A lipid nanoparticle for targeted lung delivery of broad-spectrum neutralizing antibodies and its preparation method and application

Abstract

The present invention discloses a lipid nanoparticle that can be used for targeted lung delivery of broad-spectrum neutralizing antibodies, as well as its preparation method and application. The lipid nanoparticles, whose constituent raw materials, measured by mass percentage, include 10%-70% ionizable lipid molecules of formula (a) to formula (c), 5%-60% steroidal lipids, 1%-20% lipid-polyethylene glycol, 1%-30% auxiliary lipid molecules, and 5%-60% of lipid molecules for lung-targeted delivery shown in formula (A) to formula (C), where the definitions of each group are detailed in the specification. This lung-targeted lipid nanoparticle system is applied to the broad-spectrum neutralizing antibodies of the new coronavirus to effectively improve the prevention ability of infection and reduce the respiratory viral load and lung pathological damage after exposure to the new coronavirus. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and specifically relates to a lipid nanoparticle that can be used for targeted lung delivery of broad-spectrum neutralizing antibodies, as well as a preparation method and application thereof. Background Art

[0002] In recent years, nucleic acids (including DNA and RNA) have rapidly demonstrated breakthroughs in the treatment of infectious diseases and tumors as novel pharmaceutical technologies. However, the key challenges in developing nucleic acid drugs and vaccines remain the safe and efficient delivery of nucleic acid molecules to specific target cells while protecting them from degradation.

[0003] Ideal delivery vehicles must be safe, stable, and organ-specific. Lipid nanoparticles (LNPs) are the most advanced nucleic acid carriers in clinical practice. As of June 2021, all COVID-19 mRNA vaccines approved for clinical use utilize LNP delivery systems. LNPs offer many advantages for mRNA delivery, including simple formulation, modularity, good biocompatibility, and a large mRNA payload capacity.

[0004] Unfortunately, there are ongoing reports that intramuscular injections of Pfizer / BioNTech and Moderna's COVID-19 mRNA vaccines may induce liver damage and immune hepatitis. Some people have also experienced liver dysfunction after receiving mRNA vaccines, and liver transplant patients have developed a strong immune response after vaccination. Existing LNP delivery systems can lead to severe liver accumulation, potentially causing liver damage or immune hepatitis. Therefore, most current LNP delivery systems are liver-targeted, and effective delivery to organs other than the liver (such as the lungs and kidneys) urgently needs to be addressed.

[0005] Considering that the lungs bear the brunt of COVID-19, a lung-targeted mRNA delivery system has been developed. This system delivers mRNA encoding the COVID-19 spike protein directly to the lungs, activating the body's production of corresponding antibodies to combat the viral attack. In addition to vaccines, broadly neutralizing antibodies against the coronavirus could provide effective emergency prevention or treatment in clinical practice. However, the high cost of developing and producing protein-based antibodies, and non-targeted delivery of these antibodies results in large antigen dosages, further increasing medical costs and limiting their widespread application. A lung-targeted mRNA delivery system, which delivers mRNA encoding broadly neutralizing antibodies or therapeutic proteins directly to the lungs of COVID-19 patients for treatment, could effectively reduce costs and improve prevention and treatment efficacy. This would be a powerful supplement to the current COVID-19 prevention and treatment options. Furthermore, the lung-targeted mRNA delivery system could be widely applied to prevent or treat various lung diseases, such as lung cancer, pneumonia, tuberculosis, chronic obstructive pulmonary disease, pulmonary thromboembolism, and pulmonary vasculitis. Therefore, the development of a lung-targeted mRNA delivery system is of vital importance both for the immediate COVID-19 response and for the long-term development of public health systems, safeguarding the well-being of the people and the normal operation of the economy and society.

[0006] The physicochemical properties and chemical composition of LNP delivery systems can alter their interactions with proteins in body fluids. Specifically, by manipulating the charge properties, particle size distribution, and the proportion of hydrophobic lipids in the LNP delivery system, the distribution of LNPs in the body can be altered, thereby achieving organ-targeted delivery. Summary of the Invention

[0007] The purpose of the present invention is to provide a lipid nanoparticle (LNP) that can achieve lung-targeted delivery. At the same time, the lung-targeted delivery system is applied to the prevention of novel coronavirus infection by a novel coronavirus broad-spectrum neutralizing antibody mRNA.

[0008] The lipid nanoparticles (LNPs) provided by the present invention can achieve lung-targeted delivery, and their components include: ionizable lipids, steroid lipids, lipid-polyethylene glycol (Lipid-PEG), auxiliary lipid molecules and targeting lipid molecules;

[0009] Wherein, the ionizable lipid molecule is selected from at least one of the following formulas (a) to (c):

[0010]

[0011] In the above formulas (a) to (c), L 1 、L 2 and L 3 They can be the same or different and are all hydrophobic segments.

[0012] Furthermore, the L 1 、L 2 and L3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 1-20 Fatty hydrocarbon groups, various steroid lipids, vitamin A, vitamin E or vitamin K and other hydrophobic compounds.

[0013] Preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 10-20 Hydrophobic compounds such as fatty hydrocarbon groups, cholesterol, vitamin A, vitamin E or vitamin K.

[0014] More preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 12-18 fatty hydrocarbon groups, cholesterol, vitamin A or vitamin E.

[0015] More preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, unsaturated C 12-18 fatty hydrocarbon groups, cholesterol or vitamin E.

[0016] Most preferably, L 1 、L 2 、L 3 Each independently selected from: L 1 、L 2 and L 3 Branched or unbranched, non-cyclic, unsaturated C 12-18 fatty hydrocarbon group or vitamin E.

[0017] The L 4 It is a methyl group or an ethyl group, and more preferably a methyl group.

[0018] The B 1 、B 2 and B 3 Can be the same or different; 1 、B 2 and B 3 They independently form any one of the linking groups, including carbonate group, carbamate group, ester group, ether bond, urea group / amide group or amino group, or a combination of any two of the above linking groups, with oxygen atoms or nitrogen atoms respectively.

[0019] Furthermore, the OB 1 -L 1The structure is as shown in any of the following general formulas:

[0020]

[0021] The OB 2 -L 2 The structure is as shown in any of the following general formulas:

[0022]

[0023] The NH-B 3 -L 3 The structure is as shown in any of the following general formulas:

[0024]

[0025] The NH-B 3 The structure of -N is as shown in any of the following general formulas:

[0026]

[0027] Wherein, n represents the number of methylene groups -CH2, and its value range is an integer within 0-20;

[0028] Preferably, n=an integer within the range of 1-10; more preferably, n=an integer within the range of 1-5.

[0029] Specifically, the ionizable lipid molecule is selected from at least one of the following formulas (i) to (v):

[0030]

[0031] The targeting lipid molecule is obtained by converting the above-mentioned ionizable lipid molecule into a quaternary ammonium salt (lipid quaternary ammonium salt molecule) to obtain a lipid molecule capable of achieving lung-targeted delivery.

[0032] The targeting lipid molecule is selected from at least one of the following formulas (A) to (C):

[0033]

[0034] In the above formulas (A) to (C), L 1 、L 2 and L 3 They can be the same or different and are all hydrophobic segments.

[0035] Furthermore, the L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 1-20Fatty hydrocarbon groups, various steroid lipids, vitamin A, vitamin E or vitamin K and other hydrophobic compounds.

[0036] Preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 10-20 Hydrophobic compounds such as fatty hydrocarbon groups, cholesterol, vitamin A, vitamin E or vitamin K.

[0037] More preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, saturated or unsaturated C 12-18 fatty hydrocarbon groups, cholesterol, vitamin A or vitamin E.

[0038] More preferably, L 1 、L 2 and L 3 Each independently selected from: branched or unbranched, cyclic or acyclic, unsaturated C 12-18 fatty hydrocarbon groups, cholesterol or vitamin E.

[0039] Most preferably, L 1 、L 2 and L 3 Each independently selected from: L 1 、L 2 and L 3 Branched or unbranched, non-cyclic, unsaturated C 12-18 fatty hydrocarbon group or vitamin E.

[0040] The L 4 It is a methyl group or an ethyl group, and more preferably a methyl group.

[0041] The B 1 、B 2 and B 3 Can be the same or different; 1 、B 2 and B 3 They independently form any one of the linking groups, including carbonate group, carbamate group, ester group, ether bond, urea group / amide group or amino group, or a combination of any two of the above linking groups, with oxygen atoms or nitrogen atoms respectively.

[0042] Furthermore, the OB 1 -L 1 The structure is as shown in any of the following general formulas:

[0043]

[0044] The OB 2 -L 2 The structure is as shown in any of the following general formulas:

[0045]

[0046] The NH-B 3 -L 3 The structure is as shown in any of the following general formulas:

[0047]

[0048] The NH-B 3 The structure of -N is as shown in any of the following general formulas:

[0049]

[0050] Wherein n represents the number of methylene groups -CH2, and its value range is an integer within 0-20.

[0051] For NH-B 3 -N structure, wherein 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.

[0052] The R 1 It is methyl, ethyl or benzyl, preferably methyl.

[0053] The X - Selected from various anions, X - Selected from trifluoromethanesulfonic acid anion, halogen anion (including fluoride ion, chloride ion, bromide ion and iodide ion).

[0054] For anion X - , which may be a triflate anion, a fluoride anion, a chloride anion, a bromide anion or an iodide anion.

[0055] Preferably, X - is a trifluoromethanesulfonic acid anion, a chloride ion, a bromide ion or an iodide ion.

[0056] More preferably, X - is a trifluoromethanesulfonic acid anion, a bromide ion or an iodide ion.

[0057] Most preferably, X - It is a trifluoromethanesulfonic acid anion or an iodide ion.

[0058] Specifically, the targeting lipid molecule is selected from at least one of the following formulas (I) to (V):

[0059]

[0060] The present invention converts the tertiary amine of the ionizable lipid molecular skeleton into a quaternary amine by a quaternary ammonium salt reaction, so that the ionizable lipid molecules are converted from uncharged neutral molecules into molecules in the form of quaternary ammonium salts, and realize the lung delivery effect of LNP as a lung targeting component. The lung targeting component of the present invention can be regulated to a great extent by changing the type and quantity of the quaternary ammonium salt species and the hydrophobic segment to its charge size and hydrophobicity, which can fully meet the needs of targeted lung delivery of mRNA. After being added to the LNP system, this lipid quaternary ammonium salt molecule can be enriched in the lungs within a certain period of time after intravenous injection by adjusting the surface charge and particle size distribution of LNP, completing the lung targeted delivery of the mRNA carried, and realizing the transfection of lung cells. In particular, the lung targeted delivery system directly delivers the mRNA encoding broad-spectrum neutralizing antibodies or therapeutic proteins to the lungs of new crown patients for treatment, which can effectively reduce costs and improve prevention and treatment effects.

[0061] In a second aspect, the present invention provides a method for preparing the lipid molecules represented by the above formulae (i) to (v) and (I) to (V).

[0062] The lipid molecules represented by formula (i) to formula (v) and formula (I) to formula (V) provided by the present invention all have a backbone of N-(3-aminopropyl)diethanolamine.

[0063] For the molecules represented by formula (i) and formula (v), when B 1 and / or B 2 The preparation method for forming an ester bond with an oxygen atom is exemplified, comprising the following steps:

[0064] (A1): First, a fatty acid and thionyl chloride are reacted 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 is reacted with L in formula I 1 , L 2 corresponding;

[0065]

[0066] (A2) N-(3-aminopropyl)diethanolamine is reacted with di-tert-butyl carbonic anhydride (Boc2) in ethanol to protect the amino group with Boc to obtain Boc-N-(3-aminopropyl)diethanolamine carbonate.

[0067]

[0068] (A3): The fatty acid chloride obtained in step (A1) is reacted with Boc-N-(3-aminopropyl)diethanolamine carbonate in solvent 2 at a certain temperature and for a certain time under the action of base 2 to obtain lipid molecules with different hydrophobic segments.

[0069] By controlling the feed ratio and reaction sequence, lipid molecules with different substituents can be obtained. 1 Substituted Boc-N-(3-aminopropyl)diethanolamine carbonate derivatives, and then gradually obtain L 1 , L 2 Disubstituted Boc-N-(3-aminopropyl)diethanolamine carbonate lipid molecules.

[0070]

[0071] The lipid molecule shown in formula (I) provided by the present invention, when B 1 and / or B 2 The preparation method for forming an ether bond with an oxygen atom comprises the following steps (the following reaction equation is only for illustration and is not limited to the following structure. Lipid molecules with different types and numbers of substituents can be obtained by adjusting the feed ratio and sequence):

[0072] (A4): Triethanolamine or 2-hydroxymethyl-1,3-propanediol is reacted in solvent 1 at a certain temperature for a certain time under the action of sodium hydride, and then the corresponding halogenated aliphatic hydrocarbon (XL 1 , X represents a halogen, such as a bromohydrocarbon) and react for a certain period of time to obtain a lipid molecule with an ether bond as a connecting group.

[0073] This method is a method for synthesizing ether using sodium hydride, wherein XL 1 It is a halogenated hydrocarbon. The same type of reaction will not be listed again;

[0074]

[0075] When B 1 and / or B 2 The preparation method for forming a carbonate or carbamate with an oxygen atom comprises the following steps (the following reaction equation is only for illustration and is not limited to the following structure. Lipid molecules with different types and numbers of substituents can be obtained by adjusting the feed ratio and sequence):

[0076] (A5): The skeleton Boc-N-(3-aminopropyl)diethanolamine carbonate is mixed with an activating reagent N,N'-carbonyldiimidazole or p-nitrophenyl chloroformate and reacted in solvent 3 for a period of time to obtain a Boc-N-(3-aminopropyl)diethanolamine carbonate derivative with an activated hydroxyl group;

[0077]

[0078] (A6): The hydroxyl-activated Boc-N-(3-aminopropyl)diethanolamine carbonate derivative obtained in step (A5) is reacted with an amino-containing aliphatic hydrocarbon (L 1 -NH2) for a certain period of time to obtain a lipid molecule with a corresponding carbamate connecting group;

[0079]

[0080] Alternatively, the hydroxyl-activated Boc-N-(3-aminopropyl)diethanolamine carbonate derivative obtained in step (A4) is reacted with a hydroxyl-containing aliphatic hydrocarbon (L 1 -OH) is heated in an alkaline environment for a certain period of time to obtain a lipid molecule whose corresponding linking group is a carbonate.

[0081]

[0082] (A7) The resulting Boc-N-(3-aminopropyl)diethanolamine carbonate lipid derivative is reacted with trifluoroacetic acid (TFA) to remove the Boc protecting group (see the reaction formula below). The N-(3-aminopropyl)diethanolamine lipid molecule obtained in this step is an ionizable lipid molecule and can be subsequently modified.

[0083]

[0084] (A8) The N-(3-aminopropyl)diethanolamine lipid molecule obtained in step (A7) is reacted with an amine compound in solvent 4 for a period of time under the action of catalyst 1 or an activating reagent (the activating reagent refers to step (A5), N,N'-carbonyldiimidazole or p-nitrophenyl chloroformate) and a base 3 to convert the amine group into a structure containing a tertiary amine group, and the resulting lipid molecule is used as an ionizable lipid molecule.

[0085]

[0086] (A9) After obtaining the N-(3-aminopropyl)diethanolamine lipid molecule having a tertiary amine structure through steps (A1) to (A8), the obtained tertiary amine molecule and the halogenated hydrocarbon are dissolved in solvent 3, reacted at a certain temperature for a certain time, and after the reaction, the solvent and the halogenated hydrocarbon are removed by rotary evaporation to obtain a quaternary ammonium salt lipid molecule, which can be used as a lung targeting component.

[0087]

[0088] 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 reactivity and reaction process are almost unaffected by the aliphatic hydrocarbon group, and the reaction can be carried out efficiently;

[0089] Step (A3) is the reaction of the fatty acid chloride obtained in step (A1) with Boc-N-(3-aminopropyl)diethanolamine carbonate. This 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 lipid-modified Boc-N-(3-aminopropyl)diethanolamine carbonate can be efficiently obtained.

[0090] Step (A9) 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.

[0091] 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.

[0092] 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;

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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, it is selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine or isopropylamine; more preferably, it is selected from pyridine, N,N-dimethylformamide, triethylamine or isopropylamine.

[0097] In one embodiment, the reaction temperature of step (A1) is 25-120°C; preferably, 25-80°C; more preferably, 25-60°C.

[0098] In one embodiment, the reaction time of step (A1) is 1-24 h; preferably, 2-12 h; more preferably, 4-6 h.

[0099] 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.

[0100] 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, it is selected from pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine, triethylamine, diethylamine or isopropylamine; more preferably, it is selected from pyridine, N,N-dimethylformamide, triethylamine or isopropylamine.

[0101] In one embodiment, the reaction temperature in step (A2) is 0-100°C; preferably, 0-25°C.

[0102] In one embodiment, the reaction time of step (A2) is 1-48 h; preferably, 6-24 h; more preferably, 6-12 h.

[0103] 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.

[0104] In one embodiment, in step (A8), the 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;

[0105] 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;

[0106] 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.

[0107] In one embodiment, the reaction temperature in step (A8) is 0-100°C; preferably, 25-80°C; more preferably, 25-60°C.

[0108] In one embodiment, the reaction time in step (A8) is 1-24 h; preferably, 6-24 h; more preferably, 6-12 h.

[0109] In one embodiment, the reaction temperature in step (A9) is 0-100°C; preferably, 25-80°C; more preferably, 50-80°C.

[0110] In one embodiment, the reaction time of step (A9) is 1-24 h; preferably, 6-24 h; more preferably, 12-24 h.

[0111] Furthermore, the lipid nanoparticles (LNPs) provided by the present invention that can achieve lung-targeted delivery include, in terms of mass percentage, 10%-70% of ionizable lipid molecules represented by the above-mentioned formulas (a) to (c), 5%-60% of steroidal lipids, 1%-20% of lipid-polyethylene glycol, 1%-30% of auxiliary lipid molecules, and 5%-60% of lung-targeted lipid molecules represented by the above-mentioned formulas (A) to (C).

[0112] Preferably, the mass percentage of the ionizable lipid molecules is 20%-40%; most preferably, the mass percentage of the ionizable lipid molecules is 30%.

[0113] Preferably, the mass percentage of the steroidal lipids is 10%-40%; most preferably, the mass percentage of the steroidal lipids is 15%.

[0114] Preferably, the mass percentage of the lipid-polyethylene glycol is 1%-10%; most preferably, the mass percentage of the lipid-polyethylene glycol is 5%.

[0115] Preferably, the mass percentage of the helper lipid molecules is 5%-15%; most preferably, the mass percentage of the helper lipid molecules is 8%.

[0116] Preferably, the mass percentage of the lung-targeting lipid molecules is 10%-50%; most preferably, the mass percentage of the lung-targeting lipid molecules is 42%.

[0117] The present invention also provides a lung-targeted lipid nanoparticle (LNP) encapsulating a nucleic acid drug.

[0118] The lung-targeted lipid nanoparticles (LNPs) encapsulating nucleic acid drugs include the above-mentioned lipid nanoparticles capable of achieving lung-targeted delivery and nucleic acid drugs of the present invention.

[0119] The nucleic acid drugs include but are not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA, etc.

[0120] In a specific embodiment of the present invention, the nucleic acid drug may be mRNA, including mRNAs of different sequences and lengths, specifically Firefly Luciferase mRNA or mRNA encoding a broad-spectrum neutralizing antibody (such as broad-spectrum neutralizing antibody 8-9D mRNA).

[0121] The present invention also provides a method for preparing the lung-targeted lipid nanoparticles (LNPs) containing the nucleic acid drug.

[0122] The method for preparing lung-targeted lipid nanoparticles containing nucleic acid drugs provided by the present invention specifically comprises the following steps:

[0123] (B1) mixing the ionizable lipid molecules, the helper lipid molecules, the lipid-polyethylene glycol, the steroid lipids, and the lung-targeting lipid molecules in appropriate proportions, and dissolving them in a solvent to obtain an organic phase liposome solution;

[0124] (B2) dissolving the nucleic acid drug in a buffer solution of appropriate pH to obtain an aqueous nucleic acid drug solution;

[0125] (B3) The organic phase liposome solution and the aqueous phase nucleic acid drug solution are uniformly mixed using a microfluidic device at a certain mass ratio and volume ratio to prepare lung-targeted lipid nanoparticles encapsulating the nucleic acid drug.

[0126] The method further comprises: ultrafiltration or dialysis of the lung-targeted lipid nanoparticles containing nucleic acid drugs obtained in step (B3) to obtain lung-targeted lipid nanoparticles containing nucleic acid drugs that can be used in biological experiments.

[0127] Preferably, the solvent used to dissolve the lipid molecules in step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide. More preferably, the solvent used to dissolve the lipid molecules in step (B1) is ethanol, tetrahydrofuran, or acetone. Most preferably, the solvent used to dissolve the lipid molecules in step (B1) is ethanol.

[0128] Preferably, the buffer solution in step (B2) is acetic acid / sodium acetate solution or citric acid / sodium citrate solution; most preferably, the buffer solution in step (B2) is citric acid / sodium citrate solution.

[0129] Preferably, the pH of the buffer solution in step (B2) is 3-9; more preferably, the pH of the buffer solution in step (B2) is 4-6; most preferably, the pH of the buffer solution in step (B2) is 5.

[0130] Preferably, the concentration of the buffer solution in step (B2) is 1 mM-1 M; more preferably, the concentration of the buffer solution in step (B2) is 20 mM-500 mM; most preferably, the concentration of the buffer solution in step (B2) is 100 mM.

[0131] Preferably, the mass ratio of all lipid molecules to nucleic acid drugs in step (B3) is 5:1-50:1; more preferably, the mass ratio of lipid molecules to nucleic acid drugs in step (B3) is 10:1-30:1.

[0132] The nucleic acid drug is mRNA. Preferably, the mass ratio of the lipid molecule to the mRNA is 25:1.

[0133] Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution in 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 (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 (B3) or step (B3) is 1:3.

[0134] In the present invention, the steroidal lipids are 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, fuccasterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid.

[0135] In the present invention, the lipid-polyethylene glycol is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0136] 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), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0137] The present invention also claims protection for the use of the above-mentioned ionizable lipid molecules, lipid quaternary ammonium salt molecules (lung-targeted lipid molecules) and lipid nanoparticles capable of lung-targeted delivery in the preparation of a drug delivery system.

[0138] The drug is a nucleic acid drug, including but not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, circular RNA, etc.

[0139] In a specific embodiment of the present invention, the nucleic acid drug may be mRNA, including mRNAs of different sequences and lengths, and specifically may be mRNA encoding broad-spectrum neutralizing antibodies.

[0140] The present invention claims protection for the above-mentioned ionizable lipid molecules, lipid quaternary ammonium salt molecules (lung-targeted lipid molecules) and lipid nanoparticles capable of lung-targeted delivery for RNA delivery and their use.

[0141] 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.

[0142] Compared with the prior art, the present invention has the following beneficial effects:

[0143] 1. It binds tightly to mRNA, enabling high encapsulation rate and stable protection of mRNA.

[0144] 2. The formed LNP has good biocompatibility and is more stable.

[0145] 3. After intravenous injection, it can be specifically enriched in the lungs without being distributed in other organs.

[0146] 4. This 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.

[0147] 5. The application of this lung-targeted LNP system to broad-spectrum neutralizing antibodies against the new coronavirus can effectively improve the ability to prevent infection and reduce the respiratory viral load and lung pathological damage after exposure to the new coronavirus.

[0148] 6. 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

[0149] Figure 1 The structural formula of Boc-N-(3-aminopropyl)diethanolamine carbonate (Boc-N-2OH) prepared in Example 1 is shown.

[0150] Figure 2 The Boc-N-(3-aminopropyl)diethanolamine carbonate (Boc-N-2OH) prepared in Example 1 is shown. 1 HNMR spectrum.

[0151] Figure 3 The structural formula of the ionizable lipid molecule (i) prepared in Example 2 is shown.

[0152] Figure 4 The ionizable lipid molecules (i) prepared in Example 2 are shown. 1 H NMR spectrum.

[0153] Figure 5 The structural formula of the ionizable lipid molecule (Boc-ii) prepared in Example 3 is shown.

[0154] Figure 6 The ionizable lipid molecule (Boc-ii) prepared in Example 3 is shown. 1 H NMR spectrum.

[0155] Figure 7 The particle size distribution of I-LNP@mRNA (a) and V-LNP@mRNA (b) prepared in Example 7 is shown.

[0156] Figure 8 TEM images of I-LNP@mRNA (a) and V-LNP@mRNA (b) prepared in Example 7 are shown.

[0157] Figure 9 The in vivo imaging effects of the lung-targeted LNP@mRNA and basic (liver-targeted) LNP@mRNA prepared in Example 7 are shown in mice.

[0158] Figure 10 The cytotoxicity experiments of I-LNP@mRNA (a) and V-LNP@mRNA (b) prepared in Example 7 are shown.

[0159] Figure 11 The blood biochemical indicators of mice treated with I-LNP@mRNA and V-LNP@mRNA prepared in Example 7 are shown.

[0160] Figure 12 The H&E staining results of the main organs of mice after treatment with I-LNP@mRNA and V-LNP@mRNA prepared in Example 7 are shown, where 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 after injection of I-LNP@mRNA, and c is the H&E staining result of the main organs of mice after injection of V-LNP@mRNA.

[0161] Figure 13 The distribution of the liver-targeted-8-9D and lung-targeted-8-9D antibodies prepared in Example 11 in mice after injection is shown, and the antibody concentrations in serum, bronchoalveolar lavage fluid, liver lysate, and lung lysate were detected respectively.

[0162] Figure 14 The results of viral load tests of the lungs and trachea after challenge with the novel coronavirus after injection of the liver-targeted-8-9D and lung-targeted-8-9D prepared in Example 11 are shown. a is the viral load result after challenge with the South African strain of the novel coronavirus (Beta strain), and b is the viral load result after challenge with the Omicron BA2 strain of the novel coronavirus.

[0163] Figure 15 The results of lung pathological damage after the injection of liver-targeted-8-9D and lung-targeted-8-9D prepared in Example 11 are shown. Among them, a is the lung pathological damage and pathological score results after the challenge with the South African strain of the new coronavirus (Beta strain), and b is the lung pathological damage and pathological score results after the challenge with the Omicron BA2 strain of the new coronavirus. DETAILED DESCRIPTION

[0164] I. Definition

[0165] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0166] Unless expressly 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 other components.

[0167] 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 forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present disclosure.

[0168] In this disclosure, as well as It refers to the position where a substituent is bonded.

[0169] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring.

[0170] As used herein, numerical ranges refer to the individual integers within the given range. For example, "C1-C6" 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; "C3-C6" means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0171] 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, or a nitrogen-containing amino group.

[0172] 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 compound after substitution 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 practicability.

[0173] When any variable (such as 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, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0174] The term "aliphatic hydrocarbon group" includes saturated or unsaturated, linear or branched chain or cyclic hydrocarbon groups, and aliphatic hydrocarbon groups containing or not containing heteroatoms; the heteroatoms refer to nitrogen atoms, oxygen atoms, fluorine atoms, phosphorus atoms, sulfur atoms, and selenium atoms. The type of the aliphatic hydrocarbon group can be selected from alkyl groups, alkenyl groups, alkynyl groups, etc. For example, the term "C 1-6 The term "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.

[0175] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, having the indicated number of carbon atoms. 1-6 "Alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 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.

[0176] The term "substituted" or "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., =O), 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 practicability. The substituents 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 are not limited thereto.

[0177] The term "pharmaceutical composition" means a composition comprising a compound of 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.

[0178] The medicine or pharmaceutical composition of the present disclosure can be delivered parenterally, that is, administered intravenously (iv), intracerebroventricularly (icv), subcutaneously (sc), intraperitoneally (ip), intramuscularly (im), subcutaneously (sd) or intradermally (id), by direct injection, for example, through rapid bolus injection or continuous infusion. The formulation for injection can be presented in unit dosage form, for example, in an ampoule or multi-dose container with an added preservative. The composition can be in the form of an excipient, a suspension, a solution or an 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 with a suitable carrier (e.g., sterile pyrogen-free water) before use in powder form.

[0179] The term "inflammatory disease" includes 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 particularly selected from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, berylliosis. The respiratory disorder may be selected from among 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.

[0180] The term "viral infection" includes but is not limited to retroviral infection, hepatitis virus infection, new coronavirus infection, Zika virus infection, dengue virus infection, etc.

[0181] II. Specific Examples

[0182] The present invention is described in detail below through examples. These 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 those skilled in the art based on the contents of the above invention are all within the scope of protection of the present invention.

[0183] Example 1: Preparation of Boc-N-(3-aminopropyl)diethanolamine carbonate

[0184] Dissolve N-(3-aminopropyl)diethanolamine and triethylamine in ethanol, add Boc2 ethanol solution dropwise at low temperature, react at room temperature for six hours after the addition is complete, and remove the solvent and Boc2 by rotary evaporation to obtain Boc-N-(3-aminopropyl)diethanolamine carbonate. The structural formula of the obtained product is as follows: Figure 1 As shown, the hydrogen spectrum is Figure 2 Its ESI-TOF MS [M+H] + The theoretical value is m / z = 263.1965, and the measured value is m / z = 263.1908.

[0185] Example 2: Preparation of ionizable lipid molecules (i)

[0186] Tetradecanoic acid was dissolved in toluene and reacted with thionyl chloride at 60°C for 6 hours. The solvent and thionyl chloride were then removed in vacuo to yield oleic acid chloride. Oleic acid chloride and Boc-N-(3-aminopropyl)diethanolamine carbonate were dissolved in dichloromethane, triethylamine was added, and the reaction was allowed to proceed overnight. The resulting reaction solution was washed, dried, and concentrated, and then purified by column chromatography. By controlling the molar ratio of oleic acid chloride to triethanolamine to 2:1, ditetradecanoic acid-substituted Boc-N-(3-aminopropyl)diethanolamine carbonate was obtained.

[0187] The obtained ditetradecanoic acid-substituted Boc-N-(3-aminopropyl)diethanolamine carbonate was then reacted with trifluoroacetic acid in dichloromethane for 4 hours to remove the Boc protecting group. After the reaction, the ionizable lipid molecule (i) was obtained after washing, drying and concentration. The structural formula of the obtained product is as follows: Figure 3 As shown, the hydrogen spectrum is Figure 4 Its ESI-TOF MS [M+H] + The theoretical value is m / z = 555.5095, and the measured value is m / z = 555.5108.

[0188] Example 3: Preparation of ionizable lipid molecules (ii)

[0189] Dissolve oleic acid in toluene and react with thionyl chloride at 60°C for 6 hours, then remove the solvent and thionyl chloride in vacuo to obtain oleic acid chloride. Dissolve oleic acid chloride and Boc-N-(3-aminopropyl)diethanolamine carbonate in dichloromethane, add triethylamine, and react overnight. The resulting reaction solution is washed, dried, and concentrated, and then separated and purified by a chromatographic column. By controlling the molar ratio of oleic acid chloride to triethanolamine to 2:1, dioleic acid-substituted Boc-N-(3-aminopropyl)diethanolamine carbonate is obtained. The structural formula of the obtained product is as follows: Figure 5 As shown in the figure, the hydrogen spectrum related characterization includes 1 H NMR ( Figure 6 ).

[0190] The obtained dioleic acid-substituted Boc-N-(3-aminopropyl)diethanolamine carbonate was then reacted with trifluoroacetic acid in dichloromethane for 4 hours to remove the Boc protecting group. After the reaction, the ionizable lipid molecule (ii) was obtained after washing, drying and concentration.

[0191] The structure was confirmed as follows: 1H NMR(500MHz,Chloroform-d)δ5.33(ddd,J=4.6,3.1,1.3Hz,2H),4.21(t,J=6.1Hz,2H),2.82(t,J=6.0Hz,2H),2.79–2.72(m,1H),2.56(t, J=5.8Hz,1H),2.31(t,J=8.5Hz,2H),2.06–1.96(m,4H),1.67–1.55(m,3H),1.53(t,J=6.5Hz,1H),1.36–1.23(m,20H),0.94–0.85(m,3H). [M+H] of its ESI-TOF MS + The theoretical value is m / z = 691.6347, and the measured value is m / z = 691.6335.

[0192] Example 4: Preparation of lung-targeted lipid molecules (I)

[0193] The ionizable lipid molecule (i) was dissolved in acetonitrile, and iodomethane was added, and the mixture was reacted at 70 degrees for 24 hours. After the reaction was completed, the solvent and iodomethane were removed by rotary evaporation to obtain the lung-targeting lipid molecule (I).

[0194]

[0195] The structure was confirmed as follows: 1 H NMR (500 MHz, Chloroform-d) δ 4.40 (t, J = 4.7 Hz, 2H), 3.87–3.78 (m, 3H), 3.61 (t, J = 9.5 Hz, 1H), 3.27 (d, J = 8.4 Hz, 5H), 2.35–2.24 (m, 3H), 1.60 (ddd, J = 16.0, 8.6, 7.4 Hz, 2H), 1.36–1.25 (m, 17H), 0.92–0.85 (m, 3H). Its ESI-TOF MS [M+H] + The theoretical value is m / z = 748.7046, and the measured value is m / z = 748.7040.

[0196] Example 5: Preparation of ionizable lipid molecules (v)

[0197] Ionizable lipid molecule (i) is dissolved in dichloromethane and CDI is added to react at room temperature for 24 hours. The resulting reaction solution is washed and dried, and N,N-dimethylethylenediamine is added to react for 24 hours. The resulting reaction solution is washed, dried, concentrated, and then separated and purified using a chromatographic column to obtain ionizable lipid molecule (v).

[0198]

[0199] The structure was confirmed as follows:1 H NMR(500MHz,Chloroform-d)δ4.20(t,J=6.0Hz,4H),3.26(td,J=6.2,4.4Hz,2H),3.17(td,J=6.2,5.1Hz,2H),2.82(t,J=6.0Hz,4H),2.57(dt,J=6. 8,6.1Hz,4H),2.31(t,J=8.5Hz,4H),2.27(s,4H),1.69(p,J=6.0Hz,2H),1 .60(ddd,J=16.0,8.6,7.4Hz,4H),1.36–1.25(m,34H),0.94–0.85(m,6H). [M+H] of its ESI-TOF MS + The theoretical value is m / z = 669.5888, and the measured value is m / z = 669.5870.

[0200] Example 6: Preparation of lung-targeted lipid molecules (V)

[0201] The ionizable lipid molecule (V) was dissolved in acetonitrile, and methyl iodide was added, and the reaction was carried out at 70 degrees Celsius for 24 hours. After the reaction was completed, the solvent and methyl iodide were removed by rotary evaporation to obtain the lung-targeting lipid molecule (V).

[0202]

[0203] The structure was confirmed as follows: 1 H NMR(500MHz,Chloroform-d)δ4.40(t,J=4.7Hz,1H),3.83(t,J=4.6Hz,1H),3.62–3.52(m,1H),3.28(s,0H),3.24(s,1H),3.21(td,J= 6.7, 4.2Hz, 0H), 2.31 (t, J = 8.5Hz, 1H), 1.60 (ddd, J = 16.0, 8.6, 7.4Hz, 1H), 1.36–1.27 (m, 1H), 1.31–1.25 (m, 6H), 0.92–0.86 (m, 1H). [M+H] of its ESI-TOF MS + The theoretical value is m / z = 698.6274, and the measured value is m / z = 698.6265.

[0204] Example 7: Preparation of Lung-Targeted LNP@mRNA Based on Ionizable Lipid Molecule (i) and Lung-Targeted Lipid Molecule (I), and Ionizable Lipid Molecule (v) and Lung-Targeted Lipid Molecule (V)

[0205] Ionizable lipid molecule (i), DSPE-PEG2000, cholesterol, DOPE, and lung-targeted lipid molecule (I) (or ionizable lipid molecule (v), DSPE-PEG2000, cholesterol, DOPE, and lung-targeted lipid molecule (V)) were mixed in a mass ratio of 30%:5%:15%:8%:42% and dissolved in an ethanol solution. Firefly luciferase mRNA was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The organic phase solution and the aqueous phase solution were mixed in a volume ratio of 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. Lung-targeted LNP@mRNA encapsulated with mRNA was obtained.

[0206] Separately, ionizable lipid molecules (i) and (v) were used to prepare a base (liver-targeted) LNP@mRNA. The mass ratio of ionizable lipid molecule (i) or ionizable lipid (v), DSPE-PEG2000, cholesterol, and DOPE was 52%:8.6%:25.8%:13.6%. The remaining preparation methods were identical to those for the lung-targeted LNP@mRNA.

[0207] 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 that ( Figure 7 ), the hydrated particle size of the lung-targeted I-LNP@mRNA prepared based on (i) and (I) was 78.6 nm, and the hydrated particle size of the lung-targeted V-LNP@mRNA prepared based on (i) and (I) was 80.3 nm. TEM results showed that ( Figure 8 ), I-LNP@mRNA (a) and V-LNP@mRNA (b) are both spherical with a particle size of 60-100 nm.

[0208] Example 8: Organ targeting experiment of LNP@mRNA

[0209] The two lung-targeted I-LNP@mRNA and V-LNP@mRNA obtained in Example 7 were administered to C57BL / 6G mice via the tail vein at a dose of 5 μg mRNA per mouse. In addition, liver-targeted i-LNP@mRNA and v-LNP@mRNA were injected into mice as controls. Six hours later, the mice were intraperitoneally injected with luciferin substrate, and bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system. The results are shown in Figure 2. Figure 9The results showed that both the lung-targeted delivery systems I-LNP@mRNA and V-LNP@mRNA achieved targeted delivery, accumulation, and expression in the lungs, with excellent expression. No accumulation or expression was observed in other organs, including the liver, kidneys, heart, spleen, intestines, stomach, muscle, and bone, demonstrating a significant lung-targeting effect. In contrast, liver-targeted LNP@mRNA, which does not contain a lung-targeting component, was enriched in the liver.

[0210] Example 9: Cytotoxicity experiments of lung-targeted I-LNP@mRNA and V-LNP@mRNA

[0211] 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% CO2. Fresh culture medium was replaced after 24 hours of cell incubation. Different concentrations of lung-targeted I-LNP@mRNA and V-LNP@mRNA (0-200 μg / mL, prepared in Example 7) were added, and the cells were incubated for 24 hours, and then the original culture medium was replaced with 0.5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) culture medium. After further incubation for 4 hours, the culture 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 BioTek Synergy H4 reader. The cell viability for 24 hours was calculated to verify the cytotoxicity of the delivery system obtained by the present invention.

[0212] Depend on Figure 10 It can be seen that the cell survival rate after incubation for 24 hours was above 90%, indicating that the lung-targeted nucleic acid delivery system of the present invention has low cytotoxicity and exhibits good biosafety performance.

[0213] Example 10: Biosafety Analysis of LNP@mRNA

[0214] BALB / c mice (4-6 weeks old, female, weighing approximately 18-20 g) were divided into three groups, each containing five mice. PBS, I-LNP@mRNA, and V-LNP@mRNA (10 μg mRNA) were injected via the tail vein. Five days after injection, venous blood was collected from the mice, the supernatant was centrifuged, and various liver and kidney function indicators were measured. H&E staining was performed on the hearts, livers, spleens, lungs, and kidneys of the PBS and LNP@mRNA groups five days after injection to observe the presence of pathological changes.

[0215] The results of liver and kidney function test showed that ( Figure 11), the liver and kidney functions of mice in the I-LNP@mRNA and V-LNP@mRNA groups did not show significant changes, and their indicators were comparable to those of PBS. The results of tissue section experiments showed that ( Figure 12 ), no obvious lesions were found in the major organs of mice treated with I-LNP@mRNA or V-LNP@mRNA, indicating that LNP@mRNA nanoparticles have good biosafety.

[0216] Example 11: Application of the targeted LNP system in the delivery of broadly neutralizing antibody 8-9D mRNA

[0217] Based on the above results, lung-targeted and liver-targeted delivery systems were constructed for the delivery of broadly neutralizing antibody 8-9D mRNA. Among them, liver-targeted LNPs were constructed using ionizable lipid (i), and lung-targeted LNPs were constructed using ionizable lipid (i) and lung-targeted lipid (I).

[0218] Liver-targeted LNP (liver-targeted-8-9D) was constructed using ionizable lipid (i) and prepared according to the ratio of basic LNP@mRNA in Example 8. Meanwhile, the Firefly luciferase mRNA in I-LNP@mRNA was replaced with 8-9D mRNA;

[0219] Lung-targeted LNP (lung-targeted-8-9D) was constructed using ionizable lipids (i) and lung-targeted lipids (I).

[0220] Lung-targeted LNP@8-9D mRNA was prepared according to the method of Example 7, except that the Firefly luciferase mRNA in I-LNP@mRNA was replaced with 8-9D mRNA;

[0221] The 8-9D mRNA is encoded by the following sequence A:

[0222] The nucleotide sequence of Sequence A consists of the nucleic acid encoding the light chain of the 8-9D antibody (Sequence 2, L chain nucleic acid sequence) and the nucleic acid encoding the heavy chain of the 8-9D antibody (Sequence 1, H chain nucleic acid sequence);

[0223] C57BL / 6 mice were divided into three groups, with 5 mice in each group. The mice (4-6 weeks old, female, weighing about 18-20g) were injected with PBS, liver-targeted 8-9D, and lung-targeted 8-9D (5μg mRNA) through the fundus vein. Venous blood, bronchoalveolar lavage fluid, lungs, and livers were collected from the mice 24 hours after injection. The concentrations of 8-9D antibodies in serum, bronchoalveolar lavage fluid, lung lysates, and liver lysates were measured by ELISA ( Figure 13The results showed that in the lung-targeted-8-9D group, antibody levels were significantly elevated in bronchoalveolar lavage fluid and lung tissue, while antibody levels in serum and liver were low and not significantly different from those in the PBS group. In the liver-targeted-8-9D group, 8-9D antibody levels were significantly elevated in serum and liver lysate, demonstrating the targeting properties of both LNPs.

[0224] Five mice (4-6 weeks old, female, weighing about 18-20 g) were injected intravenously with PBS, liver-targeted-8-9D, and lung-targeted-8-9D (5 μg mRNA) in each group of K18-hACE2. Twenty-four hours after injection, the mice were challenged with the South African strain of SARS-CoV-2 (Beta strain) and Omicron BA2 strain (2×10 4 TCID 50 ), 4 days after infection, the mice were killed, and the lungs and trachea were taken to detect the subgenomic viral load of the new coronavirus using the qRT-PCR method ( Figure 14 ), lung tissue was fixed and stained with HE, pathological changes were analyzed, and pathological damage was scored ( Figure 15 The results showed that in Beta strain-challenged mice, the viral load in both the liver-targeted-8-9D and lung-targeted-8-9D groups decreased significantly compared with the control group, and the viral load in the lung-targeted-8-9D group was close to the detection limit, indicating a better immune protection effect ( Figure 14 a) In terms of pathological damage, the control group showed severe bleeding, inflammatory cell infiltration and alveolar tissue structure destruction, while the pathological damage of the mice in the liver-targeted-8-9D group was alleviated, and the mice in the lung-targeted-8-9D group did not show significant pathological damage in the lungs, which was consistent with the viral load results ( Figure 15 a). In Omicron BA2 strain-challenged mice, the lung-targeted-8-9D group achieved complete protection against COVID-19 infection, with no viral sgRNA detected. The liver-targeted-8-9D group showed a significant decrease in viral load, but did not achieve complete protection ( Figure 14 b), pathological results showed that the mice in the lung targeting-8-9D group had almost no pathological damage, effectively preventing the new coronavirus infection ( Figure 15 b).

[0225] The foregoing descriptions of specific exemplary embodiments of the present disclosure are for purposes of illustration and description. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is apparent that many modifications and variations are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to make and utilize the various exemplary embodiments and various options of the present disclosure.

Claims

1. A lipid molecule for lung-targeted delivery, selected from at least one of the following formulas (A) and (C): In the formula (A) or formula (C): L 1 and L 2 The same or different, each independently selected from: saturated or unsaturated C 10-20 aliphatic hydrocarbon groups; L 4 is methyl or ethyl; The R 1 is methyl or ethyl; The X - Halogen anions include fluoride, chloride, bromide and iodide; The OB 1 -L 1 The structure is shown in the following general formula: The OB 2 -L 2 The structure is shown in the following general formula: The NH-B 3 The structure of -N is shown in the following general formula: in, n represents the number of methylene groups -CH2, and its value range is an integer within 1-5.

2. The lipid molecule for lung targeted delivery according to claim 1, characterized in that: The targeting lipid molecule is selected from at least one of the following formulas (I) and (V): （I） 。 3. A lipid nanoparticle capable of lung-targeted delivery, comprising, by weight, 10%-70% ionizable lipid molecules, 5%-60% steroidal lipids, 1%-20% lipid-polyethylene glycol, 1%-30% helper lipid molecules, and 5%-60% of the lipid molecule for lung-targeted delivery according to claim 1 or 2; The ionizable lipid molecule is selected from at least one of the following formulas (i) to (v): 。 4. The lipid nanoparticle capable of lung-targeted delivery according to claim 3, characterized in that: 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, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid; The lipid-polyethylene glycol 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-phosphoethyl Alkylamine-N-[amino(polyethylene glycol)], PEG-disterylglycerol, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine, and PEG-1,2-dimyristoyloxypropyl-3-amine; 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.

5. The lipid nanoparticle capable of lung-targeted delivery according to claim 3, characterized in that: The mass percentage of the ionizable lipid molecules is 20%-40%; The mass percentage of the steroidal lipid is 10%-40%; The mass percentage of the lipid-polyethylene glycol is 1%-10%; The mass percentage of the auxiliary lipid molecules is 5%-15%; The mass percentage of the lipid molecules for lung targeted delivery is 10%-50%.

6. The lipid nanoparticle capable of lung-targeted delivery according to claim 5, characterized in that: The mass percentage of the ionizable lipid molecules is 30%; The mass percentage of the steroidal lipid is 15%; The mass percentage of the lipid-polyethylene glycol is 5%; The mass percentage of the auxiliary lipid molecules is 8%; The mass percentage of the lung-targeting lipid molecules is 42%.

7. A lung-targeted lipid nanoparticle encapsulating a nucleic acid drug, comprising the lipid nanoparticle capable of lung-targeted delivery according to any one of claims 3 to 6 and a nucleic acid drug; The nucleic acid drugs include but are not limited to DNA, mRNA, siRNA, microRNA, antisense nucleic acid, and circular RNA.

8. The method for preparing the lung-targeted lipid nanoparticles carrying nucleic acid drugs according to claim 7, comprising the following steps: (B1) mixing the ionizable lipid molecule, auxiliary lipid molecule, lipid-polyethylene glycol, steroid lipid and the lipid molecule for lung targeted delivery according to claim 1 or 2 in proportion, and dissolving them in a solvent to obtain 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 using a microfluidic device according to a certain mass ratio and volume ratio to prepare lung-targeted lipid nanoparticles encapsulating the nucleic acid drug.

9. The preparation method according to claim 8, characterized in that: The method further comprises: ultrafiltration or dialysis of the lung-targeted lipid nanoparticles containing nucleic acid drugs obtained in step (B3) to obtain lung-targeted lipid nanoparticles containing nucleic acid drugs that can be used in biological experiments.

10. The preparation method according to claim 8 or 9, characterized in that: The solvent used to dissolve the lipid molecules in step (B1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide; The buffer solution in step (B2) is acetic acid / sodium acetate solution or citric acid / sodium citrate solution; The pH of the buffer solution in step (B2) is 3-9; The concentration of the buffer solution in step (B2) is 1 mM-1 M; In step (B3), the mass ratio of all lipid molecules to nucleic acid drugs is 5:1-50:1; The nucleic acid drug is mRNA, and the mass ratio of the lipid molecule to the mRNA is 25:1; In the step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:1-1:

10.

11. The preparation method according to claim 10, characterized in that: The solvent used to dissolve the lipid molecules in step (B1) is ethanol, tetrahydrofuran, or acetone; The buffer solution in step (B2) is a citric acid / sodium citrate solution; The pH of the buffer solution in step (B2) is 4-6; The concentration of the buffer solution in step (B2) is 20 mM-500 mM; In step (B3), the mass ratio of lipid molecules to nucleic acid drugs is 10:1-30:1; In the step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:1-1:

5.

12. The preparation method according to claim 11, characterized in that: The solvent used to dissolve the lipid molecules in step (B1) is ethanol; The pH of the buffer solution in step (B2) is 5; The concentration of the buffer solution in step (B2) is 100 mM; In step (B3), the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution is 1:3.

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