An ionizable lipid for nucleic acid delivery, its LNP composition and vaccine

Abstract

The present invention provides a novel cationic lipid, lipid nanoparticle and nucleic acid vaccine. The lipid nanoparticle mRNA vaccine prepared by selecting a specific cationic lipid in the present invention is found to have better in vitro stability and can stimulate a stronger immune response compared with the LNP prepared by the cationic lipid in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a novel ionizable lipid for nucleic acid delivery, its LNP composition, and a vaccine. Background Art

[0002] Currently, the clinically validated system for delivering mRNA is the lipid nanoparticle (LNP), which is a nanoparticle formed by lipids. The principle thereof includes cationic lipids. Existing technical research shows that after mRNA is delivered into cells, the mRNA expression rate is relatively low. For example, when Dlin-MC3-DMA is used as a cationic lipid to construct LNP, the mRNA expression level is 0.63% (Maugeri, Marco et al. “Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.” Nature Communications, 2019). Therefore, the structure of the cationic lipid is a key factor affecting the mRNA expression level, and the structure of the cationic lipid needs to be further optimized.

[0003] Varicella - varicella-zoster virus (VZV) can cause two different diseases: varicella and herpes zoster. The herpes zoster vaccine is for people who have been infected with the VZV virus, are immune to varicella, but have latent VZV virus in their bodies. Therefore, compared with the varicella vaccine for VZV virus-susceptible people, the herpes zoster vaccine functions similar to a therapeutic vaccine and needs to elicit a stronger immune response to prevent the reactivation of latent VZV virus.

[0004] The two currently marketed herpes zoster vaccines are the live attenuated vaccine ZOSTAVAX and the subunit vaccine SHINGRIX. ZOSTAVAX is the attenuated Oka strain obtained by low-temperature passage and is stored in freeze-dried form at -15°C to -50°C; the target population approved by the FDA is adults 50 years of age and older, but the Advisory Committee on Immunization Practices in the United States recommends use in people 60 years of age and older because the protective effect of this vaccine lasts for up to 8 years. The components of SHINGRIX include the extracellular region of the GE protein of the VZV virus and the AS01B adjuvant, and are stored at 2°C to 8°C in the form of freeze-dried GE protein and liquid adjuvant; the target population approved by the FDA is adults 50 years of age and older and adults 18 years of age and older with immunodeficiency or immunosuppression, but the Advisory Committee on Immunization Practices in the United States recommends use in people 50 years of age and older and people who have received ZOSTAVAX immunization for 8 weeks or more; the protective effect of this vaccine can last for 10 years or more (currently studied up to 10 years). In addition to the above two vaccines, the clinical R & D progress of domestic herpes zoster vaccines is shown in the following table.

[0005] Table 1 Clinical R & D Progress of Domestic Herpes Zoster Vaccines

[0006]

[0007]

[0008] Although the principle of VZV virus reactivation is not clear at present, it is known that VZV-specific cellular immunity is the key to restricting virus reactivation and replication. The frequency of T cells secreting IFNγ is currently considered the best surrogate indicator for examining the protective effect of herpes zoster vaccines, while there is more controversy about the correlation between the level of specific antibody response and the protective effect.

[0009] The GE protein is the main protein in the VZV virus that can cause CD4 + T cell responses; the QS-21 component in the AS01B adjuvant is also a natural saponin that promotes CD4 + T cell responses. However, the adjuvant itself has certain toxicity. Summary of the Invention

[0010] The term "neutral lipid" in the present invention refers to an uncharged lipid molecule that is not a phosphoglyceride.

[0011] The term "polyethylene glycol (PEG)-lipid conjugate" in the present invention refers to a molecule containing a lipid moiety and a polyethylene glycol moiety.

[0012] The term "lipid nanoparticle" in the present invention refers to a particle having at least one nanoscale dimension and containing at least one lipid.

[0013] The term "vaccine" in the present invention refers to a composition suitable for administration to animals (including humans), which induces an immune response after administration, and the intensity of the immune response is sufficient to at least minimally help prevent, ameliorate or cure clinical diseases caused by microbial infections.

[0014] The term "delivery system" in the present invention refers to a preparation or composition that regulates the distribution of bioactive components in space, time and dose in an organism.

[0015] In the terms of the present invention, N / P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.

[0016] The term "hydrocarbyl group" in the present invention refers to the group remaining after removing one hydrogen atom from the corresponding hydrocarbon, and in the present invention, it particularly refers to an aliphatic hydrocarbyl group, such as an alkyl group, an alkenyl group, an alkynyl group, especially an alkyl group.

[0017] The present invention relates to a cationic lipid having the following structural formula I:

[0018]

[0019] Wherein:

[0020] At least one of L1 and L2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)- or -NRa-;

[0021] And,

[0022] The other one of L1 or L2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)-, -NRa-, -O(C=O)-, -(C=O)O-, -C(=O)-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-;

[0023] G1 and G2 are each independently an unsubstituted C1-C 12 alkylene group or C1-C 12 alkenylene group;

[0024] G3 is a C1-C 24 alkylene group, a C1-C 24 alkenylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkenylene group;

[0025] Ra is H or a C1-C 12 hydrocarbyl group;

[0026] R1 and R2 are each independently a C6-C 24 alkyl group or a C6-C 24 alkenyl group;

[0027] R3 is H, OH, OR4, CN, -C(=O)OR4, -OC(=O)R4 or –NR5C(=O)R4;

[0028] R4 is a C1-C 12 hydrocarbyl group;

[0029] R5 is H or a C1-C6 hydrocarbyl group;

[0030] x is 0, 1 or 2.

[0031] Specifically, in the cationic lipid of formula I, L1 and L2 are each independently selected from -O-, -O(C=O)O-, -(C=O)NH-, -NH(C=O)- and -NH-.

[0032] Specifically, in the cationic lipid of formula I, both L1 and L2 are -O-, or both L1 and L2 are -O(C=O)O-, or both L1 and L2 are -NH-, or L1 is -NH(C=O)- and L2 is -(C=O)NH-.

[0033] Specifically, the cationic lipid has the following structure (IA):

[0034]

[0035] Wherein:

[0036] R6 is independently H, OH or a C1-C 24 hydrocarbyl group each time it appears;

[0037] n is an integer from 1 to 15.

[0038] Specifically, the cationic lipid has the following structure (IB):

[0039]

[0040] Wherein y and z are each independently an integer from 1 to 12.

[0041] Specifically, in the cationic lipid structure, n is an integer from 2 to 12, preferably n is 2, 3, 4, 5 or 6; wherein y and z are each independently an integer from 2 to 10, preferably from 4 to 9.

[0042] Specifically, in the cationic lipid structure, R1 and R2 each independently have the following structure:

[0043]

[0044] Wherein:

[0045] R7a and R 7b is independently H or a C1-C 12 hydrocarbyl group each time it appears; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;

[0046] wherein R 7a , R 7b and a are each selected such that R1 and R2 each independently contain 6 to 20 carbon atoms.

[0047] Specifically, in the cationic lipid structure, R 7a is H at least once, preferably, R 7a is H each time it appears.

[0048] Specifically, in the cationic lipid structure, R 7b is a C1-C8 hydrocarbyl group; preferably, the C1-C8 hydrocarbyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0049] Specifically, in the cationic lipid structure, R1 or R2 or both have one of the following structures:

[0050]

[0051]

[0052] Specifically, the cationic lipid compound has the following structure:

[0053]

[0054]

[0055]

[0056] The present invention provides a lipid nanoparticle comprising: the above-mentioned cationic lipid, non-cationic lipid and / or polyethylene glycol (PEG)-lipid conjugate, preferably, comprising: cationic lipid, neutral phospholipid, sterol lipid and / or polyethylene glycol (PEG)-lipid conjugate.

[0057] Specifically, the polyethylene glycol (PEG)-lipid conjugate is selected from one or more combinations of: 2-[(polyethylene glycol)-2000]-N,N-tetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerolamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA) or DMG-PEG2000, preferably DMG-PEG2000.

[0058] Specifically, the neutral lipid is selected from one or more combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), preferably DSPC.

[0059] Specifically, the sterol lipid is selected from one or more combinations of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, marasmanol, epicholesterol, ergosterol, fucosterol, hexahydroprovitamin D3, hydroxycholesterol and cholesterol modified by polypeptide; lanosterol, provitamin D3, fucosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholanic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.

[0060] Specifically, the molar percentage content of the cationic lipid in the lipid component is 20-60%, the molar percentage content of the neutral phospholipid in the lipid component is 5%-25%, and the molar percentage content of the sterol lipid in the lipid component is 25%-55%; the molar percentage content of the polyethylene glycol (PEG)-lipid conjugate in the lipid component is 0.5%-15%.

[0061] Specifically, the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 30-60:1-20:20-50:0.1-10. Preferably, the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 40-60:10-20:30-50:1-5. More preferably, the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 45:10:43:2 or 40:10:48:2.

[0062] Specifically, the vaccine further contains other excipients, and the excipients are one or a combination of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

[0063] Specifically, the average particle size of the nanoparticles is 50-200 nm, or the nanoparticles have a net neutral charge at neutral pH, or the nanoparticles have a polydispersity less than 0.4.

[0064] The present invention provides a method for preparing lipid nanoparticles, which includes the step of dissolving a cationic lipid, a non-cationic lipid, and a polyethylene glycol (PEG)-lipid conjugate in a solvent and then mixing them with mRNA.

[0065] Specifically, it is prepared by dissolving a cationic lipid, a neutral phospholipid, a sterol lipid, and a polyethylene glycol (PEG)-lipid conjugate in ethanol, mixing them with a diluted mRNA diluent, and then performing ultrafiltration, dilution, and filtration; preferably, it is prepared by dissolving a cationic lipid, a neutral phospholipid, a sterol lipid, and a polyethylene glycol (PEG)-lipid conjugate in ethanol, mixing them with a diluted mRNA diluent at a certain flow rate ratio, and then performing ultrafiltration, dilution, and filtration; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent mixing, laminar mixing, or microfluidic mixing.

[0066] Specifically, the diluent is acetate buffer, citrate buffer, phosphate buffer, or tris buffer.

[0067] Specifically, the pH of the buffer is 3-6, and the concentration is 6.25-200 mM.

[0068] Specifically, the flow rate ratio of the lipid mixed solution obtained by dissolving a cationic lipid, a non-cationic lipid, and a polyethylene glycol (PEG)-lipid conjugate in a solvent to the diluted mRNA solution is 1-5:1.

[0069] Specifically, when using lipid-encapsulated mRNA, the N / P is 2 - 10, preferably 3 - 8, and more preferably 3, 4, 5, 6, 7, 8. The N / P is the molar ratio of N in the cationic lipid to P in the single nucleotide of mRNA.

[0070] Specifically, the ultrafiltrate is selected from the group consisting of: sodium salts and tris(hydroxymethyl)aminomethane (Tris) salts. Preferably, the pH of the ultrafiltrate is 6.5 - 8.5.

[0071] Specifically, the dosage form of the vaccine is an oral preparation, intramuscular injection preparation, intravenous injection preparation, inhalation preparation, liquid preparation, lyophilized powder preparation, nebulized inhalation preparation or dry powder inhalation preparation.

[0072] The present invention provides a varicella - zoster virus lipid nanoparticle mRNA vaccine, comprising: mRNA encoding the varicella - zoster virus GE protein; the mRNA is encapsulated by the lipid nanoparticles.

[0073] Specifically, the amino acid sequence of the GE protein encoded by the mRNA is the sequence shown in SEQ ID NO:1, or an amino acid sequence having 80% or more identity with the sequence shown in SEQ ID NO:1, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identity.

[0074] The present invention provides the use of a varicella - zoster virus lipid nanoparticle mRNA vaccine in the preparation of a prophylactic drug for preventing varicella - zoster virus infection.

[0075] The varicella - zoster virus lipid nanoparticle mRNA vaccine of the present invention comprises mRNA encoding the varicella - zoster virus GE protein, cationic lipid, non - cationic lipid and polyethylene glycol (PEG) - lipid conjugate. The present invention selects specific cationic lipids and combines them with non - cationic lipids and polyethylene glycol (PEG) - lipids to prepare lipid nanoparticles, and it is found through experiments that they have good in vitro stability and can stimulate stronger immune responses.

[0076] The beneficial effects of the present invention compared with the prior art are as follows:

[0077] 1. The lipid nanoparticles prepared with the cationic lipid described in the present invention have a significantly better encapsulation rate than the commercially available cationic lipids;

[0078] 2. The varicella - zoster virus lipid nanoparticle mRNA vaccine prepared with the lipid nanoparticles described in the present invention induces significantly better humoral immune responses and cellular immune responses than the commercially available cationic lipids;

[0079] 3. The varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention can effectively promote antigen presentation cells to phagocytose and efficiently deliver antigens, and achieve slow release of the vaccine to continuously stimulate the body to generate a specific cellular immune response against VZV-gE;

[0080] 4. Compared with the marketed herpes zoster vaccine SHINGRIX, the varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention can not only induce CD8+4 cell responses, but also significantly induce CD8+ T cell responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Shown is the immunization schedule of BALB / c mice.

[0082] Figure 2 Detection results of lipid nanoparticle mRNA vaccines encapsulated with different cationic lipids.

[0083] Figure 3 Serum IgG antibody titer (Log value).

[0084] Figure 4 Shown is the frequency of IFNγ-secreting T cells detected by ICS method in BALB / c mouse model.

[0085] Figure 5 Shown is the frequency of IFNγ-secreting T cells detected by ELISPOT method in BALB / c mouse model.

[0086] Figure 6 Shown is the immunization schedule of C57BL / 6 mice.

[0087] Figure 7 Shown is the detection of gE-specific IgG titer by ELISA method in C57BL / 6 mouse model.

[0088] Figure 8 Shown is the frequency of IFNγ-secreting T cells detected by ICS method in C57BL / 6 mouse model.

[0089] Figure 9 Shown is the frequency of IFNγ-secreting T cells detected by ELISPOT method in C57BL / 6 mouse model.

[0090] Figure 10 Shown is the frequency of CD4+ T cells secreting specific TNFα, IFNγ, IL-2, IL-4 and IL-5 detected by ICS method in C57BL / 6 mouse model.

[0091] Figure 11Shown are the frequencies of CD8+ T cells that specifically secrete TNFα, IFNγ, IL-2, IL-4, and IL-5 detected by the ICS method in a C57BL / 6 mouse model. Detailed implementation manner

[0092] Next, in combination with the drawings of the present invention, the technical solutions in the present invention will be clearly and completely described. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0093] Example 1

[0094] Synthesis of Compound 1

[0095]

[0096] Synthesis of 6-bromohexyl (2-hexyldecyl) carbonate (1a)

[0097]

[0098] Dissolve 6-bromohexanol (0.91 g, 5.0 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (0.90 g, 7.5 mmol), and then add phenyl p-nitrophenyl carbonate (1.20 g, 6.0 mmol) in batches. Stir the reaction at room temperature for 3 h. Add 2-hexyldecanol (1.36 g, 5.6 mmol) to this reaction solution, and stir the mixture at room temperature overnight. After TLC shows that the reaction is complete, add 20 mL of dichloromethane for dilution, then wash with 30 mL of saturated brine. Dry the organic phase with anhydrous sodium sulfate, filter and concentrate. Column chromatography separation gives 6-bromohexyl (2-hexyldecyl) carbonate 1a (1.53 g, pale yellow oil), with a yield of 68%.

[0099] MS m / z (ESI): 449.3 [M+1]

[0100] Synthesis of Compound 1

[0101]

[0102] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 1 (454 mg, pale yellow oil), with a yield of 55%.

[0103] MS m / z(ESI):826.9[M+1]

[0104] 1 H NMR(300MHz,CDCl3):δ4.13(t,4H,J=6.6Hz),4.05(d,4H,J=5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J=6.2Hz)

[0105] Example 2

[0106] Synthesis of Compound 2

[0107]

[0108] Synthesis of 7-bromoheptyl heptadec-9-yl carbonate (2a)

[0109]

[0110] Dissolve 7-bromoheptanol (0.98 g, 5.0 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (1.22 g, 10 mmol), and then add phenyl 4-nitrophenyl carbonate (1.11 g, 5.5 mmol) in batches. Stir the reaction at room temperature for 3 h. Add 9-hydroxyheptadecane (1.44 g, 5.6 mmol) to this reaction solution, stir the mixture at room temperature overnight. After TLC shows that the reaction is complete, add 20 mL of dichloromethane for dilution, then wash with 30 mL of saturated brine, dry the organic phase over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain 7-bromoheptyl heptadec-9-yl carbonate 2a (1.50 g, pale yellow oil), with a yield of 65%.

[0111] MS m / z(ESI):477.3[M+1]

[0112] Synthesis of nonadec-9-yl (7-((2-hydroxyethyl)amino)heptyl) carbonate (2b)

[0113]

[0114] Under room temperature conditions, dissolve 7-bromoheptyl nonadec-9-yl carbonate (2a) (1.38 g, 3 mmol) in 20 mL of ethanol, add ethanolamine (2.75 g, 45 mmol), heat up to 50 °C, stir for 8 h, monitor the reaction progress. After the raw materials are completely consumed, cool down to 45 °C and rotary evaporate to remove ethanol. Dissolve the crude product with dichloromethane, wash it three times with saturated brine, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain the product nonadec-9-yl (7-((2-hydroxyethyl)amino)heptyl) carbonate 2b (1.35 g, light yellow oil).

[0115] MS m / z(ESI): 458.4 [M+1]

[0116] Synthesis of 5-bromopentyl undecyl carbonate (2c)

[0117]

[0118] Dissolve 5-bromopentanol (0.84 g, 5.0 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (1.22 g, 10 mmol), and then add phenyl chloroformate (1.11 g, 5.5 mmol) in batches. Stir the reaction at room temperature for 3 h. Add undecanol (0.97 g, 5.6 mmol) to this reaction solution, stir the mixture at room temperature overnight. After TLC shows that the reaction is complete, add 20 mL of dichloromethane for dilution, then wash with 30 mL of saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain 5-bromopentyl undecyl carbonate 2c (1.20 g, light yellow oil), with a yield of 66%.

[0119] MS m / z(ESI): 365.2 [M+1]

[0120] Synthesis of Compound 2

[0121]

[0122] Dissolve heptadec-9-yl (7-((2-hydroxyethyl)amino)heptyl) carbonate (457 mg, 1.0 mmol) in tetrahydrofuran, add acetonitrile, 5-bromopentyl undecyl carbonate (437 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 2 (440 mg, pale yellow oil), with a yield of 57%.

[0123] MS m / z(ESI):742.8[M+1]

[0124] 1 H NMR(300MHz,CDCl3):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J=5.4Hz),2.94(br,1H),2.58(t,2H,J=5.4Hz),2.45(t,4H,J=5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J=6.3Hz)

[0125] Example 3

[0126] Synthesis of Compound 3

[0127]

[0128] Synthesis of 6-bromohexyl undecyl carbonate (3a)

[0129]

[0130] Dissolve 6-bromohexanol (0.91 g, 5.0 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (0.90 g, 7.5 mmol), and then add phenyl chloroformate (1.20 g, 6.0 mmol) in batches. Stir the reaction at room temperature for 3 h. Add undecanol (0.97 g, 5.6 mmol) to this reaction solution, stir the mixture at room temperature overnight. After TLC shows that the reaction is complete, dilute with 20 mL of dichloromethane, then wash with 30 mL of saturated brine. Dry the organic phase over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain 6-bromohexyl undecyl carbonate 3a (1.25 g, pale yellow oil), with a yield of 66%.

[0131] MS m / z(ESI):379.2[M+1]

[0132] Synthesis of Compound 3

[0133]

[0134] Dissolve 6-bromohexyl undecyl carbonate (948 mg, 2.5 mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 3 (412 mg, pale yellow oil), with a yield of 60%.

[0135] MS m / z(ESI): 686.8[M + 1]

[0136] 1 H NMR(300 MHz, CDCl3): δ 4.13(t, 8H, J = 6.6 Hz), 3.58(t, 2H, J = 5.7 Hz), 2.52(t, 6H, J = 8.4 Hz), 1.74 - 1.64(m, 12H), 1.63 - 1.53(m, 5H), 1.52 - 1.39(m, 39H), 0.86(t, 6H, J = 6.2 Hz)

[0137] Example 4

[0138] Synthesis of Compound 4

[0139]

[0140] Synthesis of 6-bromohexyl heptadec-9-yl carbonate (4a)

[0141]

[0142] Dissolve 6-bromohexanol (0.91 g, 5.0 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (0.90 g, 7.5 mmol), and then add phenyl chloroformate p-nitro (1.20 g, 6.0 mmol) in batches. Stir the reaction at room temperature for 3 h. Add 9-heptadecanol (1.44 g, 5.6 mmol) to this reaction solution, stir the mixture at room temperature overnight. After TLC shows the reaction is complete, add 20 mL of dichloromethane for dilution, then wash with 30 mL of saturated brine. Dry the organic phase over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain 6-bromohexyl heptadec-9-yl carbonate 4a (1.53 g, pale yellow oil), with a yield of 66%.

[0143] MS m / z (ESI): 464.3 [M+1]

[0144] Synthesis of Compound 4

[0145]

[0146] Dissolve 6-bromohexyl heptadec-9-yl carbonate (1.16 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 4 (502 mg, pale yellow oil), with a yield of 59%.

[0147] MS m / z (ESI): 855.4 [M+1]

[0148] 1 H NMR (300 MHz, CDCl3): δ 4.71 - 4.68 (m, 2H), 4.13 (t, 4H, J = 6.6 Hz), 3.57 (t, 2H, J = 5.4 Hz), 2.49 - 2.44 (m, 6H), 1.74 - 1.28 (m, 76H), 0.90 (t, 12H, J = 6.3 Hz)

[0149] Example 5

[0150] Synthesis of Compound 5

[0151]

[0152] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 5 (487 mg, pale yellow oil), with a yield of 61%.

[0153] MS m / z (ESI): 798.9 [M+1]

[0154] 11H NMR (300 MHz, CDCl3): δ 4.14 (t, 4H, J = 6.6 Hz), 4.04 (d, 4H, J = 5.7 Hz), 3.54 (t, 2H, J = 5.4 Hz), 2.58 (t, 2H, J = 5.4 Hz), 2.46 (t, 4H, J = 7.2 Hz), 1.72 - 1.65 (m, 6H), 1.49 - 1.28 (m, 61H), 0.69 (t, 12H, J = 6.2 Hz)

[0155] Example 6

[0156] Synthesis of Compound 6

[0157]

[0158] Dissolve 5-bromopentyl undecyl carbonate (910 mg, 2.5 mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 6 (410 mg, pale yellow oil), with a yield of 65%.

[0159] MS m / z (ESI): 630.7 [M + 1]

[0160] 1 1H NMR (300 MHz, CDCl3): δ 4.10 (t, 8H, J = 6.6 Hz), 3.52 (d, 2H, J = 5.4 Hz), 2.83 (br, 1H), 2.57 (t, 2H, J = 5.4 Hz), 2.45 (t, 4H, J = 7.2 Hz), 1.73 - 1.62 (m, 8H), 1.52 - 1.39 (m, 40H), 0.69 (t, 6H, J = 6.2 Hz)

[0161] Example 7

[0162] Synthesis of Compound 7

[0163]

[0164] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, 3-methoxypropylamine (89 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 7 (495 mg, pale yellow oil), with a yield of 60%.

[0165] MS m / z(ESI): 826.7[M + 1]

[0166] Example 8

[0167] Synthesis of Compound 8

[0168]

[0169] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, 3-aminopropionitrile (70 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 8 (469 mg, pale yellow oil), with a yield of 58%.

[0170] MS m / z(ESI): 807.7[M + 1]

[0171] Example 9

[0172] Synthesis of Compound 9

[0173]

[0174] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, ethyl 4-aminobutyrate hydrochloride (167 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 9 (546 mg, pale yellow oil), with a yield of 63%.

[0175] MS m / z(ESI):868.8[M+1]

[0176] Example 10

[0177] Synthesis of Compound 10

[0178]

[0179] Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12 g, 2.5 mmol) in tetrahydrofuran, add acetonitrile, N-(4-aminobutyl)-acetamide hydrochloride (167 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83 °C for 16 - 20 h. Cool to room temperature, filter, wash the residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain product 10 (560 mg, pale yellow oil), with a yield of 69%.

[0180] MS m / z(ESI):867.8[M+1]

[0181] Example 11

[0182] Synthesis of Compound 11

[0183]

[0184] Synthesis of 8-bromo-N-(nonadec-9-yl)octanamide (11a)

[0185]

[0186] 8-Bromooctanoic acid (1.12 g, 5.0 mmol) was dissolved in 50 mL of dichloromethane. At 0 °C, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.05 g, 5.5 mmol) was added portionwise. After stirring for 30 min, 9-aminopalmitane (1.28 g, 5.0 mmol) was added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred overnight at room temperature. After TLC showed the reaction was complete, it was washed twice with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 11a (1.95 g, yellow oil), with a yield of 82%.

[0187] MS m / z (ESI): 461.3 [M+1].

[0188] Synthesis of compound 11b

[0189] 8-Bromo-N-(nonadec-9-yl)octanamide (1.15 g, 2.5 mmol) was dissolved in tetrahydrofuran. Acetonitrile, 4-amino-1-butanol (89.2 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added, and the mixture was stirred at 83 °C for 16 - 20 h. It was cooled to room temperature, filtered, and the filter residue was washed with dichloromethane. Saturated sodium bicarbonate solution was added to the obtained filtrate, and it was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain product 11b (534 mg, pale yellow oil), with a yield of 63%.

[0190] MS m / z (ESI): 848.8 [M+1];

[0191] 1 H NMR (300 MHz, CDCl3): δ 8.10 (s, 2H), 4.21 (s, 1H), 3.46 - 3.4 (m, 4H), 3.02 (t, 6H, J = 6.2 Hz), 2.14 (t, 4H, J = 4.8 Hz), 1.57 - 1.47 (t, 14H, J = 6.3 Hz), 1.36 - 1.26 (m, 66H), 0.90 (t, 12H, J = 6.3 Hz).

[0192] Synthesis of compound 11

[0193] At 0 °C, compound 11b (1.70 g, 2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg, 10 mmol) in anhydrous tetrahydrofuran (10 ml). The mixture was heated under reflux for 5 hours. After the reaction was complete, the temperature was lowered, and water was added to the system to completely decompose the excess reducing agent. The mixture was filtered, and the filter cake was washed with ethyl acetate. The resulting filtrate was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 11 (1.45 g, yellow oil), with a yield of 90%.

[0194] MS m / z(ESI): 820.8[M+1];

[0195] 1 H NMR(300 MHz, CDCl3): δ 4.11(s, 1H), 3.44(t, 2H, J = 4.8 Hz), 3.32(s, 2H), 3.00(t, 6H, J = 6.3 Hz), 2.52(t, 4H, J = 6.3 Hz), 2.48 - 2.43(m, 2H), 1.61 - 1.56(m, 2H), 1.36 - 1.26(m, 82H), 0.86(t, 12H, J = 4.8 Hz).

[0196] Example 12 Lipid Nanoparticle Encapsulated mRNA Antigen

[0197] In the present invention, cationic lipids I - XIV were used to prepare lipid nanoparticle nucleic acid vaccines respectively. The structures of the 14 cationic lipids are shown in the following table.

[0198] Table 2 Structural Formulas of Cationic Lipids

[0199]

[0200]

[0201]

[0202] Dilute the varicella-zoster virus mRNA vaccine stock solution to a concentration of 150 μg / ml with 100 mM sodium acetate buffer (pH 4.0). The vaccine stock solution contains the amino acid sequence of the mRNA antigen encoding the GE protein of varicella-zoster virus, and the antigen sequence is as shown in SEQ ID NO: 1. Prepare a lipid mixed solution according to a molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 45:10:43:2; set the total flow rate of the nano-drug manufacturing equipment to 12 ml / min, the flow rate ratio of the mRNA solution to the lipid mixed solution to 3:1, and start encapsulation. After encapsulation is completed, collect the sample by ultrafiltration and buffer exchange using a tangential flow filtration system, and add a sucrose solution. Conduct experiments under different conditions of the N / P (ionizable cationic lipid to nucleotide phosphate) molar ratio (N / P molar ratios are 3, 6, and 9 respectively). Take samples to detect the encapsulation rate ( Figure 2 ), average particle size, PDI, and Zeta potential. The results are shown in Table 3 below.

[0203] Table 3 Detection results of lipid nanoparticle mRNA vaccines after encapsulation with different cationic lipids

[0204]

[0205]

[0206]

[0207] It can be seen from the above results that under the same N / P conditions, the encapsulation rates of the lipid nanoparticle mRNA vaccines prepared with cationic lipids I, II, VI - XIV are all higher than those of cationic lipids III, IV, and V. The encapsulation rate of cationic lipid III is slightly higher than that of IV and V.

[0208] Example 13 Humoral Immunity Evaluation of Herpes Zoster Lipid Nanoparticle mRNA Vaccine

[0209] Evaluate the humoral immunity of samples 1 - 14 (A, B, C) prepared in Example 12 on the BALB / c mouse model, and set different N / P (3, 6, 9) to study the effect on the immunogenicity of the lipid nanoparticle mRNA vaccine.

[0210] As Figure 1 shown, BALB / c mice were immunized with 5 μg of mRNA-LNP on day 0 and day 14. Blood was collected on day 28 for antibody titer detection, and the detection results are shown in Table 4 below and Figure 3 shown.

[0211] Table 4 Antibody titers of lipid nanoparticle mRNA vaccines after encapsulation with different cationic lipids

[0212]

[0213]

[0214]

[0215] As can be seen from the antibody titer detection results in the above table, the titers of the lipid nanoparticle mRNA vaccines prepared from cationic lipids I, II, VI-XIV are higher than those of cationic lipids III, IV, and V. Cationic lipid III is slightly higher than IV and V.

[0216] Example 14 Immunization and Detection of Herpes Zoster Lipid Nanoparticle mRNA Vaccine in Mice

[0217] 1. Evaluation of Cellular Immune Response in BALB / c Mouse Model

[0218] Samples B-1, B-2, B-3, and B-4 (numbered mRNA-LNP1, mRNA-LNP2, mRNA-LNP3, and mRNA-LNP4) prepared in Example 12 were respectively evaluated for cellular immune response in the BALB / c mouse model.

[0219] As Figure 1 shown, BALB / c mice were immunized with 5 μg of mRNA-LNP on day 0 and day 14. On day 28, the mice were sacrificed and splenocytes were harvested and stimulated with an overlapping peptide library of VZV gE antigen. Cells producing IFN were measured by intracellular cytokine staining flow cytometry (ICS) method and enzyme-linked immunospot (ELISpot) method.

[0220] The frequency of IFN-secreting T cells is currently recognized as the best surrogate marker for the protective effect of herpes zoster vaccines. As Figure 4 and Figure 5 shown, the results of the two detection methods are consistent, and the cellular immune response induced by the mRNA vaccine using the formula of this patent can produce a higher frequency of IFN-secreting T cells.

[0221] In summary, the mRNA vaccine prepared by the present invention shows good potential for preventing herpes zoster, and the cellular immune response of the lipid nanoparticle mRNA vaccines prepared from cationic lipids I and II is better than that of cationic lipids III and IV.

[0222] 2. Evaluation of Immunological Response in Comparison with Positive Vaccine in C57BL / 6 Mouse Model

[0223] mRNA-LNP1 is an mRNA vaccine prepared with formulation (B-1) containing cationic lipid I; mRNA-LNP2 is an mRNA vaccine prepared with formulation (B-2) containing cationic lipid II; SHINGRIX is a positive commercially available subunit vaccine (varicella-zoster virus glycoprotein E and AS01B adjuvant). As Figure 6 shown, C57BL / 6 mice were immunized with 5 μg of the immunized mRNA-LNP or 5 μg of SHINGRIX on days 0 and 30. On day 44, the mice were sacrificed and splenocytes were harvested for evaluating the cellular immune response by the ICS method and the ELISpot method. Serum was collected on days 30 and 44 for detecting the gE-specific IgG antibody titer.

[0224] As Figure 7 shown, after the booster injection, the gE-specific IgG titers were comparable between the mRNA vaccines and SHINGRIX. As Figure 8 and Figure 9 shown, the results of the two detection methods were consistent, and the percentage of IFN-γ-producing cells induced by the mRNA vaccines was significantly higher than that induced by SHINGRIX. As Figure 10 and Figure 11 shown, both the mRNA vaccines and SHINGRIX induced a Th1-biased response. As shown by the published data, SHINGRIX can only activate CD4+ T cells; while the mRNA vaccines can not only activate CD4+ T cells, but also induce CD8+ T cell responses.

[0225] The AS01 adjuvant is a liposomal adjuvant containing the immunostimulant monophosphoryl lipid A (MPL) and Quillaja saponaria saponin QS-21, which can stimulate cellular immunity and humoral immunity. The high protection rate of Shingrix benefits from the addition of the AS01 adjuvant. Although the AS01 adjuvant greatly improves the effectiveness of the vaccine, it also increases the proportion of vaccine adverse reactions. The mRNA vaccine of the present invention does not contain an adjuvant and has significant advantages.

[0226] In summary, the mRNA vaccine prepared by the present invention shows better potential for preventing herpes zoster than the commercially available positive vaccine.

[0227] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0228] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A cationic lipid, characterized in that, The structure of the cationic lipid compound described above is as follows:

2. A lipid nanoparticle, characterized in that, Comprising: the cationic lipid, non-cationic lipid, and / or polyethylene glycol (PEG)-lipid conjugate as described in claim 1.

3. The lipid nanoparticle according to claim 2, wherein, The lipid nanoparticles comprise: cationic lipid, neutral phospholipid, sterol lipid, and / or polyethylene glycol (PEG)-lipid conjugate.

4. The lipid nanoparticle according to claim 3, characterized in that, The polyethylene glycol (PEG)-lipid conjugate selected from: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycerolamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or DMG-PEG2000, or a combination of one or more thereof.

5. The lipid nanoparticle according to claim 4, wherein The polyethylene glycol (PEG)-lipid conjugate is DMG-PEG2000.

6. The lipid nanoparticle according to claim 3, wherein, The neutral phospholipid selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), or a combination of one or more thereof.

7. The lipid nanoparticle according to claim 6, wherein, The neutral phospholipid is DSPC.

8. The lipid nanoparticle according to claim 3, wherein The sterol lipid selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, marasusterol, epicholesterol, ergosterol, fucosterol, hexahydroprovitamin D3, hydroxy cholesterol, and cholesterol modified with a polypeptide; lanosterol, provitamin D3, fucosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholanic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, or a combination of one or more thereof.

9. The lipid nanoparticle according to claim 8, wherein, The sterol lipid is cholesterol.

10. The lipid nanoparticle according to any one of claims 3-9, characterized in that, The molar percentage content of the cationic lipid in the lipid component is 20-60%, the molar percentage content of the neutral phospholipid in the lipid component is 5%-25%, the molar percentage content of the sterol lipid in the lipid component is 25%-55%; the molar percentage content of the polyethylene glycol (PEG)-lipid conjugate in the lipid component is 0.5%-15%.

11. The lipid nanoparticle according to any one of claims 3-9, characterized in that, The molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 30 - 60:1 - 20:20 - 50:0.1 - 10.

12. The lipid nanoparticle according to claim 11, wherein, The molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 40 - 60:10 - 20:30 - 50:1 - 5.

13. The lipid nanoparticle according to claim 12, wherein The molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 45:10:43:2 or 40:10:48:

2.

14. The lipid nanoparticle according to any one of claims 2-9, characterized in that, The average particle size of the nanoparticles is 50 - 200 nm, or the nanoparticles have a net neutral charge at neutral pH, or the nanoparticles have a polydispersity less than 0.

4.

15. A method for preparing a lipid nanoparticle according to any one of claims 2-14, characterized in that, It includes the step of dissolving the cationic lipid, non-cationic lipid, and polyethylene glycol (PEG)-lipid conjugate in a solvent and then mixing with mRNA.

16. The preparation method according to claim 15, wherein It is prepared by dissolving the cationic lipid, neutral phospholipid, sterol lipid, and polyethylene glycol (PEG)-lipid conjugate in ethanol, mixing with the diluted mRNA diluent, and then performing ultrafiltration, dilution, and filtration.

17. The preparation method according to claim 16, wherein It is prepared by dissolving the cationic lipid, neutral phospholipid, sterol lipid, and polyethylene glycol (PEG)-lipid conjugate in ethanol, mixing with the diluted mRNA diluent at a certain flow rate ratio, and then performing ultrafiltration, dilution, and filtration.

18. The preparation method according to claim 17, wherein, The ultrafiltration method is tangential flow filtration.

19. The preparation method according to claim 17, characterized in that, The mixing method can be turbulent mixing, laminar mixing, or microfluidic mixing.

20. The preparation method according to claim 17, wherein, The diluent is acetate buffer, citrate buffer, phosphate buffer, or tris buffer.

21. The preparation method according to claim 20, characterized in that, The pH of the buffer is 3 - 6, and the concentration is 6.25 - 200 mM.

22. The preparation method according to claim 17, characterized in that, The flow rate ratio of the lipid mixed solution obtained by dissolving the cationic lipid, non-cationic lipid, and polyethylene glycol (PEG)-lipid conjugate in a solvent to the diluted solution of mRNA is 1 - 5:

1.

23. The preparation method according to claim 22, characterized in that, When using lipids to encapsulate mRNA, the N / P is 2 - 10, and the N / P is the molar ratio of N in the cationic lipid to P in the single nucleotide of mRNA.

24. The preparation method according to claim 23, characterized in that, When using lipids to encapsulate mRNA, the N / P is 3 - 8.

25. The preparation method according to claim 24, characterized in that, When using lipids to encapsulate mRNA, the N / P is 3, 4, 5, 6, 7, or 8.

26. The preparation method according to claim 17, characterized in that, The ultrafiltrate is selected from the group consisting of: sodium salt and tris(hydroxymethyl)aminomethane (Tris) salt, and the pH of the ultrafiltrate is 6.5 - 8.5.

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