Ionizable cationic lipid compound, and preparation method and application thereof

Abstract

The application provides a novel 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 2-4; R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2NHCOCH3; R2 is -H; R3, R4 and R5 are or X is O, S or N heteroatom, n1 is selected from an integer of 1-10, m1 is selected from an integer of 1-10; and R6 is C6-25 alkyl, alkenyl or alkynyl containing or not containing a heteroatom. The novel compound provided by the application has better nucleic acid encapsulation rate and cell or in-vivo transfection rate, and the lipid nanoparticle thereof has the functions of specifically targeting liver, spleen and / or lung.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds, and particularly relates to a novel ionizable cationic lipid compound and a preparation method and application thereof. BACKGROUND

[0002] Nucleic acids are important components of living organisms and play a key role in life activities. Nucleic acid-based nanomedicine is a method for treating or preventing diseases through nucleic acid molecules (including DNA and RNA). DNA includes, for example, plasmids and antisense oligonucleotides (ASO); RNA includes, for example, small interfering RNA (siRNA), endogenous microRNA (miRNA), messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR / Cas9), and RNA aptamer. It can be achieved through gene inhibition, addition, replacement or editing. Benefiting 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 treatment methods have been widely developed and used, such as mRNA-based vaccines, mRNA-1273 (Moderna) and BNT162b2 (Pfizer / BioNTech) applied to prevent the 2019 coronavirus (COVID-19). However, there are still many problems in the current application of nucleic acid therapy, for example, mRNA is a negatively charged hydrophilic macromolecule, its large molecular weight and large number of negative charges make it difficult to penetrate the cell membrane; mRNA also has enzymatic instability, and its single-stranded structure is easily degraded by RNase. Therefore, how to effectively deliver mRNA to target organs or target cells is a key technology to realize its in vivo application. Therefore, there is an urgent need to develop a low-toxicity, high-efficiency, organ / tissue cell-targeting delivery system, especially an ionizable cationic lipid compound and related methods and compositions for non-liver-targeted delivery, to facilitate the delivery of various therapeutic or prophylactic agents outside or inside cells for therapeutic and / or prophylactic purposes. SUMMARY

[0003] To solve the above technical problems, the present application provides a novel ionizable cationic lipid compound and a preparation method and application thereof. The novel ionizable cationic lipid compound provided by the present application can be used for delivering biologically active molecules (such as DNA, siRNA, miRNA, mRNA, polypeptide, protein, etc.), and is particularly suitable for transporting nucleic acid molecules with negative charges, such as DNA, siRNA, mRNA, etc. It provides more choices for the development and application of biologically active molecule delivery and nucleic acid prophylactic and therapeutic agents.

[0004] The novel ionizable cationic lipid compound provided in the first aspect of the present application has the structure shown in Formula I:

[0005]

[0006] wherein n is an integer from 2 to 4; R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH3CH3, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2NHCOCH3; R2 is -H; R3, R4, R5 are X is O, S or N heteroatom, n1 is an integer from 1 to 10, m1 is an integer from 1 to 10; R6 is C6-25 alkyl, alkenyl or alkynyl with or without heteroatom. The novel ionizable cationic lipid compound of the present application has three hydrophobic tail chain structures, compared with the traditional four hydrophobic tail chain structure compounds, it can improve the nucleic acid encapsulation rate in the spatial structure, better cell or in vivo transfection rate, the present application finds the internal law of ionizable cationic lipid compound spatial structure for promoting the transfection rate, most importantly, by changing the unsaturation of the hydrophobic tail chain, heteroatom, branched structure or length can adjust the ionizable cationic lipid compound spatial structure, and make its lipid nanoparticles have the function of specifically targeting liver, spleen and / or lung.

[0007] In a preferred embodiment of the present application, R3, R4, R5 are X is O, S or N heteroatom, n1 is an integer from 1 to 10, m1 is an integer from 1 to 10, n1 and m1 are independent of each other, which can be the same or different; R6 is linear or branched C6-25 alkyl, linear or branched C6-25 alkenyl, linear or branched C6-25 alkynyl, at least one C atom of the alkyl, alkenyl or alkynyl is optionally replaced by a heteroatom independently selected from O, S or N.

[0008] In a preferred embodiment of the present application, n is 2, R1 is -CH3, R2 is -H; preferably, the compound of formula I is wherein R3, R4, R5 are X is O, S or N heteroatom, n1 is an integer from 1 to 10, m1 is an integer from 1 to 10, n1 and m1 are independent of each other, which can be the same or different; R6 is linear or branched C6-25 alkyl, linear or branched C6-25 alkenyl, linear or branched C6-25 alkynyl, at least one C atom of the alkyl, alkenyl or alkynyl is optionally replaced by a heteroatom independently selected from O, N, S, S-S or Se.

[0009] As some preferred embodiments of the present application, the compound of formula I is n is 2, R1 is -CH3, R2 is -H; wherein R3, R4, R5 are R6 is a straight or branched C6-25 alkyl, straight or branched C6-25 alkenyl, at least one C atom of said alkyl, alkenyl is optionally replaced by a heteroatom independently selected from O, S or N; preferably, X is O or N heteroatom; further preferably, n1 is an integer selected from 4 to 8, m1 is an integer selected from 4 to 8, R6 is a straight or branched C6-18 alkyl, straight or branched C6-18 alkenyl, at least one C atom of said alkyl, alkenyl is optionally replaced by a heteroatom independently selected from S or S-S.

[0010] In a preferred embodiment of the present application, 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:

[0011]

[0012] wherein each n2 in each branch is independently selected from an integer from 1 to 8, preferably from 4 to 8, each m2 is independently selected from an integer from 1 to 8, preferably from 4 to 8, and each Y is independently selected from an integer from 0 to 3; preferably, each n2 is selected from an integer from 4 to 8, each m2 is selected from an integer from 4 to 8, and each Y is independently selected from an integer from 0 to 2.

[0013]

[0014] wherein each n3 in each branch is independently selected from an integer from 1 to 18, preferably from 6 to 18; preferably, each n3 is selected from an integer from 6 to 18.

[0015]

[0016] wherein each n4 in each branch is independently selected from an integer from 1 to 18, preferably from 2 to 18; preferably, each n4 is selected from an integer from 2 to 18.

[0017]

[0018] wherein each n5 in each branch is independently selected from an integer from 1 to 18, preferably from 4 to 18, and each m5 is independently selected from an integer from 1 to 18, preferably from 1 to 14; preferably, each n5 is selected from an integer from 4 to 18, and each m5 is selected from an integer from 1 to 14.

[0019]

[0020] The present application provides a new ionizable lipid compound with R2 being -H while optimizing the remaining three hydrophobic tail chains. This specific new compound has a higher cell transfection rate than the existing four hydrophobic tail chain molecular structure compound, which may be related to the different configuration / conformation, stereochemical structure, etc. For example, the hydrophilic head group is more likely to form an ionizable positively charged hydrophilic plane to interact with the negatively charged mRNA to increase the loading capacity of the lipid nanoparticle for mRNA. In the slightly acidic lysosomal microenvironment, the compound with three hydrophobic tail chains tends to form a conical molecular structure, which can promote the hexagonal conversion of the cell membrane and lysosomal escape. And on this basis, optimizing the X group or hydrophobic tail chain (including unsaturation, heteroatom, branched structure and / or alkyl chain length) can provide a non-hepatic targeting delivery lipid compound and delivery system with more excellent specific targeting effect, and more efficient delivery of nucleic acids to target organs.

[0021] The present application also provides a method for synthesizing the new ionizable cationic lipid compound. The new ionizable cationic lipid compound of the present application can be synthesized by using existing methods in the art, for example, by reacting one equivalent or more of an amine (hydrophilic polar head containing an amine group) with three equivalents or more of a hydrophobic lipid tail chain compound under suitable conditions. The synthesis of the ionizable lipid compound can be carried out with or without a solvent, and the synthesis can be carried out at a relatively high temperature in the range of 25-120°C. The ionizable cationic lipid compound obtained can be optionally purified. For example, a mixture of ionizable cationic lipid compounds can be purified to obtain a specific ionizable cationic lipid compound, such as a product containing three hydrophobic lipid tail chains. The hydrophobic lipid tail chain compound can be purchased commercially or synthesized.

[0022] In some embodiments of the present application, a method for preparing the new ionizable cationic lipid compound is provided, comprising:

[0023] Compound synthesis route:

[0024]

[0025] (wherein each n2 on each branch is independently selected from an integer of 1-8, preferably an integer of 4-8, each m2 is independently selected from an integer of 1-8, preferably an integer of 4-8, and each Y is independently selected from an integer of 0-3; preferably, each n2 is selected from an integer of 4-8, each m2 is selected from an integer of 4-8, and each Y is independently selected from an integer of 0-2)

[0026] Specifically comprising the following steps:

[0027] 1) esterification: esterification of the hydroxyl group of compound B1 into an ester group in the presence of acryloyl chloride to obtain compound B2;

[0028] 2) Michael addition: Michael addition of compound B2 with an amine (e.g., N-methyl-2,2'-diaminodiethylamine) to obtain the novel ionizable cationic lipid compound.

[0029] In some embodiments of the present application, the provided method for preparing the novel ionizable cationic lipid compound comprises:

[0030] Compound synthesis route:

[0031]

[0032] (wherein each n3 on each branch is independently selected from an integer of 1 to 18, preferably an integer of 6 to 18; preferably, each n3 is selected from an integer of 6 to 18.)

[0033] Specifically comprising the following steps:

[0034] 1) esterification: esterification of the hydroxyl group of compound B1 into an ester group in the presence of acryloyl chloride to obtain compound B2;

[0035] 2) Michael addition: Michael addition of compound B2 with an amine (e.g., N-methyl-2,2'-diaminodiethylamine) to obtain the novel ionizable cationic lipid compound.

[0036] In some embodiments of the present application, the provided method for preparing the novel ionizable cationic lipid compound comprises:

[0037] Compound synthesis route:

[0038]

[0039] (wherein each n4 on each branch is independently selected from an integer of 1 to 18, preferably an integer of 2 to 18; preferably, each n4 is selected from an integer of 2 to 18.)

[0040] Specifically comprising the following steps:

[0041] 1) esterification: esterification of the hydroxyl group of compound B1 into an ester group in the presence of acryloyl chloride to obtain compound B2;

[0042] 2) Michael addition: Michael addition of compound B2 with an amine (e.g., N-methyl-2,2'-diaminodiethylamine) to obtain the novel ionizable cationic lipid compound.

[0043] In some embodiments of the present application, the preparation method of the novel ionizable cationic lipid compound provided comprises:

[0044] Compound synthesis route:

[0045]

[0046] (wherein each n5 in each branch is independently selected from an integer of 1-18, preferably an integer of 4-18, and each m5 is independently selected from an integer of 1-18, preferably an integer of 1-14; preferably, each n5 is selected from an integer of 4-18, and each m5 is selected from an integer of 1-14)

[0047] Specifically comprising the following steps:

[0048] 1) esterification: esterifying the hydroxyl group of compound D1 into an ester group in the presence of acryloyl chloride to obtain compound D2;

[0049] 2) Michael addition: subjecting compound D2 to Michael addition with an amine (for example, N-methyl-2,2'-diaminodiethylamine) to obtain the novel ionizable cationic lipid compound.

[0050] According to the present application, examples of the solvent used in the esterification reaction include, but are not limited to, halogenated hydrocarbons (such as dichloromethane, dichloroethane, and trichloromethane, etc.), hydrocarbons (such as n-pentane, benzene, and toluene, etc.), nitriles (such as acetonitrile, etc.), and mixed solvents formed by two or more of these solvents. The Michael addition reaction can be selected to use or not to use a solvent, and examples of the solvent used in the reaction include, but are not limited to, isopropyl alcohol, tert-butyl alcohol, tetrahydrofuran, etc. The amine can be N-methyl-2,2'-diaminodiethylamine.

[0051] According to the present application, the raw materials in the preparation method can be commercially available or synthesized by conventional methods.

[0052] According to the present application, the novel ionizable cationic lipid molecule structure provided contains two adjacent cis double bonds, or a disulfide bond (S-S), a branched structure, or a straight chain structure, which makes it have a higher encapsulation rate and a better cell transfection rate when subsequently applied to a delivery system for wrapping active substances (such as mRNA); in addition, the presence of two adjacent cis double bonds, a disulfide bond (S-S), or a branched structure in the tail chain can make the obtained lipid nanoparticles more uniform in size. The ionizable lipid compound of the present application is particularly suitable for preparing nanoparticles with a solid structure.

[0053] The present application also provides the use of the novel ionizable cationic lipid compound in the preparation of a biological active substance delivery system; preferably, the delivery system is a microbubble, a microparticle, a nanoparticle, a liposome, or a lipid nanoparticle.

[0054] In some preferred embodiments of the present application, when the novel ionizable cationic lipid compound is a compound of Formula I wherein, R3, R4, R5are R6is a linear or branched C6-25alkyl, a linear or branched C6-25alkenyl, at least one C atom of the alkyl, alkenyl is optionally replaced by a heteroatom independently selected from O, S or N; preferably, X is O or N heteroatom; further preferably, n1is an integer selected from 4-8, m1is an integer selected from 4-8, R6is a linear or branched C6-18alkyl, a linear or branched C6-18alkenyl, 2 C atoms of the alkyl, alkenyl are optionally replaced by a heteroatom independently selected from S atom. Preferably, X is O atom; further preferably, n1is an integer selected from 4-8, m1is an integer selected from 4-8, R6is a linear or branched C6-18alkyl, a linear or branched C6-18alkenyl, 2 C atoms of the alkyl, alkenyl are optionally replaced by a heteroatom independently selected from S atom.

[0055] In the present application, the intrinsic law of the spatial structure of the novel ionizable cationic lipid compound for promoting transfection rate is unexpectedly discovered by optimizing the structure of the compound, and most importantly, the spatial structure of the ionizable cationic lipid compound can be adjusted to promote its in vivo cell transfection rate by changing the unsaturation, heteroatom, branched structure or length of the hydrophobic tail chain, and correspondingly make the lipid nanoparticles have the function of specifically targeting liver, spleen and / or lung.

[0056] In a preferred embodiment of the present application, when the novel ionizable cationic lipid compound is a compound of Formula A, Formula B, the novel ionizable cationic lipid compound is used in the preparation of a specific biological active substance delivery system for specifically targeting the spleen; 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 biological active substance delivery system for specifically targeting the spleen and lung; 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 biological active substance delivery system for specifically targeting the spleen and liver.

[0057] According to the preferred embodiments of the present application, the present application employs preferred compounds of Formula A, especially with a hydrophobic tail length of 18C and containing a structure of adjacent cis double bonds, the presence of which can make the hydrophobic tail begin to bend at the C9 position to form a relatively long alkane chain of about 9C, and the structure of the three hydrophobic tails makes the compound of Formula A form a unique trigonal pyramidal spatial structure, which can more favorably bind the ionizable lipid compound to mRNA and improve the lysosomal escape rate. For example, compound N24-O18-2(3T), compared with N24-O18(3T) compound, can greatly improve the effect of delivering mRNA and specifically target the lipid nanoparticles to the spleen.

[0058] Further preferably, in some embodiments of the present application, the novel ionizable cationic lipid compound is preferably:

[0059] N24-O18-2(3T)

[0060] According to the preferred embodiments of the present application, the present application employs preferred compounds of Formula B, which can more favorably target the lipid nanoparticles to the spleen when the length of the hydrophobic tail is between 8-10C. For example, compound N24-O10(3T), compared with N24-O18(3T) compound, can greatly improve the transfection efficiency of mRNA and improve the effect of targeting the spleen.

[0061] Further preferably, in some embodiments of the present application, the novel ionizable cationic lipid compound is preferably:

[0062] N24-O6(3T)

[0063] N24-O8(3T)

[0064] N24-O10(3T) N24-O12(3T) N24-O14(3T) N24-O16(3T) N24-O18(3T)

[0065] Further preferably, in some embodiments of the present application, the novel ionizable cationic lipid compound is further preferably:

[0066] N24-O10(3T)

[0067] According to the preferred embodiments of the present application, the introduction of the heteroatom S-S in the hydrophobic tail chain can make the hydrophobic tail chain bend at the S-S position. This structure makes the plane formed by the hydrophilic head group larger, which can more easily increase the loading of mRNA molecules. For example, compound N24-O18-SS(3T) can greatly improve the delivery of mRNA compared to compound N24-O18(3T), and can be beneficial for simultaneous targeted delivery to the spleen and lungs. The lipid nanoparticles of N24-O18-SS(3T)-LNP have better effects.

[0068] Further preferably, in some embodiments of the present application, the novel ionizable cationic lipid compound is:

[0069] N24-O6-SS(3T) N24-O8-SS(3T) N24-O10-SS(3T) N24-O12-SS(3T)

[0070] N24-O14-SS(3T)

[0071] N24-O16-SS(3T)

[0072] N24-O18-SS(3T)

[0073] N24-O6-S(3T)

[0074] N24-O8-S(3T)

[0075] N24-O10-S(3T)

[0076] N24-O12-S(3T)

[0077] N24-O14-S(3T)

[0078] N24-O16-S(3T)

[0079] N24-O18-S(3T)

[0080] N24-O6-O(3T)

[0081] N24-O8-O(3T)

[0082] N24-O10-O(3T)

[0083] N24-O12-O(3T)

[0084] N24-O14-O(3T)

[0085] N24-O16-O(3T)

[0086] N24-O18-O(3T)

[0087] N24-O6-N(3T)

[0088] N24-O8-N(3T)

[0089] N24-O10-N(3T)

[0090] N24-O12-N(3T)

[0091] N24-O14-N(3T)

[0092] N24-O16-N(3T)

[0093] N24-O18-N(3T)

[0094] N24-O6-Se(3T)

[0095] N24-O8-Se(3T)

[0096] N24-O10-Se(3T)

[0097] N24-O12-Se(3T)

[0098] N24-O14-Se(3T)

[0099] N24-O16-Se(3T)

[0100] N24-O18-Se(3T)

[0101] According to a preferred embodiment of the present application, the present application employs preferred compound of Formula D, the branched structure in the hydrophobic tail chain of which makes the spatial stereostructure of the molecule more rigid, so that the hydrophilic head group exposed at one end can better contact and load mRNA. As compound N24-O11B(3T), compared with N24-O18(3T) compound, it can greatly improve the effect of delivering mRNA, and can be beneficial to simultaneously target delivery to the liver and spleen.

[0102] Further preferably, in some specific embodiments of the present application, the novel ionizable cationic lipid compound is preferably:

[0103]

[0104]

[0105] In the present application, it can be found that by adjusting the hydrophobic tail chain of the compound of the present application, such as unsaturation (the length of the hydrophobic tail chain is 18C and contains three adjacent, two cis double bonds or a single double bond), straight chain length (the length of the hydrophobic tail chain is 8C-18C straight chain alkane), heteroatom (-O-, -N-, -SS-, -S-, -Se-) and branched structure (one chain is C5-C18 length, the other branched chain is C1-C14 length), so that it obtains a specific spatial stereostructure, which can more favorably promote its ability to deliver mRNA and the ability to specifically target organs and tissues. The novel compounds provided by the above specific embodiments of the present application have better nucleic acid encapsulation rate and cell or in vivo transfection rate, and the lipid nanoparticles thereof have better functions of specifically targeting liver, spleen and / or lung.

[0106] In the present application, it has been further found through research that when the compound of the present application adopts a specific stereostructure, under the action of unsaturation, heteroatom, branched structure and straight chain length of the compound, it can more favorably promote its ability to deliver RNA.

[0107] In a preferred embodiment of the present application, the delivery system is a lipid nanoparticle.

[0108] In certain embodiments, all of the amines of the amine are fully reacted with the hydrophobic lipid tail compound to form a tertiary amine. In other embodiments, not all of the amines of the amine are fully reacted with the hydrophobic lipid tail compound, thereby resulting in primary or secondary amines in the ionizable cationic lipid compound. These primary or secondary amines can be left as is or can be reacted with another electrophile, such as a different hydrophobic lipid tail compound. It is within the skill in the art to react excess amines with hydrophobic lipid tail compounds to result in a variety of different ionizable lipid compounds having a variety of tail numbers. For example, a diamine or a polyamine can include one, two, three, or four tail compounds on various amine moieties of the molecule, thereby resulting in primary, secondary, and tertiary amines. In certain embodiments, the same identical tail compound is used; or two of the same type of tail compound is used. In other embodiments, two or more different tail compounds are used.

[0109] The present application also provides a bioactive agent delivery system comprising the novel ionizable cationic lipid compound. Preferably, the delivery system is a microvesicle, microparticle, nanoparticle, liposome, or a lipid nanoparticle.

[0110] In one embodiment of the present application, the delivery system is a lipid nanoparticle. Such lipid nanoparticles can efficiently deliver a bioactive agent (e.g., mRNA) into a cell, tissue, or organ, enabling efficient modulation of the bioactive agent. In the present application, the novel ionizable cationic lipid compound is combined with a bioactive agent (e.g., mRNA) targeted for delivery to a cell or an organ, or further comprises other agents (e.g., other anionic, cationic, or ionizable lipid compounds, synthetic or natural polymers, proteins, phospholipids, cholesterol, carbohydrates, surfactants, etc.) to form a microvesicle, microparticle, nanoparticle, liposome, or a lipid nanoparticle. The bioactive agent can be in a gaseous, liquid, or solid form, and can be a nucleotide, a small molecule compound, a polypeptide, or a protein. In the present application, the delivery system can then be optionally combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0111] The present application also provides a pharmaceutical composition comprising the bioactive agent delivery system.

[0112] In another aspect, the present application also provides a lipid nanoparticle composition comprising a lipid nanoparticle comprising the novel ionizable cationic lipid compound.

[0113] According to the present application, the lipid nanoparticle composition further comprises other lipid molecules. The other lipid molecules can be lipid molecules known or conventionally used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, lipid-like molecules, cholesterol, PEGylated lipid molecules.

[0114] According to the present application, the lipid nanoparticle composition, when used in a drug delivery system, can encapsulate a pharmaceutical agent, including nucleotides, small molecule compounds, polypeptides, proteins, or metals, etc.

[0115] The nucleic acid includes but is not limited to DNA, antisense oligonucleotide (ASO), small interfering RNA (siRNA), endogenous microRNA (miRNA), messenger RNA (mRNA), RNA aptamer, small activating RNA (saRNA), etc. The novel ionizable cationic lipid compound has several properties suitable for the preparation of a drug delivery system: 1) the ability to neutralize the charge on a negatively charged active substance; 2) the ability to complex and protect unstable pharmaceutical agents; 3) the ability to buffer the pH in the body; 4) the ability to act as a "proton sponge" and cause dissolution in the body.

[0116] According to some preferred embodiments of the present application, in the lipid nanoparticle composition, the lipid nanoparticle contains: 20-60 mol% of the novel ionizable cationic lipid compound of formula I, 5-30 mol% of neutral lipid molecules, 30-60 mol% of cholesterol lipid molecules, 0.5-5 mol% of PEGylated lipid molecules; preferably contains 20-50 mol% of ionizable cationic lipid molecules, 5-20 mol% of neutral lipid molecules, 30-50 mol% of cholesterol lipid molecules, 0.5-2.5 mol% of PEGylated lipid molecules; more preferably contains 35-48 mol% of ionizable cationic lipid molecules of formula I, 10-15 mol% of neutral lipid molecules, 35-50 mol% of cholesterol lipid molecules, 1.0-2.0 mol% of PEGylated lipid molecules.

[0117] According to some preferred embodiments of the present application, the molar percentage of the ionizable lipid molecules of formula I in the lipid of the lipid nanoparticle is 20-60 mol%.

[0118] According to some preferred embodiments of the present application, the neutral lipid molecules are uncharged lipid molecules or zwitterionic lipid molecules, such as phosphatidylcholine compounds, or / and phosphatidylethanolamine compounds.

[0119] According to some preferred embodiments of the present application, the neutral lipid molecules are selected from phosphatidylcholine compounds and / or phosphatidylethanolamine compounds.

[0120] According to some preferred embodiments of the present application, examples of neutral lipid molecules include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (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-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, and combinations thereof.

[0121] In one embodiment, the neutral lipid molecule can be selected from the group consisting of distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and dioleoylphosphatidylethanolamine (DOPE). In another embodiment, the neutral lipid molecule can be dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule can be dimyristoylphosphatidylcholine (DMPC).

[0122] According to the present application, the molar percentage of the neutral lipid molecule in the lipid of the lipid nanoparticle is 5-30 mol%.

[0123] According to the present application, the cholesterol lipid molecule includes, among others, steroids and sterols, examples of which include, but are not limited to, cholesterol and cholesterol hemisuccinate.

[0124] According to some preferred embodiments of the present application, the cholesterol lipid molecule is selected from one or more of cholesterol and cholesterol hemisuccinate.

[0125] In one embodiment, the cholesterol lipid molecule is cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate.

[0126] According to the present application, the molar percentage of the cholesterol lipid molecule in the lipid of the lipid nanoparticle is 30-60 mol%.

[0127] According to some preferred embodiments of the present application, the PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymeric moiety, denoted as the number average molecular weight of the lipid moiety-PEG, the lipid moiety comprises one or more of a diacylglycerol and / or a diacylglycamide, preferably selected from the group consisting of dilauric glycerol, dimyristic glycerol, dilauric glycerol amide, dimyristic glycerol amide, l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the PEG has a number average molecular weight of 100 to 50,000, preferably 200 to 10,000, further preferably 500 to 3,000, most preferably 1,500 to 2,500.

[0128] According to the present application, the PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymeric moiety. In some embodiments, the lipid moiety can be derived from a diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently comprising from about C4 to about C30 saturated or unsaturated carbon atoms, wherein the chain can comprise one or more functional groups, such as amide or ester. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain length can be symmetric or asymmetric. In one embodiment, the PEG moiety comprises a PEG copolymer, such as a 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 comprise a PEG copolymer, e.g., it can be a PEG monomer. In one embodiment, the PEG has a molecular weight of from about 100 to about 50,000. In certain embodiments, the PEG is "PEG 2000", which has an average molecular weight of about 2,000 Daltons.

[0129] In some embodiments of the present application, the PEG is represented by the following formula PEG-2000, wherein n is 45, means that the index average degree of polymerization comprises about 45 subunits; other PEG embodiments known in the art can also be used, including, for example, those wherein the number average degree of polymerization comprises about 23 subunits (n = 23) and / or 68 subunits (n = 68). In some embodiments, n can range from about 30 to about 60. In some embodiments, n can range from about 35 to about 55. In some embodiments, n can range from about 40 to about 50. In some embodiments, n can range from about 42 to about 48. In some embodiments, n can be 45. In some embodiments, R can be selected from the group consisting of H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R can be an unsubstituted C1-C30 alkyl, for example, a C1-C20 alkyl, a C1-C10 alkyl, a C1-C6 alkyl. In some embodiments, R can be H, methyl, or ethyl.

[0130] In some embodiments, a PEGylated lipid molecule can be represented as "lipid moiety-PEG-number average molecular weight" or "PEG-number average molecular weight-lipid moiety" or "PEG-lipid moiety". The lipid moiety is a diacylglycerol or diacylglycerol amide selected from the group consisting of dilauric glycerol, dimyristyl glycerol, dilauric glycerol amide, dimyristyl glycerol amide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of the PEG is from about 100 to about 50,000.

[0131] In some embodiments, the PEGylated lipid molecule can be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dilaurylglyceroamide, PEG-dimyristylglyceroamide, PEG-distearylglycerol (PEG-DSPE) and PEG-distearylglyceroamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]-yloxy)formamido-3',6'-dioxa octyl]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-PEG2000), 1,2-distearylsn-glycero-methoxypolyethylene glycol (DSG-PEG2000), 1,2-distearylsn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), poly(ethylene glycol)-2000-dimethacrylate (DMA-PEG2000), and 1,2-distearyloxypropyl-3-amine-N- [methoxy(polyethylene glycol)-2000] (DSA-PEG2000). In one embodiment, 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 can 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.

[0132] According to the present application, the PEGylated lipid molecule is present in the lipid of the lipid nanoparticle in a molar percentage of 0.5-5 mol%.

[0133] In some embodiments of the present application, the lipid nanoparticle contains an ionizable cationic lipid molecule of formula D, a neutral lipid molecule, a cholesterol lipid molecule, a PEGylated lipid molecule, wherein:

[0134]

[0135] wherein each n5 is independently selected from an integer of 1 to 18, preferably an integer of 4 to 18, and each m5 is independently selected from an integer of 1 to 18, preferably an integer of 1 to 14; preferably, each n5 is selected from an integer of 4 to 18, and each m5 is selected from an integer of 1 to 14; the ionizable cationic lipid molecule of Formula D is present in the lipid nanoparticle at a molar percentage of 20-60 mol%, preferably 35-50 mol% of the lipid;

[0136] the neutral lipid molecule is selected from the group consisting of a phosphatidylcholine compound, a phosphatidylethanolamine compound; the neutral lipid molecule is present in the lipid nanoparticle at a molar percentage of 5-30 mol%, preferably 5-20 mol%, more preferably 10-15 mol% of the lipid;

[0137] the cholesterolic lipid molecule is selected from the group consisting of cholesterol, cholesterol hemisuccinate; the cholesterolic lipid molecule is present in the lipid nanoparticle at a molar percentage of 30-60 mol%, preferably 30-50 mol%, more preferably 35-48 mol% of the lipid;

[0138] The PEGylated lipid molecule represents a "lipid moiety-PEG-number average molecular weight", the lipid moiety is a diacylglycerol or a diacylglycerol amide selected from the group consisting of dilaurin, dimyristin, distearin, dilaurinamide, dimyristinamide, distearinamide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of the PEG is from about 100 to about 50,000. The PEGylated lipid molecule is present in the lipid nanoparticle at a molar percentage of 0.5-5 mol%, preferably 0.5-2.5 mol%, more preferably 1.0-2.0 mol% of the lipid.

[0139] In some embodiments of the present application, the ionizable cationic lipid molecule of Formula D, the neutral lipid molecule, the cholesterolic lipid molecule, and the PEGylated lipid molecule are present in a molar ratio of 35:15:48.5:1.5.

[0140] In some embodiments of the present application, the ionizable cationic lipid molecule of Formula D, the neutral lipid molecule, the cholesterolic lipid molecule, and the PEGylated lipid molecule are present in a molar ratio of 45:15:38.5:1.5.

[0141] In some embodiments of the present application, the ionizable cationic lipid molecule of Formula D, the neutral lipid molecule, the cholesterolic lipid molecule, and the PEGylated lipid molecule are present in a molar ratio of 40:10:48.5:1.5.

[0142] In one embodiment of the present application, the ionizable cationic lipid molecule of Formula D is compound N24-O11B(3T).

[0143] In one embodiment of the present application, 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.

[0144] The tri-hydrophobic tail chain ionizable lipid compounds with adjacent cis double bond structure, or disulfide bond (S-S), or branched structure provided by the present application can provide higher active substance encapsulation rate, better cell or in vivo transfection rate, especially suitable for preparing solid structure nanoparticles, and in the lipid nanoparticle composition, the molar % ratio of the ionizable cationic lipid molecule of Formula I, the neutral lipid molecule, the cholesterol lipid molecule, and the PEGylated lipid molecule in the total lipid molecule is optimized, and most importantly, the above-mentioned lipid nanoparticles have better specific targeting function to the spleen and / or lung.

[0145] The present application also provides a method for preparing a lipid nanoparticle composition, comprising: dissolving each of the above lipid molecules in an organic solvent to form a mixed lipid solution according to the molar ratio, using the mixed lipid solution as the organic phase, using an aqueous solution of the delivery substance (e.g., mRNA) as the aqueous phase, mixing the organic phase and the aqueous phase to prepare the lipid nanoparticles. The lipid nanoparticles can be prepared by methods known to those skilled in the art, including but not limited to spray drying, solvent extraction, phase separation, nanoprecipitation, single and double emulsion solvent evaporation, microfluidics, simple and complex coacervation, and other methods.

[0146] In some embodiments, the organic solvent is an alcohol, such as ethanol.

[0147] In some embodiments, the volume ratio of the organic phase and the aqueous phase is (2-5): 1, for example 3:1.

[0148] In some embodiments, the nanoparticles are prepared using a microfluidic platform.

[0149] According to the present application, the preparation method further comprises the steps of isolating and purifying the lipid nanoparticles.

[0150] According to the present application, the preparation method further comprises the step of lyophilizing the lipid nanoparticles.

[0151] The particle size of the lipid nanoparticles in the present application ranges from 1 nm to 1000 nm.

[0152] The delivery system formed by the novel ionizable cationic lipid compound of the present application can also modify the targeting molecule to make it a targeting agent capable of targeting specific cells, tissues or organs. The targeting molecule can be included in the entire delivery system or can be located only on the surface thereof. The targeting molecule can be a small molecule, a nucleic acid, a polypeptide, a protein, a glycoprotein, a lipid, etc., examples of which include, but are not limited to, an antibody, an antibody fragment, low density lipoprotein (LDL), sialic acid, an aptamer, transferrin, asialoglycoprotein, a receptor ligand, etc.

[0153] The active substance delivered by the delivery system formed by the ionizable lipid compound of the present application can be a therapeutic agent, a diagnostic agent or a prophylactic agent. The nature of the active substance can be a small molecule compound, an isotope-labeled compound, a nucleic acid, a polypeptide, a protein, a vaccine, a metal, etc.

[0154] The delivery system formed by the novel ionizable cationic lipid compound of the present application can 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, celluloses and derivatives thereof, 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; 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; coloring, sweetening, flavoring and perfuming agents, preservatives and antioxidants; buffers, such as phosphate buffered saline, citrate buffer, etc.

[0155] The nucleic acid drug delivery system provided by the present application can efficiently and specifically deliver nucleic acid drug molecules to the spleen and / or lung and effectively translate them into target molecules, while reducing the side effects of liposome accumulation in the liver, and has important significance for the targeted administration of nucleic acid drugs and the development and application thereof.

[0156] The term "about" is used in connection with a value to indicate an interval of accuracy familiar to and acceptable by one of ordinary skill in the art. Typically, such an interval of accuracy is ±10%. BRIEF DESCRIPTION OF DRAWINGS

[0157] In order to more clearly illustrate the technical solutions of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0158] Figure 1 The hydrogen spectrum of compound acrylic acid-1-ethyl nonyl ester (a2) in the embodiments of the present application;

[0159] Figure 2 The hydrogen spectrum of compound N24-O11B (3T) in the embodiments of the present application;

[0160] Figure 3 The mass spectrum of compound N24-O11B (3T) in the embodiments of the present application;

[0161] Figure 4 The hydrogen spectrum of compound N24-O11B (4T) in the embodiments of the present application;

[0162] Figure 5 The mass spectrum of compound N24-O11B (4T) in the embodiments of the present application;

[0163] Figure 6 The dissociation constant (pKa) diagram of compound N24-O11B (3T) (A) and N24-O11B (4T) (B) in the embodiments of the present application;

[0164] Figure 7 The expression amount of Luciferase protein after transfection of 293T cells with LNP encapsulating LuciferasemRNA (LucRNA) prepared by N24-O11B (3T) and N24-O11B (4T) in the embodiments of the present application;

[0165] Figure 8 The expression amount of Luciferase protein after transfection of 293T cells with LNP encapsulating LuciferasemRNA (LucRNA) prepared by N24-O11B (3T) and ALC-0315 in the embodiments of the present application;

[0166] Figure 9 The fluorescence expression of each organ after intravenous injection of N24-O18-2 (3T) / LucRNA, N24-O10 (3T) / LucRNA, N24-O18-SS (3T) / LucRNA and N24-O11B (3T) / LucRNA in mice in the embodiments of the present application;

[0167] Figure 10 Hydrogen spectrum of compound N24-O10 (3T) in the embodiment of the present application;

[0168] Figure 11 Mass spectrum of compound N24-O10 (3T) in the embodiment of the present application;

[0169] Figure 12 Mass spectrum of compound N24-O18-2 (3T) in the embodiment of the present application;

[0170] Figure 13 Hydrogen spectrum of compound N24-O18-2 (3T) in the embodiment of the present application;

[0171] Figure 14 Hydrogen spectrum of compound N24-O18-SS (3T) in the embodiment of the present application;

[0172] Figure 15 Mass spectrum of compound N24-O18-SS (3T) in the embodiment of the present application;

[0173] Figure 16 Luciferase protein expression amount chart after 293T cells are transfected with LNP encapsulated Luciferase mRNA (LucRNA) prepared in the embodiment of the present application N24-O18 (3T), N24-O16 (3T), N24-O14 (3T), N24-O12 (3T), N24-O10 (3T), N24-O8 (3T), N24-O6 (3T);

[0174] Figure 17 Luciferase protein expression amount chart after 293T cells are transfected with LNP encapsulated Luciferase mRNA (LucRNA) prepared in the embodiment of the present application N24-O10 (3T), N24-O18-2 (3T), N24-O18-SS (3T), N24-O11B (3T). DETAILED DESCRIPTION

[0175] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below with reference to the drawings in the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0176] It should be understood that the following examples are merely illustrative and explanatory in nature and are not to be construed as limiting the scope of the present application. Any technical based on the above description of the present application is included in the scope intended to be protected by the present application. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Unless otherwise specified, the specific techniques or conditions in the examples are carried out according to the conventional methods or the techniques or conditions described in the literature of the art or according to the product instructions. The reagents and instruments used are not specified by the manufacturer, which are conventional products that can be purchased through regular channels.

[0177] Synthesis of Example 1 ionizable cationic lipid, N24-O11B(3T)

[0178]

[0179] Synthesis of 1-ethylnonyl acrylate (a2): To 30.0 mL of dichloromethane was added 3-undecanol (3.0 g), triethylamine (5.28 g) at 0 °C, followed by dropwise addition of a solution of acryloyl chloride (2.36 g) in dichloromethane (10.0 mL) to the reaction system, and the reaction solution was stirred at 20 °C for 2 h. The triethylamine salt precipitated in the reaction solution was filtered with a Buchner funnel, and the filtrate was washed with water, 5% hydrochloric acid by mass fraction, and water in turn, and the organic phase was dried over anhydrous sodium sulfate. Evaporated to dryness, and then the residue was purified by flash column chromatography eluted with petroleum ether / ethyl acetate to obtain the target product 1-ethylnonyl acrylate (a2) (2.8 g), yield 70%, the hydrogen spectrum of compound a2 is shown in Figure 1 .

[0180] 1 H NMR (400 MHz, CDC13) δ 6.39 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.80 (dd, J = 10.4, 1.5 Hz, 1H), 4.94 - 4.84 (m, 1H), 1.69 - 1.48 (m, 4H), 1.26 (s, 12H), 0.88 (q, J = 7.2 Hz, 6H).

[0181] Synthesis of N24-011B(3T), N24-011B(4T) (for comparison): To a solution of 250 mg N-methyl-2,2'-diaminodiethylamine was added 1-ethyl nonyl acrylate (1.7 g) at room temperature, then the mixture was heated to 120 °C and stirred for 24 h. When the reaction was finished, the crude product 2.0 g was obtained by TLC plate test, then the target product was purified by flash column method eluted with dichloromethane / methanol to give N24-011B(3T) 276 mg, N24-011B(4T) 195 mg. The hydrogen spectrum of compound N24-011B(3T) is shown in Figure 2 , the mass spectrum is shown in Figure 3 , the hydrogen spectrum of compound N24-011B(4T) is shown in Figure 4 , the mass spectrum is shown in Figure 5 .

[0182] N24-011B(3T):

[0183] 1 H NMR (400 MHz, CDC13) δ 4.90 - 4.72 (m, 3H), 2.92 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 7.3 Hz, 4H), 2.71 (t, J = 5.9 Hz, 2H), 2.61 - 2.49 (m, 6H), 2.45 (dd, J = 16.1, 8.6 Hz, 6H), 2.23 (s, 3H), 1.55 (ddd, J = 14.2, 9.7, 5.1 Hz, 12H), 1.26 (s, 36H), 0.95 - 0.82 (m, 18H).

[0184] MALDI-TOF MS: m / z 796.753 [M+H] + .

[0185] N24-011B(4T):

[0186] 1 H NMR (400 MHz, CDC13) δ 4.81 (p, J = 6.3 Hz, 4H), 2.81 (dd, J = 17.9, 10.6 Hz, 8H), 2.56 (s, 4H), 2.41 (dd, J = 32.9, 25.7 Hz, 12H), 2.24 (s, 2H), 1.63 - 1.46 (m, 16H), 1.28 (d, J = 14.6 Hz, 48H), 0.91 - 0.84 (m, 24H).

[0187] MALDI-TOF MS: m / z 1022.924 [M+H] + .

[0188] Dissociation constant (pKa) of ionizable cationic lipid N24-O11B(3T) of Example 2

[0189] Ionizable lipids have two main roles: binding nucleic acids and allowing the release of nucleic acid molecules in cells. The pKa of the lipid is an important factor because the lipid needs to be positively charged at low pH to bind to nucleic acids, but not charged at neutral pH, so that the LNP does not cause toxicity. As Figure 6 The pKa of ionizable lipid N24-O11B(3T) was determined by TNS dye binding assay at 6.68 (A), and the pKa of N24-O11B(3T) was 5.93 (B). It can be seen that both molecules can be positively charged under acidic conditions and RNA loading, and not charged at neutral pH (pH = 7.4).

[0190] Example 3 Preparation of mRNA encapsulated lipid nanoparticles with N24-O11B(3T)

[0191] Ionizable cationic lipid N24-O11B(3T) or N24-O11B(4T), DOPE, Cholesterol and DMG-PEG2000 were prepared as an organic phase in ethanol solution according to the molar ratio of 45%:15%:38.5%:1.5%, and Luciferase mRNA (LucRNA) was dissolved in water solution at pH = 4 as an aqueous phase. According to the volume ratio of the aqueous phase and the organic phase of 3:1, the nanoparticle suspension was prepared by microfluidic technology on the nanomedicine manufacturing instrument (Mianana). After preparation, the final LucRNA-LNP lipid nanoparticles were obtained by ultrafiltration concentration and stored at 2-8℃ for standby.

[0192] Zetasizer Pro Nanoparticle Size Potential Instrument (Malvern Panalytical) was used to characterize the particle size and Zeta potential of LucRNA-LNP. The F-280 fluorescence spectrophotometer (Tianjin Gangdong) Ribogreen method was used to detect the encapsulation efficiency of LucRNA-LNP. The detection results of Example 3 are shown in Table 1.

[0193] Table 1 Physicochemical quality control data of lipid nanoparticles

[0194]

[0195] From the results of Example 3, it can be seen that the lipid nanoparticles LucRNA-LNP prepared from the new lipid compound N24-O11B(3T) have a particle size of about 140 nm, a narrow particle size distribution (small PDI), and an encapsulation efficiency of up to 96.68%.

[0196] In addition, the transfection efficiency of the prepared LucRNA-LNP cells was detected by the luciferase reporter gene method using a multifunctional enzyme label instrument (BioTek, model SLXFATS). The method for in vitro transcription of LucRNA was as follows: 293T cells were plated at a cell density of 1 x 10 4 cells / well, and transfection was performed when the cell confluence was 30%-50%. 1.0 μg of LucRNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was performed according to the product instructions of the transfection reagent. The protein expression amount was detected using a multifunctional enzyme label instrument 24 h after transfection. The negative control was a cell culture medium without the addition of LucRNA-LNP. The in vitro cell transfection efficiency is shown in Figure 7 Figure 6, which shows that the LucRNA encapsulated by the LNP prepared from the ionizable lipid N24-O11B(3T) has very high cell transfection efficiency, and the cell transfection efficiency of the LucRNA encapsulated by the LNP prepared from the traditional ionizable lipid N24-O11B(4T) containing four hydrophobic tail chains is much lower, with a decrease of about an order of magnitude. It can be seen that the ionizable lipid N24-O11B(3T) containing three hydrophobic tail chains has higher cell transfection efficiency than the traditional ionizable lipid N24-O11B(4T) containing four hydrophobic tail chains, and the advantage is very obvious. The phenomenon that such a three-hydrophobic-tail-chain lipid compound has higher cell transfection efficiency than the traditional four-hydrophobic-tail-chain lipid compound is also applicable to other lipid compounds of the general structure in the present application.

[0197] It can also be seen from the results of Example 3 that the physicochemical characteristics of the lipid nanoparticles LucRNA-LNP prepared from the novel lipid compound N24-O11B(3T) are good, and the in vitro cell transfection efficiency is about 2-3 times higher than that of the commercial Lipofectamine 2000 (Lipo2000).

[0198] Comparative Example 1 (commercial ionizable cationic lipid molecule ALC-0315)

[0199] The molecular formula of ALC-0315 is ((4-hydroxybutyl)azabicycloalkyl)bis(hexane-6,1-diyl)bis(2-hexyldecyl ester). It was purchased from AvantiPolar Lipids (Shanghai) Co., Ltd.

[0200] The structural formula of ALC-0315 is:

[0201]

[0202] Example 4 N24-O11B(3T)

[0203] This example provides a comparison of the effects of N24-O11B(3T) and commercial ionizable cationic lipid molecule ALC-0315

[0204] Lipid nanoparticles were prepared using N24-O11B(3T) and ALC-0315 respectively according to the method described in Example 3, with the specific molar ratio of N24-O11B(3T):DOPE:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5; ALC-0315:DOPE:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5.

[0205] The physicochemical quality control data of the prepared lipid nanoparticles are shown in the following table (Table 2):

[0206] Table 2 Physicochemical quality control data of lipid nanoparticles

[0207]

[0208] As can be seen from the above table, the encapsulation efficiency of the lipid nanoparticles prepared by N24-O11B(3T) is as high as 96%, which is similar to that of the lipid nanoparticles of ALC-0315.

[0209] The prepared lipid nanoparticles were transfected into cells using the same transfection method as in Example 3, and the protein expression was observed. The results are shown in Figure 8 As shown in Table 3, after the mRNA carried by the lipid nanoparticles prepared by N24-O11B(3T) was transfected into cells, the protein expression in the cells was higher than that of Lipofectamine 2000, and the protein expression in the cells transfected with the corresponding mRNA concentration was also higher than that of ALC-0315, indicating that the cell transfection efficiency of the lipid nanoparticles prepared by N24-O11B(3T) was very high.

[0210] Example 5 Transfection experiment of N24-O18-2(3T)-LNP, N24-O10(3T)-LNP, N24-O18-SS(3T)-LNP, N24-O11B(3T)-LNP lipid nanoparticles in animals

[0211] N24-O18-2(3T), N24-O10(3T), N24-O18-SS(3T) or N24-O11B(3T)-LNP were prepared according to the method described in Example 3 by using the mouse tail vein injection of nano-lipid particles, and the specific molar ratio was: N24-O18-2(3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio was 10: 1, N24-O10(3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio was 10: 1, N24-O18-SS(3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio was 10: 1, N24-O11B(3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio was 10: 1. The mRNA was the mRNA expressing Luciferase fluorescent protein, the amount of mRNA was 10 μg, the total amount of N24-O18-2(3T), N24-O10(3T), N24-O18-SS(3T), or N24-O11B(3T)-LNP, DOPE, Cholesterol, DMG-PEG2000 was 100 μg, 200 μL of neutral PBS buffer solution was used to quickly convert the liposome environment, and quickly injected into 6-8 weeks old female C57 mice through the tail vein, and the intravenous injection of 10 μg mRNA was controlled.

[0212] The fluorescence expression of each organ of the mouse after tail vein injection of N24-O18-2(3T)-LNP, PBS (blank control) for 6 hours is shown in Figure 9 The results show that after intravenous injection (IV) of N24-O18-2(3T)-LNP lipid nanoparticles, the fluorescence expression in each organ of the mouse is mainly distributed in the spleen about 95%, liver 5%, heart 0%, lung 0%, kidney 0%, which can be specifically targeted to the spleen. The fluorescence expression of each organ of the mouse after tail vein injection of N24-O10(3T)-LNP, PBS (blank control) for 6 hours is shown in Figure 9 The results show that after intravenous injection (IV) of N24-O10(3T)-LNP lipid nanoparticles, the fluorescence expression in each organ of the mouse is mainly distributed in the spleen about 100%, heart 0%, liver 0%, lung 0%, kidney 0%, which can be specifically targeted to the spleen. The fluorescence expression of each organ of the mouse after tail vein injection of N24-O18-SS(3T)-LNP, PBS (blank control) for 6 hours is shown in Figure 9The results show that after intravenous injection (IV) of the lipid nanoparticles of N24-O18-SS(3T)-LNP, the fluorescence expression in each organ of the mouse is mainly distributed in the spleen about 60%, the liver 0%, the lung 40%, the heart 0%, and the kidney 0%, and it can be seen that it can specifically target the spleen and the lung. The fluorescence expression of each organ of the mouse after tail vein injection of N24-O11B(3T)-LNP, PBS (blank control) is shown in Figure 9 The results show that after intravenous injection (IV) of the lipid nanoparticles of N24-O18-SS(3T)-LNP, the fluorescence expression in each organ of the mouse is mainly distributed in the spleen about 60%, the liver 0%, the lung 40%, the heart 0%, and the kidney 0%, and it can be seen that it can specifically target the spleen and the lung. The fluorescence expression of each organ of the mouse after tail vein injection of N24-O11B(3T)-LNP, PBS (blank control) is shown in

[0213] Example 6 N24-O10(3T)

[0214] The same synthesis and purification method as in Example 1 is adopted, except that the compound 3-undecanol in Example 1 is replaced by n-decanol, and the structure is as follows:

[0215]

[0216] The preparation method of the compound is also provided in this embodiment.

[0217]

[0218] Synthesis of N24-O10(3T): 250 mg of N-methyl-2,2'-diaminodiethylamine solution is added with decyl acrylate (1.6 g) at room temperature, and then the mixture is heated to 120°C and continuously stirred for 24 h. When the reaction is detected by thin layer chromatography plate, 1.9 g of crude product is obtained, and then the target product is purified by fast column method eluted with dichloromethane / methanol to obtain N24-O10(3T) 300 mg. The hydrogen spectrum of the compound N24-O11B(3T) is shown in Figure 10 , and the mass spectrum is shown in Figure 11 .

[0219] N24-O10(3T):

[0220] 1H NMR (400 MHz, CDC13) δ 4.06 (dt, J = 13.9, 6.9 Hz, 6H), 2.93 (t, J = 6.6 Hz, 2H), 2.79 (t, J = 7.2 Hz, 4H), 2.73 (t, J = 5.6 Hz, 2H), 2.60 - 2.49 (m, 6H), 2.45 (dd, J = 15.6, 8.3 Hz, 6H), 2.24 (s, 3H), 1.61 (dd, J = 13.2, 6.5 Hz, 6H), 1.28 (d, J = 15.3 Hz, 42H), 0.88 (t, J = 6.7 Hz, 9H).

[0221] MALDI-TOF MS: m / z 754.319 [M+H] + .

[0222] This example also provides the dissociation constant (pKa = 7.09) of the compound N24-011B (3T) and the preparation of the LucRNA lipid nanoparticle N24-010 (3T)-LNP (N24-010 (3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio is 10: 1). The distribution of N24-010 (3T)-LNP in vivo was studied by transfection experiments of N24-010 (3T)-LNP lipid nanoparticles in animals, and it can be seen that N24-010 (3T)-LNP mainly specifically targets the spleen, such as Figure 9 Compared with the compound N24-011B (3T) of Example N24-011B (3T), the lipid compound N24-010 (3T) of this example can realize the specific targeting of the lipid nanoparticle from the spleen and liver to the single spleen site by adjusting the branched structure to a straight chain structure in the hydrophobic tail chain and keeping the length of 10 carbon atoms.

[0223] Example 7 N24-018-2 (3T)

[0224] The same synthesis and purification method as in Example 1 is used, except that the compound 3-undecanol in Example 1 is replaced by (9Z, 12Z)-9, 12-octadecadien-1-ol (linoleyl alcohol), and its structure is as follows:

[0225]

[0226] This example also provides a preparation method of the compound:

[0227]

[0228] Synthesis of N24-O18-2(3T): To a solution of 300 mg N-methyl-2,2'-diaminodiethylamine was added (9Z,12Z)-9,12-octadecadienyl acrylate (2.9 g) at room temperature, then the mixture was heated to 120 °C and stirred for 24 h. When the reaction was finished, the crude product 3.2 g was obtained by thin layer chromatography, then the target product was purified by flash column method eluted with dichloromethane / methanol to obtain N24-O18-2(3T) 480 mg. The hydrogen spectrum of compound N24-O18-2(3T) is shown in Figure 12 , and the mass spectrum is shown in Figure 13 .

[0229] N24-O18-2(3T):

[0230] 1 H NMR (400 MHz, CDC13) δ 5.45 - 5.25 (m, 12H), 4.06 (dd, J = 14.8, 7.4 Hz, 6H), 2.93 (t, J = 6.5 Hz, 2H), 2.83 - 2.68 (m, 12H), 2.55 (dd, J = 14.7, 6.7 Hz, 6H), 2.45 (dd, J = 15.6, 8.3 Hz, 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 = 6.7 Hz, 9H).

[0231] MALDI-TOF MS: m / z 1079.008 [M+H] + .

[0232] This example also provides the dissociation constant (pKa = 6.70) of the compound N24-O18-2(3T) and the preparation of the LucRNA lipid nanoparticle N24-O18-2(3T)-LNP (N24-O18-2(3T): DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5; N / P ratio is 10: 1). The distribution of N24-O18-2(3T)-LNP in vivo was studied by transfection experiments of N24-O18-2(3T)-LNP lipid nanoparticles in animals, and it can be seen that N24-O18-2(3T)-LNP can specifically target the spleen, as shown in Figure 9As shown, compared with the compound of Example N24-O11B (3T), the N24-O18-2 (3T) lipid compound of the present embodiment adjusts the branched chain structure in the hydrophobic tail chain to a straight chain structure containing unsaturation, such as adding adjacent cis double bonds between C9-C13, which can make the hydrophobic tail chain begin to bend at the C9 position to form a relatively long 9C alkane chain, and the structure of the three hydrophobic tail chains makes the N24-O18-2 (3T) compound form a unique trigonal pyramidal spatial structure, which can more favorably bind the ionizable lipid compound to mRNA and improve the lysosomal escape rate, greatly improving the effect of delivering mRNA and specifically targeting the lipid nanoparticle to the spleen alone.

[0233] Example 8 N24-O18-SS (3T)

[0234] The same synthesis and purification method as in Example 1 is used, except that the compound 3-undecanol in Example 1 is replaced by 2-(hexadecyl disulfide) ethanol, which has the following structure:

[0235]

[0236] The present embodiment also provides a preparation method of the compound:

[0237]

[0238] Synthesis of N24-O18-2 (3T): 300 mg of N-methyl-2,2'-diamino-diethylamine solution was added to 2-(hexadecyl disulfide) ethyl acrylate (3.2 g) at room temperature, and then the mixture was heated to 120°C and continuously stirred for 24 h. When the reaction was completed, 3.5 g of crude product was obtained by thin layer chromatography, and then the target product was purified by rapid column method eluted with dichloromethane / methanol to obtain N24-O18-2 (3T) 525 mg. The hydrogen spectrum of the compound N24-O18-SS (3T) is shown in Figure 14 , and the mass spectrum is shown in Figure 15 .

[0239] N24-O18-SS (3T):

[0240] 1H NMR (400 MHz, CDC13) δ 4.36 (ddd, J = 14.7, 13.4, 7.2 Hz, 6H), 2.98 (t, J = 6.4 Hz, 2H), 2.89 (dd, J = 12.3, 5.6 Hz, 6H), 2.81 (dd, J = 12.7, 5.8 Hz, 6H), 2.69 (dd, J = 16.4, 9.0 Hz, 8H), 2.57 (d, J = 6.7 Hz, 4H), 2.48 (dd, J = 14.0, 7.1 Hz, 6H), 2.27 (s, 3H), 1.74 - 1.61 (m, 6H), 1.37 (dd, J = 14.1, 7.1 Hz, 6H), 1.28 (d, J = 16.6 Hz, 60H), 0.88 (t, J = 6.8 Hz, 9H).

[0241] MALDI-TOF MS: m / z 1198.799 [M+H] + .

[0242] This example also provides the dissociation constant (pKa = 7.21) of the compound N24-018-SS(3T) and the preparation of the LucRNA lipid nanoparticle N24-018-SS(3T)-LNP (N24-018-SS(3T):DOPE:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5; N / P ratio of 10:1). The distribution of N24-018-SS(3T)-LNP in vivo was studied by transfection experiments of N24-018-SS(3T)-LNP lipid nanoparticles in animals, and it can be seen that N24-018-SS(3T)-LNP can specifically target the spleen and lungs, as shown in Figure 9 compared with the compound of example N24-011B(3T), by adjusting the branched chain structure in the hydrophobic tail chain to a straight chain structure containing S heteroatoms, such as replacing S-S at two C-C positions at C3 and C4, the introduction of S-S heteroatoms in the hydrophobic tail chain can cause the hydrophobic tail chain to bend at the S-S position. The characteristics of this structure make the plane formed by the hydrophilic head group larger, which is more likely to increase the loading of mRNA molecules. The effect of delivering mRNA is greatly improved and its lipid nanoparticles specifically target the spleen and lungs instead of the liver.

[0243] Effect of the length of the hydrophobic tail chain of the lipid compound of Comparative Example 2 on cell transfection of lipid nanoparticles

[0244] A series of lipid compounds with different hydrophobic tail chain lengths were designed and synthesized, and lipid nanoparticles were prepared using N24-O18(3T), N24-O16(3T), N24-O14(3T), N24-O12(3T), N24-O10(3T), N24-O8(3T), N24-O6(3T) respectively according to the method described in Example 3, and the specific molar ratio was: lipid compound: DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5.

[0245] The physicochemical quality control data of the prepared lipid nanoparticles are shown in the following table (Table 3):

[0246] Table 3 Physicochemical quality control data of lipid nanoparticles

[0247]

[0248]

[0249] The prepared lipid nanoparticles were transfected into cells by the same transfection method as in Example 3, and the protein expression was analyzed, and the results are shown in Figure 16 As the number of carbon atoms in the hydrophobic tail chain decreases, the cell transfection efficiency of the lipid nanoparticles prepared from the corresponding lipid compounds has an increasing and then decreasing trend, and when the tail chain length is C10, N24-O10(3T)-LNP has the highest transfection efficiency, and can realize specific targeting of the spleen site of the lipid nanoparticles.

[0250] Effect of spatial structure of hydrophobic tail chain of lipid compound on cell transfection and organ / tissue targeting of lipid nanoparticles

[0251] The spatial structure of the ionizable cationic lipid compound is mainly adjusted by changing the unsaturation, heteroatom, or branched structure of the hydrophobic tail chain, so that it can better load mRNA, and make the lipid nanoparticles have the function of specifically targeting the liver, spleen and / or lung.

[0252] A series of lipid compounds with different hydrophobic tail chain spatial structures were designed and synthesized, and lipid nanoparticles were prepared using N24-O18-2(3T), N24-O18-SS(3T), N24-O11B(3T) respectively according to the method described in Example 3, and the specific molar ratio was: lipid compound: DOPE: Cholesterol: DMG-PEG2000 = 45: 15: 38.5: 1.5.

[0253] The physicochemical quality control data of the prepared lipid nanoparticles are shown in the following table (Table 4):

[0254] Table 4 Physicochemical quality control data of lipid nanoparticles

[0255]

[0256]

[0257] Using the same transfection method as in Example 3, the prepared lipid nanoparticles were transfected into cells, and the protein expression was analyzed. The results are as follows: Figure 17 As shown, compared to example N24-O10(3T)-LNP, the introduction of unsaturation, heteroatoms, or branched structures in the hydrophobic tail chain of the lipid compound can increase the transfection efficiency of its corresponding lipid nanoparticles into cells. This result may be because the introduction of unsaturation, heteroatoms, or branched structures in the hydrophobic tail chain of the lipid compound gives the ionizable cationic lipid compound a specific spatial structure, increasing the amount of mRNA loaded and its lysosomal escape rate, ultimately improving the efficiency of cell transfection. The novel ionizable cationic lipid compound provided by this invention can effectively load mRNA, and in particular, can better achieve the specific targeting of lipid nanoparticles to specific organs.

[0258] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an ionizable cationic lipid compound in the manufacture of a delivery system for a biologically active material, characterized in that, The ionizable cationic lipid compound is N24-O18-SS(3T), and the bioactive substance delivery system specifically targets the spleen and lung; Or, the ionizable cationic lipid compound is N24-O11B(3T), and the bioactive substance delivery system specifically targets the spleen and liver. N24-O18-SS(3T), N24-O11B(3T).

2. Use according to claim 1, characterized in that, The bioactive substance delivery system is a microbubble, a microparticle, a nanoparticle, a liposome or a lipid nanoparticle.

3. A bioactive substance delivery system, characterized by The ionizable cationic lipid compound is N24-O18-SS(3T), and the bioactive substance delivery system specifically targets the spleen and lung; Or, the ionizable cationic lipid compound is N24-O11B(3T), and the bioactive substance delivery system specifically targets the spleen and liver. N24-O18-SS(3T), N24-O11B(3T).

4. A pharmaceutical composition, characterized by, The bioactive substance delivery system of claim 3.

5. The pharmaceutical composition according to claim 4, wherein The bioactive substance delivery system contains 20-60 mol% of the ionizable cationic lipid compound, 5-30 mol% of a neutral lipid molecule, 30-60 mol% of a cholesterolic lipid molecule, and 0.5-5 mol% of a PEGylated lipid molecule.

6. The pharmaceutical composition according to claim 5, wherein The bioactive substance delivery system contains 20-50 mol% of the ionizable cationic lipid compound, 5-20 mol% of a neutral lipid molecule, 30-50 mol% of a cholesterolic lipid molecule, and 0.5-2.5 mol% of a PEGylated lipid molecule.

7. The pharmaceutical composition according to claim 6, wherein The bioactive substance delivery system contains 35-48 mol% of the ionizable cationic lipid compound, 10-15 mol% of a neutral lipid molecule, 35-50 mol% of a cholesterolic lipid molecule, and 1.0-2.0 mol% of a PEGylated lipid molecule.

8. The pharmaceutical composition according to any one of claims 5 to 7, wherein The neutral lipid molecule is selected from the group consisting of a phosphatidylcholine compound and / or a phosphatidylethanolamine compound; and / or, the cholesterolic lipid molecule is selected from one or more of cholesterols and cholesteryl hemisuccinate; and / or, the PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, and the number average molecular weight of the lipid moiety-PEG is 100-50000.

9. The pharmaceutical composition according to claim 8, wherein The lipid moiety is selected from one or more of dilaurin, dimyristin, dilaurinamide, dimyristinamide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; and the number average molecular weight of the PEG is 100-50000.

10. The pharmaceutical composition according to claim 9, wherein The number average molecular weight of the PEG is 200-10000.

11. The pharmaceutical composition according to claim 10, wherein The number average molecular weight of the PEG is 500-3000.

12. The pharmaceutical composition of claim 11, wherein, The number average molecular weight of the PEG is 1500-2500.

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