CN115745788A - Ionizable lipid compound and preparation method and application thereof - Google Patents
Ionizable lipid compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an ionizable cationic lipid compound and a preparation method and application thereof. The compound has a structure shown in formula I; wherein n is an integer of 1 to 5; r 1 is-CH 3 ，‑CH 2 CH 3 ，‑CH 2 CH 2 CH 3 ，‑CH 2 OH，‑CH 2 CH 2 OH，‑CH 2 CH 2 CH 2 OH，‑CH 2 CH 2 CH 2 CH 2 OH or-CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r 3 Is composed of
X is O, N or S heteroatom, N1 is selected from integer of 1-8, m1 is selected from integer of 1-8; r 4 、R 5 、R 6 Each independently being containing or not containing a hetero atomA C10-20 alkyl, alkenyl or alkynyl group of a subgroup. The compounds provided by the invention can be better suitable for delivering bioactive molecules, especially nucleic acid molecules with negative charges, and provide better choices for bioactive molecule delivery and development and application of nucleic acid preventive and therapeutic agents.
Description
Technical Field
The invention belongs to the technical field of medicinal compounds, and particularly relates to an ionizable lipid compound as well as a preparation method and application thereof.
Background
Nucleic acids are important components of living bodies, including DNA species (e.g., antisense oligonucleotides (ASOs), plasmids) and RNA species (small interfering RNAs (sirnas), messenger RNAs (mrnas), micro RNAs (mirnas), and play a key role in life activities.as an example, mRNA is transcribed from DNA, carries corresponding genetic information, and provides a template for further translation into protein.nucleic acids have potential for use in prophylactic/therapeutic vaccines, gene therapy, protein replacement therapy, and other genetic disease therapies, since 2000, the design, modification of nucleic acid molecules, and methods for their delivery have made significant breakthrough advances, siRNA drugs and mRNA vaccines continue to enter clinical applications.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides an ionizable lipid compound, a method for preparing the same, and use thereof. The present invention provides novel ionizable lipid compounds that can be used to deliver biologically active molecules (e.g., DNA, mRNA, siRNA, miRNA, proteins, polypeptides, etc.), and are particularly useful for transporting nucleic acid molecules having negative charges, such as DNA, mRNA, siRNA, etc. Provides more choices for bioactive molecule delivery and the development and application of nucleic acid preventive and therapeutic agents.
The ionizable cationic lipid compound provided by the first aspect of the invention has a structure shown in formula I:
wherein n is an integer of 1 to 5; r 1 is-CH 3 ，-CH 2 CH 3 ，-CH 2 CH 2 CH 3 ，-CH 2 OH， -CH 2 CH 2 OH，-CH 2 CH 2 CH 2 OH，-CH 2 CH 2 CH 2 CH 2 OH or-CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r is 3 Is composed of
X is a heteroatom of O, N or S, N1 is an integer from 1 to 8, and m1 is an integer from 1 to 8; r is 4 、R 5 、R 6 Each independently is a C10-20 alkyl, alkenyl or alkynyl group, with or without heteroatoms.
In a preferred embodiment of the invention, R 4 、R 5 、R 6 The group is a linear or branched C10-20 alkyl, linear or branched C10-20 alkenyl, linear or branched C10-20 alkynyl, said alkyl, alkenyl or alkynyl having 1 or more C atoms optionally replaced by a heteroatom independently selected from O, S and N, provided that R is a heteroatom 3 、
At least one of which is
When R is 3 、
At least two of which are
In the case where n1 and m1 in each of the groups are independent of each other, they may be the same or different.
In a preferred embodiment of the invention, n is 3,R 1 is-CH 3 ，R 2 is-H; preferably, the compounds of the formula I are
Wherein R is 3 The radical is
Wherein X is O, N or S heteroatom, N1 is selected from an integer of 1-8, m1 is selected from an integer of 1-8, N1 and m1 are independent of each other, and can be the same or different; r 4 、R 5 、R 6 Each of which is independently a linear or branched C10-20 alkyl, linear or branched C10-20 alkenyl, linear or branched C10-20 alkynyl, at least 1C atom of said alkyl, alkenyl or alkynyl being optionally replaced by a heteroatom independently selected from O, S or N.
As a preferred embodiment of the present invention, the compound of formula I is
n is 3,R 1 is-CH 3 ，R 2 is-H; wherein R is 3 Is composed of
R 4 、R 5 Is composed of
Preferably, X is an O or N heteroatom; more preferably, n1 is an integer selected from 4 to 8, and m1 is an integer selected from 4 to 8.
In a preferred embodiment of the invention, the ionizable cationic lipid compound is selected from a compound of formula a or a compound of formula B:
wherein n is present on each branch 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers from 4 to 8;
wherein n is present on each branch 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n 3 Are all selected from integers of 4 to 8, each m 3 Are selected from integers from 4 to 8.
R provided by the invention 2 A novel ionizable lipid compound that optimizes the remaining three hydrophobic tails for-H, the specific novel compound having a higher cell transfection efficiency effect than the compounds having the existing four hydrophobic tail molecular structures, which is probably also associated with different configurations/conformations, etc. of the compound; for example, in a slightly acidic lysosomal microenvironment, compounds containing three hydrophobic tails tend to form a tapered molecular structure, which can promote hexagonal turnover of cell membranes and lysosomal escape. And on the basis, the X group is optimized simultaneously, so that the non-liver targeting delivery lipid compound with more excellent specific targeting effect and a delivery system can be provided, and the nucleic acid can be delivered to a target organ more efficiently.
The invention also provides methods for the synthesis of these novel ionizable lipid compounds. The ionizable lipid compounds of the present invention can be synthesized using methods known in the art, for example, by reacting one or more equivalents of amine (hydrophilic polar head containing amine groups) with three or more equivalents of hydrophobic lipid tail compounds under suitable conditions. The synthesis of ionizable lipid compounds is performed with or without solvent and can be performed at higher temperatures in the range of 25-120 ℃. The produced ionizable lipid compound may optionally be purified. For example, a mixture of ionizable lipid compounds can be purified to yield specific ionizable lipid compounds, such as a product containing three hydrophobic lipid tails. The hydrophobic lipid tail chain compounds may be purchased commercially, or prepared synthetically.
In some embodiments of the invention, there is provided a method of preparing the ionizable cationic lipid compound, comprising:
the synthesis route of the compound is as follows:
(wherein n is present in each branch 4 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 4 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably each n 4 Are all selected from integers of 4 to 8, each m 4 Are all selected from integers of 4 to 8)
The method specifically comprises the following steps:
1) Reduction: reducing the carboxyl group of compound A1 to a hydroxyl group in the presence of a reducing agent to obtain compound A2;
2) Esterification: esterifying the hydroxyl group of compound A2 to an ester group in the presence of acryloyl chloride to obtain compound A3;
3) Michael addition: compound A3 is reacted with an amine (e.g., N-bis (3-aminopropyl) methylamine) via michael addition to give the ionizable cationic lipid compound.
In some embodiments of the present invention, there is provided a method for preparing the ionizable cationic lipid compound, comprising:
(wherein n is present in each branch 4 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 4 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably each n 4 Are all selected from integers of 4 to 8, each m 4 Are all selected from integers of 4 to 8)
1) Acyl chlorination: acylating chlorination of the carboxylic group of compound B1 in presence of oxalyl chloride to obtain compound B2;
2) And (3) substitution: converting the acid chloride group of compound B2 to an amide group in the presence of ammonium hydroxide to obtain compound B3;
3) Reduction: reducing the amide of compound B3 to an amine in the presence of a reducing agent to obtain compound B4;
4) Amidation: amidating an amine group of compound B4 to an amide group in the presence of acryloyl chloride to obtain compound B5;
3) Michael addition: reacting compound B5 with an amine via michael addition to give the ionizable cationic lipid compound.
According to the present invention, examples of the reducing agent include, but are not limited to, diisobutylaluminum hydride, lithium aluminum hydride, and the like. Examples of the solvent used for the reaction include, but are not limited to, halogenated hydrocarbons (e.g., chloroform, dichloromethane, dichloroethane, etc.), ethers (e.g., diethyl ether, tetrahydrofuran, etc.), hydrocarbons (e.g., n-pentane, benzene, toluene, etc.), and mixed solvents of two or more of these solvents. Examples of the solvent used for the esterification reaction include, but are not limited to, halogenated hydrocarbons (e.g., chloroform, methylene chloride, dichloroethane, etc.), hydrocarbons (e.g., n-pentane, benzene, toluene, etc.), nitriles (e.g., acetonitrile, etc.), and mixed solvents of two or more of these solvents. The Michael addition reaction may be carried out with or without the use of a solvent, and examples of the solvent for the reaction include, but are not limited to, isopropanol, t-butanol, tetrahydrofuran, and the like. The amine may be N, N-bis (3-aminopropyl) methylamine.
According to the present invention, the raw material A1 in the preparation method may be commercially available or may be synthesized by a conventional method.
The ionizable lipid provided by the invention has a molecular structure containing two adjacent cis-double bonds, so that the ionizable lipid has a high encapsulation rate and a good cell transfection rate when being subsequently applied to a delivery system for encapsulating active substances (such as mRNA); in addition, in the preparation of lipid nanoparticles, the presence of two adjacent cis double bonds in the tail chain may make the particle size of the resulting lipid nanoparticles more uniform. The ionizable lipid compound of the present invention is particularly suitable for the preparation of solid structured nanoparticles.
The invention also provides the use of said ionizable cationic lipid compound for the preparation of a biologically active substance delivery system; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.
In a preferred embodiment of the present invention, when the ionizable cationic lipid compound is a compound of formula a, the use of said ionizable cationic lipid compound for the preparation of a specific spleen-targeting bioactive substance delivery system; use of said ionizable cationic lipid compound for the manufacture of a specific lung targeting bioactive substance delivery system when said ionizable cationic lipid compound is a compound of formula B;
wherein n is present on each branch 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers from 4 to 8;
wherein n is present on each branch 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n 3 Are all selected from integers of 4 to 8, each m 3 Are selected from integers from 4 to 8.
In a preferred embodiment of the invention, the delivery system is a lipid nanoparticle.
In certain embodiments, all amino groups of the amine are fully reacted with the hydrophobic lipid tail compound to form a tertiary amine. In other embodiments, not all of the amino groups of the amine are fully reacted with the hydrophobic lipid tail compound, thereby producing a primary or secondary amine in the ionizable lipid compound. These primary or secondary amines are left as such or can be reacted with another electrophile such as a different hydrophobic lipid tail compound. It is known in the art that reacting an excess of amine with a hydrophobic lipid tail compound will yield a variety of different ionizable lipid compounds with varying numbers of tails. For example, a diamine or polyamine can include one, two, three, or four tail chain compounds on various amino moieties of the molecule, thereby producing primary, secondary, and tertiary amines. In certain embodiments, the same tail chain compound is used; or two of the same type of tail chain compounds may be used. In other embodiments, two or more different tail chain compounds are used.
The invention also provides a bioactive substance delivery system containing the ionizable cationic lipid compound, preferably, the delivery system is a microparticle, a nanoparticle, a liposome, a lipid nanoparticle or a microbubble.
In one embodiment of the invention, the delivery system is a lipid nanoparticle. Such lipid nanoparticles can deliver bioactive substances (such as mRNA) into cells, tissues or organs with high efficiency, enabling efficient regulation of bioactive substances. In the present invention, the ionizable lipid compound is combined with a biologically active substance (e.g., mRNA) targeted for cell or organ delivery or further comprising other substances (e.g., other anionic, cationic or ionizable lipid compounds, synthetic or natural polymers, proteins, phospholipids, cholesterol, carbohydrates, surfactants, etc.) to form microbubbles, liposomes, lipid nanoparticles, or microparticles. The bioactive substance can be in gas, liquid or solid form, and can be protein, polypeptide, small molecule compound or nucleotide. In the present invention, the delivery system may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.
The invention also provides a pharmaceutical composition containing the bioactive substance delivery system.
In another aspect, the present invention also provides a lipid nanoparticle composition comprising a lipid nanoparticle comprising the ionizable cationic lipid compound.
According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The other lipid molecules may be lipid molecules known or conventionally used in the art for the construction of lipid nanoparticles, including but not limited to neutral lipid molecules, lipid-like molecules, cholesterol, pegylated lipid molecules.
According to the present invention, the lipid nanoparticle composition, when used in a drug delivery system, may encapsulate pharmaceutical agents, including nucleotides, small molecule compounds, proteins, polypeptides, metals, and the like. The nucleic acids include, but are not limited to, DNA, antisense nucleic Acids (ASO), small interfering RNA (siRNA), microrna (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamers (aptamer), and the like. The ionizable lipid compound has several properties suitable for the preparation of a drug delivery system: 1) The ability to neutralize the charge on the negatively charged active species; 2) The ability of lipids to complex and "protect" labile agents; 3) The ability to buffer the pH in vivo; 4) The ability to act as a "proton sponge" and cause dissolution in vivo.
According to some preferred embodiments of the present invention, in the lipid nanoparticle composition, the lipid nanoparticle comprises: ionizable cationic lipid compounds of formula I in an amount of 30-60mol% of the total lipid molecules, 5-20mol% of neutral lipid molecules, 30-50mol% of cholesterol lipid molecules, 0.5-5mol% of PEGylated lipid molecules; preferably contains 30-50mol% of ionizable cationic lipid molecules, 8-18mol% of neutral lipid molecules, 35-50mol% of cholesterol lipid molecules, and 0.5-2.5mol% of pegylated lipid molecules; more preferably, the lipid composition comprises 35-48mol% of ionizable cationic lipid molecules of formula I, 9-16mol% of neutral lipid molecules, 36-48mol% of cholesterol lipid molecules, and 1.2-1.8mol% of PEGylated lipid molecules.
According to some preferred embodiments of the invention, the mole percentage of ionizable lipid molecules of formula I in the lipid of the lipid nanoparticle is 30-60mol%, e.g. may be 30mol%,31mol%,32mol%,33 mol%,34mol%,35mol%,36mol%,37mol%,38mol%,39mol%,40 mol%,41mol%,42mol%,43mol%,44mol%,45mol%,46mol%,47 mol%,48mol%,49mol%,50mol%,51mol%, 52mol%,53mol%,54mol%,55mol%,56mol%,57mol%,58mol%, 59mol%,60mol%.
According to some preferred embodiments of the invention, the neutral lipid molecule is an uncharged lipid molecule or a zwitterionic lipid molecule, such as a phosphatidylcholine compound, or/and a phosphatidylethanolamine compound.
According to some preferred embodiments of the invention, the neutral lipid molecule is selected from the group consisting of phosphatidylcholine compounds and/or phosphatidylethanolamine compounds.
According to some preferred embodiments of the present invention, examples of neutral lipid molecules include, but are not limited to Dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), lysophosphatidylethanolamine, distearoylphosphatidylcholine (DSPC), phosphorylcholine (DOPC), 5-heptadecylphenyl-1,3-diol (resorcinol), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoylphosphatidylcholine-sn-glycero-3-phosphorylcholine (DAPC), phosphatidylethanolamine (PE), egg Phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diacylphosphatidylcholine-sn-glycero-sn-phosphatidylcholine (dbxft), lysophosphatidylcholine (spxcol-3425), and combinations thereof.
In one embodiment, the neutral lipid molecule may be selected from the group consisting of: distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and Dioleoylphosphatidylethanolamine (DOPE). In another embodiment, the neutral lipid molecule can be dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule can be Dimyristoylphosphatidylcholine (DMPC).
According to the invention, the molar percentage of neutral lipid molecules in the lipid of the lipid nanoparticle is 5-20mol%, for example 5mol%,6mol%,7mol%,8mol%,9mol%, 10mol%,11mol%,12mol%,13mol%,14mol%,15mol%,16mol%, 17mol%,18mol%,19mol%,20mol%.
According to the present invention, the cholesterol-based lipid molecule includes steroids, sterols, alkylresorcinols and the like, and examples include, but are not limited to, cholesterol hemisuccinate, and 5-heptadecylresorcinol.
According to some preferred embodiments of the invention, the cholesterol-based lipid molecule is selected from one or more of cholesterol, cholesterol hemisuccinate and 5-heptadecylresorcinol.
In one embodiment, the cholesterol-based lipid molecule is Cholesterol (CHOL). In one embodiment, the cholesterol-based lipid molecule is cholesterol hemisuccinate.
According to the invention, the molar percentage of the cholesterol lipid molecules in the lipid of the lipid nanoparticle is 30-50mol%, for example 30mol%,31mol%,32mol%,33 mol%,34mol%,35mol%,36mol%,37mol%,38mol%,39mol%,40 mol%,41mol%,42mol%,43mol%,44mol%,45mol%,46mol%,47 mol%,48mol%,49mol%,50mol%.
According to some preferred embodiments of the invention, the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, expressed as the number average molecular weight of the lipid moiety-PEG, the lipid moiety comprising a diacylglycerol and/or a diacylglycerol amide, preferably selected from one or more of dilauroyl glycerol, dimyristoyl glycerol, dipalmitoyl glycerol, dimyristoyl glycerol amide, dipalmitoyl glycerol amide, dilauroyl glycerol amide, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine; the PEG has a number average molecular weight of 130 to 50,000, preferably 150 to 10,000, more preferably 300 to 3,000, and most preferably 1,500 to 2,500.
According to the present invention, the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety. In some embodiments, the lipid moiety may be derived from diacylglycerols or diacyloleamides (diacylglycamides), including those comprising a dialkylglycerol or dialkylglyceramide group having an alkyl chain length independently comprising from about C4 to about C30 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as an amide or an ester. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglyceroamide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical. As used herein, unless otherwise indicated, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety may be substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the PEG moiety comprises a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J.Milton Harris, poly (ethylene glycol) chemistry: biological and biological applications (1992)); alternatively, the PEG moiety does not include a PEG copolymer, e.g., it can be a PEG homopolymer. In one embodiment, the molecular weight of the PEG is from about 130 to about 50,000, in sub-embodiments from about 150 to about 30,000, in sub-embodiments from about 150 to about 20,000, in sub-embodiments from about 150 to about 15,000, in sub-embodiments from about 150 to about 10,000, in sub-embodiments from about 150 to about 6,000, in sub-embodiments from about 150 to about 5,000, in sub-embodiments from about 150 to about 4,000, in sub-embodiments from about 150 to about 3,000, in sub-embodiments from about 300 to about 3,000, in sub-embodiments from about 1,000 to about 3,000, and in sub-embodiments from about 1,500 to about 2,500. In certain embodiments, the PEG is "PEG 2000", which isThe average molecular weight is about 2,000 daltons. In some embodiments of the invention, the PEG is represented by the formula
Expressed, for PEG-2000 where n is 45, means that the number average degree of polymerization comprises about 45 subunits; other PEG embodiments known in the art may also be used, including, for example, those in which the number average degree of polymerization comprises about 23 subunits (n = 23) and/or 68 subunits (n = 68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R can be an unsubstituted C1-C30 alkyl group, such as a C1-C20 alkyl group, a C1-C10 alkyl group, a C1-C6 alkyl group. In some embodiments, R may be H, methyl or ethyl.
In some embodiments, the pegylated lipid molecule may be represented as a "lipid moiety-PEG-number average molecular weight" or a "PEG-number average molecular weight-lipid moiety" or a "PEG-lipid moiety". The lipid moiety is a diacylglycerol or a diacylglycinamide selected from the group consisting of dilauroyl glycerol, dimyristoyl glycinamide, distearoyl glycerol, dilauryl glycinamide, distearoyl glycinamide, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine; the number average molecular weight of the PEG is from about 130 to about 50,000, e.g., from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, from about 1,500 to about 2,500, e.g., about 2000.
In some embodiments, the pegylated lipid molecule may be selected from PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dilauryl glycerol amide, PEG-dimyristyl glycerol amide, PEG-distearoyl glycerol (PEG-DSPE) 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-ditetradecyloxybenzyl- [ ω ] -methyl-poly (ethylene glycol) ether), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DMG-PEG 2000), 1,2-distearoyl-sn-glycerol-methoxy polyethylene glycol (DSG-PEG 2000), 3245-distearoyl-sn-glycerol-2000 (DMG-2000), polyethylene glycol 3732-di-methoxy-propyl-polyethylene glycol (PEG-2000), PEG-2000, PEG-2000-di-methoxy-2000 (PEG-32), the pegylated lipid molecule can be DMG-PEG2000. In one embodiment, the pegylated lipid molecule can be C-DMA-PEG2000. In one embodiment, the pegylated lipid molecule can be DSA-PEG2000. In one embodiment, the pegylated lipid molecule can be PEG2000-C11. In some embodiments, the pegylated lipid molecule can be DSG-PEG2000. In one embodiment, the pegylated lipid molecule may be DSPE-PEG2000. In one embodiment, the pegylated lipid molecule can be DMA-PEG2000. In some embodiments, the pegylated lipid molecule can be PEG2000-C14. In some embodiments, the pegylated lipid molecule can be PEG2000-C16. In some embodiments, the pegylated lipid molecule can be PEG2000-C18.
According to the present invention, the mole percentage of the pegylated lipid molecule in the lipid of the lipid nanoparticle is 0.5-5mol%, for example may be 0.5mol%,0.6mol%,0.7mol%, 0.8mol%,0.9mol%,1.0mol%,1.1mol%,1.2mol%,1.3mol%,1.4mol%, 1.5mol%,1.6mol%,1.7mol%,1.8mol%,1.9mol%,2.0mol%,2.1mol%, 2.2mol%,2.3mol%,2.4mol%,2.5mol%,2.6mol%,2.7mol%,2.8mol%, 2.9mol%,3.0mol%,3.1mol%,3.2mol%,3.3mol%,3.4mol%,3.5mol%, 3.6mol%,3.7mol%,3.8mol%,3.9mol%,4.0mol%,4.1mol%, 4.4mol%,4.5mol%, 4.4mol%, 4mol%,4.5mol%,4.6mol%, 3.7mol%,3.8mol%,3.9mol%, 4mol%,4.5mol%, etc.
In some embodiments of the present invention, the lipid nanoparticle comprises ionizable cationic lipid molecules represented by formula a, neutral lipid molecules, cholesterol-based lipid molecules, pegylated lipid molecules, wherein:
formula A, wherein each n2 is independent of each other and may be the same or different, each n2 is selected from an integer of 1 to 8, each m2 is independent of each other and may be the same or different, each m2 is selected from an integer of 0 to 8; preferably, each n2 is selected from integers from 4 to 8, each m2 is selected from integers from 4 to 8; preferably, each n2 is the same as each other, and each m2 is the same as each other; the ionizable cationic lipid molecule represented by the formula A accounts for 30-50mol%, preferably 35-48mol% of the lipid in the lipid nanoparticle;
the neutral lipid molecule is selected from phosphatidyl choline compounds and phosphatidyl ethanolamine compounds; the mole percentage of the neutral lipid molecules in the lipid nanoparticle is 8-20mol%, preferably 8-18mol%, and more preferably 9-16mol%;
the cholesterol lipid molecule is selected from cholesterol, cholesterol hemisuccinate; the mole percentage of the cholesterol lipid molecules in the lipid nanoparticle is 30-50mol%, preferably 35-50mol%, and more preferably 36-48mol%;
the pegylated lipid molecule is represented by "lipid moiety-PEG-number average molecular weight", said lipid moiety being a diacylglycerol or a diacylglycerol amide selected from the group consisting of dilauroyl glycerol, dimyristoyl glycerol, distearoyl glycerol, dilauryl glycerol amide, dimyristoyl glycerol amide, distearoyl glycerol amide, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine; PEG has a number average molecular weight of 130 to 50,000, for example 150 to 30,000, 150 to 20,000, 150 to 15,000, 150 to 10,000, 150 to 6,000, 150 to 5,000, 150 to 4,000, 150 to 3,000, 300 to 3,000,1,000 to 3,000,1,500 to 2,500, about 2000; the mole percentage of the PEGylated lipid molecules to the lipid in the lipid nanoparticle is 0.5-5mol%, preferably 0.5-2.5mol%, more preferably 1.2-1.8mol%.
In some embodiments of the invention, the ionizable cationic lipid molecule, the neutral lipid molecule, the cholesterol, and the pegylated lipid molecule represented by formula a are present in a molar ratio of 35.5.
In some embodiments of the invention, the mole ratio of ionizable cationic lipid molecules, neutral lipid molecules, cholesterol, and pegylated lipid molecules represented by formula a is 45.
In some embodiments of the invention, the ionizable cationic lipid molecule, the neutral lipid molecule, the cholesterol, and the pegylated lipid molecule represented by formula a are present in a molar ratio of 40.5.
In one embodiment of the invention, the ionizable cationic lipid molecule of formula A is the compound N34-O18-2 (3T).
In one embodiment of the invention, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DMG-PEG2000.
In one embodiment of the invention, the neutral lipid molecule is DOPE and the pegylated lipid molecule is DMG-PEG2000.
In one embodiment of the invention, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DSPE-PEG2000.
In one embodiment of the invention, the neutral lipid molecule is DOPE and the pegylated lipid molecule is DSPE-PEG2000.
The three hydrophobic tail chain ionizable lipid compounds with adjacent cis-double bond structures provided by the invention can provide higher active substance encapsulation efficiency and better transfection efficiency in cells or in vivo, and are particularly suitable for preparing nanoparticles with solid structures, and in the lipid nanoparticle composition, the ionizable cationic lipid molecules, the neutral lipid molecules, the cholesterol lipid molecules and the PEG lipid molecules in the lipid nanoparticles are most preferably in a molar ratio of the total lipid molecules, and most importantly, the lipid nanoparticles have better functional effects of specifically targeting spleen and/or lung.
The present invention also provides a method of preparing a lipid nanoparticle composition, comprising: the lipid nanoparticles are prepared by dissolving the lipid molecules in an organic solvent in a molar ratio to prepare a solution of mixed lipids, using the solution of mixed lipids as an organic phase, using an aqueous solution of a substance to be delivered (e.g., mRNA) as an aqueous phase, and mixing the organic phase and the aqueous phase. Lipid nanoparticles can be prepared using methods including, but not limited to, spray drying, solvent extraction, phase separation, nano-precipitation, single and double emulsion solvent evaporation, microfluidics, simple and complex coacervation, and others well known to those of ordinary skill in the art.
In some embodiments, the organic solvent is an alcohol, such as ethanol.
In some embodiments, the volume ratio of the organic phase to the aqueous phase is (2-4): 1, e.g., 3:1.
In some embodiments, the nanoparticles are prepared using a microfluidic platform.
According to the present invention, the preparation method further comprises the step of isolating and purifying the lipid nanoparticle.
According to the present invention, the preparation method further comprises a step of lyophilizing the lipid nanoparticle.
The particle size of the lipid nanoparticles of the present invention ranges from 1nm to 1000 nm.
The delivery system formed by the ionizable lipid compound of the present invention can also be modified with targeting molecules that can be targeted to specific cells, tissues or organs as targeting agents. The targeting molecule may be included in the entire delivery system or may be located only on its surface. The targeting molecule can be a protein, small molecule, nucleic acid, polypeptide, glycoprotein, lipid, and the like, examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), sialic acid, aptamers, transferrin (transferrin), asialoglycoprotein (asialycoprotein), receptor ligands, and the like.
The active substance delivered by the delivery system formed by the ionizable lipid compound of the present invention may be a therapeutic, diagnostic or prophylactic agent. The active substance may be in the nature of a nucleic acid, protein, polypeptide, small molecule compound, metal, isotopically-labelled compound, vaccine, etc.
The delivery system formed by the ionizable lipid compound of the present invention may be combined with one or more pharmaceutical excipients to form a pharmaceutical composition suitable for administration to an animal, including a human. The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, or the like, including, but not limited to, cellulose and its derivatives, such as sodium carboxymethylcellulose and cellulose acetate; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; gelatin; talc; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; oils such as peanut oil, cottonseed oil, corn oil, and soybean oil; surfactants such as Tween 80 (Tween 80); coloring agents, sweetening, flavoring and perfuming agents, preserving and anti-oxidants; buffers such as phosphate buffer solution, citrate buffer solution, and the like.
The pharmaceutical compositions of the present invention can be administered to humans and/or animals orally, rectally, intravenously, intramuscularly, intranasally, intraperitoneally, intravaginally, buccally, or in the form of an oral or nasal spray, and the like.
The nucleic acid drug delivery system provided by the invention can efficiently and specifically deliver nucleic acid drug molecules to spleen and/or lung and effectively translate the nucleic acid drug molecules into target molecules, and simultaneously reduce the side effect of liposome accumulation in liver, and has important significance for targeted drug delivery, development and application of nucleic acid drugs.
The terms in the present invention describe: the term "alkyl" refers to a saturated hydrocarbon group derived from a hydrocarbon moiety containing 1 to 30 carbon atoms by the removal of a single hydrogen atom. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, n-dodecyl and the like. The term "alkenyl" denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. The term "alkynyl" refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. "and/or" will be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, use of the term "and/or" in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone) and "B" (alone). "comprising" and "comprises" have the same meaning, are intended to be open-ended and allow, but do not require, the inclusion of additional elements or steps. When the term "comprising" or "including" is used herein, the term "consisting of and/or" consisting essentially of … … "is therefore also included and disclosed. "about": the term "about" used in connection with a numerical value denotes an interval of accuracy familiar and acceptable to a person skilled in the art. Typically, this accuracy is in the range of ± 10%.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a hydrogen spectrum of compound (9Z, 12Z) -9,12-octadecadien-1-ol (linoleol, a 2) in an example of the present invention;
FIG. 2 is a chart of the hydrogen spectrum of compound (9Z, 12Z) -9,12-dienoctadecanoacrylate (a 3) in the examples of the present invention;
FIG. 3 is a hydrogen spectrum of compound N34-O18-2 (3T) in the example of the present invention;
FIG. 4 is a mass spectrum of compound N34-O18-2 (3T) in the example of the present invention;
FIG. 5 is a hydrogen spectrum of compound N34-O18-2 (4T) in the example of the present invention;
FIG. 6 is a mass spectrum of compound N34-O18-2 (4T) in the example of the present invention;
FIG. 7 is a hydrogen spectrum of compound N34-N18-2 (3T) in the example of the present invention;
FIG. 8 is a mass spectrum of compound N34-N18-2 (3T) in the example of the present invention;
FIG. 9 is a hydrogen spectrum of compound N34-N18-2 (4T) in the examples of the present invention;
FIG. 10 is a mass spectrum of compound N34-N18-2 (4T) in the present example;
FIG. 11 is a graph showing dissociation constants (pKa) of compounds N34-O18-2 (3T) (A), N34-O18-2 (4T) (B) in examples of the present invention;
FIG. 12 is a graph showing the expression level of Luciferase protein 24h after transfection of 293T cells with LNP-encapsulated Luciferase mRNA (LucRNA) prepared from N34-O18-2 (3T), N34-O18-2 (4T) in example of the present invention;
FIG. 13 is a graph showing the expression levels of Luciferase protein 24h after transfection of 293T cells with LNP encapsulated Luciferase DNA (pDNA) prepared from N34-O18-2 (3T) in accordance with the present invention;
FIG. 14 is a graph showing the expression level of Luciferase protein 24h after transfection of 293T cells with LNP encapsulated Luciferase siRNA (siRNA) prepared from N34-O18-2 (3T) in example of the present invention;
FIG. 15 is a graph showing the expression level of Luciferase protein 24h after 293T cells were transfected with LNP-encapsulated Luciferase RNA (LucRNA) prepared by N34-O18-2 (3T), ALC-0315 in example of the present invention;
FIG. 16 is a graph of cytotoxicity of N34-O18-2 (3T) -LNP and ALC-0315-LNP against 293T in an example of the present invention;
FIG. 17 shows fluorescence expression of organs of mice injected with N34-O18-2 (3T) -LucRNA for 6 h;
FIG. 18 shows fluorescence expression of organs of mice after intravenous injection of N34-N18-2 (3T) -LucRNA for 6h in the present example.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
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. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The specific techniques or conditions not indicated in the examples are all conventional methods or techniques or conditions described in the literature of the field or according to the product specifications. The reagents and instruments used are conventional products which are not indicated by manufacturers and are available from normal distributors.
EXAMPLE 1 Synthesis of ionizable lipid N34-O18-2 (3T), N34-O18-2 (4T)
Synthesis of (9Z, 12Z) -9,12-octadecadien-1-ol (linoleol, a 2): liAlH was added to 950mL of tetrahydrofuran at 0 deg.C 4 (7.0 g), linoleic acid (50g, a 1), then the mixture was stirred at 25 ℃ for 2h. After completion of the reaction as shown by Thin Layer Chromatography (TLC), the reaction mixture was quenched by adding water (8.0 mL), aqueous NaOH (8.0 mL, 15% by mass) and water (25 mL) in this order, and adding Na as appropriate 2 SO 4 After stirring for 15 minutes, filtration through a Buchner funnel and washing of the filter cake with ethyl acetate, the filtrate was collected and concentrated by evaporation to give 51g, 100% yield of the desired product, linoleol (a 2), the hydrogen spectrum of compound a2 is shown in FIG. 1.
1 H NMR(400MHz,Chloroform-d)δ5.47-5.26(m,4H),3.64(t,J＝ 6.6Hz,2H),2.77(t,J＝6.5Hz,2H),2.08-2.01(m,4H),1.57(p,J＝6.6 Hz,2H),1.39-1.25(m,16H),0.89(t,J＝6.7Hz,3H).
Synthesis of (9Z, 12Z) -9,12-dienoctadecanoacrylate (a 3): to 30.0mL of methylene chloride were added (9Z, 12Z) -9,12-octadecadien-1-ol (3.2 g), and triethylamine (3.64 g) at 0 deg.C, and then a solution of acryloyl chloride (1.65 g) in methylene chloride (10.0 mL) was added dropwise to the reaction system, and the reaction solution was stirred at 20 deg.C for 2h. And filtering triethylamine salt separated out from the reaction solution by using a Buchner funnel, collecting filtrate, washing the filtrate by using water, hydrochloric acid with the mass fraction of 5% and water in sequence, and drying an organic phase by using magnesium sulfate. The residue was purified by flash column chromatography eluting with EtOAc/petroleum ether (0% -60%) to give the desired product (9Z, 12Z) -9,12-dienoctadecanoacrylate (2.7 g) in 70% yield and the hydrogen spectrum of compound a3 is shown in FIG. 2.
1 H NMR(400MHz,Chloroform-d)δ6.40(dd,J＝17.3,1.5Hz, 1H),6.12(dd,J＝17.4,10.4Hz,1H),5.81(dd,J＝10.4,1.5Hz,1H), 5.32-5.40(m,4H),4.15(t,J＝6.7Hz,2H),2.77(t,J＝6.5Hz,2H),2.05 (q,J＝6.9Hz,4H),1.67(p,J＝6.8Hz,2H),1.38-1.27(m,16H),0.89(t, J＝6.7Hz,3H).
Synthesis of N34-O18-2 (3T), N34-O18-2 (4T): to 320mg of N, N-bis (3-aminopropyl) methylamine solution was added (9Z, 12Z) -9,12-dienoctadecanoacrylate (2.5 g) at room temperature, after which the mixture was heated to 120 ℃ and stirring was continued for 48h. When the TLC plate detection reaction was completed, 2.65g of crude product was obtained, and then the objective product was purified by elution with methylene chloride/methanol by flash column chromatography to obtain 45mg of N34-O18-2 (3T), 66mg of N34-O18-2 (4T), a hydrogen spectrum of the compound N34-O18-2 (3T) shown in FIG. 3, a mass spectrum shown in FIG. 4, and a hydrogen spectrum of the compound N34-O18-2 (4T) shown in FIG. 5, and a mass spectrum shown in FIG. 6.
N34-O18-2(3T)：
1 H NMR(400MHz,Chloroform-d)δ5.45-5.26(m,12H),4.12-3.98 (m,6H),2.89(t,J＝6.5Hz,2H),2.79-2.74(m,10H),2.68(s,2H),2.54 (s,2H),2.47-2.28(m,10H),2.21(s,3H),2.05(q,J＝6.8Hz,12H), 1.74-1.54(m,10H),1.40-1.25(m,48H),0.89(t,J＝6.7Hz,9H).
MALDI-TOFMS：m/z 1107.030[M+H] + .
N34-O18-2(4T)：
1 H NMR(400MHz,Chloroform-d)δ5.42-5.29(m,16H),4.04(t,J ＝6.8Hz,8H),2.76(q,J＝7.5,7.0Hz,16H),2.44-2.41(m,J＝7.1Hz, 12H),2.29–2.16(m,5H),2.05(q,J＝6.8Hz,16H),1.63-1.60(m,12H), 1.38-1.26(m,66H),0.89(t,J＝6.7Hz,12H).
MALDI-TOFMS：m/z 1427.310[M+H] + .
Example 2 Synthesis of ionizable lipid N34-N18-2 (3T), N34-N18-2 (4T)
Synthesis of N34-N18-2 (3T), N34-N18-2 (4T): to 400mg of N, N-bis (3-aminopropyl) methylamine solution was added (9Z, 12Z) -9,12-dienoctadecanoacrylamide (3.08 g) at room temperature, after which the mixture was heated to 120 ℃ and stirring was continued for 48h. When the TLC plate detection reaction is finished, 3.10g of crude product is obtained, and then the target product is purified by a flash column chromatography method using dichloromethane/methanol for elution, so that 100mg of N34-N18-2 (3T), 120mg of N34-N18-2 (4T) and the hydrogen spectrum of the compound N34-N18-2 (3T) are shown in figure 7, and the mass spectrum is shown in figure 8; the hydrogen spectrum of compound N34-N18-2 (4T) is shown in FIG. 9, and the mass spectrum is shown in FIG. 10.
N34-N18-2(3T)：
1 H NMR (400MHz,Chloroform-d)δ7.11(br,3H),5.50-5.23(m, 12H),3.28-3.14(m,6H),2.99(s,3H),2.77(t,J＝6.5Hz,6H),2.70(t,J ＝6.3Hz,4H),2.50(s,5H),2.37(d,J＝6.4Hz,6H),2.24(s,3H),2.05 (q,J＝7.4,6.7Hz,12H),1.79(s,2H),1.63-1.40(m,10H),1.36-1.24(m, 48H),0.92-0.86(m,9H).
MALDI-TOFMS：m/z 1104.177[M+H] + .
N34-N18-2(4T)：
1 H NMR(400MHz,Chloroform-d)δ7.14(br,3H),5.40-5.29(m, 16H),3.18(q,J＝6.8Hz,8H),2.77(t,J＝6.5Hz,8H),2.68(t,J＝6.1 Hz,8H),2.47(t,J＝6.5Hz,4H),2.35(t,J＝6.0Hz,8H),2.05(q,J＝7.0 Hz,16H),1.70(s,3H),1.49(q,J＝7.2Hz,8H),1.37-1.25(m,72H),0.89 (t,J＝6.7Hz,12H).
MALDI-TOFMS：m/z 1423.553[M+H] + .
Example 3 dissociation constants (pKa) of ionizable lipids N34-O18-2 (3T), N34-O18-2 (4T)
Ionizable lipids have two main roles: binding nucleic acids and allowing the release of nucleic acid molecules in the cell. The pKa of the lipid is an important factor because lipids need to be positively charged at low pH to bind to nucleic acids, but are uncharged at neutral pH, so LNPs do not cause toxicity. As shown in FIG. 11, the ionizable lipid N34-O18-2 (3T) has a pKa of 6.72 (A) and N34-O18-2 (4T) of 5.92 (B), as determined by TNS dye binding assay. It can be seen that both molecules are positively charged and RNA-loaded under acidic conditions and uncharged at neutral pH (pH = 7.4)
Example 4N34-O18-2 (3T), N34-O18-2 (4T) encapsulation of mRNA to prepare lipid nanoparticles
Ionizable lipid N34-O18-2 (3T) or N34-O18-2 (4T), DSPC, cholesterol and DMG-PEG2000 were prepared in a molar ratio of 45%:15%:38.5%:1.5% ethanol solution as an organic phase and LuciferaemRNA (LucRNA) in pH =4 aqueous solution as an aqueous phase, respectively. According to the volume ratio of the water phase to the organic phase of 3:1, the nanoparticle suspension is prepared by the micro-fluidic technology on a nano-drug manufacturing instrument (Mianan). And after the preparation, performing ultrafiltration concentration to obtain the final LucRNA-LNP lipid nanoparticle, and storing at 2-8 ℃ for later use.
Characterization of the LucRNA-LNP particle size and Zeta potential was performed using a Zetasizer Pro nanometer particle size potentiometer (Malverapaneceae). The encapsulation efficiency of LucRNA-LNP was determined by F-280 fluorescence spectrophotometer (Tianjin Hongkong) Ribogreen. The results of the test in example 2 are shown in table 1.
TABLE 1 test results
From the results of example 4, it can be seen that the particle size of the lipid nanoparticle lucRNA-LNP prepared from the novel lipid compound N34-O18-2 (3T) is about 120nm, the particle size distribution of lucRNA-LNP is narrow (PDI is small), and the entrapment rate is as high as 98%.
In addition, the transfection efficiency of the prepared LucRNA-LNP cells was examined by the fluorescein reporter method using a multifunctional microplate reader (BioTek, model SLXFATS). The method for in vitro transcription of LucRNA is as follows: 293T cells plated at 1X 10 cell density 4 Individual cells/well, when the degree of cell confluence is 30% -50% were transfected. 1.0. Mu.g of LucRNA was transfected using the transfection reagent Lipofectamine2000 (ThermoFisher Scientific) and the transfection procedure was performed according to the transfection reagent product instructions. And detecting the protein expression level by using a multifunctional microplate reader 24h after transfection. The negative control is cell culture medium without addition of LucRNA-LNP. In vitro cell transfection efficiency as shown in fig. 12, LNP-coated mRNA prepared from ionizable lipid N34-O18-2 (3T) has extremely high cell transfection efficiency, which is much lower than that of LNP-coated mRNA prepared from ionizable lipid N34-O18-2 (4T) having four hydrophobic tails, which is conventionally used, and has about an order of magnitude of reduction in transfection efficiency. Compared with the conventional commonly used ionizable lipid N34-O18-2 (4T) containing four hydrophobic tail chains, the ionizable lipid N34-O18-2 (3T) containing three hydrophobic tail chains has higher cell transfection efficiency and obvious advantages.
From the results of example 4, it can also be seen that the lipid nanoparticle LucRNA-LNP prepared from the novel lipid compound N34-O18-2 (3T) has better physicochemical characteristics and the in vitro cell transfection efficiency is about 3-5 times higher than that of the commercial Lipofectamine 2000.
Example 5 preparation of lipid nanoparticles by DNA-encapsulated in N34-O18-2 (3T)
An ethanol solution is prepared as an organic phase by ionizable lipid N34-O18-2 (3T), DSPC, cholesterol and DMG-PEG2000 according to molar ratio of 45%:15%:38.5%:1.5%, and LuciferaseDNA (pDNA) is dissolved in an aqueous solution with pH =4 to be used as an aqueous phase. The nanoparticle suspension was prepared by microfluidics on a nano-drug manufacturing apparatus (PNI, canada, ignite model) according to a volume ratio of aqueous phase to organic phase of 3:1. After the preparation, the pDNA-LNP lipid nanoparticles are obtained by ultrafiltration and concentration, and are preserved at the temperature of 2-8 ℃ for later use.
Characterization of pDNA-LNP particle size and Zeta potential was performed using a Zetasizer Pro nanometer particle size potentiometer (Malverapanecae). The results of the measurement in example 5 are shown in Table 2, and the particle size of the lipid nanoparticle pDNA-LNP prepared by compounding the novel lipid compound is about 303nm, and the particle size distribution of pDNA-LNP is narrow (PDI is small).
TABLE 2 test results
The transfection efficiency of the prepared pDNA-LNP 293T cells was examined by the multifunctional microplate reader (BioTek, model SLXFATS) fluorescein reporter gene method. The in vitro transcription method is as follows: 293T cells plated at a cell density of 1X 10 4 Individual cells/well, when the degree of cell confluence is 30% -50% were transfected. 2 μ g of pDNA was transfected with the transfection reagent Lipofectamine2000 (ThermoFisher Scientific) and the transfection procedure was performed according to the transfection reagent product instructions. And detecting the protein expression level by using a multifunctional microplate reader 24h after transfection. The negative control was cell culture medium without pDNA-LNP addition. In vitro cell transfection efficiency as shown in FIG. 13, LNP-coated DNA prepared from ionizable lipid N34-O18-2 (3T) was shown to have high cell transfection efficiency.
From the results of example 5, it can be seen that lipid nanoparticles pDNA-LNP prepared from the novel lipid compound have good physicochemical characteristics and high in vitro cell transfection efficiency.
Example 6N34-O18-2 (3T) encapsulation of siRNA for lipid nanoparticles
An ethanol solution is prepared as an organic phase by ionizable lipid N34-O18-2 (3T), DSPC, cholesterol and DMG-PEG2000 according to a molar ratio of 45% to 15% to 38.5% to 1.5%, and Luciferase siRNA (siRNA) is dissolved in an aqueous solution with pH =4 to be used as an aqueous phase. The nanoparticle suspension was prepared by microfluidics on a nano-drug manufacturing apparatus (PNI, canada, ignite model) according to a volume ratio of aqueous phase to organic phase of 3:1. After the preparation, the final siRNA-LNP lipid nano-particles are obtained by ultrafiltration and concentration and are stored at the temperature of 2-8 ℃ for standby.
Characterization of siRNA-LNP particle size and Zeta potential was performed using a Zetasizer Pro nanometer particle size potentiometer (Malverapaceae). The results of the test of example 6 are shown in Table 3. The particle size of the lipid nanoparticle siRNA-LNP prepared by the compatibility of the novel lipid compound is about 225 nm.
TABLE 3 test results
The transfection efficiency of the prepared siRNA-LNP 293T cells was determined by the multifunctional microplate reader (BioTek, model SLXFATS) fluorescein reporter gene method. The in vitro transcription method is as follows: stably transfected 293T cells reported by Luciferase were plated at a cell density of 1X 10 4 Individual cells/well, when the degree of cell confluence is 30% -50% were transfected. siRNA was transfected using the transfection reagent Lipofectamine2000 (ThermoFisher Scientific) and transfection procedures were performed according to the transfection reagent product instructions. And detecting the protein expression amount by using a multifunctional microplate reader 24h after transfection. The negative control is cell culture medium without siRNA-LNP. In vitro cell transfection efficiency as shown in fig. 14, LNP-encapsulated sirnas prepared from the ionizable lipid N34-O18-2 (3T) were shown to have extremely high protein knockdown efficiency.
From the results of example 6, it can be seen that the particle size of lipid nanoparticle siRNA-LNP prepared from the novel lipid compound is about 225 nm. In vitro cell transfection and knockdown efficiencies were higher than commercial Lipofectamine 2000.
Example 7 comparison of the Effect of N34-O18-2 (3T) and the commercially available ionizable cationic lipid molecule ALC-0315
The molecular formula of ALC-0315 is: ((4-hydroxybutyl) azadialkyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate).
The structural formula of ALC-0315 is:
according to the method described in example 4, the lipid nanoparticles were prepared by using N34-O18-2 (3T) and ALC-0315, respectively, at specific molar ratios: N34-O18-2 (3T) DSPC: cholesterol: DMG-PEG2000= 45; ALC-0315: dspc: cholestrol: DMG-PEG2000= 45.
The physicochemical and quality control data of the prepared lipid nanoparticles are shown in the following table (table 4):
TABLE 4 lipid nanoparticle physicochemical and quality control data
As can be seen from the above table, the encapsulation rate of the lipid nanoparticles prepared from N34-O18-2 (3T) is as high as 98.7%, compared with that of the lipid nanoparticles prepared from ALC-0315.
After the prepared lipid nanoparticles were transfected into cells by the same transfection method as in example 4, and protein expression was known, the results are shown in fig. 15, and the protein expression level in the cells after the lipid nanoparticles prepared from N34-O18-2 (3T) carried mRNA and transfected into the cells was higher than that of Lipofectamine2000, and the protein expression level in the cells transfected by the corresponding mRNA concentration was also higher than that of ALC-0315, which indicates that the cell transfection efficiency of the lipid nanoparticles prepared from N34-O18-2 (3T) was very high.
In addition, the cytotoxicity of N34-O18-2 (3T) -LNP and ALC-0315-LNP is determined by MTT method, and the influence of factors such as vector dosage and action time on the cell proliferation of normal cells (such as 293T) is examined. As shown in FIG. 16, the lipid nanoparticles prepared from N34-O18-2 (3T) still maintained good cell activity at higher dose (2 μ g/mL) after transfection of cells with mRNA for 48 hours, indicating that the lipid nanoparticles prepared from N34-O18-2 (3T) had low cytotoxicity.
From the results of example 7, it can be seen that the lipid nanoparticles prepared from the novel lipid compounds have low cytotoxicity and high mRNA transfection efficiency.
Example 8 transfection of N34-O18-2 (3T) -LNP, N34-N18-2 (3T) -LNP lipid nanoparticles in animals
Lipid nanoparticles were prepared by tail vein injection of nano-lipid particle mice according to the method described in example 3, using N34-O18-2 (3T) or N34-N18-2 (3T) in the following specific molar ratios: N34-O18-2 (3T) DSPC: cholesterol: DMG-PEG2000= 45; N/P ratio of 10, 1, N34-N18-2 (3T) DSPC: cholesterol: DMG-PEG2000= 45; the N/P ratio is 10. Wherein the mRNA is mRNA for expressing Luciferase fluorescent protein, the dosage of the mRNA is 10 mu g, the total amount of N34-O18-2 (3T) or N34-N18-2 (3T), DSPC, cholesterol and DMG-PEG2000 is 100 mu g, 200 mu L of neutral PBS buffer solution is adopted to rapidly convert liposome environment, and the liposome is rapidly injected into female C57 mice with 6-8 weeks through tail veins, and the intravenous injection of 10 mu g of the mRNA is controlled.
The fluorescence expression of each organ after 6h of mice injected by tail vein with N34-O18-2 (3T) -LNP, PBS (blank control) is shown in FIG. 17. The results showed that the fluorescence expression in the organs of mice after intravenous Injection of (IV) lipid nanoparticles of N34-O18-2 (3T) -LNP was mainly distributed in spleen of about 100%, heart 0%, liver 0%, lung 0%, kidney 0%, which was seen to be specifically targeted to the spleen. The fluorescence expression of each organ after 6h of mice injected by tail vein with N34-N18-2 (3T) -LNP, PBS (blank control) is shown in FIG. 18. The results showed that the fluorescence expression in the organs of mice after intravenous Injection of (IV) lipid nanoparticles of N34-N18-2 (3T) -LNP was mainly distributed in spleen 0%, heart 0%, liver 0%, lung 100%, kidney 0%, which was found to specifically target lung. Therefore, the invention forms an isomeric compound based on three lipid hydrophobic tail chains, and further realizes specific targeting of different organs by adjusting X heteroatom in the lipid hydrophobic tail chain to be O or N.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. An ionizable cationic lipid compound having a structure represented by formula I:
wherein n is an integer of 1 to 5; r 1 is-CH 3 ，-CH 2 CH 3 ，-CH 2 CH 2 CH 3 ，-CH 2 OH，-CH 2 CH 2 OH，-CH 2 CH 2 CH 2 OH，-CH 2 CH 2 CH 2 CH 2 OH or-CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r 3 Is composed of
X is a heteroatom of O, N or S, N1 is an integer from 1 to 8, and m1 is an integer from 1 to 8; r is 4 、R 5 、R 6 Each independently is a C10-20 alkyl, alkenyl or alkynyl group, with or without heteroatoms.
2. The ionizable cationic lipid compound of claim 1, wherein n is 3,R 1 is-CH 3 ，R 2 is-H; preferably, the compounds of the formula I are
Wherein R is 3 The radical is
Wherein X is a heteroatom of O, N or S, N1 is selected from an integer of 1 to 8, m1 is selected from an integer of 1 to 8, and N1 and m1 are independent of each other and may be the same or different; r is 4 、R 5 、R 6 Each of which is independently a linear or branched C10-20 alkyl, linear or branched C10-20 alkenyl, linear or branched C10-20 alkynyl, at least 1C atom of said alkyl, alkenyl or alkynyl being optionally replaced by a heteroatom independently selected from O, S or N.
3. The ionizable cationic lipid compound of claim 2, wherein said compound of formula I is
n is 3,R 1 is-CH 3 ，R 2 is-H; wherein R is 3 Is composed of
R 4 、R 5 Is composed of
Preferably, X is an O or N heteroatom; more preferably, n1 is an integer selected from 4 to 8, and m1 is an integer selected from 4 to 8.
4. The ionizable cationic lipid compound of any of claims 1-3, wherein said ionizable cationic lipid compound is selected from a compound of formula A or a compound of formula B:
wherein n is present on each branch 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 2 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n is 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers from 4 to 8;
wherein n is present on each branch 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8, m 3 Each independently selected from the group consisting of integers of 1 to 8, preferably integers of 4 to 8; preferably, each n 3 Are all selected from integers of 4 to 8, each m 3 Are selected from integers from 4 to 8.
5. The process for the preparation of an ionizable cationic lipid compound of any of claims 1 to 4, comprising the steps of:
1) Reduction: reducing the carboxyl group of compound A1 to a hydroxyl group in the presence of a reducing agent to obtain compound A2;
2) Esterification: esterifying the hydroxyl group of compound A2 to an ester group in the presence of acryloyl chloride to obtain compound A3;
3) Michael addition: reacting compound A3 with an amine via michael addition to give the ionizable cationic lipid compound; or:
1) Acyl chlorination: acylating chlorination of the carboxy group of compound B1 in the presence of oxalyl chloride to obtain compound B2;
2) And (3) substitution: converting the acid chloride group of compound B2 to an amide group in the presence of ammonium hydroxide to obtain compound B3;
3) Reduction: reducing the amide of compound B3 to an amine in the presence of a reducing agent to obtain compound B4;
4) Amidation: amidating an amine group of compound B4 to an amide group in the presence of acryloyl chloride to obtain compound B5;
3) Michael addition: reacting compound B5 with an amine via michael addition to give the ionizable cationic lipid compound.
6. Use of the ionizable cationic lipid compound of any of claims 1 to 4 for the preparation of a biologically active substance delivery system; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.
7. A bioactive substance delivery system comprising the ionizable cationic lipid compound of any one of claims 1 to 4; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.
8. A pharmaceutical composition comprising the bioactive substance delivery system of claim 7.
9. A lipid nanoparticle composition comprising lipid nanoparticles comprising the ionizable cationic lipid compound according to any one of claims 1 to 4; preferably, in the lipid nanoparticle composition, the lipid nanoparticle contains: ionizable cationic lipid compounds of formula I in an amount of 30-60mol%, neutral lipid molecules in an amount of 5-20mol%, cholesterol lipid molecules in an amount of 30-50mol%, PEGylated lipid molecules in an amount of 0.5-5mol% based on the total lipid molecules;
preferably contains 30-50mol% of ionizable cationic lipid molecules, 8-18mol% of neutral lipid molecules, 35-50mol% of cholesterol lipid molecules, and 0.5-2.5mol% of pegylated lipid molecules;
more preferably, the lipid composition comprises 35-48mol% of ionizable cationic lipid molecules of formula I, 9-16mol% of neutral lipid molecules, 36-48mol% of cholesterol lipid molecules, and 1.2-1.8mol% of PEGylated lipid molecules.
10. The lipid nanoparticle composition of claim 9, wherein the neutral lipid molecule is selected from the group consisting of phosphatidylcholine compounds and/or phosphatidylethanolamine compounds; and/or the presence of a gas in the gas,
the cholesterol lipid molecule is selected from one or more of cholesterol, cholesterol hemisuccinate and 5-heptadecyl resorcinol; and/or the presence of a gas in the gas,
the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, expressed as the number average molecular weight of the lipid moiety-PEG, the lipid moiety comprising diacylglycerol and/or diacylglycerol amide, preferably selected from one or more of dilauroyl glycerol, dimyristoyl glycerol, dipalmitoyl glycerol, dimyristoyl glycerol amide, dipalmitoyl glycerol amide, dilauryl glycerol amide, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine; the PEG has a number average molecular weight of 130 to 50,000, preferably 150 to 10,000, more preferably 300 to 3,000, and most preferably 1,500 to 2,500.
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