CN115925563A

CN115925563A - Lipid molecule for targeted lung delivery of nucleic acid and preparation method and application thereof

Info

Publication number: CN115925563A
Application number: CN202310174813.1A
Authority: CN
Inventors: 喻国灿; 戚少龙
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-04-07
Anticipated expiration: 2043-02-28
Also published as: CN115925563B

Abstract

The invention discloses a lung targeted lipid molecule, a preparation method thereof and an LNP system containing the lipid molecule and capable of realizing lung targeted delivery. The structural formula of the lung targeting lipid molecule is selected from the following formulas (I) to (V), and the definitions of all groups in the formulas are shown in the specification. After the lung targeting lipid molecules are added into a basic LNP system, the original liver targeting LNP delivery system can be converted into a lung targeting LNP delivery system. The lung targeting component can regulate the charge size and hydrophobicity of the lung targeting component in a large range by changing the types of the quaternary ammonium salt and the types and the number of the hydrophobic chain segments, and can fully meet the requirement of targeted lung delivery of mRNA.

Description

Lipid molecule for targeted lung delivery of nucleic acid and preparation method and application thereof

Technical Field

The invention belongs to the field of medical biology, and particularly relates to a lipid molecule for targeted lung delivery of nucleic acid, and a preparation method and application thereof.

Background

In recent years, nucleic acids (including DNA and RNA) have been developed in a breakthrough in the field of treatment of infectious diseases and tumors in a short time as a novel pharmaceutical technology. However, how to deliver nucleic acid molecules safely and efficiently to specific target cells and protect them from degradation is one of the major challenges facing 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 clinically advanced nucleic acid vectors. By 6 months 2021, all mRNA vaccines approved for clinical use employed the LNP delivery system. LNPs provide many benefits for mRNA delivery, including simple formulation, modularity, good biocompatibility, and greater mRNA payload capacity.

Unfortunately, there are reports that Pfizer/BioNTech and modern's mRNA vaccines administered intramuscularly may induce liver damage and immune hepatitis. The abnormal liver function also appears after mRNA vaccine inoculation of partial population, and the strong immune response is generated after the vaccine inoculation of liver transplantation patients. That is, the existing LNP delivery system may cause severe liver accumulation, possibly causing liver injury or immune hepatitis, so most of the current LNP delivery systems are liver-targeted, and the problem of effective delivery of organs other than liver (such as lung and kidney) is urgently needed to be solved.

In view of the first promise of the lung, a lung-targeted mRNA delivery system was developed that would deliver mRNA encoding the spike protein directly to the lungs, activate the body to produce the corresponding antibody against viral challenge, or deliver mRNA encoding a broadly neutralizing antibody or therapeutic protein directly to the lungs of the patient for treatment. In addition, the lung targeting mRNA delivery system can also be widely applied to prevention or treatment of various lung diseases, such as lung cancer, pneumonia, tuberculosis, chronic obstructive pulmonary disease, pulmonary thromboembolism, pulmonary vasculitis and the like.

The physicochemical properties and chemical composition of the LNP delivery system can alter the interaction with proteins in body fluids. In particular, by regulating and controlling the charge property, the particle size distribution and the proportion of hydrophobic lipid of an LNP delivery system, the distribution condition of LNP in vivo can be changed, thereby realizing organ targeted delivery.

Disclosure of Invention

The invention aims to provide a lipid molecule capable of realizing targeted delivery, a pharmaceutically acceptable salt thereof and a stereoisomer thereof.

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

；

after a basic LNP system is prepared by using the lipid molecules, a liver-targeted LNP delivery system can be obtained.

Further, the lipid molecules are formed into quaternary ammonium salts (lipid quaternary ammonium salt molecules), and then the lipid molecules capable of realizing targeted delivery to the lung can be obtained, and specifically at least one of the following formulas (I) to (V).

After the lipid molecules for lung targeted delivery are added into a basic LNP system, the LNP delivery system for lung targeting can be obtained.

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

x is selected from halogen atoms, X ^- Selected from the group consisting of halide anions including fluoride, chloride, bromide and iodide.

B is ¹ 、B ² And B ³ May be the same or different; b is ¹ 、B ² And B ³ Each independently form, with an oxygen atom or a nitrogen atom, any of a carbonate group, a carbamate group, an ester group, an ether linkage, a urea/amide group, or an amine group, or a combination of any two of the foregoing.

Said L ¹ 、L ² 、L ³ May be the same or different and are each independently selected from C _1-20 Aliphatic hydrocarbon group, hydrogen or various steroid lipids, vitamin A, vitamin E, vitamin K and other hydrophobic compounds, and L in the above-mentioned formulas (I) and (V) ¹ 、L ² At most one is hydrogen, L in the formulae (II) to (IV) ¹ 、L ² 、L ³ At most one is hydrogen;

the R is ¹ Each independently selected from C _1-10 An aliphatic hydrocarbon group.

Further, said O-B ¹ -L ¹ The structure of (A) is shown as any one of the following general formulas:

the O-B ² -L ² The structure of (A) is shown as any one of the following general formulas:

the NH-B ¹ -L ¹ The structure of (A) is shown as any one of the following general formulas:

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

the O-B ³ -N has the structure shown in any one of the following general formulas:

wherein n represents methylene-CH ₂ The number of (2) is an integer within the range of 0 to 20.

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

The lipid molecule is converted into quaternary amine on the tertiary amine of the lipid skeleton through quaternary ammonium salt reaction, so that the lipid molecule is converted into a quaternary ammonium salt form molecule from an uncharged neutral molecule, and the quaternary ammonium salt form molecule is used as a lung targeting component to realize the lung delivery effect of LNP. After the lipid quaternary ammonium salt molecules are added into an LNP system, LNP can be enriched to the lung within a certain time after intravenous injection by adjusting the surface charge and particle size distribution of LNP, the lung targeted delivery of the carried mRNA is completed, and the transfection of lung cells is realized.

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

Preferably, L ¹ ，L ² Is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _10-20 Fatty alkyl, cholesterol, vitaminA. Hydrophobic compounds such as vitamin E and vitamin K.

More preferably, L ¹ ，L ² Is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _12-18 Fatty alkyl, cholesterol, vitamin a and vitamin E.

Further preferably, L ¹ ，L ² Is branched or unbranched, cyclic or acyclic, unsaturated C _12-18 Fatty hydrocarbyl, cholesterol and vitamin E.

Most preferably, L ¹ ，L ² Is branched or unbranched, acyclic, unsaturated C _12-18 Or vitamin E.

For the above L ³ The methyl group and the ethyl group are preferable, and the methyl group is more preferable.

For the above R ¹ Which may be branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-10 An aliphatic hydrocarbon group of (1).

Preferably, R ¹ Is branched or unbranched, acyclic, saturated or unsaturated C _1-8 An aliphatic hydrocarbon group of (1).

Most preferably, R ¹ Is branched or unbranched, acyclic, saturated C _1-5 An aliphatic hydrocarbon group of (1).

For O-B ³ -an N structure, wherein N = an integer within 1-20; preferably, n = an integer within 1-10; more preferably, n = an integer within 1-5.

For the halogen anion X ^- It may be fluoride, chloride, bromide and iodide.

Preferably, X ^- Is chloride ion, bromide ion or iodide ion.

More preferably, X ^- Are bromide and iodide.

Most preferably, X ^- Is iodide ion.

According to the embodiment of the invention, the lung targeting lipid molecule shown in the formula (I) is a lipid molecule shown in a formula (Y1-1) and a formula (Y1-2); the lung targeting lipid molecule shown in the formula (II) is a lipid molecule shown in a formula (Y2); the lung targeting lipid molecule shown in the formula (III) is a lipid molecule shown in a formula (Y3); the lung targeting lipid molecule shown in the formula (IV) is a lipid molecule shown in a formula (Y4); the lung targeting lipid molecule shown in the formula (V) is a lipid molecule shown in a formula (Y5):

(Y1-1)

(Y1-2)

(Y2)

(Y3)/>

(Y4)

(Y5)

in a second aspect, the present invention provides a method for producing the lipid molecules represented by the above formulae (I) to (V).

The lipid molecules shown in the formula (I), the formula (II) and the formula (III) are provided by the invention, wherein the formula (I) and the formula (II) both adopt triethanolamine frameworks, and the formula (III) framework is 2-hydroxymethyl-1,3-propylene glycol. The reaction sites of the formulae (I), (II) and (III) are all hydroxyl groups, and the synthesis methods are the same, and the following description is given collectively. When B (B) is used ¹ 、B ² And/or B ³ ) The preparation method in the case of forming an ester bond with an oxygen atom is exemplified by the following steps:

(A1) The method comprises the following steps Firstly, fatty acid and thionyl chloride react in a solvent 1 for a certain time at a certain temperature under the action of alkali 1 to obtain fatty acyl chloride; the fatty acid in this step is reacted with L in formula I ¹ ，L ² Corresponding;

(A2) The method comprises the following steps And (2) reacting the fatty acyl chloride obtained in the step (A1) with triethanolamine or 2-hydroxymethyl-1,3-propylene glycol in a solvent 2 under the action of alkali 2 at a certain temperature for a certain time to obtain lipid molecules with different hydrophobic chain segments.

The procedure is exemplified by triethanolamine, and 2-hydroxymethyl-1,3-propanediol can be similarly reacted, and no additional explanation of the reaction scheme is provided below.

By controlling the feeding proportion and the reaction sequence, lipid molecules with different substituents can be obtained. If first obtaining L ¹ Substituted triethanolamine or 2-hydroxymethyl-1,3-propanediol derivative, and then gradually obtaining L ¹ ，L ² Disubstituted triethanolamine lipid molecules or L ¹ ，L ² Disubstituted 2-hydroxymethyl-1,3-propylene glycol lipid molecules.

The lipid molecule shown in formula (I) provided by the invention is represented by formula B (B) ¹ 、B ² And/or B ³ ) The preparation method for forming ether bond with 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, and lipid molecules with different substituent types and different substituent numbers can be obtained by adjusting the feeding proportion and sequence):

(A3) The method comprises the following steps Reacting triethanolamine or 2-hydroxymethyl-1,3-propylene glycol in solvent 1 under the action of sodium hydride at a certain temperature for a certain time, and adding corresponding halogenated aliphatic hydrocarbon (X-L) ¹ X represents halogen such as bromohydrocarbon) for a certain period of time to obtain lipid molecules having an ether bond as a linking group.

The method adopts a method for synthesizing ether by sodium hydride, and the reactions of the same type are not listed;

the lipid molecules shown in formula (I), formula (II) and formula (III) provided by the invention are represented by formula B (B) ¹ 、B ² And/or B ³ ) The preparation method for forming carbonate or carbamate with 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, and lipid molecules with different substituent types and different substituent numbers can be obtained by adjusting the feeding proportion and sequence):

(A4) The method comprises the following steps Mixing triethanolamine or 2-hydroxymethyl-1,3-propylene glycol with an activating reagent N, N' -carbonyldiimidazole or p-nitrophenyl chloroformate, and reacting in a solvent 3 for a period of time to obtain triethanolamine or 2-hydroxymethyl-1,3-propylene glycol derivatives with activated hydroxyl groups;

(A5) The method comprises the following steps Mixing the hydroxyl-activated triethanolamine derivative or 2-hydroxymethyl-1,3-propanediol derivative obtained in step (A4) with an amino-containing aliphatic hydrocarbon (L) ¹ -NH ₂ ) Reacting for a certain time to obtain lipid molecules of which the corresponding connecting groups are carbamate;

or, the hydroxyl group-activated triethanolamine derivative or 2-hydroxymethyl-1,3-propylene glycol derivative obtained in step (A4) is reacted with a hydroxyl group-containing aliphatic hydrocarbon (L) ¹ -OH) is heated and reacted for a certain time under the alkaline environment, and the lipid molecule with the corresponding connecting group of carbonate can be obtained.

The preparation method of the lipid molecule shown in the formula (II) comprises the following steps:

(A6) The method comprises the following steps Reacting a lipid molecule shown in formula (I) with an amine compound in a solvent 4 for a period of time under the action of a catalyst 1 or an activating reagent (the activating reagent refers to step (A4), N, N' -carbonyldiimidazole or p-nitrophenylchloroformate) and alkali 3 to convert a hydroxyl group into a structure containing an amine group.

(A7) The method comprises the following steps From the disubstituted (L) obtained in step (A2) ¹ , L ² Same or different) 2-hydroxymethyl-1,3-propanediol derivative, under the action of catalyst 1 or activating reagent (activating reagent refer to step (A4)), N' -carbonyldiimidazole or p-nitrophenylchloroformate) and base 3, reacting with amine compound in solvent 4 for a period of time to convert hydroxyl group into a structure containing amine group.

(A8) When the skeleton is tris (2-aminoethyl) amine (corresponding to the lipid molecule of formula IV or V), i.e.with B (B) ¹ 、B ² And/or B ³ ) When an amide group or an amine group is formed with a nitrogen atom, the reaction step is similar to the steps (A1) to (A3), and the triethanolamine may be replaced with tris (2-aminoethyl) amine, and the reaction step will not be described again.

(A9) Triethanolamine lipid molecules with a tertiary amine structure or 2-hydroxymethyl-1,3-propylene glycol lipid molecules or tris (2-aminoethyl) amine lipid molecules obtained through steps (A1) to (A8) are ionizable lipid molecules of a basic LNP delivery system, and the basic LNP delivery system prepared from the lipid molecules can realize liver targeted delivery of nucleic acid molecules.

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

It can be understood that, in the step (A1), thionyl chloride is used to react with different fatty acids to obtain corresponding acyl chloride, the reaction is general, the reaction activity and the reaction process are hardly influenced by aliphatic hydrocarbon groups, and the reaction can be carried out efficiently;

the step (A2) is a reaction of the fatty acyl chloride obtained in the step (A1) and triethanolamine or 2-hydroxymethyl-1,3-propylene glycol, the reaction occurs between the acyl chloride and hydroxyl, the reaction is hardly influenced by the type of the fatty alkyl, and the corresponding triethanolamine ester or 2-hydroxymethyl-1,3-propylene glycol ester can be efficiently obtained.

The hydroxyl on triethanolamine ester or 2-hydroxymethyl-1,3-propylene glycol ester is activated by a catalyst or an activating reagent and then reacts with an amine compound containing a reaction site, so that the reaction is hardly influenced by other groups, and the target lipid molecule can be efficiently obtained.

The step (a 10) is a reaction in which halogenated hydrocarbon and tertiary amine form a quaternary ammonium salt, the reaction is efficient, the target product can be obtained at a conversion rate close to a quantitative one, and the product is easy to purify.

The preparation method has universality, is suitable for different aliphatic hydrocarbon chains (including saturated aliphatic hydrocarbon chains or unsaturated aliphatic hydrocarbon chains), and can obtain quaternary ammonium salt type lipid molecules with different hydrophobicity.

In one embodiment, step (A1) comprises treating with L ¹ Esters of defined groupsFatty acids and e.g. L ² The fatty acids of the defined groups are independently selected from the corresponding alternative structures described above.

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

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

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

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

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

In one embodiment, the reaction temperature of step (A1) is 25-120 ℃; preferably, it is from 25 to 80 ℃; more preferably, it is from 25 to 60 ℃.

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

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

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

In one embodiment, the reaction temperature in step (A2) is 0-100 ℃; preferably, it is from 25 to 80 ℃; more preferably, it is from 25 to 60 ℃.

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

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

In one embodiment, the catalyst 1 in step (A6) or step (A7) is selected from one or two of Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), carbenium Hexafluorophosphate (HATU), N' N-Carbonyldiimidazole (CDI), p-nitrophenylchloroformate, triethylamine, dimethylaminopyridine;

preferably, one or two selected from Dicyclohexylcarbodiimide (DCC), and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), carbonium Hexafluorophosphate (HATU), N' N-Carbonyldiimidazole (CDI), p-nitrophenylchloroformate, triethylamine, dimethylaminopyridine;

more preferably, the compound is one or two selected from 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), N' N-Carbonyldiimidazole (CDI), p-nitrophenylchloroformate, triethylamine and dimethylaminopyridine.

In one embodiment, the reaction temperature in step (A6) or step (A7) is 0 to 100 ℃; preferably, it is from 25 to 80 ℃; more preferably, it is from 25 to 60 ℃.

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

In one embodiment, the reaction temperature in step (A8) is 0-100 ℃; preferably, it is from 25 to 80 ℃; more preferably, it is from 50 to 80 ℃.

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

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

The liposome provided by the invention comprises, by mass, 10% -70% of ionizable lipid molecules shown in formulas (A) to (F), 1% -20% of polyethylene glycol lipid molecules, 5% -60% of steroid lipid, 1% -30% of auxiliary lipid molecules and 5% -60% of lipid molecules for lung targeted delivery.

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

The raw materials of the liposome provided by the invention comprise, by mass percentage, 10% -70% of the liver targeted delivery lipid molecules (ionizable lipid molecules shown in formulas (A) to (F)), 1% -20% of polyethylene glycol lipid molecules, 5% -60% of steroid lipid and 1% -30% of auxiliary lipid molecules.

In a fifth aspect, the invention claims a lung-targeted Lipid Nanoparticle (LNP) encapsulating a nucleic acid drug.

The lung targeting Lipid Nanoparticle (LNP) coated with the nucleic acid drug comprises the lipid molecule component for lung targeting delivery, a basic LNP system and the nucleic acid drug.

The nucleic acid drug includes but is not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA and the like.

The base LNP system includes ionizable lipid molecules, helper lipid molecules, polyethylene glycol lipids, and steroidal lipids.

In a specific embodiment of the invention, the nucleic acid agent may be mRNA, including RNAs of different sequences and different lengths, in particular Firefly Luciferase mRNA.

In a sixth aspect, the invention claims a liver-targeted Lipid Nanoparticle (LNP) encapsulating a nucleic acid drug.

The liver targeting Lipid Nanoparticle (LNP) for encapsulating the nucleic acid medicament comprises the liposome containing the liver targeting lipid molecular component and the nucleic acid medicament.

In a seventh aspect, the invention claims a preparation method of the liver targeting Lipid Nanoparticle (LNP) encapsulating the nucleic acid drug.

The preparation method of the liver targeting lipid nanoparticle (namely the liver targeting basic LNP) encapsulating the nucleic acid medicament, which is provided by the invention, comprises the following steps:

(A1) Mixing the lipid molecules (ionizable lipid molecules) delivered by the liver in a targeted manner, the auxiliary lipid molecules, the polyethylene glycol lipid and the steroid lipid in proportion, and dissolving the mixture by using a solvent to obtain an organic phase liposome solution;

(A2) Dissolving a nucleic acid drug by using a buffer solution with proper pH to obtain an aqueous phase nucleic acid drug solution;

(A3) And (3) uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid drug solution according to a certain mass ratio and volume ratio by using a microfluidic device to prepare the liver targeting lipid nanoparticle for encapsulating the nucleic acid drug.

The method further comprises the following steps: and (B) carrying out ultrafiltration or dialysis on the liver targeting lipid nanoparticles encapsulating the nucleic acid medicament obtained in the step (A3) to obtain the liver targeting lipid nanoparticles encapsulating the nucleic acid medicament and being used for biological experiments.

In an eighth aspect, the invention claims a preparation method of the lung targeting Lipid Nanoparticle (LNP) encapsulating the nucleic acid drug.

The invention provides a preparation method of lung targeting lipid nanoparticles carrying nucleic acid drugs, which specifically comprises the following steps:

(B1) Mixing the ionizable lipid molecules, the auxiliary lipid molecules, the polyethylene glycol lipid, the steroid lipid and the lung targeting component in proportion, and dissolving the mixture by using a solvent to obtain an organic phase liposome solution;

(B2) Dissolving a nucleic acid drug by using a buffer solution with proper pH to obtain an aqueous phase nucleic acid drug solution;

(B3) And uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid medicine solution according to a certain mass ratio and volume ratio by using a microfluidic device to prepare the lung targeting lipid nanoparticle for encapsulating the nucleic acid medicine.

The method further comprises the following steps: and (C) carrying out ultrafiltration or dialysis on the lung targeting lipid nanoparticles coated with the nucleic acid medicament obtained in the step (B3) to obtain the lung targeting lipid nanoparticles coated with the nucleic acid medicament for biological experiments.

Preferably, the solvent used for dissolving the lipid molecules in step (A1) or step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, N-dimethylformamide. More preferably, the solvent used for dissolving 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) targeted for delivery to the liver in step (A1) is between 10% and 70%. More preferably, the proportion of ionizable lipid molecules in step (A1) is between 20% and 60%. Most preferably, the proportion of ionizable lipid molecules in step (A1) is 50%.

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

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

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

Preferably, the proportion of ionizable lipid molecules in step (B1) is between 10% and 70%. More preferably, the proportion of ionizable lipid molecules in step (B1) is between 20% and 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% to 20%. More preferably, the proportion of polyethylene glycol liposomes in step (B1) is between 1% and 10%. Most preferably, the proportion of polyethylene glycol lipid in step (B1) is 5%.

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

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

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

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

Preferably, the pH of the buffer solution in step (A2) or step (B2) is 3 to 9; more preferably, the pH of the buffer solution in step (A2) or step (B2) is 4 to 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-1M; more preferably, the concentration of the buffer solution in step (A2) or step (B2) is from 20 mM to 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 drug in step (A3) or step (B3) is 5:1-50; more preferably, the mass ratio of the lipid molecule to the nucleic acid drug in step (A3) or step (B3) is 10.

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

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. 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-targeting lipid nanoparticle may be the same as or different from the ionizable lipid molecules in the liver-targeting lipid nanoparticle, and are selected from the group consisting of:

in the present invention, the polyethylene glycol lipid is selected from at least one of the following: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylethanolamide (ALC-0159), 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearoyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (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, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydroergosterol, hypasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterol, fucosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

In the present invention, the helper 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).

In a ninth aspect, the present invention claims the use of the above ionizable lipid molecule (liver targeting lipid molecule), lipid quaternary ammonium salt molecule (lung targeting lipid molecule), and liposome containing liver targeting lipid molecule, and liposome containing lung targeting lipid molecule in the preparation of drug delivery system.

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

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

The RNA species include, but are not limited to, mRNA, siRNA, microRNA, antisense nucleic acid, and the like, and the specific application includes, but is not limited to, diagnostic application and therapeutic application.

The lung targeting component can regulate the charge size and hydrophobicity of the lung targeting component in a large range by changing the types of the quaternary ammonium salt and the types and the number of the hydrophobic chain segments, and can fully meet the requirement of targeted lung delivery of mRNA.

The lung targeting lipid molecules are obtained through quaternary ammonium salt reaction, and the targeted components are added into a basic LNP system according to a certain proportion to realize the charge and particle size distribution of LNP, so that the targeted delivery of mRNA to the lung can be realized.

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

1. the protein is tightly combined with mRNA, and can realize high entrapment rate and stable protection of the mRNA.

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

3. After intravenous injection, specific enrichment in the lungs can be achieved without distribution in other organs.

4. The liposome is suitable for mRNA delivery of different nucleic acid molecular weight lengths and different nucleic acid sequences, and has universality.

5. The invention has simple technical synthesis and low raw material price, and is suitable for large-scale production.

Drawings

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

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

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

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

FIG. 5 shows the structural formula of the lung-targeting lipid molecule (Y1-1) prepared in example 1.

FIG. 6 shows the preparation of lung targeting lipid molecule (Y1-1) of example 1 ¹ H NMR spectrum.

FIG. 7 shows preparation of lung targeting lipid molecule (Y1-1) of example 1 ¹³ C NMR spectrum.

FIG. 8 shows ESI-TOF MS spectra of cationic lipid molecules lung-targeted lipid molecules (Y1-1).

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

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

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

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

FIG. 13 shows the particle size distribution (a) of Y1-1-LNP @ mRNA having (Y1-1) as a lung targeting ingredient and the particle size distribution (b) of Y1-2-LNP @ mRNA having (Y1-2) as a lung targeting ingredient, prepared in example 3.

FIG. 14 shows photographs (a) of Y1-1-LNP @ mRNATEM with (Y1-1) as a lung targeting component and (b) of TEM of Y1-2-LNP @ mRNA with (Y1-2) as a lung targeting component, prepared in example 3.

FIG. 15 is a graph showing organ targeting effects of the basic LNP delivery system L1-1-LNP prepared from L1-1 prepared in example 3, and the Y1-1-LNP @ mRNA delivery system with (Y1-1) as the lung targeting component; the number indicates the luminous intensity.

FIG. 16 shows a graph of the lung targeting effect bioluminescence image of Y1-2-LNP @ mRNA prepared in example 3 with (Y1-2) as the lung targeting component, and the number indicates the luminescence intensity.

FIG. 17 shows cytotoxicity experiments of Y1-1-LNP @ mRNA prepared in example 3 with (Y1-1) as the lung targeting ingredient.

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

FIG. 19 shows the results of H & E staining of major organs of mice treated with LNP @ mRNA prepared in example 3 using (Y1-1 and Y1-2) as lung targeting components, wherein a is the result of H & E staining of major organs of normal mice, b is the result of H & E staining of major organs of mice after injection of Y1-1 for lung targeting LNP @ mRNA prepared based on injection of Y1-1, and c is the result of H & E staining of major organs of mice after injection of Y1-2 for lung targeting LNP @ mRNA prepared based on injection of Y1-2.

FIG. 20 is a graph showing the comparison of organ targeting effects of the basal LNP delivery system prepared from the (Y2) precursor prepared in example 7, and the Y2-LNP @ mRNA delivery system with (Y2) as the lung targeting component; and structural formulae of the precursors of (Y2) and (Y2); the number indicates the luminous intensity.

FIG. 21 shows a graph comparing the organ targeting effect of the basic LNP delivery system prepared from the (Y3) precursor prepared in example 7, and the Y3-LNP @ mRNA delivery system with (Y3) as the lung targeting component; and structural formulae of the precursors of (Y3) and (Y3); the number indicates the luminous intensity.

FIG. 22 is a graph showing the comparison of organ targeting effects of the basal LNP delivery system prepared from the (Y4) precursor prepared in example 7, and the Y4-LNP @ mRNA delivery system with (Y4) as the lung targeting component; and structural formulae of the precursors of (Y4) and (Y4); the number indicates the luminous intensity.

FIG. 23 is a graph showing a comparison of organ targeting effects of the basal LNP delivery system prepared from the (Y5) precursor prepared in example 7, and the Y5-LNP @ mRNA delivery system with (Y5) as the lung targeting component; and structural formulae of the precursors of (Y5) and (Y5); the number indicates the luminous intensity.

FIG. 24 shows the mRNA entrapment efficiency of the basic LNP @ mRNA prepared in example 7 with (Y1-1 precursor, Y1-2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) as a component.

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

FIG. 26 shows the average particle size of the basal LNP @ mRNA prepared in example 7 and having (Y2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) as a component.

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

FIG. 28 shows the Zeta potentials of the basic LNP @ mRNA composed of (Y2 precursor, Y3 precursor, Y4 precursor, Y5 precursor) prepared in example 7.

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

Detailed Description

I. Definition of

In the present disclosure, unless defined otherwise, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, the relative terms and laboratory procedures used herein are terms and conventional procedures used extensively in the relevant arts. Meanwhile, for better understanding of the present disclosure, definitions and explanations of related terms are provided below.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

The disclosed compounds may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents. Racemates, diastereomers, enantiomers are included within the scope of the present disclosure.

In this disclosure,') "

"and")>

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

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

Number in this textWord range, refers to each integer in a given range. For example, "C ₁ -C ₆ By "is meant 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 By "is meant that the group may have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

The term "hydrophobic aliphatic hydrocarbon group" means an aliphatic hydrocarbon group free from hydrophilic groups such as hydroxyl group, aldehyde group, carboxyl group, and nitrogen-containing amino group.

The term "substituted" means that any one or more hydrogen atoms on a particular atom or group is replaced with a substituent, so long as the valency of the particular atom or group is normal and the substituted compound is stable. When the substituent is keto (i.e = O), it means that two hydrogen atoms are substituted. Unless otherwise specified, the kind and number of substituents may be arbitrary on the basis that they can be chemically achieved.

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

The term "aliphatic hydrocarbon group" includes saturated or unsaturated, straight-chain or branched chain or cyclic hydrocarbon groups, heteroatom-free and heteroatom-containing aliphatic hydrocarbon groups; the hetero atom means a nitrogen atom, an oxygen atom, a fluorine atom, a phosphorus atom, a sulfur atom, and a selenium atom. The type of the aliphatic hydrocarbon group may be selected from alkyl, alkenyl, alkynyl, and the like. Such as the term "C _1-6 Aliphatic hydrocarbon groups "include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 1-ethylethenyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl2-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, cyclohexyl and the like.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight or branched chain saturated hydrocarbon groups, having the indicated number of carbon atoms. Such as the term "C _1-6 Alkyl "includes C ₁ Alkyl radical, C ₂ Alkyl radical, C ₃ Alkyl radical, C ₄ Alkyl radical, C ₅ Alkyl radical, C ₆ Examples of alkyl groups 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, and the like. It may be divalent, e.g. methylene, ethylene.

The term "substituted" or "substitution" means that any one or more hydrogen atoms on a particular atom or group is replaced with a substituent, provided that the valence of the particular atom or group is normal and the substituted compound is stable. When the substituent is keto (i.e = O), it means that two hydrogen atoms are substituted. Unless otherwise specified, the kind and number of substituents may be arbitrary on the basis that they can be chemically achieved. The substituents may be substituted with one, two or more substituents selected from: deuterium, a halogen group, a cyano group, a nitro group, -C (= O) R, -C (= O) OR ', -OC (= O) R', an imide group, an amide group, a hydroxyl group, a substituted OR unsubstituted amine group, a substituted OR unsubstituted alkyl group, a substituted OR unsubstituted cycloalkyl group, a substituted OR unsubstituted haloalkyl group, a substituted OR unsubstituted alkoxy group, a substituted OR unsubstituted alkenyl group, a substituted OR unsubstituted alkynyl group, a substituted OR unsubstituted aryl group, a substituted OR unsubstituted aryloxy group, a substituted OR unsubstituted heteroaryl group, and the like, but is not limited thereto.

The term "pharmaceutical composition" means a composition comprising a 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, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature sensitive materials, temperature regulating agents, adhesives, stabilizers, suspending agents, and the like.

The medicaments or pharmaceutical compositions of the present disclosure may be delivered parenterally, i.e., by intravenous (i.v.), intracerebroventricular (i.c.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.d.), or intradermal (i.d.), administration, by direct injection, via, for example, bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers with added preservative. The compositions may take the form of an excipient (excipient), a suspension, solution or emulsion in an oil or aqueous carrier, and may contain formulatory agents such as anti-settling agents, stabilising agents and/or dispersing agents. Alternatively, the active ingredient may be reconstituted in powder form with a suitable carrier (e.g., sterile pyrogen-free water) prior to use.

The term "inflammatory disease" includes said autoimmune, allergic and inflammatory disorders, e.g. 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, becker's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, graves ' disease, hashimoto's thyroiditis, sjogren's syndrome, and blistering disorders (e.g. pemphigus vulgaris), antibody-mediated vasculitis syndromes, including ANCA-related vasculitis, purpura, and immune complex vasculitis (cancer or infection stage one or two). The allergic condition may be selected from among contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, beryllium poisoning. The respiratory disorder may be selected in particular from 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, emphysema and the like.

The term "viral infection" includes, but is not limited to, retroviral infection, hepatitis viral infection, zika viral infection, dengue viral infection, and the like.

Specific examples II

The present invention is described in detail by the following embodiments, which are only used for further illustration of the present invention and should not be construed as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art based on the above disclosure are within the scope of the present invention.

Example 1: preparation of Lung-targeting lipid molecule (Y1-1)

Oleic acid was dissolved in toluene to react with thionyl chloride at 60 ℃ for 6h, followed by vacuum removal of the solvent and thionyl chloride to give oleoyl chloride. Oleoyl chloride and triethanolamine were dissolved in dichloromethane, triethylamine was added, and the reaction was carried out overnight. The obtained reaction solution is washed, dried and concentrated and then is separated and purified by a chromatographic column. By controlling the ratio of oleoyl chloride to triethanolamine, disubstituted triethanolamine oleate (L1-1) is obtained (figure 1). The associated characterization includes ¹ H NMR (FIG. 2, test conditions: CDCl) ₃ , 400 M, 298.15 K）、 ¹³ C NMR (FIG. 3, test conditions: CDCl) ₃ 400M, 298.15K) and ESI-TOF MS (FIG. 4), which [ M + H] ⁺ The theoretical value was m/z = 678.6031 and m/z = 678.6013 was measured.

The resulting disubstituted triethanolamine oleate (L1-1) is then reacted with methyl iodide in acetonitrile at 80 ℃ for 72 hours, after which the solvent and methyl iodide are removed in vacuo. The lung targeting lipid molecule (Y1) was obtained (fig. 5). The correlation characterization includes ¹ H NMR (FIG. 6, test conditions: CDCl) ₃ , 400 M, 298.15 K）、 ¹³ C NMR (FIG. 7, test conditions: CDCl) ₃ 400M, 298.15K) and ESI-TOF MS (FIG. 8), which [ M + H] ⁺ The theoretical value was m/z = 678.6031 and m/z = 678.6013 was measured.

Example 2: preparation of Lung-targeting lipid molecule (Y1-2)

Dissolving 2-n-hexyldecanoic acid in toluene to react with thionyl chloride at 60 ℃ for 6h, and then removing the solvent and thionyl chloride in vacuum to obtain 2-n-hexyldecanoic acid acyl chloride. Dissolving 2-n-hexyldecanoic acid acyl chloride and triethanolamine in dichloromethane, adding triethylamine, and reacting overnight. The obtained reaction solution is washed, dried and concentrated, and then is 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) is obtained (figure 9). The correlation characterization includes ¹ H NMR (FIG. 10, test conditions: CDCl) ₃ 400M, 298.15K), which [ M-I + H] ⁺ The theoretical value was m/z = 626.6 and m/z = 626.2 was measured.

And then reacting the obtained disubstituted 2-n-hexyldecanoic acid triethanolamine ester with methyl iodide in acetonitrile at 80 ℃ for 72 hours, and removing the solvent and methyl iodide in vacuum after the reaction is finished. The lung targeting lipid molecule (Y1-2) was obtained (FIG. 11). The associated characterization includes ¹ H NMR (FIG. 12, test conditions: CDCl) ₃ , 400 M, 298.15 K）。

Example 3 preparation of basic and Lung-targeting LNP @ mRNA

Mixing L1-1 (or L1-2), DSPE-PEG2000, cholesterol and DOPE according to the mass ratio of 30%:5%:15%:8%:42%, and dissolving in ethanol solution. The mRNA was Firefly luciferase mRNA, dissolved in a buffer solution of sodium citrate (100 mM) at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution is 1:3, and the contained lipid and the mRNA are mixed according to the mass ratio of 25. Followed by ultrafiltration to remove ethanol. Obtaining the mRNA-entrapped liver targeting basic LNP @ mRNA.

Mixing L1-1, DSPE-PEG2000, cholesterol, DOPE and Y1-1 (or L1-2, DSPE-PEG2000, cholesterol, DOPE and Y1-2) according to the mass ratio of 30%:5%:15%:8%:42%, and dissolving in ethanol solution. The mRNA was Firefoluciferase mRNA, dissolved in a buffer solution of sodium citrate (100 mM) at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution is 1:3, and the contained lipid and the mRNA are mixed according to the mass ratio of 25. Followed by ultrafiltration to remove ethanol. Obtaining the lung targeting LNP @ mRNA coated with mRNA.

And (3) performing particle size distribution characterization and morphology characterization on the obtained LNP by using Dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM). DLS results showed (FIG. 13) that the hydrated particle size of lung targeting Y1-1-LNP @ mRNA prepared based on L1-1 and Y1-1 was 63.8 nm, and the hydrated particle size of lung targeting Y1-2-LNP @ mRNA prepared based on L1-2 and Y1-2 was 66.5 nm. TEM results showed (FIG. 14) that Y1-1-LNP @ mRNA (a) and Y1-2-LNP @ mRNA (b) are spherical and have a particle size of 60-100 nm.

Example 4: organ targeting experiments with LNP @ mRNA

Two lung-targeted lnp @ mRNA, Y1-1-lnp @ mRNA and Y1-2-lnp @ mRNA and basal lnp @ mRNA obtained from example 3 (with Y1-1 precursor molecule as ionizable liposomes) were administered via the tail vein to C57BL/6G mice at a dose of 5 ug mRNA per mouse. The mice were injected intraperitoneally with substrate after 6 hours, followed by bioluminescence imaging using a mouse in vivo fluorescence imaging system (fig. 15, 16). The results show that the basic LNP system (taking the Y1-1 precursor molecule as the ionizable liposome) can realize the enrichment of the liver, and the liver targeting effect is obvious because no enrichment or expression is found in other organs including the lung, the kidney, the heart, the spleen, the intestinal tract, the stomach, the muscle and the skeleton.

The lung targeted delivery system Y1-1-LNP @ mRNA and Y1-2-LNP @ mRNA can realize targeted delivery, enrichment and expression to the lung, and the expression effect is excellent. And other organs including liver, kidney, heart, spleen, intestinal tract, stomach, muscle and skeleton are all found to be enriched or expressed, and the lung targeting effect is obvious.

Example 5: cytotoxicity assay for Y1-1-LNP @ mRNA

HEK293 cells were seeded in 96-well plates in DMEM medium (10% fetal bovine serum and 1% penicillin). Cell containing 5% CO at 37% ₂ Is incubated in an atmosphere of (2). Cells were incubated for 24 hours and then replaced with fresh medium. Varying concentrations of lung-targeted Y1-1-LNP @ mRNA (0-200. Mu.g/mL, prepared in example 3) were added and the cells incubated for 24 hoursThe original medium was replaced with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT) medium containing 0.5 mg/mL. After a further 4 hours of incubation, the medium containing MTT was removed and carefully washed 3 times with PBS. Then, DMSO (100 μ L) was added, absorbance at a wavelength of 570 nm was measured in a BioTek Synergy H4 reader, and the cell viability of 24H was calculated, verifying the cytotoxicity of the delivery system obtained in the present invention.

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

Example 6 Biosafety analysis of LNP @ mRNA

BALB/c mice were divided into 2 groups of 5 mice each (4-6 weeks old, female, about 18-20 g in body weight), PBS and LNP @ mRNA (10. Mu.g mRNA, Y1-1-LNP @ mRNA or Y1-2-LNP @ mRNA) were injected into the tail vein, 5 days after injection, venous blood was collected from the mice, and supernatant was centrifuged to determine various indices of liver and kidney functions. H & E staining was performed on the heart, liver, spleen, lung and kidney of the PBS and LNP @ mRNA group 5 days after injection to observe whether they had lesions.

The results of the liver and kidney function index tests (fig. 18) show that the liver and kidney functions of the mice in the group Y1-1 or Y1-2 have no obvious change, and the indexes are all equivalent to those of PBS. The tissue slice experiment result shows (figure 19), all major organs of the mice treated by the LNP @ mRNA have no obvious lesion, which indicates that the LNP @ mRNA nano-particles have good biological safety.

Example 7 organ targeting experiment of LNP @ mRNA

The synthesized quaternary ammonium salt lipid molecules shown in formulas (Y2) to (Y5) and corresponding ionizable lipid molecule precursors are used for preparing the lung targeting LNP and the basic LNP respectively. C57BL/6G mice were dosed via the tail vein with 5 ug mRNA per mouse. The mice were injected intraperitoneally with substrate after 6 hours, followed by bioluminescence imaging using a mouse in vivo fluorescence imaging system (fig. 20, 21, 22, 23). The results show that the basic LNP system based on the ionizable lipid molecule precursor can realize the enrichment and expression of the liver, while the enrichment or expression of other organs including the lung, the kidney, the heart, the spleen, the intestinal tract, the stomach, the muscle and the skeleton is not found, and the liver targeting effect is remarkable.

After lung targeting components are added into a basic LNP delivery system, Y2-LNP @ mRNA, Y3-LNP @ mRNA, Y4-LNP @ mRNA and Y5-LNP @ mRNA can realize targeted delivery, enrichment and expression to the lung, and the expression effect is excellent. Other organs including liver, kidney, heart, spleen, intestinal tract, stomach, muscle and skeleton are all found to be enriched or expressed, and the lung targeting effect is obvious.

Both the basic and lung-targeted LNP delivery systems achieved high-efficiency entrapment of mRNA, with entrapment efficiencies exceeding 93% (fig. 24, 25), demonstrating the excellent entrapment capability of these vectors. Dynamic light scattering experiments (FIGS. 26, 27) demonstrated that both the basal LNP @ mRNA (as Y1-1 precursor, Y1-2 precursor, Y3 precursor, Y4 precursor, Y5 precursor as ionizable liposomes) and the lung-targeted LNP @ mRNA (Y1-1, Y1-2, Y2, Y3, Y4, Y5) had uniform particle size and average particle size of less than 100 nm. Zeta potential experiments (FIGS. 28, 29) demonstrated that the potential of the basal LNP @ mRNA (with Y1-1 precursor, Y1-2 precursor, Y3 precursor, Y4 precursor, Y5 precursor as ionizable liposomes) was less than 0 mV, and the potential of the lung-targeted LNP @ mRNA (Y1-1, Y1-2, Y2, Y3, Y4, Y5) was greater than 0 mV.

The foregoing descriptions of specific exemplary embodiments of the present disclosure have been presented for purposes of illustration and description. These descriptions are not intended to limit the disclosure to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the disclosure and its practical application to enable one skilled in the art to make and use various exemplary embodiments and various alternatives of the disclosure.

Claims

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

；

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

x is selected from halogen atoms, X ^- Selected from halogen anions;

b is ¹ 、B ² And B ³ The same or different; b is ¹ 、B ² And B ³ Independently form any connecting group of carbonate group, carbamate group, ester group, ether linkage, carbamido/amide group or amine group or the combination of any two connecting groups with oxygen atom or nitrogen atom;

said L ¹ 、L ² 、L ³ Are the same or different and are each independently selected from C _1-20 Aliphatic hydrocarbon group, hydrogen or various steroid lipids, vitamin A, vitamin E and vitamin K, and L in the formula (I) and the formula (V) ¹ 、L ² At most one is hydrogen, L in the formulae (II) to (IV) ¹ 、L ² 、L ³ At most one is hydrogen;

2. The lung-targeted lipid molecule of claim 1, wherein:

the O-B ^1- L ¹ The structure of (A) is shown as any one of the following general formulas:

；

；

；

；

the O-B ³ The structure of-N is shown as any one of the following general formulas:

；

wherein n represents methylene-CH ₂ The number of (2) is an integer within the range of 0-20;

；

3. The lung-targeted lipid molecule according to claim 1 or 2, characterized in that:

said L ¹ ，L ² Which is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-20 A fatty hydrocarbon group of (a), various steroidal lipids, vitamin A, vitamin E or vitamin K;

said L ³ Is methyl or ethyl;

the R is ¹ Which is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _1-10 An aliphatic hydrocarbon group of (2);

the halogen anion X ^- Which is fluoride, chloride, bromide or iodide.

4. The lung-targeted lipid molecule of claim 3, wherein:

said L ¹ ，L ² Is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _10-20 The fatty alkyl, cholesterol, vitamin a, vitamin E or vitamin K;

the R is ¹ Is branched or unbranched, acyclic, saturated or unsaturated C _1-8 An aliphatic hydrocarbon group of (1).

5. The lung-targeted lipid molecule of claim 4, wherein:

said L ¹ ，L ² Is branched or unbranched, cyclic or acyclic, saturated or unsaturated C _12-18 Fatty alkyl, cholesterol, vitamin a or vitamin E;

the R is ¹ Is branched or unbranched, acyclic, saturated C _1-5 An aliphatic hydrocarbon group of (1).

6. The lung-targeted lipid molecule according to claim 1 or 2, characterized in that: the lipid molecule for lung targeted delivery is selected from lipid molecules shown in any structural formula as follows:

(Y1-1)

(Y1-2)

(Y2)

(Y3)

(Y4)

(Y5)。

7. a targeted delivery lipid molecule selected from at least one of the following formulae (a) to (F):

。

8. a liposome containing a lung targeting lipid molecule, wherein the raw material comprises, by mass percentage, 10% to 70% of the lipid molecule of claim 7, 1% to 20% of a polyethylene glycol lipid molecule, 5% to 60% of a steroid lipid, 1% to 30% of a helper lipid molecule, and 20% to 60% of the lipid molecule of any one of claims 1 to 6.

9. A liposome containing liver targeting lipid molecule, which comprises 10-70% of the lipid molecule of claim 7, 1-20% of polyethylene glycol lipid molecule, 5-60% of steroid lipid and 1-30% of auxiliary lipid molecule.

10. The liposome of claim 8 or 9, wherein:

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-glyceromethoxypolyethylene glycol, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ], PEG-distearoyl glycerol, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearoyl, PEG-diacylglycerol amide, PEG-dipalmitoyl phosphatidylethanolamine, and PEG-1, 2-dimyristoyloxypropyl-3-amine;

the steroidal lipid is selected from at least one of: avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydroergosterol, hydridoterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, fucosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid;

the helper lipid molecule is selected from at least one of: 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-phosphate- (1' -rac-glycerol), oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylethanolamine.

11. A nucleic acid drug-loaded lung-targeted lipid nanoparticle comprising the lung-targeted delivery, base LNP system of any of claims 1-6 and a nucleic acid drug;

12. A liver-targeting lipid nanoparticle encapsulating a nucleic acid drug, comprising the liposome containing a liver-targeting lipid molecule of claim 9 and a nucleic acid drug.

13. A lipid nanoparticle according to claim 11 or 12, wherein: the nucleic acid medicament comprises DNA, mRNA, siRNA, microRNA, antisense nucleic acid and circular RNA.

14. The preparation method of the liver targeting lipid nanoparticle carrying the nucleic acid drug of claim 12, comprising the following steps:

(A1) Mixing the lipid molecules, helper lipid molecules, polyethylene glycol lipid, and steroid lipid according to claim 7, and dissolving with solvent to obtain organic phase liposome solution;

(A3) Uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid drug solution by using a microfluidic device to prepare liver targeting lipid nanoparticles carrying nucleic acid drugs;

the method further comprises the following steps: and (C) carrying out ultrafiltration or dialysis on the liver targeting lipid nanoparticles carrying the nucleic acid medicament obtained in the step (A3) to obtain the liver targeting lipid nanoparticles carrying the nucleic acid medicament and capable of being used for biological experiments.

15. The method for preparing the nucleic acid drug-encapsulated lung targeted Lipid Nanoparticle (LNP) of claim 14, comprising the steps of:

(B1) Mixing the lipid molecule of claim 7, the helper lipid molecule, the polyethylene glycol lipid, the steroid lipid, and the lipid molecule of any one of claims 1 to 6 in a ratio, and dissolving the mixture with a solvent to obtain an organic phase liposome solution;

(B3) Uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid drug solution by using a microfluidic device to prepare the lung targeting lipid nanoparticle carrying the nucleic acid drug;

the method further comprises the following steps: and (C) carrying out ultrafiltration or dialysis on the nucleic acid drug-encapsulated lung targeted lipid nanoparticles obtained in the step (B3) to obtain nucleic acid drug-encapsulated lung targeted lipid nanoparticles for biological experiments.

16. The production method according to claim 14 or 15, characterized in that:

the solvent used for dissolving the lipid molecules in the step (A1) or the step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, 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 ratio of the steroid lipid is 5% -60%;

in the step (A1), the proportion of the helper 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 ratio of the steroid lipid is 5% -60%;

in the step (B1), the proportion of the helper lipid molecules is 1-30%;

in step (B1), the proportion of the lipid molecules of any one of claims 1 to 6 is between 5% and 60%;

the buffer solution in the step (A2) or the step (B2) is acetic acid/sodium acetate solution or citric acid/sodium citrate solution;

the pH of the buffer solution in the step (A2) or the step (B2) is 3-9;

the concentration of the buffer solution in the step (A2) or the step (B2) is 1 mM-1M;

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

in the step (A3) or the step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:1-1.

17. The method of manufacturing according to claim 16, wherein:

the solvent used for dissolving the lipid molecules in the step (A1) or the 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 ratio of the steroid lipid is 10% -40%;

in the step (A1), the proportion of the helper 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 ratio of the steroid lipid is 10-40%;

in the step (B1), the proportion of the helper lipid molecules is 5-15%;

in step (B1), the proportion of the lipid molecules of any one of claims 1 to 6 is 10% to 50%;

the buffer solution in the step (A2) or the step (B2) is a citric acid/sodium citrate solution;

the pH of the buffer solution in the step (A2) or the step (B2) is 4-6;

the concentration of the buffer solution in the step (A2) or the step (B2) is 20 mM-500 mM;

the mass ratio of the lipid molecule to the nucleic acid drug in the step (A3) or the step (B3) is 10;

the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid medicine solution in the step (A3) or the step (B3) is 1:1-1:5.