An inhaled preparation, iterative optimization process and its applications

Abstract

The present invention relates to the technical field of drug delivery, and particularly relates to an inhalable preparation, an iterative optimization process of the inhalable preparation, and its application in the preparation of drugs for treating respiratory and / or pulmonary diseases. By selecting specific ionizable lipids and optimizing and screening co-lipids, structural lipids, and PEG-lipids, an inhalable lipid nanoparticle formulation is obtained. Then, the buffer system and excipients are optimized and screened, and based on the optimized buffer system and excipients, the corresponding relationship between the composition and biological activity of the lipid nanoparticles is studied to obtain an optimized inhalable preparation formulation. This inhalable preparation can break through the pulmonary physiological barrier, achieve efficient delivery of nucleic acid therapeutic molecules to the lungs, and has good tolerance to atomization shear damage, biological safety, and transfection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and particularly to an inhaled preparation, an iterative optimization process and its application. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease, in which the lung tissue of patients shows varying degrees of inflammation, scarring and extensive pulmonary fibrosis characteristics. At present, the only marketed drugs for treating IPF are pirfenidone and nintedanib, and most other drugs are in the preclinical research or clinical trial stage, and the treatment effect is limited and difficult to meet the clinical needs.

[0003] An inhaled preparation refers to a preparation that delivers a drug to the respiratory tract and / or lungs through inhalation to exert a local or systemic effect, which can maximize the deposition of the drug in the lungs and promote the rapid absorption of the drug, and is particularly suitable for lung diseases.

[0004] Nucleic acid drugs are the third generation of new drugs following small molecule and antibody drugs in recent years. Theoretically, they are not restricted by the druggability of target proteins and have a wider application scenario. However, due to the deficiencies of nucleic acids themselves, such as easy degradation, difficult cell uptake and low transfection efficiency, etc., it is necessary to develop safe and effective delivery vectors. At present, lipid nanoparticles (LNPs) have become 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.

[0005] Currently, the administration of LNP nucleic acid drugs in clinical practice usually adopts the intramuscular injection method. The vast majority of LNPs are distributed at the intramuscular injection site after injection, and some LNPs are also distributed in the liver. Due to the first-pass effect in the liver, this greatly limits the access of LNPs to other organs. At present, inhaled administration is becoming a new research direction for nucleic acid drug delivery. Since the lung has a large surface area and can avoid the first-pass effect, the absorption rate of the drug can be greatly improved.

[0006] However, delivering nucleic acid drugs by inhalation poses great challenges: on the one hand, it is necessary to withstand the shear damage generated during the atomization process (shear damage will affect the nanostructure of lipid nanoparticles, resulting in the aggregation, sedimentation of nanoparticles and the leakage of the carried nucleic acid molecules); on the other hand, it is necessary to overcome the mucus barrier and evade macrophage phagocytosis in order to deliver nucleic acid therapeutic molecules to the lungs to exert therapeutic effects.

[0007] Based on the above challenges, only a few companies have conducted clinical trials on inhaled nucleic acid drugs, such as Arcturus (ARCT-032), Vertex pharmaceuticals (VX-522), and Recode Therapeutics (RCT1100). The drugs they developed are used to treat cystic fibrosis (CF) and primary ciliary dyskinesia (PCD), and no inhaled mRNA-LNP candidate drug has successfully completed clinical trials.

[0008] Therefore, there is still a need in the art to develop safer and more efficient inhaled formulations to meet clinical needs. Summary of the Invention

[0009] In view of the disadvantages of the above-mentioned prior art, the present invention provides an inhaled formulation and its iterative optimization process. This formulation can break through the pulmonary physiological barrier, achieve efficient delivery of therapeutic molecules to the lungs, and has good tolerance to atomization shear damage, biosecurity, and delivery efficiency, which has important guiding significance for the research and development of nucleic acid new drugs for treating respiratory and / or pulmonary diseases including IPF.

[0010] The first aspect of the present invention provides an inhalable lipid nanoparticle, comprising the following components in mole percentages:

[0011] Ionizable lipid: 50 - 70 mol%;

[0012] Helper lipid: 10 - 22 mol%;

[0013] Structural lipid: 19 - 30 mol%;

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

[0015] In some embodiments of the present invention, the structural lipid is cholesterol.

[0016] In some embodiments of the present invention, the helper lipid is DSPC.

[0017] In some embodiments of the present invention, the PEG-lipid is PEG-DMG.

[0018] In some embodiments of the present invention, the ionizable lipid is selected from one or more of AA3-Dlin, isomers of AA3-Dlin, ALC-0315, and isomers of ALC-0315.

[0019] In some embodiments of the present invention, the mole percentages of the ionizable lipid, helper lipid, structural lipid, and PEG-lipid are 60:20:19:1.

[0020] The second aspect of the present invention provides an inhalable preparation comprising the above-mentioned inhalable lipid nanoparticles, and the inhalable preparation further comprises a buffer solution and excipients added to the lipid nano-dispersion system.

[0021] In some embodiments of the present invention, the buffer solution is a HEPEs buffer solution; and / or, the excipients are a mixture of one or more of ethanol, propylene glycol, and poloxamer.

[0022] In some embodiments of the present invention, the pH of the HEPEs buffer solution is 6.

[0023] In some embodiments of the present invention, the excipient is ethanol, and the mass-volume percentage of ethanol in the lipid nano-dispersion system is 12%.

[0024] In some embodiments of the present invention, the excipient is poloxamer 188, and the concentration of poloxamer 188 in the lipid nano-dispersion system is 8 mg / mL.

[0025] The third aspect of the present invention provides an iterative optimization process for an inhalable preparation, comprising the following steps:

[0026] (1) Optimize and screen the molar percentages of ionizable lipids, co-lipids, structural lipids, and PEG-lipids of the lipid nanoparticles to obtain preferred lipid nanoparticles that enable the preparation to withstand atomization shear damage;

[0027] (2) Dialyze the preferred lipid nanoparticles obtained in step (1) in buffer solutions with different compositions and pH values to obtain a preferred buffer solution that further improves the preparation's tolerance to atomization shear damage;

[0028] (3) Add different types of excipients to the preferred buffer solution to obtain preferred excipients that enable the preparation to completely withstand atomization shear damage;

[0029] (4) Further optimize and screen the molar percentages of ionizable lipids, co-lipids, structural lipids, and PEG-lipids of the lipid nanoparticles using the preferred buffer solution and preferred excipients, and analyze the corresponding relationship between the lipid composition of the lipid nanoparticles and biological activity and target cell protein expression to obtain an optimized inhalable preparation.

[0030] The fourth aspect of the present invention provides the use of the above-mentioned inhalable lipid nanoparticles or inhalable preparation in the preparation of drugs for treating respiratory diseases and / or lung diseases.

[0031] In some embodiments of the present invention, the drug targets the lungs to deliver bioactive substances through the inhalation administration route; and / or, the lung disease is pulmonary fibrosis.

[0032] In some embodiments of the present invention, the bioactive substance includes one or more of nucleic acids, small molecule compounds, and proteins.

[0033] In some embodiments of the present invention, the nucleic acid is scFv antibody mRNA encoding IL-11.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention selects specific ionizable lipids and develops an inhalable preparation by optimizing the lipid nanoparticle formulation, buffer, and excipients. This preparation can break through the physiological barriers of the lungs, achieve efficient delivery of nucleic acid therapeutic molecules to the lungs, and has good tolerance to atomization shear destruction, biosafety, and transfection efficiency. Compared with antibody drugs administered via the inhalation route and intravenous injection route, the present invention uses inhalable LNP to deliver mRNA encoding IL-11 scFv to the lungs, realizing lung-targeted nucleic acid drug therapy and having better therapeutic effects.

[0036] 2. The iterative optimization process of the inhalable preparation provided by the present invention, through four-step optimization and studying the corresponding relationship between the lipid composition of lipid nanoparticles and the bioactive delivery efficacy, obtains inhalable lipid nanoparticles with the best shear destruction resistance and an inhalable preparation with the best stability and therapeutic effects, which has important guiding significance for the research and development of new nucleic acid drugs for the treatment of respiratory and / or lung diseases including IPF. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the principle of treating IPF by inhaled administration of scFv@iLNP-HP08 of the present invention LOOP

[0038] Figure 2 is the screening process of the LNP formulation and system solution of the inhalable preparation based on the "LOOP" mode in Example 1 of the present invention: a is a schematic diagram of the iterative optimization process of the inhalable preparation of the present invention; b-c are the lipid compositions of 7 LNP formulations in Example 1; d is the determination result of the mRNA encapsulation efficiency (EE) of LNP; e is the determination result of the average particle size of LNP; f is the determination result of the polydispersity index (PDI) of LNP; g is the influence of the atomization process on the stability of LNP dialyzed with different buffers.

[0039] Figure 3 ​This is the excipient screening process of the inhalable preparation based on the "LOOP" mode of the present invention: a-c show the changes in the average particle size and PDI of mLuc@iLNP with different excipient concentrations before and after atomization (n = 3); d is the IVIS (small animal imaging system) imaging of the mouse lungs and other organs (including the heart, liver, spleen, and kidney) collected six hours after atomization; mLuc@iLNP with different types and concentrations of excipients added are the experimental groups, and mLuc@iLNP without excipients added is the control group (n = 3); e is the quantitative analysis of the fluorescence signal of LNPs with excipients added in the lungs during atomization (n = 3); f is the surface tension values of the atomization buffer with different concentrations of excipients (including ethanol, poloxamer 188) added.

[0040] Figure 4 Regarding the influence of the atomization process on the stability of iLNP, iLNP was prepared using the cationic lipid ALC0315, keeping the molar percentage of the lipid components unchanged: a shows the change in the average particle size of iLNP before and after atomization measured by DLS (dynamic light scattering) (n = 3); b is the PDI of iLNP before and after atomization (n = 3); c is the representative images of the major organs of mice after inhaling mLuc@iLNP-HP08 LOOP (where the cationic lipid is ALC0315) (n = 3); iLNP was dispersed in the atomization buffer with or without 8 mg / mL poloxamer, and then atomized into the mouse lungs. The results are expressed as mean ± standard deviation, and significant differences were evaluated by two-tailed unpaired Student's t-test.

[0041] Figure 5 This is the present invention's optimization of the buffer, excipient, and LNP formulation with different lipid compositions by comparing the expression level of luciferase in the lungs after mice inhaled mLuc@iLNP-HP08 LOOP : a and b are respectively the representative images and normalized quantitative results of luciferase expression in the mouse lungs after inhaling LNPs with different formulations. The molar percentages between AA3-DLin, DSPC, cholesterol, and DMG-PEG2000 are shown in the upper left corner of the image (n = 3). The lipid ratios with the same color represent the same batch of animal experiments. For each animal experiment, the group with a lipid molar percentage of 60:20:19:1 was set as the control group. For other groups, the average luminescence intensity was normalized to the control group to obtain the relative quantitative results; c is the fold change in the average particle size of iLNP before and after atomization (n = 3). d is the composition of the three best-performing iLNP formulations; e is the composition of the three worst-performing iLNP formulations. The results are expressed as mean ± standard deviation, and significant differences were evaluated by two-tailed unpaired Student's t-test.

[0042] Figure 6 This is iLNP-HP08 of the present inventionLOOP Related performance study: a is the structure schematic diagram of mEGFP@iLNP-HP08 LOOP ; b is to incubate in PBS (pH 7.4) containing 10% FBS for 24 hours and measure the average particle size of mEGFP@iLNP-HP08 at specific time points (n = 3); c is the stability study of free mEGFP and LNP-loaded mEGFP under the condition of containing FBS (fetal bovine serum); d is to analyze the effect of 10% fetal bovine serum (FBS) on the transfection efficiency of LNP-mediated mRNA by Western blotting (n = 2); e is the dose-dependent cell transfection of mEGFP@iLNP-HP08 LOOP in MLFs (mouse lung fibroblasts) (n = 3); f is to study the endocytic pathway of mEGFP@iLNP-HP08 in MLFs (n = 3); g is the cell viability study of MLFs and A549 (human lung adenocarcinoma cells) treated with different concentrations of mEGFP@iLNP-HP08 LOOP ; h is to measure the encapsulation efficiency of mEGFP in mEGFP@iLNP-HP08 before and after atomization; i is the representative transmission electron microscope (TEM) image of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 200 μm; j is the measurement of the average particle size of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 50 μm; k is the representative image of Cyanine5 (Cy5)-mRNA@iLNP-HP08 LOOP escaping from lysosomes; l is the Pearson correlation coefficient between the mRNA fluorescence signal and lysosomes at different time points (n = 3); the data in b, i and j represent the results of two independent experiments, and the results are expressed as mean ± standard deviation; statistical significance differences are analyzed by two-tailed unpaired Student's t-test (h) or one-way analysis of variance (ANOVA) combined with Tukey test (e). LOOP ; h is to measure the encapsulation efficiency of mEGFP in mEGFP@iLNP-HP08 before and after atomization; i is the representative transmission electron microscope (TEM) image of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 200 μm; j is the measurement of the average particle size of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 50 μm; k is the representative image of Cyanine5 (Cy5)-mRNA@iLNP-HP08 LOOP escaping from lysosomes; l is the Pearson correlation coefficient between the mRNA fluorescence signal and lysosomes at different time points (n = 3); the data in b, i and j represent the results of two independent experiments, and the results are expressed as mean ± standard deviation; statistical significance differences are analyzed by two-tailed unpaired Student's t-test (h) or one-way analysis of variance (ANOVA) combined with Tukey test (e). LOOP ; h is to measure the encapsulation efficiency of mEGFP in mEGFP@iLNP-HP08 before and after atomization; i is the representative transmission electron microscope (TEM) image of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 200 μm; j is the measurement of the average particle size of mEGFP@iLNP-HP08 before and after atomization, with a scale bar of 50 μm; k is the representative image of Cyanine5 (Cy5)-mRNA@iLNP-HP08 LOOP escaping from lysosomes; l is the Pearson correlation coefficient between the mRNA fluorescence signal and lysosomes at different time points (n = 3); the data in b, i and j represent the results of two independent experiments, and the results are expressed as mean ± standard deviation; statistical significance differences are analyzed by two-tailed unpaired Student's t-test (h) or one-way analysis of variance (ANOVA) combined with Tukey test (e). LOOP escaping from lysosomes; l is the Pearson correlation coefficient between the mRNA fluorescence signal and lysosomes at different time points (n = 3); the data in b, i and j represent the results of two independent experiments, and the results are expressed as mean ± standard deviation; statistical significance differences are analyzed by two-tailed unpaired Student's t-test (h) or one-way analysis of variance (ANOVA) combined with Tukey test (e).

[0043] Figure 7 Purification of IL-11scFv (single-chain antibody fragment) using immobilized metal affinity chromatography (IMAC): a is to evaluate the purity of IL-11scFv by Coomassie Brilliant Blue staining; b is to verify the expression of IL-11scFv by Western blotting; the experiments were independently repeated three times to ensure the reliability of the results.

[0044] Figure 8 is the inhalable preparation scFv@iLNP-HP08 LOOPResults of inhibiting bleomycin-induced pulmonary fibrosis: a is the design of the animal experiment; b are representative images of lung tissues of mice in different treatment groups (n = 6); c is the calculation of lung coefficients of mice in different treatment groups (n = 6); d-f are representative immunofluorescence images of lung sections of mice in different treatment groups, quantitative analysis of COL1A1 and ACTA2 (n = 6), scale bar is 100 μm; g is the analysis of hydroxyproline content in mouse lung tissues (n = 6); h is the Western blot analysis of fibronectin, COL1A1, ACTA2 and IL-11 in mouse lung tissues (n = 3).

[0045] Figure 9 To measure the content of IL-11scFv in plasma (a) and whole blood (b) at different time points after intravenous injection of IL-11scFv by ELISA (enzyme-linked immunosorbent assay); for easy comparison, the concentration of IL-11scFv at each time point was normalized to the concentration 5 minutes after injection, and the experimental results were presented in the form of mean and standard deviation, which helped to analyze the distribution and metabolism of IL-11scFv in vivo.

[0046] Figure 10 To determine the concentration of IL-11scFv in bronchoalveolar lavage fluid at 24 hours and 48 hours after intravenous injection of IL-11scFv or inhalation of scFv@iLNP-HP08 by ELISA LOOP (n = 3); the results were expressed as mean ± standard deviation, and significant differences were analyzed by one-way analysis of variance (ANOVA) and Tukey's test.

[0047] Figure 11 For the restorative effect of the inhaled formulation scFv@iLNP-HP08 LOOP on the lung function of a bleomycin-induced pulmonary fibrosis mouse model: a is respiratory resistance (Rrs); b is lung elastance (Ers); c is inspiratory capacity (IC); d is lung compliance (Crs); e is forced vital capacity (FVC); f is static compliance (Cst); g is forced expiratory volume in 0.2 seconds (FEV0.2); h is the measurement of the pV loop (pressure-volume loop); (n = 6 for the PBS treatment group, n = 11 for the mLuc@iLNP-HP08 LOOP treatment group or scFv@iLNP-HP08 LOOP treatment group), the results were expressed as mean ± standard deviation, and significant differences were evaluated by one-way analysis of variance (ANOVA) and Tukey's test.

[0048] Figure 12 For healthy mice and fibrotic mice after inhalation of PBS, inhalation of mLuc@iLNP-HP08 LOOP, Inhaled IL-11scFv, intravenous injection of IL-11scFv, and inhaled scFv@iLNP-HP08 LOOP Organ coefficients after treatment (n = 6); results are expressed as mean ± standard deviation, and significant differences were evaluated by one-way analysis of variance (ANOVA) and Tukey's test.

[0049] Figure 13 In vivo safety assessments were performed on healthy and fibrotic mice after different treatments. Healthy C57BL / 6 mice and fibrotic mice were respectively administered inhaled PBS, inhaled scFv@iLNP-HP08 LOOP , Inhaled IL-11scFv, intravenous injection of IL-11scFv, or inhaled scFv@iLNP-HP08 LOOP Treatment: a. Tissue section staining of major organs of mice. Three days after the last treatment, major organs were collected, embedded, and cross-sectioned for hematoxylin and eosin (H&E) staining (n = 6). Data represent the results of three independent experiments; b-i. Hematological and biochemical analyses of blood samples. Parameters detected included creatinine (b), blood urea nitrogen (BUN) (c), alanine aminotransferase (ALT) (d), aspartate aminotransferase (AST) (e), white blood cell count (WBC) (f), red blood cell count (RBC) (g), hemoglobin (HGB) (h), and differential white blood cell count (W-SCC) (i) (n = 6); results are expressed as mean ± standard deviation, and significant differences were evaluated by one-way analysis of variance (ANOVA) and Tukey's test. Detailed implementation mode

[0050] The following further illustrates the inhalable lipid nanoparticles suitable for preparing inhalable preparations, the inhalable preparations containing the inhalable lipid nanoparticles, and the use of the above inhalable lipid nanoparticles and inhalable preparations in the preparation of drugs for treating respiratory diseases and / or lung diseases in combination with the accompanying drawings and specific examples.

[0051] The first aspect of the present invention provides an inhalable lipid nanoparticle, comprising the following components in mole percentages:

[0052] Ionizable lipid: 50 - 70 mol%;

[0053] Auxiliary lipid: 10 - 22 mol%;

[0054] Structural lipid: 19 - 30 mol%;

[0055] PEG-lipid: 0.5 - 2.5 mol%.

[0056] In some specific embodiments, the ionizable lipid is selected from one or more of AA3-Dlin, isomers of AA3-Dlin, ALC-0315, and isomers of ALC-0315.

[0057] As used in the present invention, "AA3-Dlin" refers to 2-[4-[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]piperazin-1-yl]ethyl (9Z,12Z)-octadeca-9,12-dienoate, CAS: 2832061-33-1. The molecular structure is shown as follows:

[0058]

[0059] As used in the present invention, "ALC-0315" refers to ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), CAS: 2036272-55-4. The molecular structure is shown as follows:

[0060]

[0061] The "isomers" include but are not limited to stereoisomers and tautomers.

[0062] The "stereoisomers" refer to isomers with the same atomic connection order but different spatial arrangements of atoms.

[0063] The "tautomers" refer to the phenomenon of equilibrium mutual conversion between the structures of two functional group isomers of a compound, and the corresponding isomers are called tautomers.

[0064] The "structural lipid" refers to a lipid containing a structure 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.

[0065] In some specific embodiments, the structural lipids include, but are not limited to: one or more combinations 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, cholestenone, 5α-cholestenone, and cholesteryl caprate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)butyl ether. This is not an exhaustive list, and the choice of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0066] In some specific embodiments, the structural lipids are one or more combinations of cholesterol, sitosterol, ergosterol, corticosteroids, and their derivatives.

[0067] In some specific embodiments, the structural lipid is cholesterol.

[0068] The types of the "auxiliary lipids" are not limited, and phospholipid lipids are preferred, including but not limited to: one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoyl phosphatidylglycerol.

[0069] In some specific embodiments, the helper lipid can be selected from one or a combination of more than one 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-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-docosanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-oleoyl-2-cholesteryl succinyl-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-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-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-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, sphingomyelin.

[0070] In some specific embodiments, the phosphatidylcholine is one or a combination of more than one of DSPC, DPPC, DMPC, DOPC, POPC.

[0071] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0072] As used herein, the "PEG-lipid" generally refers to a conjugate formed by chemically linking PEG (polyethylene glycol) with lipid molecules. It includes, but is not limited to, PEG-modified phospholipids and derived lipids, such as, by way of example, one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxypolyethylene glycol ditetradecylacetamide.

[0073] In some specific 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-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol dimethacrylate (PEG-DMA), PEG distearyl glycerol, PEG dipalmitoleyl, PEG dioleyl, PEG-based distearyl, PEG-based diacylglycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG-based phosphatidylethanol, PEG-based phosphatidylethylene glycol myristyloxypropyl-3-amine, PEG-based oxypropylolamine, 1,2-distearoyl oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159), or one or more combinations thereof.

[0074] In some specific embodiments, the PEG-lipid is PEG-DMG.

[0075] In some specific embodiments of the present invention, the weight-average molecular weight of PEG in the PEG-lipid is 1000 to 10000, such as 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, and preferably 2000.

[0076] In some specific embodiments of the present invention, the molar percentages of the ionizable lipid, co-lipid, structural lipid, and PEG-lipid are 60:20:19:1.

[0077] In the present invention, there is no special limitation on the particle size of the lipid nanoparticles, and it can be within the particle size range prepared conventionally in the art, preferably 60 to 300 nm.

[0078] In a second aspect of the present invention, there is provided an inhalable preparation comprising the above lipid nanoparticles, and the inhalable preparation further comprises a buffer solution and excipients added to the inhalable preparation dispersion system.

[0079] The "inhalable preparation dispersion system" of the present invention refers to a system that delivers a drug in the form of an aerosol or droplets to the respiratory tract and / or lungs to exert a local or systemic effect. The inhalable preparation dispersion system generally needs to have specific characteristics to ensure the effective delivery and absorption of the drug.

[0080] The "buffer solution" of the present invention refers to a solution that regulates or buffers the osmotic pressure or pH of the system, including but not limited to: physiological saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, Tris-EDTA buffer solution, phosphate buffer (PB) and phosphate-buffered saline (PBS) buffer solution, Dulbecco's phosphate-buffered saline (DPBS) buffer solution, citrate buffer solution, sulfate buffer solution, carbonate buffer solution, acetate buffer solution, Tris buffer containing Tween (TBST), buffer solution containing EDTA and its sodium salt, and a combination of one or more of the above. It should be noted that the system solutions here are not exhaustive, but only a preference. As long as a solution that regulates or buffers the osmotic pressure or pH of the system is within the protection scope of the present invention.

[0081] The "excipients" of the present invention refer to compounds or compositions that have a stability-enhancing effect and can enhance the shear tolerance of LNP atomization, including but not limited to: ethanol, propylene glycol, phenethyl alcohol, poloxamer 188, Tween-80, glycerol or a combination thereof. The above is not exhaustive. Whether known or unknown excipients, as long as they are compounds or compositions that can improve the shear damage resistance performance of LNP in the inhalable preparation, they are within the protection scope of the present invention.

[0082] In some specific embodiments, the buffer solution is HEPES buffer solution; preferably, the pH of the HEPES buffer solution is 6.

[0083] In some specific embodiments, the excipients are a mixture of one or more of ethanol, propylene glycol, and poloxamer. In some more specific embodiments, the excipient is ethanol, and the mass-volume percentage of ethanol in the inhalable preparation dispersion system is 12%. In some more specific embodiments, the excipient is poloxamer 188, and the concentration of poloxamer 188 in the inhalable preparation dispersion system is 8 mg / mL.

[0084] In some specific embodiments, the mass-volume percentage range of the lipid nanoparticles in the dispersion system is 0.0025% to 10%, and the concentration range of the buffer solution is 0 to 1000 mM.

[0085] In some specific embodiments, the inhalable preparation dispersion system further comprises a cryoprotectant. The "cryoprotectant" refers to a substance (usually a solution) that can protect cells from freezing damage; it can be classified into sugars, alcohols, amino acids, salts, etc.; specifically including but not limited to sucrose, mannitol, trehalose, lactose, glucose, maltose, polyvinylpyrrolidone (PVP), polyethylene glycol, dextran, albumin, and hydroxyethyl starch. Specifically, the mass-volume percentage range of the cryoprotectant in the dispersion system is 0-20%.

[0086] The third aspect of the present invention provides an iterative optimization process for the above-mentioned inhalable preparation, comprising the following steps:

[0087] (1) Optimize and screen the molar percentages of the ionizable lipid, co-lipid, structural lipid, and PEG-lipid of the lipid nanoparticles to obtain preferred lipid nanoparticles that enable the preparation to withstand atomization shear damage;

[0088] (2) Dialyze the preferred lipid nanoparticles obtained in step (1) in buffers with different compositions and pH values to obtain a preferred buffer that further improves the preparation's tolerance to atomization shear damage;

[0089] (3) Add different types of excipients to the preferred buffer to obtain preferred excipients that enable the preparation to completely withstand atomization shear damage;

[0090] (4) Further optimize and screen the molar percentages of the ionizable lipid, co-lipid, structural lipid, and PEG-lipid of the lipid nanoparticles using the preferred buffer and preferred excipients, and analyze the corresponding relationship between the lipid composition and ratio of the lipid nanoparticles and biological activity and target cell protein expression to obtain an optimized inhalable preparation.

[0091] In step (1), the "able to withstand atomization shear damage" specifically means being able to withstand the shear force generated during atomization, and the structure of the lipid nanoparticles remains intact, that is, after atomization, the encapsulation efficiency drops to more than 65% of that before atomization, the average particle size increases within 2 times, and the PDI increases within 2.5 times.

[0092] In step (2), the "further improve the tolerance to atomization shear damage" specifically means being able to resist aggregation caused by atomization, that is, after atomization, the encapsulation efficiency drops to more than 80% of that before atomization, the average particle size increases within 2 times, and the PDI increases within 2 times.

[0093] In step (3), the "completely withstand atomization shear damage" specifically means that after atomization, the encapsulation efficiency drops by less than 7%, the average particle size increases by less than 10 nm, and the PDI increases by less than 0.05.

[0094] In step (3), the "bioactivity" specifically refers to the ability of the bioactive substance loaded in the lipid nanoparticles to exert a biological effect in vivo. For example, when the bioactive substance is nucleic acid, the higher the nucleic acid encapsulation rate, the stronger the targeting property, and the higher the cell transfection efficiency, the higher its bioactivity.

[0095] In step (3), the "target cell protein expression" generally refers to the expression level of a specific protein in a target cell type. A drug can exert its effect by affecting the expression, activity, or function of the target cell protein.

[0096] The fourth aspect of the present invention provides the use of the above-mentioned lipid nanoparticles or inhalation formulations in the preparation of drugs for treating respiratory diseases and / or lung diseases.

[0097] The "respiratory diseases" as described in the present invention include, but are not limited to, one or more combinations of pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchitis, and emphysema.

[0098] The "lung diseases" as described in the present invention include, but are not limited to, one or more combinations of pneumonia, chronic obstructive pulmonary disease, tuberculosis, pulmonary fibrosis, and lung cancer. In some specific embodiments of the present invention, the lung disease is pulmonary fibrosis, more specifically idiopathic pulmonary fibrosis (IPF).

[0099] In some specific embodiments, the drug targets the lungs to deliver bioactive substances through the inhalation administration route. In some specific embodiments, the bioactive substances include one or more of nucleic acids, small molecule compounds, and proteins. In some specific embodiments of the present invention, the "bioactive substance" includes nucleic acids.

[0100] The "nucleic acids" as described in the present invention can be nucleotide polymers of any length. It includes, but is not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), miRNA, siRNA, telomerase RNA, snRNA, scRNA, circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotides, morpholino oligonucleotides, or one or more combinations of custom-designed oligonucleotides.

[0101] In some specific embodiments of the present invention, the nucleic acid is mRNA. By loading mRNA with lipid nanoparticles (LNP), mRNA-LNP is prepared and further formulated into an inhalable preparation, enabling mRNA-based lung-targeted protein therapy.

[0102] The "mRNA" in the present invention is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template, carrying genetic information and capable of guiding protein synthesis. mRNA can encode one protein or multiple proteins simultaneously. Preferably, mRNA is obtained by in vitro transcription synthesis.

[0103] In some preferred specific embodiments of the present invention, the nucleic acid is scFv antibody mRNA encoding IL-11. The single-chain fragment variable (scFv) antibody has several prominent advantages: including low molecular weight, enhanced permeability, high specificity and affinity for antigens, and minimal immunogenicity. The scFv single-chain antibody is usually administered intravenously. In the present invention, the mRNA technology is combined with an inhalable nucleic acid delivery system, and for the first time, an inhalable LNP delivery system is used to load scFv antibody mRNA encoding IL-11 to treat idiopathic pulmonary fibrosis (IPF), achieving mRNA-based lung-targeted protein therapy. As Figure 1 shown, the lipid nanoparticle formulation optimized through the iterative optimization process of the inhalable preparation has good stability and can withstand strong atomization shear damage. The optimized inhalable preparation (scFv@iLNP-HP08 LOOP ) is inhaled for the treatment of IPF. After inhalation, scFv@iLNP-HP08 LOOP enters the lung interstitium and is internalized by various cell types. The internalized scFv@iLNP-HPO8 LOOP escapes from the endosome, releases the therapeutic scFv mRNA into the cytoplasm, and is translated into IL-11scFv with the help of ribosomes. The secreted IL-11scFv binds to IL-11 in an autocrine or paracrine manner and blocks the activation of downstream signaling pathways, thereby producing a strong anti-fibrotic effect. And from the experimental results of Example 4, it can be seen that in a bleomycin-induced pulmonary fibrosis mouse model, intravenous injection of IL-11scFv and inhalation of scFv@iLNP-HP08 LOOP can both significantly inhibit fibroblast activation and the production of extracellular matrix (ECM), and the therapeutic effect of inhaled scFv@iLNP-HP08 LOOP is more significant. The lung function test in Example 5 shows that inhalation of scFv@iLNP-P08 LOOP can significantly improve all lung function parameters of fibrotic mice.

[0104] As used herein, 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 that is a therapeutic agent and / or a prophylactic agent, such as an antibiotic, an anti-inflammatory drug, an anti-cancer drug, an anti-viral drug, an immunosuppressant, an analgesic, an anti-fungal drug, an anti-parasitic drug, an anti-convulsant drug, an anti-depressant drug, an anti-anxiety drug, an anti-psychotic drug, a lipid-lowering drug, a hypoglycemic drug, a weight loss drug, etc.

[0105] As used herein, the "protein" of the present invention refers to a molecule or complex that contains one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are generally stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals interactions. Additionally, or alternatively, proteins can include disulfide bonds, such as between the 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.

[0106] The drug of the present invention further comprises a pharmaceutically acceptable excipient. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is typically about 4 - 8, preferably about 5 - 7, and the pH value can vary depending on the nature of the substance being formulated and the condition to be treated. The formulated drug can be administered by inhalation.

[0107] As used herein, "pharmaceutically acceptable" means that when the drugs are properly administered to an animal or a human, they do not produce adverse, allergic, or other untoward reactions.

[0108] As used herein, the "pharmaceutically acceptable excipient" should be compatible with the active ingredient, i.e., it can be blended with it without significantly reducing the effectiveness of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, 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; buffer solutions; etc.; and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help enhance the activity or its bioavailability.

[0109] The delivery methods of "targeted pulmonary delivery of bioactive substances" described in the present invention include respiratory tract delivery, including but not limited to intranasal, intratracheal or pulmonary administration for delivering bioactive substances. The drug is administered to cells, and the cells are at least one of a group of respiratory epithelial cells, nasal cells, alveolar epithelial cells, lung cells, bronchial epithelial cells, and a group of human bronchial epithelial (HBE) cells.

[0110] The drug for targeted pulmonary delivery of bioactive substances described in the present invention can be in liquid form or solid form, such as dry powder preparations, aerosol preparations, inhaled aerosol droplet preparations, nasal drop preparations, etc. Preferably, the dosage of the active ingredient 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. The drug can be administered by a nebulizer or an inhaler.

[0111] The "average particle size" described in the present invention refers to the hydrodynamic average particle size measured by the dynamic light scattering (DLS) method.

[0112] The "PDI" described in the present invention refers to the polydispersity coefficient or polydispersity index, and its English name is polydispersity index.

[0113] The "EE" described in the present invention refers to the encapsulation efficiency, and its English name is encapsulation efficiency.

[0114] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention.

[0115] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. Except for the specific methods, equipment, and materials used in the examples, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0116] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in this technical field. Unless otherwise stated, the materials and equipment used in the present invention are all commercially available.

[0117] Example 1: Screening of LNP formulations for inhalable formulations based on the "LOOP" mode

[0118] The iterative optimization process of the inhaled formulation ("LOOP" mode) is as shown in Figure 2 Figure a. The prescription screening steps of the inhaled lipid nanoparticles (iLNP) are as follows:

[0119] Step 1: The ionizable cationic lipid (AA3-Dlin), DSPC, cholesterol, and DMG-PEG were selected as the components of LNP for the experiment. Seven groups of LNP with different molar percentages (numbered LNP-1 to LNP-7) were prepared using microfluidic mixing technology. The mass ratio of AA3-Dlin to mRNA was 15:1 in all cases. After preparation, the LNP was allowed to stand at room temperature for 20 min, and then dialyzed in PBS (pH = 7.4) for 6 h ( Figure 2 as shown in Figures b and c). After dialysis, 50 μL was taken out respectively, diluted 3-fold with PBS, atomized using a vibrating mesh nebulizer device (model Aerogen solo Nebulizer System), and the average particle size, PDI, and EE before and after atomization were measured. Figure 2 Figure d shows the EE of LNP before and after atomization measured using the Ribogreen assay, while Figure 2 Figures e and f show the changes in the average particle size and PDI of LNP before and after atomization measured using dynamic light scattering (DLS), respectively. The LNP with the least decrease in EE after atomization, an increase in average particle size within 2-fold, and a PDI less than 0.15 before atomization was selected as the iLNP, namely LNP-3 (molar percentages of AA3-Dlin, DSPC, cholesterol, and DMG-PEG are 60:20:19:1).

[0120] Step 2: Screening of the system solution. LNP-3 was dialyzed using PBS (pH = 6.0 and 7.4) and HEPES buffer (pH = 6.0 and 7.4) respectively. Then, 50 μL of the dialyzed formulation was taken out respectively, diluted 3-fold with the corresponding buffer mentioned above, atomized using a vibrating mesh nebulizer device (model Aerogen solo Nebulizer System), and the changes in the average particle size, PDI, and EE before and after atomization were studied ( Figure 2 as shown in Figure g). Among them, in the system with HEPES pH = 6, the physicochemical properties of LNP-3 changed less before and after atomization. Therefore, this buffer was selected as the system solution for Step 3.

[0121] Step 3: Screening of excipients to further improve the performance of iLNP in tolerating atomization shear damage. Two alcohols approved by the FDA, ethanol and propylene glycol, and poloxamer 188 were selected to study the effect of the atomization process on the stability of iLNP. The experimental results are as shown in Figure 3As shown in a-c, compared with the control group containing only HEPEs, the introduction of these three excipients significantly reduced the average particle size and PDI change before and after atomization, indicating that the addition of excipients can improve the performance of iLNP to withstand atomization shear damage. The above-mentioned iLNP was delivered to the lungs of mice at a dose of 45 μg mRNA per three mice through a vibrating mesh nebulizer. After 6 hours, the main organs were selected, and the luciferase expression was observed using an IVIS imaging system. As Figure 3 shown in d and e, with the increase in the concentration of the excipient, the luciferase expression in the lungs also increased correspondingly, which was consistent with the enhanced stability of LNP. No chemiluminescence signal was detected in other organs.

[0122] In contrast, the atomization system containing 8 mg / mL poloxamer 188 had good safety, and the level of luciferase expression in the lungs was similar to that of the atomization system containing 12% ethanol, ensuring the good safety of the system.

[0123] Meanwhile, 8 mg / mL poloxamer 188 was further applied to another lipid preparation containing cationic ALC0315 while maintaining the molar percentage of each lipid component unchanged. The experimental results are as Figure 4 shown. Compared with the iLNP without excipient addition, the introduction of 8 mg / mL poloxamer 188 could significantly reduce the change in the average particle size of LNP ( Figure 4 a in Figure 4 ) and PDI ( Figure 4 b in

[0124] ) during atomization, and the luciferase expression in the lungs of mice increased significantly by 5 times ( Figure 4 c in

[0124] ), proving the versatility and universality of the developed atomization buffer. Step 4: Based on the established dialysis and atomization buffers, study the effect of lipid component changes on the luciferase expression in the lungs of mice and study the relationship between lipid composition and in vivo biological activity. Select the formulation with a molar percentage of 60:20:19:1 for each component lipid as the control reference, and then compare the bioluminescence intensity of the luciferase in the lungs of mice in other groups with that of the reference group to obtain the relatively quantitative results. As Figure 5As shown in a and b, experiments found that the DSPC content and the cationic lipid content are two key factors affecting the luciferase expression in the lungs, and the role of DSPC is more obvious. Specifically, the DSPC content in the three worst-performing formulations (60:5:34:1, 58:8:33:1, and 55:10:34:1) is all lower than 10 mol%. However, when the cationic lipid content is fixed, it is observed that increasing the DSPC content can significantly enhance the bioluminescence intensity of luciferase in lung tissue. Therefore, the first conclusion is drawn: the DSPC content in LNP must exceed 10 mol% to achieve ideal luciferase expression. When the DSPC content rises to 22 mol%, the luciferase expression in the lungs decreases, and at this time the cholesterol content drops to 17 mol%. By measuring the change in the average particle size before and after atomization, it is found that when the cholesterol content in LNP is lower than 19 mol%, the atomization stability of LNP decreases, and the change in the average particle size after atomization is significantly larger than that of the formulation with a lipid molar percentage of 60:20:19:1 ( Figure 5 in c). Therefore, the second conclusion is drawn: to minimize the impact of the atomization process on LNP, the cholesterol content in LNP must be maintained above 19%. Similarly, when the phospholipid content is fixed above 30 mol% and the cholesterol content is fixed above 19 mol%, adjusting the cationic lipid content between 43 mol% - 50 mol% shows no obvious change in luciferase expression. This indicates that a high cationic lipid content is also crucial for luciferase expression. Therefore, the third conclusion is drawn: maintaining the cationic lipid content above 50 mol% is a necessary condition for achieving good luciferase expression. Figure 5 Figures d and e in [reference] give the compositions of the three best-performing and three worst-performing iLNP formulations.

[0125] Example 2: Characterization of the atomized preparation LNP (iLNP-HP08 LOOP )

[0126] The iLNP structure provided by the present invention has the property of resisting atomization shear damage. According to the DLS results, there is no obvious change in the average particle size of iLNP before and after atomization ( Figure 6 in i and j). The fluorescence analysis results also show that the encapsulation rates of mRNA in iLNP before and after atomization are 94.60% and 92.77% respectively, indicating that the encapsulation rate of mRNA after atomization has not decreased significantly either ( Figure 6 in h). Transmission electron microscopy (TEM) images further confirm that the morphology of iLNP after atomization remains spherical ( Figure 6 in i). In addition, Figure 6The k in the formula indicates that the iLNP has the ability to rapidly escape from lysosomes, which is crucial for the iLNP to quickly release mRNA into the cytoplasm and subsequently translate it into proteins, demonstrating that the iLNP has excellent nucleic acid delivery ability.

[0127] Example 3: Stability and safety of the nebulized formulation (iLNP-HP08 LOOP )

[0128] To verify the protective effect of iLNP on mRNA encoding enhanced green fluorescent protein (mEGFP), mEGFP@iLNP-HP08 composed of mEGFP, AA3-DLin, DSPC, cholesterol, and DMG-PEG2000 was prepared by the microfluidic method in the experiment. LOOP ( Figure 6 Free mEGFP and mEGFP@iLNP-HP08 LOOP were incubated with fetal bovine serum (FBS) for 0 - 24 hours. The results showed that free mEGFP was rapidly degraded by FBS, while the mEGFP extracted from mEGFP@iLNP-HP08 LOOP still maintained its structural integrity after being incubated with FBS for 24 hours ( Figure 6 c) in the formula). In addition, by incubating mEGFP@iLNP-HP08 LOOP with PBS (pH 7.4) containing 10% FBS for different times, the effect of serum on the stability of iLNP was studied. The DLS results showed that the average particle size of mEGFP@iLNP-HP08 LOOP remained stable within 24 hours, indicating that iLNP-HP08 LOOP could maintain its structural stability under physiological conditions ( Figure 6 b) in the formula). The results of flow cytometry detection showed that the transfection efficiency of mEGFP@iLNP-HP08 LOOP showed a dose-dependent relationship ( Figure 6 e) in the formula).

[0129] A549 (type II alveolar epithelial cells) and MLFs were selected as model cells to verify the safety of iLNP for these cells. The results of the cell counting kit-8 (CCK8) detection showed that within the tested concentration range, even at the highest concentration of mEGFP = 1 μg / mL, no obvious cytotoxicity was exhibited ( Figure 6 g) in the formula).

[0130] Example 4: scFv@iLNP-HP08 LOOP Inhibiting bleomycin-induced pulmonary fibrosis

[0131] To verify scFv@iLNP-HP08 LOOPAnti-fibrotic effect. A mouse model of pulmonary fibrosis was established by single intratracheal injection of bleomycin. To obtain free IL-11scFv, IL-11scFv mRNA was transfected into HEK293 cells using PEI. After 3 days, the cell culture supernatant was collected and IL-11scFv was purified by nickel column affinity chromatography. Its purity was evaluated by Coomassie Brilliant Blue staining, and its expression was verified by western blotting( Figure 7 ). Mice were given different treatments every 3 days from day 10 to day 27( Figure 8 a), including intravenous injection of IL-11scFv, inhalation of PBS, inhalation of mLuc@iLNP-HP08 LOOP , inhalation of IL-11scFv, or inhalation of scFv@iLNP-HP08 LOOP . For mice in the IL-11scFv inhalation and intravenous injection groups, each mouse was given 100 μg of IL-11scFv. For mice in the scFv@iLNP hp08 LOOP group, each mouse was given 25 μg of IL-11scFv mRNA.

[0132] Preliminary histological examination showed obvious hemorrhagic necrosis in the lungs of mice after bleomycin stimulation. However, the lung injury in mice in the intravenous injection of IL-11scFv and inhalation of scFv@iLNP-HP08 LOOP groups was significantly reduced, and the latter showed more obvious improvement( Figure 8 b). Free IL-11scFv was rapidly cleared after intravenous injection( Figure 9 ), while scFv@iLNP-HP08 LOOP could still be detected in the bronchoalveolar lavage fluid within 48 hours after inhalation, and its expression level was significantly higher compared with the control group( Figure 10 ). The lung tissues of mice in each experimental group were weighed and the organ coefficient was calculated. The results showed that inhalation of scFv@iLNP-HP08 LOOP could effectively reverse the increase in wet weight of lung tissue caused by bleomycin( Figure 8 c).

[0133] Immunofluorescence staining images showed that intratracheal administration of bleomycin induced excessive myofibroblast differentiation and ECM deposition in the lungs. Fortunately, both aerosolized scFv@iLNP-HP08 LOOP and intravenous injection of IL-11scFv had significant alleviating effects, and the former had a more prominent effect( Figure 8In Fig. d-f). However, inhalation of IL-11scFv failed to produce the expected therapeutic effect, which may be due to the fact that free IL-11scFv cannot withstand the shear damage generated during the atomization process. Hydroxyproline content determination showed that bleomycin increased the hydroxyproline content in the wet lung tissue to 337.3 μg / lung, and scFv@iLNP-HP08 LOOP treatment significantly reduced it to 230.2 μg / lung ( Figure 8 In Fig. g). Consistent with the immunofluorescence results, Western blotting analysis showed that compared with the fibrotic mice treated with PBS and scFv@iLNP-HP08 LOOP , inhalation of scFv@iLNP-HP08 LOOP could significantly inhibit the expression of ACTA2, fibronectin, COL1A1 and IL-11 induced by bleomycin ( Figure 8 In Fig. h).

[0134] Example 5: Inhalation treatment with scFv@iLNP-HP08 LOOP can improve lung function

[0135] The forced oscillation technique was used in the experiment to detect the effect of scFv@iLNP-HP08 LOOP on improving lung function to explore its clinical application potential. At the end of the experiment (day 28), fibrotic mice induced by bleomycin and healthy mice were selected as negative and positive controls respectively to evaluate the recovery effect of scFv@iLNP-HP08 LOOP on lung function. After bleomycin exposure, the respiratory resistance (Rrs) and elastance (Ers) increased significantly, reflecting the impaired contraction and elastic rigidity of the respiratory system including the periphery, respiratory airways, chest wall and cellular tissues. Inhalation of scFv@iLNP-HP08 LOOP treatment significantly improved these parameters ( Figure 11 In Fig. a, b). In addition, compared with the healthy control group, the inspiratory capacity (IC), compliance (Crs), forced vital capacity (FVC), static compliance (Cst) and forced expiratory volume in 0.2 s (FEV0.2) of the mice after bleomycin exposure were significantly decreased. Inhalation of scFv@iLNP-HP08 LOOP treatment significantly restored these parameters, especially IC, Crs and Cst ( Figure 11 In Fig. c-g). After bleomycin exposure, the pressure-volume loop (PV-loop) showed a characteristic downward shift, but after inhalation of scFv@iLNP-HP08 LOOP , the pressure-volume loop (PV-loop) significantly shifted upward, indicating an increase in the intrinsic elasticity of the lungs ( Figure 11 In Fig. h).

[0136] Example 6: In vivo safety evaluation of inhaled nebulized formulations

[0137] The systemic toxicity that inhaled scFv@iLNP-HP08 LOOP might cause to bleomycin-induced pulmonary fibrosis mice was evaluated. Mice were sacrificed 3 days after the last inhalation, and their hearts, livers, spleens, kidneys and blood were collected to evaluate the safety of scFv@iLNP-HP08 LOOP . As Figure 12 shown, there were no significant differences in organ coefficients among the treatment groups. In addition, representative H&E staining images of major organs showed that inhaled scFv@iLNP-HP08 LOOP did not produce any obvious adverse reactions ( Figure 13 a) in LOOP . To further examine safety, biochemical tests were also performed, and no significant differences were observed in parameters such as creatinine (Crea), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB) and lymphocyte (W-SCC) levels between the healthy control group and fibrotic mice receiving various treatments, including inhalation of PBS, inhalation of mLuc@iLNP-HP08 LOOP , inhalation of IL-11scFv, intravenous injection of IL-11scFv and inhalation of scFv@iLNP-HP08 Figure 13 b-i) in

[0138] The above examples are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above examples without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A lipid nanoparticle for aerosol inhalation, characterized in that, It includes ionizable lipids, helper lipids, structural lipids and PEG-lipids; the structural lipid is cholesterol, the helper lipid is DSPC, and the PEG-lipid is PEG-DMG; the ionizable lipid is selected from one or more of AA3-Dlin, isomers of AA3-Dlin, ALC-0315, and isomers of ALC-0315; the molar percentages of the ionizable lipid, helper lipid, structural lipid and PEG-lipid are 60:20:19:1, 60:18:21:1, 55:20:24:1 or 55:25:19:

1.

2. An aerosol inhalation preparation comprising the aerosolizable lipid nanoparticles described in claim 1, characterized in that, The inhalable preparation further includes a buffer solution and excipients added to the dispersion system of the inhalable preparation; the buffer solution is a HEPEs buffer solution; the excipients are a mixture of one or more of ethanol, propylene glycol, and poloxamer.

3. The atomization inhalation preparation according to claim 2, characterized in that, It includes one or more of the following features: (1) The pH of the HEPEs buffer solution is 6; (2) The excipient is ethanol, and the mass-volume percentage of ethanol in the dispersion system is 12%; (3) The excipient is poloxamer 188, and the concentration of poloxamer 188 in the dispersion system is 8 mg / mL.

4. Use of the atomizable inhalable lipid nanoparticles according to claim 1 or the atomized inhalable preparation according to any one of claims 2 to 3 in the preparation of a drug for treating lung diseases.

5. The application according to claim 4, wherein The drug targets the lungs to deliver bioactive substances through the atomized inhalation administration route; and / or, the lung disease is pulmonary fibrosis.

6. The application according to claim 5, characterized in that, The bioactive substances include one or more of nucleic acids, small molecule compounds, and proteins.

7. The application according to claim 6, characterized in that The nucleic acid is scFv antibody mRNA encoding IL-11.

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