Lipid compound, lipid nanoparticle containing lipid compound, and use thereof

Abstract

The present application provides a lipid compound, a lipid nanoparticle containing the lipid compound, and a use thereof in the preparation of a drug delivery carrier. The lipid nanoparticle of the present application can deliver a therapeutic agent or prophylactic agent (especially a nucleic acid therapeutic agent or prophylactic agent) to a site of injection by means of intramuscular injection. The therapeutic agent or the prophylactic agent has a high expression level in the site of injection, but shows a relatively low expression level in viscera, so that the drug can efficiently exert activity and has a relatively low visceral toxicity. The present application is of great significance to the development and application of nucleic acid therapeutic agents or prophylactic agents.

Description

A lipid compound, lipid nanoparticles comprising the compound, and their applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202410487810.8, filed on April 23, 2024, entitled "A lipid compound, lipid nanoparticles comprising the same and applications thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of molecular biology, specifically relating to a lipid compound, lipid nanoparticles containing the same, their application in the preparation of drug delivery carriers, and pharmaceutical compositions and agents based on the lipid nanoparticles. Background Technology

[0004] Therapeutic or preventative nucleic acids hold the potential to revolutionize vaccination, gene therapy, protein replacement therapy, and other treatments for genetic diseases. Since the first clinical studies of therapeutic nucleic acids began in 2000, significant progress has been made in the design of nucleic acid molecules and their delivery methods. However, nucleic acid drugs (including therapeutic and preventative agents) still face several challenges, including the fact that most liposome formulations accumulate in the liver through biological processes, reducing the efficacy of the composition in delivering it to the target site. Therefore, highly specific, high-capacity lipid nanoparticle delivery systems that reduce liver accumulation remain to be developed. Summary of the Invention

[0005] Purpose of the invention

[0006] The purpose of this application is to provide a cationic lipid compound capable of delivering nucleic acid therapeutics or preventative agents, lipid nanoparticles containing the same, their use in the preparation of drug delivery carriers, and pharmaceutical compositions and agents based on the lipid nanoparticles.

[0007] The drug delivery carrier based on the cationic lipid compound of this application can efficiently deliver nucleic acid therapeutics or preventives to the injection site via intramuscular injection, and can achieve high expression of nucleic acid at the injection site, while the expression level of nucleic acid in the viscera is low, thereby reducing toxicity.

[0008] Solution

[0009] To achieve the objectives of this application, the following technical solutions are provided:

[0010] In a first aspect, this application provides a compound as shown in formula (I) or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0011] in:

[0012] Y1 and Y2 are each independently -OC(=O)- or -C(=O)O-;

[0013] L1 and L2 are each independently bonded, optionally substituted C1-C bonds. 18 Alkylene;

[0014] R1 and R2 are each independently H or C1-C8 straight-chain or branched alkyl groups;

[0015] R3 and R4 are each independently C1-C8 straight-chain or branched alkyl groups that are optionally substituted with hydroxyl groups;

[0016] m is an integer between 1 and 10;

[0017] n is an integer between 1 and 10.

[0018] Preferably, L1 and L2 are each independently unsubstituted C2-C. 14 Alkylene.

[0019] Preferably, R1 and R2 are each independently H or C1-C8 straight-chain alkyl; more preferably, R1 and R2 are each independently H or C6-C8 straight-chain alkyl.

[0020] Preferably, R3 and R4 are each independently a C1-C4 straight-chain or branched alkyl group optionally substituted with a hydroxyl group; more preferably, R3 and R4 are each independently methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, or hydroxybutyl; even more preferably, R3 and R4 are each independently methyl or hydroxyethyl.

[0021] Preferably, m is an integer between 5 and 7;

[0022] Preferably, n is an integer between 5 and 7.

[0023] In a preferred embodiment, the compound is selected from the following compounds:

[0024] Preferably, the compound is selected from compounds 1, 2, 3, 4, 5, 6, 7, and 10 as shown in the table above, more preferably compounds 3, 4, 5, and 7, even more preferably compounds 3, 5, or 7, and most preferably compounds 5 or 7.

[0025] In the foregoing, "pharmaceutically acceptable salt" refers to a salt of the compound of this application that is pharmaceutically acceptable and has the desired pharmacological activity. Such salts include acid addition salts formed with acids such as: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-en-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-en-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, and carbonic acid. Cinnamon acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthenic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include, but are not limited to, sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. It should be recognized that the specific anion or cation forming part of any salt described in this application is not essential, as long as the salt as a whole is pharmacologically acceptable.

[0026] In the foregoing, a “prodrug” refers to a compound, such as a therapeutic agent, that can be converted into a biologically active compound under physiological conditions or by dissolution. Prodrugs are typically rapidly converted in vivo to produce the parent compound, for example, through hydrolysis in the blood. Prodrug compounds generally possess advantages such as solubility, tissue compatibility, or delayed release in mammalian organisms. The term “prodrug” also means including any covalently bonded carrier that releases the active compound in vivo when the prodrug is administered to a mammalian subject. Prodrugs include compounds in which hydroxyl, amino, or thiol groups are bound to any group that, when the prodrug is administered to a mammalian subject, cleave to form a free hydroxyl, free amino, or free thiol group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives, or amide derivatives with amine functional groups.

[0027] In the above aspects, a “stereoisomer” is an isomer of a given compound in which identical atoms are bonded to the same other atoms, but those atoms have different three-dimensional configurations. An “enantiomer” is a stereoisomer of a given compound that is a mirror image of each other, like the left and right hands. A “diastereomer” is a stereoisomer of a given compound that is not a diastereomer. A chiral molecule contains a chiral center (also called a stereocenter or steric center), which is any point (though not necessarily an atom) in a molecule carrying multiple groups, such that the interchange of any two groups produces stereoisomers. In organic compounds, the chiral center is typically a carbon, phosphorus, or sulfur atom, although other atoms can also be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, thus producing many stereoisomers. In compounds whose stereoisomerism is attributed to a tetrahedral stereocenter (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have a smaller number than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, mixtures of enantiomers can be enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. It is anticipated that for any stereocenter or chiral axis whose stereochemistry has not yet been defined, it may exist in its R form, S form, or as a mixture of said R and S forms (including racemic and non-racemic mixtures). The phrase “substantially free of other stereoisomers” as used herein means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of one or more other stereoisomers.

[0028] In a second aspect, this application provides a lipid nanoparticle comprising the compound as described in the first aspect above, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0029] Furthermore, the lipid nanoparticles may also contain at least one auxiliary lipid, which can be mixed with a drug or a drug-active molecule / agent to achieve encapsulation and effective drug delivery.

[0030] Optionally, the auxiliary lipid is one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids.

[0031] Preferably, the phospholipid is any one or a combination of DOPE, DSPC, DPPC, DMPC, DOPC, POPC, and SM, more preferably DOPE or DSPC. In one preferred embodiment, the phospholipid is DOPE. In another preferred embodiment, the phospholipid is DSPC.

[0032] Preferably, the steroid is any one or a combination of cholesterol, sitosterol, stigmasterol and ergosterol; more preferably, the steroid is cholesterol and / or sitosterol.

[0033] Regarding the aforementioned polymer-conjugated lipids, the polymer refers to a high molecular weight compound formed by the covalent bonding of one or more small molecule repeating units; the polymer can be selected from polyethylene glycol, polylactic acid, polyamide, cationic polymers, polysarcosine (pSar), polylactic-glycolic acid copolymer (PLGA), polyamino acids, polypeptides, and polymeric peptides, etc.; preferably, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid; more preferably, the polyethylene glycol-conjugated lipid is one or more selected from ALC-0159, PEG1000-DMG, PEG5000-DMG, PEG2000-DMG, and PEG2000-DSPE.

[0034] Preferably, the modifiable lipids comprise lipids that can be modified by small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations thereof.

[0035] For the above-mentioned lipid nanoparticles, preferably, the molar ratio of the compound or its pharmaceutically acceptable salt, prodrug or stereoisomer as described in the first aspect above to the auxiliary lipid is 1:(0.5 to 2), more preferably 1:(0.6 to 1.5), and more preferably 1:(0.8 to 1.2).

[0036] The lipid nanoparticles can deliver therapeutic / preventive agents to the injection site via intramuscular injection.

[0037] Thirdly, this application provides the use of the compounds described in the first aspect above, or their pharmaceutically acceptable salts, prodrugs, or stereoisomers, or the lipid nanoparticles described in the second aspect above, in the preparation of drug delivery carriers.

[0038] Practically, the drug may be a therapeutic or preventative agent.

[0039] Further feasiblely, the therapeutic or preventative agent is a nucleic acid.

[0040] The nucleic acid includes any and all forms of nucleic acid molecules, including other forms of nucleic acid molecules known in the art or that may be discovered / prepared in the future; in a preferred embodiment, the nucleic acid is selected from: single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isoforms, plasmid DNA, complementary DNA (cDNA), antisense nucleic acid molecules (ASO), small interfering nucleic acid (siRNA), small activating nucleic acid (saRNA), asymmetric interfering nucleic acid (aiRNA), micronucleic acid (miRNA), miRNA agonist (agomir), miRNA antagonist (antagomir), Dicer enzyme substrate nucleic acid, small hairpin nucleic acid (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), circular RNA (circRNA), self-replicating mRNA (samRNA), and aptamers.

[0041] The nucleic acid molecule may be a natural nucleotide, a nucleotide analog, a functional analog, or a chemically modified nucleotide.

[0042] Functional nucleotide analogs include, but are not limited to, one or more of locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholine cyclic oligonucleotide nucleic acid mimics or functional analogs.

[0043] Nucleotide chemical modifications can be located on the backbone bonds of the nucleic acid molecule. The backbone bonds can be modified by replacing one or more oxygen atoms. Modification of the backbone bonds can include replacing at least one phosphodiester bond with a thiophosphate bond.

[0044] Nucleotide chemical modifications can also be located on nucleosides. Modifications on nucleosides can be located on the sugar and base of the nucleoside. The sugar modification on the nucleoside can be selected from one or more of the following: 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose.

[0045] The chemical modification of the nucleotide can be selected from one or more of the following: 5-methylcytosine, pseudouridine, 1-methylpseudouridine, pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-2-thio-uridine, 1-taurate methyl-4-thio-uridine, 5-methyl- Urate, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine Cytidine, pyrrolo-cytidine, pyrrolo-pseudo-cytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudo-cytidine, 4-thio-1-methyl-pseudo-cytidine, 4-thio-1-methyl-1-deazo-pseudo-cytidine, 1-methyl-1-deazo-pseudo-cytidine, zebularine, 5-aza-zabraline, 5-methyl-zabraline, 5-aza-2-thio-zabraline, 2-thio-zabraline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, 2- Aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaa-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, inosine, 1-methyl-inosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. These modifications can be random or at specific sites. ,

[0046] In a preferred embodiment, the nucleic acid is mRNA.

[0047] Practically, the mRNA encodes at least one antigen or a fragment thereof or an epitope thereof, or encodes a therapeutic protein.

[0048] Further feasiblely, the antigen is a pathogenic antigen, such as a tumor-associated antigen or a pathogenic microorganism antigen.

[0049] Furthermore, the mRNA can be either a monocistronic mRNA or a polycistronic mRNA.

[0050] Fourthly, this application provides a pharmaceutical composition comprising lipid nanoparticles as described in the second aspect above, and a therapeutic or preventative agent.

[0051] Preferably, the therapeutic or preventative agent is a nucleic acid, which is encapsulated in the lipid nanoparticles.

[0052] The optional types of the nucleic acid are as defined in the third aspect above.

[0053] Furthermore, preferably, in the pharmaceutical composition, the mass ratio of the lipid nanoparticles to the therapeutic or preventative agent is 10:1 to 100:1, more preferably 20:1 to 50:1, and even more preferably 20:1 to 30:1.

[0054] Preferably, the average particle size of the pharmaceutical composition is 90 nm to 600 nm, more preferably 200 nm to 400 nm, and even more preferably 200 nm to 300 nm.

[0055] Preferably, the polydispersity index of the pharmaceutical composition is 0.001 to 0.5, more preferably 0.001 to 0.45, and even more preferably 0.001 to 0.4.

[0056] Fifthly, this application provides the use of the pharmaceutical composition described in the fourth aspect above in the preparation of targeted delivery drugs.

[0057] Practically, the drug comprises the above-described composition and pharmaceutically acceptable excipients.

[0058] In a sixth aspect, this application provides a formulation comprising the pharmaceutical composition as described in the fourth aspect above and pharmaceutically acceptable excipients.

[0059] The aforementioned "pharmaceuticalally acceptable excipients" refer to excipients and additives used in the production of pharmaceuticals and the compounding of prescriptions; these are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in pharmaceutical formulations. Besides acting as excipients, carriers, and improving stability, "pharmaceuticalally acceptable excipients" also possess important functions such as solubilization, co-solubilization, and sustained-release, and are important components that may affect the quality, safety, and efficacy of pharmaceuticals. According to their functions and uses, pharmaceutical excipients can be classified into solvents, propellants, solubilizers, co-solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, flavoring agents, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors, etc. Beneficial effects

[0060] This application provides a cationic lipid compound, lipid nanoparticles comprising the same, their application as a drug delivery carrier, and pharmaceutical compositions and agents based on the lipid nanoparticles. The lipid nanoparticles of this application can deliver therapeutic or preventative agents (especially nucleic acid-based substances) to the injection site via intramuscular injection. The therapeutic or preventative agents are highly expressed at the injection site but expressed at lower levels in viscera. This allows the drug to not only exert its activity efficiently but also exhibit low visceral toxicity, which is of great significance for the development and application of nucleic acid-based therapeutic or preventative agents. Attached Figure Description

[0061] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0062] Figure 1 shows the proton NMR spectrum of compound 1;

[0063] Figure 2 shows the proton NMR spectrum of compound 2;

[0064] Figure 3 shows the proton NMR spectrum of compound 3;

[0065] Figure 4 shows the proton NMR spectrum of compound 4;

[0066] Figure 5 shows the proton NMR spectrum of compound 5;

[0067] Figure 6 shows the proton NMR spectrum of compound 6;

[0068] Figure 7 shows the proton NMR spectrum of compound 7;

[0069] Figure 8 shows the proton NMR spectrum of compound 8;

[0070] Figure 9 shows the proton NMR spectrum of compound 9;

[0071] Figure 10 shows the proton NMR spectrum of compound 10;

[0072] Figure 11 shows the summary data of fluorescence intensity ratios at the injection site and in viscera for different lipid nanoparticles carrying luciferase mRNA after intramuscular injection.

[0073] Figure 12 shows the fluorescence signal of the lipid nanoparticles TMF1 carrying luciferase mRNA in mice 4 hours after intramuscular injection; where Color Scale: color scale bar; Min: minimum value; Max: maximum value. Detailed Implementation

[0074] A. Definition of Chemicals and Pharmaceuticals

[0075] When used in the context of chemical groups: "hydrogen" refers to -H; "deuterium" refers to... 2H or D; "hydroxyl" refers to -OH; "oxo" refers to =O; "carbonyl" refers to -C(=O)-; "carboxyl" refers to -C(=O)OH (also written as -COOH or -CO2H); "halogen" independently refers to -F, -Cl, -Br or -I; "amino" refers to -NH2; "hydroxyamino" refers to -NHOH; "nitro" refers to -NO2; "imino" refers to =NH; "cyano" refers to -CN; "isocyanate" refers to -N=C=O; "azide" refers to -Azide. "" refers to -N3; ​​in the context of monovalent, "phosphate ester" refers to -OP(O)(OH)2 or its deprotonated form; in the context of divalent, "phosphate ester" refers to -OP(O)(OH)O- or its deprotonated form; "mercapto" refers to -SH; and "thio" refers to =S; "sulfonyl" refers to -S(O)2-; "hydroxysulfonyl" refers to -S(O)2OH; "sulfonamide" refers to -S(O)2NH2; and "sulfinyl" refers to -S(O)-. In the context of a chemical formula, the symbol "-" indicates a single bond, "=" indicates a double bond, and "≡" indicates a triple bond. The symbol "----" represents an optional bond, which, if present, is either a single or double bond. When drawn perpendicularly through the bond, the symbol... This indicates the connection point of the group. It should be noted that this method is typically used only for larger groups to identify the connection point, to help the reader clearly identify it. (Symbol) This refers to a single bond, where the group attached to the thicker end of the wedge "comes out of the paper." (Symbol) This refers to a single bond, where the group attached to the thicker end of the wedge "enters the paper." (Symbol) This refers to a single bond, where the geometry (e.g., E or Z) around the double bond is undetermined. Therefore, both options and their combinations are contemplated. Any undefined valence on an atom in the structure shown in this application implicitly represents a hydrogen atom bonded to that atom. Bold dots on carbon atoms indicate that the hydrogen atom bonded to that carbon is facing outwards from the plane of the paper.

[0076] The term "alkyl" as used without the modifier "substituted" indicates a monovalent saturated aliphatic group having a carbon atom as a linker, a straight-chain or branched acyclic structure, and no atoms other than carbon and hydrogen. Non-limiting examples of alkyl groups include -CH3(Me), -CH2CH3(Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH3)2 (i-Pr, iPr or isopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, tert-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neopentyl). The term "alkanediyl," used without the modifier "substituted," refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as connecting points, a straight-chain or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2-(methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. "Alkane" refers to a class of compounds having the formula HR, where R is an alkyl group, as defined above. When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which hydrogen substitution is limited to halogenation (i.e., -F, -Cl, -Br, or -I), such that no atoms other than carbon, hydrogen, and halogen are present. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which hydrogen substitution is limited to fluorination, such that no atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0077] The term "alkenyl," used without the modifier "substituted," refers to a monovalent unsaturated aliphatic group having a carbon atom as a linking point, a straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkene diel," used without the modifier "substituted," refers to a divalent unsaturated aliphatic group having two carbon atoms as linking points, a straight-chain or branched, straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of olefinic dimethyl groups. It should be noted that although olefinic dimethyl groups are aliphatic, once connected at both ends, it does not preclude the group from forming part of an aromatic structure. The terms "olefin" and "chain olefin" are synonymous and refer to a class of compounds having the formula HR, where R is an alkenyl group, as defined above. Similarly, the terms "terminal olefin" and "α-olefin" are synonymous and refer to an olefin having exactly one carbon-carbon double bond, where the bond is the vinyl portion at the end of the molecule. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0078] The term "alkynyl" as used without the modifier "substituted" refers to a monovalent unsaturated aliphatic group having a carbon atom as a linker, a straight-chain or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The term alkynyl as used herein does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to a class of compounds having the formula HR, where R is an alkynyl group. When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0079] "Prevention" or "preventing" includes: (1) suppressing the onset of a disease in a subject or patient who may be at risk and / or susceptible to the disease but has not yet experienced or exhibited any or all symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of the disease in a subject or patient who may be at risk and / or susceptible to the disease but has not yet experienced or exhibited any or all symptoms or signs of the disease.

[0080] "Treatment" or "treating" includes (1) suppressing the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., preventing further development of the symptoms and / or signs), (2) improving the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and / or signs), and / or (3) achieving any measurable reduction of the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.

[0081] “Protein,” “polypeptide,” or “peptide” refers to a polymer of amino acid residues, including a wide range of protein molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes.

[0082] An antigen (abbreviated as Ag) is a substance that can induce antibody production; it is any substance that can trigger an immune response. Foreign molecules can be recognized by immunoglobulins on B cells or processed by antigen-presenting cells and bind to the major histocompatibility complex to form a complex that reactivates T cells, thus triggering a continuous immune response.

[0083] Antigenic epitopes, also known as antigenic determinants, are specific chemical groups that determine antigenicity. They can be composed of continuous sequences (protein primary structures) or discontinuous three-dimensional protein structures. Most antigenic epitopes are located on the surface of antigenic substances, while some reside inside the antigenic substance and are only exposed after treatment with enzymes or other methods. A natural antigenic substance can have multiple epitopes. The larger the antigen molecule, the more epitopes it contains.

[0084] Tumor-associated antigens (TAAs) are antigen molecules present on tumor cells or normal cells, including embryonic proteins, glycoprotein antigens, and squamous cell antigens. They are commonly used in the clinical diagnosis of tumors. TAAs are not specific to tumor cells; normal cells can synthesize them in trace amounts, but they are highly expressed during tumor cell proliferation, hence the name "associated antigens." Tumors originating from the same tissue type may share the same tumor-associated antigens in different individuals.

[0085] Pathogenic microorganisms are microorganisms that can invade the human body, causing infection or even infectious diseases; they are also called pathogens. Among pathogens, bacteria and viruses are the most harmful. Pathogenic microorganisms include prions, fungi, bacteria, spirochetes, mycoplasma, rickettsiae, chlamydia, and viruses. Pathogenic microorganism antigens are substances derived from pathogens that have the function of triggering an immune response.

[0086] "Lipid encapsulation" refers to lipid nanoparticles that provide an active agent or therapeutic agent (e.g., nucleic acid (e.g., mRNA)) and are fully encapsulated, partially encapsulated, or somewhere in between. In some embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticles.

[0087] In various embodiments, the lipid nanoparticles have the following average diameters: about 90 nm to about 600 nm, about 100 nm to about 550 nm, about 150 nm to about 500 nm, about 200 nm to about 400 nm, about 250 nm to about 300 nm, and about 200 nm to about 300 nm, and are substantially non-toxic.

[0088] B. Auxiliary lipids

[0089] In some aspects of this disclosure, compositions containing one or more auxiliary lipids are mixed with cationic lipids disclosed in this application to produce lipid nanoparticles. In some embodiments, the cationic lipids are mixed with 1, 2, 3, 4, or 5 different types of auxiliary lipids. It is contemplated that the cationic lipids may be mixed with multiple different types of a single type of auxiliary lipid. In some embodiments, the auxiliary lipids comprise, but are not limited to, one or more of phospholipids, steroids or steroid derivatives, polymer-conjugated lipids, and modified lipids.

[0090] "Phospholipid" refers to any lipid containing a phosphate ester group. In some embodiments, the phospholipid is a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate ester groups, and optionally a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is DOPE, DSPC, DPPC, DMPC, DOPC, POPC, or SM. In some embodiments, the phospholipid is DOPE or DSPC.

[0091] "Steroids and steroid derivatives" encompasses any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure, which may further comprise one or more substitutions, said substitutions including alkyl, alkoxy, hydroxyl, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the cyclic structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring. In some embodiments, the steroid derivative comprises the above-described cyclic structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol. In some embodiments of this disclosure, said steroid or steroid derivative is cholesterane or a cholesterane derivative. As described above, cholesterane derivatives comprise one or more non-alkyl substitutions of the above-described cyclic system. In some embodiments, said cholesterane or cholesterane derivative is cholesterene or a cholesterene derivative or a sterol or a sterol derivative. In other embodiments, said cholesterane or cholesterane derivative is cholesterene and a sterol or a derivative thereof.

[0092] "Polymer-conjugated lipids" refer to lipids that inhibit lipid nanoparticle aggregation, enhance lipid nanoparticle stability, alter immune responses, or change circulation time in vivo. These polymer-conjugated lipids include, but are not limited to, polyethylene glycol-conjugated lipids, polylactic acid-conjugated lipids, polyamide-conjugated lipids, cationic polymer-conjugated lipids, polysarcosine (pSar)-conjugated lipids, polylactic-glycolic acid copolymer (PLGA)-conjugated lipids, polyamino acid-conjugated lipids, polypeptide-conjugated lipids, polypeptide-conjugated lipids, or mixtures thereof. In some embodiments, "polyethylene glycol-conjugated lipids" refers to any lipid linked with a PEG group. In some embodiments, the PEG lipid is a diglyceride that also contains a PEG chain linked to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl or C6-C24 fatty acid groups linked by a PEG chain and a linker group. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-conjugated ceramides, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amine, and PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearylphosphatidylethanolamine or PEG-modified myristoyl-sn-glycerol is used. In some embodiments, PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the molecular weight of the PEG used for modification is from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5000, from about 500 to about 3000, or from about 1200 to about 3000. The molecular weight of the PEG used for modification is from about 100, 200, 400, 500, 600, 800, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 12500 to about 15000. "Modified lipids" include lipids modified by small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations of the above substances.

[0093] Furthermore, unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" will be understood to include the stated elements or components without excluding other elements or other components.

[0094] List of English abbreviations

[0095] To more clearly describe the purpose, features, and advantages of this application, the following detailed description of this application will be provided in conjunction with the accompanying drawings and specific embodiments. However, the embodiments of this application described in detail below are merely illustrative examples and do not constitute any limitation on this application.

[0096] In the specific embodiments of this application, the raw materials used are commercially available.

[0097] Example 1: Compound Synthesis

[0098] Compound 1 is synthesized according to the following reaction:

[0099] Step 1: Synthesis of Compound 1-1

[0100] Benzaldehyde (5.91 g, 55.66 mmol) and ethanol (17 mL) were added to a 100 mL flask and cooled to 18 °C. Ammonia (11.97 g, 85.39 mmol) was slowly added dropwise to the solution, and the mixture was stirred at 18 °C for 2 h after the addition was complete. Epichlorohydrin (5 g, 54.04 mmol) was then weighed and stirred at 40-45 °C for 16 h. After the reaction was complete, the reaction solution was concentrated to dryness, and the crude product was dissolved in toluene (20 mL) and water (20 mL). Concentrated hydrochloric acid (8.32 g, 82.13 mmol) was added dropwise to the reaction system, and the mixture was stirred at 35-40 °C for 3 h. The mixture was separated, and the toluene phase was washed with water (10 mL). The aqueous phases were combined and concentrated to obtain 6 g of product compound 1-1.

[0101] Step 2: Synthesis of Compounds 1-2

[0102] Compound 1-1 (3 g, 20.55 mmol), di-tert-butyl dicarbonate (Boc₂O) (4.89 g, 22.40 mmol), methanol (12 mL), and water (7 mL) were added to a 100 mL flask. Potassium bicarbonate (2.26 g, 11.60 mmol) was weighed and added to the reaction solution in three portions, and the mixture was stirred for 3 h. The reaction progress was monitored by TLC (10% MeOH / DCM). After the reaction was complete, dichloromethane (15 mL) and water (6 mL) were added for extraction and separation. The organic phase was concentrated to obtain approximately 5 g of crude product. Purification was performed using a Flash column (20 g silica gel, 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 5% EtOAc + 95% n-heptane; 600 mL 10% EtOAc + 90% n-heptane) to obtain pure product compound 1-2, 800 mg, with a yield of 19%. 1H NMR (400MHz, CDCl3): δ4.94(m,1H),3.81-3,75(m,1H),3.73-3.69(m,1H),3.50-3.46(m,1H),3.25-3.20(m, 2H),3.00-2.94(m,2H),2.63-2.56(m,2H),2.53-2.48(m,1H),2.37-2.33(m,1H),2.31(m,3H),1.43(s,9H).

[0103] Step 3: Synthesis of compounds 1-4

[0104] Compounds 1-2 (700 mg, 3.34 mmol), N-methyl-2-(tert-butyldiphenylsiloxy)ethylamine (compounds 1-3, CAS: 165689-59-8) (1.05 g, 3.34 mmol), potassium carbonate (1.15 g, 8.35 mmol), and isopropanol (IPA) (8 mL) were added to a 25 mL flask. The mixture was heated to 80 °C and stirred for 16 h. The extent of the reaction was monitored by TLC (10% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the organic phase was concentrated to give approximately 2 g of crude product. Purification was performed using a Flash column (12 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 15% EtOAc + 85% n-heptane; 600 mL 30% EtOAc + 70% n-heptane) to obtain pure product compounds 1-4, 1 g, in 62% yield. 1 H NMR (400MHz, CDCl3): δ7.68-7.66(m,4H),7.43-7.37(m,6H),4.99(m,1H),3.74-3,70(m,3H),3.35-3.31(m,1H),3.03 -2.97(m,1H),2.73-2.67(m,1H),2.55-2.52(m,1H),2.41-2.38(m,2H),2.30(m,3H),1.44(s,9H),1.06-1.04(m,9H).

[0105] Step 4: Synthesis of compounds 1-5

[0106] Compound 1-4 (300 mg, 0.62 mmol) and ethyl acetate (2 mL) were added to a 25 mL flask. The mixture was cooled to 0-5 °C in an ice bath. 0.6 mL of HCl / EtOAc (4 M) was weighed, and the mixture was brought to room temperature and stirred for 3 h. The extent of the reaction was determined by TLC (10% MeOH / DCM). After the reaction was complete, the organic phase was concentrated to obtain approximately 300 mg of product compound 1-5.

[0107] Step 5: Synthesis of compounds 1-7

[0108] 2-Hexyldecanoic acid 5-carboxypentyl ester (compounds 1-6, CAS: 2805989-43-7) (496.14 mg, 1.34 mmol), 1,3-dicyclohexylcarbodiimide DCC (276.25 mg, 1.34 mmol), 4-dimethylaminopyridine DMAP (122.68 mg, 1.0 mmol), and dichloromethane DCM (5 mL) were added to a 25 mL flask and stirred at room temperature for 30 min. Then, compound 1-5 (330 mg, 0.66 mmol) was added dropwise, and the mixture was stirred at room temperature for 16 h. The extent of the reaction was detected by TLC (30% EtOAc / n-heptane). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The solution was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 1 g of crude product. Purification was performed using a Flash column (12 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 200 mL 20% EtOAc + 80% n-heptane) to obtain pure product compounds 1-7,330 mg, in yield 45%. 1 H NMR (400MHz, CDCl3): δ7.67-7.65(m,4H),7.45-7.36(m,6H),6.26(m,1H),4.98(m,1H),4.07-4.02(m,4H),3.75(m,2H),3.49-3.45( m,2H),2.59(m,4H),2.34-2.22(m,7H),2.11-2.08(m,2H),1.67-1.53(m,14H),1.46-1.25(m,60H),1.05(s,9H),0.89-0.85(m,12H).

[0109] Step 6: Synthesis of Compound 1

[0110] Compound 1-7 (330 mg, 0.31 mmol) and tetrahydrofuran (THF) (3 mL) were added to a 25 mL flask. The mixture was cooled to 0-5 °C in an ice bath, and tetrabutylammonium fluoride (TBAF) (240.19 mg, 0.92 mmol) was added dropwise while maintaining the temperature at 0-5 °C. The mixture was stirred for 3 h. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution, extracted with EtOAc 5 mL*2, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated to obtain approximately 500 mg of crude product. Purification was performed using a Flash column (4 g silica gel, loaded with 100 mL n-heptane; mobile phases were: 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 10% EtOAc + 90% n-heptane; 100 mL 30% EtOAc + 70% n-heptane; 300 mL 50% EtOAc + 50% n-heptane) to obtain pure product compound 1, 100 mg, in 39% yield. 1 H NMR (400MHz, CDCl3): δ6.14-6.11(m,1H),5.06-5.04(m,1H),4.08-4.04(m,4H),3.63-3.46(m,4H),2.64-2.60(m,4H), 2.35-2.26(m,7H),2.20-2.17(m,2H),1.70-1.53(m,12H),1.46-1.35(m,8H),1.33-1.25(m,45H),0.89-0.85(m,12H).

[0111] Compound 2 is synthesized according to the following reaction:

[0112] Step 1: Synthesis of Compound 2-2

[0113] In a 25 mL flask, 8-oxo-8-(pentadecan-7-yloxy)octanoic acid (compound 2-1, CAS: 2765518-33-8) (840.64 mg, 2.19 mmol), 1,3-dicyclohexylcarbodiimide DCC (450.99 mg, 2.19 mmol), 4-dimethylaminopyridine DMAP (133.52 mg, 1.09 mmol), and dichloromethane DCM (3 mL) were added. The mixture was stirred at room temperature for 30 min, and then 1-4 (500 mg, 1.09 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (20% EtOAc / n-heptane). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 1.6 g of crude product. Purification using a Flash column (12 g silica gel, mobile phases: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane) yielded 2-2,320 mg of the pure product compound, in 25% yield.

[0114] Step 2: Synthesis of Compound 2

[0115] Compound 2-2 (320 mg, 0.29 mmol) and tetrahydrofuran (THF) (3 mL) were added to a 25 mL flask. The mixture was cooled to 0-5 °C in an ice bath, and tetrabutylammonium fluoride (TBAF) (224.16 mg, 0.86 mmol) was added dropwise while maintaining the temperature at 0-5 °C. The mixture was stirred for 3 h. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution, extracted with EtOAc 5 mL*2, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated to obtain approximately 500 mg of crude product. Purification was performed using a Flash column (4 g silica gel, loaded with 100 mL n-heptane; mobile phases were: 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 10% EtOAc + 90% n-heptane; 100 mL 30% EtOAc + 70% n-heptane; 300 mL 50% EtOAc + 50% n-heptane) to obtain pure product compound 2, 100 mg, in 40% yield. 1H NMR (400MHz, CDCl3) δ6.09(d,J=6.4Hz,1H),5.05(q,J=5.7Hz,1H),4.85(p,J=6.2Hz,2H),3.66-3.41(m,4H),2.61(t, J=5.6Hz,4H),2.39-2.22(m,9H),2.20-2.12(m,2H),1.67-1.44(m,17H),1.40-1.16(m,53H),0.87(t,J=6.7Hz,12H).

[0116] Compound 3 was synthesized according to the following reaction:

[0117] Step 1: Synthesis of Compound 3-1

[0118] In a 250 mL flask, hexadecanoic acid (5.64 g, 19.7 mmol), 1,3-dicyclohexylcarbodiimide (DCC) (4.06 g, 19.7 mmol), 4-dimethylaminopyridine (DMAP) (802.28 mg, 6.57 mmol), and dichloromethane (DCM) (30 mL) were added. The mixture was stirred at room temperature for 30 min, and then pentadecane-7-ol (1.5 g, 6.57 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was determined by TLC (30% ethyl acetate / n-heptane). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 50 mL of water and 50 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered again, and the organic phase was concentrated to give approximately 5 g of crude product. Purification using a Flash column (40 g silica gel, mobile phases: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 800 mL 8% EtOAc + 92% n-heptane) yielded 1.4 g of pure product compound 3-1, in 31% yield. 1 H NMR (400MHz, CDCl3) δ4.86 (p, J=6.3Hz, 1H), 2.31 (dt, J=26.9, 7.5Hz, 4H), 1.62 (h ,J=7.1Hz,4H),1.50(q,J=6.1Hz,4H),1.40-1.16(m,42H),0.87(t,J=6.7Hz,6H).

[0119] Step 2: Synthesis of compound 3-2

[0120] Compound 3-1 (706.27 mg, 1.42 mmol), 1,3-dicyclohexylcarbodiimide DCC (293.32 mg, 1.42 mmol), 4-dimethylaminopyridine DMAP (86.64 mg, 0.72 mmol), and dichloromethane DCM (30 mL) were added to a 25 mL flask and stirred at room temperature for 30 min. Then, compound 1-4 (derived from the intermediate in the synthesis of compound 1) (300 mg, 0.72 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 1.2 g of crude product. Purification was performed using a Flash column (4 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 80% n-heptane; 400 mL 30% EtOAc + 70% n-heptane) to obtain pure product compound 3-2, 240 mg, yield 25%. 1 H NMR (400MHz, CDCl3) δ7.72-7.61(m,5H),7.48-7.33(m,8H),4.98(s,1H),4.87(p,J=6.3Hz,3H),3.74(s,2H),3.47(t,J=5.8Hz,3H), 2.73-2.49(m,4H),2.27(t,J=7.5Hz,12H),1.70-1.44(m,23H),1.26(td,J=7.4,6.3,3.0Hz,124H),1.05(s,9H),0.91-0.84(m,12H).

[0121] Step 3: Synthesis of Compound 3

[0122] Compound 3-2 (240 mg, 0.18 mmol) and tetrahydrofuran (THF) (3 mL) were added to a 25 mL flask. The mixture was cooled to 0-5 °C in an ice bath, and tetrabutylammonium fluoride (TBAF) (140.05 mg, 0.54 mmol) was added dropwise while maintaining the temperature at 0-5 °C. The mixture was stirred for 3 h. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution, extracted with 10 mL*2 EtOAc, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated to obtain approximately 250 mg of crude product. Purification was performed using a Flash column (4 g silica gel, loaded with 100 mL n-heptane; mobile phases were: 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 10% EtOAc + 90% n-heptane; 100 mL 30% EtOAc + 70% n-heptane; 300 mL 50% EtOAc + 50% n-heptane) to obtain 3.80 mg of pure product compound in 40% yield. 1 H NMR (400MHz, CDCl3) δ5.09 (s, 1H), 4.86 (p, J = 6.3Hz, 2H), 3.64 (t, J = 5.2Hz, 2H), 3.50 (ddd, J = 14.3, 8.9, 5.5Hz, 2H), 2.6 7(s,3H),2.44-2.11(m,12H),1.61(p,J=7.3Hz,9H),1.50(q,J=6.1Hz,8H),1.35-1.17(m,96H),0.87(t,J=6.7Hz,12H).

[0123] Compound 4 was synthesized according to the following reaction:

[0124] Step 1: Synthesis of Compound 4-1

[0125] Succinic anhydride (1.75 g, 17.51 ​​mmol), 4-dimethylaminopyridine (DMAP) (2.14 g, 17.51 ​​mmol), pentadecane-7-ol (1.5 g, 6.57 mmol), and dichloromethane (DCM) (30 mL) were added to a 250 mL flask and stirred overnight at room temperature. The extent of the reaction was detected by TLC (30% ethyl acetate / n-heptane). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 50 mL of water and 50 mL of saturated brine. The solution was dried over anhydrous Na₂SO₄, filtered again, and the organic phase was concentrated to give approximately 5 g of crude product. Purification using a Flash column (40 g silica gel, mobile phases: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 600 mL 8% EtOAc + 92% n-heptane) yielded 2.4 g of pure product compound 4-1, in 42% yield. 1 H NMR (400MHz, CDCl3) δ4.88 (p, J = 6.3Hz, 1H), 2.74-2.55 (m, 4H), 1.51 (q, J = 6.2Hz, 4H), 1.26 (d, J = 6.0Hz, 22H), 0.87 (t, J = 6.7Hz, 6H).

[0126] Step 2: Synthesis of compound 4-2

[0127] Compound 4-1 (466.98 mg, 1.42 mmol), 1,3-dicyclohexylcarbodiimide DCC (293.32 mg, 1.42 mmol), 4-dimethylaminopyridine DMAP (86.64 mg, 0.72 mmol), and dichloromethane DCM (3 mL) were added to a 25 mL flask and stirred at room temperature for 30 min. Then, compound 1-4 (derived from the intermediate in the synthesis of compound 1) (300 mg, 0.72 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 800 mg of crude product. Purification was performed using a Flash column (4 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 800 mL 8% EtOAc + 92% n-heptane; 300 mL 20% EtOAc + 80% n-heptane) to obtain pure product compound 4-2, 240 mg, yield 33.5%. 1H NMR(400MHz, CDCl3)δ7.66(d,J=6.9Hz,5H),7.41(t,J=8.6Hz,7H),5.03(s,1H),4.92-4.76(m,3H) ,2.74-2.51(m,12H),1.60-1.46(m,11H),1.35-1.17(m,64H),1.05(s,9H),0.87(t,J=6.6Hz,12H).

[0128] Step 3: Synthesis of Compound 4

[0129] Compound 4-2 (240 mg, 0.24 mmol) and tetrahydrofuran (THF) (3 mL) were added to a 25 mL flask. The mixture was cooled to 0-5 °C in an ice bath, and tetrabutylammonium fluoride (TBAF) (186.84 mg, 0.72 mmol) was added dropwise while maintaining the temperature at 0-5 °C. The mixture was stirred for 3 h. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution, extracted with 10 mL*2 EtOAc, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated to give approximately 250 mg of crude product. Purification was performed using a Flash column (4 g silica gel, loaded with 100 mL n-heptane; mobile phases were: 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 10% EtOAc + 90% n-heptane; 300 mL 30% EtOAc + 70% n-heptane) to obtain 4.80 mg of pure product compound, yield 44%. 1 H NMR (400MHz, CDCl3) δ6.56(s,1H),5.18-5.07(m,1H),4.85(dp,J=12.3,6.2Hz,2H),3.61(ddt,J=10.4,6.2,3.8Hz,3H),3.43(dt ,J=14.1,6.0Hz,1H),2.83–2.55(m,10H),2.55-2.33(m,5H),1.59-1.42(m,8H),1.26(d,J=6.7Hz,42H),0.87(t,J=6.7Hz,12H).

[0130] Compound 5 was synthesized according to the following reaction:

[0131] Step 1: Synthesis of Compound 5

[0132] In a 25 mL flask, 4-dimethylaminopyridine (DMAP) (65.9 mg, 0.54 mmol), 1,3-dicyclohexylcarbodiimide (DCC) (222.7 mg, 1.1 mmol), and dichloromethane (DCM) (1.5 mL) were added. The mixture was stirred until clear, and then 5-carboxypentyl 2-hexyldecanoate (compounds 1-6, CAS: 2805989-43-7) (400 mg, 1.1 mmol) was added. The mixture was stirred at room temperature for 30 min, and then 1-amino-3-(dimethylamino)propanol (compound 5-1, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., 63.8 mg, 0.55 mmol) was added in a single batch. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (10% MeOH / DCM). After the reaction was complete, the reaction solution was filtered to remove the solids. The filtrate was washed with 5 mL of water and 5 mL of saturated saline solution, dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated to obtain approximately 510 mg of crude product. Purification was performed using a Flash column (20 g silica gel, loaded with 100 mL n-heptane; mobile phases were: 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 3% EtOAc + 97% n-heptane; 100 mL 5% EtOAc + 95% n-heptane; 100 mL 10% EtOAc + 90% n-heptane; 100 mL 20% EtOAc + 80% n-heptane; 300 mL 50% EtOAc + 50% n-heptane) to obtain 5,300 mg of pure product compound in 67% yield. 1 H NMR (400MHz, CDCl3): δ6.32-6.29(m,1H),5.03-4.97(m,2H),4.07-4.04(m,4H),3.02(m,2H),2.49-2.39(m ,2H),2.33-2.26(m,11H),2.18-2.14(m,2H),1.69-1.53(m,12H),1.46-1.24(m,51H),0.88-0.85(m,12H).

[0133] Compound 6 was synthesized according to the following reaction:

[0134] Step 1: Synthesis of Compound 6-1

[0135] In a 25 mL flask, 2-hexyldecanoic acid 5-carboxypentyl ester (compounds 1-6, CAS: 2805989-43-7) (2.1 g, 5.67 mmol), 1,3-dicyclohexylcarbodiimide DCC (1.17 g, 5.67 mmol), 4-dimethylaminopyridine DMAP (692.63 mg, 5.67 mmol), and dichloromethane DCM (10 mL) were added. The mixture was stirred at room temperature for 30 min, and then 1-amino-3-(dimethylamino)prop-2-ol (670 mg, 5.67 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 10 mL of water and 10 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 3.5 g of crude product. Purification was performed using a Flash column (40 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 80% n-heptane; 600 mL 50% EtOAc + 50% n-heptane) to obtain 1 g of pure product compound 6-1 in 37% yield.

[0136] Step 2: Synthesis of Compound 6

[0137] In a 25 mL flask, 8-oxo-8-(pentadecan-7-yloxy)octanoic acid (compound 2-1, CAS: 2765518-33-8) (245.10 mg, 0.64 mmol), 1,3-dicyclohexylcarbodiimide DCC (131.50 mg, 0.64 mmol), 4-dimethylaminopyridine DMAP (77.86 mg, 0.64 mmol), and dichloromethane DCM (3 mL) were added. The mixture was stirred at room temperature for 30 min, and then 6-1 (300 mg, 0.64 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 700 mg of crude product. Purification was performed using a Flash column (12 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 80% n-heptane; 700 mL 30% EtOAc + 70% n-heptane) to obtain 6,240 mg of pure product compound, yield 45%. 1H NMR (400MHz, CDCl3) δ6.34(d,J=5.7Hz,1H),5.01(p,J=5.9Hz,1H),4.85(p,J=6.3Hz,1H),4.05(t,J=6.7Hz,2H),3.56-3.41 (m,2H),2.48(q,J=12.9,6.1Hz,2H),2.37-2.24(m,11H),2.17(t,J=7.7Hz,2H),1.73-1.15(m,66H),0.87(t,J=6.7Hz,12H).

[0138] Compound 7 was synthesized according to the following reaction:

[0139] Step 1: Synthesis of Compound 7

[0140] In a 25 mL flask, 8-oxo-8-(pentadecano-7-yloxy)octanoic acid (compound 2-1, CAS: 2765518-33-8) (650.87 mg, 1.69 mmol), 1,3-dicyclohexylcarbodiimide DCC (349.18 mg, 1.69 mmol), 4-dimethylaminopyridine DMAP (103.38 mg, 0.85 mmol), and dichloromethane DCM (5 mL) were added. The mixture was stirred at room temperature for 30 min, and then 1-amino-3-(dimethylamino)prop-2-ol (100 mg, 0.85 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 10 mL of water and 10 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 800 mg of crude product. Purification was performed using a Flash column (12 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 80% n-heptane; 800 mL 30% EtOAc + 70% n-heptane) to obtain 7,460 mg of pure product compound, yield 64%. 1H NMR (400MHz, CDCl3) δ6.32(s,1H),5.01(t,J=5.8Hz,1H),4.85(p,J=6.2Hz,2H),3.49(t,J=5.1Hz,2H),2.47(q,J=12.8,6.1Hz, 2H),2.36-2.23(m,12H),2.15(t,J=7.6Hz,2H),1.56(d,J=48.7,6.5,6.0Hz,17H),1.40-1.16(m,53H),0.87(t,J=6.7Hz,12H).

[0141] Compound 8 was synthesized according to the following reaction:

[0142] Step 1: Synthesis of Compound 8

[0143] In a 25 mL flask, 2-hexyldecanoic acid 6-carboxyhexyl ester (compound 8-1, CAS: 2805989-46-0) (245.10 mg, 0.64 mmol), 1,3-dicyclohexylcarbodiimide DCC (131.50 mg, 0.64 mmol), 4-dimethylaminopyridine DMAP (77.86 mg, 0.64 mmol), and dichloromethane DCM (3 mL) were added. The mixture was stirred at room temperature for 30 min, and then compound 6-1 (derived from the intermediate in the synthesis of compound 6 above) (300 mg, 0.64 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 600 mg of crude product. Purification was performed using a Flash column (12 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 200 mL 20% EtOAc + 80% n-heptane) to obtain pure product compound 8, 120 mg, in yield 22%. 1 H NMR (400MHz, CDCl3) δ6.34(d,J=5.5Hz,1H),5.01(t,J=5.9Hz,1H),4.05(t,J=6.7Hz,4H),3.49(t,J=5.6Hz,2H),2.48(dd,J=8. 6, 6.2Hz, 2H), 2.30 (d, J = 11.3Hz, 10H), 2.17 (t, J = 7.7Hz, 2H), 1.71-1.50 (m, 13H), 1.48-1.14 (m, 57H), 0.87 (t, J = 6.7Hz, 12H).

[0144] Compound 9 was synthesized according to the following reaction:

[0145] Step 1: Synthesis of Compound 9

[0146] In a 25 mL flask, 6-(decanoyloxy) acid compound (compound 9-1, CAS: 2254439-01-3) (201.21 mg, 0.70 mmol), 1,3-dicyclohexylcarbodiimide DCC (144.96 mg, 0.70 mmol), 4-dimethylaminopyridine DMAP (85.83 mg, 0.70 mmol), and dichloromethane DCM (3 mL) were added. The mixture was stirred at room temperature for 30 min, and then compound 6-1 (derived from the intermediate in the synthesis of compound 6 above) (330 mg, 0.70 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The filtrate was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 700 mg of crude product. Purification was performed using a Flash column (4 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 300 mL 80% n-heptane + 30% EtOAc + 70% n-heptane) to obtain pure product compound 9,140 mg, yield 27%. 1 H NMR (400MHz, CDCl3) δ6.33 (s, 1H), 5.02 (p, J = 5.8Hz, 1H), 4.05 (t, J = 6.7Hz, 4H), 3.57-3.42 (m, 2H), 2.49 (qd, J = 12.9 ,6.1Hz,2H),2.38-2.24(m,11H),2.17(t,J=7.7Hz,2H),1.72-1.51(m,12H),1.50-1.16(m,43H),0.96-0.78(m,9H).

[0147] Compound 10 was synthesized according to the following reaction:

[0148] Step 1: Synthesis of Compound 10

[0149] In a 25 mL flask, 6-carboxyhexyl 2-hexyldecanoate (compound 8-1, CAS: 2805989-46-0) (500 mg, 1.3 mmol), 1,3-dicyclohexylcarbodiimide DCC (268.24 mg, 1.3 mmol), 4-dimethylaminopyridine DMAP (39.71 mg, 0.33 mmol), and dichloromethane DCM (3 mL) were added. The mixture was stirred at room temperature for 30 min, and then 1-amino-3-(dimethylamino)prop-2-ol (76.82 mg, 0.65 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The extent of the reaction was detected by TLC (5% MeOH / DCM). After the reaction was complete, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated saline solution. The solution was dried over anhydrous Na₂SO₄, filtered, and the organic phase was concentrated to give approximately 700 mg of crude product. Purification was performed using a Flash column (4 g silica gel, mobile phases were: 100 mL n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 8% EtOAc + 92% n-heptane; 100 mL 20% EtOAc + 80% n-heptane + 200 mL 30% EtOAc + 70% n-heptane) to obtain 10, 120 mg of pure product compound, yield 22%. 1 H NMR(400MHz, CDCl3)δ6.40(s,1H),5.04(q,J=6.0Hz,1H),4.05(td,J=6.7,2.6Hz,4H),3.50(t,J=5.6Hz,2H),2.68-2.46(m,2H), 2.31(d,J=12.8,3.8Hz,10H),2.17(t,J=7.6Hz,2H),1.60(dt,J=22.6,7.8Hz,13H),1.50-1.15(m,61H),0.87(t,J=6.7Hz,12H).

[0150] Example 2: Preparation of lipid nanoparticles (TMF)

[0151] In this embodiment, TMF1-17 refers to lipid nanoparticles formed by using the compound prepared in Example 1 as a cationic lipid and other components. There are no restrictions on the mRNA encapsulated by these lipid nanoparticles.

[0152] Preparation of lipid nanoparticles TMF1 encapsulating luciferase mRNA (hereinafter referred to as Luc-mRNA) (containing 46.3% compound 1):

[0153] 1) Compound 1 (cationic lipid), DOPE (auxiliary lipid), cholesterol (auxiliary lipid) and PEG2000-DMG (auxiliary lipid) were dissolved in ethanol to obtain oil phase (ethanol phase) stock solutions with molar percentages of 46.3%, 9.4%, 42.7% and 1.6%, respectively.

[0154] 2) Dilute the Luc-mRNA stock solution to 0.3 mg / mL with pH 4 citrate buffer to obtain the aqueous phase;

[0155] 3) The oil phase (ethanol phase) containing the mixture of four lipids obtained in step 1) and the aqueous phase containing mRNA obtained in step 2) were rapidly mixed at a volume ratio of 1:3 to prepare TMF1 with a total lipid to mRNA weight ratio of 24.5:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100kd, followed by washing with pH 7.4 PBS buffer, and finally replaced with 120mg / mL sucrose PBS buffer to obtain the final sample.

[0156] The lipid nanoparticles TMF2-17 were prepared using the same method as TMF1, with the lipid components replaced according to the lipids listed in Table 1.

[0157] Table 1. Lipid composition of lipid nanoparticles TMF1-17

[0158] In addition, ALC-0315, DODAP, and 004 were used as cationic lipid controls, and their structures are shown below:

[0159] Example 3: Characterization and Detection of Lipid Nanoparticles

[0160] Using a NanoBrook 90plus PLAS (Brookhaven Instruments, US), dynamic light scattering (DLS) was employed to determine the particle size and polydispersity index (PDI) of lipid nanoparticles at a 90° lateral scattering angle. The test results are shown in Table 2 below.

[0161] Table 2. Characterization data of lipid nanoparticles prepared from representative cationic lipid compounds

[0162] Example 4: In vivo distribution detection of delivery system

[0163] The lipid nanoparticles containing Luc-mRNA prepared in Example 2 were administered via both intravenous and intramuscular injection.

[0164] Intravenous injection: Liposomes prepared in Example 2 were injected into 6-8 week old female ICR mice via the medial canthal venous plexus of the eye at a dose of 0.5 mg / kg. Four hours after administration, 15 mg / mL of D-luciferin potassium salt was injected intraperitoneally at a dose of 150 mg / kg. Ten minutes after the luciferase substrate injection, the mice were placed under an in vivo imaging system (IVIS Lumina XRMS Series III, PerkinElmer) to observe the fluorescence intensity and distribution in the mice. The mice were then sacrificed, and the organs (heart, liver, spleen, lung, and kidney) were isolated for in vitro imaging to observe the fluorescence intensity and distribution in different organs.

[0165] The organ distribution of Luc-mRNA delivered by representative lipid compounds is shown in Table 3. As shown in Table 3, the fluorescence intensity of lipid nanoparticle TMF5 in the spleen was higher than that of the control groups TMF15, TMF16, and TMF17. Moreover, the ratio of fluorescence intensity in the spleen to the liver (spleen / liver) was higher than that of the control groups TMF16 and TMF17, but lower than that of TMF15.

[0166] Intramuscular injection: Liposomes prepared in Example 2 were injected into the medial thigh muscle of 6-8 week old female ICR mice at a dose of 0.5 mg / kg. Four hours after administration, 15 mg / mL of D-luciferin potassium salt was injected intraperitoneally at a dose of 150 mg / kg. Ten minutes after the luciferase substrate injection, the mice were placed under an in vivo imaging system (IVIS Lumina XRMS Series III, PerkinElmer) to observe the fluorescence intensity and distribution in the mice.

[0167] The in vivo distribution of Luc-mRNA delivered by representative lipid compounds is shown in Table 4. Furthermore, Figure 11 summarizes the fluorescence intensity ratios of these lipid nanoparticles at the injection site and in viscera.

[0168] As shown in Table 4 and Figure 11, lipid nanoparticles TMF7 and TMF3, with the addition of compounds 7 and 3, exhibited fluorescence intensity ratios exceeding 30 at the injection site and in viscera 4 hours after intramuscular injection in mice, with TMF3 showing the highest ratio at 35.9. Lipid nanoparticles TMF7, TMF10, TMF12, and TMF13 also showed high fluorescence intensities at the injection site, all above 6.5E+09, with TMF7 showing the highest intensity at 8.52E+09. Comparing TMF5 and TMF11, TMF7 and TMF12, and TMF10 and TMF13, the data indicate that lipid nanoparticles prepared by combining the same cationic lipids with different phospholipids exhibited similar fluorescence intensities at the injection site, and similar fluorescence intensity ratios between the injection site and viscera.

[0169] Compared with the control lipid nanoparticles TMF14 and TMF15 using compound 004, the lipid nanoparticles TMF1-TMF13 of this application not only showed higher selectivity at the injection site than the control group, but also exhibited fluorescence intensity at the injection site that was more than three orders of magnitude higher than the control group; compared with the control lipid nanoparticles TMF16 using DODAP, the lipid nanoparticles TMF1-TMF13 of this application not only showed higher selectivity at the injection site than the control group, but also exhibited fluorescence intensity at the injection site that was more than three orders of magnitude higher than the control group; compared with the control lipid nanoparticles TMF17 using ALC-0315, the lipid nanoparticles TMF1-TMF13 of this application not only showed higher selectivity at the injection site than the control group, but also exhibited fluorescence intensity at the injection site that was more than one order of magnitude higher than the control group.

[0170] Furthermore, Figure 12 shows the fluorescence signal of the lipid nanoparticles TMF1 loaded with Luc-mRNA in mice 4 hours after intramuscular injection; as shown in Figure 11, the fluorescence signal is strongest at the injection site, while the fluorescence signal is very weak in the viscera. This further illustrates that the expression of Luc-mRNA loaded with TMF1 is significantly higher at the injection site than in the viscera.

[0171] The fluorescence intensity at different sites represents the delivery efficiency of the corresponding delivery system at different sites. The above data fully demonstrate that the lipid nanoparticles of this application can successfully deliver nucleic acid molecules to the injection site and express them at high levels. Moreover, the delivery efficiency at the injection site is significantly higher than that at the viscera, reducing potential liver toxicity.

[0172] Table 3. Expression intensity (IV) of Luc-mRNA delivered by lipid nanoparticles prepared from representative cationic lipids.

[0173] Table 4. Expression intensity (IM) of Luc-mRNA delivered by lipid nanoparticles prepared from representative cationic lipids.

[0174] The above detailed description of this application is intended to enable those skilled in the art to understand the mechanism and content of this application and to implement it. It should not be construed as limiting the scope of protection of this application. All equivalent changes or modifications made in accordance with the spirit and essence of this application should be included within the scope of protection of this application. Industrial applicability

[0175] The cationic lipid compounds and lipid nanoparticles containing them provided in this application can deliver therapeutic agents or preventive agents (especially nucleic acid substances) to the injection site via intramuscular injection. The therapeutic agents or preventive agents are highly expressed at the injection site but have low expression levels in the viscera. This allows the drugs to not only exert their activity efficiently but also have low visceral toxicity, which is of great significance for the development and application of nucleic acid therapeutic agents or preventive agents.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in: Y1 and Y2 are each independently -OC(=O)- or -C(=O)O-; L1 and L2 are each independently bonded, optionally substituted C1-C 18 Alkylene; R1 and R2 are each independently H or C1-C8 straight-chain or branched alkyl groups; R3 and R4 are each independently C1-C8 straight-chain or branched alkyl groups that are optionally substituted with hydroxyl groups; m is an integer between 1 and 10; n is an integer between 1 and 10.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, characterized in that, L1 and L2 are each independently unsubstituted C2-C 14 Alkylene; And / or, R1 and R2 are each independently H or C1-C8 straight-chain alkyl; And / or, R3 and R4 are each independently C1-C4 straight-chain or branched alkyl groups optionally substituted with hydroxyl groups; And / or, m is an integer between 5 and 7; And / or, where n is an integer between 5 and 7.

3. The compound according to claim 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, characterized in that, R1 and R2 are each independently H or C6-C8 straight-chain alkyl; And / or, R3 and R4 are each independently methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, or hydroxybutyl.

4. The compound according to claim 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, characterized in that, The compound is selected from the following compounds:

5. A lipid nanoparticle comprising the compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

6. The lipid nanoparticles according to claim 5, characterized in that, The lipid nanoparticles further comprise auxiliary lipids selected from one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids.

7. The lipid nanoparticles according to claim 6, characterized in that, The phospholipids are selected from any one or a combination of DOPE, DSPC, DPPC, DMPC, DOPC, POPC, and SM. And / or, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol, and ergosterol; And / or, the polymer-conjugated lipids are selected from polyethylene glycol, polylactic acid, polyamide, cationic polymers, polysarcosine, polylactic-co-glycolic acid copolymers, polyamino acids, polypeptides, and lipids conjugated with peptides. And / or, the modifiable lipids are selected from lipids that can be modified by small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations thereof.

8. The lipid nanoparticles according to claim 7, characterized in that, The phospholipid is DOPE or DSPC; And / or, the steroid is cholesterol and / or sitosterol; And / or, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid selected from one or more of ALC-0159, PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE.

9. The lipid nanoparticles according to any one of claims 6-8, characterized in that, The molar ratio of the compound as claimed in any one of claims 1-4 or its pharmaceutically acceptable salt, prodrug or stereoisomer to the auxiliary lipid is 1:(0.5-2).

10. The lipid nanoparticles according to claim 9, characterized in that, The molar ratio of the compound as described in any one of claims 1-4 or its pharmaceutically acceptable salt, prodrug or stereoisomer to the auxiliary lipid is 1:(0.6 to 1.5).

11. The lipid nanoparticles according to claim 10, characterized in that, The molar ratio of the compound as described in any one of claims 1-4 or its pharmaceutically acceptable salt, prodrug or stereoisomer to the auxiliary lipid is 1:(0.8 to 1.2).

12. The use of the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, or the lipid nanoparticle of any one of claims 5-11 in the preparation of a drug delivery carrier.

13. The application according to claim 12, characterized in that, The drug is a therapeutic or preventative agent.

14. The application according to claim 13, characterized in that, The therapeutic or preventative agent is a nucleic acid.

15. The application according to claim 14, characterized in that, The nucleic acids are selected from: single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isoforms, plasmid DNA, complementary DNA, antisense nucleic acid molecules, small interfering nucleic acids, small activating nucleic acids, asymmetric interfering nucleic acids, micro nucleic acids, agomir, antagomir, Dicer enzyme substrate nucleic acids, hairpin nucleic acids, transfer RNA, messenger RNA, circular RNA, self-replicating mRNA, and aptamers.

16. The application according to claim 15, characterized in that, The nucleic acid is a natural nucleotide, a nucleotide analog, a functional analog, or a chemically modified nucleotide.

17. The application according to claim 15, characterized in that, The nucleic acid is mRNA.

18. The application according to claim 17, characterized in that, The mRNA encodes at least one antigen or a fragment thereof or an epitope thereof, or encodes a therapeutic protein.

19. The application according to claim 18, characterized in that, The antigen is a pathogenic antigen.

20. The application according to claim 19, characterized in that, The antigen is a tumor-associated antigen or a pathogenic microorganism antigen.

21. A pharmaceutical composition comprising lipid nanoparticles as described in any one of claims 5-11, and a therapeutic or preventative agent.

22. The pharmaceutical composition according to claim 21, characterized in that, The therapeutic or preventative agent is a nucleic acid, which is encapsulated in the lipid nanoparticles.

23. The pharmaceutical composition according to claim 22, characterized in that, The nucleic acids are selected from: single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isoforms, plasmid DNA, complementary DNA, antisense nucleic acid molecules, small interfering nucleic acids, small activating nucleic acids, asymmetric interfering nucleic acids, micro nucleic acids, agomir, antagomir, Dicer enzyme substrate nucleic acids, hairpin nucleic acids, transfer RNA, messenger RNA, circular RNA, self-replicating mRNA, and aptamers.

24. The pharmaceutical composition according to claim 23, characterized in that, The nucleic acid is a natural nucleotide, a nucleotide analog, a functional analog, or a chemically modified nucleotide.

25. The pharmaceutical composition according to claim 23, characterized in that, The nucleic acid is mRNA.

26. The pharmaceutical composition according to claim 25, characterized in that, The mRNA encodes at least one antigen or a fragment thereof or an epitope thereof, or encodes a therapeutic protein.

27. The pharmaceutical composition according to claim 26, characterized in that, The antigen is a pathogenic antigen.

28. The pharmaceutical composition according to claim 27, characterized in that, The antigen is a tumor-associated antigen or a pathogenic microorganism antigen.

29. The pharmaceutical composition according to claim 21, characterized in that, In the pharmaceutical composition, the mass ratio of the lipid nanoparticles to the therapeutic or preventative agent is from 10:1 to 100:

1.

30. The pharmaceutical composition according to claim 29, characterized in that, In the pharmaceutical composition, the mass ratio of the lipid nanoparticles to the therapeutic or preventative agent is 20:1 to 50:

1.

31. The pharmaceutical composition according to claim 30, characterized in that, In the pharmaceutical composition, the mass ratio of the lipid nanoparticles to the therapeutic or preventative agent is 20:1 to 30:

1.

32. The pharmaceutical composition according to claim 21, characterized in that, The average particle size of the pharmaceutical composition is 90 nm to 600 nm; And / or, the polydispersity index of the pharmaceutical composition is from 0.001 to 0.

5.

33. The pharmaceutical composition according to claim 32, characterized in that, The average particle size of the pharmaceutical composition is 200 nm to 400 nm; And / or, the polydispersity index of the pharmaceutical composition is from 0.001 to 0.

45.

34. The pharmaceutical composition according to claim 33, characterized in that, The average particle size of the pharmaceutical composition is 200 nm to 300 nm; And / or, the polydispersity index of the pharmaceutical composition is from 0.001 to 0.

4.

35. Use of the pharmaceutical composition according to any one of claims 21-34 in the preparation of a targeted delivery medicament.

36. A formulation comprising the pharmaceutical composition as described in any one of claims 21-34 and a pharmaceutically acceptable excipient.

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