CN116947669B - Ionizable lipid compound with high transfection efficiency and application thereof - Google Patents

Ionizable lipid compound with high transfection efficiency and application thereof Download PDF

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CN116947669B
CN116947669B CN202310862227.6A CN202310862227A CN116947669B CN 116947669 B CN116947669 B CN 116947669B CN 202310862227 A CN202310862227 A CN 202310862227A CN 116947669 B CN116947669 B CN 116947669B
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compound
lipid
ionizable lipid
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CN116947669A (en
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章雪晴
滕以龙
陈起静
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Rongcan Biomedical Technology Shanghai Co ltd
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    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons

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Abstract

The invention discloses an ionizable lipid compound with high transfection efficiency and application thereof, belonging to the field of biological medicine, wherein the ionizable lipid compound is a compound with the following structure:n＝0‑10；G 1 、G 2 each independently is alkylene, R 1 、R 2 、R 3 、R 4 Each independently is an alkanyl, alkenyl or H, G 3 Is alkylene or

Description

Ionizable lipid compound with high transfection efficiency and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to an ionizable lipid compound with high transfection efficiency and application thereof.
Background
Gene therapy (gene therapy) is a therapeutic method for correcting or compensating diseases caused by defective or abnormal genes by introducing exogenous genes into target cells. Nucleic acid vaccine (nucleic acid vaccine), also known as genetic vaccine (genomic vaccinee), refers to a vaccine that is prepared by introducing a nucleic acid sequence (such as DNA, mRNA, etc.) encoding an immunogenic protein or polypeptide into a host, expressing the immunogenic protein or polypeptide by the host cell, and inducing the host cell to produce an immune response against the immunogen, thereby achieving the purpose of preventing and treating diseases. Among them, ensuring the smooth introduction of foreign genes is an extremely important part of gene therapy and immunization with genetic vaccines. Among the methods of gene transfer, methods of developing suitable lipid nanoparticles (Lipid Nanoparticle, LNP) to encapsulate nucleic acids, target them to target cells of interest, and delivering nucleic acids of specific genes into cells are increasingly being used.
The LNP system mainly comprises four major components: ionizable lipids, structural lipids, co-lipids, and polymer conjugated lipids. Wherein, the ionizable lipid is a lipid molecule which has positive charge under acidic pH value and is neutral under physiological pH value, and can influence the surface charge of LNP under different pH conditions. This state of charge can affect its immune recognition in the blood, blood clearance and tissue distribution, and its ability to escape endosomes within the cell, which is critical for intracellular delivery of nucleic acids.
After LNP enters organisms by different routes of administration, LNP has pH sensitivity under the influence of ionizable lipid compounds. The pH of the body fluid is 7.4, and LNP is preferably neutral, so that the stability in a biological system is required to be the best, and immune clearance is avoided; after the LNP carrying the nucleic acid (e.g., mRNA) enters the cell by endocytosis, it is trapped in an acidic vesicle (endosome), the acidic medium in the endosome changes the pH environment to acidic, the pH is about 5.5, the core component of the LNP can ionize the lipid compound in the acidic environment to damage the endosome membrane, and thus the mRNA escapes from the endosome.
Therefore, there is a need in the art to develop an ionizable lipid compound for preparing LNP that is safe and stable in a neutral environment, and that properly promotes membrane rupture in an acidic environment (ph=3-5.5), and that has a better nucleic acid endosome escape ability.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the ionizable lipid compound with high transfection efficiency and the application thereof, and the mRNA-LNP nucleic acid endosome prepared by the ionizable lipid compound with a brand new structure has good escape capability, high transfection efficiency and high stability.
In order to achieve the above object, the present invention adopts the following technical scheme:
an ionizable lipid compound with high transfection efficiency, characterized in that said compound has the following structure:
wherein n=an integer of 0 to 10;
G 1 、G 2 each independently is C 1 -C 10 An alkylene group;
R 1 、R 2 、R 3 、R 4 each independently is C 1 -C 20 Alkyl, C 2 -C 20 An alkylene group or H; when R is 1 When H is the same, R 2 Is C 2 -C 20 An alkylene group; r is R 3 When H is the same, R 4 Is C 2 -C 20 An alkylene group;
G 3 is C 1 -C 10 Alkylene, or isWherein a and b are each independently an integer of 1 to 9, and a+b=an integer of 2 to 10.
The above-mentioned ionizable lipid compound G having high transfection efficiency 1 Is C 6 Alkylene group, G 2 Is C 5 -C 7 Alkylene, preferably G 2 Is C 7 An alkylene group.
The above-mentioned ionizable lipid compound G having high transfection efficiency 3 Is C 2-4 Alkylene, or is
The foregoingIs an ionizable lipid compound with high transfection efficiency, G as an example 1 Is C 6 Alkylene group, G 2 Is C 5 -C 7 Alkylene, preferably G 2 Is C 7 Alkylene group, G 3 Is C 2-4 Alkylene group, R 1 、R 2 、R 3 、R 4 Each independently is C 1 -C 20 An alkane group.
As an example, G 1 Is C 6 Alkylene group, G 2 Is C 5 -C 7 Alkylene, preferably G 2 Is C 7 Alkylene group, G 3 Is C 2-4 Alkylene group, R 1 、R 2 Each independently is C 1 -C 20 Alkyl, R 3 Is H, R 4 Is C 2 -C 20 An alkylene group.
As an example, G 1 Is C 6 Alkylene group, G 2 Is C 5 -C 7 Alkylene, preferably G 2 Is C 7 Alkylene group, G 3 Is thatR 1 、R 2 、R 3 、R 4 Each independently is C 1 -C 20 An alkane group.
As a preferred example, the above-mentioned ionizable lipid compound with high transfection efficiency has the following structural formula:
as an example, the above-mentioned ionizable lipid compound with high transfection efficiency is represented by formula IEach independently is a structure selected from the group consisting of:
as an example, the above-mentioned ionizable lipid compound with high transfection efficiency is represented by formula IEach independently is a structure selected from the group consisting of:
as an example, the above-mentioned ionizable lipid compound with high transfection efficiency is represented by formula IEach independently is a structure selected from the group consisting of:
it should be noted that the structure of the hydrophobic group is not limited, and the number and position of substituents on the alkane group are not limited, so long as the compound is used, the number and position of substituents on the alkane group are not limited, the compound is a compound which uses an N atom as a charge center, uses a hydrophilic group as a head, uses two hydrophobic groups as tails, and introduces a- (c=o) O-, and introduces a carbonate bond as a linker between the N atom and the two hydrophobic groups, respectively.
In a preferred embodiment, the one highly transfection efficient ionizable lipid compound, its stereoisomers, its tautomers or its pharmaceutically acceptable salts may be used for preparing a pharmaceutical composition.
In a more preferred embodiment, the pharmaceutical composition may comprise: a carrier containing the ionizable lipid compound, a carried pharmaceutical agent, a pharmaceutical adjuvant, or a combination thereof.
The use of an ionizable lipid compound with high transfection efficiency, wherein the carrier further comprises: one or a combination of several of a co-lipid, a structural lipid, a polymer conjugated lipid or an amphiphilic block copolymer; it should be noted that: the components of the carrier composition are not limited, and may be any of those having a known composition of matter or those having an unknown composition of matter, and any of those having an ionizable lipid structure according to the present invention are within the scope of the present invention and are encompassed by the present invention.
Lipids include, but are not limited to: one or a combination of several of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), ceramide and charged lipid; phosphatidylcholine as one preferred includes: DSPC, DPPC, DMPC, DOPC, POPC; phosphatidylethanolamine as a preferred type is DOPE; charged lipids refer to a class of lipid compounds that exist in positively or negatively charged form; the charge is independent of the pH in the physiological range, for example pH 3-9, and is not affected by pH. Charged lipids may be of synthetic or natural origin. Examples of charged lipids include, but are not limited to DOTAP, DOTMA, 18PA. The examples herein are not exhaustive and any lipid aid may be used in the present invention.
Structured lipids include, but are not limited to: sterols and derivatives thereof, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol or corticosteroids. Sterols as a preferred cholesterol; it is not intended to be exhaustive and any structural lipid may be used in the present invention.
As one example, the polymer conjugated lipid is a pegylated lipid; the pegylated lipids include: one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol. The choice of polymer-conjugated lipid is not limited and any polymer-conjugated lipid may be used in the present invention.
As an example, the amphiphilic block copolymer may include: a combination of one or more of polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyorthoester, polyanhydride, poly (β -amino ester) (PBAE), or polyethylene glycol (PEG) modified amphiphilic block copolymer. It should be noted that: the examples herein are not exhaustive and any amphiphilic block copolymer may be used in the present invention.
As an example, when used in the preparation of a composition comprising an ionizable lipid compound, the molar ratio of the ionizable lipid compound to the co-lipid of the present invention is in the range of 0.5:1 to 10:1.
As an example, when used to prepare a formulation containing an ionizable lipid compound, the molar ratio of the ionizable lipid compound to the structural lipid of the present invention is from 0.5:1 to 5:1.
As an example, when used to prepare compositions containing ionizable lipid compounds, the molar ratio of the ionizable lipid compounds of the present invention to the polymer conjugated lipid is in the range of 10:1-250:1.
As an example, when used to prepare compositions containing ionizable lipid compounds, the molar ratio of the ionizable lipid compounds of the present invention to the amphiphilic block copolymer is in the range of 0.5:1 to 80:1.
As an example, the carrier is Lipid Nanoparticle (LNP), the average particle size of the lipid nanoparticle is 30-200nm, and the polydispersity index of the nanoparticle preparation is less than or equal to 0.5. It should be noted that: any nanoparticle prepared from one or more ionizable lipid compounds is within the scope of the present patent, and is encompassed by the present invention; such as: in addition to the lipid nanoparticle may also be a hybrid nanoparticle formed by one or more ionizable lipid compounds and a macromolecule, such as: PLGA-PEG, PLA-PEG, PCL, PBAE (Poly beta-amino acid) and the like are not exhaustive herein.
In the technical scheme of the invention, the carried pharmaceutical agent is not particularly limited, and comprises but is not limited to: one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; the choice and combination formulation of the drug to be carried is not limited, and any ionizable lipid compound employing the structure of the present invention is within the scope of the present invention and is taught by the present invention.
The small molecule compound may be an active ingredient in an agent for treatment or prophylaxis, for example: antitumor agents, antiinfectives, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents, etc., are not meant to be exhaustive.
Polypeptides are compounds formed by joining alpha-amino acids together in peptide bonds, and are proteolytic intermediates.
The protein is a substance with a certain space structure formed by the twisting and folding of a polypeptide chain consisting of amino acids in a dehydration condensation mode; the protein may be an interferon, protein hormone, cytokine, chemokine or enzyme, etc.
Pharmaceutical adjuvants include, but are not limited to: one or more of a diluent, a stabilizer, a preservative or a lyoprotectant. It is not exhaustive, and any kind of drug adjuvant is selected and compounded as long as the ionizable lipid compound adopting the structure of the invention is within the scope of the invention, and the ionizable lipid compound is taught by the invention.
Diluents are any pharmaceutically acceptable water-soluble excipients known to those skilled in the art including, but not limited to: amino acids, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, other oligosaccharides, mannitol, dextran, sodium chloride, sorbitol, polyethylene glycol, phosphates, or derivatives thereof, and the like.
The stabilizer may be any pharmaceutically acceptable adjuvant known to those skilled in the art, including but not limited to tween-80, sodium dodecyl sulfate, sodium oleate, mannitol, mannose or sodium alginate, etc.
The preservative may be any pharmaceutically acceptable preservative known to those skilled in the art, exemplary representatives being: thiomerosal.
Lyoprotectants may be any pharmaceutically acceptable lyoprotectant known to those skilled in the art, exemplary representatives being: glucose, mannitol, sucrose, lactose, trehalose, maltose, and the like.
Compared with the prior art, the invention has the following advantages:
1. the ionizable lipid compound disclosed by the invention is novel in structure, takes an N atom as a charge center, takes a hydrophilic group as a head, takes two hydrophobic groups as tail, and introduces a carbonate bond as a linker while introducing- (C=O) O-, respectively, between the N atom and the two hydrophobic groups, so that the structure has low damage to cell membranes in a neutral environment and high safety.
2. After entering cells, the ionizable lipid compound disclosed by the invention has a higher effect of destroying an endosome membrane in an endosome acidic environment, and compared with a commercialized product, the ionizable lipid compound has stronger endosome escape capacity and faster escape rate, so that stronger transfection efficiency is generated;
3. the ionizable lipid compounds of the invention have high biocompatibility.
4. The synthesis steps of the ionizable lipid compound are simple, and the method is suitable for biological medicine industrialization.
5. The ionizable lipid compound of the invention can be stably stored for a long time, the key parameter change is tiny, and the transportation and commercial storage cost is low.
Drawings
FIG. 1 is a schematic diagram of experimental results of samples in a neutral pH environment in an endosome escape ability experiment of the present invention;
FIG. 2 is a schematic diagram of experimental results of samples in an acidic pH environment in an endosome escape ability experiment according to the present invention;
FIG. 3 is a schematic representation of experimental results of samples in an acidic pH environment in an endosome escape rate experiment according to the present invention;
FIG. 4 is an electron microscope image of lipid nanoparticles prepared using the compound of sample H-1 according to the present invention;
FIG. 5 is a graph showing the comparison of LNP immune effects obtained by preparing ionizable lipid compounds of the present invention.
Term, english abbreviation interpretation:
nucleic acid is a generic term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which is a biological macromolecule composed of multiple nucleotide monomers; the nucleic acid is composed of nucleotides, and the nucleotide monomers are composed of five carbon sugars, phosphate groups, nitrogen-containing bases, or any modification groups. If the five carbon sugar is ribose, then the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.
The "nucleic acid" includes, but is not limited to, one or more of single stranded DNA, double stranded DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNAs (lncRNA), micronon-coding RNAs (miRNA and siRNA), telomerase RNA (TelomeraseRNA Component), small molecule RNAs (snRNA and scRNA), circular RNAs (circRNA), synthetic mirnas (miRNA miRNAs, miRNA agomir, miRNA antagomir), antisense RNAs, ribozymes (ribozymes), asymmetric interfering RNAs (aiRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), guide RNAs (gRNA), small guide RNAs (sgrnas), locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), morpholine antisense oligonucleotides, morpholino oligonucleotides, or biospecific oligonucleotides. The examples herein are not exhaustive and can be applied to the present invention as long as they are polymerized from nucleotide monomers.
mRNA, messenger RNA, chinese translation: messenger ribonucleic acid is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to direct protein synthesis. The mRNA may be monocistronic mRNA or polycistronic mRNA. The mRNA may also contain one or more functional nucleotide analogs, examples of which include: pseudouridine, 1-methyl-pseudouridine, 5-methylcytosine, and the like. The examples herein are also not exhaustive and any modified mRNA or derivative thereof may be used in the present invention.
In the claims of the present invention, when "C1-C20 alkanyl" is described, it means that the radical may be an alkanyl radical having 1-20 carbon atoms (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), which is a saturated alkanyl radical, which may be straight-chain, or have a branched structure, and an alkanyl radical satisfying the foregoing number of carbon atoms is within the scope of the description of the term.
When describing "C2-C20 alkenyl" it is intended that the group may be an alkenyl group having 2 to 20 carbon atoms (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), which may be straight-chain, or have a branched structure, and that the alkenyl groups satisfy the foregoing numbers of carbon atoms are within the scope of the description of the term. In various embodiments of the invention, the olefinic group may be a mono-olefin or a multi-olefin (e.g., a di-olefin).
When "C1-C10 alkylene" is described, it is intended that the group may be an alkylene group having 1 to 10 carbon atoms (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), which may be straight or branched.
Pharmaceutically usable salts refer to acid addition salts or base addition salts.
Acids in which the acid addition salts include, but are not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acid-type phosphates, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, carbonic acid, cinnamic acid, citric acid, cyclic amic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaronic acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, propionic acid, glutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, succinic acid, sulfanilic acid, tartaric acid, succinic acid, tricarboxylic acid, and quaternary ammonium acids.
Wherein the base addition salts include, for example, but are not limited to: sodium, potassium, lithium, ammonium, calcium, magnesium, ferric, cupric, manganic, and aluminum salts; organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazinaniline, choline, betaine, bennetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins; preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.
DSPC: english name: distearoyl Phosphatidylcholine,1, 2-distearoyl-sn-glycero-3-phosphaline; chinese name: distearyl lecithin, CAS number 816-94-4.
DPPC: chinese name: dipalmitin phosphatidylcholine; english name: no. 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, CAS, 63-89-8.
DMPC: chinese name: dimyristoyl phosphatidylcholine; english name: 1, 2-Dimyristonyl-sn-glycero-3-phosphonine, CAS number 18194-24-6.
DOPC: chinese name: 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine; english name: 1, 2-diolyl-sn-glycero-3-phosphaline, CAS number 4235-95-4.
POPC: chinese name: 2-oleoyl-1-palmitoyl-glycerol-3-phosphorylcholine; english name: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphacholine, CAS number 26853-31-6.
DOPE: chinese name: 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine; english name: 1, 2-Dioleyl-SN-Glycero-3-PHOSPHOETHANOLAMINE, CAS: 4004-05-1.
DOTAP: chinese name: (1, 2-dioleoxypropyl) trimethylammonium chloride; english name: 1, 2-diolyl-3-trimethyllamonium-propane (chloride salt), CAS number: 132172-61-3; the chemical structural formula is shown as follows:
DOTMA: chinese name: n, N, N-trimethyl-2, 3-bis (octadeca-9-en-1-yloxy) propan-1-ammonium chloride, CAS number 1325214-86-5, chemical structural formula shown below:
18PA: CAS number: 108392-02-5, the chemical structural formula is shown as follows:
SM: chinese name: sphingomyelin (SM); english name: sphingomyelin.
PEG: chinese name: polyethylene glycol; english name: polyethylene glycol.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available.
The ionizable lipid compound was prepared by the preparation method of example 1 below.
Example 1:
synthesis of compound a: 6-Bromohexanoic acid (10.00 g,51.27 mmol), dicyclohexylcarbodiimide (DCC, 12.69g,61.52 mmol), 4-dimethylaminopyridine (1.25 g,10.25 mmol) were dissolved in 200mL Dichloromethane (DCM), 6-2-hexyldecanol (12.43 g,51.27 mmol) was added and the reaction stirred at room temperature for 12h. After the completion of the reaction, the solvent was distilled off under reduced pressure using a rotary evaporator. 300mL of ethyl acetate was added, the mixture was washed with an equal volume of saturated sodium bicarbonate solution 2 times, the mixture was washed with an equal volume of saturated sodium chloride solution 1 time, and dried over anhydrous sodium sulfate for 30 minutes, the solvent was distilled off under reduced pressure using a rotary evaporator, and the mixture was purified by column separation (silica gel column, eluent: PE: EA=100:1 (volume ratio)), to obtain 19.78g of colorless liquid, with a yield of 92%.
Synthesis of compound b: compound a (10.00 g,23.84 mmol), 2- (2-aminoethoxy) ethanol (5.01 g,47.68 mmol), N, N-diisopropylethylamine (DIPEA, 3.70g,28.61 mmol) was dissolved in 100mL ethanol (EtOH) and reacted with heating at 60℃under stirring for 12h. After the completion of the reaction, the solvent was distilled off under reduced pressure using a rotary evaporator. 200mL of ethyl acetate was added, the mixture was washed 3 times with an equal volume of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 30min, and the solvent was removed by distillation under reduced pressure using a rotary evaporator, followed by column separation and purification (silica gel column, eluent: DCM: meOH=20:1 (volume ratio)), to give 7.51g of a pale yellow liquid in 71% yield.
Synthesis of Compound c: 6-Bromon-hexanol (10 g,55.23 mmol), 4-dimethylaminopyridine (6.75 g,55.23 mmol) were dissolved in 200mL of Dichloromethane (DCM), and after stirring in an ice bath under nitrogen for 10min, phenyl p-nitrochloroformate (13.36 g,66.27 mmol) was added in portions, gradually brought to room temperature, stirred at room temperature for 3h, and then 2-hexyldecanol (14.73 g,60.75 mmol) was added under ice bath conditions and stirred at room temperature for 12h. After the completion of the reaction, the solvent was distilled off under reduced pressure using a rotary evaporator. 400mL of ethyl acetate was added, the mixture was washed with an equal volume of saturated sodium bicarbonate solution 2 times, the mixture was washed with an equal volume of saturated sodium chloride solution 1 time, and dried over anhydrous sodium sulfate for 30 minutes, the solvent was distilled off under reduced pressure using a rotary evaporator, and the mixture was purified by column separation (silica gel column, eluent: PE: EA=100:1 (volume ratio)), to obtain 20.01g of colorless liquid, with a yield of 82%.
Synthesis of Compound H-1: compound b (2.04 g,4.59 mmol), compound c (2 g,4.59 mmol), N, N-diisopropylethylamine (DIPEA, 0.71g,5.51 mmol) was dissolved in 50mL ethanol (EtOH) and reacted with heating at 60℃under stirring for 12h. After the completion of the reaction, the solvent was distilled off under reduced pressure using a rotary evaporator. 200mL of ethyl acetate was added in equal volumeThe saturated sodium chloride solution was washed 3 times, dried over anhydrous sodium sulfate for 30min, and the solvent was distilled off under reduced pressure using a rotary evaporator, followed by column separation and purification (silica gel column, eluent: DCM: meoh=50:1 (volume ratio)) to obtain 3.01g of a pale yellow liquid, yield 82%. 1 H NMR(400MHz,Chloroform-d)δ4.43(s,1H),4.09(t,J＝6.8Hz,2H),4.00(d,J＝5.9Hz,2H),3.94(d,J＝5.8Hz,2H),3.70–3.63(m,2H),3.64–3.55(m,4H),2.63(s,2H),2.47(s,4H),2.28(t,J＝7.5Hz,2H),1.70–1.18(m,64H),0.86(t,J＝6.5Hz,12H).MS m/z(ESI)：812.8[M+H] + 。
By the method of example 1, the compounds H-3 to H-12, the comparative sample H1-2, the comparative sample H2-1 and the comparative sample H2-2 can be prepared as well, and will not be described in detail here.
The hydrogen spectrum data for some compounds are shown below:
experiment one:
mRNA-LNP was prepared for the following experiments, the preparation method being:
step one: lipid nanoparticles were prepared by mixing the ionizable Lipid compounds corresponding to H-1-H-12 and comparative samples H1-2, H2-1, and H2-2 in Table 1, DOPE, cholesterol, and PEG-Lipid in a prescribed ratio (Lipid/DOPE/Cholesterol/Lipid-PEG of 35/25/38.5/1.5 (molar ratio)). The Lipid nanoparticles were prepared by optimally mixing the ionizable compound (the dioic vaccine BNT162b 2) corresponding to the commercial comparative sample h1-1 in Table 1 with Lipid/DSPC/Cholesterol/Lipid-PEG at a ratio of 46.3/9.4/42.7/1.6 (molar ratio). Commercially available comparative sample MC3Lipid nanoparticles were prepared in the formulation ratio (50/10/38.5/1.5 (molar ratio)) by dissolving the Lipid nanoparticles in ethanol (concentration of Lipid 20 mg/mL) and thoroughly mixing to obtain an ionizable Lipid ethanol solution.
Step two: mRNA was prepared at a Lipid Nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1, diluted to 0.2mg/mL using citrate or sodium acetate buffer (ph=3 or 5).
And thirdly, fully and uniformly mixing the ionizable lipid ethanol solution obtained in the step one with the mRNA solution in a volume ratio of 1:5 to 1:1. The obtained nanoparticles were purified by ultrafiltration and dialysis, and after filtration and sterilization, the particle size of mRNA-LNP (lipid nanoparticle encapsulating mRNA) and PDI were characterized by using Malvern Zetasizer Nano ZS, and the encapsulation efficiency of mRNA was determined by using a Ribogreen RNA quantitative determination kit (Thermo Fisher).
TABLE 1
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Experiment II: transfection efficiency validation experiment:
male ICR mice (6-8 week, shanghai Jieshi laboratory animals Co., ltd.) were kept at 22.+ -. 2 ℃ and a relative humidity of 45-75% for a 12h light/dark cycle. mRNA (luciferase mRNA) encoding luciferase was used as a reporter gene. Luciferase catalyzes luciferin to generate bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence in unit time. Taking luciferase mRNA as an example, preparing an mRNA-LNP sample H1-12 obtained in experiment I, and comparing samples MC3, H1-2, H2-1 and H2-2; the above samples were administered by intramuscular injection at a dose of 150 μg/kg mRNA, respectively, with 2 mice per group of samples, two legs. At a specific time point, fluorescein (20 mug/mL) was injected into the abdominal cavity of the mice, and after 5 minutes, the mice were placed on a living body imager of the small animals to measure fluorescence intensity, and the final result is represented by average fluorescence intensity, and the experimental results of the fluorescence intensity after the intraperitoneal injection administration of the mice are shown in Table 2.
TABLE 2
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Analysis of results:
the LNP sample containing H1-H12 has an improvement range of 2-3 orders of magnitude compared with the transfection efficiency of a commercial MC3 sample, and has a remarkable difference in transfection effect compared with a compound with similar structure (compared with the comparative samples H1-2, H2-1 and H2-2, the degradable groups have small differences), and the level of 1 order of magnitude can be improved.
In conclusion, the mRNA-LNP sample prepared from the ionizable lipid compound of the specific structure of the present invention has a very excellent effect on transfection efficiency.
Experiment III: endosome escape ability experiment
mRNA escape is achieved primarily because pH-sensitive liposomes promote membrane fusion in the intracellular acidic environment (pH 3-5.5). The following experiments mimic the interaction of LNP with cell membranes in a neutral pH environment; and interaction of LNP and endosome membranes in an acidic pH environment of intracellular endosomes; thereby verifying the safety and endosome escape ability of LNP prepared from the ionizable lipid compound.
The experimental procedure was as follows: four-week-old female ICR mice, weighing 15-20 g, are raised in an experimental environment with a temperature of 22+/-2 ℃ and a relative humidity of 45-75%, and have a light/dark period of 12 hours. After the mice are purchased and adapted in animal houses for one week, formal animal tests can be carried out. After taking whole blood of the mouse, 10000g of the blood of the mouse was centrifuged in a centrifuge for 5 minutes, and after separating out red blood cells of the mouse, the blood was washed five times with PBS (pH 7.4). The isolated erythrocytes were then suspended in PBS solutions at pH 7.4 and pH5.5, respectively, and added to 96-well plates. LNP prepared in experiment one of the concentration gradients, sample H-1, comparative sample H1-1, and comparative sample H1-2 were then added. Incubating for 1 hour at 37 ℃, centrifuging 10000g of the sample in the pore plate in a centrifuge for 5min, and taking supernatant containing hemoglobin. Absorbance at 540nm (bubbles cannot appear in the well plate during detection) was detected for each well using a multifunctional microplate detector, and cells not treated with LNP were used as a negative control group.
The experimental results are shown in fig. 1 and 2.
Analysis of results: as can be seen from fig. 1: LNP prepared from the ionizable lipid with the structural characteristics has low erythrocyte dissolution rate in a neutral pH environment, shows low damage to cell membranes in the neutral environment, and shows safety. In contrast, the comparative samples (the samples prepared from the gabbroil and the compound not conforming to the structural features of the present invention) were severe in cell membrane disruption effect at high concentrations (0.12 to 0.24 mM), indicating potential cytotoxicity at high concentrations. As can be seen from fig. 2: the LNP prepared by the ionizable lipid with the structural characteristics is obviously higher than that of a comparison sample in an acidic pH environment, which shows that the ionizable lipid with the structural characteristics has higher effect of destroying endosome membranes in endosomes after entering cells, and shows stronger endosome escape effect than that of the comparison sample, thereby generating stronger transfection efficiency.
Experiment IV: endosome escape rate experiment
The erythrocytes isolated in experiment three were separately suspended in PBS solution at ph5.5 and added to 96-well plates. LNP prepared in experiment two at a fixed concentration, sample H-1, commercially available comparative sample H1-1 (pyroxene), comparative sample H1-2, was then added. Incubating at 37deg.C for 10min,20min,40min,60min and 80min respectively, centrifuging 10000g of sample in the plate in centrifuge for 5min, and collecting supernatant containing hemoglobin. Absorbance at 540nm (bubbles cannot appear in the well plate during detection) was detected for each well using a multifunctional microplate detector, and cells not treated with LNP were used as a negative control group.
The experimental results are shown in FIG. 3.
Analysis of results: as can be seen from fig. 3: LNP prepared by the ionizable lipid with the structural characteristics of the invention is obviously increased along with the increase of time before 40 minutes, and the erythrocyte dissolution rate starts to be stable after 40 minutes; LNP prepared from comparative samples (the pyroxene sample and the compound which does not meet the structural characteristics of the present invention) showed a significant increase in erythrocyte dissolution rate over time before 60 minutes, and began to remain stable after 60 minutes; therefore, the LNP prepared from the ionizable lipid can promote the fusion of endosome membranes in an acidic condition, so that the endosome escape rate is higher, more mRNA with biological activity reaches cytoplasm, and the translation efficiency and the transfection efficiency are higher.
Experiment five: lipid nanoparticle structural morphology characterization experiments
Preparation and characterization of a transmission electron microscope sample (sample H-1 is taken as an example). The prepared sample 15L of 10 mu is dripped on a copper wire, and the sample is sucked dry and dried after being placed for 10 min. Uranium acetate was stained for 5min, and after the stain was blotted with filter paper, it was dried overnight, and the morphology was observed by Transmission Electron Microscopy (TEM).
As shown in FIG. 4, the lipid nanoparticles of the present invention can form stable nanostructures, have a narrow size distribution, and have a size that varies with the structures of different lipid nanoparticles, and have an average particle diameter in the range of 30-200 nm.
Experiment six: biocompatibility experiments
Cell viability was determined using the CCK-8 (cell counting kit-8) kit. Hep3B cells (100. Mu.L, cell density 2X 10) 4 Each mL) was added to a 96-well plate, incubated in a cell culture incubator for 24 hours, then the cell culture broth was removed from each well, and 100. Mu.L of fresh cell culture broth containing LNP with mRNA 20. Mu.g/mL was added, and incubated with cells for 4 hours. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and incubation was continued for 20h. Then, the supernatant was removed, 100. Mu.L of fresh cell culture solution containing CCK-8 working solution (10. Mu.L/mL) was added, incubated for 2 hours, and blank wells were set: adding a cell culture solution containing CCK-8 working solution. Absorbance at 450nm of each well (bubbles cannot appear in the well plate during detection) was detected using a multifunctional microplate detector, and cells not treated with LNP were used as a control group, and their cell viability was set to 100%.
Cell viability (%) = [ A1-A0]/[ A2-A0] ×100;
a1 is absorbance of the dosing group, A0 is absorbance of the blank group, and A2 is absorbance of the control group. The experimental results are shown in table 3.
TABLE 3 Table 3
Experimental results showed that most cells were more than 95% viable with no apparent cytotoxicity at defined LNP concentrations.
Experiment seven: lipid nanoparticle Low temperature storage stability experiment
Taking sample H-3 as an example, lipid nanoparticles prepared according to the formulation were stored at low temperature at 4 ℃ and at different time points (0, 6, 10, 15, 30, 45, 60, 90 days), the particle Size (Size) and PDI of mRNA-LNP (mRNA-entrapped lipid nanoparticles) were characterized using Malvern Zetasizer Nano ZS, and the encapsulation efficiency of mRNA was determined using the Ribogreen RNA quantitative assay kit (Thermo Fisher).
The results show that the LNP formed by the lipid molecules of the invention can be stored for 90 days at low temperature, the particle size, PDI and encapsulation rate are hardly changed, and further the LNP formed by the lipid molecules of the invention is convenient to transport and store and is suitable for industrial production.
Experiment eight: animal test for immune Effect
Material preparation: female Balb/c mice with six weeks of age, 15-20 g of weight, 24 mice are fed in an experimental environment with the temperature of 22+/-2 ℃ and the relative humidity of 45-75%, and the light/dark period is 12 hours. After the mice are purchased and adapted in animal houses for one week, formal animal tests can be carried out. 24 mice were randomly divided into 4 groups, the first group was given an equal volume of PBS (negative control group) by intramuscular injection of the hind legs, the second group was given commercial control samples H1-1 (positive control group 1), 10. Mu.g of mRNA, and a mixture of PBS, the third group was given hind leg intramuscular injection of control samples H1-2 (positive control group 2), 10. Mu.g of mRNA, and a mixture of PBS, and the fourth group was given hind leg intramuscular injection of sample H-3 (test group), 10. Mu.g of mRNA, and a mixture of PBS, which were mRNA expressed full-length Spike synthesized by in vitro transcription based on an autonomously designed template.
The experimental process is as follows: on days 0 and 14, the mRNA-entrapped LNP mixtures were injected intramuscularly into Balb/c mice in the four groups above. Ocular blood collection was performed on days 13 and 21, and after incubation at 37℃for 1 hour, the blood samples were centrifuged at 3500rpm for 15 minutes, and the supernatants were analyzed. The titer of antibodies specific for Delta variant S1 protein from both primary and secondary mouse sera was detected by self-made ELISA kit.
The specific procedure for detecting the titre of Delta variant S1 protein-specific antibodies from both primary and secondary mouse sera was as follows: spike S1 recombinant protein was added to 96-well plates at 0.25. Mu.g per well and left overnight at 4 ℃. The next day, the fluid in the wells was discarded and blocked with 5% BSA in PBST (200 ul) for 1h at 37 ℃. Afterwards, the liquid in the holes is discarded, 200ul of PBST washing liquid is used for washing for 3 times, each time is 3min, and the plate is thrown away for airing. Mouse serum was diluted with PBS (dilution ratio listed as 1:20000) or standards were diluted with PBS to a range of concentrations (stock 1ug/ul, half-diluted, total of 14 standard curves). 100. Mu.L of diluted sample and standard were added to the air and incubated at 37℃for 2h. The liquid in the holes is discarded, 200ul of PBST washing liquid is used for washing for 3 times, each time is 3min, and the plate is thrown off for airing. Goat anti-mouse IgG HRP (PBS 1:5000 dilution) was added at 100ul per well, 37℃for 1h. The liquid in the holes is discarded, 200ul of PBST washing liquid is used for washing for 3 times, each time is 3min, and the plate is thrown off for airing. TMB substrate solution A and solution B were mixed in equal proportions, 100. Mu.L per well, and left at 37℃for several minutes (3-5 mins) protected from light. When absorbance is measured at 650nm and the highest absorbance value is around 1.5, 100ul of stop solution can be added. The absorbance at 450nm was measured within 15min after addition of the stop solution. The IgG content of each group was calculated according to the standard curve formula.
The experimental results are shown in fig. 5, and the results show that: the positive control groups 1 and 2 and the test group can generate antibodies specific to the S1 protein, the antibody titer of the test group is obviously higher than that of the positive control groups 1 and 2, and the test group can efficiently deliver the mRNA into cells to express the antigen so as to excite immune reaction in vivo to generate corresponding antibodies and play a protection function.
In summary, the structure of the novel ionizable lipid compound of the present invention has prominent substantial characteristics, and the above experiments prove that the ionizable lipid compound of the present invention uses an N atom as a charge center, uses a hydrophilic group as a head, uses two hydrophobic groups as tails, introduces- (c=o) O-at the side of the N atom and the two hydrophobic groups, and introduces a carbonate bond as a degradable functional group, so that LNP can promote endosome escape in an intracellular acidic endosome environment; compared with the commercialized product, the escape rate of the endosome is faster, so that the transfection efficiency of the nucleic acid nano-drug is better; further verification shows that: compared with the LNP prepared by the commercial products and the comparison compound with similar structure, the LNP prepared by the compound with the structural characteristics has obviously increased fluorescence intensity, even can reach the difference of 2-3 orders of magnitude, and has very excellent technical effect.
What needs to be explained here is: the ionizable lipid compound is a raw material and a product of medicines, and does not relate to any treatment method or diagnosis method of diseases, and belongs to the scope of patent rights granted. The application range of the present invention is not limited, and the present invention can be applied to the field of vaccines, protein substitution therapy, gene editing, cell therapy, etc., and the examples are not exhaustive, and any ionizable lipid compound employing the structural features of the present invention is within the scope of the present invention.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.

Claims (10)

1. An ionizable lipid compound, said compound having a structure selected from the group consisting of:
2. the ionizable lipid compound of claim 1, wherein said compound has the following structure.
3. A pharmaceutical composition, said pharmaceutical composition comprising: the ionizable lipid compound according to claim 1, a stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical composition according to claim 3, wherein the pharmaceutical composition comprises: a carrier containing the ionizable lipid compound, a carried pharmaceutical agent, a pharmaceutical adjuvant, or a combination thereof.
5. The pharmaceutical composition of claim 4, wherein the carrier further comprises: a co-lipid, a structural lipid, a polymer conjugated lipid, or an amphiphilic block copolymer, or a combination thereof.
6. The pharmaceutical composition of claim 5, wherein the molar ratio of the ionizable lipid compound to the co-lipid is from 0.5:1 to 10:1.
7. The pharmaceutical composition of claim 5, wherein the molar ratio of ionizable lipid compound to structural lipid is from 0.5:1 to 5:1.
8. The pharmaceutical composition of claim 5, wherein the molar ratio of the ionizable lipid compound to the polymer conjugated lipid is from 10:1 to 250:1.
9. The pharmaceutical composition of claim 5, wherein the molar ratio of the ionizable lipid compound to the amphiphilic block copolymer is from 0.5:1 to 80:1.
10. Use of an ionizable lipid compound according to claim 1, a stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition.
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