CN110772635B - Bionic nano-vaccine coated with influenza virus body and preparation method thereof - Google Patents

Abstract

The invention discloses a biomimetic nano-vaccine coated with influenza virus body (VI) and a preparation method thereof. The biomimetic nano-vaccine includes influenza virus body, small-diameter fluorinated particles and DNA vaccine, and the biomimetic nano-vaccine has a core-shell structure In the nano system, VI is a lipid vesicle containing the envelope protein of influenza virus, the inner core of the nano system is a small-diameter fluorinated particle loaded with DNA vaccine, and the influenza virus body is wrapped on the surface of the inner core. The virosome of the invention has receptor binding activity, lysosome membrane fusion activity and antigenic activity of influenza virus; the fluorinated inner core with small particle size can deliver DNA into the nucleus and promote protein expression. The nanosystem can realize the co-loading of protein vaccine and DNA vaccine and the site-specific delivery of each component, and finally obtain a synergistic effect to improve the immune effect. The invention belongs to the field of pharmaceutical preparations and the technical field of biomedicine, can effectively prevent influenza virus infection, and has high clinical application value.

Description

Bionic nano-vaccine coated with influenza virus body and preparation method thereof

technical field

The invention relates to the field of pharmaceutical preparations and the field of biomedicine technology, in particular to a biomimetic nano-vaccine coated with influenza virus bodies and a preparation method thereof.

Background technique

The spread of seasonal influenza virus poses a huge threat to global public health security and seriously affects human health and economic development. Vaccination is still the main means of preventing and dealing with influenza virus. The currently approved influenza vaccines are mainly inactivated vaccines (such as split vaccines) and live attenuated vaccines. However, there are still many safety hazards in the use of influenza vaccines based on modified and inactivated virus strains, mainly including adverse reactions such as back mutations of pathogens, allergies, and autoimmunity. In comparison, DNA vaccines and protein vaccines have the advantages of good safety, high purity, and strong specificity, but pure DNA and protein are difficult to be taken up by antigen-presenting cells (APCs), and can only induce short-term immunity or drive Th2 cells Humoral immunity, the application is greatly limited. In order to enhance the immune effect of DNA and protein vaccines, experts from the fields of biology, medicine and materials science are paying more and more attention to the application of nanotechnology in the field of vaccines.

VI is derived from the host cell membrane in the process of virus propagation, including the glycoprotein of the virus itself. The glycoproteins on the surface of influenza virions include HA and NA, wherein HA binds to the sialic acid receptor of host cells during virus infection and mediates the endocytosis of the virus. Studies have shown that VI prepared in vitro is a unilamellar vesicle with a spherical shape and an average particle size of less than 200nm. Compared with artificially prepared liposomes, VI contains functional glycoproteins HA and NA, but does not contain viral genetic material. VI prepared in vitro retains the receptor binding activity, lysosome membrane fusion activity and antigenicity of influenza virus. Among them, the receptor binding activity is conducive to the uptake of nano-vaccine by APC, the lysosomal membrane fusion activity is beneficial to the lysosome escape of the nanoparticle core, and the antigenic activity can be directly used as a vaccine. Therefore, Ⅵ is a very potential carrier system and vaccine material.

DNA vaccines must enter the nucleus and be transcribed and translated to induce the corresponding immune response, but various cellular barriers limit the delivery of exogenous DNA to the nucleus, mainly including cell membrane barriers, endosome/lysosome barriers, and nuclear membrane barriers. At present, there are a variety of technical means to promote the transmembrane transport of DNA, the most commonly used is to form a complex between plasmid DNA (pDNA) and cationic liposomes, and then the complex interacts with negatively charged glycoproteins on the cell membrane. Acts to promote nonspecific endocytosis of the complex. For the endosome/lysosome barrier, the lysosome bursts mainly by increasing the osmotic pressure of the endosome/lysosome, so as to realize the escape of the endosome/lysosome. For a long time, the nuclear membrane barrier has been the main factor that hinders the entry of DNA into the nucleus for transcription and expression. A commonly used strategy is to modify the plasmid with a nuclear localization sequence (NLS) peptide to increase the probability of entering the nucleus. However, the expression efficiency of pDNA modified with NLS is not high. In addition, the charge interaction between anionic DNA and cationic NLS will bury NLS in DNA, which limits the function of NLS. Studies have shown that the nuclear pore size of the nucleus is 20-70nm, and particles smaller than 50nm can enter the nucleus. In addition, fluorination modification can effectively improve the transfection efficiency and stability in vivo and in vitro of cationic polymers, and can effectively increase the nuclear distribution of genes, which is conducive to promoting the transcription and expression of pDNA.

Contents of the invention

Purpose of the invention: In order to achieve the above purpose, the technical problem to be solved by the present invention is to provide a biomimetic nano-vaccine coated with influenza virus body.

The technical problem to be solved by the present invention is to provide a preparation method of the biomimetic nano-vaccine coated with influenza virions.

Technical solution: In order to solve the above technical problems, the present invention provides a biomimetic nano-vaccine coated with influenza virions. The biomimetic nano-vaccine includes influenza virions (VI), small fluorinated particles and DNA vaccine.

Wherein, the biomimetic nano-vaccine is a nano-system with a core-shell structure, the VI is a lipid vesicle containing the envelope protein of influenza virus, the core of the nano-system is a small-sized fluorinated particle loaded with DNA vaccine, and the Influenza virions wrap around the surface of the inner core.

Wherein, the influenza virus body is derived from influenza virus, and the influenza virus includes but not limited to H1N1, H3N2, H5N1, H7N9, H9N2 and other subtypes.

Wherein, the small-diameter fluorinated particles are nanostructures formed by fluorinated cationic polymers wrapping gold particles.

Wherein, the cationic polymer modified by fluorination is a gene carrier, including but not limited to polyamines (polyamines), such as linear or branched polyethyleneimine (PEI); polyamide-amine (poly(amido amine), PAA), such as dendrimers PAMAM, hyperbranched polyamide-amine (HPAMAM); polymethacrylates, such as polydimethylaminoethyl methacrylate (pDMAEMA); poly Amino acids (polyaminoacids), such as polylysine (PLL); polyester (polyester), such as linear or branched poly (β-amino ester) (PBAE); natural polysaccharides (polysaccharides), such as shell polysaccharides etc.

Wherein, the particle size of the gold particles is 10-30 nm.

The content of the present invention also includes the preparation method of the biomimetic nano-vaccine coated with influenza virus body, comprising the following steps:

1) Preparation of VI: add 1,2-dihexanoyl lecithin to the influenza virus in an ice bath, collect the supernatant by ultracentrifugation, dialyze the supernatant in HBS buffer to remove 1,2-dihexanoyl lecithin, obtaining recombinant VI;

2) Synthesis of cationic polymer modified by fluorination: react according to the molar ratio of amino group, heptafluorobutyric anhydride and triethylamine in the cationic polymer is 1:3:1.2, the reaction medium is methanol, stirred at room temperature, after the reaction, the The reaction product was dialyzed in deionized water with a pH of 3-4, and freeze-dried to obtain a fluorinated modified cationic polymer;

3) Preparation of FAu: Take gold particles with a particle size of 10-30nm and slowly drop them into the fluorinated cationic polymer, shake slowly, centrifuge, collect and precipitate to obtain fluorinated particles with small particle size FAu;

4) Preparation of FAu loaded with DNA vaccine: take the FAu in step 3), mix it with the DNA vaccine, shake slowly, the mass ratio of the cationic polymer to the DNA is 1.5-3, and obtain small-sized fluorinated particles loaded with the DNA vaccine;

5) Preparation of ARV: Mix the recombined VI in step 1) with fluorinated particles of small and medium size in step 4), extrude back and forth with an extruder, and collect by centrifugation to obtain ARV.

Wherein, the ultracentrifugation conditions of the step 1) are 4° C., 100,000×g, and 1.5 h.

Wherein, the HBS buffer in step 1) is HEPES buffer containing 0.15M NaCl.

Wherein, the concentration of the fluorinated cationic polymer in step 3) is 5-10 mg/mL.

Wherein, the DNA vaccine of said step 4) includes but not limited to nucleoprotein (NP), matrix protein (M1), membrane protein (M2), hemagglutinin (HA) or neuraminidase (NA) of influenza virus. Full-length DNA sequence or partial DNA sequence.

Wherein, the pore size of the filter membrane of the extruder in the step 5) is 50-100 nm.

Beneficial effects: Compared with the prior art, the present invention has the advantages that simple DNA and protein vaccines are difficult to be effectively taken up by APCs, and the induced immune response is relatively weak. In addition, due to the differences in the properties of different active substances, nanosystems need to achieve the co-loading of various components and the effective release of different target sites, which has always been a difficulty in formulation research. The nano-vaccine of the present invention binds specifically to the sialic acid receptor on the surface of APC through the HA on the surface, and mediates the endocytosis of the particle into the endosome/lysosome; VI fuses with the lysosome membrane to carry the DNA The nanoparticles of the vaccine are released into the cytoplasm; the nanoparticles escaped from the lysosome enter the nucleus for transcription and expression under the advantage of fluorination and the small particle size of the particles. The nano-vaccine simulates the virus replication mechanism, realizes the site-specific sequential delivery of protein and DNA vaccines, and finally obtains a synergistic effect to improve the immune effect. The process of the nano vaccine prepared by the invention is simple and controllable, the production cost is low, the repeatability is good, and it is suitable for large-scale production; the carrier has good stability and high safety. The vaccine can effectively reduce the infection risk of influenza virus and can effectively control the infection caused by influenza virus.

Description of drawings

Fig. 1, schematic diagram of the preparation principle of the biomimetic nano-vaccine coated with influenza virus body of the present invention;

Fig. 2, the characterization of the biomimetic nano-vaccine coated with influenza virus body of the present invention;

A: the particle size of the biomimetic nano-vaccine coated with influenza virus body of the present invention;

B: the potential distribution of the influenza virus body-coated biomimetic nano-vaccine of the present invention;

C: Transmission electron micrograph of the biomimetic nano-vaccine coated with influenza virus body of the present invention;

Fig. 3: The membrane fusion of the biomimetic nano-vaccine coated with influenza virus body of the present invention and the delivery of plasmid DNA into the nucleus;

A: ARV and VI prepared in Example 1 of the present invention were incubated with erythrocytes respectively, and the OD ₅₄₀ ultraviolet absorption curve varies with pH;

B: Membrane fusion experiments of the influenza virus body-coated biomimetic nano-vaccine of the present invention at pH 5.5 and pH 7.4 respectively;

C: ARV' without fluorinated modified cationic polymer and ARV delivery plasmid DNA prepared by the present invention into the nucleus;

Figure 4: Cellular and humoral immune responses of the biomimetic nano-vaccine coated with influenza virions of the present invention;

A: The biomimetic nano-vaccine group coated with influenza virions (ARV group): the nano-vaccine was injected subcutaneously at the base of the tail at the first and third week, and the spleen was isolated at the fourth week to prepare a single-cell suspension, which was mixed with CD3 and CD8 Antibody incubation, flow cytometry to detect the content of CD8 ⁺ T cells in mice; Saline group: subcutaneous injection of normal saline at the base of the tail at the first and third week, and the rest were the same as ARV group; VI group: at the first and third week In the 3rd week, VI was subcutaneously injected at the base of the tail, and the rest were the same as in the ARV group.

B: Spleen single cell suspension was taken, incubated with CD3 and CD4 antibodies, and the content of CD4 ⁺ T cells in mice was detected by flow cytometry;

C: After the mice were immunized, serum samples were collected at the 4th week to determine the IgG titer;

Fig. 5: the protective effect of the biomimetic nano-vaccine coated with influenza virus body of the present invention on virus infection;

A: The biomimetic nano-vaccine group coated with influenza virus body, i.e. the ARV group: the nano-vaccine was subcutaneously injected at the base of the tail at the 1st and 3rd week, and the virus was challenged at the 5th week, and each mouse was infected with 1×10 ⁴ CFU PR8 Virus, the weight of the mice was continuously recorded for 14 days; Saline group: Subcutaneous injection of normal saline at the base of the tail at the first and third week, challenge at the fifth week, each mouse was infected with 1×10 ⁴ CFU PR8 virus, continuous recording The body weight of the mice was 14 days; Normal group: without treatment, the body weight of the mice was continuously recorded for 14 days.

B: After challenge, the survival rate of the mice was recorded;

C: 4 days after the challenge, the lung tissue was isolated and the virus titer was determined;

D: 4 days after the challenge, the lung tissue was separated, and the lung index was measured;

E: 4 days after challenge, the lung tissue was isolated and stained with HE.

Detailed ways

The present invention will be further described below through specific embodiments. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some modifications and improvements can also be made, and these should also be regarded as belonging to the present invention. protection scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

Materials and Equipment:

(1) Gold particles were purchased from Donna Biotech;

(2) Polyethyleneimine (PEI) was purchased from Sigma, USA;

(3) Heptafluorobutyric anhydride (HFBA) was purchased from Sigma, USA;

(4) The nuclear/cytoplasmic cell component extraction kit was purchased from Biovision, USA;

(5) The Cy5 nucleic acid labeling kit was purchased from Mirus Bio, USA;

(6) The DNA vaccine of influenza virus nucleoprotein (NP) was synthesized by Changzhou Jiyu Biotechnology Co., Ltd. (the eukaryotic expression vector obtained by inserting the NP sequence of influenza virus PR8 into PCDNA3.1(+));

(7) Influenza virus is the laboratory-preserved virus strain PR8;

(8) 1,2-Dicaproyl lecithin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

(9) The extruder and filter membrane were purchased from Mog Machinery (Shanghai) Co., Ltd.;

(10) CD3, CD4, CD8 flow antibodies were purchased from eBioscience, USA;

(11) DOPC and cholesterol were purchased from Avanti Polar Lipids, USA;

(12) FRET reagent pair NBD-PE and Rho-PE were purchased from Biotium Corporation of the United States.

Example 1

(1) Preparation of VI: Influenza virus PR8 was ultracentrifuged, and the bottom precipitate was collected. For every 5 mg of precipitated virus, 375 μL of 200 mM 1,2-dihexanoylphosphatidylcholine was added for 30 minutes on ice. Collect the supernatant by ultracentrifugation, dialyze the supernatant in HBS buffer for 24 hours, remove 1,2-dicaproyl lecithin, and obtain recombinant VI, wherein the ultracentrifugation conditions are 4°C, 100,000×g, 1.5h;

(2) Synthesis of fluorinated cationic polymer: react according to the molar ratio of amino group, heptafluorobutyric anhydride and triethylamine in the cationic polymer is 1:3:1.2, the reaction medium is methanol, and stirred at room temperature for 48 hours. After the reaction, the reaction product was dialyzed in deionized water with a pH of 3 to 4 for 2 days, and freeze-dried to obtain a fluorinated modified cationic polymer. The cationic polymer used in this example was 25kDa PEI;

(3) Preparation of FAu: Take 3× ¹⁰⁹ gold particles with a particle size of 10-30nm and slowly add them dropwise to 0.5mL of fluorinated cationic polymer, shake gently for 30min, centrifuge at 10000rpm for 10min, and collect the precipitate as small particle size of fluorinated particles FAu;

(4) Preparation of ARV: Get the FAu in step (3), mix it with the DNA vaccine of influenza virus nucleoprotein (NP), shake slowly and lightly for 30min, the mass ratio of cationic polymer to DNA is 1.5, and obtain the DNA vaccine loaded DNA vaccine. Particle Size Fluorinated Particles (pFAu). Take the VI in step (1) and mix it with pFAu, and extrude back and forth 10 times with an extruder, wherein the filter membrane pore size of the extruder is 100 nm, and centrifuge at 10,000 rpm for 10 min to collect nano-vaccine ARV.

The prepared ARV was characterized and observed by particle size analyzer and transmission electron microscope. As shown in Figure 2A, the size of ARV was 68.2nm, and the polydispersity index (PDI) was 0.21; as shown in Figure 2B, the potential of ARV was -5.6mV; as shown in Figure 2C, the shape of ARV was spherical.

Example 2

Low pH conditions in cellular endosomes/lysosomes can induce conformational changes in influenza virus hemagglutinin (HA), causing fusion of the viral envelope with endosome/lysosome membranes. Therefore, under acidic conditions, when the conformation of HA adsorbed on red blood cells changes, it will cause the red blood cell membrane to rupture, thereby releasing hemoglobin and causing hemolysis. Thus, the fusion process of the membrane can be simulated by using the hemolysis experiment. As shown in Figure 3A, when ARV and VI prepared in Example 1 were incubated with erythrocytes, the OD ₅₄₀ ultraviolet absorption increased with the decrease of pH. Both ARV and VI had hemolytic effect, indicating that membrane fusion had occurred. Further, anionic liposomes are used to simulate the membrane structure of cells and carry fluorescence resonance energy transfer (FRET) compounds, which are composed of DOPC, cholesterol and FRET reagent pair NBD-PE and Rho-PE, wherein DOPC: cholesterol: NBD- The molar ratio of PE:Rho-PE is 10:1:0.1:0.05. At pH 5.5 and 7.4, ARV was co-incubated with anionic liposomes, and the fluorescence intensity (450nm/595nm) of liposomes was detected at specific time points, and the lipid fusion rate=(I _t -I ₀ )/( II ₀ )×100%, wherein, I ₀ is the fluorescence intensity before incubation, I _t is the fluorescence intensity at each measurement time point, and I is the liposome treated with 0.1% (v:v) Tween X-100 Fluorescence intensity of liposomes. As shown in Figure 3B, after 1 hour of incubation, the membrane fusion rate of ARV was 35% at pH 5.5, but less than 10% at pH 7.4, indicating that ARV can fuse efficiently under the acidic conditions of lysosomes cell membrane. To investigate the ability of ARV to deliver plasmid DNA to the nucleus, the plasmid DNA was labeled with Cy5 nucleic acid labeling kit, incubated with mouse dendritic cells DC2.4 for different times, the nuclei were extracted, and the Cy5 fluorescence intensity was detected. As shown in Figure 3C, compared with the ARV' group without fluorinated cationic polymer, the fluorescence intensity of ARV nucleus was significantly enhanced (P<0.005), so ARV can effectively deliver plasmid DNA into the nucleus.

Example 3

The ARV prepared in Example 1 was immunized into mice (C57BL/6, 20±2 g, male) by subcutaneous injection at the base of the tail at the first week and the third week, and each dose was 40 μg of ARV protein. Isolate the spleen at the 4th week, prepare a single cell suspension, collect 2 million cells into a 1.5mL centrifuge tube, centrifuge at 1600rpm for 5min, remove the supernatant, wash gently with 1mL PBS twice, and finally resuspend in 100μl PBS, wash with CD3, CD8 Antibody and CD3, CD4 antibody labeled cells, incubated at 4°C in the dark for 30min. Subsequently, it was fixed with 4% paraformaldehyde for flow detection. As shown in Figures 4A-4B, VI, pFAu and ARV can effectively induce the increase of CD8 ⁺ T and CD4 ⁺ T cells in vivo, among which ARV has the strongest induction ability. Serum samples of immunized mice were prepared at the 4th week, and the IgG titer in the serum was determined. As shown in Figure 4C, the prepared ARV has a strong humoral immune effect on mice, and there are significant differences compared with VI and pFAu (P<0.05, P<0.01). Therefore, the nanovaccine can effectively induce humoral and cellular immune responses in vivo.

Example 4

20±2 g male C57BL/6 mice were selected, and the ARV prepared in Example 1 was subcutaneously injected at the base of the tail at the first week and the third week, and each dose was 40 μg of ARV protein. In the fifth week of challenge, each mouse was infected with 1×10 ⁴ CFU PR8 virus, and then the body weight and survival rate of the mice were recorded for two consecutive weeks; the mice in the Saline group were subcutaneously injected with ARV at the base of the tail in the first week and the third week Equal volume of normal saline, challenged at week 5, each mouse was infected with 1×10 ⁴ CFU PR8 virus, and then the body weight and survival rate of the mice were recorded for two consecutive weeks; mice in the Normal group were not treated, and then for two consecutive weeks The body weight and survival rate of the mice were recorded. As shown in Figure 5A, the weight of the mice in the normal group gradually increased over time, while the weight of the mice in the Saline group continued to decrease. After the 5th day, the body weight showed a trend of slow growth. As shown in Figure 5B, all the mice in the Saline group died at 12 days, while the mice in the ARV immunized group and the normal group did not die. The statistical results of lung virus titer and lung index showed that after ARV immunization, the lung virus content and lung damage could be significantly reduced (Fig. 5C-5D). HE staining of the lung tissue showed (Fig. 5E) that a large number of inflammatory cell infiltrations appeared in the lungs after challenge, while the normal group and the ARV immune group had good cell morphology, complete tissue structure, and no obvious inflammation. Therefore, the prepared nano-vaccine has obvious immunopreventive effect on influenza virus.

Claims (5)

1. The bionic nano vaccine coated by the influenza virosome is characterized by comprising the influenza virosome, small-particle-size fluorinated particles and a DNA vaccine, wherein the bionic nano vaccine is a nano system with a core-shell structure, the influenza virosome is a lipid vesicle containing influenza virus envelope protein, the inner core of the nano system is the small-particle-size fluorinated particles loaded with the DNA vaccine, the influenza virosome is coated on the surface of the inner core, the influenza virosome is derived from influenza virus, the influenza virus comprises H1N1, H3N2, H5N1, H7N9 or H9N2 subtypes, the small-particle-size fluorinated particles are of a nano structure formed by coating gold particles with a fluorinated and modified cationic polymer, the cationic polymer is a gene vector and comprises one or more of polyamines, polyamidoamines, hyperbranched polyamidoamines, polymethacrylates, polyaminoacids, polyesters or natural polysaccharides, and the particle size of the gold particles is 10-30 nm.

2. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 1, which is characterized by comprising the following steps:

1) VI preparation: adding 1, 2-dihexanoyl lecithin into influenza virus for ice bath, ultracentrifuging for 1.5h, collecting supernatant, dialyzing the supernatant in HBS buffer solution, and removing 1, 2-dihexanoyl lecithin to obtain recombinant VI;

2) Fluorinated modified cationic polymer synthesis: reacting according to the molar ratio of amino, heptafluorobutyric anhydride and triethylamine in the cationic polymer of 1.3, wherein the reaction medium is methanol, stirring at room temperature, dialyzing the reaction product in deionized water with pH of 3-4 after the reaction is finished, and freeze-drying to obtain a fluorinated modified cationic polymer;

3) Preparation of FAu: slowly dripping gold particles with the particle size of 10-30 nm into the fluorinated modified cationic polymer, slowly shaking gently, centrifuging, and collecting precipitates to obtain fluorinated particles FAu with small particle size;

4) Preparation of DNA vaccine-loaded FAu: mixing the FAu obtained in the step 3) with a DNA vaccine, and slowly shaking to obtain small-particle-size fluorinated particles loaded with the DNA vaccine, wherein the mass ratio of the cationic polymer to the DNA is 1.5 to 3;

5) Preparing a bionic nano vaccine coated by influenza virosome: and (3) mixing the VI recombined in the step (1) with the small-particle-size fluorinated particles in the step (4), extruding back and forth by using an extruder, and centrifugally collecting to obtain the bionic nano vaccine coated by the influenza virus corpuscles.

3. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 2, wherein the concentration of the fluorinated modified cationic polymer in the step 3) is 5 to 10mg/mL.

4. The method for preparing an influenza virosome-coated biomimetic nano-vaccine according to claim 2, wherein the DNA vaccine of step 4) comprises a full-length DNA sequence of influenza virus nucleoprotein, matrix protein, membrane protein, hemagglutinin or neuraminidase or a partial DNA sequence thereof.

5. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 2, wherein the size of the filter membrane hole of the extruder in the step 5) is 50-100 nm.

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