WO2023279836A1 - 一种用于核酸递送的可电离脂质化合物及其lnp组合物 - Google Patents

一种用于核酸递送的可电离脂质化合物及其lnp组合物 Download PDF

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WO2023279836A1
WO2023279836A1 PCT/CN2022/091881 CN2022091881W WO2023279836A1 WO 2023279836 A1 WO2023279836 A1 WO 2023279836A1 CN 2022091881 W CN2022091881 W CN 2022091881W WO 2023279836 A1 WO2023279836 A1 WO 2023279836A1
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lipid
virus
peg
lipid compound
independently
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PCT/CN2022/091881
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French (fr)
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郝婧
王庆彬
闫胜勇
王浩猛
严志红
刘健
宇学峰
邱东旭
林美娜
郭军
熊艳丽
朱涛
赵宣
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天津键凯科技有限公司
康希诺(上海)生物研发有限公司
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Priority to EP22836593.8A priority Critical patent/EP4342880A1/en
Publication of WO2023279836A1 publication Critical patent/WO2023279836A1/zh

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    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
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    • C07C215/14—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to hydrocarbon groups substituted by amino groups
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    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a novel ionizable lipid for nucleic acid delivery and its application in delivery of biologically active substances.
  • Gene therapy refers to the use of molecular biology methods to introduce target genes into patients so that they can be expressed in order to correct or compensate for diseases caused by gene defects and abnormalities. It also refers to introducing nucleic acids into cells to inhibit the expression of target genes (gene silencing) or Increase the expression of the target gene (gene activation) to achieve the purpose of treating the disease. As a new method of disease treatment combining modern medicine and molecular biology, gene therapy gradually occupies an important position in the medical field.
  • the treatment of "expressing a certain gene” and “expressing a certain protein” is mainly realized through the introduction of plasmid DNA and mRNA, while the treatment of "inhibiting a certain gene” is mainly through siRNA or microRNA (microRNA, miRNA) ( That is, RNAi technology) to achieve.
  • siRNA and mRNA Gene therapy methods based on siRNA and mRNA have unique advantages that other types of nucleic acid drugs do not have. However, the common difficulty in the development of mRNA and siRNA is how to effectively deliver them to cells at the target site.
  • LNP lipid nanoparticle
  • GalNac GalNac
  • LPP lipopolyplexes
  • LNP is generally prepared from four lipids in a certain ratio, usually these four lipids include cationic lipids, neutral lipids, steroidal lipids and polymer-conjugated lipids.
  • cationic liposomes are positively charged and electrostatically interact with negatively charged membrane lipids in endosomes.
  • Membrane lipids are flipped from the lumen of endosomes to the lumen and form neutral electron pairs with positive charges. The drug escapes from the cationic liposome and enters the nucleus.
  • Patent documents CN110352071A and CN1882693A disclose cationic lipid compounds and the use of lipids to prepare lipid nanoparticles or lipid mixtures for delivering biologically active substances into the body.
  • Patent document US20200197510A1 discloses respiratory virus ribonucleic acid vaccines and combination vaccines, and methods for using vaccines and compositions containing vaccines.
  • the first aspect of the present invention provides a lipid compound, the lipid compound has the following formula I structure:
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;
  • Ra is H or C 1 -C 12 hydrocarbon group
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 4 is C 1 -C 12 hydrocarbon group
  • R 5 is H or C 1 -C 6 hydrocarbon group
  • x 0, 1 or 2.
  • said Ra is H or C 1 -C 6 alkyl (such as methyl, ethyl, n-propyl, n-butyl), especially H.
  • R1 and R2 each independently have the following structure:
  • R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence;
  • a is an integer from 2 to 12 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), preferably, a is an integer from 8 to 12;
  • each of R 7a , R 7b and a in the structure of formula II is selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.
  • R 7a in the structure of formula II is H, preferably, R 7a is H every time it occurs.
  • R 7b that appears at least once in the structure of formula II is a C 1 -C 8 hydrocarbon group, especially a C 1 -C 8 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n- Butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 or both have one of the following structures:
  • lipid compound of the present invention has the following structure (IA):
  • R 6 is independently H, OH or C 1 -C 24 hydrocarbon group at each occurrence;
  • n is an integer from 1 to 15 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).
  • lipid compound of the present invention has the following structure (IB):
  • y and z are each independently an integer from 1 to 12.
  • n in the structure of formula IB is an integer from 2 to 12, preferably, n is 2, 3, 4, 5 or 6.
  • y and z are each independently an integer of 2 to 10 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably, 4 to 9 an integer of .
  • R 6 is independently H, OH or C 1 -C 6 alkyl (such as methyl, ethyl, n-propyl, n-butyl) at each occurrence, especially H.
  • R 5 is H or C 1 -C 6 alkyl (such as methyl, ethyl, n-propyl, n-butyl), especially H.
  • R 4 is C 1 -C 6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl.
  • the present invention also provides a lipid compound with the following structure, said compound having the following structure:
  • Another aspect of the present invention provides the application of the lipid compound in delivering biologically active substances to cells or organs.
  • Another aspect of the present invention provides the application of the lipid compound in the preparation of a bioactive substance delivery system.
  • the biologically active substances may be small molecular compounds, nucleic acids, oligopeptides and the like.
  • the biologically active substance is nucleic acid.
  • the biologically active substance is DNA or RNA.
  • the DNA includes non-coding DNA (antisense DNA) or coding DNA.
  • RNA includes antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA and the like.
  • the nucleic acid is used to prevent and/or treat cancer, inflammation, fibrotic disease, autoimmune disease, infection, mental disorder, blood disease, chromosomal disease, genetic disease, connective tissue disease, digestive disease, ear, nose and throat disease, endocrine disease, eye disease, reproductive disease, heart disease, kidney disease, lung disease, metabolic disease, oral disease, musculoskeletal disease, newborn screening, nutritional disease, parasitic disease, skin disease, etc.
  • the present invention also provides the application of the lipid compound for delivering siRNA to cells or organs.
  • the present invention also provides the use of the lipid compound in the preparation of a lipid or lipid nanoparticle delivery system.
  • the present invention also provides the application of the lipid compound for delivering mRNA vaccines to cells or organs.
  • the present invention also provides the application of the lipid compound in the preparation of mRNA vaccines.
  • the vaccine can be used to prevent cancer, viral infection, bacterial infection, fungal infection and the like.
  • the viruses include, but are not limited to: Norovirus, Ebola virus, coronavirus (including novel coronavirus SARS-CoV-2), cytomegalovirus, dengue virus, Zika virus, Coxsackie virus, enterovirus , hepatitis virus, herpes simplex virus, human papilloma virus, influenza virus, Marburg virus, measles virus, polio virus, rabies virus, rotavirus, measles virus, etc.
  • the mRNA vaccine is a SARS-CoV-2 mRNA vaccine.
  • Another aspect of the present invention provides a lipid composition, which comprises biologically active substances and the lipid compound described in the present invention.
  • the preparation method of the lipid composition can adopt conventional methods in the art, such as heating method, reverse evaporation method, or mixing method.
  • the heating method includes adding the organic solvent solution of the lipid compound to the aqueous solution of the biologically active substance to obtain a mixed solution, and heating the mixed solution at an appropriate temperature.
  • the heating temperature is 25°C-100°C.
  • the heating time is 10 minutes to 24 hours.
  • the reverse evaporation method includes mixing the aqueous solution of the biologically active substance and the organic solvent solution of the lipid compound to obtain a mixed solution.
  • Another aspect of the present invention provides a lipid nanoparticle, which comprises biologically active substances and the lipid compound described in the present invention.
  • the lipid nanoparticle further includes polyethylene glycol lipids, steroidal lipids and neutral lipids.
  • polyethylene glycol lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dicarotene Myristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)]( PEG-DSPE), PEG-Disterolylglycerol (PEG-DSG), PEG-Dipalmitoleyl, PEG-Dioleyl, PEG-Distearyl, PEG-Diacylglyceramide (PEG-DAG), PEG - dipalmitoylphosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
  • AAC-0159 2-[(polyethylene glycol)-2000]-N,
  • the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dicarnoyl Myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) , 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoy
  • the steroidal lipids are selected from the group consisting of avenasterol, ⁇ -sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydrocholesterol, Ergocalciferol, Dihydrocholesterol, Dihydroergosterol, Black Histerol, Epicholesterol, Ergosterol, Fucosterol, Hexahydrophotosterol, Hydroxycholesterol; Lanosterol, Photosterol, Seawesterol, Sitosterol, Sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.
  • polyethylene glycol lipid in the lipid nanoparticles of the present invention is ALC-0159, and/or the steroid lipid is cholesterol, and/or the neutral lipid is DSPC.
  • polyethylene glycol lipid in the lipid nanoparticles of the present invention is DMG-PEG2000, and/or the steroid lipid is cholesterol, and/or the neutral lipid is DSPC.
  • the lipid compound described in the present invention is
  • the molar ratio of the lipid compounds, neutral lipids, steroidal lipids, and polyethylene glycol lipids of the present invention is (40-60):( 5-20): (30-50): (0.5-5), preferably (45-55): (8-12): (35-45): (1-2); in an embodiment of the present invention Among them, the molar ratio is 49:10:39.5:1.5.
  • the lipid nanoparticles of the present invention can be prepared by conventional methods for preparing lipid nanoparticles in the art, such as high-pressure homogenization, emulsification and precipitation, ultrasonic dispersion, and the like.
  • Another aspect of the present invention provides a pharmaceutical composition, which includes the lipid composition or the lipid nanoparticle of the present invention, and pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable auxiliary materials described in the present invention are, for example, carriers, adjuvants, diluents and the like.
  • the lipid composition, lipid nanoparticle or pharmaceutical composition of the present invention can deliver the bioactive substance through oral administration, inhalation or injection.
  • Another aspect of the present invention provides a method of delivering biologically active substances, the method administers the lipid mixture of the present invention, the lipid nanoparticles of the present invention or the lipid nanoparticles of the present invention to a population in need. pharmaceutical composition.
  • the mRNA LNP has better stability and transfection efficiency, and can cause a higher specific antibody response in experimental animals.
  • Figure 1 shows the Zeta potential detection spectrum of sample 1.
  • Figure 2 shows the Zeta potential detection spectrum of sample 2.
  • Figure 3 shows the Zeta potential detection spectrum of sample 3.
  • Figure 4 shows the Zeta potential detection spectrum of sample 4.
  • Figure 5 shows the Zeta potential detection spectrum of sample 5.
  • Figure 6 shows the Zeta potential detection spectrum of sample 6.
  • Figure 7 shows the Zeta potential detection spectrum of sample 7.
  • Figure 8 shows the Zeta potential detection spectrum of sample 8.
  • Figure 9 shows the Zeta potential detection spectrum of sample 9.
  • Figure 10 shows the zeta potential detection spectrum of sample 10.
  • Fig. 11 shows the zeta potential detection spectrum of sample 11.
  • Figure 12 shows the zeta potential detection spectrum of sample 12.
  • Figure 13 shows the average particle size detection spectrum of sample 1.
  • Figure 14 shows the average particle size detection spectrum of sample 2.
  • Figure 15 shows the average particle size detection spectrum of sample 3.
  • Figure 16 shows the average particle size detection spectrum of sample 4.
  • Figure 17 shows the average particle size detection spectrum of sample 5.
  • Figure 18 shows the average particle size detection spectrum of sample 6.
  • Figure 19 shows the average particle size detection spectrum of sample 7.
  • Figure 20 shows the average particle size detection spectrum of sample 8.
  • Figure 21 shows the average particle size detection spectrum of sample 9.
  • Figure 22 shows the average particle size detection spectrum of sample 10.
  • Figure 23 shows the average particle diameter detection spectrum of sample 11.
  • Figure 24 shows the average particle size detection spectrum of sample 12.
  • Figure 25 shows the encapsulation efficiency of LNP-mRNA prepared by different cationic lipids.
  • Figure 26 shows the average particle size of LNP-mRNA prepared with different cationic lipids.
  • Figure 27 shows the PDI of LNP-mRNA prepared with different cationic lipids.
  • Figure 28 shows the in vitro expression results of LNP-mRNA prepared by different cationic lipids.
  • Figure 29 shows the S protein-specific antibody titer (LOG value) 14 days after the primary immunization. Data significance was analyzed by Tukey's multiple comparisons method.
  • Figure 30 shows the S protein-specific antibody titer (LOG value) 14 days after the second immunization. Data significance was analyzed by Tukey's multiple comparisons method.
  • Figure 31 shows the inhibition rates of the four groups of mixed samples diluted with different multiples detected with the original strain RBD protein 14 days after the second immunization.
  • the protein used for ELISA detection is ACE2-Fc
  • the protein used for competition is the RBD protein of the original strain.
  • Figure 32 shows the inhibition rates of the 4 groups of mixed samples diluted with different multiples detected with the RBD protein of the Beta mutant strain 14 days after the second immunization.
  • the protein used for ELISA detection is ACE2-Fc
  • the protein used for competition is the RBD protein of the Beta variant.
  • Figure 33 shows the specific CD8+ T cell immune response detected by ICS (Intracellular Cytokine Staining, intracellular cytokine staining).
  • N is the negative control group.
  • ns means that there is no significant difference after multiple comparison analysis with basic method, **** means p value is less than 0.0001.
  • Figure 34 shows the detection of specific CD4+ T cell immune response by ICS method.
  • N is the negative control group.
  • ns means that there is no significant difference after multiple comparison analysis using the basic method.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA, and hybrids thereof.
  • lipid refers to a group of organic compounds which include but are not limited to esters of fatty acids and are generally characterized as being poorly soluble in water but soluble in many organic solvents.
  • cationic lipid refers to a lipid molecule capable of being positively charged.
  • neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
  • polyethylene glycol lipid refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
  • lipid nanoparticle refers to a particle having at least one nanoscale size, which comprises at least one lipid.
  • vaccine in the present invention refers to a composition suitable for application to animals (including humans), which induces an immune response after administration, and its strength is sufficient to help prevent, ameliorate or cure clinical diseases caused by microbial infection at a minimum.
  • delivery system in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dose in a living body.
  • 6-Bromo-n-hexanol (0.91g, 5.0mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (0.90g, 7.5mmol) was added, and then phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, 2-hexyldecanol (1.36g, 5.6mmol) was added to the reaction solution, the mixture was stirred at room temperature overnight, after TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexyl (2-hexyldecyl) carbonate 1a (1.53g, light yellow oil), Yield 68%.
  • Heptadecan-9-yl (7-((2-hydroxyethyl)amino)heptyl)carbonate (457mg, 1.0mmol) was dissolved in tetrahydrofuran, acetonitrile was added, 5-bromopentylundecylcarbonate Ester (437mg, 1.2mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h.
  • 6-Bromo-n-hexanol (0.91g, 5.0mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (0.90g, 7.5mmol) was added, and then phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, and undecyl alcohol (0.97g, 5.6mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight.
  • compound 11b (1.70 g, 2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg, 10 mmol) in anhydrous THF (10 ml), and the mixture was heated to reflux for 5 hours. After the reaction is complete, lower the temperature and add water to the system to completely decompose the excess reducing agent. After filtration, the filter residue was washed with ethyl acetate, and the obtained filtrate was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 11 (1.45 g, yellow oil) with a yield of 90%.
  • Cationic lipids I-II prepared in Examples 1 and 2 of the present invention and control lipids III-IV were respectively used to prepare novel coronavirus mRNA lipid complexes.
  • the structures of the four cationic lipids are shown in the table below.
  • the preparation process of the novel coronavirus lipid nanoparticle mRNA vaccine is as follows: dilute the mRNA stock solution with sodium acetate buffer to a concentration of 135 ⁇ g/ml, according to the molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG 2000 is 49:10:39.5:1.5 Prepare the lipid mixture solution; after the encapsulation is completed on the nanomedicine manufacturing equipment, the liquid is changed by ultrafiltration and the sample is collected. Sampling was taken to test the encapsulation efficiency, average particle size, PDI and Zeta potential. The test results are shown in the table below and Figure 1-24.
  • Cationic lipid I was used for samples 1, 5, and 9; cationic lipid II was used for samples 2, 6, and 10; cationic lipid III was used for samples 3, 7, and 10; cationic lipid IV was used for samples 4, 8, and 12 .
  • the four LNP-mRNA samples 9, 10, 11, and 12 prepared in Example 12 were respectively placed in a constant temperature incubator at 25°C for 1, 2, 3, and 4 weeks to investigate their stability.
  • Cell plating prepare Hep3B cells, trypsinize them, adjust the cell density to 6.5 ⁇ 105cells/ml, inoculate 1ml/well into a 6-well cell culture plate, and add EMEM complete medium to 3ml. Place in a 37°C, 5% CO2 cell culture incubator for overnight.
  • Cell harvesting and lysis (1) Pipette the cells until they completely fall off the bottom of the culture plate, centrifuge the cell suspension at 1000 rpm for 5 minutes, and discard the supernatant. (2) Add 150 ⁇ l RIPA lysate to mix the cell pellet by pipetting, then lyse on ice for 30 minutes, and vortex 4-6 times during the period. (3) After lysing, the cells were centrifuged at 15,000 ⁇ g at 4°C for 20 minutes, and the supernatant was transferred to a new EP tube, and stored in a -20°C refrigerator for future use. (4) Take the sample in (3) and add the corresponding volume of 6 ⁇ Protein Loading Buffer to mix well, put it in a boiling water bath or 100°C for 5 minutes, and wait for the test.
  • Electrophoresis Add the sample to be tested and the protein marker into the sample tank of the protein electrophoresis gel. Constant pressure, stop the electrophoresis when the bromophenol blue indicator runs out of the bottom of the gel.
  • Transfer membrane transfer the protein on the protein electrophoresis gel to the nitrocellulose membrane.
  • Blocking Place the nitrocellulose membrane in the blocking solution and block overnight at 2-8°C or on a shaker at room temperature for 1-2 hours.
  • Primary antibody incubation immerse the nitrocellulose membrane in a certain concentration of novel coronavirus S protein-specific antibody, and incubate overnight at 2-8°C or on a shaker at room temperature for 2 hours.
  • Membrane washing Wash the membrane 3 times with PBST washing solution, 5 minutes each time.
  • Primary antibody incubation immerse the nitrocellulose membrane in a certain concentration of horseradish peroxidase-labeled goat anti-rabbit IgG, and incubate on a shaking table at room temperature for 1 hour.
  • Membrane washing Wash the membrane 6 times with PBST washing solution, 5 minutes each time.
  • Imaging Add a luminescent substrate for imaging.
  • Example 15 The mouse immunization and detection of LNP-mRNA prepared by different cationic lipids
  • mice Six to eight-week-old female BALB/c mice were randomly divided into 5 groups, and immunized by intramuscular injection in the hind legs. Among them, animals in groups 1, 2, 3, and 4 were respectively immunized with samples 9, 10, 11, and 12 (prepared in Example 12); and group 5 was used as a negative control group and immunized with physiological saline. They were immunized on day 0 and day 14 respectively, and the single immunization dose was 5 ⁇ g mRNA-LNP. Blood was collected on the 14th and 28th days of immunization and serum was separated; mice were dissected after blood collection on the 28th day of immunization and splenocytes were separated.
  • the serum samples on the 14th day of immunization were tested for the specific antibody titer against the novel coronavirus S protein by ELISA.
  • the test results are shown in Figure 29, all four samples elicited high specific antibody responses, and there was no significant difference in the levels of antibody responses among the groups.
  • the serum samples on the 28th day of immunization were also tested for specific antibody titers against the novel coronavirus S protein by ELISA.
  • the test results are shown in Figure 30, the four samples all elicited high specific antibody responses, and the antibody response level induced by group 1 (sample 9) was the highest, which was not significantly different from that of group 2 (sample 10). However, it was significantly higher than that of positive control samples in groups 3 and 4 (samples 11 and 12).
  • the neutralizing antibody titer was detected by competitive ELISA.
  • the protein used for ELISA detection is ACE2-Fc
  • the competing proteins are the RBD protein of the original strain and the RBD protein of the Beta mutant strain.
  • the neutralizing antibody titers of the 4 groups of mixed samples detected with the original strain RBD protein were 2591, 2644, 1707 and 1313, respectively.
  • the neutralizing antibody titers of the 4 groups of mixed samples detected by the Beta mutant strain RBD protein were 4144, 3542, 3534 and 2567, respectively.
  • the trend of the detection results of the two RBD proteins is consistent, and the trend of the antibody titer detection results is also consistent. It was demonstrated that formulations containing lipid compounds of the present invention elicited higher antibody responses compared to positive control samples.
  • the splenocytes isolated after mouse dissection were stimulated with the full-length overlapping peptide library of S protein, and the antigen specificity was detected by ICS method, and the CD4+ cells secreting IL-2, IFN ⁇ , TNF ⁇ , IL-4 and IL-5 Frequency of T cells and CD8+ T cells.
  • the cellular immune response of specific CD8+ T cells is shown in Figure 33.
  • the immune responses induced by all experimental groups were Th1-biased immune responses, and there were basically no CD8+ cells secreting IL-2, IL-4, and IL-5. T cells. There was no significant difference in the proportion of IFN ⁇ secretion-specific CD8+ T cells and TNF ⁇ secretion-specific CD8+ T cells among mice in each experimental group.
  • the cellular immune response of specific CD4+ T cells is shown in Figure 34.
  • the immune responses induced by all experimental groups were Th1-biased immune responses, and there were basically no CD4+ T cells secreting IL-4 and IL-5.
  • the ratios of IFN ⁇ secretion-specific CD8+ T cells, TNF ⁇ secretion-specific CD8+ T cells and IL-2 secretion-specific CD8+ T cells in groups 1 to 2 were higher than those in groups 3 to 4 ( Samples 11, 12) have no significant difference, but the mean value is higher. It was demonstrated that the specific cellular immune response elicited by the formulation containing the lipid compound of the present invention was not inferior to that of the corresponding positive control sample.

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Abstract

提供了一种用于核酸递送的式I的可电离脂质化合物及其LNP组合物，可以高效稳定的将生物活性物质递送至靶细胞或器官。以该脂质化合物作为阳离子脂质制得的mRNA LNP具有较佳的稳定性和转染效率，在实验动物体内可引起较高的特异性抗体应答。

Description

一种用于核酸递送的新型可电离脂质及其LNP组合物 技术领域
本发明属于生物医药领域，具体涉及一种用于核酸递送的新型可电离脂质以及在生物活性物质递送中的应用。
背景技术
基因治疗是指利用分子生物学的方法将目的基因导入患者体内使之表达，以纠正或补偿因基因缺陷和异常引起的疾病，也指将核酸导入细胞内抑制目的基因的表达(基因沉默)或增加目的基因的表达(基因激活)，以达到治疗疾病的目的。基因治疗作为现代医学和分子生物学相结合的疾病治疗的新方法，逐步在医疗领域占据着重要的地位。目前利用“表达某种基因”、“表达某种蛋白”进行治疗主要通过质粒DNA、mRNA的导入来实现，利用“抑制某种基因”进行治疗则主要通过siRNA或微小RNA(microRNA,miRNA)(即RNAi技术)来实现。
基于siRNA、mRNA的基因治疗手段具有其他类型核酸药物所没有的独特优势，然而mRNA和siRNA开发的共同难点就是如何将其有效地递送到靶部位的细胞中去。
目前递送核酸的系统多采用不同种类的脂质化合物递送方式，例如脂质纳米颗粒LNP(lipid nanoparticle)、GalNac、LPP(lipopolyplexes)等。LNP一般由四种脂质通过一定的比例制备而成，通常这四种脂质包含阳离子脂质、中性脂质、甾类脂质和聚合物缀合脂质。其中，阳离子脂质体带正电荷，与内涵体中带负电荷的膜脂质发生静电相互作用，膜脂质由内涵体的腔外翻转到腔内，与正电荷形成中性电子对，基因药物脱离阳离子脂质体进入细胞核。
专利文献CN110352071A和CN1882693A公开了阳离子脂质化合物及应用脂质制备得到脂质纳米颗粒或脂质混合物，用于将生物活性物质递送至体内。
专利文献US20200197510A1公开了呼吸道病毒核糖核酸疫苗和组合疫苗，以及使用疫苗和包含疫苗的组合物的方法。
尽管现有技术已经报道了一系列阳离子脂质化合物，但是仍然需要提供具有高效和稳定递送性能的脂质化合物。
发明内容
本发明的第一方面提供了一种脂质化合物，所述的脂质化合物具有如下式I结构：
Figure PCTCN2022091881-appb-000001
其中：
L 1和L 2中的至少一个为-O(C＝O)O-或-NRa-，并且
L 1或L 2中的另一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-、-NRa-、-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-S(O)x-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRaC(＝O)NRa-、-OC(＝O)NRa-或-NRaC(＝O)O-；
G 1和G 2各自独立地为未取代的C 1-C 12亚烷基或C 1-C 12亚烯基；
G 3为C 1-C 24亚烷基、C 1-C 24亚烯基、C 3-C 8亚环烷基、C 3-C 8亚环烯基；
Ra为H或C 1-C 12烃基；
R 1和R 2各自独立地为C 6-C 24烷基或C 6-C 24烯基；
R 3为H、OH、OR 4、CN、-C(＝O)OR 4、-OC(＝O)R 4或–NR 5C(＝O)R 4；
R 4为C 1-C 12烃基；
R 5为H或C 1-C 6烃基；
x为0、1或2。
进一步的，所述的Ra为H或C 1-C 6烷基(例如甲基、乙基、正丙基、正丁基)，特别是H。
进一步的，所述的式I结构中L 1和L 2各自独立地选自-O(C＝O)O-和-NH-。
进一步的，所述的式I结构中L 1和L 2为-O(C＝O)O-，或者，L 1和L 2为-NH-。
进一步的，所述的式I结构中R 1和R 2各自独立地具有以下结构：
Figure PCTCN2022091881-appb-000002
其中，R 7a和R 7b在每次出现时独立地为H或C 1-C 12烃基；
并且，a为2至12的整数(例如2、3、4、5、6、7、8、9、10、11、12)，优选的，a为8至12的整数；
进一步的，所述的式Ⅱ结构中R 7a、R 7b和a各自被选择为使得R 1和R 2各自独立地包含6至20个碳原子。
进一步的，所述的式Ⅱ结构中至少一次出现的R 7a为H，优选的，R 7a在每次出现时为H。
进一步的，所述的式Ⅱ结构中至少一次出现的R 7b为C 1-C 8烃基，特别是C 1-C 8烷基，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。
进一步的，所述的式I结构中R 1或R 2或两者具有以下结构之一：
Figure PCTCN2022091881-appb-000003
进一步的，本发明所述的脂质化合物具有以下结构(IA)：
Figure PCTCN2022091881-appb-000004
进一步的，所述的式IA结构中R 6在每次出现时独立地为H、OH或C 1-C 24烃基；
进一步的，所述的式IA结构中n为1至15的整数(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15)。
进一步的，本发明所述的脂质化合物具有以下结构(IB)：
Figure PCTCN2022091881-appb-000005
其中y和z各自独立地为1至12的整数。
进一步的，所述的式IB结构中n为2至12的整数，优选的，n为2、3、4、5或6。
进一步的，所述的式IB结构中y和z各自独立地为2至10的整数(例如2、3、4、5、6、7、8、9、10)，优选的，为4至9的整数。
进一步的，所述的式IA结构中R 6在每次出现时独立地为H、OH或C 1-C 6烷基(例如甲基、乙基、正丙基、正丁基)，特别是H。
进一步的，所述的R 5为H或C 1-C 6烷基(例如甲基、乙基、正丙基、正丁基)，特别是H。
进一步的，所述的R 4为C 1-C 6烷基，例如甲基、乙基、正丙基、异丙基、正丁基。
进一步的，所述的R 3选自：H、OH、OCH 3、OCH 2CH 3、CN、-C(＝O)OCH 3、-C(＝O)OCH 2CH 3、-OC(＝O)CH 3、-OC(＝O)CH 2CH 3、-NHC(＝O)CH 3、-NHC(＝O)CH 2CH 3。
本发明还提供了如下结构的脂质化合物，所述的化合物具有如下结构：
Figure PCTCN2022091881-appb-000006
Figure PCTCN2022091881-appb-000007
本发明的还一方面提供了所述的脂质化合物在递送生物活性物质至细胞或器官的应用。
本发明的还一方面提供了所述的脂质化合物在制备生物活性物质递送系统中的应用。
进一步的，所述的生物活性物质可以为小分子化合物、核酸、寡肽等。优选的，所述的生物活性物质为核酸。
进一步的，所述的生物活性物质为DNA或RNA。
进一步的，所述DNA包括非编码DNA(反义DNA)或编码DNA。
进一步的，所述RNA包括反义RNA、saRNA、mRNA、lncRNA、miRNA、siRNA、piRNA、gRNA、tsRNA等。
进一步的，所述核酸用于预防和/或治疗癌症、炎症、纤维化疾病、自身免疫病、感染、精神性病症、血液病、染色体疾病、遗传病、结缔组织疾病、消化性疾病、耳鼻喉疾病、内分泌疾病、眼病、生殖性疾病、心脏病、肾病、肺病、代谢性病症、口部疾病、肌肉骨骼疾病、新生儿筛查、营养性疾病、寄生虫疾病、皮肤疾病等。
进一步的，本发明还提供了所述的脂质化合物用于递送siRNA至细胞或器官的应用。
进一步，本发明还提供了所述的脂质化合物用于制备脂质或脂质纳米颗粒递送系统。
进一步，本发明还提供了所述的脂质化合物用于递送mRNA疫苗至细胞或器官的应用。
进一步，本发明还提供了所述的脂质化合物用于制备mRNA疫苗的应用。优选的，所述的疫苗可以用于预防癌症、病毒感染、细菌感染、真菌感染等。所述的病毒包括但不限于：诺如病毒、埃博拉病毒、冠状病毒(包括新型冠状病毒SARS-CoV-2)、巨细胞病毒、登革热病毒、寨卡病毒、柯萨奇病毒、肠病毒、肝炎病毒、单纯疱疹病毒、人乳头瘤病毒、流感病毒、马尔堡病毒、麻疹病毒、脊髓灰质炎病毒、狂犬病病毒、轮状病毒、麻疹病毒等。在本发明的一个实施例中，所述mRNA疫苗为SARS-CoV-2mRNA疫苗。
本发明还一方面提供了一种脂质组合物，所述的脂质组合物包含生物活性物质和本发明所述的脂质化合物。
进一步的，所述的脂质组合物的制备方法可以采用本领域的常规方法，例如加热法、逆向蒸发法，或者混合法。
进一步的，所述加热法包括将脂质化合物的有机溶剂溶液加入到生物活性物质的水溶液中得 到混合溶液，并在适当的温度下加热所述混合溶液。优选的，所述的加热温度为25℃-100℃。优选的，所述的加热时间为10分钟-24小时。
进一步的，所述的逆向蒸发法包括将生物活性物质的水溶液与脂质化合物的有机溶剂溶液进行混合以得到混合溶液。
本发明还一方面提供了一种脂质纳米颗粒，所述的脂质纳米颗粒包含生物活性物质和本发明所述的脂质化合物。
进一步的，所述的脂质纳米颗粒，所述的脂质纳米颗粒中还包含聚乙二醇脂质、甾族脂质和中性脂质。
进一步的，所述的聚乙二醇脂质选自2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)或PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)。
进一步的，所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)。
进一步的，所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸。
进一步的，本发明所述的脂质纳米颗粒中聚乙二醇脂质为ALC-0159，和/或，甾族脂质为胆固醇，和/或，中性脂质为DSPC。
进一步的，本发明所述的脂质纳米颗粒中聚乙二醇脂质为DMG-PEG2000，和/或，甾族脂质为胆固醇，和/或，中性脂质为DSPC。
在本发明的实施例中，上述脂质纳米颗粒中，本发明所述的脂质化合物为
Figure PCTCN2022091881-appb-000008
进一步的，本发明所述的脂质纳米颗粒中，本发明所述的脂质化合物、中性脂质、甾族脂质、聚乙二醇脂质的摩尔比为(40-60)：(5-20)：(30-50)：(0.5-5)，优选为(45-55)：(8-12)：(35-45)：(1-2)；在本发明的一个实施例中，该摩尔比为49：10：39.5：1.5。
本发明的脂质纳米颗粒可以采用本领域常规的脂质纳米颗粒制备方法制备得到，例如高压乳匀法、乳化沉淀法、超声分散法等。
本发明还一方面提供了一种药物组合物，所述的药物组合物包括本发明所述的脂质组合物或者本发明所述的脂质纳米颗粒，以及药学上可接受的辅料。
本发明所述的药学上可接受的辅料例如是载体、佐剂、稀释剂等。
本发明所述的脂质组合物、脂质纳米颗粒或药物组合物可以通过口服、吸入或注射的方式递送所述生物活性物质。
本发明还一方面提供了一种递送生物活性物质的方法，所述的方法向有需要的人群施用本发明所述的脂质混合物、本发明所述的脂质纳米颗粒或本发明所述的药物组合物。
采用本发明所述的脂质化合物制成PEG-脂质/阳离子脂质/中性脂质/甾族脂质-mRNA纳米颗粒 (LNP)，显示本发明所述的脂质化合物作为阳离子脂质的mRNA LNP具有较佳的稳定性和转染效率，在实验动物体内可引起较高的特异性抗体应答。
附图说明
图1所示为样品1的Zeta电位检测谱图。
图2所示为样品2的Zeta电位检测谱图。
图3所示为样品3的Zeta电位检测谱图。
图4所示为样品4的Zeta电位检测谱图。
图5所示为样品5的Zeta电位检测谱图。
图6所示为样品6的Zeta电位检测谱图。
图7所示为样品7的Zeta电位检测谱图。
图8所示为样品8的Zeta电位检测谱图。
图9所示为样品9的Zeta电位检测谱图。
图10所示为样品10的Zeta电位检测谱图。
图11所示为样品11的Zeta电位检测谱图。
图12所示为样品12的Zeta电位检测谱图。
图13所示为样品1的平均粒径检测谱图。
图14所示为样品2的平均粒径检测谱图。
图15所示为样品3的平均粒径检测谱图。
图16所示为样品4的平均粒径检测谱图。
图17所示为样品5的平均粒径检测谱图。
图18所示为样品6的平均粒径检测谱图。
图19所示为样品7的平均粒径检测谱图。
图20所示为样品8的平均粒径检测谱图。
图21所示为样品9的平均粒径检测谱图。
图22所示为样品10的平均粒径检测谱图。
图23所示为样品11的平均粒径检测谱图。
图24所示为样品12的平均粒径检测谱图。
图25所示为不同阳离子脂质制得的LNP-mRNA的包封率。
图26所示为不同阳离子脂质制得的LNP-mRNA的平均粒径。
图27所示为不同阳离子脂质制得的LNP-mRNA的PDI。
图28所示为不同阳离子脂质制得的LNP-mRNA体外表达结果。
图29所示为一免14天后的S蛋白特异性抗体滴度(LOG值)。数据显著性通过图基法多重比较分析。
图30所示为二免14天后的S蛋白特异性抗体滴度(LOG值)。数据显著性通过图基法多重比较分析。
图31所示为二免14天后用原始毒株RBD蛋白检测的4组混合样品稀释不同倍数后的抑制率。其中ELISA检测包板用蛋白为ACE2-Fc，竞争用蛋白为原始毒株的RBD蛋白。
图32所示为二免14天后用Beta变异株RBD蛋白检测的4组混合样品稀释不同倍数后的抑制率。其中ELISA检测包板用蛋白为ACE2-Fc，竞争用蛋白为Beta变异株的RBD蛋白。
图33所示为ICS(Intracellular Cytokine Staining，细胞内细胞因子染色)法检测特异性CD8+T细胞免疫应答。N为阴性对照组。ns指用基法多重比较分析后发现无显著性差异，****指p值小于0.0001。
图34所示为ICS法检测特异性CD4+T细胞免疫应答。N为阴性对照组。ns指用基法多重比较分析后发现无显著性差异。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明术语“核酸”是指呈单链或双链形式的含有至少两种脱氧核糖核苷酸或核糖核苷酸的聚合物，并且包括DNA、RNA及其杂交物。
本发明术语“脂质”是指一组有机化合物，其包括但不限于脂肪酸的酯，并且通常以难溶于水但可溶于许多有机溶剂为特征。
本发明术语“阳离子脂质”是指能够带正电的脂质分子。
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。
本发明术语“聚乙二醇脂质”是指包含脂质部分和聚乙二醇部分的分子。
本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物，在施用后诱导免疫应答，其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。
本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。
实施例1 化合物1的合成
Figure PCTCN2022091881-appb-000009
6-溴己基(2-己基癸基)碳酸酯(1a)的合成
Figure PCTCN2022091881-appb-000010
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入2-己基癸醇(1.36g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基(2-己基癸基)碳酸酯1a(1.53g，淡黄色油状物)，产率68％。
MS m/z(ESI):449.3[M+1]
化合物1的合成
Figure PCTCN2022091881-appb-000011
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物1(454mg，淡黄色油状物)，产率55％。MS m/z(ESI):826.9[M+1]
1H NMR(300MHz,CDCl 3):δ4.13(t,4H,J＝6.6Hz),4.05(d,4H,J＝5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J＝6.2Hz)
实施例2 化合物2的合成
Figure PCTCN2022091881-appb-000012
7-溴庚基十七烷-9-基碳酸酯(2a)的合成
Figure PCTCN2022091881-appb-000013
将7-溴庚醇(0.98g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入9-羟基十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到7-溴庚基十七烷-9-基碳酸酯2a(1.50g，淡黄色油状物)，产率65％。
MS m/z(ESI):477.3[M+1]
十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(2b)的合成
Figure PCTCN2022091881-appb-000014
室温条件下，将7-溴庚基十七烷-9-基碳酸酯(2a)(1.38g，3mmol)溶于20mL乙醇中，加入乙醇胺(2.75g，45mmol)，升温至50℃，搅拌8h，监控反应进程，原料消耗完全后降温至45℃旋干除去乙醇，用二氯甲烷溶解粗品，用饱和食盐水洗涤三次，有机相用无水硫酸钠干燥，浓缩得到产品十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯2b(1.35g，淡黄色油状物)。
MS m/z(ESI):458.4[M+1]
5-溴戊基十一烷基碳酸酯(2c)的合成
Figure PCTCN2022091881-appb-000015
将5-溴戊醇(0.84g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL 饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到5-溴戊基十一烷基碳酸酯2c(1.20g，淡黄色油状物)，产率66％。
MS m/z(ESI):365.2[M+1]
化合物2的合成
Figure PCTCN2022091881-appb-000016
将十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(457mg，1.0mmol)溶于四氢呋喃中，加入乙腈，5-溴戊基十一烷基碳酸酯(437mg，1.2mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物2(440mg，淡黄色油状物)，产率57％。
MS m/z(ESI):742.8[M+1]
1H NMR(300MHz,CDCl 3):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J＝5.4Hz),2.94(br,1H),2.58(t,2H,J＝5.4Hz),2.45(t,4H,J＝5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J＝6.3Hz)
实施例3 化合物3的合成
Figure PCTCN2022091881-appb-000017
6-溴己基十一烷基碳酸酯(3a)的合成
Figure PCTCN2022091881-appb-000018
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十一烷基碳酸酯3a(1.25g，淡黄色油状物)，产率66％。
MS m/z(ESI):379.2[M+1]
化合物3的合成
Figure PCTCN2022091881-appb-000019
将6-溴己基十一烷基碳酸酯(948mg，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物3(412mg，淡黄色油状物)，产率60％。
MS m/z(ESI):686.8[M+1]
1H NMR(300MHz,CDCl 3):δ4.13(t,8H,J＝6.6Hz),3.58(t,2H,J＝5.7Hz),2.52(t,6H,J＝8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J＝6.2Hz)
实施例4 化合物4的合成
Figure PCTCN2022091881-appb-000020
6-溴己基十七烷-9-基碳酸酯(4a)的合成
Figure PCTCN2022091881-appb-000021
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入9-十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十七烷-9-基碳酸酯4a(1.53g，淡黄色油状物)，产率66％。
MS m/z(ESI):464.3[M+1]
化合物4的合成
Figure PCTCN2022091881-appb-000022
将6-溴己基十七烷-9-基碳酸酯(1.16g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg,2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物4(502mg，淡黄色油状物)，产率59％。
MS m/z(ESI):855.4[M+1]
1H NMR(300MHz,CDCl 3):δ4.71-4.68(m,2H),4.13(t,4H,J＝6.6Hz),3.57(t,2H,J＝5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J＝6.3Hz)
实施例5 化合物5的合成
Figure PCTCN2022091881-appb-000023
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物5(487mg，淡黄色油状物)，产率61％。
MS m/z(ESI):798.9[M+1]
1H NMR(300MHz,CDCl 3):δ4.14(t,4H,J＝6.6Hz),4.04(d,4H,J＝5.7Hz),3.54(t,2H,J＝5.4Hz),2.58(t, 2H,J＝5.4Hz),2.46(t,4H,J＝7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J＝6.2Hz)
实施例6 化合物6的合成
Figure PCTCN2022091881-appb-000024
将5-溴戊基十一烷基碳酸酯(910mg，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物6(410mg，淡黄色油状物)，产率65％。
MS m/z(ESI):630.7[M+1]
1H NMR(300MHz,CDCl 3):δ4.10(t,8H,J＝6.6Hz),3.52(d,2H,J＝5.4Hz),2.83(br,1H),2.57(t,2H,J＝5.4Hz),2.45(t,4H,J＝7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J＝6.2Hz)
实施例7 化合物7的合成
Figure PCTCN2022091881-appb-000025
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-甲氧基丙胺(89mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物7(495mg，淡黄色油状物)，产率60％。
MS m/z(ESI):826.7[M+1]
实施例8 化合物8的合成
Figure PCTCN2022091881-appb-000026
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-氨基丙腈(70mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物8(469mg，淡黄色油状物)，产率58％。
MS m/z(ESI):807.7[M+1]
实施例9 化合物9的合成
Figure PCTCN2022091881-appb-000027
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基丁酸乙酯盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物9(546mg，淡黄色油状物)，产率63％。
MS m/z(ESI):868.8[M+1]
实施例10 化合物10的合成
Figure PCTCN2022091881-appb-000028
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，N-(4-氨基丁基)-乙酰胺盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物10(560mg，淡黄色油状物)，产率69％。
MS m/z(ESI):867.8[M+1]
实施例11 化合物11的合成
Figure PCTCN2022091881-appb-000029
8-溴-N-(十七烷-9-基)辛酰胺(11a)的合成
Figure PCTCN2022091881-appb-000030
将8-溴辛酸(1.12g，5.0mmol)溶于50mL二氯甲烷中，在0℃下分批加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1.05g，5.5mmol)，搅拌30min后，在反应液中逐滴加入9-氨基十七烷(1.28g，5.0mmol)，滴加完毕后，混合物在室温下搅拌过夜，TLC显示反应完成后，用100ml水洗涤2次，有机相用无水硫酸钠干燥，过滤并浓缩，得到化合物11a(1.95g，黄色油状物)，产率82％。
MS m/z(ESI):461.3[M+1]。
化合物11b的合成
Figure PCTCN2022091881-appb-000031
将8-溴-N-(十七烷-9-基)辛酰胺(1.15g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室 温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物11b(534mg，淡黄色油状物)，产率63％。
MS m/z(ESI):848.8[M+1]；
1H NMR(300MHz,CDCl 3):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J＝6.2Hz),2.14(t,4H,J＝4.8Hz),1.57-1.47(t,14H,J＝6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J＝6.3Hz)。
化合物11的合成
Figure PCTCN2022091881-appb-000032
在0℃下，将化合物11b(1.70g，2mmol)缓慢加入四氢铝锂(379mg，10mmol)的无水四氢呋喃(10ml)溶液中，混合物加热回流5小时。反应完全后，降温，在体系中加入水使过量的还原剂完全分解。过滤，滤渣用乙酸乙酯洗涤，得到的滤液用水洗涤，经无水硫酸钠干燥，过滤并浓缩，得到化合物11(1.45g，黄色油状物)，产率90％。
MS m/z(ESI):820.8[M+1]；
1H NMR(300MHz,CDCl 3):δ4.11(s,1H),3.44(t,2H,J＝4.8Hz),3.32(s,2H),3.00(t,6H,J＝6.3Hz),2.52(t,4H,J＝6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J＝4.8Hz)。
实施例12 新型冠状病毒LNP-mRNA的制备
分别采用本发明实施例1和2制备的阳离子脂质I～II与对照脂质III～IV制备新型冠状病毒mRNA脂质复合物，四种阳离子脂质结构如下表所示。
表1：阳离子脂质结构式
Figure PCTCN2022091881-appb-000033
新型冠状病毒脂质纳米颗粒mRNA疫苗制备过程如下：醋酸钠缓冲液稀释mRNA原液至浓度为135μg/ml，按照阳离子脂质：DSPC：胆固醇：DMG-PEG 2000摩尔比为49：10：39.5：1.5配制脂质混合溶液；在纳米药物制造设备上完成包封后，超滤换液，收集样品。取样检测包封率、平均粒径、PDI及Zeta电位，检测结果如下表及图1-24所示。
表2：不同阳离子脂质包封后新型冠状病毒脂质纳米颗粒mRNA疫苗检测结果
Figure PCTCN2022091881-appb-000034
Figure PCTCN2022091881-appb-000035
注：样品1、5、9分别采用阳离子脂质I；2、6、10分别采用阳离子脂质II；3、7、10分别采用阳离子脂质III；4、8、12分别采用阳离子脂质IV。
由以上结果可以看出，在相同N/P条件下，利用阳离子脂质I、II所制得的样品1、2、5、6、9、10的包封率均高于利用对照阳离子脂质III、IV所制得的样品3、4、7、8、11、12，初步可以得出阳离子脂质I、II对mRNA抗原具有更好的包封效果。
实施例13 不同阳离子脂质制得的LNP-mRNA稳定性考察
分别取实施例12中所制备的样品9、10、11、12四个LNP-mRNA置于25℃恒温培养箱中分别放置1、2、3、4周考察其稳定性。
检测结果如图25-27所示，由以上结果可以看出，样品9、10在加速期间，包封率、平均粒径及PDI均无明显变化，而样品11、12在加速期间包封率有明显下降，平均粒径及PDI明显上升，初步可以得出阳离子脂质I、II所制得的LNP-mRNA稳定性较阳离子脂质III、IV更优。
实施例14 不同阳离子脂质制得的LNP-mRNA体外表达考察
取实施例12中所制备的样品9、10、11、12四个LNP-mRNA采用WB法进行供试品目的基因表达检测。
细胞铺板：准备Hep3B细胞，经胰酶消化后，调整细胞密度至6.5×105cells/ml，1ml/孔接种到6孔细胞培养板中，补加EMEM完全培养基至3ml。置于37℃、5％CO2细胞培养箱中培养，过夜。
加样：(1)清除工作环境中的RNase。将EMEM完全培养基更换为EMEM无血清培养基，3ml/孔。(2)于6孔细胞培养板中加入供试品，加入量25μl/孔(约2500ng mRNA)，同时设置阴性对照(空白对照)。(3)将细胞培养板置于37℃，5％CO 2细胞培养箱中培养22～26小时。
细胞收获与裂解：(1)吹打细胞至完全从培养板底部脱落，将细胞悬液1000rpm离心5分钟，弃上清。(2)加入150μl RIPA裂解液将细胞沉淀吹打混匀后，于冰上裂解30分钟，期间涡旋震荡4～6次。(3)裂解后细胞于4℃，15000×g离心20分钟，将上清转移至新的EP管中，放入-20℃冰箱保存，备用。(4)取(3)中样品加入相应体积的6×Protein Loading Buffer混匀，置于沸水浴或100℃，5min，待测。
电泳：将待测样品和蛋白Marker加入蛋白电泳胶的样品槽内。恒压，待溴酚蓝指示剂跑出胶底停止电泳。
转膜：将蛋白电泳胶上的蛋白转移至硝酸纤维素膜上。
封闭：将硝酸纤维素膜放在封闭液中，2～8℃过夜或室温摇床封闭1～2小时。
一抗孵育：将硝酸纤维素膜浸入一定浓度的新型冠状病毒S蛋白特异性抗体中，2～8℃过夜或室温摇床孵育2小时。
洗膜：用PBST洗液洗膜3次，5分钟/次。
一抗孵育：将硝酸纤维素膜浸入一定浓度的辣根过氧化物酶标记山羊抗兔IgG中，室温摇床孵育1小时。
洗膜：用PBST洗液洗膜6次，5分钟/次。
成像：加入发光底物，进行成像。
检测结果如图28所示，样品9、10、11、12在体外均可表达出蛋白条带，但样品9、10较样品11、12的蛋白条带更深，初步可以得出阳离子脂质I、II较阳离子脂质III、IV的体外转染效率更好。
实施例15 不同阳离子脂质制得的LNP-mRNA的小鼠免疫与检测
将6-8周龄的雌性BALB/c小鼠按6只/组随机分成5组，采用后腿肌肉注射的免疫途径进行免疫。其中，第1、2、3、4组动物分别免疫样品9、10、11、12(实施例12中制备)；第5组作为阴性对照组，免疫生理盐水。分别在第0天和第14天免疫，单次免疫剂量为5μg mRNA-LNP。免疫第14天和第28天采血并分离血清；免疫第28天采血后解剖小鼠并分离脾细胞。
免疫第14天的血清样品用ELISA法检测针对新型冠状病毒S蛋白的特异性抗体滴度。检测结果如图29所示，4个样品都引起了较高的特异性抗体应答，且各组间的抗体应答水平无显著性差异。免疫第28天的血清样品也用ELISA法检测了针对新型冠状病毒S蛋白的特异性抗体滴度。检测结果如图30所示，4个样品都引起了较高的特异性抗体应答，其中第1组(样品9)引起的抗体应答水平最高，与第2组(样品10)无显著性差异，但显著高于阳性对照样品第3、4组(样品11、12)。
每组第28天的免疫血清样品混合后，用竞争性ELISA法检测中和抗体滴度。其中ELISA检测包板用蛋白为ACE2-Fc，竞争用蛋白分别为原始毒株的RBD蛋白和Beta变异株的RBD蛋白。如图31所示，用原始毒株RBD蛋白检测的4组混合样品中和抗体滴度分别是2591、2644、1707和1313。如图32所示，用Beta变异株RBD蛋白检测的4组混合样品中和抗体滴度分别是4144、3542、3534和2567。两种RBD蛋白的检测结果的趋势一致，和抗体滴度检测结果的趋势也具有一致性。证明含本发明的脂质化合物的处方相比于阳性对照样品可以引起更高的抗体应答。
小鼠解剖后分离出的脾细胞用S蛋白全长的重叠肽库进行刺激，并分别用ICS法检测抗原特异性，分泌IL-2、IFNγ、TNFα、IL-4和IL-5的CD4+T细胞和CD8+T细胞的频数。特异性CD8+T细胞的细胞免疫应答情况如图33所示，所有实验组引起的免疫反应均为Th1类偏向的免疫反应，基本没有分泌IL-2、IL-4和IL-5的CD8+T细胞。各实验组小鼠的IFNγ分泌特异性CD8+T细胞和TNFα分泌特异性CD8+T细胞占比无显著性差异。特异性CD4+T细胞的细胞免疫应答情况如图34所示，所有实验组引起的免疫反应均为Th1类偏向的免疫反应，基本没有分泌IL-4和IL-5的CD4+T细胞。第1～2组(样品9、10)的IFNγ分泌特异性CD8+T细胞、TNFα分泌特异性CD8+T细胞和IL-2分泌特异性CD8+T细胞占比分别与第3～4组(样品11、12)无显著性差异，但均值更高。证明含本发明的脂质化合物的处方引起的特异性细胞免疫应答不劣于对应阳性对照样品。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (23)

  1. 一种脂质化合物，其特征在于，所述的脂质化合物具有如下式I结构：
    Figure PCTCN2022091881-appb-100001
    其中：
    L 1和L 2中的至少一个为-O(C＝O)O-或-NRa-，并且
    L 1或L 2中的另一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-、-NRa-、-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-S(O)x-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRaC(＝O)NRa-、-OC(＝O)NRa-或-NRaC(＝O)O-；
    G 1和G 2各自独立地为未取代的C 1-C 12亚烷基或C 1-C 12亚烯基；
    G 3为C 1-C 24亚烷基、C 1-C 24亚烯基、C 3-C 8亚环烷基、C 3-C 8亚环烯基；
    Ra为H或C 1-C 12烃基；
    R 1和R 2各自独立地为C 6-C 24烷基或C 6-C 24烯基；
    R 3为H、OH、OR 4、CN、-C(＝O)OR 4、-OC(＝O)R 4或–NR 5C(＝O)R 4；
    R 4为C 1-C 12烃基；
    R 5为H或C 1-C 6烃基；
    x为0、1或2。
  2. 如权利要求1所述的脂质化合物，其特征在于，其中L 1和L 2各自独立地选自-O(C＝O)O-和-NH-。
  3. 如权利要求1所述的脂质化合物，其特征在于，其中L 1和L 2为-O(C＝O)O-，或者，L 1和L 2为-NH-。
  4. 如权利要求1所述的脂质化合物，其特征在于，其具有以下结构(IA)：
    Figure PCTCN2022091881-appb-100002
    其中：
    R 6在每次出现时独立地为H、OH或C 1-C 24烃基；
    n为1至15的整数。
  5. 如权利要求4所述的脂质化合物，其特征在于，其具有以下结构(IB)：
    Figure PCTCN2022091881-appb-100003
    其中y和z各自独立地为1至12的整数。
  6. 如权利要求5所述的脂质化合物，其特征在于，其中n为2至12的整数，优选的，n为2、3、4、5或6；
    其中y和z各自独立地为2至10的整数。
  7. 如权利要求1所述的脂质化合物，其特征在于，其中R 1和R 2各自独立地具有以下结构：
    Figure PCTCN2022091881-appb-100004
    其中：
    R 7a和R 7b在每次出现时独立地为H或C 1-C 12烃基；并且
    a为2至12的整数；
    其中R 7a、R 7b和a各自被选择为使得R 1和R 2各自独立地包含6至20个碳原子。
  8. 如权利要求7所述的脂质化合物，其特征在于，其中至少一次出现的R 7a为H。
  9. 如权利要求7所述的脂质化合物，其特征在于，其中至少一次出现的R 7b为C 1-C 8烃基。
  10. 如权利要求7所述的脂质化合物，其特征在于，其中R 1或R 2或两者具有以下结构之一：
    Figure PCTCN2022091881-appb-100005
  11. 如权利要求1所述的脂质化合物，其特征在于，所述的化合物具有如下结构：
    Figure PCTCN2022091881-appb-100006
    Figure PCTCN2022091881-appb-100007
  12. 如权利要求1-11任一项所述的脂质化合物在制备生物活性物质递送系统中的应用。
  13. 如权利要求12所述的应用，其特征在于，所述的生物活性物质为DNA或RNA，所述RNA选自反义RNA、saRNA、mRNA、lncRNA、miRNA、siRNA、piRNA、gRNA、tsRNA。
  14. 如权利要求12所述的应用，其特征在于，所述的生物活性物质递送系统为脂质或脂质纳米颗粒递送系统。
  15. 如权利要求12所述的应用，其特征在于，所述的生物活性物质递送系统为mRNA疫苗。
  16. 如权利要求15所述的应用，其特征在于，所述的疫苗为用于预防癌症、病毒感染、细菌感染、真菌感染的疫苗。
  17. 如权利要求16所述的应用，其特征在于，所述的病毒选自：诺如病毒、埃博拉病毒、冠状病毒、巨细胞病毒、登革热病毒、寨卡病毒、柯萨奇病毒、肠病毒、肝炎病毒、单纯疱疹病毒、人乳头瘤病毒、流感病毒、马尔堡病毒、麻疹病毒、脊髓灰质炎病毒、狂犬病病毒、轮状病毒、麻疹病毒。
  18. 如权利要求16所述的应用，其特征在于，所述的病毒为SARS-CoV-2。
  19. 一种脂质组合物，其特征在于，所述的脂质组合物包含生物活性物质和权利要求1-11任一项所述的脂质化合物。
  20. 一种脂质纳米颗粒，其特征在于，所述的脂质纳米颗粒包含生物活性物质和权利要求1-11任一项所述的脂质化合物。
  21. 如权利要求20所述的脂质纳米颗粒，其特征在于，所述的脂质纳米颗粒中还包含聚乙二醇脂质、甾族脂质和中性脂质。
  22. 如权利要求21所述的脂质纳米颗粒，其特征在于，所述的聚乙二醇脂质选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)或PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)；
    和/或，所述的中性脂质选自：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)；
    和/或，所述的甾族脂质选自：燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸。
  23. 一种药物组合物，其特征在于，所述的药物组合物包括权利要求19所述的脂质组合物或者权利要求20-22任一项所述的脂质纳米颗粒，以及药学上可接受的辅料。
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