Nanovaccine, preparation method and application thereof

Abstract

The application provides a preparation method of a polymer lipid nano-vaccine, comprising the following steps: dissolving a lipid material and a polymer material in organic solvents respectively, mixing uniformly as an oil phase, and taking a 10%-50% ethanol aqueous solution as a water phase; slowly dropping the oil phase into the water phase under a first stirring speed, adjusting the stirring speed to a second stirring speed after the dropping is completed, and obtaining a mixed phase; solidifying the mixed phase for 1 h to 12 h to remove the organic solvents, cleaning and freeze-drying to obtain polymer lipid nanoparticles; and incubating the polymer lipid nanoparticles and mRNA encoding a hepatitis B virus antigen protein at room temperature for 20 min to 120 min, so as to obtain a polymer lipid mRNA nano-vaccine.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a nanovaccine, its preparation method, and its application. Background Technology

[0002] Nanoparticle vaccines have shown great potential in the treatment of various chronic infectious diseases and tumors. Nucleic acid drugs, especially mRNA vaccines, have been extensively researched and applied. They induce humoral, especially cellular, immunity through in vivo transfection and expression of antigen proteins, achieving immunotherapeutic effects. Utilizing the antigen-encoding information carried by mRNA, they guide human cells to autonomously synthesize antigen proteins to initiate an immune response. Their development and production are rapid, enabling quick responses to emerging pathogen threats, and they are relatively safe as they do not contain live viruses. However, mRNA molecules are unstable and easily degraded, requiring delivery vectors for protection.

[0003] Chronic hepatitis B is a major cause of liver fibrosis, cirrhosis, and liver cancer, posing a significant threat to public health. Current treatments for chronic hepatitis B primarily rely on nucleoside analogues and interferon-alpha, but neither can achieve sufficient viral clearance. Therapeutic hepatitis B vaccines have been developed through numerous trials, including those based on recombinant proteins, adenovirus vectors, and DNA technology, which can achieve effective seroclearance of HBsAg and seroconversion of anti-HBs antibodies (Ab). Next-generation mRNA vaccines have shown superior performance compared to traditional vaccines in inducing antibody and cellular immune responses. Their strong intrinsic adjuvant properties can also activate the innate immune system, offering hope for a complete cure of chronic hepatitis B. However, for mRNA vaccines, selecting a suitable delivery vector is crucial. Summary of the Invention

[0004] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, this application provides a nano-vaccine, a method for its preparation, and its application.

[0005] According to one aspect of this application, a method for preparing a polymer lipid nanovaccine is provided, comprising: dissolving lipid materials and polymer materials separately in an organic solvent and mixing them uniformly to form an oil phase; using an aqueous solution containing 10% to 50% ethanol as an aqueous phase; slowly adding the oil phase dropwise to the aqueous phase at a first stirring speed; after the addition is complete, adjusting the stirring speed to a second stirring speed to obtain a mixed phase; solidifying the mixed phase for 1 to 12 hours to remove the organic solvent; washing and freeze-drying to obtain polymer lipid nanoparticles; and incubating the polymer lipid nanoparticles and mRNA encoding hepatitis B virus antigen protein at room temperature for 20 to 120 minutes to obtain a polymer lipid mRNA nanovaccine.

[0006] According to another aspect of this application, a nano-vaccine is provided, obtained according to the above preparation method.

[0007] According to another aspect of this application, the above-described nanovaccine is provided for use in the preparation of an mRNA vaccine for hepatitis B-related diseases.

[0008] According to the embodiments of this application, lipid materials and polymer materials are co-dissolved in an organic solvent to form an oil phase, and an aqueous solution containing 10%–50% ethanol is used as the aqueous phase. The oil phase is slowly added dropwise at a first stirring speed, and after the addition is complete, the stirring speed is adjusted to a second speed for continued stirring. This allows the polymer and lipids to self-assemble into polymer-lipid nanoparticles with uniform particle size, good dispersibility, and positively charged surfaces. The introduction of the ethanol-containing aqueous phase improves the interfacial properties, facilitating the diffusion of the organic solvent and the uniform nucleation of nanoparticles, effectively preventing particle aggregation. Curing for 1–12 hours ensures complete removal of the organic solvent, while washing and freeze-drying remove free materials and residual solvents, improving the purity and storage stability of the nanoparticles. The reconstitution performance after freeze-drying is excellent. Incubating the obtained polymer-lipid nanoparticles with mRNA encoding hepatitis B virus antigen protein at room temperature for 20–120 minutes allows for efficient mRNA transport via electrostatic adsorption, forming a structurally stable and biosafety-enhanced polymer-lipid mRNA nanovaccine.

[0009] Furthermore, the polymer lipid nanovaccine prepared by the above method can protect mRNA from enzymatic degradation, improve the uptake efficiency of mRNA in antigen-presenting cells such as dendritic cells (DCs), promote the expression of antigen proteins, and has good liver-targeted delivery capability. It can enhance the level of hepatitis B virus-specific humoral and cellular immune responses in the liver and spleen system, and induce immune memory. Attached Figure Description

[0010] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0011] Figure 1 The graph shows the effect of polymer and lipid mass ratio on nanoparticle size and PDI of different polymer lipid nanoparticles prepared in Examples 1-6 of this application.

[0012] Figure 2 The graph shows the effect of the polymer-to-lipid mass ratio on the surface morphology of the different polymer lipid nanoparticles prepared in Examples 1-6 of this application.

[0013] Figure 3The graph shows the effect of the external aqueous phase volume ratio on the nanoparticle size and PDI of the polymer lipid nanoparticles prepared in Examples 7-13 of this application; where the bar graph corresponds to the particle size and the line graph corresponds to the polydispersity index.

[0014] Figure 4 The figure shows the effect of the external aqueous phase volume ratio on the surface morphology of the polymer lipid nanoparticles prepared in Examples 7-13 of this application.

[0015] Figure 5 The graphs show the surface morphology, particle size, PDI value and Zeta potential of the polymer lipid nanoparticles prepared in Examples 3, 17 and 18 of this application.

[0016] Figure 6 This is a cytotoxicity analysis diagram of the three nanoparticles PLGA / SM-102, PLGA / DOTAP, and PLGA / DDAB in Example 31 of this application;

[0017] Figure 7 This is a cytotoxicity analysis diagram of the mRNA@PLGA / SM-102 nanovaccine in Example 32 of this application;

[0018] Figure 8 This is a diagram showing the mRNA expression results of the hepatitis B mRNA polymer lipid nanovaccine loaded in Example 33 of this application; where A is a cell nuclear staining diagram, B is a target protein expression diagram, and C is an overlay and merged diagram;

[0019] Figure 9 This is a diagram showing the activation results of DCs induced by the mRNA@PLGA / SM-102 nanovaccine in Example 34 of this application;

[0020] Figure 10 This is a diagram showing the results of mRNA@PLGA / SM-102 nanovaccine inducing DCs to secrete cytokines in Example 35 of this application;

[0021] Figure 11 This is a diagram showing the results of inducing local liver immune cell activation levels in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 36 of this application;

[0022] Figure 12 This is a diagram showing the results of inducing local liver immune memory levels in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 37 of this application.

[0023] Figure 13 This is a graph showing the results of inducing local liver cytokine secretion levels in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 38 of this application;

[0024] Figure 14 This is a graph showing the results of specific serum antibody levels induced in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 39 of this application;

[0025] Figure 15 This is a graph showing the results of the activation level of spleen system immune cells induced after mice were immunized with the mRNA@PLGA / SM-102 nanovaccine in Example 40 of this application;

[0026] Figure 16 This is a graph showing the results of inducing the level of immune memory in the spleen system after immunizing mice with the mRNA@PLGA / SM-102 nanovaccine in Example 41 of this application;

[0027] Figure 17 This is a graph showing the results of the induced cytokine secretion levels in the spleen system after mice were immunized with the mRNA@PLGA / SM-102 nanovaccine in Example 42 of this application. Detailed Implementation

[0028] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0029] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0030] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0031] The term "zeta potential" as used in this application refers to the sectional potential, which is used to measure the stability of colloidal dispersions. The surface zeta potential of the polymer lipid nanoparticles described in this application is +25 to +60, indicating that the polymer lipid nanoparticles of this application have good stability.

[0032] The term "PDI value" as used in this application refers to the polymer dispersion index, an indicator used to describe the molecular weight distribution of a polymer. The PDI value of the polymer lipid nanoparticles described in this application is 0.05-0.5, indicating that the polymer lipid nanoparticles of this application have a concentrated molecular weight distribution and uniform distribution across all positions of the nanoparticles.

[0033] The terms used in this application, such as "mRNA@PLGA / SM-102", refer to polymer lipid nanovaccines carrying target mRNA, PLGA refers to the polymer material polylactic acid-glycolic acid copolymer (PLGA), SM-102 refers to the lipid material SM-102, and so on.

[0034] Specifically, according to one aspect of this application, a method for preparing a polymer lipid nanovaccine is provided, comprising the following steps S1 to S4:

[0035] Step S1: Dissolve the lipid material and polymer material separately in an organic solvent and mix them evenly to form the oil phase, and use an aqueous solution containing 10%~50% ethanol as the aqueous phase;

[0036] Step S2: Slowly add the oil phase to the aqueous phase at the first stirring speed. After the addition is complete, adjust the stirring speed to the second stirring speed to obtain the mixed phase.

[0037] Step S3: The mixed phase is cured for 1 to 12 hours to remove the organic solvent, and then washed and freeze-dried to obtain polymer lipid nanoparticles;

[0038] Step S4: Incubate the polymer lipid nanoparticles and mRNA encoding hepatitis B virus antigen protein at room temperature for 20 min to 120 min to obtain the polymer lipid mRNA nanovaccine.

[0039] According to the embodiments of this application, lipid materials and polymer materials are co-dissolved in an organic solvent to form an oil phase, and an aqueous solution containing 10%–50% ethanol is used as the aqueous phase. The oil phase is slowly added dropwise at a first stirring speed, and after the addition is complete, the stirring speed is adjusted to a second speed for continued stirring. This allows the polymer and lipids to self-assemble into polymer-lipid nanoparticles with uniform particle size, good dispersibility, and positively charged surfaces. The introduction of the ethanol-containing aqueous phase improves the interfacial properties, facilitating the diffusion of the organic solvent and the uniform nucleation of nanoparticles, effectively preventing particle aggregation. Curing for 1–12 hours ensures complete removal of the organic solvent, while washing and freeze-drying remove free materials and residual solvents, improving the purity and storage stability of the nanoparticles. The reconstitution performance after freeze-drying is excellent. Incubating the obtained polymer-lipid nanoparticles with mRNA encoding hepatitis B virus antigen protein at room temperature for 20–120 minutes allows for efficient mRNA transport via electrostatic adsorption, forming a structurally stable and biosafety-enhanced polymer-lipid mRNA nanovaccine.

[0040] Furthermore, the polymer lipid nanovaccine prepared by the above method can protect mRNA from enzymatic degradation, improve the uptake efficiency of mRNA in antigen-presenting cells such as DCs, and promote the expression of antigen proteins.

[0041] Furthermore, when the aforementioned polymer-lipid nanovaccine is applied to the treatment of chronic hepatitis B, its liver-targeted delivery efficiency can be effectively improved. This promotes the uptake of the nanovaccine by dendritic cells (DCs), enhances the expression of hepatitis B antigen proteins, and thereby improves the level of hepatitis B virus-specific immune response and immune memory in the liver. Simultaneously, it induces systemic-specific responses and immune memory against hepatitis B virus in peripheral immune organs such as the spleen, potentially leading to a complete cure for hepatitis B. In addition, this nanovaccine has advantages such as good stability, high biosafety, and high liver-targeted delivery efficiency. This application provides new scientific evidence for the development of liver-related nanovaccines and also provides basic data and references for the treatment of chronic hepatitis B.

[0042] Furthermore, the preparation method described in this application is highly adaptable, simple to operate, and easy to scale up, and the prepared polymer lipid nanoparticles exhibit high stability. In addition, by optimizing the process conditions and the nitrogen-to-phosphorus ratio, the stability of the mRNA-loaded system can be further enhanced, while also improving the encapsulation efficiency and drug loading rate, thus improving biosafety and enhancing the immune response.

[0043] According to the embodiments of this application, in step S1, the first stirring speed is 700 rpm to 800 rpm, preferably 750 rpm; the second stirring speed is 500 rpm to 600 rpm, preferably 550 rpm.

[0044] According to the embodiments of this application, the first stirring speed can be 700 rpm, 720 rpm, 740 rpm, 760 rpm, 780 rpm, or 800 rpm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 700 rpm to 780 rpm, 720 rpm to 750 rpm, etc.

[0045] According to embodiments of this application, the second stirring speed can be 500 rpm, 520 rpm, 540 rpm, 560 rpm, 580 rpm, or 600 rpm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 500 rpm to 580 rpm, 520 rpm to 550 rpm, etc.

[0046] According to the embodiments of this application, during the slow dripping of the oil phase into the aqueous phase, a higher rotation speed can enable the oil phase to be rapidly and uniformly dispersed in the aqueous phase, forming droplets of uniform size. This avoids excessively high local oil phase concentration, which could lead to aggregation or the formation of large particles, thereby ensuring that the nanoparticles formed by the subsequent self-assembly of the polymer and lipids have uniform particle size and good dispersibility. After the dripping is completed, the stirring speed is reduced, which can maintain a mild flow state of the system, which is conducive to the gradual evaporation / diffusion of the organic solvent into the outer aqueous phase to complete the solidification. It can also avoid excessive shear force from damaging the already formed nanoparticle structure or causing agglomeration between particles, thus ensuring the integrity and stability of the nanoparticles.

[0047] According to embodiments of this application, the lipid material is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTMA), bis(octadecyldimethylammonium bromide) (DDAB), 1,2-dioleoyloxy-3-(dimethylamino)propane, 1-octylnonyl octanoate of 8-[(2-hydroxyethyl)[6-oxo-6-((decyloxy)hexyl]amino]octanoate]octanoate, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate) (SM-102), and 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP).

[0048] In one specific embodiment of this application, the lipid material is SM-102.

[0049] According to the embodiments of this application, the above-mentioned lipid materials are all cationic or ionizable lipids with a positive charge, which can efficiently bind to negatively charged mRNA through electrostatic adsorption, thereby improving the encapsulation efficiency of mRNA.

[0050] According to embodiments of this application, the polymer material is selected from at least one of polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol-polylactic acid copolymer (PELA), polycaprolactone (PCL), and polyhydroxyalkanoate (PHA).

[0051] In one specific embodiment of this application, the polymer material is polylactic acid-hydroxyacetic acid copolymer.

[0052] According to the embodiments of this application, the above polymers are all biodegradable and biocompatible polymer materials, which can endow nanoparticles with a stable hydrophobic core, protect mRNA from nuclease degradation, delay mRNA release, and improve the in vivo stability and immune durability of vaccines.

[0053] According to an embodiment of this application, in step S3, the cleaning includes centrifugation with deionized water or membrane washing.

[0054] According to the embodiments of this application, centrifugation with deionized water or membrane washing can remove residual organic solvents such as ethanol, free lipids, polymer materials, and unencapsulated small molecule impurities after curing, thus avoiding adverse effects of residues on the subsequent freeze-drying process and the biosafety of the final vaccine, and improving the purity of nanoparticles.

[0055] According to embodiments of this application, the mass ratio of polymer material to lipid material is 3:1 to 10:1.

[0056] According to embodiments of this application, the mass ratio of polymer material to lipid material can be 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 5:1 to 10:1, 3:1 to 8:1, etc.

[0057] According to embodiments of this application, polymer materials (such as PLGA) serve as a hydrophobic framework, providing mechanical strength and structural compactness to the nanoparticles, preventing early mRNA leakage and enzymatic degradation; lipid materials provide positive charge and membrane activity, endowing the nanoparticles with mRNA binding capacity and endosome escape function. Too low a polymer ratio leads to a loose nanoparticle structure and poor stability; too high a ratio results in insufficient lipids, reducing mRNA encapsulation and endosome escape efficiency.

[0058] Furthermore, within a mass ratio range of 3:1 to 10:1, and by combining parameters such as ethanol concentration and stirring speed, polymer lipid nanoparticles with uniform particle size (50–500 nm), low PDI (0.05–0.5), and suitable surface charge (+25–+60 mV) can be stably prepared, providing a good carrier basis for subsequent efficient mRNA carrying and liver-targeted delivery.

[0059] According to embodiments of this application, the organic solvent is selected from at least one of ethyl acetate, a mixture of acetone and ethanol, and N,N-dimethylformamide.

[0060] According to the embodiments of this application, the volume ratio of the oil phase to the water phase can be 1:3, 1:5, 1:10, 1:15, 1:20, 1:25, or 1:30, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 1:15 to 1:30, 1:3 to 1:20, etc.

[0061] According to the embodiments of this application, the volume ratio of the oil phase to the water phase is controlled at 1:3 to 1:30, which can effectively regulate the solvent diffusion rate and the supersaturated precipitation rate of the polymer / lipid. When the oil phase is slowly dripped into the larger volume of the water phase, the organic solvent diffuses rapidly, promoting the rapid and uniform nucleation of nanoparticles and avoiding excessively high local concentrations that could lead to particle aggregation or increased particle size. At the same time, a suitable oil-water ratio can maintain the concentration of each component in the system within a moderate range, which is beneficial for forming nanoparticles with uniform particle size and good dispersibility.

[0062] According to embodiments of this application, the addition amount of polymer lipid nanoparticles and mRNA encoding hepatitis B virus antigen protein is controlled, such that the nitrogen-to-phosphorus ratio of the nanovaccine is 1 to 10.

[0063] According to the embodiments of this application, the nitrogen-to-phosphorus ratio (N / P) of the nanovaccine is 1-10, which allows the positive charge of lipids to fully electrostatically bind with the negative charge of mRNA. This enables efficient encapsulation or adsorption of mRNA within the nanoparticles, preventing free mRNA from being degraded by nucleases and improving vaccine stability and delivery efficiency. A low N / P ratio leads to insufficient mRNA encapsulation and inadequate protection; a high ratio results in excessively strong surface positive charge, causing cytotoxicity or serum protein adsorption and accelerating in vivo clearance.

[0064] According to embodiments of this application, the polymer lipid nanoparticles satisfy at least one of the following conditions: (1) the particle size of the polymer lipid nanoparticles is 50 nm to 500 nm; (2) the zeta potential of the polymer lipid nanoparticles is +25 mV to +60 mV; (3) the PDI value of the polymer lipid nanoparticles is 0.05 to 0.5.

[0065] According to the embodiments of this application, a particle size of 50 nm to 500 nm enables the nanovaccine to effectively circumvent rapid clearance by the kidneys. Simultaneously, its suitable size facilitates accumulation in target organs such as the liver, promoting uptake by antigen-presenting cells such as dendritic cells (DCs), thereby enhancing intracellular mRNA delivery and antigen expression efficiency. A zeta potential of +25 mV to +60 mV, with an appropriate amount of positive charge on the nanoparticle surface, allows for efficient binding to negatively charged mRNA via electrostatic interactions, improving encapsulation stability and protecting mRNA from nuclease degradation. Furthermore, it facilitates interaction between the nanoparticles and negatively charged cell membranes, promoting uptake by immune cells such as DCs and endosome escape, thereby enhancing antigen protein expression levels. A PDI value of 0.05 to 0.5 indicates uniform particle size and good system dispersibility, which helps ensure consistent vaccine behavior and batch-to-batch reproducibility in vivo, avoiding differences in immune responses due to uneven particle size. It also helps maintain a stable nano-dispersion state after lyophilization and reconstitution.

[0066] Preferably, the concentration of the polymer material is 1-10 mg / mL, for example, it can be 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0067] Preferably, the concentration of the lipid material is 0.01-2.5 mg / mL, for example, it can be 0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.30 mg / mL, 0.40 mg / mL, 0.50 mg / mL, 0.60 mg / mL, 0.80 mg / mL, 1.00 mg / mL, 1.20 mg / mL, 1.40 mg / mL, 1.60 mg / mL, 1.80 mg / mL, 2.00 mg / mL, 2.20 mg / mL, 2.50 mg / mL, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0068] Preferably, the organic solvent is selected from ethyl acetate, a mixture of acetone and ethanol, and N,N-dimethylformamide. The volume ratio of the organic solvent to the external aqueous phase is 1:3 to 1:50, for example, 1:3, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0069] Preferably, the curing time of the polymer lipid nanoparticles is 1-12 h, for example, it can be 1 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, 5.0 h, 6.0 h, 7.0 h, 8.0 h, 9.0 h, 10.0 h, 11.0 h, 12.0 h, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0070] Preferably, the concentration of the nanoparticle suspension when the polymer lipid nanoparticles carry mRNA is from 0.5 mg / mL to 5 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0071] Preferably, the mRNA concentration of the polymer lipid nanoparticles carrying mRNA is from 0.05 μg / mL to 5 μg / mL, for example, it can be 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0072] As a preferred embodiment of this application, the addition amount (mass ratio) of polymer lipid nanoparticles to mRNA can be varied in the range of 1:100,000 to 1:100. Preferably, in order to significantly enhance the stability of the drug delivery system, while improving the encapsulation efficiency, drug loading rate, biosafety, and immune effect of the drug delivery system, the addition amount of polymer lipid nanoparticles to mRNA is preferably 1:50,000 to 1:5,000, more preferably 1:30,000 to 1:6,000, and even more preferably 1:20,000 to 1:10,000 (so that the nitrogen-phosphorus ratio of the nanovaccine is 4-8), for example 4.2:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, and 8.5:1.

[0073] Preferably, the incubation time at room temperature is 0.5-2 h, for example, it can be 0.5 h, 1 h, 1.5 h, 2 h, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0074] According to another aspect of this application, a nano-vaccine is provided, obtained according to the above preparation method.

[0075] According to another aspect of this application, the above-described nanovaccine is provided for use in the preparation of an mRNA vaccine for hepatitis B-related diseases.

[0076] According to the embodiments of this application, a novel nanovaccine has been developed using lipids and polymers as materials. After the particulate vaccine is taken up by dendritic cells (DCs), it expresses hepatitis B antigen protein, inducing humoral and cellular immunity to achieve the prevention and treatment of chronic hepatitis B. The in vivo and in vitro effects were studied using mice as an animal model. The mRNA-loaded polymer-lipid nanoparticles prepared by the method of this application are highly efficient and stable, have low toxicity and side effects, and exhibit high liver-targeted delivery efficiency. This can improve the effective DC uptake rate of the nanovaccine and enhance the immune level of the liver and the system against hepatitis B virus.

[0077] The following will further explain the solution of this application based on specific embodiments. Unless otherwise specified, the reagents used in this application are all commercially available reagents, and the test methods and experimental methods used are all conventional methods.

[0078] Example 1: Preparation of polymer lipid nanoparticles

[0079] Polymer lipid nanoparticles were prepared using a nanoprecipitation method. 9 mg of polymer material PLGA was accurately weighed and completely dissolved in 1.9 mL of ethyl acetate. 3 mg of lipid material SM-102 was accurately weighed and completely dissolved in 0.1 mL of ethyl acetate. The two solutions were thoroughly mixed by stirring to form the oil phase (O). 12 mL of anhydrous ethanol was accurately measured and added to 28 mL of deionized water to form a 30% (v / v) ethanol solution, which was then used as the aqueous phase (W). The oil phase was slowly added dropwise and at a uniform rate to the aqueous phase, which was being stirred at a uniform speed (750 rpm). After the addition was complete, the magnetic stirrer speed was adjusted to 550 rpm, and the mixture was cured for 8 h to remove the organic solvent. After curing, the polymer lipid nanoparticles were collected by centrifugation (10000 g, 15 min). The supernatant was discarded, and the polymer lipid nanoparticles were washed with deionized water by centrifugation (10000 g, 15 min). This process was repeated 3 times. After the last wash, the polymer lipid nanoparticle precipitate was resuspended in an appropriate amount of deionized water and the sample was collected to obtain PLGA / SM-102 nanoparticles.

[0080] Example 2: Preparation of polymer lipid nanoparticles

[0081] The same preparation method as in Example 1 was used, except that the amount of lipid material SM-102 used was 2 mg.

[0082] Example 3: Preparation of polymer lipid nanoparticles

[0083] The same preparation method as in Example 1 was used, except that the amount of lipid material SM-102 used was 1.5 mg.

[0084] Example 4: Preparation of polymer lipid nanoparticles

[0085] The same preparation method as in Example 1 was used, except that the amount of lipid material SM-102 used was 1.2 mg.

[0086] Example 5: Preparation of polymer lipid nanoparticles

[0087] The same preparation method as in Example 1 was used, except that the amount of lipid material SM-102 used was 1 mg.

[0088] Example 6: Preparation of polymer lipid nanoparticles

[0089] The same preparation method as in Example 1 was used, except that the amount of lipid material SM-102 used was 0.9 mg.

[0090] Test Example 1

[0091] The polymer lipid nanoparticles prepared in Examples 1 to 6 were characterized, and the results are as follows: Figure 1 and Figure 2 As shown.

[0092] Figure 1 The graph shows the effect of the polymer and lipid mass ratio on the nanoparticle size and PDI of the different polymer lipid nanoparticles prepared in Examples 1-6 of this application.

[0093] Figure 2 The graph shows the effect of the polymer-to-lipid mass ratio on the surface morphology of the different polymer lipid nanoparticles prepared in Examples 1-6 of this application.

[0094] The polymer lipid nanoparticles prepared in Example 1 had a particle size of 196±6.8 nm, a PDI value of 0.289±0.020, and a Zeta potential of 58.3±6.4 mV.

[0095] The polymer lipid nanoparticles prepared in Example 2 had a particle size of 290±8.2 nm, a PDI value of 0.271±0.018, and a Zeta potential of 46.8±9.6 mV.

[0096] The polymer lipid nanoparticles prepared in Example 3 had a particle size of 190±4.8 nm, a PDI value of 0.125±0.075, and a Zeta potential of 28.7±3.5 mV.

[0097] The polymer lipid nanoparticles prepared in Example 4 had a particle size of 391±4.6 nm, a PDI value of 0.422±0.098, and a Zeta potential of 25.6±4.7 mV.

[0098] The polymer lipid nanoparticles prepared in Example 5 had a particle size of 295±2.7 nm, a PDI value of 0.523±0.076, and a Zeta potential of 24.3±3.6 mV.

[0099] The polymer lipid nanoparticles prepared in Example 6 had a particle size of 562±2.9 nm, a PDI value of 0.531±0.046, and a Zeta potential of 23.8±5.9 mV.

[0100] Example 7 Preparation of polymer lipid nanoparticles

[0101] The same preparation method as in Example 1 was used, except that 4.5 mL of anhydrous ethanol was added to 10.5 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0102] Example 8: Preparation of polymer lipid nanoparticles

[0103] The same preparation method as in Example 1 was used, except that 6 mL of anhydrous ethanol was added to 14 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0104] Example 9: Preparation of Polymer Lipid Nanoparticles

[0105] The same preparation method as in Example 1 was used, except that 7.5 mL of anhydrous ethanol was added to 17.5 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0106] Example 10 Preparation of polymer lipid nanoparticles

[0107] The same preparation method as in Example 1 was used, except that 9.0 mL of anhydrous ethanol was added to 21.0 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0108] Example 11 Preparation of polymer lipid nanoparticles

[0109] The same preparation method as in Example 1 was used, except that 15.0 mL of anhydrous ethanol was added to 35.0 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0110] Example 12 Preparation of polymer lipid nanoparticles

[0111] The same preparation method as in Example 1 was used, except that 20.0 mL of anhydrous ethanol was added to 40.0 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0112] Example 13 Preparation of polymer lipid nanoparticles

[0113] The same preparation method as in Example 1 was used, except that 4.0 mL of anhydrous ethanol was added to 36.0 mL of deionized water to form a 30% concentration (v / v) ethanol solution.

[0114] Test Example 2

[0115] The polymer lipid nanoparticles prepared in Examples 7 to 13 were characterized, and the results are as follows: Figure 3 and Figure 4 As shown.

[0116] Figure 3 The graph shows the effect of the external aqueous phase volume ratio on the nanoparticle size and PDI of the polymer lipid nanoparticles prepared in Examples 7-13 of this application.

[0117] Figure 4The figure shows the effect of the external aqueous phase volume ratio on the surface morphology of the polymer lipid nanoparticles prepared in Examples 7-13 of this application.

[0118] The polymer lipid nanoparticles prepared in Example 7 had a particle size of 210±2.43 nm, a PDI value of 0.211±0.-12, and a Zeta potential of 30.6±3.8 mV.

[0119] The polymer lipid nanoparticles prepared in Example 8 had a particle size of 208±3.5 nm, a PDI value of 0.247±0.018, and a Zeta potential of 31.8±3.9 mV.

[0120] The polymer lipid nanoparticles prepared in Example 9 had a particle size of 210±2.8 nm, a PDI value of 0.255±0.021, and a Zeta potential of 34.6±2.5 mV.

[0121] The polymer lipid nanoparticles prepared in Example 10 had a particle size of 290±5.8 nm, a PDI value of 0.633±0.016, and a Zeta potential of 35.9±2.4 mV.

[0122] The polymer lipid nanoparticles prepared in Example 11 had a particle size of 318±13.5 nm, a PDI value of 0.339±0.021, and a Zeta potential of 30.8±1.9 mV.

[0123] The polymer lipid nanoparticles prepared in Example 12 had a particle size of 507±3.8 nm, a PDI value of 0.383±0.019, and a Zeta potential of 29.8±1.6 mV.

[0124] The polymer lipid nanoparticles prepared in Example 13 had a particle size of 512±6.7 nm, a PDI value of 0.587±0.023, and a Zeta potential of 30.8±3.2 mV.

[0125] Example 14 Preparation of polymer lipid nanoparticles

[0126] The same preparation method as in Example 1 was used, except that 8.0 mL of anhydrous ethanol was added to 32.0 mL of deionized water to form a 20% concentration (v / v) ethanol solution, which was used as the aqueous phase (W).

[0127] The prepared polymer lipid nanoparticles had a particle size of 480±9.8 nm, a PDI value of 0.482±0.016, and a Zeta potential of 31.8±2.7 mV.

[0128] Example 15 Preparation of polymer lipid nanoparticles

[0129] The same preparation method as in Example 1 was used, except that 16.0 mL of anhydrous ethanol was added to 24.0 mL of deionized water to form a 40% concentration (v / v) ethanol solution, which was used as the aqueous phase (W).

[0130] The prepared polymer lipid nanoparticles had a particle size of 378±8.7 nm, a PDI value of 0.386±0.024, and a Zeta potential of 32.4±3.1 mV.

[0131] Example 16 Preparation of polymer lipid nanoparticles

[0132] The same preparation method as in Example 1 was used, except that 20.0 mL of anhydrous ethanol was added to 20 mL of deionized water to form a 50% concentration (v / v) ethanol solution, which was used as the aqueous phase (W).

[0133] The prepared polymer lipid nanoparticles had a particle size of 320±8.4 nm, a PDI value of 0.236±0.018, and a Zeta potential of 32.7±2.3 mV.

[0134] Example 17 Preparation of polymer lipid nanoparticles

[0135] The same preparation method as in Example 1 was used, except that the lipid material DOTAP was used.

[0136] The prepared polymer lipid nanoparticles had a particle size of 236.3±8.2 nm, a PDI value of 0.183±0.32, and a Zeta potential of 42.72±5.8 mV.

[0137] Example 18 Preparation of polymer lipid nanoparticles

[0138] The same preparation method as in Example 1 was used, except for the lipid material DDAB.

[0139] The prepared polymer lipid nanoparticles had a particle size of 187.8±6.9 nm, a PDI value of 0.156±0.056, and a Zeta potential of 45.97±9.4 mV.

[0140] Example 19 Preparation of polymer lipid nanoparticles

[0141] The same preparation method as in Example 1 was used, except that the lipid material DPTMA was used.

[0142] The prepared polymer lipid nanoparticles had a particle size of 387.8±8.9 nm, a PDI value of 0.198±0.053, and a Zeta potential of 48.97±8.6 mV.

[0143] Example 20 Preparation of polymer lipid nanoparticles

[0144] The same preparation method as in Example 1 was used, except that the lipid material DODAP was used.

[0145] The prepared polymer lipid nanoparticles had a particle size of 432.8±7.2 nm, a PDI value of 0.198±0.046, and a Zeta potential of 41.36±8.7 mV.

[0146] Example 21 Preparation of polymer lipid nanoparticles

[0147] The same preparation method as in Example 1 was used, except that the polymer material was PLA.

[0148] The prepared polymer lipid nanoparticles had a particle size of 188±6.7 nm, a PDI value of 0.138±0.084, and a Zeta potential of 30.57±3.6 mV.

[0149] Example 22 Preparation of polymer lipid nanoparticles

[0150] The same preparation method as in Example 1 was used, except that the polymer material was PELA.

[0151] The prepared polymer lipid nanoparticles had a particle size of 198±6.4 nm, a PDI value of 0.168±0.092, and a Zeta potential of 31.56±6.9 mV.

[0152] Example 23 Preparation of polymer lipid nanoparticles

[0153] The same preparation method as in Example 1 was used, except that the polymer material was PCL.

[0154] The prepared polymer lipid nanoparticles had a particle size of 175±4.9 nm, a PDI value of 0.194±0.077, and a Zeta potential of 30.89±9.4 mV.

[0155] Example 24 Preparation of polymer lipid nanoparticles

[0156] The same preparation method as in Example 1 was used, except that the polymer material PHA was used.

[0157] The prepared polymer lipid nanoparticles had a particle size of 198±6.4 nm, a PDI value of 0.168±0.092, and a Zeta potential of 31.56±6.9 mV.

[0158] Test Example 3

[0159] The polymer lipid nanoparticles prepared in Examples 3, 17, and 18 were characterized, and the results are as follows: Figure 5 As shown.

[0160] Figure 5 The graphs show the surface morphology, particle size, PDI value, and Zeta potential of the polymer lipid nanoparticles prepared in Examples 3, 17, and 18 of this application.

[0161] Example 25: Preparation of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0162] Accurately weigh 3.0 mg of the polymer lipid nanoparticles from Example 3, suspend and disperse them in 2.0 mL of deionized water, vortex to ensure thorough dispersion, calculate and accurately measure the amount of mRNA (80 μg) according to N:P = 1:10, add it to the polymer lipid nanoparticle suspension, incubate on ice for 30 min, and obtain a hepatitis B mRNA-loaded nanovaccine. The obtained nanovaccine has a particle size of 240 ± 10.20 nm, a PDI value of 0.228 ± 0.015, and an mRNA loading rate of 95.86 ± 1.80%. The amino acid synthesis sequence of hepatitis B surface antigen mRNA (SEQ ID No. 1):

[0163] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS STTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI.

[0164] Example 26: Preparation of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0165] The same preparation method as in Example 25 was used, except that N:P = 1:8.

[0166] The obtained nanovaccine had a particle size of 238±8.7 nm, a PDI value of 0.215±0.023, and an mRNA loading rate of 96.66±2.90%.

[0167] Example 27: Preparation of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0168] The same preparation method as in Example 25 was used, except that N:P = 1:6.

[0169] The obtained nanovaccine had a particle size of 255±9.3nm, a PDI value of 0.236±0.054, and an mRNA carrying capacity of 96.87±2.78%.

[0170] Example 28: Preparation of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0171] The same preparation method as in Example 25 was used, except that N:P = 1:4.

[0172] The obtained nanovaccine had a particle size of 246±3.8 nm, a PDI value of 0.278±0.055, and an mRNA loading rate of 97.02±2.45%.

[0173] Example 29: Preparation of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0174] The same preparation method as in Example 25 was used, except that N:P = 1:4.

[0175] The obtained nanovaccine had a particle size of 262±5.7 nm, a PDI value of 0.248±0.063, and an mRNA carrying capacity of 97.23±1.98%.

[0176] Example 30: Preparation of a polymer-lipid nanovaccine loaded with hepatitis B mRNA

[0177] The same preparation method as in Example 25 was used, except that N:P = 1:1.

[0178] The obtained nanovaccine had a particle size of 251±9.2 nm, a PDI value of 0.257±0.048, and an mRNA carrying capacity of 97.92±2.37%.

[0179] Example 31 Cytotoxicity of Polymer Lipid Nanoparticles

[0180] The cytotoxicity of polymer lipid nanoparticles (PLGA / SM102, PLGA / DOTAP, and PLGA / DDAB, i.e., the polymer lipid nanoparticles of Examples 3, 17, and 18) was evaluated using the CCK-8 assay. RAW264.7 cells were cultured at 8 × 10⁻⁶ cells / day. 4Cells were seeded at a density of 50 μL per well in 96-well plates, followed by 150 μL of complete culture medium. The plates were incubated at 37°C with 5% CO2 for 24 h. Cells were then seeded with 50 μL of polymer lipid nanoparticle suspensions at gradient concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL). Negative control wells (cells without nanoparticles) and blank control wells (cell-free reagent blanks) were included, with three replicates per well. The 96-well plates were incubated at 37°C with 5% CO2 for another 24 h. After treatment, 10% CCK-8 reagent was added to each well, and the mixture was shaken to mix. Cells were incubated for another 4 h, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. The results are shown below. Figure 6 As shown.

[0181] Figure 6 This is a cytotoxicity analysis diagram of the three nanoparticles PLGA / SM-102, PLGA / DOTAP, and PLGA / DDAB in Example 31 of this application.

[0182] like Figure 6 As shown, at low concentrations (25 μg / mL and 50 μg / mL), the cell survival rate of PLGA / SM-102 NPs was relatively high, reaching 90%. At medium concentrations (100 μg / mL and 150 μg / mL), the cell survival rate of PLGA / SM-102 NPs remained relatively high, although it was lower than that at low concentrations, it was still higher than that of the other two NPs. At high concentrations (200 μg / mL), the cell survival rates of all three NPs decreased further, but the cell survival rate of PLGA / DOTAP NPs was higher than that of the other two NPs.

[0183] Example 32 Cytotoxicity of Hepatitis B mRNA-loaded polymer lipid nanovaccine

[0184] The cytotoxicity of a polymer-lipid nanovaccine loaded with hepatitis B mRNA was investigated using the CCK-8 assay. First, myeloid-derived dendritic cells (BMDCs) were extracted and cultured:

[0185] (1) Select 4-6 week old female C57BL / 6 mice, euthanize them and disinfect them. Dissect the mice under sterile conditions, remove two leg bones, soak them in 75% alcohol for 10 s, and transfer them to RPMI-1640 medium.

[0186] (2) Remove the leg muscles, rinse the bone marrow into a 15 mL sterile centrifuge tube, rinse repeatedly until the cells are clean, centrifuge (500 g, 5 min), and discard the supernatant;

[0187] (3) Add 3 mL of red blood cell lysis buffer to resuspend the cells, centrifuge (500 g, 5 min) after 1 min, discard the supernatant, and wash the cells once more.

[0188] (4) Resuspend the cells in a complete culture medium containing 10 ng / mL GM-CSF and 50 ng / mL interleukin-4 (IL-4);

[0189] (5) Dilute the cells 100 times and count them;

[0190] (6) Disperse the cells evenly, according to 1×10 6 Cells / wells were seeded in 24-well cell culture plates, and each well was filled with complete culture medium to a final volume of 1 mL.

[0191] (7) Continue culturing for 48 h, observe the cells, and change the cell culture medium;

[0192] (8) Observe the cells on day 5 and change the cell culture medium;

[0193] (9) On the 7th day, the cells were collected, which are DCs.

[0194] Collect the BMDCs cultured to day 7 into sterile centrifuge tubes, centrifuge (500 g, 5 min), resuspend the cells, dilute and count; then distribute the cells at 8 × 10⁻⁶ cells / tubes. 4 Cells were seeded at a density of 50 μL per well in 96-well plates, followed by 150 μL of complete culture medium. The plates were incubated at 37°C with 5% CO2 for 24 h. Cells were then incubated with 50 μL of a polymer lipid nanovaccine (Example 25) suspension at varying concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL). Negative control wells (cells without nanoparticles) and blank control wells (cell-free reagent blanks) were also included, with three replicates per well. The 96-well plates were incubated at 37°C with 5% CO2 for another 24 h. After treatment, 10% CCK-8 reagent was added to each well, and the mixture was shaken to mix. Cells were incubated for another 4 h, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. The results are shown below. Figure 7 As shown.

[0195] Figure 7 This is a cytotoxicity analysis diagram of the mRNA@PLGA / SM-102 nanovaccine in Example 32 of this application.

[0196] like Figure 7As shown, the cell viability of the mRNA vaccine remained at a high level (70%–90%). Even at the highest concentration (200 μg / mL), the cell viability did not decrease significantly, indicating that the vaccine has relatively low toxicity to DC cells in the concentration range of 25–200 μg / mL.

[0197] Example 33 mRNA expression of a polymer lipid nanovaccine loaded with hepatitis B mRNA

[0198] For this experiment, EZ Cap™ Firefly Luciferase mRNA (5-moUTP) labeled mRNA expressing firefly luciferase protein was selected for nanovaccine preparation. The vaccine was prepared according to the steps in Example 25, i.e., Fluc mRNA and PLGA / SM-102 nanoparticles were mixed at an N / P ratio of 10:1 and co-incubated for 30 min to obtain the Fluc mRNA-loaded nanovaccine. The vaccine was then co-incubated with dendritic cells (DCs), and the protein expression in the DCs and co-localization with the cell nucleus were observed and imaged using a resolution laser confocal microscope. The specific procedures are as follows:

[0199] (1) After adding 250 μL of 0.01 mg / mL poly-L-lysine and incubating for 24 h, wash twice and air dry;

[0200] (2) Inoculate DCs into confocal dishes and incubate at 37°C with 5% CO2 for 24 h;

[0201] (3) Add 20 μL of mRNA nanoparticles and continue culturing for 6 h;

[0202] (4) Gently aspirate the cell culture medium, add 50 μL of DAPI staining working solution, and incubate with the cells for 30 min;

[0203] (5) Remove the staining solution, wash the cells three times with PBS, and discard the PBS;

[0204] (6) Add D-fluorescein potassium salt and incubate for 15 min;

[0205] (7) Add 500 μL of 4% paraformaldehyde (PFA) and incubate for 30 min. Remove the supernatant and wash the cells 3 times.

[0206] (8) Images were acquired using a confocal microscope to visually observe the expression level of mRNA. The results are as follows: Figure 8 As shown.

[0207] Figure 8 This is a diagram showing the mRNA expression results of the hepatitis B mRNA polymer lipid nanovaccine loaded in Example 33 of this application.

[0208] like Figure 8 As shown, fluorescent proteins were observed at 6 h and were widely distributed both inside and outside the cells, indicating that within 6 h, PLGA / SM-102 NPs had delivered mRNA into DC cells, and some proteins had been secreted extracellularly. This result demonstrates that the delivery system functions well and can serve as a delivery vector for mRNA.

[0209] Example 34 Activation of DCs in a Polymer Lipid Nanovaccine Loaded with Hepatitis B mRNA

[0210] The ability of the hepatitis B mRNA-loaded polymer-lipid nanovaccine to activate dendritic cells (DCs) was investigated using the nanovaccine from Example 25. After co-incubating the nanovaccine with DCs for a certain period, flow cytometry was used to detect the expression levels of marker molecules on the surface of DCs, thereby evaluating the nanovaccine's ability to activate DCs. The specific experimental steps are as follows:

[0211] (1) Take DCs cultured to day 7, and divide them into 1×10⁻⁶ samples per well. 6 The cells were seeded into 24-well plates at the specified density and incubated at 37°C and 5% CO2 for 24 h.

[0212] (2) Add the mRNA vaccine into the corresponding well (where the mRNA dose is 3 μg / well).

[0213] (3) After culturing for another 24 h, collect the cells, centrifuge at 500 g for 5 min, discard the supernatant and resuspend the cells, repeat twice. Add 50 μL of Fc inhibitor and incubate at 4℃ in the dark for 10 min;

[0214] (4) Add 50 μL of flow cytometry antibodies (BV605-MHC II, AF700-CD11c, PE-Cyanine5-CD40, PE-Cyanine7-CD80 and APCCY7-CD86) for staining;

[0215] (5) After washing twice, resuspend the DCs in the flow cytometer tube and test them. The results are as follows: Figure 9 As shown.

[0216] Figure 9 This is a diagram showing the activation results of DCs induced by the mRNA@PLGA / SM-102 nanovaccine in Example 34 of this application.

[0217] like Figure 9As shown, the expression of DC surface markers CD40, CD80, CD86, and MHCII in the PLGA / SM-102 NPs@mRNA group was significantly increased compared to the PBS group (1.50, 1.08, 1.28, and 1.07 times, respectively) and the naked mRNA group (1.14, 1.1, 1.21, and 1.06 times, respectively), especially the expression of CD40 and CD80. This indicates that the PLGA / SM-102 NPs delivery system can effectively deliver mRNA into DC cells, enhance mRNA stability, and promote DC activation.

[0218] Example 35: Hepatitis B mRNA-loaded polymer lipid nanovaccine stimulates DCs to secrete cytokine levels

[0219] The ability of the hepatitis B mRNA-loaded polymer-lipid nanovaccine in promoting DC maturation was studied using the nanovaccine from Example 25. After co-incubating the nanovaccine with DCs for a certain period, the cell culture supernatant was collected by centrifugation. An ELISA cytokine kit was used to detect the levels of interferon-gamma (IFN-γ) for Helper T cell 1 (Th1) and IL-4 for Helper T cell 2 (Th2). The specific experimental procedures are as follows:

[0220] (1) Take BMDCs cultured to day 7, and divide them into 1×10⁻⁶ samples per well. 6 Cells were seeded at a density in 24-well plates and cultured for 24 h.

[0221] (2) Add the mRNA vaccine into the corresponding well (where the mRNA dose is 3 μg / well).

[0222] (3) After culturing for 24 h, centrifuge (500 g, 5 min), collect the supernatant, and determine the cytokine secretion level according to the detection method provided in the kit. The results are as follows: Figure 10 As shown.

[0223] Figure 10 This is a diagram showing the results of mRNA@PLGA / SM-102 nanovaccine in Example 35 of this application inducing DCs to secrete cytokines.

[0224] like Figure 10 As shown, the secretion of IFN-γ and IL-4 in the supernatant of DCs cells in the PLGA / SM-102 NPs@mRNA group was increased compared with both the PBS group and the naked mRNA group, and significantly increased compared with the PBS group, which helps to initiate and regulate the body's specific immune response.

[0225] Example 36: Liver lymphocyte activation level in mice immunized with hepatitis B mRNA-loaded polymer lipid nanovaccine

[0226] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0227] On day 35 post-immunization, the mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The livers were then removed and processed accordingly to prepare a single-cell suspension of liver cells. The specific procedures for preparing and activating the liver cell suspension are as follows:

[0228] (1) The mice were euthanized by dislocation of the neck, disinfected by soaking in 75% alcohol for 3 minutes, and the liver was removed by dissecting the mice.

[0229] (2) Rinse the liver with HBSS buffer, gently tear the liver into pieces, transfer it to a 15 mL centrifuge tube, add 10 mL of digestion solution and shake at 37°C for 15 min to digest;

[0230] (3) Add 10 mL of pre-cooled DMEM (containing 10% FBS) medium to stop digestion, pass the cells through a 70 μm cell sieve into a 15 mL sterile centrifuge tube, and rinse the cell sieve.

[0231] (4) Add 5 mL of DMEM complete culture medium, invert and mix well, then centrifuge (500 g, 5 min, 4℃) and discard the supernatant;

[0232] (5) Crack the red powder for 5 min, then centrifuge (500 g, 5 min, 4℃);

[0233] (6) Discard the supernatant, wash the cells twice, and dilute for counting;

[0234] (7) Seed the above liver cell suspension into 24-well plates at a concentration of 5 × 10⁻⁶ cells per well. 6 Cells were cultured at 37°C and 5% CO2 for 48 h after adding 6 μg / mL of HBsAg antigen and collecting the cells.

[0235] (8) Wash the cells twice with staining buffer;

[0236] (9) Add 1 mL of FVD506 to resuspend the cells and incubate at 4°C in the dark for 30 min;

[0237] (10) Centrifuge (500 g, 5 min) and discard the supernatant;

[0238] (11) Add 50 μL of Fc inhibitor to resuspend the cells and incubate at 4°C in the dark for 10 min;

[0239] (12) Add 100 μL of flow cytometry antibody (APC-CD3, eFluor 450-CD4, Brilliant Ultra Violet™ 395-CD8a, Brilliant UV Violet™ 496-CD19, APC-eFluor 780-CD69, Super Bright™ 600-CD178, PE-Cyanine7-CD44, PE-CD62L) directly to each centrifuge tube for staining, and incubate at 4°C in the dark for 30 min;

[0240] (13) Wash the cells twice, discard the supernatant, add 300 μL of 2% paraformaldehyde to each tube, incubate at 4°C in the dark for 20-30 min, wash the cells again, resuspend them, and analyze them by flow cytometry. The results are as follows: Figure 11 As shown.

[0241] Figure 11 This is a graph showing the results of the activation level of local immune cells in the liver after mice were immunized with the mRNA@PLGA / SM-102 nanovaccine in Example 36 of this application.

[0242] like Figure 11 As shown, the activation levels of T / B lymphocytes in the PLGA / SM-102@mRNA vaccine group were significantly higher than those in the PBS group and the naked mRNA group, increasing by 1.96-fold, 1.82-fold, and 1.86-fold, respectively, compared to the naked mRNA group. The naked mRNA group also showed significantly higher levels of activated B cells and CD4+ compared to the PBS group. + T cells and CD8 + T cell counts increased by 1.58-fold, 5.34-fold, and 14.25-fold, respectively. This result indicates that the PLGA / SM-102@mRNA vaccine can also induce a strong immune response locally in the liver, and significantly enhances the activation of T / B lymphocytes.

[0243] Example 37: Hepatic immune memory level in mice immunized with hepatitis B mRNA-loaded polymer lipid nanovaccine

[0244] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0245] On day 35 post-immunization, mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The livers were then removed and processed accordingly to prepare a single-cell suspension of liver cells. The specific procedures for preparing the liver cell suspension and detecting immune memory are as follows:

[0246] (1) The mice were euthanized by dislocation of the neck, disinfected by soaking in 75% alcohol for 3 minutes, and the liver was removed by dissecting the mice.

[0247] (2) Rinse the liver with HBSS buffer, gently tear the liver into pieces, transfer it to a 15 mL centrifuge tube, add 10 mL of digestion solution and shake at 37°C for 15 min to digest;

[0248] (3) Add 10 mL of pre-cooled DMEM (containing 10% FBS) medium to stop digestion, pass the cells through a 70 μm cell sieve into a 15 mL sterile centrifuge tube, and rinse the cell sieve.

[0249] (4) Add 5 mL of DMEM complete culture medium, invert and mix well, then centrifuge (500 g, 5 min, 4℃) and discard the supernatant;

[0250] (5) Crack the red powder for 5 min, then centrifuge (500 g, 5 min, 4℃);

[0251] (6) Discard the supernatant, wash the cells twice, and dilute for counting;

[0252] (7) Seed the above liver cell suspension into 24-well plates at a concentration of 5 × 10⁻⁶ cells per well. 6 Cells were cultured at 37°C and 5% CO2 for 48 h after adding 6 μg / mL of HBsAg antigen and collecting the cells.

[0253] (8) Wash the cells twice with staining buffer;

[0254] (9) Add 1 mL of FVD506 to resuspend the cells, incubate at 4°C in the dark for 30 min, centrifuge (500 g, 5 min), discard the supernatant, add 50 μL of Fc blocking agent, and incubate at 4°C for 10 min.

[0255] (10) Add 100 μL of flow cytometry antibody (APC-CD3, eFluor 450-CD4, Brilliant Ultra Violet™ 395-CD8a, Brilliant UV Violet™ 496-CD19, APC-eFluor780-CD69, Super Bright™ 600-CD178, PE-Cyanine7-CD44, PE-CD62L) directly to each centrifuge tube for staining, and incubate at 4°C in the dark for 30 min.

[0256] (11) Wash the cells twice, discard the supernatant, add 300 μL of 2% PFA to each tube, incubate at 4℃ in the dark for 20-30 min, wash the cells once, resuspend, and analyze by flow cytometry. The results are as follows: Figure 12 As shown.

[0257] Figure 12 This is a graph showing the results of inducing local hepatic immune memory levels in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 37 of this application.

[0258] like Figure 12 As shown, central CD4 was detected locally in the liver. + The PLGA / SM-102 NPs@mRNA vaccine showed significant effects on T cells and effector CD8+ T cells, indicating that it can effectively induce the accumulation or activation of these two cell types in the liver.

[0259] Example 38: Cytokine secretion levels in liver cells of mice immunized with a hepatitis B mRNA-loaded polymer lipid nanovaccine

[0260] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0261] On day 35 post-immunization, mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The livers were then removed and processed accordingly to prepare a single-cell suspension of liver cells. The specific procedures for preparing the liver cell suspension and detecting cytokine secretion levels are as follows:

[0262] (1) The mice were euthanized by dislocation of the neck, disinfected by soaking in 75% alcohol for 3 minutes, and the liver was removed by dissecting the mice.

[0263] (2) Rinse the liver with HBSS buffer, gently tear the liver into pieces, transfer it to a 15 mL centrifuge tube, add 10 mL of digestion solution and shake at 37°C for 15 min to digest;

[0264] (3) Add 10 mL of pre-cooled DMEM (containing 10% FBS) medium to stop digestion, pass the cells through a 70 μm cell sieve into a 15 mL sterile centrifuge tube, and rinse the cell sieve.

[0265] (4) Add 5 mL of DMEM complete culture medium, invert and mix well, then centrifuge (500 g, 5 min, 4℃) and discard the supernatant;

[0266] (5) Crack the red powder for 5 min, then centrifuge (500 g, 5 min, 4℃);

[0267] (6) Discard the supernatant, wash the cells twice, and dilute for counting;

[0268] (7) Seed the above liver cell suspension into 24-well plates at a concentration of 5 × 10⁻⁶ cells per well. 6 Add 6 μg / mL of HBsAg antigen to the cell / mL container and incubate at 37℃ and 5% CO2 for 48 h.

[0269] (8) Centrifuge at 500 g for 5 min to collect cells. Carefully transfer the supernatant to a 1.5 mL EP tube. Detect the amount of cytokines secreted in the cell supernatant according to the kit instructions. The results are as follows: Figure 13 As shown.

[0270] Figure 13 This is a graph showing the results of inducing local liver cytokine secretion levels in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 38 of this application.

[0271] like Figure 13 As shown, the levels of IFN-γ, IL-4, TNF-α, and IL-1β secreted by the PLGA / SM-102 NPs@mRNA vaccine group were all increased compared to the PBS group, at 1.18-fold, 1.20-fold, 1.14-fold, and 1.12-fold respectively, and 1.02-fold, 1.08-fold, 1.06-fold, and 1.08-fold respectively compared to the naked mRNA group. This indicates that the vaccine can effectively regulate the local immune microenvironment of the liver, enhance cellular and humoral immune responses, initiate inflammatory responses, and activate immune cells, providing more comprehensive and stronger immune protection for the liver.

[0272] Example 39 Serum specific antibody levels in mice immunized with hepatitis B mRNA-loaded polymer lipid nanovaccine

[0273] Mice were immunized via tail vein injection on days 0, 7, and 21. The experiment consisted of three groups: the hepatitis B mRNA-loaded polymer-lipid nanovaccine group (227 μg vaccine, containing 12 μg mRNA, injection volume 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume 300 μL), and the PBS control group (injection volume 300 μL). Immunization was performed. Blood samples were collected from the orbital cavity on days 7, 21, 28, and 35 post-immunization (blood was collected before drug administration) for serum antibody detection. The levels of immunoglobulin G (IgG) in serum samples at different time points after immunization were detected using ELISA. Specific procedures are as follows:

[0274] (1) Take HBsAg protein culture medium solution with a concentration of 2 μg / mL and add 100 μL per well to a 96-well plate. Place the plate in a humidified box and incubate overnight at 4°C.

[0275] (2) Remove the plate that has been coated with antigen and discard the coating solution. Wash the plate three times with PBST washing solution, add blocking solution, and block at 37°C for 2 h;

[0276] (3) Wash the plate 4 times and pat dry. Dilute the serum sample: 1:100 for the first well, and then use a 2-fold serial dilution method. Seal the 96-well plate with sealing film and incubate for 60 min.

[0277] (4) Dilute the horseradish peroxidase (HRP)-labeled secondary antibody, wash the plate 4 times and pat dry;

[0278] (5) Add 100 μL of enzyme-labeled secondary antibody, seal the plate with sealing film, and incubate for 60 min;

[0279] (5) Wash the plate 4 times, add TMB, and incubate for 15-20 min;

[0280] (6) Add 50 μL of stop solution to stop the color development;

[0281] (7) Absorbance detection, detection wavelength 450 nm, results are as follows Figure 14 As shown.

[0282] Figure 14 This is a graph showing the results of specific serum antibody levels induced in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 39 of this application.

[0283] like Figure 14As shown, PLGA / SM-102 NPs@mRNA can induce high levels of IgG in mice, and antibody titers gradually increase with the strengthening of immunization. At all time points, the IgG levels in the PLGA / SM-102 NPs@mRNA group were significantly higher than those in the naked mRNA group, especially after the third immunization (day 28), when serum levels were significantly higher. On day 35, the antibody level induced by the PLGA / SM-102 NPs@mRNA vaccine was twice that of the naked mRNA group. This further confirms the protective effect of PLGA / SM-102 NPs on mRNA and its promoting effect on antigen presentation, improving cellular uptake efficiency and thus enabling more stable and efficient protein expression, providing continuous antigen stimulation for antibody production, and more effectively activating B cells to stimulate antibody secretion.

[0284] Example 40: Activation level of spleen lymphocytes in mice after immunization with a hepatitis B mRNA-loaded polymer-lipid nanovaccine.

[0285] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0286] On day 35 post-immunization, mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The spleen was then removed and processed accordingly to prepare a single-cell suspension of spleen cells. The specific procedures for preparing and activating the spleen cell suspension are as follows:

[0287] (1) Under aseptic conditions, remove the mouse spleen and place it in a culture medium;

[0288] (2) Cut and grind the cells to prepare a single-cell suspension, filter it, and collect it in a 15 mL sterile centrifuge tube;

[0289] (3) Centrifuge (400 g, 5 min, 4℃), discard the supernatant;

[0290] (4) Add 3 mL of red blood cell lysis buffer to resuspend the cells and incubate for 3-5 min to fully lyse the red blood cells;

[0291] (5) Add culture medium to stop the red sclereidation and centrifuge (400 g, 5 min, 4℃);

[0292] (6) Aspirate the supernatant, resuspend the cells, add more to 5 mL, mix well, and centrifuge (400 g, 5 min, 4℃).

[0293] (7) Aspirate the supernatant, resuspend the cells, and then bring the volume to 2 mL;

[0294] (8) Dilute the cells 100 times and count them;

[0295] (9) Seed the above spleen cell suspension into 24-well plates at a concentration of 5 × 10⁻⁶ μL per well. 6 Cells were cultured at 37°C and 5% CO2 for 48 h after adding 6 μg / mL of HBsAg antigen to each cell.

[0296] (10) Wash the cells twice with staining buffer;

[0297] (11) Add 1 mL of FVD506 to resuspend the cells and incubate at 4°C in the dark for 30 min;

[0298] (12) Centrifuge (500 g, 5 min) and discard the supernatant;

[0299] (13) Add 50 μL of Fc inhibitor to resuspend the cells and incubate at 4°C in the dark for 10 min;

[0300] (14) Add 100 μL of flow cytometry antibody (APC-CD3, eFluor 450-CD4, Brilliant Ultra Violet™ 395-CD8a, Brilliant UV Violet™ 496-CD19, APC-eFluor780-CD69, Super Bright™ 600-CD178, PE-Cyanine7-CD44, PE-CD62L) directly to each centrifuge tube for staining, and incubate at 4°C in the dark for 30 min.

[0301] (15) Wash the cells twice, discard the supernatant, add 300 μL of 2% paraformaldehyde to each tube, incubate at 4°C in the dark for 20-30 min, wash the cells again, resuspend them, and analyze them using a flow cytometer. The results are as follows: Figure 15 As shown.

[0302] Figure 15 This is a graph showing the results of the activation level of spleen system immune cells induced in mice after immunization with the mRNA@PLGA / SM-102 nanovaccine in Example 40 of this application.

[0303] like Figure 15 As shown, the activation levels of T / B lymphocytes in the PLGA / SM-102 NPs@mRNA vaccine group were significantly higher than those in the PBS group and the naked mRNA group. Among them, the activated B cells and CD4+ cells were significantly higher. + T cells and CD8 +T cell counts were 2.47, 2.63, and 2.04 times higher than those in the PBS group, and 1.96, 1.82, and 1.86 times higher than those in the naked mRNA group, respectively. The naked mRNA group also showed higher levels of activated B cells and CD4+ compared to the PBS group. + T cells and CD8 + T cell counts increased by 1.25-fold, 1.44-fold, and 1.09-fold, respectively. This result indicates that the PLGA / SM-102 delivery system, upon binding to mRNA, significantly enhances lymphocyte activation and greatly improves the immunogenicity of the vaccine.

[0304] Example 41: Immunomodulatory memory level of spleen cells in mice immunized with hepatitis B mRNA-loaded polymer lipid nanovaccine

[0305] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0306] On day 35 post-immunization, mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The spleen was then removed and processed accordingly to prepare a single-cell suspension of spleen cells. The specific procedures for preparing the spleen cell suspension and detecting immune memory are as follows:

[0307] (1) Under aseptic conditions, remove the mouse spleen and place it in a culture medium;

[0308] (2) Cut and grind the cells to prepare a single-cell suspension, filter it, and collect it in a 15 mL sterile centrifuge tube;

[0309] (3) Centrifuge (400 g, 5 min, 4℃), discard the supernatant;

[0310] (4) Add 3 mL of red blood cell lysis buffer to resuspend the cells and incubate for 3-5 min to fully lyse the red blood cells;

[0311] (5) Add culture medium to stop the red sclereidation and centrifuge (400 g, 5 min, 4℃);

[0312] (6) Aspirate the supernatant, resuspend the cells, add more to 5 mL, mix well, and centrifuge (400 g, 5 min, 4℃).

[0313] (7) Aspirate the supernatant, resuspend the cells, and then bring the volume to 2 mL;

[0314] (8) Dilute the cells 100 times and count them;

[0315] (9) Wash the cells twice with staining buffer;

[0316] (10) Add 1 mL of FVD506 to resuspend the cells, incubate at 4°C in the dark for 30 min, centrifuge (500 g, 5 min), discard the supernatant, add 50 μL of Fc inhibitor, and incubate at 4°C for 10 min.

[0317] (11) Add 100 μL of flow cytometry antibody (APC-CD3, eFluor 450-CD4, Brilliant Ultra Violet™ 395-CD8a, Brilliant UV Violet™ 496-CD19, APC-eFluor780-CD69, Super Bright™ 600-CD178, PE-Cyanine7-CD44, PE-CD62L) directly to each centrifuge tube for staining, and incubate at 4°C in the dark for 30 min.

[0318] (12) Wash the cells twice, discard the supernatant, add 300 μL of 2% PFA to each tube, incubate at 4℃ in the dark for 20-30 min, wash the cells once, resuspend, and analyze by flow cytometry. The results are as follows: Figure 16 As shown.

[0319] Figure 16 This is a graph showing the results of inducing the level of immune memory in the spleen system after immunizing mice with the mRNA@PLGA / SM-102 nanovaccine in Example 41 of this application.

[0320] like Figure 16 As shown, compared to the PBS group, the PLGA / SM-102 NPs@mRNA group produced central memory CD4+. + T cells, central memory CD8 + T cells, and effector memory CD4 + T cells, effector-type memory CD8 + The number of T cells was significantly increased, with an increase in central memory CD4+. + and CD8 + T cells and effector memory CD4 + and CD8 + T cells were 1.80, 1.53, 1.11, and 2.17 times higher than in the PBS group, respectively. They were also significantly increased compared to the naked mRNA group, with an increase in central memory CD4+. + and CD8 + T cells and effector memory CD4 + and CD8 +T cells were 1.25, 1.07, 1.04, and 1.50 times that of the naked mRNA group, respectively. Among them, the naked mRNA group produced central memory CD4+. + and CD8 + The number of T cells was also significantly increased compared to the PBS group, being 1.43 times that of the PBS group. The experimental results indicate that the PLGA / SM-102 NPs@mRNA vaccine can induce the production of a large number of memory T cells. These cells are in a resting state in lymphoid tissues but can survive for a long time. Upon re-exposure to the same antigen, they can rapidly proliferate and differentiate, providing long-lasting immune protection. A large number of effector memory T cells, especially effector memory CD8 cells, were also observed. + T cells can rapidly reach the site of infection in the early stages of antigen invasion, directly killing target cells and effectively curbing the spread and diffusion of pathogens.

[0321] Example 42: Cytokine secretion levels of spleen cells after in vitro stimulation of mice immunized with a hepatitis B mRNA-loaded polymer lipid nanovaccine.

[0322] Mice were immunized by tail vein injection on days 0, 7, and 21. The experiment was divided into three groups: the hepatitis B mRNA-loaded polymer lipid nanovaccine group of Example 25 (injection dose of 227 μg vaccine, containing 12 μg mRNA, injection volume of 300 μL), the naked mRNA group (containing 12 μg mRNA, injection volume of 300 μL), and the PBS control group (injection volume of 300 μL).

[0323] On day 35 post-immunization, mice were sacrificed and then immersed in 75% alcohol for 3 minutes. The spleen was then removed and processed accordingly to prepare a single-cell suspension of spleen cells. The specific procedures for preparing the spleen cell suspension and detecting cytokine secretion levels are as follows:

[0324] (1) Under aseptic conditions, remove the mouse spleen and place it in a culture medium;

[0325] (2) Cut and grind the cells to prepare a single-cell suspension, filter it, and collect it in a 15 mL sterile centrifuge tube;

[0326] (3) Centrifuge (400 g, 5 min, 4℃), discard the supernatant;

[0327] (4) Add 3 mL of red blood cell lysis buffer to resuspend the cells and incubate for 3-5 min to fully lyse the red blood cells;

[0328] (5) Add culture medium to stop the red sclereidation and centrifuge (400 g, 5 min, 4℃);

[0329] (6) Aspirate the supernatant, resuspend the cells, add more to 5 mL, mix well, and centrifuge (400 g, 5 min, 4℃).

[0330] (7) Aspirate the supernatant, resuspend the cells, and then bring the volume to 2 mL;

[0331] (8) Dilute the cells 100 times and count them;

[0332] (7) Seed the above liver cell suspension into 24-well plates at a concentration of 5 × 10⁻⁶ μL per well. 6 Add 6 μg / mL of HBsAg antigen to the cell / mL container and incubate at 37℃ and 5% CO2 for 48 h.

[0333] (8) Centrifuge at 500 g for 5 min to collect cells. Carefully transfer the supernatant to a 1.5 mL EP tube. Detect the amount of cytokines secreted in the cell supernatant according to the kit instructions. The results are as follows: Figure 17 As shown.

[0334] Figure 17 This is a graph showing the results of the induced cytokine secretion levels in the spleen system after mice were immunized with the mRNA@PLGA / SM-102 nanovaccine in Example 42 of this application.

[0335] like Figure 17 As shown, the levels of IFN-γ, IL-4, and TNF-α secreted by the PLGA / SM-102 NPs@mRNA vaccine group were significantly increased compared to the PBS group, at 1.68-fold, 1.44-fold, and 1.31-fold respectively, and at 1.17-fold, 1.14-fold, and 1.11-fold respectively compared to the naked mRNA group. Furthermore, the secretion levels of IFN-γ and IL-4 in the naked mRNA group were also significantly increased compared to the PBS group, at 1.44-fold and 1.26-fold respectively, but both were lower than those in the PLGA / SM-102 NPs@mRNA vaccine group. This indicates that the PLGA / SM-102 NPs@mRNA vaccine can significantly enhance the immunogenicity of mRNA. In addition, the IL-1β secretion level in the PLGA / SM-102 NPs@mRNA vaccine group was 1.14 times higher than that in the naked mRNA group and 1.25 times higher than that in the PBS group. The naked mRNA group was also 1.10 times higher than that in the PBS group.

[0336] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a polymer lipid nanovaccine, comprising: The lipid material and polymer material were dissolved separately in organic solvents and mixed evenly to form the oil phase, while the aqueous solution containing 10%~50% ethanol was used as the aqueous phase. The oil phase is slowly added dropwise to the aqueous phase at a first stirring speed. After the addition is complete, the stirring speed is adjusted to a second stirring speed to obtain a mixed phase. The mixed phase was cured for 1 to 12 hours to remove the organic solvent, and then washed and freeze-dried to obtain polymer lipid nanoparticles. The polymer lipid nanoparticles and mRNA encoding hepatitis B virus antigen protein were incubated at room temperature for 20 to 120 minutes to obtain a polymer lipid mRNA nanovaccine.

2. The preparation method according to claim 1, wherein, The first stirring speed is 700 rpm to 800 rpm, preferably 750 rpm; The second stirring speed is 500 rpm to 600 rpm, preferably 550 rpm.

3. The preparation method according to claim 1, wherein, The cleaning process includes centrifugation with deionized water or membrane washing.

4. The preparation method according to claim 1, wherein, The lipid material is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleenoyloxypropylammonium chloride, dioctadecyl dimethylammonium bromide, 1,2-dioleoyloxy-3-(dimethylamino)propane, SM-102, and 1,2-dioleoyloxy-3-(dimethylamino)propane, preferably SM-102; The polymer material is selected from at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol-polylactic acid copolymer, polycaprolactone, and polyhydroxy fatty acid ester, preferably polylactic acid-glycolic acid copolymer.

5. The method according to claim 1, wherein, Also includes: The amount of polymer lipid nanoparticles and the amount of mRNA encoding hepatitis B virus antigen protein added are controlled so that the nitrogen-to-phosphorus ratio of the nanovaccine is 1 to 10.

6. The method according to claim 1, wherein, The mass ratio of the polymer material to the lipid material is 3:1 to 10:

1.

7. The method according to claim 1, wherein, The organic solvent is selected from at least one of ethyl acetate, a mixed solvent of acetone and ethanol, and N,N-dimethylformamide; The volume ratio of the oil phase to the water phase is 1:3 to 1:

30.

8. The method according to claim 1, wherein, At least one of the following conditions must be met: (1) The particle size of the polymer lipid nanoparticles is 50 nm to 500 nm; (2) The zeta potential of the polymer lipid nanoparticles is +25mV to +60mV; (3) The PDI value of the polymer lipid nanoparticles is 0.05 to 0.

5.

9. A nanovaccine, obtained by the preparation method according to any one of claims 1 to 8.

10. The use of the nanovaccine of claim 9 in the preparation of an mRNA vaccine for hepatitis B-related diseases.

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