Tissue-targeted aerosol inhalation type lipid nanoparticles as well as delivery method and application thereof

Abstract

The invention discloses a tissue-targeted aerosol inhalation type lipid nanoparticle as well as a delivery method and application thereof. The invention provides a method for regulating and controlling atomization dosage form lipid nanoparticles to target tissues, and the method aims at non-disease diagnosis and treatment and comprises the following steps: preparing atomization dosage form lipid nanoparticles with different charges by judging whether charged lipid is doped or not, so as to regulate and control the atomization dosage form lipid nanoparticles to target different tissues; the nebulization type lipid nanoparticles with different charges are electrically neutral nebulization type lipid nanoparticles or charged nebulization type lipid nanoparticles. The invention finds that mRNA (messenger ribonucleic acid) also has strong expression in the liver after the mRNA-LNPs with electrically neutral surface charges are aerosolized and administered in the trachea. Furthermore, the efficient, safe and repeatable administration aerosol inhalation type liver targeting lipid nanoparticles (called NebuLi LNP) are obtained through further optimization, and the application research of the aerosol inhalation mRNA-LNP is widened.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a tissue-targeted atomized inhalation lipid nanoparticle and a delivery method and application thereof. Background Art

[0002] Due to the flexibility of its sequence, messenger RNA (mRNA) can provide a template for the synthesis of any given protein, protein fragment or peptide, and does not need to enter the cell nucleus and has no risk of integration into the genome. Therefore, mRNA-based therapeutics have a wide range of disease applications, including infectious diseases, genetic diseases and metabolic diseases. mRNA needs to be packaged in a carrier to avoid degradation by nucleases and to be absorbed by cells across the cell membrane. At present, the carriers of most mRNA therapies are lipid nanoparticles (LNPs). The successful application of SARS-CoV-2 mRNA vaccines has also demonstrated the great potential of LNP as a delivery carrier, becoming the main advantage carrier for mRNA delivery. Traditional LNPs are mainly composed of four lipids, namely ionizable cationic lipids, cholesterol, auxiliary lipids and polyethylene glycol (PEG) lipids, which together complete the protection and delivery of mRNA. At the same time, effective delivery of mRNA-LNP needs to be designed for specific routes of administration and drug types. Different modes of administration will directly affect the target organ specificity and expected clinical results. For example, local administration has been shown to effectively deliver transfected mRNA in target organs, including eyes, muscles, brain or tumors. Intramuscular or subcutaneous injection can achieve targeted delivery and transfection of mRNA to draining lymph nodes, and intraperitoneal injection can effectively deliver mRNA to pancreatic tissue. When administered intravenously, mRNA tends to be expressed in the liver. Recently, Siegwart et al. developed an organ-selective targeting (SORT) strategy, introducing a fifth lipid component with different charges into the traditional electrically neutral four-component LNP formulation. After intravenous administration, specific delivery and expression of mRNA in the liver, spleen, and lungs of mice were achieved, and tissue-specific gene editing was also mediated.

[0003] As a non-invasive and convenient method of drug administration, nebulized inhalation has unique advantages in the treatment of lung-related diseases. Nebulized mRNA-LNP has also been widely explored for use in lung-targeted mRNA delivery systems. Summary of the invention

[0004] The technical problem solved by the present invention is how to regulate the targeting of aerosolized lipid nanoparticles to tissues and improve the transfection efficiency of mRNA in tissues.

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for regulating the targeting of nebulized lipid nanoparticles to tissues, the method is for non-disease diagnosis and treatment purposes, and comprises the following steps: preparing nebulized lipid nanoparticles with different charges by incorporating charged lipids, so as to achieve the regulation of the targeting of nebulized lipid nanoparticles to different tissues;

[0006] The aerosolized lipid nanoparticles with different charges are electrically neutral aerosolized lipid nanoparticles or charged aerosolized lipid nanoparticles.

[0007] In the above method, the different tissue is liver or lung.

[0008] In the above method, the preparation of aerosolized lipid nanoparticles with different charges by incorporating charged lipids is as follows 1) or 2):

[0009] 1) preparing electrically neutral aerosolized lipid nanoparticles without incorporating charged lipids; the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids and polyethylene glycol lipids;

[0010] The electrically neutral aerosolized lipid nanoparticles are targeted to the liver;

[0011] 2) preparing charged aerosol-type lipid nanoparticles by incorporating charged lipids; the lipids of the charged aerosol-type lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and charged lipids;

[0012] The charged aerosolized lipid nanoparticles are targeted to the lungs.

[0013] In the method described above, the charge of the electrically neutral aerosolized lipid nanoparticles is between -5 and +5 mV;

[0014] Alternatively, the charged lipid is a cationic lipid having a positive charge under neutral conditions or an anionic lipid having a negative charge.

[0015] In the method described above, the lipids of the neutrally charged aerosolized lipid nanoparticles or the charged aerosolized lipid nanoparticles further include auxiliary lipids;

[0016] The auxiliary lipid is selected from one or any combination of steroids, steroid derivatives and phospholipids.

[0017] In the method described above,

[0018] The positively charged cationic lipid is a lipid molecule having a positively charged group (such as a quaternary ammonium salt group) under neutral conditions.

[0019] And / or, the negatively charged anionic lipid is a lipid molecule having a negatively charged group (such as a carboxylic acid group or a phosphate group) under neutral conditions.

[0020] And / or, the cationic lipid molecule with a positive charge may be DOTAP, DDAB, DOTMA or EPC.

[0021] Or the anionic lipid molecule with a negative charge may be DOPG, 14PA, 18BMP, DOPS or 18PA.

[0022] And / or, the phospholipid is a lipid having a pharmaceutically acceptable amphipathic lipid phospholipid structure.

[0023] Or, the lipid having a pharmaceutically acceptable amphiphilic lipid phospholipid structure is DOPE, DSPE, POPE, 4ME, DOPC or DSPC.

[0024] The steroid is cholesterol or its analogue;

[0025] Alternatively, the steroid derivative may be β-sitosterol, 7α-hydroxycholesterol or 25-hydroxycholesterol, but is not limited thereto.

[0026] The polyethylene glycol lipid is DMG-PEG, DSG-PEG or DSPE-PEG.

[0027] The ionizable cationic lipid is ALC-0315, SM-102, C12-200 or Dlin-MC3-DMA.

[0028] In a second aspect, the present invention provides a tissue-targeted aerosolized lipid nanoparticle, which is as follows 1) or 2):

[0029] 1) The aerosolized lipid nanoparticles are electrically neutral aerosolized lipid nanoparticles;

[0030] The lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids and polyethylene glycol lipids;

[0031] The electrically neutral aerosolized lipid nanoparticles are targeted to the liver;

[0032] 2) The aerosolized lipid nanoparticles are charged aerosolized lipid nanoparticles;

[0033] The lipids of the charged aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and charged lipids;

[0034] The charged aerosolized lipid nanoparticles are targeted to the lungs.

[0035] In the above-mentioned nebulized lipid nanoparticles, the charge of the electrically neutral nebulized lipid nanoparticles is between -5 and +5 mV;

[0036] Or, the charged lipid is a cationic lipid with a positive charge under neutral conditions or an anionic lipid with a negative charge;

[0037] Or, the lipids of the neutrally charged nebulized dosage form lipid nanoparticles and the charged nebulized dosage form lipid nanoparticles further include auxiliary lipids;

[0038] The auxiliary lipid is selected from one or any combination of steroids, steroid derivatives and phospholipids.

[0039] In the above-mentioned aerosolized lipid nanoparticles, the aerosolized lipid nanoparticles further include nucleic acids encapsulated therein.

[0040] and / or, the nucleic acid is mRNA,

[0041] And / or, the mRNA expresses a protein with therapeutic function or expresses a protein with fluorescent reporter function.

[0042] In the above-mentioned aerosolized lipid nanoparticles, the protein with therapeutic function can be a protein with tumor-killing function.

[0043] And / or, the weight ratio of total lipid to mRNA in the aerosolized lipid nanoparticles is 10:1 to 50:1.

[0044] Furthermore, the weight ratio of total lipids to mRNA in the aerosolized lipid nanoparticles is specifically 40:1.

[0045] Alternatively, the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and steroids; the molar ratios of the individual lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 50%-70%, the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3% and is not 0, and the molar ratio of the steroids to the total lipids is 15-50%.

[0046] Or, the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids and phospholipids; the molar ratios of the individual lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 30%-50%, the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0, the molar ratio of the steroids to the total lipids is 20-50%, and the molar ratio of the phospholipids to the total lipids is 0-20%, and is not 0.

[0047] In an embodiment of the present invention, the electrically neutral aerosolized lipid nanoparticle is NebuLi ALC-0315LNP, and the lipids of NebuLi ALC-0315LNP include ionizable cationic lipid ALC-0315, polyethylene glycol lipid DMG-PEG, cholesterol and phospholipid DSPC; the molar ratios of each lipid are as follows: the molar ratio of the ionizable cationic lipid to the total lipids is 46.3%, the molar ratio of the polyethylene glycol lipid to the total lipids is 1.6%, the molar ratio of the steroid to the total lipids is 42.7%, and the molar ratio of the phospholipid DSPC to the total lipids is 9.4%.

[0048] Or the lipids of the charged atomized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids and charged lipids; the molar ratios of the individual lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 30%-50%, the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0, the molar ratio of the steroids to the total lipids is 20-50%, and the molar ratio of the charged lipids to the total lipids is 0-50%, and is not 0.

[0049] Or the lipids of the charged atomized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids, charged lipids and phospholipids; the molar ratios of each lipid are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 15%-60%, the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0, the molar ratio of the steroids to the total lipids is 15-50%, the molar ratio of the phospholipids to the total lipids is 0-20%, and is not 0; the molar ratio of the charged lipids to the total lipids is 0-60%, and is not 0.

[0050] The administration type of the aerosolized lipid nanoparticles described above is intratracheal aerosol administration.

[0051] In a third aspect, the present invention provides the use of the aerosolized lipid nanoparticles described in the second aspect as a carrier for drug delivery;

[0052] The drug is a nucleic acid.

[0053] Or the present invention provides the use of the electrically neutral aerosolized lipid nanoparticles in the second aspect in the preparation of a product having the following functions A1) or A3):

[0054] Or the present invention provides the use of the charged atomized lipid nanoparticles in the second aspect in the preparation of a product having the following A2) functions:

[0055] A1) Targeting the drug to the liver through aerosol inhalation;

[0056] A2) Targeting the drug to the lungs through aerosol inhalation;

[0057] A3) Treatment of chronic metabolic diseases.

[0058] Alternatively, the present invention provides a product having the following functions A1) and / or A3), comprising the electrically neutral aerosolized lipid nanoparticles in the second aspect;

[0059] A1) Targeting the drug to the liver through aerosol inhalation;

[0060] A3) Treatment of chronic metabolic diseases.

[0061] Alternatively, the present invention provides a product having the following function A2), comprising the charged atomized lipid nanoparticles described in the second aspect;

[0062] A2) Inhalation of aerosolized medication is targeted to the lungs.

[0063] In the above, the chronic metabolic disease may specifically be hereditary tyrosinemia type 1 or type 2 diabetes.

[0064] In a fourth aspect, the present invention provides a method for preparing the aerosolized lipid nanoparticles according to the second aspect, comprising the following steps:

[0065] 1) preparing lipid nanoparticles according to the lipid components and corresponding molar ratios of the aerosolized lipid nanoparticles described in the second aspect;

[0066] 2) Atomizing the lipid nanoparticles to obtain the atomized lipid nanoparticles according to the second aspect.

[0067] The above-mentioned atomization is achieved by atomization administration through an intratracheal micro-sprayer.

[0068] Alternatively, the present invention provides a method for delivering nucleic acid lipid nanoparticles targeting the liver, comprising the following steps:

[0069] 1) preparing the electrically neutral aerosolized lipid nanoparticles of the second aspect;

[0070] 2) The electrically neutral aerosolized lipid nanoparticles are delivered by aerosolization to achieve liver-targeted nucleic acid lipid nanoparticle delivery.

[0071] Or, a method for delivering nucleic acid lipid nanoparticles targeting the lungs, comprising the following steps:

[0072] 1) preparing the charged atomized lipid nanoparticles in the second aspect;

[0073] 2) The electrically neutral aerosolized lipid nanoparticles are delivered by aerosolization to achieve targeted lung delivery of nucleic acid lipid nanoparticles.

[0074] The methods described above are all non-disease diagnosis and treatment methods. As non-disease diagnosis and treatment methods, they can be simply used to study the targeting of nucleic acid lipid nanoparticles to the liver or lungs, or they can be used to study the organ-targeted expression of nucleic acid lipid nanoparticles in different charge atomization formulations.

[0075] The present invention studies whether LNPs with different charge properties will also affect the distribution and expression of mRNA in various organs after aerosol administration. To this end, a series of LNPs with different surface potentials were created by adding charged lipids in different proportions to the traditional four-component neutral LNP. After intratracheal aerosol inhalation of LNPs encapsulating luciferase (Luc) mRNA, the expression of mRNA in different organs was detected. It is worth noting that it was found that aerosol mRNA-LNPs with positive or negative charges on the surface were mainly expressed in the lungs, while LNPs with neutral surface charges were mainly concentrated in the liver after aerosol administration. These mRNA-LNPs targeting the liver after intratracheal aerosol administration are called aerosol liver-targeted LNPs (NebuLi LNPs).

[0076] Subsequently, the NebuLi LNP drug delivery platform was used to verify its therapeutic effects in two metabolic disease models: hereditary tyrosinemia type 1 (HT-1) and type 2 diabetes mellitus (T2DM). - / - In mice, NebuLi LNP successfully delivered fumarylacetoacetate hydrolase (FAH) mRNA to the liver, restored the expression of FAH protein in the liver, and effectively maintained the body weight and liver function of the diseased mice. In addition, dulaglutide mRNA was delivered to the liver via NebuLi LNP, using the liver as a protein production factory to express and secrete dulaglutide protein into the blood, effectively reducing blood sugar levels and improving glucose homeostasis in T2DM mouse models ( Figure 1 ). In conclusion, this platform provides a non-invasive, convenient, and non-invasive treatment strategy for chronic metabolic diseases and has good prospects for clinical application.

[0077] The mRNA-LNP for aerosol inhalation of the present invention has the advantages of rapid onset, good efficacy, few systemic adverse reactions, and high patient compliance, and has the potential to treat a variety of diseases. At present, research on aerosol mRNA delivery mainly focuses on lung-related delivery and treatment, but there is limited exploration of its potential for delivery to other organs. This study explores the relationship between the surface charge characteristics of LNPs and the efficiency and specificity of mRNA expression in different organs after intratracheal aerosol administration. The results showed that charged LNPs mainly delivered mRNA to the lungs, while neutral LNPs preferentially expressed mRNA to the liver. Liver mRNA expression reached a peak 10 hours after aerosol administration, and the mRNA expression efficiency was comparable after repeated administration of NebuLi LNP. In addition, NebuLi LNP has good safety, and serum biochemical indicators and organ tissue pathology analysis showed no obvious toxicity. At the same time, the therapeutic effect of NebuLi LNP was also confirmed in two metabolic disease models. Encapsulation and delivery of FAH mRNA using NebuLi LNP can effectively maintain the body weight of HT-1 disease model mice and reduce liver damage. In addition, NebuLi LNPs delivered dulaglutide mRNA to the liver, where it was expressed and secreted into the blood, effectively reducing blood glucose levels in T2DM mice. The blood glucose control effect was comparable to that of the marketed drug Trulicity, indicating that this approach may provide a promising non-invasive treatment strategy for liver-related metabolic diseases.

[0078] In summary, the present invention develops aerosolized inhalation type mRNA-LNP for tissue-targeted gene drug delivery and treatment. The main innovations and key points of the present invention are as follows:

[0079] 1) This study rationally designed and synthesized aerosolized inhalable LNPs with different charge properties. After intratracheal aerosol administration, the organ targeting and expression efficiency of the delivered mRNA were tested. It was found that the organ targeting of aerosolized LNPs with different charge properties was different in vivo, and the correlation between charge properties and in vivo targeting was established, which provided certain guidance for the subsequent design and optimization of aerosolized LNPs.

[0080] 2) The present invention found that aerosolized mRNA-LNPs with positive or negative charges on the surface are mainly expressed in the lungs, and mRNA-LNPs with neutral surface charges are also strongly expressed in the liver after intratracheal aerosol administration. And further optimization has been obtained to obtain efficient, safe and repeatable aerosolized liver-targeted lipid nanoparticles (called NebuLiLNP), which has broadened the application research of aerosolized mRNA-LNP.

[0081] 3) For the first time, FAH mRNA was delivered by aerosolized LNP for the treatment of HT-1 mouse disease model, maintaining FAH - / -The weight and liver function of mice have expanded the application scope of protein replacement therapy in genetic metabolic diseases of the liver.

[0082] 4) The present invention uses NebuLi LNP as a carrier to deliver dulaglutide mRNA to treat T2DM mice, which reduces blood glucose levels and improves glucose homeostasis, providing a new and non-invasive drug delivery method and treatment strategy for the treatment of T2DM. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram of the use of aerosolized inhaled liver-targeted lipid nanoparticles (NebuLi LNP) for the treatment of metabolic-related diseases.

[0084] Figure 2 LNP formulas with different charge properties and the proportion of each component.

[0085] Figure 3 Hydration particle size, polydispersity coefficient and encapsulation efficiency of LNPs with different charge properties.

[0086] Figure 4 Zeta potential of LNPs with different charge properties.

[0087] Figure 5 Schematic diagram of the preparation and in vivo aerosol delivery of LNPs with different charge properties.

[0088] Figure 6 This is a photo of the intratracheal aerosol drug delivery process in mice.

[0089] Figure 7 These are in vitro images of the main organs of mice (heart, liver, spleen, lung, and kidney) after aerosol administration of LNPs with different charge properties.

[0090] Figure 8 Comparison of fluorescence intensity of Luc mRNA expression in liver, lung and spleen mediated by atomized LNPs with different charge properties.

[0091] Fig. 9 This is a correlation analysis between the Zeta potential of aerosolized LNP and the liver mRNA expression efficiency.

[0092] Fig.10 The formula of electrically neutral mRNA-LNP and the lipid ratio of each component.

[0093] Fig.11 Hydration particle size, polydispersity coefficient, Zeta potential and encapsulation efficiency of electrically neutral mRNA-LNPs.

[0094] Fig.12 Whole-body and in vitro IVIS imaging of mice after aerosol administration of electrically neutral mRNA-LNP.

[0095] Fig.13 It is the fluorescence intensity of mRNA in the liver and the expression percentage in the liver, lung and spleen mediated by electrically neutral atomized LNPs.

[0096] Fig.14 These are IVIS images of the whole body and organs of mice at different time points after administration of NebuLi ALC-0315LNPs@Luc mRNA.

[0097] Fig.15 Statistics of fluorescence values ​​of the whole body, liver, lung, and spleen of mice at different time points after administration of NebuLi ALC-0315LNPs@Luc mRNA.

[0098] Fig.16 NebuLi ALC-0315LNPs@Luc mRNA formulas with different lipid and mRNA mass ratios and the lipid proportions of each component.

[0099] Fig.17 Hydration particle size, polydispersity coefficient and encapsulation efficiency of NebuLi ALC-0315LNPs@Luc mRNA with different lipid and mRNA mass ratios.

[0100] Fig.18 Bioluminescence imaging and numerical statistics of the whole body and organs of mice after administration of NebuLi ALC-0315LNPs@Luc mRNA with different lipid and mRNA mass ratios.

[0101] Fig.19 This is the whole-body bioluminescence image and quantitative analysis of mice after repeated administration of NebuLi ALC-0315LNPs@Luc mRNA.

[0102] Fig. 20 These are the serum liver function (AST, ALT) and renal function (UREA, CREA) levels of mice after administration of NebuLi ALC-0315LNP.

[0103] Fig.21 H&E staining images of the main organ sections of mice after administration of NebuLi ALC-0315LNP.

[0104] Fig. 22 Schematic diagram of the principle of NebuLi ALC-0315LNPs@FAH mRNA treatment of HT-1.

[0105] Fig.23 Fluorescence imaging of mouse liver tissue sections after administration of NebuLi ALC-0315LNPs@FAH mRNA.

[0106] Fig.24 NebuLi ALC-0315LNPs@FAH mRNA for the treatment of FAH - / - Diagram of the experimental procedure and timeline for mice.

[0107] Fig.25 Relative changes in body weight of mice during NebuLi ALC-0315LNPs@FAH mRNA treatment.

[0108] Fig.26 This is a photo of the serum appearance of mice after NebuLi ALC-0315LNPs@FAH mRNA treatment.

[0109] Fig. 27 The levels of liver injury markers (ALT, AST, TBIL) in mice after NebuLi ALC-0315LNPs@FAH mRNA treatment.

[0110] Fig.28 Fluorescence imaging of mouse liver tissue sections after NebuLi ALC-0315LNPs@FAH mRNA treatment.

[0111] Fig.29 Representative images of Sirius red staining and statistical analysis of fibrosis areas in mouse liver after NebuLi ALC-0315LNPs@FAH mRNA treatment.

[0112] Fig.30 This is the treatment plan for T2DM mice with NebuLi ALC-0315LNPs@dulaglutide mRNA LNP.

[0113] Fig.31 Western Blot was used to detect the in vitro expression of dulaglutide mRNA under reducing and non-reducing conditions.

[0114] Fig.32 After NebuLi ALC-0315LNP delivered dulaglutide mRNA, an ELISA experiment was used to detect the dulaglutide protein level in peripheral blood.

[0115] Fig.33 This is the treatment plan for T2DM mice with NebuLi ALC-0315LNPs@dulaglutide mRNA LNP.

[0116] Fig.34 Body weight changes during treatment of T2DM mice with NebuLi ALC-0315LNPs@dulaglutide mRNA LNPs.

[0117] Fig.35 Random changes in blood glucose levels during treatment of T2DM mice with NebuLi ALC-0315LNPs@dulaglutide mRNA LNPs.

[0118] Fig.36 IGTT assay in mice after treatment with NebuLi ALC-0315LNPs@dulaglutide mRNA LNPs.

[0119] Fig.37 Mouse ITT study after NebuLi ALC-0315LNPs@dulaglutide mRNA LNP treatment DETAILED DESCRIPTION

[0120] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0121] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0122] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0123] The structural formulas of the compounds in the following examples are shown in Table 1:

[0124] Table 1 shows the structural formula of the compounds

[0125]

[0126]

[0127] Table 2 shows the coding sequence of mRNA

[0128]

[0129]

[0130]

[0131] The various mRNA-LNPs in the following examples are produced by rapidly mixing an ethanol phase containing lipid molecules and an aqueous phase containing mRNA molecules. The specific synthesis method is as follows:

[0132] 1) Aqueous phase containing mRNA molecules

[0133] According to the required dose of LNP, the corresponding amount of mRNA molecules was dissolved in a citric acid-sodium citrate buffer at pH = 4.0 and 10 mM to obtain an mRNA solution, which was recorded as the aqueous phase containing mRNA molecules;

[0134] Taking the 0.25 mg / kg injection dose as an example, the concentration of the mRNA solution is 0.167 mg / mL;

[0135] 2) Ethanol phase containing lipid molecules

[0136] First, the LNP components corresponding to each example were dissolved in ethanol at a specific molar ratio and mixed, and then a corresponding volume of ethanol in which lipids were dissolved was taken according to the mass ratio of LNP lipids to mRNA to obtain an ethanol phase containing a fixed mass of lipids.

[0137] Taking a 0.25 mg / kg injection dose as an example, the concentration of lipid molecules in the ethanol phase is 31.56 mM.

[0138] 3) Preparation of LNPs encapsulating mRNA

[0139] The aqueous phase containing mRNA molecules obtained in step 1) and the ethanol phase containing lipid molecules obtained in step 2) were quickly mixed at a volume ratio of 3:1, and the mass ratio of total lipids to mRNA was fixed at 40 / 1. Incubate at room temperature for 15 minutes, then dialyze in 1× phosphate buffered saline (PBS) at pH = 7.4 for 2 to 4 hours, and then dilute with PBS or ultrafilter to finally obtain an mRNA-LNP solution (solvent is PBS solution) of corresponding concentration (if the dosage for a mouse weighing about 20 g is 0.25 mg / kg and the dosage volume is 50 microliters, the concentration is 0.1 mg / mL).

[0140] Unless otherwise specified, the PBS in the following examples refers to 1× phosphate buffered saline (1×PBS) with a pH of 7.4.

[0141] 0.1×PBS is obtained by diluting the above 1×PBS with water.

[0142] Figure 1 Schematic diagram of the use of aerosolized inhaled liver-targeted lipid nanoparticles (NebuLi LNP) for the treatment of metabolic-related diseases.

[0143] Example 1. Preparation of LNPs with different charge properties

[0144] Inspired by the SORT strategy, the inventors wanted to study whether LNPs with different charge properties would affect the organ selectivity and delivery efficiency of mRNA after aerosol administration. To this end, an LNP library based on lipids with different charges was constructed.

[0145] 1. Synthesis of lipid nanoparticles (LNPs) encapsulating mRNA

[0146] According to the aforementioned mRNA-LNP synthesis method, Luciferase mRNA (whose coding sequence is Sequence 1 shown in Table 2) was prepared using the FDA-approved ALC-0315 formula (ALC-0315: DSPC: cholesterol: DMG-PEG = 46.3 / 9.4 / 42.7 / 1.6, mol / mol) as the initial formula, and then the luciferase mRNA was added to the Figure 2 A series of mRNA-encapsulated LNPs were prepared with different molar ratios (10%-30%) of positively charged lipid trimethyl-2,3-dioleyloxypropylamine (DOTAP) or negatively charged lipid dioleoylphosphatidylglycerol (DOPG) as shown, with a mass ratio of total lipid / Luc mRNA (Luc mRNA) = 40:1. Figure 2 ), named as 30% DOPG, 20% DOPG, 10% DOPG, ALC-0315, 10% DOTAP, 20% DOTAP, 30% DOTAP LNPs respectively.

[0147] 2. Testing of physical and chemical properties

[0148] 1. Detection of particle size

[0149] The freshly prepared mRNA-LNPs solution was diluted with 1× PBS to a mRNA concentration of 1 ng / mL, and then the particle size of LNPs was measured using a nanoparticle size and potential analyzer.

[0150] The results are as follows Figure 3 As shown in the left figure, the size distribution of all LNPs is relatively uniform, ranging from 100 to 200 nm, and the polydispersity index (PDI) is less than 0.2.

[0151] 2. Encapsulation efficiency test

[0152] The mRNA encapsulation efficiency of LNPs was determined using the RiboGreen fluorescent dye detection method. The free mRNA concentration in the mRNA-LNP solution was measured separately, and the concentration of all mRNA in the solution after the mRNA-LNP was fully lysed with TritonX-100 to calculate the encapsulation efficiency of LNP for Luc mRNA. Specifically, 100 microliters of mRNA-LNP solution diluted only in TE buffer was added to a 96-well plate to quantify the concentration of free mRNA, and an equal volume of mRNA-LNP solution containing TE and TritonX-100 was added to measure the concentration of mRNA after lysis, and shaken at room temperature for 30 minutes to react. Then 100 microliters of Ribogreen solution diluted 500 times with TE buffer was added. Finally, the fluorescence reading was recorded using an ELISA reader. Then the encapsulation efficiency (EE%) was calculated = [(actual concentration of mRNA after lysis - actual concentration of free mRNA) / actual concentration of mRNA after lysis] * 100%.

[0153] The results are as follows Figure 3 As shown in the figure on the right, the encapsulation efficiency of a series of LNPs is between 90% and 100%, which indicates that LNP has a high encapsulation efficiency for mRNA and can be used as a carrier for mRNA delivery.

[0154] 3. Zeta potential detection

[0155] The freshly prepared mRNA-LNP solution was diluted to 1.5 ng / mL using 0.1× PBS, and then the surface Zeta potential of LNP was measured using a nanoparticle size and potential analyzer.

[0156] The results showed that the Zeta potential of LNPs decreased with the increase of DOPG ratio, but increased with the increase of DOTAP ratio. At the same time, the surface charge of these LNPs was relatively evenly distributed between about -10mV and +10mV, while the surface charge of ALC-0315LNPs was almost neutral ( Figure 4 ).

[0157] Thus, a series of LNPs with different charge properties were formed for in vivo aerosol delivery.

[0158] 3. Drug administration

[0159] To investigate the relationship between the charge properties of LNPs and the organ targeting and delivery efficiency of mRNA in vivo after administration.

[0160] The LNPs encapsulating Luc mRNA constructed above were administered to mice via an intratracheal microsprayer, and the mRNA expression in vivo was detected using an IVIS imaging system ( Figure 5 ).

[0161] The details are as follows:

[0162] The above mRNA-LNP solutions encapsulating Luciferase mRNA were diluted with a PBS solution at pH=7.4 to obtain LNP preparations encapsulating Luciferase mRNA at a concentration of 0.1 mg / mL.

[0163] Each LNP preparation encapsulating Luciferase mRNA was administered to mice using an intratracheal microsprayer. Specifically, C57BL / 6 mice were anesthetized with isoflurane and then administered via an intratracheal microsprayer ( Figure 6 ) 0.25 mg / kg of LNP preparations encapsulating Luciferase mRNA were administered to mice lungs, with an injection volume of 50 microliters. Ten hours after administration, 100 microliters of D-luciferin potassium salt solution (dissolve D-luciferin potassium salt dry powder in PBS aqueous solution for injection) with a concentration of 30 mg / mL were intraperitoneally injected. Two to three minutes later, bioluminescent imaging of the whole body and major organs (heart, liver, spleen, lung, kidney) of the mice was performed using the IVIS small animal in vivo imaging system.

[0164] The results are as follows Figure 7 As shown, it can be seen that the fluorescence signal is mainly seen in the lungs, liver and spleen. After the incorporation of DOPG, mRNA expression gradually migrated to the lungs. At the same time, after the addition of DOTAP, the fluorescence signal was specifically observed in the lungs (specificity ~ 90%). When the DOTAP incorporation ratio was 10%, the expression efficiency in the lungs was the highest. When the electrically neutral ALC-0315LNP was aerosolized and administered, mRNA was mainly expressed in the liver, with an expression specificity of up to 80%.

[0165] The fluorescence intensity of the three main organs of mice, liver, spleen, and lung, was compared after administration of LNPs encapsulating Luciferase mRNA. Figure 8 As shown, it can be seen that the charged aerosolized mRNA-LNP is specifically expressed in the lung, while the neutrally charged aerosolized mRNA-LNP has a strong expression in the liver.

[0166] The fluorescence intensity expressed in the three main organs of mice, liver, spleen, and lung, after administration of LNPs encapsulating Luciferase mRNA was correlated with the Zeta potential.

[0167] The results are as follows Fig. 9 As described above, it can be seen that when the Zeta potential is near zero, the higher the expression percentage of mRNA in the liver, and when the positive charge is higher or the negative charge is higher, the expression percentage of mRNA in the lung increases.

[0168] 4. Preparation of electrically neutral LNPs

[0169] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Fig.10 The LNP lipids and corresponding molar ratios shown in the figure, the weight ratio of total lipids to mRNA was 40:1 to prepare the mRNA-LNP solution, and the following electrically neutral LNPs encapsulating Luciferase mRNA were obtained: ALC-0315, SM-102, CL2-200 and Dlin-MC3-DMA LNPs.

[0170] The physical and chemical properties were tested according to the above two methods. The results are as follows Fig.11 As shown, it can be seen that LNPs with suitable particle size, uniformity, electrical neutrality and good encapsulation efficiency were obtained.

[0171] According to the above three methods, various electrically neutral LNPs encapsulating Luciferase mRNA: ALC-0315, SM-102, CL2-200 and Dlin-MC3-DMA were nebulized to mice at a dose of 0.25 mg / kg. Ten hours later, the substrate D-luciferin potassium salt solution was intraperitoneally injected, and IVIS imaging of the mouse body and major organs was performed.

[0172] The results are as follows Fig.12 , it can be seen that these LNPs mainly expressed mRNA in the liver after aerosol administration, and had less expression in other organs.

[0173] Electroneutral atomized LNPs mediated mRNA fluorescence intensity in the liver and expression percentage in the liver, lung, and spleen Fig.13 As shown, it can be seen that the mRNA expression of all these formulations after nebulization was mainly in the liver, with a specificity of more than 70%.

[0174] These results demonstrate the universality of mRNA liver targeting after intratracheal aerosol administration of electrically neutral LNPs.

[0175] Based on these characteristics, this type of electrically neutral LNPs that mediate mRNA expression in the liver after aerosol administration are uniformly named NebuLi LNP. Among them, the electrically neutral LNP with the highest liver delivery efficiency, ALC-0315 (named NebuLi ALC-0315LNP, with a charge of -1.52mV, close to neutrality), was selected for subsequent experiments.

[0176] 5. Evaluation of changes in mRNA expression over time after NebuLi ALC-0315 LNP delivery

[0177] An important advantage of mRNA-based therapeutics is that proteins are expressed transiently, do not require transcription, and are not integrated into the host genome. However, the specific duration of protein expression depends on the route of administration. Therefore, the changes in mRNA expression over time in vivo after delivery by NebuLi ALC-0315LNP were evaluated.

[0178] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Fig.10 The individual LNP lipids and corresponding molar ratios of ALC-0315 are shown, and NebuLi ALC-0315LNP@LucmRNA was prepared with a weight ratio of total lipid to mRNA of 40:1.

[0179] According to the above three methods, mice were administered 0.25 mg / kg of NebuLi ALC-0315LNP@Luc mRNA by intratracheal aerosolization. At different time points after administration, the mice were euthanized and bioluminescence imaging of the whole body and major organs was performed.

[0180] The results are as follows Fig.14 As shown in the figure, it can be seen that mRNA expression can be detected as early as 3 hours after administration, and liver expression reaches a peak at 10 hours. Fig.15 As shown, after the peak, the expression gradually decreased and was negligible after 48 hours, which provides certain guidance for subsequent disease treatment.

[0181] 6. Exploration of the mass ratio of lipids and mRNA in NebuLi ALC-0315LNP

[0182] The mass ratio of lipids and mRNA affects the delivery efficiency of LNP, so the weight ratio of total lipids and mRNA of NebuLi ALC-0315LNP was further optimized.

[0183] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Fig.16 The mRNA-LNP solution was prepared by using the respective LNP lipids of the NebuLi ALC-0315LNP and the corresponding molar ratio and the weight ratio of total lipid to mRNA of 40:1 or 20:1, and the electrically neutral NebuLi ALC-0315LNP (NebuLi ALC-0315LNP@Luc mRNA) encapsulating Luciferase mRNA with a total lipid to mRNA mass ratio of 40:1 and 20:1 was obtained, respectively.

[0184] The physicochemical properties were tested according to the above two methods. The freshly prepared mRNA-LNPs solution was diluted with 1×PBS to an mRNA concentration of 1 ng / mL. The particle size and polydispersity coefficient of LNPs were then determined using a nanoparticle size and potentiometric analyzer. The mRNA encapsulation efficiency of LNPs was determined using the RiboGreen fluorescent dye detection method.

[0185] The results are as follows Fig.17 As described above, it can be seen that the physicochemical properties of the two LNPs are similar.

[0186] According to the above three methods, the two LNPs encapsulating Luciferase mRNA were administered to mice by aerosol at a dose of 0.25 mg / kg. Ten hours later, the substrate D-luciferin potassium salt solution was intraperitoneally injected, and IVIS imaging of the whole body and major organs of the mice was performed.

[0187] The results are as follows Fig.18 As shown, the in vivo mRNA expression results indicated that when the weight ratio was 40:1, the mRNA expression level in the liver was higher.

[0188] Therefore, LNPs with a total lipid to mRNA weight ratio of 40:1 were selected for subsequent experiments.

[0189] VII. Evaluation of the Effect of Repeated Dosing

[0190] One of the advantages of LNPs is that they allow repeated administration without causing severe immune responses. However, the PEG in the LNP component may trigger an immunogenic response. Some studies have found that PEG-modified LNPs can induce anti-PEG antibodies in vivo after the first injection. These antibodies may accelerate the blood clearance of LNPs during subsequent administration, thereby reducing the efficiency of administration. Therefore, the changes in the mRNA expression efficiency of mouse livers after repeated aerosol administration of NebuLi ALC-0315LNPs were evaluated.

[0191] According to the above method, C57BL / 6 mice were administered 0.25 mg / kg of the above-mentioned six-constructed NebuLi ALC-0315LNP@Luc mRNA (40:1) twice, with an interval of 48 hours, and the in vivo bioluminescence was detected at different times after each administration.

[0192] The results are as follows Fig.19 As shown, it can be seen that the expression of luciferase in the liver after two doses is comparable, indicating that multiple aerosol administrations do not affect its liver delivery efficiency, which is beneficial for the continuous treatment of subsequent diseases.

[0193] 8. Safety Testing

[0194] In addition to efficacy, safety and immunogenicity are also key factors affecting the clinical performance of lipid nanoparticles. Uncontrolled immune responses may lead to serious adverse events, including tissue verification, allergic reactions, and organ damage. Therefore, the in vivo safety of Nebuli ALC-0315LNPs was tested.

[0195] According to the above method, C57BL / 6 mice were administered 0.25 mg / kg PBS and NebuLi ALC-0315 LNPs (NebuLi ALC-0315LNP@Luc mRNA constructed in the above method; 40:1) by aerosolization.

[0196] 48 hours after administration, the levels of liver function markers (aspartate aminotransferase, AST; alanine aminotransferase, ALT) and renal function markers (blood urea, UREA; blood creatinine, CREA) in serum were detected.

[0197] The results are as follows Fig. 20 As shown, there was no significant difference after LNP administration compared with the PBS group, indicating that it was well tolerated in vivo.

[0198] H&E staining of tissue sections was also used to evaluate the damage to major organs after aerosol administration. The results showed that the NebuLi ALC-0315LNP treatment group did not produce obvious toxicity in vivo ( Fig.21 ), indicating that NebuLi ALC-0315LNP has good safety.

[0199] Example 2: Application of NebuLi LNP in the treatment of metabolic liver-related diseases

[0200] After verifying the high liver targeting efficiency and good in vivo safety of NebuLi LNP, we want to further explore its potential for metabolic liver-related diseases through protein replacement therapy. This approach could provide a more convenient, safer and more patient-friendly strategy for treating liver diseases.

[0201] 1. Application of NebuLi LNP in the treatment of HT-1 disease

[0202] Hereditary tyrosinemia type 1 (HT-1) is a liver-related metabolic genetic disease caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene. This mutation can lead to severe complications, including renal insufficiency, liver failure, neurological disorders, and ultimately death. Therefore, the HT-1 disease mouse model (FAH - / -The therapeutic effect of NebuLiALC-0315LNP@FAH mRNA was evaluated in mice. In this model, the FAH gene was knocked out and the FAH protein was inactivated, leading to liver-related phenotypic changes such as weight loss and liver fibrosis. By administering exogenous FAH mRNA, it is intended to restore FAH protein expression in the liver, thereby promoting the last step of tyrosine metabolism and reducing the accumulation of toxic substances ( Fig. 22 ).

[0203] 1. Preparation of NebuLi ALC-0315LNPs@FAH mRNA

[0204] FAH mRNA was successfully synthesized by in vitro transcription, and its coding sequence is Sequence 2 shown in Table 2. According to the aforementioned mRNA-LNP synthesis method, FAH mRNA and Fig.10 The lipids in the ALC-0315LNP and the corresponding molar ratio and the weight ratio of total lipids to mRNA of 40:1 were used to prepare the mRNA-LNP solution, and NebuLi ALC-0315LNPs@FAH mRNA encapsulating FAH mRNA was obtained.

[0205] According to the aforementioned mRNA-LNP synthesis method, mCherry mRNA (whose coding sequence is sequence 3 shown in Table 2) and Fig.10 The lipids in the ALC-0315LNP and the corresponding molar ratio and the weight ratio of total lipids to mRNA of 40:1 were used to prepare the mRNA-LNP solution, and NebuLi ALC-0315LNPs@control mRNA encapsulating the control mRNA was obtained.

[0206] 2. Verification of FAH mRNA expression delivered by NebuLi ALC-0315LNPs in vivo

[0207] According to the method of Example 1, FAH - / - Mouse (strain name: ICR-Fah eml Rag2 eml Iq eml / IOZ; sold by: Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences; product catalog number: IOZLAC-200010) were administered 0.3 mg / kg of NebuLi ALC-0315LNPs@FAH mRNA via tracheal atomization.

[0208] Ten hours after administration, mice were killed to prepare frozen liver tissue sections, and then immunofluorescence staining was performed to detect the expression of FAH mRNA in the liver. Specifically, the liver tissue was cut into 10 μm slices, fixed with 4% paraformaldehyde, and permeabilized with 0.125% Triton X-100. The tissue was then washed 4 times with PBS and incubated with FAH primary antibody (Company: Proteintec; Product No.: 67986-1-Ig,) at 4°C overnight. After washing 4 times with PBS, the FAH mRNA was detected by immunofluorescence staining. TM The cells were incubated with 647-conjugated secondary antibody (Company: Invitrogen; Product No.: A-21235), and then washed repeatedly with PBS. Images were collected using a laser confocal microscope (Leica Stellaris).

[0209] The results are as follows Fig.23 As shown, it can be seen that FAH mRNA is efficiently expressed in the liver.

[0210] These data indicate that NebuLi ALC-0315LNP can effectively deliver and express FAH mRNA in mouse liver.

[0211] 3. NebuLi ALC-0315LNP-mediated aerosol therapy for FAH - / - Therapeutic effects in mice

[0212] Further study of NebuLi ALC-0315LNP-mediated aerosol therapy for FAH - / - Therapeutic effects of FAH in mice. - / - Mice usually need to drink nitisinone (NTBC) water to relieve HT-1 symptoms. Once NTBC water is stopped, they immediately lose weight and accumulate toxic substances in the liver, leading to liver failure in a short period of time. Therefore, in order to evaluate the therapeutic effect of NebuLi ALC-0315LNP@FAH mRNA, FAH - / - The mice were randomly divided into four groups. Three groups stopped using NTBC water and were given PBS (G1), NebuLi ALC-0315LNPs@Control mRNA (G2), and NebuLi ALC-0315LNPs@FAH mRNA (G3) by aerosol every three days. One group continued to be given NTBC water (G4) as a positive control ( Fig.24 ).

[0213] The body weight of these mice was continuously monitored throughout the treatment period. Fig.25As shown in the figure, it can be seen that after discontinuation of NTBC, the body weight of G1 and G2 groups decreased significantly, while the body weight of FAH treated with NebuLi ALC-0315LNPs@FAH mRNA decreased significantly. - / - The mice maintained about 95% of their body weight, indicating that NebuLi ALC-0315LNPs@FAH mRNA can mediate the maintenance of mouse body weight.

[0214] When the body weight of the mice in the control group dropped to 80%, the drug administration was stopped, all mice were killed, and the serum and liver were collected for further evaluation. When the mouse serum was collected, it was found that the serum color of the G1 and G2 groups turned dark yellow, indicating that the liver was severely damaged. The serum of the G3 group was normal, similar to the G4 group fed with NTBC water. This shows that the G3 group effectively alleviated the progression of the disease ( Fig.26 ).

[0215] This observation prompted the evaluation of hematological indices related to liver function, including AST, ALT, and total bilirubin (TBIL), and the levels of these indices were analyzed using a fully automatic biochemical analyzer (Company: Hitachi, Japan; Product Model: Hitachi 7600). Fig. 27 The results showed that the levels of liver injury markers in groups G1 and G2 were significantly increased, while there was no significant difference between group G3 and the positive control group (G4).

[0216] The expression of FAH mRNA was verified by the immunofluorescence method of liver tissue sections as described above. Fig.28 As shown in Figure 2, FAH protein was successfully expressed in the liver of the G3 treatment group 18 hours after the last administration, confirming that NebuLi ALC-0315LNP effectively delivered FAH mRNA to FAH - / - mice, causing the liver to produce functional FAH protein.

[0217] In addition, in order to evaluate the alleviating effect of NebuLi ALC-0315LNPs@FAH mRNA on liver fibrosis in mice, liver sections were stained with Sirius red after treatment. Specifically, the liver tissue was first embedded in paraffin, and after the tissue was cut into 10μm slices, it was rehydrated with a gradient of dewaxing solution and anhydrous ethanol, and then stained with a modified Sirius red dye solution. After dehydration and sealing, a panoramic tissue cell analyzer (company: Leica; instrument model: Leica Aperio VESA8) was used for image acquisition and analysis. Compared with the G1 and G2 groups, the red staining area of ​​the G3 group was significantly reduced, indicating that the degree of liver fibrosis was effectively alleviated ( Fig.29 ).

[0218] These results indicate that aerosol treatment with NebuLi ALC-0315LNP@FAH mRNA can significantly reduce liver damage and maintain normal body weight in mice, providing a promising therapeutic strategy for the noninvasive treatment of HT-1 disease.

[0219] 2. Application of NebuLi LNP in the treatment of T2DM

[0220] As a natural protein synthesis factory, the liver produces and secretes a large amount of protein into the blood to perform various physiological functions. Its dysfunction is associated with a variety of chronic metabolic diseases, which usually require frequent injections for treatment, which is a great burden for patients and medical staff. This prompted the consideration of whether mRNA encoding functional proteins can be targeted to the liver for expression through aerosol inhalation and subsequently secreted into the blood, which may provide a more convenient and patient-friendly treatment strategy for chronic diseases. Therefore, T2DM was selected as a disease model. T2DM is a chronic metabolic disorder characterized by insulin resistance and impaired β-cell function. As the disease progresses, multiple oral medications or insulin injections are often required, which can lead to side effects such as weight gain or hypoglycemia. At the same time, glucagon-like peptide-1 receptor agonists (GLP-1RAs) have become a promising treatment for T2DM and have achieved remarkable success. However, GLP-1RAs are mainly produced by genetically engineered mammalian cell culture, which increases production costs. In addition, frequent subcutaneous injections are burdensome to patients. Therefore, the delivery of GLP-1RA mRNA by aerosolized LNP may provide a convenient, effective, and non-invasive treatment for diabetes. Dulaglutide is a long-acting GLP-1RA, whose protein structure is a protein dimer formed by combining a GLP-1 analog with the human IgG4 Fc part. Compared with natural GLP-1, dulaglutide significantly prolongs its half-life.

[0221] Therefore, NebuLi ALC-0315LNP was used as a carrier to deliver dulaglutide mRNA, aiming to explore its therapeutic potential for the treatment of T2DM.

[0222] 1. Preparation of NebuLi ALC-0315LNPs@dulaglutide mRNA

[0223] When designing dulaglutide mRNA, a signal peptide was added to the original dulaglutide to enable it to be secreted after liver expression ( Fig.30 ).

[0224] Dulaglutide mRNA (whose coding sequence is Sequence 4 shown in Table 2) was synthesized by in vitro transcription and transfected into MDA-MB-231 cells, and total cell protein was extracted after 24 hours. The expression of dulaglutide was verified by western blot.

[0225] Trulicity (a commercially available dulaglutide drug) was used as a positive control. Considering that dulaglutide protein is a dimer formed by disulfide bonds, the expression of monomers and dimers was detected under reducing and non-reducing conditions, respectively, indicating that dulaglutide mRNA can be correctly expressed ( Fig.31 ).

[0226] The ability of NebuLi ALC-0315LNP-encapsulated dulaglutide mRNA to be expressed in vivo and secreted into the blood was also evaluated.

[0227] According to the above mRNA-LNP synthesis method, FAH mRNA and Fig.10 The lipids in the ALC-0315LNP and the corresponding molar ratio and the weight ratio of total lipids to mRNA of 40:1 were used to prepare an mRNA-LNP solution, thereby obtaining NebuLi ALC-0315LNPs@dulaglutide mRNA encapsulating dulaglutide mRNA.

[0228] 2. In vivo expression of dulaglutide mRNA

[0229] According to the method of Example 1, 0.5 mg / kg of NebuLiALC-0315LNPs@dulaglutide mRNA was administered to C57BL / 6 mice by tracheal aerosolization. The same volume (ie, 50 μl) of PBS was administered as a control.

[0230] Peripheral blood serum samples were collected from mice at different times after administration (6 hours, 1 to 7 days, 9, 12, 17, 24, and 30 days), and the Dulaglutide protein content in the serum was detected by enzyme-linked immunosorbent assay (ELISA) using a Human IgG ELISA kit (company: Solarbio; product number: SEKH-0208).

[0231] The results are as follows Fig.32 As shown, compared with the PBS group, the dulaglutide content in serum increased significantly after aerosol administration of LNP. At the same time, the protein can be maintained in the blood for more than 2 weeks, showing a metabolic kinetic curve similar to that after subcutaneous injection (sc) of Trulicity.

[0232] These findings demonstrate successful expression of dulaglutide mRNA in vitro and in vivo.

[0233] 3. Treatment of T2DM mouse model with aerosolized mRNA-LNP

[0234] To further evaluate the therapeutic effect of NebuLi ALC-0315LNP delivery of dulaglutide mRNA on T2DM mice, a T2DM mouse model was constructed by high-fat diet combined with streptozotocin (STZ, company: Sigma-Aldrich; product number: V900890) injection. Specifically, 4-week-old male C57BL / 6 mice were fed a 60% high-fat diet (company: ResearchDiets; product number: D14292) for 21 weeks to induce insulin resistance, and then STZ was intraperitoneally injected (30 mg / kg, for 7 consecutive days). Random blood glucose (RBG) levels were measured one week after the last STZ injection. Mice with blood glucose levels greater than 16.7 mmol / L were T2DM mice, and the rest of the mice that did not meet the modeling criteria were eliminated.

[0235] Then, a NebuLi ALC-0315LNPs@dulaglutide mRNA treatment experiment was conducted: T2DM mice were administered 0.35 mg / kg of NebuLi ALC-0315LNPs@dulaglutide mRNA by tracheal aerosolization every week according to the method of Example 1. The same volume (ie, 50 μl) of PBS was administered as a control.

[0236] Body weight and random blood glucose (RBG) levels were monitored throughout the treatment period, and intraperitoneal glucose tolerance (IGTT) and insulin tolerance (ITT) tests were performed in the late treatment period to evaluate its effect on regulating glucose homeostasis. Specifically, in the IGTT experiment, all mice were fasted overnight, and 0.8 g / kg glucose solution (Company: Sigma-Aldrich, Product No.: D9434; the glucose powder was dissolved in sterile water at a concentration of 20%) was injected intraperitoneally the next day. Tail vein blood was collected at 0, 30, 60, 90 and 120 minutes after injection, and blood glucose levels were measured using a blood glucose meter (Company: Roche; Product Model: Active Blood Glucose Meter); in the ITT experiment, all mice were fasted for 4 hours, and mice were intraperitoneally injected with 0.5 U / kg insulin solution (Company: Coolaber; Product No.: SL9710), and blood glucose levels were measured at 0, 15, 30, 45, 60, and 90 minutes after injection. Finally, the changes in each mouse were statistically analyzed, and the area under the curve (AUC) was analyzed using Graphpad Pism9.5 software.

[0237] Mice administered with PBS and Trulicity (subcutaneous injection, 0.6 mg / kg) were used as controls, named NC and PC groups, respectively, and healthy wild-type (WT) C57BL / 6 mice were used as positive controls ( Fig.33 ).

[0238] During the treatment, compared with the NC group, the NebuLi ALC-0315LNPs@dulaglutide mRNA (mRNA) group and the PC group could reduce the body weight of mice ( Fig.34 ) and significantly reduced the RBG level in T2DM mice ( Fig.35 ), these results suggest that aerosolized dulaglutide mRNA-LNP has an effective glucose-lowering potential. In the IGTT ( Fig.36 ) and ITT experiments ( Fig.37 ) also further confirmed that blood glucose homeostasis was improved in T2DM mice.

[0239] These results suggest that NebuLi ALC-0315LNP-mediated delivery of dulaglutide mRNA provides a promising and convenient non-invasive treatment for T2DM.

[0240] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for regulating the targeting of nebulized lipid nanoparticles to tissues, the method being for non-disease diagnosis and treatment purposes, comprising the following steps: preparing nebulized lipid nanoparticles with different charges by incorporating charged lipids, thereby regulating the targeting of nebulized lipid nanoparticles to different tissues; The aerosolized lipid nanoparticles with different charges are electrically neutral aerosolized lipid nanoparticles or charged aerosolized lipid nanoparticles.

2. The method according to claim 1, characterized in that: The different tissue is liver or lung.

3. The method according to claim 1 or 2, characterized in that: The method of preparing lipid nanoparticles in the form of aerosolized dosage forms with different charges by incorporating charged lipids is as follows 1) or 2): 1) preparing electrically neutral aerosolized lipid nanoparticles without incorporating charged lipids; the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids and polyethylene glycol lipids; The electrically neutral aerosolized lipid nanoparticles are targeted to the liver; 2) preparing charged aerosol-type lipid nanoparticles by incorporating charged lipids; the lipids of the charged aerosol-type lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and charged lipids; The charged aerosolized lipid nanoparticles are targeted to the lungs.

4. The method according to any one of claims 1 to 3, characterized in that: The charge of the electrically neutral aerosolized lipid nanoparticles is between -5 and +5 mV; Alternatively, the charged lipid is a cationic lipid having a positive charge under neutral conditions or an anionic lipid having a negative charge.

5. The method according to claim 3 or 4, characterized in that: The lipids of the neutrally charged aerosolized lipid nanoparticles or the charged aerosolized lipid nanoparticles further include auxiliary lipids; The auxiliary lipid is selected from one or any combination of steroids, steroid derivatives and phospholipids.

6. A tissue-targeted aerosolized lipid nanoparticle, which is as follows 1) or 2): 1) The aerosolized lipid nanoparticles are electrically neutral aerosolized lipid nanoparticles; The lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids and polyethylene glycol lipids; The electrically neutral aerosolized lipid nanoparticles are targeted to the liver; 2) The aerosolized lipid nanoparticles are charged aerosolized lipid nanoparticles; The lipids of the charged aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and charged lipids; The charged aerosolized lipid nanoparticles are targeted to the lungs.

7. The aerosolized lipid nanoparticle according to claim 6, characterized in that: The charge of the electrically neutral aerosolized lipid nanoparticles is between -5 and +5 mV; Or, the charged lipid is a cationic lipid with a positive charge under neutral conditions or an anionic lipid with a negative charge; Or, the lipids of the neutrally charged nebulized dosage form lipid nanoparticles and the charged nebulized dosage form lipid nanoparticles further include auxiliary lipids; The auxiliary lipid is selected from one or any combination of steroids, steroid derivatives and phospholipids.

8. The aerosolized lipid nanoparticle according to claim 7, characterized in that: The aerosolized lipid nanoparticles also include nucleic acids encapsulated therein. and / or, the nucleic acid is mRNA, And / or, the mRNA expresses a protein with therapeutic function or expresses a protein with fluorescent reporter function; and / or, the weight ratio of total lipid to mRNA in the aerosolized lipid nanoparticles is 10:1 to 50:1; Or, the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids and steroids; the molar ratios of the lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 50%-70%; the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0; the molar ratio of the steroids to the total lipids is 15-50%; Or, the lipids of the electrically neutral aerosolized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids and phospholipids; the molar ratios of the lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 30%-50%; the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0; the molar ratio of the steroids to the total lipids is 20-50%; the molar ratio of the phospholipids to the total lipids is 0-20%, and is not 0; Or the lipids of the charged atomized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids and charged lipids; the molar ratios of the lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 30%-50%; the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0; the molar ratio of the steroids to the total lipids is 20-50%; the molar ratio of the charged lipids to the total lipids is 0-50%, and is not 0; Or the lipids of the charged atomized lipid nanoparticles include ionizable cationic lipids, polyethylene glycol lipids, steroids, charged lipids and phospholipids; the molar ratios of each lipid are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 15%-60%; the molar ratio of the polyethylene glycol lipids to the total lipids is 0-3%, and is not 0; the molar ratio of the steroids to the total lipids is 15-50%; the molar ratio of the phospholipids to the total lipids is 0-20%, and is not 0; the molar ratio of the charged lipids to the total lipids is 0-60%, and is not 0.

9. Use of the aerosolized lipid nanoparticles according to any one of claims 6 to 8 as a carrier for drug delivery; The drug is a nucleic acid; Or the use of the electrically neutral aerosolized lipid nanoparticles in any one of claims 6 to 8 in the preparation of a product having the following functions A1) or A3): Or the use of the charged atomized lipid nanoparticles in any one of claims 6 to 8 in the preparation of a product having the following function A2): A1) Targeting the drug to the liver through aerosol inhalation; A2) Targeting the drug to the lungs through aerosol inhalation; A3) Treatment of chronic metabolic diseases; Or, a product having the following functions A1) and / or A3), comprising the electrically neutral aerosolized lipid nanoparticles according to any one of claims 6 to 8; A1) Targeting the drug to the liver through aerosol inhalation; A3) Treatment of chronic metabolic diseases; Or, a product having the following function A2), comprising the charged atomized lipid nanoparticles according to any one of claims 6 to 8; A2) Inhalation of aerosolized medication is targeted to the lungs.

10. A method for preparing the aerosolized lipid nanoparticles according to any one of claims 6 to 8, comprising the following steps: 1) preparing lipid nanoparticles according to the lipid components and corresponding molar ratios of the aerosolized lipid nanoparticles according to any one of claims 6 to 8; 2) atomizing the lipid nanoparticles to obtain the atomized lipid nanoparticles according to any one of claims 6 to 8; Or, a method for delivering nucleic acid lipid nanoparticles targeting the liver, comprising the following steps: 1) preparing the electrically neutral aerosolized lipid nanoparticles according to any one of claims 6 to 8; 2) atomizing and delivering the electrically neutral atomized lipid nanoparticles to achieve liver-targeted nucleic acid lipid nanoparticle delivery; Or, a method for delivering nucleic acid lipid nanoparticles targeting the lungs, comprising the following steps: 1) preparing the charged atomized lipid nanoparticles according to any one of claims 6 to 8; 2) The charged atomized lipid nanoparticles are delivered by atomization to achieve targeted lung delivery of nucleic acid lipid nanoparticles.

Images