WO2024109612A1 - Method for preparing lipid nanoparticle for efficiently delivering nucleic acid drug and use thereof - Google Patents

Method for preparing lipid nanoparticle for efficiently delivering nucleic acid drug and use thereof Download PDF

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WO2024109612A1
WO2024109612A1 PCT/CN2023/131882 CN2023131882W WO2024109612A1 WO 2024109612 A1 WO2024109612 A1 WO 2024109612A1 CN 2023131882 W CN2023131882 W CN 2023131882W WO 2024109612 A1 WO2024109612 A1 WO 2024109612A1
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lipid
nucleic acid
ionizable
glyceryl
lnp
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PCT/CN2023/131882
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French (fr)
Chinese (zh)
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吕雪光
林佳奇
程星迪
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中国科学院化学研究所
深圳近邻生物科技有限公司
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Publication of WO2024109612A1 publication Critical patent/WO2024109612A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • 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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the technical field to which the present invention belongs is the field of medical technology, and specifically relates to a lipid nanoparticle (LNP) capable of efficiently delivering nucleic acid drugs, and a preparation method and application thereof.
  • LNP lipid nanoparticle
  • mRNA messenger RNA
  • mRNA vaccines have the advantages of high safety, short R&D cycle, high production efficiency, ability to encode multiple proteins, and no need for adjuvants.
  • mRNA itself is difficult to enter cells, and mRNA itself has poor stability and is easily degraded by nucleases in the body. Therefore, a suitable delivery vehicle is needed to assist mRNA in entering target cells to work.
  • Lipid nanoparticles are currently the only clinically approved mRNA vaccine carriers, which are composed of ionizable lipids, auxiliary phospholipids, cholesterol and pegylated lipids. LNP can effectively deliver mRNA into cells after intramuscular injection, activate cellular immunity and humoral immune responses, and produce effective immune protection.
  • the delivery efficiency of LNP is the key to the efficacy of mRNA vaccines, and the low lysosomal escape efficiency of LNP after entering the cell is the key to limiting the delivery efficiency of LNP. Therefore, it is urgent to develop new carriers that can increase the lysosomal escape efficiency of LNP and improve the delivery efficiency of LNP.
  • the present invention provides a lipid nanoparticle (hereinafter referred to as LNP), comprising a carrier and an encapsulated nucleic acid, wherein the carrier comprises a composite ionizable lipid, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid; the nucleic acid comprises but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and a plasmid.
  • LNP lipid nanoparticle
  • the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP, such as 30 mol%, 40 mol%, 50 mol%, 60 mol%.
  • the complex ionizable lipid comprises a first ionizable lipid and a second ionizable lipid.
  • the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2, such as 3:7, 4:6, 5:5, 6:4, 7:3, and more preferably 6:4.
  • the first ionizable lipid and the second ionizable lipid are different and are independently selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102), [(4-hydroxybutyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin MC3DMA), 3,6-bis ⁇ 4-[bis(2-hydroxydodecyl)amino]butyl ⁇ piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butyryloxy)heptadecanedioic
  • the complex ionizable lipid includes 3,6-bis ⁇ 4-[bis(2-hydroxydodecyl)amino]butyl ⁇ piperazine-2,5-dione (cKK-E12) and 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), and the molar ratio thereof is 1:99 to 99:1, preferably 2:8 to 8:2, for example 3:7, 4:6, 5:5, 6:4, 7:3.
  • the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.
  • the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DS
  • the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.
  • the cholesterol-like substance is selected from cholesterol and its derivatives.
  • the cholesterol and its derivatives include but are not limited to one or more of cholesterol, ⁇ -sitosterol, cholestanol, cholestanone, cholestenone, 7 ⁇ -hydroxycholesterol, 7 ⁇ -hydroxycholesterol, preferably cholesterol.
  • the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.
  • the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.
  • DMG-PEG 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol
  • DSG-PEG 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol
  • DPG-PEG 1,2-dipalmitoyl-rac-glyceryl-3-me
  • total lipids refers to the sum of complex ionizable lipids, auxiliary phospholipids, cholesterol substances and PEGylated lipids.
  • the nucleic acid selected by the present invention includes at least one of CpG-FAM, mFLuc (mRNA encoding firefly luciferase), mOVA (mRNA encoding chicken ovalbumin), and mVZV (mRNA encoding herpes zoster gE protein).
  • the encapsulation rate of the nucleic acid is 30% to 99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.
  • the molar ratio of the nitrogen element contained in the complex ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.
  • the hydrated particle size of the lipid nanoparticles is less than 200 nm, such as 80 to 180 nm.
  • the polydispersity coefficient of the lipid nanoparticles is less than 0.2, for example, 0.119, 0.102, or 0.093.
  • the molar ratio of the complex ionizable lipid: helper phospholipid: cholesterol-like substance: polyethylene glycol lipid is 50:10:38.5:1.5.
  • the composite ionizable lipid comprises a first ionizable lipid and a second ionizable lipid, the two being Examples are 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, or 2:8.
  • the nucleic acid is CpG-FAM, mFLuc, mOVA or mVZV.
  • the present invention also provides a method for preparing the lipid nanoparticles, which comprises preparing the lipid nanoparticles by blending an aqueous phase containing nucleic acids and a lipid organic phase through a blending method or a microfluidics method.
  • the lipid organic phase is prepared as follows: the composite ionizable lipid, auxiliary phospholipid, cholesterol-like substance and pegylated lipid are dissolved in an organic solvent in proportion to obtain an organic phase.
  • the organic solvent is an organic solvent that can dissolve the above substances, such as anhydrous ethanol.
  • the preparation method of the aqueous phase containing nucleic acid is as follows: dissolving the nucleic acid in a buffer solution to obtain an aqueous phase.
  • the buffer solution can be a buffer solution with a pH of 4 to 6, for example, a sodium citrate buffer solution with a pH of 4 or 5.5.
  • the blending method includes the following steps: using a pipette to absorb the aqueous phase containing nucleic acids, quickly adding it to the lipid organic phase, and then quickly blowing the mixed liquid for more than 30 times, blowing it evenly and letting it stand at room temperature for 10 minutes, and dialyzing the resulting product after standing, for example, dialyzing it in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.
  • the microfluidic method includes the following steps: fixing syringes containing an aqueous phase and a lipid organic phase on a syringe pump respectively, injecting the aqueous phase: lipid organic phase into a microfluidic chip at a volume ratio of 3:1 and mixing thoroughly, collecting the mixed solution after mixing and standing at room temperature for 10 minutes, and dialyzing the resulting product with a PBS buffer solution after standing, for example, dialyzing in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.
  • the present invention also provides the use of the lipid nanoparticles in the preparation of pharmaceutical preparations or biological preparations.
  • the biologic is an injectable biologic.
  • the biologic is a vaccine, preferably an mRNA vaccine.
  • the administration method of the biological preparation is intramuscular injection.
  • the biological agent is used to prevent herpes zoster.
  • the present invention also provides a pharmaceutical preparation or a biological preparation having the meaning as described above.
  • the present invention also provides a method for preventing and/or treating a disease, comprising administering a preventively and/or therapeutically effective amount of the above-mentioned pharmaceutical preparation or biological product to a subject; for example, the disease is herpes zoster.
  • the term "effective amount" can be determined according to the method mastered by a physician with clinical practice qualifications in the field to determine the amount of the pharmaceutical preparation or biological product of the present invention that is sufficient to achieve the intended application (including but not limited to the treatment of diseases defined above). Determining the effective dose for prevention and/or treatment is within the capabilities of clinicians or researchers in the field and may be changed due to the following factors: the intended application (in vitro or in vivo), or the subject and disease condition to be treated, such as the subject's weight and age, general health, severity of the disease condition, mode of administration, and other factors that affect the efficacy, such as a history of drug allergies.
  • the specific dosage will vary depending on the following factors: the specific biological product selected, the dosage regimen based on, whether it is co-administered with other drugs, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.
  • the "subject" of the present invention refers to a specific human or other warm-blooded mammal.
  • the human as “subject” of the present invention includes adults, infants, and children, and other warm-blooded mammals include but are not limited to non-human primates, such as chimpanzees, other apes or monkeys, and other zoo animals, domestic mammals or laboratory animals, such as cats, pigs, dogs, cows, sheep, mice, rats, and guinea pigs.
  • the LNP prepared by the present invention has uniform particle size, good dispersibility, high encapsulation efficiency, good biocompatibility, low toxicity and side effects, and can be highly
  • the LNP preparation method of the present invention has simple operation steps and is easy to mass produce.
  • the present invention combines a first ionizable lipid and a second ionizable lipid to form a composite ionizable lipid, which has an ionizable lipid with high efficiency in entering cells and an ionizable lipid with a long alkyl chain and high efficiency in membrane fusion ability.
  • the composite ionizable lipid of the present invention can significantly improve the mRNA expression efficiency of the commercial four-component LNP through synergistic effect.
  • the present invention uses composite ionizable lipids to prepare five-component LNPs, which have a higher activation efficiency on antigen-presenting cells in draining lymph nodes and spleen than commercial four-component LNPs after intramuscular injection.
  • the five-component LNP prepared by the composite ionizable lipids of the present invention can efficiently deliver the herpes zoster mRNA vaccine. After intramuscular injection, it significantly activates the cellular immunity and humoral immune response in the body, produces a large number of antigen-specific binding antibodies in the blood, and increases the antigen-specific T cell content in the spleen, which has an excellent protective effect. It is expected to achieve a breakthrough in the field of herpes zoster mRNA vaccines and has great application prospects.
  • FIG. 1 Chemical structures of the first ionizable lipid and the second ionizable lipid used in the examples of the present invention.
  • FIG. 2 Hydration particle size distribution curve of LNP obtained in Example 1B of the present invention.
  • FIG3 Cryo-TEM image of the five-component LNP obtained in Example 1A of the present invention.
  • FIG. 4 shows the bioluminescence intensity of DC2.4 cells transfected with LNPs loaded with mFluc in Example 3 of the present invention.
  • FIG5 The membrane fusion ability of the five-component LNP was detected using mouse red blood cells in Example 4 of the present invention.
  • Example 5 of the present invention in vivo imaging of small animals was used to verify the bioluminescence intensity of the five-component LNP loaded with mFLuc at the injection site of mice; (a) In vivo imaging image and statistical results of bioluminescence intensity at the injection site 4 hours after intramuscular injection; (b) In vivo imaging image and statistical results of bioluminescence intensity at the injection site 24 hours after intramuscular injection.
  • Figure 7 Flow chart of animal experiment in Example 6 of the present invention.
  • FIG8 Activation efficiency of different LNPs loaded with mOVA on DC cells in draining lymph nodes and spleen in Example 6 of the present invention.
  • Figure 9 Flow chart of animal experiment in Example 7 of the present invention.
  • Figure 10A Antigen-specific Total IgG antibody titers in the serum of mice on days 14, 28, and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
  • Figure 11 The number of antigen-specific splenocytes secreting IFN- ⁇ in the spleen of mice on the 42nd day after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention (a) ELISpot spot number statistics; (b) optical photograph of the ELISpot well.
  • Figure 12 The number of antigen-specific CD4 + T cells secreting IFN- ⁇ in the spleen of mice on day 42 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
  • Figure 13 Concentrations of IL-2 and IFN- ⁇ secreted by antigen-specific spleen cells on day 42 after the first intramuscular injection of LNPs encapsulating mVZV in mice in Example 7 of the present invention.
  • Figure 14 Body weight monitoring curve of mice during immunization in Example 8 of the present invention.
  • FIG. 15 H&E staining of tissue and organ sections on day 42 after the first intramuscular injection of LNPs loaded with mVZV in mice in Example 8 of the present invention.
  • Figure 16 Flow chart of animal experiment in Example 9 of the present invention.
  • Figure 17 Total serum levels at 2, 4 and 6 weeks after the first intramuscular injection of LNPs loaded with mVZV in mice in Example 9 of the present invention Antibody titers of IgG, IgG1, IgG2a, and IgG2c.
  • Figure 18 Total IgG antibody titer in the serum of mice during 8 months after the first intramuscular injection of LNPs loaded with mVZV in Example 9 of the present invention.
  • Figure 19 The number of specific splenocytes secreting IFN- ⁇ in the spleen of mice on the 42nd day after the first intramuscular injection of LNPs loaded with mVZV in Example 9 of the present invention (a) optical photograph of ELISpot wells; (b) statistical graph of the number of ELISpot spots.
  • Figure 20 Body weight monitoring curve of mice during immunization process in Example 9 of the present invention.
  • Figure 21 Schematic diagram of the deglycosylation site in Example 10 of the present invention.
  • Figure 22 Total IgG, IgG1, IgG2a and IgG2c antibody titers in the serum of mice at 2, 4 and 6 weeks after the first intramuscular injection of LNPs loaded with mVZV in Example 10 of the present invention.
  • Figure 23 Flow chart of animal experiments in Example 11 of the present invention.
  • Figure 24 A diagram of the skin changes on the back of a guinea pig after infection with a poison in Example 11 of the present invention.
  • Figure 25 Guinea pig skin lesion scoring table in Example 11 of the present invention.
  • Figure 26 Skin lesion scores on the back of guinea pigs after poison challenge in Example 11 of the present invention.
  • Figure 27 H&E staining of the skin tissue of the guinea pig's back 7 days after infection in Example 11 of the present invention.
  • Figure 28 Skin viral load on the back of guinea pigs 7 days after infection in Example 11 of the present invention.
  • Figure 29 Body weight monitoring curve of guinea pigs during immunization in Example 11 of the present invention.
  • MC in the above figures represents the product of Comparative Example 1, i.e., a four-component LNP with MC3 as the ionizable lipid;
  • cKK-E12 represents the product of Comparative Example 2, i.e., a four-component LNP with cKK-E12 as the ionizable lipid;
  • L319 represents the product of Comparative Example 3, i.e., a four-component LNP with L319 as the ionizable lipid.
  • the composite ionizable lipid includes a first ionizable lipid 3,6-bis ⁇ 4-[bis(2-hydroxydodecyl)amino]butyl ⁇ piperazine-2,5-dione (abbreviated as cKK-E12), and a second ionizable lipid 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (abbreviated as L319);
  • the auxiliary phospholipid is 1,2-distearoyl-sn-glyceryl-3-phosphocholine, abbreviated as DSPC;
  • the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glyceryl-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000.
  • the structural formulas of the first ionizable lipid and the second ionizable lipid
  • Five-component lipid nanoparticles (referred to as five-component LNPs) were prepared as follows:
  • the preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, wherein the nucleic acid drug is mRNA encoding firefly luciferase (mFLuc), and the molar ratio of cKK-E12 and L319 is 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 or 8:2.
  • the nucleic acid drug is mRNA encoding firefly luciferase (mFLuc)
  • molar ratio of cKK-E12 and L319 is 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 or 8:2.
  • the preparation method of the five-component lipid nanoparticles of this example is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding chicken ovalbumin (mOVA).
  • the nucleic acid drug is mRNA encoding chicken ovalbumin (mOVA).
  • the preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding the herpes zoster virus gE protein (mVZV).
  • the nucleic acid drug is mRNA encoding the herpes zoster virus gE protein (mVZV).
  • Comparative Example 1 the composite ionizable lipid was replaced with MC3 (i.e., 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester), i.e., a four-component LNP with MC3 as the ionizable lipid;
  • MC3 i.e., 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester
  • Comparative Example 2 the composite ionizable lipid was replaced with cKK-E12 (i.e., the molar ratio of cKK-E12 to L319 was 1:0), i.e., a four-component LNP with cKK-E12 as the ionizable lipid;
  • the hydrated particle size and polydispersity index of the five-component LNP of Example 1A are 140 nm and 0.09, respectively, the hydrated particle size and polydispersity index of the five-component LNP of Example 1C are 110 nm and 0.1, respectively, and the hydrated particle size and polydispersity index of the five-component LNP of Example 1D are 130 nm and 0.1, respectively.
  • the hydrated particle sizes of the five-component lipid nanoparticles prepared in Examples 1A to 1D are all less than 200 nm, and the polydispersity coefficient is less than 0.2, indicating that the preparation method of the present invention can form stable lipid nanoparticles with uniform particle size.
  • the five-component LNPs loaded with mFLuc prepared in Example 1B were used, and the four-component LNPs of Comparative Examples 1 to 3 were selected as a control group to screen different ratios of cKK-E12 and L319 as ionizable lipids to act synergistically.
  • the experimental steps are as follows: DC2.4 cells were incubated overnight in a 96-well plate at 20,000 cells per well. When the cell confluence reached 50%, LNPs of Example 1B and Comparative Examples 1 to 3 were added respectively, and cultured in a saturated humidity incubator at 37°C and 5% CO2 concentration for 24 hours. An Untreated cells group (referring to cells without any treatment after plating) and a Free mRNA group (referring to mRNA without a carrier being added directly to the cell culture medium) were also set up. The culture medium was removed, cell lysate and firefly luciferase substrate were added, and after shaking for 10 minutes, the bioluminescence value was read using an ELISA reader. The test results are shown in Figure 4.
  • Example 1A The mFLuc-encapsulated five-component LNP prepared in Example 1A was used, and the four-component LNP of Comparative Examples 1 to 3 was selected as a control group to detect the membrane fusion ability of the five-component LNP.
  • mice erythrocytes are placed in a transparent 96-well plate, sodium citrate buffer solution of pH 5.5 and PBS buffer solution of pH 7.4 are added respectively, and then LNPs of Example 1 and Comparative Examples 1 to 3 are added correspondingly and incubated at 37°C for 1 hour.
  • a negative control group i.e., only buffer solution and erythrocytes are added to the well plate, recorded as Untreated cells
  • a positive control group i.e., buffer solution, erythrocytes and 0.5% Triton X-100 are added to the well plate, recorded as 0.5% Triton X-100 are incubated at 37°C for 1 hour.
  • Example 1A The mFLuc-encapsulated five-component LNP prepared in Example 1A was used, and the four-component LNP of Comparative Examples 1 to 3 were selected as a control group to detect the mRNA expression efficiency of the five-component LNP of the present invention in vivo.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected, and 2 ⁇ g of LNP of Example 1A and Comparative Examples 1 to 3 were administered to each mouse in each group by intramuscular injection. 200 ⁇ L of 200 mg/mL firefly luciferase substrate was injected intraperitoneally into each mouse 4 hours and 24 hours after administration, respectively. After 15 minutes, the bioluminescence intensity in the mice was detected using small animal in vivo imaging.
  • the five-component LNP of the present invention has a high mRNA expression efficiency in vivo.
  • Example 1C The mOVA-encapsulated five-component LNP prepared in Example 1C was used, wherein the four-component LNP of Comparative Example 1 was selected as a control group, and the DC cell activation efficiency of the five-component LNP of the present invention in C57BL/6 mice was detected.
  • mice 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection on day 0 and day 7, and each mouse in each group was administered 10 ⁇ g of LNP of Example 1C and Comparative Example 1, respectively. A PBS group was also set up in which only PBS buffer solution was injected; on day 8, the mice were killed, and the draining lymph nodes and spleen at the injection site were removed in vitro. For the experimental process, see Figure 7.
  • lymph node cells and spleen cells were processed separately, a single cell suspension was obtained, and the number of activated DC cells (CD11c + CD80 + , CD11c + CD86 + ) in the spleen and draining lymph nodes was detected by flow cytometry, see Figure 8.
  • the mRNA-loaded five-component LNP of the present invention has a high DC cell activation efficiency in C57BL/6 mice in the draining lymph nodes and spleen of mice.
  • Example 7 Verification of the immune efficacy of five-component LNP in C57BL/6 mice
  • the five-component LNPs encapsulating mVZV prepared in Example 1D were used, wherein the four-component LNPs of Comparative Example 1, the attenuated VZV virus vaccine (hereinafter referred to as the attenuated vaccine) and the herpes zoster recombinant gE protein subunit vaccine plus AS01 adjuvant (hereinafter referred to as the subunit vaccine) were selected as the control groups of this experiment to detect the immune efficacy of the five-component LNPs of the present invention in C57BL/6 mice.
  • the attenuated vaccine the attenuated VZV virus vaccine
  • AS01 adjuvant hereinafter referred to as the subunit vaccine
  • mice 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection at week 0 and week 3. Each mouse in each group was administered 10 ⁇ g of LNP, attenuated vaccine and subunit vaccine of Example 1D and Comparative Example 1, respectively. A PBS group was also set up to be injected with only PBS buffer solution. Blood was collected from the mice at week 2, 4 and 5, and serum was separated. The mice were killed at week 6. The experimental process is shown in Figure 9. The ELISA was used to measure the 14th, 28th and 35th day of the mice. The test results of the antibody titers of herpes zoster-specific IgG, IgG1, and IgG2c in serum are shown in FIG. 10A to FIG. 10C .
  • the number of herpes zoster-specific T cells (CD3 + CD4 + CD45 + IFN- ⁇ + ) was detected by flow cytometry, and the test results are shown in Figure 12.
  • Splenocytes were then placed in a 96-well plate with 1 million cells per well, stimulated with the herpes zoster gE peptide library for 48 hours, centrifuged to obtain the cell supernatant, and the amount of IL-2 and IFN- ⁇ secreted by spleen cells in the cell supernatant was measured by ELISA, and the test results are shown in Figure 13.
  • the mRNA-loaded five-component LNP of the present invention has the ability to efficiently activate specific cellular immunity and humoral immunity in C57BL/6 mice.
  • Example 8 Verification of the toxicity of five-component LNP in vivo
  • the five-component LNPs loaded with mVZV prepared in Example 1D were used, and the four-component LNPs, attenuated vaccine and subunit vaccine of Comparative Example 1 were selected as the control groups of this experiment to detect the vaccine efficacy of the five-component LNPs in vivo.
  • mice 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection at week 0 and week 3. Each mouse in each group was administered 10 ⁇ g of the five-component LNP in Example 1D and the four-component LNP, attenuated vaccine and subunit vaccine in Comparative Example 1, respectively.
  • a PBS group was set up in which only PBS buffer solution was injected. The weight of the mice was monitored during the immunization period. See Figure 14 for the experimental process. The mice were killed in week 6, and the heart, liver, spleen, lung, kidney, brain and injection site muscles of the mice were embedded in paraffin for H&E staining sections. See Figure 15 for the test results.
  • the five-component LNPs loaded with mVZV prepared in Example 1D were used, wherein the four-component LNPs of Comparative Example 1, the attenuated vaccine and the subunit vaccine were selected as the control groups of this experiment to detect the vaccine efficacy of the five-component LNPs in BALB/c mice.
  • mice 8-week-old female BALB/c mice were selected, with 3 mice in each experimental group, and the mice were immunized by intramuscular injection at week 0 and week 3, respectively.
  • Each mouse was administered 10 ⁇ g of the five-component LNP in Example 1D and the four-component LNP, attenuated vaccine and subunit vaccine in Comparative Example 1, respectively.
  • Orbital blood was drawn from the mice at weeks 2, 4 and 6, the serum was separated, and the mice were killed at week 6. See Figure 16 for the experimental process.
  • ELISA was used to measure the antibody titers of Total IgG, IgG1, IgG2a and IgG2c specific to herpes zoster in the serum of mice at 2, 4 and 6 weeks, see Figure 17.
  • ELISA was used to measure the changes in the titers of Total IgG specific to herpes zoster in the serum of mice within 8 months after immunization, see Figure 18.
  • the spleen of the mice was processed to obtain a single cell suspension, and 300,000 cells were placed in each well of the ELISpot plate and stimulated with the herpes zoster gE peptide library for 24 hours, and then the number of spleen cells that secreted IFN- ⁇ specifically for herpes zoster was detected by color development, see Figure 19 (a) ELISpot well optical photograph, (b) ELISpot spot number statistical chart. The weight of the mice was monitored during the immunization period, see Figure 20.
  • Example 10 Verification of the effect of five-component LNPs loaded with mVZV of different deglycosylation designs on humoral immunity
  • mVZV in Example 1D was used to perform different deglycosylation site designs, including: two nitrogen-glycosylation site mutations (abbreviated as mMUTE-N), six oxygen-glycosylation site mutations (abbreviated as mMUTE-O), and a combination of two glycosylation site mutations (abbreviated as mMUTE-N+O), see Figure 21.
  • mMUTE-N nitrogen-glycosylation site mutations
  • mMUTE-O oxygen-glycosylation site mutations
  • mMUTE-N+O a combination of two glycosylation site mutations
  • Example 9 Referring to the immune experiment process and experimental steps in Example 9, specifically including: selecting 8-week-old female BALB/c mice, 6 mice in each experimental group, immunizing the mice by intramuscular injection at week 0 and week 3, and administering 10 ⁇ g of the five-component LNP in Example 1D and the five-component LNP containing mMUTE-N, the five-component LNP containing mMUTE-O, and the five-component LNP containing mMUTE-N+O prepared above, respectively, which are recorded as VZV, mMUTE-N, mMUTE-O, and mMUTE-N+O, respectively, and collecting blood from the eye sockets of the mice at weeks 2, 4, and 6 to separate the serum.
  • Example 11 Verification of the five-component LNP in guinea pigs against VZV virus invasion
  • the five-component LNPs containing mVZV prepared in Example 1D were used, and subunit vaccines and PBS were selected as the control group of this experiment to detect the efficacy of the five-component LNPs in guinea pigs against the virus.
  • the experimental process is shown in Figure 23. The experimental steps are specifically as follows: guinea pigs weighing 200-250g were selected, and the guinea pigs were immunized by intramuscular injection on days 0 and 14. The guinea pigs were administered 10 ⁇ g of the five-component LNPs, subunit vaccines and PBS in Example 1D, respectively. The guinea pigs were scratched on the back on days 21 and 22 for the challenge experiment.
  • Example 1D The body weight of guinea pigs was monitored during the immunization period, see Figure 29. It was verified that the five-component LNP encapsulated with mVZV in Example 1D can effectively protect guinea pigs from VZV virus invasion.

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Abstract

Provided are a lipid nanoparticle for efficiently delivering a nucleic acid drug, a method for preparing same, and use thereof. The lipid nanoparticle comprises a vector and an encapsulated nucleic acid. The vector comprises a composite ionizable lipid, an auxiliary phospholipid, a cholesterol substance, and a pegylated lipid. The nucleic acid comprises one or more of mRNA, circular RNA, siRNA, microRNA, an antisense nucleic acid, and a plasmid. The composite ionizable lipid comprises an ionizable lipid with the capability of efficiently entering cells, and an ionizable lipid with a long alkyl chain and the capability of efficient membrane fusion. The composite ionizable lipid can significantly improve the mRNA expression efficiency of commercial four-component LNPs by means of synergism. The lipid nanoparticle facilitates the efficient expression of the nucleic acid drug entrapped therein after intramuscular administration, thus efficiently activating humoral and cell-mediated immune responses.

Description

一种用于高效递送核酸药物的脂质纳米颗粒的制备方法和应用Preparation method and application of lipid nanoparticles for efficient delivery of nucleic acid drugs
本申请要求享有2022年11月23日向中国国家知识产权局提交的，专利申请号为202211477861.X，发明名称为“一种用于高效递送核酸药物的脂质纳米颗粒的制备方法和应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。This application claims the priority benefit of a prior application filed with the State Intellectual Property Office of China on November 23, 2022, with patent application number 202211477861.X and invention name “A preparation method and application of lipid nanoparticles for efficient delivery of nucleic acid drugs”. The entire text of the prior application is incorporated into this application by reference.
技术领域Technical Field
本发明所属技术领域为医药技术领域，具体涉及一种可以高效递送核酸药物的脂质纳米颗粒(LNP)及其制备方法和应用。The technical field to which the present invention belongs is the field of medical technology, and specifically relates to a lipid nanoparticle (LNP) capable of efficiently delivering nucleic acid drugs, and a preparation method and application thereof.
背景技术Background technique
新冠疫情爆发后，美国食品药品管理局(FDA)迅速批准了Moderna和Pfizer两家公司的信使RNA(mRNA)新冠疫苗，获批的两款mRNA疫苗均采用脂质纳米颗粒作为递送载体。在短短两年内，全球已经接种超过30亿剂新冠mRNA疫苗。与传统的蛋白质、病毒、脱氧核糖核酸(DNA)疫苗相比，mRNA疫苗具有安全性高、研发周期短、生产效率高、可编码多种蛋白和无需佐剂等优势。然而，由于mRNA分子量大、带负电导致其自身难以进入细胞内，且mRNA自身稳定性差，在体内易被核酸酶降解。因此，需要合适的递送载体协助mRNA进入靶细胞才能发挥作用。After the outbreak of the COVID-19 pandemic, the U.S. Food and Drug Administration (FDA) quickly approved messenger RNA (mRNA) COVID-19 vaccines from Moderna and Pfizer. Both approved mRNA vaccines use lipid nanoparticles as delivery vehicles. In just two years, more than 3 billion doses of COVID-19 mRNA vaccines have been administered worldwide. Compared with traditional protein, virus, and deoxyribonucleic acid (DNA) vaccines, mRNA vaccines have the advantages of high safety, short R&D cycle, high production efficiency, ability to encode multiple proteins, and no need for adjuvants. However, due to its large molecular weight and negative charge, mRNA itself is difficult to enter cells, and mRNA itself has poor stability and is easily degraded by nucleases in the body. Therefore, a suitable delivery vehicle is needed to assist mRNA in entering target cells to work.
脂质纳米颗粒(lipid nanoparticle，LNP)是目前唯一临床获批的mRNA疫苗载体，由可离子化脂质，辅助磷脂，胆固醇和聚乙二醇化脂质四部分组成。LNP通过肌肉注射后可以有效递送mRNA到细胞中，激活细胞免疫和体液免疫应答，产生有效的免疫保护。LNP的递送效率是影响mRNA疫苗效力的关键，而LNP进入细胞后的溶酶体逃逸效率低是限制LNP递送效率的关键。因此，亟需发展可以增加LNP溶酶体逃逸效率、提高LNP递送效率的新型载体。Lipid nanoparticles (LNP) are currently the only clinically approved mRNA vaccine carriers, which are composed of ionizable lipids, auxiliary phospholipids, cholesterol and pegylated lipids. LNP can effectively deliver mRNA into cells after intramuscular injection, activate cellular immunity and humoral immune responses, and produce effective immune protection. The delivery efficiency of LNP is the key to the efficacy of mRNA vaccines, and the low lysosomal escape efficiency of LNP after entering the cell is the key to limiting the delivery efficiency of LNP. Therefore, it is urgent to develop new carriers that can increase the lysosomal escape efficiency of LNP and improve the delivery efficiency of LNP.
发明内容Summary of the invention
本发明提供一种脂质纳米颗粒(以下简写为LNP)，包括载体和被包封的核酸，所述载体包括复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质；所述核酸包括但不限定于mRNA、环状RNA、siRNA、microRNA、反义核酸和质粒中的一种或多种。The present invention provides a lipid nanoparticle (hereinafter referred to as LNP), comprising a carrier and an encapsulated nucleic acid, wherein the carrier comprises a composite ionizable lipid, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid; the nucleic acid comprises but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and a plasmid.
根据本发明的实施方案，所述复合可离子化脂质占所述LNP中总脂质的20mol％～70mol％，例如30mol％、40mol％、50mol％、60mol％。According to an embodiment of the present invention, the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP, such as 30 mol%, 40 mol%, 50 mol%, 60 mol%.
根据本发明的实施方案，所述复合可离子化脂质包括第一可离子化脂质和第二可离子化脂质。According to an embodiment of the present invention, the complex ionizable lipid comprises a first ionizable lipid and a second ionizable lipid.
根据本发明的实施方案，第一可离子化脂质和第二可离子化脂质的摩尔比为1:99～99:1，优选为2:8～8:2，例如3:7、4:6、5:5、6:4、7:3，更优选为6:4。According to an embodiment of the present invention, the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2, such as 3:7, 4:6, 5:5, 6:4, 7:3, and more preferably 6:4.
根据本发明的实施方案，所述第一可离子化脂质和第二可离子化脂质不同，彼此独立的选自8-[(2-羟乙基)(6-氧代-6-癸氧基己基)氨基]辛酸(十七烷-9-基)酯(SM-102)、[(4-羟基丁基)氮杂二基]双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)、4(N,N二甲基氨基)丁酸(二亚油基)甲酯(DLin MC3DMA)、3,6-双{4-[双(2-羟基十二烷基)氨基]丁基}哌嗪-2,5-二酮(cKK-E12)、9-(4-(二甲氨基)丁酰氧基)十七烷二酸二((Z)-壬-2-烯-1-基)酯(L319)、N2,2-二亚油基-4-二甲氨基乙基-[1,3]-二氧戊环(DLin-KC2-DMA)、8-[(2-羟乙基)(8-壬氧基-8-氧代辛基)氨基]辛酸(十七烷-9-基)酯(Lipid5)、1,1'-[(2-{4-[2-({2-[双(2-羟基十二烷基)氨基]乙基}(2-羟基十二烷基)氨基)乙基]哌嗪-1-基}乙基)氮杂二烷基]双(十二烷-2-醇)(C12-200)、(2,3二油酰基丙基)三甲基氯化铵(DOTAP)、双甲基双十八烷基溴化铵(DDAB)、四(8-甲基壬基)3',3',3”,3”-{[(甲基 氮杂二烷基)双(丙烷-3,1二基)]双(氮杂三基)}四丙酸酯(306Oi10)中的一种或多种；优选为8[(2羟乙基)(6氧代6癸氧基己基)氨基]辛酸(十七烷9基)酯(SM-102)或[(4羟基丁基)氮杂二基]双(己烷6,1二基)双(2己基癸酸酯)(ALC-0315)。According to an embodiment of the present invention, the first ionizable lipid and the second ionizable lipid are different and are independently selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102), [(4-hydroxybutyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin MC3DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecanedioic acid 9-yl) ester (Lipid5), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azadialkyl]bis(dodecane-2-ol)(C12-200), (2,3 dioleoylpropyl)trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium bromide (DDAB), tetra(8-methylnonyl) 3',3',3",3"-{[(methyl One or more of [(4-hydroxybutyl) azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); preferably 8[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecanyl) ester (SM-102) or [(4-hydroxybutyl) azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).
在一些实施方案中，所述复合可离子化脂质包括3,6-双{4-[双(2-羟基十二烷基)氨基]丁基}哌嗪-2,5-二酮(cKK-E12)和9-(4-(二甲氨基)丁酰氧基)十七烷二酸二((Z)-壬-2-烯-1-基)酯(L319)，其摩尔比为1:99～99:1，优选为2:8～8:2，例如3:7、4:6、5:5、6:4、7:3。In some embodiments, the complex ionizable lipid includes 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12) and 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), and the molar ratio thereof is 1:99 to 99:1, preferably 2:8 to 8:2, for example 3:7, 4:6, 5:5, 6:4, 7:3.
根据本发明的实施方案，所述辅助磷脂占所述LNP中总脂质的2mol％～20mol％，例如4mol％、8mol％、10mol％、12mol％、4mol％、16mol％、18mol％。According to an embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.
根据本发明的实施方案，所述辅助磷脂包括但不限于1,2-二硬脂酰基-sn-甘油基-3-磷脂酰胆碱(DSPC)、1,2-二油酰基-sn-甘油基-3-磷脂酰胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油基-3-磷脂酰胆碱(DPPC)、2-油酰基-1-棕榈酰基-sn-甘油基-3-磷脂酰胆碱(POPC)、1,2-二油酰基-sn-甘油基-3-磷脂酰乙醇胺(DOPE)、2-油酰基-1-棕榈酰基-sn-甘油基-3-磷脂酰乙醇胺(POPE)、1,2-二硬脂酰基-sn-甘油基-3-磷脂酰乙醇胺(DSPE)、1,2-二棕榈酰基-sn-甘油基-3-磷脂酰乙醇胺(DPPE)中的一种或多种，优选为DSPC。According to an embodiment of the present invention, the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably DSPC.
根据本发明的实施方案，所述胆固醇类物质占所述LNP中总脂质的10mol％～60mol％，例如20mol％、30mol％、40mol％、50mol％。According to an embodiment of the present invention, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.
根据本发明的实施方案，所述胆固醇类物质选自胆固醇及其衍生物。According to an embodiment of the present invention, the cholesterol-like substance is selected from cholesterol and its derivatives.
根据本发明的实施方案，所述胆固醇及其衍生物包括但不限于胆固醇、β-谷甾醇、胆甾烷醇、胆甾烷酮、胆甾烯酮、7β-羟基胆固醇、7α-羟基胆固醇中的一种或多种，优选为胆固醇。According to an embodiment of the present invention, the cholesterol and its derivatives include but are not limited to one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, preferably cholesterol.
根据本发明的实施方案，所述聚乙二醇化脂质占所述LNP中总脂质的0.3mol％～30mol％，优选为0.5mol％～2.5mol％，例如1.0mol％、1.5mol％、2.0mol％。According to an embodiment of the present invention, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.
根据本发明的实施方案，所述聚乙二醇化脂质包括但不限于1,2-二肉豆蔻酰基-rac-甘油基-3-甲氧基聚乙二醇(DMG-PEG)、1,2-二硬脂酰基-rac-甘油基-3-甲氧基聚乙二醇(DSG-PEG)、1,2-二棕榈酰基-rac-甘油基-3-甲氧基聚乙二醇(DPG-PEG)、1,2-二硬脂酰基-sn-甘油基-3-磷脂酰乙醇胺-甲氧基聚乙二醇(DSPE-PEG)中的一种或多种，优选为DMG-PEG。According to an embodiment of the present invention, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.
本发明中，上述“总脂质”指的是复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质的总和。In the present invention, the above-mentioned "total lipids" refers to the sum of complex ionizable lipids, auxiliary phospholipids, cholesterol substances and PEGylated lipids.
根据本发明的实施方案，本发明选用的核酸例如包括CpG-FAM、mFLuc(编码萤火虫荧光素酶的mRNA)、mOVA(编码鸡卵清蛋白的mRNA)、mVZV(编码带状疱疹gE蛋白的mRNA)中的至少一种。According to an embodiment of the present invention, the nucleic acid selected by the present invention includes at least one of CpG-FAM, mFLuc (mRNA encoding firefly luciferase), mOVA (mRNA encoding chicken ovalbumin), and mVZV (mRNA encoding herpes zoster gE protein).
根据本发明的实施方案，所述核酸的包封率为30～99％，优选为70％-90％，比如70％、75％、80％、85％、90％。According to an embodiment of the present invention, the encapsulation rate of the nucleic acid is 30% to 99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.
根据本发明的实施方案，所述复合可离子化脂质含有的氮元素与核酸含有的磷元素的摩尔比例为(1～50):1；优选为(5～10):1，示例性为5.67:1。According to an embodiment of the present invention, the molar ratio of the nitrogen element contained in the complex ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.
根据本发明的实施方案，所述脂质纳米颗粒的水合粒径小于200nm，例如80～180nm。According to an embodiment of the present invention, the hydrated particle size of the lipid nanoparticles is less than 200 nm, such as 80 to 180 nm.
根据本发明的实施方案，所述脂质纳米颗粒的多分散系数小于0.2，例如为0.119、0.102、0.093。According to an embodiment of the present invention, the polydispersity coefficient of the lipid nanoparticles is less than 0.2, for example, 0.119, 0.102, or 0.093.
在一些实施方案中，所述复合可离子化脂质:辅助磷脂:胆固醇类物质:聚乙二醇脂质的摩尔比为50:10:38.5:1.5。In some embodiments, the molar ratio of the complex ionizable lipid: helper phospholipid: cholesterol-like substance: polyethylene glycol lipid is 50:10:38.5:1.5.
在一些实施方案中，所述复合可离子化脂质包括第一可离子化脂质和第二可离子化脂质，两者比 例为8:2、7:3、6:4、5:5、4:6、3:7或2:8。In some embodiments, the composite ionizable lipid comprises a first ionizable lipid and a second ionizable lipid, the two being Examples are 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, or 2:8.
所述的核酸为CpG-FAM、mFLuc、mOVA或mVZV。The nucleic acid is CpG-FAM, mFLuc, mOVA or mVZV.
本发明还提供上述脂质纳米颗粒的制备方法，所述制备方法包括将含有核酸的水相与脂质有机相通过共混法或者微流控法制备得到所述脂质纳米颗粒。The present invention also provides a method for preparing the lipid nanoparticles, which comprises preparing the lipid nanoparticles by blending an aqueous phase containing nucleic acids and a lipid organic phase through a blending method or a microfluidics method.
根据本发明的实施方案，所述脂质有机相的制备方法如下：将复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质按比例溶解在有机溶剂中得到有机相。优选地，所述有机溶剂选用可使上述物质溶解的有机溶剂，例如为无水乙醇。According to an embodiment of the present invention, the lipid organic phase is prepared as follows: the composite ionizable lipid, auxiliary phospholipid, cholesterol-like substance and pegylated lipid are dissolved in an organic solvent in proportion to obtain an organic phase. Preferably, the organic solvent is an organic solvent that can dissolve the above substances, such as anhydrous ethanol.
根据本发明的实施方案，所述含有核酸的水相的制备方法如下：将所述核酸溶解在缓冲溶液中得到水相。优选地，所述缓冲溶液可选用pH为4～6的缓冲溶液，例如选用pH为4或5.5的柠檬酸钠缓冲溶液。According to an embodiment of the present invention, the preparation method of the aqueous phase containing nucleic acid is as follows: dissolving the nucleic acid in a buffer solution to obtain an aqueous phase. Preferably, the buffer solution can be a buffer solution with a pH of 4 to 6, for example, a sodium citrate buffer solution with a pH of 4 or 5.5.
根据本发明的实施方案，所述共混法包括如下步骤：使用移液枪吸取含有核酸的水相，快速加入至所述脂质有机相中，然后迅速吹打混合液体大于30次，吹匀后室温静置10分钟，静置后对所得产物进行透析，比如在大于1000倍体积的PBS缓冲溶液中透析6h以上，得到所述脂质纳米颗粒。According to an embodiment of the present invention, the blending method includes the following steps: using a pipette to absorb the aqueous phase containing nucleic acids, quickly adding it to the lipid organic phase, and then quickly blowing the mixed liquid for more than 30 times, blowing it evenly and letting it stand at room temperature for 10 minutes, and dialyzing the resulting product after standing, for example, dialyzing it in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.
根据本发明的实施方案，所述微流控法包括如下步骤：将装有水相和脂质有机相的注射器分别固定在注射泵上，以水相：脂质有机相体积比3:1注入微流控芯片中充分混合，混合完毕后收集混合液室温静置10分钟，静置后使用PBS缓冲溶液对所得产物进行透析，比如在大于1000倍体积的PBS缓冲溶液中透析6h以上，得到所述脂质纳米颗粒。According to an embodiment of the present invention, the microfluidic method includes the following steps: fixing syringes containing an aqueous phase and a lipid organic phase on a syringe pump respectively, injecting the aqueous phase: lipid organic phase into a microfluidic chip at a volume ratio of 3:1 and mixing thoroughly, collecting the mixed solution after mixing and standing at room temperature for 10 minutes, and dialyzing the resulting product with a PBS buffer solution after standing, for example, dialyzing in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.
本发明还提供上述脂质纳米颗粒在制备药物制剂或生物制剂中的应用。The present invention also provides the use of the lipid nanoparticles in the preparation of pharmaceutical preparations or biological preparations.
根据本发明的实施方案，所述生物制剂为可注射的生物制剂。According to an embodiment of the present invention, the biologic is an injectable biologic.
在一些实施方案中，所述生物制剂为疫苗，优选为mRNA疫苗。In some embodiments, the biologic is a vaccine, preferably an mRNA vaccine.
根据本发明的实施方案，所述生物制剂的给药方式为肌肉注射给药。According to an embodiment of the present invention, the administration method of the biological preparation is intramuscular injection.
根据本发明的实施方案，所述生物制剂用于预防带状疱疹。According to an embodiment of the present invention, the biological agent is used to prevent herpes zoster.
本发明还提供一种药物制剂或生物制剂，其具有如上文所述的含义。The present invention also provides a pharmaceutical preparation or a biological preparation having the meaning as described above.
本发明还提供一种预防和/或治疗疾病的方法，包括将预防和/或治疗有效量的上述药物制剂或生物制品施用于受试者；例如所述疾病为带状疱疹。The present invention also provides a method for preventing and/or treating a disease, comprising administering a preventively and/or therapeutically effective amount of the above-mentioned pharmaceutical preparation or biological product to a subject; for example, the disease is herpes zoster.
术语定义与解释Definition and Explanation of Terms
术语“多种”代表两种或者三种以上。The term "plurality" means two or more than three.
术语“有效量”可以根据本领域具有临床执业资格的医生所掌握的方法确定足以实现预期应用(包括但不限于上述定义的疾病治疗)的本发明所述药物制剂或生物制品的量。确定预防和/或治疗有效剂量是本领域临床医生或研究人员力所能及的，可以因以下因素而改变：预期应用(体外或者体内)，或者所治疗的受试者和疾病病症如受试者的体重和年龄、一般健康状况、疾病病症的严重性、给药方式以及其他影响疗效的因素例如药物过敏史等。具体施用剂量将取决于以下因素而改变：所选择的特定生物制品、所依据的给药方案、是否与其它药物联合给药、给药的时间安排、所给药的组织和所承载的物理递送系统。The term "effective amount" can be determined according to the method mastered by a physician with clinical practice qualifications in the field to determine the amount of the pharmaceutical preparation or biological product of the present invention that is sufficient to achieve the intended application (including but not limited to the treatment of diseases defined above). Determining the effective dose for prevention and/or treatment is within the capabilities of clinicians or researchers in the field and may be changed due to the following factors: the intended application (in vitro or in vivo), or the subject and disease condition to be treated, such as the subject's weight and age, general health, severity of the disease condition, mode of administration, and other factors that affect the efficacy, such as a history of drug allergies. The specific dosage will vary depending on the following factors: the specific biological product selected, the dosage regimen based on, whether it is co-administered with other drugs, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.
本发明的“受试者”指具体的人或其他温血哺乳动物。本发明作为“受试者”的人包括成人和婴幼儿、儿童，其他温血哺乳动物包括但不限于非人类的灵长类动物、例如黑猩猩、其他类人猿或猴，以及其他动物园动物、家养哺乳动物或实验室动物，例如猫、猪、狗、牛、羊、小鼠、大鼠和豚鼠等。The "subject" of the present invention refers to a specific human or other warm-blooded mammal. The human as "subject" of the present invention includes adults, infants, and children, and other warm-blooded mammals include but are not limited to non-human primates, such as chimpanzees, other apes or monkeys, and other zoo animals, domestic mammals or laboratory animals, such as cats, pigs, dogs, cows, sheep, mice, rats, and guinea pigs.
本发明的有益效果如下：The beneficial effects of the present invention are as follows:
本发明制备的LNP，粒径均一，分散性好，包封率高，生物相容性好，毒副作用低，并且可以高 效递送核酸药物。本发明的LNP的制备方法操作步骤简单，易大量生产。The LNP prepared by the present invention has uniform particle size, good dispersibility, high encapsulation efficiency, good biocompatibility, low toxicity and side effects, and can be highly The LNP preparation method of the present invention has simple operation steps and is easy to mass produce.
本发明将第一可离子化脂质和第二可离子化脂质结合形成复合可离子化脂质，其具有高效进入细胞能力的可离子化脂质和带有长烷基链、具有高效膜融合能力的可离子化脂质。本发明的复合可离子化脂质通过协同作用可以显著提高商品化四组分LNP的mRNA表达效率。The present invention combines a first ionizable lipid and a second ionizable lipid to form a composite ionizable lipid, which has an ionizable lipid with high efficiency in entering cells and an ionizable lipid with a long alkyl chain and high efficiency in membrane fusion ability. The composite ionizable lipid of the present invention can significantly improve the mRNA expression efficiency of the commercial four-component LNP through synergistic effect.
本发明使用复合可离子化脂质制备得到五组分LNP，经肌肉注射给药后，对引流淋巴结和脾脏中的抗原呈递细胞的激活效率优于商品化四组分LNP。The present invention uses composite ionizable lipids to prepare five-component LNPs, which have a higher activation efficiency on antigen-presenting cells in draining lymph nodes and spleen than commercial four-component LNPs after intramuscular injection.
本发明使用复合可离子化脂质制备的五组分LNP可以高效递送带状疱疹mRNA疫苗，经肌肉注射给药后，显著激活生物体内的细胞免疫和体液免疫应答，在血液中产生大量抗原特异性结合抗体，提高脾脏中抗原特异性的T细胞含量，起到优异的保护效果，有望在带状疱疹mRNA疫苗领域实现突破，有重大的应用前景。The five-component LNP prepared by the composite ionizable lipids of the present invention can efficiently deliver the herpes zoster mRNA vaccine. After intramuscular injection, it significantly activates the cellular immunity and humoral immune response in the body, produces a large number of antigen-specific binding antibodies in the blood, and increases the antigen-specific T cell content in the spleen, which has an excellent protective effect. It is expected to achieve a breakthrough in the field of herpes zoster mRNA vaccines and has great application prospects.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：本发明实施例中所用的第一可离子化脂质和第二可离子化脂质的化学结构。FIG. 1 : Chemical structures of the first ionizable lipid and the second ionizable lipid used in the examples of the present invention.
图2：本发明实施例1B中所得LNP的水合粒径分布曲线。FIG. 2 : Hydration particle size distribution curve of LNP obtained in Example 1B of the present invention.
图3：本发明实施例1A中所得五组分LNP的冷冻透射电镜图。FIG3 : Cryo-TEM image of the five-component LNP obtained in Example 1A of the present invention.
图4：本发明实施例3中使用DC2.4细胞转染包载mFluc的LNP后的生物发光强度。FIG. 4 shows the bioluminescence intensity of DC2.4 cells transfected with LNPs loaded with mFluc in Example 3 of the present invention.
图5：本发明实施例4中使用小鼠红细胞检测五组分LNP的膜融合能力。FIG5 : The membrane fusion ability of the five-component LNP was detected using mouse red blood cells in Example 4 of the present invention.
图6：本发明实施例5中使用小动物活体成像验证注射包载mFLuc的五组分LNP在小鼠注射部位的生物发光强度；(a)肌肉注射4小时后活体成像图及注射部位生物发光强度统计值结果；(b)肌肉注射24小时后活体成像图及注射部位生物发光强度统计值结果。Figure 6: In Example 5 of the present invention, in vivo imaging of small animals was used to verify the bioluminescence intensity of the five-component LNP loaded with mFLuc at the injection site of mice; (a) In vivo imaging image and statistical results of bioluminescence intensity at the injection site 4 hours after intramuscular injection; (b) In vivo imaging image and statistical results of bioluminescence intensity at the injection site 24 hours after intramuscular injection.
图7：本发明实施例6中动物实验流程图。Figure 7: Flow chart of animal experiment in Example 6 of the present invention.
图8：本发明实施例6中包载mOVA的不同LNP对引流淋巴结和脾脏中DC细胞的激活效率。FIG8 : Activation efficiency of different LNPs loaded with mOVA on DC cells in draining lymph nodes and spleen in Example 6 of the present invention.
图9：本发明实施例7中动物实验流程图。Figure 9: Flow chart of animal experiment in Example 7 of the present invention.
图10A：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第14、28和35天的血清中抗原特异性Total IgG的抗体滴度。Figure 10A: Antigen-specific Total IgG antibody titers in the serum of mice on days 14, 28, and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
图10B：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第14、28和35天的血清中抗原特异性IgG1的抗体滴度。10B : Antigen-specific IgG1 antibody titers in the serum of mice on days 14, 28 and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
图10C：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第14、28和35天的血清中抗原特异性IgG2c的抗体滴度。10C : Antigen-specific IgG2c antibody titers in the serum of mice on days 14, 28 and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
图11：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第42天脾脏中分泌IFN-γ的抗原特异性脾细胞数量(a)ELISpot斑点数量统计图；(b)ELISpot孔的光学照片。Figure 11: The number of antigen-specific splenocytes secreting IFN-γ in the spleen of mice on the 42nd day after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention (a) ELISpot spot number statistics; (b) optical photograph of the ELISpot well.
图12：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第42天脾脏中分泌IFN-γ的抗原特异性CD4+的T细胞数量。Figure 12: The number of antigen-specific CD4 + T cells secreting IFN-γ in the spleen of mice on day 42 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.
图13：本发明实施例7中小鼠第一次肌肉注射包载mVZV的LNP后，第42天抗原特异性的脾脏细胞分泌IL-2和IFN-γ的浓度。Figure 13: Concentrations of IL-2 and IFN-γ secreted by antigen-specific spleen cells on day 42 after the first intramuscular injection of LNPs encapsulating mVZV in mice in Example 7 of the present invention.
图14：本发明实施例8中小鼠免疫过程中体重监测曲线。Figure 14: Body weight monitoring curve of mice during immunization in Example 8 of the present invention.
图15：本发明实施例8中小鼠第一次肌肉注射包载mVZV的LNP后，第42天组织器官切片H&E染色图。FIG. 15 : H&E staining of tissue and organ sections on day 42 after the first intramuscular injection of LNPs loaded with mVZV in mice in Example 8 of the present invention.
图16：本发明实施例9中动物实验流程图。Figure 16: Flow chart of animal experiment in Example 9 of the present invention.
图17：本发明实施例9中小鼠第一次肌肉注射包载mVZV的LNP后，第2、4和6周的血清中Total  IgG、IgG1、IgG2a和IgG2c的抗体滴度。Figure 17: Total serum levels at 2, 4 and 6 weeks after the first intramuscular injection of LNPs loaded with mVZV in mice in Example 9 of the present invention Antibody titers of IgG, IgG1, IgG2a, and IgG2c.
图18：本发明实施例9中小鼠第一次肌肉注射包载mVZV的LNP后，8个月期间的血清中Total IgG的抗体滴度。Figure 18: Total IgG antibody titer in the serum of mice during 8 months after the first intramuscular injection of LNPs loaded with mVZV in Example 9 of the present invention.
图19：本发明实施例9中小鼠第一次肌肉注射包载mVZV的LNP后，第42天脾脏中分泌IFN-γ的特异性脾细胞数量(a)ELISpot孔的光学照片图；(b)ELISpot斑点数量统计图。Figure 19: The number of specific splenocytes secreting IFN-γ in the spleen of mice on the 42nd day after the first intramuscular injection of LNPs loaded with mVZV in Example 9 of the present invention (a) optical photograph of ELISpot wells; (b) statistical graph of the number of ELISpot spots.
图20：本发明实例9中小鼠免疫过程中体重监测曲线。Figure 20: Body weight monitoring curve of mice during immunization process in Example 9 of the present invention.
图21：本发明实施例10中，去糖基化位点示意图。Figure 21: Schematic diagram of the deglycosylation site in Example 10 of the present invention.
图22：本发明实施例10中小鼠第一次肌肉注射包载mVZV的LNP后，第2、4和6周的血清中Total IgG、IgG1、IgG2a和IgG2c的抗体滴度。Figure 22: Total IgG, IgG1, IgG2a and IgG2c antibody titers in the serum of mice at 2, 4 and 6 weeks after the first intramuscular injection of LNPs loaded with mVZV in Example 10 of the present invention.
图23：本发明实施例11中动物实验流程图。Figure 23: Flow chart of animal experiments in Example 11 of the present invention.
图24：本发明实施例11中豚鼠背部攻毒后皮肤变化图。Figure 24: A diagram of the skin changes on the back of a guinea pig after infection with a poison in Example 11 of the present invention.
图25：本发明实施例11中豚鼠皮肤病变评分表。Figure 25: Guinea pig skin lesion scoring table in Example 11 of the present invention.
图26：本发明实施例11中豚鼠背部攻毒后皮肤病变评分。Figure 26: Skin lesion scores on the back of guinea pigs after poison challenge in Example 11 of the present invention.
图27：本发明实施例11中豚鼠背部攻毒7天后皮肤组织H&E染色图。Figure 27: H&E staining of the skin tissue of the guinea pig's back 7 days after infection in Example 11 of the present invention.
图28：本发明实施例11中豚鼠背部攻毒7天后皮肤病毒载量。Figure 28: Skin viral load on the back of guinea pigs 7 days after infection in Example 11 of the present invention.
图29：本发明实例11中豚鼠免疫过程中体重监测曲线。Figure 29: Body weight monitoring curve of guinea pigs during immunization in Example 11 of the present invention.
以上附图中的MC代表对比例1产品，即以MC3作为可离子化脂质的四组分LNP；cKK-E12代表对比例2产品，即以cKK-E12作为可离子化脂质的四组分LNP；L319代表对比例3产品，即以L319作为可离子化脂质的四组分LNP。MC in the above figures represents the product of Comparative Example 1, i.e., a four-component LNP with MC3 as the ionizable lipid; cKK-E12 represents the product of Comparative Example 2, i.e., a four-component LNP with cKK-E12 as the ionizable lipid; L319 represents the product of Comparative Example 3, i.e., a four-component LNP with L319 as the ionizable lipid.
具体实施方式Detailed ways
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
下述实施例所制备的LNP中，复合可离子化脂质中的氮元素与包载的核酸药物中的磷元素的摩尔比为5.67:1，水相与有机相的体积比为3:1；其中，水相是指将核酸药物溶于pH＝4的50mM柠檬酸钠缓冲溶液得到的，其中，核酸药物的浓度为0.17mg/mL；有机相是指复合可离子化脂质：辅助磷脂：胆固醇类物质：聚乙二醇化脂质以摩尔比为50:38.5:10:1.5溶于无水乙醇中得到的，其中，第一可离子化脂质和第二可离子化脂质的摩尔比为1:99～99:1，优选为2:8～8:2，例如为2:8、3:7、4:6、5:5、6:4、7:3、8:2，更优选为6:4。In the LNP prepared in the following example, the molar ratio of nitrogen element in the composite ionizable lipid to phosphorus element in the encapsulated nucleic acid drug is 5.67:1, and the volume ratio of the aqueous phase to the organic phase is 3:1; wherein the aqueous phase refers to the nucleic acid drug dissolved in a 50mM sodium citrate buffer solution at pH = 4, wherein the concentration of the nucleic acid drug is 0.17mg/mL; the organic phase refers to the composite ionizable lipid: auxiliary phospholipid: cholesterol substance: PEGylated lipid dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5, wherein the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2, for example, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and more preferably 6:4.
所述复合可离子化脂质包括第一可离子化脂质3,6-双{4-[双(2-羟基十二烷基)氨基]丁基}哌嗪-2,5-二酮(缩写为cKK-E12)，和第二可离子化脂质9-(4-(二甲氨基)丁酰氧基)十七烷二酸二((Z)-壬-2-烯-1-基)酯(缩写为L319)；辅助磷脂为1,2-二硬脂酰基-sn-甘油基-3-磷酸胆碱，缩写为DSPC；聚乙二醇脂质为1,2-二肉豆蔻酰基-sn-甘油基-3-甲氧基聚乙二醇2000，缩写为DMG-PEG2000。第一可离子化脂质和第二可离子化脂质的结构式如图1所示。The composite ionizable lipid includes a first ionizable lipid 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (abbreviated as cKK-E12), and a second ionizable lipid 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (abbreviated as L319); the auxiliary phospholipid is 1,2-distearoyl-sn-glyceryl-3-phosphocholine, abbreviated as DSPC; and the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glyceryl-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000. The structural formulas of the first ionizable lipid and the second ionizable lipid are shown in Figure 1.
实施例1AExample 1A
制备五组分的脂质纳米颗粒(记为五组分LNP)，具体如下：Five-component lipid nanoparticles (referred to as five-component LNPs) were prepared as follows:
(1)将带有FAM荧光修饰的CPG DNA(FAM-CPG)的核酸药物按上述浓度溶于柠檬酸钠缓冲溶液中构成水相； (1) dissolving a nucleic acid drug with FAM fluorescently modified CPG DNA (FAM-CPG) in a sodium citrate buffer solution at the above concentration to form an aqueous phase;
(2)再将复合可离子化脂质，辅助磷脂，胆固醇和聚乙二醇化脂质按上述比例溶于无水乙醇中构成有机相，其中，cKK-E12和L319的摩尔比为6:4；通过共混法或微流控法将水相和有机相混合，混合完毕后使用PBS缓冲溶液对所得产物进行透析，制备得到脂质纳米颗粒。(2) The composite ionizable lipid, auxiliary phospholipid, cholesterol and pegylated lipid are dissolved in anhydrous ethanol in the above proportions to form an organic phase, wherein the molar ratio of cKK-E12 to L319 is 6:4; the aqueous phase and the organic phase are mixed by a blending method or a microfluidics method, and after the mixing is completed, the obtained product is dialyzed using a PBS buffer solution to prepare lipid nanoparticles.
实施例1BExample 1B
本实施例的五组分的脂质纳米颗粒的制备方法基本同实施例1A，其中核酸药物为编码萤火虫荧光素酶的mRNA(mFLuc)，cKK-E12和L319的摩尔比为2:8、3:7、4:6、5:5、6:4、7:3或8:2。The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, wherein the nucleic acid drug is mRNA encoding firefly luciferase (mFLuc), and the molar ratio of cKK-E12 and L319 is 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 or 8:2.
实施例1CExample 1C
本实施例的五组分的脂质纳米颗粒的制备方法基本同实施例1A，不同在于，核酸药物为编码鸡卵清蛋白的mRNA(mOVA)。The preparation method of the five-component lipid nanoparticles of this example is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding chicken ovalbumin (mOVA).
实施例1DExample 1D
本实施例的五组分的脂质纳米颗粒的制备方法基本同实施例1A，不同在于，核酸药物为编码带状疱疹病毒gE蛋白的mRNA(mVZV)。The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding the herpes zoster virus gE protein (mVZV).
对比例1～3Comparative Examples 1 to 3
对比例1～3的脂质纳米颗粒的制备方法基本同实施例1B，不同在于，The preparation methods of the lipid nanoparticles of Comparative Examples 1 to 3 are basically the same as those of Example 1B, except that:
对比例1中，将复合可离子化脂质替换为MC3(即4(N,N二甲基氨基)丁酸(二亚油基)甲酯)，即为以MC3作为可离子化脂质的四组分LNP；In Comparative Example 1, the composite ionizable lipid was replaced with MC3 (i.e., 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester), i.e., a four-component LNP with MC3 as the ionizable lipid;
对比例2中，将复合可离子化脂质替换为cKK-E12(即cKK-E12和L319的摩尔比为1:0)，即为以cKK-E12作为可离子化脂质的四组分LNP；In Comparative Example 2, the composite ionizable lipid was replaced with cKK-E12 (i.e., the molar ratio of cKK-E12 to L319 was 1:0), i.e., a four-component LNP with cKK-E12 as the ionizable lipid;
对比例3中，将复合可离子化脂质替换为L319(即cKK-E12和L319的摩尔比为0:1)，即为以L319作为可离子化脂质的四组分LNP。In Comparative Example 3, the composite ionizable lipid was replaced with L319 (ie, the molar ratio of cKK-E12 to L319 was 0:1), which resulted in a four-component LNP with L319 as the ionizable lipid.
实施例2：脂质纳米颗粒水合粒径的表征Example 2: Characterization of Hydrated Particle Size of Lipid Nanoparticles
取100μL实施例1A～1D中制备的五组分的脂质纳米颗粒，使用动态光散射法测定脂质纳米颗粒在PBS缓冲溶液中的水合粒径，其中图2为实施例1B的五组分LNP(其中，cKK-E12和L319摩尔比为6:4)和对比例1的四组分LNP(记为MC3)。实施例1A的五组分LNP的水合粒径和多分散系数分别为140nm和0.09，实施例1C的五组分LNP的水合粒径和多分散系数分别为110nm和0.1，实施例1D的五组分LNP的水合粒径和多分散系数分别为130nm和0.1。Take 100 μL of the five-component lipid nanoparticles prepared in Examples 1A to 1D, and use dynamic light scattering to measure the hydrated particle size of the lipid nanoparticles in PBS buffer solution, where Figure 2 shows the five-component LNP of Example 1B (wherein the molar ratio of cKK-E12 and L319 is 6:4) and the four-component LNP of Comparative Example 1 (referred to as MC3). The hydrated particle size and polydispersity index of the five-component LNP of Example 1A are 140 nm and 0.09, respectively, the hydrated particle size and polydispersity index of the five-component LNP of Example 1C are 110 nm and 0.1, respectively, and the hydrated particle size and polydispersity index of the five-component LNP of Example 1D are 130 nm and 0.1, respectively.
取10μL实施例1A中制备的五组分的脂质纳米颗粒，使用冷冻透射电镜拍摄脂质纳米颗粒的形貌如图3为所示。10 μL of the five-component lipid nanoparticles prepared in Example 1A was taken and the morphology of the lipid nanoparticles was photographed using a cryo-transmission electron microscope as shown in FIG3 .
实施例1A～1D中制备得到的五组分的脂质纳米颗粒的水合粒径均小于200nm，多分散系数小于0.2，说明采用本发明的制备方法可以形成稳定且粒径均一的脂质纳米颗粒。The hydrated particle sizes of the five-component lipid nanoparticles prepared in Examples 1A to 1D are all less than 200 nm, and the polydispersity coefficient is less than 0.2, indicating that the preparation method of the present invention can form stable lipid nanoparticles with uniform particle size.
实施例3：使用体外转染的方法筛选五组分LNPExample 3: Screening of five-component LNPs using in vitro transfection
使用实施例1B中制备的包载mFLuc的五组分LNP，选取对比例1～3的四组分LNP作为对照组，筛选不同比例的cKK-E12与L319作为可离子化脂质协同发挥作用。The five-component LNPs loaded with mFLuc prepared in Example 1B were used, and the four-component LNPs of Comparative Examples 1 to 3 were selected as a control group to screen different ratios of cKK-E12 and L319 as ionizable lipids to act synergistically.
实验步骤如下：将DC2.4细胞以20000细胞每孔在96孔板中孵育过夜，待细胞汇合度达到50％时，分别加入实施例1B和对比例1～3的LNP，在37℃、5％CO2浓度的饱和湿度培养箱中培养24小时，另设Untreated cells组(指的是细胞铺板后未做任何处理)，Free mRNA组(指的是直接向细胞培养基中加入没有载体的mRNA)。移除培养基，加入细胞裂解液和萤火虫荧光素酶底物，震荡10分钟后，使用酶标仪读取生物发光值，测试结果参见图4。The experimental steps are as follows: DC2.4 cells were incubated overnight in a 96-well plate at 20,000 cells per well. When the cell confluence reached 50%, LNPs of Example 1B and Comparative Examples 1 to 3 were added respectively, and cultured in a saturated humidity incubator at 37°C and 5% CO2 concentration for 24 hours. An Untreated cells group (referring to cells without any treatment after plating) and a Free mRNA group (referring to mRNA without a carrier being added directly to the cell culture medium) were also set up. The culture medium was removed, cell lysate and firefly luciferase substrate were added, and after shaking for 10 minutes, the bioluminescence value was read using an ELISA reader. The test results are shown in Figure 4.
由此验证可知，以6/4摩尔比的cKK-E12和L319协同作用的五组分LNP具有最高的mRNA表 达效率。This showed that the five-component LNP with a 6/4 molar ratio of cKK-E12 and L319 had the highest mRNA expression. Achieve efficiency.
实施例4：验证五组分LNP的膜融合能力Example 4: Verification of the membrane fusion ability of the five-component LNP
使用实施例1A制备的包载mFLuc的五组分LNP，其中选取对比例1～3的四组分LNP作为对照组，检测五组分LNP的膜融合能力。The mFLuc-encapsulated five-component LNP prepared in Example 1A was used, and the four-component LNP of Comparative Examples 1 to 3 was selected as a control group to detect the membrane fusion ability of the five-component LNP.
实验步骤如下：取小鼠红细胞置于透明96孔板中，分别加入pH5.5的柠檬酸钠缓冲溶液和pH7.4的PBS缓冲溶液，再对应加入实施例1和对比例1～3的LNP在37℃共孵育1h，另设阴性对照组(即仅在孔板中加入缓冲溶液和红细胞，记为Untreated cells)、阳性对照组(即在孔板中加入缓冲溶液、红细胞和0.5％的曲拉通X-100，记为0.5％Triton X-100)在37℃共孵育1h。共孵育完毕后离心取上清液，测上清液在540nm处的吸光值，通过吸光度的值来判断红细胞溶血的程度，测试结果参见图5。The experimental steps are as follows: mouse erythrocytes are placed in a transparent 96-well plate, sodium citrate buffer solution of pH 5.5 and PBS buffer solution of pH 7.4 are added respectively, and then LNPs of Example 1 and Comparative Examples 1 to 3 are added correspondingly and incubated at 37°C for 1 hour. A negative control group (i.e., only buffer solution and erythrocytes are added to the well plate, recorded as Untreated cells) and a positive control group (i.e., buffer solution, erythrocytes and 0.5% Triton X-100 are added to the well plate, recorded as 0.5% Triton X-100) are incubated at 37°C for 1 hour. After the incubation is completed, the supernatant is centrifuged and the absorbance value of the supernatant at 540nm is measured. The degree of hemolysis of erythrocytes is judged by the absorbance value. The test results are shown in Figure 5.
由此验证可知，本发明的五组分LNP的膜融合能力位于仅含cKK-E12的LNP或仅含L319的LNP之间。This demonstrates that the membrane fusion ability of the five-component LNP of the present invention is between that of the LNP containing only cKK-E12 or the LNP containing only L319.
实施例5：验证五组分LNP在生物体内的mRNA表达效率Example 5: Verification of mRNA expression efficiency of five-component LNP in vivo
使用实施例1A中制备的包载mFLuc的五组分LNP，并选取对比例1～3的四组分LNP作为对照组，检测本发明的五组分LNP在生物体内的mRNA表达效率。The mFLuc-encapsulated five-component LNP prepared in Example 1A was used, and the four-component LNP of Comparative Examples 1 to 3 were selected as a control group to detect the mRNA expression efficiency of the five-component LNP of the present invention in vivo.
实验步骤如下：选取6-8周龄的雌性C57BL/6小鼠，通过肌肉注射的方式对各组中每只小鼠分别给药2μg实施例1A和对比例1～3的LNP，分别在给药4小时和24小时后向每只小鼠腹腔注射200μL的200mg/mL的萤火虫荧光素酶底物，15分钟后，使用小动物活体成像检测小鼠体内生物发光强度。The experimental steps are as follows: female C57BL/6 mice aged 6-8 weeks were selected, and 2 μg of LNP of Example 1A and Comparative Examples 1 to 3 were administered to each mouse in each group by intramuscular injection. 200 μL of 200 mg/mL firefly luciferase substrate was injected intraperitoneally into each mouse 4 hours and 24 hours after administration, respectively. After 15 minutes, the bioluminescence intensity in the mice was detected using small animal in vivo imaging.
通过上述实验验证可知，使用实施例1的五组分LNP后，注射部位在4小时和24小时均具有最强的生物发光强度，参见图6，其中(a)4小时生物发光强度，(b)24小时生物发光强度。The above experiments verified that after using the five-component LNP of Example 1, the injection site had the strongest bioluminescence intensity at both 4 hours and 24 hours, see Figure 6, where (a) is the bioluminescence intensity at 4 hours, and (b) is the bioluminescence intensity at 24 hours.
由此可知，本发明的五组分LNP在活体上具有高效的mRNA表达效率。It can be seen from this that the five-component LNP of the present invention has a high mRNA expression efficiency in vivo.
实施例6：验证五组分LNP在C57BL/6小鼠体内的DC细胞激活效率Example 6: Verification of the DC cell activation efficiency of the five-component LNP in C57BL/6 mice
使用实施例1C中制备的包载mOVA的五组分LNP，其中选取对比例1的四组分LNP作为对照组，检测本发明的五组分LNP在C57BL/6小鼠体内的DC细胞激活效率。The mOVA-encapsulated five-component LNP prepared in Example 1C was used, wherein the four-component LNP of Comparative Example 1 was selected as a control group, and the DC cell activation efficiency of the five-component LNP of the present invention in C57BL/6 mice was detected.
实验步骤如下：选取8周龄的雌性C57BL/6小鼠，每个实验组4只小鼠，在第0天和第7天通过肌肉注射的方式分别对各组小鼠进行免疫，各组中每只小鼠分别给药10μg实施例1C和对比例1的LNP，另设一组仅注射PBS缓冲溶液的PBS组；在第8天将小鼠处死，离体注射部位的引流淋巴结和脾脏，实验流程参见图7。将淋巴结细胞和脾脏细胞分别处理之后，得到单细胞悬液，使用流式细胞术检测检测脾脏和引流淋巴结中激活的DC细胞(CD11c+CD80+，CD11c+CD86+)的数量，参见图8。The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection on day 0 and day 7, and each mouse in each group was administered 10 μg of LNP of Example 1C and Comparative Example 1, respectively. A PBS group was also set up in which only PBS buffer solution was injected; on day 8, the mice were killed, and the draining lymph nodes and spleen at the injection site were removed in vitro. For the experimental process, see Figure 7. After the lymph node cells and spleen cells were processed separately, a single cell suspension was obtained, and the number of activated DC cells (CD11c + CD80 + , CD11c + CD86 + ) in the spleen and draining lymph nodes was detected by flow cytometry, see Figure 8.
由此验证可知，在小鼠引流淋巴结和脾脏中，本发明的负载mRNA的五组分LNP在C57BL/6小鼠体内具有高效的DC细胞激活效率。This demonstrates that the mRNA-loaded five-component LNP of the present invention has a high DC cell activation efficiency in C57BL/6 mice in the draining lymph nodes and spleen of mice.
实施例7：验证五组分LNP在C57BL/6小鼠体内的免疫效力Example 7: Verification of the immune efficacy of five-component LNP in C57BL/6 mice
使用实施例1D中制备的包载mVZV的五组分LNP，其中选取对比例1的四组分LNP、减毒的VZV病毒疫苗(以下简称减毒疫苗)和带状疱疹重组gE蛋白亚单位疫苗加AS01佐剂(以下简称亚单位疫苗)作为本实验的对照组，检测本发明的五组分LNP在C57BL/6小鼠体内的免疫效力。The five-component LNPs encapsulating mVZV prepared in Example 1D were used, wherein the four-component LNPs of Comparative Example 1, the attenuated VZV virus vaccine (hereinafter referred to as the attenuated vaccine) and the herpes zoster recombinant gE protein subunit vaccine plus AS01 adjuvant (hereinafter referred to as the subunit vaccine) were selected as the control groups of this experiment to detect the immune efficacy of the five-component LNPs of the present invention in C57BL/6 mice.
实验步骤如下：选取8周龄的雌性C57BL/6小鼠，每个实验组4只小鼠，在第0周和第3周通过肌肉注射的方式对各组小鼠进行免疫，各组中每只小鼠分别给药10μg实施例1D和对比例1的LNP、减毒疫苗和亚单位疫苗，另设一组仅注射PBS缓冲溶液的PBS组；在第2、4和5周对小鼠进行眼眶取血，分离血清，在第6周将小鼠处死，实验流程参见图9。使用ELISA测小鼠第14、28和35天的 血清中带状疱疹特异性的IgG、IgG1和IgG2c的抗体滴度，测试结果参见图10A～图10C。The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection at week 0 and week 3. Each mouse in each group was administered 10 μg of LNP, attenuated vaccine and subunit vaccine of Example 1D and Comparative Example 1, respectively. A PBS group was also set up to be injected with only PBS buffer solution. Blood was collected from the mice at week 2, 4 and 5, and serum was separated. The mice were killed at week 6. The experimental process is shown in Figure 9. The ELISA was used to measure the 14th, 28th and 35th day of the mice. The test results of the antibody titers of herpes zoster-specific IgG, IgG1, and IgG2c in serum are shown in FIG. 10A to FIG. 10C .
将小鼠脾脏处理后得到单细胞悬液，取30万细胞每孔置于ELISpot孔板中加入带状疱疹gE肽库刺激24h，然后进行显色检测带状疱疹特异性分泌IFN-γ的脾细胞数量，测试结果参见图11，其中，(a)ELISpot斑点数量统计图，(b)ELISpot孔光学照片图。将脾细胞以每孔100万细胞置于96孔板中，加入带状疱疹gE肽库刺激5h，加入蛋白转运抑制剂。使用流式细胞术检测带状疱疹特异性的T细胞(CD3+CD4+CD45+IFN-γ+)的数量，测试结果参见图12。再将脾细胞以每孔100万细胞置于96孔板中，加入带状疱疹gE肽库刺激48h，离心取细胞上清液，使用ELISA测细胞上清液中脾细胞分泌的IL-2和IFN-γ的量，测试结果参见图13。After the mouse spleen was processed, a single cell suspension was obtained, and 300,000 cells were placed in each well of the ELISpot plate and stimulated with the herpes zoster gE peptide library for 24 hours, and then the number of spleen cells that specifically secreted IFN-γ was detected by color development. The test results are shown in Figure 11, where (a) ELISpot spot number statistics, (b) ELISpot well optical photo. Splenocytes were placed in a 96-well plate with 1 million cells per well, stimulated with the herpes zoster gE peptide library for 5 hours, and protein transport inhibitors were added. The number of herpes zoster-specific T cells (CD3 + CD4 + CD45 + IFN-γ + ) was detected by flow cytometry, and the test results are shown in Figure 12. Splenocytes were then placed in a 96-well plate with 1 million cells per well, stimulated with the herpes zoster gE peptide library for 48 hours, centrifuged to obtain the cell supernatant, and the amount of IL-2 and IFN-γ secreted by spleen cells in the cell supernatant was measured by ELISA, and the test results are shown in Figure 13.
由此验证可知，本发明的负载mRNA的五组分LNP具有高效的激活C57BL/6小鼠体内特异性的细胞免疫和体液免疫的能力。This demonstrates that the mRNA-loaded five-component LNP of the present invention has the ability to efficiently activate specific cellular immunity and humoral immunity in C57BL/6 mice.
实施例8：验证五组分LNP在生物体内的毒性Example 8: Verification of the toxicity of five-component LNP in vivo
使用实施例1D中制备的包载mVZV的五组分LNP，并选取对比例1的四组分LNP、减毒疫苗和亚单位疫苗作为本实验的对照组，检测五组分LNP在生物体内的疫苗效力。The five-component LNPs loaded with mVZV prepared in Example 1D were used, and the four-component LNPs, attenuated vaccine and subunit vaccine of Comparative Example 1 were selected as the control groups of this experiment to detect the vaccine efficacy of the five-component LNPs in vivo.
实验步骤如下：选取8周龄的雌性C57BL/6小鼠，每个实验组4只小鼠，在第0周和第3周通过肌肉注射的方式对各组小鼠进行免疫，各组中每只小鼠分别给药10μg实施例1D中的五组分LNP和对比例1的四组分LNP、减毒疫苗和亚单位疫苗，另设一组仅注射PBS缓冲溶液的PBS组。在免疫期间监测小鼠体重，实验流程参见图14。在第6周处死小鼠，取小鼠心、肝、脾、肺、肾、脑和注射部位肌肉进行石蜡包埋做H&E染色切片，测试结果参见图15。The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection at week 0 and week 3. Each mouse in each group was administered 10 μg of the five-component LNP in Example 1D and the four-component LNP, attenuated vaccine and subunit vaccine in Comparative Example 1, respectively. A PBS group was set up in which only PBS buffer solution was injected. The weight of the mice was monitored during the immunization period. See Figure 14 for the experimental process. The mice were killed in week 6, and the heart, liver, spleen, lung, kidney, brain and injection site muscles of the mice were embedded in paraffin for H&E staining sections. See Figure 15 for the test results.
由此验证可知，本发明的包载mRNA的五组分LNP在生物体内的毒性较低。This demonstrates that the five-component LNP containing mRNA of the present invention has low toxicity in vivo.
实施例9：验证五组分LNP在BALB/c小鼠体内的疫苗效力Example 9: Verification of the vaccine efficacy of five-component LNP in BALB/c mice
使用实施例1D中制备的包载mVZV的五组分LNP，其中选取对比例1的四组分LNP，减毒疫苗和亚单位疫苗作为本实验的对照组，检测五组分LNP在BALB/c小鼠体内的疫苗效力。The five-component LNPs loaded with mVZV prepared in Example 1D were used, wherein the four-component LNPs of Comparative Example 1, the attenuated vaccine and the subunit vaccine were selected as the control groups of this experiment to detect the vaccine efficacy of the five-component LNPs in BALB/c mice.
实验步骤如下：选取8周龄的雌性BALB/c小鼠，每个实验组3只小鼠，分别在第0周和第3周通过肌肉注射的方式对小鼠进行免疫，每只小鼠分别给药10μg实施例1D中的五组分LNP和对比例1的四组分LNP、减毒疫苗和亚单位疫苗，在第2、4和6周对小鼠进行眼眶取血，分离血清，并在第6周将小鼠处死，实验流程参见图16。The experimental procedures are as follows: 8-week-old female BALB/c mice were selected, with 3 mice in each experimental group, and the mice were immunized by intramuscular injection at week 0 and week 3, respectively. Each mouse was administered 10 μg of the five-component LNP in Example 1D and the four-component LNP, attenuated vaccine and subunit vaccine in Comparative Example 1, respectively. Orbital blood was drawn from the mice at weeks 2, 4 and 6, the serum was separated, and the mice were killed at week 6. See Figure 16 for the experimental process.
使用ELISA测小鼠第2、4和6周的血清中带状疱疹特异性的Total IgG、IgG1、IgG2a和IgG2c的抗体滴度，参见图17。使用ELISA测小鼠免疫后8个月内血清中带状疱疹特异性的Total IgG滴度变化，参见图18。将小鼠脾脏处理后得到单细胞悬液，取30万细胞每孔置于ELISpot孔板中加入带状疱疹gE肽库刺激24h，然后进行显色检测带状疱疹特异性分泌IFN-γ的脾细胞数量，参见图19(a)ELISpot孔光学照片图，(b)ELISpot斑点数量统计图。在免疫期间监测小鼠体重，参见图20。ELISA was used to measure the antibody titers of Total IgG, IgG1, IgG2a and IgG2c specific to herpes zoster in the serum of mice at 2, 4 and 6 weeks, see Figure 17. ELISA was used to measure the changes in the titers of Total IgG specific to herpes zoster in the serum of mice within 8 months after immunization, see Figure 18. The spleen of the mice was processed to obtain a single cell suspension, and 300,000 cells were placed in each well of the ELISpot plate and stimulated with the herpes zoster gE peptide library for 24 hours, and then the number of spleen cells that secreted IFN-γ specifically for herpes zoster was detected by color development, see Figure 19 (a) ELISpot well optical photograph, (b) ELISpot spot number statistical chart. The weight of the mice was monitored during the immunization period, see Figure 20.
由此验证可知，本发明的包载mRNA的五组分LNP具有高效的激活BALB/c小鼠体内特异性的细胞免疫和体液免疫的能力。This demonstrates that the five-component LNPs encapsulating mRNA of the present invention have the ability to efficiently activate specific cellular immunity and humoral immunity in BALB/c mice.
实施例10：验证包载不同去糖基化设计的mVZV的五组分LNP对体液免疫效力的影响Example 10: Verification of the effect of five-component LNPs loaded with mVZV of different deglycosylation designs on humoral immunity
(1)制备包载不同去糖基化设计的mVZV的五组分LNP：使用实施例1D中的mVZV进行不同的去糖基化位点设计，包括：含有两个氮-糖基化位点突变(缩写为mMUTE-N)、六个氧-糖基化位点突变(缩写为mMUTE-O)和两个糖基化位点突变的组合(缩写为mMUTE-N+O)，参见图21。参照实施例1D中所述的方法分别制备得到包载mMUTE-N的五组分LNP、包载mMUTE-O的五组分LNP和包载mMUTE-N+O的五组分LNP。 (1) Preparation of five-component LNPs containing mVZV with different deglycosylation designs: mVZV in Example 1D was used to perform different deglycosylation site designs, including: two nitrogen-glycosylation site mutations (abbreviated as mMUTE-N), six oxygen-glycosylation site mutations (abbreviated as mMUTE-O), and a combination of two glycosylation site mutations (abbreviated as mMUTE-N+O), see Figure 21. Five-component LNPs containing mMUTE-N, five-component LNPs containing mMUTE-O, and five-component LNPs containing mMUTE-N+O were prepared according to the method described in Example 1D.
(2)参照实施例9中的免疫实验流程和实验步骤，具体包括：选取8周龄的雌性BALB/c小鼠，每个实验组6只小鼠，在第0周和第3周通过肌肉注射的方式对小鼠进行免疫，小鼠分别给药10μg实施例1D中的五组分LNP和上述制备得到的包载mMUTE-N的五组分LNP、包载mMUTE-O的五组分LNP和包载mMUTE-N+O的五组分LNP，分别记为VZV、mMUTE-N、mMUTE-O、mMUTE-N+O，在第2、4和6周对小鼠进行眼眶取血，分离血清，具体流程参见图17。使用ELISA测小鼠第2、4和6周的血清中带状疱疹特异性的Total IgG、IgG1、IgG2a和IgG2c的抗体滴度，参见图22。经验证可知，实施例1D中包载mVZV的五组分LNP具有最高效的激活BALB/c小鼠体内特异性体液免疫的能力。(2) Referring to the immune experiment process and experimental steps in Example 9, specifically including: selecting 8-week-old female BALB/c mice, 6 mice in each experimental group, immunizing the mice by intramuscular injection at week 0 and week 3, and administering 10 μg of the five-component LNP in Example 1D and the five-component LNP containing mMUTE-N, the five-component LNP containing mMUTE-O, and the five-component LNP containing mMUTE-N+O prepared above, respectively, which are recorded as VZV, mMUTE-N, mMUTE-O, and mMUTE-N+O, respectively, and collecting blood from the eye sockets of the mice at weeks 2, 4, and 6 to separate the serum. For the specific process, see Figure 17. ELISA was used to measure the antibody titers of Total IgG, IgG1, IgG2a, and IgG2c specific to herpes zoster in the serum of mice at weeks 2, 4, and 6, see Figure 22. It has been verified that the five-component LNP containing mVZV in Example 1D has the most efficient ability to activate specific humoral immunity in BALB/c mice.
实施例11：验证五组分LNP在豚鼠体内抵抗VZV病毒入侵Example 11: Verification of the five-component LNP in guinea pigs against VZV virus invasion
使用实施例1D制备的包载mVZV的五组分LNP，选取亚单位疫苗和PBS作为本实验的对照组，检测五组分LNP在豚鼠体内抵御病毒的效力，实验流程参见图23。实验步骤具体为：选取体重为200-250g的豚鼠，在第0天和第14天通过肌肉注射的方式对豚鼠进行免疫，豚鼠分别给药10μg实施例1D中的五组分LNP、亚单位疫苗和PBS，在第21天和第22天将豚鼠背部划伤进行攻毒实验，在第23天到第29天中，每两天对豚鼠背部皮肤进行拍照，参见图24。在第29天将豚鼠处死，提取其背部皮肤组织。豚鼠经病毒感染后背部皮肤发生病变，其病变评分参见图25，豚鼠的病变评分结果参见图26。免疫后第29天，将提取的豚鼠背部皮肤进行H&E染色分析，参见图27。并使用qPCR定量豚鼠背部皮肤中的VZV病毒载量，参见图28。在免疫期间监测豚鼠体重，参见图29。经验证可知，实施例1D中包载mVZV的五组分LNP能够高效的保护豚鼠抵御VZV病毒的侵害。The five-component LNPs containing mVZV prepared in Example 1D were used, and subunit vaccines and PBS were selected as the control group of this experiment to detect the efficacy of the five-component LNPs in guinea pigs against the virus. The experimental process is shown in Figure 23. The experimental steps are specifically as follows: guinea pigs weighing 200-250g were selected, and the guinea pigs were immunized by intramuscular injection on days 0 and 14. The guinea pigs were administered 10 μg of the five-component LNPs, subunit vaccines and PBS in Example 1D, respectively. The guinea pigs were scratched on the back on days 21 and 22 for the challenge experiment. From day 23 to day 29, the guinea pigs were photographed every two days on the back skin, see Figure 24. The guinea pigs were killed on day 29 and their back skin tissue was extracted. After the guinea pigs were infected with the virus, lesions occurred on the back skin, and the lesion scores were shown in Figure 25, and the lesion score results of the guinea pigs were shown in Figure 26. On day 29 after immunization, the extracted guinea pig back skin was subjected to H&E staining analysis, see Figure 27. The VZV viral load in the dorsal skin of guinea pigs was quantified using qPCR, see Figure 28. The body weight of guinea pigs was monitored during the immunization period, see Figure 29. It was verified that the five-component LNP encapsulated with mVZV in Example 1D can effectively protect guinea pigs from VZV virus invasion.
以上对本发明示例性的实施方式进行了说明。但是，本申请的保护范围不拘囿于上述实施方式。本领域技术人员在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。 The above is a description of the exemplary embodiments of the present invention. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

  1. 一种脂质纳米颗粒，其特征在于，所述脂质纳米颗粒包括载体和被包封的核酸，所述载体包括复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质；所述核酸包括mRNA、环状RNA、siRNA、microRNA、反义核酸和质粒中的一种或多种。A lipid nanoparticle, characterized in that the lipid nanoparticle comprises a carrier and an encapsulated nucleic acid, the carrier comprises a complex ionizable lipid, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid; the nucleic acid comprises one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and a plasmid.
  2. 根据权利要求1所述的脂质纳米颗粒，其特征在于，所述复合可离子化脂质占所述LNP中总脂质的20mol％～70mol％。The lipid nanoparticle according to claim 1, characterized in that the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP.
    优选地，所述复合可离子化脂质包括第一可离子化脂质和第二可离子化脂质。Preferably, the complex ionizable lipid comprises a first ionizable lipid and a second ionizable lipid.
    优选地，第一可离子化脂质和第二可离子化脂质的摩尔比为1:99～99:1，优选2:8～8:2。Preferably, the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2.
    和/或，所述第一可离子化脂质和第二可离子化脂质不同，彼此独立的选自8-[(2-羟乙基)(6-氧代-6-癸氧基己基)氨基]辛酸(十七烷-9-基)酯、[(4-羟基丁基)氮杂二基]双(己烷-6,1-二基)双(2-己基癸酸酯)、4(N,N二甲基氨基)丁酸(二亚油基)甲酯、3,6-双{4-[双(2-羟基十二烷基)氨基]丁基}哌嗪-2,5-二酮、9-(4-(二甲氨基)丁酰氧基)十七烷二酸二((Z)-壬-2-烯-1-基)酯、N2,2-二亚油基-4-二甲氨基乙基-[1,3]-二氧戊环、8-[(2-羟乙基)(8-壬氧基-8-氧代辛基)氨基]辛酸(十七烷-9-基)酯、1,1'-[(2-{4-[2-({2-[双(2-羟基十二烷基)氨基]乙基}(2-羟基十二烷基)氨基)乙基]哌嗪-1-基}乙基)氮杂二烷基]双(十二烷-2-醇)、(2,3二油酰基丙基)三甲基氯化铵、双甲基双十八烷基溴化铵、四(8-甲基壬基)3',3',3”,3”-{[(甲基氮杂二烷基)双(丙烷-3,1二基)]双(氮杂三基)}四丙酸酯中的一种或多种，优选为8[(2羟乙基)(6氧代6癸氧基己基)氨基]辛酸(十七烷9基)酯或[(4羟基丁基)氮杂二基]双(己烷6,1二基)双(2己基癸酸酯)。and/or, the first ionizable lipid and the second ionizable lipid are different and are independently selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester, [(4-hydroxybutyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 4(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 8-[(2-hydroxyethyl)(8-nonoxy-8-oxooctyl) )amino] octanoic acid (heptadecan-9-yl) ester, 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazine-1-yl}ethyl)azadialkyl]bis(dodecane-2-ol), (2,3 dioleoylpropyl)trimethylammonium chloride, dimethyldioctadecylammonium bromide, tetra(8-methylnonyl) 3',3',3",3"-{[(methylazadialkyl)bis(propane-3,1-diyl)]bis(azatriyl)}tetrapropionate, preferably one or more of 8[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino] octanoic acid (heptadecan-9-yl) ester or [(4-hydroxybutyl)azadiyl]bis(hexane 6,1-diyl)bis(2-hexyldecanoate).
  3. 根据权利要求1或2所述的脂质纳米颗粒，其特征在于，所述辅助磷脂占所述LNP中总脂质的2mol％～20mol％。The lipid nanoparticle according to claim 1 or 2, characterized in that the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP.
    优选地，所述辅助磷脂包括1,2-二硬脂酰基-sn-甘油基-3-磷脂酰胆碱、1,2-二油酰基-sn-甘油基-3-磷脂酰胆碱、1,2-二棕榈酰基-sn-甘油基-3-磷脂酰胆碱、2-油酰基-1-棕榈酰基-sn-甘油基-3-磷脂酰胆碱、1,2-二油酰基-sn-甘油基-3-磷脂酰乙醇胺、2-油酰基-1-棕榈酰基-sn-甘油基-3-磷脂酰乙醇胺、1,2-二硬脂酰基-sn-甘油基-3-磷脂酰乙醇胺、1,2-二棕榈酰基-sn-甘油基-3-磷脂酰乙醇胺中的一种或多种。Preferably, the auxiliary phospholipids include one or more of 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine, 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine.
  4. 根据权利要求1-3任一项所述的脂质纳米颗粒，其特征在于，所述胆固醇类物质占所述LNP中总脂质的10mol％～60mol％。The lipid nanoparticle according to any one of claims 1-3, characterized in that the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP.
    优选地，所述胆固醇类物质选自胆固醇及其衍生物。Preferably, the cholesterol-like substance is selected from cholesterol and its derivatives.
    优选地，所述胆固醇及其衍生物包括胆固醇、β-谷甾醇、胆甾烷醇、胆甾烷酮、胆甾烯酮、7β-羟基胆固醇、7α-羟基胆固醇中的一种或多种。Preferably, the cholesterol and its derivatives include one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol.
  5. 根据权利要求1-4任一项所述的脂质纳米颗粒，其特征在于，所述聚乙二醇化脂质占所述LNP中总脂质的0.3mol％～30mol％。The lipid nanoparticle according to any one of claims 1 to 4, characterized in that the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP.
    优选地，所述聚乙二醇化脂质包括1,2-二肉豆蔻酰基-rac-甘油基-3-甲氧基聚乙二醇、1,2-二硬脂酰基-rac-甘油基-3-甲氧基聚乙二醇、1,2-二棕榈酰基-rac-甘油基-3-甲氧基聚乙二醇、1,2-二硬脂酰基-sn-甘油基-3-磷脂酰乙醇胺-甲氧基聚乙二醇中的一种或多种。Preferably, the PEGylated lipid comprises one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol, and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol.
    所述“总脂质”指的是复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质的总和。The term "total lipids" refers to the sum of complex ionizable lipids, auxiliary phospholipids, cholesterol-like substances and PEGylated lipids.
  6. 根据权利要求1-5任一项所述的脂质纳米颗粒，其特征在于，所述核酸的包封率为30～99％。The lipid nanoparticle according to any one of claims 1-5, characterized in that the encapsulation efficiency of the nucleic acid is 30-99%.
    优选地，所述复合可离子化脂质含有的氮元素与核酸含有的磷元素的摩尔比例为(1～50):1。Preferably, the molar ratio of the nitrogen element contained in the composite ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1.
    优选地，所述脂质纳米颗粒的水合粒径小于200nm。Preferably, the hydrated particle size of the lipid nanoparticles is less than 200 nm.
    优选地，所述脂质纳米颗粒的多分散系数小于0.2。 Preferably, the polydispersity coefficient of the lipid nanoparticles is less than 0.2.
  7. 权利要求1-6任一项所述的脂质纳米颗粒的制备方法，其特征在于，所述制备方法包括将含有核酸的水相与脂质有机相通过共混法或者微流控法制备得到所述脂质纳米颗粒。The method for preparing lipid nanoparticles according to any one of claims 1 to 6 is characterized in that the preparation method comprises preparing the lipid nanoparticles by blending an aqueous phase containing nucleic acids and a lipid organic phase through a blending method or a microfluidic method.
    优选地，所述脂质有机相的制备方法如下：将复合可离子化脂质、辅助磷脂、胆固醇类物质和聚乙二醇化脂质按比例溶解在有机溶剂中得到有机相。Preferably, the lipid organic phase is prepared as follows: the composite ionizable lipid, auxiliary phospholipid, cholesterol substance and PEGylated lipid are dissolved in an organic solvent in proportion to obtain an organic phase.
    优选地，所述含有核酸的水相的制备方法如下：将所述核酸溶解在缓冲溶液中得到水相。优选地，所述缓冲溶液可选用pH为4～6的缓冲溶液。Preferably, the preparation method of the aqueous phase containing nucleic acid is as follows: dissolving the nucleic acid in a buffer solution to obtain an aqueous phase. Preferably, the buffer solution can be a buffer solution with a pH of 4 to 6.
  8. 根据权利要求7所述的制备方法，其特征在于，所述共混法包括如下步骤：使用移液枪吸取含有核酸的水相，快速加入至所述脂质有机相中，然后迅速吹打混合液体大于30次，吹匀后室温静置10分钟，静置后对所得产物进行透析，得到所述脂质纳米颗粒。The preparation method according to claim 7 is characterized in that the blending method comprises the following steps: using a pipette to absorb the aqueous phase containing the nucleic acid, quickly adding it to the lipid organic phase, and then quickly blowing the mixed liquid for more than 30 times, blowing it evenly and letting it stand at room temperature for 10 minutes, and dialyzing the resulting product after standing to obtain the lipid nanoparticles.
    优选地，所述微流控法包括如下步骤：将装有水相和脂质有机相的注射器分别固定在注射泵上，以水相：脂质有机相体积比3:1注入微流控芯片中充分混合，混合完毕后收集混合液室温静置10分钟，静置后使用PBS缓冲溶液对所得产物进行透析，得到所述脂质纳米颗粒。Preferably, the microfluidic method comprises the following steps: respectively fixing syringes containing an aqueous phase and a lipid organic phase on a syringe pump, injecting the aqueous phase: lipid organic phase into a microfluidic chip at a volume ratio of 3:1 and mixing thoroughly, collecting the mixed solution after mixing and standing at room temperature for 10 minutes, and dialyzing the obtained product with a PBS buffer solution after standing to obtain the lipid nanoparticles.
  9. 权利要求1-6任一项所述的脂质纳米颗粒在制备药物制剂或生物制剂中的应用。Use of the lipid nanoparticles according to any one of claims 1 to 6 in the preparation of pharmaceutical preparations or biological preparations.
    优选地，所述生物制剂为可注射的生物制剂。Preferably, the biologic is an injectable biologic.
    优选地，所述生物制剂的给药方式为肌肉注射给药。Preferably, the biological preparation is administered by intramuscular injection.
    优选地，所述生物制剂用于预防带状疱疹。Preferably, the biologic is for use in preventing herpes zoster.
  10. 一种药物制剂或生物制剂，其为权利要求9中的药物制剂或生物制品。 A pharmaceutical preparation or biological preparation, which is the pharmaceutical preparation or biological preparation in claim 9.
PCT/CN2023/131882 2022-11-23 2023-11-15 Method for preparing lipid nanoparticle for efficiently delivering nucleic acid drug and use thereof WO2024109612A1 (en)

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CN114099699A (en) * 2021-11-22 2022-03-01 华南理工大学 Nano delivery system and preparation method and application thereof
CN114306279A (en) * 2021-12-30 2022-04-12 复旦大学 Lipid nanoparticle system based on corosolic acid or analogues thereof, and preparation method and application thereof
WO2022207862A2 (en) * 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
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CN111467321A (en) * 2020-03-26 2020-07-31 深圳市新合生物医疗科技有限公司 Intracellular delivery system of mRNA nucleic acid medicine, preparation method and application
WO2022207862A2 (en) * 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
CN114099699A (en) * 2021-11-22 2022-03-01 华南理工大学 Nano delivery system and preparation method and application thereof
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