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WO2023273364A1 - 聚乙二醇脂质及其应用 - Google Patents

聚乙二醇脂质及其应用 Download PDF

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WO2023273364A1
WO2023273364A1 PCT/CN2022/076994 CN2022076994W WO2023273364A1 WO 2023273364 A1 WO2023273364 A1 WO 2023273364A1 CN 2022076994 W CN2022076994 W CN 2022076994W WO 2023273364 A1 WO2023273364 A1 WO 2023273364A1
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lipid
polyethylene glycol
group
virus
biologically active
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French (fr)
Inventor
林美娜
胡树振
朱长有
王庆彬
王杰
贾宏丽
陈晓萌
赵宣
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Tianjin Jenkem Technology Co Ltd
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Tianjin Jenkem Technology Co Ltd
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Priority to US18/573,699 priority Critical patent/US20240350640A1/en
Priority to EP22831196.5A priority patent/EP4342929A4/en
Publication of WO2023273364A1 publication Critical patent/WO2023273364A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33306—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a polyethylene glycol lipid compound and its application in the delivery of biologically active substances.
  • RNAi drugs have been developed rapidly, which has brought great development prospects to the field of biopharmaceuticals.
  • Alnylam of the United States has developed a series of gene drugs using RNAi technology and has successfully launched four RNAi drugs: Onpattro (patisiran), Givlaari (givosiran), Oxlumo (lumasiran) and Leavio (inclisiran), among which Leavio (inclisiran) is a drug for the treatment of hyperlipidemia
  • Onpattro patisiran
  • Givlaari givosiran
  • Oxlumo lumasiran
  • Leavio inclisiran
  • nucleic acid drugs Due to their own properties, such as negative charge, easy to be degraded by nucleases, etc., nucleic acid drugs cannot effectively penetrate the cell membrane into cells and are quickly degraded in vivo, so a good delivery system is required to stabilize the nucleic acid. delivered to the target site and take effect.
  • the common difficulty in the development of mRNA and siRNA is how to effectively deliver it to cells at the target site. During the delivery process, it is also necessary to consider how to avoid rapid clearance, avoid degradation by nucleases, improve escape after endocytosis, etc.
  • LNP lipid nanoparticle
  • GalNac GalNac
  • LPP lipopolyplexes
  • LNP is generally prepared from four kinds of lipids in a certain ratio, usually these four kinds of lipids include cationic lipids, neutral lipids, steroidal lipids and polymer-conjugated lipids, in which polymer-conjugated lipids Refers to polyethylene glycol lipids.
  • the preparation process of LNP is relatively complicated, and simply includes the preparation of lipid nanoparticles, RNA encapsulation and cultivation, dialysis, freeze-drying and other processes.
  • PEG Polyethylene glycol
  • patent documents CN106795096A, WO2005026372A1, WO2020061295A1, CN102665685A, US20210154148A1, WO2012099755A1, etc. all disclose polyethylene glycol lipid compounds and the preparation of lipid nanoparticles using lipids or lipid mixtures for the delivery of 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.
  • Patent document CN102068701A discloses the application of cleavable polyethylene glycol lipid derivatives in the preparation of PEGylated preparations that reduce or avoid accelerated blood clearance.
  • the present invention provides a novel polyethylene glycol lipid, which does not contain breakable bonds (such as ester bonds, amide bonds, etc.) The substance is delivered to the target cell or organ.
  • the novel polyethylene glycol lipid can be positively charged in a specific pH environment, and is more likely to form stable particles with biologically active substances, so that the biologically active substances can play a role in target cells or organs.
  • the first aspect of the present invention provides a kind of polyethylene glycol lipid, described polyethylene glycol lipid has following formula I structure:
  • R 1 and R 2 are the same or different long-chain hydrocarbon groups, preferably, the long-chain hydrocarbon groups contain 8-25 carbon atoms;
  • PEG is a polyethylene glycol chain
  • L 1 , L 2 and L 3 are absent or are non-cleavable linkers in vivo.
  • the long-chain hydrocarbon group in the present invention contains 11-17 carbon atoms, for example, the long-chain hydrocarbon group contains 11, 12, 13, 14, 15, 16 or 17 carbon atoms. In an embodiment of the present invention, the long-chain hydrocarbon group contains 14 carbon atoms.
  • the polyethylene glycol chain described in the present invention comprises -(OCH 2 CH 2 ) n - repeating unit, and the n is selected from integers ranging from 20 to 300, more preferably, the n is selected from 20- An integer of 228, particularly preferably said n is selected from an integer of 30-60, for example n is selected from 30, 35, 40, 45, 50, 55, 60.
  • the polyethylene glycol chain described in the present invention is -(OCH 2 CH 2 ) n -X, wherein X is the terminal group of the polyethylene glycol chain, selected from methyl, methoxy, hydroxyl , amino, mercapto, maleimide, etc.
  • X is the terminal group of the polyethylene glycol chain, selected from methyl, methoxy, hydroxyl , amino, mercapto, maleimide, etc.
  • the X is methoxy
  • the polyethylene glycol chain is -(OCH 2 CH 2 ) n -OCH 3 .
  • the in vivo non-cleavable linker of the present invention refers to a relatively stable linker that does not contain in vivo cleavable bonds, so it is not easy to degrade in the in vivo environment.
  • the in vivo unbreakable linker can be a thioether linker or an alkyl linker
  • the alkyl linker is, for example, a C1-C10 alkylene group, preferably a C2-C8 alkylene group, more It is preferably a C2-C5 alkylene group.
  • the alkylene group can be a straight chain or branched chain alkylene group. More preferably, the said alkylene group is a straight chain alkylene group, such as -CH 2 -, -CH2CH2- , -CH2CH2CH2- . _
  • Still another aspect of the present invention provides a kind of polyethylene glycol lipid, described polyethylene glycol lipid has following formula II structure:
  • R 1 , R 2 , X and n are as defined above,
  • i is selected from an integer of 2-7, for example, i is selected from 2, 3, 4, 5, 6 or 7, preferably, i is selected from 2 or 3.
  • polyethylene glycol lipid of the present invention has the following structure:
  • the polyethylene glycol lipid has the following structure:
  • X and n are as defined above, preferably, X is -OCH 3 .
  • the polyethylene glycol chain molecular weight of the present invention can be 1000 to 20000, preferably 1000 to 10000, such as 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000 , 7500, 8000, 8500, 9000, 9500, 10000, etc.
  • the polyethylene glycol lipid of the present invention is in a low pH solution, and the lipid will be ionized and positively charged, and is more likely to combine with negatively charged phosphate groups in RNA or mRNA.
  • Another aspect of the present invention provides a method for preparing polyethylene glycol lipids, said method comprising: obtaining the compound of formula II of the present invention by reducing the carbonyl group in the compound of the following formula IV,
  • R 1 , R 2 , X, i and n are as defined above.
  • the reducing agent in the reduction reaction is LiAlH 4 .
  • the reduction reaction includes: under nitrogen atmosphere, LiAlH 4 is added to the reaction device containing the first solvent, the compound of formula IV is dissolved in the second solvent, and added dropwise to the reaction device for reaction to obtain the compound of formula II compound.
  • the first solvent is preferably tetrahydrofuran.
  • the second solvent is preferably tetrahydrofuran.
  • the reduction reaction includes: under a nitrogen atmosphere, LiAlH 4 is slowly added to a reaction device containing tetrahydrofuran, the compound of formula IV is dissolved in tetrahydrofuran, and slowly added dropwise to the reaction device, 3 Hours later, water was added dropwise to the reaction system, filtered, and the filtrate was concentrated, and isopropanol and ethyl acetate were added, heated to dissolve, and cooled to crystallize.
  • the compound of formula IV is composed of the compound of formula III and Condensation reaction is carried out to obtain:
  • R 1 , R 2 , X, i and n are as defined above.
  • condensation reagent of the condensation reaction is preferably HATU (N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate) .
  • the condensation reaction includes: adding the compound of formula III and X-(OCH 2 CH 2 ) n -COOH into a reaction device containing a third solvent, adding DIEA (N,N-diisopropylethyl Amine) and HATU reaction, obtain formula IV compound.
  • DIEA N,N-diisopropylethyl Amine
  • the third solvent is preferably dichloromethane.
  • condensation reaction time is 3-8 hours. Preferably, the condensation reaction time is 5 hours.
  • the condensation reaction includes: adding the compound of formula III and X-(OCH 2 CH 2 ) n -COOH into a reaction device containing dichloromethane, stirring to dissolve, adding DIEA and HATU, and stirring at room temperature for 5 hours , filter the reaction system, concentrate, add water after concentration, wash with ethyl acetate after stirring and dissolving, add sodium chloride to the water phase after washing and stir to dissolve, extract with dichloromethane, combine organic phases, and dry over anhydrous sodium sulfate, Filter with suction, concentrate, add isopropanol and ethyl acetate, heat to dissolve, cool down to crystallize.
  • Another aspect of the present invention provides the application of the polyethylene glycol lipid in delivering biologically active substances to cells or organs.
  • Another aspect of the present invention provides the application of the polyethylene glycol lipid 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, 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 polyethylene glycol lipid for delivering siRNA to cells or organs.
  • the present invention also provides the use of the polyethylene glycol lipid in the preparation of a lipid or lipid nanoparticle delivery system.
  • the present invention also provides the application of the polyethylene glycol lipid for delivering mRNA vaccines to cells or organs.
  • the present invention also provides the application of the polyethylene glycol lipid in the preparation of mRNA vaccines.
  • the vaccine can be used to prevent cancer, viral infection, bacterial infection, fungal infection and the like.
  • the virus infection includes but not limited to: Norovirus, Ebola virus, coronavirus (including new coronavirus SARS-CoV-2), cytomegalovirus, dengue fever virus, Zika virus, Coxsackie virus, intestinal Viruses, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, polio virus, rabies virus, rotavirus, measles virus, etc.
  • Another aspect of the present invention provides a lipid mixture, which comprises biologically active substances and the polyethylene glycol lipid described in the present invention.
  • the preparation method of the lipid mixture includes a heating method, a reverse evaporation method, or a mixing method.
  • the heating method includes adding the organic solvent solution of polyethylene glycol lipid to the aqueous solution of the bioactive 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 bioactive substance and the organic solvent solution of the polyethylene glycol lipid compound to obtain a mixed solution.
  • the biologically active substance in the lipid mixture is mixed with polyethylene glycol lipid particles at a molar ratio of 1:1-100, preferably, the biologically active substance in the lipid mixture is mixed with polyethylene glycol
  • the lipid particles are mixed according to the molar ratio of 1:5-30, such as 1:5, 1:10, 1:15, 1:20, 1:25, 1:30.
  • Another aspect of the present invention provides a lipid nanoparticle, which comprises biologically active substances and the polyethylene glycol lipid compound of the present invention.
  • the lipid nanoparticles also contain one or more of cationic lipids, steroidal lipids or neutral lipids.
  • the cationic lipids include but are not limited to: ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) ( ALC-0315), octadecyl-9-yl 8-((2-hydroxyethyl)(6-oxO-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 1 ,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 313-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyl octadecyl ammonium (DDA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane ( DPTAP), distea
  • the neutral lipids include but are not limited to: 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3 -phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine alkali (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and the like.
  • DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine
  • DPPC
  • the steroidal lipids include, but are not limited to, avenasterol, ⁇ -sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, Dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, nigrosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterol, seawesterol, sitostane alcohol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, etc.
  • the lipid nanoparticle of the present invention comprises biologically active substances, the polyethylene glycol lipid compound of the present invention, cationic lipids, steroidal lipids and neutral lipids.
  • the molar ratio of polyethylene glycol lipid compound, cationic lipid, steroidal lipid and neutral lipid in the lipid nanoparticles of the present invention is (0.5-5):(30-55): (30-55): (5-20).
  • the molar ratio of polyethylene glycol lipid compound, cationic lipid, steroidal lipid and neutral lipid is (1-5):(35-50):(40-50):(8-15 ). More preferably, the molar ratio of polyethylene glycol lipid compound, cationic lipid, steroidal lipid and neutral lipid is 1.5:50:38.5:10.
  • the lipid nanoparticle cationic lipid of the present invention is AC-0315, and/or the steroidal lipid is cholesterol, and/or the neutral lipid is DSPC.
  • the lipid nanoparticle cationic lipid of the present invention is SM-102, and/or the steroidal lipid is cholesterol, and/or the neutral lipid is DSPC.
  • 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.
  • the present invention also provides a pharmaceutical composition, which comprises the lipid mixture 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 mixture, lipid nanoparticle or pharmaceutical composition of the present invention can deliver the bioactive substance through oral administration, inhalation or injection.
  • the present invention also provides a method for delivering biologically active substances, said method administering the lipid mixture, lipid nanoparticle or pharmaceutical composition of the present invention to a population in need.
  • Figure 1 shows the histograms of the remaining GFP fluorescence intensity percentages of the samples in each group after 3 days and 6 days of transfection.
  • Figure 2 shows the expression of GFP protein observed by fluorescence microscope.
  • Figure 3 is a flow cytometric representation of the ratio of GFP expression-positive cells after transfection.
  • 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.
  • Example 3 Fluorescence inhibition effect of PEG-lipid/cationic lipid/neutral lipid/steroidal lipid-siRNA nanoparticles after delivery of siRNA in MDA-MB-231-GFP cells
  • PEG-lipid/cationic lipid/neutral lipid/steroidal lipid-siRNA nanoparticle formula and preparation method are as follows:
  • ALC-0315/M-DTDA-2000 prepared in Example 1
  • siRNA-GFP target sequence such as SEQ ID NO: 1: 5'-GGCUACGUCCAGGAGCGCACC-3'
  • lipid-free 30% ethanol-citric acid solution at a concentration of 2mg/ml
  • siRNA-GFP target sequence such as SEQ ID NO: 1: 5'-GGCUACGUCCAGGAGCGCACC-3'
  • lipid-free 30% ethanol-citric acid solution at a concentration of 2mg/ml
  • the 30% ethanol-citric acid solution of lipid was mixed according to the mass ratio of siRNA-GFP:lipid at 0.06:1.
  • LNP M-DMG-2000-siRNA-GFP (sample 3) was prepared from the above solution using a microfluidic device (model MPE-L2, Suzhou Atsen Pharmaceutical Equipment Co., Ltd.).
  • M-DMG-2000 The structure of M-DMG-2000 is: MW 2000.
  • MDA-MB-231-GFP cell culture uses RPMI-1640 medium containing 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin, in a 37°C, 5% CO2 incubator Cultured and subcultured every 3 days. 24 hours before transfection, the MDA-MB-231-GFP cells were digested and plated on a 24-well plate at a plate density of 1.5 ⁇ 10 5 cells/well, and the cells were cultured for another 24 hours.
  • Preparation of reagents for each group of lipofectamine RNAiMAX first mix lipofectamine RNAiMAX with 1/2 volume of opti-MEM according to the recommended amount in the instruction manual, and mix 20 ⁇ M siGFP/siNC with 1/2 volume of opti-MEM according to the calculated volume, and let stand for 5 minutes. Mix them together to prepare a 10 ⁇ working solution, and let it stand at room temperature. In the lipofectamine RNAiMAX group alone, mix the same amount of lipofectamine RNAiMAX and opti-MEM directly, and let it stand for later use.
  • Preparation of each group of LNP Dilute the prepared LNP to 10 ⁇ working concentration with opti-MEM, and let it stand at room temperature for 30 minutes.
  • Transfection Discard the medium in the cell well, and then add 400 ⁇ l RPMI-1640+10% FBS medium. Then add 100 ⁇ l each of the above transfection reagents. Take samples 3d and 6d after transfection, remove the culture medium of each well, wash with PBS first, then add 300 ⁇ l cell lysate, 37°C for 10min, then take out the samples from each well, centrifuge at 10000 ⁇ g4°C for 10min, and take the supernatant For detection of protein concentration and fluorescence intensity.
  • Preparation of working solution According to the number of standards and samples, prepare BCA working solution by adding 50 volumes of BCA reagent and 1 volume of Cu reagent (50:1), and mix well (there may be turbidity during mixing, but it will disappear after mixing. ). BCA working solution is stable at room temperature for 24 hours.
  • Dilute the standard Take 10 ⁇ l of BSA standard and dilute to 100 ⁇ l with PBS (the sample can generally be diluted with PBS), so that the final concentration is 0.5 mg/mL. Add 0, 2, 4, 6, 8, 12, 16, 20 ⁇ L of the standard to the protein standard wells of the 96-well plate, and add PBS to make up to 20 ⁇ L.
  • Example 4 Fluorescence enhancement effect of PEG-lipid/cationic lipid/neutral lipid/steroidal lipid-mRNA nanoparticles after delivering mRNA in 293T cells
  • LNP composition cationic lipid (ALC0315)/PEG-lipid component (M-DMG-2000, M-DTDA-2000 or M-DTDAM-2000)/DSPC/cholesterol, the molar ratio of which is 50%/1.5% /10%/38.5%.
  • ALC0315 cationic lipid
  • PEG-lipid component M-DMG-2000, M-DTDA-2000 or M-DTDAM-2000
  • DSPC/cholesterol the molar ratio of which is 50%/1.5% /10%/38.5%.
  • Both ALC0315 and M-DMG-2000 are commercially available from Beijing Jiankai Technology Co., Ltd.
  • M-DTDA-2000 and M-DTDAM-2000 were prepared from Example 1.
  • LNP solutions A total of 3 kinds of LNP solutions were prepared, the composition of which is as follows (DMG stands for M-DMG-2000, DTDA stands for M-DTDA-2000, DTDAM stands for M-DTDAM-2000, all are PEG-lipid): DSPC+cholesterol+ALC0315+DMG /DTDA/DTDAM;
  • the ratio of LNP solution to GFP mRNA dilution is 1:3.
  • the total volume of GFP mRNA diluent is 150uL: 140uL pH4.0 PBS citric acid is used to dilute 10ug GFP mRNA (i.e. 10uL GFP mRNA).
  • 150uL GFP mRNA dilution and 51.5uL LNP solution were vortexed and mixed, and left at room temperature for 30min.
  • Plating Inoculate 293T cells in a 24-well plate one day in advance. The appropriate number of cells at the time of transfection is about 12,000-50,000 cells/well. ml/well. In a 24-well plate, three duplicate wells were cultured at 37°C, 5% CO 2 for one day.
  • Figure 3 shows the proportion of GFP positive cells after transfection characterized by flow cytometry
  • test number Sample code mean fluorescence intensity Proportion of positive cells (%) 1 blank control 1 0 2 positive control 51.44 90.23 3 naked mRNA control 1 0 4 ALC-0315&DMG 5.55 80.33 5 ALC-0315&DTDA 11.51 86.99 6 ALC-0315&DTDAM 5.11 81.99

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Abstract

一种新型聚乙二醇脂质及其应用,该脂质不含有在体内可断裂键，可以更稳定的将生物活性物质递送至靶细胞或器官。另外，所述的新型聚乙二醇脂质在特定pH环境下可带有正电荷，更易于与生物活性物质形成稳定的颗粒,使生物活性物质在靶细胞或器官内发挥作用。

Description

聚乙二醇脂质及其应用 技术领域
本发明属于生物医药领域，具体涉及一种聚乙二醇脂质化合物以及在生物活性物质递送中的应用。
背景技术
近年来随着生物技术进步，基因治疗药物得到了快速的发展，给生物药物领域带来了巨大的发展前景。美国Alnylam公司采用RNAi技术研发了系列基因药物并成功的上市了四种RNAi药物：Onpattro(patisiran)、Givlaari(givosiran)、Oxlumo(lumasiran)和Leavio(inclisiran)，其中Leavio(inclisiran)是治疗高血脂症的生物药物，该药的成功上市将基因治疗药物从传统的治疗罕见性遗传病的领域拓宽到治疗大众病领域，为RNAi药物带来了新的机遇。
核酸类药物由于其本身的性质，如负电性、易于被核酸酶所降解等导致其不能有效的透过细胞膜进入细胞并且在体内很快就被降解掉，所以需要很好的递送系统将核酸稳定的递送至靶位置并起效。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。在递送过程中，还需要考虑如何避免被快速清除、避免被核酸酶降解、改善内吞后逃逸等。
目前递送核酸的系统多采用不同种类的脂质体递送方式，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，其中聚合物缀合脂质指的是聚乙二醇脂质。LNP的制备过程相对来说比较复杂，简单来说包括脂质纳米颗粒的制备、RNA的包裹培育、透析、冷冻干燥等过程。
聚乙二醇(PEG)脂质已经被广泛报道，例如，专利文献CN106795096A、WO2005026372A1、WO2020061295A1、CN102665685A、US20210154148A1、WO2012099755A1等均公开了聚乙二醇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。
专利文献CN102068701A公开了可断裂聚乙二醇脂质衍生物在制备减轻或避免加速血液清除PEG化制剂中的应用。
然而，包括如上所述文献公开的现有聚乙二醇脂质，例如PEG-DMG、ALC-0159(M-DTDAM)，其结构中均含有在体内会断裂的连接键，在体内具有不稳定性，从而影响生物活性物质递送效率。
发明内容
为克服现有技术中的缺陷，本发明提供了一种新型聚乙二醇脂质,该脂质不含有在体内可断裂键(例如酯键、酰胺键等)，可以更稳定的将生物活性物质递送至靶细胞或器官。另外，所述的新型聚乙二醇脂质在特定pH环境下可带有正电荷，更易于与生物活性物质形成稳定的颗粒,使生物活性物质在靶细胞或器官内发挥作用。
本发明第一方面提供一种聚乙二醇脂质，所述的聚乙二醇脂质具有如 下式I结构：
Figure PCTCN2022076994-appb-000001
其中，R 1和R 2为相同或不同的长链烃基，优选的，所述的长链烃基含有8-25个碳原子；
PEG为聚乙二醇链；
L 1、L 2和L 3不存在或为在体内不可断裂的连接子。
进一步的，本发明所述的长链烃基含有11-17个碳原子，例如，所述的长链烃基含有11、12、13、14、15、16或17个碳原子。在本发明的实施方式中，所述的长链烃基含有14个碳原子。
进一步的，本发明所述的聚乙二醇链包含-(OCH 2CH 2) n-重复单元，所述的n选自20-300的整数，更优选的，所述的n选自20-228的整数，特别优选的所述的n选自30-60的整数，例如n选自30、35、40、45、50、55、60。
进一步的，本发明所述的聚乙二醇链为-(OCH 2CH 2) n-X,其中，X为聚乙二醇链的端基基团，选自甲基、甲氧基、羟基、氨基、巯基、马来酰亚胺基等。优选的，所述的X为甲氧基，所述的聚乙二醇链为-(OCH 2CH 2) n-OCH 3。
进一步的，本发明所述的在体内不可断裂连接子是指相对比较稳定的连接子，不含有在体内可断裂键，因此在体内环境下不易降解。例如，所述的在体内不可断裂连接子可以是硫醚连接子或烷基连接子，所述烷基连接子例如是C1-C10的亚烷基，优选是C2-C8的亚烷基，更优选是C2-C5的亚烷基，所述的亚烷基可以是直链或支链亚烷基，更优选的，所述的亚烷基是直链亚烷基，例如-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-。
本发明的还一方面提供了一种聚乙二醇脂质，所述的聚乙二醇脂质具有 如下式II结构：
Figure PCTCN2022076994-appb-000002
其中,R 1、R 2、X和n如前述定义，
i选自2-7的整数，例如，i选自2、3、4、5、6或7，优选的，i选自2或3。
进一步的，本发明所述的聚乙二醇脂质具有如下结构：
在发明的具体实施方式中，所述的聚乙二醇脂质具有如下结构：
Figure PCTCN2022076994-appb-000003
其中,X和n如前述定义,优选的，X为-OCH 3。
本发明所述的聚乙二醇链分子量可以为1000至20000，优选为1000至10000，例如是1000、1500、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10000等。
本发明所述的聚乙二醇脂质在低PH溶液中，所述的脂质会离子化，带正电荷，更易于和RNA或mRNA中带负电荷的磷酸基结合。
本发明的还一方面提供一种聚乙二醇脂质的制备方法，所述的方法包括：将如下式IV化合物中羰基通过还原反应得到本发明式II的化合物，
Figure PCTCN2022076994-appb-000004
其中，R 1、R 2、X、i和n如前述定义。
进一步的，所述的还原反应的还原剂为LiAlH 4。
进一步的，所述的还原反应包括：氮气氛围下，将LiAlH 4加入含有第一溶剂的反应装置中，将式IV化合物溶解至第二溶剂中，滴加至反应装置中反应，得到式II的化合物。
进一步的，所述的第一溶剂优选为四氢呋喃。
进一步的，所述的第二溶剂优选为四氢呋喃。
在本发明的具体实施方式中，所述的还原反应包括：氮气氛围下，将LiAlH 4缓慢加入含有四氢呋喃的反应装置中，将式IV化合物溶解四氢呋喃中，并缓慢滴加至反应装置中，3小时后向反应体系滴加水，过滤，将滤液浓缩，加入异丙醇和乙酸乙酯，加热溶解，降温析晶。
进一步的，所述的式IV化合物是由如下式III的化合物与
Figure PCTCN2022076994-appb-000005
进行缩合反应得到：
Figure PCTCN2022076994-appb-000006
其中，R 1、R 2、X、i和n如前述定义。
进一步的，所述的缩合反应的缩合试剂优选为HATU(N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲)。
进一步的，所述的缩合反应包括：将式III的化合物与X-(OCH 2CH 2) n-COOH加入到含有第三溶剂的反应装置中，加入DIEA(N,N-二异丙基乙胺)和HATU反应，得到式IV化合物。
进一步的，所述的第三溶剂优选为二氯甲烷。
进一步的，所述的缩合反应时间为3-8小时。优选的，所述的缩合反应时间为5小时。
进一步的，所述的缩合反应包括：将式III的化合物与X-(OCH 2CH 2) n-COOH加入到含有二氯甲烷的反应装置中，搅拌溶解，加入DIEA和HATU，室温搅拌5小时，将反应体系过滤，浓缩，浓缩后加入水，搅拌溶解后用乙酸乙酯洗涤，洗涤后向水相加入氯化钠搅拌溶解，使用二氯甲烷萃取，合并有机相，无水硫酸钠干燥，抽滤，浓缩，加入异丙醇和乙酸乙酯，加热溶解，降温析晶。
本发明的还一方面提供了所述的聚乙二醇脂质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聚乙二醇脂质、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、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聚乙二醇脂质用于递送siRNA至细胞或器官的应用。
进一步，本发明还提供了所述的聚乙二醇脂质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聚乙二醇脂质用于递送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聚乙二醇脂质用于制备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。所述的病毒感染包括但不限于：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。
本发明的还一方面提供了一种脂质混合物，所述的脂质混合物包含生物活性物质和本发明所述的聚乙二醇脂质。
进一步的，所述的脂质混合物的制备方法包括加热法、逆向蒸发法，或者混合法。
进一步的，所述加热法包括将聚乙二醇脂质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聚乙二醇脂质化合物的有机溶剂溶液进行混合以得到混合溶液。
进一步的，所述的脂质混合物中生物活性物质与聚乙二醇脂质颗粒按照 摩尔比为1：1-100混合，优选的，所述的脂质混合物中生物活性物质与聚乙二醇脂质颗粒按照摩尔比为1：5-30，例如1：5、1：10、1：15、1：20、1：25、1：30混合。
本发明的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聚乙二醇脂质化合物。
进一步的，所述的脂质纳米颗粒中还包含阳离子脂质、甾族脂质或中性脂质中的一种或两种以上。
进一步的，所述的阳离子脂质包括但不限于：((4-羟基丁基)氮杂二烷基)双(己烷-6，1-二基)双(2-己基癸酸酯)(ALC-0315)、十八烷-9-基8-((2-羟乙基)(6-oxO-6-(十一烷氧基)己基)氨基)辛酸酯(SM-102)、1,2-二油酰基氧基-3-(三甲基铵基)丙烷(DOTAP)、313-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇(DC胆固醇)、二甲基二十八烷基铵(DDA)、1,2-二肉豆蔻酰基-3-三甲基铵丙烷(DMTAP)、二棕榈酰(C16:0)三甲基铵丙烷(DPTAP)、二硬脂酰基三甲基铵丙烷(DSTAP)、N-[1-(2,3-二烯丙氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、N,N-二油酰基-N,N-二甲基氯化铵(DODAC)、1,2-二油酰基-sn-丙三氧基-3-乙基磷酸胆碱(DOEPC)、1,2-二油酰基-3-二甲基铵丙烷(DODAP)、1,2-二亚油基氧基-3-二甲基氨基丙烷(DLinDMA)等。
进一步的，所述的中性脂质包括但不限于：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)等。
进一步的，所述的甾族脂质包括但不限于燕麦甾醇、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、脱氧胆酸和石胆酸等。
进一步的，本发明所述的脂质纳米颗粒包含生物活性物质、本发明所述的聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质。
进一步的，本发明所述的脂质纳米颗粒中聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质的摩尔比为(0.5-5):(30-55)：(30-55):(5-20)。优选的，聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质的摩尔比为(1-5):(35-50)：(40-50):(8-15)。更优选的，聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质的摩尔比为1.5：50：38.5：10。
进一步的，本发明所述的脂质纳米颗粒阳离子脂质为AC-0315，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阳离子脂质为SM-102，和/或，甾族脂质为胆固醇，和/或，中性脂质为DSPC。
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混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。
本发明所述的脂质混合物、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、脂质纳米颗粒或药物组合物。
附图说明
图1所示为各组样品在转染3天和6天后，剩余GFP荧光强度百分比柱状图。
图2所示为荧光显微镜观察GFP蛋白的表达情况。
图3所示为流式细胞术表征转染后GFP表达阳性细胞的比例图。
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聚乙二醇脂质(M-DTDA-2000)的制备
1)将M-CM-2000(Methoxy PEG Acetic Acid,mPEG-COOH，2g，1mmol)和双十四烷基胺(0.576g，1.4mmol)加入到含有二氯甲烷(20ml)的100ml单口瓶中，搅拌溶解，再依次加入DIEA(260mg，2mmol)和HATU(760mg，2mmol)，室温搅拌5小时。将反应体系过滤，母液真空浓缩，浓缩后加入40ml净化水，搅拌溶解后用乙酸乙酯洗涤3次，每次使用40ml。洗涤后向水相加入6g氯化钠搅拌溶解，使用二氯甲烷萃取水相二次，每次使用20ml。合并有机相，无水硫酸钠干燥，抽滤，将滤液真空浓缩，加入30ml异丙醇和10ml乙酸乙酯，加热至40度条件下溶解，降温至零下15度析晶，抽滤。滤饼真空干燥得1.4g白色固体(M-DTDAM-2000)，收率58.3％。
1H-NMR(400MHz,DMSO)δ:4.1(2H,s),3.55(180H,m),3.26(3H,s),3.18(4H,t),1.46(4H,m),1.24(44H,m),0.86(6H,t)。
Figure PCTCN2022076994-appb-000007
M-DTDAM-2000(MW 2000)
2)氮气氛围下，将LiAlH 4(380mg,10mmol)缓慢加入冰水浴中装有30ml四氢呋喃的三口瓶中。将M-DTDAM-2000(2g,1mmol)溶解在10ml四氢呋喃当 中，并缓慢滴加至反应瓶中，3小时后向反应体系滴加10ml净化水，使用硅藻土过滤体系，将滤液真空浓缩，加入20ml异丙醇，加热至40度条件下溶解，冰水浴降温至析晶，抽滤。滤饼真空干燥得1.1g白色固体(M-DTDA-2000)，收率55％。
1H-NMR(400MHz,DMSO)δ:3.55(180H,m),3.24(3H,s),2.36(4H,t),1.34(4H,m),1.24(44H,m),0.86(6H,t)。
Figure PCTCN2022076994-appb-000008
M-DTDA-2000(MW 2000)
实施例2聚乙二醇脂质(M-DTDA-2000(p))的制备
1)将M-PA-2000(Methoxy PEG Propionic Acid,mPEG-CH2CH2COOH，2g，1mmol)和双十四烷基胺(0.617g，1.5mmol)加入到含有二氯甲烷(20ml)的100ml单口瓶中，搅拌溶解，再依次加入DIEA(260mg，2mmol)和HATU(760mg，2mmol)，室温搅拌5小时。将反应体系过滤，母液真空浓缩，浓缩后加入40ml净化水，搅拌溶解后用乙酸乙酯洗涤3次，每次使用40ml。洗涤后向水相加入6g氯化钠搅拌溶解，使用二氯甲烷萃取水相二次，每次使用20ml。合并有机相，无水硫酸钠干燥，抽滤，将滤液真空浓缩，加入30ml异丙醇和10ml乙酸乙酯，加热至40度条件下溶解，降温至零下15度析晶，抽滤。滤饼真空干燥得1.32g白色固体(M-DTDAM-2000(p))，收率55％。
1H-NMR(400MHz,DMSO)δ:3.55(180H,m),3.26(3H,s),3.18(4H,t),2.45(2H,t)1.46(4H,m),1.24(44H,m),0.86(6H,t)。
Figure PCTCN2022076994-appb-000009
M-DTDAM-2000(p)(MW 2000)
2)氮气氛围下，将LiAlH 4(380mg,10mmol)缓慢加入冰水浴中装有30ml四氢呋喃的三口瓶中。将M-DTDAM-2000(p)(2g,1mmol)溶解在10ml四氢呋喃当中，并缓慢滴加至反应瓶中，3小时后向反应体系滴加10ml净化水，使用硅藻土过滤体系，将滤液真空浓缩，加入20ml异丙醇，加热至40度条件下溶解，冰水浴降温至析晶，抽滤。滤饼真空干燥得1.15g白色固体(M-DTDA-2000(p))，收率57.5％。
1H-NMR(400MHz,DMSO)δ:3.55(180H,m),3.24(3H,s),2.36(4H,t),1.34(4H,m),1.24(44H,m),0.86(6H,t)。
Figure PCTCN2022076994-appb-000010
M-DTDA-2000(p)(MW 2000)
实施例3 PEG-lipid/阳离子脂质/中性脂质/甾族脂质-siRNA纳米颗粒在MDA-MB-231-GFP细胞中递送siRNA后荧光抑制效果
各组PEG-lipid/阳离子脂质/中性脂质/甾族脂质-siRNA纳米颗粒配方及制备方法如下：
将ALC-0315/M-DTDA-2000(实施例1制备得到)/DSPC/胆固醇按照摩尔比50％/1.5％/10％/38.5％的比例溶解在无水乙醇中，配成10mmol/L混合液，然后加入PH＝4的柠檬酸缓冲液，制成含有四种脂质的30％乙醇-柠檬酸溶液，用0.1um的过滤膜过滤备用。将siRNA-GFP(靶序列如SEQ ID NO:1：5′-GGCUACGUCCAGGAGCGCACC-3′)按照2mg/ml的浓度溶解在不含脂质的30％乙醇-柠檬酸溶液中，然后与上述含有四种脂质的30％乙醇-柠檬酸溶液按照siRNA-GFP：脂质的质量比为0.06：1进行混合，孵育30分钟，用PH＝7.4的PBS透析16小时以上，制备得到LNP(M-DTDA-2000)-siRNA-GFP(样品1)。
将ALC-0315/M-DMG-2000(Methoxy PEG Dimyristoyl-rac-glycero，北京键凯科技股份有限公司市售)/DSPC/胆固醇按照摩尔比50％/1.5％/10％/38.5％的比例溶解在无水乙醇中，配成10mmol/L混合液，然后加入PH＝4的柠檬酸缓冲液，制成含有四种脂质的30％乙醇-柠檬酸溶液，用0.22um的过滤膜过滤备用。将siRNA-GFP(靶序列如SEQ ID NO:1：5′-GGCUACGUCCAGGAGCGCACC-3′)按照2mg/ml的浓度溶解在不含脂质的30％乙醇-柠檬酸溶液中，然后与上述含有四种脂质的30％乙醇-柠檬酸溶液按照siRNA-GFP：脂质的质量比为0.06：1进行混合。
将上述溶液孵育30分钟，用PH＝7.4的PBS透析16小时以上，制备得到LNP(M-DMG-2000)-siRNA-GFP(样品2)。
将上述溶液采用微流控设备(型号MPE-L2，苏州艾特森制药设备有限公司)制备LNP(M-DMG-2000)-siRNA-GFP(样品3)。
M-DMG-2000结构为：
Figure PCTCN2022076994-appb-000011
MW 2000。
细胞培养：MDA-MB-231-GFP细胞培养使用含10％胎牛血清、100U/ml青霉素、0.1mg/ml链霉素的RPMI-1640培养基，于37℃、5％CO 2培养箱中培养，每3天传代一次。在转染前24h，将MDA-MB-231-GFP细胞消化铺24孔板，铺板密度1.5×10 5细胞/孔，将细胞继续培养24h。
lipofectamine RNAiMAX各组试剂配制：先将lipofectamine RNAiMAX按说明书推荐量与1/2体积的opti-MEM混合，20μM siGFP/siNC按计算的体积与1/2体积opti-MEM混合，静置5min后，两者混合制备成10×工作液，室温静置。单独lipofectamine RNAiMAX组中，将同等量的lipofectamine  RNAiMAX与opti-MEM直接混合，静置备用。
LNP各组配制：用opti-MEM将制备好的LNP稀释至10×工作浓度，室温静置30min。
转染：弃除细胞孔中的培养基，再加入400μl RPMI-1640+10％FBS培养基。然后加入上述转染试剂各100μl。转染后3d、6d分别取样，去除各孔的培养基，先用PBS洗一遍，再加入300μl细胞裂解液，37℃10min，然后将各孔样品取出，10000×g4℃离心10min，取上清用于蛋白浓度和荧光强度的检测。
蛋白标准曲线制备和蛋白浓度检测：
配制工作液：根据标准品和样品数量，按50体积BCA试剂加1体积Cu试剂(50:1)配制成BCA工作液，充分混匀(混合时可能会有浑浊，但混匀后就会消失)。BCA工作液室温24小时内稳定。
稀释标准品：取10μl BSA标准品用PBS稀释至100μl(样品一般可用PBS稀释)，使终浓度为0.5mg/mL。将标准品按0,2,4,6,8,12,16,20μL加到96孔板的蛋白标准品孔中，加PBS补足至20μL。
将20μL样品到96孔板的样品孔中。同时设置空白对照，取20μl细胞裂解液加入孔板中。各孔加入200μl BCA工作液，37℃放置15-30分钟。用酶标仪测定A562nm，根据标准曲线计算出蛋白浓度。
荧光强度检测，取各样品100μl加入到黑色不透光96孔板中，同时设置空白对照，取20μl细胞裂解液加入孔板中。使用酶标仪检测，设置激发光波长488nm，发射光波长509nm。
数据计算：各样品在计算时，首先减去空白孔读值，以减少背景干扰。各样品的荧光值用蛋白浓度进行标准化后，再对blank组均值进行标准化，得到剩余GFP荧光强度百分比数据。结果如表1和图1。
表1
Figure PCTCN2022076994-appb-000012
在转染后3天和6天，LNP各组和lipofectamine RNAiMAX+5nM siGFP组的GFP荧光强度均显著下降，显示出良好的抑制作用。其中，LNP(M-DTDA-2000)-siRNA-GFP组(样品1)作用达到了与lipofectamine RNAiMAX+5nMsiRNA-GFP组相似的效果，优于LNP(M-DMG-2000)-siRNA-GFP组(样品2和样品3)的抑制效果。
结果表明，采用M-DTDA-2000/ALC-0315/DSPC/胆固醇与siRNA-GFP制成的各组脂质纳米颗粒递送siRNA-GFP入胞后，对GFP mRNA有明显而持续的抑制作用，并且，M-DTDA-2000制备得到脂质纳米颗粒组的总体基因抑制效果好于其他组别。
实施例4 PEG-lipid/阳离子脂质/中性脂质/甾族脂质-mRNA纳米颗粒在293T细胞中递送mRNA后荧光增强效果
LNP组合成分：阳离子脂质(ALC0315)/PEG-lipid组分(M-DMG-2000、M-DTDA-2000或M-DTDAM-2000)/DSPC/胆固醇，其摩尔比比例为 50％/1.5％/10％/38.5％。现配制浓度均为10mM的各组分溶液，无水乙醇溶解。取阳离子脂质溶液258uL，PEG组分溶液7.68uL，DSPC溶液51.6uL，胆固醇溶液198.6uL进行混合。涡旋振荡混匀后，用0.22uM滤膜过滤，配制成LNP溶液备用。
ALC0315和M-DMG-2000均为北京键凯科技股份有限公司市售。
M-DTDA-2000和M-DTDAM-2000由本实施例1制备得到。
共配制了3种LNP溶液，其组成如下(其中DMG代表M-DMG-2000、DTDA代表M-DTDA-2000、DTDAM代表M-DTDAM-2000,均为PEG-lipid)：DSPC+胆固醇+ALC0315+DMG/DTDA/DTDAM；
LNP溶液与GFP mRNA稀释液比例1：3。GFP mRNA稀释液总体积150uL：140uL pH4.0PBS柠檬酸稀释10ug GFP mRNA(即10uL GFP mRNA)。150uL GFP mRNA稀释液与51.5uL LNP溶液涡旋振荡混匀，室温静置30min。用40uL无血清稀释液(基础培养基DMEM)稀释0.5ug GFP mRNA(即10uL GFP mRNA-LNP混合液)，涡旋10s充分混匀制成50uL GFP mRNA转染混合物。
阳性对照：
按说明书比例0.5ug GFP mRNA加50uLmRNA Buffer，涡旋10s充分混匀，制成GFP mRNA稀释液；加入1ul的jetMESSENGER reagent，(mRNA/jetMESSENGER ratio为1:2)涡旋10s，旋转下来并在室温下孵育15分钟。
裸mRNA对照：
0.5ug GFP mRNA加50uL DEME培养基涡旋10s充分混匀，制成GFP mRNA稀释液。
空白对照：50uL DEME培养基。
铺板：提前一天将293T细胞接种在24孔板中，以转染时细胞数量在12000-50000个/孔左右为宜，转染前全培养基(无抗生素，含10％血清)总量为0.5ml/孔。24孔板中，3个复孔，37℃，5％CO 2培养一天。
转染：将50uL GFP mRNA转染混合物滴加到有0.5ml全培养基(无抗生素，含10％血清)的细胞上，前后轻轻移动培养皿，混合均匀，37℃，5％CO 2培养箱培养。
荧光显微镜下观察荧光蛋白表达：转染后4小时观察细胞状态，继续培养24-72小时，在荧光显微镜下观察GFP蛋白表达情况并拍照记录(图2)。
流式细胞术表征转染后GFP表达阳性细胞的比例图见图3
荧光显微镜及流式细胞术结果见表2：
表2
测试编号 样品代号 平均荧光强度 阳性细胞比例(％)
1 空白对照 1 0
2 阳性对照 51.44 90.23
3 裸mRNA对照 1 0
4 ALC-0315&DMG 5.55 80.33
5 ALC-0315&DTDA 11.51 86.99
6 ALC-0315&DTDAM 5.11 81.99
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 (19)

  1. 一种聚乙二醇脂质，其特征在于，所述的聚乙二醇脂质具有如下式I结构：
    Figure PCTCN2022076994-appb-100001
    其中，R 1和R 2为相同或不同的长链烃基；
    PEG为聚乙二醇链；
    L 1、L 2和L 3不存在或为在体内不可断裂的连接子。
  2. 权利要求1所述的聚乙二醇脂质，其特征在于，所述的长链烃基含有11-17个碳原子。
  3. 权利要求1所述的聚乙二醇脂质，其特征在于，所述的聚乙二醇链包含-(OCH 2CH 2) n-重复单元，所述的n选自20-300的整数。
  4. 权利要求3所述的聚乙二醇脂质，其特征在于，所述的聚乙二醇链为-(OCH 2CH 2) n-X,其中，X为聚乙二醇链的端基基团，选自甲基、甲氧基、羟基、氨基、巯基和马来酰亚胺基。
  5. 权利要求1所述的聚乙二醇脂质，其特征在于，所述的在体内不可断裂连接子是C1-C10的亚烷基。
  6. 权利要求1所述的聚乙二醇脂质，其特征在于，所述的聚乙二醇脂质具有如下式II结构：
    Figure PCTCN2022076994-appb-100002
    其中，R 1和R 2为相同或不同的长链烃基；
    X为聚乙二醇链的端基基团，选自甲基、甲氧基、羟基、氨基、巯基和马来酰亚胺基；
    n选自20-300的整数；
    i选自2-7的整数。
  7. 权利要求1所述的聚乙二醇脂质，其特征在于，所述的聚乙二醇脂质具有如下结构：
    Figure PCTCN2022076994-appb-100003
    其中,X为聚乙二醇链的端基基团，选自甲基、甲氧基、羟基、氨基、巯基和马来酰亚胺基；
    n选自20-300的整数；
    i选自2-7的整数。
  8. 权利要求1-7所述的聚乙二醇脂质在制备生物活性物质递送系统中的应用。
  9. 权利要求8所述的应用，其特征在于，所述的生物活性物质为核酸。
  10. 权利要求8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。
  11. 权利要求8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。
  12. 权利要求11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。
  13. 权利要求12所述的应用，其特征在于，所述的病毒感染选自诺如病毒、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。
  14. 一种脂质混合物，其特征在于，所述的脂质混合物包含生物活性物质和权利要求1-7所述的聚乙二醇脂质。
  15. 权利要求14所述的脂质混合物，其特征在于，所述的脂质混合物中生物活性物质与聚乙二醇脂质颗粒按照摩尔比为1：1-100混合。
  16. 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1-7所述的聚乙二醇脂质化合物。
  17. 权利要求16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阳离子脂质、甾族脂质和中性脂质。
  18. 权利要求17所述的脂质纳米颗粒，其特征在于，所述的阳离子脂质选自((4-羟基丁基)氮杂二烷基)双(己烷-6，1-二基)双(2-己基癸酸酯)(ALC-0315)、十八烷-9-基8-((2-羟乙基)(6-oxO-6-(十一烷氧基)己基)氨基)辛酸酯(SM-102)、1,2-二油酰基氧基-3-(三甲基铵基)丙烷(DOTAP)、313-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇(DC胆固醇)、二甲基二十八烷基铵(DDA)、1,2-二肉豆蔻酰基-3-三甲基铵丙烷(DMTAP)、二棕榈酰(C16:0)三甲基铵丙烷(DPTAP)、二硬脂酰基三甲基铵丙烷(DSTAP)、N-[1-(2,3-二烯丙氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、N,N-二油酰基-N,N-二甲基氯化铵(DODAC)、1,2-二油酰基-sn-丙三氧基-3-乙基磷酸胆碱(DOEPC)、1,2-二油酰基-3-二甲基铵丙烷(DODAP)、1,2-二亚油基氧基-3-二甲基氨基丙烷(DLinDMA)；
    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)；
    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。
  19. 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14-15任意一项脂质混合物或者权利要求16-18任意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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