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WO2023143601A1 - Novel ionizable lipid used for nucleic acid delivery as well as lnp composition and vaccine thereof - Google Patents

Novel ionizable lipid used for nucleic acid delivery as well as lnp composition and vaccine thereof Download PDF

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Publication number
WO2023143601A1
WO2023143601A1 PCT/CN2023/073791 CN2023073791W WO2023143601A1 WO 2023143601 A1 WO2023143601 A1 WO 2023143601A1 CN 2023073791 W CN2023073791 W CN 2023073791W WO 2023143601 A1 WO2023143601 A1 WO 2023143601A1
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lipid
peg
cationic
cationic lipid
mrna
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PCT/CN2023/073791
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French (fr)
Chinese (zh)
Inventor
王浩猛
严志红
李荩
原晋波
史建明
邓捷
刘健
宇学峰
邱东旭
朱涛
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康希诺生物股份公司
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Publication of WO2023143601A1 publication Critical patent/WO2023143601A1/en

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    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
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    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/14—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to hydrocarbon groups substituted by amino groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/16—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/24—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the same saturated acyclic carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a novel ionizable lipid for nucleic acid delivery and its LNP composition and vaccine.
  • the clinically proven system for delivering mRNA is lipid nanoparticle (Lipid Nanoparticle, LNP), which belongs to lipid-formed nanoparticles, and its principle includes cationic lipids.
  • LNP lipid nanoparticle
  • the mRNA expression rate is low.
  • Dlin-MC3-DMA is used as a cationic lipid to construct LNP, and 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 cationic lipids is a key factor affecting the expression of mRNA, and the structure of cationic lipids needs further optimization.
  • VZV Varicella-varicella-zoster virus
  • the shingles vaccine is aimed at people who have been infected with the VZV virus and are immune to chickenpox, but the VZV virus is dormant in the body.
  • a shingles vaccine would function like a therapeutic vaccine, requiring a stronger immune response to prevent reactivation of latent VZV, than the varicella vaccine for VZV-susceptible populations.
  • the two currently marketed herpes zoster vaccines are the live attenuated vaccine ZOSTAVAX and the subunit vaccine SHINGRIX.
  • ZOSTAVAX is an attenuated Oka strain obtained by low-temperature passaging, stored in a lyophilized form at -15°C to -50°C; the FDA-approved target population is adults 50 years old and above, but the US Advisory Committee on Immunization recommends 60 years old and above The population of the population, because the protection of the vaccine lasts up to 8 years.
  • SHINGRIX include the extracellular region of the GE protein of the VZV virus and the AS01B adjuvant, which is stored in the form of freeze-dried GE protein and adjuvant liquid at 2°C to 8°C; the FDA-approved target population is adults aged 50 and above and immunocompromised, suppressed adults over 18 years of age, but the U.S. Advisory Committee on Immunization recommends people 50 years of age and older and those who have been immunized with ZOSTAVAX for 8 weeks or more; the vaccine’s protection can last 10 years or more (current study to 10 years). In addition to the above two vaccines, the current clinical research and development progress of herpes vaccines in China is shown in the table below.
  • VZV-specific cellular immunity is the key to limiting viral reactivation and replication.
  • the frequency of T cells secreting IFN ⁇ is currently considered to be the best surrogate indicator to investigate the protective effect of herpes zoster vaccine, but the correlation between the level of specific antibody response and the protective effect is more controversial.
  • GE protein is the main protein in VZV virus that can cause CD4 + T cell response; and the QS-21 component in AS01B adjuvant is also a natural saponin that promotes CD4 + T cell response. But the adjuvant itself has certain toxicity.
  • neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
  • polyethylene glycol (PEG)-lipid conjugate refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.
  • lipid nanoparticle refers to a particle having at least one nanoscale size, which comprises at least one lipid.
  • vaccine in the present invention refers to a composition suitable for application to animals (including humans), which induces an immune response after administration, and its strength is sufficient to help prevent, ameliorate or cure clinical diseases caused by microbial infection at a minimum.
  • delivery system in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dose in a living body.
  • N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
  • hydrocarbyl in the present invention refers to the remaining group after the corresponding hydrocarbon loses a hydrogen atom, especially refers to aliphatic hydrocarbon groups in the present invention, such as alkyl, alkenyl, alkynyl, especially alkyl.
  • the present invention relates to a cationic lipid having the following formula I structure:
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;
  • Ra is H or C 1 -C 12 hydrocarbon group
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 4 is C 1 -C 12 hydrocarbon group
  • R 5 is H or C 1 -C 6 hydrocarbon group
  • x 0, 1 or 2.
  • the cationic lipid wherein has the following structure (IA):
  • R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence;
  • n is an integer of 1 to 15.
  • the cationic lipid wherein has the following structure (IB):
  • y and z are each independently an integer from 1 to 12.
  • n in the cationic lipid structure 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, is an integer from 4 to 9.
  • R 1 and R 2 each independently have the following structure in the cationic lipid structure:
  • R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;
  • R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.
  • R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs.
  • R 7b that occurs at least once in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein the C 1 -C 8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-propyl Butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
  • R1 or R2 or both have one of the following structures:
  • the structure of the cationic lipid compound is as follows:
  • 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, medium phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates.
  • PEG polyethylene glycol
  • the polyethylene glycol (PEG)-lipid conjugate is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159 ), 1,2-Dimyristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino (Polyethylene Glycol)](PEG-DSPE), PEG-Disteryl Glycerol (PEG-DSG), PEG-Dipalmityl, PEG-Dioleyl, PEG-Distearyl, PEG-Diacylglycerol Amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or DMG-PEG2000
  • AAC-0159 1,2-
  • the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-di Myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC ), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), preferably DSPC.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmit
  • the steroidal lipids are selected from the group consisting of avenasterol, ⁇ -sitosterol, brassicasterol, ergocalcidol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydrocholesterol, Ergocalciferol, dihydrocholesterol, dihydroergosterol, nigrosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol and cholesterol modified by peptides; lanosterol, photosterol, seaweed One or more combinations of sterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.
  • the molar percentage of the cationic lipid in the lipid component is 20-60%
  • the molar percentage of the neutral phospholipid in the lipid component is 5%-25%
  • the steroidal lipid The molar percentage of the lipid in the lipid component is 25% to 55%; the molar percentage of the polyethylene glycol (PEG)-lipid conjugate in the lipid component is 0.5% to 15%.
  • the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 30-60:1-20:20-50:0.1-10
  • the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 40-60:10-20:30-50:1-5
  • the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 45:10:43:2 or 40:10:48:2 .
  • the vaccine also contains other adjuvants, and the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.
  • the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.
  • 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 of less than 0.4.
  • the invention provides a preparation method of lipid nanoparticles, comprising the steps of dissolving cationic lipids, non-cationic lipids and polyethylene glycol (PEG)-lipid conjugates into a solvent and then mixing them with mRNA.
  • cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and mixed with diluted mRNA dilutions, followed by ultrafiltration, dilution, Prepared after filtration; preferably, cationic lipids, neutral phospholipids, steroidal lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and diluted mRNA diluent at a certain flow rate Prepared by ultrafiltration, dilution, and filtration after mixing; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent flow mixing, laminar flow mixing or microfluidic mixing .
  • the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer.
  • the buffer solution has a pH of 3-6 and a concentration of 6.25-200 mM.
  • the solution flow rate ratio of the lipid mixed solution obtained after dissolving cationic lipids, non-cationic lipids, and polyethylene glycol (PEG)-lipid conjugates into a solvent and mRNA after dilution is 1 to 5: 1.
  • the N/P when using lipid-encapsulated mRNA is 2-10, preferably N/P is 3-8, more preferably, N/P is 3, 4, 5, 6, 7, 8, so Said N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
  • the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris) salt, preferably, the pH of the ultrafiltrate is 6.5-8.5.
  • the dosage form of the vaccine is an oral preparation, an intramuscular injection preparation, an intravenous injection preparation, an inhalation preparation, a liquid preparation, a freeze-dried powder, an aerosol inhalation or a dry powder inhalation.
  • the invention provides a varicella-zoster virus lipid nanoparticle mRNA vaccine, comprising: mRNA encoding varicella-zoster virus GE protein; the mRNA is encapsulated by the lipid nanoparticle.
  • amino acid sequence of the mRNA encoding GE protein 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 85%, 90% , 95%, 96%, 97%, 98%, 99% or more or 100% amino acid sequence identity.
  • the invention provides the application of a varicella-zoster virus lipid nanoparticle mRNA vaccine in the preparation of preventive medicine for preventing varicella-zoster virus infection.
  • the varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention comprises mRNA encoding varicella-zoster virus GE protein, cationic lipids, non-cationic lipids and polyethylene glycol (PEG)-lipid conjugates things.
  • the present invention selects specific cationic lipids and Lipid nanoparticles prepared in combination with non-cationic lipids and polyethylene glycol (PEG)-lipids were found to have good in vitro stability and stimulate stronger immune responses.
  • the lipid nanoparticle prepared by the cationic lipid of the present invention has an encapsulation efficiency significantly better than that of the listed cationic lipid;
  • the varicella-zoster virus lipid nanoparticle mRNA vaccine prepared by using the lipid nanoparticle of the present invention has significantly better humoral and cellular immune responses than cationic lipids already on the market;
  • the varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention can effectively promote the phagocytosis of antigen-presenting cells and efficiently deliver antigens, and realize the sustained release of the vaccine to continuously stimulate the body to produce specific cellular immunity against VZV-gE answer;
  • 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 .
  • FIG 1 shows the immunization procedure for BALB/c mice.
  • Fig. 2 Detection results of lipid nanoparticle mRNA vaccine after encapsulation with different cationic lipids.
  • Figure 4 shows the frequency of IFN ⁇ -secreting T cells detected by ICS on the BALB/c mouse model.
  • Figure 5 shows the frequency of IFN ⁇ -secreting T cells detected by ELISPOT on the BALB/c mouse model.
  • Figure 6 shows the immunization procedure for C57BL/6 mice.
  • Figure 7 shows the titer of gE-specific IgG detected by ELISA on the C57BL/6 mouse model.
  • Figure 8 shows the frequency of IFN ⁇ -secreting T cells detected by ICS on the C57BL/6 mouse model.
  • Figure 9 shows the frequency of IFN ⁇ -secreting T cells detected by ELISPOT on the C57BL/6 mouse model.
  • Figure 10 shows the frequency of CD4+T cells specifically secreting TNF ⁇ , IFN ⁇ , IL-2, IL-4 and IL-5 detected by ICS on the C57BL/6 mouse model.
  • Figure 11 shows the frequency of CD8+ T cells specifically secreting TNF ⁇ , IFN ⁇ , IL-2, IL-4 and IL-5 detected by ICS on the C57BL/6 mouse model.
  • Heptadecan-9-yl (7-((2-hydroxyethyl)amino)heptyl)carbonate (457mg, 1.0mmol) was dissolved in tetrahydrofuran, acetonitrile was added, 5-bromopentylundecylcarbonate Ester (437mg, 1.2mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h.
  • compound 11b (1.70 g, 2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg, 10 mmol) in anhydrous THF (10 ml), and the mixture was heated to reflux for 5 hours. After the reaction is complete, lower the temperature and add water to the system to completely decompose the excess reducing agent. After filtration, the filter residue was washed with ethyl acetate, and the obtained 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%.
  • the present invention uses cationic lipids I to XIV to prepare lipid nanoparticle nucleic acid vaccines respectively, and the structures of 14 kinds of cationic lipids are shown in the following table.
  • 100mM sodium acetate buffer dilutes the stock solution of varicella-zoster virus mRNA vaccine to a concentration of 150 ⁇ g/ml, which contains the amino acid sequence of the mRNA antigen encoding the GE protein of varicella-zoster virus, and the antigen sequence is as SEQID NO: 1.
  • cationic lipid DSPC: cholesterol: DMG-PEG2000 molar ratio is 45:10:43:2 to prepare lipid mixed solution; set the total flow rate of nano drug manufacturing equipment 12ml/min, mRNA solution and lipid mixed solution flow rate ratio 3 : 1 and start the encapsulation, after the encapsulation is completed, the tangential flow filtration system ultrafiltration change liquid to collect samples, and add sucrose solution.
  • N/P ionizable cationic lipid to nucleotide phosphate
  • N/P molar ratios were 3, 6, 9, respectively.
  • Sampling was carried out to detect encapsulation efficiency (Figure 2), average particle size, PDI and Zeta potential, and the results are shown in Table 3 below.
  • the encapsulation efficiency of the lipid nanoparticle mRNA vaccine prepared by cationic lipids I, II, VI-XIV is higher than that of cationic lipids III, IV, V .
  • the encapsulation efficiency of cationic lipid III was slightly higher than that of IV and V.
  • Samples 1 to 14 (A, B, C) prepared in Example 12 were evaluated for humoral immunity on the BALB/c mouse model, and different N/P (3, 6, 9) pairs of lipid nanoparticles were set. Immunogenicity impact of mRNA vaccines.
  • mice were immunized with 5 ⁇ g of mRNA-LNP on days 0 and 14. On the 28th day, blood was collected for antibody titer detection, and the test results are shown in Table 4 and Figure 3 below.
  • the titer of the lipid nanoparticle mRNA vaccine prepared by cationic lipids I, II, VI-XIV is higher than that of cationic lipids III, IV, V.
  • Cationic lipid III is slightly higher than IV, V.
  • the samples B-1, B-2, B-3, B-4 (numbered as mRNA-LNP1, mRNA-LNP2, mRNA-LNP3, mRNA-LNP4) prepared in Example 12 were respectively placed on the BALB/c mouse model Evaluation of the cellular immune response.
  • mice were immunized with 5 ⁇ g of mRNA-LNP on days 0 and 14. On day 28, mice were sacrificed and splenocytes were harvested and stimulated with an overlapping peptide pool of VZV gE antigen. IFN-producing cells were measured by intracellular cytokine staining flow cytometry (ICS) method and enzyme-linked immunospot (ELISpot) method.
  • ICS cytokine staining flow cytometry
  • ELISpot enzyme-linked immunospot
  • the frequency of IFN-secreting T cells is currently recognized as the best surrogate index for the protective effect of herpes zoster vaccine. As shown in Figure 4 and Figure 5, the results of the two detection methods are consistent, and the cellular immune response induced by the mRNA vaccine using the patented formula can generate a higher frequency of IFN-secreting T cells.
  • the mRNA vaccine prepared by the present invention shows a better potential for preventing herpes zoster, and the cellular immune response of the lipid nanoparticle mRNA vaccine prepared by cationic lipid I and II is better than that of cationic lipid III, IVs are better.
  • mRNA-LNP1 is the mRNA vaccine prepared with the formulation (B-1) containing cationic lipid I
  • mRNA-LNP2 is the mRNA vaccine prepared with the formulation (B-2) containing cationic lipid II
  • SHINGRIX is the positive commercially available sub Unit vaccine (varicella-zoster virus glycoprotein E and AS01B adjuvant).
  • C57BL/6 mice were immunized with 5 immunized mRNA-LNP or 5RN SHINGRIX on day 0 and 30.
  • mice were sacrificed and splenocytes were harvested for evaluation of cellular immune responses by ICS method and ELISpot method. Serum was collected on day 30 and day 44 for detection of gE-specific IgG antibody titers.
  • gE-specific IgG titers were comparable between the mRNA vaccine and SHINGRIX after the booster injection.
  • the results of the two detection methods were consistent, the percentage of cells producing IFN induced by mRNA vaccine was significantly higher than that induced by SHINGRIX.
  • both the mRNA vaccine and SHINGRIX induced Th1-biased responses According to public data, SHINGRIX can only activate CD4+ T cells; while mRNA vaccines can not only activate CD4+ T cells, but also induce CD8+ T cell responses.
  • AS01 adjuvant is a liposomal adjuvant containing immunostimulant monophosphoryl lipid A (MPL) and saponin QS-21, which can stimulate Cellular and humoral immunity.
  • MPL monophosphoryl lipid A
  • saponin QS-21 saponin QS-21
  • the high protection rate of Shingrix is due to the addition of AS01 adjuvant.
  • AS01 adjuvant greatly improves the effectiveness of the vaccine, it also increases the proportion of adverse reactions of the vaccine.
  • the mRNA vaccine of the present invention does not contain adjuvants and has significant advantages.
  • the mRNA vaccine prepared by the present invention shows a better potential for preventing herpes zoster than the commercially available positive vaccine.

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Abstract

Provided in the present invention are a novel cationic lipid, a lipid nanoparticle and a nucleic acid vaccine. In the present invention, a specific cationic lipid is selected for preparing a lipid nanoparticle mRNA vaccine, which is found to have better in-vitro stability and can stimulate a stronger immunoreaction compared with LNPs prepared from cationic lipids in the prior art.

Description

一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗A Novel Ionizable Lipid for Nucleic Acid Delivery and Its LNP Composition and Vaccine 技术领域technical field

本发明涉及生物医药技术领域，具体涉及一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗。The invention relates to the technical field of biomedicine, in particular to a novel ionizable lipid for nucleic acid delivery and its LNP composition and vaccine.

背景技术Background technique

目前递送mRNA在临床上经过验证的系统为脂质纳米粒(Lipid Nanoparticle，LNP)，属于脂质形成的纳米微粒，其中原理包括阳离子脂质，而现有技术研究表明，mRNA递送至细胞内后，mRNA表达率偏低，例如Dlin-MC3-DMA作为阳离子脂质构建LNP，mRNA的表达量为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)，因此，阳离子脂质的结构是影响mRNA表达量的关键因素，对于阳离子脂质的结构需要进一步的优化。At present, the clinically proven system for delivering mRNA is lipid nanoparticle (Lipid Nanoparticle, LNP), which belongs to lipid-formed nanoparticles, and its principle includes cationic lipids. , the mRNA expression rate is low. For example, Dlin-MC3-DMA is used as a cationic lipid to construct LNP, and 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 cationic lipids is a key factor affecting the expression of mRNA, and the structure of cationic lipids needs further optimization.

水痘－水痘-带状疱疹病毒(varicella-zoster virus，VZV)会导致两种不同的疾病：水痘和带状疱疹。带状疱疹疫苗针对已经感染过VZV病毒、对水痘具有免疫力，但体内潜伏着的VZV病毒的人群。因此，相比于针对VZV病毒易感人群的水痘疫苗，带状疱疹疫苗的功能类似于治疗性疫苗，需要引起更强的免疫反应以防止潜伏VZV病毒的重新激活。Varicella-varicella-zoster virus (VZV) causes two different diseases: chickenpox and shingles. The shingles vaccine is aimed at people who have been infected with the VZV virus and are immune to chickenpox, but the VZV virus is dormant in the body. Thus, a shingles vaccine would function like a therapeutic vaccine, requiring a stronger immune response to prevent reactivation of latent VZV, than the varicella vaccine for VZV-susceptible populations.

目前上市的两种带状疱疹疫苗为减毒活疫苗ZOSTAVAX和亚单位疫苗SHINGRIX。ZOSTAVAX为通过低温传代获得的减毒Oka株，冻干形式在-15℃到-50℃保存；FDA批准的目标人群为50岁及以上的成年人，但美国免疫接种咨询委员会推荐60岁及以上的人群使用，因为该疫苗保护力最多持续8年。SHINGRIX的组分包括VZV病毒的GE蛋白胞外区和AS01B佐剂，以GE蛋白冻干、佐剂液体的形式在2℃到8℃保存；FDA批准的目标人群为50岁及以上的成年人和免疫缺陷、抑制的18岁以上的成年人，但美国免疫接种咨询委员会推荐50岁及以上的人群和ZOSTAVAX免疫8周及以上的人群使用；该疫苗保护力可以持续10年或以上(目前研究到10年)。除上述两款疫苗外，目前国内的带疱疫苗临床研发进展如下表所示。The two currently marketed herpes zoster vaccines are the live attenuated vaccine ZOSTAVAX and the subunit vaccine SHINGRIX. ZOSTAVAX is an attenuated Oka strain obtained by low-temperature passaging, stored in a lyophilized form at -15°C to -50°C; the FDA-approved target population is adults 50 years old and above, but the US Advisory Committee on Immunization recommends 60 years old and above The population of the population, because the protection of the vaccine lasts up to 8 years. The components of SHINGRIX include the extracellular region of the GE protein of the VZV virus and the AS01B adjuvant, which is stored in the form of freeze-dried GE protein and adjuvant liquid at 2°C to 8°C; the FDA-approved target population is adults aged 50 and above and immunocompromised, suppressed adults over 18 years of age, but the U.S. Advisory Committee on Immunization recommends people 50 years of age and older and those who have been immunized with ZOSTAVAX for 8 weeks or more; the vaccine’s protection can last 10 years or more (current study to 10 years). In addition to the above two vaccines, the current clinical research and development progress of herpes vaccines in China is shown in the table below.

表1国内带状疱疹疫苗临床研发进展表

Table 1 Progress of clinical research and development of herpes zoster vaccine in China

虽然VZV病毒重新激活的原理现在并不清楚，但已知VZV特异性细胞免疫是限制病毒重新激活和复制的关键。分泌IFNγ的T细胞频数目前被认为是考察带状疱疹疫苗保护作用的最佳替代性指标，而关于特异性抗体应答水平与保护作用相关性的争议性则较大。Although the mechanism of VZV viral reactivation is not yet clear, it is known that VZV-specific cellular immunity is the key to limiting viral reactivation and replication. The frequency of T cells secreting IFNγ is currently considered to be the best surrogate indicator to investigate the protective effect of herpes zoster vaccine, but the correlation between the level of specific antibody response and the protective effect is more controversial.

GE蛋白是VZV病毒当中可引起CD4+T细胞反应的主要蛋白；而AS01B佐剂当中的QS-21组分也是一种促进CD4+T细胞反应的天然皂苷。但是佐剂本身具有一定毒性。GE protein is the main protein in VZV virus that can cause CD4 + T cell response; and the QS-21 component in AS01B adjuvant is also a natural saponin that promotes CD4 + T cell response. But the adjuvant itself has certain toxicity.

发明内容Contents of the invention

本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。The term "neutral lipid" as used herein refers to uncharged, non-phosphoglyceride lipid molecules.

本发明术语“聚乙二醇(PEG)-脂质缀合物”是指包含脂质部分和聚乙二醇部分的分子。The term "polyethylene glycol (PEG)-lipid conjugate" according to the present invention refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。The term "lipid nanoparticle" according to the present invention refers to a particle having at least one nanoscale size, which comprises at least one lipid.

本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物，在施用后诱导免疫应答，其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。The term "vaccine" in the present invention refers to a composition suitable for application to animals (including humans), which induces an immune response after administration, and its strength is sufficient to help prevent, ameliorate or cure clinical diseases caused by microbial infection at a minimum.

本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。The term "delivery system" in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dose in a living body.

本发明术语，N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。In terms of the present invention, N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.

本发明术语“烃基”是指相应的烃失去一个氢原子后剩余的基团，在本发明中特别指脂烃基，例如烷基、烯基、炔基，特别是烷基。The term "hydrocarbyl" in the present invention refers to the remaining group after the corresponding hydrocarbon loses a hydrogen atom, especially refers to aliphatic hydrocarbon groups in the present invention, such as alkyl, alkenyl, alkynyl, especially alkyl.

本发明涉及一种阳离子脂质具有如下式I结构：
The present invention relates to a cationic lipid having the following formula I structure:

其中： in:

L1和L2至少一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-或-NRa-，At least one of L 1 and L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)- or -NRa-,

并且，and,

L1或L2中的另一个为-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-或-NRaC(＝O)O-；The other of L 1 or L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)-, -NRa-, -O(C =O)-, -(C=O)O-, -C(=O)-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)- , -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-;

G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基；G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;

G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基；G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;

Ra为H或C1-C12烃基；Ra is H or C 1 -C 12 hydrocarbon group;

R1和R2各自独立地为C6-C24烷基或C6-C24烯基；R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl;

R3为H、OH、OR4、CN、-C(＝O)OR4、-OC(＝O)R4或–NR5C(＝O)R4；R 3 is H, OH, OR 4 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

R4为C1-C12烃基；R 4 is C 1 -C 12 hydrocarbon group;

R5为H或C1-C6烃基；R 5 is H or C 1 -C 6 hydrocarbon group;

x为0、1或2。x is 0, 1 or 2.

具体地，其中的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C＝O)O-、-(C＝O)NH-、-NH(C＝O)-和-NH-。Specifically, L 1 and L 2 in the cationic lipid formula I structure are each independently selected from -O-, -O(C=O)O-, -(C=O)NH-, -NH(C= O)- and -NH-.

具体地，其中的阳离子脂质式I结构中，L1和L2均为-O-，或者，L1和L2均为-O(C＝O)O-，或者，L1和L2均为-NH-，或者，L1为-NH(C＝O)-，L2为-(C＝O)NH-。Specifically, in the cationic lipid formula I structure, L 1 and L 2 are both -O-, or, L 1 and L 2 are both -O(C=O)O-, or, L 1 and L 2 Both are -NH-, or, L 1 is -NH(C=O)-, and L 2 is -(C=O)NH-.

具体地，其中的阳离子脂质其有以下结构(IA)：
Specifically, the cationic lipid wherein has the following structure (IA):

其中：in:

R6在每次出现时独立地为H、OH或C1-C24烃基；R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence;

n为1至15的整数。 n is an integer of 1 to 15.

具体地，其中的阳离子脂质其有以下结构(IB)：
Specifically, the cationic lipid wherein has the following structure (IB):

其中y和z各自独立地为1至12的整数。wherein y and z are each independently an integer from 1 to 12.

具体地，其中的阳离子脂质结构中n为2至12的整数，优选的，n为2、3、4、5或6；其中y和z各自独立地为2至10的整数，优选的，为4至9的整数。Specifically, n in the cationic lipid structure 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, is an integer from 4 to 9.

具体地，其中的阳离子脂质结构中R1和R2各自独立地具有以下结构：
Specifically, R 1 and R 2 each independently have the following structure in the cationic lipid structure:

其中：in:

R7a和R7b在每次出现时独立地为H或C1-C12烃基；并且a为2至12的整数，优选的，a为8至12的整数；R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;

其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.

具体地，其中的阳离子脂质结构中至少一次出现的R7a为H，优选的，R7a在每次出现时为H。Specifically, at least one occurrence of R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs.

具体地，其中的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基；优选的，其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。Specifically, R 7b that occurs at least once in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein the C 1 -C 8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-propyl Butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

具体地，其中的阳离子脂质结构中R1或R2或两者具有以下结构之一：

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

具体地，其中的阳离子脂质化合物下结构如下：


Specifically, the structure of the cationic lipid compound is as follows:


本发明提供一种脂质纳米颗粒，包含：上述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物，优选地，包含：阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。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, medium phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates.

具体地，所述的聚乙二醇(PEG)-脂质缀合物选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合，优选的为 DMG-PEG2000。Specifically, the polyethylene glycol (PEG)-lipid conjugate is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159 ), 1,2-Dimyristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino (Polyethylene Glycol)](PEG-DSPE), PEG-Disteryl Glycerol (PEG-DSG), PEG-Dipalmityl, PEG-Dioleyl, PEG-Distearyl, PEG-Diacylglycerol Amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or DMG-PEG2000 One or more combinations of, preferably DMG-PEG2000.

具体地，所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合，优选的为DSPC。Specifically, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-di Myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC ), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), preferably DSPC.

具体地，所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。Specifically, the steroidal lipids are selected from the group consisting of avenasterol, β-sitosterol, brassicasterol, ergocalcidol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydrocholesterol, Ergocalciferol, dihydrocholesterol, dihydroergosterol, nigrosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol and cholesterol modified by peptides; lanosterol, photosterol, seaweed One or more combinations of sterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.

具体地，所述的阳离子脂质在脂质组分中的摩尔百分含量为20～60％、中性磷脂在脂质组分中的摩尔百分含量为5％～25％、甾族脂质在脂质组分中的摩尔百分含量为25％～55％；聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5％～15％。Specifically, the molar percentage of the cationic lipid in the lipid component is 20-60%, the molar percentage of the neutral phospholipid in the lipid component is 5%-25%, and the steroidal lipid The molar percentage of the lipid in the lipid component is 25% to 55%; the molar percentage of the polyethylene glycol (PEG)-lipid conjugate in the lipid component is 0.5% to 15%.

具体地，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10，优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为40-60:10-20:30-50:1-5，更优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为45:10:43:2或40:10:48:2。Specifically, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 30-60:1-20:20-50:0.1-10, Preferably, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 40-60:10-20:30-50:1-5, More preferably, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar ratio is 45:10:43:2 or 40:10:48:2 .

具体地，所述疫苗中还包含其他辅料，所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。Specifically, the vaccine also contains other adjuvants, and the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

具体地，所述纳米颗粒的平均粒径为50～200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。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 of less than 0.4.

本发明提供一种脂质纳米颗粒的制备方法，包括将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。 The invention provides a preparation method of lipid nanoparticles, comprising the steps of dissolving cationic lipids, non-cationic lipids and polyethylene glycol (PEG)-lipid conjugates into a solvent and then mixing them with mRNA.

具体地，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得；优选的，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得；优选的，所述的超滤方式为切向流过滤；更优选的，所述的混合方式可为湍流混合、层流混合或微流体混合。Specifically, cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and mixed with diluted mRNA dilutions, followed by ultrafiltration, dilution, Prepared after filtration; preferably, cationic lipids, neutral phospholipids, steroidal lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and diluted mRNA diluent at a certain flow rate Prepared by ultrafiltration, dilution, and filtration after mixing; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent flow mixing, laminar flow mixing or microfluidic mixing .

具体地，稀释液为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。Specifically, the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer.

具体地，所述缓冲液pH为3～6，浓度为6.25～200mM。Specifically, the buffer solution has a pH of 3-6 and a concentration of 6.25-200 mM.

具体地，将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1～5:1。Specifically, the solution flow rate ratio of the lipid mixed solution obtained after dissolving cationic lipids, non-cationic lipids, and polyethylene glycol (PEG)-lipid conjugates into a solvent and mRNA after dilution is 1 to 5: 1.

具体地，采用脂质包封mRNA时的N/P为2-10，优选的N/P为3-8，更优选的，N/P为3、4、5、6、7、8，所述N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。Specifically, the N/P when using lipid-encapsulated mRNA is 2-10, preferably N/P is 3-8, more preferably, N/P is 3, 4, 5, 6, 7, 8, so Said N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.

具体地，所述的超滤液选自由以下组成的组：钠盐和三(羟甲基)氨基甲烷(Tris)盐，优选的，超滤液pH为6.5～8.5。Specifically, the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris) salt, preferably, the pH of the ultrafiltrate is 6.5-8.5.

具体地，疫苗的剂型为口服制剂、肌肉注射制剂、静脉注射制剂、吸入制剂、液体制剂、冻干粉剂、雾化吸入剂或干粉吸入剂。Specifically, the dosage form of the vaccine is an oral preparation, an intramuscular injection preparation, an intravenous injection preparation, an inhalation preparation, a liquid preparation, a freeze-dried powder, an aerosol inhalation or a dry powder inhalation.

本发明提供一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗，包含：编码水痘-带状疱疹病毒GE蛋白的mRNA；所述mRNA被所述的脂质纳米颗粒包裹。The invention provides a varicella-zoster virus lipid nanoparticle mRNA vaccine, comprising: mRNA encoding varicella-zoster virus GE protein; the mRNA is encapsulated by the lipid nanoparticle.

具体的，其中所述mRNA编码GE蛋白的氨基酸序列为SEQID NO:1所示序列，或者与SEQ ID NO:1所示序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上或100％同一性的氨基酸序列。Specifically, wherein the amino acid sequence of the mRNA encoding GE protein 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 85%, 90% , 95%, 96%, 97%, 98%, 99% or more or 100% amino acid sequence identity.

本发明提供一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗在制备用于预防水痘-带状疱疹病毒感染的预防性药物中的应用。The invention provides the application of a varicella-zoster virus lipid nanoparticle mRNA vaccine in the preparation of preventive medicine for preventing varicella-zoster virus infection.

本发明的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗包含编码水痘-带状疱疹病毒GE蛋白的mRNA、阳离子脂质、非-阳离子脂质及聚乙二醇(PEG)-脂质缀合物。本发明选择特定的阳离子脂质并 与非-阳离子脂质及聚乙二醇(PEG)-脂质组合制备脂质纳米颗粒，实验发现具有良好的体外稳定性并能激发更强的免疫反应。The varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention comprises mRNA encoding varicella-zoster virus GE protein, cationic lipids, non-cationic lipids and polyethylene glycol (PEG)-lipid conjugates things. The present invention selects specific cationic lipids and Lipid nanoparticles prepared in combination with non-cationic lipids and polyethylene glycol (PEG)-lipids were found to have good in vitro stability and stimulate stronger immune responses.

本发明相比现有技术的有益效果为：The beneficial effect of the present invention compared with prior art is:

1、采用本发明所述的阳离子脂质制备的脂质纳米颗粒，其包封率显著优于已上市的阳离子脂质；1. The lipid nanoparticle prepared by the cationic lipid of the present invention has an encapsulation efficiency significantly better than that of the listed cationic lipid;

2、采用本发明所述的脂质纳米颗粒制得的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗，其引起的体液免疫反应及细胞免疫反应显著优于已上市的阳离子脂质；2. The varicella-zoster virus lipid nanoparticle mRNA vaccine prepared by using the lipid nanoparticle of the present invention has significantly better humoral and cellular immune responses than cationic lipids already on the market;

3、本发明所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗可有效促进抗原提呈细胞吞噬和高效递送抗原，并实现疫苗的缓释持续刺激机体产生针对VZV-gE特异的细胞免疫应；3. The varicella-zoster virus lipid nanoparticle mRNA vaccine of the present invention can effectively promote the phagocytosis of antigen-presenting cells and efficiently deliver antigens, and realize the sustained release of the vaccine to continuously stimulate the body to produce specific cellular immunity against VZV-gE answer;

4、本发明所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗与已上市的带状疱疹疫苗SHINGRIX相比，除了可诱导CD8+4细胞反应外，还可显著诱导CD8+T细胞反应。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 .

附图说明Description of drawings

图1所示为BALB/c小鼠免疫程序。Figure 1 shows the immunization procedure for BALB/c mice.

图2不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果。Fig. 2 Detection results of lipid nanoparticle mRNA vaccine after encapsulation with different cationic lipids.

图3血清IgG抗体滴度(Log值)。Figure 3 Serum IgG antibody titer (Log value).

图4所示为BALB/c小鼠模型上ICS法检测IFNγ分泌T细胞频数。Figure 4 shows the frequency of IFNγ-secreting T cells detected by ICS on the BALB/c mouse model.

图5所示为BALB/c小鼠模型上ELISPOT法检测IFNγ分泌T细胞频数。Figure 5 shows the frequency of IFNγ-secreting T cells detected by ELISPOT on the BALB/c mouse model.

图6所示为C57BL/6小鼠免疫程序。Figure 6 shows the immunization procedure for C57BL/6 mice.

图7所示为C57BL/6小鼠模型上ELISA法检测gE特异性IgG滴度。Figure 7 shows the titer of gE-specific IgG detected by ELISA on the C57BL/6 mouse model.

图8所示为C57BL/6小鼠模型上ICS法检测IFNγ分泌T细胞频数。Figure 8 shows the frequency of IFNγ-secreting T cells detected by ICS on the C57BL/6 mouse model.

图9所示为C57BL/6小鼠模型上ELISPOT法检测IFNγ分泌T细胞频数。Figure 9 shows the frequency of IFNγ-secreting T cells detected by ELISPOT on the C57BL/6 mouse model.

图10所示为C57BL/6小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD4+T细胞频数。Figure 10 shows the frequency of CD4+T cells specifically secreting TNFα, IFNγ, IL-2, IL-4 and IL-5 detected by ICS on the C57BL/6 mouse model.

图11所示为C57BL/6小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD8+T细胞频数。 Figure 11 shows the frequency of CD8+ T cells specifically secreting TNFα, IFNγ, IL-2, IL-4 and IL-5 detected by ICS on the C57BL/6 mouse model.

具体实施方式Detailed ways

下面将结合本发明的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solution in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

实施例1Example 1

化合物1的合成
Synthesis of compound 1

6-溴己基(2-己基癸基)碳酸酯(1a)的合成
Synthesis of 6-bromohexyl(2-hexyldecyl)carbonate (1a)

将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入2-己基癸醇(1.36g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基(2-己基癸基)碳酸酯1a(1.53g，淡黄色油状物)，产率68％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, 2-hexyldecanol (1.36g, 5.6mmol) was added to the reaction solution, the mixture was stirred at room temperature overnight, after TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexyl (2-hexyldecyl) carbonate 1a (1.53 g, pale yellow oil), Yield 68%.

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

化合物1的合成
Synthesis of compound 1

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物1(454mg，淡黄色油状物)，产率55％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 1 (454 mg, pale yellow oil) in 55% yield.

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

1H 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) 1 H NMR (300MHz, CDCl 3 ): 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)

实施例2Example 2

化合物2的合成
Synthesis of Compound 2

7-溴庚基十七烷-9-基碳酸酯(2a)的合成
Synthesis of 7-bromoheptylheptadecan-9-yl carbonate (2a)

将7-溴庚醇(0.98g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入9-羟基十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到7-溴庚基十七烷-9-基碳酸酯2a(1.50g，淡黄色油状物)，产率65％。Dissolve 7-bromoheptanol (0.98g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (1.22g, 10mmol), then add phenyl p-nitrochloroformate (1.11g , 5.5mmol), the reaction was stirred at room temperature for 3h, 9-hydroxyheptadecanol (1.44g, 5.6mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight. After TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 7-bromoheptyl heptadecan-9-yl carbonate 2a (1.50g, light yellow oil) , yield 65%.

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

十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(2b)的合成
Synthesis of Heptadecan-9-yl(7-((2-Hydroxyethyl)amino)heptyl)carbonate (2b)

室温条件下，将7-溴庚基十七烷-9-基碳酸酯(2a)(1.38g，3mmol)溶于20mL乙醇中，加入乙醇胺(2.75g，45mmol)，升温至50℃，搅拌8h，监控反应进程，原料消耗完全后降温至45℃旋干除去乙醇，用二氯甲烷溶解粗品，用饱和食盐水洗涤三次，有机相用无水硫酸钠干燥，浓缩得到产品十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯2b(1.35g，淡黄色油状物)。At room temperature, 7-bromoheptylheptadecan-9-yl carbonate (2a) (1.38g, 3mmol) was dissolved in 20mL of ethanol, and ethanolamine (2.75g, 45mmol) was added, heated to 50°C, and stirred for 8h , monitor the reaction process, after the raw materials are completely consumed, cool down to 45°C and spin dry to remove ethanol, dissolve the crude product in dichloromethane, wash with saturated brine three times, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain the product heptadecane-9- (7-((2-Hydroxyethyl)amino)heptyl)carbonate 2b (1.35 g, pale yellow oil).

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

5-溴戊基十一烷基碳酸酯(2c)的合成
Synthesis of 5-bromopentylundecyl carbonate (2c)

将5-溴戊醇(0.84g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL 饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到5-溴戊基十一烷基碳酸酯2c(1.20g，淡黄色油状物)，产率66％。Dissolve 5-bromopentanol (0.84g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (1.22g, 10mmol), and then add phenyl p-nitrochloroformate (1.11g , 5.5mmol), the reaction was stirred at room temperature for 3h, undecyl alcohol (0.97g, 5.6mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. After washing with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 5-bromopentylundecyl carbonate 2c (1.20 g, light yellow oil) with a yield of 66%.

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

化合物2的合成
Synthesis of Compound 2

将十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(457mg，1.0mmol)溶于四氢呋喃中，加入乙腈，5-溴戊基十一烷基碳酸酯(437mg，1.2mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物2(440mg，淡黄色油状物)，产率57％。Heptadecan-9-yl (7-((2-hydroxyethyl)amino)heptyl)carbonate (457mg, 1.0mmol) was dissolved in tetrahydrofuran, acetonitrile was added, 5-bromopentylundecylcarbonate Ester (437mg, 1.2mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 2 (440 mg, pale yellow oil) in 57% yield.

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

1H 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) 1 H NMR (300MHz, CDCl 3 ): 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)

实施例3Example 3

化合物3的合成
Synthesis of compound 3

6-溴己基十一烷基碳酸酯(3a)的合成
Synthesis of 6-bromohexylundecyl carbonate (3a)

将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十一烷基碳酸酯3a(1.25g，淡黄色油状物)，产率66％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, undecyl alcohol (0.97g, 5.6mmol) was added to the reaction solution, the mixture was stirred at room temperature overnight, after TLC showed that the reaction was complete, 20mL of dichloromethane was added for dilution, and then 30 mL of saturated brine was washed, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexylundecyl carbonate 3a (1.25 g, light yellow oil), with a yield of 66%.

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

化合物3的合成
Synthesis of Compound 3

将6-溴己基十一烷基碳酸酯(948mg，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物3(412mg，淡黄色油状物)，产率60％。Dissolve 6-bromohexylundecyl carbonate (948mg, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), Potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 3 (412 mg, pale yellow oil) in 60% yield.

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

1H NMR(300MHz,CDCl3):δ4.13(t,8H,J＝6.6Hz),3.58(t,2H,J＝5.7Hz),2.52(t,6H,J＝8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.13(t, 8H, J=6.6Hz), 3.58(t, 2H, J=5.7Hz), 2.52(t, 6H, J=8.4Hz), 1.74- 1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J＝6.2Hz)

实施例4Example 4

化合物4的合成
Synthesis of Compound 4

6-溴己基十七烷-9-基碳酸酯(4a)的合成
Synthesis of 6-bromohexylheptadecan-9-yl carbonate (4a)

将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入9-十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十七烷-9-基碳酸酯4a(1.53g，淡黄色油状物)，产率66％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, 9-heptadecanol (1.44g, 5.6mmol) was added to the reaction solution, the mixture was stirred at room temperature overnight, after TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexyl heptadecan-9-yl carbonate 4a (1.53 g, pale yellow oil), Yield 66%.

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

化合物4的合成
Synthesis of Compound 4

将6-溴己基十七烷-9-基碳酸酯(1.16g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg,2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物4(502mg，淡黄色油状物)，产率 59％。Dissolve 6-bromohexylheptadecan-9-yl carbonate (1.16g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 4 (502 mg, light yellow oil), yield 59%.

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

1H NMR(300MHz,CDCl3):δ4.71-4.68(m,2H),4.13(t,4H,J＝6.6Hz),3.57(t,2H,J＝5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J＝6.3Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.71-4.68(m, 2H), 4.13(t, 4H, J=6.6Hz), 3.57(t, 2H, J=5.4Hz), 2.49-2.44(m ,6H),1.74-1.28(m,76H),0.90(t,12H,J＝6.3Hz)

实施例5Example 5

化合物5的合成Synthesis of compound 5

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物5(487mg，淡黄色油状物)，产率61％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg , 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 5 (487 mg, pale yellow oil) in 61% yield.

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

1H NMR(300MHz,CDCl3):δ4.14(t,4H,J＝6.6Hz),4.04(d,4H,J＝5.7Hz),3.54(t,2H,J＝5.4Hz),2.58(t,2H,J＝5.4Hz),2.46(t,4H,J＝7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.14(t, 4H, J=6.6Hz), 4.04(d, 4H, J=5.7Hz), 3.54(t, 2H, J=5.4Hz), 2.58( t, 2H, J=5.4Hz), 2.46(t, 4H, J=7.2Hz), 1.72-1.65(m, 6H), 1.49-1.28(m, 61H), 0.69(t, 12H, J=6.2Hz )

实施例6Example 6

化合物6的合成
Synthesis of compound 6

将5-溴戊基十一烷基碳酸酯(910mg，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至 室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物6(410mg，淡黄色油状物)，产率65％。Dissolve 5-bromopentylundecyl carbonate (910mg, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol ), stirred at 83°C for 16-20h. cooled to Filter at room temperature, wash the filter 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. Product 6 (410 mg, pale yellow oil) was obtained in 65% yield.

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

1H NMR(300MHz,CDCl3):δ4.10(t,8H,J＝6.6Hz),3.52(d,2H,J＝5.4Hz),2.83(br,1H),2.57(t,2H,J＝5.4Hz),2.45(t,4H,J＝7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.10(t, 8H, J=6.6Hz), 3.52(d, 2H, J=5.4Hz), 2.83(br, 1H), 2.57(t, 2H, J =5.4Hz), 2.45(t, 4H, J=7.2Hz), 1.73-1.62(m, 8H), 1.52-1.39(m, 40H), 0.69(t, 6H, J=6.2Hz)

实施例7Example 7

化合物7的合成
Synthesis of compound 7

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-甲氧基丙胺(89mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物7(495mg，淡黄色油状物)，产率60％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 3-methoxypropylamine (89mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol) , potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 7 (495 mg, pale yellow oil) in 60% yield.

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

实施例8Example 8

化合物8的合成
Synthesis of Compound 8

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-氨基丙腈(70mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物8(469mg，淡黄色油状物)，产率58％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 3-aminopropionitrile (70mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), Potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 8 (469 mg, pale yellow oil) in 58% yield.

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

实施例9Example 9

化合物9的合成
Synthesis of compound 9

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基丁酸乙酯盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物9(546mg，淡黄色油状物)，产率63％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethyl 4-aminobutyrate hydrochloride (167mg, 1.0mmol), potassium carbonate (550mg , 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 9 (546 mg, pale yellow oil) in 63% yield.

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

实施例10Example 10

化合物10的合成
Synthesis of Compound 10

将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，N-(4-氨基丁基)-乙酰胺盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物10(560mg，淡黄色油状物)，产率69％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, N-(4-aminobutyl)-acetamide hydrochloride (167mg, 1.0mmol) , potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 10 (560 mg, pale yellow oil) in 69% yield.

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

实施例11Example 11

化合物11的合成
Synthesis of compound 11

8-溴-N-(十七烷-9-基)辛酰胺(11a)的合成
Synthesis of 8-bromo-N-(heptadecan-9-yl)octylamide (11a)

将8-溴辛酸(1.12g，5.0mmol)溶于50mL二氯甲烷中，在0℃下分批加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1.05g，5.5mmol)，搅拌30min后，在反应液中逐滴加入9-氨基十七烷(1.28g，5.0mmol)，滴加完毕后，混合物在室温下搅拌过夜，TLC显示反应完成后，用100ml水洗涤2次，有机相用无水硫酸钠干燥，过滤并浓缩，得到化合物11a(1.95g，黄色油状物)，产率82％。Dissolve 8-bromooctanoic acid (1.12g, 5.0mmol) in 50mL of dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in batches at 0°C (1.05g, 5.5mmol), after stirring for 30min, 9-aminoheptadecane (1.28g, 5.0mmol) was added dropwise to the reaction solution. After the addition was complete, the mixture was stirred overnight at room temperature, and TLC showed that the reaction was complete , washed twice with 100ml of water, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 11a (1.95g, yellow oil), with a yield of 82%.

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

化合物11b的合成Synthesis of Compound 11b

将8-溴-N-(十七烷-9-基)辛酰胺(1.15g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物11b(534mg，淡黄色油状物)，产率63％。Dissolve 8-bromo-N-(heptadecan-9-yl)octylamide (1.15g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), carbonic acid Potassium (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter 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, column chromatography Isolation afforded product 11b (534 mg, pale yellow oil) in 63% yield.

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

1H NMR(300MHz,CDCl3):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J＝6.2Hz),2.14(t,4H,J＝4.8Hz),1.57-1.47(t,14H,J＝6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J＝6.3Hz)。 1 H NMR (300MHz, CDCl 3 ): δ8.10(s, 2H), 4.21(s, 1H), 3.46-3.4(m, 4H), 3.02(t, 6H, J=6.2Hz), 2.14(t , 4H, J=4.8Hz), 1.57-1.47(t, 14H, J=6.3Hz), 1.36-1.26(m, 66H), 0.90(t, 12H, J=6.3Hz).

化合物11的合成Synthesis of Compound 11

在0℃下，将化合物11b(1.70g，2mmol)缓慢加入四氢铝锂(379mg，10mmol)的无水四氢呋喃(10ml)溶液中，混合物加热回流5小时。反应完全后，降温，在体系中加入水使过量的还原剂完全分解。过滤，滤渣用乙酸乙酯洗涤，得到的滤液用水洗涤，经无水硫酸钠干燥，过滤并浓缩，得到化合物11(1.45g，黄色油状物)，产率90％。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 THF (10 ml), and the mixture was heated to reflux for 5 hours. After the reaction is complete, lower the temperature and add water to the system to completely decompose the excess reducing agent. After filtration, the filter residue was washed with ethyl acetate, and the obtained 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%.

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

1H NMR(300MHz,CDCl3):δ4.11(s,1H),3.44(t,2H,J＝4.8Hz),3.32(s,2H),3.00(t,6H,J＝6.3Hz),2.52(t,4H,J＝6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J＝4.8Hz)。 1 H NMR (300MHz, CDCl 3 ): δ4.11(s, 1H), 3.44(t, 2H, J=4.8Hz), 3.32(s, 2H), 3.00(t, 6H, J=6.3Hz), 2.52(t, 4H, J=6.3Hz), 2.48-2.43(m, 2H), 1.61-1.56(m, 2H), 1.36-1.26(m, 82H), 0.86(t, 12H, J=4.8Hz) .

实施例12脂质纳米颗粒包裹mRNA抗原Example 12 Lipid Nanoparticle Encapsulated mRNA Antigen

本发明分别使用阳离子脂质I～ⅩⅣ制备脂质纳米颗粒核酸疫苗，14种阳离子脂质结构如下表。The present invention uses cationic lipids I to XIV to prepare lipid nanoparticle nucleic acid vaccines respectively, and the structures of 14 kinds of cationic lipids are shown in the following table.

表2阳离子脂质结构式


Table 2 cationic lipid structural formula


100mM醋酸钠缓冲液(pH 4.0)稀释水痘-带状疱疹病毒mRNA疫苗原液至浓度为150μg/ml，该疫苗原液含有编码水痘-带状疱疹病毒GE蛋白的mRNA抗原的氨基酸序列，抗原序列如SEQID NO：1。按照阳离子脂质：DSPC：胆固醇：DMG-PEG2000摩尔比为45:10:43:2配制脂质混合溶液；设定纳米药物制造设备总流速12ml/min、mRNA溶液与脂质混合溶液流速比3：1并开始包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶液。在N/P(可电离的阳离子脂质与核苷酸磷酸盐)摩尔比不同条件下进行试验(N/P摩尔比分别为3、6、9)。取样检测包封率(图2)、平均粒径、PDI及Zeta电位，结果如下表3。100mM sodium acetate buffer (pH 4.0) dilutes the stock solution of varicella-zoster virus mRNA vaccine to a concentration of 150 μg/ml, which contains the amino acid sequence of the mRNA antigen encoding the GE protein of varicella-zoster virus, and the antigen sequence is as SEQID NO: 1. According to cationic lipid: DSPC: cholesterol: DMG-PEG2000 molar ratio is 45:10:43:2 to prepare lipid mixed solution; set the total flow rate of nano drug manufacturing equipment 12ml/min, mRNA solution and lipid mixed solution flow rate ratio 3 : 1 and start the encapsulation, after the encapsulation is completed, the tangential flow filtration system ultrafiltration change liquid to collect samples, and add sucrose solution. Experiments were carried out under different conditions of N/P (ionizable cationic lipid to nucleotide phosphate) molar ratio (N/P molar ratios were 3, 6, 9, respectively). Sampling was carried out to detect encapsulation efficiency (Figure 2), average particle size, PDI and Zeta potential, and the results are shown in Table 3 below.

表3不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果


Table 3 Detection results of lipid nanoparticle mRNA vaccine after encapsulation of different cationic lipids


由以上结果可以看出，在相同N/P条件下，阳离子脂质I、II、VI-XIV所制得的脂质纳米颗粒mRNA疫苗的包封率均高于阳离子脂质III、IV、V。阳离子脂质III的包封率略高于IV、V。As can be seen from the above results, under the same N/P conditions, the encapsulation efficiency of the lipid nanoparticle mRNA vaccine prepared by cationic lipids I, II, VI-XIV is higher than that of cationic lipids III, IV, V . The encapsulation efficiency of cationic lipid III was slightly higher than that of IV and V.

实施例13带状疱疹脂质纳米颗粒mRNA疫苗体液免疫评价Example 13 Evaluation of Humoral Immunity of Herpes Zoster Lipid Nanoparticle mRNA Vaccine

将实施例12制备的样品1～14(A、B、C)分别在BALB/c小鼠模型上的进行体液免疫的评价，设置不同N/P(3、6、9)对脂质纳米颗粒mRNA疫苗的免疫原性影响。Samples 1 to 14 (A, B, C) prepared in Example 12 were evaluated for humoral immunity on the BALB/c mouse model, and different N/P (3, 6, 9) pairs of lipid nanoparticles were set. Immunogenicity impact of mRNA vaccines.

如图1所示，BALB/c小鼠在第0天和14天免疫5μg的mRNA-LNP。在第28天采血进行抗体滴度检测，检测结果如下表4及图3所示。 As shown in Figure 1, BALB/c mice were immunized with 5 μg of mRNA-LNP on days 0 and 14. On the 28th day, blood was collected for antibody titer detection, and the test results are shown in Table 4 and Figure 3 below.

表4不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗抗体滴度

Lipid nanoparticle mRNA vaccine antibody titer after table 4 different cationic lipid encapsulation

由上表的抗体滴度检测结果可以看出，阳离子脂质I、II、VI-XIV所制得的脂质纳米颗粒mRNA疫苗的滴度高于阳离子脂质III、IV、V。阳离子脂质III略高于IV、V。 As can be seen from the antibody titer test results in the above table, the titer of the lipid nanoparticle mRNA vaccine prepared by cationic lipids I, II, VI-XIV is higher than that of cationic lipids III, IV, V. Cationic lipid III is slightly higher than IV, V.

实施例14带状疱疹脂质纳米颗粒mRNA疫苗小鼠免疫与检测Example 14 Immunization and Detection of Herpes Zoster Lipid Nanoparticle mRNA Vaccine Mice

1、在BALB/c小鼠模型上的细胞免疫反应评价1. Evaluation of cellular immune response on BALB/c mouse model

将实施例12制备的样品B-1、B-2、B-3、B-4(编号为mRNA-LNP1、mRNA-LNP2、mRNA-LNP3、mRNA-LNP4)分别在BALB/c小鼠模型上的进行细胞免疫反应的评价。The samples B-1, B-2, B-3, B-4 (numbered as mRNA-LNP1, mRNA-LNP2, mRNA-LNP3, mRNA-LNP4) prepared in Example 12 were respectively placed on the BALB/c mouse model Evaluation of the cellular immune response.

如图1所示，BALB/c小鼠在第0天和14天免疫5μg的mRNA-LNP。在第28天，处死小鼠并收获脾细胞并使用VZV gE抗原的重叠肽库进行刺激。通过细胞内细胞因子染色流式细胞术(ICS)方法和酶联免疫斑点(ELISpot)方法测量产生IFN的细胞。As shown in Figure 1, BALB/c mice were immunized with 5 μg of mRNA-LNP on days 0 and 14. On day 28, mice were sacrificed and splenocytes were harvested and stimulated with an overlapping peptide pool of VZV gE antigen. IFN-producing cells were measured by intracellular cytokine staining flow cytometry (ICS) method and enzyme-linked immunospot (ELISpot) method.

IFN分泌T细胞频数是目前公认的带状疱疹疫苗保护作用的最佳替代性指标。如图4和图5所示，两种检测方法的结果一致，使用本专利配方的mRNA疫苗引发的细胞免疫反应可以产生较高的IFN分泌T细胞频数。The frequency of IFN-secreting T cells is currently recognized as the best surrogate index for the protective effect of herpes zoster vaccine. As shown in Figure 4 and Figure 5, the results of the two detection methods are consistent, and the cellular immune response induced by the mRNA vaccine using the patented formula can generate a higher frequency of IFN-secreting T cells.

综上，本发明制备的mRNA疫苗显示出较好的用于预防带状疱疹的潜力，且阳离子脂质I、II所制得的脂质纳米颗粒mRNA疫苗的细胞免疫反应较阳离子脂质III、IV更好。In summary, the mRNA vaccine prepared by the present invention shows a better potential for preventing herpes zoster, and the cellular immune response of the lipid nanoparticle mRNA vaccine prepared by cationic lipid I and II is better than that of cationic lipid III, IVs are better.

2、在C57BL/6小鼠模型上对比阳性疫苗的免疫反应评价2. Evaluation of the immune response of the positive vaccine on the C57BL/6 mouse model

mRNA-LNP1为用含阳离子脂质I的配方(B-1)制备的mRNA疫苗；mRNA-LNP2为用含阳离子脂质II的配方(B-2)制备的mRNA疫苗；SHINGRIX为阳性市售亚单位疫苗(水痘-带状疱疹病毒糖蛋白E及AS01B佐剂)。如图6所示，C57BL/6小鼠在第0天和第30天免疫5免疫的mRNA-LNP或5RN的SHINGRIX。在第44天，处死小鼠并收获脾细胞用于通过ICS方法和ELISpot方法评估细胞免疫反应。在第30天和第44天收集血清用于检测gE特异性IgG抗体滴度。mRNA-LNP1 is the mRNA vaccine prepared with the formulation (B-1) containing cationic lipid I; mRNA-LNP2 is the mRNA vaccine prepared with the formulation (B-2) containing cationic lipid II; SHINGRIX is the positive commercially available sub Unit vaccine (varicella-zoster virus glycoprotein E and AS01B adjuvant). As shown in Figure 6, C57BL/6 mice were immunized with 5 immunized mRNA-LNP or 5RN SHINGRIX on day 0 and 30. On day 44, mice were sacrificed and splenocytes were harvested for evaluation of cellular immune responses by ICS method and ELISpot method. Serum was collected on day 30 and day 44 for detection of gE-specific IgG antibody titers.

如图7所示，在加强注射后，mRNA疫苗和SHINGRIX之间的gE特异性IgG滴度具有可比性。如图8和图9所示，两种检测方法的结果一致，mRNA疫苗诱导的产生IFN导的细胞的百分比显着高于SHINGRIX诱导的细胞百分比。如图10和图11所示，mRNA疫苗和SHINGRIX都诱导了Th1偏向反应。SHINGRIX如公开数据显示，只能激活CD4+T细胞；而mRNA疫苗除可以激活CD4+T细胞外，还可以诱导CD8+T细胞反应。As shown in Figure 7, gE-specific IgG titers were comparable between the mRNA vaccine and SHINGRIX after the booster injection. As shown in Figure 8 and Figure 9, the results of the two detection methods were consistent, the percentage of cells producing IFN induced by mRNA vaccine was significantly higher than that induced by SHINGRIX. As shown in Figures 10 and 11, both the mRNA vaccine and SHINGRIX induced Th1-biased responses. According to public data, SHINGRIX can only activate CD4+ T cells; while mRNA vaccines can not only activate CD4+ T cells, but also induce CD8+ T cell responses.

AS01佐剂是一种包含免疫刺激剂单磷酸酰脂质A(MPL)和皂树皂苷QS-21的脂质体佐剂，能够激 发细胞免疫和体液免疫。Shingrix高保护率得益于添加了AS01佐剂，AS01佐剂虽然很大程度提升了疫苗的有效性，但同时也增加了疫苗不良反应比例。本发明mRNA疫苗不含佐剂，具有显著优势。AS01 adjuvant is a liposomal adjuvant containing immunostimulant monophosphoryl lipid A (MPL) and saponin QS-21, which can stimulate Cellular and humoral immunity. The high protection rate of Shingrix is due to the addition of AS01 adjuvant. Although AS01 adjuvant greatly improves the effectiveness of the vaccine, it also increases the proportion of adverse reactions of the vaccine. The mRNA vaccine of the present invention does not contain adjuvants and has significant advantages.

综上，本发明制备的mRNA疫苗显示出比市售阳性疫苗更好的用于预防带状疱疹的潜力。In summary, the mRNA vaccine prepared by the present invention shows a better potential for preventing herpes zoster than the commercially available positive vaccine.

以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but 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. These simple modifications All belong to the protection scope of the present invention.

另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。 In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

Claims (29)

一种阳离子脂质，其特征在于，所述的阳离子脂质具有如下式I结构：
A kind of cationic lipid, is characterized in that, described cationic lipid has following formula I structure:
其中：in: L1和L2至少一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-或-NRa-，At least one of L 1 and L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)- or -NRa-, 并且，and, L1或L2中的另一个为-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-或-NRaC(＝O)O-；The other of L 1 or L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)-, -NRa-, -O(C =O)-, -(C=O)O-, -C(=O)-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)- , -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-; G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基；G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene; G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基；G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene; Ra为H或C1-C12烃基；Ra is H or C 1 -C 12 hydrocarbon group; R1和R2各自独立地为C6-C24烷基或C6-C24烯基；R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl; R3为H、OH、OR4、CN、-C(＝O)OR4、-OC(＝O)R4或–NR5C(＝O)R4；R 3 is H, OH, OR 4 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R4为C1-C12烃基；R 4 is C 1 -C 12 hydrocarbon group; R5为H或C1-C6烃基；R 5 is H or C 1 -C 6 hydrocarbon group; x为0、1或2。x is 0, 1 or 2. 根据权利要求1所述的阳离子脂质，其特征在于，所述的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C＝O)O-、-(C＝O)NH-、-NH(C＝O)-和-NH-。The cationic lipid according to claim 1, wherein L1 and L2 are each independently selected from the group consisting of -O-, -O(C=O)O-, -(C =O)NH-, -NH(C=O)- and -NH-. 根据权利要求1-2任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质式I结构中所述L1和L2均为-O-，或者，L1和L2均为-O(C＝O)O-，或者，L1和L2均为-NH-，或者，L1为-NH(C＝O)-，L2为-(C＝O)NH-。 The cationic lipid according to any one of claims 1-2, characterized in that, the L1 and L2 in the cationic lipid formula I structure are both -O-, or, L1 and L2 are both -O (C=O)O-, or, both L1 and L2 are -NH-, or, L1 is -NH(C=O)-, and L2 is -(C=O)NH-. 根据权利要求1-3任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质其有以下结构(IA)：
According to the cationic lipid described in any one of claims 1-3, it is characterized in that, it has following structure (IA) of described cationic lipid:
其中：in: R6在每次出现时独立地为H、OH或C1-C24烃基；R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence; n为1至15的整数。n is an integer of 1 to 15. 根据权利要求1-4任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质其有以下结构(IB)：
According to the cationic lipid described in any one of claims 1-4, it is characterized in that, it has following structure (IB) of described cationic lipid:
其中y和z各自独立地为1至12的整数。wherein y and z are each independently an integer from 1 to 12. 根据权利要求1-5任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中n为2至12的整数，优选的，n为2、3、4、5或6；其中y和z各自独立地为2至10的整数，优选的，为4至9的整数。The cationic lipid according to any one of claims 1-5, characterized in that, 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 an integer from 4 to 9. 根据权利要求1-6任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中R1和R2各自独立地具有以下结构：
According to the cationic lipid according to any one of claims 1-6, it is characterized in that, in the described cationic lipid structure, R 1 and R 2 each independently have the following structure:
其中：in: R7a和R7b在每次出现时独立地为H或C1-C12烃基；并且a为2至12的整数，优选的，a为8至12的 整数；R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer of 2 to 12, preferably, a is 8 to 12 integer; 其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms. 根据权利要求1-7任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中至少一次出现的R7a为H，优选的，R7a在每次出现时为H。The cationic lipid according to any one of claims 1-7, characterized in that, at least one occurrence of R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs. 根据权利要求1-8任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基；优选的，其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。The cationic lipid according to any one of claims 1-8, characterized in that, at least one occurrence of R 7b in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein C 1 -C 8 Hydrocarbyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-hexyl or n-octyl. 根据权利要求1-9任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中R1或R2或两者具有以下结构之一：
According to the cationic lipid according to any one of claims 1-9, it is characterized in that, in the described cationic lipid structure, R 1 or R 2 or both have one of the following structures:
根据权利要求1-10任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质化合物下结构如下：


The cationic lipid according to any one of claims 1-10, wherein the structure of the cationic lipid compound is as follows:


一种脂质纳米颗粒，其特征在于，包含：如权利要求1-11任一项所述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物，优选地，包含：阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。A lipid nanoparticle, characterized in that, comprising: cationic lipid, non-cationic lipid and/or polyethylene glycol (PEG)-lipid conjugate as described in any one of claims 1-11 , preferably, comprising: cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates. 根据权利要求12所述的脂质纳米颗粒，其特征在于，所述的聚乙二醇(PEG)-脂质缀合物选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合，优选的为DMG-PEG2000。The lipid nanoparticle according to claim 12, wherein the polyethylene glycol (PEG)-lipid conjugate is selected from: 2-[(polyethylene glycol)-2000]-N, N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn -Glyceryl-3-phosphoethanolamine-N-[Amino(polyethylene glycol)](PEG-DSPE), PEG-Disterylglycerol (PEG-DSG), PEG-Dipalmitoleyl, PEG-Dioleyl , PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3 - One or more combinations of amine (PEG-c-DMA) or DMG-PEG2000, preferably DMG-PEG2000. 根据权利要求12-13任一项所述的脂质纳米颗粒，其特征在于，所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合，优选的为DSPC。The lipid nanoparticle according to any one of claims 12-13, wherein the neutral lipid is 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-phosphate-(1 One or more combinations of '-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), preferably DSPC. 根据权利要求12-14任一项所述的脂质纳米颗粒，其特征在于，所述的甾族脂质选自燕麦甾醇、β- 谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。The lipid nanoparticle according to any one of claims 12-14, wherein the steroidal lipid is selected from the group consisting of avenasterol, β- Sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, black hesterol, Epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, and cholesterol modified by peptides; lanosterol, photosterol, algalsterol, sitostanol, sitosterol, stigmasterol, stigmasterol , cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid in one or more combinations, preferably cholesterol. 根据权利要求12-15任一项所述的脂质纳米颗粒，其特征在于，所述的阳离子脂质在脂质组分中的摩尔百分含量为20～60％、中性磷脂在脂质组分中的摩尔百分含量为5％～25％、甾族脂质在脂质组分中的摩尔百分含量为25％～55％；聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5％～15％。The lipid nanoparticle according to any one of claims 12-15, characterized in that, the molar percentage of the cationic lipid in the lipid component is 20% to 60%, and the neutral phospholipid in the lipid The molar percentage of the component is 5% to 25%, and the molar percentage of the steroidal lipid in the lipid component is 25% to 55%; polyethylene glycol (PEG)-lipid conjugate The mole percentage in the lipid component is 0.5%-15%. 根据权利要求12-16任一项所述的脂质纳米颗粒，其特征在于，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10，优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为40-60:10-20:30-50:1-5，更优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为45:10:43:2或40:10:48:2。The lipid nanoparticle according to any one of claims 12-16, wherein the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate mole The ratio is 30-60:1-20:20-50:0.1-10, preferably, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar The ratio is 40-60:10-20:30-50:1-5, more preferably, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate The molar ratio is 45:10:43:2 or 40:10:48:2. 根据权利要求12-17任一项所述的脂质纳米颗粒，其特征在于，所述疫苗中还包含其他辅料，所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。According to the lipid nanoparticle according to any one of claims 12-17, it is characterized in that, other adjuvants are also included in the vaccine, and the adjuvants are sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride , disodium hydrogen phosphate, and one or more combinations of sucrose. 根据权利要求12-18任一项所述的脂质纳米颗粒，其特征在于，所述纳米颗粒的平均粒径为50～200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。The lipid nanoparticle according to any one of claims 12-18, characterized in that, the average particle diameter of the nanoparticle is 50-200nm or the nanoparticle has a net neutral charge at neutral pH or the The nanoparticles have a polydispersity of less than 0.4. 一种根据权利要求1-19任一项所述的脂质纳米颗粒的制备方法，其特征在于，包括将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。A method for preparing lipid nanoparticles according to any one of claims 1-19, characterized in that comprising conjugating cationic lipids, non-cationic lipids, polyethylene glycol (PEG)-lipids After the substance is dissolved in the solvent, it is mixed with the mRNA. 根据权利要求20所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗的制备方法，其特征在于，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得；优选的，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得；优选的，所述的超滤方式为切向流过滤；更优选的，所述的混合方式可为湍流混合、层流混合或微流 体混合。The preparation method of varicella-zoster virus lipid nanoparticle mRNA vaccine according to claim 20, is characterized in that, cationic lipid, neutral phospholipid, steroidal lipid, polyethylene glycol (PEG)-lipid The protein conjugate is dissolved in ethanol and mixed with the diluted mRNA diluent, and then obtained by ultrafiltration, dilution, and filtration; preferably, cationic lipids, neutral phospholipids, steroidal lipids, polyethylene glycol After the (PEG)-lipid conjugate is dissolved in ethanol, it is mixed with the diluted mRNA diluent at a certain flow rate ratio and then prepared by ultrafiltration, dilution, and filtration; preferably, the ultrafiltration method is tangential Flow filtration; more preferably, the mixing method can be turbulent mixing, laminar mixing or micro flow body mix. 根据权利要求20-21任一项所述的脂质纳米颗粒的制备方法，其特征在于，稀释液为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。The method for preparing lipid nanoparticles according to any one of claims 20-21, wherein the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer. 根据权利要求20-22任一项所述的脂质纳米颗粒的制备方法，其特征在于，所述缓冲液pH为3～6，浓度为6.25～200mM。The preparation method of lipid nanoparticles according to any one of claims 20-22, characterized in that the buffer solution has a pH of 3-6 and a concentration of 6.25-200 mM. 根据权利要求20-23任一项所述的脂质纳米颗粒的制备方法，其特征在于，将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1～5:1。The preparation method of lipid nanoparticles according to any one of claims 20-23, is characterized in that cationic lipids, non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved to The flow rate ratio of the obtained lipid mixed solution after the solvent to the diluted mRNA is 1-5:1. 根据权利要求20-24任一项所述的脂质纳米颗粒的制备方法，其特征在于，采用脂质包封mRNA时的N/P为2-10，优选的N/P为3-8，更优选的，N/P为3、4、5、6、7、8，所述N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。The preparation method of lipid nanoparticles according to any one of claims 20-24, characterized in that the N/P when using lipid-encapsulated mRNA is 2-10, and the preferred N/P is 3-8, More preferably, N/P is 3, 4, 5, 6, 7, 8, and said N/P is the molar ratio of N in cationic lipid to P in mRNA single nucleotide. 根据权利要求20-25任一项所述的脂质纳米颗粒的制备方法，其特征在于，所述的超滤液选自由以下组成的组：钠盐和三(羟甲基)氨基甲烷(Tris)盐，优选的，超滤液pH为6.5～8.5。The method for preparing lipid nanoparticles according to any one of claims 20-25, wherein the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris ) salt, preferably, the pH of the ultrafiltrate is 6.5-8.5. 根据权利要求20-26任一项所述的脂质纳米颗粒的制备方法，其特征在于，疫苗的剂型为口服制剂、肌肉注射制剂、静脉注射制剂、吸入制剂、液体制剂、冻干粉剂、雾化吸入剂或干粉吸入剂。The preparation method of lipid nanoparticles according to any one of claims 20-26, wherein the dosage form of the vaccine is oral preparation, intramuscular injection preparation, intravenous injection preparation, inhalation preparation, liquid preparation, freeze-dried powder, mist inhalers or dry powder inhalers. 一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗，其特征在于，包含：编码水痘-带状疱疹病毒GE蛋白的mRNA；所述mRNA被权利要求12-27任一项所述的脂质纳米颗粒包裹或被ALC-0315制备的脂质纳米颗粒包裹。A varicella-zoster virus lipid nanoparticle mRNA vaccine, characterized in that it comprises: mRNA encoding varicella-zoster virus GE protein; said mRNA is replaced by the lipid described in any one of claims 12-27 Nanoparticles encapsulated or encapsulated by lipid nanoparticles prepared with ALC-0315. 一种权利要求1-19、28任一项所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗在制备用于预防水痘-带状疱疹病毒感染的预防性药物中的应用。 A use of the varicella-zoster virus lipid nanoparticle mRNA vaccine according to any one of claims 1-19 and 28 in the preparation of preventive medicine for preventing varicella-zoster virus infection.
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