Suction type bionic nano particle for monkey pox prevention and preparation method and application thereof

Abstract

The invention discloses a suction type bionic nano particle for monkey pox prevention and a preparation method and application of the suction type bionic nano particle. According to the suction type bionic nano particles, lipid simulating lung surface active substances and macrophage membranes are mixed to form a carrier of the nano particles, monkey pox antigens and cGAMP are wrapped, the suction type bionic nano particles are synthesized, and the suction type bionic nano particles can effectively enhance humoral immunity and cellular immunity of anti-monkey pox viruses. The invention also provides an application of the inhalation type bionic nanoparticle in preparation of a nasal inhalation type vaccine preparation, and the nasal inhalation type vaccine preparation can induce mucosal immunity in lungs and mucosal tissues, effectively protect orthopoxvirus from invading through vagina and enteric cavity, and prevent sexual transmission of the orthopoxvirus; the nasal inhalation type vaccine can completely prevent mice from death caused by infection of attenuated strains of orthopoxvirus VACV by immunizing, rash is completely eliminated, and the nasal inhalation type vaccine has significance on effective prevention of monkey pox.

Description

Technical Field

[0001] The present invention belongs to the technical field of infectious disease treatment and prevention and control, and specifically relates to an inhalable bionic nanoparticle for monkeypox prevention, and a preparation method and application thereof. Background Art

[0002] Monkeypox is a zoonotic disease caused by infection with the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus. Although monkeypox is generally considered a self-limited disease, severe MPXV infection can cause cytokine storm and death, especially in infants, young children (less than 8 years old), pregnant women, and those with compromised immune systems (e.g., those with human immunodeficiency virus infection). Therefore, there is an urgent need to develop effective diagnostic, therapeutic, and preventive strategies.

[0003] Vaccines are an important tool for preventing viral infections and reducing their prevalence. However, there is currently a lack of effective vaccines specifically for monkeypox. Smallpox vaccines such as ACAM2000, JYNNEOS, and LC16m8 may indirectly protect against monkeypox virus infection by inducing cross-reactive antibodies, and are therefore authorized as alternative vaccines to control the spread of monkeypox virus. However, the resources of these smallpox vaccines are very scarce worldwide. Moreover, these vaccines have certain risks of side effects and decreased protection rates in the vaccinated population, which limits their large-scale application. Therefore, the development of new vaccines that are more accessible, less toxic, and more efficient is of great clinical significance.

[0004] The continuous development of nanotechnology has provided a lot of support for the development of new vaccines. Nano vaccines have inherent advantages in promoting high-density arrangement of antigens, protecting active ingredients from degradation, improving the targeting of antigens to lymph nodes, and enhancing the uptake of antigens by antigen-presenting cells. Liposomal nanoparticles have high biocompatibility but lack the ability to target antigen-presenting cells. Therefore, it is of great significance to develop a unique inhaled biomimetic nano vaccine with strong targeting against monkeypox virus to achieve effective prevention of monkeypox. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an inhalable bionic nanoparticle for monkeypox prevention. The bionic nanoparticle penetrates the alveolar surfactant (PS) barrier and targets alveolar macrophages through inhalation inoculation, promotes the effective absorption of encapsulated antigens and adjuvants and subsequent anti-monkeypox virus immune activation, thereby achieving effective prevention of monkeypox.

[0006] The second object of the present invention is to provide a method for preparing the inhalable bionic nanoparticles.

[0007] The third object of the present invention is to provide the application of the inhalable bionic nanoparticles.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention firstly provides an inhalable bionic nanoparticle, which uses a hybrid bionic nanovesicle composed of liposomes simulating alveolar surfactant and macrophage membrane fusion as a carrier to encapsulate monkeypox virus specific antigen and 2',3'-cyclic guanosine monophosphate (cGAMP).

[0010] The inhalable bionic nanoparticles of the present invention can achieve effective penetration of the alveolar surfactant barrier, while targeting respiratory antigen presenting cells, thereby effectively activating lung and distal mucosal tissue anti-monkeypox virus immunity. Wherein, the liposomes simulating alveolar surfactant in the carrier of the inhalable bionic nanoparticles can assist water-soluble small molecules to penetrate the alveolar surfactant barrier, thereby achieving inhalation inoculation of nanoparticles; bionic nanoparticles (BNPs) based on macrophage membranes can specifically target homologous alveolar macrophages. Therefore, the present invention combines the advantages of liposomes simulating lung surfactant and BNPs at the same time, can effectively penetrate the lung surfactant barrier and accurately target alveolar macrophages, and achieve efficient presentation of monkeypox antigens and effective anti-monkeypox immune activation. Furthermore, the research of the present invention shows that the inhalable bionic nanoparticles have better immune activation effect than free monkeypox antigen and adjuvant cGAMP, inhalable bionic nanoparticles without macrophage membrane components, and inhalable bionic nanoparticles without adjuvant cGAMP. They can completely prevent the death of mice caused by infection with the attenuated strain of the orthopoxvirus VACV and completely eliminate the occurrence of rash, indicating that the inhalable bionic nanoparticle components of the present invention synergistically enhance the immune effect against monkeypox virus and have a strong adjuvant effect.

[0011] Furthermore, the mass ratio of the liposome simulating alveolar surfactant to the macrophage membrane is 2-3:1.

[0012] Preferably, the mass ratio of the liposomes simulating alveolar surfactant to the macrophage membrane is 3:1.

[0013] Furthermore, the mass ratio of the liposome simulating alveolar surfactant to the monkeypox virus specific antigen and 2',3'-cyclic guanosine monophosphate is 200-250:3:10.

[0014] Preferably, the mass ratio of the liposome simulating alveolar surfactant to the monkeypox virus-specific antigen and 2',3'-cyclic guanosine monophosphate is 250:3:10.

[0015] Furthermore, the monkeypox virus-specific antigen is A29L and / or A35R.

[0016] Preferably, the monkeypox virus-specific antigens are A29L and A35R, and their mass ratio is 1:1.

[0017] Furthermore, the components of the liposome simulating alveolar surfactant are dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), cholesterol (Chol) and distearoylphosphatidyl acetamide-polyethylene glycol (DSPE-PEG).

[0018] Preferably, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol is 9-10:1:1:1.

[0019] Preferably, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol is 10:1:1:1.

[0020] Preferably, the distearoylphosphatidylacetamide-polyethylene glycol (DSPE-PEG) is DSPE-PEG2000.

[0021] Preferably, the preparation method of the liposome simulating alveolar surfactant is to mix dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol and then prepare it by ultrasonic method.

[0022] Preferably, the ultrasonic method is to dissolve dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol in an organic solvent, mix them by ultrasonication, remove the solvent, and then add water phase for hydration to obtain liposomes simulating alveolar surfactant.

[0023] Preferably, the organic solvent is chloroform, and the aqueous phase is PBS buffer solution.

[0024] Preferably, the ultrasonic treatment time is 30 min.

[0025] Preferably, the solvent is removed by rotary evaporation, and the hydration is performed by rotary evaporation for 5 minutes.

[0026] The present invention provides a method for preparing any of the above-mentioned inhalable bionic nanoparticles, comprising mixing liposomes simulating alveolar surfactant with monkeypox virus specific antigen and 2',3'-cyclic guanosine monophosphate, ultrasonicating, adding macrophage membranes and mixing, and obtaining by membrane extrusion.

[0027] Furthermore, the preparation method of the inhalable bionic nanoparticles is to first dissolve dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol in an organic solvent, perform ultrasonic mixing, remove the solvent to form a phospholipid film, add an aqueous phase for hydration, then add monkeypox virus-specific antigen and 2',3'-cyclic guanosine adenosine phosphate, perform water bath ultrasonication, and obtain liposomes encapsulating the monkeypox virus-specific antigen and 2',3'-cyclic guanosine adenosine phosphate; the synthesized liposomes are then mixed with macrophage membranes, and the membranes are extruded to prepare the bionic nanoparticles.

[0028] Furthermore, the monkeypox virus-specific antigens are A29L and A35R.

[0029] Preferably, in the preparation method, the mass ratio of lipid, A29L, A35R and 2',3'-cyclic guanosine monophosphate is 400-500:3:3:20; the lipid is dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol.

[0030] Preferably, in the preparation method, the mass ratio of lipid, A29L, A35R and 2',3'-cyclic guanosine monophosphate is 500:3:3:20; the lipid is dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol.

[0031] Furthermore, the macrophage membrane is extracted by freeze-thaw method; specifically, the freeze-thaw method is: culturing macrophages, collecting cell precipitates, resuspending in homogenization buffer, repeatedly freezing and thawing, grinding and lysing cells, gradient centrifugation to remove cell debris, ultracentrifuging the supernatant, collecting precipitates, and obtaining macrophage membrane.

[0032] Preferably, the gradient centrifugation is 800-1000 g for 10 min, 6000-8000 g for 15 min, and 12000-15000 g for 30 min.

[0033] Preferably, the gradient centrifugation is 1000g, 10 min, 6000g, 15 min, 15000g, 30 min.

[0034] Preferably, the ultracentrifugation is 100000 g ultracentrifugation for 2 h.

[0035] Furthermore, the ultrasound is water bath ultrasound for 30 to 60 minutes.

[0036] Furthermore, the membrane extrusion is sequentially extruded through membranes with pore sizes of 800 nm, 400 nm and 200 nm.

[0037] Preferably, the film extrusion temperature is 37-45°C.

[0038] Preferably, the film extrusion temperature is 40°C.

[0039] The present invention provides use of any of the above-mentioned inhalable bionic nanoparticles in preparing a drug for preventing and / or treating monkeypox virus infection.

[0040] Furthermore, the medicine is a vaccine preparation.

[0041] Preferably, the vaccine formulation is a nasal inhalation vaccine formulation.

[0042] The present invention provides a vaccine preparation containing any of the above-mentioned inhalable bionic nanoparticles. The inhalable bionic nanoparticles have the function of penetrating the alveolar surface active barrier and targeting alveolar macrophages, thereby enhancing the presentation of monkeypox antigens by alveolar macrophages and enhancing the activation of the body's anti-monkeypox virus immunity.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention first provides an inhaled bionic nanoparticle for monkeypox prevention, which is a mixed bionic nanovesicle formed by mixing lipids and macrophage membranes that simulate lung surfactant as a carrier, encapsulating monkeypox virus-specific antigens and interferon receptor agonist cGAMP as an immune adjuvant to form an inhaled bionic nanoparticle, and the inhaled bionic nanoparticle has the function of penetrating the alveolar surfactant barrier and targeting alveolar macrophages, which can effectively enhance the humoral immunity and cellular immunity against monkeypox virus. The present invention also provides the inhaled bionic nanoparticle for preparing a nasal inhalation vaccine preparation for preventing monkeypox virus infection. Compared with the injection of traditional vaccines, inhalation vaccination has the advantages of simple portability and high non-invasive acceptance; more importantly, inhalation vaccination can induce strong mucosal immunity in the lungs and distant mucosal tissues, effectively protect orthopoxviruses from invading through the vagina and intestinal cavity, and prevent the sexual transmission of monkeypox virus (the main transmission route of monkeypox virus at present), which provides new insights for the development of feasible prevention strategies for monkeypox virus. Immunization with the inhalable bionic nanoparticles as a vaccine can completely prevent mice from dying from infection with the attenuated strain of the orthopoxvirus VACV, and completely eliminate the occurrence of skin rashes. The present invention is of great significance for achieving effective prevention of monkeypox. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Flow chart of the preparation of AAG@AS-M NVs.

[0046] Figure 2 Confocal images (A) and photoluminescence (PL) spectra (B) of the fusion between AS and macrophage membrane during the synthesis of AAG@AS-M NVs.

[0047] Figure 3 The hydrated particle size and zeta potential diagram of AAG@AS-M NVs.

[0048] Figure 4 Transmission electron microscopy image of AAG@AS-M NVs.

[0049] Figure 5 Confocal images of RB@AS-M NVs taken up by macrophages (A) and representative confocal images of lung sections showing their distribution in the lungs of mice after nasal inhalation (B).

[0050] Figure 6 The innate immune activation effect of AAG@AS-M biomimetic nanoparticles. The activation levels of macrophages (A) and DC cells (B) in the alveoli of mice, the levels of resident classical DC cells (C), and the levels of proinflammatory cytokines TNF-α (D), IFN-γ (E), and an IFN-Is (IFN-β) (F) in lung tissue after nasal inhalation of different vaccines.

[0051] Figure 7 Figure 3 is the humoral immune activation effect of AAG@AS-M bionic nanoparticles; A and B are the levels of B cell activation and memory B cells in the lung tissue of mice after nasal inhalation of different vaccines, and C and D are the levels of serum anti-A29L and A35R specific IgG antibodies, respectively.

[0052] Figure 8 is the cellular immune activation effect of AAG@AS-M bionic nanoparticles; A to C are the levels of CD8+T cells, memory effector T cells (TEM) and tissue-resident memory T cells (TRM) in the lung tissue of mice after nasal inhalation of different vaccines.

[0053] Fig. 9 is the mucosal immune activation effect of AAG@AS-M bionic nanoparticles; A~B are the levels of anti-A29L and A35R specific IgA antibodies in the alveoli of mice after nasal inhalation of different vaccines, C~D are the levels of anti-A29L and A35R specific IgA antibodies in the vagina, and E~F are the levels of anti-A29L and A35R specific IgA antibodies in the intestinal cavity.

[0054] Fig.10 Schematic diagram of the principle of inducing anti-monkeypox virus immunity and blocking monkeypox virus infection through mucosal invasion based on AAG@AS-M bionic nanoparticles.

[0055] Fig.11Figure 2 is the result of in vitro and in vivo biosafety assessment of AAG@AS-M NVs; A to C are the effects of AAG@AS-M NVs on the survival of L929, HUVEC and HSF cells, respectively; D is the effect of AAG@AS-M NVs on mouse body weight; and E is the effect of AAG@AS-M NVs on alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN) in mouse plasma.

[0056] Fig.12 This is an evaluation of the anti-vaccinia virus protection effect based on AAG@AS-M bionic nanoparticles; A is the mouse survival curve, and B is the mouse tail rash and monkeypox virus immunohistochemistry section results. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0058] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0059] Example 1 Preparation of AAG@AS-M biomimetic nanoparticles (AAG@AS-M NVs)

[0060] Figure 1 The preparation flow chart of AAG@AS-M NVs is shown in Figure 1. The two monkeypox virus-specific antigens (A29L and A35R) and the immune adjuvant adjuvant 2',3'-cyclic guanosine monophosphate (cGAMP) are encapsulated into simulated alveolar surfactant (AS) liposomes, and then the simulated alveolar surfactant (AS) liposomes are fused with macrophage membranes to form hybrid biomimetic nanovesicles, and finally the biomimetic nanoparticles of AAG@AS-M NVs are formed. The specific steps include the following:

[0061] 1. When RAW 264.7 cells were grown to complete confluence in a T-75 culture flask, the cells were scraped from the culture flask, centrifuged at 1000 rpm for 5 min, and washed with PBS three times. The cell pellets were collected and resuspended in a homogenization buffer containing 25 mM sucrose, 10 mM Tris·HCl (pH=7.5), 1 mM MgCl2 (Sigma), 1 mM KCl (Sigma), and 2 mM PMSF (Thermo Fisher). After the cell suspension was repeatedly frozen and thawed three times, it was placed in a buffered homogenizer and ground 15 to 20 times to lyse the cells. Subsequently, the cell suspension was subjected to gradient centrifugation. The first centrifugation was 1000 g for 10 min to remove the cell pellet; the second centrifugation was 6000 g for 15 min to remove the cell pellet; the third centrifugation was 15000 g for 30 min to remove the cell pellet. Finally, the supernatant was ultracentrifuged at 100000 g for 2 h (ultracentrifuge CP100WX, Hitachi, Japan) to collect the precipitate as macrophage cell membranes. The obtained macrophage cell membranes were stored at -80°C, resuspended in PBS before use, and the concentration was determined using a BCA kit.

[0062] 2. Weigh DPPC, DPPG, Chol and DSPE-PEG2000 in a mass ratio of 10:1:1:1 and dissolve them in 3 mL of chloroform. Ultrasonic treatment was performed at 40°C for 30 min. The lipid solution was subjected to rotary evaporation at 220 rpm / min and 40°C to remove the solvent, forming a phospholipid film at the bottom of the flask. An appropriate amount of PBS was added to the flask, and after continuing the rotary evaporation for 5 min, A29L, A35R proteins, cGAMP were added, and water bath ultrasound was performed for 30 min. Liposomes were obtained. Among them, the amount of A29L, A35R and cGAMP added was determined according to the amount of lipid (DPPC, DPPG, Chol and DSPE-PEG2000). In this embodiment, 30 μg A29L, 30 μg A35R and 200 μg cGAMP were added to about 5 mg of lipid.

[0063] 3. The synthesized liposomes and macrophage membranes were mixed in a mass ratio of 3:1 and extruded through membranes of 800 nm, 400 nm and 200 nm in sequence at 40°C. Each membrane pore size was extruded 15 to 20 times.

[0064] Example 2

[0065] The preparation steps of biomimetic nanoparticles of AAG@AS-M NVs are as follows:

[0066] 1. When RAW 264.7 cells were grown to complete confluence in a T-75 culture flask, the cells were scraped from the culture flask, centrifuged at 1000 rpm for 5 min, and washed with PBS three times. The cell pellets were collected and resuspended in a homogenization buffer containing 25 mM sucrose, 10 mM Tris·HCl (pH=7.5), 1 mM MgCl2 (Sigma), 1 mM KCl (Sigma), and 2 mM PMSF (Thermo Fisher). After the cell suspension was repeatedly frozen and thawed three times, it was placed in a buffered homogenizer and ground 15 to 20 times to lyse the cells. Subsequently, the cell suspension was subjected to gradient centrifugation. The first centrifugation was 800 g for 10 min to remove the cell pellet; the second centrifugation was 8000 g for 15 min to remove the cell pellet; the third centrifugation was 12000 g for 30 min to remove the cell pellet. Finally, the supernatant was ultracentrifuged at 100000 g for 2 h (ultracentrifuge CP100WX, Hitachi, Japan) to collect the precipitate as macrophage cell membranes. The obtained macrophage cell membranes were stored at -80°C, resuspended in PBS before use, and the concentration was determined using a BCA kit.

[0067] 2. Weigh DPPC, DPPG, Chol and DSPE-PEG2000 in a mass ratio of 9:1:1:1 and dissolve them in 3 mL of chloroform. Ultrasonic treatment was performed at 40°C for 30 min. The lipid solution was subjected to rotary evaporation at 220 rpm / min and 40°C to remove the solvent, forming a phospholipid film at the bottom of the flask. An appropriate amount of PBS was added to the flask, and after continuing the rotary evaporation for 5 min, A29L, A35R proteins, cGAMP were added, and water bath ultrasound was performed for 60 min. Liposomes were obtained. Among them, the amount of A29L, A35R and cGAMP added was determined according to the amount of lipid (DPPC, DPPG, Chol and DSPE-PEG2000). In this embodiment, 30 μg A29L, 30 μg A35R and 200 μg cGAMP were added to about 4 mg of lipid.

[0068] 3. The synthesized liposomes and macrophage membranes were mixed in a mass ratio of 3:1 and extruded through 800 nm, 400 nm and 200 nm membranes at 45 °C in sequence. Each membrane with a pore size was extruded 15 to 20 times to prepare AAG@AS-M NVs.

[0069] Example 3 Performance Characterization of AAG@AS-M Biomimetic Nanoparticles

[0070] The performance of the AAG@AS-M bionic nanoparticles prepared in Example 1 was characterized.

[0071] 1. AAG@AS LPs (liposome nanoparticles without cell membrane components) and MNVs (macrophage membrane nanoparticles) were stained with cell membrane dyes DiI and DiO, respectively. Figure 2 As shown in A, DiI-labeled AAG@AS LPs were co-extruded through a 200 nm filter and basically overlapped with DiI-labeled M NVs, indicating that AAG@AS LPs and M NVs were fused and AAG@AS-M NVs were successfully synthesized. In contrast, no membrane fusion was observed when AAG@AS LPs and M NVs were directly mixed.

[0072] DiO and DiI are a pair of fluorescence resonance energy transfer (FRET) dyes. In theory, the fluorescence emitted by DiO (wavelength is about 501nm) can be accurately absorbed by DiI, which increases the fluorescence intensity of DiI at 565nm and reduces the fluorescence intensity of DiO. Figure 2 As shown in (B), compared with AAG@AS LPs, the fluorescence of AAG@AS-M NVs prepared under 483 nm laser stimulation was enhanced at 565 nm and weakened at 501 nm, which strongly demonstrated the successful membrane integration of AAG@AS LPs and M NVs.

[0073] 2. Zetasizer Nano particle size / zeta potential detector: Dynamic light scattering (DLS) showed that the average size of AAG@ASLPs (NV-1), M NV (NV-2) and AAG@AS-M NVs (NV-3) was about 200 nm, and the zeta potential (ζ) was -52.8, -33.3 and -44.5 mV, respectively. Figure 3 ). It is noteworthy that the ζ of AAG@AS-M NVs is located between the other two nanoparticles, which further proves the fusion of liposomes and macrophage membranes. The high ζ value of AAG@AS LPs is attributed to the introduction of the strongly negatively charged lipid DPPG.

[0074] 3. Transmission electron microscopy (TEM): Transmission electron microscopy (TEM) showed that the three nanoparticles all exhibited a typical spherical vesicle structure with a diameter of about 190nm ( Figure 4 ).

[0075] 4. The ability of AAG@AS-M NVs to penetrate the AS barrier and be taken up by AMs: Rhodamine B (RB) was used as a substitute for monkeypox virus antigen / adjuvant (forming RB@AS-M NVs) as shown in Figure 5 As shown in A, RB@AS-M NVs were more efficiently engulfed by macrophages than free RB (Free RB) and RB@ASs LPs (without macrophage membrane components) after co-incubation with RAW264.7 for 1 h. RB fluorescence was mainly distributed in the cytoplasm, which may be the homologous targeting effect of macrophage membrane on macrophages. After intranasal administration in mice, lung tissue sections showed that the inhaled free RB was almost confined to the AS layer on the alveolar surface, and lung cells took up very little ( Figure 5B). In contrast, both RB@AS LPs and RB@AS-M NVs were able to break through the AS barrier and were taken up by lung cells, and the number of lung cells taking up RB@AS-M NVs was significantly higher than that taking up RB@AS LPs.

[0076] 5. AAG@AS-M NVs innate immune activation effect: AAG@AS-M NVs (Vac-3) was used as a nanovaccine to intranasally immunize healthy mice. After 24 hours, bronchoalveolar lavage fluid (BALFs) was collected, and the percentage of activated M1 AMs (M1-AMs) and mature dendritic cells (DCs) in the alveoli was detected by flow cytometry. At the same time, the lung tissue was separated and ground, and the secretion levels of proinflammatory cytokines and IFN-Is were detected by enzyme-linked immunosorbent assay (ELISA). For comparison, free monkeypox virus antigen plus adjuvant (A29L+A35R+cGAMP, Vac-1) and AAG@AS LPs (Vac-2) were used as control vaccines (each mouse was vaccinated with a vaccine equivalent to 3μg A29L, 3μg A35R and 20μg cGAMP), and PBS was used as a negative control. Figure 6 As shown in the figure, the M1-AMs counts after AAG@AS-MNVs treatment were much higher than those of the control vaccine, which strongly confirmed their adjuvant effect. In addition, the activation efficiency of AAG@AS LPs on AMs was better than that of free antigens and adjuvants, because the AS-mimicking components they carried promoted the penetration of the AS barrier and entry into AMs. Similarly, the percentage of mature dendritic cells after stimulation with AAG@AS-M NVs was significantly higher than that of other groups. In addition, the number of conventional dendritic cells (cDCs) in BALF increased significantly after vaccination with AAG@AS-M NVs, indicating that a large number of circulating dendritic cells migrated and resided in lung tissues to participate in antiviral immunity. Measurements of TNF-α, INF-γ, and IFN-β concentrations in lung tissues showed that AAG@AS-M NVs had the strongest ability to promote inflammation and IFN-β production compared with the control group, demonstrating that this biomimetic nanovaccine has a strong adjuvant effect.

[0077] 6. AAG@AS-M NVs adaptive immune activation effect:

[0078] Healthy mice were immunized intranasally with AAG@AS-M NVs (G5) on day 0 (start) and boosted on day 28. On day 31, the mice were euthanized, and their lung tissues, bronchoalveolar lavage fluid (BALF) and serum were collected to detect adaptive immunity-related parameters, including humoral immunity, cellular immunity, mucosal immunity and immune memory. For comparison, free MPXV antigen plus adjuvant (A29L+A35R+cGAMP, G2), AAG@AS LPs (without macrophage membrane components, G3) and AA@AS-M NVs (without encapsulated cGAMP, G4) were used as experimental controls, and PBS was used as a negative control (G1).

[0079] 7. AAG@AS-M NVs humoral immune activation effect: Flow cytometry was used to detect activated B cells and memory B cells in mouse lung tissue. The results are as follows: Figure 7 A, Figure 7 As shown in B, AAG@AS-M NVs can significantly promote the maturation and differentiation of B cells into plasma cells (PC) and memory B cells (MBC). Using A29L and A35R as coating antigens on the ELISA plate, the specific neutralizing antibodies in the sera of vaccinated mice were measured. Figure 7 C, Figure 7 As shown in D, the anti-A29L and anti-A35R IgG titers in the serum of mice immunized with AAG@AS-M NVs were significantly higher than those of the control vaccine, indicating the effective induction of monkeypox virus-specific humoral immunity.

[0080] 8. AAG@AS-M NVs cell immune activation effect: Flow cytometry was used to detect the levels of CD8+T cells, memory effector T cells (TEM) and tissue-resident memory T cells (TRM) in mouse lung tissue. Figure 8 As shown in the figure, the proportion of CD8+T cells in the lungs of mice treated with AAG@AS-M NVs was significantly higher than that in the control group, indicating that the monkeypox virus-specific cellular immune response was successfully stimulated. On the other hand, the number of effector memory T cells (TEMs) and tissue-resident memory T cells (TRMs) increased significantly in lung tissue, indicating that the vaccination of biomimetic nanovaccines induced long-lasting immune memory.

[0081] 9. AAG@AS-M NVs mucosal immune activation effect: ELISA was used to detect IgA antibodies in mouse BALF to evaluate the ability of AAG@AS-MNVs to activate mucosal immunity (production of sIgA) in situ in the lung. Fig. 9As shown. AAG@AS-M NVs immunization resulted in significantly higher A29L and A35R-specific sIgA titers in BALF than the control vaccine, which was consistent with the trend of MPXV-specific IgG in serum and provided the possibility of preventing MPXV invasion through the respiratory tract. Similarly, anti-A29L and anti-A35R IgA in the vaginal and rectal mucosa of mice were significantly superior to other control vaccines and also superior to AAG@AS-M NVs (G6) vaccinated by intramuscular injection. In contrast, intramuscular injection of AAG@AS-M NVs induced very low MPXV-specific IgA in the vagina and rectum, implying that intranasal vaccination is a better route to induce distal mucosal immune protection. Fig.10 Schematic diagram of the principle of anti-monkeypox virus immunity induction and blocking of monkeypox virus infection through mucosal invasion based on AAG@AS-M bionic nanoparticles.

[0082] 10. Biosafety of AAG@AS-M: Fig.11 A. Fig.11 B and Fig.11 As shown in Figure 3, after incubation with L929, HUVEC, and HSF for 48 h, AAG@AS-M NVs had almost no inhibitory effect on cell viability and proliferation, indicating that the nanoparticles had almost no cytotoxicity to normal cells. Fig.11 D and Fig.11 As shown in Figure E, high concentration (in vivo dose) of AAG@AS-M NVs was intranasally administered to healthy mice, and AAG@AS-M NVs did not cause significant body weight changes. Seven days after administration, peripheral blood of mice was collected for plasma biochemical testing. The biochemical indicators of plasma liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN), did not change significantly, indicating that AAG@AS-M NVs had almost no acute liver and kidney toxicity.

[0083] Example 3 Application of AAG@AS-M NVs in preventing monkeypox virus infection

[0084] The AAG@AS-M NVs prepared in Example 1 were used as nanovaccines to investigate their role in preventing monkeypox virus infection.

[0085] Since the highly virulent monkeypox virus is extremely harmful to humans, an attenuated strain of VACV of the genus Orthopoxvirus was selected as an alternative virus. Healthy mice were intranasally inoculated with AAG@AS-M NVs or control vaccines on day -35 (starting) and boosted on day -7. After intravenous injection of a lethal titer of VACV on day 0, the survival rate and symptoms of the immunized mice were monitored. Fig.12As shown, survival analysis showed that AAG@AS-M NVs immunization completely prevented the death of mice due to VACV infection, while the mortality rates of mice vaccinated with other vaccines were 70%, 50%, 20%, and 30%, respectively. In addition, the skin lesions of the mouse tails showed that the unvaccinated mice (PBS-treated group) developed a large number of characteristic rashes similar to monkeypox lesions on the skin 7 days after VACV challenge. Vaccination with A29L+A35R+cGAMP, AAG@AS LPs, and AA@AS-M NVs resulted in different reductions in the number of tail rashes, while AAG@AS-MNVs immunization completely eliminated the production of rashes.

[0086] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An inhalable bionic nanoparticle, characterized in that: Hybrid biomimetic nanovesicles composed of liposomes simulating alveolar surfactant and macrophage membranes were used as carriers to encapsulate monkeypox virus-specific antigens and 2',3'-cyclic guanosine monophosphate.

2. The inhalable bionic nanoparticle according to claim 1, characterized in that: The mass ratio of the liposome simulating alveolar surfactant to the macrophage membrane is 2-3:

1.

3. The inhalable bionic nanoparticle according to claim 1, characterized in that: The components of the liposome simulating alveolar surfactant are dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol.

4. The inhalable bionic nanoparticle according to claim 3, characterized in that: The mass ratio of the dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol is 9-10:1:1:

1.

5. The inhalable bionic nanoparticle according to claim 3, characterized in that: The preparation method of the liposome simulating alveolar surfactant is to mix dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol and then prepare the liposome by ultrasonic method.

6. The inhalable bionic nanoparticle according to claim 1, characterized in that: The monkeypox virus specific antigen is A29L and / or A35R.

7. The method for preparing inhalable bionic nanoparticles according to any one of claims 1 to 6, characterized in that: The method comprises mixing liposomes simulating alveolar surfactant with monkeypox virus specific antigen and 2',3'-cyclic guanosine monophosphate adenosine, ultrasonicating, adding macrophage membranes for mixing, and obtaining the mixture by membrane extrusion.

8. Use of the inhalable bionic nanoparticles according to any one of claims 1 to 6 in the preparation of a drug for preventing and / or treating monkeypox virus infection.

9. The use according to claim 8, characterized in that: The medicine is a vaccine preparation.

10. A vaccine preparation, characterized in that Contains the inhalable bionic nanoparticles according to any one of claims 1 to 6.

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