WO2023186149A1 - 一种脂质化合物、包含其的组合物及应用 - Google Patents

一种脂质化合物、包含其的组合物及应用 Download PDF

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WO2023186149A1
WO2023186149A1 PCT/CN2023/085724 CN2023085724W WO2023186149A1 WO 2023186149 A1 WO2023186149 A1 WO 2023186149A1 CN 2023085724 W CN2023085724 W CN 2023085724W WO 2023186149 A1 WO2023186149 A1 WO 2023186149A1
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lipid compound
lipid
peg
formula
modified
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French (fr)
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张元�
付正强
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华南理工大学
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/12—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of biomedicine technology, and specifically relates to a lipid compound, a composition containing the same and its application.
  • Gene therapy refers to the delivery of a type of nucleic acid drugs such as siRNA, mRNA, pDNA and other exogenous genes with therapeutic purposes into target cells through carrier materials to exert therapeutic effects.
  • nucleic acid drugs such as siRNA, mRNA, pDNA and other exogenous genes with therapeutic purposes into target cells through carrier materials to exert therapeutic effects.
  • gene therapy has achieved good results in clinical treatment and can effectively treat congenital and acquired diseases, such as cancer, autoimmune diseases, infectious diseases, heart disease, etc.
  • gene therapy is not difficult to understand, that is, replacing abnormal genes to compensate.
  • gene therapy also faces many problems that need to be solved urgently. Among them, how nucleic acid drugs use carriers to enter specific cells of the human body to work is a key link in gene therapy. Therefore, developing an efficient and safe nucleic acid delivery system is the cornerstone of gene therapy.
  • nucleic acid delivery systems are mainly viral vectors and non-viral vectors.
  • Viral vectors have relatively high transfection efficiency, but there are problems such as safety and poor targeting.
  • non-viral vectors have developed rapidly, among which liposomes have the advantages of low immunogenicity, good biocompatibility, and high transfection efficiency and are regarded as ideal nucleic acid delivery systems.
  • ionizable cationic lipids have pH-dependent charge variable characteristics and are relatively safe, but their transfection efficiency needs to be improved.
  • the present invention aims to solve the technical problems existing in the above-mentioned prior art.
  • the present invention proposes a lipid compound, a composition containing the same, and applications.
  • the lipid compound can be used alone or in combination with other lipid compounds, such as cholesterol, auxiliary phospholipids, and polyethylene glycol-modified lipids, to efficiently deliver active pharmaceutical ingredients to cells or tissues to exert specific biological functions. For example, delivering siRNA to cells in vivo to exert gene silencing effect or delivering mRNA or pDNA to cells to promote gene expression.
  • the reaction pathway of this lipid compound is simple and the yield is high, so it has broad application prospects.
  • a first aspect of the present invention provides a lipid compound, or a pharmaceutically acceptable salt or a stereoisomer thereof.
  • the lipid compound has a structure represented by formula (I):
  • the R 2 is selected from the structure shown below:
  • n is selected from an integer from 0 to 3, preferably 1 or 2;
  • the lipid compound has a structure represented by formula (II);
  • y is selected from 1 or 2;
  • R 3 is chosen from
  • R 5 is selected from hydroxyl, unsubstituted or methyl-substituted tetrahydropyrrolyl, piperidinyl, piperazinyl, dimethylamino, dihydroxymethylamino, diethylamino, dihydroxyethylamino; preferably hydroxyl , unsubstituted or methyl-substituted tetrahydropyrrolyl, piperidinyl, piperazinyl;
  • R 3 is chosen from
  • X is selected from Methylene, oxygen atom, or X is connected to any N in the formula (II) to form a five-membered heterocyclic ring or a six-membered heterocyclic ring; or R 3 and two Ns in the formula (II) are combined to form a five-membered heterocyclic ring. or six-membered heterocycle;
  • the X is selected from methylene
  • Each p is independently selected from 0 or 1, and,
  • R 4 is selected from C 7 to C 17 linear alkyl or linear alkenyl
  • the R 4 directly connected to the N atom is selected from hydrogen, and the remaining R 4 is independently selected from C 7 to C 17 linear alkyl or linear alkenyl;
  • R 4 is selected from the structure shown below:
  • R2 is selected from the structure shown below:
  • n is selected from 1 or 2;
  • y is selected from 1 or 2;
  • R 3 is chosen from Wherein, R 5 is selected from hydroxyl, unsubstituted or methyl-substituted tetrahydropyrrolyl;
  • R 3 is chosen from where X is selected from
  • Each p is independently selected from 0 or 1, and,
  • R 4 is selected from C 7 to C 17 linear alkyl or linear alkenyl
  • the R 4 directly connected to the N atom is selected from hydrogen, and the remaining R 4 is independently selected from C 7 to C 17 linear alkyl or linear alkenyl;
  • R 4 is selected from the structure shown below:
  • the lipid compound is selected from the structure shown below:
  • the lipid compound is selected from: A1, A2, B1, B2, B3, B4, B5, C2, C4, C6, 1-O15B, 2-O15B, 3-O15B, 2-O19B, 1-O25B ,2-O25B.
  • the ionizable lipid compounds of the present invention are divided into two major categories: (1) The structure shown in formula (I), in the disulfide bond (SS)
  • the two ends are connected to an ester group, a tertiary amine, and an alkyl chain in sequence.
  • An ester group can also be introduced between the alkyl chain and the tertiary amine, or into the alkyl chain to improve the biodegradability of the lipid compound; and, formula (2)
  • the structure shown in (2) connects the hydrophobic tail with an ester group and a disulfide bond to an organic amine.
  • the ionizable lipid compound of the present invention is electrically neutral or electronegative when transported in the blood, can reduce the cytotoxicity caused by the positive charge in traditional cationic lipids used for gene delivery, and can prolong circulation in the body time to improve pharmacokinetic characteristics.
  • the lipid compound of the present invention can obtain a positive charge under acidic conditions, and then combine with negatively charged RNA, DNA and small molecule drugs through electrostatic interaction to self-assemble into lipid nanoparticles (Lipid Nanoparticles, LNP).
  • the delivery of active ingredients can efficiently deliver DNA and mRNA in mammalian cells, express targeted genes, and efficiently transfect siRNA to specifically silence the expression of targeted genes. Since reducing substances such as glutathione that are upregulated in cells can quickly degrade the disulfide bonds in ionizable lipid compounds, causing the nanocarriers to rupture and the contained nucleic acid molecules to be released into the cytoplasm, thereby achieving endosomal escape and improving transfer. dyeing efficiency. In addition, the biodegradability of disulfide and ester bond structures makes ionizable lipid compounds easier to be metabolized in the body.
  • a second aspect of the present invention provides a method for preparing the above-mentioned lipid compound:
  • the synthetic route of the lipid compound is as follows:
  • the synthetic route of the lipid compound is as follows:
  • R 2 and n are respectively as defined above.
  • the compound is obtained by a Michael addition reaction between an acrylate derivative and an organic amine; this reaction can be achieved by controlling the chemistry of the organic amine and the acrylate derivative.
  • the equivalent ratio controls the degree of substitution of free hydrogen atoms on the organic amine nitrogen.
  • the acrylate has the structure shown below:
  • R 4 is as defined above.
  • the organic amine is selected from the following structures:
  • the ionizable lipid provided by the present invention has a streamlined structure, a simple reaction pathway, and a high yield.
  • a third aspect of the present invention provides a composition comprising the above-mentioned lipid compound, or a pharmaceutically acceptable salt or stereoisomer thereof.
  • the composition further includes other lipid compounds.
  • the other lipid compounds include at least one of cholesterol or ⁇ -sitosterol and their derivatives, auxiliary phospholipids and polyethylene glycol modified lipids.
  • the auxiliary phospholipids include egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine, dimelanine Myristoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl lecithin (DOPC), dioleoylphosphatidylcholine At least one kind selected from choline and dilauroylphosphatidylcholine.
  • the polyethylene glycol (PEG)-modified lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG At least one of modified diacylglycerol and PEG-modified dialkylglycerol.
  • composition when the composition further includes cholesterol or ⁇ -sitosterol and their derivatives, auxiliary phospholipids and PEG modified lipids, the lipid compound, or a pharmaceutically acceptable compound thereof
  • auxiliary phospholipid: PEG modified lipid is 30 ⁇ 50:30 ⁇ 50:5 ⁇ 20:1 ⁇ 2.5, for example, 40 ⁇ 50: 35 ⁇ 50: 5 ⁇ 10: 1.5 ⁇ 2, 40 ⁇ 50: 40 ⁇ 50: 5 ⁇ 10: 1.5 ⁇ 2, 40 ⁇ 45: 45 ⁇ 48: 8 ⁇ 10: 1.5 ⁇ 2, 40:48 :10:2, preferably 40:48:10:2.
  • the composition is lipid nanoparticles, liposomes.
  • the lipid nanoparticles or liposomes of the present invention can be used to prepare cell transfection reagents and have high transfection efficiency.
  • the composition further includes a pharmaceutically active ingredient.
  • the molar ratio of the lipid compound, or its pharmaceutically acceptable salt or its stereoisomer to the pharmaceutically active ingredient is 1 to 100:1.
  • the pharmaceutically active ingredient includes at least one of nucleic acid molecules, polypeptides, proteins and small molecule compounds.
  • the nucleic acid molecule includes at least one of siRNA, mRNA, modRNA, miRNA, circRNA, antisense RNA, CRISPR guide RNA, repRNA, CDN, poly IC, CpG ODN, pDNA, and microcircle DNA. species, preferably siRNA or pDNA.
  • the protein includes at least one of colony-stimulating factors, interleukins, interferon proteins, tumor necrosis factors, antibodies, and protein antigens.
  • the pharmaceutically active ingredient when the pharmaceutically active ingredient includes a nucleic acid molecule, the nitrogen-to-phosphorus ratio of the lipid compound, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and the nucleic acid molecule is 4 to 4 32:1, preferably 16 to 32:1, more preferably 32:1.
  • the composition of the present invention can carry nucleic acid molecules through the cell membrane, so it can be used as a transfection reagent, especially when it is transfected with siRNA, it can effectively inhibit the expression of the target gene; or when it is transfected with pDNA, it can effectively increase the target gene. expression.
  • the preparation method of a composition loaded with pharmaceutical active ingredients includes the following steps:
  • the composition also includes one or more pharmaceutically acceptable excipients; the composition can be in any pharmaceutically acceptable dosage form.
  • Pharmaceutically acceptable excipients are substances that are nontoxic, compatible with the active ingredient, and otherwise biologically compatible with the organism. The selection of specific excipients will depend on the mode of administration or the type and condition of disease used to treat the particular patient.
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, conventional solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, adhesives, lubricants, Stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, etc.
  • flavoring agents, preservatives, sweeteners, etc. can also be added to the pharmaceutical composition.
  • the pharmaceutical compositions described above may be administered to a patient or subject in need of such treatment by oral, parenteral, rectal or pulmonary administration.
  • oral administration the pharmaceutical composition can be made into oral preparations, for example, it can be made into conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be made into oral liquid preparations, such as Oral solutions, oral suspensions, syrups, etc.
  • oral preparations appropriate fillers, binders, disintegrants, lubricants, etc. can be added.
  • parenteral administration the above pharmaceutical composition can also be made into injections, including injection liquid, sterile powder for injection and concentrated solution for injection.
  • the pharmaceutical composition When making injections, conventional methods in the existing pharmaceutical field can be used. When preparing injections, no additives may be added, or appropriate additives may be added according to the properties of the drug.
  • the pharmaceutical composition When used for rectal administration, the pharmaceutical composition can be made into suppositories and the like. When used for transpulmonary administration, the pharmaceutical composition can be made into inhalation preparations, aerosols, powder mist or sprays, etc.
  • the fourth aspect of the present invention provides the above-mentioned lipid compound, or a pharmaceutically acceptable salt or a stereoisomer thereof, or the use of the above-mentioned composition in the preparation of nucleic acid drugs and vaccines, gene vaccines, polypeptide or protein drugs and vaccines, small Applications in molecular medicine.
  • the lipid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer or the above composition can exert an immunotherapeutic effect by activating innate immunity.
  • nucleic acid drugs and vaccines, gene vaccines, polypeptide or protein drugs and vaccines, and small molecule drugs can be used to treat other related diseases caused by genetic abnormalities, including single-gene diseases, such as Marfan Syndrome, hereditary methemoglobinemia, sickle cell anemia, hemochromatosis; polygenic diseases, for example, tumors (such as leukemia blood diseases, breast cancer, thyroid tumors, lung cancer, bone tumors, skin cancer, multiple myeloma, lymphoma, etc.), cardiovascular diseases (such as heart failure, cardiomyopathy, familial hypercholesterolemia, Danon disease, etc.) , metabolic diseases (such as glycogen storage disease, mucopolysaccharidosis, galactosemia, phenylketonuria, congenital hyperammonemia, Gaucher disease, hepatolenticular degeneration, etc.), neurological and psychiatric diseases (such as Parkinson's disease); and acquired genetic diseases, such as AIDS.
  • single-gene diseases such as Marfan
  • the hydrophobic ends of lipids are composed of long carbon chain alkanes or alkenes, which are difficult to be degraded by enzymes and are relatively difficult to metabolize in the body.
  • the lipid compound of the present invention introduces a biodegradable ester bond into the structure, which can be degraded by esterase in the body, making the lipid compound easy to be metabolized and cleared.
  • the ionizable lipid compound of the present invention is electrically neutral under neutral conditions, which can increase the stability of nanoparticles, thereby reducing the cytotoxicity caused by excessive positive charges, and can avoid interactions with positively charged cells in the body.
  • the ionizable lipid compound of the present invention can be ionized into cations, and can be combined with negatively charged pharmaceutical active ingredients through charge interactions, and can be combined with auxiliary lipids such as dioleoylphosphatidylethanolamine (DOPE) ) or distearoylphosphatidylcholine (DSPC), cholesterol (Cholesterol), distearoylphosphatidylacetamide-polyethylene glycol (DSPE-PEG), etc. to form LNP, which can effectively deliver active pharmaceutical ingredients into cells.
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC distearoylphosphatidylcholine
  • cholesterol Choesterol
  • DSPE-PEG distearoylphosphatidylacetamide-polyethylene glycol
  • the ionizable lipid compound of the present invention has a reducing environment-sensitive disulfide bond structure in its head or tail structure.
  • GSH glutathione
  • the disulfide bond can be broken and promote The release of drugs helps nanomedicines escape from endosomes, thereby enhancing the transfection effect.
  • the data in the examples also show that the lipid compound prepared by the present invention has high transfection efficiency.
  • the present invention has the advantages of easy availability of raw materials, simple reaction and high yield.
  • linear alkyl refers to a group containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more carbon atoms) linear saturated hydrocarbon group.
  • carbon atoms e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more carbon atoms
  • C 8 to C 12 linear alkyl group "C 9 to C 17 linear alkyl group” and “C 7 to C 17 linear alkyl group” respectively refer to 8 to 12 carbon atoms, 9 to 17 carbon atoms, straight-chain saturated hydrocarbon groups of 7 to 17 carbon atoms, such as
  • linear alkenyl refers to a linear hydrocarbon radical containing two or more carbon atoms and at least one carbon-carbon double bond (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms).
  • Alkenyl groups may include one to four carbon-carbon double bonds.
  • C 9 to C 17 linear alkenyl group refers to a linear hydrocarbon group including 9 to 17 carbon atoms and one to four carbon ⁇ carbon double bonds.
  • C 7 to C 17 linear alkenyl refers to a linear hydrocarbon group including 7 to 17 carbon atoms and one to four carbon ⁇ carbon double bonds, for example,
  • the "five-membered ring or six-membered ring” includes the following structures:
  • salt may be selected from the group consisting of sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, and dihydrogen phosphates prepared from inorganic acids. Salt, metaphosphate, pyrophosphate, chloride, bromide, iodide, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc.
  • Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate Acid, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, Methanesulfonate, glucoheptonate, lactobionate, lauryl sulfonate and isethionate, etc.
  • Salts may also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • Representative salts include: acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, Maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluate, benzene Acetate, citrate, lactate, maleate, tartrate, methanesulfonate, etc.
  • Pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethyl Ammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also covered are salts of amino acids, such as arginine salts, gluconates, galacturonates, etc.
  • isomers refers to different compounds having the same molecular formula. Stereoisomers are particularly preferred in the present invention, with the term “stereoisomer” being isomers that differ only in the spatial arrangement of the atoms.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • stereoisomers described in the present invention include cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, non- Enantiomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomeric or diastereomerically enriched mixtures, all of which Mixtures are within the scope of this invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • composition includes products containing an effective amount of a compound of the present invention, as well as any product resulting, directly or indirectly, from a combination of compounds of the present invention.
  • Figure 1 is a proton nuclear magnetic resonance spectrum of intermediate 1 of the present invention.
  • Figure 2 is a hydrogen nuclear magnetic resonance spectrum of B2 of the present invention.
  • Figure 3 is a hydrogen nuclear magnetic resonance spectrum of B4 of the present invention.
  • Figure 4 is a hydrogen nuclear magnetic resonance spectrum of B6 of the present invention.
  • Figure 5 is a hydrogen nuclear magnetic resonance spectrum of C2 of the present invention.
  • Figure 6 is a proton nuclear magnetic resonance spectrum of C-VA of the present invention.
  • Figure 7 is a proton nuclear magnetic resonance spectrum of intermediate 4 of the present invention.
  • Figure 8 is a hydrogen nuclear magnetic resonance spectrum of 2-O15B of the present invention.
  • Figure 9 shows the firefly fluorescence after LNPs constructed by A1, A2, B1-B6, C2, C4, C6, 1-O15B and 2-O15B of the present invention are encapsulated with firefly luciferase small interfering RNA (Luciferase siRNA, siLuc). Table of Luciferase Quantitative results chart.
  • Figure 11 shows the transfection of A1, A2, B1-B6, C2, C4, C6, 1-O15B, 2-O15B, 3-O15B, 4-O15B, 2-O19B and 2-O25B of the present invention into LNP.
  • Green fluorescent protein plasmid DNA relative luciferase activity results after 48 hours; among them, polyethylenimine (PEI) is a commercial transfection reagent.
  • PEI polyethylenimine
  • FIG. 12 After A1, A2, B1-B6, C2, C4, C6, 1-O15B, 2-O15B, 3-O15B, 4-O15B, 2-O19B and 2-O25B of the present invention are made into LNP, they are transfected with green Fluorescent protein plasmid DNA, fluorescence microscope picture after 48 hours; among them, PEI is a commercial transfection reagent.
  • Figure 13 shows the ability of LNP to condense DNA at different nitrogen to phosphorus ratios.
  • Figure 14 is a comparison chart of the stability of LNP condensed DNA under different conditions.
  • Figure 15 shows A1, A2, B1-B6, C2, C4, C6, C-VA, C-VE, 1-O15B, 2-O15B, 3-O15B, 4-O15B, 5-O15B, 6- Picture of the activation effect of O15B, 1-O19B, 2-O19B, 1-O25B, and 2-O25B on innate immunity after being made into LNP.
  • the hydrogen spectrum of the obtained product is shown in Figure 6.
  • the reaction mechanism of the lipid compound of the present invention can be summarized as the alcoholysis reaction of acid chloride and the Michael addition reaction of olefin and organic amine. This reaction mechanism is very mature and the reaction process is well known in the art. Therefore, the specific type and reaction degree of the compounds generated by the above reaction mechanism are completely predictable.
  • the above are the reaction conditions and structural characterization of some of the compounds synthesized by the present invention.
  • the synthesis of other compounds of the present invention is the same as the above-mentioned compounds, and their structural formulas and structural characterization data will not be described in detail here.
  • a melanoma cell line expressing firefly luciferase (B16F10-Luciferase, B16F10-Luc) was used to verify the efficiency of gene knockout by delivering siLuc via LNP containing ionizable lipids.
  • Ionizable lipids 1-O15B, 2-O15B, A1, A2, B1-B6, C2, C4, and C6 were used as carrier materials for siLuc delivery respectively.
  • B16F10-Luc cells were seeded in a 96-well cell culture plate, and the cells were transfected until the cell density grew to about 70-80%.
  • the volume of the sodium buffer is twice the total volume of the lipid mixture), quickly mix the buffer containing siLuc with the lipid mixture to form LNP, and incubate at room temperature for 15 min. After diluting the prepared LNP with PBS, add it to the cell culture medium for transfection (each well is transfected with LNP containing 50ng siLuc).
  • the nitrogen-phosphorus ratio (N/P ratio) of the ionizable lipid and siRNA can be The optimal ratio is a molar ratio of 32:1 between the protonatable amino groups on the ionized lipid and the phosphate groups on the RNA.
  • Lipofectamine 2000 lipo2000 commercial transfection reagent was used to transfect siLuc. According to the instructions for use of lipo2000, add 50ng siLuc to 5 ⁇ L Opti-MEM transfection solution, then add 0.3 ⁇ L lipo2000 to another 5 ⁇ L Opti-MEM transfection solution, and then mix the siRNA Opti-MEM solution with lipo2000 The Opti-MEM solution was mixed thoroughly, incubated at room temperature for 15 minutes, and then added to the B16F10-Luc cell culture plate for transfection.
  • Negative control group B16F10-Luc cells were cultured normally without transfection.
  • the results are shown in Figure 9.
  • the negative control group expressed the highest relative amount of firefly luciferase, and the positive control lipo2000 transfection efficiency was 30% (the relative amount of firefly luciferase expressed was 0.7, and 30% of gene expression was knocked out relative to the negative control).
  • the ionizable lipid compound A2 was used to optimize the ratio between the four materials for preparing nanoparticles and deliver siLuc in melanoma cell lines (B16F10-Luciferase, B16F10-Luc).
  • Example 14 the experimental group is: ionizable lipid A2, DSPC, Cholesterol, and DSPE-PEG are dissolved in absolute ethanol at a certain concentration and stored in a -20°C refrigerator.
  • Ratio B is ionizable lipid A2: Cholesterol: DSPC: DSPE-PEG.
  • Plasmid DNA (pDNA-GFP-Luc) encoding both green fluorescent protein (GFP) and firefly luciferase (Luc) was transfected into the 293T cell line using ionizable lipid delivery. Ionizable lipids 1-O15B, 2-O15B, 3-O15B, 4-O15B, 2-O19B, 2-O25B, A1, A2, B1-B6, C2, C4, and C6 lipids were used as pDNA-GFP- Carrier material delivered by Luc.
  • 293T cells were seeded in a 96-well cell culture plate, and the cells were transfected until the cell density grew to about 70-80%.
  • Experimental group combine ionizable lipids 1-O15B, 2-O15B, 3-O15B, 4-O15B, 2-O19B, 2-O25B, A1, A2, B1-B6, C2, C4, C6 and DOPE, Cholesterol, Dissolve DSPE-PEG in absolute ethanol at a certain concentration and store it in a -20°C refrigerator.
  • the prepared LNPs were diluted with PBS and then added to the cell culture medium for transfection (each well was transfected with LNP containing 200ng pDNA-GFP-Luc).
  • the nitrogen-phosphorus ratio of the ionizable lipid to pDNA (N/ P ratio (the molar ratio between the protonatable amino groups on the ionizable lipid and the phosphate groups on the DNA) 32:1 is the optimal ratio.
  • Positive control group pDNA-GFP-Luc was transfected using PEI commercial transfection reagent and cationic lipid DOTAP.
  • PEI commercial transfection reagent and cationic lipid DOTAP.
  • dissolve 200ng DNA in 5 ⁇ L DEPC water and mix well then dissolve 2 ⁇ L PEI in 5 ⁇ L DEPC, add the diluted PEI to the DNA DEPC water solution, incubate at room temperature for 15 minutes, and add it to the 96-well cells of 293T Transfection was performed in a culture plate;
  • DOTAP-LNP was prepared in the same way as the ionizable lipid preparation method described above.
  • To prepare DOTAP-LPR nanoparticles first prepare DOTAP liposomes.
  • the specific operation is to air-dry the chloroform mixture of DOTAP and cholesterol (1:1, molar ratio) and dry it under a vacuum pump overnight; add preheated DEPC water (50°C ) was added to the lipid membrane, vortexed for 1 min, and ultrasonicated in a 55°C water bath for 10 min for hydration; then the liposomes were extruded through polycarbonate membranes (Whatman) with pore sizes of 200 nm and 100 nm in order to form 80-100 nm Unilamellar liposomes.
  • the steps for preparing DOTAP-LPR nanoparticles are as follows: mix the DEPC aqueous solution containing 200ng pDNA-GFP-Luc and 150ng protamine (0.1mg/mL) and incubate at room temperature for 10min, and then add 1.939 ⁇ L of premade DOTAP Liposomes (10mM) were mixed with the above solution and incubated at room temperature for 10 min. Finally, 1.11 ⁇ g DSPE-PEG was mixed with the above solution, placed in a 50°C metal bath for 12 min, and then transfected.
  • Negative control group 293T cells were cultured normally without transfection.
  • the negative control expresses the lowest firefly luciferase
  • the positive control PEI transfection expresses the relative amount of firefly luciferase up to 1.6 million values.
  • the commercial cationic lipid DOTAP is prepared by either DOTAP-LNP or DOTAP- Neither LPR can efficiently transfect pDNA-GFP-Luc.
  • the partially ionizable lipids of the present invention have strong transfection efficiency, among which 2-O15B expresses the highest relative amount of firefly luciferase, reaching a value of 1.9 million.
  • LNPs containing pDNA-GFP-Luc use 2-O15B, 2-O19B, and 2-O25B to prepare nitrogen and phosphorus ratios of 8:1, 16:1, 24:1, and 32:1 respectively (i.e. The molar ratio between the protonatable amino group on the ionizable lipid and the phosphate group on the DNA) LNP.
  • the LNP is mixed with an appropriate amount of PBS and then added to a 1% agarose gel at a voltage of 100V. Electrophoresis was carried out for 50 minutes under the conditions, and the results were observed using a gel imager. See Figure 13 for details.
  • LNP containing 2-O15B can still completely condense DNA when the nitrogen to phosphorus ratio is as low as 8:1, while LNP containing 2-O19B and 2-O25B can condense DNA when the nitrogen to phosphorus ratio is 24:1.
  • DNA can be completely condensed only at 32:1, indicating that Within a certain range, as the length of the alkyl chain increases, the ability of LNP to condense DNA gradually decreases.
  • the condensation of LNP and DNA is mainly due to electrostatic interaction, and an alkyl chain that is too long may affect the protonation of the amino head. ability, so the ability to package DNA decreases.
  • LNP containing 2-O15B can remain stable in PBS solution, and the stability of LNP can still be maintained in a solution containing 10% serum for 2 hours without DNA release.
  • 5Mm GSH simulated cytoplasmic environmental conditions
  • a certain amount of DNA was released after 30 minutes, which shows that this LNP can trigger the degradation of disulfide bonds in cells, thereby effectively releasing the wrapped genetic drugs.
  • LNP prepared from ionizable lipids The activation effect of LNP prepared from ionizable lipids on the innate immunity of RAW-Luc cells was detected.
  • ISG interferon-stimulated gene
  • RAW-Luc cells were seeded in a 96-well cell culture plate, and the cells were transfected until the cell density grew to about 70-80%.
  • lipids 1-O15B, 2-O15B, 3-O15B, 4-O15B, 5-O15B, 6-O15B, 1-O19B, 2-O19B, 1-O25B, 2-O25B, A1, A2, B1-B6, C2, C4, C6, C-VA, C-VE and DOPE, Cholesterol, DSPE-PEG are dissolved in absolute ethanol at a certain concentration and stored in a -20°C refrigerator.
  • the prepared LNPs were diluted with PBS and then added to the cell culture medium for transfection.
  • Negative control group RAW-Luc cells were cultured normally without transfection.

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Abstract

本发明涉及生物医药技术领域，具体公开了一种脂质化合物、包含其的组合物及应用。该脂质化合物具有式(I)或式(II)所示的结构。该脂质化合物在酸性条件下可电离成阳离子化合物，与带负电的药物活性成分通过静电相互作用结合，从而组装成载药脂质纳米颗粒，进行药物活性成分的递送。本发明所提供的脂质化合物结构简单，反应路径简单，产率高，构建得到的载药脂质纳米颗粒可用于制备核酸药物和疫苗、基因疫苗、多肽或蛋白质药物和疫苗、小分子药物，具备广泛的应用前景；

Description

一种脂质化合物、包含其的组合物及应用 技术领域
本发明属于生物医药技术领域，具体涉及一种脂质化合物、包含其的组合物及应用。
背景技术
基因治疗是指将一类核酸药物如siRNA、mRNA、pDNA等一些具有治疗目的的外源基因通过载体材料递送到靶细胞中从而发挥治疗效果。几十年来基因治疗已经在临床治疗上取得了较好的效果，可以有效的治疗先天和后天的疾病，如，癌症、自身免疫性疾病、传染病、心脏病等。事实上，基因治疗并不难理解，即替换补偿异常基因。但是基因治疗也面临着许多亟需解决的难题，其中核酸药物如何借助载体进入人体特定细胞发挥作用是基因治疗的关键环节。因此，开发一种高效安全的核酸递送系统是基因治疗的基石。
目前运用最广泛的核酸递送系统主要是病毒载体和非病毒载体，其中病毒载体转染效率相对较高，但是存在安全性、靶向性差等问题。近年来，非病毒载体发展迅速，其中脂质体具有免疫原性低、生物相容性好，转染效率高等优势而被看作理想的核酸递送系统。尤其是，可电离阳离子脂质具有pH依赖性电荷可变特性，安全性较高，但其转染效率有待提高。
发明内容
本发明旨在解决上述现有技术中存在的技术问题。为此，本发明提出一种脂质化合物、包含其的组合物及应用。该脂质化合物可单独使用或与其它脂质化合物，如胆固醇、辅助磷脂、聚乙二醇修饰脂质组合使用，用于高效地将药物活性成分递送至细胞或者组织，发挥特定的生物功能，如递送siRNA至体内细胞发挥基因沉默作用或者递送mRNA、pDNA至细胞促进基因表达。另外，该脂质化合物的反应路径简单，产率高，因此，具有广泛的应用前景。
本发明的第一方面，提供一种脂质化合物，或其药学上可接受的盐或其立体异构体，所述脂质化合物具有式(I)所示的结构：

优选的，所述R2选自如下所示的结构：
n选自0～3的整数，优选1或2；
或，所述脂质化合物具有式(II)所示的结构；
y选自1或2；其中，
当y选自1时，R3选自
其中，R5选自羟基、未取代或甲基取代的四氢吡咯基、哌啶基、哌嗪基、二甲氨基、二羟甲基氨基、二乙氨基、二羟乙基氨基；优选羟基、未取代或甲基取代的四氢吡咯基、哌啶基、哌嗪基；
当y选自2时，R3选自
其中X选自亚甲基、氧原子、或者X与式(II)中的任意一个N相连形成五元杂环或六元杂环；或R3与式(II)中的两个N一起形成五元杂环或六元杂环；
优选的，所述X选自亚甲基；
每个p各自独立的选自0或1，且，
式(II)中至多存在一个p为0，且，
当p均为1时，R4选自C7～C17直链烷基或直链烯基；
或，当其中一个p为0、其余p均为1时，与N原子直接相连的R4选自氢，其余R4独立选自C7～C17直链烷基或直链烯基；
优选的，R4选自如下所示的结构：
在本发明的一些实施方式中，
R2选自如下所示的结构：
n选自1或2；
y选自1或2；其中，
当y选自1时，R3选自其中，R5选自羟基、未取代或甲基取代的四氢吡咯基；
当y选自2时，R3选自其中X选自
每个p各自独立的选自0或1，且，
式(II)中至多存在一个p为0，且，
当p均为1时，R4选自C7～C17直链烷基或直链烯基；
或，当其中一个p为0、其余p均为1时，与N原子直接相连的R4选自氢，其余R4独立选自C7～C17直链烷基或直链烯基；
优选的，R4选自如下所示的结构：
在本发明的一些实施方式中，所述脂质化合物选自如下所示的结构：

优选的，所述脂质化合物选自：A1、A2、B1、B2、B3、B4、B5、C2、C4、C6、1-O15B、2-O15B、3-O15B、2-O19B、1-O25B、2-O25B。
本发明的可电离脂质化合物分为两个大类：(1)式(I)所示结构，在二硫键(S-S)

两端依次连接酯基、叔胺、烷基链，还可在烷基链与叔胺之间、或烷基链中引入酯基以提高脂质化合物的生物降解性；以及，(2)式(2)所示结构，将带有酯基和二硫键的疏水尾部与有机胺连接。本发明的可电离脂质化合物在血液中运输时呈电中性或者电负性，可以减小用于基因递送的传统阳离子脂质中的正电荷所带来的细胞毒性，并且可以延长体内循环时间，改善药物代谢动力学特征。本发明的脂质化合物可在酸性条件下获得正电荷，再与带有负电荷的RNA、DNA以及小分子药物通过静电相互作用结合，自组装为脂质纳米颗粒(Lipid Nanoparticle,LNP)，进行活性成分的递送，如，可以在哺乳动物细胞内高效递送DNA、mRNA，表达靶向基因，以及高效转染siRNA，特异性沉默靶向基因表达。由于细胞内上调的谷胱甘肽等还原性物质可以快速降解可电离脂质化合物中的二硫键，导致纳米载体破裂及包载的核酸分子释放到细胞质中，从而实现内涵体逃逸，提升转染效率。除此之外，二硫键和酯键结构的可生物降解性使得可电离脂质化合物更容易在体内被代谢。
本发明的第二方面，提供上述脂质化合物的制备方法：
当所述脂质化合物具有式(I)所示的结构，且其中所述R1选自C8～C12直链烷基时，所述脂质化合物的合成路线如下：
当所述脂质化合物具有式(I)所示的结构，且其中所述R1选自时，所述脂质化合物的合成路线如下：
R2、n分别如前文所定义。
当所述脂质化合物具有式(II)所示的结构时，该所述化合物由丙烯酸酯衍生物与有机胺通过迈克尔加成反应得到；该反应可以通过控制有机胺与丙烯酸酯衍生物的化学当量比来控制有机胺氮上游离氢原子的取代程度。
所述丙烯酸酯具有如下所示的结构：
R4如前文所定义。
优选的，所述有机胺选自如下所示的结构：
与传统阳离子脂质的合成需要复杂的化学合成路线不同，本发明提供的可电离脂质结构精简，反应途径简单，产率较高。
本发明的第三方面，提供一种组合物，所述组合物包括上述的脂质化合物，或其药学上可接受的盐或其立体异构体。
在本发明的一些实施方式中，所述组合物还包括其他脂质化合物。
在本发明的一些实施方式中，所述其他脂质化合物包括胆固醇或β-谷甾醇以及它们的衍生物、辅助磷脂和聚乙二醇修饰脂质中的至少一种。
在本发明的一些实施方式中，所述辅助磷脂包括蛋黄卵磷脂、氢化蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、鞘磷脂、磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰基卵磷脂(DOPC)、二油酰磷脂酰胆碱、二月桂酰磷脂酰胆碱中的至少一种。
在本发明的一些实施方式中，所述聚乙二醇(PEG)修饰脂质包括PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油中的至少一种。
在本发明的一些实施方式中，在所述组合物还包括胆固醇或β-谷甾醇以及它们的衍生物、辅助磷脂和PEG修饰脂质时，所述脂质化合物、或其药学上可接受的盐或其立体异构体：胆固醇或β-谷甾醇以及它们的衍生物：辅助磷脂：PEG修饰脂质的摩尔比为30～50：30～50：5～20：1～2.5，例如，40～50：35～50：5～10：1.5～2、40～50：40～50：5～10：1.5～2、40～45：45～48：8～10：1.5～2、40：48：10：2，优选40：48：10：2。
在本发明的一些实施方式中，所述组合物为脂质纳米颗粒、脂质体。本发明所述的脂质纳米颗粒或脂质体可用于制备细胞转染试剂，具有很高的转染效率。
在本发明的一些实施方式中，所述组合物还包括药物活性成分。
在本发明的一些实施方式中，所述脂质化合物，或其药学上可接受的盐或其立体异构体与药物活性成分的摩尔比为1～100：1。
在本发明的一些实施方式中，所述药物活性成分包括核酸分子、多肽、蛋白质和小分子化合物中的至少一种。
在本发明的一些实施方式中，所述核酸分子包括siRNA、mRNA、modRNA、miRNA、circRNA、antisense RNA、CRISPR guide RNA、repRNA、CDN、poly IC、CpG ODN、pDNA、微环DNA中的至少一种，优选siRNA、pDNA。
在本发明的一些实施方式中，所述蛋白质包括细胞集落刺激因子、白介素类、干扰素类蛋白、肿瘤坏死因子、抗体类、蛋白抗原中的至少一种。
在本发明的一些实施方式中，当所述药物活性成分包括核酸分子时，所述脂质化合物、或其药学上可接受的盐或其立体异构体与核酸分子的氮磷比为4～32：1，优选16～32：1，更优选32：1。
具体的，本发明的组合物可携带核酸分子透过细胞膜，因此可以作为转染试剂，尤其当转染siRNA后，能够有效抑制靶基因的表达；或当转染pDNA后，能够有效增加靶基因的表达。
在本发明的一些实施方式中，负载药物活性成分的组合物的制备方法包括以下步骤：
将所述脂质化合物与所述药物活性成分按比例溶解于缓冲盐溶液(pH＝4～6)中；若存在其他脂质化合物，在所述混合过程中加入所述其他脂质化合物；室温孵育15-60min，即得。
所述组合物还包括一种或多种药学上可接受的辅料；该组合物可为药学上可接受的任一剂型。药学上可接受的辅料是无毒性、与活性成分相容且其他方面生物学性质上适用于生物体的物质。特定辅料的选择将取决于用于治疗特定患者的给药方式或疾病类型和状态。药学上可接受的辅料其实例包括但不限于药学领域常规的溶剂、稀释剂、分散剂、助悬剂、表面活性剂、等渗剂、增稠剂、乳化剂、粘合剂、润滑剂、稳定剂、水合剂、乳化加速剂、缓冲剂、吸收剂、着色剂、离子交换剂、脱模剂、涂布剂、矫味剂、和抗氧化剂等。必要时，还可以在药物组合物中加入香味剂、防腐剂和甜味剂等。
在某些实施方案中，上述药物组合物可以以口服、肠胃外、直肠或经肺给药等方式施用于需要这种治疗的患者或受试者。用于口服给药时，所述药物组合物可制成口服制剂，例如可以制成常规的口服固体制剂，如片剂、胶囊剂、丸剂、颗粒剂等；也可制成口服液体制剂，如口服溶液剂、口服混悬剂、糖浆剂等。制成口服制剂时，可以加入适宜的填充剂、粘合剂、崩解剂、润滑剂等。用于肠胃外给药时，上述药物组合物也可制成注射剂、包括注射液、注射用无菌粉末与注射用浓溶液。制成注射剂时，可采用现有制药领域中的常规方法生产，配置注射剂时，可以不加入附加剂，也可以根据药物的性质加入适宜的附加剂。用于直肠给药时，所述药物组合物可制成栓剂等。用于经肺给药时，所述药物组合物可制成吸入制剂、气雾剂、粉雾剂或喷雾剂等。
本发明的第四方面，提供上述脂质化合物、或其药学上可接受的盐或其立体异构体，或上述组合物在制备核酸药物和疫苗、基因疫苗、多肽或蛋白质药物和疫苗、小分子药物中的应用。
特别的，本发明所述的脂质化合物、或其药学上可接受的盐或其立体异构体或上述组合物可通过激活先天性免疫发挥免疫治疗作用。
除此之外，所述的核酸药物和疫苗、基因疫苗、多肽或蛋白质药物和疫苗、小分子药物为可用于治疗其它由基因异常引起的相关疾病，所述疾病包括单基因疾病，例如马凡氏综合征、遗传性高铁血红蛋白血症、镰刀红细胞贫血、血色病；多基因疾病，例如，肿瘤(如白 血病、乳腺癌、甲状腺肿瘤、肺癌、骨肿瘤、皮肤癌、多发性骨髓瘤、淋巴瘤等)、心血管疾病(如心力衰竭、心肌病、家族性高胆固醇血症、达农病等)、代谢性疾病(如糖原累积病、黏多糖病、半乳糖血症、苯丙酮尿症、先天性高氨血症、戈谢病、肝豆状核变性等)、神经和精神类疾病(如帕金森症)；以及获得性基因病，例如，艾滋病。
根据本发明实施方式的脂质化合物或组合物，至少具备如下有益效果：
在现有的技术中，脂质的疏水端由长碳链烷烃或烯烃构成，难以被酶降解，体内代谢相对困难。而本发明的脂质化合物在结构中引入了生物可降解的酯键，在体内可被酯酶降解，使得脂质化合物易于代谢清除。本发明的可电离脂质化合物在中性条件下呈电中性，可以增加纳米颗粒的稳定性，从而减小因正电荷过多而带来的细胞毒性，而且能避免在体内与带正电的血红蛋白结合而迅速降解，并有助于延长所负载的基因药物的体内循环时间、改善其药物动力学特征。但在酸性pH条件下，本发明的可电离脂质化合物可电离成阳离子，能够与带负电的药物活性成分通过电荷的相互作用进行结合，与辅助性脂质如二油酰磷脂酰乙醇胺(DOPE)或二硬脂酰基磷脂酰胆碱(DSPC)、胆固醇(Cholesterol)、二硬脂酰磷脂酰乙酰胺-聚乙二醇(DSPE-PEG)等组成LNP，有效递送药物活性成分进入细胞内。本发明的可电离脂质化合物，其头部或尾部结构中具有还原性环境敏感的二硫键结构，在胞内高浓度谷胱甘肽(GSH)存在下，可以使二硫键断裂，促进药物的释放，帮助纳米药物从内涵体中逃脱，进而增强转染效果。实施例中的数据也表明本发明制备得到的脂质化合物具有很高的转染效率。除此之外，本发明具有原料易得、反应简单、产率高的优点。
本申请中术语：
术语“直链烷基”是指包括一个或多个碳原子(例如、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个碳原子)的直链饱和烃基。具体地，“C8～C12直链烷基”、“C9～C17直链烷基”、“C7～C17直链烷基”分别指包括8～12个碳原子、9～17个碳原子、7～17个碳原子的直链饱和烃基，例如
术语“直链烯基”是指包含两个或更多个碳原子和至少一个碳□碳双键的直链烃基(例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个碳原子)。烯基可以包括一个至四个碳□碳双键。具体地，“C9～C17直链烯基”是指包括9～17个碳原子和一个至四个碳□碳双键的直链烃基。“C7～C17直链烯基”是指包括7～17个碳原子和一个至四个碳□碳双键的直链烃基，例如，
所述“五元环或六元环”包括以下结构：

术语“药学上可接受的盐”可选自无机酸制备的硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、硝酸盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物，酸例如盐酸、硝酸、硫酸、氢溴酸、氢碘酸、磷酸等。代表性盐包括：氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、硝酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、甲苯磺酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、萘甲酸盐、甲磺酸盐、葡庚糖酸盐、乳糖酸盐、月桂基磺酸盐和羟乙磺酸盐等。盐也可以是从有机酸制备的，例如脂肪族一元与二元羧酸、苯基取代的烷酸、羟基烷酸、烷二酸、芳香族酸、脂肪族与芳香族磺酸等。代表性盐包括：乙酸盐、丙酸盐、辛酸盐、异丁酸盐、草酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、扁桃酸盐、苯甲酸盐、氯苯甲酸盆、甲基苯甲酸盐、二硝基苯甲酸盐、萘甲酸盐、苯磺酸盐、甲苯奭酸盐、苯乙酸盐、柠檬酸盐、乳酸盐、马来酸盐、酒石酸盐、甲磺酸盐等。药学上可接受的盐可以包括基于碱金属与碱土金属的阳离子，例如钠、锂、钾、钙、镁等，以及无毒的铵、季铵和胺阳离子，包括但不限于铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、乙胺等。还涵盖氨基酸的盐，例如精氨酸盐、葡糖酸盐、半乳糖醛酸盐等。
术语“异构体”为具有同样分子式的不同化合物。本发明尤其优选立体异构体，术语“立体异构体”为只是原子空间排列不同的异构体。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明所述的这类立体异构体，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
术语“组合物”包括含有效量的本发明的化合物的产品，以及直接地或间接地由本申请化合物的组合产生的任何产品。
附图说明
下面结合附图和实施例对本发明作进一步的说明，其中：
图1为本发明中间体1的核磁共振氢谱图。
图2为本发明B2的核磁共振氢谱图。
图3为本发明B4的核磁共振氢谱图。
图4为本发明B6的核磁共振氢谱图。
图5为本发明C2的核磁共振氢谱图。
图6为本发明C-VA的核磁共振氢谱图。
图7为本发明中间体4的核磁共振氢谱图。
图8为本发明2-O15B的核磁共振氢谱图。
图9为本发明A1、A2、B1-B6、C2、C4、C6、1-O15B、2-O15B所构建的LNP包载萤火虫萤光素酶小干扰RNA(Luciferase siRNA,siLuc)后，萤火虫荧光素酶(Luciferase)的表 达量结果图。
图10为本发明A2所构建的LNP包载siLuc后，Luciferase的表达量结果图；其中，比例A为可电离脂质化合物A2：Cholesterol：DSPC：DSPE-PEG＝30:28.5:10:0.75；比例B为可电离脂质化合物A2：Cholesterol：DSPC：DSPE-PEG＝50:38.5:10:1.5；比例C为可电离脂质化合物A2：Cholesterol：DSPC：DSPE-PEG＝35:46.5:16:2.5；比例D为可电离脂质化合物A2：Cholesterol：DSPC：DSPE-PEG＝40:48:10:2。
图11为本发明的A1、A2、B1-B6、C2、C4、C6、1-O15B、2-O15B、3-O15B、4-O15B、2-O19B、2-O25B制成LNP后，转染绿色荧光蛋白质粒DNA，48h后的相对荧光素酶活性结果图；其中，聚乙烯亚胺(PEI)为商业化转染试剂。
图12本发明的A1、A2、B1-B6、C2、C4、C6、1-O15B、2-O15B、3-O15B、4-O15B、2-O19B、2-O25B制成LNP后，转染绿色荧光蛋白质粒DNA，48h后的荧光显微镜图；其中，PEI为商业化转染试剂。
图13为LNP在不同氮磷比固缩DNA的能力图。
图14为LNP在不同条件下固缩DNA的稳定性对比图。
图15为本发明的A1、A2、B1-B6、C2、C4、C6、C-VA、C-VE、1-O15B、2-O15B、3-O15B、4-O15B、5-O15B、6-O15B、1-O19B、2-O19B、1-O25B、2-O25B制成LNP后，对先天性免疫的激活作用结果图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例
实施例1中间体1的合成
向装有磁子的三口烧瓶中加入17mmol 2,2-二硫二乙醇、51mmol三乙胺(TEA)、25mL四氢呋喃(THF)，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加40.8mmol丙烯酰氯，反应在室温下过夜，然后用20mL二氯甲烷(DCM)稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯体积比为10：1)，得到二硫烷二基双(乙烷-2,1-二基)二丙烯酸酯(中间体1)，收率达92％。所得产物的氢谱如图1所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ6.36(d,J＝17.2Hz,2H),6.07(t,J＝10.4Hz,2H),5.79(d,J＝13.6Hz,2H),4.37-4.33(m,4H),2.92-2.90(m,4H)。
实施例2中间体2的合成
向装有磁子的5mL反应瓶内加入5mmol中间体1和11mmol 2-甲氨基乙醇，在室温搅拌24h，使用旋转蒸发仪除去溶剂，得到产物为中间体2，进行下一步反应。
实施例3中间体3的合成
向装有磁子的5mL反应瓶内加入5mmol中间体1，11mmol 2-甲氨基丙醇，在室温搅拌24h，使用旋转蒸发仪除去溶剂，得到产物为中间体3，即可进行下一步反应。
实施例4脂质化合物B2的合成
向装有磁子的三口烧瓶中加入5mmol中间体2、12mmol TEA、25mL DCM，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加12mmol月桂酰氯，反应在室温下过夜，然后用20mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝1：1)，得到可电离脂质化合物B2，收率达86％。所得产物氢谱如图2所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ4.29(t,J＝6.8Hz,4H),4.10(t,J＝6.0Hz,4H),2.87(t,J＝6.8Hz,4H),2.72(t,J＝7.2Hz,4H),2.60(t,J＝6.0Hz,4H),2.44(t,J＝7.2Hz,4H),2.25(t,J＝8.8Hz,10H),1.57-1.54(m,4H),1.26-1.20(m,32H),0.82(t,J＝6.4Hz,6H)。
实施例5脂质化合物B4的合成
向装有磁子的三口烧瓶中加入5mmol中间体2、12mmol TEA、25mL DCM，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加12mmol棕榈酰氯，反应在室温下过夜，然后用20mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝1：1)，得到可电离脂质化合物B4，收率达81％。所得产物氢谱如图3所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ4.33(t,J＝6.4Hz,4H),4.14(t,J＝6.0Hz,4H),2.91(t,J＝6.4Hz,4H),2.76(t,J＝7.2Hz,4H),2.64(t,J＝6.0Hz,4H),2.48(t,J＝7.2Hz,4H),2.28(t,J＝4.8Hz,10H),1.61-1.58(m,4H),1.28-1.24(m,46H),0.87(t,J＝6.4Hz,6H)。
实施例6脂质化合物B6的合成
向装有磁子的三口烧瓶中加入5mmol中间体2、12mmol TEA、25mL DCM，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加12mmol油酰氯，反应在室温下过夜，然后用20mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝1：1)，得到可电离脂质化合物B6，收率达67％。所得产物氢谱如图4所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ5.37-5.31(m,4H),4.33(t,J＝6.4Hz,4H),4.15(t,J＝6.0Hz,4H),2.90(t,J＝6.4Hz,4H),2.77(t,J＝7.2Hz,4H),2.66(t,J＝6.0Hz,4H),2.49(t,J＝7.2Hz,4H),2.29(t,J＝7.2Hz,10H),2.04-1.98(m,8H),1.59(t,J＝7.2Hz,4H),1.29-1.24(m,40H),,0.87-0.85(m,6H)。
实施例7脂质化合物C2的合成
向装有磁子的三口烧瓶中加入5mmol中间体3、12mmol TEA、25mL DCM，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加12mmol月桂酰氯，反应在室温下过夜，然后用20
mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝2：1)，得到可电离脂质化合物C2，收率达80％。所得产物氢谱如图5所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ4.33(t,J＝6.4Hz,4H),4.08(t,J＝6.4Hz,4H),2.91(t,J＝6.4Hz,4H),2.68(t,J＝7.2Hz,4H),2.49-2.40(m,8H),2.29-2.21(m,10H),1.77(t,J＝7.2Hz,4H),1.61-1.58(m,4H),1.30-1.24(m,32H),0.86(t,J＝6.4Hz,6H)。
实施例8脂质化合物C4的合成
向装有磁子的三口烧瓶中加入5mmol中间体3、12mmol TEA、25mL DCM，在冰浴下搅拌20min，然后使用恒压漏斗缓慢滴加12mmol棕榈酰氯，反应在室温下过夜，然后用20mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发 仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝2：1)，得到可电离脂质C4，收率达78％。
实施例9脂质化合物C-VA的合成
向装有磁子的三口烧瓶中加入2mmol中间体3、4.8mmol视黄酸(Retinoic Acid)，再依次加入0.83mmol 4-二甲氨基吡啶(DMAP)，6.3mmol 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)，25mL DCM，反应在室温下过夜，通过薄层色谱(Thin Layer Chromatography,TLC)检测原料反应完毕，用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(DCM：甲醇＝20：1)，得到可电离脂质化合物C-VA，收率达64％。所得产物氢谱如图6所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ6.97-6.91(m,2H)，6.33-6.06(m,10H)，3.78(t,J＝7.6Hz,4H)，3.33-3.29(m,4H)，2.34-2.28(m,5H)，2.22-2.16(m,18H)，2.03-1.95(m,11H)，1.81-1.78(m,4H)，1.71-1.59(m,14H)，1.47-1.44(m,4H)，1..01(t,J＝6.4Hz,12H)。
实施例10中间体4的合成
向装有磁子的三口烧瓶中加入8.87mmol巯基乙醇、9.67mmol TEA、20mL三氯甲烷，在冰浴下搅拌15min，然后使用恒压漏斗缓慢滴加8mmol辛酰氯，反应在室温下过夜，然后用20mL DCM稀释并用50mL HCl(1mol/L)洗涤，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，得到产物2-巯基辛酸乙酯，可进行下一步反应。
向装有磁子的三口烧瓶中加入13.65mmol 2,2’-二硫二吡啶、催化当量的冰乙酸、12mL甲醇，在室温搅拌下缓慢滴加6.75mmol 2-巯基辛酸乙酯(溶于10mL DCM溶液中)，反应在室温下搅拌3h，用DCM和饱和食盐水萃取，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝15：1)，得到产物2-(吡啶-2-基二硫烷基)辛酸乙酯，收率达85％。
向装有磁子的三口烧瓶中加入5mmol 2-(吡啶-2-基二硫烷基)辛酸乙酯、催化当量的冰乙酸、10mL甲醇，在室温搅拌下缓慢滴加5.5mmol巯基乙醇(溶于10mL甲醇溶液中)，反应在室温下搅拌6h，用DCM和饱和食盐水萃取，有机层用Na2SO4干燥并过滤，使用旋转蒸发仪除去溶剂，得到产物2-((2-羟乙基)二硫烷基)辛酸乙酯，进行下一步反应。
向装有磁子的三口烧瓶中加入5mmol 2-(吡啶-2-基二硫烷基)辛酸乙酯、7.5mmol TEA、20mL DCM，在冰浴下搅拌20min，然后用恒压漏斗缓慢滴加6.25mmol丙烯酰氯，反应在室温下过夜，用DCM(20mL)稀释并用50mL HCl(1mol/L)洗涤，，有机层用Na2SO4干燥
并过滤，使用旋转蒸发仪除去溶剂，产物通过薄层色谱柱分离(石油醚：乙酸乙酯＝15：1)，得到中间体4，收率达76％。产物氢谱如图7所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ6.43(dd,J＝16.0,4.6Hz,1H),6.12(q,J＝6.8Hz,1H),5.85(dd,J＝8.8,1.6Hz,1H),4.37(dt,J＝35.6,6.8Hz,4H),2.94(td,J＝13.2,6.8Hz,4H),2.31(t,J＝7.2Hz,,2H),1.65-1.59(m,2H),1.29-1.26(m,8H),0.88-0.85(m,3H)。
实施例11脂质化合物1-O15B的合成
向装有磁子的5mL反应瓶内加入200mg 2,2'-二氨基-N-甲基二乙胺，3倍化学当量的中间体4，90℃搅拌48h，经过薄层色谱柱分离(DCM：甲醇＝10：1)，即可得到可电离脂质化合物1-O15B。
实施例12脂质化合物2-O15B的合成
向装有磁子的5mL反应瓶内加入200mg N,N-双(3-氨丙基)甲胺，4倍化学当量的中间体4，90℃搅拌48h，经过薄层色谱柱分离(DCM：甲醇＝10：1)，即可得到可电离脂质化合物2-O15B。所得产物氢谱如图8所示，氢谱数据如下：1H NMR(400MHz,CDCl3):δ4.32(t,J＝6.8Hz,14H),2.92(t,J＝6.4Hz,16H),2.76(t,J＝7.2Hz,6H),2.47-2.08(m,29H),1.61(t,J＝7.2Hz,14H),1.32-1.26(m,32H),0.87(t,J＝6.4Hz,12H)。
实施例13脂质化合物3-O15B的合成
向装有磁子的5mL反应瓶内加入200mg 1-(2-氨乙基)吡咯烷，2倍化学当量的中间体4，90℃搅拌48h，经过薄层色谱柱分离(DCM：甲醇＝10：1)，即可得到可电离脂质化合物3-O15B。
本发明的脂质化合物的反应机理可以概括为酰氯的醇解反应，烯烃和有机胺的迈克尔加成反应。该反应机理十分成熟，反应过程也为本领域熟知，因此利用上述反应机理生成的化合物，其具体类型以及反应程度都是完全可以预料的。上述为本发明合成的部分化合物的反应条件以及结构表征，本发明其余化合物的合成同上述化合物，其结构式及结构表征数据在此不再详述。
实施例14
使用能表达萤火虫荧光素酶的黑色素瘤细胞系(B16F10-Luciferase,B16F10-Luc)验证含有可电离脂质的LNP递送siLuc进行基因敲除的效率。分别使用可电离脂质1-O15B、2-O15B、A1、A2、B1-B6、C2、C4、C6作为siLuc递送的载体材料。
具体步骤如下：
1.细胞培养
实验前一天将B16F10-Luc细胞种于96孔细胞培养板中，待细胞密度生长至70～80％左右，进行细胞转染。
2.制备包裹siLuc的LNP并进行细胞转染
实验组：将可电离脂质1-O15B、2-O15B、A1、A2、B1-B6、C2、C4、C6与DSPC，Cholesterol，DSPE-PEG按照一定浓度溶于无水乙醇中，放置于-20℃冰箱保存，将可电离脂质：Cholesterol：DSPC：DSPE-PEG按照比例40：48：10：2均匀混合，同时吸取适量siLuc溶于柠檬酸钠缓冲液(pH＝4)中(柠檬酸钠缓冲液体积为脂质混合物总体积的两倍)，将含有siLuc的缓冲液与脂质混合液快速混合以形成LNP，在室温孵育15min。将制得的LNP用PBS稀释后，加入到细胞培养液中进行转染(每孔转染含50ng siLuc的LNP)，其中可电离脂质与siRNA的氮磷比例(N/P ratio，即可电离脂质上的可质子化氨基与RNA上的磷酸基团之间的摩尔比)32:1为最优比例。
阳性对照组：使用Lipofectamine 2000(lipo2000)商业化转染试剂对siLuc进行转染。按照lipo2000的使用说明书，取50ng siLuc加入到5μL Opti-MEM转染液中，再取0.3μL lipo2000置于另一5μL Opti-MEM转染液中，然后将siRNA Opti-MEM溶液与lipo2000 Opti-MEM溶液充分混合，室温孵育15min后，加入到B16F10-Luc细胞培养板中进行转染。
阴性对照组：正常培养B16F10-Luc细胞，不进行转染。
3.细胞转染效率分析
转染24h后，裂解细胞，离心后取上清液体，加入萤火虫荧光素酶底物，用酶标仪检测萤火虫荧光素酶含量(化学发光)。结果如图9所示，其中阴性对照组表达萤火虫荧光素酶相对量最高，阳性对照lipo2000转染效率为30％(表达萤火虫荧光素酶相对量为0.7，相对于阴性对照敲除了30％基因表达)，而本发明所合成的可电离脂质化合物大部分都有较强的转染效率，其中1-O15B和2-O15B转染效率为70％～75％左右，明显高于商业化转染试剂Lipofectamine 2000。
实施例15
使用可电离脂质化合物A2对制备纳米颗粒四种物质之间的比例进行优化实验，在黑色素瘤细胞系(B16F10-Luciferase,B16F10-Luc)中递送siLuc。
具体步骤参考实施例14，区别在于实施例15中，实验组为：可电离脂质A2以及DSPC，Cholesterol，DSPE-PEG按照一定浓度溶于无水乙醇中，放置-20℃冰箱保存。使用四种不同摩尔比例进行混合，比例A为可电离脂质A2：Cholesterol：DSPC：DSPE-PEG＝30:28.5:10:0.75；比例B为可电离脂质A2：Cholesterol：DSPC：DSPE-PEG＝50:38.5:10:1.5；比例C为可电离脂质A2：Cholesterol：DSPC：DSPE-PEG＝35:46.5:16:2.5；比例D为可电离脂质A2：Cholesterol：DSPC：DSPE-PEG＝40:48:10:2。
3.细胞转染效率分析
转染24h后，裂解细胞，离心后取上清液体，加入萤火虫荧光素酶底物，用酶标仪检测萤火虫荧光素酶含量(化学发光)。结果如图10所示，这四种比例组合制备的LNP对B16F10-Luc细胞都有一定的敲除效果，其中比例D为最优比例。
实施例16
使用可电离脂质递送同时编码绿色荧光蛋白(GFP)和萤火虫荧光素酶(Luc)的质粒DNA(pDNA-GFP-Luc)转染至293T细胞系中。分别使用可电离脂质1-O15B、2-O15B、3-O15B、4-O15B、2-O19B、2-O25B、A1、A2、B1-B6、C2、C4、C6脂质作为pDNA-GFP-Luc递送的载体材料。
具体步骤如下：
1.细胞培养
实验前一天将293T细胞种于96孔细胞培养板中，待细胞密度生长至70～80％左右，进行细胞转染。
2.制备包裹pDNA-GFP-Luc的LNP并进行细胞转染
实验组：将可电离脂质1-O15B、2-O15B、3-O15B、4-O15B、2-O19B、2-O25B、A1、A2、B1-B6、C2、C4、C6以及DOPE，Cholesterol，DSPE-PEG按照一定浓度溶于无水乙醇中，放置-20℃冰箱保存，按照比例可电离脂质：Cholesterol：DOPE：DSPE-PEG＝40：48：10：2均匀混合，同时吸取适量pDNA-GFP-Luc溶于醋酸钠缓冲液(pH＝5.3)中(醋酸钠缓冲液体积为脂质混合物总体积的两倍)，将含有pDNA-GFP-Luc的缓冲液与脂质混合液快速 混合以形成LNP，在室温孵育15min。将制得的LNP用PBS稀释后，分别加入到细胞培养液中进行转染(每孔转染含200ng pDNA-GFP-Luc的LNP)，其中可电离脂质与pDNA的氮磷比例(N/P ratio，即可电离脂质上的可质子化氨基与DNA上的磷酸基团之间的摩尔比)32:1为最优比例。
阳性对照组：使用PEI商业化转染试剂和阳离子脂质DOTAP对pDNA-GFP-Luc进行转染。其中PEI按照使用说明书，取200ng DNA溶于5μL DEPC水中混匀，再取2μL PEI溶于5μL DEPC中，将稀释的PEI加入到DNA的DEPC水溶液中，室温孵育15min，加入到293T的96孔细胞培养板中进行转染；DOTAP-LNP的制备方法与上述可电离脂质制备方法相同。为制备DOTAP-LPR纳米颗粒，先制备DOTAP脂质体，具体操作是将DOTAP和胆固醇(1:1，摩尔比)的氯仿混合物风干并在真空泵下干燥过夜；将预热的DEPC水(50℃)加入到脂质膜中，涡旋1min，并在55℃水浴下超声10min进行水化；然后将脂质体依次挤出通过200nm和100nm孔径的聚碳酸酯膜(Whatman)，形成80～100nm单层脂质体。随后，制备DOTAP-LPR纳米颗粒的步骤如下：将含200ng pDNA-GFP-Luc的DEPC水溶液和150ng鱼精蛋白(0.1mg/mL)混合，并在室温下孵育10min，然后将1.939μL预制的DOTAP脂质体(10mM)和上述溶液混合，并在室温下孵育10min，最后，将1.11μg DSPE-PEG与上述溶液混合，置于50℃金属浴中12min，然后进行转染。
阴性对照组：正常培养293T细胞，不进行转染。
3.细胞转染效率分析
转染48h后，使用荧光显微镜检测绿色荧光蛋白的表达；然后，裂解细胞，离心后取上清液体，加入萤火虫荧光素酶底物，用酶标仪检测萤火虫荧光素酶含量(化学发光)。相对荧光素酶活性结果如图11所示，荧光显微镜结果如图12所示。
其中阴性对照表达萤火虫荧光素酶为最低，阳性对照PEI转染表达荧火虫荧光素酶相对量可达160万数值，而商业化阳离子脂质DOTAP，其制备方式无论是DOTAP-LNP还是DOTAP-LPR都无法有效转染pDNA-GFP-Luc。而本发明的部分可电离脂质有很强的转染效率，其中2-O15B表达萤火虫荧光素酶相对量最高，达到190万数值。
实施例17凝胶迁移实验考察LNP固缩DNA的能力
1.制备pDNA-GFP-Luc的LNP进行凝胶迁移实验
依照上述制备包载pDNA-GFP-Luc的LNP的方法，使用2-O15B，2-O19B，2-O25B分别制备氮磷比例为8:1，16:1，24:1，32:1(即可电离脂质上的可质子化氨基与DNA上的磷酸基团之间的摩尔比)的LNP，制备完成后，LNP与适量的PBS混合后加样于1％琼脂糖凝胶中，100V电压条件下电泳50min，采用凝胶成像仪观察结果，具体见图13。
依照上述制备包载pDNA-GFP-Luc的LNP的方法，使用2-O15B制备三份氮磷比例为32:1的LNP，制备完成后，分别将LNP置于PBS，10％血清(serum)，5mM GSH的溶液中，分别孵育一定的时间后加样于1％琼脂糖凝胶中，100V电压条件下电泳50min，采用凝胶成像仪观察结果，具体见图14。
2.凝胶迁移结果分析
如图13所示，含有2-O15B的LNP在氮磷比例最小为8:1时依然能完全固缩DNA，而含有2-O19B，2-O25B的LNP则在氮磷比例为24:1，32:1时候才能完全固缩DNA，说明在 一定的范围内随着烷基链长度的增加，LNP固缩DNA的能力在逐步下降，LNP和DNA的固缩主要是因为静电相互作用，而烷基链太长可能会影响氨基头部质子化的能力，所以包裹DNA的能力下降。
如图14所示，含有2-O15B的LNP在PBS溶液中能保持稳定，在含有10％血清的溶液中2个小时依然能保持LNP的稳定性，DNA并没有释放。而在模拟细胞质内环境条件下(5Mm GSH)，30min后就有一定量的DNA完成了释放，这说明此LNP在细胞内可以触发二硫键的降解，从而有效的释放出所包裹的基因药物。
实施例18
检测由可电离脂质制备的LNP对RAW-Luc细胞的先天性免疫的激活作用。这种RAW-Luc细胞的干扰素刺激基因(interferon-stimulated gene，ISG)的信号通路被激活时，会分泌萤火虫荧光素酶到胞外的细胞培养液中，通过酶标仪检测萤火虫荧光素酶的分泌情况(化学发光)，从而定量检测由可电离脂质制备的LNP对先天性免疫的激活作用。
具体步骤如下：
1.细胞培养
实验前一天将RAW-Luc细胞种于96孔细胞培养板，待细胞密度生长至70～80％左右，进行细胞转染。
2.制备空包载的LNP并进行细胞转染
实验组：将可电离脂质1-O15B、2-O15B、3-O15B、4-O15B、5-O15B、6-O15B、1-O19B、2-O19B、1-O25B、2-O25B、A1、A2、B1-B6、C2、C4、C6、C-VA、C-VE与DOPE，Cholesterol，DSPE-PEG按照一定浓度溶于无水乙醇中，放置于-20℃冰箱保存，按照比例可电离脂质：Cholesterol：DOPE：DSPE-PEG＝40:48:10:2均匀混合。随后，将一定量的醋酸钠缓冲液(pH＝5.3，醋酸钠缓冲液体积为脂质混合物总体积的两倍)与脂质混合液快速混合以形成LNP，在室温孵育15min。将制得的LNP用PBS稀释后，分别加入到细胞培养液中进行转染。
阴性对照组：正常培养RAW-Luc细胞，不进行转染。
3.结果分析
转染12，24，36小时后，取细胞培养液上清，加入萤火虫荧光素酶底物，用酶标仪检测萤火虫荧光素酶含量(化学发光)。结果如图15所示，第二种类可电离脂质对RAW-Luc细胞激活效果明显，在36h后，含有2-O19B和2-O25B的LNP有8～10倍的激活效果。表明2-O19B和2-O25B可能具有免疫治疗作用。
上面结合附图对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 脂质化合物，或其药学上可接受的盐或其立体异构体，其特征在于，
    所述脂质化合物具有式(I)所示的结构：
    R1选自C8～C12直链烷基或
    其中，R2选自C9～C17直链烷基或直链烯基、
    n选自0～3的整数；
    或，所述脂质化合物具有式(II)所示的结构；
    y选自1或2；其中，
    当y选自1时，R3选自其中，R5选自羟基、未取代或甲基取代的四氢吡咯基、哌啶基、哌嗪基、二甲氨基、二羟甲基氨基、二乙氨基、二羟乙基氨基；
    当y选自2时，R3选自其中X选自亚甲基、氧原子、或者X与式(II)中的任意一个N相连形成五元杂环或六元杂环；或R3与式(II)中的两个N一起形成五元杂环或六元杂环；
    每个p各自独立的选自0或1，且，
    式(II)中至多存在一个p为0，且，
    当p均为1时，R4选自C7～C17直链烷基或直链烯基；
    或，当其中一个p为0、其余p均为1时，与N原子直接相连的R4选自氢或C1～C4直链烷基，其余R4独立选自C7～C17直链烷基或直链烯基。
  2. 根据权利要求1所述的脂质化合物，其特征在于，
    R2选自如下所示的结构：
    n选自1或2。
  3. 根据权利要求1所述的脂质化合物，其特征在于，
    y选自1或2；其中，
    当y选自1时，R3选自其中，R5选自羟基、未取代或甲基取代的四氢吡咯基、哌啶基、哌嗪基；
    当y选自2时，R3选自其中X选自亚甲基；
    每个p各自独立的选自0或1，且，
    式(II)中至多存在一个p为0，且，
    当p均为1时，R4选自C7～C17直链烷基或直链烯基；
    或，当其中一个p为0、其余p均为1时，与N原子直接相连的R4选自氢，其余R4独立选自C7～C17直链烷基或直链烯基；
    所述C7～C17直链烷基或直链烯基选自如下所示的结构：
  4. 根据权利要求1所述的脂质化合物，其特征在于，所述脂质化合物选自如下所示的结构：


  5. 一种如权利要求1-4中任一项所述的脂质化合物的制备方法，其特征在于：
    当所述脂质化合物具有式(I)所示的结构，且其中所述R1选自C8～C12直链烷基时，所述脂质化合物的合成路线如下：
    当所述脂质化合物具有式(I)所示的结构，且其中所述R1选自时，所述脂质化合物的合成路线如下：
    R2、n如权利要求1-4中任一项所定义；
    当所述脂质化合物具有式(II)所示的结构时，该所述脂质化合物由丙烯酸酯衍生物与有机胺通过迈克尔加成反应得到；
    其中，所述丙烯酸酯具有如下所示的结构：
    R4选自C7～C17直链烷基或直链烯基。
  6. 一种组合物，其特征在于，所述组合物包括权利要求1-4中任一项所述的脂质化合物，或其药学上可接受的盐或其立体异构体。
  7. 根据权利要求6所述组合物，其特征在于，所述组合物还包括胆固醇或β-谷甾醇以及它们的衍生物，辅助磷脂和聚乙二醇修饰脂质中的至少一种。
  8. 根据权利要求7所述组合物，其特征在于，所述辅助磷脂包括蛋黄卵磷脂、氢化蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、鞘磷脂、磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰基卵磷脂(DOPC)、二油酰磷脂酰胆碱、二月桂酰磷脂酰胆碱中的至少一种；
    任选地，所述聚乙二醇(PEG)修饰脂质包括PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油、ALC-0159以及以上化合物的PEG化学修饰产物(例如-Maleimide、-COOH、-NH2)中的至少一种；
    任选地，在所述组合物还包括胆固醇或β-谷甾醇以及它们的衍生物、辅助磷脂和聚乙二醇修饰脂质时，所述脂质化合物、或其药学上可接受的盐或其立体异构体：胆固醇或β-谷甾醇以及它们的衍生物：辅助磷脂：聚乙二醇修饰脂质的摩尔比为30～50：30～50：5～20：1～2.5。
  9. 根据权利要求6-8中任一项所述的组合物，其特征在于，还包括药物活性成分；进一步，所述药物活性成分包括核酸分子和蛋白类药物，所述核酸分子包括小干扰RNA(Small interfering RNA,siRNA)、微小(microRNA,miRNA)、信使RNA(Messenger RNA,mRNA)、化学修饰mRNA(Synthetic chemically modified mRNA,modRNA)、环状RNA(circRNA)、反义RNA(antisense RNA)、CRISPR guide RNA、可复制型RNA(Self-replicating RNA,repRNA)、环状二核苷酸(cyclic dinucleotide,CDN)、poly IC、CpG ODN、质粒DNA(plasmid DNA,pDNA)、微环DNA；所述蛋白质类药物包括细胞集落刺激因子、白介素类、淋巴毒素、干扰素类蛋白、肿瘤坏死因子、抗体类、蛋白抗原。
    更进一步，当所述药物活性成分包括核酸分子时，所述脂质化合物，或其药学上可接受的盐或其立体异构体中的氮与核酸分子中的磷的氮磷比为4～32：1。
  10. 权利要求1-4中任一项所述的脂质化合物，或其药学上可接受的盐或其立体异构体，或权利要求6-9中任一项所述的组合物在制备核酸药物和疫苗、基因疫苗、多肽或蛋白质药物和疫苗、小分子药物中的应用。
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