WO2025218536A1 - Lipid nanoparticles for targeted high-efficiency delivery of nucleic acid to lung, inhaled formulation, and use - Google Patents

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 nanoparticles, inhalation formulations and applications for efficient lung-targeted delivery of nucleic acids Technical Field

The present invention relates to the technical field of nucleic acid drugs, and in particular to lipid nanoparticles, inhalation preparations and applications for efficiently delivering nucleic acids to the lungs.

Background Art

Inhalation preparations are designed to deliver drugs to the respiratory tract and/or lungs via inhalation for local or systemic effects. Traditional inhalation preparations primarily consist of small-molecule drugs. However, recent advances in biopharmaceuticals and genetic engineering have led to the emergence of a large number of large-molecule drugs such as peptides, proteins, and nucleic acids, providing a broader application space for inhalation preparations.

Nucleic acids are electronegative macromolecules that are susceptible to degradation and have low cellular uptake and transfection efficiency. Therefore, the development of safe and effective delivery vehicles is crucial to protect nucleic acid drugs from degradation and promote cellular uptake. Currently, lipid nanoparticles (LNPs), due to their unique structure and physicochemical properties, exhibit high delivery efficiency and good safety in vivo, making them a mainstream delivery vehicle for nucleic acid drugs.

Currently, LNPs are typically administered clinically via intramuscular injection. While the vast majority of LNPs are distributed locally at the injection site, some are also distributed in the liver. The liver's first-pass effect significantly limits LNPs' ability to reach other organs. In contrast, the lungs have a large surface area and avoid the first-pass effect, significantly enhancing drug absorption. Therefore, pulmonary delivery of nucleic acid drugs has become a key breakthrough area for LNPs.

The lung delivery of nucleic acid drugs is extremely challenging: on the one hand, it is necessary to withstand the shear damage generated during the atomization process (shear damage will destroy the nanostructure of lipid nanoparticles, leading to aggregation and sedimentation of nanoparticles and leakage of the carried nucleic acid molecules); on the other hand, it is necessary to overcome multiple physiological barriers (such as mucus barrier, ciliary clearance, and macrophage phagocytosis) in order to deliver nucleic acid therapeutic molecules to the lungs to exert their therapeutic effects.

Faced with these challenges, some companies in this field are developing solutions, such as Recode's SORT delivery system. Its LNP formulation consists of ionizable lipids, DOTAP, cholesterol, auxiliary phospholipids, and PEGylated lipids to achieve targeted lung delivery. However, DOTAP is a permanent cation and has certain toxicity to humans. Cremaide's nebulized LNP formulation is formulated with the ionizable lipid AX4/DSPC/cholesterol/DMG-PEG. However, LNPs prepared with a high cholesterol content achieve a high encapsulation efficiency. Furthermore, the evaluation of the nebulized LNP is primarily based on in vitro physicochemical properties of the LNP. The in vivo expression of the mRNA-LNP after nebulization in mice has not been tested, and its in vivo efficacy cannot be proven.

Therefore, there is still a need in this field to develop safer and more efficient inhalation formulations to meet the clinical needs of lung-targeted nucleic acid drugs.

Summary of the Invention

In view of the shortcomings of the prior art mentioned above, the present invention provides a new lung-targeted inhalation preparation by optimizing the formulation of lipid nanoparticles containing specific ionizable lipids. This preparation not only has good resistance to atomization shear damage, but also can break through the physiological barrier of the lungs, efficiently deliver nucleic acids to the lungs, and has high transfection efficiency.

To achieve the above-mentioned objectives and other related objectives, the present invention provides lipid nanoparticles for targeted lung delivery of nucleic acids, an inhalation formulation comprising the lipid nanoparticles for targeted lung delivery of nucleic acids, and the use of the lipid nanoparticles and inhalation formulation in preparing a pharmaceutical composition for targeted lung delivery of nucleic acids.

The first aspect of the present invention provides the lipid nanoparticles for targeted lung delivery of nucleic acids, wherein the lipid nanoparticles include the following components in molar percentages:

Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 45-65 mol%;

Helper lipids: 10-30 mol%;

Structural lipids: 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;

G ₁ and G ₂ are each independently a C1-C10 alkylene group;

R ₁ , R ₂ , R ₃ , and R ₄ are each independently H, a C1-C20 linear or branched alkane group, or a C2-C20 linear or branched alkene group;

G ₃ is a C1-C10 alkylene group; or G ₃ is (CH ₂ ) _a -O-(CH ₂ ) _b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer of 2-10;

L ₁ is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

L ₂ is -NH(C=O)O- or -O(C=O)NH-.

The second aspect of the present invention provides an inhalation preparation containing the lipid nanoparticles for targeted lung delivery of nucleic acids, wherein the inhalation preparation includes a lipid nanoparticle dispersion system, and the lipid nanoparticle dispersion system is composed of the following components: the lipid nanoparticles for targeted lung delivery of nucleic acids, a cryoprotectant, a system solution; and a surface tension regulator added to the lipid nanoparticle dispersion system.

A third aspect of the present invention provides use of the lipid nanoparticles or inhalation formulations described above in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids.

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

1. The present invention selects specific ionizable lipids and optimizes and screens ionizable lipids, auxiliary lipids, structural lipids and PEG-lipids to prepare lipid nanoparticles that efficiently deliver nucleic acids to the lungs. The lipid nanoparticles have good atomization stability, and the particle size and encapsulation efficiency of the nanoparticles do not change significantly before and after atomization. The experimental results of administering the drug to animals via inhalation show that the lipid nanoparticles can break through the physiological barrier of the lungs and can efficiently mediate the expression of target proteins in the lungs.

2. The inhalation preparation provided by the present invention comprises lipid nanoparticles for efficiently delivering nucleic acids to the lungs. The inhalation preparation contains a specific surface tension regulator, which can withstand the shear damage generated during the atomization process, overcome multiple physiological barriers, deliver lipid nanoparticles to the lungs, and have a high transfection efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a fluorescence imaging result of an in vivo atomization study experiment of mRNA-LNP prepared in Example 1 of the present invention.

FIG2 is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in Example 2 of the present invention.

FIG3 is the fluorescence imaging result of the in vivo atomization study of mRNA-LNP prepared in the comparative experiment in Example 2 of the present invention.

FIG4 is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in Example 3 of the present invention.

FIG5 is the fluorescence imaging result of the in vivo atomization study experiment of the mRNA-LNP prepared in 4.1 in Example 4 of the present invention.

FIG6 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 invention.

DETAILED DESCRIPTION

To address the deficiencies of the prior art, the present invention aims to provide a lipid nanoparticle for targeted lung delivery of nucleic acids, an inhalation formulation comprising the lipid nanoparticle for targeted lung delivery of nucleic acids, and the use of the lipid nanoparticle and inhalation formulation in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

The first aspect of the present invention provides lipid nanoparticles for targeted lung delivery of nucleic acids, wherein the lipid nanoparticles include the following molar percentages of each component:

Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 45-65 mol%;

Helper lipids: 10-30 mol%;

Structural lipids: 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;

G ₁ and G ₂ are each independently a C1-C10 alkylene group;

R ₁ , R ₂ , R ₃ , and R ₄ are each independently H, a C1-C20 linear or branched alkane group, or a C2-C20 linear or branched alkene group;

G ₃ is a C1-C10 alkylene group; or G ₃ is (CH ₂ ) _a -O-(CH ₂ ) _b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer of 2-10;

L ₁ is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

L ₂ is -NH(C=O)O- or -O(C=O)NH-.

The "C1-C20 straight-chain or branched alkane group", "C2-C20 straight-chain or branched olefin group" and "C1-C10 alkylene group" described in the present invention are as described in [0047], [0048] and [0049] of the specification of Chinese patent application CN115947671A, respectively.

In some specific embodiments of the present invention, -CH(R ₁ )R ₂ and -CH(R ₃ )R ₄ in the ionizable lipid are as described in [0019], [0020] and [0021] of the specification of Chinese patent application CN115947671A.

In some specific embodiments of the present invention, the ionizable lipid is as described in [0022], [0023], [0024] and [0025] of the specification of Chinese patent application CN115947671A.

In some specific embodiments of the present invention, the ionizable lipid is selected from the group consisting of:

The "isomers" include stereoisomers and tautomers.

The term "stereoisomer" refers to isomers that have the same sequence of atoms connected but different arrangements of atoms in space.

The term "tautomer" refers to a phenomenon in which the structure of a compound undergoes equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

The term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention that exist in the form of a free base or free acid can be converted into their pharmaceutically acceptable salts by treating with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of the present invention can be formed by converting them into their free base or acid form using standard techniques.

Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used 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, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconoheptate, 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, and the like. The salt derived from suitable base comprises alkali metal salt, alkaline earth metal salt, ammonium salt.Representational alkali metal salt or alkaline earth metal salt comprises sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt etc.In appropriate cases, other pharmaceutically acceptable salt comprises the non-toxic ammonium, quaternary ammonium and amine cation formed using the counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Other pharmaceutically acceptable salt comprises the salt formed by the quaternization of amine, and this quaternization is carried out using suitable electrophilic reagent (for example, alkyl halide), to form quaternized alkylated amino salt.

The "structured lipids" mentioned above refer to lipids containing structures that can stabilize the composition, including but not limited to one or more combinations of sterols and their derivatives and non-sterols and their derivatives.

In some specific embodiments, the structured lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, coprosterol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprosterol, cholesteryl-(2′-hydroxy) ethyl ether, cholesteryl-(4′-hydroxy) butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestenone, and cholesterol decanoate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog, such as cholesteryl-(4′-hydroxy) butyl ether. This is not exhaustive, and the selection of structured lipids is not limited, and any structured lipid can be applied to the present invention.

In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

In some embodiments, the structured lipid is cholesterol.

There is no limitation on the type of the “helper lipid”, and phospholipids are preferred, including but not limited to: a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoylphosphatidylglycerol.

In some embodiments, the helper lipid can be selected from: 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-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPPC), ), 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), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1- Oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docohexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl A combination of one or more of acyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docohexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin.

In some embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

In some embodiments, the helper lipid is 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 phosphatidylethanolamine, specifically DOPE.

The lipid nanoparticles provided by the present invention do not contain (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP) in the lipids, thereby ensuring that the lipid nanoparticles deliver nucleic acids in a targeted manner to the lungs and avoiding the cytotoxicity caused by permanent cationic lipids.

The term "PEG-lipid" as used herein generally refers to a conjugate formed by chemically linking PEG (polyethylene glycol) to a lipid molecule. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by combinations of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxypolyethylene glycol ditetradecylacetamide.

In some embodiments, the PEG-lipids include but are not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol A combination of one or more of PEG-2,4-DISTEARYL GLYRIDE (PEG-DMA), PEG-DISTEARYL glycerol, PEG-DIPALMITOYL, PEG-DISOLENE, PEG-DISTEARYL, PEG-DIACYLGLYCAMIDE, PEG-DIPALMITYL PHOSPHATIDYL ETHANOLAMINE, PEG-PHOSPHATIDYL ETHANOL, PEG-PHOSPHATIDYL ETHYL DIMYRISTYL OXYPROPYL-3-AMINE, PEG-OXYPROPYL OLAMINE, 1,2-DISTEARYLOXYPROPYL-3-AMINE-N[METHOXY(POLYETHYLENE GLYCOL)](PEG-DSA), methoxypolyethylene glycol laurate, and methoxypolyethylene glycol ditetradecyl acetamide (ALC0159).

In some embodiments, the PEG-lipid is PEG-DMG.

In some specific embodiments of the present invention, the weight average molecular weight of PEG in the PEG-lipid is 1000-10000, for example, 1000-2000, 2000-4000, 4000-6000, 6000-8000, 8000-10000, preferably 2000.

In some specific embodiments of the present invention, the lipid nanoparticles for targeted lung delivery of nucleic acids comprise the following molar percentages of the components:

Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 50-65 mol%;

Helper lipids: 10-30 mol%;

Structural lipids: 15-25 mol%;

PEG-lipid: 0.5-2 mol%.

In some specific embodiments of the present invention, the particle size of the lipid nanoparticles ranges from 60 to 300 nm, preferably from 60 to 200 nm, and more preferably from 70 to 160 nm.

The second aspect of the present invention provides an inhalation preparation containing lipid nanoparticles for targeted lung delivery of nucleic acids, the inhalation preparation comprising a lipid nanoparticle dispersion system, the lipid nanoparticle dispersion system consisting of the following components: the above-mentioned lipid nanoparticles for efficient lung delivery of nucleic acids, a cryoprotectant, a system solution; and a surface tension regulator added to the lipid nanoparticle dispersion system.

The "cryoprotectant" mentioned in the present invention 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 include: sucrose, mannitol, trehalose, lactose, glucose, maltose, polyvinylpyrrolidone (PVP), polyethylene glycol, dextran, albumin and hydroxyethyl starch.

The term "surface tension modifier" as used herein refers to a compound or composition that can adjust the surface tension of a formulation and enhance the shear resistance of LNP during aerosolization, including but not limited to ethanol, propylene glycol, phenylethyl alcohol, poloxamer 188, Tween-80, glycerol, or a combination thereof. This list is not exhaustive; any surface tension modifier, whether known or unknown, that can improve the shear resistance of an inhalation formulation is within the scope of this invention.

In some specific embodiments of the present invention, the surface tension regulator is ethanol, and the mass volume percentage of ethanol in the lipid nanoparticle dispersion system ranges from 0.05% to 30%.

In some specific embodiments of the present invention, the surface tension regulator is propylene glycol, and the mass volume percentage of propylene glycol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%.

In some specific embodiments of the present invention, the surface tension regulator is a composition of propylene glycol and ethanol, the mass volume percentage of propylene glycol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%, and the mass volume percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%.

In some specific embodiments of the present invention, 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 to 10 mg/mL.

In some specific embodiments of the present invention, the surface tension regulator is a mixture of poloxamer 188 and ethanol, the concentration of poloxamer 188 in the lipid nanoparticle dispersion system is in the range of 0.5 to 10 mg/mL, and the mass volume percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%.

In some specific embodiments of the present invention, the surface tension regulator is Tween-80, and the mass volume percentage of Tween-80 in the lipid nanoparticle dispersion system is in the range of 0.01% to 2%.

In some specific embodiments of the present invention, the surface tension regulator is a mixture of Tween-80 and ethanol, the mass volume percentage of Tween-80 in the lipid nanoparticle dispersion system is in the range of 0.01% to 2%, and the mass volume percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%.

In some specific embodiments of the present invention, the mass volume percentage of the lipid nanoparticles in the dispersed 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 cryoprotectant ranges from 0% to 20%.

In some specific embodiments of the present invention, the system solution of the present invention includes, but is not limited to, physiological saline, 4-hydroxyethylpiperazineethanesulfonic acid (HEPEs) buffer, tris (hydroxymethylaminomethane) Tris buffer, TrisEDTA buffer, phosphate PB and phosphate PBS buffer, Dulbecco's phosphate (DPBS) buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tris buffer containing Tween (TBST), buffer containing EDTA and its sodium salt, and combinations of one or more of the above. It should be noted that the system solutions herein are not exhaustive, but are merely preferred. Any solution that regulates or buffers the osmotic pressure or pH of the system is within the scope of the present invention.

The third aspect of the present invention provides the use of the above-mentioned lipid nanoparticles or inhalation preparations in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids, wherein the pharmaceutical composition further comprises the drug and pharmaceutically acceptable excipients.

The "carried drug" described in the present invention includes one or more of nucleic acids, small molecule compounds, and proteins; in some specific embodiments of the present invention, the "carried drug" includes nucleic acids.

The "nucleic acid" of the present invention may be a nucleotide polymer of any length, including but not limited to single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), miRNA, siRNA, telomerase RNA, small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme, asymmetric interfering 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 a combination of one or more of a custom oligonucleotide.

In some specific embodiments of the present invention, the nucleic acid is mRNA. mRNA is a type of single-stranded RNA transcribed from a single strand of DNA as a template, carrying genetic information and guiding protein synthesis. mRNA can encode a single protein or multiple proteins simultaneously. Preferably, the mRNA is synthesized by in vitro transcription.

As used herein, "small molecules" refer to compounds that are not proteins or nucleic acid molecules. Small molecules can be therapeutic and/or prophylactic agents, such as antibiotics, anti-inflammatory drugs, anticancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungals, antiparasitics, anticonvulsants, antidepressants, anxiolytics, antipsychotics, lipid-lowering drugs, hypoglycemic drugs, and weight-loss drugs.

As used herein, "protein" refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary and/or quaternary structures. The secondary, tertiary and/or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions and/or van der Waals interactions. Additionally or alternatively, proteins may 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, and the like.

The pharmaceutical compositions of the present invention also contain pharmaceutically acceptable excipients. These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, typically at a pH of about 4-8, preferably about 5-7, although the pH may vary depending on the nature of the substance being formulated and the condition being treated. The formulated drug can be administered by inhalation.

The term "pharmaceutically acceptable" as used herein means that the drugs will not produce adverse, allergic or other untoward reactions when appropriately administered to animals or humans.

The "pharmaceutically acceptable excipients" described herein should be compatible with the active ingredient, meaning they can be blended with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of pharmaceutically acceptable excipients include 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; lyoprotectants; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; buffers, and combinations thereof. These substances are used to improve the stability of the formulation or to enhance the activity or bioavailability of the formulation, as needed.

The delivery method of "targeted lung delivery of nucleic acid" described in the present invention includes delivery through the respiratory tract, including but not limited to delivery of nucleic acid via inhalation or nasal drops.

The pharmaceutical composition for targeted lung delivery of nucleic acids of the present invention can be prepared as an inhalation formulation, such as a dry powder formulation, an aerosol formulation, an inhalation mist drop formulation, a nasal drop formulation, etc. The amount of the active ingredient administered is a therapeutically effective amount, for example, about 10 μg/kg to about 50 mg/kg body weight per day.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, any prior art methods, devices, and materials similar or equivalent to those in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

Unless otherwise noted, the experimental methods, detection methods, and preparation methods disclosed herein utilize conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise noted, the materials and equipment used herein are commercially available.

Example 1: Ionizable lipid screening

1. Preparation of mRNA-LNP

Ionizable lipids E-1, E-5, E-6, and E-11 are exemplarily selected, and their structural formulas are shown in Table 1. mRNA-LNPs are prepared according to the formula in Table 2. The mRNA-LNP preparation method comprises the following steps:

Step 1: The ionizable lipids in Table 1, DSPC (Avitor (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avitor (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG ₂₀₀₀ were dissolved in ethanol according to the ratio in Table 2 to prepare a lipid ethanol solution with a concentration of 20 mg/mL of Lipid.

Step 2: Prepare mRNA at a lipid nanoparticle (LNP) to mRNA mass ratio 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: The lipid ethanol solution obtained in step 1 and the mRNA solution are thoroughly mixed at a volume ratio of 1:5 to 1:1, and incubated for 20 minutes to obtain mRNA-LNP (lipid nanoparticles encapsulating mRNA).

Table 1 Structural formula of ionizable lipids

Table 2 mRNA-LNP formulation

2. In vitro atomization study

The mRNA-LNP prepared above was subjected to a nebulization study. The experimental method was as follows: the mRNA-LNP prepared above was dialyzed in a 20 mM HEPEs buffer solution (buffer) at pH 6 containing 10% sucrose, and then 50 μL was taken out respectively. The buffer was diluted 3 times with 6% g/ml ethanol (surface tension regulator), and atomized using a vibrating mesh nebulizer (model Aerogen solo Nebulizer System). The particle size and PDI of the mRNA-LNP before and after nebulization were characterized using a Malvern Zetasizer Nano ZS. The encapsulation efficiency (EE%) of mRNA before and after nebulization was determined using a Ribogreen RNA quantification kit (Thermo Fisher). The results are shown in Table 3 below.

Table 3 Particle size, PDI and EE of mRNA-LNP before and after atomization

Note: “pre” and “post” refer to samples before and after atomization, respectively.

The experimental results in Table 3 show that the LNPs prepared above can withstand aerosol shear damage before and after aerosolization. However, the particle size change before and after aerosolization of formulation 1 (E-1-1, E-5-1, E-6-1, E-11-1) is greater than that of formulation 2 (E-1-2, E-5-2, E-6-2, E-11-2), and the encapsulation efficiency is also slightly lower. However, the different ionizable lipids have no substantial effect on the aerosolization performance of the LNP formulations.

3. In vivo atomization study

E-1-1, E-5-1, E-6-1, E-11-1, E-5-2, and E-11-2 were selected to continue the in vivo mouse nebulization inhalation study. Bal/c mice, 6 to 8 weeks old, were selected. Luciferase mRNA was encapsulated in the preparation and administered by nebulization using a vibrating mesh nebulizer. The dosage was 20 μg/mouse. Mice were imaged 6 hours later. Before imaging, mice were injected with D-luciferin sodium substrate. 5 minutes after the injection, the mice were dissected, and their hearts, livers, spleens, lungs, and kidneys were removed for fluorescence imaging, as shown in Figure 1.

Experimental results showed that while E-1-1, E-5-1, E-6-1, and E-11-1 could withstand aerosol shear damage in vitro, they had difficulty breaking through the lung's physiological barrier in vivo, resulting in low protein expression. However, corresponding LNP formulations prepared using E-5-2 and E-11-2 were able to withstand aerosol shear damage while also breaking through the lung's physiological barrier, effectively mediating target protein expression in the lungs. This suggests that, after inhalation administration, these LNP inhalation formulations can achieve high local concentrations of nucleic acid drugs in the lungs, thereby effectively mediating gene transfection in the lungs.

Example 2: Screening of helper lipids

1. In vivo atomization study

Using E-1 as the ionizable lipid, mRNA-LNP was prepared according to the prescription in Table 4 and the mRNA-LNP preparation method in Example 1. The obtained product was subjected to an in vivo atomization study according to the in vivo atomization study method in Example 1. The results are shown in FIG2 .

Table 4 mRNA-LNP formulation

From the experimental results shown in FIG2 , it can be seen that different auxiliary lipids have no substantial effect on the atomization effect of mRNA-LNP.

2. Comparative experiment

Using E-1 as the ionizable lipid, mRNA-LNP was prepared according to the formulation in Table 5 and the mRNA-LNP preparation method in Example 1, and the LNP particle size, PDI and encapsulation efficiency were measured as shown in Table 6.

Table 5 mRNA-LNP formulation

Table 6 Particle size, PDI and EE of mRNA-LNP before and after atomization

In vivo nebulization studies were conducted according to the experimental method in Example 1, and the results are shown in Figure 3. Experimental Conclusion: After replacing DSPC with DOTAP or adding additional DOTAP, the corresponding protein was almost not expressed in the lungs after nebulized administration. In the comparative experimental formulation, the introduction of DOTAP not only affected the LNP's ability to break through the physiological barrier of the lungs, but also affected the LNP's escape from lysosomes, thus preventing efficient delivery of mRNA to the lungs and affecting the expression of the corresponding protein in the lungs.

Example 3: PEG lipid content screening

3.1 Preparation of mRNA-LNPs

mRNA-LNPs were prepared according to the formulation in Table 7 and the preparation method in Example 1, and the particle size, PDI, and encapsulation efficiency of mRNA-LNPs were measured as shown in Table 8.

Table 7 mRNA-LNP formulation

Table 8 Particle size, PDI and EE of mRNA-LNP before and after atomization

3.2 In vivo aerosolization studies

The obtained mRNA-LNP product was subjected to in vivo aerosolization study according to the in vivo aerosolization study method in Example 1, and the results are shown in Figure 4. As can be seen from the experimental results shown in Figure 4, within a certain molar percentage range, as the PEG lipid content increases, the expression level in the mouse lungs is increased to a certain extent. However, exceeding a certain molar percentage, the lung expression level decreases instead. The increase in PEG content can help LNP break through the lung mucus layer, but it also affects cell endocytosis. Therefore, a suitable PEG lipid content is more conducive to the lung delivery of LNP.

Example 4: Screening of ionizable lipids, cholesterol, and auxiliary lipid contents

Preparation of mRNA-LNP and in vivo aerosolization study

4.1 E-11 was selected as the ionizable lipid, and mRNA-LNPs were prepared according to the formulation in Table 9. In vivo aerosolization studies were performed. The results are shown in Figure 5 : Formulations E-11-1, E-11-2, and E-11-4 had high transfection efficiencies and could all highly express the corresponding proteins. Formulation E-11-3 had poor transfection efficiency and low protein expression.

Table 9 mRNA-LNP formulation

4.2 E-5 was selected as the ionizable lipid, and mRNA-LNPs were prepared according to the formulation in Table 10. The particle size, PDI, and EE were measured as shown in Table 11. In vivo aerosolization studies were performed, and the results are shown in Figure 6. All formulations can express a certain amount of protein, but the mRNA-LNP preparations prepared by E5-1-1, E5-1-2, and E5-1-3 have smaller PDI, more uniform particle size distribution, higher encapsulation efficiency, and higher protein expression.

Table 10 mRNA-LNP formulation

Table 11 Particle size, PDI and EE of mRNA-LNP before and after atomization

The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims (9)

A lipid nanoparticle for targeted lung delivery of nucleic acids, characterized in that the lipid nanoparticle comprises the following components in molar percentages: Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 45-65 mol%; Helper lipids: 10-30 mol%; Structural lipids: 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; G ₁ and G ₂ are each independently a C1-C10 alkylene group; R ₁ , R ₂ , R ₃ , and R ₄ are each independently H, a C1-C20 linear or branched alkane group, or a C2-C20 linear or branched alkene group; G ₃ is a C1-C10 alkylene group; or G ₃ is (CH ₂ ) _a -O-(CH ₂ ) _b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer of 2-10; L ₁ is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L ₂ is -NH(C=O)O- or -O(C=O)NH-. The lipid nanoparticle according to claim 1, characterized in that it comprises one or more of the following features: (1) The structural lipid is cholesterol; (2) The helper lipid is DSPC, DOPE, or a combination of DSPC and DPPC; (3) the PEG-lipid is PEG-DMG; (4) The particle size of the lipid nanoparticles ranges from 60 to 300 nm. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle for targeted lung delivery of nucleic acid comprises the following molar percentages of each component: Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 50-65 mol%; Helper lipids: 10-30 mol%; Structural lipids: 15-25 mol%; PEG-lipid: 0.5-2 mol%. An inhalation preparation comprising the lipid nanoparticles for targeted lung delivery of nucleic acids according to claim 1, characterized in that the inhalation preparation comprises a lipid nanoparticle dispersion system, which is composed of the following components: the lipid nanoparticles for targeted lung delivery of nucleic acids, a cryoprotectant, a system solution; and a surface tension regulator added to the lipid nanoparticle dispersion system. The inhalation preparation according to claim 4, characterized in that the surface tension modifier comprises: a mixture of one or more of ethanol, propylene glycol, phenylethanol, poloxamer 188, Tween-80, and glycerol; the preferred surface tension modifier is ethanol, and further preferably, the mass volume percentage of ethanol in the lipid nanoparticle dispersion system is in the range of 0.05% to 30%. The inhalation preparation according to claim 4, characterized in that it includes one or more of the following features: (1) The mass volume percentage of the lipid nanoparticles in the dispersion system is in the range of 0.0025% to 10%, the concentration of the system solution is in the range of 0 to 1000 mM, and the mass volume percentage of the cryoprotectant is in the range of 0% to 20%; (2) The system solution includes: physiological saline, 4-hydroxyethylpiperazineethanesulfonic acid HEPEs buffer, tris (hydroxymethylaminomethane) Tris buffer, TrisEDTA buffer, phosphate PB and phosphate PBS buffer, Dulbecco's phosphate DPBS buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tris buffer containing Tween (TBST), buffer containing EDTA and its sodium salt, and a combination of one or more of the above. Use of the lipid nanoparticles according to any one of claims 1 to 3 or the inhalation formulation according to any one of claims 4 to 6 in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids; the pharmaceutical composition further comprises the drug and a pharmaceutically acceptable excipient. The use according to claim 7, characterized in that the pharmaceutical composition delivers nucleic acid to the lungs by inhalation or nasal drops. The use according to claim 7, wherein the carried drug comprises one or more of nucleic acids, small molecule compounds, and proteins.