Saccharide-containing lipid composition and application thereof in enhancing nucleic acid transfection

Abstract

The invention discloses a lipid composition containing saccharides or derivatives thereof and application of the lipid composition in enhancing nucleic acid transfection and disease treatment. The composition comprises (a) an active agent or a therapeutic agent containing nucleic acid; (b) a saccharide or a derivative thereof added in an amount of about 0.5-60 wt% of the total weight of the composition; (c) a cationic lipid; (d) a non-cationic lipid; and (e) a lipid conjugate. The composition can significantly improve the biological effect of the nucleic acid therapeutic agent and improve the biological effect of the nucleic acid therapeutic agent for treating or preventing diseases. The pharmaceutical composition can be applied to prevention or treatment of immune system diseases, cancers, virus infection, bacterial infection, metabolic diseases or hereditary diseases and the like, and the dosage forms comprise inhalant, nasal spray, freeze-dried powder, injection or solution.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery and biological preparation, and in particular to a lipid composition containing sugars, and also to a preparation method and application of the lipid composition containing sugars. Background Art

[0002] Lipid nanoparticles (LNP) are a carrier system widely used in nucleic acid delivery and have been successfully applied to a variety of mRNA vaccines and gene therapy products. However, conventional LNPs still have significant limitations in terms of epithelial mucosal barriers, intracellular transport efficiency and transfection effect. The present invention unexpectedly discovered that the host metabolic state (such as glycolysis) and the intracellular ion channel environment (such as the secretion of calcium ions and chloride ions in bronchial epithelial cells) and the host immune microenvironment have an important influence on the performance of LNP delivery nucleic acid molecules. As natural metabolic factors, carbohydrate substances not only have good biocompatibility and permeability, but may also indirectly affect the cellular uptake, intracellular transport and transfection efficiency of nanoparticles (such as LNPs) by regulating the metabolic pathways and mucus microenvironment of host cells.

[0003] Through research, the inventors discovered that carbohydrates can significantly enhance the efficiency of LNP-based nucleic acid delivery through multiple biological mechanisms: First, carbohydrates can activate the "Warburg effect" to promote glycolysis, significantly increasing intracellular ATP levels, thereby accelerating nucleic acid endocytosis and protein translation. Second, the increased ATP induces calcium ion release and chloride ion efflux via the P2Y2-IP3-CLCA1 / TMEM16A signaling axis, remodeling the respiratory mucus barrier and overcoming the physiological barriers that traditional nanoparticles struggle to penetrate. Furthermore, carbohydrates can also upregulate CXCL10 gene expression in animals, promoting the recruitment of regulatory T cells (Tregs) to lung tissue via the CXCL10–CXCR3 axis, thereby enhancing the IL-10-mediated immunosuppressive pathway. This significantly reduces local inflammation at the site of administration, improves the immune microenvironment shaped by innate immunity, prevents the rapid clearance of exogenous nucleic acid drugs, and thus enhances the bioavailability and biological efficacy of nucleic acid therapeutics.

[0004] Despite this, there is currently a lack of research or technical solutions for systematically integrating carbohydrates into LNP formulations to enhance their delivery performance. Therefore, the development of a structurally stable, safe, and efficient carbohydrate-lipid combination is of great significance for enhancing the biological effects of nucleic acid drugs such as mRNA. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide a lipid composition comprising carbohydrates or their derivatives; a second object of the present invention is to provide a method for preparing the composition; and a third object of the present invention is to provide the use of the lipid composition comprising carbohydrates or their derivatives in the preparation of drugs for treating or preventing diseases.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A lipid composition comprising a carbohydrate or a derivative thereof, comprising the following components:

[0008] (a) an active or therapeutic agent comprising a nucleic acid;

[0009] (b) sugars or their derivatives;

[0010] (c) cationic lipids;

[0011] (d) non-cationic lipids; and

[0012] (e) lipid conjugates,

[0013] The nucleic acid comprises at least one of messenger RNA, self-amplifying RNA, small interfering RNA, circular RNA, antisense oligonucleotide, plasmid DNA, and single-stranded DNA;

[0014] The saccharide or its derivatives account for about 0.5% to 60% of the total weight of the composition.

[0015] Preferably, the chemical structure of the sugar or its derivative is as follows:

[0016] (a)C m (H2O) n , where m and n are positive integers, m is 5-12, n is 5-12; or

[0017] (b)(C6H 10 O5) m , wherein m is an integer from 2 to 500; or

[0018] (c)R-(CHOH) n -CH2OH, wherein R is a hydrogen atom, a hydroxyl group or a modifying group, and n is an integer from 3 to 6;

[0019] The sugar or its derivative comprises at least one selected from the group consisting of glucose, idose, fructose, galactose, mannose, sucrose, trehalose, cyclodextrin, lactose, lactulose, xylose, arabinose, ribose, maltose, chitosan, pullulan, starch, mannitol, sorbitol, erythritol, xylitol, maltitol, microcrystalline cellulose, hydroxypropyl methylcellulose, lentinan, astragalus polysaccharide, hyaluronic acid or its derivative.

[0020] Preferably, the sugar or its derivatives account for about 1% to 30% of the total weight of the composition.

[0021] Preferably, the cationic lipid comprises at least one selected from ionizable cationic lipids, cholesterol-derived cationic lipids, permanent cationic lipids, dendritic cationic polymers or dendritic blocks;

[0022] Preferably, it is an ionizable cationic lipid; the cationic lipid accounts for 23 mol%-80 mol% of the total lipids in the composition, preferably 40.0 mol%-60 mol%;

[0023] The molar ratio of nitrogen (amine) groups in the cationic lipid in the composition to the phosphate groups of the nucleic acid active agent (N / P ratio) is about 1.0-about 30.0, about 3.0-about 15.0, about 4.0-about 10.0, about 6.0-about 8.0; the cationic lipid in the composition comprises at least one selected from the group consisting of DOTAP, DLin-MC3-DMA, Acuitas ALC-0315 (ALC-0315), Moderna Lipid H (SM-102), C12-200, cKK-E12, imidazole cholesterol ester (ICE), N4-arginine cholesterol carbonylamide (GL67) and derivatives thereof.

[0024] Preferably, the non-cationic lipid comprises at least one selected from the group consisting of a neutral lipid, a zwitterionic lipid, and an anionic lipid; preferably, the non-cationic lipid comprises a neutral lipid, preferably, the neutral lipid accounts for 19.0 mol% to 75.0 mol% of the total lipids present in the composition;

[0025] Preferably, the neutral lipid comprises: cholesterol or a neutral lipid derived from cholesterol; phospholipid; or a mixture of cholesterol or a neutral lipid derived from cholesterol and phospholipid;

[0026] wherein the cholesterol or cholesterol-derived neutral lipids account for 14.0 mol%-70.0 mol% of the total lipids in the composition, and the cholesterol-derived neutral lipids comprise at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide / protein, and derivatives thereof, preferably the cholesterol-derived neutral lipids comprise β-sitosterol;

[0027] The phospholipids account for 5.0 mol% to 65.0 mol% of the total lipids in the composition, and include at least one selected from the group consisting of dioleoylphosphatidylserine, egg yolk sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylethanolamine, palmitoyloleoyl-phosphatidylglycerol, dipalmitoyl-phosphatidylethanolamine, dimyristoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, di-antioleoyl-phosphatidylethanolamine, stearoyloleoyl-phosphatidylethanolamine, and derivatives thereof.

[0028] Preferably, the carbohydrate-lipid composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: poly(ethylene glycol)-lipid conjugates (PEG-lipid conjugates), polyamide oligomer-lipid conjugates, polysarcosine-lipid conjugates, polyglycerol-lipid conjugates, polypeptide / protein-lipid conjugates, cation-polymer-lipid conjugates, and derivatives thereof;

[0029] Preferably, the lipid conjugate comprises a PEG-lipid conjugate, comprising at least one selected from the group consisting of: DMG-PEG2000, ALC-0159, DSPE-PEG2000, DMG-PEG5000, PEG1000-PE, PEG3000-PE and PEG5000-PE and derivatives thereof; wherein the lipid conjugate accounts for 0.1 mol%-15.0 mol%, preferably 0.3 mol%-5.0 mol% of the total lipids in the composition.

[0030] The present invention is preferred, wherein the composition comprises the following components:

[0031] (1) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) or dimyristoylglycerol-polyethylene glycol 5000 (DMG-PEG5000), wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 10% by weight of the composition;

[0032] (2) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 29.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 10% by weight of the composition;

[0033] (3) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 19.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.0 mol% of the total lipids, and the carbohydrates and their derivatives account for 25% by weight of the composition;

[0034] (4) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 15% by weight of the composition;

[0035] (5) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 29.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 20% by weight of the composition;

[0036] (6) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or β-sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 5% by weight of the composition;

[0037] (7) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 30% by weight of the composition;

[0038] Preferably, the composition further comprises an excipient, and preferably the excipient comprises at least one selected from the group consisting of Tween, Span, poloxamer, poloxamine, polyethylene glycol, polyvinyl pyrrolidone, hyaluronate, and poly-β-amino ester.

[0039] 2. A method for preparing the composition, comprising:

[0040] (A) in the presence of the saccharide and its derivative (i.e., the saccharide and its derivative is added to the solution containing the active agent or therapeutic agent and / or the solution containing the lipid in advance), mixing the aqueous phase containing the active agent or therapeutic agent with the solution containing the lipid to form the composition; or

[0041] (B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the lipid nanoparticle solution with a solution of saccharides and their derivatives to form the composition; or

[0042] (C) preforming the lipids into lipid nanoparticles without an active agent or therapeutic agent in a solution containing the lipids, and then mixing a solution containing the active agent or therapeutic agent, the saccharide and its derivatives with the lipid nanoparticle solution to form the composition; or

[0043] (D) Preforming the saccharide and its derivatives and lipid into lipid nanoparticles without active agent or therapeutic agent, and then mixing a solution containing the active agent or therapeutic agent with the lipid nanoparticle solution to form the composition.

[0044] The solution used to prepare the above composition includes: physiological saline, 4-hydroxyethylpiperazineethanesulfonic acid HEPEs buffer, tris (hydroxymethylaminomethane) Tris buffer, Tris-EDTA buffer, phosphate PB and phosphate PBS buffer, Dulbecco's phosphate DPBS buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tris buffer containing Tween (TBST), buffer containing EDTA and its sodium salt, and a combination of one or more of the above.

[0045] 3. Use of the lipid composition comprising carbohydrates or their derivatives in the preparation of a drug for treating or preventing diseases, wherein the diseases include immune system diseases, cancer, viral infections, bacterial infections, metabolic diseases or genetic diseases; the formulations include sprays, aerosols, lyophilized powders, dry powders, microneedle patches, capsules, solutions or injections; the drug is delivered through the respiratory tract, lungs, trachea, bronchi, nasal cavity, ocular mucosa, oral mucosa or administered through injection routes such as intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, and intraperitoneal injection; preferably, the drug is delivered through the respiratory tract by intranasal instillation, intranasal spray, intratracheal instillation or inhalation.

[0046] The beneficial effects of the present invention are:

[0047] The carbohydrate-lipid composition provided by the present invention can significantly improve the transfection efficiency of nucleic acid drugs such as mRNA, and is particularly suitable for tissues with rich mucus barriers such as the lungs; the preparation process of the composition is simple, compatible with a variety of carbohydrate addition strategies, and has good operational flexibility and potential for industrial scale-up; carbohydrates have good biocompatibility and metabolic safety, and are suitable for a variety of clinical application scenarios such as inhalation administration, nasal administration, intramuscular injection, intravenous injection, and subcutaneous injection.

[0048] The technology of the present invention can be expanded to various treatment modes such as vaccines, gene editing, antibody therapy, immunotherapy, protein replacement, etc., and has broad application prospects and industrial transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0050] Figure 1 The expression of mRNA encoding firefly luciferase (Fluc-mRNA) mediated by carbohydrate-lipid compositions prepared for different types of carbohydrates in cultured cells (human bronchial epithelial 16HBE cells) in vitro;

[0051] Figure 2 The expression of Fluc-mRNA mediated by trehalose-lipid compositions prepared with different trehalose contents in 16HBE cells cultured in vitro;

[0052] Figure 3 The expression of Fluc-mRNA mediated by arabinose-lipid compositions prepared with different arabinose contents in 16HBE cells cultured in vitro;

[0053] Figure 4 The expression of Fluc-mRNA mediated by maltose-lipid compositions prepared with different maltose contents in 16HBE cells cultured in vitro;

[0054] Figure 5 The expression of Fluc-mRNA mediated by pullulan-lipid compositions prepared with different pullulan contents in 16HBE cells cultured in vitro;

[0055] Figure 6 The expression of Fluc-mRNA mediated by glycerol-lipid compositions prepared with different glycerol contents in 16HBE cells cultured in vitro;

[0056] Figure 7 The expression of Fluc-mRNA mediated by sorbitol-lipid compositions prepared with different sorbitol contents in 16HBE cells cultured in vitro;

[0057] Figure 8 Comparison of Fluc-mRNA transfection in mouse lungs mediated by glucose-lipid compositions (Glu-LNPs) prepared with different PEG-conjugated lipid (DMG-PEG2000) contents and different N / P conditions and LNPs without added sugar;

[0058] Figure 9 To compare the expression of Fluc-mRNA in mouse lungs mediated by Glu-LNPs prepared with different contents of phospholipids (DSPC) and different ratios of cationic lipids (Dlin-MC3-DMA) and LNPs without sugar.

[0059] Figure 10 Comparison of Fluc-mRNA expression in mouse lungs mediated by Glu-LNPs prepared with different types of phospholipids and cationic lipids and LNPs without added sugars;

[0060] Figure 11 Comparison of Fluc-mRNA expression in mouse lungs mediated by Glu-LNPs prepared with different types of PEG lipids and LNPs without sugar;

[0061] Figure 12 The expression of Fluc-mRNA mediated by glucose-lipid composite Glu-LNP prepared in different ways in 16HBE cells cultured in vitro;

[0062] Figure 13 To investigate the effect of glucose-lipid composite Glu-LNP on mRNA uptake in different in vitro cultured cells by flow cytometry;

[0063] Figure 14 The results show that the glucose-lipid composite Glu-LNP mediated the transfection of mRNA encoding enhanced green fluorescent protein (EGFP-mRNA) in cultured cells and organoids in vitro;

[0064] Figure 15 Glucose can enhance the transfection efficiency of Fluc-mRNA mediated by lipid delivery system LNP in various cells;

[0065] Figure 16 Glucose can enhance the transfection efficiency of Fluc-mRNA mediated by the polymer-lipid delivery system PolixNano in various cells;

[0066] Figure 17 Glucose can enhance the cationic polymer delivery system poly (β-amino ester) (PBAE) mediated Fluc-mRNA transfection efficiency in various cells;

[0067] Figure 18 In vitro evaluation of the toxicity of glucose-lipid composition Glu-LNP to various cell lines (MTT method);

[0068] Figure 19 The expression of Fluc-mRNA in mice mediated by carbohydrate-lipid compositions prepared from different types of carbohydrates after intranasal administration;

[0069] Figure 20 The expression of Fluc-mRNA in mice mediated by Glu-LNP, a glucose-lipid composite prepared with different glucose contents, after intranasal administration;

[0070] Figure 21 The expression of Fluc-mRNA mediated by the glucose-lipid composition Glu-LNP in living mice after administration via different routes of administration;

[0071] Figure 22 The distribution of glucose-lipid composition Glu-LNP in different organs of mice after nasal administration;

[0072] Figure 23 The nasal administration of the glucose-lipid composition Glu-LNP mediates the transfection efficiency of Fluc-mRNA in living mice and isolated organs;

[0073] Figure 24To analyze the protein expression dynamics of Fluc-mRNA in mice after intranasal delivery of glucose-lipid composite Glu-LNP;

[0074] Figure 25 The expression of mRNA mediated by the glucose-lipid composition Glu-LNP in different animal models after intranasal delivery;

[0075] Figure 26 This is an investigation of the histopathology of major organs in mice after intranasal delivery of the glucose-lipid composition Glu-LNP;

[0076] Figure 27 Systemic toxicity evaluation of glucose-lipid composite Glu-LNP after intranasal delivery - mouse serum biochemical analysis;

[0077] Figure 28 To investigate the immunological characteristics of Th1 / Th17 mucosal immune responses induced by glucose-lipid composite Glu-LNP-mediated OVA-mRNA (mRNA encoding ovalbumin) vaccine;

[0078] Figure 29 To investigate the immunological characteristics of Th1 / Th17 mucosal immune responses induced by OVA-mRNA vaccine mediated by glucose-lipid composite Glu-LNP;

[0079] Figure 30 To evaluate the therapeutic effect of glucose-lipid combination Glu-LNP mediated IL-12 mRNA in melanoma lung metastasis model;

[0080] Figure 31 To evaluate the preventive protective effect of glucose-lipid composition Glu-LNP mediated OVA-mRNA vaccine in a melanoma lung metastasis model. DETAILED DESCRIPTION

[0081] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0082] Example 1. Preparation of in vitro transcribed mRNA delivery system

[0083] The preparation method of the delivery system for stably encapsulating in vitro transcribed mRNA is as follows:

[0084] 1. Preparation of lipid nanoparticles (LNP):

[0085] Preparation of the organic phase: Weigh lipids using a 100,000 microbalance. Dissolve the cationic lipid SM-102, phospholipid DSPC, cholesterol, and PEG-lipid conjugate DMG-PEG2000 in ethanol at a molar ratio of 49:20.1:30:0.9, mix them, and dilute the total lipid concentration with ethanol to 33.3 mg / mL. Incubate the mixture in a 37°C metal bath for at least 20 min.

[0086] Preparation of the aqueous phase: Dilute in vitro transcribed mRNA encoding firefly luciferase (Fluc-mRNA), enhanced green fluorescent protein (EGFP-mRNA), ovalbumin (OVA-mRNA), or IL-12 (IL-12mRNA) to a final concentration of 0.17 mg / mL in 50 mM citrate buffer (pH 4.0) before use (the Fluc mRNA sequence is shown in SEQ ID NO. 1, the EGFP-mRNA sequence is shown in SEQ ID NO. 2, the OVA-mRNA sequence is shown in SEQ ID NO. 3, and the IL-12 mRNA sequence is shown in SEQ ID NO. 4);

[0087] The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 using a microfluidic device at a total flow rate of 12 mL / min. The mixture was added to a dialysis bag (MWCO: 8000-14000) and dialyzed in 1× PBS (pH 7.4) for 18 hours (ambient temperature: room temperature or 4°C) to obtain lipid nanoparticles (LNPs) that stably encapsulated mRNA.

[0088] 2. Preparation of DiR Fluorescently Labeled Lipid Nanoparticle Composition LNP

[0089] (1) Preparation of organic phase: Weigh lipid materials, including SM-102, DSPC, Cholesterol, and DMG-PEG2000, and mix them in a molar ratio of 49:20.1:30:0.9 in ethanol. Add DiR dye at the desired final concentration (the recommended addition amount is 0.5-1% w / w of the total lipid mass). Mix well and incubate in a 37°C metal bath for at least 20 minutes to allow for full dissolution and formation of a uniform lipid-DiR organic phase mixture.

[0090] (2) Preparation of aqueous phase: Dilute the mRNA to be encapsulated (e.g., Fluc-mRNA) to a final concentration of 0.17 mg / mL using 50 mM citrate buffer (pH 4.0) and set aside;

[0091] (3) Nanoparticle preparation and dialysis: The aqueous phase and the organic phase were rapidly mixed at a volume ratio of 3:1 (aqueous phase: organic phase) using a microfluidic chip device, and the total flow rate was set to 12 mL / min. The crude LNP solution obtained after mixing was immediately placed in a dialysis bag (MWCO: 8000-14000 Da) and dialyzed in 1× PBS buffer (pH 7.4) for 18 h (room temperature or 4°C) to remove ethanol and free DiR. Finally, lipid nanoparticles (DiR-LNPs) with stable mRNA encapsulation and DiR fluorescent labeling were obtained.

[0092] 3. Preparation of carbohydrate-lipid composition (sugar-LNP):

[0093] Method 1: Add sugar to the water phase in advance

[0094] (a) Prepare the aqueous phase: Dilute Fluc-mRNA to a final concentration of 0.17 mg / mL in 50 mM citrate buffer (pH 4.0). Then, add sterile 50% (w / v) sugar solution and adjust the total volume to a final sugar concentration of 13.3% (w / v) in the aqueous phase. Gently mix and set aside.

[0095] (b) Preparation of the organic phase: cationic lipid SM-102, phospholipid DSPC, cholesterol, and PEG-lipid conjugate DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:20.1:30:0.9. The total lipid concentration was adjusted to 33.3 mg / mL and incubated in a metal bath at 37°C for 20 min.

[0096] (c) The aqueous phase and the organic phase were rapidly mixed at a volume ratio of 3:1 at 12 mL / min using a microfluidic chip. The mixture was dialyzed against PBS for 18 h to obtain a carbohydrate-lipid nanoparticle composition.

[0097] Method 2: Add sugar after LNP preparation

[0098] (a) preparing mRNA-loaded lipid nanoparticles (LNPs) according to the method described in "Preparing Lipid Nanoparticles (LNPs)" in Example 1, and performing dialysis to remove impurities;

[0099] (b) The purified LNPs were then mixed with sterile 50% (w / v) sugar solution at a 9:1 volume ratio to a final concentration of 10% (w / v) sugar solution. The mixture was shaken at 800 rpm at 4°C for 5 minutes, incubated at room temperature for 20 minutes, and filtered through a 0.22 μm filter before use in subsequent experiments.

[0100] Method 3: Empty LNP mixed with mRNA + sugar solution

[0101] (a) The organic phase was prepared as above, but the aqueous phase contained only 50 mM citrate buffer (pH 4.0) and no mRNA. The two were mixed in a 3:1 ratio using a microfluidic chip and dialyzed for 18 h to obtain unloaded lipid nanoparticles.

[0102] (b) The empty LNPs were mixed with a solution containing Fluc-mRNA (final concentration 0.17 mg / mL) and 20% (w / v) sugar at a volume ratio of 1:1 and incubated at 4°C for 30 min to form sugar (10% w / v)-modified LNPs encapsulating the active substance.

[0103] Method 4: Sugar + lipid first makes empty LNP, then adds mRNA

[0104] (a) The organic phase was prepared by dissolving SM-102, DSPC, Cholesterol, and DMG-PEG2000 in ethanol at a molar ratio of 49:20.1:30:0.9 and diluting with 50% (w / v) sugar solution. The volume ratio was adjusted to achieve a final sugar concentration of 40% (w / v) in the organic phase.

[0105] (b) Using 50 mM citrate buffer as the aqueous phase, glycolipid-unloaded LNPs were prepared and then dialyzed to remove ethanol;

[0106] (c) The Fluc-mRNA solution (0.17 mg / mL) was then slowly added to the glycolipid empty LNPs, incubated at 4°C for 30 min, and filtered to obtain a preparation with a final sugar concentration of 10% (w / v).

[0107] 4. Preparation of Polymer-Lipid Hybrid Nanoparticles (Polixnano)

[0108] (1) Preparation of organic phase: lipids were weighed using a 100,000 micrometer balance. SM-102, DSPC, Cholesterol, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:20.1:30:0.9 and mixed. The total lipid concentration was diluted with ethanol to 33.3 mg / mL. The mixture was incubated in a 37°C metal bath for more than 20 min.

[0109] (2) Preparation of aqueous phase: Fluc-mRNA and poloxamer 188 (P188) solutions were prepared into 0.34 mg / mL and 24 mg / mL stock solutions, respectively, using 50 mM citrate buffer (pH 4.0). The above-mentioned Fluc-mRNA stock solution and P188 stock solution were mixed evenly in a volume ratio of 1:1 using a microfluidic device, purified, dialyzed, and filtered for use.

[0110] The aqueous and organic phases were rapidly mixed using a microfluidic device at a volume ratio of 3:1 at a total flow rate of 12 mL / min. The mixture was added to a dialysis bag (MWCO: 8000-14000) and dialyzed against 1× PBS (pH 7.4) for 18 hours (at room temperature or 4°C) to obtain the Polixnano formulation.

[0111] 5. Preparation of Cationic Polymer Nanoparticles (PBAE)

[0112] (1) Synthesis of poly (β-amino ester) (PBAE) polymer: Select a suitable diol monomer (1,4-butanediol diacrylate, BDA) and a primary amine monomer (4,4′-trimethylenedihexylamine, TMDA) for addition reaction, dissolve the diol and amine in dry anhydrous DMSO at a molar ratio of 1.2:1 (BDA:amine); stir the reaction in an oil bath at 60°C for 24 hours to generate a PBAE prepolymer; after cooling, precipitate the polymer three times with anhydrous ether to purify it; after vacuum drying, dissolve the PBAE powder in anhydrous DMSO (concentration 10 mg / mL) or ethanol for later use;

[0113] (2) Preparation of PBAE nanoparticles: PBAE polymer and mRNA solution were mixed at a molar ratio of PBAE to mRNA of 50, and mRNA was diluted to the desired concentration (e.g., 0.1-0.2 mg / mL) in 50 mM citric acid buffer (pH 5.5); the PBAE solution was slowly added dropwise to the mRNA solution while gently vortexing to mix; and the mixture was allowed to stand at room temperature for 15-30 minutes to form PBAE nanoparticles.

[0114] Example 2: Expression of Firefly Luciferase-Encoding mRNA (Fluc-mRNA) in Cultured Cells (Human Bronchial Epithelial Cells 16HBE) Mediated by Carbohydrate-Lipid Compositions Prepared from Different Types of Sugar

[0115] (1) Preparation of preparation: A carbohydrate-lipid composition containing Fluc-mRNA was prepared by referring to the method described in Item 3 "Method 2" of Example 1, wherein the carbohydrates are fructose, galactose, mannose, sucrose, trehalose, lactose, lactulose, xylose, arabinose, ribose, pullulan, starch, mannitol, sorbitol, xylitol, maltose, chitosan, maltitol, hyaluronic acid, and glycerol. Corresponding to general formula (a) C m (H2O) n , where m and n are 5-12, and the minimum value structure is selected to represent arabinose (C5H 10 O5) and the maximum structure represent trehalose (C 12 H 22 O 11 ); corresponding to general formula (b)(C6H 10 O5)m , where m is 2-500, the minimum value structure represents maltose (m=2) and the maximum value structure represents pullulan (m≈500); corresponding to the general formula (c) R-(CHOH) n -CH2OH, where n is 3-6 and R is hydrogen or hydroxyl, with the minimum structure representing glycerol (n=3) and the maximum structure representing sorbitol (n=6), respectively. The final concentration of these sugars in the formulation was 10% (w / v). Simultaneously, a control LNP formulation containing Fluc-mRNA was prepared according to the method described in Example 1, item 1.

[0116] (2) Human bronchial epithelial cells (16HBE) were prepared and cultured under appropriate conditions until they were stable, and then seeded into 6-well plates and cultured according to conventional culture conditions until they reached 70-80% confluency.

[0117] (3) Carbohydrate-LNP or LNP preparation containing 1 μg Fluc-mRNA was added to each well for incubation for 6 h. After transfection, the luciferase activity level of each group of cells was measured using a luciferase detection kit to reflect the transfection efficiency of Fluc-mRNA.

[0118] The results are as follows Figure 1 As shown in the results, most sugars can significantly improve the cell transfection level of Fluc-mRNA, and its in vitro transfection level is significantly higher than that of the control LNP group without sugar. Sugars of different structural types show good synergistic effects in regulating the LNP-mediated mRNA transfection efficiency.

[0119] Example 3: Expression of Fluc-mRNA in 16HBE Cells Cultured in Vitro by Carbohydrate-Lipid Compositions Prepared from Different Types of Carbohydrates and Different Contents

[0120] (1) Preparation: A carbohydrate-lipid composition containing Fluc-mRNA was prepared according to the method described in Example 1, Item 3, "Method 2," except that the amount of added carbohydrate varied, namely 0.1%, 1%, 5%, 20%, 30%, 40%, 50%, and 60% (w / v). Simultaneously, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in Example 1, Item 1.

[0121] (2) 16HBE cells were prepared and cultured under appropriate conditions until they were stable, and then seeded into 6-well plates and cultured according to conventional culture conditions until they reached 70-80% confluency.

[0122] (3) Sugar-LNP or LNP preparation containing 1 μg of Fluc-mRNA was added to each well for incubation for 6 h. After transfection, the luciferase activity level of each group of cells was measured using a luciferase detection kit to investigate the transfection efficiency of Fluc-mRNA.

[0123] The results are as follows Figure 2-Figure 7 As shown in the figure, under the sugar concentration gradient of 0.1% to 60%, the cell transfection efficiency of the sugar-LNP group containing sugar was significantly better than that of the LNP group without sugar at each concentration. The luciferase activity reached a peak when the sugar concentration was 10%, indicating that different sugars can enhance the transfection efficiency of mRNA at different concentrations.

[0124] Example 4. Comparison of Fluc-mRNA expression in mouse lungs mediated by glucose-lipid compositions (Glu-LNP) prepared with different PEG-conjugated lipid (DMG-PEG2000) contents and different N / P conditions and LNP without sugar

[0125] Preparation of Formulations: A series of glucose-lipid composition (Glu-LNP) formulations were prepared by referring to the method described in Example 1, Item 3, "Method 2," by further adjusting the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups of the nucleic acid active agent (N / P ratio) to 4, 6, 8, 10, 12, and 14, and also adjusting the content of the PEG-conjugated lipid (DMG-PEG2000). A control LNP formulation containing Fluc-mRNA was prepared according to the method described in Example 1, Item 1. This formulation, along with the aforementioned Glu-LNP formulation, was administered intranasally to mice (2.5 μg Fluc-mRNA / mouse) to compare the transfection efficiency of the two formulations in mediating Fluc-mRNA expression in vivo.

[0126] The results are as follows Figure 8 Under different DMG-PEG2000 contents (0.5-3%) and N / P ratios (4-14), Glu-LNP showed better mRNA expression levels than LNP in all groups, indicating that sugars can enhance mRNA transfection efficiency in vivo under various formulation parameters.

[0127] Example 5. Comparison of the expression of Fluc-mRNA in mouse lungs mediated by Glu-LNPs prepared under conditions of different phospholipid contents (DSPC) and different ratios of cationic lipids (Dlin-MC3-DMA) and LNPs without sugar

[0128] Referring to the method described in Example 1, Item 3, "Method 2," the PEG-conjugated lipid (DMG-PEG2000) ratio was fixed at 1 mol%, with the remaining 99 mol% of the lipid components consisting of a cationic lipid (Dlin-MC3-DMA), a phospholipid (DSPC), and cholesterol. A series of glucose-lipid composition (Glu-LNP) formulations were prepared by adjusting the molar ratio of Dlin-MC3-DMA to 40%, 45%, 50%, 55%, and 60% of the total lipid content, while the DSPC ratio was set to 5%, 10%, 15%, 20%, 25%, and 30%, with the remainder being cholesterol to make up 99% of the total lipid content. A control LNP formulation containing Fluc-mRNA was prepared according to the method described in Example 1, Item 1. This formulation, along with the aforementioned Glu-LNP formulation, was administered intranasally to mice (2.5 μg Fluc-mRNA / mouse) to compare the transfection efficiency of the two formulations in mediating Fluc-mRNA expression in vivo.

[0129] The results are as follows Figure 9 When the ratio of Dlin-MC3-DMA to DSPC was adjusted (Dlin: 40-60%, DSPC: 5-30%), the transfection efficiency of the Glu-LNP group was better than that of the LNP group in all combinations, indicating that the carbohydrate enhancement strategy has good adaptability to lipid formulations.

[0130] Example 6. Comparison of Fluc-mRNA Expression in Mouse Lungs Mediated by Glu-LNPs Prepared from Different Types of Phospholipids and Different Kinds of Cationic Lipids and LNPs Without Sugar

[0131] Referring to the method described in "Method 2" in Section 3 of Example 1, the cationic lipid in the Glu-LNP was selected from Dlin-MC3-DMA, SM-102, or ALC-0315, and the phospholipid type was selected from DOPE, DOPC, DSPC, DPPC, or SOPC. A series of glucose-lipid composition (Glu-LNP) formulations were prepared using a ratio of cationic lipid:phospholipid:cholesterol:PEG-conjugated lipid = 50%:20%:29%:1% in each group. A control LNP formulation containing Fluc-mRNA was prepared according to the method described in Section 1 of Example 1. These formulations, along with the aforementioned Glu-LNP formulations, were administered intranasally to mice (2.5 μg Fluc-mRNA / mouse) to compare the transfection efficiency of the two formulations in mediating Fluc-mRNA expression in vivo.

[0132] The results are as follows Figure 10Regardless of whether the cationic lipid was Dlin-MC3-DMA, SM-102, or ALC-0315, and the phospholipid was DOPE, DOPC, DSPC, DPPC, or SOPC, the transfection efficiency of Glu-LNP was higher than that of the LNP group in all combinations, further verifying that carbohydrates are suitable for delivery systems composed of various types of lipid structures.

[0133] Example 7. Comparison of Fluc-mRNA Expression in Mouse Lungs Mediated by Glu-LNPs Prepared with Different Types of PEG Lipids and LNPs Without Sugar

[0134] Referring to the method described in Example 1, Item 3, "Method 2," the molar ratio of the cationic lipid (SM-102), DSPC, and cholesterol was fixed at 50:20:29, respectively, and the PEG lipid content was fixed at 1 mol%. Specific PEG lipids selected included ALC-0159, DSPE-PEG2000, DMG-PEG2000, PEG1000-PE, PEG3000-PE, and PEG5000-PE. A series of glucose-lipid composition (Glu-LNP) formulations were prepared using the above method. A control LNP formulation containing Fluc-mRNA was prepared according to the method described in Example 1, Item 1. These formulations were administered intranasally (2.5 μg Fluc-mRNA / mouse) to mice, along with the aforementioned Glu-LNP formulations, to compare the transfection efficiency of the two formulations in mediating Fluc-mRNA expression in vivo.

[0135] The results are as follows Figure 11 As shown in the results, when the PEG lipid was ALC-0159, DSPE-PEG2000, DMG-PEG2000, PEG1000-PE, PEG3000-PE or PEG5000-PE, Glu-LNP was superior to LNP in all groups, verifying that this carbohydrate modification strategy has no obvious adaptation limitation with the PEG structure and has good universal synergistic potential.

[0136] Example 8. Preparation of comparison preparations for the expression of Fluc-mRNA mediated by glucose-lipid compositions Glu-LNP prepared by different methods in 16HBE cultured cells in vitro: Four different glucose-lipid composition (Glu-LNP) preparations were prepared according to the method described in Item 3 of Example 1, "Preparation of carbohydrate-lipid compositions (Glu-LNP)". A LNP control preparation containing Fluc-mRNA was prepared according to the method described in Item 1 of Example 1. These preparations were administered to mice via nasal drops (2.5 μg Fluc-mRNA / mouse) along with the above-mentioned Glu-LNP preparations to compare the transfection efficiency of the two preparations in mediating Fluc-mRNA expression in vivo.

[0137] The results are as follows Figure 12 As shown in the results, after respiratory administration, Glu-LNPs prepared using various preparation methods significantly improved mRNA transfection efficiency in mouse lungs, demonstrating that glucose effectively enhances LNP-mediated mRNA transfection. The preparation obtained using "preparing LNP nanoparticles first and then adding glucose" (Method 2) performed best, with superior transfection efficiency compared to other preparation methods. Subsequent studies will utilize this method to prepare carbohydrate-lipid compositions unless otherwise specified.

[0138] Example 9: Glucose-lipid composition Glu-LNP enhances mRNA uptake efficiency in different in vitro cultured cells

[0139] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing fluorescently labeled mRNA (FITC-mRNA) was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing FITC-mRNA was prepared according to the method described in item 1 of Example 1.

[0140] (2) Mouse dendritic cell line (DC2.4), human bronchial epithelial cells (16HBE) and human lung cancer cells (A549) were seeded into 6-well plates, and cultured to 70-80% confluency according to conventional culture conditions.

[0141] (3) Preparation treatment: Add LNP or Glu-LNP preparation containing 1 μg Fluc-mRNA / well to the above cell culture medium for 6 h. After removing the excess preparation, prepare a single cell suspension for flow cytometry detection.

[0142] (4) Flow cytometry analysis: The mRNA uptake efficiency of each cell was analyzed by calculating the percentage of FITC-positive cells by flow cytometry, and the fluorescence intensity (MFI) of positive cells was further calculated.

[0143] The results are as follows Figure 13 Flow cytometry analysis showed that the cellular uptake efficiency of the Glu-LNP group was significantly higher than that of the LNP group in all tested cell types, indicating that the addition of glucose significantly enhanced the cellular uptake of mRNA preparations.

[0144] Example 10: Glucose-lipid composition Glu-LNP enhances EGFP-mRNA transfection efficiency in different in vitro cultured cells and organoids

[0145] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing enhanced green fluorescent protein mRNA (EGFP-mRNA) was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing EGFP-mRNA was prepared according to the method described in item 1 of Example 1.

[0146] (2) 16HBE, DC2.4 cells, and human gastric organoids were prepared and cultured under appropriate conditions until they were stable. They were seeded into 6-well plates and cultured to 70-80% confluency according to conventional culture conditions.

[0147] (3) Add LNP or Glu-LNP preparation containing 1 μg Fluc-mRNA / well and incubate for 6 h.

[0148] (4) Use confocal microscopy to observe the transfection of EGFP fluorescently labeled mRNA in different cells and organoids, calculate the percentage of FITC-positive cells by flow cytometry, and further count the fluorescence intensity of positive cells.

[0149] The results are as follows Figure 14 As shown, the results showed that Glu-LNP significantly improved the transfection efficiency of EGFP-mRNA in different cells compared to the LNP control. In the EGFP-mRNA transfection test in the organoid model, the Glu-LNP treatment group showed stronger EGFP expression and the transfection efficiency in the organoid was significantly better than that of the LNP group. These results show that the addition of glucose can not only effectively enhance the cell transfection effect, but also significantly improve the transfection efficiency of mRNA in more complex three-dimensional organoid models.

[0150] Example 11: Glucose can enhance the transfection efficiency of Fluc-mRNA mediated by various delivery systems in different cell types

[0151] (1) Preparation of preparations: LNP, PolixNano and PBAE preparations containing Fluc-mRNA were prepared according to the method described in Example 1, and Glu-LNP, Glu-PolixNano and Glu-PBAE preparations were prepared respectively by adding glucose solution with a final concentration of 10% (w / v).

[0152] (2) Mouse bone marrow-derived dendritic cells (BMDCs), DC2.4 cells, 16HBE cells, A549 cells, and mouse macrophages (RAW264.7) were seeded in 96-well plates and cultured to 70-80% confluency according to conventional culture conditions.

[0153] (3) Add LNP, PolixNano, PBAE preparations containing 1 μg Fluc-mRNA / well or the corresponding Glu-LNP, Glu-PolixNano and Glu-PBAE preparations (containing equal amounts of Fluc-mRNA) and incubate for 6 h.

[0154] (4) Luciferase activity in each group of cells was measured 24 h after transfection using a luciferase assay kit to reflect the expression efficiency of Fluc-mRNA. The results were expressed in relative luminescence units (RLU).

[0155] Test results such as Figures 15 to 17 The results showed that in all five cell types tested, various types of delivery systems containing glucose (Glu-LNP, Glu-PolixNano, and Glu-PBAE) significantly improved the transfection efficiency of Fluc-mRNA compared to the control formulation without sugar, demonstrating that sugars have the universality to significantly enhance mRNA transfection mediated by different types of delivery systems.

[0156] Example 12: In vitro evaluation of the toxicity of the glucose-lipid composition Glu-LNP on various cell lines (MTT method)

[0157] (1) Preparation of formulations: A carbohydrate-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0158] (2) Cell preparation: 16HBE, DC2.4, and A549 cells were prepared and cultured under appropriate conditions until they were stable, and then seeded into 96-well plates, respectively, and cultured under conventional culture conditions until they reached 70-80% confluency.

[0159] (3) Sample treatment: Various treatment groups were added to the cells for incubation, including: Lipofectamine The cells were treated with Lipo3000 (positive control), LNP (LNP), Glu-LNP (Glu-LNP), naked mRNA (Nude mRNA), glucose-only control (Glucose-only), and PBS (Negative control) for 6 hours, followed by replacement with fresh complete medium and continued culture for 24 hours.

[0160] (4) MTT assay: After treatment, MTT working solution (5 mg / mL, 20 μL) was added to each well and incubated at 37°C for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the crystals. The absorbance was measured at a wavelength of 570 nm using a microplate reader.

[0161] The results are as follows Figure 18The results showed that the Glu-LNP-treated group showed no significant toxicity in any of the three cell types, with cell viability similar to that of the PBS group and significantly superior to that of the Lipo3000 positive control group. This indicates that the sugar-containing delivery system has good biocompatibility with cells.

[0162] Example 13: Comparison of Fluc-mRNA expression in mice mediated by carbohydrate-lipid compositions prepared from different types of carbohydrates after nasal administration

[0163] (1) Preparation of formulations: A carbohydrate-lipid composition formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0164] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, individual mice were intranasally administered with sugar-LNP or LNP formulation containing 2.5 μg Fluc-mRNA.

[0165] (3) Six hours after administration, the luciferase luminescence signal in the mouse lungs was measured using an IVIS optical imaging system for characterization.

[0166] The results are as follows Figure 19 The results showed that most sugars significantly enhanced the level of mRNA transfection mediated by the formulation in vivo. Among them, a monosaccharide combination represented by glucose, fructose, galactose, and mannose exhibited the highest luciferase activity, with expression levels far exceeding those of the unsweetened control LNP group. Sugars of different structural types demonstrated a synergistic effect in regulating the efficiency of LNP-mediated mRNA transfection.

[0167] Example 14: Expression of Fluc-mRNA in Mice Mediated by Nasal Administration of Glucose-Lipid Compositions Glu-LNP Prepared with Different Glucose Contents

[0168] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0169] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered intranasally to each mouse.

[0170] (3) Six hours after administration, the luciferase luminescence signal in the mouse lungs was measured using an IVIS optical imaging system for characterization.

[0171] The results are as follows Figure 20 The results showed that the Glu-LNP group had significantly better mouse lung transfection efficiency than the unsweetened LNP group at each concentration of glucose, ranging from 0.1% to 60%. Luciferase activity peaked at 10% glucose concentration, indicating that the preparation had optimal lung mRNA delivery efficiency at this concentration.

[0172] Example 15. Comparison of the expression of Fluc-mRNA mediated by the glucose-lipid composition Glu-LNP in mice after administration via different routes of administration

[0173] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0174] (2) Balb / C mice (6-8 weeks old) were randomly divided into groups of 3.

[0175] The composition was administered intratracheally (it): During isoflurane inhalation anesthesia, Glu-LNP or LNP formulation containing 2.5 μg Fluc-mRNA was administered via intratracheal spraying into a single mouse using a high-pressure spray needle.

[0176] The composition was administered intranasally (in): During isoflurane inhalation anesthesia, a single mouse was administered with Glu-LNP or LNP formulation containing 2.5 μg Fluc-mRNA in the form of one droplet to the nostril of the animal, and the formulation was naturally inhaled.

[0177] The composition was administered by intramuscular injection (im): the skin surface of the outer thigh muscle of Balb / c mice was disinfected with 75% alcohol cotton balls, the muscle was quickly punctured with a syringe, and a single mouse was injected with Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA.

[0178] The composition was administered via nebulization: Glu-LNP or LNP formulation containing 100 μg of Fluc-mRNA was added to a nebulizer, and a single animal was placed in a nebulizer chamber to inhale the nebulized aerosol particles for 30 minutes, during which the ventilation flow rate and formulation concentration were kept stable.

[0179] The composition was administered intravenously (iv): Balb / c mice were disinfected by wiping their tails with 75% alcohol cotton balls. Glu-LNPs or LNP formulations containing 2.5 μg of Fluc-mRNA were then administered to each mouse via slow injection into the tail vein, ensuring no fluid leakage during the injection. After the injection, the injection site was pressed with a dry cotton ball to stop bleeding.

[0180] Six hours after administration, the luciferase luminescence signal of living mice was measured using an IVIS optical imaging system for characterization.

[0181] The results are as follows Figure 21 As shown. The results showed that Glu-LNPs demonstrated superior mRNA transfection efficiency compared to LNPs under various routes of administration, as determined by in vivo luciferase activity assays. The effects were particularly pronounced when administered via intratracheal (it) and intranasal (in). The it-administered Glu-LNPs demonstrated superior mRNA delivery to the lungs compared to the LNP control. Following intramuscular (im) and intravenous (iv) administration, the mRNA transfection efficiency of Glu-LNPs in mice was also superior to that of the unsweetened LNP group, suggesting that the glucose co-delivery strategy has strong versatility and enhanced effects.

[0182] Example 16: Distribution of glucose-lipid composition Glu-LNP in different organs of mice after nasal administration

[0183] (1) Preparation of preparations: DiR fluorescently labeled LNP preparations containing Fluc-mRNA and DiR fluorescently labeled glucose-lipid composition (Glu-LNP) preparations containing Fluc-mRNA were prepared according to the method described in item 2 of Example 1.

[0184] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered to each mouse via nasal drops.

[0185] (3) 6 h after administration, mice were killed by cervical dislocation, and the main organs (brain, lungs, heart, liver, spleen, and kidneys) were removed. The fluorescence intensity of each organ was analyzed in vitro using the IVIS optical imaging system, and the luciferase luminescence signal in the lungs was measured as a characterization.

[0186] The results are as follows Figure 22 The results showed that the fluorescence accumulation signal of the Glu-LNP group in the mouse lungs was stronger than that of the LNP group, while there was almost no obvious fluorescence signal in other organs, indicating that the Glu-LNP after intranasal administration had a stronger distribution and enrichment ability in the lungs.

[0187] Example 17: Glucose-lipid composition Glu-LNP enhances the transfection efficiency of Fluc-mRNA in living mice and isolated organs

[0188] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0189] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered intranasally to each mouse.

[0190] (3) Six hours after administration, bioluminescence imaging of different organs of mice was performed using an IVIS imaging system to record the luciferase luminescence signal.

[0191] The results are as follows Figure 23 The results showed that the luciferase expression levels in the lungs and whole tissues of mice in the Glu-LNP group were significantly higher than those in the LNP group, indicating that glucose can enhance the transfection efficiency of Fluc-mRNA in mice.

[0192] Example 18: Kinetic Analysis of Fluc-mRNA Protein Expression in Mice Mediated by Intranasal Delivery of Glucose-Lipid Composition Glu-LNP

[0193] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0194] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered intranasally to each mouse.

[0195] (3) The bioluminescence signals of mice were monitored using an IVIS optical in vivo imaging system at different time points (3, 6, 12, 24, 48, and 72 h) after administration, and the luciferase luminescence signals were recorded to evaluate the mRNA expression levels at different time points.

[0196] The experimental results are as follows Figure 24The results showed that the LNP group produced only a weak transient fluorescence signal early on, while the Glu-LNP group reached peak mRNA expression at 6 hours and maintained a significant bioluminescent signal for 72 hours, demonstrating stronger and more sustained mRNA expression. This suggests that the Glu-LNP formulation possesses superior transfection persistence in vivo.

[0197] Example 19: Comparison of the expression of Fluc-mRNA in different animal models after nasal administration of the glucose-lipid composition Glu-LNP

[0198] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0199] (2) Male SD rats, C57BL / 6 mice, and Syrian hamsters (n=3) were selected. After anesthesia, LNPs or Glu-LNPs containing 10 μg Fluc-mRNA were administered intranasally to each SD rat, 10 μg Fluc-mRNA to each Syrian hamster, and 2.5 μg Fluc-mRNA to each C57BL / 6 mouse.

[0200] (3) Six hours after administration, the luciferase signal in the lungs of mice, Syrian golden hamsters, and rats was detected in vivo and in vitro using the IVIS optical in vivo imaging system to evaluate the transfection efficiency.

[0201] The results are as follows Figure 25 As shown, compared with the LNP control group, Glu-LNP showed significantly enhanced mRNA transfection ability in all three animal models, indicating that Glu-LNP has good cross-species adaptability and lung transfection enhancement effect.

[0202] Example 20 Histopathological investigation of major organs of mice after nasal administration of the glucose-lipid composition Glu-LNP

[0203] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0204] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered intranasally to each mouse. The PBS group was set as a negative control.

[0205] (3) Organ collection and processing: 6 h after administration, the mice were sacrificed and the lung, liver, spleen, kidney, heart and intestinal tissues were removed. The tissues were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin. 5 μm thick sections were prepared.

[0206] (4) Tissue staining and observation: The tissue sections of each organ were stained using the hematoxylin and eosin (H&E) staining method, and the changes in the tissue structure were observed using an optical microscope.

[0207] The results are as follows Figure 26 The results showed that the tissue structure of the main organs (lungs, liver, spleen, kidneys, heart, and intestines) of mice in the Glu-LNP treatment group remained intact, with no obvious inflammatory infiltration, cell necrosis, or organ damage, and there was no significant difference compared to the PBS control group, indicating that Glu-LNP intranasal delivery has good biocompatibility and tissue safety in the short term. Notably, the inflammatory infiltration of lung tissue sections in the Glu-LNP treatment group was somewhat reduced compared to the LNP control group, suggesting that the addition of glucose helps to alleviate the local inflammation induced by LNP in the mouse lungs.

[0208] Example 21 Systemic toxicity evaluation of glucose-lipid composition Glu-LNP after nasal administration - mouse serum biochemical analysis

[0209] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing Fluc-mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing Fluc-mRNA was prepared according to the method described in item 1 of Example 1.

[0210] (2) Balb / C mice (6-8 weeks old) were randomly divided into 3 groups. During isoflurane inhalation anesthesia, Glu-LNP or LNP preparation containing 2.5 μg Fluc-mRNA was administered intranasally to each mouse. The PBS group was set as a negative control.

[0211] (3) Blood collection and processing: Six hours after administration, whole blood was collected from mice by retroorbital blood collection. After standing at 4°C, the blood was centrifuged (3000 rpm, 10 minutes) to obtain serum for subsequent biochemical analysis.

[0212] (4) Serum biochemical index detection: The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU) and glycosylated serum protein (GSP) in serum were measured.

[0213] The results are as follows Figure 27 As shown in the figures, no significant differences were observed among the LNP, Glu-LNP and PBS control groups for all test indicators, and all values ​​were within the normal reference range (gray area), indicating that intranasal delivery of Glu-LNP did not cause significant systemic toxicity reactions and had good systemic biosafety.

[0214] Example 22: Immunological Characteristics of Th1 / Th17 Mucosal Immune Responses Induced by Glucose-Lipid Composition Glu-LNP-Mediated Ovalbumin-Encoding mRNA Vaccine (OVA-mRNA)

[0215] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing OVA-mRNA was prepared according to the method described in item 3, “Method 2” of Example 1. Simultaneously, a LNP control formulation containing OVA-mRNA was prepared according to the method described in item 1 of Example 1.

[0216] (2) Animal immunization and sample collection: C57BL / 6 mice (6–8 weeks old, either sex) were selected and immunized intranasally with the following three preparations on day 0 and day 21, respectively (n = 9): control group: PBS; positive control group: LNP vaccine (encapsulated with 2.5 μg OVA-mRNA / mouse); experimental group: Glu-LNP vaccine (encapsulated with 2.5 μg OVA-mRNA / mouse).

[0217] (3) The animals were killed on day 28, and the secretion of antigen-specific IFN-γ, IL-17, and IL-4 was detected using an ELISpot kit after isolating lung tissue lymphocytes.

[0218] (4) Flow cytometry detection of CD4 + IFN-γ + and CD8 + TIFN-γ + The proportion of cell subpopulations.

[0219] The results are as follows Figures 28-29 The results showed that compared with the PBS and LNP groups, the ELISpot in the lung tissue lymphocytes of the mice in the Glu-LNP immunization group showed a significant increase in IFN-γ + IL-17 + and IL-4 spot-forming units (SFU) ( Figure 28 ), further flow cytometry analysis showed that the CD4 + and CD8 + A higher proportion of antigen-specific IFN-γ positive cells were detected in T cells ( Figure 29 ), indicating that Glu-LNP has a better effect than LNP control in inducing cellular immune response.

[0220] Example 23: Evaluation of the therapeutic effect of glucose-lipid composition Glu-LNP mediated IL-12 mRNA on a melanoma lung metastasis model

[0221] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing IL-12 mRNA was prepared according to the method described in item 3 "Method 2" of Example 1. At the same time, a LNP control formulation containing IL-12 mRNA was prepared according to the method described in item 1 of Example 1.

[0222] (2) Animal model establishment and treatment method: C57BL / 6 mice were selected and injected with 2*10 6 B16-F10-Luc melanoma cells were used to establish a lung metastasis model. The following three groups (n=9) were treated by intranasal instillation on days 6, 9, and 12 after modeling: control group: PBS solution; positive control group: LNP formulation encapsulated with 2.5 μg IL-12 mRNA / mouse; experimental group: Glu-LNP formulation encapsulated with 2.5 μg IL-12 mRNA / mouse ( Figure 30 a).

[0223] (3) Tumor growth monitoring: On days 9, 12, 15, 18, and 21, luciferase bioluminescence imaging was performed on the tumor burden in mice using an in vivo imaging system (IVIS), and the tumor fluorescence signal intensity was quantified.

[0224] (4) Lung tissue analysis: The animals were sacrificed on day 21, and the lung tissues were removed for macroscopic observation and weighed.

[0225] (5) qPCR detection: Total RNA was extracted from lung tissue, reverse transcribed, and then subjected to real-time fluorescence quantitative PCR to analyze the expression level of the melanoma-related gene Tyrp1.

[0226] (6) Survival monitoring: record the survival status of mice, draw survival curves, and evaluate the effects of different treatments on animal survival.

[0227] The results are as follows Figure 30As shown in b, the tumor growth progression of the Glu-LNP treatment group was significantly delayed from day 9 to day 18, and the tumor bioluminescence intensity was always lower than that of the LNP group and the PBS group. The lung tissue weight of the Glu-LNP group was significantly reduced compared with the LNP group ( Figure 30 c), qPCR analysis results showed that the expression level of Tyrp1 gene in lung tissue of Glu-LNP group was significantly lower than that of LNP group and PBS group ( Figure 30 d), further indicating a reduction in tumor burden. The survival rate of mice in the Glu-LNP group was significantly improved compared to the LNP group, with more than 50% of the animals still alive after day 30, while all animals in the PBS group died within day 30 ( Figure 30 e).

[0228] Example 24: Evaluation of the preventive protective effect of glucose-lipid composition Glu-LNP mediated OVA-mRNA vaccine in a melanoma lung metastasis model

[0229] (1) Preparation of formulations: A glucose-lipid composition (Glu-LNP) formulation containing OVA-mRNA was prepared according to the method described in item 3, “Method 2” of Example 1. Simultaneously, a LNP control formulation containing OVA-mRNA was prepared according to the method described in item 1 of Example 1.

[0230] (2) Animal modeling and treatment: C57BL / 6 mice were selected and immunized with the following preparations (n=9) via the “prime-boost” immunization program via intranasal route on days 0 and 14: experimental group: Glu-LNP preparation encapsulating 2.5 μg OVA-mRNA / mouse; positive control group: LNP preparation encapsulating 2.5 μg OVA-mRNA / mouse; negative control group: PBS. On day 21, 2*10 6 The mouse lung metastasis model was established by using B16-F10-OVA melanoma cells ( Figure 31 a).

[0231] (3) Lung tissue analysis: The animals were sacrificed on day 50, and the lung tissues were dissected and weighed.

[0232] (4) qPCR detection: Total RNA was extracted from lung tissue and the mRNA expression level of melanoma-related gene Tyrp1 was detected.

[0233] (5) Survival monitoring: Record the survival status of each group of mice, draw a survival curve, and evaluate the effect of different vaccine treatments on survival time.

[0234] The results showed that compared with the PBS and LNP groups, the lung tissue weight of the Glu-LNP group was significantly lower than that of the LNP group ( Figure 31b). qPCR test results showed that the expression of Tyrp1 gene in lung tissue of Glu-LNP group was significantly lower than that of control group ( Figure 31 c). Survival analysis showed that the survival rate of mice in the Glu-LNP group was significantly higher than that in the LNP group, with more than half of the mice still alive after day 30, while all mice in the PBS group had died before day 30 ( Figure 31 d).

[0235] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A lipid composition comprising a carbohydrate or a derivative thereof, characterized in that Includes the following components: (a) an active or therapeutic agent comprising a nucleic acid; (b) sugars or their derivatives; (c) cationic lipids; (d) non-cationic lipids; and (e) lipid conjugates, The nucleic acid comprises at least one of messenger RNA, self-amplifying RNA, small interfering RNA, circular RNA, antisense oligonucleotide, plasmid DNA, and single-stranded DNA; The saccharide or its derivatives account for about 0.5% to 60% of the total weight of the composition.

2. The lipid composition comprising carbohydrates or derivatives thereof according to claim 1, wherein: The general chemical structure formula of the saccharide or its derivative is as follows: (a)C m (H2O) n , where m and n are positive integers, m is 5-12, n is 5-12; or (b)(C6H 10 O5) m , wherein m is an integer from 2 to 500; or (c)R-(CHOH) n -CH2OH, wherein R is a hydrogen atom, a hydroxyl group or a modifying group, and n is an integer from 3 to 6; The sugar or its derivative comprises at least one selected from the group consisting of glucose, idose, fructose, galactose, mannose, sucrose, trehalose, cyclodextrin, lactose, lactulose, xylose, arabinose, ribose, maltose, chitosan, pullulan, starch, mannitol, sorbitol, erythritol, xylitol, maltitol, microcrystalline cellulose, hydroxypropyl methylcellulose, lentinan, astragalus polysaccharide, hyaluronic acid or its derivative.

3. The lipid composition comprising carbohydrates or derivatives thereof according to claim 2, wherein: The sugar or its derivatives account for about 1% to 30% of the total weight of the composition.

4. The lipid composition comprising carbohydrates or derivatives thereof according to claim 1, wherein: The cationic lipid comprises at least one selected from ionizable cationic lipids, cholesterol-derived cationic lipids, permanent cationic lipids, dendritic cationic polymers or dendrimers; Preferably, the cationic lipid is an ionizable cationic lipid; The cationic lipid accounts for 23 mol% to 80 mol% of the total lipids in the composition, preferably 40.0 mol% to 60 mol%; The molar ratio (N / P) of nitrogen (amine) groups in the cationic lipid in the composition to phosphate groups of the nucleic acid active agent is about 1.0-30.0, about 3.0-15.0, about 4.0-10.0, about 6.0-8.0; the cationic lipid in the composition comprises at least one selected from the group consisting of DOTAP, DLin-MC3-DMA, Acuitas ALC-0315 (ALC-0315), Moderna LipidH (SM-102), C12-200, cKK-E12, imidazole cholesterol ester (ICE), N4-arginine cholesterol carbonylamide (GL67) and derivatives thereof.

5. The lipid composition comprising carbohydrates or derivatives thereof according to claim 1, characterized in that: The non-cationic lipid comprises at least one selected from the group consisting of: a neutral lipid, a zwitterionic lipid, and an anionic lipid; Preferably, the non-cationic lipid comprises a neutral lipid, and the neutral lipid accounts for 19.0 mol% to 75.0 mol% of the total lipids present in the composition; Preferably, the neutral lipid comprises: cholesterol or a neutral lipid derived from cholesterol; phospholipid; or a mixture of cholesterol or a neutral lipid derived from cholesterol and phospholipid; wherein the cholesterol or cholesterol-derived neutral lipids account for 14.0 mol%-70.0 mol% of the total lipids in the composition, and the cholesterol-derived neutral lipids comprise at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide / protein, and derivatives thereof, preferably the cholesterol-derived neutral lipids comprise β-sitosterol; The phospholipids account for 5.0 mol% to 65.0 mol% of the total lipids in the composition, and include at least one selected from the group consisting of dioleoylphosphatidylserine, egg yolk sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylethanolamine, palmitoyloleoyl-phosphatidylglycerol, dipalmitoyl-phosphatidylethanolamine, dimyristoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, di-antioleoyl-phosphatidylethanolamine, stearoyloleoyl-phosphatidylethanolamine, and derivatives thereof.

6. The lipid composition comprising a carbohydrate or a derivative thereof according to claim 1, wherein: The carbohydrate-lipid composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: poly(ethylene glycol)-lipid conjugates, polyamide oligomer-lipid conjugates, polysarcosine-lipid conjugates, polyglycerol-lipid conjugates, polypeptide / protein-lipid conjugates, cation-polymer-lipid conjugates, and derivatives thereof; Preferably, the lipid conjugate comprises a poly(ethylene glycol)-lipid conjugate, comprising at least one selected from the group consisting of: DMG-PEG2000, ALC-0159, DSPE-PEG2000, DMG-PEG5000, PEG1000-PE, PEG3000-PE and PEG5000-PE and derivatives thereof; The lipid conjugate accounts for 0.1 mol% to 15.0 mol%, preferably 0.3 mol% to 5.0 mol% of the total lipids in the composition.

7. The lipid composition comprising a carbohydrate or a derivative thereof according to any one of claims 1 to 6, characterized in that: The composition comprises the following components: (1) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) or dimyristoylglycerol-polyethylene glycol 5000 (DMG-PEG5000), wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 10% by weight of the composition; (2) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 29.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 10% by weight of the composition; (3) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 19.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.0 mol% of the total lipids, and the carbohydrates and their derivatives account for 25% by weight of the composition; (4) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 15% by weight of the composition; (5) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 29.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 20% by weight of the composition; (6) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or β-sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 0.9 mol% of the total lipids, and the carbohydrates and their derivatives account for 5% by weight of the composition; (7) carbohydrates and their derivatives; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2000 or DMG-PEG5000, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2000 or DMG-PEG5000 account for 1.5 mol% of the total lipids, and the carbohydrates and their derivatives account for 30% by weight of the composition.

8. A method for preparing the composition according to any one of claims 1 to 7, characterized in that: The method comprises: (A) mixing an aqueous phase containing the active agent or therapeutic agent with a solution containing the lipid in the presence of the saccharide and its derivatives to form the composition; or (B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the lipid nanoparticle solution with a solution of saccharides and their derivatives to form the composition; or (C) preforming the lipids into lipid nanoparticles without an active agent or therapeutic agent in a solution containing the lipids, and then mixing a solution containing the active agent or therapeutic agent, the saccharide and its derivatives with the lipid nanoparticle solution to form the composition; or (D) Preforming the saccharide and its derivatives and lipid into lipid nanoparticles without active agent or therapeutic agent, and then mixing a solution containing the active agent or therapeutic agent with the lipid nanoparticle solution to form the composition. The solution used to prepare the above composition includes: physiological saline, 4-hydroxyethylpiperazineethanesulfonic acid HEPEs buffer, tris (hydroxymethylaminomethane) Tris buffer, Tris-EDTA buffer, phosphate PB and phosphate PBS buffer, Dulbecco's phosphate DPBS buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tris buffer containing Tween (TBST), buffer containing EDTA and its sodium salt, and a combination of one or more of the above.

9. The lipid composition comprising a carbohydrate or a derivative thereof according to any one of the preceding claims, characterized in that: The composition further comprises an excipient, preferably the excipient comprises at least one selected from the group consisting of Tween, Span, poloxamer, poloxamine, polyethylene glycol, polyvinyl pyrrolidone, hyaluronate, and poly-β-amino ester.

10. Use of the lipid composition comprising carbohydrates or derivatives thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating or preventing a disease, characterized in that: The diseases include immune system diseases, cancer, viral infection, bacterial infection, metabolic disease or genetic disease; the preparation forms include spray, aerosol, lyophilized powder, dry powder, microneedle patch, capsule, solution or injection; the purpose is to deliver through the respiratory tract, lungs, trachea, bronchi, nasal cavity, eye mucosa, oral mucosa or administer through injection routes such as intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intraperitoneal injection; preferably, it is delivered through the respiratory tract by intranasal instillation, intranasal spray, intratracheal instillation or inhalation.

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