Ionizable cationic lipid-mediated pulmonary targeting nucleic acid delivery system and uses thereof

Abstract

The present application relates to ionizable cationic lipid-mediated pulmonary targeting nucleic acid delivery system and application. Specifically, the present application provides a kind of lipid nanoparticles, the lipid part of the lipid nanoparticles includes ionizable cationic lipid, phospholipid and polyethylene glycol lipid, wherein ionizable cationic lipid includes compound 1, and wherein the content of compound 1 is 80 mol% or more based on the total amount of lipid in the lipid nanoparticle. The lipid nanoparticle significantly improves the lung targeting efficiency and simplifies the formulation design process.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a lung-targeted nucleic acid delivery system mediated by ionizable cationic lipids and its applications. Background Technology

[0002] Messenger RNA (mRNA) therapy can be transiently translated into target proteins within the cytoplasm, avoiding genome integration, and has shown transformative potential in areas such as vaccines, protein replacement, and gene editing. Lipid nanoparticles (LNPs), serving as their core delivery carrier, are synergistically assembled from ionizable cationic lipids, helper phospholipids, cholesterol, and polyethylene glycol (PEG) lipids, efficiently encapsulating and protecting mRNA while mediating endosome escape. Currently, marketed or late-stage clinical mRNA-LNP products are still limited to liver and muscle tissue; the therapeutic potential of mRNA-LNPs in other organs has not yet been fully explored.

[0003] Extrahepatic organ delivery, especially lung delivery, holds significant promise for the treatment of lung infections, primary lung cancer, and metastatic lesions. As a complex and crucial target organ, the lungs require efficient and specific mRNA delivery, which can significantly improve therapeutic efficacy and minimize systemic side effects. Currently, various strategies for lung targeting have been explored: from a drug delivery perspective, in addition to local inhalation, systemic delivery strategies include: 1) incorporating additional cationic lipids (such as DOTAP) into lung nuclei (LNPs) to modulate surface charge and promote LNP accumulation in the lungs; 2) conjugating antibodies to the LNP surface for targeted uptake via receptor-mediated endocytosis; and 3) designing novel ionizable lipids to enhance LNP targeting selectivity for the lungs. However, both ionizable and permanent cationic lipids present safety challenges: ionizable lipids administered via the respiratory tract can easily induce local inflammatory responses; while permanent cationic lipids such as DOTAP, when injected intravenously, may cause systemic toxicity, making their safety profile unsuitable for clinical needs. Therefore, developing a biocompatible LNP system that combines high delivery efficiency with excellent safety remains a key issue that urgently needs to be addressed in the field.

[0004] The inventors are dedicated to further improving the safety and efficacy of lung-targeted mRNA delivery. First, based on the diazid compound lipid family, a novel ionizable lipid compound 1 was designed. Second, the inventors also established a simplified three-component lipid nanoparticle (stLNP, simplified targeted LNP) platform, which significantly improves lung-targeting efficiency while simplifying the formulation design process. Combining stLNP with mRNA engineering strategies enables selective expression of mRNA in specific cell types, thereby achieving precise treatment of lung tumors. The novel ionizable lipid compound 1, combined with the stLNP optimization platform, constructs a lung-targeted delivery system. The compound 1 stLNP disclosed in this invention is a new generation three-component formulation containing the aforementioned novel ionizable lipid compound 1. After system optimization, the lung-targeting efficiency of this formulation is more than 100 times higher than the unoptimized control, with a lung-targeting specificity exceeding 99%, and it exhibits excellent biocompatibility: even at a high dose of 12 mg / kg, no animal deaths were reported. Compared to DOTAP-based lung-targeting LNP systems, compound 1 stLNP exhibits superior lung delivery efficiency and lower toxicity. Furthermore, compound 1 stLNP demonstrates versatility across multiple RNA forms, enabling functional mRNA expression, base editing, and small interfering RNA (siRNA)-mediated gene silencing. Finally, therapeutic efficacy evaluation in a mouse model of acute lung injury (ALI) indicates that compound 1 stLNP holds significant potential as a clinical translational platform for lung mRNA therapy. Summary of the Invention

[0005] In a first aspect, the present invention provides lipid nanoparticles, wherein the lipid portion of the lipid nanoparticles comprises ionizable cationic lipids, phospholipids, and polyethylene glycol lipids, wherein the ionizable cationic lipids comprise compound 1, and wherein the content of compound 1 is 80 mol% or more based on the total amount of lipids in the lipid nanoparticles. In some embodiments, the lipid nanoparticles are lung-targeting. In some embodiments, a lung-targeting system comprising the lipid nanoparticles of the present invention is provided.

[0006] In some embodiments, the content of compound 1 is 80 mol% or more, 81 mol% or more, 82 mol% or more, 83 mol% or more, 84 mol% or more, 85 mol% or more, 86 mol% or more, 87 mol% or more, 88 mol% or more, 89 mol% or more, 90 mol% or more, 91 mol% or more, 92 mol% or more, 93 mol% or more, 94 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more.

[0007] In some embodiments, the content of compound 1 is less than 100 mol, less than 99 mol%, less than 98 mol%, less than 97 mol%, less than 96 mol%, less than 95 mol%, less than 94 mol%, less than 93 mol%, less than 92 mol%, less than 91 mol%, or less than 90 mol%.

[0008] In some embodiments, the content of compound 1 is 85 mol% to 95 mol%, 86 mol% to 94 mol%, 87 mol% to 93 mol%, 88 mol% to 92 mol%, 89 mol% to 91 mol%, or 90 mol%.

[0009] In some embodiments, the lipid portion of the lipid nanoparticles is steroid-free. In some embodiments, the lipid portion of the lipid nanoparticles is cholesterol-free.

[0010] In some embodiments, the lipid portion of the lipid nanoparticles is composed of ionizable cationic lipids, phospholipids, and polyethylene glycol lipids.

[0011] In some embodiments, the phospholipid content is from 5 mol% to 20 mol%, based on the total amount of lipids in the lipid nanoparticles. In some embodiments, the phospholipid content is 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, or 20 mol%, based on the total amount of lipids in the lipid nanoparticles.

[0012] In some embodiments, the phospholipids comprise 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-diphydanoyl-sn-glycerol-3-phosphate choline (4ME). 16:0PC or 4ME), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate glycerol (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), or any mixture thereof.

[0013] In some embodiments, the phospholipid comprises POPE. In some embodiments, the phospholipid is POPE.

[0014] In some embodiments, the content of the polyethylene glycol lipids is from 0.1 mol% to 10 mol%, based on the total amount of lipids in the lipid nanoparticles. In some embodiments, the content of the polyethylene glycol lipids is 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 3.0 mol%, 4.0 mol%, 5.0 mol%, 6.0 mol%, 7.0 mol%, 8.0 mol%, 9.0 mol%, or 10.0 mol%, based on the total amount of lipids in the lipid nanoparticles.

[0015] In some embodiments, the polyethylene glycol lipid comprises 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), N-(methylpolyoxyethylene carbonyl)-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (PEG-DSPE), 1,2-oleoyl-sn-glycerol-3-phosphate ethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), or any mixture thereof.

[0016] In some embodiments, the polyethylene glycol lipoprotein comprises DMG-PEG. In some embodiments, the polyethylene glycol lipoprotein is DMG-PEG. In some embodiments, the polyethylene glycol lipoprotein is DMG-PEG2000.

[0017] In some embodiments, the lipid nanoparticles further comprise nucleic acids, wherein the N / P ratio of the lipid nanoparticles is between 1 and 20, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the N / P ratio of the lipid nanoparticles is between 5 and 15, between 8 and 12, or between 9 and 11.

[0018] In some embodiments, the nucleic acid is RNA, such as siRNA, sgRNA, or mRNA. In some embodiments, the nucleic acid encodes a therapeutic agent, such as a therapeutic agent for treating lung diseases.

[0019] In some embodiments, the nucleic acid encodes a cytokine, such as interleukin-10 (IL-10), interleukin-4 (IL-4), or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0020] In some embodiments, the lipid nanoparticles are prepared by dissolving nucleic acids in an acetate buffer. In some embodiments, the concentration of the acetate buffer is from 0 to 40 mM, for example, 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM. In some embodiments, the pH of the acetate buffer is between 2.0 and 6.0, for example, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, or 6.0.

[0021] In a second aspect, the present invention provides the use of the lipid nanoparticles according to the first aspect of the present invention in the preparation of a medicament for treating lung diseases.

[0022] In some embodiments, the lung disease is selected from lung infections, lung tumors, airway lung diseases, interstitial lung diseases, and pulmonary circulation diseases.

[0023] In some embodiments, the lung disease is a lung infection, such as pneumonia, bronchitis, or tuberculosis. In some embodiments, the lung disease is a lung tumor, such as lung cancer, including non-small cell lung cancer and small cell lung cancer. In some embodiments, the lung disease is an airway-lung disease, such as asthma, chronic obstructive pulmonary disease, or chronic bronchitis. In some embodiments, the lung disease is an interstitial lung disease, such as pulmonary fibrosis or pulmonary nodules. In some embodiments, the lung disease is a pulmonary circulation disease, such as pulmonary embolism or pulmonary hypertension.

[0024] In some implementations, the lung disease is acute lung injury.

[0025] Thirdly, the present invention provides a method for treating lung diseases, the method comprising administering lipid nanoparticles according to the first aspect of the present invention to a patient in need. Attached Figure Description

[0026] Figure 1A : Structural formula of compound 1.

[0027] Figure 1B Synthetic route of compound 1.

[0028] Figure 1C Preparation route of M-1.

[0029] Figure 1D Preparation route of M-2.

[0030] Figure 1E The reaction route for synthesizing compound 1 from M-2 and M-3.

[0031] Figure 1F HPLC chromatogram of compound 1.

[0032] Figure 1G Physicochemical properties of LNPs in each group during the lipid ratio optimization screening of compound 1.

[0033] Figure 2 The lung-targeting effect of each group of LNPs in mice during the lipid ratio optimization screening of compound 1.

[0034] Figure 3 Biocompatibility of four different formulations of LNP: G5, G6, G7, and G8.

[0035] Figure 4 Physicochemical properties of LNP formulations G8-1 to G8-5.

[0036] Figure 5 Lung-targeting effect of LNP in mice in groups G8-1 to G8-5.

[0037] Figure 6Physicochemical properties of different auxiliary lipids (LNPs).

[0038] Figure 7 The lung-targeting effects of different auxiliary lipids (LNPs) in mice.

[0039] Figure 8 Physicochemical properties of LNPs with different N / P ratios.

[0040] Figure 9 Lung-targeting effects of different N / P groups in mice.

[0041] Figure 10 Toxicity test of empty LNP at different doses of compound 1 stLNP.

[0042] Figure 11 H&E-stained tissue pathological sections were used to detect the toxicity of compound 1 stLNP.

[0043] Figure 12 Physicochemical properties of compound 1 stLNP prepared under different buffer conditions.

[0044] Figure 13 Lung-targeting effects of compound 1 stLNP prepared with different preparation buffers (B1-B6).

[0045] Figure 14 pK of compound 1 stLNP a Measurement.

[0046] Figure 15 Structure of compound 1 stLNP under cryo-electron microscopy.

[0047] Figure 16 Stability monitoring of compound 1 stLNP.

[0048] Figure 17 Comparison of lung delivery efficiency and organ specificity of compound 1 stLNP and DOTAP-5-LNP.

[0049] Figure 18 Comparison of in vivo toxicity of compound 1 stLNP and DOTAP-5-LNP.

[0050] Figure 19 Histopathological comparison of in vivo toxicity of compound 1 stLNP and DOTAP-5-LNP.

[0051] Figure 20 Analysis of the distribution of Cy5-mRNA delivered by compound 1 stLNP in mice and the proportion of different cell groups in the lungs.

[0052] Figure 21The effect of compound 1 stLNP delivering Cre mRNA on lung editing in Ai14 mice.

[0053] Figure 22 : Expression of compound 1 stLNP under single and multiple administrations.

[0054] Figure 23 Compound 1 stLNP mediates adenine base editor (ABE) to achieve gene editing.

[0055] Figure 24 The therapeutic effect of compound 1 stLNP@IL-10 mRNA on LPS-induced acute lung injury in mice.

[0056] Figure 25 HE-stained pathological sections of lung tissue from different treatment groups after acute lung injury mouse modeling.

[0057] Figure 26 Compound 1 stLNP efficiently delivered siRNA in mice, successfully mediating the lung knockdown of Tie2.

[0058] Figure 27 Compound 1 stLNP effectively silences the expression of the EGFP gene in the lungs of transgenic mice that stably express EGFP. Detailed Implementation

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the definitions herein shall prevail. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0060] In this invention, the terms "comprising," "including," or "having" mean that any of the listed elements must be included, and other elements may optionally be included. The term "consisting of" means that all unlisted elements are excluded. The terms "comprising," "including," or "having" cover "consisting of" or "substantially consisting of."

[0061] In this invention, the term "about" or "approximately," when applied to one or more values ​​of interest, refers to a value that is close to or within an acceptable error range of the stated reference value, the acceptable error range being determined by those skilled in the art and depending in part on how the value is measured or determined, such as limitations of the measurement system. In one instance, the term "about" refers to any value within a variation of up to ±10% of the value modified by the term "about," including both integer and fractional components. Alternatively, according to practice in the art, "about" may mean within three or more standard deviations. Or, for example, for biological systems or processes, the term "about" may mean within an order of magnitude of the value, within five times in some embodiments, and within two times in others.

[0062] In this invention, the terms "lung-targeted" or "lung-specific" are used interchangeably, both referring to the expression level of nucleic acid in the lungs exceeding 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% when the delivery system of this invention is used to deliver nucleic acid. In other words, the lung targeting specificity of the lipid nanoparticles or lung-targeting system of the present invention is 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more.

[0063] lipid nanoparticles

[0064] In this invention, the term "lipid nanoparticle" or "LNP" refers to particles containing one or more lipids and having a nanoscale size (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticles of this invention comprise ionizable cationic lipids, auxiliary lipids, and / or PEG lipids.

[0065] In some embodiments, the pKa value of the lipid nanoparticles of the present invention is between 7.0 and 8.0, for example, between 7.50 and 7.80, such as 7.50, 7.51, 7.52, 7.53, 7.54, 7.55, 7.56, 7.57, 7.58, 7.59, 7.60, 7.61, 7.62, 7.63, 7.64, 7.65, 7.66, 7.67, 7.68, 7.69, 7.70, 7.71, 7.72, 7.73, 7.74, 7.75, 7.76, 7.77, 7.78, 7.79, and 7.80.

[0066] In some embodiments, the lipid nanoparticles of the present invention have a particle size of 50 nm to 200 nm, for example, 100 nm to 200 nm. In some embodiments, the polydispersity index (PDI) of the lipid nanoparticles of the present invention is less than 0.3, for example, less than 0.2, less than 0.15, or less than 0.1. In some embodiments, the encapsulation efficiency (EE) of the lipid nanoparticles of the present invention is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the zeta potential of the lipid nanoparticles of the present invention is between 0 and 20 mV, for example, between 5 and 15 mV.

[0067] In some embodiments, the lipid nanoparticles of the present invention further comprise nucleic acids. In some embodiments, LNP@nucleic acid indicates that the LNP comprises the nucleic acid.

[0068] N / P ratio

[0069] In this invention, the N / P ratio refers to the molar ratio between positively charged lipid amine (N) groups and negatively charged nucleic acid phosphate (P) groups in lipid nanoparticles. In some embodiments, the N / P ratio of the lipid nanoparticles is between 1 and 20, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the N / P ratio of the lipid nanoparticles is between 5 and 15, between 8 and 12, or between 9 and 11.

[0070] Ionizable cationic lipids

[0071] In this invention, ionizable cationic lipids refer to lipids that are protonated and become positively charged under specific conditions, such as in an acidic environment. In some embodiments, ionizable cationic lipids include compound 1. In some embodiments, ionizable cationic lipids are compound 1. In this invention, compound 1 refers to... Figure 1AThe compound with the shown structural formula. In this invention, compound 1 is also named NNCC14.

[0072] assist lipids

[0073] In this invention, the auxiliary lipids may include phospholipids and steroids. In some embodiments, the auxiliary lipids in the lipid nanoparticles are only phospholipids. In some embodiments, the lipid nanoparticles do not contain steroids. In some embodiments, the lipid nanoparticles do not contain cholesterol.

[0074] In some embodiments, the phospholipid may be selected from 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and 1,2-diphydanoyl-sn-glycerol-3-phosphocholine (4ME). 16:0PC or 4ME), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate glycerol (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine (DSPE), or any mixture thereof.

[0075] polyethylene glycol lipids

[0076] In this invention, "polyethylene glycol lipid" or "PEG lipid" may be used interchangeably and refer to lipids modified with polyethylene glycol (PEG). Examples of polyethylene glycol lipids include, but are not limited to, PEG-modified ethanolamine phosphate, PEG-modified phosphoric acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and any mixture thereof.

[0077] In some embodiments, the polyethylene glycol lipid may be selected from 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol (DSG-PEG), N-(methylpolyoxyethylene carbonyl)-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (PEG-DSPE), 1,2-oleoyl-sn-glycerol-3-phosphate ethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), or any mixture thereof.

[0078] In some embodiments, the molecular weight of the PEG-modified material is from about 100 to about 15,000. In some embodiments, the molecular weight of the PEG-modified material is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, or about 15,000. In some embodiments, the PEG is PEG200, PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900, or PEG3000. In some embodiments, the PEG is PEG2000. In some embodiments, the polyethylene glycol lipoprotein is DMG-PEG200, DSG-PEG200, DMG-PEG2000, or DSG-PEG2000.

[0079] In some embodiments, the polyethylene glycol lipoprotein may further include a linker portion to couple one or more modification portions. In some embodiments, the modification portion may be an antibody.

[0080] Example

[0081] Example 1: Structure and Synthesis of Compound 1

[0082] The structural formula of compound 1 is shown in Figure 1A Its molecular formula is C. 74 H 147 N9O7, with a molecular weight of 1275.05, and an exact molecular weight of 1274.14. The synthetic route for compound 1 is shown below. Figure 1B .

[0083] The preparation route of M-1 used in the synthetic route of compound 1 is as follows: Figure 1C As shown, the specific preparation process is as follows:

[0084] 50.0 g of methyl palmitate was added to a 2 L single-necked flask, followed by 800 mL of methanol. The mixture was stirred at 70 °C until dissolved. 92.5 mL of 50% hydrazine hydrate was added, and the mixture was heated to 75 °C and refluxed for 24 ± 0.5 h. The reaction was monitored by TLC (PE / EA = 15 / 1), and the starting material disappeared. The mixture was cooled to 25 ± 5 °C and stirred for 0.5 h. The mixture was filtered, washed with 3 x 50 mL methanol, and dried under vacuum. The filter cake was added to a 1 L single-necked flask, followed by 500 mL of ethanol. The mixture was heated to 85 °C for 0.5 h, cooled to 25 ± 5 °C, and stirred for 0.5 h. The mixture was filtered and dried under vacuum. The filter cake was added to a 1 L single-necked flask, followed by 500 mL of methanol. The mixture was heated to 75 °C for 0.5 h, cooled to 25 ± 5 °C, and stirred for 0.5 h. The mixture was filtered, washed with 3 x 50 mL methanol, and dried under vacuum. Drying under reduced pressure at 50°C yielded 45.3 g of bright crystalline solid, with a yield of 90.6%.

[0085] The preparation route of M-2 used in the synthetic route of compound 1 is as follows: Figure 1D As shown, the specific preparation process is as follows:

[0086] Palmitoyl hydrazide (19.0 g) and anhydrous THF (380.0 mL) were added to a 500 mL three-necked flask. Under N2 protection, the temperature was lowered to -5 °C. DIEA (1) (10.90 g) was added, and acryloyl chloride THF solution (6.28 g / 20 mL) was slowly added dropwise, controlling the internal temperature to -5 to 0 °C. The reaction was maintained at this temperature for 5 h. DIEA (2) (1.82 g) and acryloyl chloride (2) (0.64 mL) were added, and the reaction was maintained at this temperature for another 1 h. TLC monitoring (DCM / MeOH = 10 / 1) showed that the starting material had essentially disappeared. The reaction solution was transferred to a 5 L measuring cup, and water (1) (1.14 L) was slowly added while stirring. After the addition was complete, stirring was continued for 0.5 h. The mixture was filtered, and the THF / water (1 / 3 v / v) (130 mL) was obtained. 3) Wash and dry.

[0087] Add the filter cake to a 1L single-necked flask, add ethanol (390ml), reflux to dissolve, and then filter while hot (without diatomaceous earth).

[0088] Cool to 25±5℃, stir for 0.5 h, filter, and dry under vacuum. Add the resulting filter cake to a 1L single-necked flask, add methanol (570 ml), reflux to dissolve, filter while hot, cool to 25±5℃, stir for 0.5 h, filter, and dry under vacuum. Dry under reduced pressure at 50℃ to obtain 9.4 g of white solid, yield: 41.3%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.06 (s, 1H), 9.89 (s,1H), 6.33 – 6.12 (m, 2H), 5.69 (dd, J= 9.8, 2.4 Hz, 1H), 2.13 (t, J = 7.4Hz, 2H), 1.57 – 1.41 (m, 2H), 1.24 (s, 24H), 0.85 (t, J = 6.7 Hz, 3H).

[0089] The reaction route for synthesizing compound 1 from M-2 and M-3 is as follows: Figure 1E As shown, the specific preparation process is as follows:

[0090] 1-(3-(3-(3-aminopropyl)(methyl)propyl)amino)decanediol (0.50 g, 1.0 equivalent) and N'-acryloylpalmitoylhydrazide (1.94 g, 3.6 equivalent) were added to a 20 mL reaction flask, along with BHT (100 mg), AcOH (70 μL), and 5.0 mL of anhydrous ethanol. The mixture was stirred at 80 °C for 24 h. The reaction mixtures were combined, and approximately 50 mL of dichloromethane was added. The mixture was filtered, and the filtrate was purified by rapid column chromatography (DCM ~ DCM / MeOH = 28 / 1 + 0.5% ammonia) to obtain 3.20 g of the product, yield: 37.8%. The product was characterized by HPLC. Figure 1F ).

[0091] HPLC(ELSD): 96.30%. 1H NMR (400 MHz, chloroform-d) δ 3.69 (tt, J = 7.1, 3.3Hz, 1H), 3.02 – 2.88 (m, 1H), 2.79 – 2.49 (m, 7H), 2.41 (dt, J = 18.8, 7.0Hz, 9H), 2.27 (qd, J = 9.7, 7.9, 3.2 Hz, 10H), 2.10 (s, 3H), 1.73 – 1.54 (m,8H), 1.53 (s, 2H), 1.25 (d, J = 6.0 Hz, 86H), 0.87 (t, J = 6.7 Hz, 12H). HR-ESI-MS: m / z Chemical formula: C 74 H 147 N9O7, calculated molecular weight [M+H]+ 1275.1423, measured molecular weight 1275.25391.

[0092] Example 2: Optimization process and toxicity assessment of compound 1 stLNP

[0093] Universal preparation and luciferase detection method for RNA-LNP

[0094] The general preparation method of RNA-LNP according to this invention is as follows: The lipid component of the LNP formulation is dissolved in ethanol solvent at a predetermined molar ratio, while the RNA is dissolved in a specific buffer solution. Subsequently, the RNA-containing buffer solution and the lipid-ethanol solution are rapidly mixed at a volume ratio (v / v) of 3:1. After mixing, the resulting solution is allowed to stand at room temperature for 10 minutes. Finally, the solution is diluted to the target concentration using phosphate-buffered saline (PBS) and administered to experimental mice via tail vein injection.

[0095] The specific steps for the in vivo detection method of luciferase are as follows: At a set time point, 100 μL of a 30 mg / mL D-luciferin potassium salt solution is injected intraperitoneally into mice. The mice are placed in an in vivo imaging system, and the fluorescence intensity data of each mouse is detected and recorded. The mice are dissected, and target tissue and organ samples are collected. The expression of luciferase at the organ level is further evaluated by photographing and quantitative analysis of fluorescence intensity. The above-described RNA-LNP preparation method and in vivo luciferase detection method are general technical means in the subsequent embodiments of this invention. Unless otherwise specified, the above methods are used in all embodiments and will not be repeated hereafter.

[0096] This study focuses on the formulation optimization of novel lipid compound 1 in LNP. By systematically adjusting the proportion of core lipids, the proportion and types of auxiliary lipids, the N / P ratio, and the type of buffer solution, an optimized formulation with both high lung targeting efficiency and low toxicity was screened.

[0097] 1. Proportion testing of key lipid compound 1

[0098] Given that compound 1 is a novel ionizable cationic lipid, its molar proportion in LNPs, as a key lipid, has a decisive impact on nucleic acid loading efficiency and gene expression. This study systematically optimized the proportion of the core lipid based on the classic formulation (G4 in Table 1, with a molar ratio of compound 1:DSPC:Chol:DMG-PEG2000 of 50:10:38.5:1.5).

[0099] A screening library containing nine LNP samples (G1-G9) was constructed. The experimental design followed these principles: ① The molar percentage of DMG-PEG2000 was fixed at 1.5% (except for the G9 group); ② The molar ratio of DSPC to Chol was maintained at 10:38.5 (except for the G9 group); ③ The molar percentage of each compound was gradually adjusted to cover the range of 0-100%. For each LNP sample, the physicochemical properties such as particle size, polydispersity index (PDI), encapsulation efficiency (EE), and zeta potential were measured (results are shown in [link to results]). Figure 1GThe mice were injected via tail vein at a dose of 0.1 mg / kg (Luc mRNA, SEQ ID NO: 1) to screen for in vivo targeting efficiency.

[0100] In vivo experimental results showed that ( Figure 2 The lung targeting efficiency of LNPs showed a significant positive correlation with the percentage of compound 1 mole. Specifically, the lung targeting efficiency of group G8 (compound 1 comprising 90%) was approximately 12-fold higher than that of the initial group G4. Further comparison of the organ targeting specificity of LNPs across groups confirmed that samples from groups G5, G6, G7, and G8 all possessed excellent lung tissue targeting performance and low non-specific organ distribution.

[0101] To evaluate the biosafety of different LNP formulations, empty LNPs corresponding to RNA delivery doses of 5 mg / kg were prepared. After tail vein injection into mice, blood samples were collected at 6 and 24 hours to detect liver function indicators (alanine aminotransferase ALT, aspartate aminotransferase AST), kidney function indicators (urea, creatinine CREA), and inflammatory markers (interleukin IL-6, IL-1β, tumor necrosis factor TNF-α, chemokine CCL-2). PBS and intraperitoneal injection of lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Experimental data showed (…). Figure 3 No animal deaths were observed in the G5-G8 groups at an empty dose of 5 mg / kg, indicating that all formulations exhibited good biological tolerability, and there were no significant differences in liver and kidney function indicators and inflammatory factor levels among the groups. Based on the comprehensive evaluation results of targeting efficiency and biosafety, the G8 formulation (compound 1 mole, 90% concentration) was selected as the base formulation for subsequent optimization. This formulation demonstrated the dual advantages of highly efficient lung targeting and low toxicity.

[0102] Table 1. Specific formulations of each LNP for lipid percentage screening of compound 1

[0103]

[0104] 2. Optimization of lipid proportions, types, and N / P ratios, and toxicity testing.

[0105] Based on the G8 formulation (compound 1:DSPC:Chol:DMG-PEG2000=90:1.8:6.7:1.5), this stage further optimized parameters such as the proportion of auxiliary lipids. The specific research content is as follows:

[0106] 2.1 Optimization of auxiliary lipid ratio

[0107] Five derivative formulations (G8-1 to G8-5) were designed (Table 2). The proportions of compound 1 and DMG-PEG2000 were fixed, and the molar ratio of DSPC and Chol was systematically adjusted. The physicochemical properties of the LNPs prepared from each formulation were characterized. Figure 4 Mice were administered a tail vein injection of 0.1 mg / kg. In vivo screening results showed ( Figure 5 The G8-5 group (compound 1:DSPC:Chol:DMG-PEG2000=90:8.5:0:1.5) showed good fluorescence signal intensity and organ specificity in the lungs. Among them, G8-5 showed an improvement of about 1.9 times compared with the G8-2 group, which is similar to the original G8 component, indicating that appropriately increasing the proportion of DSPC helps to enhance lung targeting.

[0108] Table 2. Specific formulations of each LNP based on further optimization of G8 components.

[0109]

[0110] 2.2 Assisted lipid type screening

[0111] Based on the G8-5 formulation, the auxiliary lipids were expanded from DSPC to phospholipids with different head structures and fatty acid chains, including DOPC, 4ME, DOPE, and POPE (Table 3). The physicochemical properties of the five LNP formulations were analyzed. Figure 6 ) and in vivo expression assays ( Figure 7 The results showed that the formulation using POPE as an auxiliary lipid exhibited the highest fluorescence expression in the lungs, approximately 1.7 times higher than the initial DSPC group.

[0112] Table 3. Specific formulations of each LNP for screening auxiliary lipids

[0113]

[0114] 2.3 Optimization of N / P ratio

[0115] After determining POPE as the optimal auxiliary lipid, the effect of the N / P ratio on the performance of LNPs was investigated. Four LNP formulations with N / P ratios of 5, 10, 15, and 20 were prepared (Table 4), and their physicochemical properties were tested. Figure 8 And perform in vivo screening () Figure 9 The results showed that formulations with N / P ratios of 10 and 20 exhibited good lung targeting and organ specificity. Considering that a high N / P value might increase carrier toxicity, the formulation with N / P = 10 was ultimately selected as the candidate and named compound 1 stLNP (simplified targeted LNP).

[0116] Table 4. Specific formulations of each LNP for screening N / P

[0117]

[0118] 2.4 Safety Evaluation

[0119] To assess the biosafety of compound 1 stLNP, empty LNP vectors with corresponding RNA doses of 5, 8, 10, and 12 mg / kg were prepared and injected into mice via the tail vein. Serum biochemical parameters were measured at 6 h, 24 h, and 48 h, respectively. Figure 10 Results showed that liver function indicators, ALT and AST, slightly increased within 24 hours after administration, but returned to the levels of the PBS control group by 48 hours. Inflammatory factor detection showed that IL-6, IL-1β, TNF-α, and CCL-2 levels exhibited a similar transient increase followed by a return to normal. Histopathological examination revealed no significant pathological damage in heart, liver, spleen, lung, and kidney tissue sections after 48 hours. Figure 11 The above results confirm that compound 1stLNP maintains good biocompatibility at doses up to 12 mg / kg, providing a safety basis for its subsequent application.

[0120] 3. Screening of LNP preparation buffer systems

[0121] Previous studies have reported that the buffer system has a certain impact on LNP performance. This invention conducts a buffer type screening experiment to optimize LNP preparation. Six different buffer formulations (B1-B6) were designed (Table 5). The physicochemical properties of LNPs prepared by dissolving RNA in these buffers were characterized, including particle size, polydispersity index (PDI), encapsulation efficiency (EE), and zeta potential (results are shown in [Table 5]). Figure 12 To further evaluate the effects of different buffer solutions on the in vivo performance of LNP, mice were administered the drug via tail vein injection at a dose of 0.2 mg / kg for in vivo screening experiments. The experimental results showed that ( Figure 13 The LNP prepared using buffer group B6 (20 mM acetic acid, pH 4.0) showed the best performance in terms of lung targeting efficiency and organ specificity, with an effect of nearly 8 times compared to the initially used buffer group B1 (10 mM citric acid, pH 3.0). Based on this, 20 mM acetic acid buffer was selected as the preparation buffer for compound 1 stLNP.

[0122] In summary, through the systematic optimization of all the above steps, compound 1 stLNP has improved lung targeting efficiency by more than 100 times compared to the original formulation (G4).

[0123] Table 5. Different buffer solutions for preparing compound 1 stLNP

[0124]

[0125] Example 3 Physicochemical properties of compound 1 stLNP

[0126] This invention systematically studies the physicochemical properties of compound 1stLNP. First, its pKa value was determined using the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) method. The pKa value has a crucial influence on the stability, cellular uptake, and in vivo distribution of LNP. In its free state, TNS is almost non-luminescent in aqueous solution; however, as the solution pH gradually decreases, LNP is gradually protonated and becomes positively charged. Upon binding with TNS, it emits strong fluorescence, and the higher the degree of protonation of LNP, the stronger the TNS fluorescence. Based on this principle, LNP solutions with pH values ​​of 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11 were prepared, and the fluorescence intensity of TNS in each group of solutions was measured. Correlation curves were plotted for fitting analysis. The results showed that when the pH value was 7.67, the fluorescence intensity of TNS reached 50% of the maximum value. This pH value is the pKa value of compound 1stLNP. Figure 14 Compared to conventional liver-targeting LNPs, compound 1 stLNP exhibits a significantly higher pKa value, suggesting that its particle surface may carry more positive charge. This experimental result aligns with existing theories regarding lung-targeting LNPs, namely that the positive charge enriched on the LNP surface can promote its interaction with specific proteins in serum, forming a protein crown that is more conducive to LNP targeting the lungs in vivo, thereby achieving more precise drug delivery.

[0127] The morphology and structure of compound 1 stLNP were further observed using cryo-transmission electron microscopy (Cryo-TEM). Figure 15 The results showed that compound 1 stLNP exhibited regular and uniform spherical shapes with a particle size smaller than 100 nm as shown by the scale bar in the figure. The particles were well dispersed and no obvious agglomeration or aggregation was observed. The uniform spherical shape and suitable particle size range help to improve its in vivo circulation time and cellular uptake efficiency, while good dispersibility ensures the uniformity of drug loading and stability during delivery, providing a solid structural basis for achieving efficient and safe drug delivery.

[0128] In addition, to evaluate the long-term stability of compound 1 stLNP, the prepared compound 1 stLNP was stored at room temperature, and samples were taken periodically at set time intervals to dynamically detect particle size, polydispersity index (PDI), and encapsulation efficiency (EE). Figure 16Experimental data show that, during a test period exceeding 7 days of storage, the performance indicators of compound 1 stLNP remained stable. Although the particle size increased slightly, it remained within the normal range for LNPs; the polydispersity index (PDI) remained consistently around 0.2; and the encapsulation efficiency (EE) remained essentially constant without significant fluctuations.

[0129] Example 4: Compound 1 stLNP exhibited high efficiency, high specificity, and low toxicity in lung targeting.

[0130] To verify the performance advantages of compound 1stLNP as a novel lung-targeted delivery system, it was compared with previously reported DOTAP-5-LNP. The DOTAP-5-LNP formulation was MC3 / DSPC / Chol / DMG-PEG2000 / DOTAP = 25 / 5 / 19.25 / 0.75 / 50; both LNPs were loaded with Luc mRNA and formulated at a uniform N / P 10 ratio (including the available nitrogen of MC3 and DOTAP in DOTAP-5-LNP), and were injected intravenously into C57BL / 6 mice at a dose of 0.25 mg / kg mRNA. Six hours after injection, in vivo fluorescence imaging and lung tissue quantification were performed: the lung fluorescence signal in the compound 1stLNP group was significantly stronger than that in the DOTAP-5-LNP group, with a delivery efficiency approximately 4.93 times higher; lung-specific expression was >99%, and liver and spleen combined expression was <1%, while spleen leakage was observed in the DOTAP-5-LNP group, indicating significantly lower targeting specificity than compound 1stLNP. Figure 17 ).

[0131] In the in vivo toxicity evaluation, mice were injected intravenously with 1 mg / kg Luc mRNA-LNP, with a PBS blank control. Blood samples were collected at 6, 24, and 48 h to measure liver and kidney function (ALT, AST, UREA, CREA) and inflammatory factors (IL-6, IL-1β, TNF-α, CCL-2), with 5 mice in each group. Results: In the DOTAP-5-LNP group, ALT / AST significantly increased from 6 h and persisted until 48 h, indicating hepatotoxicity; the inflammatory factors in this group increased acutely at 6 h, significantly higher than those in the PBS and compound 1 stLNP groups, while the inflammation levels in the latter were similar to those in the PBS group. Figure 18 After blood biochemistry testing, histopathological analysis was performed. 48 hours later, heart, kidney, liver, lung, and spleen sections were prepared using hematoxylin and eosin (HE) technology. Extensive damage was observed in the LPS group, while the morphology was normal in the PBS group. Pathological changes were observed in some tissues of the DOTAP-5-LNP group. The tissues of the compound 1stLNP group were highly similar to those of the PBS group, with no abnormalities. Figure 19 ).

[0132] In summary, when high-dose mRNA is delivered in vivo, compound 1stLNP significantly reduces acute liver injury and systemic inflammation compared to DOTAP-5-LNP, has less impact on the pathology of major organs, and exhibits superior biocompatibility and safety, providing a reliable basis for its subsequent translation into nucleic acid drug delivery applications.

[0133] Example 5: Compound 1 stLNP achieves efficient and specific distribution and expression in lung tissue.

[0134] 1. In vivo distribution study

[0135] To clarify the distribution characteristics of compound 1stLNP in mice, this invention uses Cy5-labeled mRNA as a tracer and analyzes its organ distribution and cell targeting using fluorescence imaging combined with flow cytometry. The specific experimental method is as follows: Cy5-labeled mRNA (prepared by introducing 25% Cy5-U to replace ordinary U) was loaded into compound 1stLNP (dose: 0.5 mg / kg) and administered to experimental mice via tail vein injection. Six hours after administration, in vivo imaging of major mouse organs (heart, liver, lung, spleen, and kidney) was performed using the IVIS Lumina system. Imaging results showed that Cy5 fluorescence signals were enriched in the lung, liver, and spleen tissues, indicating that compound 1stLNP was mainly distributed in these organs. To further understand its cell targeting in the lungs, lung tissue was enzymatically digested to prepare a single-cell suspension, and specific fluorescent staining was performed on lung endothelial cells, epithelial cells, and immune cells, followed by quantitative analysis by flow cytometry. The results showed ( Figure 20 The positive rate of Cy5 in lung endothelial cells and epithelial cells reached approximately 80%, while the positive rate in immune cells was approximately 10%, resulting in an overall cellular positive rate of about 50%. These results indicate that compound 1 stLNP has a highly efficient delivery capability to lung cells, particularly endothelial cells and epithelial cells, providing experimental evidence for targeted delivery strategies in the treatment of lung diseases.

[0136] 2. In vivo gene editing effect study

[0137] Based on the above distribution characteristics, this invention further utilizes a gene reporter mouse model (Ai14 mouse) to evaluate the mRNA delivery and gene editing efficiency mediated by compound 1 stLNP. In Ai14 mice, tdTomato red fluorescent protein expression can be induced at the Cre recombinase expression site, thus directly reflecting the gene editing effect. The specific experimental method is as follows: Cre mRNA (SEQ ID NO: 2) was loaded into compound 1 stLNP (dose: 0.3 mg / kg) and injected intravenously into Ai14 mice. Forty-eight hours after administration, the mice were euthanized and major organs were collected. tdTomato fluorescence expression was detected using an IVIS imaging system. To detect the main cell types and proportions edited in the mouse lungs by compound 1 stLNP, lung tissue was used to prepare a single-cell suspension. After staining endothelial cells, epithelial cells, and immune cells, the proportion of tdTomato-positive cells was detected by flow cytometry. The results showed ( Figure 21 Compound 1 stLNP efficiently mediates Cre mRNA delivery, achieving targeted gene editing in lung endothelial cells (68% positive rate) and epithelial cells (70% positive rate), while the editing efficiency of immune cells is 32%, with an overall cell editing positive rate exceeding one-third. These results confirm that compound 1 stLNP can effectively deliver functional mRNA to target cells and achieve efficient expression, providing a reference for further in-depth research into the application of compound 1 stLNP in the treatment of lung diseases.

[0138] Example 6: Expression monitoring of compound 1 stLNP after single and multiple administrations

[0139] To evaluate the delivery efficiency and expression timeliness of compound 1stLNP, this invention uses luciferase (Luc) mRNA as a reporter gene to systematically study its expression dynamics after a single dose. The specific experimental method is as follows: Luc mRNA was loaded into compound 1stLNP to prepare a formulation of 0.75 mg / kg, which was administered via a single tail vein injection. Mice were monitored in real-time using an IVIS in vivo imaging system at 0.5, 2, 6, 12, 24, and 48 hours after administration, and the fluorescence signal intensity was recorded and quantitatively analyzed. The experimental results showed ( Figure 22 Significant fluorescence signals were detected rapidly within 0.5 hours after administration, indicating that compound 1 stLNP can quickly deliver mRNA to the target organ and initiate expression, with a noticeable signal remaining detectable up to 48 hours later. These results confirm that compound 1 stLNP-mediated gene expression is characterized by rapid initiation, high-efficiency expression, and sustained action, and a single dose can meet the needs of short-term, high-intensity gene expression.

[0140] Based on the data characteristics of a single dose, this invention further designed a multiple-dose regimen to evaluate the delivery effect of compound 1stLNP with repeated administration. The specific experimental method is as follows: Compound 1stLNP@Luc mRNA formulation at a dose of 0.75 mg / kg was administered via tail vein injection at 0, 48, and 96 hours. Throughout the experimental period (0-144 hours), fluorescence signals were monitored at preset time points (0.5, 2, 6, 12, 24, 48, 50, 72, 96, 98, 120, and 144 hours). The results showed that the fluorescence signal rapidly recovered to a similar peak level after each dose, and the signal decline curves were basically consistent within the dosing intervals, exhibiting a typical "peak-decline" periodicity. This phenomenon indicates that compound 1stLNP, through multiple administrations, can effectively replenish the mRNA consumed by metabolism in the body, maintain stable gene expression levels, and provide a reliable guarantee for long-term treatment. In summary, compound 1 stLNP exhibits rapid and efficient gene delivery capabilities with a single dose, making it suitable for short-term, high-intensity gene expression needs; while the multiple-dose strategy fully leverages its long-term advantages, maintaining stable gene expression and showing significant application potential in long-term gene therapy for chronic diseases.

[0141] Example 7: Gene editing achieved using compound 1 stLNP-mediated adenine base editor (ABE).

[0142] This invention verifies the gene-editing function of compound 1stLNP in ABE reporter mice. In this mouse strain, green fluorescent protein (GFP) is normally not expressed due to the presence of a stop codon (TAG) upstream of the gene encoding GFP. When the adenine base editor successfully corrects the expression in mice, the stop codon TAG is replaced by CAG, and GFP expression is restored. In the experiment, ABE mRNA (SEQ ID NO: 3) and sgRNA (SEQ ID NO: 4) were loaded into compound 1stLNP at a 2:1 mass ratio, and mice were injected in vivo at a dose of 1 mg / kg of total RNA. Five days after injection, lung tissue was collected from the mice for tissue sectioning and fluorescence detection. The results showed (…). Figure 23 Compared with the PBS control group, the mouse lungs of the compound 1stLNP group showed extensive and significant fluorescence signals. Next-generation sequencing analysis revealed a gene editing efficiency of 3.24% in the mouse lungs. These experimental results demonstrate that the compound 1stLNP developed in this invention can efficiently deliver adenine base editor mRNA and sgRNA to the target organ (lung) in vivo, successfully achieving gene editing, thus strongly highlighting its translational application potential in the field of gene editing delivery vectors.

[0143] Example 8: Compound 1 stLNP for the treatment of acute lung injury in mice

[0144] This invention validates the mRNA delivery potential of compound 1stLNP in the treatment of acute lung injury (ALI). The experiment employed a classic LPS-induced ALI model, designed and prepared murine his-IL-10 mRNA (SEQ ID NO: 5), and first validated its protein expression at the cellular level. In ALI model construction, mice were anesthetized and infused with 50 μL of PBS or LPS (2 mg / kg) via tracheal infusion. Two hours later, pre-prepared compound 1stLNP@IL-10 mRNA or empty LNP was injected via tail vein at a dose of 1 mg / kg mRNA. Twenty-four hours after modeling, mouse lung tissue was collected for HE sectioning, or bronchoalveolar lavage was performed to obtain BALF supernatant and ground lung tissue supernatant. The levels of inflammatory factors were detected by ELISA. Results showed that the levels of inflammatory factors such as TNF-α and IL-6 in the lung tissue and BALF of mice in the empty LNP group were significantly increased. Figure 24 HE staining of lung tissue revealed significant airway inflammatory cell infiltration; while in mice in the compound 1stLNP@IL-10 mRNA group, the levels of inflammatory factors in lung tissue and BALF were significantly reduced, and lung tissue pathological damage was also alleviated. Figure 25 The above data confirm that the compound 1stLNP described in this invention can efficiently deliver IL-10 mRNA to lung tissue and express functional IL-10, effectively inhibit LPS-induced inflammatory response, significantly reduce ALI pathological damage, and demonstrate its translational application prospects in the field of mRNA therapeutic vectors for lung injury.

[0145] Example 9: Application of compound 1 stLNP in siRNA delivery

[0146] 1. Determination of siRNA delivery efficiency targeting the Tie2 gene

[0147] To verify the delivery capability of compound 1 stLNP as a multifunctional delivery system for siRNA, this invention targets the Tie2 gene specifically expressed in mouse lung endothelial cells and systematically evaluates its delivery efficiency and dose-response relationship. siTie2 (sequence shown in Table 6) was loaded into compound 1 stLNP and prepared into formulations with different concentration gradients of 0, 0.02, 0.05, 0.1, 0.25, 0.75, and 1.5 mg / kg, which were injected into mice via tail vein. Three days after administration, lung tissue samples were collected, and RNA and protein were extracted separately. Figure 26At the RNA level, the expression level of Tie2 mRNA was quantitatively analyzed using reverse transcription combined with real-time quantitative PCR (qPCR). The calculated half-maximal effective dose (ED50) for compound 1stLNP@siTie2 to successfully inhibit Tie2 mRNA was 0.056 mg / kg. At the protein level, Tie2 protein expression was detected by Western blot. The results showed that Tie2 protein expression was significantly downregulated at a dose of 0.05 mg / kg; at doses of 0.1 mg / kg and above, Tie2 protein expression was almost completely inhibited, indicating that compound 1stLNP can efficiently mediate siRNA delivery and achieve gene silencing.

[0148] Table 6

[0149]

[0150] The meanings of the symbols in the table are as follows:

[0151] A, G, U, C: RNA nucleotides;

[0152] u, c: 2'-O-methylated nucleotides;

[0153] dT: deoxythymidine;

[0154] s: Phosphothiophosphate bond;

[0155] P: 5'-phosphate.

[0156] 2. Validation of siRNA delivery efficacy targeting the EGFP gene

[0157] Furthermore, using transgenic mice stably expressing green fluorescent protein (EGFP) as a model, the silencing efficiency of compound 1stLNP on EGFP was evaluated. siEGFP (sequence shown in Table 6) was loaded into compound 1stLNP and formulated into a 2 mg / kg dose, which was injected intravenously into transgenic mice. Three days after administration, lung tissue was collected, RNA was extracted and reverse transcribed, and the expression level of EGFP mRNA was detected using qPCR. Experimental data showed ( Figure 27 The expression of EGFP mRNA was effectively suppressed, with a knockdown exceeding 50%. Simultaneously, lung tissue sections were prepared, and EGFP expression was observed using fluorescence microscopy. The results showed that, compared to the control group, the experimental group exhibited a significantly reduced green fluorescence intensity, directly confirming that compound 1 stLNP-mediated siEGFP effectively silences EGFP gene expression. These experimental results indicate that compound 1 stLNP has significant application potential in the field of siRNA delivery, particularly suitable for targeted therapy of lung-related gene diseases.

Claims

1. A lipid nanoparticle, wherein the lipid portion of the lipid nanoparticle comprises an ionizable cationic lipid, a phospholipid, and a polyethylene glycol lipid, wherein the ionizable cationic lipid comprises compound 1. ， The total amount of lipids in the lipid nanoparticles includes: compound 1 having a content of 80 mol% or more; phospholipids having a content of 5 mol% to 20 mol%; and polyethylene glycol lipids having a content of 0.1 mol% to 10 mol%. The lipid nanoparticles further contain nucleic acids, and the N / P ratio of the lipid nanoparticles is between 5 and 15.

2. The lipid nanoparticles according to claim 1, wherein the lipid portion of the lipid nanoparticles is composed of ionizable cationic lipids, phospholipids, and polyethylene glycol lipids.

3. The lipid nanoparticles according to claim 1, wherein the phospholipid comprises POPE.

4. The lipid nanoparticles according to claim 1, wherein the polyethylene glycol lipid comprises DMG-PEG.

5. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles are prepared by dissolving nucleic acids in an acetate buffer.

6. Use of the lipid nanoparticles according to any one of claims 1-5 in the preparation of a medicament for the treatment of acute lung injury.

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