CN116143707A - Base ionizable lipid and preparation method and application thereof - Google Patents

Base ionizable lipid and preparation method and application thereof Download PDF

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CN116143707A
CN116143707A CN202310184973.4A CN202310184973A CN116143707A CN 116143707 A CN116143707 A CN 116143707A CN 202310184973 A CN202310184973 A CN 202310184973A CN 116143707 A CN116143707 A CN 116143707A
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lipid
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姜新义
孙唯一
赵坤
付志鹏
赵晓天
荆卫强
韩茂森
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Abstract

The invention provides a base ionizable lipid, a preparation method and application thereof. The structure of the base ionizable lipid is shown as a formula (I). The method for preparing the base ionizable lipid has the advantages of low-cost and easily-obtained raw materials, simple steps, mild reaction conditions, easy realization and convenient and fast product separation. The prepared base ionizable lipid can effectively deliver nucleic acid drugs, and has high transfection efficiency and high biosafety.

Description

Base ionizable lipid and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a base-ionizable lipid, and a preparation method and application thereof.
Background
The gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate diseases caused by gene defects or abnormal gene expression, and the treated subjects are gradually expanded from single genetic diseases to malignant tumors, infectious diseases, cardiovascular diseases, autoimmune diseases, metabolic diseases and other serious diseases, so that the gene therapy has wide application prospect. However, nucleic acids are relatively sensitive to nucleases, they are degraded into small molecule nucleotides before entering target cells, losing therapeutic effect, and the larger molecular weight and negative charge characteristics of nucleic acid drugs prevent them from entering target cells. Therefore, the development of safe and effective nucleic acid delivery vehicles to improve the stability of nucleic acid drugs and the ability to penetrate cell membranes is critical to the potential of gene therapy applications.
Currently, the more common nucleic acid delivery vectors are largely divided into two categories, viral vectors and non-viral vectors. The virus vector has the advantages of early development and high transfection efficiency, but has the problems of low safety, off-target effect and the like. Most of the non-viral vectors are synthesized artificially, have various types and more controllable structural properties, and comprise ionizable liposome, cationic polymer, nano particles and the like, wherein the ionizable liposome is most widely applied. Under acidic conditions, the ionizable lipid can be protonated to obtain positive charges, and the negative charges of the ionizable lipid are combined with the nucleic acid drug through electrostatic action, so that the nucleic acid drug is protected from nuclease degradation, and meanwhile, negative charges of the nucleic acid drug are masked to facilitate the uptake of the nucleic acid drug by target cells, so that the transfection efficiency is remarkably improved. In addition, under the physiological pH condition, the ionizable lipid is electrically neutral, so that cytotoxicity is remarkably reduced, and huge clinical transformation potential is shown.
However, the number of ionizable lipids currently on the market is small, and further screening and optimization are required, severely restricting the development of nucleic acid pharmaceutical formulations and gene therapy. Therefore, development of ionizable lipid compounds having both high transfection efficiency and high biosafety is urgent.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a base ionizable lipid, and a preparation method and application thereof. The method for preparing the base ionizable lipid has the advantages of low-cost and easily-obtained raw materials, simple steps, mild reaction conditions, easy realization and convenient and fast product separation. The prepared base ionizable lipid can effectively deliver nucleic acid drugs, and has high transfection efficiency and high biosafety.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a base-ionizable lipid having a structure represented by formula (I);
Figure BDA0004103405110000021
wherein, the Base is a Base group, and the Base group is a purine Base or a pyrimidine Base; r is R 1 、R 2 Independently selected from substituted or unsubstituted C 8-24 Alkyl, or, substituted or unsubstituted C 8-24 Alkenyl, or, substituted or unsubstituted C 8-24 Alkynyl; r is R 3 Is substituted or unsubstituted C 1-6 Alkyl, or hydrogen; x is oxygen or nitrogen; l (L) 1 、L 2 Independently selected from substituted or unsubstituted C 1-2 Alkyl, or-CH 2 CH 2 COO-, or-CH 2 CH 2 CONH-, or-CH 2 CH 2 OCO-, or-CH 2 CH 2 NHCO-; n is a positive integer from 1 to 8; m selectingA positive integer from 1 to 8.
According to the invention, in formula (I), the base group is an adenine (A) group, a guanine (G) group, a cytosine (C) group, a thymine (T) group or a uracil (U) group. Preferably, in formula (I), the base group is a thymine (T) group or a uracil (U) group.
According to the invention, R in formula (I) 1 、R 2 Independently selected from C 8-24 An alkyl group; r is R 3 Is hydrogen; x is nitrogen; l (L) 1 、L 2 Independently selected from C 1-2 Alkyl, n is 1, m is 1; preferably, in formula (I), R 1 、R 2 Independently selected from C 8-12 Alkyl, L 1 、L 2 Is ethyl.
Preferably, the base ionizable lipid is selected from one of the following compounds:
Figure BDA0004103405110000031
in a second aspect, the present invention provides a method for preparing a base-ionizable lipid according to the first aspect of the present invention, comprising the steps of: in an organic solvent, under the action of a catalyst, base carboxylic acid (II) and organic amine (III) react to obtain base ionizable lipid;
Figure BDA0004103405110000032
wherein in the formulas (II), (III), base, n and R 1 、R 2 、R 3 、L 1 、L 2 X and m have the same meaning as in the compounds of formula (I).
According to the invention, the base carboxylic acids (II) and the organic amines (III) are preferably commercially available or are prepared by the known methods.
Preferably, when R 1 、R 2 Independently selected from C 8-24 Alkyl, R 3 Is hydrogen, X is nitrogen, L 1 、L 2 Independently selected from C 1-2 Alkyl, when m is 1, organic amineThe preparation method of (III) comprises the following steps: in acetonitrile, under the action of potassium carbonate, N-tert-butoxycarbonyl-1, 2-ethylenediamine and 1-bromoalkane react to obtain an intermediate 1; under the action of 1, 4-dioxane solution of hydrochloric acid, the intermediate 1 is reacted to obtain an intermediate 2, namely organic amine (III).
Further preferably, the volume ratio of the molar quantity of the N-t-butoxycarbonyl-1, 2-ethylenediamine to acetonitrile is 0.01-1mol/L; the molar ratio of the potassium carbonate to the N-tert-butyloxycarbonyl-1, 2-ethylenediamine is 1-3:1; the mol ratio of the N-tert-butyloxycarbonyl-1, 2-ethylenediamine to the 1-bromoalkane is 1:2-2.5; the reaction temperature of the N-tert-butoxycarbonyl-1, 2-ethylenediamine and 1-bromoalkane is 60-100 ℃ and the reaction time is 60-80h; the volume ratio of the mol quantity of the intermediate 1 to the 1, 4-dioxane solution of the hydrochloric acid is 0.1-10mol/L; the concentration of the 1, 4-dioxane solution of the hydrochloric acid is 1-5mol/L; the reaction temperature of the intermediate 1 is room temperature, and the reaction time is 1-5h.
Preferably, according to the present invention, the organic solvent is selected from one or more of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, methylene chloride, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, N-dimethylformamide or triethanolamine; the molar amount of the base carboxylic acid (II) and the volume ratio of the organic solvent are 0.01-10mol/L.
Preferably according to the present invention, the catalyst is selected from one or a combination of two or more of N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), O-benzotriazol-tetramethylurea Hexafluorophosphate (HBTU) or O-benzotriazol-N, N' -tetramethylurea tetrafluoroboric acid (TBTU); the molar ratio of the catalyst to the base carboxylic acid (II) is 2-3:1.
According to the invention, the molar ratio of base carboxylic acid (II) to organic amine (III) is preferably from 1:1 to 1.1.
According to the invention, the reaction temperature is preferably room temperature and the reaction time is from 10 to 30 hours.
According to the present invention, a method for post-treating a reaction solution obtained by reacting a base carboxylic acid (II) with an organic amine (III) preferably comprises the steps of: adding saturated NaCl aqueous solution into the reaction solution, extracting with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, filtering, evaporating to dryness under reduced pressure, and separating by silica gel column chromatography to obtain base ionizable lipid; the eluent used in the silica gel column chromatographic separation is a mixed solution of methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 1:50.
In a third aspect the present invention provides the use of a base ionizable lipid according to the first aspect of the invention in a drug delivery vehicle.
Preferably, according to the present invention, the drug comprises one or a combination of two or more of a biological drug or a chemical drug; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; further preferred, the nucleic acid agent comprises one or more than two of Small interfering RNA (Small interfering RNA; siRNA), messenger RNA (mRNA), microRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotides (Antisense Oligonucleotides, ASOs), small activating RNA (saRNA) or Aptamer (Aptamer); the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer. Most preferably, the drug is Messenger RNA (mRNA).
In a fourth aspect, the present invention provides a lipid nanoparticle comprising a base ionizable lipid according to the first aspect of the invention, said lipid nanoparticle comprising: base ionizable lipids, helper lipids, sterols, PEG lipids, and drugs.
According to a preferred embodiment of the present invention, the auxiliary lipid is selected from one or a combination of two or more of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), diethyl pyrocarbonate (DEPC), phosphatidylcholine (POPC) or dimyristoyl phosphatidylcholine (DMPC); preferably, the helper lipid is dioleoyl phosphatidylethanolamine (DOPE).
According to a preferred embodiment of the invention, the sterol is cholesterol.
According to the invention, preferably, the PEG lipid is selected from one or more than two of DSPC-PEG, DMG-PEG, DPPE-PEG or DMA-PEG; preferably, the PEG lipid is DMG-PEG.
According to the preferred invention, the molar ratio of base ionizable lipid, helper lipid, sterol, and PEG lipid is 20-50:10-40:30-60:0.5-10; the mass ratio of the base ionizable lipid to the medicine is 1-100:1.
According to the present invention, the lipid nanoparticle preferably has a diameter in the range of 1nm to 1000 nm. For example, the particle diameter is in the range of 20nm to 800nm, or in the range of 50nm to 500nm, or in the range of 80nm to 200nm, or in the range of 1nm to 100nm, or in the range of 1nm to 10 nm. When the diameter of the lipid nanoparticle is in the range of 1nm to 1000nm, it is a nanoparticle generally described in the art.
According to the present invention, the lipid nanoparticle may be prepared using any method known in the art. These methods include, but are not limited to, liposome extrusion, thin film hydration, nano-precipitation, microfluidic, and impingement jet mixing, as well as other methods known to those of ordinary skill in the art. Preferably, the preparation method of the lipid nanoparticle comprises the steps of: dissolving base ionizable lipid, auxiliary lipid, sterol and PEG lipid in ethanol to obtain lipid ethanol solution; fully dispersing the medicine in potassium hydrogen phthalate-sodium hydroxide buffer solution with pH less than 5 to obtain medicine solution; rapidly mixing the lipid ethanol solution and the drug solution by utilizing micro-flow control to prepare a solution containing lipid nanoparticles; then removing ethanol through dialysis to obtain lipid nanoparticles; the concentration of the base ionizable lipid in the lipid ethanol solution is 5-150mg/mL; the concentration of the drug solution is 1-1000 ng/. Mu.L. The above-mentioned ethanol removal by dialysis may further comprise a drying step such as lyophilization or the like to obtain lipid nanoparticles.
Preferably, according to the present invention, the drug comprises one or a combination of two or more of a biological drug or a chemical drug; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; further preferred, the nucleic acid agent comprises one or more than two of Small interfering RNA (Small interfering RNA; siRNA), messenger RNA (mRNA), microRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotides (Antisense Oligonucleotides, ASOs), small activating RNA (saRNA) or Aptamer (Aptamer); the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer. Most preferably, the drug is Messenger RNA (mRNA).
According to the invention, the targeting molecule can be further modified in the lipid nanoparticle to have a targeting function so as to target specific cells, tissues or organs. The targeting molecule may be in the whole lipid nanoparticle or may be located only on the surface of the lipid nanoparticle. The targeting molecule may be a protein, peptide, glycoprotein, lipid, small molecule, nucleic acid, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), transferrin (transferrin), asialoglycoprotein (asialoglycoprotein), receptor ligands, sialic acids, aptamers, etc.
The lipid nanoparticle of the present invention can be used for the prevention and treatment of various diseases of human and/or animals by oral, rectal, intravenous, intramuscular, intravaginal, intranasal, intradermal, intraperitoneal, buccal, or in the form of oral or nasal spray, etc.
In a fifth aspect the present invention provides the use of a base ionizable lipid according to the first aspect of the invention and a lipid nanoparticle according to the fourth aspect for in vitro construction of an engineered cell; the engineered cell comprises one of a T cell, NK cell or macrophage.
In a sixth aspect, the invention provides the use of a base ionizable lipid according to the first aspect of the invention, a lipid nanoparticle according to the fourth aspect, and an engineered cell according to the fifth aspect for preventing, treating or alleviating a disease.
The invention has the technical characteristics and beneficial effects that:
1. the invention provides a base-ionizable lipid compound which comprises a hydrophilic base head part and a hydrophobic fatty chain tail part, has amphipathy and can form a liposome structure in a water phase. The charge of the compound can be changed along with the change of pH, the compound can be ionized into cations under the acidic condition, and the cations are combined with negatively charged nucleic acid molecules through electrostatic action, so that the base heads of the lipid compound can be further combined with nucleic acid through hydrogen bonding and pi-pi stacking action, and nucleic acid drugs can be effectively delivered into cells. The lipid compound has amide bond, can be rapidly hydrolyzed by enzyme in vivo, is easy to be metabolically cleared, and has biodegradability.
2. The synthesis of the base ionizable lipid compound provided by the invention is based on common base carboxylic acid and organic amine compounds, and is prepared through a simple reaction, the raw materials are low in price and easy to obtain, the synthesis route is scientific, the method is simple in steps, the reaction condition is mild, the realization is easy, and the product separation is convenient.
3. In the lipid nanoparticle formula provided by the invention, the structure and the proportion of the ionizable lipid influence the lysosome escape capacity of the nucleic acid; the auxiliary lipid and the ionizable lipid form a hexagonal phase tissue form, and the selection of different phospholipids and the proportion of the ionizable lipid also influence the transfection efficiency of nucleic acid; in order to realize efficient nucleic acid transfection, the molar ratio of the base ionizable lipid, the auxiliary lipid, the sterol and the PEG lipid is 20-50:10-40:30-60:0.5-10; the mass ratio of the base ionizable lipid to the medicine is 1-100:1.
4. The base-ionizable lipid compound provided by the invention solves the problem in nucleic acid delivery, can effectively deliver nucleic acid drugs, can realize efficient transfection of nucleic acid in vitro and in vivo, has the transfection effect equivalent to that of a commercial transfection reagent, has high biological safety, and can be widely applied to efficient delivery of nucleic acid drugs such as mRNA and the like, and the development of the nucleic acid drugs is promoted.
Term interpretation:
alkyl "refers to saturated aliphatic hydrocarbon groups, including straight chainsAnd branched alkyl groups. In some embodiments, the alkyl group has 1-6 carbons, also known as C 1-6 An alkyl group. In some embodiments, the alkyl group has 8 to 24 carbons, also known as C 8-24 An alkyl group. The alkyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
Alkenyl "refers to unsaturated aliphatic hydrocarbon groups, including straight chain and branched alkenyl groups. In some embodiments, the alkenyl group has 8 to 24 carbons, also known as C 8-24 Alkenyl groups. Alkenyl groups include, for example, ethenyl, propenyl, n-butenyl, isobutenyl, and the like. The alkenyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
Alkynyl "refers to unsaturated aliphatic hydrocarbon groups, including straight-chain and branched alkynyl groups. In some embodiments, alkynyl groups have 8 to 24 carbons, also known as C 8-24 Alkynyl groups. Alkynyl groups include, for example, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. The alkynyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy, thio and thioalkyl.
Substitution "means that one or more hydrogen atoms in the group are substituted independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) the possible substitutions without undue effort.
Drawings
FIG. 1 shows the synthetic routes of base-ionizable lipids of examples 4 to 8 of the present invention.
FIG. 2 shows the encapsulation efficiency of lipid nanoparticles in example 9 of the present invention.
FIG. 3 is a graph showing the particle diameter and Zeta potential characteristics of lipid nanoparticles in example 9 of the present invention.
FIG. 4 is a transmission electron microscope characterization of lipid nanoparticles in example 9 of the present invention.
FIG. 5 is an evaluation of transfection efficiency of lipid nanoparticles in test example 1 of the present invention.
Detailed Description
The invention is further described below in connection with examples, which are not intended to limit the scope of the invention.
The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.
Example 1
Intermediate 2a, N 1 ,N 1 Synthesis of Didecyl-1, 2-ethylenediamine
Into a 250mL round bottom flask equipped with a magneton, t-butoxycarbonyl-1, 2-ethylenediamine (10 mmol), 1-bromodecane (22 mmol), potassium carbonate (20 mmol) and acetonitrile (60 mL) were added and heated under reflux at 80℃for 72h. The reaction solution was filtered by suction, the solid was discarded, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by column chromatography (eluent: methanol: dichloromethane volume ratio=1:20), to obtain intermediate 1a, yield 75%.
Intermediate 1a (5 mmol) was dissolved in 1, 4-dioxane (10 mL), and 4mol/L hydrochloric acid 1, 4-dioxane solution (12.5 mL) was added and stirred at room temperature for 2h. The reaction mixture was dried under reduced pressure, extracted with dichloromethane (3X 30 mL), and the organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was removed using a rotary evaporator to give intermediate 2a in 98% yield.
Example 2
Intermediate 2b, N 1 ,N 1 The synthesis of didodecyl ethane-1, 2-diamine is as described in example 1, with the difference that: 1-bromodecane was replaced with 1-bromododecane (22 mmol); other steps and conditions were consistent with example 1.
The single step yield of intermediate 1b was 72% and that of intermediate 2b was 94%.
Example 3
Intermediate 2c, N 1 ,N 1 The synthesis of ditetradecyl ethane-1, 2-diamine is as described in example 1, with the difference that: 1-bromodecane was replaced with 1-bromotetradecane (22 mmol); other steps and conditions were consistent with example 1.
The single step yield of intermediate 1c was 83% and that of intermediate 2c was 95%.
Example 4
Preparation of N- (2- (didecylamino) ethyl) -2- (2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 1)
2- (2, 4-Dioxopyrimidin-1-yl) acetic acid (5 mmol) was dissolved in N, N-dimethylformamide (45 mL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (5.5 mmol) and N-hydroxysuccinimide (5.5 mmol) were added to intermediate 2a (5 mmol) prepared in example 1, and stirred overnight (12 h) at room temperature. The reaction mixture was added with an appropriate amount of saturated aqueous NaCl, extracted with ethyl acetate (3×60 mL), the organic phase was dried over anhydrous sodium sulfate and filtered, evaporated to dryness under reduced pressure, and the residual mixture was separated by column chromatography (eluent: methanol: dichloromethane volume ratio=1:50) to give N- (2- (didecylamino) ethyl) -2- (2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (compound 1) (yellow solid, yield 54%). Product nuclear magnetic resonance data were as follows: 1 H NMR(400MHz,CDCl 3 )δ6.75(s,1H),5.67(d,J＝7.9Hz,1H),4.26(s,2H),3.27(q,J＝5.5Hz,2H),2.52(t,J＝5.9Hz,2H),2.39(t,J＝7.7Hz,4H),1.20(s,32H),0.81(t,J＝6.7Hz,6H)。
example 5
Preparation of 2- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -N- (2- (bistetradecylamino) ethyl) acetamide (compound 2), as described in example 4, except that: intermediate 2a (5 mmol) prepared in example 1 was replaced by intermediate 2c (5 mmol) prepared in example 3; other steps and conditions were consistent with example 4. To give 2- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -N- (2- (bistetradecylamino) ethyl) acetamide (Compound 2) (yellow solid,yield 43%). The nuclear magnetic data of the product are as follows: 1 H NMR(400MHz,CDCl 3 )δ8.45(s,1H),5.64(d,J＝7.9Hz,1H),4.43(s,2H),3.60(q,J＝5.3Hz,2H),3.08(t,J＝5.3Hz,2H),2.94(t,J＝8.4Hz,4H),1.19(s,48H),0.81(t,J＝6.7Hz,6H)。
example 6
Preparation of N- (2- (didecylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 3) as described in example 4, except that 2- (2, 4-dioxopyrimidin-1-yl) acetic acid (5 mmol) was replaced with thymine-1-acetic acid (5 mmol), and the other steps and conditions were the same as in example 4. N- (2- (didecylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 3) (yellow oily liquid, yield 50%). Product was obtained as follows: 1 H NMR(400MHz,CDCl 3 )δ8.24(s,1H),7.01(d,J＝1.4Hz,1H),4.37(s,2H),3.55(q,J＝5.7Hz,2H),3.01(t,J＝5.5Hz,2H),2.86(t,J＝8.3Hz,4H),1.84(s,3H),1.24–1.13(m,32H),0.81(t,J＝6.7Hz,6H).
example 7
Preparation of N- (2- (didodecylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 4) as described in example 4, except that 2- (2, 4-dioxopyrimidin-1-yl) acetic acid (5 mmol) was replaced with thymine-1-acetic acid (5 mmol), intermediate 2a (5 mmol) prepared in example 1 was replaced with intermediate 2b (5 mmol) prepared in example 2, and the other steps and conditions were the same as in example 4. N- (2- (didodecylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 4) (yellow oily liquid, yield 33%). Nuclear magnetic data of the product were as follows: 1 H NMR(400MHz,CDCl 3 )δ8.27–8.22(m,1H),7.09(d,J＝1.5Hz,1H),4.43(s,2H),3.61(q,J＝5.5Hz,2H),3.06(t,J＝5.5Hz,2H),3.00–2.87(m,4H),1.92(s,3H),1.37–1.20(m,40H),0.88(t,J＝6.7Hz,6H)。
example 8
Preparation of N- (2- (Bitetradecylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 5) as described in example 4, except that 2- (2, 4-Di)Oxo-pyrimidin-1-yl) acetic acid (5 mmol) was replaced with thymine-1-acetic acid (5 mmol), intermediate 2a (5 mmol) prepared in example 1 was replaced with intermediate 2c (5 mmol) prepared in example 3; other steps and conditions were consistent with example 4. N- (2- (Bistyristoylamino) ethyl) -2- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H) -acetamide (Compound 5) (yellow oily liquid, yield 41%) the nuclear magnetic data of the product are as follows: 1 H NMR(400MHz,CDCl 3 )δ7.87(s,1H),7.00(d,J＝1.5Hz,1H),4.33(s,2H),3.47(q,J＝5.4Hz,2H),2.87(d,J＝6.4Hz,2H),2.74(t,J＝8.2Hz,4H),1.85(s,3H),1.19(s,48H),0.81(t,J＝6.7Hz,6H)。
example 9
Preparation and characterization of lipid nanoparticles
The base ionizable lipid prepared in example 7, cholesterol, DMG-PEG, DOPE were dissolved in ethanol at a molar ratio of 33:44:1:22 to prepare a lipid ethanol solution (wherein the concentration of base ionizable lipid was 10 mg/mL). mRNA was dissolved in potassium hydrogen phthalate-sodium hydroxide buffer at pH=4 to give mRNA solution (10 ng/. Mu.L). Lipid ethanol solution and mRNA solution were base-ionizable lipids using nanoAsssembrmicrofluidic device (Precision Nanosystem Co.): and (3) rapidly mixing the mRNA in a weight ratio of 10:1 to prepare a solution containing the lipid nanoparticles.
Encapsulation efficiency was determined using a Quant-iT RiboGreen RNA Assay Kit RNA quantitative detection kit. As shown in fig. 2, the encapsulation efficiency was 89.68%, and the lipid nanoparticle composed of the base-ionizable lipid can effectively encapsulate mRNA.
After removing ethanol by dialysis of the solution containing the lipid nanoparticles, the lipid nanoparticles were characterized using dynamic light scattering and transmission electron microscopy. As shown in fig. 3 and 4, the lipid nanoparticle has a uniformly dispersed spherical structure, the particle size is about 110nm, and the surface charge is close to electric neutrality.
Test example 1
Lipid nanoparticle in vitro delivery mRNA performance test
Preparation of lipid nanoparticles: the base-ionizable lipids (compounds 1 to 4), cholesterol, DMG-PEG, DOPE prepared in examples 4 to 7 were dissolved in ethanol at a molar ratio of 33:44:1:22 to prepare a lipid ethanol solution (wherein the molar concentration of the base-ionizable lipid was 10 mg/mL). mRNA of Green Fluorescent Protein (GFP) was dissolved in potassium hydrogen phthalate-sodium hydroxide buffer at pH=4 to give mRNA solution (10 ng/. Mu.L). Lipid ethanol solution and mRNA solution were base-ionizable lipids using nanoAsssembrmicrofluidic device (Precision NanoSystems Co.): rapidly mixing the mRNA in a weight ratio of 10:1 to prepare a solution containing lipid nanoparticles; and (3) dialyzing the solution containing the lipid nanoparticles to remove ethanol, thereby obtaining the lipid nanoparticles. Lipid nanoparticles prepared from the base-ionizable lipids prepared in examples 4-7 were designated LNP-1, LNP-2, LNP-3, LNP-4 in order.
Transfection efficiency of lipid nanoparticles in Hep 3B cell lines was evaluated:
hep 3B cells in logarithmic growth phase were taken at 1X 10 4 Density of wells/wells were inoculated in 24-well plates with DMEM medium, incubated overnight at 37℃with fresh medium containing lipid nanoparticles (300 ng of mRNA) added to each well, control group added the same volume of PBS, incubated at 37℃for 12h, and the transfection efficiencies were examined by flow cytometry and were 62.36%, 73.27%, 72.75% and 80.68% for LNP-1, LNP-2, LNP-3 and LNP-4, respectively, as shown in FIG. 5.

Claims (10)

1. A base-ionizable lipid, characterized in that the structure of the base-ionizable lipid is represented by formula (i);
Figure FDA0004103405100000011
wherein, the Base is a Base group, and the Base group is a purine Base or a pyrimidine Base; r is R 1 、R 2 Independently selected from substituted or unsubstituted C 8-24 Alkyl, or, substituted or unsubstituted C 8-24 Alkenyl, or, substituted or unsubstituted C 8-24 Alkynyl; r is R 3 Is substituted or unsubstituted C 1-6 Alkyl, or hydrogen; x is oxygen or nitrogen; l (L) 1 、L 2 Independently selected from substituted or unsubstitutedSubstituted C 1-2 Alkyl, or-CH 2 CH 2 COO-, or-CH 2 CH 2 CONH-, or-CH 2 CH 2 OCO-, or-CH 2 CH 2 NHCO-; n is a positive integer from 1 to 8; m is selected from positive integers of 1-8.
2. The base ionizable lipid of claim 1, wherein in formula (i), the base group is an adenine (a) group, a guanine (G) group, a cytosine (C) group, a thymine (T) group, or a uracil (U) group; preferably, in formula (I), the base group is a thymine (T) group or a uracil (U) group.
3. The base ionizable lipid of claim 2, wherein in formula (i), R 1 、R 2 Independently selected from C 8-24 An alkyl group; r is R 3 Is hydrogen; x is nitrogen; l (L) 1 、L 2 Independently selected from C 1-2 Alkyl, n is 1, m is 1; preferably, in formula (I), R 1 、R 2 Independently selected from C 8-12 Alkyl, L 1 、L 2 Is ethyl.
4. A base ionizable lipid according to claim 3, characterized in that the base ionizable lipid is selected from one of the following compounds:
Figure FDA0004103405100000012
Figure FDA0004103405100000021
5. the method for producing a base-ionizable lipid according to any one of claims 1 to 4, comprising the steps of: in an organic solvent, under the action of a catalyst, base carboxylic acid (II) and organic amine (III) react to obtain base ionizable lipid;
Figure FDA0004103405100000022
wherein in the formulas (II), (III), base, n and R 1 、R 2 、R 3 、L 1 、L 2 X and m have the same meaning as in the compounds of formula (I).
6. The method of preparing a base ionizable lipid according to claim 5, comprising one or more of the following conditions:
i. the organic solvent is selected from one or more than two of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, N-dimethylformamide or triethanolamine; the volume ratio of the molar quantity of the base carboxylic acid (II) to the organic solvent is 0.01-10mol/L;
ii. The catalyst is selected from one or more than two of N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), O-benzotriazole-tetramethylurea Hexafluorophosphate (HBTU) or O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid (TBTU); the molar ratio of the catalyst to the base carboxylic acid (II) is 2-3:1;
iii, the molar ratio of the base carboxylic acid (II) to the organic amine (III) is 1:1-1.1;
iv, the reaction temperature is room temperature, and the reaction time is 10-30h;
the post-treatment method of the reaction liquid obtained by the reaction of v, the base carboxylic acid (II) and the organic amine (III) comprises the following steps: adding saturated NaCl aqueous solution into the reaction solution, extracting with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, filtering, evaporating to dryness under reduced pressure, and separating by silica gel column chromatography to obtain base ionizable lipid; the eluent used for chromatographic separation of the silica gel column is a mixed solution of methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 1:50.
7. Use of a base ionizable lipid according to any one of claims 1-4 in a drug delivery vehicle;
preferably, the drug comprises one or a combination of two or more of a biological drug or a chemical drug; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; further preferred, the nucleic acid agent comprises one or more than two of Small interfering RNA (Small interfering RNA; siRNA), messenger RNA (mRNA), microRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotides (Antisense Oligonucleotides, ASOs), small activating RNA (saRNA) or Aptamer (Aptamer); the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer; most preferably, the drug is Messenger RNA (mRNA).
8. A lipid nanoparticle comprising the base-ionizable lipid of any one of claims 1-4, said lipid nanoparticle comprising: base ionizable lipids, helper lipids, sterols, PEG lipids, and drugs;
preferably, the auxiliary lipid is selected from one or more of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), diethyl pyrocarbonate (DEPC), phosphatidylcholine (POPC) or dimyristoyl phosphatidylcholine (DMPC); further preferably, the helper lipid is dioleoyl phosphatidylethanolamine (DOPE);
preferably, the sterol is cholesterol;
preferably, the PEG lipid is selected from one or more than two of DSPC-PEG, DMG-PEG, DPPE-PEG or DMA-PEG; further preferably, the PEG lipid is DMG-PEG;
preferably, the molar ratio of the base ionizable lipid, the auxiliary lipid, the sterol and the PEG lipid is 20-50:10-40:30-60:0.5-10; the mass ratio of the base ionizable lipid to the medicine is 1-100:1;
preferably, the lipid nanoparticle has a diameter in the range of 1nm to 1000 nm;
preferably, the preparation method of the lipid nanoparticle comprises the steps of: dissolving base ionizable lipid, auxiliary lipid, sterol and PEG lipid in ethanol to obtain lipid ethanol solution; fully dispersing the medicine in potassium hydrogen phthalate-sodium hydroxide buffer solution with pH less than 5 to obtain medicine solution; rapidly mixing the lipid ethanol solution and the drug solution by utilizing micro-flow control to prepare a solution containing lipid nanoparticles; then removing ethanol through dialysis to obtain lipid nanoparticle solution; the concentration of the base ionizable lipid in the lipid ethanol solution is 5-150mg/mL; the concentration of the drug solution is 1-1000 ng/. Mu.L.
9. Use of the base ionizable lipid of any one of claims 1-4 and the lipid nanoparticle of claim 8 for in vitro construction of an engineered cell; the engineered cell comprises one of a T cell, NK cell or macrophage.
10. Use of a base ionizable lipid according to any one of claims 1-4, a lipid nanoparticle according to claim 8 and an engineered cell according to claim 9 for preventing, treating or alleviating a disease.
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