CN115504945A - Ionizable heterocyclic lipid molecule and application thereof in preparation of lipid nanoparticles - Google Patents
Ionizable heterocyclic lipid molecule and application thereof in preparation of lipid nanoparticles Download PDFInfo
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- 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
- C07D295/08—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 bound oxygen or sulfur atoms
- C07D295/084—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 bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/088—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 bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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- A61P35/00—Antineoplastic agents
Abstract
The invention relates to the technical field of nucleic acid drug delivery, in particular to an ionizable heterocyclic-containing lipid molecule and application thereof in preparation of lipid nanoparticles. Compared with the lipid nanoparticles adopting MC3 lipid on the market at present, the lipid nanoparticles obtained by the ionizable heterocyclic lipid molecule have the advantages that the formed mRNA LNP particles are more uniform and stable, the cell transfection advantages are more obvious, the efficient and low-frequency drug administration can be realized, and the potential toxicity risk is further reduced.
Description
Technical Field
The invention relates to the technical field of nucleic acid drug delivery, in particular to an ionizable heterocyclic-containing lipid molecule and application thereof in preparation of lipid nanoparticles.
Background
RNA-based therapies currently gain widespread interest in treating a variety of diseases, including diseases associated with "non-treatable" targets. The RNA drugs mainly comprise antisense oligonucleotides (ASOs), small interfering RNA (siRNA), micro RNA (miRNA) and mRNA. During the past decade, a range of RNA-based therapeutic drugs have been approved for the treatment of different types of diseases, such as macular degeneration, spinal muscular atrophy, hypercholesterolemia, and degenerative amyloidosis. In 2020, two encapsulated mRNA drugs BNT162b2 and mRNA-1273 have obtained FDA and EMA's Emergency Use Authority (EUA) as a SARS-CoV-2 vaccine against 2019 coronavirus disease (COVID-19).
Although RNA drugs have great potential in the treatment of various diseases, RNA drugs are easily degraded by enzymes and have extremely low cellular uptake rate of free RNA drugs, so that some carriers need to be introduced to carry out package delivery of RNA so as to expand the application of RNA.
Of such delivery vehicles, lipid Nanoparticles (LNPs) are currently the most prominent non-viral delivery vehicles used for nucleic acid based drugs. siRNA drugs currently on the market
The mRNA vaccines developed by BioNTech/Pfizer and Moderna are delivered by lipid nanoparticles, and the lipid complex in the lipid nanoparticles consists of cationic lipid, auxiliary neutral phospholipid, cholesterol and PEG lipid.
Cationic lipids can form complexes by ionic interactions with negatively charged RNA through their positively charged charges. Cationic lipids are in turn divided into lipids with a permanent positive charge and ionizable lipids. Cationic lipids with a permanent positive charge include DOTAP and DOTMA. Early attempts to only use cationic lipids with permanent positive charges to encapsulate nucleic acids showed high transfection efficiency in vitro, but the cationic lipids had permanent positive charges and non-degradable characteristics, which limited the in vivo efficacy and possibly led to cellsAnd (4) toxicity problem. Lipofectin is a mixture of DOTMA and DOPE, and the addition of the helper lipid DOPE reduces cytotoxicity, so that mRNA can be loaded and expressed in vivo. The other cationic lipid, namely the ionized lipid, is a lipid which is almost uncharged under the condition of normal neutral physiological pH and positively charged under the condition of acidic pH, and comprises DODMA and siRNA lipid nanoparticle medicines
Dlin-MC3-DMA used, and ALC0315 and SM102 used in the mRNA vaccines currently developed by BioNTech/Pfizer and Moderna. The pH sensitive nature of ionizable lipids prevents their metabolism by the reticuloendothelial system (RES), thus prolonging their half-life, while also being less likely to cause immune activation or interaction with serum proteins, thus improving their safety.
In addition to cationic lipids, other lipids contained in the lipid nanoparticle formulation also have their own role, assisting neutral lipids, such as DSPC, to support the lipid bilayer structure; cholesterol is used as a stabilizer for enhancing the stability of the preparation; PEG-lipid can prolong the half-life of the preparation, reduce non-specific interaction with plasma protein, improve the stability of the preparation and prevent aggregation.
Although the current RNA delivery system is based on lipid nanoparticles, lipid nanoparticles themselves have some disadvantages. The key lipids in the current formulation of nucleic acid-like lipid nanoparticles on the market are ionizable cationic lipids that, after uptake by the cell, can be protonated in the acidic endosome and interact with the anionic endosomal phospholipid to form cone-shaped ion pairs that are incompatible with the bilayer, these cation-anion lipid pairs drive the transition from the bilayer structure to the inverted hexagonal HII phase, which contributes to membrane fusion/disruption, endosomal escape and drug release into the cytoplasm. Also, these ionizable cationic lipids affect the stability of RNA formulations, including expression stability. In order to maintain good stability and drug effect of the prior ionizable lipid, the content of ionizable cationic lipid in the lipid nanoparticle formula needs to be kept above 50%, as disclosed in chinese patent CN 102119217B. Thus, there remains a question whether ionizable cationic lipids in lipid nanoparticles currently on the market are already the optimal choice for delivering RNA.
In order to overcome the above problems, the present invention:
1. by improving the structure of ionizable amido lipid molecules, saturated heterocyclic system piperazine groups, hydroxyl hydrophilic heads, double alcohol connecting functional groups and/or asymmetric long tail chains are introduced to obtain novel ionizable amido lipid;
2. the lipid nanoparticles are obtained by combining the novel ionizable amino lipid with neutral phospholipid, cholesterol and PEG lipid, and the obtained lipid nanoparticles can well wrap mRNA (messenger ribonucleic acid) and can improve the translation expression level of the mRNA on cells; and
3. compared with the lipid nanoparticles adopting MC3 lipid on the market at present, the formed mRNA LNP particles are more uniform and stable, which indicates that the novel ionizable lipid has better stability and adaptability, and is less influenced by the prescription content factor compared with MC 3; meanwhile, compared with MC3 lipid, the novel ionizable lipid has more remarkable cell transfection advantage in a low-content prescription, and can realize high-efficiency low-frequency administration, thereby reducing potential toxicity risk.
Disclosure of Invention
The invention provides an ionizable heterocyclic lipid molecule and application thereof in preparing lipid nanoparticles based on the problems in the prior art.
The present invention provides a lipid compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
wherein the content of the first and second substances,
R 1 and R 2 Each independently is C 6 -C 20 Alkyl radical, C 6 -C 20 Alkenyl or C 6 -C 20 An alkynyl group;
x is independently-C (= O) O-, -OC (= O) O-, -C (= S))O-、-OC(＝S)-、-OC(＝S)O-、-C(＝O)S-、-SC(＝O)-、-OC(＝O)S-、-O-、-S-、-C 1 -C 6 alkylene-O-, -O-C 1 -C 6 Alkylene-, -C 1 -C 6 alkylene-S-or-S-C 1 -C 6 Alkylene-;
R 3 independently a 5-6 membered saturated heterocyclyl comprising 1 or 2 ring heteroatoms independently selected from N, O and S, optionally substituted with R 4 Substitution;
R 4 independently is C 1 -C 6 Alkyl, optionally substituted with-OH;
m and n are each independently selected from integers of 1 to 10.
In one embodiment of the present invention, the lipid compound represented by formula (I) has a structure represented by the following formula (II):
in one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), R 1 Is C 6 -C 10 Linear alkyl radical, and R 2 Is C 10 -C 20 A branched alkyl group.
In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), R 1 Is C 9 Linear alkyl radical, and R 2 Is composed of
In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), X is independently-C (= O) O-, -OC (= O) -, -C 1 -C 6 alkylene-O-or-O-C 1 -C 6 Alkylene-.
In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), X is independently-OC (= O) -or-O-CH 2 -。
In one embodiment of the inventionIn the lipid compound represented by the above formula (I) or formula (II), R 4 Independently is-CH 3 、-CH 2 CH 2 OH、-CH 2 CH 2 CH 2 OH or-CH 2 CH 2 CH 2 CH 2 OH。
In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), m and n are each independently 6.
In one embodiment of the present invention, the lipid compound represented by formula (I) has a structure represented by the following formula (III):
in one embodiment of the present invention, in the lipid compound represented by the above formula (III), X is independently-OC (= O) -or-O-CH 2 -, and R 4 Independently is-CH 3 、-CH 2 CH 2 OH、-CH 2 CH 2 CH 2 OH or-CH 2 CH 2 CH 2 CH 2 OH。
In one embodiment of the present invention, in the lipid compound represented by the above formula (I), formula (II) or formula (III), the compound is selected from:
the present invention also provides a lipid nanoparticle composition comprising a lipid compound represented by the above formula (I), formula (II), or formula (III), or a pharmaceutically acceptable salt thereof, as described herein.
In one embodiment of the present invention, in the above lipid nanoparticle composition, a neutral lipid, cholesterol, and a PEG lipid are further included.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is selected from the group consisting of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1, 2-dipentanyl-sn-glycerol-3-phosphoethanolamine (trans-DOPE), and the PEG lipid is selected from the group consisting of PEG-DMG, PEG-dipalmitoyl glycerol, PEG-DSPE, PEG-dilauryl glycerol amide, PEG-dimyristyl glycerol amide, PEG-dipalmitoyl glycerol amide and PEG-distearoyl glycerol amide, PEG-cholesterol (1- [8' - (cholest-5-en-3 [ β ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ ω ] -methyl-poly (ethylene glycol), PEG-DMB, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2 k-dsk-DMA and PEG2k-DSA.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is DSPC and the PEG lipid is PEG2k-DMG.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the lipid compound described herein accounts for 30-50% of the total lipid component of the composition by mole.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the lipid compound described herein accounts for 40-50% of the total lipid component of the composition by mole.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the mole percentages of each lipid component described herein in relation to the total lipid components of the composition are respectively: 30-50% of the lipid compound, 8-12% of the neutral lipid, 35-60% of the cholesterol and 1-3% of the PEG lipid.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the mole percentages of each lipid component described herein in relation to the total lipid components of the composition are respectively: 31.5% of the lipid compound, 10% of the neutral lipid, 56% of the cholesterol and 2.5% of the PEG lipid.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the mole percentages of each lipid component described herein in relation to the total lipid components of the composition are respectively: 43.3% of the lipid compound, 8.7% of the neutral lipid, 46.5% of the cholesterol and 1.5% of the PEG lipid.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the mole percentages of each lipid component described herein in relation to the total lipid components of the composition are respectively: 46.3% of the lipid compound, 9.4% of the neutral lipid, 42.7% of the cholesterol and 1.6% of the PEG lipid.
In one embodiment of the present invention, in the above lipid nanoparticle composition, the mole percentages of each lipid component described herein in relation to the total lipid components of the composition are respectively: 50% of the lipid compound, 10% of the neutral lipid, 38.5% of the cholesterol and 1.5% of the PEG lipid.
In one embodiment of the present invention, in the above lipid nanoparticle composition, further comprising a nucleic acid molecule selected from the group consisting of mRNA, siRNA, antisense oligonucleotide (ASO), saRNA, and miRNA.
The present invention also provides a method of delivering a nucleic acid into a cell comprising delivering into said cell the above lipid nanoparticle as described herein.
In one embodiment of the invention, the cell is a mammalian cell, preferably the cell is a human cell.
In one embodiment of the invention, the cell is a mammalian precancerous germ cell, preferably the cell is a human precancerous germ cell.
In one embodiment of the invention, the cell is a tumor cell, preferably the cell is a human tumor cell.
The invention also provides the application of the lipid nanoparticles in preparation of medicines for treating diseases.
The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.
The term "pharmaceutically acceptable" as used herein, is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" as used herein refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have the specified substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Examples of the pharmaceutically acceptable acid addition Salts include inorganic acid Salts, organic acid Salts, and also Salts of amino acids such as arginine and the like, and Salts of organic acids such as glucuronic acid and the like (see Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science 66 (1977). Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, the salt is prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.
As used herein, the term "alkyl" or "alkylene" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the indicated number of carbon atoms. For example, "C 1 -C 10 Alkyl "(or alkylene) is intended to include C 1 、C 2 、C 3 、C4、C 5 、C 6 、C 7 、C 8 、C 9 And C 10 Alkyl (or alkylene). An alkyl group may be unsubstituted or substituted, wherein at least one hydrogen is replaced by another chemical group. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.
The term "alkenyl" as used herein is intended to include hydrocarbon chains having a straight or branched configuration and having one or more carbon-carbon double bonds which may be present at any stable point along the chain. For example, "C 2 -C 6 Alkenyl "is intended to include C 2 、C 3 、C 4 、C 5 And C 6 An alkenyl group; such as ethenyl, propenyl, butenyl, pentenyl and hexenyl.
The term "alkynyl", as used herein, is intended to include hydrocarbon chains having a straight or branched configuration and having one or more carbon-carbon triple bonds which may occur at any stable point along the chain. For example, "C 2 -C 6 Alkynyl "is intended to include C 2 、C 3 、C 4 、C 5 And C 6 Alkynyl; such as ethynyl, propynyl, butynyl, pentynyl and hexynyl.
As used herein, the term "heterocycle" or "heterocyclyl" means a stable mono-, bi-or tricyclic ring containing a heteroatom or group of heteroatoms, which may be saturated, partially unsaturated, or unsaturated (aromatic), which comprises carbon atoms and 1,2, 3, or 4 ring heteroatoms independently selected from N, O, and S. The nitrogen atom may be substituted or unsubstituted. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, azanyl, azaazepinyl, 1-pyridonyl, 4-piperidinonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1, 3-dioxolane, and tetrahydro-1, 1-dioxothienyl, and the like. Exemplary bicyclic heterocyclyl groups include quinuclidinyl. The "heterocycle" employed in the present invention is preferably a 5-6 membered saturated heterocyclic group comprising 1 or 2 ring heteroatoms independently selected from N, O and S, more preferably a piperazinyl group.
The term "neutral lipid" as used herein refers to any of a variety of lipids that exist in the uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. Furthermore, the neutral lipid of the present invention may also be selected from Distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE) and oleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans-PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1, 2-dipentanyl-sn-glycerol-3-phosphoethanolamine (trans-DOPE), preferably, the neutral lipid is selected from DSPC.
As used herein, the term "PEG lipid" may be selected from the group consisting of PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol (PEG-DSPE), PEG-dilauryl glycerol amide, PEG-dimyristylyl glycerol amide, PEG-dipalmitoyl glycerol amide and PEG-distearoyl glycerol amide, PEG-cholesterol (1- [8' - (cholest-5-ene-3 [ β ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ ω ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecylphenylmethyl- [ ω ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DMG), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DSar) (available from catalog number 120, avasa, 2-Derma, avaI-Derma, ab, 2-glycerol-2-D, avaR, polyethylene glycol catalog; GS-020, NOF Tokyo, japan), poly (ethylene glycol) -2000-dimethacrylate (PEG 2 k-DMA) and 1, 2-distearoyloxypropyl- 3-amine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DSA), preferably, the PEG lipid is selected from PEG2k-DMG.
The term "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those of ordinary skill in the art, e.g., intended to mean a molecule comprising, preferably consisting of, a nucleic acid component. The term nucleic acid molecule preferably refers to a DNA or RNA molecule. Preferably used synonymously with the term polynucleotide. Preferably, the nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. Preferably, the nucleic acid molecule is selected from mRNA, siRNA, antisense oligonucleotide (ASO), saRNA or miRNA.
The ionizable heterocyclic lipid molecule and the lipid nanoparticle prepared from the same provided by the invention have the following beneficial effects:
1. by improving the structure of ionizable amido lipid molecules, saturated heterocyclic system piperazine groups, hydroxyl hydrophilic heads, double alcohol connecting functional groups and/or asymmetric long tail chains are introduced to obtain novel ionizable amido lipid;
2. the novel ionizable amino lipid is combined with neutral phospholipid, cholesterol and PEG lipid to obtain lipid nanoparticles, and the obtained lipid nanoparticles can well wrap mRNA and can improve the translation expression level of the mRNA on cells; and
3. compared with the lipid nanoparticles adopting MC3 lipid on the market at present, the formed mRNA LNP particles are more uniform and stable, which indicates that the novel ionizable lipid has better stability and adaptability, and is less influenced by the prescription content factor compared with MC 3; meanwhile, compared with MC3 lipid, the novel ionizable lipid has a more remarkable cell transfection advantage in a low-content prescription, and can realize high-efficiency low-frequency administration, so that potential toxicity risk is reduced.
Drawings
FIG. 1: a preparation flow chart of the lipid nanoparticle carrying nucleic acid;
FIG. 2: in-vitro transfection experiment results of different lipid nanoparticles carrying mRNA, wherein luciferase mRNA is carried by the lipid nanoparticles prepared from different ionizable amino lipids, then Hela cells are transfected in vitro, and the transfection efficiency is detected 24h after transfection. The prescription of the lipid nanoparticle is based on the prescription of Dlin-MC 3-DMA;
FIG. 3: in-vitro transfection experiment results of different lipid nanoparticles encapsulating mRNA, wherein MC3-LNP with different formulas and the lipid 8-LNP of the invention are adopted to encapsulate luciferase mRNA, hela cells are transfected in vitro, and the transfection efficiency is detected 24h after transfection. The formulation of lipid nanoparticles is based on the formulation of Dlin-MC 3-DMA.
FIG. 4 is a schematic view of: in-vitro transfection experiment results of different lipid nanoparticles encapsulating mRNA, wherein MC3-LNP with different formulas and the lipid 11-LNP of the invention are adopted to encapsulate luciferase mRNA, hela cells are transfected in vitro, and the transfection efficiency is detected 24h after transfection. The prescription of the lipid nanoparticles is based on the prescription of Dlin-MC 3-DMA.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. Any technical solutions implemented on the basis of the present disclosure are covered in the scope of the present invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
EXAMPLES Synthesis of different ionizable lipids
Target: a series of ionizable amino lipid compounds were synthesized for corresponding evaluation and lipid screening.
Example 1: preparation of lipid 1
80g (1 mol,1.0 eq) of diethyl malonate and 300g (1.75mol, 2.5 eq) of p-toluenesulfonic acid monohydrate were dissolved in 150ml of acetonitrile, stirred until clear, and 9g (0.2mol, 1.08eq) of N-bromosuccinimide was added, and the mixture was heated and refluxed for 12 hours. After the reaction is finished, decompressing, concentrating, desolventizing, adding a proper amount of water and dichloromethane, stirring, standing, layering, collecting an organic layer, and drying by using anhydrous sodium sulfate. Suction filtration, cake washing with a small amount of dichloromethane, desolventization under reduced pressure gave 130g of crude product, which was further purified on a silica gel column, eluent from petroleum ether to petroleum ether ethyl acetate =1, giving 65.2g of oil (0.27 mol, compound 1) in 34.6% yield.
65.2g (0.27mol, 1.0eq) of Compound 1, 2.2g (0.27mol, 1.0eq) of N-methylpiperazine and 156.4g (0.41mol, 10.05eq) of potassium carbonate were dissolved in 80ml of acetonitrile and reacted at room temperature for 48 hours. After the reaction is finished, concentrating under reduced pressure for desolventizing, adding a proper amount of water and ethyl acetate, stirring, standing, layering, collecting an organic layer, and drying with anhydrous sodium sulfate; suction filtration, cake washing with a small amount of ethyl acetate, desolventization under reduced pressure gave 100g of crude product, which was further purified on a silica gel column eluting from petroleum ether to dichloromethane alcohol =1:20, giving 39g of oil (0.15 mol, compound 2) in 55.5% yield. 1H NMR (400MHz, CDCl) 3 )δ4.28(q,J＝7.2Hz,4H),4.07(s,1H),2.84(t,J＝4.7Hz,4H),2.64–2.36(m,4H),2.34(s,3H),1.33(t,J＝7.1Hz,6H)。
19.5g (0.25mol, 12.5eq) of LAH was dissolved in 40ml of THF, and then 66g (0.3mol, 3.0eq) of Compound 2 (dissolved in 10ml of THF) was gradually added dropwise under low temperature conditions, and reacted under the same conditions for 12 hours. After the reaction, 200ml of NaOH was slowly added dropwise to the reaction solution, the heat was strongly released and the gas was evolved, and a large amount of solid was precipitated under constant temperature control. Then 100ml dichloromethane was added for dilution, suction filtration was carried out, the filtrate was concentrated and eluted with pillared ether/ethyl acetate to give 3.5g oil (0.02 mol, compound 3) in 20% yield. 1H NMR (400MHz, DMSO). Delta.4.26 (brs, 2H), 3.71-3.22 (m, 6H), 2.62 (t, J =4.8Hz, 4H), 2.46 (t, J =6.1Hz, 1H), 2.28 (s, 2H), 2.14 (s, 3H).
0.87g (0.005mol, 1.0 eq) of Compound 3, 2.8g (0.01mol, 2.0 eq) of linoleic acid, 10.93g (0.15mol, 3.0 eq) of DIPEA, 4.49g (0.04mol, 0.8eq) of DMAP, and 12.39g (0.125mol, 2.5eq) of EDCI were dissolved in 30ml of DCM and reacted at room temperature for 48 hours. After the reaction is finished, decompressing, concentrating and desolventizing, further purifying the crude product by using a silica gel column, and eluting from petroleum ether to petroleum ether: dichloromethane =1, 100, yielding 170mg of oil (0.00024 mol, lipid 1) with a yield of 4.86%.1H NMR (400MHz, CDCl) 3 )δ5.39(td,J＝11.9,5.0Hz,8H),4.32(dd,J＝11.7,6.2Hz,2H),4.13(dd,J＝11.8,5.5Hz,2H),2.78(dt,J＝22.3,5.7Hz,8H),2.54–2.21(m,9H),2.09(q,J＝6.9Hz,8H),1.65(t,J＝7.3Hz,4H),1.36(d,J＝13.6Hz,32H),0.93(t,J＝6.5Hz,6H)。
Example 2: preparation of lipid 2
15.12g (0.02mol, 1.0eq) of 9-heptadecanol, 2.4g (0.02mol, 1.0eq) of suberic acid, 13.9g (0.03mol, 1.5eq) of DIPEA, 11g (0.008mol, 0.4eq) of DMAP and 4.16g (0.024mol, 1.2eq) of EDCI were dissolved in 150ml of DCM and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolvation, and the crude product was further purified by a silica gel column, eluent was changed from dichloromethane to dichloromethane: methanol =1 to 100, whereby 1.1g of an oil (0.0027 mol, compound 4) was obtained in a yield of 13.75%.1H NMR (400MHz, CDCl) 3 )δ4.92(q,J＝6.3Hz,1H),2.40(dd,J＝26.3,6.3Hz,4H),1.75(p,J＝3.5Hz,4H),1.57(q,J＝6.2Hz,4H),1.32(s,26H),0.94(t,J＝6.7Hz,6H)。
0.98g (0.002,2.0eq) of Compound 4, 0.12g (0.001,1.0eq) of Compound 3, 1.39g (0.003mol, 3.0eq) of DIPEA, 1.1g (0.0008mol, 0.8eq) of DMAP, and 1.5g (0.0025mol, 2.5eq) of EDCI were reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluting from dichloromethane to dichloromethane: methanol =1, 100 to obtain 100mg of an oil (0.0001 mol, lipid 2) with a yield of 10%.1H NMR (400MHz, CDCl) 3 )δ4.90(p,J＝6.3Hz,2H),4.31(dd,J＝11.6,6.3Hz,2H),4.12(dd,J＝11.6,5.6Hz,2H),2.75(t,J＝4.8Hz,4H),2.45(s,3H),2.38–2.23(m,13H),1.65(h,J＝7.4Hz,10H),1.53(t,J＝6.2Hz,9H),1.43–1.31(m,24H),1.29(s,31H),0.91(t,J＝6.7Hz,15H)。
Example 3: preparation of lipid 3
In 350ml of DCM, 12.5g (0.02mol, 2.0eq) of Compound 3, 21.8g (0.02mol, 3.0eq) of linoleic acid, 3.19g (0.03mol, 1.5eq) of DIPEA, 11g (0.008mol, 0.4eq) of DMAP, and 41.6g (0.024mol, 1.2eq) of EDCI were dissolved and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluting from dichloromethane to dichloromethane: methanol =1, 100 to obtain 2.2g of an oil (0.005 mol, compound 5) in a yield of 25%.1H NMR (400MHz, CDCl) 3 )δ5.39(p,J＝6.6Hz,4H),4.27(dd,J＝11.7,6.6Hz,1H),4.07(dd,J＝11.7,5.6Hz,1H),3.57(dd,J＝10.8,5.1Hz,1H),3.45(t,J＝10.2Hz,1H),3.00(dq,J＝10.6,5.7Hz,1H),2.90(d,J＝9.5Hz,2H),2.80(q,J＝8.4Hz,3H),2.65(t,J＝8.5Hz,2H),2.51(s,4H),2.33(d,J＝10.0Hz,5H),2.09(q,J＝6.9Hz,4H),1.76–1.53(m,1H),1.38(d,J＝7.6Hz,4H),1.34(s,11H),0.93(t,J＝6.6Hz,3H)。
1.94g (0.00229mol, 1.0eq) of Compound 4, 11g (0.00229mol, 2.0eq) of Compound 5, 10.44g (0.00343mol, 1.15eq) DIPEA, 10.11g (0.00009mol, 1.4eq) DMAP and 0.53g (0.00275mol, 1.2eq) EDCI were dissolved in 100ml DCM and reacted at room temperature for 48 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, and the eluent was purified with dichloromethane: ethanol =1 = 100 to obtain 200mg of an oil (0.00024 mol, lipid 3) with a yield of 10.5%.1H NMR (400MHz, CDCl) 3 )δ5.38(tt,J＝9.8,5.9Hz,4H),4.32(dd,J＝11.6,6.3Hz,2H),4.13(dd,J＝11.7,5.5Hz,2H),3.70(ddt,J＝36.7,13.8,4.5Hz,3H),2.78(dt,J＝22.5,5.7Hz,6H),2.45(s,2H),2.33(d,J＝10.9Hz,10H),2.09(q,J＝6.9Hz,5H),1.64(dt,J＝15.0,7.5Hz,8H),1.36(d,J＝13.9Hz,25H),1.30(s,21H),0.93(q,J＝6.1Hz,11H)。
Example 4: preparation of lipid 4
12g (0.0115mol, 3.0eq) of Compound 3, 31.27g (0.0115mol, 4.0eq) stearic acid, 12.22g (0.0172mol, 1.0eq) DIPEA, 0.516g (0.0046 mol, 0.14eq) DMAP, and 2.614g (0.0138mol, 11.2eq) EDCI were dissolved in 330ml DCM and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolvation, and the crude product was further purified by a silica gel column, and the eluent was separated from dichloromethane: ethanol =1:100 to obtain 0.5g of an oil (0.0012 mol, compound 6) in a yield of 10.4%.
0.46g (0.0011mol, 1.0eq) of Compound 4, 10.5g (0.0011mol, 11.0eq) of Compound 6, 20.22g (0.0017mol, 1.25eq) of DIPEA, 6.05g (0.00004mol, 0.8mol) of DMAP and 10.26g (0.0014mol, 9.2eq) of EDCI were dissolved in 1000ml of DCM and reacted at 40 ℃ for 48 hours. After the reaction is finished, decompressing, concentrating and desolventizing, and further purifying the crude product by using a silica gel column, wherein an eluent is dichloromethane: ethanol =1, 100, yielding 120mg of oil (0.00014 mol, lipid 4) in 13.06% yield. 1H NMR (400MHz, CDCl) 3 )δ4.36–4.23(m,2H),4.13(dd,J＝11.8,5.5Hz,1H),3.81–3.59(m,4H),3.50(t,J＝6.6Hz,1H),2.80(s,1H),2.36(d,J＝8.4Hz,4H),2.34–2.28(m,3H),1.69–1.50(m,11H),1.48–1.35(m,7H),1.33(s,14H),1.30(s,36H),0.94(dt,J＝13.6,7.1Hz,11H)。
Example 5: preparation of lipid 5
10.8g (0.002mol, 1.0eq) of Compound 4, 0.14g (0.002mol, 11.0eq) of Compound 3, 20.39g (0.003mol, 3.02eq) of DIPEA, 10.1g (0.0008mol, 3.8eq) of DMAP, and 7.5g (0.0025mol, 21.5eq) of EDCI were dissolved in 1000ml of DCM and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluting from petroleum ether to dichloromethane: ethyl acetate = 150, to obtain 0.6g of an oil (0.0011 mol, compound 7) in 54.54% yield.
15g (0.0287mol, 1.0eq) of suberic acid, 112.12g (0.043mol, 32.0eq) of DIPEA, 12.4g (0.0115mol, 10.4eq) of DMAP and 36.6g (0.0344mol, 1.2eq) of EDCI were dissolved in 1500ml of DCM, and the reaction mixture was stirred for 24 hours, then 24.14g (0.0287mol, 1.0eq) of n-nonanol was added thereto and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluting from petroleum ether to dichloromethane: ethyl acetate =1, 80 to obtain 1g of an oil (0.003 mol, compound 8) in 10.45% yield.
0.93g (0.001mol, 1.0eq) of Compound 8, 10.55g (0.001mol, 1.0eq) of Compound 7, 2.26g (0.0015mol, 2.0eq) of DIPEA, 7.05g (0.0004mol, 0.49eq) of DMAP and 10.23g (0.0012mol, 1.2eq) of EDCI were dissolved in 1000ml of DCM and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolvation, and the crude product was further purified by a silica gel column, eluent was changed from petroleum ether to dichloromethane: ethyl acetate =1, to obtain 200mg of an oil (0.00024 mol, lipid 5) in a yield of 23.5%.1H NMR (400MHz, CDCl) 3 )δ4.90(p,J＝6.4Hz,1H),4.32(dd,J＝11.7,6.7Hz,2H),4.10(q,J＝5.8Hz,4H),3.03(s,5H),2.63(s,3H),2.34(dt,J＝20.9,7.2Hz,8H),1.67(q,J＝7.1Hz,10H),1.54(d,J＝6.9Hz,2H),1.45–1.19(m,48H),0.92(t,J＝6.6Hz,9H)。
Example 6: preparation of lipid 6
40g (0.1mol, 1.0 eq) of NaH was dissolved in 40ml of THF, 4.4g (0.1mol, 1.0 eq) of n-nonanol (dissolved in 500ml of THF) was added dropwise, and the mixture was stirred for 48 hours, then 93.3g (0.1mol, 1.0 eq) of 8-bromooctanoic acid was added dropwise and reacted at room temperature for 48 hours. After the completion of the reaction, the reaction solution was adjusted to pH =6, and the organic layer was concentrated and purified by a silica gel column, eluent dichloromethane: methanol = 150, to obtain 20g of oil (0.067 mol, compound 9) in 66.67% yield.
10.3g (0.001mol, 1.0eq) of Compound 9, 10.55g (0.001mol, 1.0eq) of Compound 7, 0.216g (0.0015mol, 1.5eq) of DIPEA, 0.095g (0.0004mol, 0.4eq) of DMAP and 0.93g (0.0012mol, 1.2eq) of EDCI were dissolved in 10ml of DCM, and the mixture was cooled at room temperatureThe reaction was carried out for 48 hours. After completion of the reaction, the reaction was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluent was purified from dichloromethane: ethanol =1 100 to give 200mg of oil (0.00024 mol, lipid 6) with a yield of 23.89%.1H NMR (400MHz, CDCl) 3 )δ4.32(dd,J＝11.6,6.3Hz,1H),4.12(dd,J＝11.8,5.0Hz,1H),3.77(d,J＝11.3Hz,1H),3.68(t,J＝5.1Hz,1H),3.55(dt,J＝15.9,6.0Hz,2H),3.43(t,J＝6.7Hz,3H),2.80(t,J＝4.8Hz,2H),2.55(s,5H),2.49–2.33(m,7H),2.32(dd,J＝11.9,5.6Hz,3H),1.73–1.45(m,1H),1.42–1.21(m,33H),0.92(t,J＝6.6Hz,6H)。
Example 7: preparation of lipid 7
55.85g (0.15mol, 1.0eq.) of Compound 1 was dissolved in 40mL of acetonitrile, and 61.85g (0.225mol, 1.5eq.) of N-BOC-piperazine and 91.05g (0.225mol, 1.5eq.) of a solid potassium carbonate were added at room temperature, and the mixture was heated at 40 ℃ for 48 hours after completion of the addition. Sampling reaction liquid, detecting by TLC (thin layer chromatography) to detect that the raw material is reacted completely, concentrating the reaction liquid to remove acetonitrile, adding 30mL of ethyl acetate to dissolve the reaction liquid, washing the reaction liquid with saturated citric acid aqueous solution until the reaction liquid is acidic, separating an organic phase, washing the organic phase with saturated sodium chloride aqueous solution once, separating the liquid, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating the filtrate to obtain 47.0g of colorless oily liquid (0.1366 mol, compound 10), wherein the crude product yield is as follows: 90.7 percent. The crude product is directly put into the next reaction.
47.0g (0.14mol, 1.0eq.) of compound 10 was dissolved in ethanol, and 121g (0.56mol, 4.0eq.) of sodium borohydride was added in portions at room temperature, and the mixture was heated to 40 ℃ for reaction for 48 hours. Sampling a reaction solution, detecting by TLC (thin layer chromatography) that the reaction of the raw materials is finished, concentrating the reaction solution to remove ethanol, adding ethyl acetate to dissolve the reaction solution, filtering, adding silica gel into filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 20.0g of a light yellow oily liquid (0.077 mol, compound 11), wherein the yield is as follows: 56.3 percent. 1H NMR (400MHz, CDCl) 3 )δ3.86(s,2H),3.74(d,J＝4.9Hz,4H),3.50(s,4H),2.82(d,J＝7.3Hz,1H),2.78(s,4H),1.49(s,9H).ESI-MS:m/z 261.0(M+1)+；m/z 283.0(M+Na)+。
22.6g (0.01mol, 1.0eq) of Compound 11, 15.6g (0.02mol, 2.0eq) of linoleic acid, 33.9g (0.03mol, 3.0eq) of DIEA, 10g (0.008mol, 0.8eq) of DMAP and 34.8g (0.025mol, 2.5eq) of EDCI were dissolved in 10ml of DCM and reacted at room temperature for 48 hours. After the reaction is finished, concentrating under reduced pressure for desolventizing, further purifying the crude product by using a silica gel column, and eluting dichloromethane: ethanol =1, 50, yielding 5.4g of oil (0.007 mol, compound 12) in 70% yield.
25.4g (0.007mol, 1.0 eq) of Compound 12 and 50ml of TFA were dissolved in 10ml of DCM, and reacted at room temperature for 48 hours, after completion of the reaction, the crude compound 13 was concentrated under reduced pressure and desolventized, and then the crude compound was directly subjected to the next reaction.
4.7g (0.007mol, 1.0eq) of Compound 13, 10.1g (0.008mol, 1.2eq) of potassium carbonate and 18g (0.008mol, 1.2eq) of 2-bromoethanol were dissolved in 10ml of THF and reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure for desolventization, and the crude product was further purified by a silica gel column, eluting from petroleum ether to dichloromethane: ethanol =1, 80 to obtain 700mg of an oil (0.00096 mol, lipid 7) with a yield of 13.72%.1H NMR (400MHz, CDCl) 3 )δ5.40(tt,J＝11.3,4.7Hz,8H),4.33(dd,J＝11.5,6.2Hz,2H),4.14(dd,J＝11.5,5.6Hz,2H),3.65(t,J＝5.3Hz,2H),3.04(q,J＝6.0Hz,1H),2.78(dt,J＝25.0,5.6Hz,8H),2.56(dd,J＝12.1,6.3Hz,6H),2.35(t,J＝7.6Hz,5H),2.08(p,J＝7.8Hz,9H),1.66(p,J＝7.0Hz,5H),1.37(dd,J＝16.5,5.6Hz,34H),0.93(t,J＝6.6Hz,6H)。
Example 8: preparation of lipid 8
121.38g (0.03mol, 1.0eq.) of Compound 4 was dissolved in methylene chloride, and 18.9g (0.0315mol, 1.05eq.) of Compound 11, 19.3g (0.09mol, 3.0eq.) of DIPEA, 10.8g (0.015mol, 0.5eq.) of DMAP, and 28.3g (0.045mol, 1.5eq.) of EDCI were added in this order at room temperature, and the addition was completed and reacted at room temperature for 48 hours. Sampling reaction liquid, detecting the reaction of the raw materials by TLC (thin layer chromatography), directly adding 10mL of 90% citric acid aqueous solution into the reaction liquid, stirring, separating liquid, washing organic phase with water and saturated sodium chloride aqueous solution once respectively, separating liquid, adding organic phase into anhydrous sodium sulfate, drying, filtering, adding silica gel into filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 7.66g of light yellow oily liquid (0.0117 mol, a compound 14), wherein the yield is as follows: 39.8 percent.
ESI-MS:m/z 655.2(M+1) + 。
13.67g (0.0056mol, 1.0eq.) of compound 14 was dissolved in methylene chloride, and 21.52g (0.0084mol, 1.5eq.) of compound 8, 11.81g (0.014 mol, 2.5eq.) of DIPEA, 21.34g (0.0028mol, 0.5eq.) of DMAP, and 12.15g (0.0112, 2.0eq.) of EDCI were sequentially added at room temperature, and the reaction was carried out at room temperature for 48 hours after the addition was completed. Sampling the reaction solution, detecting the reaction of the raw materials by TLC (thin layer chromatography), directly adding 300mL of citric acid aqueous solution into the reaction solution, stirring, separating, washing the organic phase with water and saturated sodium chloride aqueous solution once, separating, adding organic phase, drying with anhydrous sodium sulfate, filtering, adding silica gel into the filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 5.10g of light yellow oily liquid (0.005 mol, compound 15), wherein the yield is as follows: 89.2 percent.
ESI-MS:m/z 938.2(M+1)+。
55.10g (0.005mol, 1.0 eq.) of Compound 15 was dissolved in methylene chloride, added with TFA at room temperature, and reacted at room temperature for 48 hours. Sampling reaction liquid, detecting by TLC (thin layer chromatography) to finish the reaction of the raw materials, directly concentrating and drying the reaction liquid, adding EA (ethylene imine) and water to dissolve and clarify, adjusting the pH to 6 by using a sodium carbonate solid, separating an organic phase, washing the aqueous solution once, separating liquid, drying the organic phase by adding anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain 4.8g of yellow oily liquid (0.005 mol, compound 16), and obtaining the crude product yield: 100 percent.
24.8g (0.005mol, 1.0eq.) of compound 16 was dissolved in acetonitrile, and 20.94g (0.0075mol, 1.5eq.) of 2-bromoethanol, 12.07g (0.015mol, 3.0eq.) of potassium carbonate, and 10.36g (0.0025mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the reaction was carried out at room temperature for 48 hours after the addition was completed. Sampling reaction liquid, detecting the reaction of the raw materials by TLC (thin layer chromatography), directly adding water into the reaction liquid, extracting a product by ethyl acetate, washing an organic phase aqueous solution once, separating the solution, adding an organic phase into anhydrous sodium sulfate for drying, filtering, adding silica gel into filtrate for mixing the sample, and separating and purifying by using a silica gel column to obtain 1.10g of light yellow oily liquid (lipid 8, 0.00125mol), wherein the yield is as follows: 42.6 percent. 1H NMR (400MHz, CDCl) 3 )δ4.98–4.83(m,1H),4.33(dd,J＝11.5,6.3Hz,2H),4.18–4.05(m,4H),3.70(t,J＝5.0Hz,2H),3.08–3.00(m,1H),2.90(s,4H),2.79(s,4H),2.63(d,J＝4.6Hz,6H),2.40–2.29(m,10H),1.67(s,12H),1.39(s,12H),1.30(s,32H),0.92(t,J＝6.3Hz,9H).
ESI-MS:m/z 881.3(M+1)+。
Example 9: preparation of lipid 9
22.5g (0.003mol, 1.0eq.) of Compound 16 was dissolved in acetonitrile, and 10.63g (0.0045mol, 1.5eq.) of 3-bromopropanol, 13.24g (0.009mol, 3.0eq.) of potassium carbonate, and 10.23g (0.0015mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the addition was completed and reacted at room temperature for 48 hours. Sampling reaction liquid, detecting by TLC (thin layer chromatography), detecting the reaction of the raw materials, directly adding water into the reaction liquid, extracting a product by ethyl acetate, washing the reaction liquid once by using an organic phase aqueous solution, separating liquid, adding organic phase anhydrous sodium sulfate, drying, filtering, adding silica gel into filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 1.20g of light yellow oily liquid (lipid 9, 0.00134mol), wherein the yield is as follows: 45.8 percent. 1H NMR (400MHz, CDCl) 3 )δ4.95–4.84(m,1H),4.31(dd,J＝11.4,6.2Hz,2H),4.09(dd,J＝13.2,6.2Hz,4H),3.60(s,2H),3.05–2.97(m,1H),2.77(s,4H),2.55(s,4H),2.43(s,2H),2.37–2.28(m,9H),1.67(d,J＝18.3Hz,16H),1.37(s,12H),1.29(s,34H),0.91(t,J＝6.2Hz,9H).ESI-MS:m/z 895.3(M+1)+。
Example 10: preparation of lipid 10
10.5g (0.0003 mol, 1.0eq.) of Compound 16 was dissolved in 3mL of acetonitrile, 10.07g (0.00045mol, 1.5eq.) of 4-bromobutanol, 20.12g (0.0009mol, 3.0eq.) of potassium carbonate, and 40.02g (0.00015mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the reaction was carried out at room temperature for 48 hours after the addition. Sampling reaction liquid, detecting by TLC, adding water, extracting with ethyl acetate to obtain product, washing with organic sodium hydroxide solution, separating, drying with organic anhydrous sodium sulfate, filtering, mixing filtrate with silica gel, and filteringSilica gel column separation and purification gave 915mg of orange-yellow oily liquid (lipid 10, 0.00005mol), yield: 17.1 percent. 1H NMR (400MHz, CDCl) 3 )δ4.95–4.83(m,1H),4.29(dd,J＝11.5,6.3Hz,2H),4.09(dd,J＝14.1,6.7Hz,4H),3.82(t,J＝4.9Hz,2H),3.02(dd,J＝11.5,5.7Hz,2H),2.73(s,4H),2.62(dd,J＝22.3,17.0Hz,6H),2.37–2.28(m,10H),1.79–1.71(m,2H),1.65(s,12H),1.37(s,12H),1.29(s,34H),0.91(t,J＝6.2Hz,9H).ESI-MS:m/z 909.3(M+1)+。
Example 11: preparation of lipid 11
3.67g (0.0056mol, 1.0eq.) of compound 14 was dissolved in methylene chloride, and 2.40g (0.0084mol, 1.5eq.) of compound 9, 2.17g (0.0168mol, 3.0eq.) of DIPEA, 0.34g (0.0028mol, 0.5eq.) of DMAP, and 2.15g (0.0112mol, 2.0eq.) of EDCI were added in this order at room temperature, and the reaction was completed at room temperature for 8 hours. Sampling a reaction solution, detecting by TLC (thin layer chromatography), detecting the reaction of the raw materials, directly adding a citric acid aqueous solution into the reaction solution, stirring, separating, washing an organic phase aqueous solution once, separating, drying an organic phase anhydrous sodium sulfate, filtering, adding silica gel into a filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 3.62g of a light yellow oily liquid (a compound 17,0.0039 mol), wherein the yield is as follows: 70.1 percent. ESI-MS, M/z 923.3 (M + 1) +.
295mg (0.00032mol, 1.0 eq.) of compound 17 (1.0 eq.) was dissolved in dichloromethane, added with TFA at room temperature, and reacted at room temperature for 4 hours. Sampling reaction liquid, detecting by TLC (thin layer chromatography) to detect that the reaction of the raw materials is finished, directly concentrating and drying the reaction liquid, adding EA (ethylene oxide) and water to dissolve and clarify, adjusting the pH to 8-9 by using sodium hydroxide solid, separating an organic phase, washing once by using water solution, separating liquid, drying the organic phase by adding anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain 260mg of yellow oily liquid (compound 18,0.0003 mol), and obtaining the crude product yield: 97.3 percent.
260mg (0.0003 mol, 1.0eq.) of compound 18 was dissolved in acetonitrile, 56mg (0.00045mol, 1.5eq.) of 2-bromoethanol, 124mg (0.0009 mol, 3.0eq.) of potassium carbonate, and 23mg (0.00015mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the addition was completed at room temperature for 12 hours.Sampling reaction liquid, detecting by TLC (thin layer chromatography), detecting the reaction of the raw materials, directly adding 10mL of water into the reaction liquid, extracting a product by using ethyl acetate, washing an organic phase by using an aqueous solution once, separating liquid, adding organic phase and anhydrous sodium sulfate for drying, filtering, adding silica gel into filtrate for mixing samples, and separating and purifying by using a silica gel column to obtain 115.0mg of light yellow oily liquid (lipid 11, 0.00013mol), wherein the yield is as follows: 42.9 percent. 1H NMR (400MHz, CDCl) 3 )δ4.93–4.85(m,1H),4.38–4.24(m,2H),4.12(dd,J＝11.0,4.9Hz,2H),3.65(s,2H),3.42(t,J＝6.4Hz,4H),3.07–2.99(m,1H),2.75(s,4H),2.57(d,J＝5.5Hz,6H),2.39–2.29(m,7H),1.63–1.48(m,10H),1.36(s,8H),1.29(s,40H),0.91(d,J＝6.0Hz,9H).ESI-MS:m/z 867.7(M+1)+。
Example 12: preparation of lipid 12
260mg (0.0003 mol, 1.0eq.) of compound 18 was dissolved in acetonitrile, 63mg (0.00045mol, 1.5eq.) of 3-bromopropanol, 124mg (0.0009mol, 3.0eq.) of potassium carbonate, and 23mg (0.00015mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the addition was completed at room temperature for 12 hours. Sampling reaction liquid, detecting by TLC (thin layer chromatography), detecting the reaction of the raw materials, directly adding 10mL of water into the reaction liquid, extracting a product by using ethyl acetate, washing an organic phase by using an aqueous solution once, separating liquid, adding an organic phase, drying by using anhydrous sodium sulfate, filtering, adding silica gel into a filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 118.0mg of light yellow oily liquid (lipid 12, 0.00013mol), wherein the yield is as follows: 44.5 percent.
Example 13: preparation of lipid 13
260mg (0.0003 mol, 1.0eq.) of compound 18 was dissolved in acetonitrile, 69mg (0.00045mol, 1.5eq.) of 4-bromobutanol, 124mg (0.0009 mol, 3.0eq.) of potassium carbonate, and 23mg (0.00015mol, 0.5eq.) of sodium iodide were sequentially added at room temperature, and the addition was completed at room temperature for 12 hours. Sampling reaction liquid, detecting by TLC (thin layer chromatography), when the reaction liquid is completely reacted, directly adding 10mL of water into the reaction liquid, extracting a product by using ethyl acetate, washing an organic phase by using an aqueous solution once, separating liquid, adding an organic phase, drying by using anhydrous sodium sulfate, filtering, adding silica gel into a filtrate, mixing the filtrate with a sample, and separating and purifying by using a silica gel column to obtain 60.0mg of light yellow oily liquid (lipid 13, 0.00007mol), wherein the yield is as follows: 22.3 percent.
Test examples
Test example 1: physicochemical Properties and in vitro transfection efficiency of mRNA LNP prepared with different ionizable amino lipid Compounds
1.1 test object
The physical and chemical properties and in vitro transfection efficiency of mRNA LNP prepared from different ionizable amino lipid compounds are examined.
1.2 test materials and instruments
Luc mRNA used in the experiment is provided by Nanjing Gimey Biotechnology Co. The lipid materials used in the experiments were as follows: lipids 1-10 are all made by oneself, dlin-MC3-DMA (shanghai aviveto technologies, ltd.), DSPC (sienna millennium biotechnology, ltd.), cholesterol (sigma-aldrich shanghai trade, ltd.), PEG2k-DMG (Avanti Polar Lipids, inc.), tf-PEG2k-DMG (sienna millennium biotechnology, ltd.).
The equipment used for LNP preparation was michanan lab scale microfluidic equipment, model: INano L;
particle size detection was performed using a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern Panalytical Ltd).
Encapsulation efficiency is obtained by
(Thermo Fisher Scientific Inc) fluorescence method.
1.3 test methods
1.3.1 preparation of LNPs Using different ionizable lipid Compounds
Each ionizable amino lipid mRNA LNP was prepared as shown in FIG. 1. The following solutions were prepared separately: A. ethanol solution of lipids, the molar ratio of each lipid being: dlin-MC3-DMA or lipid 1-10: cholesterol: PEG2k-DMG = 50; B. malate buffer containing mRNA, pH =4.0; and (2) putting the solution A and the solution B in a micro-fluidic chip according to the volume ratio of 1Mixing, and then transferring the LNP intermediate to a dialysis cassette (Slide-A-Lyzer) TM MWCO =20 k), dialyzed against PBS pH =7.4 for 16h, and ethanol removed to give LNP product.
1.3.2 test of physicochemical Properties
The product particle size, particle size distribution (PDI) and Zeta potential were measured by a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern). In the detection, the sample was diluted 50-fold with 5mM NaCl solution and transferred to a DTS1070 cuvette. The detection mode was 173 ° back-scattered light, and detection was resumed after each sample was equilibrated in the apparatus for 120s to reach 25 ℃.
The encapsulation efficiency of the product is detected by a Ribogreen-based nucleic acid fluorescence method. The test solution obtained by diluting the LNP sample 100-fold with 1X PBS was used to determine the free and total amount of mRNA in the sample. A series of concentration gradient standard solutions are prepared by diluting a free mRNA solution with 1 XPBS, and an LNP sample test solution and the mRNA standard solution are added into a 96-hole blackboard. TritonX100 and Ribogreen working solution are prepared according to the instruction, added into a 96-hole blackboard and mixed with LNP sample test solution and mRNA standard solution, and then slowly shaken for 10min in dark at room temperature. And measuring the fluorescence of the sample at 480nm of excitation light and 520nm of emission light by using a microplate reader, and calculating the encapsulation efficiency of the sample according to the fluorescence value of the sample.
1.3.3 cell assay
The transfection efficiency of each lipid was evaluated in vitro on Hela cells. Hela cells were routinely cultured in DMEM +10% FBS medium to ensure that the cells were in logarithmic growth phase; one day prior to transfection, 96-well plates were seeded at the appropriate cell density and grown overnight. When transfection is carried out, 70-90% of cells are fused; we prepared LNP-mRNA at different concentrations in serum-free medium, added 10ul to 96 cell culture plates to achieve concentrations of 100, 50, 25ng per well, and used firefly luciferase mRNA at the corresponding concentration as a positive reference
Transfection of mRNA was performed by MessengerMax (Thermo). At 37 deg.C, 5% CO 2 After incubation in an incubator for 24h, an equal volume of ONE GLO (Promega) detection assay was added to the wellsAnd (3) uniformly blowing the reagent, transferring the reagent into a 96-hole white board, and detecting the light intensity in a Lumi mode of an enzyme labeling instrument.
1.4 test results
The results of particle size, particle size distribution (PDI), zeta potential, encapsulation efficiency and transfection efficiency versus MC3 for the product are shown in table 1:
TABLE 1 particle size, PDI, zeta potential, encapsulation efficiency and transfection efficiency results for mRNA LNPs prepared with different ionizable amino lipids
Results of the cell assay for in vitro evaluation of transfection efficiency of each lipid on Hela cells are shown in fig. 2.
1.5 conclusion of the test
This experiment evaluated the in vitro transfection efficiency of mRNA LNP prepared with different new ionizable amino lipids, where MC3 is a commercially available RNA delivering ionizable amino lipid, lipid 1 is the lipid disclosed in patent WO2010030739A1, lipid 7 is a simple structural modification of lipid 1, with hydroxyethyl functional groups added, and lipids 6, 8, 9, 10 are new structural ionizable amino lipids that we prepared according to this design protected lipid structure. The results show that the LNPs prepared with lipids 6, 8, 9, 10 all have a particle size around 100nm or below, similar to the MC3 LNP; the PDI is less than 0.1, is smaller than that of LNP of MC3, lipid 1 and lipid 7, and is uniform in particles; the encapsulation efficiency is more than 90 percent and is higher than the encapsulation efficiency of LNP of lipid 1 and lipid 7; the transfection efficiency is more than or equal to that of MC3 LNP, wherein the expression of LNP of lipid 6 and 8 is more than 5 times of that of MC3 LNP.
Test example 2: in vitro transfection efficiency of mRNA LNP prepared under different prescription conditions using ionizable amino lipid compounds
1.1 test object
The differences in vitro transfection efficiency of lipids designed by us (e.g., lipid 8) with MC3 lipids under different prescription conditions were examined.
1.2 test materials and instruments
The materials and equipment used in this test were the same as those in test example 1.
1.3 test methods
1.3.1 preparation of LNP for ionizable lipid compounds
Each ionizable amino lipid mRNA LNP was prepared as shown in FIG. 1. The following solutions were prepared separately:
A. ethanol solutions of lipids, wherein the molar ratio of each lipid in different samples was formulated as in table 2 below.
TABLE 2 molar ratio of lipids in the samples
| Sample numbering | Molar ratio of lipids in the |
| 1 | Dlin-MC 3-DMA/DSPC/cholesterol/PEG 2k-DMG =31.5/10/56/2.5 |
| 2 | Dlin-MC 3-DMA/DSPC/cholesterol/PEG 2k-DMG =43.3/8.7/46.5/1.5 |
| 3 | Dlin-MC 3-DMA/DSPC/cholesterol/PEG 2k-DMG =46.3/9.4/42.7/1.6 |
| 4 | Dlin-MC 3-DMA/DSPC/cholesterol/PEG 2k-DMG =50/10/38.5/1.5 |
| 5 | Lipid 8/DSPC/cholesterol/PEG 2k-DMG =31.5/10/56/2.5 |
| 6 | Lipid 8/DSPC/cholesterol/PEG 2k-DMG =43.3/8.7/46.5/1.5 |
| 7 | Lipid 8/DSPC/cholesterol/PEG 2k-DMG =46.3/9.4/42.7/1.6 |
| 8 | Lipid 8/DSPC/cholesterol/PEG 2k-DMG =50/10/38.5/1.5 |
B. Malate buffer containing mRNA, pH =4.0.
Mixing the solution A and the solution B in a volume ratio of 1 TM MWCO =20 k), dialyzed against PBS pH =7.4 for 16h, and ethanol removed to give LNP product.
1.3.2 physicochemical Properties test and cell assay
The product particle size, particle size distribution (PDI) and Zeta potential were measured by a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern). The encapsulation efficiency of the product is detected by a Ribogreen-based nucleic acid fluorescence method. The transfection efficiency of each lipid was evaluated in vitro on Hela cells. The procedure of the test was the same as in test example 1.
1.4 test results
The results of particle size, particle size distribution (PDI), zeta potential, encapsulation efficiency and transfection efficiency versus MC3 for the product are shown in table 3:
TABLE 3 particle size, PDI, zeta potential, encapsulation efficiency and transfection efficiency results for mRNA LNP prepared with MC3 and lipid 8 under different prescription conditions
Cell assay the results of in vitro evaluation of the transfection efficiency of each lipid on Hela cells are shown in figure 3.
1.5 conclusion of the test
The results show that the particle size was <100nm for different formulations of LNP prepared with lipid 8, consistent with MC3 LNP particle size; the PDI of the lipid particles is less than 0.1, and the PDI of MC3 LNP is gradually larger and reaches more than 0.2 along with the reduction of the MC3 lipid ratio, which shows that the particles of MC3 LNP are gradually inhomogeneous and unstable along with the increase of the content of the ionizable amino lipid, while the LNP of lipid 8 does not have the problem and is more stable than the MC3 LNP particles. In addition, as can be seen from cell transfection experiments, when the content of the ionizable amino lipid in the LNP is reduced, the LNP formed by lipid 8 shows a more obvious transfection advantage in cell transfection compared with MC3 LNP; especially at doses of 50 ng/well, LNP increased 18.05 fold over MC3 LNP cell transfection when ionizable amino lipids were reduced to 43.3%, and this advantage was more pronounced at lower doses, up to 35.02 fold. This indicates that the formed mRNA LNP particles of the lipid (such as lipid 8) designed by us are more uniform and stable than the lipid nanoparticles currently marketed using MC3 lipid, indicating that the novel ionizable lipid has better stability and suitability, and is less affected by the formulation content factor than MC 3; meanwhile, compared with MC3 lipid, the novel ionizable lipid has more remarkable cell transfection advantage in a low-content prescription, and can realize high-efficiency low-frequency administration, thereby reducing potential toxicity risk.
Test example 3: in vitro transfection efficiency of mRNA LNP prepared under different prescription conditions using ionizable amino lipid compounds
1.1 test object
The difference in transfection efficiency of the lipid designed by us (e.g. lipid 11) with MC3 lipids in vitro under different prescription conditions was examined.
1.2 test materials and instruments
The materials and apparatus used in this test were the same as those in test example 1.
1.3 test methods
1.3.1 preparation of LNP for ionizable lipid compounds
Each ionizable amino lipid mRNA LNP was prepared as shown in FIG. 1. The following solutions were prepared separately:
A. ethanol solutions of lipids, wherein the molar ratio of each lipid in different samples was formulated as in table 4 below.
TABLE 4 molar ratio of lipids in the samples
B. Malate buffer containing mRNA, pH =4.0.
Mixing the solution A and the solution B in a volume ratio of 1 TM MWCO =20 k), dialyzed against PBS pH =7.4 for 16h, and ethanol was removed to give LNP product.
1.3.2 physicochemical Properties testing and cell assay
The product particle size, particle size distribution (PDI) and Zeta potential were measured by a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern). The encapsulation efficiency of the product is detected by a Ribogreen-based nucleic acid fluorescence method. The transfection efficiency of each lipid was evaluated in vitro on Hela cells. The procedure of the test was the same as in test example 1.
1.4 test results
The results of particle size, particle size distribution (PDI), zeta potential, encapsulation efficiency and transfection efficiency versus MC3 for the product are shown in table 5:
TABLE 5 particle size, PDI, zeta potential, encapsulation efficiency and transfection efficiency results for mRNA LNP prepared with MC3 and lipid 11 under different prescription conditions
Cell assay results of in vitro evaluation of transfection efficiency of each lipid on Hela cells are shown in fig. 4.
1.5 conclusion of the test
The results show that the particle size was <100nm for different formulations of LNP prepared with lipid 11, consistent with MC3 LNP particle size; the PDI of the lipid is less than 0.13, and the PDI of MC3 LNP is gradually larger and reaches more than 0.2 along with the reduction of the MC3 lipid ratio, which shows that the particles of MC3 LNP are gradually inhomogeneous and unstable along with the increase of the content of the ionizable amino lipid, while the LNP of lipid 11 does not have the problem and is more stable than the MC3 LNP particles. In addition, it can be seen from cell transfection experiments that when the content of ionizable amino lipid in LNP is reduced, LNP formed by lipid 11 shows more obvious transfection advantage in cell transfection than MC3 LNP; especially at doses of 50 ng/well, LNP increased 85.29 fold over MC3 LNP cell transfection when ionizable amino lipids were reduced to 43.3%, and this advantage was more pronounced at lower doses, increasing by a factor of 124.76. This indicates that the lipid (such as lipid 11) designed by the inventor is more uniform and stable than the lipid nanoparticles using MC3 lipid on the market at present, and indicates that the novel ionizable lipid has better stability and suitability, and is less influenced by the prescription content factor compared with MC 3; meanwhile, compared with MC3 lipid, the novel ionizable lipid has more remarkable cell transfection advantage in a low-content prescription, and can realize high-efficiency low-frequency administration, thereby reducing potential toxicity risk.
Claims (23)
1. A lipid compound represented by the formula (I) or a pharmaceutically acceptable salt thereof:
wherein the content of the first and second substances,
R 1 and R 2 Each independently is C 6 -C 20 Alkyl radical, C 6 -C 20 Alkenyl or C 6 -C 20 Alkynyl;
x is independently-C (= O) O-, -OC (= O) O-, -C (= S) O-, -OC (= S) O-, -C (= O) S-, -SC (= O) -, -OC (= O) S-, -O-, -S-, -C 1 -C 6 alkylene-O-, -O-C 1 -C 6 Alkylene-, -C 1 -C 6 alkylene-S-or-S-C 1 -C 6 Alkylene-;
R 3 independently is a 5-6 membered saturated heterocyclyl containing 1 or 2 ring heteroatoms independently selected from N, O and S, optionally substituted with R 4 Substitution;
R 4 independently is C 1 -C 6 Alkyl, optionally substituted with-OH;
m and n are each independently selected from integers of 1 to 10.
2. A lipid compound according to claim 1, having the structure shown in formula (II) below:
3. a lipid compound according to any one of claims 1 to 2, wherein R 1 Is C 6 -C 10 Linear alkyl radical, and R 2 Is C 10 -C 20 A branched alkyl group.
4. A lipid compound according to any one of claims 1 to 3, wherein R 1 Is C 9 Linear alkyl radical, and R 2 Is composed of
5. A lipid compound according to any one of claims 1 to 4, wherein X is independently-C (= O) O-, -OC (= O) -, -C 1 -C 6 alkylene-O-or-O-C 1 -C 6 An alkylene group-.
6. A lipid compound according to any one of claims 1 to 5, wherein X is independently-OC (= O) -or-O-CH 2 -。
7. A lipid compound according to any one of claims 1 to 6, wherein R 4 Independently is-CH 3 、-CH 2 CH 2 OH、-CH 2 CH 2 CH 2 OH or-CH 2 CH 2 CH 2 CH 2 OH。
8. A lipid compound according to any one of claims 1 to 7, wherein m and n are each independently 6.
9. A lipid compound according to any one of claims 1 to 8, having a structure represented by the following formula (III):
10. a lipid compound according to any one of claims 1 to 9, wherein X is independently-OC (= O) -or-O-CH 2 -, and R 4 Independently is-CH 3 、-CH 2 CH 2 OH、-CH 2 CH 2 CH 2 OH or-CH 2 CH 2 CH 2 CH 2 OH。
11. A lipid compound according to any one of claims 1 to 10, wherein the compound is selected from:
12. a lipid nanoparticle composition comprising the lipid compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.
13. The lipid nanoparticle composition of claim 12, further comprising a neutral lipid, cholesterol, and a PEG lipid.
14. The lipid nanoparticle composition according to claim 13, wherein the neutral lipid is selected from the group consisting of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1, 2-dipentanyl-sn-glycero-3-phosphoethanolamine (trans DOPE), and the PEG lipid is selected from the group consisting of PEG-DMG, PEG-dipalmitoyl glycerol, PEG-DSPE, PEG-dilauryl glycerol amide, PEG-dimyristyl glycerol amide, PEG-dipalmitoyl glycerol amide and PEG-distearoyl glycerol amide, PEG-cholesterol (1- [8' - (cholest-5-ene-3 [ β ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ ω ] -methyl-poly (ethylene glycol), PEG-DMB, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA and PEG2k-DSA.
15. The lipid nanoparticle composition of claim 13 or 14, wherein the neutral lipid is DSPC and the PEG lipid is PEG2k-DMG.
16. The lipid nanoparticle composition of any one of claims 12 to 15, wherein the lipid compound is present in a molar percentage of 30-50% of the total lipid component of the composition.
17. The lipid nanoparticle composition of any one of claims 12 to 16, wherein the lipid compound comprises 40-50% mole percent of the total lipid component of the composition.
18. The lipid nanoparticle composition of any one of claims 12-17, further comprising a nucleic acid molecule selected from the group consisting of mRNA, siRNA, antisense oligonucleotide (ASO), saRNA, and miRNA.
19. A method of delivering a nucleic acid into a cell, comprising delivering into the cell the lipid nanoparticle composition of any one of claims 12 to 18.
20. The method according to claim 19, wherein the cell is a mammalian cell, preferably the cell is a human cell.
21. The method according to claim 19, wherein said cell is a mammalian precancerous lesion cell, preferably said cell is a human precancerous lesion cell.
22. The method according to claim 19, wherein the cell is a tumor cell, preferably the cell is a human tumor cell.
23. Use of a lipid nanoparticle composition of any one of claims 12 to 18 in the manufacture of a medicament for the treatment of a disease.
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