 μg / mL) or not, the supernatant was collected after 72 h, and the levels of IFN-γ, IL-4 and IL-17A were detected using mouse IFN-γ, IL-4 and IL-17A ELISA kits.

[0083] 16. Flow Cytometry Analysis

[0084] One week after the last immunization, single cell suspension of inguinal lymph nodes was prepared. The number of cells was 1×10 6 / mL, Fc blocking antibody was added and blocked at 4°C for 15 min, washed and stained with fluorescent dye-conjugated antibodies against lymphocyte surface receptors, APC-Mouse I-Ab, PerCP-Cy5.5-CD11c, PE-F4 / 80, PE-CD3, and APC-CD19 (BD Biosciences) at 4°C in the dark for 30 min, then the cells were washed with 2% paraformaldehyde solution and fixed for 20 min, and the data were acquired on a BD FACS Canto II flow cytometer (BD Biosciences), and at least 1 × 10 were included in each sample. 5 Events were analyzed using FlowJo software.

[0085] 17. HE staining of lung tissue

[0086] 24 hours after ATCC17978 challenge, the lungs were removed under sterile conditions and fixed with 4% formalin. After being stained with paraffin-eosin (HE), the paraffin-embedded sections were examined histopathologically under a microscope. Using Image Promacro, lung injury was estimated by the percentage of lesion area to total lung area.

[0087] 18. Statistical analysis

[0088] We used SPSS25.0 statistical software to analyze the collected data. When comparing the means between the two groups of samples, we used t-test, one-way ANOVA analysis of variance for inter-group comparison, and variance homogeneity analysis. P<0.05 indicated that the difference was statistically significant.

[0089] result:

[0090] Preparation of neutrophil membrane and Dcpep

[0091] Neutrophils were extracted from the peripheral blood of healthy adults using a peripheral blood neutrophil separation kit, and neutrophils were identified using a CDiff-Ouik Stain kit. Figure 1 ), and the neutrophil membrane was obtained by ultracentrifugation. Nanjing Putao Biotechnology Co., Ltd. was commissioned to chemically synthesize DCpep, which has 12 amino acids. The molecular weight of DCpep was identified as 1479.63Da by mass spectrometry, and the purity of the synthetic polypeptide was identified as 98.23% by HPLC ( Figure 2 ).

[0092] Characterization of Nanoparticles

[0093] The average particle size of PLGA-rOmpW detected by particle size analyzer was 131.33±8.22 ( Figure 3 A), PDI was 33.33±0.47%, and the average particle size of DCpep-NM-PLGA-rOmpW was 135.67±11.26 ( Figure 3 B), PDI was 28.33±4.50%, and the Zeta potential of PLGA-rOmpW and DCpepNM-PLGA-rOmpW were 0.35±0.22 mV ( Figure 3 C) and -10.37±0.17mV( Figure 3 D).

[0094] Transmission electron microscopy was used to observe the PLGA, NM and DCpep-NM-PLGA ( Figure 4 ), the neutrophil membrane can be well attached to the PLGA surface without affecting the size of the particles, and DCpep-NM-PLGA is spherical, uniform in size, and evenly dispersed.

[0095] SDS-PAGE: DCpep-NM-PLGA, NM-PLGA and NM showed similar electrophoretic bands, indicating that DCpep-NM-PLGA nanoparticles prepared by extrusion method can better maintain the integrity of neutrophil membrane surface proteins ( Figure 5 ).

[0096] We modified DCpep with FITC. The UV-visible spectrum showed that pure FITC-DCpep had an obvious absorption peak at 495nm, and the aqueous solution of FITC-DCpep-NM-PLGA also had an obvious absorption peak at 495nm ( Figure 6), indicating that DCpep can be successfully modified on the surface of human neutrophil membrane. 50μl of PLGA, NM-PLGA and FITC-DCpep-NM-PLGA were co-cultured with RAW264.7 cells and observed by laser scanning confocal microscopy 1 hour after uptake. It can be clearly observed that there is a significant co-localization phenomenon between DCpep, neutrophil membrane and PLGA ( Figure 7 ).

[0097] Encapsulation efficiency and in vitro release test

[0098] The standard curve was drawn by HPLC with rOmpW concentration as the horizontal axis and the peak area at the corresponding concentration as the vertical axis. The equation of the standard curve is y=11385x-217.23, R 2 The value is 0.9998, indicating a good linear relationship ( Figure 8 A). The encapsulation efficiency of DCpep-NM-PLGA-rOmpW was indirectly calculated by HPLC to be 43.25%, which was in line with expectations. DCpep-NM-PLGA-rOmpW was dissolved in 5 ml of pH 7.4 saline, in a shaker at 37°C, with the speed set at 100 rpm, and the rOmpW content in the supernatant was determined by HPLC. The cumulative rOmpW released by DCpep-NM-PLGA-rOmpW within 1, 2, 4, 8, 13, 24, 48 and 72 h was 17.7%, 23.3%, 27.7%, 32.7%, 33.8%, 34.3%, 38.8% and 44.3% ( Figure 8 B). It shows that rOmpW has an obvious burst release at 1 hour, followed by a gradual and slow release, and can be continuously released for 72 hours, indicating that the nanovaccine has a long protection time.

[0099] Immune efficacy of nanovaccines

[0100] BALB / c mice were subcutaneously injected with DCpep-NM-PLGA-rOmpW containing rOmpW 50μg / 200μl, an equal volume of DCpep-NM-PLGA-PBS, rOmpW 50μg and an equal volume of saline. The mice were immunized subcutaneously once every 2 weeks for a total of three times. The same volume of PBS was used once every two weeks for a total of three times. Peripheral blood and spleen were collected one week after the last immunization, and rOmpW-specific IgG ( Fig. 9 A), IgG1( Fig. 9 B) and IgG2a( Fig. 9C), the OD values ​​of serum specific IgG, IgG1 and IgG2a in the nanovaccine group were significantly higher than those in the NS, rOmpW and DCpep-NM-PLGA-PBS groups (all P<0.001), indicating that the nanovaccine can induce a stronger humoral immune response. The expression levels of IFN-γ, IL-4 and IL-17A in the supernatant of spleen cells were detected by ELISA ( Fig. 9 DF), the expression levels of IFN-γ and IL-17A in the supernatant of spleen cells of mice in the nanovaccine group were significantly higher than those in the rOmpW, NS and DCpep-NM-PLGA-PBS groups (P<0.001), while the level of IL-4 was significantly lower (P<0.001), indicating that the nanovaccine can induce stronger Th1 and Th17 type cellular immune responses.

[0101] One week after the last immunization, flow cytometry was used to detect the CD3 + 、CD19 + 、CD11c + and F4 / 80 + The percentage of cells ( Fig. 9 GH), nano vaccine group CD3 + T lymphocytes, CD19 + B lymphocytes, CD11c + DCs and F4 / 80 + The percentage of macrophages was significantly higher than that in the NS, rOmpW and DCpep-NM-PLGA-PBS groups (P<0.001), indicating that the nanovaccine can promote the differentiation of more B lymphocytes and T lymphocytes in the lymph nodes, and can induce more DCs and macrophages to accumulate in the lymph nodes.

[0102] Immunoprotective effect of nanovaccines

[0103] BALB / c mice were challenged with a lethal dose of ATCC17978 intratracheally to establish an acute lethal pneumonia model. Peripheral blood and right lung tissue were collected 24 hours later for colony counts ( Fig.10 AB), the colony counts in peripheral blood and lung tissues of mice in the nanovaccine group were significantly lower than those in the NS, rOmpW and DCpep-NM-PLGA-PBS groups (P<0.001); the left lung tissue was taken for HE staining ( Fig.10 C), the results showed that: NS and DCpep-NM-PLGA-PBS groups showed a large number of inflammatory cell infiltration (mainly neutrophils), lung tissue necrosis, and alveolar cavity structure destruction; rOmpW group showed a small amount of inflammatory cell infiltration, mild thickening of the lung interstitium, and intact alveolar cavity structure; nano vaccine group had only a very small amount of inflammatory cell infiltration, intact alveolar cavity structure, and no edema and thickening of the lung interstitium. Image pro plus software analysis showed ( Fig.10 D): The inflammation area of ​​the nanovaccine group was significantly lower than that of the NS, rOmpW and DCpep-NM-PLGA-PBS groups (all P<0.001).

[0104] The survival status, weight changes and symptom scores of mice in each group were recorded for 7 consecutive days after ATCC17978 attack; the survival rate of the nanovaccine group was 83.33% (5 / 6) at 1 week, the rOmpW group was 66.67% (4 / 6), and all the mice in the DCpep-NM-PLGA-PBS and NS groups died within 3 days ( Fig.10 E). The body weight of mice in each group dropped to the lowest level about 2 days after Ab attack, and then the body weight of the surviving mice in the nanovaccine group and rOmpW group gradually increased, and basically returned to the pre-attack level about 7 days later ( Fig.10 F). The symptom scores dropped to the lowest level 2-3 days after Ab challenge, and then the symptom scores of the surviving mice in the nanovaccine group and rOmpW group gradually increased and basically returned to normal in about 7 days.

[0105] Serum rOmpW-specific IgG duration

[0106] The medial canthus venous blood of mice was collected at five time points, 1, 3, 5, 7, and 9 after the last immunization, and the specific IgG level in the mouse serum was detected by indirect ELISA method ( Fig.11 ), compared with the NS, rOmpW and DCpep-NM-PLGA-PBS groups, the OD values ​​of serum specific IgG in the nanovaccine group increased significantly at the five time points (P<0.001), and the nanovaccine group still had a high level at the 9th week, while the serum specific IgG level in the rOmpW group gradually decreased at the 5th week, and there was no significant difference between the rOmpW group and the NS group at the 9th week (P>0.05).

[0107] Safety of Nanovaccines

[0108] A549 cells were incubated with different concentrations of DCpep-NM-PLGA-rOmpW for 24 h and 48 h, and the effects on the proliferation level of A549 cells were detected by CCK-8 method ( Fig.12 The results showed that the nanovaccine had no effect on the proliferation level of A549 cells. One week after the last immunization, the serum and major organs of the mice were collected to detect the changes in serum levels of BUN, Cre, ALT and AST. HE staining was used to evaluate the pathological changes in the heart, liver, spleen, lung and kidney of the mice. Fig.12 ), the nano vaccine had no effect on liver and kidney function and the pathology of major organs (heart, liver, spleen, lung and kidney). This fully proves that the nano vaccine has good safety.

Claims

1. A DC targeting peptide modified human neutrophil membrane coated with PLGA rOmpW nano vaccine, characterized in that Contains rOmpW, PLGA, PVA, human neutrophil membrane and DCpep peptide.

2. The rOmpW nanovaccine of a DC targeting peptide modified human neutrophil membrane coated with PLGA according to claim 1, characterized in that: The DCpep peptide was DSPE-PEG2000-DCpep.

3. The method for preparing a DC targeting peptide modified human neutrophil membrane coated with PLGA rOmpW nano vaccine according to claim 1, characterized in that: Prepared by the following steps: A. Preparation of PLGA-rOmpW: (1) Preparation of the internal aqueous phase: 2 mg of rOmpW was dissolved in a solution containing 0.003% sodium alginate; (2) Preparation of organic phase: 80 mg of PLGA was dissolved in 2 ml of dichloromethane; (3) Preparation of external aqueous phase: Mix 10 ml of 2% PVA solution with 1 ml of 5% anhydrous calcium chloride and an appropriate amount of emulsifier Tween; (4) Preparation of colostrum: adding the inner aqueous phase solution containing the polypeptide and sodium alginate to the organic phase, and treating it with an ultrasonic crusher for 2 minutes under ice bath conditions to form water-in-oil colostrum; (5) Emulsion formation: Then, colostrum was added to the external aqueous phase and mixed thoroughly, and then ultrasonic treatment was performed again in an ice bath for 2 min to form a water / oil / water emulsion structure to prepare PLGA-rOmpW. B. Preparation of NM-PLGA-rOmpW After mixing PLGA-rOmpW and human neutrophil membrane at a volume ratio of 5:1, NM-PLGA-rOmpW was obtained by extrusion; C. Preparation of Nanovaccine DCpep-NM-PLGA-rOmpW DSPE-PEG2000-DCpep was added to the NM-PLGA-rOmpW solution, the volume ratio was 1:5, and the mixture was stirred to obtain the nanovaccine DCpep-NM-PLGA-rOmpW.

4. Use of the rOmpW nanovaccine containing a DC targeting peptide modified human neutrophil membrane and PLGA coated according to claim 1 in the preparation of a drug for treating pulmonary tuberculosis.

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