Lipid nanoparticles for targeted high-efficiency delivery of nucleic acid to lung, inhaled formulation, and use

Abstract

The present invention relates to lipid nanoparticles for targeted high-efficiency delivery of nucleic acid to the lung, an inhaled formulation comprising the lipid nanoparticles, and use of the lipid nanoparticles and the inhaled formulation in the preparation of a pharmaceutical composition for targeted delivery of the nucleic acid to the lung. The inhaled formulation of the lipid nanoparticles for targeted high-efficiency delivery of the nucleic acid to the lung can withstand shearing damage generated during atomization and overcome multiple physiological barriers to deliver the lipid nanoparticles to the lung, and it has high transfection efficiency.

Description

Lipid nanoparticle for efficient delivery of nucleic acid to the lung, inhalation formulation and use thereof TECHNICAL FIELD The present application relates to the technical field of nucleic acid drugs, in particular to a lipid nanoparticle for efficient delivery of nucleic acid to the lung, an inhalation formulation and use thereof. BACKGROUND Inhalation formulation refers to a formulation for delivering drugs to the respiratory tract and / or lung through inhalation route to exert local or systemic effect. Traditional inhalation formulation is mainly small molecule drugs. With the development of biopharmaceutical and genetic engineering technologies in recent years, a large number of polypeptide, protein and nucleic acid macromolecular drugs have emerged, providing a broader application space for inhalation formulation. Nucleic acid is a kind of macromolecule with electronegativity, which is easy to be degraded, low in cell uptake and transfection efficiency, so it is necessary to develop safe and effective delivery carriers to protect nucleic acid drugs from degradation and promote cell uptake. At present, lipid nanoparticle (LNP) has become one of the mainstream delivery carriers for nucleic acid drugs due to its unique structure and physicochemical properties, which exhibit high delivery efficiency and good safety in vivo. At present, the LNP administration method in clinical practice usually adopts intramuscular injection. In addition to the majority of LNP distributed in the local injection site, part of LNP is also distributed in the liver. Due to the first-pass effect of the liver, this greatly limits the LNP to reach other organs. In contrast, the lung has a large surface area and can avoid the first-pass effect, greatly improving drug absorption. Therefore, lung delivery of nucleic acid drugs has become a key breakthrough direction for LNP. However, there are great challenges in using lung delivery of nucleic acid drugs. On the one hand, it is necessary to tolerate the shear damage generated during atomization (shear damage can destroy the nanostructure of lipid nanoparticles, leading to aggregation, sedimentation of nanoparticles and leakage of nucleic acid molecules carried); on the other hand, it is necessary to overcome multiple physiological barriers (such as mucus barrier, cilia clearance, macrophage phagocytosis) to deliver nucleic acid therapeutic molecules to the lung to exert efficacy. In the face of the above challenges, some companies in the field have developed corresponding solutions. For example, the SORT delivery system of Recode, which is composed of ionizable lipids, DOTAP, cholesterol, auxiliary phospholipids and polyethylene glycol lipids, to achieve targeted delivery to the lung. However, DOTAP is a permanent cation, which has certain toxicity to the human body. The atomized LNP formulation of Corima is composed of ionizable lipids AX4 / DSPC / cholesterol / DMG-PEG, but only LNP prepared with high content of cholesterol has high encapsulation efficiency. Moreover, the evaluation of LNP after atomization is mainly based on the physicochemical properties of LNP detected in vitro, and there is no detection of the in vivo expression effect of mRNA-LNP after mouse lung administration by the atomization device, which cannot prove its in vivo effectiveness. Therefore, there is still a need in the art to develop safer and more efficient inhalation formulations to meet the clinical needs of lung-targeted nucleic acid drugs. SUMMARY In view of the above-mentioned shortcomings of the prior art, the present application provides a novel inhalation formulation for lung-targeted delivery of nucleic acids by optimizing the formulation of lipid nanoparticles containing specific ionizable lipids, which not only has good resistance to shear-induced damage, but also can break through the physiological barrier of the lung and efficiently deliver nucleic acids to the lung. To achieve the above-mentioned objects and other related objects, the present application provides a lipid nanoparticle for lung-targeted delivery of nucleic acids, an inhalation formulation comprising the lipid nanoparticle for lung-targeted delivery of nucleic acids, and use of the above-mentioned lipid nanoparticle and inhalation formulation in the preparation of a pharmaceutical composition for lung-targeted delivery of nucleic acids. The first aspect of the present application provides the lipid nanoparticle for lung-targeted delivery of nucleic acids, which comprises the following components in the following molar percentages: ionizable lipid or isomer thereof or pharmaceutically acceptable salt thereof: 45-65 mol%; co-lipid: 10-30 mol%; structural lipid: 15-30 mol%; PEG-lipid: 0.5-2.5 mol%; The ionizable lipid has the following structure: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1, G2 are each independently C1-C10 alkylene; R1, R2, R3, R4 are each independently H, C1-C20 linear or branched alkyl, or C2-C20 linear or branched alkenyl; G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b wherein a, b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is -NH(C=O)O- or -O(C=O)NH-. The second aspect of the present application provides an inhalation preparation comprising the lipid nanoparticles for targeted pulmonary delivery of nucleic acids, the inhalation preparation comprising a lipid nanoparticle dispersion system consisting of the lipid nanoparticles for targeted pulmonary delivery of nucleic acids, a cryoprotective agent, a system solution; and a surface tension regulator added in the lipid nanoparticle dispersion system. The third aspect of the present application provides the use of the above-mentioned lipid nanoparticles or inhalation preparation in the preparation of a pharmaceutical composition for targeted pulmonary delivery of nucleic acids. Compared with the prior art, the present application has the following beneficial effects: 1. The present application selects specific ionizable lipids, and optimizes and screens ionizable lipids, auxiliary lipids, structural lipids and PEG-lipids, to prepare the lipid nanoparticles for targeted pulmonary high-efficiency delivery of nucleic acids, which have good atomization stability, and the particle size and encapsulation efficiency of the nanoparticles do not change significantly before and after atomization. The test results of animal administration by inhalation show that the lipid nanoparticles can break through the physiological barrier of the lung and mediate the expression of target proteins in the lung with high efficiency. 2. The inhalation preparation comprising the lipid nanoparticles for targeted pulmonary high-efficiency delivery of nucleic acids provided by the present application contains a specific surface tension regulator, which can withstand the shear damage generated during atomization, overcome multiple physiological barriers, deliver the lipid nanoparticles to the lung, and has high transfection efficiency. BRIEF DESCRIPTION OF DRAWINGS Fig. 1 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in Example 1 of the present application. Fig. 2 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in Example 2 of the present application. Fig. 3 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in the comparative experiment in Example 2 of the present application. Fig. 4 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in Example 3 of the present application. Fig. 5 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in 4.1 of Example 4 of the present application. Fig. 6 is the fluorescence imaging result of the in-vivo atomization study experiment of the mRNA-LNP prepared in 4.2 of Example 4 of the present application. DETAILED DESCRIPTION To solve the problems of the prior art, the present application aims to provide a lipid nanoparticle for targeted pulmonary delivery of nucleic acids, an inhalation preparation comprising the lipid nanoparticle for targeted pulmonary delivery of nucleic acids, and the use of the above-mentioned lipid nanoparticle and inhalation preparation in the preparation of a pharmaceutical composition for targeted pulmonary delivery of nucleic acids. To achieve the above object, the present application adopts the technical solutions as follows: The present application provides, in a first aspect, a lipid nanoparticle for targeted pulmonary delivery of a nucleic acid, the lipid nanoparticle comprising each component in the following molar percentages: an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof: 45-65 mol%; a helper lipid: 10-30 mol%; a structural lipid: 15-30 mol%; a PEG-lipid: 0.5-2.5 mol%; The ionizable lipid has the following structure: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; G1, G2 are each independently a C1-C10 alkylene group; R1, R2, R3, R4 are each independently H, a C1-C20 linear or branched alkyl group, or a C2-C20 linear or branched alkenyl group; G3 is a C1-C10 alkylene group; or G3 is (CH2) a -O-(CH2) b wherein a, b are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is -NH(C=O)O- or -O(C=O)NH-. The "C1-C20 linear or branched alkyl group", "C2-C20 linear or branched alkenyl group", "C1-C10 alkylene group" in the present application are respectively as described in the specification of Chinese patent application CN115947671A

[0047] ,

[0048] and

[0049] as described in the specification of Chinese patent application CN115947671A In some embodiments of the present application, -CH(R1)R2 and -CH(R3)R4 in the ionizable lipid are as described in the specification of Chinese patent application CN115947671A

[0019] ,

[0020] and

[0021] as described in the specification of Chinese patent application CN115947671A In some embodiments of the present application, the ionizable lipid is selected from the group consisting of:

[0022] ,

[0023] ,

[0024] and

[0025] . In some embodiments of the present application, the ionizable lipid is selected from the group consisting of: The "isomers" include stereoisomers and tautomers. The "stereoisomers" refer to isomers that have the same sequence of atoms but differ in the arrangement of atoms in space. The "tautomers" refer to the phenomenon of interconversion between two functional groups isomers of a compound that produce an equilibrium interconversion of structure, and the corresponding isomers are called tautomers. The "pharmaceutically acceptable salts" refer to either acid or base addition salts. All compounds of the present application exist in free base or free acid form. Such free bases and acids have the ability to form pharmaceutically acceptable salts by treatment with inorganic or organic acids or bases as is known to those skilled in the art. Salts of the compounds of the present application can be converted into their free bases or acids by standard techniques. Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and quaternary amine salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and arylsulfonate. Further pharmaceutically acceptable salts include salts formed from the quaternization of an amine using an appropriate electrophile (e.g., an alkyl halide) to form a quaternary alkylated ammonium salt. The term "structural lipid" refers to a combination comprising one or more of a sterol and derivatives thereof and a non-sterol and derivatives thereof that can stabilize the composition. In some embodiments, the structural lipid includes, but is not limited to, a combination of one or more of a sterol and its derivatives, a non-sterol, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, coprostanol, alpha-tocopherol, or a corticosteroid. The sterol is preferably cholesterols and its derivatives; non-limiting examples of cholesterols derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)ethyl ether, cholesteryl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestan, cholestanone, 5α-cholestenone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterols derivative is a polar analog such as cholesteryl-(4'-hydroxy)butyl ether. The selection of structural lipids is not limited and any structural lipid can be used in the present application, without limitation. In some embodiments, the structural lipid is a combination of one or more of cholesterols, sitosterol, ergosterol, corticosteroids, and its derivatives. In some embodiments, the structural lipid is cholesterols. The class of the "auxiliary lipid" is not limited, and is preferably a phospholipid, including, but not limited to, a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol. In some embodiments, the helper lipid can be selected from the group consisting of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn glycero phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1-oleoyl-2-cholesterylhemisuccinoyl- sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn- glycero-3-phosphocholine, 1,2-didocosahexanoyl-sn-glycero 3-phosphocholine, 1,2- diphytanyloyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexanoyl-sn-glycero 3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl- phosphatidyl ethanolamine (DEPE), stearoyl-phosphatidyl ethanolamine (SOPE), lysophosphatidylcholine, sphingomyelin, or a combination thereof. In some embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, POPC. In some embodiments, the helper lipid is a phosphatidylcholine, specifically DSPC. In some embodiments, the helper lipid is a phosphatidylcholine, specifically a combination of DSPC and DPPC. In some embodiments, the helper lipid is a phosphatidylethanolamine, specifically DOPE. The lipid nanoparticles provided by the present application do not include (2,3-dioleyloxypropyl) trimethylammonium chloride (DOTAP) in the lipid, which can ensure the targeted lung delivery of nucleic acid by the lipid nanoparticles, and avoid the cytotoxicity caused by permanent cationic lipids. The "PEG-lipid" of the present application generally refers to a conjugate formed by linking PEG (polyethylene glycol) and a lipid molecule by a chemical bond. It includes but is not limited to PEG-modified phospholipids and derivatized lipids, such as one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol dimyristyl acetyl amide, or a combination thereof. In some embodiments, the PEG-lipid includes but is not limited to one or more of the following: PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearylphosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, methoxypolyethylene glycol phosphatidylethanolamine, methoxypolyethylene glycol ceramide, methoxypolyethylene glycol dimethacrylate (PEG-DMA), PEG distearoylglycerol, PEG dipalmitoyloleyl, PEG dioleyl, PEG distearoyl, PEG diacylglycerol amide, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl dimyristyl oxyl propyl-3-amine, PEG oxyl propyl ethanol amine, 1,2-distearyloxypropyl-3-amine-N[methoxyl (polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol laurate, methoxypolyethylene glycol dimyristyl acetyl amide (ALC0159). In some embodiments of the present application, the PEG-lipid is PEG-DMG. In some embodiments of the present application, the PEG-lipid has a weight average molecular weight of PEG of 1000-10000, such as 1000-2000, 2000-4000, 4000-6000, 6000-8000, 8000-10000, preferably 2000. In some embodiments of the present application, the lipid nanoparticles for targeted lung delivery of nucleic acid comprise the following molar percentages of each component: ionizable lipid or isomer thereof or pharmaceutically acceptable salt thereof: 50-65 mol%; helper lipid: 10-30 mol%; Structural lipid: 15-25 mol%; PEG-lipid: 0.5-2 mol%. In some embodiments of the present application, the particle size of the lipid nanoparticle is in the range of 60-300 nm, preferably 60-200 nm, and further preferably 70-160 nm. The second aspect of the present application provides an inhalation formulation comprising the lipid nanoparticle for targeting lung delivery of nucleic acid, which comprises a lipid nanoparticle dispersion system consisting of the above-mentioned lipid nanoparticle for targeting lung efficient delivery of nucleic acid, a cryoprotective agent, a system solution; and a surface tension regulator added in the lipid nanoparticle dispersion system. The "cryoprotective agent" in the present application refers to a substance (usually a solution) that can protect cells from freezing damage, including but not limited to sugars, alcohols, amino acids, salts, etc.; exemplary ones are: sucrose, mannitol, trehalose, lactose, glucose, maltose, polyvinylpyrrolidone (PVP), polyethylene glycol, dextran, albumin, and hydroxyethyl starch. The "surface tension regulator" in the present application refers to a compound or composition that can adjust the surface tension of the formulation, which is used to enhance the shear tolerance of LNP atomization, including but not limited to: ethanol, propylene glycol, phenylethanol, poloxamer 188, Tween-80, glycerol or a combination thereof. The above is not exhaustive, and any surface tension regulator known or unknown is within the scope of the present application, as long as it is a compound or composition that can improve the shear damage tolerance of the inhalation formulation. In some embodiments of the present application, the surface tension regulator is ethanol, and the mass percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05%-30%. In some embodiments of the present application, the surface tension regulator is propylene glycol, and the mass percentage of propylene glycol in the lipid nanoparticle dispersion system is in the range of 0.05%-30%. In some embodiments of the present application, the surface tension regulator is a combination of propylene glycol and ethanol, the mass percentage of propylene glycol in the lipid nanoparticle dispersion system is in the range of 0.05%-30%, and the mass percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05%-30%. In some embodiments of the present application, the surface tension regulator is poloxamer 188, and the concentration of poloxamer 188 in the lipid nanoparticle dispersion system is in the range of 0.5-10 mg / mL. In some embodiments of the present application, the surface tension regulator is a mixture of poloxamer 188 and ethanol, the concentration of poloxamer 188 in the lipid nanoparticle dispersion system ranges from 0.5 to 10 mg / mL, and the mass percentage of ethanol in the lipid nanoparticle dispersion system ranges from 0.05% to 30%. In some embodiments of the present application, the surface tension regulator is Tween-80, and the mass percentage of Tween-80 in the lipid nanoparticle dispersion system ranges from 0.01% to 2%. In some embodiments of the present application, the surface tension regulator is a mixture of Tween-80 and ethanol, the mass percentage of Tween-80 in the lipid nanoparticle dispersion system ranges from 0.01% to 2%, and the mass percentage of ethanol in the lipid nanoparticle dispersion system ranges from 0.05% to 30%. In some embodiments of the present application, the mass percentage of the lipid nanoparticle in the dispersion system ranges from 0.0025% to 10%, the concentration of the system solution ranges from 0 to 1000 mM, and the mass percentage of the cryoprotectant ranges from 0% to 20%. In some embodiments of the present application, the system solution of the present application includes, but is not limited to, physiological saline, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPEs) buffer, tris-hydroxymethyl aminomethane (Tris) buffer, Tris-EDTA buffer, phosphate (PB) and phosphate (PBS) buffer, Dulbecco's phosphate (DPBS) buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tween-containing Tris buffer (TBST), EDTA and its sodium salt-containing buffer, and combinations of one or more of the above. It should be noted that the system solution herein is not exhaustive, but is a preferred one, and any solution that can regulate or buffer the osmotic pressure or pH of the system is within the scope of the present application. The third aspect of the present application provides the use of the above-mentioned lipid nanoparticle or inhalation preparation in the preparation of a pharmaceutical composition for targeted pulmonary delivery of nucleic acids, wherein the pharmaceutical composition further comprises a drug carried and pharmaceutically acceptable excipients. The "drug carried" of the present application includes one or more of nucleic acids, small molecule compounds, and proteins; in some embodiments of the present application, the "drug carried" includes nucleic acids. The "nucleic acid" of the present application can be a nucleotide polymer of any length. It includes, but is not limited to, one or more of a combination of single-stranded DNA, double-stranded DNA, plasmid DNA, short isoform, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), miRNA, siRNA, telomerase RNA (Telomerase RNA), small nuclear RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme, asymmetric interference RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide, or biologically customized oligonucleotide. In some embodiments of the present application, the nucleic acid is mRNA. The mRNA is a single-stranded ribonucleic acid that carries genetic information capable of directing protein synthesis, which is transcribed from one strand of DNA as a template. The mRNA can encode one protein or multiple proteins at the same time. Preferably, the mRNA is synthesized by in vitro transcription. The "small molecule" of the present application refers to a compound that is not a protein or a nucleic acid molecule. The small molecule can be a small molecule therapeutic agent and / or prophylactic agent, such as an antibiotic, an anti-inflammatory drug, an anticancer drug, an antiviral drug, an immunosuppressant, an analgesic, an antifungal drug, an antiparasitic drug, an anticonvulsant, an antidepressant, an anxiolytic, an antipsychotic, a lipid-lowering drug, a hypoglycemic drug, a weight loss drug, etc. The "protein" of the present application refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary, and / or quaternary structure. The secondary, tertiary, and / or quaternary structure of a protein is typically stabilized using noncovalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals forces. Additionally, or alternatively, a protein can include disulfide bonds, for example, between thiol groups of cysteine residues. Exemplary proteins include, but are not limited to, antibodies, antigens, or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, glycoproteins, lipoproteins, ribosomal proteins, chemically modified proteins, etc. The pharmaceutical composition of the present application further comprises a pharmaceutically acceptable excipient. Generally, these substances are formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably the pH is about 5-7, and the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated drug can be administered by inhalation. The term "pharmaceutically acceptable" as used herein means that the drugs are not adversely, allergically or otherwise adversely reacted when administered to an animal or human in the proper manner. The term "pharmaceutically acceptable excipient" as used herein means that the excipient is compatible with the active ingredient, i.e., it can be blended therewith and will not, in general, substantially reduce the effect of the drug. Specific examples of some of the substances that can be used as pharmaceutically acceptable excipients are alcohols such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protecting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffers; and the like, and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help improve the activity or its bioavailability. The term "targeted pulmonary delivery of nucleic acids" as used herein means that the delivery of nucleic acids to the lung via the respiratory tract, including but not limited to delivery of nucleic acids via inhalation or nasal instillation. The pharmaceutical composition for targeted pulmonary delivery of nucleic acids according to the present application can be prepared as an inhalation formulation such as a dry powder formulation, an aerosol formulation, an inhalation mist droplet formulation, a nasal instillation formulation, and the like. The amount of the active ingredient to be administered is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. Before further description of the specific embodiments of the present application, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as to the scope of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Other specifically identified materials, equipment, procedures, etc. which are used to implement or test the application can also be used for implementing or testing the application, unless otherwise indicated. Any patents and publications referred to in this application are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Other specifically identified materials, equipment, procedures, etc. which are used to implement or test the application can also be used for implementing or testing the application, unless otherwise indicated. Any patents and publications referred to in this application are incorporated by reference in their entirety. Example 1: Screening of ionizable lipids 1. Preparation of mRNA-LNP Exemplary ionizable lipids E-1, E-5, E-6, E-11 were selected, and mRNA-LNP was prepared according to the formulations in Table 2. The mRNA-LNP preparation method comprises the following steps: Step 1: Dissolve the ionizable lipids in Table 1, DSPC (Avanti (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avanti (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG 2000 According to the prescription in Table 2, ethanol was dissolved in ethanol to prepare a lipid ethanol solution (the concentration of Lipid was 20 mg / mL). Step 2: Prepare mRNA according to the mass ratio of lipid nanoparticles (LNP) to mRNA of 10:1 to 30:1, and dilute the mRNA to 0.2 mg / mL using citrate or sodium acetate buffer (pH = 3 or 5). Step 3: Mix the lipid ethanol solution obtained in step 1 with the mRNA solution at a volume ratio of 1:5 to 1:1, incubate for 20 min, and obtain mRNA-LNP (lipid nanoparticles loaded with mRNA). Table 1 Ionizable Lipid Structure Table 2 mRNA-LNP Formulation 2. In vitro nebulization study The mRNA-LNP prepared above was subjected to nebulization study, and the experimental method was as follows: the mRNA-LNP prepared above was dialyzed in a 20 mM HEPEs buffer containing 10% sucrose at pH = 6, then 50 μL was taken out, the above buffer was diluted 3 times with ethanol (surface tension regulator) containing 6% g / ml, and the vibration mesh nebulization device (model Aerogen solo Nebulizer System) was used for nebulization. The particle size and PDI of the mRNA-LNP before and after nebulization were determined using Malvern Zetasizer Nano ZS, and the encapsulation efficiency EE% of the mRNA before and after nebulization was determined using Ribogreen RNA quantitative determination kit (Thermo Fisher). The results are shown in Table 3 below. Table 3 Particle size, PDI and EE of mRNA-LNP before and after nebulization Note: "pre" and "post" represent samples before and after nebulization, respectively. The experimental results in Table 3 show that the LNP prepared above can withstand nebulization shear damage before and after nebulization, but the particle size change of prescription 1 (E-1-1, E-5-1, E-6-1, E-11-1) is higher than that of prescription 2 (E-1-2, E-5-2, E-6-2, E-11-2) before and after nebulization, and the encapsulation efficiency is also slightly lower. However, different ionizable lipids have no substantial effect on the nebulization effect of LNP formulations. 3. In vivo nebulization study Select E-1-1, E-5-1, E-6-1, E-11-1, E-5-2, E-11-2 to continue the in vivo mouse inhalation study, select Bal / c mice, 6-8 weeks old, preparation of Luciferase mRNA, using a vibrating mesh nebulizer device for nebulization, the dose is 20 μg per mouse, 6 hours later, the mice were imaged, and the mice were dissected 5 minutes after injection of D-luciferin sodium substrate, and the heart, liver, spleen, lung and kidney were imaged, as shown in Figure 1. The experimental results show that: E-1-1, E-5-1, E-6-1, E-11-1 can withstand nebulization shear damage in vitro, but it is difficult to break through the lung physiological barrier in vivo, and the protein expression is low. The corresponding LNP prepared by using E-5-2, E-11-2 can withstand nebulization shear damage while breaking through the lung physiological barrier, and can efficiently mediate target protein expression in the lung. Thus, it is shown that the LNP inhalation preparation can achieve high local concentration of nucleic acid drugs in the lung after inhalation administration, and then efficiently mediate gene transfection in the lung. Example 2: Screening of auxiliary lipids 1. In vivo nebulization study Using E-1 as an ionizable lipid, mRNA-LNP was prepared according to the prescription in Table 4 and the mRNA-LNP preparation method in Example 1, and the obtained product was subjected to in vivo nebulization study according to the in vivo nebulization study method in Example 1, and the results are shown in Figure 2. Table 4 mRNA-LNP formulation As shown by the experimental results in Figure 2, different auxiliary lipids have no substantial effect on the nebulization effect of mRNA-LNP. 2. Comparative experiment Using E-1 as an ionizable lipid, mRNA-LNP was prepared according to the prescription in Table 5 and the mRNA-LNP preparation method in Example 1, and the LNP particle size, PDI and encapsulation efficiency were determined as shown in Table 6. Table 5 mRNA-LNP formulation Table 6 Particle size, PDI and EE of mRNA-LNP before and after nebulization According to the experimental method in Example 1, in vivo nebulization study was carried out, and the results are shown in Figure 3. Experimental conclusion: after nebulization administration, almost no corresponding protein is expressed in the lung when DSPC is replaced by DOTAP or DOTAP is additionally added. In the prescription of the comparative experiment, the introduction of DOTAP affects the LNP in breaking through the lung physiological barrier, and also affects the escape of LNP from lysosomes, so that mRNA cannot be efficiently delivered to the lung, affecting the expression of the corresponding protein in the lung. Example 3: PEG lipid content screening 3.1 Preparation of mRNA-LNP The mRNA-LNP was prepared according to the prescription in Table 7 and the preparation method in Example 1, and the particle size, PDI and encapsulation efficiency of the mRNA-LNP were determined as shown in Table 8. Table 7 mRNA-LNP formulation Table 8 Particle size, PDI and EE of mRNA-LNP before and after nebulization 3.2 In vivo nebulization study The obtained mRNA-LNP product was subjected to in vivo nebulization study according to the in vivo nebulization study method in Example 1, and the results are shown in Figure 4. As shown by the experimental results in Figure 4, within a certain molar percentage range, the expression in the lung of the mouse is increased with the increase of the PEG lipid content. However, when the molar percentage exceeds a certain value, the lung expression is decreased, and the increase of the PEG content can help the LNP to break through the lung mucus layer, but at the same time, it can also affect the endocytosis. Therefore, a suitable PEG lipid content is more conducive to the lung delivery of LNP. Example 4: Ionizable lipid, cholesterol, helper lipid content screening Preparation of mRNA-LNP and in vivo nebulization study 4.1 E-11 was selected as the ionizable lipid, mRNA-LNP was prepared according to the prescription in Table 9, and in vivo nebulization study was carried out, and the results are shown in Figure 5: the formulations E-11-1, E-11-2 and E-11-4 have high transfection efficiency, and can express corresponding proteins at high levels, and the formulation E-11-3 has poor transfection efficiency and low protein expression. Table 9 mRNA-LNP formulation 4.2 E-5 was selected as the ionizable lipid, mRNA-LNP was prepared according to the prescription in Table 10, and the particle size, PDI and EE were determined as shown in Table 11, and in vivo nebulization study was carried out, and the results are shown in Figure 6: all the formulations can express a certain amount of protein, but the mRNA-LNP formulations prepared by E5-1-1, E5-1-2 and E5-1-3 have small PDI, uniform particle size distribution, high encapsulation efficiency and higher protein expression. Table 10 mRNA-LNP formulation Table 11 Particle size, PDI and EE of mRNA-LNP before and after nebulization The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A lipid nanoparticle for targeted pulmonary delivery of a nucleic acid, characterized in that, The lipid nanoparticle comprises the following molar percentages of each component: ionizable lipid or isomers thereof or pharmaceutically acceptable salts thereof: 45-65 mol%; co-lipid: 10-30 mol%; structural lipid: 15-30 mol%; PEG-lipid: 0.5-2.5 mol%; The ionizable lipid has the following structure: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1, G2 are each independently C1-C10 alkylene; R1, R2, R3, R4 are each independently H, C1-C20 linear or branched alkyl, or C2-C20 linear or branched alkenyl; G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b wherein a, b are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is -NH(C=O)O- or -O(C=O)NH-.

2. The lipid nanoparticle of claim 1, wherein, One or more of the following features are included: (1) the structural lipid is cholesterol; (2) the co-lipid is DSPC, DOPE or a combination of DSPC and DPPC; (3) the PEG-lipid is PEG-DMG; (4) the lipid nanoparticle has a particle size ranging from 60 to 300 nm.

3. The lipid nanoparticle of claim 1, wherein, The lipid nanoparticle for targeted pulmonary delivery of nucleic acids comprises the following molar percentages of each component: ionizable lipid or isomers thereof or pharmaceutically acceptable salts thereof: 50-65 mol%; co-lipid: 10-30 mol%; structural lipid: 15-25 mol%; PEG-lipid: 0.5-2 mol%.

4. An inhaled formulation comprising the lipid nanoparticle of claim 1 for targeted pulmonary delivery of a nucleic acid, wherein, The inhalation formulation comprises a lipid nanoparticle dispersion system, which is composed of the lipid nanoparticle for targeted pulmonary delivery of nucleic acids, a cryoprotective agent, a system solution; and a surface tension regulator added in the lipid nanoparticle dispersion system.

5. The inhalation formulation of claim 4, wherein the amount of the compound is about 0.1 mg to about 1 mg. The surface tension regulator comprises a mixture of one or more of the following: ethanol, propylene glycol, phenyl ethanol, poloxamer 188, Tween-80, glycerol; preferably the surface tension regulator is ethanol, and further preferably, the mass / volume percentage of ethanol in the lipid nanoparticle dispersion system ranges from 0.05% to 30%.

6. The inhalation formulation of claim 4, wherein the amount of the compound is about 0.1 mg. One or more of the following features are included: (1) the mass / volume percentage of the lipid nanoparticle in the dispersion system ranges from 0.0025% to 10%, the concentration of the system solution ranges from 0 to 1000 mM, and the mass / volume percentage of the cryoprotective agent ranges from 0% to 20%; (2) the system solution comprises one or more of the following: physiological saline, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPEs) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, Tris-EDTA buffer, phosphate (PB) and phosphate (PBS) buffer, Dulbecco's phosphate (DPBS) buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tween-containing Tris buffer (TBST), EDTA and its sodium salt-containing buffer, and a combination of one or more of the above.

7. Use of the lipid nanoparticle of any one of claims 1 to 3 or the inhalable formulation of any one of claims 4 to 6 in the manufacture of a pharmaceutical composition for targeted pulmonary delivery of a nucleic acid; the pharmaceutical composition further comprising a drug cargo and pharmaceutically acceptable excipients.

8. Use according to claim 7, wherein the compound is ###0002### The pharmaceutical composition is for targeted pulmonary delivery of a nucleic acid by an inhalation or nasal drop administration route.

9. The use according to claim 7, wherein the compound is ###0006### The drug cargo comprises one or more of a nucleic acid, a small molecule compound, a protein.

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