CN117534585A - Novel ionizable cationic lipid compound, and preparation method and application thereof - Google Patents

Novel ionizable cationic lipid compound, and preparation method and application thereof Download PDF

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CN117534585A
CN117534585A CN202311260962.6A CN202311260962A CN117534585A CN 117534585 A CN117534585 A CN 117534585A CN 202311260962 A CN202311260962 A CN 202311260962A CN 117534585 A CN117534585 A CN 117534585A
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lipid
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integers
ionizable cationic
cationic lipid
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林耀新
苏林嘉
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National Center for Nanosccience and Technology China
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Abstract

The invention provides a novel ionizable cationic lipid compound, and a preparation method and application thereof. Has a structure shown in a formula I; wherein n is an integer of 2 to 4; r is R 1 is-CH 3 ，‑CH 2 CH 3 ，‑CH 2 CH 2 CH 3 ，‑CH 2 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，‑CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r is R 3 、R 4 、R 5 Is thatOr (b)X is O, S or N heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10; r is R 6 Is a C6-25 alkyl, alkenyl or alkynyl group with or without heteroatoms. The novel compound provided by the invention has better nucleic acid encapsulation efficiency and cell or in-vivo transfection efficiency, and the lipid nanoparticle has the function of specifically targeting the liver, spleen and/or lung.

Description

Novel ionizable cationic lipid compound, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicinal compounds, and particularly relates to a novel ionizable cationic lipid compound, and a preparation method and application thereof.
Background
Nucleic acids are important components of life and play a critical role in life activities. Nucleic acid-based nanomedicine is a method of treating or preventing diseases by means of nucleic acid molecules, including DNA-like and RNA-like. Wherein the DNA class includes, for example, plasmids and antisense oligonucleotides (ASOs) and the like; the RNAs include, for example, small interfering RNAs (sirnas), endogenous microRNA (miRNA), messenger RNAs (mrnas), clustered regularly interspaced short palindromic repeats and their nuclease 9 (CRISPR/Cas 9), and RNA aptamers, among others. It can be achieved by suppression, addition, substitution or editing of genes. Benefit from long-term research in the fields of nucleic acid biology, nucleic acid therapy, and nucleic acid-based nanomedicine delivery systems. These nucleic acid-based therapies have been widely developed and used, such as mRNA-based vaccines, mRNA-1273 (Moderna) and BNT162b2 (Pfizer/BioNTech) for use in the prevention of coronavirus (covd-19) in 2019. However, there are a number of problems in nucleic acid therapeutic applications today, for example, mRNA is a negatively charged hydrophilic macromolecule, whose large molecular weight and large negative charge make it difficult to permeate the cell membrane; mRNA is enzymatically labile and its single-stranded structure is extremely susceptible to degradation by RNase. Therefore, how to efficiently deliver mRNA to a target organ or target cell is a key technology to achieve its in vivo application. Thus, there is an urgent need to develop low toxicity, high efficiency, organ/tissue cell targeted delivery systems, particularly ionizable cationic lipid compounds for non-liver targeted delivery and related methods and compositions to facilitate extracellular or intracellular delivery of various therapeutic or prophylactic agents for therapeutic and/or prophylactic purposes.
Disclosure of Invention
In order to solve the technical problems, the invention provides a novel ionizable cationic lipid compound, and a preparation method and application thereof. The present invention provides novel ionizable cationic lipid compounds that are useful for delivering biologically active molecules (e.g., DNA, siRNA, miRNA, mRNA, polypeptides, proteins, etc.), particularly useful for transporting nucleic acid molecules having a negative charge, such as DNA, siRNA, mRNA, etc. Providing more options for delivery of bioactive molecules and development and use of nucleic acid prophylactic and therapeutic agents.
The novel ionizable cationic lipid compound provided by the first aspect of the invention has a structure shown in a formula I:
wherein n is an integer of 2 to 4; r is R 1 is-CH 3 ，-CH 2 CH 3 ，-CH 2 CH 2 CH 3 ，-CH 2 CH 2 CH 3 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，-CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r is R 3 、R 4 、R 5 Is thatX is O, S or N heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10; r is R 6 Is a C6-25 alkyl, alkenyl or alkynyl group with or without heteroatoms. The novel ionizable cationic lipid compound of the invention has three hydrophobic tail structures, and compared with the traditional compounds with four hydrophobic tail structures, the novel ionizable cationic lipid compound can improve the encapsulation rate of nucleic acid on space structure, and has better cell or in vivo transfection efficiencyThe steric space structure of the ionizable cationic lipid compound is provided with an inherent rule for promoting the transfection efficiency, and the steric space structure of the ionizable cationic lipid compound can be regulated by changing the unsaturation degree, the heteroatom, the branched chain structure or the length of the hydrophobic tail chain of the steric space structure, so that the lipid nanoparticle has the function of specifically targeting the liver, the spleen and/or the lung.
In a preferred embodiment of the invention, R 3 、R 4 、R 5 Is that X is O, S or N heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10, N1 and m1 are independent from each other and can be the same or different; r is R 6 Is a linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, a linear or branched C6-25 alkynyl group, at least 1C atom of said alkyl, alkenyl or alkynyl group being optionally replaced by a heteroatom independently selected from O, S or N.
In a preferred embodiment of the invention, n is 2, R 1 is-CH 3 ，R 2 is-H; preferably, the compound of formula I isWherein R is 3 、R 4 、R 5 Is->X is O, S or N heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10, N1 and m1 are independent from each other and can be the same or different; r is R 6 Is a linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, a linear or branched C6-25 alkynyl group, at least 1C atom of said alkyl, alkenyl or alkynyl group being optionally replaced by a heteroatom independently selected from O, N, S, S-S or Se.
As some preferred embodiments of the present invention, the compounds of formula I aren is 2, R 1 is-CH 3 ，R 2 is-H; wherein R is 3 、R 4 、R 5 Is->R 6 A linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, at least 1C atom of said alkyl, alkenyl group being optionally replaced by a heteroatom independently selected from O, S or N; preferably, X is an O or N heteroatom; more preferably, n1 is an integer selected from 4 to 8, m1 is an integer selected from 4 to 8, R 6 Is a linear or branched C6-18 alkyl group, a linear or branched C6-18 alkenyl group, at least 1C atom of said alkyl, alkenyl groups being optionally replaced by a heteroatom independently selected from S or S-S.
In a preferred embodiment of the present invention, the novel ionizable cationic lipid compound is selected from a compound of formula a, a compound of formula B, a compound of formula C, or a compound of formula D:
wherein n on each branch 2 Each independently selected from integers of 1 to 8, preferably 4 to 8, m 2 Each Y is independently selected from integers of 1 to 8, preferably 4 to 8, and each Y is independently selected from integers of 0 to 3; preferably, each n 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers of 4-8, and each Y is independently selected from integers of 0-2.
Wherein n on each branch 3 Each independently selected from integers from 1 to 18, preferably from 6 to 18; preferably, each n 3 Are all selected from integers of 6 to 18.
Wherein n on each branch 4 Each independently selected from integers from 1 to 18, preferably integers from 2 to 18; preferably, each n 4 Are all selected from integers from 2 to 18.
Wherein n on each branch 5 Each independently selected from integers of 1 to 18, preferably 4 to 18, m 5 Each independently selected from integers from 1 to 18, preferably from 1 to 14; preferably, each n 5 Are all selected from integers of 4 to 18, each m 5 Are all selected from integers of 1 to 14.
R provided by the invention 2 Novel ionizable lipid compounds that are-H while optimizing the remaining three hydrophobic tails, the particular novel compounds having a higher cell transfection efficiency effect than existing compounds of four hydrophobic tail molecular structures, perhaps also associated with the different configurations/conformations, steric structures, etc. that the compounds have; for example, hydrophilic head groups more readily form an ionizable positively charged hydrophilic plane that interacts with negatively charged mRNA to increase the loading of mRNA by the lipid nanoparticle. In the meta-acidic lysosome microenvironment, compounds containing three hydrophobic tails tend to form tapered molecular structures, which can promote hexagonal switching of cell membranes and lysosome escape. And on this basis, simultaneously optimizing the X group or the hydrophobic tail (including unsaturation, hetero atom, branched structure and/or alkyl chain length) can provide a lipid compound and a delivery system for non-liver targeted delivery with more excellent specific targeting effect, and more efficiently deliver nucleic acid to a target organ.
The invention also provides a synthesis method of the novel ionizable cationic lipid compounds. The novel ionizable cationic lipid compounds of the present invention can be synthesized using methods known in the art, for example, using one or more equivalents of an amine (hydrophilic polar head containing an amine group) reacted with three or more equivalents of a hydrophobic lipid tail compound under suitable conditions. The synthesis of the ionizable lipid compounds is performed with or without a solvent, and the synthesis may be performed at a higher temperature in the range of 25-120 ℃. The resulting ionizable cationic lipid compound may optionally be purified. For example, a mixture of ionizable cationic lipid compounds can be purified to yield a particular ionizable cationic lipid compound, such as a product containing three hydrophobic lipid tails. The hydrophobic lipid tail compounds may be commercially available or synthetically prepared.
In some embodiments of the invention there is provided a method of preparing the novel ionizable cationic lipid compounds comprising:
compound synthesis route:
(wherein n on each branched chain 2 Each independently selected from integers of 1 to 8, preferably 4 to 8, m 2 Each Y is independently selected from integers of 1 to 8, preferably 4 to 8, and each Y is independently selected from integers of 0 to 3; preferably, each n 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers of 4 to 8, each Y is independently selected from integers of 0 to 2)
The method specifically comprises the following steps:
1) Esterification: esterifying the hydroxyl group of compound A1 into an ester group in the presence of acryloyl chloride to obtain compound A2;
2) Michael addition: the novel ionizable cationic lipid compounds are obtained by subjecting compound A2 to a michael addition reaction with an amine (e.g., N-methyl-2, 2' -diaminodiethylamine).
In some embodiments of the present invention, provided are methods of preparing the novel ionizable cationic lipid compounds comprising:
compound synthesis route:
(wherein n on each branched chain 3 Each independently selected from integers from 1 to 18, preferably from 6 to 18; preferably, each n 3 Are all selected from integers of 6 to 18. )
The method specifically comprises the following steps:
1) Esterification: esterifying the hydroxyl group of compound B1 to an ester group in the presence of acryloyl chloride to obtain compound B2;
2) Michael addition: the novel ionizable cationic lipid compounds are obtained by subjecting compound B2 to a michael addition reaction with an amine (e.g., N-methyl-2, 2' -diaminodiethylamine).
In some embodiments of the present invention, provided are methods of preparing the novel ionizable cationic lipid compounds comprising:
compound synthesis route:
(wherein n on each branched chain 4 Each independently selected from integers from 1 to 18, preferably integers from 2 to 18; preferably, each n 4 Are all selected from integers from 2 to 18)
The method specifically comprises the following steps:
1) Esterification: esterifying the hydroxyl group of compound C1 to an ester group in the presence of acryloyl chloride to obtain compound C2;
2) Michael addition: the novel ionizable cationic lipid compounds are obtained by subjecting compound C2 to a michael addition reaction with an amine (e.g., N-methyl-2, 2' -diaminodiethylamine).
In some embodiments of the present invention, provided are methods of preparing the novel ionizable cationic lipid compounds comprising:
compound synthesis route:
(wherein n on each branched chain 5 Each independently selected from integers of 1 to 18, preferably 4 to 18, m 5 Each independently selected from integers from 1 to 18, preferably from 1 to 14; preferably, each n 5 Are all selected from integers of 4 to 18, each m 5 Are all selected from integers from 1 to 14)
The method specifically comprises the following steps:
1) Esterification: esterifying the hydroxyl group of compound D1 to an ester group in the presence of acryloyl chloride to obtain compound D2;
2) Michael addition: the novel ionizable cationic lipid compounds are obtained by subjecting compound D2 to a michael addition reaction with an amine (e.g., N-methyl-2, 2' -diaminodiethylamine).
Examples of the solvent used in the esterification reaction according to the present invention include, but are not limited to, halogenated hydrocarbons (such as methylene chloride, dichloroethane, chloroform, etc.), hydrocarbons (such as n-pentane, benzene, toluene, etc.), nitriles (such as acetonitrile, etc.), and mixed solvents of two or more of these solvents. The Michael addition reaction may optionally be carried out without a solvent, examples of which include, but are not limited to, isopropanol, t-butanol, tetrahydrofuran, and the like. The amine may be N-methyl-2, 2' -diaminodiethylamine.
According to the invention, the starting materials in the preparation method are commercially available or can be synthesized by conventional methods.
The novel ionizable cationic lipid provided by the invention contains two adjacent cis double bonds or disulfide bonds (S-S), and has a branched chain structure or a straight chain structure, so that the novel ionizable cationic lipid has higher encapsulation efficiency and better cell transfection efficiency when being subsequently applied to a delivery system for wrapping active substances (such as mRNA); in addition, the presence of two adjacent cis double bonds, disulfide bonds (S-S) or branched structures in the tail chain may allow for a more uniform particle size of the resulting lipid nanoparticle when preparing the lipid nanoparticle. The ionizable lipid compounds of the invention are particularly suitable for preparing nanoparticles of solid structure.
The invention also provides application of the novel ionizable cationic lipid compound in preparing a bioactive substance delivery system; preferably, the delivery system is a microbubble, microparticle, nanoparticle, liposome or lipid nanoparticle.
In some preferred embodiments of the present invention, when the novel ionizable cationic lipid compound is a compound of formula IWherein R is 3 、R 4 、R 5 Is->R 6 A linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, at least 1C atom of said alkyl, alkenyl group being optionally replaced by a heteroatom independently selected from O, S or N; preferably, X is an O or N heteroatom; more preferably, n1 is an integer selected from 4 to 8, m1 is an integer selected from 4 to 8, R 6 Is a linear or branched C6-18 alkyl group, a linear or branched C6-18 alkenyl group, at least 1C atom of said alkyl, alkenyl groups being optionally replaced by a heteroatom independently selected from S. Preferably, X is an O atom; more preferably, n1 is an integer selected from 4 to 8, m1 is an integer selected from 4 to 8, R 6 Is a linear or branched C6-18 alkyl group, a linear or branched C6-18 alkenyl group, 2C atoms of said alkyl, alkenyl groups being optionally replaced by an independently selected S atom.
In the invention, the internal rule of the novel ionisable cationic lipid compound steric space structure for promoting the transfection efficiency is unexpectedly discovered through optimizing the structure of the compound, and the steric space structure of the ionisable cationic lipid compound can be regulated to promote the in vivo cell transfection efficiency by changing the unsaturation degree, the hetero atom, the branched chain structure or the length of the hydrophobic tail chain of the novel ionisable cationic lipid compound, and the lipid nano particles have the function of specifically targeting the liver, the spleen and/or the lung.
In a preferred embodiment of the present invention, when the novel ionizable cationic lipid compound is a compound of formula a, formula B, the use of said novel ionizable cationic lipid compound in the preparation of a specific spleen-targeted bioactive substance delivery system; when the novel ionizable cationic lipid compound is a compound of formula C, the novel ionizable cationic lipid compound is used in the preparation of a specific spleen-and lung-targeted bioactive substance delivery system; when the novel ionizable cationic lipid compound is a compound of formula D, the novel ionizable cationic lipid compound is used in the preparation of a specific spleen-and liver-targeted bioactive substance delivery system.
According to a preferred embodiment of the invention, the invention employs a preferred compound of formula a, in particular a hydrophobic tail of length 18C and containing an adjacent cis double bond structure, the presence of which allows the hydrophobic tail to begin to bend at the C9 position to form an alkane chain length of about 9C in relative length, the three hydrophobic tail structures allowing the compound of formula a to form a unique triangular pyramid-shaped spatial steric structure, which can facilitate more binding of the ionizable lipid compound to mRNA and increase lysosomal escape rate, such as the compound N24-O18-2 (3T), which has a greatly increased mRNA delivery effect compared to the N24-O18 (3T) compound and which lipid nanoparticles can specifically target the spleen.
Further preferably, in some embodiments of the present invention, the novel ionizable cationic lipid compound is preferably:
N24-O18-2(3T)
according to a preferred embodiment of the invention, the compound of formula B is preferably used, and when the chain length of the hydrophobic tail is between 8 and 10C, the targeted transportation of the lipid nanoparticle to spleen organs is more facilitated, such as the compound N24-O10 (3T), and compared with the N24-O18 (3T) compound, the transfection efficiency of mRNA is greatly improved, and the targeted spleen effect is improved.
Further preferably, in some embodiments of the present invention, the novel ionizable cationic lipid compound is preferably:
N24-O6(3T)
N24-O8(3T)
N24-O10(3T)
N24-O12(3T)
N24-O14(3T)
N24-O16(3T)
N24-O18(3T)
further preferably, in some embodiments of the present invention, the novel ionizable cationic lipid compounds are further preferably:
N24-O10(3T)
according to the preferred embodiment of the invention, the preferred compound of the formula C is adopted, the introduction of the hetero atom S-S in the hydrophobic tail chain can lead the hydrophobic tail chain to bend at the S-S position, and the characteristics of the structure lead the plane formed by the hydrophilic head group to be larger, so that the loading on mRNA molecules is easier to increase. Such as the compound N24-O18-SS (3T), which can greatly enhance the effect of delivering mRNA compared to N24-O18 (3T) compounds, and can facilitate simultaneous targeted delivery to the spleen and lung. The lipid nanoparticle effect of N24-O18-SS (3T) -LNP is better.
Further preferred, in some embodiments of the invention, the novel ionizable cationic lipid compounds:
N24-O6-SS(3T)
N24-O8-SS(3T)
N24-O10-SS(3T)
N24-O12-SS(3T)
N24-O14-SS(3T)
N24-O16-SS(3T)
N24-O18-SS(3T)
N24-O6-S(3T)
N24-O8-S(3T)
N24-O10-S(3T)
N24-O12-S(3T)
N24-O14-S(3T)
N24-O16-S(3T)
N24-O18-S(3T)
N24-O6-O(3T)
N24-O8-O(3T)
N24-O10-O(3T)
N24-O12-O(3T)
N24-O14-O(3T)
N24-O16-O(3T)
N24-O18-O(3T)
N24-O6-N(3T)
N24-O8-N(3T)
N24-O10-N(3T)
N24-O12-N(3T)
N24-O14-N(3T)
N24-O16-N(3T)
N24-O18-N(3T)
N24-O6-Se(3T)
N24-O8-Se(3T)
N24-O10-Se(3T)
N24-O12-Se(3T)
N24-O14-Se(3T)
N24-O16-Se(3T)
N24-O18-Se(3T)
according to a preferred embodiment of the invention, the invention employs a preferred compound of formula D, the branched structure in the hydrophobic tail of which results in an increase in the spatial steric structural rigidity of the molecule, allowing the hydrophilic head group exposed at one end to better contact and support mRNA. Such as the compound N24-O11B (3T), which can greatly enhance the effect of delivering mRNA compared to the N24-O18 (3T) compound, and can facilitate simultaneous targeted delivery to the liver and spleen.
Further preferably, in some embodiments of the present invention, the novel ionizable cationic lipid compound is preferably:
in the present invention, it has been found that by adjusting the hydrophobic tail of the compounds of the present invention, such as unsaturation (hydrophobic tail length 18C and containing adjacent three, two cis double bonds or a single double bond), linear length (linear alkane with hydrophobic tail length 8C-18C), heteroatom (-O-, -N-, -SS-, -S-, -Se-) and branched structure (wherein one chain is C5-C18 length and the other branched chain is C1-C14 length), specific steric structure can be obtained, which can more advantageously promote its mRNA delivery ability and organ tissue specific targeting ability. The novel compound provided by the embodiment of the invention has better nucleic acid encapsulation rate and cell or in-vivo transfection efficiency, and the lipid nanoparticle has better function of specifically targeting liver, spleen and/or lung.
In the invention, it is further found through research that when the compound adopts a specific stereo structure, the compound can more favorably promote the capability of delivering RNA under the actions of compound unsaturation degree, hetero atom, branched structure and straight-chain length.
In a preferred embodiment of the invention, the delivery system is a lipid nanoparticle.
In certain embodiments, all of the amino groups of the amine are fully reacted with the hydrophobic lipid tail compound to form the 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 cationic lipid compound. These primary or secondary amines are left intact or may 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 produce a variety of different ionizable lipid compounds having a variety of tail numbers. For example, a diamine or polyamine may include one, two, three, or four tail compounds on various amino moieties of the molecule, thereby producing primary, secondary, and tertiary amines. In certain embodiments, the same tail compound is used; or two tail compounds of the same type are used. In other embodiments, two or more different tail compounds are used.
The present invention also provides a bioactive substance delivery system comprising the novel ionizable cationic lipid compounds, preferably, microbubbles, microparticles, nanoparticles, liposomes or lipid nanoparticles.
In one embodiment of the invention, the delivery system is a lipid nanoparticle. Such lipid nanoparticles can efficiently deliver bioactive substances (e.g., mRNA) into cells, tissues, or organs, enabling efficient regulation of bioactive substances. In the present invention, the novel ionizable cationic lipid compounds are combined with biologically active substances (e.g., mRNA) that are targeted for cellular or organ delivery or further comprise other substances (e.g., other anionic, cationic or ionizable lipid compounds, synthetic or natural polymers, proteins, phospholipids, cholesterol, carbohydrates, surfactants, etc.) to form microbubbles, microparticles, nanoparticles, liposomes, or lipid nanoparticles. The bioactive substance may be in the form of a gas, liquid or solid, and may be a nucleotide, a small molecule compound, a polypeptide or a protein. In the present invention, the delivery system may then optionally be combined with pharmaceutical excipients to form a pharmaceutical composition.
The invention also provides a pharmaceutical composition comprising the bioactive substance delivery system.
In another aspect, the present invention also provides a lipid nanoparticle composition comprising lipid nanoparticles comprising the novel ionizable cationic lipid compounds.
According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The additional lipid molecules may be lipid molecules known or conventionally used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, cholesterol, pegylated lipid molecules.
According to the present invention, the lipid nanoparticle composition, when used in a drug delivery system, can encapsulate a pharmaceutical agent, including a nucleotide, a small molecule compound, a polypeptide, a protein, a metal, or the like.
Such nucleic acids include, but are not limited to, DNA, antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), endogenous microRNA (miRNA), messenger RNAs (mrnas), RNA aptamers (rnaaptamers), small activating RNAs (sarnas), and the like. The novel ionizable cationic lipid compounds have several properties suitable for preparing drug delivery systems: 1) The ability to neutralize the charge on the negatively charged active species; 2) Lipid complexing and the ability to protect labile agents; 3) The ability to buffer pH in vivo; 4) Acting as a "proton sponge" and causing dissolution in vivo.
According to some preferred embodiments of the invention, the lipid nanoparticle composition comprises: 20-60mol% of the total lipid molecules of the novel ionizable cationic lipid compound of formula I, 5-30mol% of the neutral lipid molecule, 30-60mol% of the cholesterol lipid molecule, 0.5-5mol% of the pegylated lipid molecule; preferably contains 20-50 mole% of ionizable cationic lipid molecules, 5-20 mole% of neutral lipid molecules, 30-50 mole% of cholesterol lipid molecules, 0.5-2.5 mole% of PEGylated lipid molecules; more preferably contains 35-48 mole% of ionizable cationic lipid molecules of formula I, 10-15 mole% of neutral lipid molecules, 35-50 mole% of cholesterol lipid molecules, 1.0-2.0 mole% of PEGylated lipid molecules.
According to some preferred embodiments of the invention, the mole percentage of the ionizable lipid molecules of formula I in the lipid of the lipid nanoparticle is 20-60mol%.
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-like compound, or/and a phosphatidylethanolamine-like compound.
According to some preferred embodiments of the invention, the neutral lipid molecule is selected from 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, dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1, 2-ditolyl-sn-glycero-3-phosphocholine (DBPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (MPPC), 1-palmitoyl phosphatidylcholine (SPPC), and combinations thereof.
In one embodiment, the neutral lipid molecule may be selected from the group consisting of: distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), and dioleoyl phosphatidylethanolamine (DOPE). In another embodiment, the neutral lipid molecule may be dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule may be dimyristoyl phosphatidylcholine (DMPC).
According to the invention, the mole percentage of neutral lipid molecules in the lipid of the lipid nanoparticle is 5-30mol%.
According to the present invention, cholesterol lipid molecules include steroids and sterols, examples include, but are not limited to, cholesterol and cholesterol hemisuccinate.
According to some preferred embodiments of the invention, the cholesterol lipid molecule is selected from one or more of cholesterol and cholesterol hemisuccinate.
In one embodiment, the cholesterol lipid molecule is Cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate.
According to the invention, the molar percentage of cholesterol lipid molecules in the lipid of the lipid nanoparticle is 30-60mol%.
According to some preferred embodiments of the present invention, the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, expressed as a lipid moiety-the number average molecular weight of PEG, the lipid moiety comprising diacylglycerol and/or diacylglycerol amide, preferably one or more selected from dilauroylglycerol, dimyristoylglycerol, dilauryl glyceramide, dimyristoylglycerol amide, 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine; the number average molecular weight of the PEG is 100 to 50,000, preferably 200 to 10,000, more preferably 500 to 3,000, and most preferably 1,500 to 2,500.
According to the 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 diacylglycerols amides, 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 about C10 to C20. The dialkylglycerol or dialkylglyceramide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical. 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: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not include a PEG copolymer, e.g., it may be a PEG monopolymer. In one embodiment, the molecular weight of the PEG is from about 100 to about 50,000. In certain embodiments, the PEG is "PEG 2000" having an average molecular weight of about 2,000 daltons.
In some embodiments of the invention, PEG is represented by the formulaMeaning that for PEG-2000 where n is 45, the meaning of index-averaged 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 be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of 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 implementationsIn embodiments, R may be unsubstituted C1-C30 alkyl, such as C1-C20 alkyl, C1-C10 alkyl, C1-C6 alkyl. In some embodiments, R may be H, methyl or ethyl.
In some embodiments, the pegylated lipid molecule may be expressed as a "lipid fraction-PEG-number average molecular weight" or "PEG-number average molecular weight-lipid fraction" or "PEG-lipid fraction". The lipid moiety is a diacylglycerol or diacylglycerol amide selected from dilauroyl glycerol, dimyristoyl glycerol, dilauryl glycerol amide, dimyristoyl glycerol amide, 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine; the PEG has a number average molecular weight of about 100 to about 50,000.
In some embodiments, the pegylated lipid molecule may be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dilauroylglycerol amide, PEG-dimyristoylglycerol amide, PEG-distearylglycerol (PEG-DSPE) and PEG-distearylglycerol amide, PEG-cholesterol (1- [8' - (cholest-5-en-3 [ beta ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecyloxybenzyl- [ omega ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DMG-PEG 2000), 1, 2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG 2000), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG-pe), poly (ethylene glycol) -2000-dimethacrylate (DMA-PEG 2000) and 1, 2-distearoyloxypropyl-3-amine-N- [ methoxy (polyethylene glycol) -2000] (DSA-PEG 2000). In one embodiment, the pegylated lipid molecule may be DMG-PEG2000. In one embodiment, the pegylated lipid molecule may be C-DMA-PEG2000. In one embodiment, the pegylated lipid molecule may be DSA-PEG2000. In one embodiment, the pegylated lipid molecule may be PEG2000-C11. In some embodiments, the pegylated lipid molecule may be DSG-PEG2000. In one embodiment, the pegylated lipid molecule may be DSPE-PEG2000. In one embodiment, the pegylated lipid molecule may be DMA-PEG2000. In some embodiments, the pegylated lipid molecule may be PEG2000-C14. In some embodiments, the pegylated lipid molecule may be PEG2000-C16. In some embodiments, the pegylated lipid molecule may be PEG2000-C18.
According to the invention, the mole percentage of pegylated lipid molecules in the lipid of the lipid nanoparticle is 0.5-5mol%.
In some embodiments of the invention, the lipid nanoparticle comprises an ionizable cationic lipid molecule of formula D, a neutral lipid molecule, a cholesterol lipid molecule, a pegylated lipid molecule, wherein:
wherein n on each branch 5 Each independently selected from integers of 1 to 18, preferably 4 to 18, m 5 Each independently selected from integers from 1 to 18, preferably from 1 to 14; preferably, each n 5 Are all selected from integers of 4 to 18, each m 5 Are all selected from integers from 1 to 14; the ionizable cationic lipid molecules represented by formula D comprise 20-60mol%, preferably 35-50mol%, of the lipid in the lipid nanoparticle;
the neutral lipid molecule is selected from phosphatidylcholine compounds and phosphatidylethanolamine compounds; the neutral lipid molecules account for 5-30mol%, preferably 5-20mol%, more preferably 10-15mol% of the lipid in the lipid nanoparticle;
the cholesterol lipid molecule is selected from cholesterol, cholesterol hemisuccinate; the cholesterol lipid molecules account for 30-60mol%, preferably 30-50mol%, more preferably 35-48mol% of the lipid in the lipid nanoparticle;
The pegylated lipid molecule means "lipid fraction-PEG-number average molecular weight", said lipid fraction being a diacylglycerol or diacylglycerol amide selected from dilauryl 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; the PEG has a number average molecular weight of about 100 to about 50,000. The PEGylated lipid molecules comprise from 0.5 to 5 mole%, preferably from 0.5 to 2.5 mole%, more preferably from 1.0 to 2.0 mole% of the lipids in the lipid nanoparticle.
In some embodiments of the invention, the molar ratio of the ionizable cationic lipid molecule represented by formula D, the neutral lipid molecule, the cholesterol, and the pegylated lipid molecule is 35:15:48.5:1.5.
In some embodiments of the invention, the molar ratio of ionizable cationic lipid molecules represented by formula D, neutral lipid molecules, cholesterol, and pegylated lipid molecules is 45:15:38.5:1.5.
In some embodiments of the invention, the molar ratio of ionizable cationic lipid molecules represented by formula D, neutral lipid molecules, cholesterol, and pegylated lipid molecules is 40:10:48.5:1.5.
In one embodiment of the invention, the ionizable cationic lipid molecule of formula D is the compound N24-O11B (3T).
In one embodiment of the invention, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DMG-PEG2000. Alternatively, the neutral lipid molecule is DOPE and the pegylated lipid molecule is DMG-PEG2000. Alternatively, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DSPE-PEG2000. Alternatively, 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, or disulfide bonds (S-S), or branched structures, provided by the invention, can provide higher active substance encapsulation efficiency and better cell or in vivo transfection efficiency, are especially suitable for preparing solid-structure nanoparticles, and in the lipid nanoparticle composition, the ionizable cationic lipid molecules of the formula I, neutral lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules in the lipid nanoparticle are most preferably in the molar percentage of the total lipid molecules, and most importantly, the lipid nanoparticle has better specific spleen and/or lung targeting function.
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 to prepare a lipid-mixed solution, mixing the lipid-mixed solution with an aqueous solution of the delivered substance (for example, mRNA) as an aqueous phase, and mixing the organic phase and the aqueous phase. Lipid nanoparticles may be prepared using other methods including, but not limited to, spray drying, solvent extraction, phase separation, nano-precipitation, single and double emulsion solvent evaporation, microfluidic, 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 (2-5): 1, e.g., 3:1.
In some embodiments, the nanoparticle is prepared using a microfluidic platform.
According to the present invention, the preparation method further comprises the step of separating 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 nanoparticle in the present invention ranges from 1nm to 1000 nm.
The delivery system formed by the novel ionizable cationic lipid compounds of the present invention may also be modified with targeting molecules that render them targeted to specific cells, tissues or organs. The targeting molecule may be included in the entire delivery system or may be located only on its surface. The targeting molecule may be a small molecule, nucleic acid, polypeptide, protein, glycoprotein, lipid, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), sialic acid, aptamers, transferrin (transferrin), asialoglycoprotein (asialoglycoprotein), receptor ligands, etc.
The active substance delivered by the delivery system formed by the ionizable lipid compounds of the present invention may be a therapeutic, diagnostic, or prophylactic agent. The nature of the active substance may be small molecule compounds, isotopically labeled compounds, nucleic acids, polypeptides, proteins, vaccines, metals, etc.
The delivery system formed from the novel ionizable cationic lipid compounds of the present invention may be combined with one or more pharmaceutical excipients to form a pharmaceutical composition suitable for administration to animals, including humans. The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, etc., including, but not limited to, cellulose and its derivatives, such as sodium carboxymethyl cellulose and cellulose acetate; sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; gelatin; talc; diols 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 Tween80 (Tween 80); coloring agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants; buffers such as phosphate buffer, citrate buffer, and the like.
The nucleic acid drug delivery system provided by the invention can be used for efficiently and specifically delivering nucleic acid drug molecules into spleen and/or lung and effectively translating the nucleic acid drug molecules into target molecules, and simultaneously reducing the accumulated side effects of liposome in liver, thereby having important significance for targeted drug administration, development and application of nucleic acid drugs.
Description of terms in the present invention: "about": the term "about" used in conjunction with a numerical value means the range of accuracy familiar to and acceptable to those skilled in the art. Typically, this accuracy is in the interval of + -10%.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a hydrogen spectrum of 1-ethyl nonyl acrylate (a 2) as a compound in the examples of the present invention;
FIG. 2 is a hydrogen spectrum of the compound N24-O11B (3T) in the examples of the present invention;
FIG. 3 is a mass spectrum of a compound N24-O11B (3T) in an embodiment of the invention;
FIG. 4 is a hydrogen spectrum of the compound N24-O11B (4T) in the examples of the present invention;
FIG. 5 is a mass spectrum of a compound N24-O11B (4T) in an embodiment of the invention;
FIG. 6 is a graph of dissociation constants (pKa) for compounds N24-O11B (3T) (A), N24-O11B (4T) (B) in examples of the invention;
FIG. 7 is a graph showing the expression level of the Luciferase protein after transfection of 293T cells with N24-O11B (3T), N24-O11B (4T) prepared LNP-encapsulated Luciferase mRNA (LucRNA) for 24 hours in examples of the present invention;
FIG. 8 is a graph showing the expression level of the Luciferase protein after transfection of 293T cells with LNP-encapsulated Luciferase mRNA (LucRNA) prepared by ALC-0315 for 24 hours in examples of the present invention;
FIG. 9 shows the fluorescence expression of the organs of mice according to the examples of the present invention after 6h of intravenous injection of N24-O18-2 (3T)/LucRNA, N24-O10 (3T)/LucRNA, N24-O18-SS (3T)/LucRNA and N24-O11B (3T)/LucRNA;
FIG. 10 is a hydrogen spectrum of compound N24-O10 (3T) in the examples of the present invention;
FIG. 11 is a mass spectrum of the compound N24-O10 (3T) in the examples of the present invention;
FIG. 12 is a mass spectrum of compound N24-O18-2 (3T) in the examples of the present invention;
FIG. 13 is a hydrogen spectrum of compound N24-O18-2 (3T) in the examples of the present invention;
FIG. 14 is a hydrogen spectrum of the compound N24-O18-SS (3T) in the examples of the present invention;
FIG. 15 is a mass spectrum of the compound N24-O18-SS (3T) in the examples of the present invention;
FIG. 16 is a graph showing the expression level of the Lucifer protein after transfection of 293T cells with LNP-encapsulated Lucifer mRNA (LucRNA) prepared from N24-O18 (3T), N24-O16 (3T), N24-O14 (3T), N24-O12 (3T), N24-O10 (3T), N24-O8 (3T) and N24-O6 (3T) for 24 hours in examples of the present invention;
FIG. 17 is a graph showing the expression level of the Lucifer protein after transfection of 293T cells with N24-O10 (3T), N24-O18-2 (3T), N24-O18-SS (3T), and N24-O11B (3T) prepared LNP-encapsulated Luciferase mRNA (LucRNA) for 24 hours in examples of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention. Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.
EXAMPLE 1 Synthesis of ionizable cationic lipids, N24-O11B (3T)
Synthesis of 1-ethyl-nonyl acrylate (a 2): 3-undecanol (3.0 g), triethylamine (5.28 g) were added to 30.0mL of methylene chloride at 0℃and then a methylene chloride (10.0 mL) solution containing acryloyl chloride (2.36 g) was added dropwise to the reaction system, and the reaction solution was stirred at 20℃for 2 hours. The triethylamine salt precipitated in the reaction solution was filtered through a buchner funnel, and the filtrate was collected, washed with water, 5% by mass of hydrochloric acid, and then with water, and the organic phase was dried over anhydrous sodium sulfate. Evaporation to dryness followed by purification of the residue by flash column chromatography eluting with petroleum ether/ethyl acetate gave the desired product 1-ethyl nonyl acrylate (a 2) (2.8 g), yield 70%, hydrogen profile of compound a2 as shown in FIG. 1.
1 H NMR(400MHz,CDCl3)δ6.39(dd,J＝17.3,1.5Hz,1H),6.12(dd,J＝17.3,10.4Hz,1H),5.80(dd,J＝10.4,1.5Hz,1H),4.94–4.84(m,1H),1.69–1.48(m,4H),1.26(s,12H),0.88(q,J＝7.2Hz,6H).
Synthesis of N24-O11B (3T), N24-O11B (4T) (for comparison): to a solution of 250mg of N-methyl-2, 2' -diaminodiethylamine was added 1-ethyl nonyl acrylate (1.7 g) at room temperature, after which the mixture was heated to 120℃and stirring continued for 24h. When the thin layer chromatography plate detection reaction was completed, 2.0g of a crude product was obtained, and then the target product was purified by eluting with methylene chloride/methanol by a rapid column chromatography to obtain 195mg of N24-O11B (3T), 195mg of N24-O11B (4T), the hydrogen spectrum of the compound N24-O11B (3T) was shown in FIG. 2, the mass spectrum was shown in FIG. 3, the hydrogen spectrum of the compound N24-O11B (4T) was shown in FIG. 4, and the mass spectrum was shown in FIG. 5.
N24-O11B(3T)：
1 H NMR(400MHz,CDCl 3 )δ4.90–4.72(m,3H),2.92(t,J＝6.7Hz,2H),2.80(t,J＝7.3Hz,4H),2.71(t,J＝5.9Hz,2H),2.61–2.49(m,6H),2.45(dd,J＝16.1,8.6Hz,6H),2.23(s,3H),1.55(ddd,J＝14.2,9.7,5.1Hz,12H),1.26(s,36H),0.95–0.82(m,18H).
MALDI-TOF MS：m/z 796.753[M+H] + .
N24-O11B(4T)：
1 H NMR(400MHz,CDCl 3 )δ4.81(p,J＝6.3Hz,4H),2.81(dd,J＝17.9,10.6Hz,8H),2.56(s,4H),2.41(dd,J＝32.9,25.7Hz,12H),2.24(s,2H),1.63–1.46(m,16H),1.28(d,J＝14.6Hz,48H),0.91–0.84(m,24H).
MALDI-TOF MS：m/z 1022.924[M+H] + .
Example 2 dissociation constant (pKa) of ionizable cationic lipid N24-O11B (3T)
Ionizable lipids have two main roles: bind nucleic acids and allow release of nucleic acid molecules in cells. The pKa of lipids is an important factor because lipids need to be positively charged at low pH to bind nucleic acids, but not charged at neutral pH, so LNP does not cause toxicity. As shown in FIG. 6, the ionizable lipid N24-O11B (3T) had a pKa of 6.68 (A) and N24-O11B (3T) had a pKa of 5.93 (B) as determined by the TNS dye binding assay. It can be seen that both molecules are positively charged under acidic conditions and RNA-loaded, and are uncharged at neutral pH (ph=7.4).
EXAMPLE 3 preparation of lipid nanoparticles by encapsulation of mRNA with N24-O11B (3T)
The ionizable cationic lipids N24-O11B (3T) or N24-O11B (4T), DOPE, cholesterol and DMG-PEG2000 were each prepared as an organic phase by preparing an ethanol solution at a molar ratio of 45%:15%:38.5%:1.5%, and a water solution of Luciferase mRNA (LucRNA) in ph=4 as an aqueous phase. According to the volume ratio of the water phase to the organic phase of 3:1, preparing the nanoparticle suspension by using a microfluidic technology on a nano-drug manufacturing instrument (Micana). 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 LucRNA-LNP particle size and Zeta potential was performed using a zetasizer pro nanoparticle size potentiometer (malverpanaceae). The encapsulation efficiency of LucRNA-LNP was measured by the method of F-280 fluorescence spectrophotometer (Tianjin harbor east) Ribogreen. The test results of example 3 are shown in Table 1.
TABLE 1 physical and chemical control data for lipid nanoparticles
From the results of example 3, it can be seen that the lipid nanoparticle LucRNA-LNP prepared from the novel lipid compound N24-O11B (3T) has a particle size of about 140nm, a relatively narrow LucRNA-LNP particle size distribution (relatively small PDI), and an encapsulation efficiency as high as 96.68%.
In addition, transfection efficiency of the prepared LucRNA-LNP cells was examined by the multifunctional enzyme-labeled instrument (BioTek, model SLXFATS) fluorescein reporter method. The method for in vitro transcription of LucRNA is as follows: 293T cells were plated at a cell density of 1X10 4 Individual cells/well, transfected when the cell fusion was 30% -50%. 1.0 μg of LucRNA was transfected with Lipofectamine 2000 (ThermoFisher Scientific) and the transfection procedure was performed according to the instructions of the transfection reagent product. And (5) detecting the protein expression quantity by using a multifunctional enzyme-labeled instrument after 24 hours of transfection. The negative control was cell culture medium without addition of LucRNA-LNP. In vitro cell transfection efficiency as shown in FIG. 7, LNP-coated mRNA prepared from the ionizable lipid N24-O11B (3T) was shown to have a very high cell transfection efficiency, which was significantly lower, about an order of magnitude lower, than that of the conventional ionizable lipid N24-O11B (4T) containing four hydrophobic tail chains. It can be seen that the ionizable lipid N24-O11B (3T) containing three hydrophobic tails has higher cell transfection efficiency than the conventional ionizable lipid N24-O11B (4T) containing four hydrophobic tails, and the advantage is very obvious. Compared with the traditional common lipid compounds containing four hydrophobic tail chains, the three hydrophobic tail chain lipid compounds have higher cell transfection efficiency and are applicable to other lipid compounds with the general structure in the invention.
From the results of example 3, it can also be seen that the lipid nanoparticle LucRNA-LNP prepared from the novel lipid compound N24-O11B (3T) has better physicochemical characteristics and in vitro cell transfection efficiency is about 2-3 times higher than that of commercial Lipofectamine 2000 (Lipo 2000).
Comparative example 1 (commercial ionizable cationic lipid molecule ALC-0315)
ALC-0315 has the formula: ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate). Yu Aiwei Tuo (Shanghai) pharmaceutical technologies Co., ltd.
The structural formula of ALC-0315 is:
example 4N24-O11B (3T)
This example provides a comparison of the effects of N24-O11B (3T) and the commercial ionizable cationic lipid molecule ALC-0315
Lipid nanoparticles were prepared according to the method described in example 3, using N24-O11B (3T) and ALC-0315, respectively, in the following molar ratios: N24-O11B (3T) DOPE Cholesterol DMG-PEG 2000=45:15:38.5:1.5; ALC-0315:DOPE:Cholesterol:DMG-PEG 2000=45:15:38.5:1.5.
The physicochemical quality control data of the prepared lipid nanoparticle are shown in the following table (table 2):
table 2 physical and chemical control data for lipid nanoparticles
From the above table, it can be seen that the encapsulation efficiency of the lipid nanoparticle prepared with N24-O11B (3T) is as high as 96%, compared with the lipid nanoparticle similar to ALC-0315.
By adopting the same transfection method as in example 3, the prepared lipid nanoparticle is transfected into cells, the protein expression condition is known, and the result is shown in fig. 8, after the lipid nanoparticle prepared by N24-O11B (3T) carries mRNA to transfect the cells, the protein expression amount in the cells is higher than that of Lipofectamine 2000, and the protein expression amount in the cells transfected by the corresponding mRNA concentration is also higher than ALC-0315, which indicates that the cell transfection efficiency of the lipid nanoparticle prepared by N24-O11B (3T) is very high.
Example 5 transfection experiments of N24-O18-2 (3T) -LNP, N24-O10 (3T) -LNP, N24-O18-SS (3T) -LNP, N24-O11B (3T) -LNP lipid nanoparticles in animals
Lipid nanoparticles were prepared by intravenous injection of lipid nanoparticles into the tail of mice using N24-O18-2 (3T), N24-O10 (3T), N24-O18-SS (3T) or N24-O11B (3T) -LNP according to the method described in example 3, in the following molar ratios: N24-O18-2 (3T) DOPE Cholesterol DMG-PEG 2000=45:15:38.5:1.5; N/P ratio is 10:1, N24-O10 (3T) DOPE: cholesterol: DMG-PEG 2000=45:15:38.5:1.5; N/P ratio is 10:1, N24-O18-SS (3T) DOPE: cholesterol: DMG-PEG 2000=45:15:38.5:1.5; N/P ratio is 10:1, N24-O11B (3T) DOPE: cholesterol: DMG-PEG 2000=45:15:38.5:1.5; the N/P ratio was 10:1. Wherein mRNA is mRNA expressing Luciferase fluorescent protein, the amount of mRNA is 10 μg, N24-O18-2 (3T), N24-O10 (3T), N24-O18-SS (3T), or N24-O11B (3T) -LNP, DOPE, cholesterol, DMG-PEG2000 is 100 μg, the liposome environment is rapidly converted by 200 μl of neutral PBS buffer solution, and the liposome environment is rapidly injected into 6-8 week female C57 mice via tail vein, and intravenous injection of 10 μg mRNA is controlled.
Mice were injected with N24-O18-2 (3T) -LNP, PBS (blank) via the tail vein, and fluorescent expression of each organ after 6h is shown in FIG. 9. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N24-O18-2 (3T) -LNP, the fluorescent expression levels in various organs of mice were mainly distributed in spleen about 95%, liver 5%, heart 0%, lung 0%, kidney 0%, which can be seen to specifically target spleen. Mice were injected with N24-O10 (3T) -LNP, PBS (blank) via the tail vein, and fluorescent expression of each organ after 6h is shown in FIG. 9. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N24-O10 (3T) -LNP, the fluorescence expression levels in various organs of mice were mainly distributed in about 100% of spleen, 0% of heart, 0% of liver, 0% of lung, and 0% of kidney, which was seen to target spleen specifically. Mice were injected with N24-O18-SS (3T) -LNP, PBS (blank) via the tail vein, and fluorescent expression of each organ after 6h is shown in FIG. 9. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N24-O18-SS (3T) -LNP, the fluorescent expression levels in various organs of mice were mainly distributed in spleen about 60%, liver 0%, lung 40%, heart 0%, kidney 0%, which can be seen to specifically target spleen and lung. Mice were injected with N24-O11B (3T) -LNP, PBS (blank) via the tail vein, and fluorescent expression of each organ after 6h is shown in FIG. 9. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N24-O11B (3T) -LNP, the fluorescent expression levels in various organs of mice were mainly distributed in spleen about 50%, liver 50%, heart 0%, lung 0%, kidney 0%, which can be seen to specifically target spleen and liver. It can be seen that the present invention forms an isomerised compound based on three lipid hydrophobic tails, further achieving specific targeting of different organs by modulating lipid X groups or hydrophobic tails (including unsaturation, heteroatoms, branched structures and/or alkyl chain length).
Example 6N24-O10 (3T)
The same synthesis and purification procedure as in example 1 was used, except that the compound 3-undecanol in example 1 was changed to n-decanol, which had the following structure:
the present example also provides a process for the preparation of the compound:
synthesis of N24-O10 (3T): decyl acrylate (1.6 g) was added to a solution of 250mg of N-methyl-2, 2' -diaminodiethylamine at room temperature, after which the mixture was heated to 120℃and stirring continued for 24h. When the reaction was completed by thin layer chromatography, 1.9g of a crude product was obtained, and then the objective product was purified by rapid column chromatography eluting with methylene chloride/methanol to obtain 300mg of N24-O10 (3T). The hydrogen spectrum of the compound N24-O11B (3T) is shown in FIG. 10, and the mass spectrum is shown in FIG. 11.
N24-O10(3T)：
1 H NMR(400MHz,CDCl 3 )δ4.06(dt,J＝13.9,6.9Hz,6H),2.93(t,J＝6.6Hz,2H),2.79(t,J＝7.2Hz,4H),2.73(t,J＝5.6Hz,2H),2.60–2.49(m,6H),2.45(dd,J＝15.6,8.3Hz,6H),2.24(s,3H),1.61(dd,J＝13.2,6.5Hz,6H),1.28(d,J＝15.3Hz,42H),0.88(t,J＝6.7Hz,9H).
MALDI-TOF MS：m/z 754.319[M+H] + .
This example also provides the preparation of the compound N24-O11B (3T) with dissociation constant (pKa=7.09) and entrapped LucRNA lipid nanoparticle N24-O10 (3T) -LNP (N24-O10 (3T): DOPE: cholesterol: DMG-PEG 2000=45:15:38.5:1.5, N/P ratio of 10:1). The in vivo distribution of the N24-O10 (3T) -LNP lipid nanoparticle was studied by transfection experiments in animals, and it is seen that N24-O10 (3T) -LNP mainly specifically targets spleen, as shown in FIG. 9, the N24-O10 (3T) lipid compound of this example can realize specific targeting of lipid nanoparticle by spleen and liver into individual spleen sites by adjusting the branched structure in the hydrophobic tail chain to be a linear structure and keeping the length of the branched structure to be 10 carbon atoms compared with the N24-O11B (3T) compound of the example.
Example 7N24-O18-2 (3T)
The same synthesis and purification procedure as in example 1 was followed, except that the compound 3-undecanol in example 1 was changed to (9Z, 12Z) -9, 12-octadecadien-1-ol (linoleyl alcohol) having the following structure:
the present example also provides a process for the preparation of the compound:
synthesis of N24-O18-2 (3T): to 300mg of N-methyl-2, 2' -diaminodiethylamine solution was added (9Z, 12Z) -9, 12-dienearyl acrylate (2.9 g) at room temperature, after which the mixture was heated to 120℃and stirred continuously for 24h. When the reaction was completed by thin layer chromatography, 3.2g of a crude product was obtained, and then the objective product was purified by rapid column chromatography eluting with methylene chloride/methanol to obtain 480mg of N24-O18-2 (3T). The hydrogen spectrum of the compound N24-O18-2 (3T) is shown in FIG. 12, and the mass spectrum is shown in FIG. 13.
N24-O18-2(3T)：
1 H NMR(400MHz,CDCl 3 )δ5.45–5.25(m,12H),4.06(dd,J＝14.8,7.4Hz,6H),2.93(t,J＝6.5Hz,2H),2.83–2.68(m,12H),2.55(dd,J＝14.7,6.7Hz,6H),2.45(dd,J＝15.6,8.3Hz,6H),2.24(s,3H),2.13–1.98(m,12H),1.67–1.56(m,6H),1.42–1.23(m,48H),0.89(t,J＝6.7Hz,9H).
MALDI-TOF MS：m/z 1079.008[M+H] + .
This example also provides the preparation of the dissociation constant (pka=6.70) of this compound N24-O18-2 (3T) and the entrapped LucRNA lipid nanoparticle N24-O18-2 (3T) -LNP (N24-O18-2 (3T): DOPE: cholestenol: DMG-pe2000=45:15:38.5:1.5, N/P ratio 10:1). The in vivo distribution of the N24-O18-2 (3T) -LNP lipid nanoparticle in an animal body is studied through a transfection experiment on the N24-O18-2 (3T) -LNP lipid nanoparticle in the animal body, and as shown in fig. 9, the N24-O18-2 (3T) lipid compound of the embodiment can specifically target the spleen, compared with the N24-O11B (3T) compound of the embodiment, the N24-O18-2 (3T) lipid compound of the embodiment can further facilitate the combination of the ionizable lipid compound and mRNA, improve the escape rate of lysosomes, greatly improve the effect of mRNA delivery and convert lipid nanoparticles into specific targeting sites of the liver by singly by adjusting the branched chain structure of the hydrophobic tail chain to a linear structure containing unsaturation degree, such as adding adjacent cis double bonds between C9 and C13, the hydrophobic tail chain can start bending at the C9 position to form an alkane chain length with the relative length of about 9C, and the three hydrophobic tail chains can lead the N24-O18-2 (3T) compound to form a unique triangular conical space three-dimensional structure.
Example 8N24-O18-SS (3T)
The same synthesis and purification procedure as in example 1 was used, except that the compound 3-undecanol in example 1 was changed to 2- (hexadecyldithio) ethanol, which had the following structure:
the present example also provides a process for the preparation of the compound:
synthesis of N24-O18-2 (3T): to a solution of 300mg of N-methyl-2, 2' -diaminodiethylamine was added 2- (hexadecyldithio) ethyl acrylate (3.2 g) at room temperature, after which the mixture was heated to 120℃and stirred for 24 hours. When the reaction was completed by thin layer chromatography, 3.5g of a crude product was obtained, and then the objective product was purified by rapid column chromatography eluting with methylene chloride/methanol to obtain 525mg of N24-O18-2 (3T). The hydrogen spectrum of the compound N24-O18-SS (3T) is shown in FIG. 14, and the mass spectrum is shown in FIG. 15.
N24-O18-SS(3T)：
1 H NMR(400MHz,CDCl 3 )δ4.36(ddd,J＝14.7,13.4,7.2Hz,6H),2.98(t,J＝6.4Hz,2H),2.89(dd,J＝12.3,5.6Hz,6H),2.81(dd,J＝12.7,5.8Hz,6H),2.69(dd,J＝16.4,9.0Hz,8H),2.57(d,J＝6.7Hz,4H),2.48(dd,J＝14.0,7.1Hz,6H),2.27(s,3H),1.74–1.61(m,6H),1.37(dd,J＝14.1,7.1Hz,6H),1.28(d,J＝16.6Hz,60H),0.88(t,J＝6.8Hz,9H).
MALDI-TOF MS：m/z 1198.799[M+H] + .
This example also provides the preparation of the dissociation constant (pka=7.21) of the compound N24-O18-SS (3T) and the entrapped LucRNA lipid nanoparticle N24-O18-SS (3T) -LNP (N24-O18-SS (3T): DOPE: cholestenol: DMG-pe2000=45:15:38.5:1.5, N/P ratio 10:1). The in vivo distribution of the N24-O18-SS (3T) -LNP lipid nanoparticle is studied by transfection experiments on the N24-O18-SS (3T) -LNP lipid nanoparticle in animals, and the N24-O18-SS (3T) -LNP can specifically target spleen and lung, as shown in fig. 9, the N24-O18-SS (3T) lipid compound of the embodiment is more enlarged in plane formed by hydrophilic head groups and easier to increase the load on mRNA molecules compared with the N24-O11B (3T) compound by adjusting the branched chain structure in the hydrophobic tail chain to be a linear structure containing S heteroatoms, such as S-S is replaced at two C-C positions of C3 and C4, and the introduction of the heteroatom S-S in the hydrophobic tail chain can lead the hydrophobic tail chain to bend at the S-S position. Its effect of delivering mRNA is greatly enhanced and its lipid nanoparticle specific targeting is transformed from spleen and liver to spleen and lung.
Comparative example 2 Effect of hydrophobic tail chain Length of lipid Compounds on lipid nanoparticle cell transfection
The invention designs and synthesizes a series of lipid compounds with different hydrophobic tail chain lengths, and according to the method described in the example 3, N24-O18 (3T), N24-O16 (3T), N24-O14 (3T), N24-O12 (3T), N24-O10 (3T), N24-O8 (3T) and N24-O6 (3T) are respectively used for preparing lipid nano particles, wherein the specific molar ratios are as follows: lipid compound DOPE Cholesterol DMG-PEG 2000=45:15:38.5:1.5.
The physicochemical quality control data of the prepared lipid nanoparticle are shown in the following table (table 3):
TABLE 3 physical and chemical control data for lipid nanoparticles
The prepared lipid nanoparticle was transfected into cells in the same transfection manner as in example 3, and the protein expression was analyzed, and the result is shown in fig. 16, in which the cell transfection efficiency of the lipid nanoparticle prepared by the lipid compound corresponding thereto tended to increase and then decrease with the decrease of the carbon number of the hydrophobic tail chain, and N24-O10 (3T) -LNP had the highest transfection efficiency with the tail chain length of C10, and specific targeting of the lipid nanoparticle to the spleen site was enabled.
Comparative example 3 Effect of lipid Compound hydrophobic Tail space Structure on lipid nanoparticle cell transfection and organ/tissue targeting
The space structure of the ionizable cationic lipid compound is regulated mainly by changing the unsaturation degree, the heteroatom or the branched chain structure of the hydrophobic tail chain, so that mRNA can be better loaded, and the lipid nanoparticle has the function of specifically targeting the liver, spleen and/or lung.
The invention designs and synthesizes a series of lipid compounds with different hydrophobic tail chain space structures, and according to the method described in the example 3, N24-O18-2 (3T), N24-O18-SS (3T) and N24-O11B (3T) are respectively used for preparing lipid nano particles, wherein the specific molar ratio is as follows: lipid compound DOPE Cholesterol DMG-PEG 2000=45:15:38.5:1.5.
The physicochemical quality control data of the prepared lipid nanoparticle are shown in the following table (table 4):
TABLE 4 physical and chemical control data for lipid nanoparticles
Using the same transfection procedure as in example 3, the prepared lipid nanoparticle was transfected into cells, and the protein expression was analyzed, and the results are shown in FIG. 17, wherein the introduction of the unsaturation, heteroatom, or branched structure in the hydrophobic tail of the lipid compound increased the efficiency of the cell transfection corresponding to the lipid nanoparticle, compared with the introduction of the unsaturation, heteroatom, or branched structure in the hydrophobic tail of the lipid compound, which may be that the ionizable cationic lipid compound had a specific spatial structure after the introduction of the hydrophobic tail of the lipid compound, increased the amount of mRNA loaded, and increased the lysosome escape rate, and finally improved the efficiency of cell transfection. The novel ionizable cationic lipid compound provided by the invention can well load mRNA, and particularly can better realize specific targeting of lipid nanoparticles to specific organs.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A novel ionizable cationic lipid compound characterized by having a structure represented by formula I:
wherein n is an integer of 2 to 4; r is R 1 is-CH 3 ，-CH 2 CH 3 ，-CH 2 CH 2 CH 3 ，-CH 2 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，-CH 2 CH 2 NHCOCH 3 ；R 2 is-H; r is R 3 、R 4 、R 5 Is thatX is O, S or N heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10; r is R 6 Is a C6-25 alkyl, alkenyl or alkynyl group with or without heteroatoms.
2. The novel ionizable cationic lipid compound of claim 1, wherein n is 2, r 1 is-CH 3 ，R 2 is-H;
preferably, the compound of formula I isWherein R is 3 、R 4 、R 5 Is thatX is O, N or S heteroatom, N1 is selected from integers from 1 to 10, m1 is selected from integers from 1 to 10, N1 and m1 are independent from each other and can be the same or different; r is R 6 Is a linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, a linear or branched C6-25 alkynyl group, at least 1C atom of said alkyl, alkenyl or alkynyl group being optionally replaced by a heteroatom independently selected from O, N, S, S-S or Se.
3. The novel ionizable cationic lipid compound of claim 1 or 2, wherein n is 2, r 1 is-CH 3 ，R 2 is-H; preferably, the compound of formula I isWherein R is 3 、R 4 、R 5 Is->R 6 A linear or branched C6-25 alkyl group, a linear or branched C6-25 alkenyl group, at least 1C atom of said alkyl, alkenyl group being optionally replaced by a heteroatom independently selected from O, S or N; preferably, X is an O or N heteroatom; more preferably, n1 is an integer selected from 4 to 8, m1 is an integer selected from 4 to 8, R 6 Is a linear or branched C6-18 alkyl group, a linear or branched C6-18 alkenyl group, at least 1C atom of said alkyl, alkenyl groups being optionally replaced by a heteroatom independently selected from S or S-S.
4. A novel ionizable cationic lipid compound according to any of claims 1-3, characterized in that said novel ionizable cationic lipid compound is selected from a compound of formula a or a compound of formula B or a compound of formula C or a compound of formula D:
wherein n on each branch 2 Each independently selected from integers of 1 to 8, preferably 4 to 8, m 2 Each Y is independently selected from integers of 1 to 8, preferably 4 to 8, and each Y is independently selected from integers of 0 to 3; preferably, each n 2 Are all selected from integers of 4 to 8, each m 2 Are all selected from integers of 4-8, and each Y is independently selected from integers of 0-2;
wherein n on each branch 3 Each independently selected from integers from 1 to 18, preferably from 6 to 18; preferably, each n 3 Are all selected from integers of 6 to 18；
Wherein n on each branch 4 Each independently selected from integers from 1 to 18, preferably integers from 2 to 18; preferably, each n 4 Are all selected from integers from 2 to 18;
wherein n on each branch 5 Each independently selected from integers of 1 to 18, preferably 4 to 18, m 5 Each independently selected from integers from 1 to 18, preferably from 1 to 14; preferably, each n 5 Are all selected from integers of 4 to 18, each m 5 Are all selected from integers of 1 to 14.
5. A process for the preparation of novel ionizable cationic lipid compounds according to any of claims 1-4, characterized by comprising the steps of:
1) Esterification: esterifying the hydroxyl group of compound A1 into an ester group in the presence of acryloyl chloride to obtain compound A2;
2) Michael addition: subjecting compound A2 to michael addition reaction with an amine to obtain the novel ionizable cationic lipid compound; or:
1) Esterification: esterifying the hydroxyl group of compound B1 to an ester group in the presence of acryloyl chloride to obtain compound B2;
2) Michael addition: subjecting compound B2 to michael addition reaction with an amine to obtain the novel ionizable cationic lipid compound; or:
1) Esterification: esterifying the hydroxyl group of compound C1 to an ester group in the presence of acryloyl chloride to obtain compound C2;
2) Michael addition: subjecting compound C2 to a michael addition reaction with an amine to obtain the novel ionizable cationic lipid compound; or:
1) Esterification: esterifying the hydroxyl group of compound D1 to an ester group in the presence of acryloyl chloride to obtain compound D2;
2) Michael addition: and (3) allowing the compound D2 to react with amine through Michael addition reaction to obtain the novel ionizable cationic lipid compound.
6. Use of a novel ionizable cationic lipid compound according to any of claims 1-4 for the preparation of a bioactive substance delivery system; preferably, the delivery system is a microbubble, microparticle, nanoparticle, liposome or lipid nanoparticle.
7. A bioactive substance delivery system comprising the novel ionizable cationic lipid compound of any one of claims 1-4; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.
8. A pharmaceutical composition comprising the bioactive agent delivery system of claim 7.
9. A lipid nanoparticle composition comprising lipid nanoparticles comprising the novel ionizable cationic lipid compound of any one of claims 1-4; preferably, in the lipid nanoparticle composition, the lipid nanoparticle comprises: 20-60mol% of the total lipid molecules of the novel ionizable cationic lipid compound of formula I, 5-30mol% of the neutral lipid molecule, 30-60mol% of the cholesterol lipid molecule, 0.5-5mol% of the pegylated lipid molecule;
preferably contains 20-50 mole% of ionizable cationic lipid molecules, 5-20 mole% of neutral lipid molecules, 30-50 mole% of cholesterol lipid molecules, 0.5-2.5 mole% of PEGylated lipid molecules;
more preferably contains 35-48 mole% of ionizable cationic lipid molecules of formula I, 10-15 mole% of neutral lipid molecules, 35-50 mole% of cholesterol lipid molecules, 1.0-2.0 mole% of PEGylated lipid molecules.
10. The lipid nanoparticle composition of claim 9, wherein the neutral lipid molecule is selected from phosphatidylcholine compounds and/or phosphatidylethanolamine compounds; and/or the number of the groups of groups,
The cholesterol lipid molecules are selected from one or more of cholesterol and cholesterol hemisuccinate; and/or the number of the groups of groups,
the pegylated lipid molecule comprises a lipid moiety comprising diacylglycerol and/or diacylglycerol amide, preferably one or more selected from dilauroylglycerol, dimyristoylglycerol, dilauryl glyceramide, dimyristoylglycerol amide, 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, and a PEG-based polymer moiety, expressed as a number average molecular weight of the lipid moiety-PEG; the number average molecular weight of the PEG is 100 to 50,000, preferably 200 to 10,000, more preferably 500 to 3,000, and most preferably 1,500 to 2,500.
CN202311260962.6A 2023-09-27 2023-09-27 Novel ionizable cationic lipid compound, and preparation method and application thereof Pending CN117534585A (en)

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