Compounds, compositions, methods of making compositions and use in medicine

Abstract

The embodiment of the application discloses a kind of compound, composition, preparation method of composition and application in pharmacy, the compound can form lipid nanoparticle, by adjacent hydrophilic head and located at both ends by ester bond connected hydrophobic tail make the particle size of the lipid nanoparticle formed smaller, one hydrophilic head of multistage ionizing amine plays stronger proton sponge effect, hydrophilic head and nucleic acid molecule cooperate to cover nucleic acid molecule in particle interior.And ester bond connected fatty acid is non-toxic to cell, can improve the antigen expression amount of cell.Therefore, more efficient, reliable lipid nanoparticle is constructed.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to a compound, a composition, a method for preparing the composition, and its application in pharmaceutical manufacturing. Background Technology

[0002] Lipid nanoparticles (LNPs) are a highly efficient nucleic acid delivery system, in which ionizable lipids are the core component. However, current ionizable lipids suffer from low nucleic acid delivery efficiency and high biotoxicity, severely limiting the application of lipid nanoparticles.

[0003] Therefore, the need for more efficient and reliable ionizable lipids has become an urgent problem to be solved. Summary of the Invention

[0004] This application provides a compound that can serve as an ionizable lipid in lipid nanoparticles. Compared to existing ionizable lipids, the ionizable lipid provided in this application is more efficient and reliable.

[0005] In a first aspect, a compound is provided having the formula: ; Wherein, R1 and R2 are independently selected from substituted or unsubstituted saturated C2. 14 To C 25 The alkyl acyl group; the L group includes an ionizable amine linker, the ionizable amine linker including at least one primary amine and at least one tertiary amine; the A group includes a targeting ligand, the targeting ligand including at least one hydroxyl group, the targeting ligand being used to target receptors on the surface of immune cells.

[0006] The compound provided in this application has a hydrophilic head formed by a targeting ligand and an ionizable amine linker. This compound self-assembles into stable particles in an aqueous phase. Because the hydrophilic head is compatible with the aqueous phase, while the hydrophobic tail repels water molecules, the compound spontaneously arranges itself into a spherical structure for easy delivery of nucleic acid molecules. The head contains adjacent hydroxyl and amino groups, resulting in stronger hydrophilicity, smaller spherical particle size, and longer blood circulation time, enabling long-term antigen expression. R1 and R2 are independently alkyl acyl groups, and the ester bond between the hydrophobic tail and the glycerol backbone is specifically hydrolyzed by esterases within the cell, thereby rapidly releasing nucleic acid molecules. Furthermore, the metabolites of this compound are fatty acids and glycosyl groups, exhibiting no significant cytotoxicity and no inhibition of translation, thus increasing antigen expression levels. The compound head contains multiple ionizable amino groups, including at least one primary amine and at least one tertiary amine. It is neutral under physiological conditions, has a wider pKa response range, can achieve multi-level protonation in acidic endosomes, exhibits a strong proton sponge effect, high endosome escape efficiency, and improved cytoplasmic delivery efficiency. These properties combine to enable efficient delivery of nucleic acid molecules and maintain long-term, high-volume antigen expression, thereby enhancing the immune response mediated by biological agents made from this compound.

[0007] In some embodiments, the pKa of the compound is in the range of 6.0-6.6.

[0008] The compounds provided in this application have pKa values ​​of 6.0-6.6 due to multiple amine groups. They exhibit high protonation in acidic endosome environments, strong proton sponge effect, and high endosome escape efficiency, thereby improving cytoplasmic delivery efficiency.

[0009] In some embodiments, the A group is selected from 2-acetamido-2-deoxy-D-mannose-6-oxy, CLEC9A affinity peptide, or folic acid.

[0010] In the compounds provided in this application, 2-acetamino-2-deoxy-D-mannose-6-oxy is a natural building block of human glycoproteins. While efficiently delivering nucleic acid molecules, its degradation products do not have significant cytotoxicity and do not cause complement activation-related allergic reactions. Furthermore, 2-acetamino-2-deoxy-D-mannose-6-oxy makes the compound more likely to accumulate in the spleen during long-term blood circulation, thereby reducing liver accumulation, which can reduce the burden on the liver and reduce inflammatory responses.

[0011] In some embodiments, R1 and R2 are each independently selected from substituted or unsubstituted saturated C. 14 To C 25 Straight-chain alkyl acyl group.

[0012] In some embodiments, R1 and R2 are each independently selected from unsubstituted saturated C. 14 To C25 Straight-chain alkyl acyl group.

[0013] In the compounds provided in the embodiments of this application, saturated C 14 To C 25 The strong hydrophobicity of straight-chain alkyl acyl groups can promote the formation of spherical coatings on nucleic acid molecules, further reducing the diameter of the formed spheres, allowing for longer blood circulation and enabling long-term antigen expression.

[0014] In some embodiments, R1 and R2 are independently selected from hexadecylacyl and octadecylacyl, respectively.

[0015] In some embodiments, R1 and R2 are the same group.

[0016] In some embodiments, the L group is selected from N,N-di(2-aminoethyl)-N-methylamino or N-(2-aminoethyl)-N-methylethanolamino.

[0017] The number and position of amino groups in N,N-di(2-aminoethyl)-N-methylamino and N-(2-aminoethyl)-N-methylethanolamino groups are suitable. Experiments have shown that the inter-amino group spacing is appropriate, the number of amino groups is moderate, and the distance between the L group branches and the adjacent A group is also appropriate. This can reduce the possibility of in vivo aggregation or aggregation with other proteins due to charge concentration, thereby enabling the compound to remain in a stable state in vivo for a long time and improving delivery efficiency.

[0018] In some embodiments, the compound has the formula: or .

[0019] In some embodiments, the C atom attached to the L group has an S configuration.

[0020] In a second aspect, a composition is provided, comprising: a carrier comprising a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as described in any of the first aspects; a nucleic acid molecule; wherein the carrier is used to deliver the nucleic acid molecule.

[0021] In some embodiments, the carrier further includes: auxiliary lipids, cholesterol substances and PEGylated lipids; the molar ratio of the ionizable lipid molecules, auxiliary lipids, cholesterol substances and PEGylated lipids is (10-20):(30-55):(20-45):(0.5-3).

[0022] In some embodiments, the auxiliary lipid is selected from 1,2-distearyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. The cholesterol-like substance is selected from one or more of 3-phosphatidylethanolamine; the cholesterol-like substance is selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; the PEGylated lipid is selected from one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-distearatel-rac-glycero-3-methoxy polyethylene glycol, 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol, and 1,2-distearatel-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol.

[0023] In some embodiments, the nucleic acid molecule is one or more of mRNA, siRNA, miRNA, circular RNA, and plasmid DNA; the mass ratio of the nucleic acid molecule to the ionizable lipid molecule is 1:(10-25).

[0024] In some embodiments, the composition is a nanoparticle formulation.

[0025] In some embodiments, the average particle size of the nanoparticle formulation is 70-120 nm; the polydispersity index of the nanoparticle formulation is ≤0.1.

[0026] In some embodiments, the composition has a zeta potential of 2.0 mV to 0 mV in PBS buffer at pH 7.4.

[0027] In some embodiments, when the composition is co-incubated with HEK 293T cells for 24 hours, the survival rate of the HEK 293T cells is greater than or equal to 90%.

[0028] In some embodiments, the composition, after injection into mice, accumulates in the spleen at a rate greater than or equal to 80% and in the liver at a rate less than or equal to 10%.

[0029] Thirdly, a method for preparing a nanoparticle formulation is provided, comprising: preparing an oil phase containing ionizable lipid molecules, auxiliary lipids, cholesterol-like substances, and PEGylated lipids, wherein the ionizable lipid molecules include compounds as described in any one of the first aspects, their stereoisomers, or pharmaceutically acceptable salts thereof; preparing an aqueous phase containing nucleic acid molecules; mixing an oil phase with a concentration of 7.5-20 mM and an aqueous phase with a concentration of 0.1 mg / mL at a volume ratio of 1:(2-4) using a microfluidic chip to obtain a mixture; and dialyzing the mixture to obtain the nanoparticle formulation.

[0030] Fourthly, the invention provides the use of lipid nanoparticles in the preparation of pharmaceuticals or biological agents, the lipid nanoparticles comprising compounds as described in any of the first aspects, their stereoisomers, or pharmaceutically acceptable salts thereof; the pharmaceutical is a gene therapy drug for treating hereditary diseases, tumors, or infectious diseases; the biological agent is a vaccine formulation for preventing and / or treating viral infections or tumors. Attached Figure Description

[0031] Figure 1 The particle size and zeta potential tests of the compositions provided in this application are shown; Figure 2 A schematic diagram showing the fluorescence intensity of transfected EGFP qualitatively observed using a fluorescence microscope is shown. Figure 3 A schematic diagram showing cell viability after transfection with different compositions is presented; Figure 4 A schematic diagram showing the distribution of different compositions in mice after transfection is presented. Figure 5 The study shows the trends in body weight changes in mice after transfection with different compositions; Figure 6 The levels of alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), albumin (ALB), creatinine (CRE), and urea (UREA) in the serum of mice after transfection with different compositions are shown. Figure 7 The results of hematoxylin-eosin (HE) staining of different organs in mice after transfection with different compositions are shown. Detailed Implementation

[0032] The abbreviations used in this article have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas listed in this article are constructed according to the standard rules of chemical valence known in the field of chemistry.

[0033] Where substituent groups are designated by their conventional chemical formula written from left to right, they similarly cover chemically identical substituents that would be obtained by writing the structure from right to left, for example, -CH2O- is equivalent to -OCH2-.

[0034] Unless otherwise stated, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain (i.e., unbranched) or branched carbon chain (or carbon) or combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include monovalent, divalent, and polyvalent groups having a specified number of carbon atoms (i.e., C1-C10 means one to ten carbons). An alkyl group is an uncyclic chain. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkyl group is an alkyl group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologues and isomers. An alkoxy group is an alkyl group linked to the rest of the molecule via an oxygen-linking group (-O-).

[0035] Unless otherwise stated, the terms “halogenated” or “halogen” on their own or as part of another substituent refer to a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “halogenated alkyl” are intended to include both monohalogenated and polyhalogenated alkyl groups. For example, the term “halogenated (C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0036] Unless otherwise stated, the term "acyl" means -C(O)R, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0037] Each of the above terms includes both the substituted and unsubstituted forms of the indicated base.

[0038] It will be apparent to those skilled in the art that some of the compounds of the present invention may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the present invention.

[0039] Unless otherwise stated, the structures described herein are intended to include all stereochemical forms of the structures; that is, the R and S configurations for each asymmetry center. Therefore, single stereochemical isomers of the compounds of the present invention, as well as enantiomers and diastereomeric mixtures, are within the scope of the present invention.

[0040] This application provides a compound having the formula: ; Wherein, R1 and R2 are independently selected from substituted or unsubstituted saturated C2. 14 To C 25 The alkyl acyl group; the L group includes an ionizable amine linker, the ionizable amine linker including at least one primary amine and at least one tertiary amine; the A group includes a targeting ligand, the targeting ligand including at least one hydroxyl group, the targeting ligand being used to target receptors on the surface of immune cells.

[0041] Current ionizable lipid molecules suffer from short in vivo circulation time and low nucleic acid delivery efficiency due to their large particle size and low protonation.

[0042] In the compounds provided in this application, the L group and the A group are attached to adjacent C atoms of the main chain. The ionizable amino group of the L group and the hydroxyl group of the A group can work together to form a hydrophilic end, which makes the compound more polar than a single hydrophilic end, thereby resulting in smaller particle size of the lipid nanoparticles with the compound as the main structure. R1 and R2 are independently selected from substituted or unsubstituted saturated C atoms. 14 To C 25 Alkyl acyl groups and saturated alkyl groups are hydrophobic, thus forming a spherical structure with hydrophobic groups on the outside and hydrophilic groups on the inside. Simultaneously, the hydrophilic head encapsulates nucleic acid molecules within the formed paper-like nanoparticles, serving to deliver the nucleic acid molecules.

[0043] The L group includes an ionizable amine linker comprising at least one primary amine and at least one tertiary amine. Under acidic conditions, the compound can undergo multi-stage protonation with a stronger proton sponge effect, facilitating the release of nucleic acid molecules. R1 and R2 are independently selected from substituted or unsubstituted saturated C groups. 14 To C 25 Alkyl acyl groups, within cells, are hydrolyzed by esterases, which degrade the hydrophobic tail, thereby removing the coating from nucleic acid molecules and releasing them more quickly.

[0044] The compound degrades into fatty acids within cells, which are not significantly toxic to cells and therefore do not inhibit the process of cellular antigen expression, thereby promoting stronger immune protection in the organism.

[0045] The compound provided in this application targets a nucleic acid molecule via a ligand, and self-assembles into stable particles in an aqueous phase. Due to the hydrophilic head being compatible with the aqueous phase and the hydrophobic tail repelling water molecules, the compound spontaneously arranges itself into a spherical structure for easy delivery of nucleic acid molecules. The head contains adjacent hydroxyl and amino groups, resulting in stronger hydrophilicity, smaller spherical particle size, and longer blood circulation time, enabling long-term antigen expression. R1 and R2 are independently alkyl acyl groups, and the ester bond between the hydrophobic tail and the glycerol backbone is specifically hydrolyzed by esterases within the cell, thereby rapidly releasing nucleic acid molecules. Furthermore, the compound's metabolites are fatty acids and glycosyl groups, exhibiting no significant cytotoxicity and no inhibition of translation, thus increasing antigen expression levels. The compound's head contains multiple ionizable amino groups, including at least one primary amine and at least one tertiary amine, and is neutral under physiological conditions, possessing a wider pKa response range. It can achieve multi-level protonation in acidic endosomes, exhibiting a strong proton sponge effect, high endosome escape efficiency, and improved cytoplasmic delivery efficiency. This enables efficient delivery of nucleic acid molecules and maintains long-term, high-volume antigen expression, thereby enhancing the immune response mediated by biological agents made from this compound.

[0046] In some embodiments, the pKa of the compound is in the range of 6.0-6.6.

[0047] The pKa is located at 6.0-6.6 through multiple amine groups, resulting in a high degree of protonation in the acidic endosome environment, a strong proton sponge effect, high endosome escape efficiency, and improved cytoplasmic delivery efficiency.

[0048] In some embodiments, the A group is selected from 2-acetamido-2-deoxy-D-mannose-6-oxy, CLEC9A affinity peptide, or folic acid.

[0049] 2-Acetamino-2-deoxy-D-mannose-6-oxy group exhibits high specificity for the spleen, targeting immune cells such as dendritic cells and macrophages, and is particularly well-suited for spleen accumulation, thus reducing the burden on the liver. The 2-acetamino substitution enhances the sugar ring's resistance to glycosidases, prolonging its circulation time in vivo and improving bioavailability. The presence of the 6-oxy group increases the molecule's hydrophilicity, facilitating dispersion and transport in the physiological environment, and its lipid solubility can be adjusted through chemical modification to optimize transmembrane capacity.

[0050] In the compounds provided in this application, 2-acetamino-2-deoxy-D-mannose-6-oxy is a natural building block of human glycoproteins. While efficiently delivering nucleic acid molecules, its degradation products do not have significant cytotoxicity and do not cause complement activation-related allergic reactions. Furthermore, 2-acetamino-2-deoxy-D-mannose-6-oxy makes the compound more likely to accumulate in the spleen during long-term blood circulation, thereby reducing liver accumulation, which can reduce the burden on the liver and reduce inflammatory responses.

[0051] In some embodiments, R1 and R2 are each independently selected from substituted or unsubstituted saturated C. 14 To C 25 Straight-chain alkyl acyl group.

[0052] In some embodiments, R1 and R2 are each independently selected from unsubstituted saturated C. 14 To C 25 Straight-chain alkyl acyl group.

[0053] In the compounds provided in the embodiments of this application, saturated C 14 To C 25 The strong hydrophobicity of straight-chain alkyl acyl groups can promote the formation of spherical coatings on nucleic acid molecules, further reducing the diameter of the formed spheres, allowing for longer blood circulation and enabling long-term antigen expression.

[0054] In some embodiments, R1 and R2 are independently selected from hexadecylacyl and octadecylacyl, respectively.

[0055] In some embodiments, R1 and R2 are the same group.

[0056] In the compounds provided in this application, R1 and R2 are the same group, thereby making the morphology of the formed composition more stable.

[0057] In some embodiments, the L group is selected from N,N-di(2-aminoethyl)-N-methylamino or N-(2-aminoethyl)-N-methylethanolamino.

[0058] The number and position of amino groups in N,N-di(2-aminoethyl)-N-methylamino and N-(2-aminoethyl)-N-methylethanolamino groups are suitable. Experiments have shown that the inter-amino group spacing is appropriate, the number of amino groups is moderate, and the distance between the L group branches and the adjacent A group is also appropriate. This can reduce the possibility of in vivo aggregation or aggregation with other proteins due to charge concentration, thereby enabling the compound to remain in a stable state in vivo for a long time and improving delivery efficiency.

[0059] The aforementioned groups contain multiple functional sites, which facilitate chemical modification to optimize the biodegradability, metabolic stability, and tissue specificity of lipids, making them suitable for the design of novel delivery systems targeting extrahepatic organs (such as the lungs, spleen, and immune cells).

[0060] Compared to traditional cationic lipids, this group is more easily metabolized and cleared after delivery, reducing inflammatory responses and cell damage caused by long-term accumulation, and has better safety potential.

[0061] In some embodiments, the compound has the formula: or .

[0062] In some embodiments, the C atom attached to the L group has an S configuration.

[0063] The following uses compound 1 (lipid01) provided in the embodiments of this application as an example to illustrate the synthesis process of the compound in the embodiments of this application. The chemical names used in this process are merely exemplary. When synthesizing similar compounds, the intermediate can be replaced with the corresponding group of the target compound.

[0064] The structural formula of compound 1 (lipid01) is as follows:

[0065] The compounds in the embodiments of this application can be prepared in the following manner: 1. Bihydrophobic tail esterification to synthesize intermediate 1 ((2S)-2-(hydroxymethyl)-3-hydroxy-1,4-bis(octadecyloxy)butane).

[0066] (1) Add (2S)-2-(hydroxymethyl)-1,3,4-butanetriol and 30 mL of anhydrous DCM to a 100 mL three-necked flask, stir to dissolve, and cool to 0°C in an ice bath; add imidazole, stir for 5 min, and then add TBSCl in batches, keeping the temperature ≤5°C throughout the process. After the addition is complete, stir at 0°C for 1 h, and then raise the temperature to room temperature for 4 h. Monitor the reaction by TLC.

[0067] (2) The reaction solution was washed twice with saturated sodium bicarbonate solution and saturated NaCl solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain colorless oily (2S)-2-(TBS-oxymethyl)-1,3,4-butanetriol, which can be used directly in the next step without additional purification.

[0068] (3) Add the above protected product and 20 mL of anhydrous DCM to a 250 mL three-necked flask, stir to dissolve, add TEA and DMAP, and cool to 0°C in an ice bath; slowly add freshly distilled octadecyl chloride, keeping the temperature ≤5°C throughout the process, and stir in an ice bath for 30 min after the addition is complete, then heat to 25°C and stir for 8 h, and monitor the reaction by TLC.

[0069] (4) Slowly add saturated sodium bicarbonate solution to quench excess acyl chloride under ice bath until no bubbles are generated; separate the liquid and extract the aqueous phase three times with DCM, combine the organic phases, wash three times with saturated NaCl solution, and dry with anhydrous sodium sulfate; after filtration and concentration, purify by silica gel column chromatography to obtain white waxy solid intermediate 1 (white waxy solid).

[0070] 2. The ionizable head can be coupled to synthesize intermediate 2 ((2S)-2-(hydroxymethyl)-3-(N,N-bis(2-Boc-aminoethyl)-N-methylamino)-1,4-bis(octadecyloxy)butane).

[0071] (1) Argon gas was passed through a 100 mL three-necked flask to replace the air three times. 1 and 20 mL of anhydrous THF intermediate were added and stirred to dissolve. The mixture was then cooled to 0°C in an ice bath. NaH was added in batches, with the temperature controlled at ≤5°C throughout. After the addition was complete, the mixture was stirred at 0°C for 15 min to complete the deprotonation of the hydroxyl group.

[0072] (2) Dilute N-(2-chloroethyl)-N,N-bis(2-Boc-aminoethyl)-N-methylamine with 5 mL of anhydrous THF and slowly add it dropwise to the reaction solution over 30 min, while keeping the temperature ≤5℃. After the addition is complete, raise the temperature to 40℃ and stir for 16 h. Monitor the reaction by TLC.

[0073] (3) Slowly add saturated ammonium chloride solution to quench excess NaH under ice bath until no bubbles are generated; add DCM to dilute, separate the liquid and extract the aqueous phase with DCM 3 times, combine the organic phases, wash 3 times with saturated NaCl solution, and dry with anhydrous sodium sulfate; filter and concentrate, then purify by silica gel column chromatography to obtain intermediate 2 (pale yellow waxy solid).

[0074] 3. Mannose ligand coupling to synthesize intermediate 3 ((2S)-1-(2-acetamido-2-deoxy-D-mannose-6-oxy)-3-(N,N-bis(2-Boc-aminoethyl)-N-methylamino)-2,4-bis(octadecyloxy)butane).

[0075] (1) Add intermediate 2 and 20 mL of anhydrous THF to a 100 mL three-necked flask, stir to dissolve, and cool to 0°C in an ice bath; slowly add 1 M TBAF solution dropwise over 10 min, and after the addition is complete, heat to 25°C and stir for 2 h, and monitor the deprotection reaction by TLC.

[0076] (2) The reaction solution was washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain a colorless oily deprotected product, which was directly used in the next step.

[0077] (3) Argon gas was passed through a 100 mL three-necked flask to replace the air three times. The deprotected product and 15 mL of anhydrous DMF were added, stirred and dissolved, and cooled to 0°C in an ice bath. NaH was added in batches, and the temperature was controlled at ≤5°C. The mixture was stirred for 15 min to complete the deprotonation. 2-acetamido-2-deoxy-D-mannose-6-OTs were added, and the temperature was raised to 50°C and stirred for 14 h. The reaction was monitored by TLC.

[0078] (4) Slowly add saturated ammonium chloride solution to quench the liquid under ice bath, separate the liquid and extract the aqueous phase three times with DCM, combine the organic phases, wash five times with saturated NaCl solution, and dry with anhydrous sodium sulfate; after filtration and concentration, purify by silica gel column chromatography to obtain intermediate 3 (white waxy solid).

[0079] 4. Remove Boc protection to obtain the target molecule.

[0080] (1) Dissolve intermediate 3 (8.26 g, 0.01 mol) in anhydrous DCM (20 mL), cool to 0°C in an ice bath, and slowly add TFA (5.7 g, 0.05 mol). (2) The reaction solution was concentrated under reduced pressure in a water bath at 40°C to remove most of the TFA and DCM. 50 mL of DCM was added to dissolve the solution. Slowly dropwise saturated sodium carbonate solution was added under ice bath to neutralize the pH to 8-9. The solution was separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, washed twice with saturated NaCl solution, and dried with anhydrous sodium sulfate.

[0081] (3) After filtration, the solvent was removed by vacuum concentration and dried at 40°C under high vacuum (0.1MPa) for 4 hours to obtain the target molecule (white waxy solid).

[0082] Example 1: Preparation of the lipid01 composition LNP-Fluc (lipid01).

[0083] The following example uses mRNA as the nucleic acid molecule loaded in the composition to illustrate the preparation method of the composition. However, the embodiments of this application are not limited to this. For example, the composition can be loaded with one or more of siRNA, miRNA, circular RNA, and plasmid DNA. The mRNA can be Firefly luciferase mRNA (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 17101ES80); or commonly used EGFP (enhanced green fluorescent protein) mRNA. Those skilled in the art can select nucleic acid molecules with corresponding functions or therapeutic effects according to actual application needs.

[0084] Taking Firefly luciferase mRNA as an example, the specific preparation method of the mRNA-LNP composition is as follows: (1) Preparation of LNP (microfluidic method) Oil phase preparation: The compound lipid01 (10 μmol), sphingomyelin (50 μmol), cholesterol (38 μmol), and 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol (2 μmol) were dissolved in anhydrous ethanol (1 mL).

[0085] The above are merely exemplary embodiments, and the embodiments of this application are not limited thereto. For example, the compound can be other compounds, and the molar amount of the compound can be any or any combination of 10.0 μmol, 10.5 μmol, 11.0 μmol, 11.5 μmol, 12.0 μmol, 12.5 μmol, 13.0 μmol, 13.5 μmol, 14.0 μmol, 14.5 μmol, 15.0 μmol, 15.5 μmol, 16.0 μmol, 16.5 μmol, 17.0 μmol, 17.5 μmol, 18.0 μmol, 18.5 μmol, 19.0 μmol, 19.5 μmol, and 20.0 μmol. Sphingomyelin can be replaced by other auxiliary lipids, such as one or more selected from 1,2-distearyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. The molar amount of the auxiliary lipid can be any one or any combination of 30.0 μmol, 32.0 μmol, 34.0 μmol, 36.0 μmol, 38.0 μmol, 40.0 μmol, 42.0 μmol, 44.0 μmol, 46.0 μmol, 48.0 μmol, 50.0 μmol, 52.0 μmol, 54.0 μmol, and 55.0 μmol. Cholesterol can be replaced by cholesterol-like substances, such as one or more of β-sitosterol, cholesterol, cholesterol ketone, cholesterol, 7β-hydroxycholesterol, and 7α-hydroxycholesterol. The molar amount of cholesterol-like substances can be any one or any combination of 20.0 μmol, 21.0 μmol, 22.0 μmol, 23.0 μmol, 24.0 μmol, 25.0 μmol, 26.0 μmol, 27.0 μmol, 28.0 μmol, 29.0 μmol, 30.0 μmol, 31.0 μmol, 32.0 μmol, 33.0 μmol, 34.0 μmol, 35.0 μmol, 36.0 μmol, 37.0 μmol, 38.0 μmol, 39.0 μmol, 40.0 μmol, 41.0 μmol, 42.0 μmol, 43.0 μmol, 44.0 μmol, and 45.0 μmol.DMG-PEG2000 can be replaced with other PEGylated lipids, such as one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-distearyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol, and 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol. The molar amount of the PEGylated lipid can be any or any combination of 0.5 μmol, 0.7 μmol, 0.9 μmol, 1.1 μmol, 1.3 μmol, 1.5 μmol, 1.7 μmol, 1.9 μmol, 2.1 μmol, 2.3 μmol, 2.5 μmol, 2.7 μmol, 2.9 μmol, and 3.0 μmol.

[0086] (2) Aqueous phase preparation: Firefly luciferase mRNA (10 μg) was dissolved in 50 mM citrate buffer (pH 4.0, 1 mL).

[0087] The above are merely exemplary embodiments, and the embodiments of this application are not limited thereto. For example, the mass of mRNA can be any one or any two of 10.0 μg, 11.0 μg, 12.0 μg, 13.0 μg, 14.0 μg, 15.0 μg, 16.0 μg, 17.0 μg, 18.0 μg, 19.0 μg, 20.0 μg, 21.0 μg, 22.0 μg, 23.0 μg, 24.0 μg, and 25.0 μg.

[0088] (3) Mixing: The oil phase and water phase are mixed at a suitable volume ratio and flow rate using a microfluidic chip (channel width can be 100 μm), for example, at a volume ratio of 1:3, a flow rate of 24 mL / min, and incubated at room temperature for 30 min.

[0089] (4) Dialysis: Dialyze with PBS buffer (pH 7.4) for 2 h to remove ethanol and obtain LNP-Fluc (lipid01) composition.

[0090] Example 2: LNP-mRNA was prepared by replacing the compound in Example 1 of this application with lipid02 to obtain LNP-Fluc (lipid02). The preparation method is the same as in Example 1. Comparative Example 1: In this comparative example, commercially available SM102 lipid was used to replace the compounds in the examples of this application to prepare LNP-mRNA, resulting in LNP-Fluc (SM102). The preparation method is the same as in Example 1.

[0091] SM102 lipid is a synthetic cationic lipid possessing both a hydrophobic tail chain and a hydrophilic cationic head group, enabling it to self-assemble into lipid bilayers or spherical lipid nanoparticles in aqueous systems. It has the following structural formula:

[0092] Comparative Example 2: In this comparative example, commercially available MC3 lipid (DLin-MC3-DMA) was used to replace the compounds in the examples of this application to prepare LNP-mRNA, resulting in LNP-Fluc (MC3). The preparation method is the same as in Example 1.

[0093] DLin-MC3-DMA has the following structure:

[0094] Comparative Example 3: In this comparative example, commercially available EGFP mRNA was used to replace the Firefly luciferase mRNA in the examples of this application to obtain LNP-EGFP mRNA. The preparation method is the same as in Example 1.

[0095] Figure 1 The particle size and zeta potential tests of the compositions provided in this application are shown. Among them, Figure 1 The top left of the image shows the particle size and zeta potential tests of the LNP-Fluc (lipid01) composition. Figure 1 The upper right of Figure 1 shows the particle size and zeta potential test results of the LNP-Fluc (lipid02) composition; the lower left of Figure 1 shows the particle size and zeta potential test results of the LNP-Fluc (SM102) composition. Figure 1 The lower right of the image shows the particle size and zeta potential tests of the LNP-Fluc (MC3) composition.

[0096] The particle size and zeta potential can be determined by using dynamic light scattering (DLS) to measure the particle size of each mRNA-LNP composition and to measure the zeta potential of the above mRNA-LNP compositions in PBS buffer (pH 7.4).

[0097] In some embodiments, the average particle size of the composition provided in this application is 70-120 nm; the polydispersity index of the nanoparticle formulation is ≤0.1. Further, the average particle size of the composition is 75-110 nm. Preferably, the average particle size of the composition is 80-105 nm.

[0098] The average particle size of the composition may also be any of the following values ​​or a combination thereof: 70.0 nm, 72.5 nm, 75.0 nm, 77.5 nm, 80.0 nm, 82.5 nm, 85.0 nm, 87.5 nm, 90.0 nm, 92.5 nm, 95.0 nm, 97.5 nm, 100.0 nm, 101.0 nm, 102.5 nm, 105.0 nm, 107.5 nm, 110.0 nm, 112.5 nm, 115.0 nm, 117.5 nm, and 120.0 nm.

[0099] like Figure 1 As shown, the average particle size of LNP-Fluc (lipid01) is approximately 85 nm. The average particle size of LNP-Fluc (lipid02) is approximately 101 nm. Referring to the particle size distribution diagrams of LNP-Fluc (SM102) and LNP-Fluc (MC3), the average particle size of LNP-Fluc (SM102) is approximately 103 nm, and the average particle size of LNP-Fluc (MC3) is approximately 119 nm. It can be seen that the average particle size of LNP-Fluc (lipid01) provided in this embodiment is smaller than that of LNP-Fluc (SM102) and LNP-Fluc (MC3).

[0100] In some embodiments, the composition has a zeta potential of 2.0 mV to 0 mV in PBS buffer at pH 7.4.

[0101] In PBS buffer at pH 7.4, the zeta potential of the LNP-Fluc (lipid01) composition was 1.3 mV, indicating that it is almost electroneutrally neutral in neutral solution. Similarly, the zeta potential of the LNP-Fluc (lipid01) composition was 1.2 mV. In contrast, the zeta potentials of LNP-Fluc (SM102) and LNP-Fluc (MC3) in PBS buffer at pH 7.4 were above 2.2 mV. For example, the zeta potential of LNP-Fluc (SM102) reached 5.0 mV and above, while that of LNP-Fluc (MC3) reached 10 mV. The decrease in zeta potential indicates that the compositions of this application are in a more stable state in vivo, while currently used lipid molecules have short circulation periods in vivo and are prone to aggregation.

[0102] The encapsulation efficiency of mRNA in the mRNA-LNP composition was determined using the RiboGreen kit. The results showed that the encapsulation efficiency of LNP-Fluc (lipid01) was 93.8 ± 1.2%.

[0103] Figure 2A schematic diagram showing the fluorescence intensity of transfected EGFP qualitatively observed using a fluorescence microscope is presented.

[0104] In vitro cell delivery efficiency can be determined in the following ways: HEK 293T cells were seeded in 96-well plates (1×10⁴ cells / well) and cultured at 37°C and 5% CO₂ for 24 h. LNP-EGFP (lipid01) and LNP-EGFP (lipid02) mRNA were added to each well for transfection. Control groups LNP-EGFP (SM102) and LNP-EGFP (MC3) were also included. Cells were cultured for 24 h. The intensity of EGFP green fluorescence in LNP-EGFP mRNA was observed using a fluorescence microscope.

[0105] from Figure 2 It can be seen that the fluorescence intensity of LNP-EGFP (lipid01) and LNP-EGFP (lipid02) is significantly stronger than that of LNP-EGFP (SM102) and LNP-EGFP (MC3), indicating that LNP-EGFP (lipid01) has a higher in vitro cell delivery efficiency.

[0106] Figure 3 A schematic diagram showing the cell survival rates after transfection with different compositions is presented.

[0107] Cell viability can be determined using the following methods: HEK 293T cells were seeded in 96-well plates (1×10⁴ cells / well) and cultured at 37 °C with 5% CO₂ for 24 h. LNP-Fluc mRNA was added to each well at a concentration of 0.1 μg / well for co-incubation. Control groups of LNP-Fluc (SM102) and LNP-Fluc (MC3) were also included. Cell viability was then assessed using a CCK-8 assay.

[0108] In some embodiments, when the composition is co-incubated with HEK 293T cells for 24 hours, the survival rate of the HEK 293T cells is greater than or equal to 90%.

[0109] like Figure 3 The cell survival rate results show that the cell survival rate of LNP-Fluc (lipid01) prepared in Example 1 of this application is 92%, and the cell survival rate of lipid02 is 91%, which is significantly higher than 80% of LNP-Fluc (SM102) and 81.5% of LNP-Fluc (MC3).

[0110] The following is combined with Figures 4-7 This document describes the animal experiment results of the compositions provided in the embodiments of this application.

[0111] Figure 4 A schematic diagram showing the distribution of different compositions in mice after transfection is presented. Figure 5 The study shows the trends in body weight changes in mice after transfection with different compositions; Figure 6 The levels of alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), albumin (ALB), creatinine (CRE), and urea (UREA) in the serum of mice after transfection with different compositions are shown. Figure 7 The results of hematoxylin-eosin (HE) staining of different organs in mice after transfection with different compositions are shown.

[0112] In vivo animal experiments can be conducted using the following methods: C57BL / 6 mice (6-8 weeks old, female) were selected, with 6 mice in each group. Mice injected with LNP-Fluc (lipid01) (mRNA dose: 10 μg / mouse) via tail vein were designated LNP (lipid01), and mice injected with LNP-Fluc (lipid02) (mRNA dose: 10 μg / mouse) via tail vein were designated LNP (lipid02). The control groups were the LNP-Fluc (SM102) group and the LNP-Fluc (MC3) group, designated LNP (SM102) group and LNP (MC3) group, respectively.

[0113] Mice were sacrificed 24 hours after drug administration, and heart, liver, spleen, lung, and kidney tissues were collected. The luminescence intensity was detected using a luciferase assay kit (Promega, catalog number: 12352207). Fourteen days after drug administration, changes in mouse body weight were monitored, serum ALT, AST, ALB, CRE, and UREA levels were measured, and organs were observed by HE staining.

[0114] In some embodiments, the composition, after injection into mice, accumulates in the spleen at a rate greater than or equal to 80% and in the liver at a rate less than or equal to 10%.

[0115] like Figure 4 As shown, the spleen luciferase activity of the LNP (lipid01) group in the examples was 9.08 × 10⁻⁶. 8 RLU / g tissue, is in the LNP (SM102) group (1.5×10). 5 The RLU / g tissue ratio was 6053 times higher than that of the LNP (lipid02) group, and the spleen luciferase activity was 8.03 × 10⁻⁶. 8RLU / g tissue, is in the LNP (SM102) group (1.5×10). 5 The ratio of RLU / g tissue is 5353 times, indicating that the LNP-Fluc (lipid01) and LNP-Fluc (lipid02) prepared in Example 1 of this application have superior spleen enrichment and targeting compared to LNP-Fluc (SM102) and LNP-Fluc (MC3).

[0116] like Figure 5 As shown, the body weight of mice in the LNP (lipid01), LNP (lipid02), LNP (SM102), and LNP (MC3) groups in the examples did not change significantly, indicating that the injection of LNP had no significant effect on the body weight of mice.

[0117] like Figure 6 As shown in the results, serum ALT, AST, ALB, CRE, and UREA levels did not significantly increase 7 days after administration of the LNP-Fluc (lipid01) / LNP-Fluc (lipid02) prepared in this application (P>0.05). In contrast, LNP-Fluc (SM102) showed varying degrees of increase in ALT, ALB, CRE, and UREA, while LNP-Fluc (MC3) showed varying degrees of increase in ALB, CRE, and UREA. This indicates that the LNP-mRNA prepared in this invention has excellent biocompatibility.

[0118] like Figure 7 As shown in the HE staining results, 7 days after administration of the LNP-Fluc(lipid01) / LNP-Fluc(lipid02) prepared in the embodiments of this application, all organs stained red, indicating no obvious organ damage. In contrast, the livers of the LNP(SM102) and LNP(MC3) groups showed a small number of blue-purple granules, indicating a certain degree of inflammatory response in the LNP(SM102) and LNP(MC3) groups. These indicators demonstrate that the compositions prepared using the compounds from the embodiments of this application possess excellent in vivo biocompatibility.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application.

Claims

1. A compound, characterized in that, Having the following formula: ； Wherein, R1 and R2 are independently selected from substituted or unsubstituted saturated C2. 14 To C 25 Alkyl acyl; The L group includes an ionizable amine linker, wherein the ionizable amine linker includes at least one primary amine and at least one tertiary amine; Group A includes a targeting ligand, the targeting ligand comprising at least one hydroxyl group, the targeting ligand being used to target receptors on the surface of immune cells.

2. The compound according to claim 1, characterized in that, The pKa of the compound is in the range of 6.0-6.

6.

3. The compound according to claim 1 or 2, characterized in that, The A group is selected from 2-acetamido-2-deoxy-D-mannose-6-oxy, CLEC9A affinity peptide, or folic acid.

4. The compound according to any one of claims 1 to 3, characterized in that, R1 and R2 are each independently selected from substituted or unsubstituted saturated C. 14 To C 25 Straight-chain alkyl acyl group.

5. The compound according to claim 4, characterized in that, R1 and R2 are each independently selected from unsubstituted saturated C. 14 To C 25 Straight-chain alkyl acyl group.

6. The compound according to claim 5, characterized in that, R1 and R2 are independently selected from hexadecyl acyl and octadecyl acyl, respectively.

7. The compound according to any one of claims 1 to 6, characterized in that, R1 and R2 are the same group.

8. The compound according to any one of claims 1 to 7, characterized in that, The L group is selected from -N,N-di(2-aminoethyl)-N-methylamino or N-(2-aminoethyl)-N-methylethanolamino.

9. The compound according to any one of claims 1 to 8, characterized in that, The compound has the formula: or .

10. The compound according to any one of claims 1 to 9, characterized in that, The C atom attached to the L group has an S configuration.

11. A composition, characterized in that, include: The carrier comprises a compound as described in any one of claims 1 to 10, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; Nucleic acid molecules; The carrier is used to deliver the nucleic acid molecule.

12. The composition according to claim 11, characterized in that, The carrier also includes: Supporting lipids, cholesterol substances, and PEGylated lipids; The molar ratio of the ionizable lipid molecules, auxiliary lipids, cholesterol substances and PEGylated lipids is (10-20):(30-55):(20-45):(0.5-3).

13. The composition according to claim 12, characterized in that, The auxiliary lipid is selected from one or more of the following: 1,2-distearyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. The cholesterol-like substances are selected from one or more of cholesterol, β-sitosterol, cholesterol, cholesterol ketone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; The PEGylated lipid is selected from one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol, 1,2-distearate-rac-glycero-3-methoxy polyethylene glycol, 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol, and 1,2-distearate-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol.

14. The composition according to any one of claims 11 to 13, characterized in that, The nucleic acid molecule is one or more of mRNA, siRNA, miRNA, circular RNA, and plasmid DNA; The mass ratio of the nucleic acid molecules to the ionizable lipid molecules is 1:(10-25).

15. The composition according to any one of claims 11 to 14, characterized in that, The composition is a nanoparticle formulation.

16. The composition according to claim 15, characterized in that, The average particle size of the nanoparticle formulation is 70-120 nm. The polydispersity index of the nanoparticle formulation is ≤0.

1.

17. The composition according to claim 15 or 16, characterized in that, The composition exhibits a Zeta potential of 2.0 mV to 0 mV in PBS buffer at pH 7.

4.

18. The composition according to any one of claims 15 to 17, characterized in that, When the composition is co-incubated with HEK 293T cells for 24 hours, the survival rate of the HEK 293T cells is greater than or equal to 90%.

19. The composition according to any one of claims 15 to 18, characterized in that, When the composition was injected into mice, it showed an enrichment of ≥80% in the spleen and ≤10% in the liver.

20. A method for preparing a nanoparticle formulation, characterized in that, include: An oil phase containing ionizable lipid molecules, auxiliary lipids, cholesterol-like substances and PEGylated lipids is prepared, wherein the ionizable lipid molecules include the compounds, stereoisomers thereof, or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 10. Preparation of an aqueous phase containing nucleic acid molecules; A mixture was prepared by mixing an oil phase with a concentration of 7.5-20 mM and an aqueous phase with a concentration of 0.1 mg / mL at a volume ratio of 1:(2-4) using a microfluidic chip. The nanoparticle formulation was obtained by dialysis of the mixture.

21. The application of a lipid nanoparticle in the preparation of a drug or biological agent, characterized in that, The lipid nanoparticles comprise the compound as described in any one of claims 1 to 10, its stereoisomer, or a pharmaceutically acceptable salt thereof; The drug is a gene therapy drug, which is used to treat hereditary diseases, tumors, or infectious diseases. The biological agent is a vaccine preparation, and the biological agent is used to prevent and / or treat viral infections or tumors.

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