Lipid nanoparticles, inhalation preparations and applications for efficient delivery of nucleic acids to the lungs

Abstract

The present invention relates to the field of drug delivery technology, and in particular to lipid nanoparticles for efficiently delivering nucleic acids to the lungs, inhalation preparations containing the lipid nanoparticles, and the use of the lipid nanoparticles and inhalation preparations in preparing pharmaceutical compositions for efficiently delivering nucleic acids to the lungs. The present invention selects specific ionizable lipids, and optimizes and screens ionizable lipids, auxiliary lipids, structural lipids and PEG-lipids to prepare lipid nanoparticles for efficiently delivering nucleic acids to the lungs. The particle size and encapsulation rate of the nanoparticles before and after atomization do not change significantly. 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 efficiently mediate the expression of target proteins in the lungs; the inhalation preparation containing the lipid nanoparticles for efficiently delivering nucleic acids to the lungs can withstand the shear damage generated during the atomization process, overcome multiple physiological barriers, deliver the lipid nanoparticles to the lungs, and have a high transfection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid drug technology, and in particular to lipid nanoparticles, inhalation preparations and applications for efficiently delivering nucleic acids to the lungs. Background Art

[0002] Inhalation preparations refer to preparations that deliver drugs to the respiratory tract and / or lungs through inhalation to exert local or systemic effects. Traditional inhalation preparations are mainly small molecule drugs. With the development of biopharmaceuticals and genetic engineering technology in recent years, a large number of peptide, protein, and nucleic acid macromolecular drugs have emerged, providing a broader application space for inhalation preparations.

[0003] Nucleic acids are a type of electronegative macromolecule that is easily degraded and has low cellular uptake and transfection efficiency, so it is necessary to develop safe and effective delivery vectors to protect nucleic acid drugs from degradation and promote cellular uptake. Currently, lipid nanoparticles (LNPs) have become one of the mainstream delivery vectors for nucleic acid drugs due to their unique structure and physicochemical properties, showing high delivery efficiency and good safety in vivo.

[0004] At present, LNP is usually administered clinically by intramuscular injection. In addition to the vast majority of LNPs distributed in the local intramuscular injection site, some LNPs are also distributed in the liver. Due to the first-pass effect in the liver, this greatly limits the LNP from reaching other organs. In contrast, the lungs have a large surface area and can avoid the first-pass effect, greatly improving the absorption of drugs. Therefore, the lung delivery of nucleic acid drugs has become a key breakthrough direction for LNP.

[0005] The lung delivery of nucleic acid drugs is full of great challenges: 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 barriers, ciliary clearance, and macrophage phagocytosis) in order to deliver nucleic acid therapeutic molecules to the lungs to exert their therapeutic effects.

[0006] In the face of the above challenges, some companies in this field have been developing corresponding solutions, such as Recode's SORT delivery system. The LNP preparation is composed of ionizable lipids, DOTAP, cholesterol, auxiliary phospholipids and PEGylated lipids to achieve targeted delivery to the lungs, but DOTAP is a permanent cation and has certain toxicity to the human body. The formula of the atomized LNP preparation of Cremaide is ionizable lipid AX4 / DSPC / cholesterol / DMG-PEG, but LNPs prepared with high cholesterol content must have a high encapsulation rate. In addition, the evaluation of the atomized LNP is mainly based on the physical and chemical properties of the LNP detected in vitro, and the in vivo expression effect of mRNA-LNP after mouse lung administration through the atomization device has not been tested, and its in vivo effectiveness cannot be proved.

[0007] Therefore, there is still a need in this field to develop safer and more efficient inhalation preparations to meet the clinical needs of lung-targeted nucleic acid drugs. Summary of the invention

[0008] 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. The 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.

[0009] To achieve the above objectives and other related objectives, the present invention provides lipid nanoparticles for targeted lung delivery of nucleic acids, an inhalation preparation comprising the lipid nanoparticles for targeted lung delivery of nucleic acids, and the use of the lipid nanoparticles and inhalation preparation in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids.

[0010] The first aspect of the present invention provides the lipid nanoparticles for targeted lung delivery of nucleic acid, wherein the lipid nanoparticles include the following components in molar percentages:

[0011] Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 45-65 mol%;

[0012] Helper lipids: 10-30 mol%;

[0013] Structural lipids: 15-30 mol%;

[0014] PEG-lipid: 0.5-2.5 mol%;

[0015] The ionizable lipid has the following structure:

[0016]

[0017] wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0018] G1 and G2 are each independently a C1-C10 alkylene group;

[0019] R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group;

[0020] G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0021] L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

[0022] L2 is -NH(C=O)O- or -O(C=O)NH-.

[0023] 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.

[0024] 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.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 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 for efficient delivery of nucleic acids to the lungs. The lipid nanoparticles have good atomization stability, and there is no obvious change in the particle size and encapsulation rate of the nanoparticles 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.

[0027] 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 has a high transfection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in Example 1 of the present invention.

[0029] Figure 2 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in Example 2 of the present invention.

[0030] Figure 3 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in the comparative experiment in Example 2 of the present invention.

[0031] Figure 4 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in Example 3 of the present invention.

[0032] Figure 5 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in 4.1 in Example 4 of the present invention.

[0033] Figure 6 This is the fluorescence imaging result of the in vivo atomization research experiment of the mRNA-LNP prepared in 4.2 of Example 4 of the present invention. DETAILED DESCRIPTION

[0034] To address the deficiencies of the prior art, the object of the present invention is to provide a lipid nanoparticle for targeted lung delivery of nucleic acids, an inhalation preparation comprising the lipid nanoparticle for targeted lung delivery of nucleic acids, and the use of the above lipid nanoparticles and inhalation preparation in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids.

[0035] In order to achieve the above object, the present invention adopts the following technical solution:

[0036] The first aspect of the present invention provides a lipid nanoparticle for targeted lung delivery of nucleic acid, wherein the lipid nanoparticle comprises the following components in molar percentages:

[0037] Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 45-65 mol%;

[0038] Helper lipids: 10-30 mol%;

[0039] Structural lipids: 15-30 mol%;

[0040] PEG-lipid: 0.5-2.5 mol%;

[0041] The ionizable lipid has the following structure:

[0042]

[0043] wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0044] G1 and G2 are each independently a C1-C10 alkylene group;

[0045] R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group;

[0046] G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0047] L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

[0048] L2 is -NH(C=O)O- or -O(C=O)NH-.

[0049] The "C1-C20 straight-chain or branched alkane group", "C2-C20 straight-chain or branched olefin group" and "C1-C10 alkylene group" mentioned in the present invention are as described in

[0047] ,

[0048] and

[0049] of the specification of Chinese patent application CN115947671A, respectively.

[0050] In some specific embodiments of the present invention, -CH(R1)R2 and -CH(R3)R4 in the ionizable lipid are as described in

[0019] ,

[0020] and

[0021] of the specification of Chinese patent application CN115947671A.

[0051] 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.

[0052] In some specific embodiments of the present invention, the ionizable lipid is selected from the group consisting of:

[0053]

[0054] The "isomers" include stereoisomers and tautomers.

[0055] The term "stereoisomer" refers to isomers that have the same sequence of atoms connected but different arrangements of atoms in space.

[0056] 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.

[0057] The "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 free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of compounds of the present invention can be converted into their free bases or acids by standard techniques.

[0058] Pharmaceutically acceptable salts of the compounds of the 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 such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate heptanoate, 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. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and aryl sulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using suitable electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0059] The "structured lipids" mentioned above refer to lipids containing structures that can stabilize the composition, including but not limited to a combination of one or more of sterols and their derivatives and non-sterols and their derivatives.

[0060] In some specific embodiments, the structural lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterols, ergosterols, cholestanones, cholestenones, campesterols, stigmasterols, brassicasterols, tomatine, ursolic acid, coprosterols, α-tocopherols, 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 decanoic acid cholesterol ester; 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, the selection of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0061] In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0062] In some embodiments, the structured lipid is cholesterol.

[0063] The type of the "auxiliary lipid" is not limited, 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.

[0064] In some specific 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 (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), 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-docosahexanoyl-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-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexanoyl-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.

[0065] In some specific embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

[0066] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0067] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically a combination of DSPC and DPPC.

[0068] In some specific embodiments, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0069] The lipid nanoparticles provided by the present invention do not include (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP) in the lipids, which can ensure that the lipid nanoparticles deliver nucleic acids in a targeted manner to the lungs and avoid the cytotoxicity caused by permanent cationic lipids.

[0070] The "PEG-lipid" described in the present invention generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds, including but not limited to PEG-modified phospholipids and derived lipids, such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecyl acetamide, or a combination of one or more thereof.

[0071] 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-DMA, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanol, PEG-phosphatidylethylmyristyloxypropyl-3-amine, PEG-oxypropyl alcoholamine, 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)](PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159).

[0072] In some embodiments, the PEG-lipid is PEG-DMG.

[0073] 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.

[0074] In some specific embodiments of the present invention, the lipid nanoparticles for targeted lung delivery of nucleic acid include the following molar percentages of each component:

[0075] Ionizable lipid or its isomer or pharmaceutically acceptable salt thereof: 50-65 mol%;

[0076] Helper lipids: 10-30 mol%;

[0077] Structural lipids: 15-25 mol%;

[0078] PEG-lipid: 0.5-2 mol%.

[0079] 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.

[0080] 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.

[0081] 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, polyvinyl pyrrolidone (PVP), polyethylene glycol, dextran, albumin and hydroxyethyl starch.

[0082] The "surface tension regulator" of the present invention refers to a compound or composition that can adjust the surface tension of the preparation and 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 known or unknown surface tension regulator, as long as it is a compound or composition that can improve the shear damage tolerance of the inhalation preparation, is within the scope of protection of the present invention.

[0083] 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%.

[0084] 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%.

[0085] 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%.

[0086] In some specific embodiments of the present invention, the surface tension regulator is poloxamer 188, and the concentration range of poloxamer 188 in the lipid nanoparticle dispersion system is 0.5-10 mg / mL.

[0087] In some specific embodiments of the present invention, the surface tension regulator is a mixture of poloxamer 188 and ethanol, the concentration range of poloxamer 188 in the lipid nanoparticle dispersion system is 0.5-10 mg / mL, and the mass volume percentage range of ethanol in the lipid nanoparticle dispersion system is 0.05%-30%.

[0088] 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%.

[0089] 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%.

[0090] 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 range of the system solution ranges from 0 to 1000 mM, and the mass volume percentage range of the cryoprotectant ranges from 0% to 20%.

[0091] 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 a combination of one or more of the above. It should be noted that the system solution here is not exhaustive, but only a preferred one. As long as the solution plays a role in regulating or buffering the osmotic pressure or pH of the system, it is within the protection scope of the present invention.

[0092] 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 carried drug and a pharmaceutically acceptable excipient.

[0093] The "carried drug" described in the present invention includes one or more of nucleic acid, small molecule compound, and protein; in some specific embodiments of the present invention, the "carried drug" includes nucleic acid.

[0094] The "nucleic acid" of the present invention can 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 (Telomerase RNA), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNAmimics, 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 biological custom oligonucleotide one or more combinations.

[0095] In some specific embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information and can guide protein synthesis. The mRNA can encode one protein or multiple proteins at the same time. Preferably, the mRNA is synthesized by in vitro transcription.

[0096] The "small molecule" of the present invention refers to a compound that is not a protein or nucleic acid molecule. The small molecule can be a small molecule of a therapeutic agent and / or a preventive 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 glucose-lowering drug, a weight-loss drug, etc.

[0097] "Protein" of the present invention 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. In addition, or alternatively, proteins may include disulfide bonds, such as 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.

[0098] The pharmaceutical composition of the present invention further comprises pharmaceutically acceptable excipients. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, and the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated drug can be administered by inhalation.

[0099] The term "pharmaceutically acceptable" as used herein means that when the drugs are properly administered to animals or humans, they will not produce adverse, allergic or other untoward reactions.

[0100] The "pharmaceutically acceptable excipients" described in the present invention should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable excipients can be alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers, such as Tween, etc.; wetting agents, such as sodium lauryl sulfate, etc.; surfactants; freeze-drying protectants; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; buffer solutions, etc., and combinations thereof. These substances are used to improve the stability of the formulation or help improve the activity or its biological effectiveness as needed.

[0101] 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.

[0102] The pharmaceutical composition for targeted lung delivery of nucleic acid of the present invention can be prepared into an inhalation preparation, such as a dry powder preparation, an aerosol preparation, an inhalation mist drop preparation, a nasal drop preparation, etc. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day.

[0103] 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.

[0104] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0105] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. Unless otherwise specified, the materials and equipment used in the present invention are all commercially available.

[0106] Example 1: Ionizable lipid screening

[0107] 1. Preparation of mRNA-LNP

[0108] Ionizable lipids E-1, E-5, E-6, and E-11 are selected as examples, 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:

[0109] Step 1: The ionizable lipids in Table 1, DSPC (Avito (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avito (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG 2000 Dissolve in ethanol according to the ratio in Table 2 to prepare a lipid ethanol solution (Lipid concentration is 20 mg / mL).

[0110] 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).

[0111] 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).

[0112] Table 1 Structural formula of ionizable lipids

[0113]

[0114] Table 2 mRNA-LNP formulation

[0115]

[0116] 2. In vitro atomization study

[0117] 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, and the buffer was diluted 3 times with 6% g / ml ethanol (surface tension regulator), and nebulized 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, and the encapsulation efficiency EE% of mRNA before and after nebulization was determined using a Ribogreen RNA quantitative assay kit (Thermo Fisher). The results are shown in Table 3 below.

[0118] Table 3 Particle size, PDI and EE of mRNA-LNP before and after atomization

[0119] Serial number Z-Ave(d.nm) PDI EE(%) E-1-1-pre 105.6 0.1253 95.6 E-1-1-post 123.6 0.2324 80.6 E-5-1-pre 108.2 0.1197 94.8 E-5-1-post 128.7 0.1972 81.2 E-6-1-pre 107.9 0.1093 95.3 E-6-1-post 127.2 0.1896 80.1 E-11-1-pre 106.1 0.1154 95.1 E-11-1-post 125.8 0.1895 80.8 E-1-2-pre 100.5 0.0872 98.5 E-1-2-post 104.7 0.1023 92.1 E-5-2-pre 102.6 0.0798 98.6 E-5-2-post 107.8 0.1124 91.8 E-6-2-pre 101.3 0.0833 98.7 E-6-2-post 105.9 0.1202 92.3 E-11-2-pre 102.1 0.0721 98.5 E-11-2-post 104.7 0.1193 91.9

[0120] Note: “pre” and “post” refer to samples before and after atomization, respectively.

[0121] The experimental results in Table 3 show that the LNPs prepared above can withstand atomization shear damage before and after atomization, but the particle size change of prescription 1 (E-1-1, E-5-1, E-6-1, E-11-1) before and after atomization is higher than that of prescription 2 (E-1-2, E-5-2, E-6-2, E-11-2), and the encapsulation efficiency is also slightly lower. However, different ionizable lipids have no substantial effect on the atomization effect of LNP preparations.

[0122] 3. In vivo atomization study

[0123] 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 aerosol inhalation study. Bal / c mice, 6 to 8 weeks old, were selected. Luciferase mRNA was encapsulated in the preparation and aerosolized with a vibrating mesh atomizer. The dosage was 20 μg / mouse. Mice were imaged 6 hours later. Before imaging, the 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 taken for fluorescence imaging. Figure 1 shown.

[0124] The experimental results show that although E-1-1, E-5-1, E-6-1, and E-11-1 can withstand atomization shear damage in vitro, it is difficult to break through the physiological barrier of the lung in vivo, and the protein expression is low. The corresponding LNP formulations prepared using E-5-2 and E-11-2 can break through the physiological barrier of the lung while withstanding atomization shear damage, and efficiently mediate the expression of the target protein in the lung, which shows that after inhalation administration, the LNP inhalation preparation can make the nucleic acid drug have a higher local concentration in the lung, and then efficiently mediate gene transfection in the lung.

[0125] Example 2: Screening of helper lipids

[0126] 1. In vivo atomization study

[0127] 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, and the obtained product was subjected to in vivo atomization study according to the in vivo atomization study method in Example 1. The results are as follows: Figure 2 shown.

[0128] Table 4 mRNA-LNP formulation

[0129] Serial number <![CDATA[E-1:DSPC:DPPC:Cholesterol:DMG-PEG 2000 ]]> E-1-D-1 60:20:0:19:1 E-1-D-2 60:15:5:19:1 E-1-D-3 60:10:10:19:1 E-1-D-4 60:5:15:19:1 E-1-D-5 60:2.5:17.5:19:1 E-1-D-6 60:0:20:19:1

[0130] Depend on Figure 2 The experimental results show that different auxiliary lipids have no substantial effect on the atomization effect of mRNA-LNP.

[0131] 2. Comparative experiment

[0132] Using E-1 as the 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 measured as shown in Table 6.

[0133] Table 5 mRNA-LNP formulation

[0134] Serial number <![CDATA[(Molar ratio) E-1: DSPC: Cholesterol: DMG-PEG 2000 : DOTAP]]> E-1-T-1 60:20:19:1:0 E-1-T-2 60:20:19:1:100 E-1-T-3 60:20:19:1:42.9 E-1-T-4 60:20:19:1:11.1 E-1-T-5 46.3:9.4:42.7:1.6:0 E-1-T-6 46.3:9.4:42.7:1.6:100 E-1-T-7 46.3:9.4:42.7:1.6:42.9 E-1-T-8 46.3:9.4:42.7:1.6:11.1 Serial number <![CDATA[E1:DOTAP:Cholesterol:DMG-PEG 2000 ]]> E-1-T-9 60:20:19:1 E-1-T-10 30:39:30:1

[0135] Table 6 Particle size, PDI and EE of mRNA-LNP before and after atomization

[0136] Serial number Z-Ave(d.nm) PDI EE(%) E-1-T-1 81.84 0.07081 95.2 E-1-T-2 99.96 0.2604 98.5 E-1-T-3 96.99 0.192 97.1 E-1-T-4 96.81 0.07916 79.6 E-1-T-5 62.95 0.09412 96.5 E-1-T-6 82.65 0.251 98.2 E-1-T-7 111.1 0.1867 96.6 E-1-T-8 96.03 0.1187 85.2 E-1-T-9 102.4 0.2283 94.7 E-1-T-10 51.07 0.2929 98.7

[0137] The in vivo atomization study was carried out according to the experimental method in Example 1. The results are as follows Figure 3As shown. Experimental conclusion: After replacing DSPC with DOTAP or adding additional DOTAP, the corresponding protein is almost not expressed in the lungs after aerosol administration. In the prescription of the comparative experiment, the introduction of DOTAP affects the LNP's breakthrough of the lung's physiological barrier on the one hand, and the escape of LNP from the lysosome on the other hand, thus failing to achieve efficient delivery of mRNA to the lungs and affecting the expression of the corresponding protein in the lungs.

[0138] Example 3: PEG lipid content screening

[0139] 3.1 Preparation of mRNA-LNP

[0140] 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 mRNA-LNP were measured as shown in Table 8.

[0141] Table 7 mRNA-LNP formulation

[0142] Serial number <![CDATA[E-6:DSPC:Cholesterol:DMG-PEG 2000 ]]> E-6-P-0.5 60:20:19.5:0.5 E-6-P-1 59:21:19:1 E-6-P-2 60:19:18.5:2.5 E-6-P-3 58:20:19:3

[0143] Table 8 Particle size, PDI and EE of mRNA-LNP before and after atomization

[0144] Serial number Z-Ave(d.nm) PDI EE(%) E-6-P-0.5 72.28 0.09952 84.60 E-6-P-1 79.57 0.04219 95.51 E-6-P-2 77.32 0.07564 88.45 E-6-P-3 71.08 0.1495 77.15

[0145] 3.2 In vivo aerosolization study

[0146] The obtained mRNA-LNP product was subjected to in vivo aerosolization study according to the in vivo aerosolization study method in Example 1. The results are as follows: Figure 4 As shown. Figure 4 The experimental results show that within a certain molar percentage range, as the PEG lipid content increases, the expression level in the mouse lungs increases to a certain extent. However, when it exceeds a certain molar percentage, the lung expression level decreases. Although the increase in PEG content can help LNP break through the lung mucus layer, it will also affect cell endocytosis. Therefore, the appropriate PEG lipid content is more conducive to the lung delivery of LNP.

[0147] Example 4: Screening of ionizable lipids, cholesterol, and auxiliary lipid contents

[0148] Preparation of mRNA-LNP and in vivo aerosolization study

[0149] 4.1 E-11 was selected as the ionizable lipid, and mRNA-LNP was prepared according to the prescription in Table 9, and an in vivo atomization study was performed. The results are as follows Figure 5 As shown: Prescriptions E-11-1, E-11-2, and E-11-4 have high transfection efficiency and can highly express the corresponding proteins, while prescription E-11-3 has poor transfection efficiency and low protein expression.

[0150] Table 9 mRNA-LNP formulation

[0151]

[0152]

[0153] 4.2 E-5 was selected as the ionizable lipid, and mRNA-LNP was prepared according to the formulation in Table 10. The particle size, PDI, and EE were measured as shown in Table 11, and an in vivo atomization study was performed. The results are shown in Figure 6 As shown: 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, uniform particle size distribution, higher encapsulation efficiency, and higher protein expression.

[0154] Table 10 mRNA-LNP formulation

[0155] Serial number Molar ratio: E5:DSPC:Cholesterol:DMG-PEG2000 E5-1-1 60:20:19:1 E5-1-2 55:25:19:1 E5-1-3 53:30:15:2 E5-1-4 47:33:19:1 E5-1-5 45:35:19:1 E5-1-6 50:22:25:3

[0156] Table 11 Particle size, PDI and EE of mRNA-LNP before and after atomization

[0157] Sample name Z-Ave(d.nm) PDI EE(%) E5-1-1 95.6 0.03089 96.0 E5-1-2 102.7 0.04968 96.2 E5-1-3 120.2 0.04332 89.3 E5-1-4 122 0.05773 88.3 E5-1-5 127 0.08739 79.7 E5-1-6 132.8 0.06228 85.3

[0158] 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 familiar with 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A lipid nanoparticle for targeted lung delivery of nucleic acid, characterized in that: The lipid nanoparticles include the following components in molar percentages: Ionizable lipid 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 lipids are selected from: The helper lipid is DSPC or a combination of DSPC and DPPC.

2. The lipid nanoparticle according to claim 1, characterized in that Includes one or more of the following characteristics: (1) The structural lipid is cholesterol; (2) the PEG-lipid is PEG-DMG; (3) The particle size of the lipid nanoparticles is in the range of 60 to 300 nm.

3. The lipid nanoparticle according to claim 1, characterized in that The lipid nanoparticles for targeted lung delivery of nucleic acid include the following components in molar percentages: 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%.

4. An inhalation preparation comprising the lipid nanoparticles for targeted lung delivery of nucleic acid according to claim 1, characterized in that: The inhalation preparation comprises a lipid nanoparticle dispersion system, which is composed of the following components: lipid nanoparticles for targeted lung delivery of nucleic acid, a cryoprotectant, a system solution; and a surface tension regulator added to the lipid nanoparticle dispersion system.

5. The inhalation preparation according to claim 4, characterized in that The surface tension regulator includes: a mixture of one or more of ethanol, propylene glycol, phenylethyl alcohol, poloxamer 188, Tween-80 and glycerol.

6. The inhalation preparation according to claim 5, characterized in that The surface tension regulator is ethanol.

7. The inhalation preparation according to claim 6, characterized in that The mass volume percentage range of ethanol in the lipid nanoparticle dispersion system is 0.05% to 30%.

8. The inhalation preparation according to claim 4, characterized in that Includes one or more of the following characteristics: (1) The mass volume percentage of the lipid nanoparticles in the dispersed 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.

9. Use of the lipid nanoparticles according to any one of claims 1 to 3 or the inhalation preparation according to any one of claims 4 to 8 in the preparation of a pharmaceutical composition for targeted lung delivery of nucleic acids; the pharmaceutical composition further comprises the drug contained therein and a pharmaceutically acceptable excipient.

10. The use according to claim 9, characterized in that The pharmaceutical composition delivers nucleic acid to the lungs through inhalation or nasal administration.

11. The use according to claim 9, characterized in that The drugs carried include one or more of nucleic acids, small molecule compounds, and proteins.

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