WO2024037575A1 - Nanoparticle adjuvant loaded with anionic and hydrophobic immunologic adjuvants, method for preparing same, and use thereof - Google Patents

Abstract

A nanoparticle adjuvant loaded with anionic and hydrophobic immunologic adjuvants and use thereof. The nanoparticle adjuvant comprises an ionizable lipid, an anionic immunologic adjuvant and/or a hydrophobic immunologic adjuvant, and an auxiliary lipid, the auxiliary lipid comprising a neutral auxiliary lipid, cholesterol, and a polyethylene glycol lipid. An ionizable lipid material encapsulates the anionic adjuvant and the hydrophobic adjuvant with different functions to prepare a nanoparticle adjuvant system, which breaks through limitations in the traditional practice that cationic lipids are used to load an anionic adjuvant and a hydrophobic adjuvant. The prepared immunologic adjuvant in the form of the nanoparticle adjuvant has a relatively high encapsulation rate and can generate relatively high humoral immunity after being administrated to an animal and thereby enhance cellular immunity.

Description

A nanoparticle adjuvant co-loaded with anionic and hydrophobic immune adjuvants and its preparation method and application Technical field

The invention belongs to the field of medical technology, and more specifically, relates to lipid nano-adjuvants formed by using ionizable lipids to co-load anionic immune adjuvants and/or hydrophobic immune adjuvants and preparation methods thereof, and their combination with varicella-zoster Application of vaccines using virus (VZV) related protein gE as antigen.

Background technique

Immune adjuvants are also called immune agonists. They are non-specific immune-enhancing substances that can enhance the immunogenicity of the antigen and change the type of immune response by immunizing the body with the antigen at the same time or in advance. Among the adjuvants currently approved for use in human vaccines, aluminum salts and MF59 can enhance humoral immunity and Th2 immune responses, but they cannot induce sufficient cytotoxic T lymphocyte immune responses. The production process of virus-like particles is complicated and the ingredients are unclear. The research and development of tumor therapeutic vaccines urgently requires new adjuvants with clear structures, safe and effective, and easy to produce.

In recent years, some new cellular Toll-like receptor agonists can produce strong cellular immune responses, such as TLR4 agonist MPLA, TLR9 agonist CpG-ODN, TLR3 agonist Poly(I:C), TLR7/8 agonist IMQ, etc. Molecular adjuvants have been widely used in preventive and therapeutic vaccines. However, due to the small molecular weight of such adjuvants, they can easily cause systemic inflammation and toxicity, and are easily metabolized quickly. Molecular adjuvants are wrapped with biological materials to form nano-adjuvants (usually less than 100 nm), which can be carried to draining lymph nodes through targeted lymph nodes or through antigen-presenting cells. Nanoadjuvants can avoid adjuvant-induced systemic toxicity, and by targeting lymph nodes, they can remain in lymph nodes rich in antigen-presenting cells (APCs) for a long time and continuously activate immune responses. And because the biological material wrapping the adjuvant can greatly increase the density of the adjuvant in the lymph nodes, it can effectively activate the TLR pathway; in addition, because different molecular adjuvants have different immune characteristics, two or more molecular adjuvants are usually used together. ; Currently, the only clinically approved vaccine is the Shingrix recombinant vaccine developed by GSK, which uses the AS01 adjuvant to stimulate strong humoral and cellular immune responses. AS01-loaded MPLA and QS21 are embedded in liposome vesicles through hydrophobic interactions. This method has limitations for loading some anionic adjuvants with good adjuvant effects. Moreover, the AS01 nanoparticle adjuvant is prepared through the traditional membrane hydration method, which has a complicated preparation process and poor controllability. Even though some existing cationic lipids, such as DOTAP, DOTMA, etc., can simultaneously load anionic molecule adjuvants and hydrophobic small molecule adjuvants, their strong positive charges will bind the anionic molecule adjuvants too strongly, which will lead to adjuvant release efficiency. Low, the adjuvant effect is reduced; in addition, the permanent cationic lipid particles will interact with the body due to their strong positive electricity. Serum protein binding in fluids results in rapid clearance from the humoral system, resulting in a shortened drug half-life. Moreover, liposomes composed of cationic lipids have problems such as thermodynamic instability, which greatly limits their application. At the same time, the high cytotoxicity of such cationic adjuvants prevents such cationic lipids from being used to simultaneously deliver anionic and hydrophobic adjuvants. Therefore, there is a need for a nanoparticle adjuvant system that can load anionic adjuvants and hydrophobic adjuvants with different functions and has better loading efficiency and immune efficiency.

Contents of the invention

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory procedures involved in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "ionizable lipid" refers to a lipid-like molecule having a hydrophilic group of both a tertiary or tertiary amine and a hydrophobic alkyl chain, with a pKa between 5.0 and 7.4.

As used herein, the term "particulate adjuvant" refers to a state of matter characterized by the presence of discrete particles, pellets, beads or pellets that is loaded with a corresponding molecular adjuvant and has a specific shape within a size range. Regardless of its size, shape or form.

As used herein, the term "nanoparticle adjuvant" refers to particles that are less than 200 nanometers in one dimension (i.e., diameter in the longest dimension of the particle).

As used in this article, the term "particle size" or "equivalent particle size" refers to when a certain physical characteristic or physical behavior of the measured particle is most similar to a homogeneous sphere (or combination) of a certain diameter. The diameter (or combination) of the sphere is regarded as the equivalent particle size (or particle size distribution) of the measured particles.

As used herein, the term "average particle size" refers to the difference between an actual particle population consisting of particles of different sizes and shapes compared to an imaginary particle population consisting of uniform spherical particles. If the diameter and total length are the same, the diameter of this spherical particle is called the average particle diameter of the actual particle group. The measurement method of the average particle size is known to those skilled in the art, such as the light scattering method; the measurement instrument of the average particle size includes but is not limited to a Malvern particle sizer.

As used herein, the term "room temperature" refers to 25±5°C.

As used herein, the term "immune adjuvant" refers to a substance that is administered together with an antigen or in advance into the body and can enhance immunogenicity or change the type of immune response. The immune adjuvant itself may be immunogenic (eg, BCG vaccine), or may not be immunogenic (eg, aluminum hydroxide adjuvant). The "anionic immune adjuvant" refers to an immune adjuvant that is negatively charged after ionization in water; the "hydrophobic immune adjuvant" "Immune adjuvant" refers to an immune adjuvant that is insoluble in water and can only be dissolved in neutral and non-polar solutions (such as organic solvents).

As used herein, the term "antigen" or "immunogen" refers to a substance capable of inducing a specific immune response in a host. Antigens may include whole organisms (e.g., inactivated, attenuated, or live organisms); subunits or portions of an organism; recombinant vectors containing immunogenic inserts; capable of inducing immunity upon presentation to a host Responsive DNA portion or fragment; protein, glycoprotein, lipoprotein, polypeptide, peptide, epitope, hapten, toxin, antitoxin, or any combination thereof.

The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art and provide a nanoparticle adjuvant.

Another object of the present invention is to provide a preparation method of the nanoparticle adjuvant.

Another object of the present invention is to provide the application of the nanoparticle adjuvant.

A kind of nanoparticle adjuvant, comprising ionizable lipid, anionic immune adjuvant and/or hydrophobic immune adjuvant and auxiliary lipid, the auxiliary lipid includes neutral auxiliary lipid, cholesterol and polyethylene glycol (PEG). ) lipids.

Ionizable lipids are a type of substance with a pKa between 5 and 7. Their structure has ionizable tertiary ammonia or swollen ammonia, hydrophobic alkyl chains and functional structural groups. They show different charge properties in different pH environments. , has been successfully used for the delivery of siRNA and mRNA in the existing technology. The present invention has shown that this type of ionizable lipid can load hydrophobic molecule adjuvants, and is negatively charged when the pH is greater than pKa, and positively charged when the pH is less than pKa. Different from traditional cationic lipids such as DOTAP and DOTMA, ionizable lipids can form lipid nanoparticle adjuvants by wrapping negatively charged molecular adjuvants (anionic immune adjuvants) at low pH. After the pH of the buffer solution is reached, the surface charge of the lipid nanoparticles will be reduced to uncharged or weakly negative. In the body fluid environment, the lipid nanoparticles have a slight negative charge, so that the lipid nanoparticle adjuvant ultimately has good biocompatibility in the body fluid environment and can release the immune adjuvant well, ensuring that the immune adjuvant is The efficient loading of weakly charged adjuvants can avoid burst release or no release of molecular adjuvants. Eventually injected simultaneously with the antigen to elicit a strong immune response.

Specifically, ionizable lipids behave neutrally at physiological pH but are positively charged in the acidic environment of endosomes. Positively charged in pH 4 conditions to efficiently encapsulate negatively charged adjuvants, nearly neutral at physiological conditions of pH 7.4 after injection to prevent non-specific interactions with serum proteins and improve circulation time, and based on ionizable lipids After the formed particles are endocytosed, they are protonated in the acidic environment of the endosome and interact with negatively charged endogenous lipids, causing endosomal membrane instability and escaping to the cytoplasm. Ionizable lipids greatly improve the efficacy and toxicity characteristics.

The neutral auxiliary lipid is used to support the formation of the lipid bilayer structure and stabilize its structural arrangement; cholesterol with membrane fusion adjusts the integrity and hardness of the lipid membrane and enhances the stability of nanoparticles; it can improve hydrophilicity The unique PEGylated lipid is located on the surface of the nanoparticles, which can not only prevent the rice particles from being quickly cleared by the immune system to prolong the circulation time, but also prevent the aggregation of the rice particles to increase stability. The lipid components undergo intermolecular interactions and spontaneously organize into core-shell nanostructured entities. The PEG lipid will form a shell structure in the outermost layer to wrap around the outer layer of the core.

The "ionizable lipid" is not limited to only containing carbon, hydrogen, and nitrogen elements. Its basic structure has an ionizable tertiary ammonia or swollen ammonia hydrophilic head and a hydrophobic alkyl chain tail, with a final pKa of 5~ 7.4, and is not limited to chemical groups containing ester groups, aldehyde groups, carbonyl groups, disulfide bonds, hydrazone bonds, unsaturated bonds and other chemical groups. The hydrophobic alkyl chain is not limited to one.

Preferably, the ionizable lipid is a lipid with an acid dissociation constant (pKa) between 5.0 and 7.4, and its structure has a tertiary or tertiary ammonia, a hydrophobic alkyl chain and a functional structural group. The ionizable lipid is selected from FDA-approved highly biocompatible 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester (Dlin-MC3-DMA), 1-octylnonane 8-[(2-Hydroxyethyl)[6-O-6-(Undecyloxy)hexyl]amino]-octanoate (SM102), ((4-hydroxybutyl)azadiakyl ) bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC0315); and the ionizable lipid (10Z)-N-[3-(dimethylamino) independently designed by the present invention )propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadec-9-enoyl{R2-1,N-(3 -(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide},(10Z,12Z)-N-[3-(dimethylamino)propyl ]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienoamide{R2-2,(9Z,12Z )-N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9,12-dienamide},(10Z,12Z)-N -[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadec-9,12- Dienoamide {R3-1,N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide}, (10Z,12Z)-N -[3-(diethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadec-9,12- Dienoamide{R3-2,(9Z,12Z)-N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9, Any one or more of 12-dienamide}.

Further preferably, the ionizable lipid is R3-2.

Preferably, the anionic immune adjuvant is selected from one or more of natural immune agonists, plant-derived adjuvants or cytokine adjuvants.

Preferably, the natural immune agonist is a pattern recognition receptor (Pattern recognition receptors, PRRs) agonist.

Further preferably, the pattern recognition receptors (PRRs) agonist is selected from the group consisting of Toll-like receptors (TLRs) agonists, nucleotide-binding oligomerization domain NOD-like receptors (NOD-like receptors) , NLRs) agonist, retinoic acid-inducible gene I (RIG-1)-like receptor (RIG-1like receptors, RLRs) agonist, C-type lectin receptor (C-type lectin receptor, CLRs) agonist Or one or more agonists of intracellular nucleic acid sensor STING.

Further preferably, the Toll-like receptors (TLRs) agonist is selected from one of CPG ODNs, ssRNA, 23S rRNA, Pam2csk4, Pam3csk4, FLA, ssPoly(U) or poly(I:C) Kind or variety.

Further preferably, the CpG ODNs include but are not limited to CpG-ODN M362, CpG-ODN2216, CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN2395, CpG-ODN 1668, CpG-ODN 2007, CpG- ODN BW006, CpG-ODN SL01, CpG-ODN 1585, CpG-ODN 2336, CpG-ODN SL03, etc.

Further preferably, the nucleotide-binding oligomerization domain NOD-like receptors (NOD-like receptors, NLRs) agonist is selected from C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, Tri -One or more of DAP, M-TriDAP, Gram-PGNs, MDP or Murabutide.

Further preferably, the retinoic acid-inducible gene I (RIG-1)-like receptors (RIG-1like receptors, RLRs) agonist is selected from the group consisting of 3p-hpRNA, 5'ppp-dsRNA, Poly(dA:dT), One or more poly(I:C)LyoVec.

Further preferably, the C-type lectin receptor (C-type lectin receptor, CLRs) agonist is selected from Beta-glucan peptide or Dectin-I.

Further preferably, the intracellular nucleic acid sensor STING agonist is selected from one or more of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP or cAIMP.

Preferably, the plant-derived adjuvant is selected from one or more of QS-21, quinine or lectin.

Preferably, the cytokine adjuvant is selected from one or more of IFN-α, IL-2, TNF, IFN-γ or GM-CSF.

Preferably, the hydrophobic immune adjuvant is selected from the group consisting of imiquimod (IMQ), monophosphoryl Monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), muramyl dipeptide (moradin ester), Loxoribine, Gardiquinod, Resiquimod; bacterial-derived adjuvant tetanus toxoid (tetanus toxoid (TT), E. coli heat labile enterotoxin (LT) and Salmonella pilin (Flagellin); one or more of squalene, heat shock protein 70 or heat shock protein 90.

Preferably, the neutral auxiliary lipid is selected from dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylchol Base (DSPC), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl lecithin (DPPC), dioleoylphosphatidylcholine (DOPC), dimyristoyl lecithin (DMPC), dilauroyl phosphatidylcholine Phospholipids (DLPC), di-erucoylphosphatidylcholine (DEPC), 1-palmitoyl-2-oleoyl lecithin (POPC), distearoylphosphatidylcholine (DSPC), palmitoylphosphatidylserine (DPPS) ), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (EPG), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG-Na), 1,2 - Palmitoylphosphatidylglycerol (DPPG-NA), distearoylphosphatidylglycerol (DSPG-Na), dimyristoylphosphatidylglycerol (DMPG-Na), distearoylphosphatidic acid (DSPA), dipalmitoyl One or more acylphosphatidic acids (DPPA).

Preferably, the polyethylene glycol (PEG) lipid is selected from the group consisting of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycerol-3 -Methoxypolyethylene glycol 2000 (DMG-PEG2000), 2-[(Polyethylene glycol)-2000]-N,N-tetradecyl acetamide (ALC-0159), dipalmitoyl phosphatidyl One or more of ethanolamine-methoxypolyethylene glycol 2000 (DPPE-MPEG2000), dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 (DPPE-MPEG5000).

Further preferably, the anionic immune adjuvant is CpG oligodeoxynucleotide and/or plant-derived adjuvant QS21; the hydrophobic immune adjuvant is monophosphoryl lipid A (monophosphoryl lipid A, MPLA)) and/or Or imiquimod IMQ (Imiquimod, IMQ).

Preferably, the molar mass ratio of the ionizable lipid, anionic immune adjuvant, hydrophobic immune adjuvant and auxiliary lipid is 35-65:10-30:10-30:35-65.

Further preferably, the molar mass ratio of the ionizable lipid, anionic immune adjuvant, hydrophobic immune adjuvant and auxiliary lipid is 3:1:1:3 or 2:1:1:2.

Preferably, the mass ratio of the ionizable lipid to each adjuvant is 1-2:1-2.

Further preferably, the mass ratio of the ionizable lipid to each adjuvant is 1:1 or 1.5:1 or 2:1 or 1.5:2.

Preferably, the molar mass ratio of the ionizable lipid, neutral auxiliary lipid, cholesterol and PEGylated lipid is 44-55:9.4-10:38.5-45:1.5-1.6.

Further preferably, the molar mass ratio of the ionizable lipid, neutral auxiliary lipid, cholesterol and PEGylated lipid is 55:10:38.5:1.5 or 45.5:10:43:1.5.

Specifically, the nanoparticle adjuvant is a liposome core-shell structure, the core is an anionic immune adjuvant, and the shell is ionizable lipids and auxiliary lipids and hydrophobic immune adjuvants wrapped on the core; or the core is It is a partially anionic immune adjuvant. The shell is ionizable lipids, auxiliary lipids and hydrophobic immune adjuvants wrapped around the core. The particle surface is loaded with other anionic immune adjuvants.

Preferably, the nanoparticles are approximately spherical.

Preferably, the particle size of the nanoparticles is 30-200 nm, such as 30-50 nm, 50-80 nm, 80-100 nm, 100-150 nm or 150-200 nm.

Preferably, the zeta potential of the nanoparticles is -10 to +20mV, such as -10 to -5mV, -5 to -2mV, -2 to +2mV, +2 to +5mV, +5 to +10mV, +10 to +15mV, +15 to +20mV.

Preferably, the encapsulation rate of the immune adjuvant in the nanoparticles is 70% to 100%, such as 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% ~95% or 95%~100%.

The present invention also provides a method for preparing any of the above-mentioned nanoparticle adjuvants, which includes the following steps:

S1. Provide a solution containing ionizable lipids, auxiliary lipids and hydrophobic immune adjuvants, and a solution containing anionic immune adjuvants;

S2. Pass the solution containing ionizable lipids, auxiliary lipids and hydrophobic immune adjuvants through the first channel. Three tubes of solutions containing anionic immune adjuvants pass through the second channel, the third channel and the fourth channel respectively. Four The solution in the channel reaches the mixing area and is mixed to obtain a nanoparticle adjuvant solution;

S3. Stepwise dialyze the solvent to obtain a nanoparticle adjuvant aqueous solution.

Preferably, the pH of the solution containing the anionic immune adjuvant is 3-5.

Further preferably, the pH of the solution containing the anionic immune adjuvant is 4.

Preferably, the method is carried out in an apparatus comprising a first channel, a second channel, a third channel, a fourth pass and a mixing zone. In a preferred embodiment, the device is a multi-inlet vortex mixer, such as a four-inlet vortex mixer.

The multi-inlet vortex mixer in the present invention includes a first component located in the upper part, a second component located in the middle and a third component located in the lower part. The first component, the second component and the third component have the same straight diameter. diameter cylinder. The first component is provided with a plurality of channels, the second component is provided with a vortex mixing area and a plurality of flow guide areas, and the third component is provided with channels. The channel of the first component is in fluid communication with the flow-direction area of the second component. The flow guide areas of the second component are all in fluid communication with the vortex mixing area. The vortex mixing region of the second component is in fluid communication with the channel of the third component. The first part, the second part and the third part may be sealingly connected using a threaded connection device.

In some embodiments, the first component is provided with a plurality of channels, and the upper and lower ends of the channels are respectively located on the upper surface and the lower surface of the first component. In certain embodiments, the plurality of channels are circular in cross-section. In some embodiments, the plurality of channels are respectively connected to external pipes through connecting components.

In some embodiments, the upper surface of the second component is recessed to provide a plurality of flow guide areas and a vortex mixing area. In certain embodiments, the plurality of flow-direction regions are in fluid communication with the vortex mixing region through grooves disposed on the upper surface of the second component. In certain embodiments, the turbulent mixing region of the second component is in fluid communication with the channels of the third component through channels parallel to the axial direction of the second component.

In certain embodiments, the cross-section of the vortex mixing zone is circular and has a common center with the cross-section of the second component. In certain embodiments, the plurality of flow-direction regions are circular in cross-section. In certain embodiments, the second component has the same number of flow areas as the first component has channels. In certain embodiments, the plurality of flow-direction areas of the second component are each located directly beneath the plurality of channels of the first component.

In some embodiments, the upper and lower ends of the channel of the third component are respectively located on the upper surface and the lower surface of the third component. In certain embodiments, the channels of the third component are circular in cross-section. In certain embodiments, the channel of the third component is connected to the external conduit via a connecting component.

In certain embodiments, the multi-entry vortex mixer is made of rigid material (eg, stainless steel).

The above-mentioned device has the characteristics of high throughput and strong controllability. The prepared nanoparticles are evenly distributed and have small particle size, and the difference between batches is small.

The above technology (FNC) and device are described in the inventor's previous patent application number PCT/US2017/014080. The dispersion of the prepared nanoparticles can be made more uniform.

Preferably, the flow rate of each channel is the same, ranging from 1 to 40 mL/min, such as 1 mL/min, 5 mL/min, 8 mL/min, 10 mL/min, 15 mL/min, 20 mL/min, 30 mL/min or 40 mL/min. .

More preferably, the flow rate of each channel is 10 mL/min.

Preferably, the method further includes step S4: freeze-drying and concentrating the aqueous solution containing the nanoparticles, for example, by adding a freeze-drying protective agent.

Primary infection caused by varicella-zoster virus (VZV) manifests as water Varicella, and lurks in the host's sensory ganglia. When VZV is re-infected, the virus travels down the sensory nerve axon to the skin cells innervated by the nerve and proliferates, and then cascades along the sensory nerve pathway on the skin. The vesicular rash is band-like in shape, hence the name herpes zoster. Herpes zoster (HZ) is more common in adults and the elderly. There are currently no specific therapeutic drugs for chickenpox and shingles, and vaccination is currently the most effective way to prevent and control VZV. In addition to live attenuated vaccines, the chickenpox and shingles vaccines currently on the market also include subunit vaccines and DNA vaccines. The safety of traditional inactivated or attenuated vaccines and the systemic immune storm they cause cannot be avoided in many vaccine systems. Subunit vaccines have attracted much attention due to their high safety profile. gE glycoprotein is one of the most important structural proteins of VZV. It has abundant B cell and T cell epitopes and can stimulate the body to generate an immune response against VZV at the serum immune level and cellular immune level. It has been successfully used in subunit herpes zoster vaccine and has shown good results in clinical trials. .

The nanoparticles of the present invention can induce an immune response, and the nanoparticle adjuvant obtained above can be combined with VZV antigen to prepare an immunogenic composition for preventing and/or treating diseases related to varicella-zoster virus infection. In one aspect, the present invention claims the use of the nanoparticle adjuvant in the preparation of immunogenic compositions for diseases associated with VZV infection.

Preferably, the disease associated with VZV infection is one or more of chickenpox and herpes zoster.

The present invention also provides an immunogenic composition, which comprises any of the above-mentioned nanoparticle adjuvants of the present invention.

The immunogenic compositions of the invention may be formulated for any suitable mode of administration, including, for example, topical, oral, intranasal, mucosal, intravenous, intradermal, intraperitoneal, subcutaneous and intramuscular administration.

Preferably, the immunogenic compositions of the invention may be used in vaccine compositions, optionally in combination with adjuvants and/or (other) suitable carriers.

Preferably, the immunogenic composition further comprises pharmaceutically acceptable auxiliary materials, such as excipients, preservatives, antibacterial agents and/or additional immune adjuvants.

Preferably, the immunogenic composition is a vaccine.

Preferably, the immunogenic composition also includes VZV antigen, which is a VZV inactivated/inactivated virus strain, VZV glycoprotein such as VZV gE glycoprotein, VZV gB glycoprotein, VZV gH glycoprotein, VZV gL glycoprotein, etc. , as well as VZV lipoproteins, polypeptides, peptides, epitopes, haptens, toxins, antitoxins, or any combination thereof.

Preferably, the selected antigens are VZV gE and OKA strains;

Preferably, the VZV gE glycoprotein is a recombinant protein.

Among them, the amount of VZV antigen is selected to induce an immune protective response in a typical vaccine without obvious adverse side effects. The amount of antigen can change with the change of the specific immunogen used. Generally speaking, each dose of vaccine contains 5 to 1000 μg of protein, such as 5 to 200 μg or 20 to 100 μg. For VZV gE recombinant protein, the dosage used in mice is 1-25 μg, preferably 2 μg, 5 μg or 20 μg; the dosage used in humans is 10-100 μg, preferably 20 μg, 50 μg or 80 μg. For the OKA strain, the dosage is 100 to 100000pfu/0.5mL, preferably 10000pfu/0.5mL, 30000pfu/0.5mL, 50000pfu/0.5mL, 70000pfu/0.5mL, and 100000pfu/0.5mL.

Preferably, the immunogenic composition is used to prevent and/or treat diseases associated with VZV infection in a subject, such as chickenpox, herpes zoster.

Preferably, the subject is a mammal, such as a bovine, an equine, a bovine, a porcine, a canine, a feline, a rodent, a primate; for example, the The subjects are humans.

Preferably, the immunogenic composition further comprises a second immunogenic substance. For example, the immunogenic composition further includes other proteins of VZV other than the VZV gE protein. For example, the immunogenic composition further includes inactivated and inactivated VZV. For example, the immunogenic composition also includes other pathogenic microorganisms (including live, inactivated or attenuated) other than VZV. For example, the immunogenic composition may also comprise parts of other pathogenic microorganisms other than VZV.

Preferably, the VZV antigens of the invention and attenuated VZV may be used together in a composition to elicit an immune response against VZV, or used alone - simultaneously or sequentially in a challenge-boost regimen. Useable vaccine components can be delivered simultaneously or sequentially in any order. In one embodiment, the VZV antigen or immunogenic derivative thereof is delivered following delivery of live attenuated VZN or fully inactivated VZV. In another embodiment, live attenuated VZV or fully inactivated VZV is delivered following delivery of VZV antigen or an immunogenic derivative thereof.

Preferably, the invention further relates to a method of preventing and/or reducing the severity of herpes zoster and/or post-herpetic neuralgia, comprising delivering to an individual at risk of herpes zoster a vaccine containing live attenuated VZV and VZV Immunogenic compositions of antigens.

Preferably, in another embodiment, the present invention relates to a method of preventing and/or reducing the severity of herpes zoster and/or post-herpetic neuralgia, comprising sequential or simultaneous delivery to a patient at risk of herpes zoster Individual live attenuated VZV and VZV antigens.

In one aspect, the invention also provides a method for preventing and/or treating VZV infection-related A method of treating a disease, comprising administering to a subject a nanoparticle or an immunogenic composition (eg, a vaccine) of the invention.

Preferably, the diseases related to VZV infection are chickenpox and herpes zoster.

Preferably, the subject is a mammal, such as a bovine, an equine, a bovine, a porcine, a canine, a feline, a rodent, a primate; for example, the The subjects are humans.

In one aspect, the invention provides a method of inducing or enhancing an immune response to VZV in a subject, comprising administering to the subject a nanoparticle or immunogenic composition (eg, a vaccine) of the invention.

Preferably, the subject is a mammal, such as a bovine, an equine, a bovine, a porcine, a canine, a feline, a rodent, a primate; the subject The subject was a C57BL/6 mouse.

The nanoparticle adjuvant system of the present invention breaks through the traditional limitations of relying on cationic lipids to load anionic adjuvants and hydrophobic adjuvants, allowing the adjuvant loading efficiency and immune efficiency to reach the highest level, which lies in VZV gE antigen or VZV attenuated/killed strains. Combined use shows a strong immune effect.

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

(1) The present invention prepares a new nanoparticle adjuvant system by using ionizable lipid materials to package anionic adjuvants and hydrophobic adjuvants with different functions. This nanoparticle adjuvant system breaks through the traditional limitations of relying on cationic lipids to load anionic adjuvants and hydrophobic adjuvants, greatly improving the adjuvant release efficiency and adjuvant effect.

(2) The nanoparticles prepared by the present invention have the characteristics of high throughput and strong controllability. The prepared nanoparticles have regular shape, round shape, smooth surface, good dispersion, and no obvious adhesion, damage, collapse, etc.; The distribution is uniform and the particle size is small (30~200nm), with little difference between batches.

(3) The immune adjuvant loaded in the nanoparticles of the present invention has a high encapsulation rate (70% to 100%); after the nanoparticle adjuvant is applied to animals, it can produce strong humoral immunity and make cells Immunity is significantly enhanced, and the immune effect is better than the free form of antigen/adjuvant mixed injection and the existing aluminum-adjuvanted vaccine and other VZV vaccines currently on the market; it is compatible with the existing AS01 developed by GSK and traditional cationic lipid loading Compared with hydrophobic adjuvants, anionic adjuvants can produce higher specific antibodies and can stimulate stronger humoral vaccines and cellular immunity.

(4) The nanoparticles of the present invention have the function of targeting lymph nodes, improving the enrichment of vaccines in lymph nodes and the uptake of antigen-presenting cells;

(5) The nanoparticle adjuvant of the present invention can be continuously prepared by a simple method, has stable quality, and is easy for industrial production.

Description of drawings

Figure 1 is a schematic diagram of the immunization scheme of the nanoadjuvant of the present invention.

Figure 2 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with D01-D11 nanoadjuvant (day 34). Experimental results show that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 3 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with S01-S11 nanoadjuvant (day 34). Experimental results show that after immunizing mice with the S01-S11 nanoadjuvant of the present invention, it can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells, thereby enhancing the cellular immune effect mediated by T cells.

Figure 4 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R3201-R3211 nanoadjuvant (day 34). Experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 5 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with A01-A11 nanoadjuvant (day 34). Experimental results show that after immunizing mice with the A01-A11 nanoadjuvant of the present invention, it can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells, thereby enhancing the cellular immune effect mediated by T cells.

Figure 6 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R2101-R2111 nanoadjuvant (day 34). Experimental results show that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 7 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R2201-R2211 nanoadjuvant (day 34). Experimental results show that the R2201-R2211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 8 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R3101-R3111 nanoadjuvant (day 34). Experimental results show that R3101-R3111 of the present invention After immunizing mice, nanoadjuvants can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells, thereby enhancing the cellular immunity mediated by T cells.

Figure 9 shows the percentage of IFN-γ+ and TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with F00-F11 (day 34). Experimental results show that after immunizing mice with free adjuvants, they can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells to a certain extent. However, compared with the nanoadjuvants shown in Figures 2 to 8, Free adjuvants are not as effective as nano-adjuvants in enhancing T cell-mediated cellular immunity.

Figure 10 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in spleen cells stimulated with VZV gE and immune D01-D11 nanoadjuvant (day 42). Experimental results show that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 11 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune S01-S11 nanoadjuvant (day 42). Experimental results show that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 12 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R3201-R3211 nanoadjuvant (day 42). Experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 13 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune A01-A11 nanoadjuvant (day 42). Experimental results show that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 14 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R2101-R2111 nanoadjuvant (day 42). Experimental results show that after immunizing mice with the R2101-R2111 nanoadjuvant of the present invention, it can increase the expression of IFN-γ in lymphocytes, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 15 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immunized with R2201-R2211 nanoadjuvant (day 42). Experimental results show that the R2201-R221 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing T cell mediation. induced cellular immune effects.

Figure 16 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R3101-R3111 nanoadjuvant (day 42). Experimental results show that after immunizing mice with the R3101-R3111 nanoadjuvant of the present invention, it can increase the expression of IFN-γ in lymphocytes, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 17 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in spleen cells stimulated with VZV gE to stimulate immune F00-F11 (day 42). Experimental results show that free adjuvants can increase the expression of IFN-γ in lymphocytes after immunizing mice. However, compared with the nano-adjuvants shown in Figures 10-16, free adjuvants are less effective in T cell-mediated cellular immunity. The enhancement effect is not as good as that of nanoadjuvants.

Figure 18 shows the fluorescence signal intensity in mouse lymph nodes of each group of nanoadjuvant particles loaded with MPLA and CpG and their corresponding free adjuvant group F02.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

The following Examples 45-77 use four ionizable lipids independently designed by the present invention. Their chemical structures and preparation methods are as follows:

1. Ionizable lipid R21: (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidene Hept-2-yl]octadecy-9-enoyl{R2-1,N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl) Preparation method of oleamide};

Add 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N, N-dimethyl-1,3-propanediamine to 0.5 mL of methanol solution at room temperature. After reacting at room temperature for 60 min, add 1.0 mmol of oleic acid. After reacting at room temperature for 60 minutes, 0.5 mmol of octadecyl isonitrile was added, and the reaction was carried out at 40°C for 24 hours. After the reaction, the product was separated and purified through a chromatography column. The mobile phase was a mixture of methanol and dichloromethane.

2. Ionizable lipid R22: (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidene Hept-2-yl]octadecy-9-enoyl{R2-1,N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl) Preparation method of oleamide};

Add 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N, N-dimethyl-1,3-propanediamine to 0.5 mL of methanol solution at room temperature. After reacting at room temperature for 60 min, add 1.0 mmol of linoleic acid. , add 0.5 mmol octadecyl isonitrile after reacting at room temperature for 60 minutes, and react at 40°C for 24 hours. After the reaction is completed, the product is separated and purified through a chromatography column. The mobile phase is a mixture of methanol and dichloromethane.

3. Ionizable lipid R31: (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxo Yethylenehept-2-yl]octadecy-9,12-dienoamide{R3-1,N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan Preparation method of -2-yl)oleamide};

Add 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N, N-diethyl-1,3-propanediamine to 0.5 mL of methanol solution at room temperature. After reacting at room temperature for 60 min, add 1.0 mmol of oleic acid. After reacting at room temperature for 60 minutes, 0.5 mmol of octadecyl isonitrile was added, and the reaction was carried out at 40°C for 24 hours. After the reaction, the product was separated and purified through a chromatography column. The mobile phase was a mixture of methanol and dichloromethane.

4. Ionizable lipid R32: (10Z,12Z)-N-[3-(diethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxo Yethylenehept-2-yl]octadeca-9,12-dienoamide{R3-2,(9Z,12Z)-N-(3-(diethylamino)propyl)-N-(3-ethyl-1-( octadecylamino)-1-oxoheptan-2- Preparation method of yl)octadeca-9,12-dienamide};

Add 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N, N-diethyl-1,3-propanediamine to 0.5 mL of methanol solution at room temperature. After reacting at room temperature for 60 min, add 1.0 mmol of linoleic acid. , add 0.5 mmol octadecyl isonitrile after reacting at room temperature for 60 minutes, and react at 40°C for 24 hours. After the reaction is completed, the product is separated and purified through a chromatography column. The mobile phase is a mixture of methanol and dichloromethane.

In addition, in the following examples of the present invention, unless otherwise specified, for the combination of multiple adjuvants, the final mass of each adjuvant is the same, and the mass ratio between any two adjuvants is 1:1.

The CpG used in the following examples of the present invention is CpG ODN. The CpG ODN is CpG-ODN1826.

Example 1 Preparation of Adjuvant D01

(1) Dissolve the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, and DMG-PEG2000 in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution, a 10 mg/mL DSPC, and Chol solution. Ethanol solution, 5mg/mL DMG-PEG2000 ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, and DMG-PEG2000 into the No. 1 syringe, and add it to the other three syringes. For CpG/QS21 solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 2 Preparation of adjuvant D02

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into the No. 1 syringe, and add the CpG solution to the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 3 Preparation of adjuvant D03

(1) Dissolve the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL DLin-MC3-DMA ethanol. solution, 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution, 1 mg/mL MPLA ethanol solution and 1 mg/mL IMQ ethanol solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvants MPLA and IMQ into the No. 1 syringe, and add pH to the other three syringes. =4 50mM CA buffer solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant.

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 4 Preparation of adjuvant D04

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into the No. 1 syringe, and add the QS21 solution to the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 5 Preparation of Adjuvant D05

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10mg/mL DSPC, Chol ethanol solution, 5mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into the No. 1 syringe, and add the CpG solution to the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 6 Preparation of Adjuvant D06

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into the No. 1 syringe, and add the QS21 solution to the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 7 Preparation of Adjuvant D07

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into the No. 1 syringe, and add CpG/QS21 to the other three syringes. solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant.

(Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 8 Preparation of Adjuvant D08

(1) Dissolve the ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL DLin-MC3-DMA ethanol solution. 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, and add CpG/QS21 to the other three syringes. solution, place the four syringes on the high-pressure pump respectively, and each syringe is Through channels 1 to 4, nanoparticle adjuvants are obtained. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 9 Preparation of Adjuvant D09

(1) Dissolve the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL DLin-MC3-DMA ethanol. solution, 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution, and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into the No. 1 syringe, and add CpG to the other three syringes. solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 10 Preparation of Adjuvant D10

(1) Dissolve the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL DLin-MC3-DMA ethanol. solution, 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution, and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvants MPLA, and IMQ into the No. 1 syringe, and add QS21 to the other three syringes. solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 11 Preparation of Adjuvant D11

(1) Dissolve the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL DLin-MC3-DMA ethanol. solution, 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution, and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG, S21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvants MPLA, and IMQ into the No. 1 syringe, and add CpG to the other three syringes. /QS21 solution, place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 12 Preparation of Adjuvant S01

(1) Dissolve the ionizable lipid SM102, auxiliary lipid DSPC, Chol, and DMG-PEG2000 in ethanol to obtain a 50 mg/mL SM102 ethanol solution and a 10 mg/mL DSPC, Chol B Alcohol solution, 5 mg/mL DMG-PEG2000 ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, and DMG-PEG2000 into the No. 1 syringe, add the CpG/QS21 solution to the other three syringes, and place the four syringes respectively. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 13 Preparation of adjuvant S02

(1) Dissolve the ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and immune adjuvant MPLA in ethanol to obtain 50 mg/mL SM102 ethanol solution, 10 mg/mL DSPC, and Chol ethanol solution, 5 mg. /mL DMG-PEG2000 ethanol solution and 1mg/mL MPLA ethanol solution. Dissolve CpG in a 50mM CA solution with pH=4 to obtain a 200μg/mL CpG solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add CpG solution to the other three syringes, and add the CpG solution to the four syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 14 Preparation of adjuvant S03

(1) Dissolve the ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution and 1 mg/mL IMQ ethanol solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA and IMQ into No. 1 syringe, and add 50mM pH=4 to the other three syringes. CA buffer solution, place four syringes on the high-pressure pump, each syringe Pass through channels 1 to 4 respectively to obtain nanoparticle adjuvants. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 15 Preparation of adjuvant S04

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL SM102 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add QS21 solution to the other three syringes, and add the QS21 solution to the other three syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400 rpm and 4°C for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 16 Preparation of Adjuvant S05

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into No. 1 syringe, add CpG solution to the other three syringes, and add the CpG solution to the four syringes. They are placed on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 17 Preparation of Adjuvant S06

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant IMQ into the No. 1 syringe, add the QS21 solution to the other three syringes, and add the QS21 solution to the four syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to the PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 18 Preparation of Adjuvant S07

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 19 Preparation of Adjuvant S08

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL SM102 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 20 Preparation of Adjuvant S09

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe and the other three syringes. Add CpG solution, place four syringes on the high-pressure pump, and pass each syringe through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 21 Preparation of Adjuvant S10

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe, add QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400 rpm and 4°C for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 22 Preparation of Adjuvant SM11

(1) Dissolve the ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL SM102 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe, and add CpG/QS21 solution to the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 23 Preparation of adjuvant R3201

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, and DMG-PEG2000 in ethanol to obtain 50 mg/mL R32 ethanol solution, 10 mg/mL DSPC, Chol ethanol solution, and 5 mg/mL DMG. -PEG2000 ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, and DMG-PEG2000 into the No. 1 syringe, add the CpG/QS21 solution to the other three syringes, and place the four syringes respectively. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 24 Preparation of adjuvant R3202

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and immune adjuvant MPLA in ethanol to obtain 50 mg/mL R32 ethanol solution, 10 mg/mL DSPC, and Chol ethanol solution, 5 mg. /mL DMG-PEG2000 ethanol solution and 1mg/mL MPLA ethanol solution. Dissolve CpG in a 50mM CA solution with pH=4 to obtain a 200μg/mL CpG solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add CpG solution to the other three syringes, and add the CpG solution to the four syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 25 Preparation of adjuvant R3203

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution and 1 mg/mL IMQ ethanol solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA and IMQ into No. 1 syringe, and add Add 50mM CA buffer solution with pH=4, place four syringes on the high-pressure pump, and pass each syringe through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 26 Preparation of adjuvant R3204

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL R32 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add QS21 solution to the other three syringes, and add the QS21 solution to the other three syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to the PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 27 Preparation of adjuvant R3205

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of R322:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into No. 1 syringe, add CpG solution to the other three syringes, and put the four syringes Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 28 Preparation of adjuvant R3206

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into the No. 1 syringe, add the QS21 solution to the other three syringes, and put the four syringes Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 29 Preparation of adjuvant R3207

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 30 Preparation of adjuvant R3208

(1) Dissolve the ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL R32 ethanol solution, a 10 mg/mL DSPC, and Chol ethanol solution. solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of R322:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 31 Preparation of adjuvant R3209

(1) Dissolve the ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe, add CpG solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 32 Preparation of adjuvant R3210

(1) Dissolve the ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe, add QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 33 Preparation of adjuvant R3211

(1) Dissolve the ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain a 50 mg/mL R32 ethanol solution and a 10 mg/mL DSPC, respectively. Chol ethanol solution, 5 mg/mL DMG-PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic adjuvant MPLA, IMQ into No. 1 syringe, and add CpG/QS21 solution into the other three syringes. Place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 34 Preparation of Adjuvant A01

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipid DSPC, Chol, and DMG-PEG2000/ALC0159 in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, and Chol ethanol solution, 5 mg/mL. :DMG-PEG2000/ALC0159 ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) Configure according to different proportions, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, and PEG2000 into the No. 1 syringe, add the CpG/QS21 solution to the other three syringes, and place the four syringes on the high-pressure pump respectively. On the top, each syringe passes through channels 1 to 4 respectively to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400 rpm and 4°C for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 35 Preparation of Adjuvant A02

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipid DSPC, Chol, PEG2000, and immune adjuvant MPLA in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, and Chol ethanol solution, 5 mg/mL. PEG2000 ethanol solution and 1mg/mL MPLA ethanol solution. Dissolve CpG in a 50mM CA solution with pH=4 to obtain a 200μg/mL CpG solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant MPLA into the No. 1 syringe, add the CpG solution to the other three syringes, and place the four syringes separately. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 36 Preparation of Adjuvant A03

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, and Chol ethanol respectively. solution, 5 mg/mL PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution and 1 mg/mL IMQ ethanol solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution of ionizable lipid ALC0315, auxiliary lipid DSPC, Chol, PEG2000, hydrophobic adjuvant MPLA and IMQ into No. 1 syringe, and add 50mM CA buffer solution with pH=4 to the other three syringes. , place four syringes on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant.

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 37 Preparation of Adjuvant A04

(1) Dissolve the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL ALC0315 ethanol solution, a 10 mg/mL DSPC, and a Chol ethanol solution. 5 mg/mL PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant MPLA into the No. 1 syringe, add the QS21 solution to the other three syringes, and place the four syringes separately. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 38 Preparation of Adjuvant A05

(1) Dissolve the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL ALC0315 ethanol solution, a 10 mg/mL DSPC, and a Chol ethanol solution. 5 mg/mL PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant IMQ into the No. 1 syringe, add the CpG solution to the other three syringes, and place the four syringes separately. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 39 Preparation of Adjuvant A06

(1) Dissolve the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL ALC0315 ethanol solution, a 10 mg/mL DSPC, and a Chol ethanol solution. 5 mg/mL PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant IMQ into the No. 1 syringe, add the QS21 solution to the other three syringes, and place the four syringes separately. On the high-pressure pump, each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 40 Preparation of Adjuvant A07

(1) Dissolve the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ in ethanol to obtain a 50 mg/mL ALC0315 ethanol solution and a 10 mg/mL DSPC, Chol ethanol solution. 5 mg/mL PEG2000 ethanol solution and 1 mg/mL IMQ ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant IMQ into the No. 1 syringe, add the CpG/QS21 solution to the other three syringes, and add the CpG/QS21 solution to the four syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 41 Preparation of Adjuvant A08

(1) Dissolve the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant MPLA in ethanol to obtain a 50 mg/mL ALC0315 ethanol solution and a 10 mg/mL DSPC, Chol ethanol solution, 5 mg/mL PEG2000 ethanol solution and 1 mg/mL MPLA ethanol solution. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution of the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic adjuvant MPLA into the No. 1 syringe. Add the CpG/QS21 solution to the other three syringes. Place the four syringes respectively. Place it on the high-pressure pump, and each syringe passes through channels 1 to 4 respectively to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 42 Preparation of Adjuvant A09

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, Chol ethanol. solution, 5 mg/mL PEG2000 ethanol solution and 1 mg/mL ethanol solutions of MPLA and IMQ. The immune adjuvant CpG was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, hydrophobic adjuvant MPLA, and IMQ into the No. 1 syringe, add the CpG solution to the other three syringes, and add the CpG solution to the four syringes. They are placed on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to the PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 43 Preparation of Adjuvant A10

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, Chol ethanol. solution, 5 mg/mL PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing the ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, the hydrophobic adjuvant MPLA, and IMQ into the No. 1 syringe, add the QS21 solution to the other three syringes, and add the QS21 solution to the four syringes. Place them on high-pressure pumps, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 44 Preparation of Adjuvant A11

(1) Dissolve the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, hydrophobic immune adjuvants MPLA and IMQ in ethanol to obtain 50 mg/mL ALC0315 ethanol solution, 10 mg/mL DSPC, Chol ethanol. solution, 5 mg/mL PEG2000 ethanol solution and 1 mg/mL MPLA and IMQ ethanol solutions. The immune adjuvant CpG, QS21 was dissolved in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) According to the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid ALC0315, auxiliary lipid DSPC, Chol, PEG2000, hydrophobic adjuvant MPLA, and IMQ into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add the four Each syringe is placed on a high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

Example 45-55 Preparation of Adjuvants R2101-R2111

(1) Dissolve ionizable lipid R21, auxiliary lipid DSPC, Chol, and DMG-PEG2000 in ethanol to obtain 50 mg/mL R21 ethanol solution, 10 mg/mL DSPC, Chol ethanol solution, and 5 mg/mL DMG. -PEG2000 ethanol solution. Dissolve the hydrophobic immune adjuvant MPLA/IMQ in ethanol to obtain 1 mg/mL MPLA/IMQ. Dissolve the immune adjuvant CpG and QS21 in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) Configure according to different proportions, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R21, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add four The syringes are respectively placed on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

(The preparation method of adjuvant R2101-R2111 nano-adjuvant, the ratio of ionizable lipid to adjuvant, and the composition of adjuvant are similar to those in S01-S11)

Example 56-66 Preparation of Adjuvants R2201-R2211

(1) Dissolve the ionizable lipid R22, auxiliary lipid DSPC, Chol, and DMG-PEG2000 in ethanol to obtain 50 mg/mL R22 ethanol solution, 10 mg/mL DSPC, Chol ethanol solution, and 5 mg/mL DMG. -PEG2000 ethanol solution. Dissolve the hydrophobic immune adjuvant MPLA/IMQ in ethanol to obtain 1 mg/mL MPLA/IMQ. Dissolve the immune adjuvant CpG and QS21 in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) Configure according to different proportions, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R22, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add four The syringes are respectively placed on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

(The preparation method of adjuvant R2201-R2211 nano-adjuvant, the ratio of ionizable lipid to adjuvant, and the composition of adjuvant are similar to those in S01-S11)

Example 67-77 Preparation of Adjuvants R3101-R3111

(1) Dissolve ionizable lipid R31, auxiliary lipid DSPC, Chol, and DMG-PEG2000 in ethanol to obtain 50 mg/mL R31 ethanol solution, 10 mg/mL DSPC, Chol ethanol solution, and 5 mg/mL DMG. -PEG2000 ethanol solution. Dissolve the hydrophobic immune adjuvant MPLA/IMQ in ethanol to obtain 1 mg/mL MPLA/IMQ. Dissolve the immune adjuvant CpG and QS21 in a 50mM CA solution with pH=4 to obtain a 200 μg/mL CpG/QS21 solution.

(2) Configure according to different proportions, add ethanol solution to make up to 1 mL of mixed phospholipid solution.

(3) Put the mixed phospholipid solution containing ionizable lipid R31, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant into No. 1 syringe, add CpG/QS21 solution to the other three syringes, and add four The syringes are respectively placed on the high-pressure pump, and each syringe passes through channels 1 to 4 to obtain the nanoparticle adjuvant. (Use a 5mL syringe, remove air bubbles, and set the flow rate to 10mL/min)

(4) Let it sit for 2-3 minutes and then perform gradient dialysis:

1) Add the obtained nanoadjuvant to a 1000D dialysis bag (record the volume in detail), place it in a 50mM CA buffer solution (1000 volumes) with pH=6.7±0.1, and dialyze at 400rpm and 4℃ for 4 hours.

2) Transfer the nanoadjuvant to PBS buffer solution with pH=7.4 (1000 volume), and dialyze at 400 rpm and 4°C for 6 hours.

(5) Let it stand for 2-3 minutes and then measure the particle size again.

(The preparation method of adjuvant R3101-R3111 nano-adjuvant, the ratio of ionizable lipid to adjuvant, and the composition of adjuvant are similar to those in S01-S11).

Test Example 1 Particle Size Test

The particle size of the nano-adjuvant of Example 1 to Example 77 was tested using a Malvern particle sizer (with a dynamic light scattering detector), and the results are shown in Tables 1-7, (each example only shows The optimal proportion of adjuvant particle size data, the optimal proportion is determined by the particle size and dispersion coefficient of nanoparticles, where CLP is an ionizable phospholipid).

Table 1 Physicochemical properties of D01-D11 nanoadjuvant

Results are presented as mean±SD(n＝3)

*Polydispersity index.

Table 2 Physicochemical properties of S01-S11 nanoadjuvant

Table 3 Physicochemical properties of R3201-R3211 nanoadjuvant

Table 4 Physicochemical properties of A01-A11 nanoadjuvants

Table 5 Physical and chemical properties of R2101-R2111 nanoadjuvant

Table 6 Physicochemical properties of R2201-R2211 nanoadjuvants

Table 7 Physicochemical properties of R3101-R3111 nanoadjuvants

It can be seen from the results in Tables 1-7 that the present invention prepares a new nanoparticle adjuvant system by using ionizable lipid materials to wrap anionic adjuvants and/or hydrophobic adjuvants with different functions. The prepared nanoparticles have high It has the characteristics of high flux, strong controllability, uniform distribution, small particle size (30~200nm), and small difference between batches.

Test Example 2 Calculation of encapsulation efficiency of immune adjuvants in nanoparticles

Take 1mL of the nanoparticle adjuvant solution into a 300kDa ultrafiltration tube, centrifuge for 30 minutes at 4°C and 3000rpm, take the filtrate below, and use a Limulus test kit to detect the content of free MPLA in the filtrate. Quant-iT ^TM OliGreen ^TM ssDNA Assay Kit was used to detect the CpG content in the filtrate, and HPLC was used to detect the QS21 and IMQ contents in the filtrate. The encapsulation rate of the adjuvant in the nanoparticles was calculated according to the following formula. The encapsulation rate of adjuvant = w ₀ -w ₁ /w ₀ ×100%, where w ₀ is the total amount of adjuvant added; w ₁ is the total amount of free adjuvant in the filtrate.

The measurement results of the encapsulation efficiency of the immune adjuvant in the nanoparticles of Example 1 to Implementation 77 are shown in Tables 8-14. (The encapsulation rate here is the encapsulation rate of the nanoadjuvant selected from the optimal ratio screening in Table 1-Table 7)

Table 8 Encapsulation efficiency of adjuvants in D01-D11 nanoparticle adjuvants

Table 9 Encapsulation efficiency of adjuvants in S01-S11 nanoparticle adjuvants

Table 10 Encapsulation efficiency of adjuvants in R3201-R3211 nanoparticle adjuvants

Table 11 Encapsulation efficiency of adjuvants in A01-A11 nanoparticle adjuvants

Table 12 Encapsulation efficiency of adjuvants in R2101-R2111 nanoparticle adjuvants

Table 13 Encapsulation efficiency of adjuvants in R2201-R2211 nanoparticle adjuvants

Table 14 Encapsulation efficiency of adjuvants in R3101-R3111 nanoparticle adjuvants

It can be seen from the results in Tables 8-14 that the present invention prepares a new nanoparticle adjuvant system by using ionizable lipid materials to wrap anionic adjuvants and/or hydrophobic adjuvants with different functions. The nanoparticle adjuvant system It breaks through the traditional limitations of relying on cationic lipids to load anionic adjuvants and hydrophobic adjuvants. Among the above-mentioned various nanoparticles of the present invention, the encapsulation rate of various adjuvants is relatively high, reaching 70% to 100%, which is beneficial to nanoparticles. The particles induce a strong immune effect.

Test Example 3 Evaluation of the immune effect of nanoparticles in mice

1. Immunization method

Female C57BL/6 mice aged 5 to 8 weeks were randomly divided into groups, with 5 mice in each group. First, mix the adjuvant with the VZV gE antigen, use subcutaneous injection/muscular injection at the base of the tail (dose: VZV gE = 5 μg/mouse, each adjuvant = 5 μg/mouse), and inoculate mice according to the immunization scheme in Figure 1 Immunize and boost the vaccine once every 4 weeks for a total of 2 immunizations. Aluminum adjuvant and free adjuvant were used as controls, and the groups were as described in Table 15. Similarly, each group of adjuvants was mixed with VZV gE antigen and the mice were immunized twice according to the immunization schedule in Figure 1 (dose: VZV gE = 5 μg/mouse, each adjuvant = 5 μg/mouse).

Table 15 List of naming of aluminum adjuvants and other free adjuvants

2. Evaluation of humoral immunity effect

(1) Detection of IgG in mouse serum

On the 28th and 42nd days after the first immunization, blood was collected from the orbit, the serum was separated, and the IgG titer in the serum was detected by Elisa.

Detection process:

1) Coat 5 μg/mL VZV gE recombinant protein antigen in a 96-well plate, 100 μL per well, and coat overnight at 4°C.

2) Coated plate overnight, wash 3 times with PBST, 200 μL each time, with 200 μL 3% BSA at 37°C Closed for 2 hours.

3) Take 2 μL of immune serum or negative control serum, dilute it to 200 μL, then dilute it several times in sequence, add it to the antigen-coated well, and incubate at room temperature for 2 hours.

4) Wash 5 times, add working concentration of IgG-HRP, 100 μL per well, and incubate at room temperature for 2 hours.

5) Wash 5 times, add 100 μL TMB substrate to each well, incubate in the dark for 20 minutes, terminate the reaction with 200 μL 2M H ₂ SO ₄ , and detect the OD value at 450 nm.

6) Calculate the titer. If the ratio of the average absorption value (P) of the specimen hole to the average absorption value (N) of the negative control (group A) (i.e. P/N) is greater than 2.1, the specimen hole is judged to be positive.

(2) Detection of IgG1 in mouse serum

On the 28th and 42nd days after the first immunization, blood was collected from the orbit, the serum was separated, and the IgG1 titer in the serum was detected by Elisa.

(3) Detection of IgG2c in mouse serum

On the 28th and 42nd days after the first immunization, blood was collected from the orbit, the serum was separated, and the IgG2c titer in the serum was detected by Elisa.

The humoral immune effects of the nanoparticle adjuvants described in Example 1 to Implementation 77 are shown in Tables 16-22; the humoral immune effects of Al adjuvants and other free adjuvants are shown in Table 23.

Table 16 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with D01-D11 nanoparticle adjuvant

Table 17 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with S01-S11 nanoparticle adjuvant

Table 18 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with R3201-R3211 nanoparticle adjuvant

Table 19 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with A01-A11 nanoparticle adjuvant

Table 20 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with R2101-R2111 nanoparticle adjuvant

Table 21 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with R2201-R2211 nanoparticle adjuvant

Table 22 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 28 and day 42 after the first immunization with R3101-R3111 nanoparticle adjuvant

Table 23 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd days after the first immunization with aluminum adjuvant and free adjuvant (F01-F11)

At the same time, the cationic lipid trimethyl-2,3-dioleoyloxypropyl ammonium bromide (DOTAP) was used to replace the ionizable lipid + cholesterol to prepare cationic nano-adjuvants loaded with different adjuvants and the AS01 adjuvant developed by GSK. As a comparison, the immune effect was compared with the D01-D04 nanoparticle adjuvant. The results are shown in Table 24:

Table 24 VZV gE-specific IgG antibody titers in serum of mice in each group on day 42

It can be seen from the results in Tables 15-23 that after immunization with the above-mentioned various nanoparticle adjuvants of the present invention is applied to animals, strong humoral immunity can be generated and sufficiently strong VZV gE-specific antibodies can be produced; and the immune effect is excellent Vaccines containing a free form antigen/adjuvant mixture for injection and an aluminum adjuvant.

At the same time, it can be seen from the results in Table 24 that the nanoparticle adjuvant of the present invention can produce higher specific antibodies than the existing AS01 developed by GSK (the average titer produced by AS01 in 42 days is 9.8×10 ⁵ ); Compared with traditional cationic lipid-loaded cationic nano-adjuvants with different adjuvants, it can produce higher specific antibodies.

3. Evaluation of cellular immune effects

(1) On the 34th day after the first immunization, collect blood from the orbit, culture it, do flow cytometry, and measure INF-gamma, TNF-alpha and other cytokines.

(2) On the 42nd day after the first immunization, the mice were euthanized, the spleens were taken to isolate lymphocytes, and drugs were added to stimulate them. The ability of lymphocytes in the spleen to secrete INF-gamma was detected by elispot.

The cellular immune effect of the nanoparticle adjuvant is shown in Figure 2-17:

Figure 2 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with D01-D11 nanoadjuvant (day 34). Experimental results show that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 3 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with S01-S11 nanoadjuvant (day 34). Experimental results show that after immunizing mice with the S01-S11 nanoadjuvant of the present invention, it can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells, thereby enhancing the cellular immune effect mediated by T cells.

Figure 4 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R3201-R3211 nanoadjuvant (day 34). Experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 5 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with A01-A11 nanoadjuvant (day 34). Experimental results show that after immunizing mice with the A01-A11 nanoadjuvant of the present invention, it can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells, thereby enhancing the cellular immune effect mediated by T cells.

Figure 6 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R2101-R2111 nanoadjuvant (day 34). Experimental results show that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 7 shows the percentage of IFN-γ+/TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with R2201-R2211 nanoadjuvant (day 34). Experimental results show that the R2201-R2211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 8 shows CD4+T cells and CD8+T cells in peripheral blood after immunization with R3101-R3111 nanoadjuvant. The percentage of IFN-γ+/TNF-α+ in cells (day 34). Experimental results show that the R3101-R3111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells after immunizing mice, thereby enhancing the cellular immune effect mediated by T cells.

Figure 9 shows the percentage of IFN-γ+ and TNF-α+ in CD4+T cells and CD8+T cells in peripheral blood after immunization with F00-F11 (day 34). Experimental results show that after immunizing mice with free adjuvants, they can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphoid T cells to a certain extent. However, compared with the nanoadjuvants shown in Figures 2 to 8, Free adjuvants are not as effective as nano-adjuvants in enhancing T cell-mediated cellular immunity.

Figure 10 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in spleen cells stimulated with VZV gE and immune D01-D11 nanoadjuvant (day 42). Experimental results show that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 11 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune S01-S11 nanoadjuvant (day 42). Experimental results show that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 12 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R3201-R3211 nanoadjuvant (day 42). Experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 13 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune A01-A11 nanoadjuvant (day 42). Experimental results show that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunizing mice, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 14 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R2101-R2111 nanoadjuvant (day 42). Experimental results show that after immunizing mice with the R2101-R2111 nanoadjuvant of the present invention, it can increase the expression of IFN-γ in lymphocytes, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 15 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immunized with R2201-R2211 nanoadjuvant (day 42). Experimental results show that R2201-R221 of the present invention After immunizing mice, nanoadjuvants can increase the expression of IFN-γ in lymphocytes, thereby enhancing the cellular immunity mediated by T cells.

Figure 16 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in splenocytes stimulated with VZV gE and immune R3101-R3111 nanoadjuvant (day 42). Experimental results show that after immunizing mice with the R3101-R3111 nanoadjuvant of the present invention, it can increase the expression of IFN-γ in lymphocytes, thereby enhancing the cellular immunity effect mediated by T cells.

Figure 17 shows the ELISPOT detection of the number of cells secreting specific IFN-γ in spleen cells stimulated with VZV gE to stimulate immune F00-F11 (day 42). Experimental results show that free adjuvants can increase the expression of IFN-γ in lymphocytes after immunizing mice. However, compared with the nano-adjuvants shown in Figures 10-16, free adjuvants are less effective in T cell-mediated cellular immunity. The enhancement effect is not as good as that of nanoadjuvants.

It can be seen from the results in Figure 2-17 above that after the above-mentioned multiple nanoparticle adjuvants of the present invention are administered to animals, the cellular immunity is significantly enhanced, more IFN-γ and TNF-α can be expressed, and the immune effect is better than Free form antigen/adjuvant mixture injection and aluminum adjuvanted vaccine. In addition, the eslispot experiment confirmed that the nanoparticle adjuvant loaded with MPLA and QS21 prepared by the present invention can express more IFN-γ, which shows that the nanoparticle adjuvant prepared by the present invention is more effective than AS01 adjuvant and traditional cationic nanoadjuvant. Can stimulate stronger cellular immunity.

4. Lymph node imaging analysis

In order to study the accumulation behavior of different adjuvant groups in lymph nodes, mice were randomly divided into groups (3 mice per group), and the adjuvant and antigen were mixed and injected subcutaneously into the root of the tail (equal dose for each mouse: VZV gE =5μg/animal, each group of adjuvant =5μg/animal). Animals in each group were euthanized at specific time points, and inguinal and axillary lymph nodes were removed for in vitro imaging. The fluorescence intensity of lymph nodes at different time points in various animals was then plotted.

Figure 18 shows the fluorescence signal intensity in the lymph nodes of mice injected with each group of nanoadjuvant particles loaded with MPLA and CpG and their corresponding free adjuvant group F02. It can be seen from the results in Figure 18 that the nanoparticles of the present invention have the function of targeting lymph nodes and improve the enrichment of vaccines in lymph nodes.

Claims (34)

A nanoparticle adjuvant, characterized by comprising ionizable lipids, anionic immune adjuvants and/or hydrophobic immune adjuvants and auxiliary lipids, the auxiliary lipids including neutral auxiliary lipids, cholesterol and polyethylene Diolated lipids. The nanoparticle adjuvant according to claim 1, wherein the ionizable lipid is a lipid with an acid dissociation constant pKa between 5.0 and 7.4. The nanoparticle adjuvant according to claim 2, characterized in that the ionizable lipid is selected from the group consisting of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1-octylnonyl 8-[(2-Hydroxyethyl)[6-O-6-(Undecyloxy)hexyl]amino]-octanoate, ((4-hydroxybutyl)azadialkyl)bis(hexyl) Alk-6,1-diyl)bis(2-hexyldecanoate), (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(decanoate) Octadecylamino)-1-oxyylidenehept-2-yl]octadec-9-enoyl{R2-1,N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-( octadecylamino)-1-oxoheptan-2-yl)oleamide}, (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecyl) Amino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienoamide{R2-2,(9Z,12Z)-N-(3-(dimethylamino)propyl)-N-(3 -ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9,12-dienamide}, (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[ 3-Ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienoamide{R3-1,N-(3-(diethylamino)propyl )-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide}, (10Z,12Z)-N-[3-(diethylamino)propyl]-N-[ 3-Ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienoamide{R3-2,(9Z,12Z)-N-( Any one or more of 3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9,12-dienamide}. The nanoparticle adjuvant according to claim 1, characterized in that the anionic immune adjuvant is selected from one or more of natural immune agonists, plant-derived adjuvants or cytokine adjuvants. The nanoparticle adjuvant according to claim 4, wherein the natural immune agonist is a pattern recognition receptor agonist. The nanoparticle adjuvant according to claim 5, wherein the pattern recognition receptor agonist is selected from the group consisting of Toll-like receptor agonists, nucleotide-binding oligomerization domain NOD-like receptor agonists, and retinoids. One or more of an acid-inducible gene I-like receptor agonist, a C-type lectin receptor agonist, or an intracellular nucleic acid sensor STING agonist. The nanoparticle adjuvant according to claim 6, characterized in that the Toll-like receptor agonist is selected from CPG ODNs, ssRNA, 23S rRNA, Pam2csk4, Pam3csk4, FLA, ssPoly(U) or poly(I:C) one or more of them. The nanoparticle adjuvant according to claim 6, wherein the nucleotide-binding oligomerization domain NOD-like receptor agonist is selected from the group consisting of C12-iE-DAP, C14-Tri-LAN-Gly, iE- One or more of DAP, Tri-DAP, M-TriDAP, Gram-PGNs, MDP or Murabutide. The nanoparticle adjuvant according to claim 6, wherein the retinoic acid-inducible gene I-like receptor agonist is selected from the group consisting of 3p-hpRNA, 5'ppp-dsRNA, Poly(dA:dT), poly(I :C) One or more of LyoVec. The nanoparticle adjuvant according to claim 6, wherein the C-type lectin receptor agonist is selected from Beta-glucan peptide or Dectin-I. The nanoparticle adjuvant according to claim 6, wherein the intracellular nucleic acid sensor STING agonist is selected from the group consisting of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di- One or more of GMP or cAIMP. The nanoparticle adjuvant according to claim 4, characterized in that the plant-derived adjuvant is selected from one or more of QS-21, quinine or phytohemagglutinin. The nanoparticle adjuvant according to claim 4, characterized in that the cytokine adjuvant is selected from one or more of IFN-α, IL-2, TNF, IFN-γ or GM-CSF. The nanoparticle adjuvant according to claim 1, characterized in that the hydrophobic immune adjuvant is selected from the group consisting of imiquimod, monophosphoryl lipid A, lipopolysaccharide, muramyl dipeptide, Loxoribine, Gardiquinod, Rui Quinimod; bacterially derived adjuvant tetanus toxoid, E. coli heat-labile toxin and Salmonella pilin; one or more of squalene, heat shock protein 70 or heat shock protein 90. The nanoparticle adjuvant according to claim 1, characterized in that the neutral auxiliary lipid is selected from dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, distearoyl Phosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoyl lecithin, dioleoylphosphatidylcholine, dimyristoyl lecithin, dilauroyl lecithin, dierucoylphosphatidylcholine, 1- Palmitoyl-2-oleoyl lecithin, distearoylphosphatidylcholine, palmitoylphosphatidylserine, dioleoylphosphatidylserine, dioleoylphosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2 One or more of oleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, distearoylphosphatidic acid, and dipalmitoylphosphatidic acid kind. The nanoparticle adjuvant according to claim 1, characterized in that the pegylated lipid is selected from the group consisting of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 1,2-dimyristoyl-rac- Glycerin-3-methoxypolyethylene glycol 2000, 2-[(polyethylene glycol)-2000]-N,N-tetradecyl acetamide, dipalmitoylphosphatidylethanolamine-methoxypolyethylene Diol 2000, one or more of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000. The nanoparticle adjuvant according to claim 1, characterized in that the anionic immune adjuvant is CpG oligodeoxynucleotide and/or plant-derived adjuvant QS21; the hydrophobic immune adjuvant is monophosphoryl lipid A and/or imiquimod. The nanoparticle adjuvant according to any one of claims 1 to 17, characterized in that the molar mass ratio of the ionizable lipid, anionic immune adjuvant, hydrophobic immune adjuvant and auxiliary lipid is 35 to 65:10 ~30:10~30:35~65. The nanoparticle adjuvant according to claim 18, wherein the molar mass ratio of the ionizable lipid, anionic immune adjuvant, hydrophobic immune adjuvant and auxiliary lipid is 3:1:1:3 or 2 :1:1:2. The nanoparticle adjuvant according to claim 1, wherein the molar mass ratio of the ionizable lipid, neutral auxiliary lipid, cholesterol and PEGylated lipid is 44-55:9.4-10:38.5 ~45:1.5~1.6. The nanoparticle adjuvant according to claim 1, wherein the pH of the solution containing the anionic immune adjuvant is 3-5. The nanoparticle adjuvant according to claim 1, characterized in that the nanoparticle adjuvant is a liposome core-shell structure, the core is an anionic immune adjuvant, and the shell is an ionizable lipid and an auxiliary substance wrapped around the core. Lipids and hydrophobic immune adjuvants; or the core contains part of the anionic immune adjuvant, the shell consists of ionizable lipids and auxiliary lipids and hydrophobic immune adjuvants wrapped around the core, and the surface of the particle is loaded with another part of the anionic immune adjuvant. . The nanoparticle adjuvant according to claim 1, characterized in that the nanoparticle adjuvant is approximately spherical. The nanoparticle adjuvant according to claim 1, characterized in that the particle size of the nanoparticle adjuvant is 30-200 nm. The nanoparticle adjuvant according to claim 1, wherein the zeta potential of the nanoparticle adjuvant is -10 to +20 mV. The nanoparticle adjuvant according to claim 1, wherein the encapsulation rate of the anionic immune adjuvant and/or hydrophobic immune adjuvant is 70% to 100%. The preparation method of the nanoparticle adjuvant according to any one of claims 1 to 26, characterized in that it includes the following steps: S1. Provide a solution containing ionizable lipids, auxiliary lipids and hydrophobic immune adjuvants, and a solution containing anionic immune adjuvants; S2. Let the solution containing ionizable lipids, auxiliary lipids and hydrophobic immune adjuvants pass through the first channel, and the solution containing anionic immune adjuvants pass through the second channel, the third channel and the fourth channel respectively. The four channels The solution reaches the mixing area and is mixed to obtain a nanoparticle adjuvant solution; S3. Stepwise dialyze the solvent to obtain a nanoparticle adjuvant aqueous solution. The method for preparing nanoparticle adjuvant according to claim 27, characterized in that the flow rate of each channel is the same, ranging from 1 to 40 mL/min. The method for preparing nanoparticle adjuvant according to claim 27, wherein the flow rate of each channel is 10 mL/min. Application of the nanoparticle adjuvant according to any one of claims 1 to 26 in the preparation of an immunogenic composition for diseases related to varicella-zoster virus infection. An immunogenic composition, characterized in that the immunogenic composition contains the nanoparticle adjuvant according to any one of claims 1 to 26. The immunogenic composition according to claim 31, wherein the immunogenic composition further contains pharmaceutically acceptable excipients. The immunogenic composition according to claim 31, wherein the immunogenic composition is a vaccine. The immunogenic composition according to claim 31, wherein the immunogenic composition further comprises VZV antigen and attenuated VZV.