WO2023029928A1 - Amino lipid and application thereof - Google Patents

Amino lipid and application thereof Download PDF

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WO2023029928A1
WO2023029928A1 PCT/CN2022/111656 CN2022111656W WO2023029928A1 WO 2023029928 A1 WO2023029928 A1 WO 2023029928A1 CN 2022111656 W CN2022111656 W CN 2022111656W WO 2023029928 A1 WO2023029928 A1 WO 2023029928A1
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amino lipid
cancer
preparation
lipid
amino
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Chinese (zh)
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查高峰
彭星华
夏恒
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深圳虹信生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/31—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atoms of the sulfonamide groups bound to acyclic carbon atoms
    • C07C311/32—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atoms of the sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P39/02—Antidotes
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
    • C07C303/02—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of sulfonic acids or halides thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
    • C07C303/36—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids
    • C07C303/38—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids by reaction of ammonia or amines with sulfonic acids, or with esters, anhydrides, or halides thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the invention relates to the technical field of medicinal chemistry, in particular to an amino lipid and its application.
  • Gene medicine refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities to achieve therapeutic purposes; or the genes are expressed to produce corresponding antigens, thereby inducing memory immune responses.
  • gene medicine has pushed the research from preclinical to clinical in many disease treatment fields, and has irreplaceable advantages for diseases caused by genetic abnormalities that are difficult to solve in the medical field, such as tumors.
  • Common gene medicines include plasmid DNA (plasmid DNA, pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA) and messenger RNA (mRNA).
  • the gene carrier has to go through multiple complex processes when transporting the gene: reaching the target cell through blood circulation, cell uptake, escape of endosomes, intracellular movement, and release of gene material by the carrier. Its main barriers are mainly extracellular barriers in the complex blood environment and intracellular barriers in the degradation of lysosomal enzymes. Therefore, it is an urgent problem for gene carrier researchers to find a good gene carrier to make the target gene reach the target site and play a role.
  • viral vector system a natural carrier resource.
  • Viral genome has simple structure, high transfection efficiency, and strong target cell specificity.
  • its disadvantages such as poor orientation, low carrying capacity, and immunogenicity limit its use. Therefore, the non-viral vector system with diversity, non-immunogenicity and easy-to-control production has attracted much attention in recent years and has been applied in many therapeutic fields.
  • Commonly used non-viral vector systems are mainly cationic lipids vectors.
  • Cationic lipids have three important structural regions: a positively charged hydrophilic polar head gene, a connecting chain in the middle responsible for connecting the polar and nonpolar ends, and a hydrophobic lipid chain.
  • the polar head containing amine groups plays a role in the interaction between liposome and RNA, liposome/RNA complex and cell membrane, affects the charge of lipid, and plays a major role in the process of lysosome escape.
  • Linking chains determine the chemical and biological stability of cationic liposomes, especially the resulting cytotoxicity.
  • the hydrophobic region can be in the form of a carbon chain or a variety of structures such as steroids, and the length of the carbon chain, whether it is saturated or not, and the specific type will affect the behavior of the lipid, which not only provides sufficient fluidity for the lipid bilayer, but also promotes the formation of cationic liposomes. Lipid fusion in vivo.
  • Cationic liposomes form liposome/gene complexes through electrostatic interactions with negatively charged groups.
  • the complex is positively charged due to the excess of cationic liposomes, and the positively charged liposome/gene complex is adsorbed on the negatively charged cell surface due to electrostatic interaction. It then enters the cell by fusion with the cell membrane or by endocytosis.
  • the main feature of cationic lipids for gene therapy is charge-influenced membrane fusion during endosomal escape. But at the same time, the excess positive charge of the cationic lipid/gene complex and the refractory characteristics of some cationic lipids also lead to cytotoxicity. Therefore lower transfection efficiency and cytotoxicity are the main disadvantages limiting the application of cationic lipids.
  • the present invention attempts to design ionizable cationic lipids to solve the above problems, so as to achieve better gene therapy effect,
  • the invention provides an amino lipid and its application.
  • a kind of amino lipid, its structure is as shown in formula (I):
  • L is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R2, R3, R4 and R5 are the same or different from each other, and each independently selected from H, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl; said C1-C24 alkyl, C2-C24 alkenyl Base, C2-C24 alkynyl can be optionally substituted by C1-C6 hydrocarbon group;
  • R2 and R3 are connected to form a 4-10-membered heterocycle, wherein the multi-membered heterocycle contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
  • said R2 is selected from C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl; said C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl can be optionally Substituted by C1-C6 hydrocarbyl.
  • L and R1 are connected to form NH2-L-R1 selected from A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14 , A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39 , A40 in one.
  • L and R1 are linked to N atom after linking.
  • the position substituted by NH2 in the above NH2-L-R1 is the free radical position connected to the compound of formula (I).
  • R2, R3 and adjacent N atoms form R2R3-NH, wherein H is the position of a free radical;
  • R2R3-NH is selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, One of N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23.
  • R2 and R3 are connected to the same N atom, and the N atom is connected to the S atom on the sulfonyl group.
  • the H atom in the above R2R3-NH is the position connected to the S atom in the formula (I).
  • the preparation method of described amino lipid comprises the following steps:
  • step S2 Add R2R3NH to the reaction system in step S1, and heat the reaction in the presence of a base.
  • the method comprises the steps of:
  • the aminolipid compound of formula I is obtained.
  • the heating temperature in the above step S2 is 50-120°C.
  • the base used in the above preparation method is an organic base or an inorganic base, such as: triethylamine, DIPEA, pyridine, DMAP, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and the like.
  • amino lipid and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof in the preparation of medicaments for gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA.
  • the above application is an application in the preparation of therapeutic drugs for cancer or genetic diseases.
  • the above-mentioned application is the application in the preparation of medicines for lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer, and the genetic disease is hemophilia, Mediterranean One or more of anemia, Gaucher's disease.
  • the above application is the application in the preparation of medicines for treating cancer, allergy, toxicity and pathogenic infection.
  • the above application is the application in the preparation of medicines for nucleic acid transfer.
  • the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA ( siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small inhibitory RNA
  • snRNA small nuclear RNA
  • An aminolipid compound disclosed by the present invention uses vinyl sulfonyl fluoride (ESF), a bifunctional electrophile, to construct an amino head group and a hydrophobic chain into the aminolipid, making full use of the advantages of ESF Click chemistry reaction characteristics, mild reaction conditions in the process of constructing amino lipids, no need for protection and deprotection, high atom economy. In in vitro and in vivo delivery studies, it shows excellent ability to deliver nucleic acid to cells.
  • the above-mentioned amino lipid compound has two sulfonamides, and the introduction of this group significantly enhances the stability of the lipid nanoparticle, improves the circulation time in vivo, and thus improves the delivery efficiency in vivo.
  • the preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements for instruments and equipment and simple operation.
  • OVA mRNA stimulates BMDC in Fig. 1 embodiment 10, differentiate into the ratio figure of the cell population that presents OVA antigen;
  • test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
  • PEG2000-DMG (1-(Monomethoxypolyethylene glycol)-2,3 Dimyristoylglycerol
  • Embodiment 1 Parallel synthesis and characterization of A1Ny series amino amino lipid compound library
  • Step I reaction solution In a 250mL reaction flask, add n-hexylamine (25.3mg, 0.25mmol), vinylsulfonyl fluoride (55mg, 0.5mmol), anhydrous tetrahydrofuran 2.5mL, and stir at room temperature for 5min to obtain the Step I reaction solution (2.5 mL, 0.1M).
  • Table 1 MW/z values of A1Ny series amino amino lipid compound library
  • HeLa cell line HeLa cell line (ATCC)
  • DMEM Human fetal bovine serum
  • Detection Percentage of GFP fluorescent cell number relative to total cell number (determined using nuclear dye Hoechst - see Figure 2). Lipofectamine 2000 (Invitrogen) was used as a positive control group according to the manufacturer's instructions.
  • Method Use an 8-channel pipette to add samples. Amounts shown are for a single well of a 96-well plate.
  • the amino lipid compound described in Example 1 is mixed with dioleoylphosphatidylethanolamine (DOPE), cholesterol, and the molar ratio of PEG2000-DMG is 45:10:42.5:2.5 and dissolved in absolute ethanol ;
  • the mass ratio of aminolipid compound to green fluorescent protein mRNA (EGFP mRNA) is about 8:1, and the amount of mRNA per hole is 100ng.
  • lipid nanoparticle solution was incubated at room temperature for 30 min, 90 ⁇ L of freshly resuspended HeLa cells (3-5 ⁇ 10 4 cells) were added and mixed with a pipette. Transfer 100 ⁇ L of cells+lipid nanoparticles to separate wells of a 96-well culture plate and place in an incubator at 37 °C with 5% CO2 .
  • Hoechst33258 (Invitrogen) was added to the cells at a final concentration of 0.2 ⁇ g/ml, and incubated at 37° C. for 15 minutes in the dark. Then the cells were washed once with PBS solution, and culture medium was added for 20 to 24 hours.
  • Embodiment 8 Transfection of lipid nanoparticles prepared by aminolipid compounds on BMDC primary cells
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • lipid nanoparticles encapsulating luciferase mRNA to the 96-well all-white ELISA plate covered with primary cells, and control the amount of luciferase mRNA lipid nanoparticles added in each well to 3ug . Then place it in an incubator at 37° C. with 5% CO 2 concentration for 12 hours to fully express luciferase mRNA.
  • Detection of transfection efficiency Add 10ul of 10mg/ml D-luciferin potassium salt to each well of a 96-well all-white microplate plate, and immediately place it in a microplate reader to detect the luminescence intensity.
  • the expression intensity of Fluc mRNA transfected by representative aminolipid compounds on BMDC is shown in Table 3.
  • DLin-MC3 was used as a control, and the expression intensity of many amino lipids was similar to that of MC3, and some of them were significantly better than the positive control.
  • Example 9 In vivo delivery performance evaluation of luciferase mRNA of lipid nanoparticles prepared from amino lipid compounds
  • the amino lipid compound of the present invention is mixed with DOPE, cholesterol, (1-(monomethoxypolyethylene glycol)-2,3 dimyrisyl glycerol (PEG2000-DMG) according to the ratio of 45:10:42.5:2.5
  • the molar ratio is mixed and dissolved in absolute ethanol, so that the molar concentration of the amino lipid compound is 0.001-0.01mmol/L.
  • TriLink FLuc-mRNA
  • the particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 40uL of LNP solution for particle size measurement, and cycle three times, each cycle 30s.
  • Encapsulation rate detection use The RNA HS Assay kit was used to detect the concentration of LNP RNA. Theoretical RNA concentration is the amount of total RNA input divided by the total volume of the final solution.
  • the preparation method is the same as the preparation method 1, except that amino lipid compound, DSPC, cholesterol and PEG2000-DMG are used in a molar ratio of 50:10:38.5:1.5.
  • the resulting lipid nanoparticle (LNP) solution was administered to the test animals by tail vein and intramuscular injection.
  • In vivo delivery 9 C57BL/6 mice were randomly selected in each group, and the dosage of 0.5mg/kg mRNA was injected into the lipid nanoparticle solution by subcutaneous, intramuscular and tail vein injection respectively (each administration method 3 mice). After 12 hours, 200 ⁇ L of 10 mg/mL D-luciferin potassium salt was injected into each mouse through the tail vein, and after 10 minutes, the mouse was placed under an in vivo imaging system (IVIS-200, Xenogen), and each mouse was observed. The total fluorescence intensity of each mouse was recorded by taking pictures. The expression intensities of Fluc mRNA delivered by representative amino lipid compounds through the three administration methods are shown in Table 6-8. DLin-MC3 served as a control.
  • Table 8 Expression intensity of Fluc mRNA delivered by tail vein administration of representative aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • Activation of immune cells add 1ug of ovalbumin mRNA lipid nanoparticles to each well of a 12-well plate, and place in a 37°C, 5% CO 2 incubator for 24 hours.
  • the cells were blown down with PBS solution, washed and centrifuged (100g, 5 minutes) with PBS three times, and then incubated with CD11c-APC antibody and SIINFEKL-H-2Kb-PE antibody, CD11c-APC antibody and MHC-II-PE antibody for 30 Minutes, then washed with PBS and centrifuged (100g, 5 minutes) once to remove unbound antibodies, and then detected by flow cytometry (Beckman cytoflex LX).
  • CD11 is a marker of BMDC
  • the CD11c-APC antibody is used for the labeling of the DC population
  • the SIINFEKL-H-2Kb-PE antibody is used for labeling the cell population presenting the OVA antigen in the cell population
  • the MHC-II-PE antibody is used for labeling Mature DC population.
  • Example 11 Evaluation of in vivo delivery performance of luciferase mRNA of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg. Six hours later, each mouse was injected with 200 ⁇ L of 10 mg/mL D-luciferin potassium salt through the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe The total fluorescence intensity of each mouse was taken and recorded.
  • IVIS-200, Xenogen in vivo imaging system
  • the expression intensity of Fluc mRNA delivered by representative aminolipid compounds is shown in Table 10, and DLin-MC3 was used as a control.
  • the expression intensity of the multiple amino lipids is similar to that of Dlin-MC3, and some of them are significantly better than the positive control.
  • Example 12 In vivo delivery of ovalbumin mRNA and immune performance evaluation of lipid nanoparticles prepared from amino lipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg (Day 0). After 7 days, use the same amount for another booster (Day 7). On the 21st day, blood was collected from the tail vein for serological analysis, and DLin-MC3 was used as a control.
  • Enzyme-linked immunosorbent assay (ELISA): flat-bottomed 96-well plates (Nunc) were pre-coated in 50 mM carbonate buffer, the concentration of OVA protein was 0.5 ⁇ g protein per well (pH 9.6) overnight at 4 ° C, and then used 5% glycine blocked. The sera of the immunized animals were diluted from 10 ⁇ 2 to 10 ⁇ 6 using PBS-0.05% Tween (PBS-T, pH 7.4), added to the wells and incubated at 37° C. for 1 hour. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was labeled at a dilution of 1:10,000 in PBS-T-1% BSA.
  • HRP horseradish peroxidase
  • the absorbance at 450 nm was detected in an ELISA plate reader (Bio-Rad) to determine the optical density at one wavelength.
  • the IgG antibody titers produced by A14N13 and MC3 were comparable, while the IgG antibody titers of A32N22, A34N22, and A35N20 were significantly better than the MC3 control group.
  • Example 13 In vivo immune and tumor therapeutic effect evaluation of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • B16-OVA melanoma cells (1.5 x 105 ) were injected subcutaneously into the right side of 4-6 week old mice. Vaccination was started when the tumor size was less than 50 mm3 (approximately on day 4 or day 5 after tumor inoculation). Animals were immunized by intramuscular injection of LNP preparations containing 15 ⁇ g of OVA-mRNA. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5 ⁇ length ⁇ width. Euthanize the mice when the tumor volume reaches 2,000 mm. Tumor suppression was compared to mice bearing freshly inoculated tumors. The median survival period of the non-vaccinated group was 29 weeks. After vaccination, the corresponding median survival periods were: 38 weeks (MC3), 49 weeks (A32N22), and 52 weeks (A35N20), as shown in Figure 4.

Abstract

An amino lipid and an application thereof: using ethenesulfonyl fluoride (ESF), a dual-function electrophile, to build an amine head group and a hydrophobic chain into an amino lipid, fully utilizing the click chemical reaction properties of ESF; in the process of constructing the amino lipid, reaction conditions are mild, protection and deprotection are not needed, and atom economy is high. In vitro and in vivo delivery studies show an excellent ability to deliver nucleic acid into cells. The amino lipid compound is provided with two sulfonamides, and the introduction of the group significantly enhances the stability of lipid nanoparticles and improves the in-vivo cycle time, thereby improving in-vivo delivery efficiency. The preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements for instruments and equipment, and simple operations.

Description

一种氨基脂质及其应用A kind of amino lipid and its application 技术领域technical field
本发明医药化学技术领域，特别涉及一种氨基脂质及其应用。The invention relates to the technical field of medicinal chemistry, in particular to an amino lipid and its application.
背景技术Background technique
基因药物是指将外源正常基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，以达到治疗目的；或者基因得到表达，产生相应的抗原，进而诱导记忆性的免疫反应。在过去的二十多年间，基因药物在很多疾病治疗领域将研究从临床前推向了临床，对于由基因异常引起的，医药界至今难以解决的疾病如肿瘤等具有无可替代的优势。常见的基因药物有质粒DNA(plasmid DNA，pDNA)、反义寡核昔酸(antisense ODN)、小干扰RNA(siRNA)和信使RNA(mRNA)。Gene medicine refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities to achieve therapeutic purposes; or the genes are expressed to produce corresponding antigens, thereby inducing memory immune responses. In the past two decades, gene medicine has pushed the research from preclinical to clinical in many disease treatment fields, and has irreplaceable advantages for diseases caused by genetic abnormalities that are difficult to solve in the medical field, such as tumors. Common gene medicines include plasmid DNA (plasmid DNA, pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA) and messenger RNA (mRNA).
然而，将外源基因引入体内，其会被体内的核酸酶降解，在未进入靶细胞之前，便被降解成小分子核苷酸，从而失去治疗作用。因此，实现基因治疗的关键是高效、安全的基因递送***。基因载体在运送基因的时候要经历多个复杂的过程：通过血液循环到达靶细胞，细胞摄取，内涵体的逃逸，胞内运动，载体释放基因物质。其主要障碍主要是复杂血液环境的细胞外障碍和溶酶体酶降解的细胞内障碍。因此寻找良好的基因载体，使得靶基因到达靶点发挥效用，是基因载体研究者亟待解决的问题。However, when exogenous genes are introduced into the body, they will be degraded by nucleases in the body, and before they enter the target cells, they will be degraded into small molecule nucleotides, thus losing their therapeutic effect. Therefore, the key to realizing gene therapy is an efficient and safe gene delivery system. The gene carrier has to go through multiple complex processes when transporting the gene: reaching the target cell through blood circulation, cell uptake, escape of endosomes, intracellular movement, and release of gene material by the carrier. Its main barriers are mainly extracellular barriers in the complex blood environment and intracellular barriers in the degradation of lysosomal enzymes. Therefore, it is an urgent problem for gene carrier researchers to find a good gene carrier to make the target gene reach the target site and play a role.
目前，在基因输送载体***方面主要分为两大类：一是病毒载体***，二是非病毒载体***。病毒载体是一种天然的载体资源，病毒基因组结构简单，转染效率高，靶细胞特异性强，但其导向性差、携带能力低、免疫原性等缺点限制了其使用。因此多样性、无免疫原性及易于控制生产的非病毒载体***近年来备受关注，并在很多治疗领域有所应用。常用的非病毒载体***主要是阳离子脂质(cationic lipids)载体。Currently, gene delivery vector systems are mainly divided into two categories: one is viral vector system, and the other is non-viral vector system. Viral vector is a natural carrier resource. Viral genome has simple structure, high transfection efficiency, and strong target cell specificity. However, its disadvantages such as poor orientation, low carrying capacity, and immunogenicity limit its use. Therefore, the non-viral vector system with diversity, non-immunogenicity and easy-to-control production has attracted much attention in recent years and has been applied in many therapeutic fields. Commonly used non-viral vector systems are mainly cationic lipids vectors.
阳离子脂质有三个重要的结构区域:带正电荷的亲水极性头部基因，中间负责连接极性和非极性的两端的连接链和疏水脂质链。含胺类基团的极性头部起着脂质体与RNA，脂质体/RNA复合物与细胞膜相互结合的作用，影响脂质带电情况，在溶酶体逃逸过程起主要作用。连接链决定了阳离子脂质体的化学和生物稳定性，特别是因此而产生的细胞毒性。疏水区可以为碳链形式或类固醇等多种结构，并且碳链的长度、是否饱和和具体类型将影响脂质行为，其既为脂双层提供足够的流动性，又能促使阳离子脂质体在体内的脂质融合。Cationic lipids have three important structural regions: a positively charged hydrophilic polar head gene, a connecting chain in the middle responsible for connecting the polar and nonpolar ends, and a hydrophobic lipid chain. The polar head containing amine groups plays a role in the interaction between liposome and RNA, liposome/RNA complex and cell membrane, affects the charge of lipid, and plays a major role in the process of lysosome escape. Linking chains determine the chemical and biological stability of cationic liposomes, especially the resulting cytotoxicity. The hydrophobic region can be in the form of a carbon chain or a variety of structures such as steroids, and the length of the carbon chain, whether it is saturated or not, and the specific type will affect the behavior of the lipid, which not only provides sufficient fluidity for the lipid bilayer, but also promotes the formation of cationic liposomes. Lipid fusion in vivo.
阳离子脂质体与带负电的基团通过静电作用形成脂质体/基因复合物。复合物因阳离子脂质体的过剩带正电，带正电的脂质体/基因复合物由于静电作用吸附于带负电的细胞表面。然 后通过与细胞膜融合或细胞的内吞作用进入细胞内。阳离子脂质用于基因治疗的主要特点是在核内体逃逸过程中的电荷影响的膜融合作用。但同时，阳离子脂质/基因复合物过剩的正电以及部分阳离子脂质难降解的特性也导致了细胞毒性。因此较低的转染效率和细胞毒性是限制阳离子脂质应用的主要缺点。Cationic liposomes form liposome/gene complexes through electrostatic interactions with negatively charged groups. The complex is positively charged due to the excess of cationic liposomes, and the positively charged liposome/gene complex is adsorbed on the negatively charged cell surface due to electrostatic interaction. It then enters the cell by fusion with the cell membrane or by endocytosis. The main feature of cationic lipids for gene therapy is charge-influenced membrane fusion during endosomal escape. But at the same time, the excess positive charge of the cationic lipid/gene complex and the refractory characteristics of some cationic lipids also lead to cytotoxicity. Therefore lower transfection efficiency and cytotoxicity are the main disadvantages limiting the application of cationic lipids.
技术问题technical problem
目前阳离子脂质作为基因载体因其结构简单、操作简便、生物安全性高等特点成为了目前应用最为广泛的非病毒载体，但其转染的效率低、正电荷所导致的细胞毒性问题仍待解决。At present, cationic lipids as gene carriers have become the most widely used non-viral vectors due to their simple structure, easy operation, and high biological safety. However, the problems of low transfection efficiency and cytotoxicity caused by positive charges still need to be solved. .
技术解决方案technical solution
针对现有技术中阳离子脂质体的转染的效率低、正电荷所导致的细胞毒性等技术问题，本发明尝试设计可电离阳离子脂质来解决上述问题，以达到较好的基因治疗效果，本发明提供了一种氨基脂质及其应用。Aiming at technical problems such as low transfection efficiency of cationic liposomes and cytotoxicity caused by positive charges in the prior art, the present invention attempts to design ionizable cationic lipids to solve the above problems, so as to achieve better gene therapy effect, The invention provides an amino lipid and its application.
本发明的目的通过以下技术方案予以实现：The purpose of the present invention is achieved through the following technical solutions:
一种氨基脂质，其结构如式(I)所示：A kind of amino lipid, its structure is as shown in formula (I):
Figure PCTCN2022111656-appb-000001
Figure PCTCN2022111656-appb-000001
其中，L为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基；Wherein, L is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
R1为H、OR5、CN、-C(＝O)OR4、-OC(＝O)R4、-C(＝O)N R4R5、-NR5C(＝O)R4或N R4R5；R1 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4, -C(=O)N R4R5, -NR5C(=O)R4 or N R4R5;
R2、R3、R4和R5彼此相同或不同，并且各自独立地选自H,C1-C24烷基、C2-C24烯基、C2-C24炔基；所述C1-C24烷基、C2-C24烯基、C2-C24炔基可任选地被C1-C6烃基取代；R2, R3, R4 and R5 are the same or different from each other, and each independently selected from H, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl; said C1-C24 alkyl, C2-C24 alkenyl Base, C2-C24 alkynyl can be optionally substituted by C1-C6 hydrocarbon group;
或R2和R3相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。Or R2 and R3 are connected to form a 4-10-membered heterocycle, wherein the multi-membered heterocycle contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
优选地，所述R2选自C6-C24烷基、C6-C24烯基、C6-C24炔基；所述被C6-C24烷基、C6-C24烯基、C6-C24炔基可任选地被C1-C6烃基取代。Preferably, said R2 is selected from C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl; said C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl can be optionally Substituted by C1-C6 hydrocarbyl.
优选地，以NH2作为自由基的位置，L和R1相连形成NH2-L-R1选自A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A11、A12、A13、A14、A15、A16、A17、A18、A19、 A20、A21、A22、A23、A24、A25、A26、A27、A28、A29、A30、A31、A32、A33、A34、A35、A36、A37、A38、A39、A40中的一种。Preferably, with NH2 as the position of the free radical, L and R1 are connected to form NH2-L-R1 selected from A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14 , A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39 , A40 in one.
在式(I)的化合物中L和R1相连以后与N原子连接。上述NH2-L-R1中NH2取代的位置即为与式(I)化合物相接的自由基位置。In compounds of formula (I) L and R1 are linked to N atom after linking. The position substituted by NH2 in the above NH2-L-R1 is the free radical position connected to the compound of formula (I).
Figure PCTCN2022111656-appb-000002
Figure PCTCN2022111656-appb-000002
优选地，R2、R3与相邻的N原子形成R2R3-NH，其中H作为自由基的位置；R2R3-NH选自N1、N2、N3、N4、N5、N6、N7、N8、N9、N10、N11、N12、N13、N14、N15、N16、N17、N18、N19、N20、N21、N22、N23中的一种。Preferably, R2, R3 and adjacent N atoms form R2R3-NH, wherein H is the position of a free radical; R2R3-NH is selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, One of N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23.
在式(I)的化合物中R2、R3连接在同一N原子上，N原子再与磺酰基上的S原子连接。上述R2R3-NH中H原子即为与式(I)中S原子连接的位置。In the compound of formula (I), R2 and R3 are connected to the same N atom, and the N atom is connected to the S atom on the sulfonyl group. The H atom in the above R2R3-NH is the position connected to the S atom in the formula (I).
Figure PCTCN2022111656-appb-000003
Figure PCTCN2022111656-appb-000003
所述氨基脂质的制备方法，包括以下步骤：The preparation method of described amino lipid comprises the following steps:
S1.化合物NH2-L-R1与乙烯基磺酰氟在溶剂中搅拌反应；S1. Compound NH2-L-R1 reacts with vinylsulfonyl fluoride with stirring in a solvent;
S2.在步骤S1反应体系中加入R2R3NH，并在碱存在的条件下加热反应即得。S2. Add R2R3NH to the reaction system in step S1, and heat the reaction in the presence of a base.
反应的流程如下：The flow of the reaction is as follows:
Figure PCTCN2022111656-appb-000004
Figure PCTCN2022111656-appb-000004
优选地，所述方法包括以下步骤：Preferably, the method comprises the steps of:
(1)在-20℃至40℃的温度下，在乙烯基磺酰氟(ESF)与由R1-L-NH2表示的化合物进行第一反应，得到第一中间体；(1) performing a first reaction between vinylsulfonyl fluoride (ESF) and a compound represented by R1-L-NH2 at a temperature of -20°C to 40°C to obtain a first intermediate;
(2)在分离或不分离所述第一中间体的情况下，在作为缚酸剂的碱的存在下，使所述第一中间体与由HNR2R3表示的胺在加热条件进行第二反应，得到所述式I的氨基脂质化合物。优选地，上述步骤S2中加热温度为50～120℃。上述制备方法中使用的碱为有机碱或无机碱，如：三乙胺、DIPEA、吡啶、DMAP、氢氧化钠、氢氧化钾、碳酸钠、碳酸钾等。(2) under the condition of isolating or not isolating the first intermediate, in the presence of a base as an acid-binding agent, causing the first intermediate to undergo a second reaction with an amine represented by HNR2R3 under heating conditions, The aminolipid compound of formula I is obtained. Preferably, the heating temperature in the above step S2 is 50-120°C. The base used in the above preparation method is an organic base or an inorganic base, such as: triethylamine, DIPEA, pyridine, DMAP, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and the like.
所述氨基脂质及其药物可接受的盐、前药或立体异构体在制备用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。Use of the amino lipid and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof in the preparation of medicaments for gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA.
优选地，上述应用为在制备用于癌症或遗传疾病的治疗药物中的应用。Preferably, the above application is an application in the preparation of therapeutic drugs for cancer or genetic diseases.
优选地，上述应用为在制备用于肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或***癌药物中的应用，所述遗传疾病为血友病，地中海贫血、高雪氏病中的一种或多种。Preferably, the above-mentioned application is the application in the preparation of medicines for lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer, and the genetic disease is hemophilia, Mediterranean One or more of anemia, Gaucher's disease.
优选地，上述应用为在制备用于治疗癌症、过敏、毒性和病原体感染药物中的应用。Preferably, the above application is the application in the preparation of medicines for treating cancer, allergy, toxicity and pathogenic infection.
优选地，上述应用为在制备用于核酸转移的药物中的应用。Preferably, the above application is the application in the preparation of medicines for nucleic acid transfer.
优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。Preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA ( siRNA) and small nuclear RNA (snRNA).
有益效果Beneficial effect
本发明公开的一种氨基脂质化合物，通过乙烯基磺酰氟(ESF)这一双功能的亲电试剂，将胺基头部基团和疏水链构建至氨基脂质中，充分利用了ESF的点击化学反应特性，构建氨基脂质的过程中反应条件温和，不需要保护和脱保护，原子经济性高。在体外、体内的递送研究中，显示了优良的递送核酸至细胞中的能力。上述氨基脂质化合物具备两个磺酰胺，该基团的引入显著增强了脂质纳米颗粒的稳定性，改善了体内循环时间，从而提高了体内递送效率。所述氨基脂质化合物的制备方法具有原料易得、反应条件温和、反应选择性好、反应产率高、仪器设备要求低和操作简单的优点。An aminolipid compound disclosed by the present invention uses vinyl sulfonyl fluoride (ESF), a bifunctional electrophile, to construct an amino head group and a hydrophobic chain into the aminolipid, making full use of the advantages of ESF Click chemistry reaction characteristics, mild reaction conditions in the process of constructing amino lipids, no need for protection and deprotection, high atom economy. In in vitro and in vivo delivery studies, it shows excellent ability to deliver nucleic acid to cells. The above-mentioned amino lipid compound has two sulfonamides, and the introduction of this group significantly enhances the stability of the lipid nanoparticle, improves the circulation time in vivo, and thus improves the delivery efficiency in vivo. The preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements for instruments and equipment and simple operation.
附图说明Description of drawings
图1实施例10中代表性氨基脂质化合物递送OVA mRNA刺激BMDC后，分化成呈递OVA抗原的细胞群体的比例图；After representative aminolipid compound delivery OVA mRNA stimulates BMDC in Fig. 1 embodiment 10, differentiate into the ratio figure of the cell population that presents OVA antigen;
图2实施例10中代表性氨基脂质化合物递送OVA mRNA刺激BMDC后，分化为成熟DC细胞群体的比例图；After representative aminolipid compound delivery OVA mRNA stimulates BMDC in the embodiment 10 of Fig. 2, it is differentiated into the ratio diagram of mature DC cell population;
图3实施例12中代表性氨基脂质化合物皮下给药递送OVA mRNA所产生的体液抗体滴度图；The humoral antibody titer figure that representative aminolipid compound subcutaneous administration delivers OVA mRNA in Fig. 3 embodiment 12 produces;
图4实施例13中肌肉注射OVA mRNA疫苗后的荷瘤小鼠生存曲线图。The survival curve of tumor-bearing mice after intramuscular injection of OVA mRNA vaccine in Example 13 in Fig. 4.
本发明的实施方式Embodiments of the present invention
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.
下述实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
本发明所用的术语“任选地取代的”意指与原子或基团连接的一个或多个氢原子独立地未被取代，或被一个或多个例如一、二、三或四个取代基取代。当一个原子或基团被多个取代基取代时，所述多个取代基可以相同或不同。The term "optionally substituted" as used herein means that one or more hydrogen atoms attached to the atom or group are independently unsubstituted, or are substituted by one or more, eg one, two, three or four substituents replace. When an atom or group is substituted by multiple substituents, the multiple substituents may be the same or different.
文中的缩写：Abbreviations in the text:
DNA             脱氧核糖核酸DNA Deoxyribonucleic acid
RNA             核糖核酸RNA ribonucleic acid
DOPE            二油酰基磷脂酰乙醇胺DOPE Dioleoyl Phosphatidylethanolamine
DSPC            二硬脂酰磷脂酰胆碱DSPC Distearoylphosphatidylcholine
PEG2000-DMG     (1-(单甲氧基聚乙二醇)-2,3二肉豆寇酰基甘油PEG2000-DMG (1-(Monomethoxypolyethylene glycol)-2,3 Dimyristoylglycerol
kD              千道尔顿kD Kilodalton
PBS             磷酸盐缓冲溶液。PBS Phosphate buffered saline.
实施例1 A1Ny系列氨基胺基脂质化合物库的平行合成与表征 Embodiment 1 Parallel synthesis and characterization of A1Ny series amino amino lipid compound library
Figure PCTCN2022111656-appb-000005
Figure PCTCN2022111656-appb-000005
在250mL的反应瓶中依次加入正已胺(25.3mg,0.25mmol)，乙烯基磺酰氟(55mg,0.5mmol)，无水四氢呋喃2.5mL,室温下搅拌反应5min，得Step I反应液(2.5mL,0.1M)。In a 250mL reaction flask, add n-hexylamine (25.3mg, 0.25mmol), vinylsulfonyl fluoride (55mg, 0.5mmol), anhydrous tetrahydrofuran 2.5mL, and stir at room temperature for 5min to obtain the Step I reaction solution (2.5 mL, 0.1M).
用移液枪将每个Step I反应液分别转移至22个1.5mL的EP管中(每个0.1mL,0.01mmol)，相应的EP管中各加入二胺的THF溶液(0.12mL,0.024mmol，0.2M)、DIPEA的THF溶液(0.2mL,0.04mmol,0.2M)，于加热型振摇反应器(Thermo-Shaker)中于78℃反应1h，TLC检测无Step I原料。反应结束后，将反应管内的溶剂常温挥干，即得到23个胺基脂质化合物A1Ny。进行质谱检测，结果见下面的表1。Use a pipette gun to transfer each Step I reaction solution to 22 1.5mL EP tubes (each 0.1mL, 0.01mmol), and add a THF solution of diamine (0.12mL, 0.024mmol , 0.2M), DIPEA in THF (0.2mL, 0.04mmol, 0.2M), reacted at 78°C for 1h in a heated shaking reactor (Thermo-Shaker), and no Step I raw material was detected by TLC. After the reaction, the solvent in the reaction tube was evaporated to dryness at room temperature to obtain 23 amino lipid compounds A1Ny. Mass spectrometry was performed, and the results are shown in Table 1 below.
表1：A1Ny系列氨基胺基脂质化合物库的MW/z值Table 1: MW/z values of A1Ny series amino amino lipid compound library
Figure PCTCN2022111656-appb-000006
Figure PCTCN2022111656-appb-000006
Figure PCTCN2022111656-appb-000007
Figure PCTCN2022111656-appb-000007
Figure PCTCN2022111656-appb-000008
Figure PCTCN2022111656-appb-000008
实施例2 2,2'-(壬基氮杂二基)双(N-十一烷基乙烷-1-磺酰胺)Example 2 2,2'-(nonylazadiyl)bis(N-undecylethane-1-sulfonamide)
Figure PCTCN2022111656-appb-000009
Figure PCTCN2022111656-appb-000009
在250mL的反应瓶中依次加入正壬胺(1.43g,10mmol)，乙烯基磺酰氟(2.2g,20mmol)，无水四氢呋喃80mL,室温下搅拌反应5min，再加入正十一烷基胺(13.7g,80mmol)，二异丙基乙基胺(20.6g,160mmol)升温至75℃反应4h。浓缩后，使用快速柱层析***纯化(二氯甲烷：甲醇＝20：1至5：1)得到化合物A4N4(5.06g,76％)。 1HNMR(400MHz,DMSO-d 6)：δ3.62(m,4H),3.03(m,2H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,50H),0.89(m,9H).ESI-MS calculated for C 35H 76N 3O 4S 2 +[M+H] +667.1,found 667.3。 Add n-nonylamine (1.43g, 10mmol), vinylsulfonyl fluoride (2.2g, 20mmol) and 80mL anhydrous tetrahydrofuran successively into a 250mL reaction flask, stir and react at room temperature for 5min, then add n-undecylamine ( 13.7g, 80mmol), diisopropylethylamine (20.6g, 160mmol) was heated to 75°C for 4h. After concentration, it was purified by flash column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain compound A4N4 (5.06 g, 76%). 1 HNMR (400MHz, DMSO-d 6 ): δ3.62(m,4H),3.03(m,2H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,50H), 0.89(m,9H). ESI-MS calculated for C 35 H 76 N 3 O 4 S 2 + [M+H] + 667.1, found 667.3.
实施例3 2,2'-(十一碳六烷基-氮杂二烷基)双(N-壬基乙烷-1-磺酰胺)Example 3 2,2'-(Undecahexaalkyl-azadialkyl)bis(N-nonylethane-1-sulfonamide)
Figure PCTCN2022111656-appb-000010
Figure PCTCN2022111656-appb-000010
在250mL的反应瓶中依次加入6-胺基十一烷(1.71g,10mmol)，乙烯基磺酰氟(2.2g,20mmol)，无水四氢呋喃80mL,室温下搅拌反应5min，再加入正壬胺(11.4g,80mmol)，二异丙基乙基胺(20.6g,160mmol)升温至75℃反应4h。浓缩后，使用快速柱层析***纯化(二氯甲烷：甲醇＝20：1至5：1)得到化合物A12N2(5.06g,76％)。 1H NMR(400MHz,DMSO-d 6)：δ3.62(m,4H),3.03(m,2H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,50H),0.89(m,9H).ESI-MS calculated for C 35H 76N 3O 4S 2 +[M+H] +667.1,found 667.3。 Add 6-aminoundecane (1.71g, 10mmol), vinylsulfonyl fluoride (2.2g, 20mmol) and 80mL anhydrous tetrahydrofuran to a 250mL reaction flask in sequence, stir at room temperature for 5min, then add n-nonylamine (11.4g, 80mmol), diisopropylethylamine (20.6g, 160mmol) was heated to 75°C for 4h. After concentration, it was purified by flash column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain compound A12N2 (5.06 g, 76%). 1 H NMR (400MHz, DMSO-d 6 ): δ3.62(m,4H),3.03(m,2H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,50H) , 0.89 (m, 9H). ESI-MS calculated for C 35 H 76 N 3 O 4 S 2 + [M+H] + 667.1, found 667.3.
实施例4 2,2'-(壬基氮杂二基)双(N-十一烷基乙烷-1-磺酰胺)Example 4 2,2'-(nonylazadiyl)bis(N-undecylethane-1-sulfonamide)
Figure PCTCN2022111656-appb-000011
Figure PCTCN2022111656-appb-000011
在250mL的反应瓶中依次加入乙醇胺(0.61g,10mmol)，乙烯基磺酰氟(2.2g,20mmol)，无水四氢呋喃80mL,室温下搅拌反应5min，再加入正十五烷基甲基胺(14.5g,60mmol)，二异丙基乙基胺(20.6g,160mmol)升温至75℃反应4h。浓缩后，使用快速柱层析***纯化(二氯甲烷：甲醇＝20：1至5：1)得到化合物A14N13(5.06g,76％)。 1H NMR(400MHz,DMSO-d 6)：δ3.62(m,4H),2.49(m,1H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,44H),0.89(m,12H).ESI-MS calculated for C 33H 72N 3O 4S 2 +[M+H] +667.1,found 667.3。 Add ethanolamine (0.61g, 10mmol), vinylsulfonyl fluoride (2.2g, 20mmol) and 80mL of anhydrous tetrahydrofuran successively into a 250mL reaction flask, stir and react at room temperature for 5min, then add n-pentadecylmethylamine ( 14.5g, 60mmol), diisopropylethylamine (20.6g, 160mmol) was heated to 75°C for 4h. After concentration, it was purified by flash column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain compound A14N13 (5.06 g, 76%). 1 H NMR (400MHz, DMSO-d 6 ): δ3.62(m,4H),2.49(m,1H),2.94(m,4H),2.89(m,4H),1.54-1.23(m,44H) , 0.89 (m, 12H). ESI-MS calculated for C 33 H 72 N 3 O 4 S 2 + [M+H] + 667.1, found 667.3.
实施例5 2,2'-(壬基氮杂二基)双(N-十一烷基乙烷-1-磺酰胺)Example 5 2,2'-(nonylazadiyl)bis(N-undecylethane-1-sulfonamide)
Figure PCTCN2022111656-appb-000012
Figure PCTCN2022111656-appb-000012
在250mL的反应瓶中依次加入4-氨基四氢吡喃(1.01g,10mmol)，乙烯基磺酰氟(2.2g,20mmol)，无水四氢呋喃80mL,室温下搅拌反应5min，再加入二已胺(14.8g,80mmol)，二异丙基乙基胺(20.6g,160mmol)升温至75℃反应4h。浓缩后，使用快速柱层析***纯化(二氯甲烷：甲醇＝20：1至5：1)得到化合物A21N16(4.43g,68％)。 1H NMR(400MHz,DMSO-d 6)：δ3.69-3.61(m,4H),3.52(m,4H),2.94(m,4H),2.72(m,1H),2.39(m,8H),1.78-1.52(m,4H),1.39-1.23(m,32H),0.89(m,12H).ESI-MS calculated for C 33H 70N 3O 5S 2 +[M+H] +652.5,found 652.9。 Add 4-aminotetrahydropyran (1.01g, 10mmol), vinylsulfonyl fluoride (2.2g, 20mmol) and 80mL anhydrous tetrahydrofuran to a 250mL reaction flask in sequence, stir for 5min at room temperature, then add diethylamine (14.8g, 80mmol), diisopropylethylamine (20.6g, 160mmol) was heated to 75°C for 4h. After concentration, it was purified by flash column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain compound A21N16 (4.43 g, 68%). 1 H NMR (400MHz, DMSO-d 6 ): δ3.69-3.61(m,4H),3.52(m,4H),2.94(m,4H),2.72(m,1H),2.39(m,8H) ,1.78-1.52(m,4H),1.39-1.23(m,32H),0.89(m,12H).ESI-MS calculated for C 33 H 70 N 3 O 5 S 2 + [M+H] + 652.5, Found 652.9.
实施例6 2,2'-(壬基氮杂二基)双(N-十一烷基乙烷-1-磺酰胺)Example 6 2,2'-(nonylazadiyl)bis(N-undecylethane-1-sulfonamide)
Figure PCTCN2022111656-appb-000013
Figure PCTCN2022111656-appb-000013
在250mL的反应瓶中依次加入N,N-二甲基丙二胺(204mg,2mmol)，乙烯基磺酰氟(440mg,4mmol)，无水四氢呋喃40mL,室温下搅拌反应5min，再加入甲基二烯十八烷基胺(3.36g,12mmol)，二异丙基乙基胺(4.12g,32mmol)升温至75℃反应4h。浓缩后，使用快速柱层析***纯化(二氯甲烷：甲醇＝20：1至5：1)得到化合物A32N23(1.41g,84％)。 1H NMR(400MHz,DMSO-d 6)：δ5.42-5.28(m,8H),3.52(m,4H),2.94(m,4H),2.89(s,6H),2.80(m,4H),2.39-2.35(m,8H),2.16(m,8H),2.14(m,6H),1.54-1.23(m,38H),0.89(m,6H).ESI-MS calculated for C 47H 93N 4O 4S 2 +[M+H] +841.7,found 841.9。 Add N,N-dimethylpropylenediamine (204mg, 2mmol), vinylsulfonyl fluoride (440mg, 4mmol), 40mL of anhydrous tetrahydrofuran to a 250mL reaction flask in sequence, stir the reaction at room temperature for 5min, and then add methyl Dienyl octadecylamine (3.36g, 12mmol) and diisopropylethylamine (4.12g, 32mmol) were heated to 75°C for 4h. After concentration, it was purified by flash column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain compound A32N23 (1.41 g, 84%). 1 H NMR (400MHz, DMSO-d 6 ): δ5.42-5.28(m,8H),3.52(m,4H),2.94(m,4H),2.89(s,6H),2.80(m,4H) ,2.39-2.35(m,8H),2.16(m,8H),2.14(m,6H),1.54-1.23(m,38H),0.89(m,6H).ESI-MS calculated for C 47 H 93 N 4 O 4 S 2 + [M+H] + 841.7, found 841.9.
实施例7 氨基脂质化合物作为mRNA载体的体外评价Example 7 In vitro evaluation of aminolipid compounds as mRNA carriers
细胞系：HeLa细胞系(ATCC)Cell line: HeLa cell line (ATCC)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)Medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum
筛选形式：96孔板细胞转染Screening format: 96-well plate cell transfection
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数－参见图2)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine2000(Invitrogen)用作阳性对照组。Detection (readout): Percentage of GFP fluorescent cell number relative to total cell number (determined using nuclear dye Hoechst - see Figure 2). Lipofectamine 2000 (Invitrogen) was used as a positive control group according to the manufacturer's instructions.
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。Method: Use an 8-channel pipette to add samples. Amounts shown are for a single well of a 96-well plate.
1.将实施例1中所述的氨基脂质化合物与二油酰基磷脂酰乙醇胺(DOPE),胆固醇，PEG2000-DMG的摩尔比为45：10：42.5：2.5的比例混合溶解在无水乙醇中；EGFP mRNA(TriLink)溶解在醋酸钠溶液(50mM,pH＝4.0)中，使用排枪取出上述的混合脂质溶液，加入至EGFP-mRNA溶液中，充分混合，控制其配比为乙醇溶液与醋酸钠溶液(50mM,pH＝4.0)的比例为1:3，制得脂质纳米颗粒溶液。氨基脂质化合物与绿色荧光蛋白mRNA(EGFP mRNA)的质量比约为8:1，每孔mRNA的用量为100ng。1. The amino lipid compound described in Example 1 is mixed with dioleoylphosphatidylethanolamine (DOPE), cholesterol, and the molar ratio of PEG2000-DMG is 45:10:42.5:2.5 and dissolved in absolute ethanol ; EGFP mRNA (TriLink) was dissolved in sodium acetate solution (50mM, pH=4.0), and the above-mentioned mixed lipid solution was taken out with a discharge gun, added to the EGFP-mRNA solution, mixed fully, and the ratio was controlled to be ethanol solution and acetic acid The ratio of sodium solution (50 mM, pH=4.0) was 1:3 to prepare lipid nanoparticle solution. The mass ratio of aminolipid compound to green fluorescent protein mRNA (EGFP mRNA) is about 8:1, and the amount of mRNA per hole is 100ng.
2.脂质纳米颗粒溶液在室温下孵育30min后，加入90μL新鲜重悬浮的HeLa细胞(3-5×10 4细胞)，并用移液管混合。将100μL的细胞+脂质纳米颗粒转移至96-孔培养平板的分开孔中，并且置于37℃的含有5％CO 2的培养箱中。 2. After the lipid nanoparticle solution was incubated at room temperature for 30 min, 90 μL of freshly resuspended HeLa cells (3-5×10 4 cells) were added and mixed with a pipette. Transfer 100 μL of cells+lipid nanoparticles to separate wells of a 96-well culture plate and place in an incubator at 37 °C with 5% CO2 .
3.细胞初始转染后20至24小时，将Hoechst33258(Invitrogen)以0.2μg/ml的终浓度加入细胞中，在37℃黑暗下培养15min。然后用PBS溶液清洗细胞1次，再加入培养基培养20至24小时。3. 20 to 24 hours after the initial transfection of the cells, Hoechst33258 (Invitrogen) was added to the cells at a final concentration of 0.2 μg/ml, and incubated at 37° C. for 15 minutes in the dark. Then the cells were washed once with PBS solution, and culture medium was added for 20 to 24 hours.
4.将细胞置于高通量共聚焦显微镜(Molecular Devices ImageXpress)中，从每个孔捕获细胞的4个图像视野，对于每个样品，捕获3种激光波长图像：细胞的亮视野图像，显示总细胞核的Hoechst染色图像和显示用质粒DNA成功转染并表达GFP的GFP图像。用MetaXpress软件对获得的Hoechst染色图像和GFP图像分别进行细胞计数，再将表达GFP的细胞数除以总细胞核数，即为细胞绝对转染效率。绝对转染效率如下计算：4. Place the cells in a high-throughput confocal microscope (Molecular Devices ImageXpress) and capture 4 image fields of the cells from each well. For each sample, capture 3 laser wavelength images: bright field images of the cells, showing Hoechst-stained images of total nuclei and GFP images showing successful transfection with plasmid DNA and expression of GFP. Cells were counted on the Hoechst stained image and GFP image obtained by MetaXpress software, and then the number of cells expressing GFP was divided by the total number of nuclei, which was the absolute transfection efficiency of the cells. Absolute transfection efficiencies were calculated as follows:
Figure PCTCN2022111656-appb-000014
Figure PCTCN2022111656-appb-000014
结果：部分化合物库对HeLa细胞的eGFP-mRNA的转染效率示于表2中。Results: The eGFP-mRNA transfection efficiencies of some compound libraries to HeLa cells are shown in Table 2.
表2：部分化合物库对HeLa细胞的eGFP-mRNA的转染效率Table 2: The eGFP-mRNA transfection efficiency of some compound libraries to HeLa cells
 the A1A1 A3A3 A4A4 A12A12 A14A14 A15A15 A21A21 A26A26 A32A32 A34A34 A35A35
N1N1 8.0％8.0% 23.0％23.0% 12.9％12.9% 0.3％0.3% 2.1％2.1% 7.0％7.0% 9.8％9.8% 0.0％0.0% 1.5％1.5% 0.3％0.3% 6.0％6.0%
N2N2 9.2％9.2% 2.7％2.7% 33.2％33.2% 0.0％0.0% 4.3％4.3% 0.2％0.2% 12.0％12.0% 0.4％0.4% 2.6％2.6% 5.5％5.5% 9.0％9.0%
N3N3 3.1％3.1% 35.0％35.0% 23.0％23.0% 0.3％0.3% 5.9％5.9% 9.6％9.6% 14.8％14.8% 0.2％0.2% 2.5％2.5% 1.4％1.4% 13.9％13.9%
N4N4 35.0％35.0% 0.8％0.8% 68.0％68.0% 67.0％67.0% 2.0％2.0% 0.3％0.3% 19.7％19.7% 12.9％12.9% 0.9％0.9% 1.2％1.2% 1.0％1.0%
N5N5 51.0％51.0% 56.0％56.0% 0.7％0.7% 0.9％0.9% 5.3％5.3% 12.9％12.9% 8.0％8.0% 69.0％69.0% 11.9％11.9% 4.0％4.0% 9.0％9.0%
N6N6 27.9％27.9% 23.0％23.0% 45.0％45.0% 0.7％0.7% 4.4％4.4% 3.5％3.5% 60.0％60.0% 34.3％34.3% 4.9％4.9% 9.6％9.6% 0.9％0.9%
N7N7 41.2％41.2% 60.0％60.0% 7.7％7.7% 2.1％2.1% 7.2％7.2% 34.0％34.0% 3.1％3.1% 21.7％21.7% 6.6％6.6% 22.7％22.7% 12.0％12.0%
N8N8 32.5％32.5% 23.0％23.0% 14.4％14.4% 0.4％0.4% 16.4％16.4% 12.1％12.1% 35.0％35.0% 2.8％2.8% 11.9％11.9% 7.9％7.9% 8.0％8.0%
N9N9 36.2％36.2% 44.0％44.0% 37.3％37.3% 76.0％76.0% 35.4％35.4% 40.4％40.4% 51.0％51.0% 0.6％0.6% 29.6％29.6% 41.5％41.5% 9.2％9.2%
N10N10 32.7％32.7% 67.0％67.0% 32.5％32.5% 1.8％1.8% 16.6％16.6% 60.1％60.1% 27.9％27.9% 0.7％0.7% 24.0％24.0% 41.4％41.4% 3.1％3.1%
N11N11 11.6％11.6% 23.6％23.6% 0.4％0.4% 2.2％2.2% 0.3％0.3% 0.0％0.0% 41.2％41.2% 0.0％0.0% 0.1％0.1% 12.8％12.8% 35.0％35.0%
N12N12 45.7％45.7% 3.5％3.5% 8.0％8.0% 34.9％34.9% 34.9％34.9% 12.0％12.0% 56.0％56.0% 12.9％12.9% 8.0％8.0% 23.6％23.6% 51.0％51.0%
N13N13 45.0％45.0% 45.7％45.7% 9.2％9.2% 18.8％18.8% 68.0％68.0% 41.8％41.8% 23.8％23.8% 69.0％69.0% 9.2％9.2% 8.0％8.0% 27.9％27.9%
N14N14 8.0％8.0% 3.8％3.8% 3.1％3.1% 33.9％33.9% 23.9％23.9% 44.9％44.9% 33.0％33.0% 34.3％34.3% 3.1％3.1% 9.2％9.2% 41.2％41.2%
N15N15 9.2％9.2% 1.4％1.4% 35.0％35.0% 31.8％31.8% 12.9％12.9% 22.8％22.8% 26.9％26.9% 21.7％21.7% 35.0％35.0% 3.1％3.1% 56.0％56.0%
N16N16 3.1％3.1% 41.9％41.9% 51.0％51.0% 8.0％8.0% 69.0％69.0% 34.9％34.9% 67.0％67.0% 2.8％2.8% 51.0％51.0% 35.0％35.0% 23.0％23.0%
N17N17 35.0％35.0% 2.2％2.2% 27.9％27.9% 9.2％9.2% 34.3％34.3% 23.8％23.8% 33.9％33.9% 3.8％3.8% 27.9％27.9% 51.0％51.0% 45.9％45.9%
N18N18 51.0％51.0% 3.0％3.0% 41.2％41.2% 3.1％3.1% 21.7％21.7% 33.6％33.6% 23.8％23.8% 12.9％12.9% 41.2％41.2% 27.9％27.9% 41.7％41.7%
N19N19 27.9％27.9% 34.9％34.9% 56.0％56.0% 35.0％35.0% 2.8％2.8% 36.8％36.8% 22.1％22.1% 69.0％69.0% 56.0％56.0% 41.2％41.2% 34.9％34.9%
N20N20 41.2％41.2% 54.1％54.1% 56.3％56.3% 51.0％51.0% 34.8％34.8% 21.3％21.3% 33.9％33.9% 34.3％34.3% 34.8％34.8% 56.0％56.0% 56.0％56.0%
N21N21 56.0％56.0% 34.7％34.7% 45.0％45.0% 27.9％27.9% 23.8％23.8% 12.8％12.8% 12.3％12.3% 21.7％21.7% 43.0％43.0% 45.7％45.7% 85.0％85.0%
N22N22 34.0％34.0% 26.8％26.8% 34.8％34.8% 41.2％41.2% 12.9％12.9% 82.0％82.0% 33.9％33.9% 2.8％2.8% 23.2％23.2% 75.0％75.0% 23.8％23.8%
N23N23 38.0％38.0% 21.0％21.0% 34.0％34.0% 56.0％56.0% 33.7％33.7% 45.8％45.8% 27.9％27.9% 23.9％23.9% 82.0％82.0% 78.9％78.9% 34.9％34.9%
实施例8：氨基脂质化合物制备的脂质纳米颗粒在BMDC原代细胞上的转染Embodiment 8: Transfection of lipid nanoparticles prepared by aminolipid compounds on BMDC primary cells
制剂方法：同实施例7。Preparation method: with embodiment 7.
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
细胞获取：把C57BL/6小鼠进行脱颈臼除死，并置于75％酒精中浸泡5分钟进行消毒，解刨获取小鼠大腿胫骨，并把附着的肌肉剔除露出骨质，然后用1ml吸有PBS的注射器把胫骨中的骨髓吹出，把所得骨髓吹散后通过50um滤网滤去杂质，往所得过滤物中加入红细胞裂解液后放置5分钟后进行100g、5分钟的离心除去上清液，将所得细胞置于1640培养基(含10％胎牛血清、20ng/ml GMCSF、10ng/ml IL4)中重悬，并接种于6孔板中，接种密度为100000个细胞/毫升培养基，放置于37℃、5％CO 2细胞培养箱中，每2天进行半换液一次，于第七天收集悬浮细胞和松散贴壁的细胞，并接种到96孔全白酶标板接种密度为每孔10000个细胞，培养基体积为100ul。 Cell acquisition: C57BL/6 mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes for disinfection, dissected to obtain the mouse thigh tibia, and removed the attached muscles to expose the bone, and then used 1ml Blow out the bone marrow in the tibia with a syringe sucked with PBS, blow out the obtained bone marrow and filter out impurities through a 50um filter, add red blood cell lysate to the obtained filtrate, let it stand for 5 minutes, then centrifuge at 100g for 5 minutes to remove the supernatant The resulting cells were resuspended in 1640 medium (containing 10% fetal bovine serum, 20ng/ml GMCSF, 10ng/ml IL4), and seeded in a 6-well plate at a seeding density of 100,000 cells/ml medium , placed in a 37°C, 5% CO 2 cell culture incubator, half-change the medium every 2 days, collect suspension cells and loosely adherent cells on the seventh day, and inoculate them into 96-well all-white microplates at the inoculation density For 10000 cells per well, the medium volume is 100ul.
细胞转染：往铺有原代细胞的96孔全白酶标板中加入包裹萤光素酶mRNA的脂质纳米颗粒，控制每孔中的萤光素酶mRNA脂质纳米颗粒加入量为3ug。随后放置在37℃、5％CO 2浓度的培养箱中12小时，使萤光素酶mRNA充分表达。 Cell transfection: Add lipid nanoparticles encapsulating luciferase mRNA to the 96-well all-white ELISA plate covered with primary cells, and control the amount of luciferase mRNA lipid nanoparticles added in each well to 3ug . Then place it in an incubator at 37° C. with 5% CO 2 concentration for 12 hours to fully express luciferase mRNA.
转染效率检测：往96孔全白酶标板中每孔加入10ul的10mg/ml的D-萤光素钾盐，马上放置于酶标仪中检测发光强度。代表性氨基脂质化合物在BMDC上转染Fluc mRNA的表达强度见表3。DLin-MC3作为对照,所述的氨基脂质多个与MC3表达强度相似，并有多个显著优于阳性对照。Detection of transfection efficiency: Add 10ul of 10mg/ml D-luciferin potassium salt to each well of a 96-well all-white microplate plate, and immediately place it in a microplate reader to detect the luminescence intensity. The expression intensity of Fluc mRNA transfected by representative aminolipid compounds on BMDC is shown in Table 3. DLin-MC3 was used as a control, and the expression intensity of many amino lipids was similar to that of MC3, and some of them were significantly better than the positive control.
表3：代表性氨基脂质化合物在BMDC上的转染的表达强度Table 3: Expression Intensities of Transfection of Representative Aminolipid Compounds on BMDCs
Figure PCTCN2022111656-appb-000015
Figure PCTCN2022111656-appb-000015
Figure PCTCN2022111656-appb-000016
Figure PCTCN2022111656-appb-000016
Figure PCTCN2022111656-appb-000017
Figure PCTCN2022111656-appb-000017
Figure PCTCN2022111656-appb-000018
Figure PCTCN2022111656-appb-000018
实施例9 胺基脂质化合物制备的脂质纳米颗粒的萤光素酶mRNA体内递送性能评价Example 9 In vivo delivery performance evaluation of luciferase mRNA of lipid nanoparticles prepared from amino lipid compounds
1.脂质纳米颗粒的制备1. Preparation of Lipid Nanoparticles
制剂方法1：Preparation method 1:
将本发明的胺基脂质化合物与DOPE、胆固醇、(1-(单甲氧基聚乙二醇)-2,3二肉豆寇酰基甘油(PEG2000-DMG)按45:10:42.5:2.5的摩尔比混合并溶解在无水乙醇中，使胺基脂质化合物的摩尔浓度为0.001-0.01mmol/L。使用微量注射泵，使所得的乙醇溶液和溶解有FLuc-mRNA(TriLink)的醋酸钠溶液(50mM,pH＝4.0)以1:3的体积比在微流道芯片中混合以制得脂质纳米颗粒的粗溶液，然后将其用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，在使用前通过0.22μm的微孔滤膜过滤。胺基脂质化合物与萤光素酶mRNA(Fluc mRNA)的质量比约为10:1。所得的脂质纳米颗粒(LNP)溶液通过皮下给药方式施用于受试动物。The amino lipid compound of the present invention is mixed with DOPE, cholesterol, (1-(monomethoxypolyethylene glycol)-2,3 dimyrisyl glycerol (PEG2000-DMG) according to the ratio of 45:10:42.5:2.5 The molar ratio is mixed and dissolved in absolute ethanol, so that the molar concentration of the amino lipid compound is 0.001-0.01mmol/L.Using a microsyringe pump, make the resulting ethanol solution and acetic acid dissolved with FLuc-mRNA (TriLink) Sodium solution (50 mM, pH = 4.0) was mixed in a microfluidic chip at a volume ratio of 1:3 to prepare a crude solution of lipid nanoparticles, which was then used in a dialysis cassette (Fisher, MWCO 20,000) in 1X PBS, Dialyze 6h under temperature control 4 ℃, pass through the microporous membrane filtration of 0.22 μm before use.The mass ratio of amino lipid compound and luciferase mRNA (Fluc mRNA) is about 10:1. Gained lipid nano The granule (LNP) solution was administered to the test animals by subcutaneous administration.
脂质纳米颗粒的表征：Characterization of Lipid Nanoparticles:
粒径的表征：所制备的脂质纳米颗粒的粒径和PDI通过Nano-ZSZEN3600(Malvern)测定。取LNP溶液40uL进行粒径测量，循环三次，每次循环30s。Characterization of particle size: The particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 40uL of LNP solution for particle size measurement, and cycle three times, each cycle 30s.
包封率检测：使用
Figure PCTCN2022111656-appb-000019
RNA HS Assay试剂盒检测LNP RNA浓度。理论RNA浓度为投入的总RNA量除以最终溶液的总体积。 Encapsulation rate detection: use
Figure PCTCN2022111656-appb-000019
The RNA HS Assay kit was used to detect the concentration of LNP RNA. Theoretical RNA concentration is the amount of total RNA input divided by the total volume of the final solution.
Figure PCTCN2022111656-appb-000020
Figure PCTCN2022111656-appb-000020
表4：使用制剂方法1用代表性胺基脂质化合物制备的LNP的表征数据Table 4: Characterization Data for LNPs Prepared with Representative Amino Lipid Compounds Using Formulation Method 1
Figure PCTCN2022111656-appb-000021
Figure PCTCN2022111656-appb-000021
制剂方法2：Preparation method 2:
制备方法同制剂方法1，除了使用摩尔比为50:10:38.5:1.5的胺基脂质化合物、DSPC、胆固醇和PEG2000-DMG。所得的脂质纳米颗粒(LNP)溶液通过尾静脉和肌肉注射的给药方式施用于受试动物。The preparation method is the same as the preparation method 1, except that amino lipid compound, DSPC, cholesterol and PEG2000-DMG are used in a molar ratio of 50:10:38.5:1.5. The resulting lipid nanoparticle (LNP) solution was administered to the test animals by tail vein and intramuscular injection.
表5：使用制剂方法2用代表性胺基脂质化合物制备的LNP的表征数据Table 5: Characterization Data for LNPs Prepared with Representative Amino Lipid Compounds Using Formulation Method 2
Figure PCTCN2022111656-appb-000022
Figure PCTCN2022111656-appb-000022
Figure PCTCN2022111656-appb-000023
Figure PCTCN2022111656-appb-000023
2.动物实验2. Animal experiments
动物准备：选取体重约20g的6周龄的雌性C57BL/6小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice weighing about 20 g were selected and raised in an SPF-grade breeding room. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.
体内递送：每组随机选取9只C57BL/6小鼠，按0.5mg/kg mRNA的用量，分别使用皮下、肌肉和尾静脉注射三种给药方式注射脂质纳米颗粒溶液(每种给药方式3只小鼠)。12小时后，往每只小鼠体内通过尾静脉注射200μL 10mg/mL的D-萤光素钾盐，10分钟后，将小鼠放置于活体成像***(IVIS-200,Xenogen)下，观察每只小鼠总的萤光强度，并拍照记录下来。代表性胺基脂质化合物通过3种给药方式递送的Fluc mRNA的表达强度见表6-8。DLin-MC3作为对照。In vivo delivery: 9 C57BL/6 mice were randomly selected in each group, and the dosage of 0.5mg/kg mRNA was injected into the lipid nanoparticle solution by subcutaneous, intramuscular and tail vein injection respectively (each administration method 3 mice). After 12 hours, 200 μL of 10 mg/mL D-luciferin potassium salt was injected into each mouse through the tail vein, and after 10 minutes, the mouse was placed under an in vivo imaging system (IVIS-200, Xenogen), and each mouse was observed. The total fluorescence intensity of each mouse was recorded by taking pictures. The expression intensities of Fluc mRNA delivered by representative amino lipid compounds through the three administration methods are shown in Table 6-8. DLin-MC3 served as a control.
表6：代表性胺基脂质化合物皮下给药递送的Fluc mRNA的表达强度Table 6: Expression intensity of Fluc mRNA delivered by subcutaneous administration of representative aminolipid compounds
 the 氨基脂质编号amino lipid number 萤光强度Fluorescence intensity
11 A4N4A4N4 2.7E+062.7E+06
22 A3N10A3N10 1.9E+071.9E+07
33 A12N4A12N4 7.6E+067.6E+06
44 A12N9A12N9 1.4E+071.4E+07
55 A14N13A14N13 3.7E+083.7E+08
66 A15N22A15N22 4.8E+084.8E+08
77 A14N16A14N16 3.1E+073.1E+07
88 A26N16A26N16 1.1E+071.1E+07
99 A21N16A21N16 8.2E+068.2E+06
1010 A32N23A32N23 9.7E+069.7E+06
1111 A34N22A34N22 1.9E+071.9E+07
1212 A35N21A35N21 6.8E+066.8E+06
1313 DLin-MC3DLin-MC3 3.1E+063.1E+06
表7：代表性胺基脂质化合物肌注给药递送的Fluc mRNA的表达强度Table 7: Expression intensity of Fluc mRNA delivered by intramuscular injection of representative aminolipid compounds
 the 氨基脂质编号amino lipid number 萤光强度Fluorescence intensity
11 A4N4A4N4 4.1E+064.1E+06
22 A3N10A3N10 2.7E+062.7E+06
33 A12N4A12N4 1.2E+071.2E+07
44 A12N9A12N9 4.3E+074.3E+07
55 A14N13A14N13 2.8E+072.8E+07
66 A15N22A15N22 2.7E+072.7E+07
77 A14N16A14N16 4.7E+064.7E+06
88 A26N16A26N16 4.7E+064.7E+06
99 A21N16A21N16 8.2E+068.2E+06
1010 A32N23A32N23 4.2E+064.2E+06
1111 A34N22A34N22 9.1E+069.1E+06
1212 A35N21A35N21 1.8E+061.8E+06
1313 DLin-MC3DLin-MC3 8.5E+068.5E+06
表8：代表性胺基脂质化合物尾静脉给药递送的Fluc mRNA的表达强度Table 8: Expression intensity of Fluc mRNA delivered by tail vein administration of representative aminolipid compounds
 the 氨基脂质编号amino lipid number 萤光强度Fluorescence intensity
11 A4N4A4N4 4.6E+064.6E+06
22 A3N10A3N10 5.2E+065.2E+06
33 A12N4A12N4 3.6E+063.6E+06
44 A12N9A12N9 5.1E+075.1E+07
55 A14N13A14N13 7.2E+067.2E+06
66 A15N22A15N22 5.1E+075.1E+07
77 A14N16A14N16 5.2E+065.2E+06
88 A26N16A26N16 2.1E+062.1E+06
99 A21N16A21N16 6.5E+066.5E+06
1010 A32N23A32N23 3.9E+063.9E+06
1111 A34N22A34N22 2.1E+062.1E+06
1212 A35N21A35N21 8.2E+068.2E+06
1313 DLin-MC3DLin-MC3 2.7E+072.7E+07
实施例10：氨基脂质化合物制备的脂质纳米颗粒在BMDC原代细胞上免疫评价Example 10: Immunological Evaluation of Lipid Nanoparticles Prepared by Aminolipid Compounds on BMDC Primary Cells
制剂方法：将本发明中所述的氨基脂质化合物与DOPE,胆固醇，PEG2000-DMG的摩尔比为45：10：42.5：2.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用两个微量注射泵，控制其配比为乙醇溶液与醋酸钠溶液(50mM,pH＝4.0)的比例为1：3，在微流道芯片中制得脂质纳米颗粒的粗溶液，再 使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为8:1。Preparation method: mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in sodium acetate solution (50mM, pH=4.0). Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH=4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous membrane before use. The mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
细胞获取：同实施例8。Cell acquisition: same as Example 8.
免疫细胞的激活：往12孔板中每孔加入1ug卵清蛋白mRNA脂质纳米颗粒，置于37℃，5％CO 2培养箱中培养24小时。用PBS溶液将细胞吹打下来，并用PBS洗涤离心(100g、5分钟)三次，之后用CD11c-APC抗体和SIINFEKL-H-2Kb-PE抗体、CD11c-APC抗体和MHC-II-PE抗体进行孵育30分钟，然后用PBS洗涤离心(100g、5分钟)一次，除去没有结合上的抗体，然后用流式细胞仪(贝克曼cytoflex LX)检测。其中CD11是BMDC的标记物，因此CD11c-APC抗体用于DC群体的标记，SIINFEKL-H-2Kb-PE抗体用于标记细胞群中呈递OVA抗原的细胞群体，MHC-II-PE抗体用于标记成熟DC细胞群体。结果如图1、2所示，A14N13和A15N12与MC3刺激BMDC成熟和呈递OVA抗原能力相当，而A32N22，A34N22和A35N20免疫激活效果显著优于MC3对照组。 Activation of immune cells: add 1ug of ovalbumin mRNA lipid nanoparticles to each well of a 12-well plate, and place in a 37°C, 5% CO 2 incubator for 24 hours. The cells were blown down with PBS solution, washed and centrifuged (100g, 5 minutes) with PBS three times, and then incubated with CD11c-APC antibody and SIINFEKL-H-2Kb-PE antibody, CD11c-APC antibody and MHC-II-PE antibody for 30 Minutes, then washed with PBS and centrifuged (100g, 5 minutes) once to remove unbound antibodies, and then detected by flow cytometry (Beckman cytoflex LX). Among them, CD11 is a marker of BMDC, so the CD11c-APC antibody is used for the labeling of the DC population, the SIINFEKL-H-2Kb-PE antibody is used for labeling the cell population presenting the OVA antigen in the cell population, and the MHC-II-PE antibody is used for labeling Mature DC population. The results were shown in Figures 1 and 2. A14N13 and A15N12 were comparable to MC3 in stimulating BMDC maturation and presenting OVA antigen, while A32N22, A34N22 and A35N20 had significantly better immune activation effects than the MC3 control group.
实施例11：氨基脂质化合物制备的脂质纳米颗粒的萤光素酶mRNA体内递送性能评价Example 11: Evaluation of in vivo delivery performance of luciferase mRNA of lipid nanoparticles prepared from aminolipid compounds
制剂方法：将本发明中所述的氨基脂质化合物与DOPE,胆固醇，PEG2000-DMG的摩尔比为45：10：42.5：2.5的比例混合溶解在无水乙醇中。萤光素酶mRNA(Fluc mRNA)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用两个微量注射泵，控制其配比为乙醇溶液与醋酸钠溶液(50mM,pH＝4.0)的比例为1：3，在微流道芯片中制得脂质纳米颗粒的粗溶液，再使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与萤光素酶mRNA(Fluc mRNA)的质量比约为8:1。Preparation method: mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol. Luciferase mRNA (Fluc mRNA) was dissolved in sodium acetate solution (50 mM, pH=4.0). Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH=4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous membrane before use. The mass ratio of aminolipid compound to luciferase mRNA (Fluc mRNA) is about 8:1.
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
体内递送：每组随机选取3只小鼠，按0.5mg/kg的用量，使用皮下注射脂质纳米颗粒。6小时后，分别往每只小鼠体内通过尾静脉注射200μL 10mg/mL的D-萤光素钾盐，10分钟后，将小鼠放置于活体成像***(IVIS-200,Xenogen)下，观察每只小鼠总的萤光强度，并拍照记录下来。代表性氨基脂质化合物递送Fluc mRNA的表达强度见表10，DLin-MC3作为对照。所述的多个氨基脂质与Dlin-MC3表达强度相似，并有多个显著优于阳性对照。In vivo delivery: 3 mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg. Six hours later, each mouse was injected with 200 μL of 10 mg/mL D-luciferin potassium salt through the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe The total fluorescence intensity of each mouse was taken and recorded. The expression intensity of Fluc mRNA delivered by representative aminolipid compounds is shown in Table 10, and DLin-MC3 was used as a control. The expression intensity of the multiple amino lipids is similar to that of Dlin-MC3, and some of them are significantly better than the positive control.
表9：代表性胺基脂质化合物制备的LNP的表征数据Table 9: Characterization data for LNPs prepared from representative aminolipid compounds
Figure PCTCN2022111656-appb-000024
Figure PCTCN2022111656-appb-000024
表10：代表性氨基脂质化合物递送Fluc mRNA的表达强度Table 10: Expression intensity of Fluc mRNA delivered by representative aminolipid compounds
编号serial number 胺基脂质编号Amino Lipid ID 萤光强度Fluorescence intensity
11 A4N4A4N4 2.8E+062.8E+06
22 A3N10A3N10 1.8E+061.8E+06
33 A12N4A12N4 2.1E+052.1E+05
44 A12N9A12N9 4.5E+064.5E+06
55 A14N13A14N13 5.1E+065.1E+06
66 A15N22A15N22 2.8E+062.8E+06
77 A14N16A14N16 8.1E+068.1E+06
88 A26N16A26N16 7.8E+067.8E+06
99 A21N16A21N16 6.1E+056.1E+05
1010 A32N23A32N23 4.1E+064.1E+06
1111 A34N22A34N22 1.2E+071.2E+07
1212 A35N21A35N21 6.4E+066.4E+06
1313 DLin-MC3DLin-MC3 4.2E+064.2E+06
实施例12：氨基脂质化合物制备的脂质纳米颗粒的卵清蛋白mRNA体内递送及免疫性能评价Example 12: In vivo delivery of ovalbumin mRNA and immune performance evaluation of lipid nanoparticles prepared from amino lipid compounds
制剂方法：将本发明中所述的氨基脂质化合物与DOPE,胆固醇，PEG2000-DMG的摩尔比为45：10：42.5：2.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用两个微量注射泵，控制其配比为乙醇溶液与醋酸钠溶液(50mM,pH＝4.0)的比例为1：3，在微流道芯片中制得脂质纳米颗粒的粗溶液，再使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为8:1。Preparation method: mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in sodium acetate solution (50mM, pH=4.0). Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH=4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous membrane before use. The mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
体内递送：每组随机选取3只小鼠，按0.5mg/kg的用量，使用皮下注射脂质纳米颗粒(Day0)。7天后，使用相同的量再加强一次(Day 7)。在第21天尾静脉取血进行血清学分析，DLin-MC3作为对照。In vivo delivery: 3 mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg (Day 0). After 7 days, use the same amount for another booster (Day 7). On the 21st day, blood was collected from the tail vein for serological analysis, and DLin-MC3 was used as a control.
酶联免疫吸附测定(ELISA)：对平底96孔板(Nunc)进行预涂在50mM碳酸盐缓冲液中，OVA蛋白的浓度为每孔0.5μg蛋白(pH 9.6)在4℃过夜，然后用5％甘氨酸封闭。使用PBS-0.05％Tween(PBS-T,pH 7.4)将免疫动物的血清从10 -2稀释至10 -6，添加到孔中并在37℃下孵育1小时。辣根过氧化物酶(HRP)偶联的山羊抗小鼠IgG在PBS-T-1％BSA中以1：10,000的稀释度进行标记。添加HRP基板后，在一个波长下确定光密度ELISA酶标仪(Bio-Rad)中检测450nm下的吸光度。如图3所示，A14N13与MC3所产生的IgG抗体滴定相当，而A32N22，A34N22，A35N20的IgG抗体滴定显著优于MC3对照组。 Enzyme-linked immunosorbent assay (ELISA): flat-bottomed 96-well plates (Nunc) were pre-coated in 50 mM carbonate buffer, the concentration of OVA protein was 0.5 μg protein per well (pH 9.6) overnight at 4 ° C, and then used 5% glycine blocked. The sera of the immunized animals were diluted from 10 −2 to 10 −6 using PBS-0.05% Tween (PBS-T, pH 7.4), added to the wells and incubated at 37° C. for 1 hour. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was labeled at a dilution of 1:10,000 in PBS-T-1% BSA. After adding the HRP substrate, the absorbance at 450 nm was detected in an ELISA plate reader (Bio-Rad) to determine the optical density at one wavelength. As shown in Figure 3, the IgG antibody titers produced by A14N13 and MC3 were comparable, while the IgG antibody titers of A32N22, A34N22, and A35N20 were significantly better than the MC3 control group.
实施例13：氨基脂质化合物制备的脂质纳米颗粒的体内免疫和肿瘤治疗效果评价Example 13: In vivo immune and tumor therapeutic effect evaluation of lipid nanoparticles prepared from aminolipid compounds
制剂方法：将本发明中所述的氨基脂质化合物与DOPE,胆固醇，PEG2000-DMG的摩尔比为45：10：42.5：2.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用两个微量注射泵，控制其配比为乙醇溶液与醋酸钠溶液(50mM,pH＝4.0)的比例为1：3，在微流道芯片中制得脂质纳米颗粒的粗溶液，再使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为8:1。Preparation method: mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in sodium acetate solution (50mM, pH=4.0). Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH=4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous membrane before use. The mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲 养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。Animal preparation: 6-week-old female C57BL/6 mice were selected, weighing about 20g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
体内递送：将B16-OVA黑色素瘤细胞(1.5×10 5)皮下注射到4-6周龄的小鼠右侧。当肿瘤大小小于50mm 3时(约在肿瘤接种后第4天或第5天)开始接种疫苗。通过肌肉注射含有15μg OVA-mRNA的LNP制剂对动物进行免疫。使用数显卡尺每周测量肿瘤生长3次，计算公式为0.5×长度×宽度。当肿瘤体积达到2,000mm 3时对小鼠实施安乐死。将肿瘤抑制与携带新鲜接种肿瘤的小鼠进行比较。未注射疫苗组的中位生存期为29周，注射疫苗后，对应的中位生存期分别为：38周(MC3)，49周(A32N22)，52周(A35N20)，如图4所示。 In vivo delivery: B16-OVA melanoma cells (1.5 x 105 ) were injected subcutaneously into the right side of 4-6 week old mice. Vaccination was started when the tumor size was less than 50 mm3 (approximately on day 4 or day 5 after tumor inoculation). Animals were immunized by intramuscular injection of LNP preparations containing 15 μg of OVA-mRNA. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5×length×width. Euthanize the mice when the tumor volume reaches 2,000 mm. Tumor suppression was compared to mice bearing freshly inoculated tumors. The median survival period of the non-vaccinated group was 29 weeks. After vaccination, the corresponding median survival periods were: 38 weeks (MC3), 49 weeks (A32N22), and 52 weeks (A35N20), as shown in Figure 4.
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。The above is only a preferred embodiment of the patent of the present invention, and is not intended to limit the patent of the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the patent of the present invention shall be included in this patent. within the protection scope of the invention patent.

Claims (10)

  1. 一种氨基脂质，其特征在于，其结构如式(I)所示：A kind of amino lipid, is characterized in that, its structure is as shown in formula (I):
    Figure PCTCN2022111656-appb-100001
    Figure PCTCN2022111656-appb-100001
    其中，L为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基；Wherein, L is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
    R1为H、OR5、CN、-C(＝O)OR4、-OC(＝O)R4、-C(＝O)N R4R5、-NR5 C(＝O)R4或N R4R5；R1 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4, -C(=O)N R4R5, -NR5 C(=O)R4 or N R4R5;
    R2、R3、R4和R5彼此相同或不同，并且各自独立地选自H,C1-C24烷基、C2-C24烯基、C2-C24炔基；所述C1-C24烷基、C2-C24烯基、C2-C24炔基可任选地被C1-C6烃基取代；R2, R3, R4 and R5 are the same or different from each other, and each independently selected from H, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl; said C1-C24 alkyl, C2-C24 alkenyl Base, C2-C24 alkynyl can be optionally substituted by C1-C6 hydrocarbon group;
    或R2和R3相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。Or R2 and R3 are connected to form a 4-10-membered heterocycle, wherein the multi-membered heterocycle contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
  2. 根据权利要求1所述氨基脂质，其特征在于，所述R2选自C6-C24烷基、C6-C24烯基、C6-C24炔基；所述被C6-C24烷基、C6-C24烯基、C6-C24炔基可任选地被C1-C6烃基取代。The amino lipid according to claim 1, wherein said R2 is selected from C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl; said C6-C24 alkyl, C6-C24 alkenyl The group, the C6-C24 alkynyl group can be optionally substituted by the C1-C6 hydrocarbon group.
  3. 根据权利要求1或2所述氨基脂质，其特征在于，以NH2作为自由基的位置，L和R1相连形成NH2-L-R1选自A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A11、A12、A13、A14、A15、A16、A17、A18、A19、A20、A21、A22、A23、A24、A25、A26、A27、A28、A29、A30、A31、A32、A33、A34、A35、A36、A37、A38、A39、A40中的一种：According to the amino lipid described in claim 1 or 2, it is characterized in that, with NH2 as the position of free radical, L and R1 are connected to form NH2-L-R1 is selected from A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, One of A33, A34, A35, A36, A37, A38, A39, A40:
    Figure PCTCN2022111656-appb-100002
    Figure PCTCN2022111656-appb-100002
  4. 根据权利要求1或2所述氨基脂质，其特征在于，R2、R3与相邻的N原子形成R2R3-NH，其中H作为自由基的位置；R2R3-NH选自N1、N2、N3、N4、N5、N6、N7、N8、N9、N10、N11、N12、N13、N14、N15、N16、N17、N18、N19、N20、N21、N22、N23中的一种：According to the amino lipid described in claim 1 or 2, it is characterized in that R2, R3 form R2R3-NH with adjacent N atoms, wherein H is the position of free radical; R2R3-NH is selected from N1, N2, N3, N4 , N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23:
    Figure PCTCN2022111656-appb-100003
    Figure PCTCN2022111656-appb-100003
  5. 根据权利要求1～4任一项所述氨基脂质的制备方法，其特征在于，包括以下步骤：According to the preparation method of amino lipid according to any one of claims 1 to 4, it is characterized in that it comprises the following steps:
    S1.化合物NH2-L-R1与乙烯基磺酰氟在溶剂中搅拌反应；S1. Compound NH2-L-R1 reacts with vinylsulfonyl fluoride with stirring in a solvent;
    S2.在步骤S1反应体系中加入R2R3NH，并在碱存在的条件下加热反应即得。S2. Add R2R3NH to the reaction system in step S1, and heat the reaction in the presence of a base.
  6. 权利要求1至4任一项所述氨基脂质及其药物可接受的盐在制备用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。The application of the amino lipid and the pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of medicines for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA.
  7. 根据权利要求6所述应用，其特征在于，在制备用于癌症或遗传疾病的治疗药物中的应用。The use according to claim 6, characterized in that it is used in the preparation of therapeutic drugs for cancer or genetic diseases.
  8. 根据权利要求7所述应用，其特征在于，在制备用于肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或***癌药物中的应用。The application according to claim 7, characterized in that it is used in the preparation of drugs for lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or prostate cancer.
  9. 根据权利要求7所述应用，其特征在于，在制备用于治疗癌症、过敏、毒性和病原体感染药物中的应用。The use according to claim 7, characterized in that it is used in the preparation of medicines for treating cancer, allergy, toxicity and pathogen infection.
  10. 根据权利要求6所述应用，其特征在于，在制备用于核酸转移的药物中的应用。The application according to claim 6, characterized in that it is used in the preparation of medicines for nucleic acid transfer.
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