CN115925563B - Lipid molecule for targeted pulmonary delivery of nucleic acid, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a lung targeting lipid molecule, a preparation method thereof and an LNP system containing the lipid molecule and capable of realizing lung targeting delivery. The structural formula of the lung-targeted lipid molecule is selected from the following formulas (I) to (V), wherein each group is defined in the specification. After the above-described lung-targeted lipid molecules are added to the basal LNP system, the original liver-targeted LNP delivery system can be converted to a lung-targeted LNP delivery system. The lung targeting component can greatly regulate and control the charge size and the hydrophobicity of the quaternary ammonium salt by changing the types of the quaternary ammonium salt and the types and the quantity of the hydrophobic chain segments, and can fully meet the requirement of targeted lung delivery of mRNA.

Description

A lipid molecule that can be used for targeted lung delivery of nucleic acids and its preparation method and application

Technical Field

The present invention belongs to the field of medicine and biology, and specifically relates to a lipid molecule that can be used for targeted lung delivery of nucleic acid, and a preparation method and application thereof.

Background Art

In recent years, nucleic acids (including DNA and RNA) have made breakthrough progress in the treatment of infectious diseases and tumors as a new pharmaceutical technology in a short period of time. However, how to safely and efficiently deliver nucleic acid molecules to specific target cells and protect them from degradation is one of the main challenges currently faced in the development of nucleic acid drugs and vaccines.

The ideal delivery vehicle must be safe, stable, and organ-specific. Lipid nanoparticles (LNPs) are the most advanced nucleic acid carriers in the clinic. As of June 2021, all mRNA vaccines approved for clinical use use LNP delivery systems. LNPs offer many benefits for mRNA delivery, including simple formulation, modularity, good biocompatibility, and large mRNA payload capacity.

Unfortunately, there have been reports that intramuscular injections of Pfizer/BioNTech and Moderna's mRNA vaccines may induce liver damage and immune hepatitis. Some people have also experienced abnormal liver function after receiving mRNA vaccines, and liver transplant patients have produced a very strong immune response after vaccination. That is, the existing LNP delivery system will cause severe liver accumulation, which may cause liver damage or immune hepatitis. Therefore, most of the current LNP delivery systems are liver-targeted, and the problem of effective delivery to organs other than the liver (such as the lungs and kidneys) needs to be solved urgently.

Considering that the lungs are the first to be affected, a lung-targeted mRNA delivery system has been developed to deliver mRNA encoding the spike protein directly to the lungs, activating the body to produce corresponding antibodies to resist viral attacks, or to deliver mRNA encoding broad-spectrum neutralizing antibodies or therapeutic proteins directly to the patient's lungs for treatment. In addition, the lung-targeted mRNA delivery system can also be widely used in various lung diseases, such as prevention or treatment of lung cancer, pneumonia, tuberculosis, chronic obstructive pulmonary disease, pulmonary thromboembolism and pulmonary vasculitis.

The physicochemical properties and chemical composition of LNP delivery systems can change the interaction with proteins in body fluids. Specifically, by regulating the charge properties, particle size distribution, and the proportion of hydrophobic lipids in the LNP delivery system, the distribution of LNP in the body can be changed, thereby achieving organ-targeted delivery.

Summary of the invention

The object of the present invention is to provide a lipid molecule capable of achieving targeted delivery, a pharmaceutically acceptable salt thereof and a stereoisomer thereof.

The lipid molecule capable of achieving targeted delivery is selected from at least one of the following formulas (A) to (F):

；

;

After preparing the basic LNP system using the above lipid molecules, a liver-targeted LNP delivery system can be obtained.

Furthermore, after the above-mentioned lipid molecules are formed into quaternary ammonium salts (lipid quaternary ammonium salt molecules), lipid molecules capable of achieving lung targeted delivery can be obtained, which are specifically selected from at least one of the following formulas (I) to (V).

After adding the above-mentioned lipid molecules for lung targeted delivery into the basic LNP system, a lung targeted LNP delivery system can be obtained.

In the above formulae (I) to (V):

The X is selected from halogen atoms, and ^X- is selected from halogen anions, including fluoride ion, chloride ion, bromide ion and iodide ion.

^B1 , ^B2 and ^B3 may be the same or different; ^B1 , ^B2 and ^B3 may independently form any one of carbonate, carbamate, ester, ether, urea/amide or amine linking groups or a combination of any two of the above linking groups with oxygen or nitrogen atoms.

The L ¹ , L ² , and L ³ may be the same or different, and are independently selected from C _1-20 aliphatic hydrocarbon groups, hydrogen, or hydrophobic compounds such as various steroidal lipids, vitamin A, vitamin E, and vitamin K, and at most one of L ¹ and L ² in the formula (I) and formula (V) is hydrogen, and at most one of L ¹ , L ² , and L ³ in the formulas (II) to (IV) is hydrogen;

The R ¹ is independently selected from a C _1-10 aliphatic hydrocarbon group.

Furthermore, the structure of ^OB1 - ^L1 is as shown in any of the following general formulas:

The structure of OB ² -L ² is shown in any of the following general formulas:

The structure of NH-B ¹ -L ¹ is shown in any of the following general formulas:

The NH-B ² -L ² structure is shown in any of the following general formulas:

The structure of OB ³ -N is shown in any of the following general formulas:

Wherein, n represents the number of methylene groups -CH ₂ , and its value range is an integer within 0-20.

The structure of NH-B ³ -N is shown in any of the following general formulas:

Wherein n represents the number of methylene groups -CH ₂ , and its value range is an integer within 0-20.

The present invention converts the tertiary amine of the lipid skeleton into a quaternary amine through a quaternary ammonium salt reaction, so that the lipid molecule is converted from a neutral molecule without charge into a molecule in the form of a quaternary ammonium salt, and the pulmonary delivery effect of LNP is achieved as a lung targeting component. After being added to the LNP system, the surface charge and particle size distribution of the LNP can be adjusted, so that the LNP is enriched in the lungs within a certain period of time after intravenous injection, and the lung-targeted delivery of the mRNA carried is completed, and the transfection of lung cells is achieved.

The above L ¹ and L ² may be branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-20 aliphatic hydrocarbon groups, various steroid lipids, vitamin A, vitamin E, vitamin K and other hydrophobic compounds.

Preferably, L ¹ and L ² are branched or unbranched, cyclic or non-cyclic, saturated or unsaturated C _10-20 aliphatic hydrocarbon groups, cholesterol, vitamin A, vitamin E, vitamin K and other hydrophobic compounds.

More preferably, L ¹ and L ² are branched or unbranched, cyclic or acyclic, saturated or unsaturated C _12-18 aliphatic hydrocarbon groups, cholesterol, vitamin A and vitamin E.

More preferably, L ¹ and L ² are branched or unbranched, cyclic or non-cyclic, unsaturated C _12-18 aliphatic hydrocarbon groups, cholesterol and vitamin E.

Most preferably, L ¹ and L ² are branched or unbranched, non-cyclic, unsaturated C _12-18 aliphatic hydrocarbon groups or vitamin E.

The above-mentioned L ³ is preferably a methyl group or an ethyl group, and more preferably a methyl group.

The above R ¹ may be a branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-10 aliphatic hydrocarbon group.

Preferably, R ¹ is a branched or unbranched, acyclic, saturated or unsaturated C _1-8 aliphatic hydrocarbon group.

Most preferably, R ¹ is a branched or unbranched, non-cyclic, saturated C _1-5 aliphatic hydrocarbon group.

For the OB ³ -N structure, n=an integer within the range of 1-20; preferably, n=an integer within the range of 1-10; more preferably, n=an integer within the range of 1-5.

As the halogen anion X ⁻ , there may be mentioned fluoride ion, chloride ion, bromide ion and iodide ion.

Preferably, X- ^is chloride, bromide or iodide.

More preferably, ^X- is bromide and iodide.

Most preferably, ^X- is iodide ion.

According to an embodiment of the present invention, the lung-targeted lipid molecule represented by formula (I) is a lipid molecule represented by formula (Y1-1) and formula (Y1-2); the lung-targeted lipid molecule represented by formula (II) is a lipid molecule represented by formula (Y2); the lung-targeted lipid molecule represented by formula (III) is a lipid molecule represented by formula (Y3); the lung-targeted lipid molecule represented by formula (IV) is a lipid molecule represented by formula (Y4); the lung-targeted lipid molecule represented by formula (V) is a lipid molecule represented by formula (Y5):

(Y1-1)

(Y1-1)

(Y1-2)

(Y1-2)

(Y2)

(Y2)

(Y3)

(Y3)

(Y4)

(Y4)

(Y5)

(Y5)

In a second aspect, the present invention provides a method for preparing the lipid molecules represented by the above formula (I) to (V).

The lipid molecules shown in formula (I), formula (II) and formula (III) provided by the present invention, wherein formula (I) and formula (II) are both based on triethanolamine skeletons, and the skeleton of formula (III) is 2-hydroxymethyl-1,3-propanediol. The reaction sites of formula (I), formula (II) and formula (III) are all hydroxyl groups, and the synthesis methods involved are the same, which are described uniformly below. When the preparation method is taken as an example when B (B ¹ , B ² and/or B ³ ) forms an ester bond with an oxygen atom, it includes the following steps:

(A1): First, a fatty acid is reacted with thionyl chloride in a solvent 1 at a certain temperature for a certain time under the action of a base 1 to obtain a fatty acid chloride; the fatty acid in this step corresponds to L ¹ and L ² in formula I;

(A2): The fatty acid chloride obtained in step (A1) is reacted with triethanolamine or 2-hydroxymethyl-1,3-propanediol in solvent 2 at a certain temperature for a certain time under the action of base 2 to obtain lipid molecules with different hydrophobic segments.

The operation steps are taken as an example of triethanolamine. 2-Hydroxymethyl-1,3-propanediol can also undergo a similar reaction, and the reaction formula will not be further explained below.

By controlling the feed ratio and reaction sequence, lipid molecules with different substituents can be obtained. For example, first obtain L ¹ -substituted triethanolamine or 2-hydroxymethyl-1,3-propanediol derivatives, and then gradually obtain L ¹ , L ² disubstituted triethanolamine lipid molecules or L ¹ , L ² disubstituted 2-hydroxymethyl-1,3-propanediol lipid molecules.

The preparation method of the lipid molecule represented by formula (I) provided by the present invention, when B ( ^B1 , ^B2 and/or ^B3 ) forms an ether bond with an oxygen atom, comprises the following steps (the following reaction equation is only used for illustration of the method, and is not limited to the following structure. Lipid molecules with different types of substituents and different numbers of substituents can be obtained by adjusting the feed ratio and sequence):

(A3): Triethanolamine or 2-hydroxymethyl-1,3-propanediol is reacted in solvent 1 under the action of sodium hydride at a certain temperature for a certain time, and then the corresponding halogenated aliphatic hydrocarbon (XL ¹ , X represents a halogen, such as a bromohydrocarbon) is added and reacted for a certain time to obtain a lipid molecule with an ether bond as a connecting group.

This method is a method of synthesizing ether using sodium hydride, and similar reactions are not listed here;

The preparation method of the lipid molecules represented by formula (I), formula (II) and formula (III) provided by the present invention, when B ( ^B1 , ^B2 and/or ^B3 ) forms carbonate or carbamate with oxygen atom, comprises the following steps (the following reaction equation is only used for method illustration, and is not limited to the following structure. Lipid molecules with different types of substituents and different numbers of substituents can be obtained by adjusting the feeding ratio and sequence):

(A4): The backbone triethanolamine or 2-hydroxymethyl-1,3-propanediol is mixed with an activation reagent N,N'-carbonyldiimidazole or p-nitrophenyl chloroformate, and reacted in solvent 3 for a period of time to obtain a triethanolamine or 2-hydroxymethyl-1,3-propanediol derivative with an activated hydroxyl group;

(A5): reacting the triethanolamine derivative or 2-hydroxymethyl-1,3-propanediol derivative with activated hydroxyl group obtained in step (A4) with an amino group-containing aliphatic hydrocarbon (L ¹ -NH ₂ ) for a certain period of time to obtain a lipid molecule with a corresponding connecting group being a carbamate;

Alternatively, the triethanolamine derivative or 2-hydroxymethyl-1,3-propanediol derivative with activated hydroxyl groups obtained in step (A4) is heated and reacted with a hydroxyl-containing aliphatic hydrocarbon (L ¹ -OH) in an alkaline environment for a certain period of time to obtain a lipid molecule with a corresponding carbonate connecting group.

The method for preparing the lipid molecule represented by formula (II) provided by the present invention comprises the following steps:

(A6): The lipid molecule represented by formula (I) is reacted with an amine compound in a solvent 4 for a period of time in the presence of a catalyst 1 or an activating agent (the activating agent refers to step (A4), N,N'-carbonyldiimidazole or p-nitrophenyl chloroformate) and a base 3 to convert the hydroxyl group into a structure containing an amine group.

The method for preparing the lipid molecule represented by formula (II) provided by the present invention comprises the following steps:

(A7): The disubstituted ( ^L1 and ^L2 are the same or different) 2-hydroxymethyl-1,3-propanediol derivative obtained in step (A2) is reacted with an amine compound in solvent 4 for a period of time in the presence of catalyst 1 or an activating agent (the activating agent is referred to step (A4), N,N'-carbonyldiimidazole or p-nitrophenyl chloroformate) and base 3 to convert the hydroxyl group into a structure containing an amine group.

(A8) When the backbone is tris(2-aminoethyl)amine (corresponding to the lipid molecule of Formula IV or Formula V), that is, when B ( ^B1 , ^B2 and/or ^B3 ) forms an amide group or an amine group with the nitrogen atom, the reaction steps are similar to steps (A1) to (A3), except that triethanolamine is replaced by tris(2-aminoethyl)amine, and the reaction steps are not repeated.

(A9) The triethanolamine lipid molecule or 2-hydroxymethyl-1,3-propanediol lipid molecule or tri(2-aminoethyl)amine lipid molecule with a tertiary amine structure obtained through steps (A1) to (A8) is the ionizable lipid molecule of the basic LNP delivery system. The basic LNP delivery system prepared from these lipid molecules can achieve liver-targeted delivery of nucleic acid molecules.

(A10) After obtaining triethanolamine lipid molecules or 2-hydroxymethyl-1,3-propanediol lipid molecules or tri(2-aminoethyl)amine lipid molecules with a tertiary amine structure through steps (A1) to (A8), the obtained tertiary amine molecules and halogenated hydrocarbons are dissolved in solvent 3, reacted at a certain temperature for a certain time, and after the reaction, the solvent and halogenated hydrocarbons are removed by rotary evaporation to obtain quaternary ammonium salt lipid molecules, which can be used as lung targeting components.

It can be understood that step (A1) is to use thionyl chloride to react with different fatty acids to obtain the corresponding acyl chlorides. This reaction is universal, and its reaction activity and reaction process are almost not affected by the aliphatic hydrocarbon group, and the reaction can be carried out efficiently;

Step (A2) is a reaction between the fatty acid chloride obtained in step (A1) and triethanolamine or 2-hydroxymethyl-1,3-propanediol. The reaction occurs between the acid chloride and the hydroxyl group and is also almost unaffected by the type of the fatty hydrocarbon group. The corresponding triethanolamine ester or 2-hydroxymethyl-1,3-propanediol ester can be efficiently obtained.

Step (A6) and step (A7) are steps in which the hydroxyl groups on triethanolamine ester or 2-hydroxymethyl-1,3-propanediol ester react with amine compounds containing reaction sites under the action of a catalyst or after being activated by an activation reagent. The reaction is hardly affected by other groups, and the target lipid molecules can be obtained efficiently.

Step (A10) is a reaction of a halogenated hydrocarbon with a tertiary amine to form a quaternary ammonium salt. The reaction is highly efficient and can obtain the target product with a nearly quantitative conversion rate. The product is also easy to purify.

The above preparation method is universal and suitable for different fatty hydrocarbon chains (including saturated fatty hydrocarbon chains or unsaturated fatty hydrocarbon chains), and can obtain quaternary ammonium salt lipid molecules with different hydrophobicity.

In one embodiment, the fatty acid comprising the group defined as ^L1 and the fatty acid comprising the group defined as ^L2 in step (A1) are independently selected from the corresponding optional structures described above.

The solvent 1, solvent 2, solvent 3 and solvent 4 of the present invention are independently selected from dichloromethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran;

Base 1, Base 2 and Base 3 are all independently selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine, isopropylamine, potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide.

In the above method, the reaction temperature of step (A1) is 25-120°C and the reaction time is 1-24h; the reaction temperature of step (A2) is 0-100°C and the reaction time is 1-24h; the reaction temperature of step (A3) is 0-100°C and the reaction time is 1-24h.

In one embodiment, solvent 1 is selected from dichloromethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran; preferably, selected from dichloromethane, toluene or N,N-dimethylformamide; more preferably, selected from dichloromethane or toluene.

In one embodiment, base 1 is selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine, isopropylamine, potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide; preferably, selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine or isopropylamine; more preferably, selected from pyridine, N,N-dimethylformamide, triethylamine or isopropylamine.

In one embodiment, the reaction temperature of step (A1) is 25-120°C; preferably, 25-80°C; more preferably, 25-60°C.

In one embodiment, the reaction time of step (A1) is 1-24 h; preferably, 2-12 h; more preferably, 4-6 h.

In one embodiment, solvent 2 is selected from dichloromethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran; preferably, selected from dichloromethane, tetrahydrofuran or N,N-dimethylformamide; more preferably, selected from dichloromethane or N,N-dimethylformamide.

In one embodiment, base 2 is selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine, isopropylamine, potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide; preferably, selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine or isopropylamine; more preferably, selected from pyridine, N,N-dimethylformamide, triethylamine or isopropylamine.

In one embodiment, the reaction temperature in step (A2) is 0-100°C; preferably, 25-80°C; more preferably, 25-60°C.

In one embodiment, the reaction time of step (A2) is 1-48 h; preferably, 6-24 h; more preferably, 6-12 h.

In one embodiment, solvent 3 is selected from dichloromethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran; preferably, selected from dichloromethane, tetrahydrofuran or N,N-dimethylformamide; more preferably, selected from dichloromethane or N,N-dimethylformamide.

In one embodiment, in step (A6) or step (A7), catalyst 1 is selected from one or two of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), carbonium hexafluorophosphate (HATU), N'N-carbonyldiimidazole (CDI), p-nitrophenyl chloroformate, triethylamine, and dimethylaminopyridine;

Preferably, one or two selected from dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), carbonium hexafluorophosphate (HATU), N'N-carbonyldiimidazole (CDI), p-nitrophenyl chloroformate, triethylamine, and dimethylaminopyridine;

More preferably, it is one or two selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), N'N-carbonyldiimidazole (CDI), p-nitrophenyl chloroformate, triethylamine, and dimethylaminopyridine.

In one embodiment, the reaction temperature in step (A6) or step (A7) is 0-100°C; preferably, 25-80°C; more preferably, 25-60°C.

In one embodiment, the reaction time in step (A6) or step (A7) is 1-24 h; preferably, 6-24 h; more preferably, 6-12 h.

In one embodiment, the reaction temperature in step (A8) is 0-100°C; preferably, 25-80°C; more preferably, 50-80°C.

In one embodiment, the reaction time of step (A8) is 1-24 h; preferably, 6-24 h; more preferably, 12-24 h.

In a third aspect, the present invention claims a liposome containing the above-mentioned lung targeting component lipid molecules.

The liposomes provided by the present invention contain, in terms of mass percentage, 10%-70% of ionizable lipid molecules represented by formula (A) to formula (F), 1%-20% of polyethylene glycol lipid molecules, 5%-60% of steroid lipids, 1%-30% of auxiliary lipid molecules and 5%-60% of the lipid molecules for lung targeted delivery.

In a fourth aspect, the present invention claims protection for a liposome containing the above-mentioned liver targeting component lipid molecule.

The liposomes provided by the present invention contain, in terms of mass percentage, 10%-70% of the lipid molecules for liver targeted delivery (ionizable lipid molecules represented by formula (A) to formula (F)), 1%-20% of polyethylene glycol lipid molecules, 5%-60% of steroid lipids and 1%-30% of auxiliary lipid molecules.

In a fifth aspect, the present invention claims a lung-targeted lipid nanoparticle (LNP) encapsulating a nucleic acid drug.

The lung-targeted lipid nanoparticle (LNP) encapsulating the nucleic acid drug comprises the above-mentioned lung-targeted delivery lipid molecule component of the present invention, a basic LNP system and the nucleic acid drug.

The nucleic acid drugs include but are not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA, etc.

The basic LNP system includes ionizable lipid molecules, auxiliary lipid molecules, polyethylene glycol lipids and steroidal lipids.

In a specific embodiment of the present invention, the nucleic acid drug may be mRNA, including RNA of different sequences and lengths, specifically Firefly Luciferase mRNA.

In a sixth aspect, the present invention claims protection for a liver-targeted lipid nanoparticle (LNP) encapsulating a nucleic acid drug.

The liver-targeted lipid nanoparticle (LNP) encapsulating the nucleic acid drug comprises the liposome containing the liver-targeted lipid molecule component and the nucleic acid drug.

The nucleic acid drugs include but are not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA, etc.

In a specific embodiment of the present invention, the nucleic acid drug may be mRNA, including RNA of different sequences and lengths, specifically Firefly Luciferase mRNA.

In a seventh aspect, the present invention claims a method for preparing the liver-targeted lipid nanoparticles (LNPs) encapsulating nucleic acid drugs.

The preparation method of liver-targeted lipid nanoparticles (i.e., liver-targeted basic LNPs) containing nucleic acid drugs provided by the present invention specifically comprises the following steps:

(A1) mixing the liver-targeted delivery lipid molecule (ionizable lipid molecule), auxiliary lipid molecule, polyethylene glycol lipid, and steroid lipid in proportion, dissolving them in a solvent, and obtaining an organic phase liposome solution;

(A2) dissolving the nucleic acid drug in a buffer solution of appropriate pH to obtain an aqueous nucleic acid drug solution;

(A3) The organic phase liposome solution and the aqueous phase nucleic acid drug solution are uniformly mixed according to a certain mass ratio and volume ratio using a microfluidic device to prepare liver-targeted lipid nanoparticles encapsulating nucleic acid drugs.

The method further comprises: ultrafiltration or dialysis of the liver-targeted lipid nanoparticles encapsulating the nucleic acid drug obtained in step (A3) to obtain the liver-targeted lipid nanoparticles encapsulating the nucleic acid drug that can be used in biological experiments.

In an eighth aspect, the present invention claims a method for preparing the above-mentioned lung-targeted lipid nanoparticles (LNPs) encapsulating nucleic acid drugs.

The method for preparing lung-targeted lipid nanoparticles encapsulating nucleic acid drugs provided by the present invention specifically comprises the following steps:

(B1) mixing the ionizable lipid molecules, auxiliary lipid molecules, polyethylene glycol lipids, steroid lipids and lung targeting components in proportion, dissolving them with a solvent, and obtaining an organic phase liposome solution;

(B2) dissolving the nucleic acid drug in a buffer solution of appropriate pH to obtain an aqueous nucleic acid drug solution;

(B3) The organic phase liposome solution and the aqueous phase nucleic acid drug solution are uniformly mixed according to a certain mass ratio and volume ratio using a microfluidic device to prepare lung-targeted lipid nanoparticles encapsulating nucleic acid drugs.

The method further comprises: ultrafiltration or dialysis of the lung-targeted lipid nanoparticles encapsulating nucleic acid drugs obtained in step (B3) to obtain lung-targeted lipid nanoparticles encapsulating nucleic acid drugs that can be used in biological experiments.

Preferably, the solvent used to dissolve the lipid molecules in step (A1) or step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, N,N-dimethylformamide. More preferably, the solvent used to dissolve the lipid molecules in step (A1) or step (B1) is ethanol, tetrahydrofuran, acetone. Most preferably, the solvent used to dissolve the lipid molecules in step (A1) or step (B1) is ethanol.

Preferably, the proportion of lipid molecules (ionizable lipid molecules) for liver-targeted delivery in step (A1) is 10%-70%. More preferably, the proportion of ionizable lipid molecules in step (A1) is 20%-60%. Most preferably, the proportion of ionizable lipid molecules in step (A1) is 50%.

Preferably, the proportion of polyethylene glycol lipids in step (A1) is 1%-20%. More preferably, the proportion of polyethylene glycol liposomes in step (A1) is 5%-15%. Most preferably, the proportion of polyethylene glycol liposomes in step (A1) is 10%.

Preferably, the proportion of steroidal lipids in step (A1) is 5%-60%. More preferably, the proportion of cholesterol in step (A1) is 10%-40%. Most preferably, the proportion of cholesterol in step (A1) is 30%.

Preferably, the proportion of the helper lipid molecule in step (A1) is 1%-30%. More preferably, the proportion of DSPC in step (A1) is 5%-15%. Most preferably, the proportion of the helper lipid molecule in step (A1) is 10%.

Preferably, the proportion of ionizable lipid molecules in step (B1) is 10%-70%. More preferably, the proportion of ionizable lipid molecules in step (B1) is 20%-40%. Most preferably, the proportion of ionizable lipid molecules in step (B1) is 30%.

Preferably, the proportion of polyethylene glycol lipid in step (B1) is 1%-20%. More preferably, the proportion of polyethylene glycol liposome in step (B1) is 1%-10%. Most preferably, the proportion of polyethylene glycol lipid in step (B1) is 5%.

Preferably, the proportion of steroidal lipids in step (B1) is 5%-60%. More preferably, the proportion of cholesterol in step (B1) is 10%-40%. Most preferably, the proportion of cholesterol in step (B1) is 15%.

Preferably, the proportion of the helper lipid molecule in step (B1) is 1%-30%. More preferably, the proportion of DSPC in step (B1) is 5%-15%. Most preferably, the proportion of the helper lipid molecule in step (B1) is 8%.

Preferably, the proportion of lung targeting lipids in step (B1) is 5%-60%. More preferably, the proportion of lung targeting lipids in step (B1) is 10%-50%. Most preferably, the proportion of lung targeting lipids in step (B1) is 42%.

Preferably, the buffer solution in step (A2) or step (B2) is acetic acid/sodium acetate solution or citric acid/sodium citrate solution; most preferably, the buffer solution in step (A2) or step (B2) is citric acid/sodium citrate solution.

Preferably, the pH of the buffer solution in step (A2) or step (B2) is 3-9; more preferably, the pH of the buffer solution in step (A2) or step (B2) is 4-6; most preferably, the pH of the buffer solution in step (A2) or step (B2) is 5.

Preferably, the concentration of the buffer solution in step (A2) or step (B2) is 1 mM-1 M; more preferably, the concentration of the buffer solution in step (A2) or step (B2) is 20 mM-500 mM; most preferably, the concentration of the buffer solution in step (A2) or step (B2) is 100 mM.

Preferably, the mass ratio of lipid molecules to nucleic acid drugs in step (A3) or step (B3) is 5:1-50:1; more preferably, the mass ratio of lipid molecules to nucleic acid drugs in step (A3) or step (B3) is 10:1-30:1.

The nucleic acid drug is mRNA, and preferably, the mass ratio of the lipid molecule to the mRNA is 25:1.

Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution in step (A3) or step (B3) is 1:1-1:10. More preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution in step (A3) or step (B3) is 1:1-1:5. Most preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution in step (A2) or step (B2) is 1:3.

In the present invention, the ionizable lipid molecules in the lung-targeted lipid nanoparticles may be the same as or different from the ionizable lipid molecules in the liver-targeted lipid nanoparticles, and are selected from:

In the present invention, the polyethylene glycol lipid is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

In the present invention, the steroidal lipid is selected from at least one of the following: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid.

In the present invention, the auxiliary lipid molecule is selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

Ninthly, the present invention claims protection for the use of the above-mentioned ionizable lipid molecules (liver-targeted lipid molecules), lipid quaternary ammonium salt molecules (lung-targeted lipid molecules), and liposomes containing liver-targeted lipid molecules and liposomes containing lung-targeted lipid molecules in the preparation of drug delivery systems.

The drug is a nucleic acid drug, including but not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA, etc.

In the tenth aspect, the present invention claims protection for the above-mentioned ionizable lipid molecules (liver-targeted lipid molecules), lipid quaternary ammonium salt molecules (lung-targeted lipid molecules), and liposomes containing liver-targeted lipid molecules, and methods and applications of liposomes containing lung-targeted lipid molecules for RNA delivery.

Among them, RNA types include but are not limited to mRNA, siRNA, microRNA, antisense nucleic acid, etc., and specific applications include but are not limited to diagnostic applications and therapeutic applications.

The lung-targeted component of the present invention can regulate its charge size and hydrophobicity over a wide range by changing the type of quaternary ammonium salt and the type and quantity of hydrophobic segments, which can fully meet the needs of targeted lung delivery of mRNA.

The present invention obtains lung-targeted lipid molecules through a quaternary ammonium salt reaction. After the targeting component is added into a basic LNP system in a certain proportion, the charge and particle size distribution of the LNP can be achieved, thereby achieving targeted delivery of mRNA to the lungs.

Compared with the prior art, the present invention has the following beneficial effects:

1. It binds tightly to mRNA and can achieve high encapsulation rate and stable protection of mRNA.

2. The formed LNP has good biocompatibility and is more stable.

3. After intravenous injection, it can be specifically enriched in the lungs without being distributed in other organs.

4. The liposome is suitable for the delivery of mRNA with different nucleic acid molecular weights and lengths and different nucleic acid sequences, and has universal applicability.

5. The technology of the present invention is simple to synthesize, the raw materials are cheap, and it is suitable for large-scale production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the structural formula of the cationic lipid molecule (L1-1) prepared in Example 1.

FIG. 2 shows the ¹ H NMR spectrum of the cationic lipid molecule (L1-1) prepared in Example 1.

FIG. 3 shows the ¹³ C NMR spectrum of the cationic lipid molecule (L1-1) prepared in Example 1.

FIG. 4 shows the ESI-TOF MS spectrum of the cationic lipid molecule (L1-1) prepared in Example 1.

FIG5 shows the structural formula of the lung-targeting lipid molecule (Y1-1) prepared in Example 1.

FIG. 6 shows the ¹ H NMR spectrum of the lung-targeting lipid molecule (Y1-1) prepared in Example 1.

FIG. 7 shows the ¹³ C NMR spectrum of the lung-targeting lipid molecule (Y1-1) prepared in Example 1.

FIG. 8 shows the ESI-TOF MS spectrum of the cationic lipid molecule lung-targeting lipid molecule (Y1-1).

FIG. 9 shows the structural formula of the cationic lipid molecule (L1-2) prepared in Example 2.

FIG. 10 shows the ¹ H NMR spectrum of the cationic lipid molecule (L1-2) prepared in Example 2.

FIG. 11 shows the structural formula of the lung-targeting lipid molecule (Y1-2) prepared in Example 2.

FIG. 12 shows the ¹ H NMR spectrum of the lung-targeting lipid molecule (Y1-2) prepared in Example 2.

FIG. 13 shows the particle size distribution of Y1-1-LNP@mRNA prepared in Example 3 with (Y1-1) as the lung targeting component (a) and the particle size distribution of Y1-2-LNP@mRNA with (Y1-2) as the lung targeting component (b).

FIG. 14 shows a TEM image (a) of Y1-1-LNP@mRNA prepared in Example 3 with (Y1-1) as the lung targeting component and a TEM image (b) of Y1-2-LNP@mRNA with (Y1-2) as the lung targeting component.

Figure 15 shows a comparison of the organ targeting effects of the basic LNP delivery system L1-1-LNP prepared with L1-1 prepared in Example 3, and the Y1-1-LNP@mRNA delivery system with (Y1-1) as the lung targeting component; the numbers represent the luminescence intensity.

FIG. 16 shows the bioluminescent imaging of the lung targeting effect of Y1-2-LNP@mRNA prepared in Example 3 with (Y1-2) as the lung targeting component, and the numbers represent the luminescent intensity.

FIG. 17 shows the cytotoxicity experiment of Y1-1-LNP@mRNA prepared in Example 3 with (Y1-1) as the lung targeting component.

FIG. 18 shows the blood biochemical indices of mice of Y1-1-LNP@mRNA prepared in Example 3 with (Y1-1) as the lung targeting component.

Figure 19 shows the H&E staining results of the main organs of mice treated with LNP@mRNA with (Y1-1 and Y1-2) as lung-targeted components prepared in Example 3, wherein a is the H&E staining result of the main organs of normal mice, b is the H&E staining result of the main organs of mice injected with lung-targeted LNP@mRNA prepared based on Y1-1, and c is the H&E staining result of the main organs of mice injected with lung-targeted LNP@mRNA prepared based on Y1-2.

Figure 20 shows a comparison of the organ targeting effects of the basic LNP delivery system prepared with the (Y2) precursor prepared in Example 7, and the Y2-LNP@mRNA delivery system with (Y2) as the lung targeting component; as well as the structural formulas of (Y2) and the (Y2) precursor; the numbers represent the luminescence intensity.

Figure 21 shows a comparison of the organ targeting effects of the basic LNP delivery system prepared with the (Y3) precursor prepared in Example 7, and the Y3-LNP@mRNA delivery system with (Y3) as the lung targeting component; as well as the structural formulas of (Y3) and the (Y3) precursor; the numbers represent the luminescence intensity.

Figure 22 shows a comparison of the organ targeting effects of the basic LNP delivery system prepared with the (Y4) precursor prepared in Example 7, and the Y4-LNP@mRNA delivery system with (Y4) as the lung targeting component; as well as the structural formulas of (Y4) and the (Y4) precursor; the numbers represent the luminescence intensity.

Figure 23 shows a comparison of the organ targeting effects of the basic LNP delivery system prepared with the (Y5) precursor prepared in Example 7, and the Y5-LNP@mRNA delivery system with (Y5) as the lung targeting component; as well as the structural formulas of (Y5) and (Y5) precursors; the numbers represent the luminescence intensity.

FIG. 24 shows the mRNA encapsulation efficiency of the basic LNP@mRNA prepared in Example 7 with (Y1-1 precursor, Y1-2 precursor, Y2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) as components.

FIG. 25 shows the mRNA encapsulation efficiency of the lung-targeted LNP@mRNA prepared in Example 7 with (Y1-1, Y1-2, Y2, Y3, Y4, Y5) as components.

FIG. 26 shows the average particle size of the basic LNP@mRNA prepared in Example 7 with (Y2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) as components.

Figure 27 shows the average particle size of the lung-targeted LNP@mRNA prepared in Example 7 with (Y1-1, Y1-2, Y2, Y3, Y4, Y5) as components.

FIG. 28 shows the Zeta potential of the basic LNP@mRNA prepared in Example 7 with (Y2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) as components.

FIG. 29 shows the Zeta potential of the lung-targeted LNP@mRNA prepared in Example 7 with (Y1-1, Y1-2, Y2, Y3, Y4, Y5) as components.

DETAILED DESCRIPTION

I. Definition

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present disclosure.

”以及“

”是指取代基键合的位置。In this disclosure, “

"as well as"

” refers to the position to which a substituent is bonded.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both the occurrence of said event or circumstance and the non-occurrence of said event or circumstance.

Numeric ranges herein refer to each integer in the given range. For example, "C ₁ -C ₆ " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms; "C ₃ -C ₆ " means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

The term "hydrophobic aliphatic hydrocarbon group" refers to an aliphatic hydrocarbon group that does not contain a hydrophilic group such as a hydroxyl group, an aldehyde group, a carboxyl group, and a nitrogen-containing amino group.

The term "substituted" means that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, as long as the valence state of the particular atom or group is normal and the substituted compound is stable. When the substituent is a keto group (i.e., =0), it means that two hydrogen atoms are replaced. Unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

When any variable (e.g., R _n ) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 R, the group may be optionally substituted with up to 5 R, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

The term "aliphatic group" includes saturated or unsaturated, linear or branched chain or cyclic hydrocarbon groups, heteroatom-free and heteroatom-containing aliphatic groups; the heteroatom refers to nitrogen atoms, oxygen atoms, fluorine atoms, phosphorus atoms, sulfur atoms, and selenium atoms. The type of the aliphatic group can be selected from alkyl, alkenyl, alkynyl, etc. For example, the term "C _1-6 aliphatic hydrocarbon group" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 1-butenyl, 1-ethylvinyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, etc.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including a linear or branched saturated hydrocarbon group having the indicated number of carbon atoms. For example, the term "C _1-6 alkyl" includes C ₁ alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. It may be divalent, such as methylene, ethylene.

The term "substituted" or "substituted" means that any one or more hydrogen atoms on a specific atom or group are replaced by a substituent, as long as the valence state of the specific atom or group is normal and the substituted compound is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are replaced. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical achievable. The substituent can be selected from one, two or more of the following substituents: deuterium, halogen group, cyano group, nitro group, -C(=O)R, -C(=O)OR', -OC(=O)R", imide group, amide group, hydroxyl group, substituted or unsubstituted amine group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted haloalkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted alkenyl group, substituted or unsubstituted alkynyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aryloxy group, substituted or unsubstituted heteroaryl group, etc., but not limited thereto.

The term "pharmaceutical composition" means a composition comprising the compound described in the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

The drugs or pharmaceutical compositions of the present disclosure can be delivered parenterally, i.e., administered intravenously (i.v.), intracerebroventricularly (i.c.v.), subcutaneously (s.c.), intraperitoneally (i.p.), intramuscularly (i.m.), subcutaneously (s.d.), or intradermally (i.d.), by direct injection, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. The composition can be in the form of an excipient, a suspension, solution, or emulsion in an oil or aqueous vehicle, and can include formulation agents such as anti-settling agents, stabilizers, and/or dispersants. Alternatively, the active ingredient can be reconstituted in powder form with a suitable carrier (e.g., sterile pyrogen-free water) before use.

The term "inflammatory disease" includes such autoimmune, allergic and inflammatory disorders, for example selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allograft rejection, Behcet's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, Sjogren's syndrome, and blistering disorders (e.g. pemphigus vulgaris), antibody-mediated vasculitis syndromes, including ANCA-associated vasculitis, purpura, and immune complex vasculitis (cancer or infection primary or secondary). The allergic disorder may be selected in particular from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollens and related allergens, berylliosis. The respiratory disorder may be selected from, inter alia, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, pulmonary fibrosis, respiratory failure, acute respiratory distress syndrome, primary pulmonary hypertension and emphysema, among others.

The term "viral infection" includes, but is not limited to, retroviral infection, hepatitis virus infection, Zika virus infection, dengue virus infection, etc.

II. Specific Examples

The present invention is specifically described below through examples. The examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the contents of the above invention all belong to the scope of protection of the present invention.

Example 1: Preparation of lung-targeted lipid molecule (Y1-1)

Oleic acid was dissolved in toluene and reacted with thionyl chloride at 60°C for 6h, and then the solvent and thionyl chloride were removed in vacuo to obtain oleoyl chloride. Oleoyl chloride and triethanolamine were dissolved in dichloromethane, triethylamine was added, and the reaction was allowed to proceed overnight. The obtained reaction solution was washed, dried, and concentrated, and then separated and purified by a chromatographic column. By controlling the ratio of oleoyl chloride to triethanolamine, disubstituted oleic acid triethanolamine ester (L1-1) was obtained (Figure 1). The relevant characterizations included ¹ H NMR (Figure 2, test conditions: CDCl ₃ , 400 M, 298.15K), ¹³ C NMR (Figure 3, test conditions: CDCl ₃ , 400 M, 298.15 K) and ESI-TOF MS (Figure 4), and the theoretical value of [M+H] ⁺ was m/z = 678.6031, and the measured m/z = 678.6013.

The obtained disubstituted oleic acid triethanolamine ester (L1-1) was then reacted with iodomethane in acetonitrile at 80 degrees Celsius for 72 hours. After the reaction, the solvent and iodomethane were removed in vacuo. The lung-targeted lipid molecule (Y1) was obtained (Figure 5). The relevant characterizations included ¹ H NMR (Figure 6, test conditions: CDCl ₃ , 400 M, 298.15 K), ¹³ C NMR (Figure 7, test conditions: CDCl ₃ ,400 M, 298.15 K) and ESI-TOF MS (Figure 8). The theoretical value of [M+H] ⁺ was m/z = 678.6031, and the measured m/z = 678.6013.

Example 2: Preparation of lung-targeted lipid molecules (Y1-2)

2-n-Hexyldecanoic acid was dissolved in toluene and reacted with thionyl chloride at 60°C for 6h, and then the solvent and thionyl chloride were removed in vacuo to obtain 2-n-hexyldecanoic acid chloride. 2-n-Hexyldecanoic acid chloride and triethanolamine were dissolved in dichloromethane, triethylamine was added, and the reaction was allowed to proceed overnight. The obtained reaction solution was washed, dried, and concentrated, and then separated and purified by a chromatographic column. By controlling the ratio of 2-n-hexyldecanoic acid chloride to triethanolamine, disubstituted 2-n-hexyldecanoic acid triethanolamine ester (L1-2) was obtained (Figure 9). Related characterizations include ¹ H NMR (Figure 10, test conditions: CDCl ₃ , 400 M, 298.15 K), whose [M-I+H] ⁺ theoretical value is m/z = 626.6, and the measured m/z = 626.2.

The obtained disubstituted 2-n-hexyldecanoic acid triethanolamine ester was then reacted with iodomethane in acetonitrile at 80 degrees Celsius for 72 hours, and the solvent and iodomethane were removed in vacuo after the reaction. The lung-targeted lipid molecule (Y1-2) was obtained (Figure 11). Related characterizations include ¹ H NMR (Figure 12, test conditions: CDCl ₃ , 400 M, 298.15 K).

Example 3: Preparation of basic LNP@mRNA and lung-targeted LNP@mRNA

L1-1 (or L1-2), DSPE-PEG2000, cholesterol and DOPE were mixed in a mass ratio of 30%:5%:15%:8%:42% and dissolved in an ethanol solution. The mRNA was Firefly luciferase mRNA, which was dissolved in a sodium citrate (100mM) buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipids and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration. The liver-targeted basic LNP@mRNA encapsulating mRNA was obtained.

L1-1, DSPE-PEG2000, cholesterol, DOPE and Y1-1 (or L1-2, DSPE-PEG2000, cholesterol, DOPE and Y1-2) were mixed in a mass ratio of 30%:5%:15%:8%:42% and dissolved in an ethanol solution. The mRNA was Fireflyluciferase mRNA, which was dissolved in a sodium citrate (100 mM) buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipids and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration. The lung-targeted LNP@mRNA encapsulated with mRNA was obtained.

Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to characterize the particle size distribution and morphology of the obtained LNPs. DLS results showed (Figure 13) that the hydrated particle size of the lung-targeted Y1-1-LNP@mRNA prepared based on L1-1 and Y1-1 was 63.8 nm, and the hydrated particle size of the lung-targeted Y1-2-LNP@mRNA prepared based on L1-2 and Y1-2 was 66.5 nm. TEM results showed (Figure 14) that both Y1-1-LNP@mRNA (a) and Y1-2-LNP@mRNA (b) were spherical with a particle size of 60-100 nm.

Example 4: Organ targeting experiment of LNP@mRNA

The two lung-targeted LNP@mRNAs obtained in Example 3, Y1-1-LNP@mRNA and Y1-2-LNP@mRNA, and the basic LNP@mRNA (with the Y1-1 precursor molecule as an ionizable liposome) were administered to C57BL/6G mice via the tail vein at a dose of 5 ug mRNA per mouse. Six hours later, the substrate was injected into the mouse intraperitoneally, and then bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system (Figures 15, 16). The results showed that the basic LNP system (with the Y1-1 precursor molecule as an ionizable liposome) can achieve enrichment in the liver, while no enrichment or expression was found in other organs, including the lungs, kidneys, heart, spleen, intestines, stomach, muscles, and bones, and the liver targeting effect was significant.

The lung-targeted delivery systems Y1-1-LNP@mRNA and Y1-2-LNP@mRNA can achieve targeted delivery, enrichment and expression in the lungs, and the expression effect is excellent. Other organs, including liver, kidney, heart, spleen, intestine, stomach, muscle and bone, were not found to be enriched or expressed, and the lung-targeted effect was significant.

Example 5: Cytotoxicity experiment of Y1-1-LNP@mRNA

HEK293 cells were seeded in DMEM medium (10% fetal bovine serum and 1% penicillin) in 96-well plates. The cells were incubated at 37°C in an atmosphere containing 5% CO _2. Fresh medium was replaced after 24 hours of cell incubation. Different concentrations of lung-targeted Y1-1-LNP@mRNA (0-200 μg/mL, prepared in Example 3) were added, and the cells were incubated for 24 hours, and then the original medium was replaced with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) medium. After further incubation for 4 hours, the medium containing MTT was removed and carefully washed 3 times with PBS. Then, DMSO (100 μL) was added, and the absorbance at a wavelength of 570 nm was measured in a BioTekSynergy H4 reader, and the cell survival rate for 24 h was calculated to verify the cytotoxicity of the delivery system obtained by the present invention.

As can be seen from FIG. 17 , the cell survival rate after 24 h of incubation was above 90%, indicating that the lung-targeted nucleic acid delivery system of the present invention has low cytotoxicity and exhibits good biosafety performance.

Example 6: Biosafety Analysis of LNP@mRNA

BALB/c mice were divided into 2 groups, 5 mice in each group. BALB/c mice (4-6 weeks old, female, weighing about 18-20 g) were injected with PBS and LNP@mRNA (10 μg mRNA, Y1-1-LNP@mRNA or Y1-2-LNP@mRNA) through the tail vein, respectively. Five days after the injection, venous blood was collected from the mice, and the supernatant was centrifuged to measure various indicators of liver and kidney function. H&E staining was performed on the heart, liver, spleen, lung and kidney of the mice in the PBS and LNP@mRNA groups 5 days after the injection to observe whether there were any lesions.

The results of liver and kidney function test showed (Figure 18) that there were no significant changes in liver and kidney functions of mice in the Y1-1 or Y1-2 groups, and their indicators were comparable to those of PBS. The results of tissue section experiments showed (Figure 19) that no obvious lesions were found in the major organs of mice treated with LNP@mRNA, indicating that LNP@mRNA nanoparticles have good biosafety.

Example 7: Organ targeting experiment of LNP@mRNA

The synthesized quaternary ammonium lipid molecules shown in formula (Y2) to formula (Y5) and their corresponding ionizable lipid molecule precursors were used to prepare lung-targeted LNPs and basic LNPs encapsulating mRNA, respectively. C57BL/6G mice were administered via the tail vein at a dose of 5 ug mRNA per mouse. Six hours later, the substrate was injected intraperitoneally into the mice, and then bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system (Figures 20, 21, 22, 23). The results showed that the basic LNP system based on ionizable lipid molecule precursors could achieve enrichment and expression in the liver, while no enrichment or expression was found in other organs, including the lungs, kidneys, heart, spleen, intestines, stomach, muscles, and bones, and the liver targeting effect was significant.

After adding lung targeting components to the basic LNP delivery system, Y2-LNP@mRNA, Y3-LNP@mRNA, Y4-LNP@mRNA and Y5-LNP@mRNA can achieve targeted delivery, enrichment and expression in the lungs, and the expression effect is excellent. No enrichment or expression was found in other organs, including liver, kidney, heart, spleen, intestine, stomach, muscle and bone, and the lung targeting effect is significant.

Both the basic LNP delivery system and the lung-targeted LNP delivery system achieved efficient mRNA encapsulation, with an encapsulation efficiency of more than 93% (Figures 24 and 25), demonstrating the excellent encapsulation capacity of these carriers. Dynamic light scattering experiments (Figures 26 and 27) demonstrated that the basic LNP@mRNA (with Y1-1 precursor, Y1-2 precursor, Y2 precursor, Y3 precursor, Y4 precursor, and Y5 precursor as ionizable liposomes) and lung-targeted LNP@mRNA (Y1-1, Y1-2, Y2, Y3, Y4, and Y5) had uniform particle sizes, with an average particle size of less than 100 nm. Zeta potential experiments (Figures 28 and 29) demonstrated that the potential of basic LNP@mRNA (with Y1-1 precursor, Y1-2 precursor, Y2 precursor, Y3 precursor, Y4 precursor, and Y5 precursor as ionizable liposomes) was less than 0 mV, and the potential of lung-targeted LNP@mRNA (Y1-1, Y1-2, Y2, Y3, Y4, and Y5) was greater than 0 mV.

The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments and various different options of the present disclosure.

Claims (17)

1. A lipid molecule for lung targeted delivery, wherein the structural formula is selected from any of the following formulas (I) to (V):

In the above formulae (I) to (V): The X is selected from halogen atoms, and ^X- is selected from halogen anions; The B ¹ , B ² and B ³ are the same or different; the B ¹ , B ² and B ³ form a carbamate group, an ester group, an ether bond or an amide group independently with the oxygen atom or the nitrogen atom; The L ¹ , L ² , and L ³ are the same or different, and are independently selected from C _1-20 aliphatic hydrocarbon groups and various steroidal lipids; The R ¹ is independently selected from a C _1-10 aliphatic hydrocarbon group. 2. The lipid molecule for lung targeted delivery according to claim 1, characterized in that: The structure of OB1 ^{- L1} is shown in any of the following general formulas:

The structure of OB ² -L ² is shown in any of the following general formulas:

The structure of NH-B ¹ -L ¹ is shown in any of the following general formulas:

The NH-B ² -L ² structure is shown in any of the following general formulas:

The structure of OB ³ -N is shown in any of the following general formulas:

Wherein, n represents the number of methylene -CH ₂ , and the value range is an integer within 0-20; The structure of NH-B ³ -N is shown in any of the following general formulas:

Wherein n represents the number of methylene groups -CH ₂ , and its value range is an integer within 0-20. 3. The lipid molecule for lung targeted delivery according to claim 1 or 2, characterized in that: The L ¹ and L ² are branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-20 aliphatic hydrocarbon groups, and various steroidal lipids; The ^L3 is a methyl group or an ethyl group; The R ¹ is a branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-10 aliphatic hydrocarbon group; The halogen anion X ⁻ is a fluoride ion, a chloride ion, a bromide ion or an iodide ion. 4. The lipid molecule for lung targeted delivery according to claim 3, characterized in that: The L ¹ and L ² are branched or unbranched, cyclic or non-cyclic, saturated or unsaturated C _10-20 aliphatic hydrocarbon groups or cholesterol; The R ¹ is a branched or unbranched, non-cyclic, saturated or unsaturated C _1-8 aliphatic hydrocarbon group. 5. The lipid molecule for lung targeted delivery according to claim 4, characterized in that: The L ¹ and L ² are branched or unbranched, cyclic or non-cyclic, saturated or unsaturated C _12-18 aliphatic hydrocarbon groups, or cholesterol; The R ¹ is a branched or unbranched, non-cyclic, saturated C _1-5 aliphatic hydrocarbon group. 6. The lipid molecule for lung-targeted delivery according to claim 1 or 2, characterized in that: the lipid molecule for lung-targeted delivery is selected from the lipid molecules represented by any of the following structural formulas:

(Y1-1)

(Y1-2)

(Y2)

(Y3)

(Y4)

(Y5). 7. The lipid molecule for targeted delivery is selected from at least one of the following formulas (A) to (F):

. 8. A liposome containing lung-targeted lipid molecules, wherein the raw materials thereof, in terms of mass percentage, comprise 10%-70% of the lipid molecules described in claim 7, 1%-20% of polyethylene glycol lipid molecules, 5%-60% of steroid lipids, 1%-30% of auxiliary lipid molecules, and 20%-60% of the lipid molecules described in any one of claims 1-6. 9. A liposome containing liver-targeted lipid molecules, wherein the raw materials thereof comprise, by weight percentage, 10%-70% of the lipid molecules according to claim 7, 1%-20% of polyethylene glycol lipid molecules, 5%-60% of steroid lipids and 1%-30% of auxiliary lipid molecules. 10. The liposome according to claim 8 or 9, characterized in that: The polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide, 1,2-dimyristoyl-sn-glyceromethoxy polyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoyl phosphatidylethanolamine and PEG-1,2-dimyristoyloxypropyl-3-amine; The steroidal lipid is selected from at least one of the following: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid; The auxiliary lipid molecule is selected from at least one of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), oleoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine. 11. A lung-targeted lipid nanoparticle encapsulating a nucleic acid drug, comprising the lung-targeted delivery, basic LNP system and a nucleic acid drug according to any one of claims 1 to 6; The basic LNP system includes ionizable lipid molecules, auxiliary lipid molecules, polyethylene glycol lipids and steroidal lipids. 12. A liver-targeted lipid nanoparticle encapsulating a nucleic acid drug, comprising the liposome containing the liver-targeted lipid molecule according to claim 9 and the nucleic acid drug. 13. The lipid nanoparticle according to claim 11 or 12, characterized in that the nucleic acid drug comprises DNA, mRNA, siRNA, microRNA, antisense nucleic acid, and circular RNA. 14. The method for preparing liver-targeted lipid nanoparticles carrying nucleic acid drugs according to claim 12, comprising the following steps: (A1) mixing the lipid molecule, auxiliary lipid molecule, polyethylene glycol lipid and steroid lipid according to claim 7 in a certain proportion, dissolving them in a solvent, and obtaining an organic phase liposome solution; (A2) dissolving the nucleic acid drug in a buffer solution of appropriate pH to obtain an aqueous nucleic acid drug solution; (A3) uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid drug solution using a microfluidic device to prepare liver-targeted lipid nanoparticles encapsulating nucleic acid drugs; The method further comprises: ultrafiltration or dialysis of the liver-targeted lipid nanoparticles encapsulating the nucleic acid drug obtained in step (A3) to obtain the liver-targeted lipid nanoparticles encapsulating the nucleic acid drug that can be used in biological experiments. 15. The method for preparing the lung-targeted lipid nanoparticles (LNP) containing nucleic acid drugs according to claim 14, comprising the following steps: (B1) mixing the lipid molecule of claim 7, the auxiliary lipid molecule, the polyethylene glycol lipid, the steroid lipid and the lipid molecule of any one of claims 1 to 6 in proportion, dissolving them in a solvent, and obtaining an organic phase liposome solution; (B2) dissolving the nucleic acid drug in a buffer solution of appropriate pH to obtain an aqueous nucleic acid drug solution; (B3) uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid drug solution using a microfluidic device to prepare lung-targeted lipid nanoparticles encapsulating nucleic acid drugs; The method further comprises: ultrafiltration or dialysis of the lung-targeted lipid nanoparticles encapsulating nucleic acid drugs obtained in step (B3) to obtain lung-targeted lipid nanoparticles encapsulating nucleic acid drugs that can be used in biological experiments. 16. The preparation method according to claim 14 or 15, characterized in that: The solvent used to dissolve the lipid molecules in step (A1) or step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, N,N-dimethylformamide; In the step (A1), the proportion of the lipid molecules is 10%-70%; In the step (A1), the proportion of the polyethylene glycol lipid is 1%-20%; In the step (A1), the proportion of the steroidal lipid is 5%-60%; In the step (A1), the ratio of the auxiliary lipid molecules is 1%-30%; In the step (B1), the proportion of the lipid molecules is 10%-70%; In the step (B1), the proportion of the polyethylene glycol lipid is 1%-20%; In the step (B1), the proportion of the steroidal lipid is 5%-60%; In the step (B1), the ratio of the auxiliary lipid molecules is 1%-30%; In the step (B1), the proportion of the lipid molecules according to any one of claims 1 to 6 is 5% to 60%; The buffer solution in step (A2) or step (B2) is acetic acid/sodium acetate solution or citric acid/sodium citrate solution; The pH of the buffer solution in step (A2) or step (B2) is 3-9; The concentration of the buffer solution in step (A2) or step (B2) is 1 mM-1 M; In the step (A3) or step (B3), the mass ratio of lipid molecules to nucleic acid drugs is 5:1-50:1; In the step (A3) or step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:1-1:10. 17. The preparation method according to claim 16, characterized in that: The solvent used to dissolve the lipid molecules in step (A1) or step (B1) is ethanol, tetrahydrofuran, or acetone; In the step (A1), the proportion of the lipid molecules is 20%-60%; In the step (A1), the proportion of the polyethylene glycol lipid is 5%-15%; In the step (A1), the proportion of the steroidal lipid is 10%-40%; In the step (A1), the ratio of the auxiliary lipid molecules is 5%-15%; In the step (B1), the proportion of the lipid molecules is 20%-40%; In the step (B1), the proportion of the polyethylene glycol lipid is 1%-10%; In the step (B1), the proportion of the steroidal lipid is 10%-40%; In the step (B1), the ratio of the auxiliary lipid molecules is 5%-15%; In the step (B1), the proportion of the lipid molecules according to any one of claims 1 to 6 is 10% to 50%; The buffer solution in step (A2) or step (B2) is a citric acid/sodium citrate solution; The pH of the buffer solution in step (A2) or step (B2) is 4-6; The concentration of the buffer solution in step (A2) or step (B2) is 20 mM-500 mM; In the step (A3) or step (B3), the mass ratio of lipid molecules to nucleic acid drugs is 10:1-30:1; In the step (A3) or step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:1-1:5.

Images