Lipid molecules for nucleic acid delivery and liver microenvironment repair and applications thereof

Abstract

The application discloses a kind of lipid molecules for nucleic acid delivery and liver microenvironment repair and application, belong to medical biotechnology field, and the lipid molecule is the compound shown in formula (1) or its pharmaceutically acceptable salt, it contains tocopherol or tocol trienol fragment and zinc ion coordination group in its structure, with the chemical and biological function of ionizable lipid and the antioxidant, anti-inflammatory activity of vitamin E and zinc.The lipid nanoparticle comprising the compound can efficiently deliver various nucleic acids, and while releasing in liver target, by eliminating active oxygen, inhibiting inflammatory pathways, actively improving liver microenvironment, reducing oxidative stress and fibrosis damage.The delivery system realizes the synergistic treatment concept of "carrier is medicine", and has wide application prospect in nucleic acid drug treatment and gene vaccine development in chronic liver disease, liver fibrosis, liver cancer and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biology, specifically relating to a method for preparing lipid nanoparticles that can deliver nucleic acids and remodel the liver microenvironment, and their applications. Background Technology

[0002] In recent years, with the rapid breakthroughs in biotechnology, nucleic acid drugs based on small interfering RNA (siRNA), messenger RNA (mRNA), and antisense oligonucleotides (ASO) have been regarded as a revolutionary strategy for treating refractory liver diseases because they can precisely regulate the expression of pathogenic proteins at the post-transcriptional level.

[0003] In the clinical translation of nucleic acid drugs, lipid nanoparticles (LNPs) have become the dominant non-viral delivery carriers due to their superior nucleic acid loading capacity, high cellular uptake efficiency, and ease of large-scale production. Utilizing the ability of LNPs to readily adsorb apolipoprotein E (ApoE) from plasma and enter hepatocytes via low-density lipoprotein receptors (LDLRs), researchers have achieved highly efficient targeted delivery of nucleic acid drugs to the liver. However, existing commercially available LNP formulations still have significant limitations. Their main component—ionizable lipids—while achieving endosome escape, often exhibits dose-limiting toxicity. Numerous studies have confirmed that after accumulation in the liver, these exogenous lipid carriers disrupt cell membrane integrity, stimulate Toll-like receptor (TLR) pathways, and induce non-specific innate immune responses. For pathological livers that are already in a fragile state, the additional inflammatory storm and oxidative stress (elevated ROS) caused by traditional LNPs are like "adding insult to injury." This not only offsets some of the treatment effects but also exacerbates the damage to the liver microenvironment, severely limiting the widespread application and long-term safety of nucleic acid drugs in the treatment of chronic liver diseases.

[0004] Addressing the shortcomings of existing technologies, the development of a functional delivery system that can efficiently deliver nucleic acids while actively repairing or improving the liver microenvironment is particularly urgent. Against this backdrop, Vitamin E and Zinc, as natural substances with clearly defined pharmacological activities, have entered the research field. Vitamin E and its derivatives (mainly α-tocopherol) are the most important lipid-soluble antioxidants in the body's cell membrane system. They can block the lipid peroxidation chain reaction by providing hydrogen atoms, effectively scavenging free radicals such as superoxide anions, thereby maintaining cell membrane fluidity and stability and protecting hepatocytes from oxidative damage. Meanwhile, zinc, as an essential trace element for the human body, is a cofactor for many key antioxidant enzymes such as superoxide dismutase (SOD) and is widely involved in the regulation of cell signal transduction, cell proliferation, and apoptosis. Zinc not only has significant anti-inflammatory properties, inhibiting the activation of inflammatory pathways such as NF-κB, but has also been shown to maintain the integrity of the epithelial barrier and inhibit the progression of liver fibrosis. Introducing vitamin E and zinc into the construction of lipid nanoparticles not only promises to optimize the physical stability of LNPs by utilizing their structural properties, but more importantly, it endows the delivery carrier with additional biological functions—that is, while delivering therapeutic nucleic acids, it can also utilize the anti-inflammatory and antioxidant activities of the carrier components themselves to synergistically reshape the liver microenvironment, achieving the adjuvant therapeutic effect of "carrier as drug," and providing a brand-new technical approach for the comprehensive treatment of liver diseases. Summary of the Invention

[0005] Based on the limitations of existing technologies and clinical needs, this invention designs and develops a novel class of lipid molecules with liver microenvironment remodeling capabilities, and successfully applies them to the construction of nucleic acid delivery vectors. Studies have confirmed that LNPs prepared using these lipid molecules can not only significantly improve the delivery efficiency of nucleic acid drugs and the expression level of target genes in vivo, but also simultaneously achieve effective remodeling of the damaged liver microenvironment. This provides a novel solution integrating "efficient delivery" and "environmental remediation" for the development of next-generation nucleic acid drugs and gene vaccines.

[0006] The main objective of this invention is to provide an ionizable lipid molecule containing tocopherol and its derivatives and a zinc ion coordination structure as shown in formula (1), lipid nanoparticles containing the same, a method for preparing the same, and its uses. Compared with conventionally used ionizable lipid molecules in the art, the lipid nanoparticles prepared from the ionizable lipid molecule shown in formula (1) of this invention can achieve highly efficient delivery and expression of nucleic acids and remodeling of the liver microenvironment.

[0007] [Ionizable lipid molecules - compounds of formula (1) or pharmaceutically acceptable salts thereof] This invention provides a compound of formula (1) or a pharmaceutically acceptable salt thereof.

[0008] in, X is selected from N or CH; Indicates a single bond or a double bond; R a R b and R c Independently selected from H or methyl L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2 - or combinations of these groups with amino acid residues; The amino acid residues are selected from divalent groups derived from twenty common amino acids, or combinations thereof; The twenty common amino acids are well-known to those skilled in the art.

[0009] The amino acid residues are selected from divalent groups derived from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine, or combinations thereof. Each R 2 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10-membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is unsubstituted or substituted by one or more OH, NH2 or halogens; Each L a Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; L 4 L 5 and L 6 Independently selected from single bonds, C 1-20 Alkylene, C 2-10 Alkenyl group or -(OCH2CH2) n - The alkylene or alkenylene group is unsubstituted or substituted with one or more OH, NH2 or halogens; wherein n is an integer from 1 to 10.

[0010] R 1 Selected from C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 The alkynyl group, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or is surrounded by one or more OH, NH2, halogen, or -OC groups. 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10 heteroaryl substitutions; In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0011] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds or C 1-6 Alkylene, wherein the alkylene is unsubstituted or substituted with one or more OH groups.

[0012] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, -CH2-, -CH2CH2-, , , or .

[0013] In some implementations, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2 - or combinations of these groups with amino acid residues; the amino acid residues are selected from divalent groups derived from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine, or combinations thereof.

[0014] In some implementations, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2- or combinations of these groups with amino acid residues; the amino acid residues are selected from leucine, threonine, glutamic acid, phenylalanine, tryptophan, arginine, serine, histidine, alanine, or glycine by removing H and / or OH to obtain divalent groups.

[0015] In some implementations, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -NHC(=O)-, -OC(=O)NH-, , , , , or .

[0016] In some implementations, each R 2 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10-membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0017] In some implementations, each R 2 Independently selected from H and C 1-6 Alkyl or C 2-6 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0018] In some implementations, each R 2 H stands for H independently.

[0019] In some implementations, each L a Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0020] In some implementations, each L a Independently selected from single bonds or C 1-6 Alkylene, wherein the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0021] In some implementations, each L a Independently selected from single bonds, -CH2-, -CH2CH2-, , , or .

[0022] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, C 1-20 Alkylene, C 2-10 Alkenyl group or -(OCH2CH2) n - The alkylene or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0023] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, C 1-16 Alkylene or -(OCH2CH2) n - The alkylene group is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0024] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, -CH2-, -CH2CH2-, , , , , , or .

[0025] In some implementations, n is an integer from 1 to 10; or, n is an integer from 1 to 6; or, n is an integer from 1 to 3.

[0026] In some implementation schemes, Selected from , , , , , , or .

[0027] In some implementation schemes, R 1Selected from vitamin E and its derivatives, including but not limited to α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, and the monovalent group of δ-tocotrienol, C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 The alkynyl group, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or is surrounded by one or more OH, NH2, halogen, or -OC groups. 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10 heteroaryl substitutions.

[0028] In some implementation schemes, R 1 Selected from C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 The alkynyl group, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or is surrounded by one or more OH, NH2, halogen, or -OC groups. 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10 heteroaryl substitutions.

[0029] In some implementation schemes, R 1 Selected from , , , , , , , C 8-20 Alkyl or C 8-20 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or converted by one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6 Alkyl substitution. In some embodiments, R 1 Selected from , , , , , , , , , , , , , , , , , , or .

[0030] In some embodiments, the present invention provides a compound of formula (1) or a pharmaceutically acceptable salt thereof, said compound being selected from: .

[0031] [Lipid carrier] The present invention provides a lipid carrier comprising ionizable lipid molecules, polyethylene glycol lipid molecules, steroidal lipid molecules and auxiliary lipid molecules, wherein the ionizable lipid molecules include compounds represented by formula (1) above or pharmaceutically acceptable salts thereof.

[0032] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0033] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE).

[0034] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159) or 1,2-distearate-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE).

[0035] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0036] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: cholesterol, cholesterol, ergocalciferol, dihydrocholesterol, phycosterol, taurine, and deoxycholic acid.

[0037] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol, cholesterol, dihydrocholesterol, phycosterol, and deoxycholic acid.

[0038] In some implementations, the steroidal lipid molecule is selected from at least one of cholesterol and dihydrocholesterol.

[0039] In some embodiments, the auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)DOPG, oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0040] In some embodiments, the auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE).

[0041] In some embodiments, the auxiliary lipid molecule is selected from at least one of 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC) and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0042] In some embodiments, the lipid carrier comprises, by molar percentage, 10%-70% of the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof (ionizable lipid molecule), 1%-30% of polyethylene glycol lipid molecules, 5%-60% of steroidal lipid molecules and 1%-60% of auxiliary lipid molecules.

[0043] [Nucleic Acid Lipid Nanoparticle Composition] The present invention provides a nucleic acid lipid nanoparticle composition comprising the above-mentioned lipid carrier and nucleic acid.

[0044] In some embodiments, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, and circular RNA.

[0045] In some implementations, the nucleic acid is mRNA or siRNA.

[0046] In some embodiments, the nucleic acid is firefly luciferase mRNA, green fluorescent protein (GFP) mRNA, ovalbumin (OVA) mRNA, negative control siRNA (si-NC), or heat shock protein 47 siRNA (siHSP47).

[0047] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 5:1 to 50:1.

[0048] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 10:1 to 30:1.

[0049] In some embodiments, the nucleic acid is mRNA or siRNA, and the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1-30:1.

[0050] In some embodiments, the nucleic acid is mRNA or siRNA, and the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1, 25:1, or 30:1.

[0051] In some embodiments, the particle size of the nucleic acid lipid nanoparticle composition is 30-500 nm.

[0052] In some embodiments, the particle size of the nucleic acid lipid nanoparticle composition is 30-200 nm.

[0053] In some implementations, the particle size can be 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, etc.

[0054] In some embodiments, the encapsulation efficiency of nucleic acids in the nucleic acid lipid nanoparticle composition is greater than 50%. Exemplarily, the encapsulation efficiency can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0055] [Nucleic Acid Lipid Nanoparticle Formulation] The present invention provides a nucleic acid lipid nanoparticle formulation comprising the above-mentioned nucleic acid lipid nanoparticle composition and pharmaceutically acceptable excipients.

[0056] The present invention also provides the use of the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof, the lipid carrier above or the nucleic acid lipid nanoparticle composition above in the preparation of nucleic acid drugs or gene vaccines.

[0057] The present invention also provides a method for in vivo delivery of nucleic acid drugs or gene vaccines, the method comprising administering the above-described nucleic acid lipid nanoparticle composition or the above-described nucleic acid lipid nanoparticle formulation to a subject in need.

[0058] The present invention also provides compounds of formula (1) above or pharmaceutically acceptable salts thereof, lipid carriers above or nucleic acid lipid nanoparticle compositions above for delivery of nucleic acid drugs or gene vaccines.

[0059] The present invention also provides a method for treating or preventing diseases (e.g., inflammatory diseases, viral infections, and cancer) or conditions in a subject by delivering nucleic acids, the method comprising administering the above-described nucleic acid lipid nanoparticle composition or the above-described nucleic acid lipid nanoparticle formulation to a subject in need.

[0060] The term "inflammatory disease" includes autoimmune diseases, allergic diseases, and inflammatory conditions, such as those selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allogeneic graft rejection, Bechtel's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Sjögren's syndrome, and blistering diseases (e.g., pemphigus vulgaris), antibody-mediated vasculitis syndromes including ANCA-associated vasculitis, purpura, and immune complex vasculitis (stage I or II cancer or infection). Allergic diseases may include contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, and beryllium poisoning.

[0061] The term "viral infection" includes, but is not limited to, retroviral infection, hepatitis virus infection, Zika virus infection, dengue virus infection, etc.

[0062] The term "cancer" includes, but is not limited to, primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer (lung metastasis of liver cancer, lung metastasis of breast cancer, lung metastasis of colon cancer, lung metastasis of melanoma, etc.), as well as cancers in other parts of the body.

[0063] In some embodiments, the above-described nucleic acid lipid nanoparticle composition or formulation is administered via one of the following routes: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the above-described nucleic acid lipid nanoparticle composition or formulation is administered, for example, via enteral or parenteral administration. In some embodiments, the nucleic acid lipid nanoparticle composition or formulation is administered to the subject at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0064] [Preparation method of nucleic acid-loaded lipid nanoparticles] This invention provides a method for preparing lipid nanoparticles loaded with nucleic acids, comprising the following steps: (A1) The compound represented by formula (1) or its pharmaceutically acceptable salt, polyethylene glycol lipid molecules, steroidal lipid molecules and auxiliary lipid molecules are mixed in the proportions described above and dissolved in a solvent to obtain an organic phase liposome solution; (A2) Dissolve the nucleic acid and zinc salt in a buffer solution of appropriate pH to obtain an aqueous nucleic acid solution; (A3) The organic phase liposome solution and the aqueous phase nucleic acid solution are uniformly mixed using a microfluidic device according to the mass ratio and a certain volume ratio described above to prepare a lipid nanoparticle solution loaded with nucleic acid; In some embodiments, the solvent used to dissolve lipid molecules in step (A1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide.

[0065] In some embodiments, the solvent in step (A1) is ethanol, tetrahydrofuran, or acetone.

[0066] In some implementations, the solvent in step (A1) is ethanol.

[0067] In some embodiments, the zinc salt in step (A2) is zinc chloride, zinc acetate, zinc nitrate, zinc sulfate, zinc carbonate, or zinc gluconate.

[0068] In some embodiments, the zinc salt in step (A2) is zinc chloride, zinc acetate, or zinc nitrate.

[0069] In some implementations, the zinc salt in step (A2) is zinc chloride.

[0070] In some embodiments, the buffer solution in step (A2) is an acetic acid / sodium acetate solution or a citric acid / sodium citrate solution.

[0071] In some embodiments, the buffer solution in step (A2) is a citric acid / sodium citrate solution.

[0072] In some implementations, the pH of the buffer solution in step (A2) is 3-9.

[0073] In some implementations, the pH of the buffer solution in step (A2) is 4-6.

[0074] In some implementations, the pH of the buffer solution in step (A2) is 5.

[0075] In some embodiments, the concentration of the buffer solution in step (A2) is 1 mM-1 M.

[0076] In some embodiments, the concentration of the buffer solution in step (A2) is 20 mM-500 mM.

[0077] In some embodiments, the concentration of the buffer solution in step (A2) is 100 mM.

[0078] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1 to 1:10.

[0079] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1 to 1:5.

[0080] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:3.

[0081] In some implementations, the microfluidic device in step (A3) can be a microfluidic device conventionally used in the art, such as INano™ L / L+, Myanna, or NanoAssemblr. ® BT, Precision NanoSystems.

[0082] The present invention has the following beneficial effects: 1. A lipid carrier containing the compound of formula (1) or a pharmaceutically acceptable salt thereof binds tightly to nucleic acids, thereby achieving high loading rate and stable protection of nucleic acids.

[0083] 2. The LNPs formed have good biocompatibility and are more stable; they can improve the efficiency of nucleic acid delivery in vivo, and realize the efficient utilization and expression of nucleic acids.

[0084] 3. Due to the special physicochemical properties and bioactive functions of tocopherol and its derivatives as well as zinc ions, the LNP of this invention can remodel the liver microenvironment and protect the liver.

[0085] 4. This LNP is suitable for nucleic acid delivery with different molecular weights and sequences, and has universality.

[0086] 5. The technology of this invention is simple to synthesize, uses inexpensive raw materials, and is suitable for large-scale production. Attached Figure Description

[0087] Figure 1 This study demonstrates a Basic LNP@mRNA with ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) as the ionizable lipid component. Luc The I-1 LNP@mRNA of the present invention, which uses compounds I-1, II-1, and V-2 as ionizable lipid components, and Luc II-1 LNP@mRNA Luc and V-2 LNP@mRNA Luc Particle size distribution.

[0088] Figure 2 This demonstrates a basic LNP@mRNA with ALC-0315 as the ionizable lipid component. LucThe I-1 LNP@mRNA of the present invention, which uses compounds I-1, II-1, and V-2 as ionizable lipid components, and Luc II-1 LNP@mRNA Luc and V-2 LNP@mRNA Luc TEM photographs.

[0089] Figure 3 The particle size distributions of Basic LNP@si-NC with ALC-0315 as the ionizable lipid component and the I-1 LNP@si-NC, II-1 LNP@si-NC and V-2 LNP@si-NC of the present invention with compounds I-1, II-1 and V-2 as the ionizable lipid components are shown.

[0090] Figure 4 TEM images are shown of Basic LNP@si-NC with ALC-0315 as the ionizable lipid component, and of the present invention, I-1 LNP@si-NC, II-1 LNP@si-NC and V-2 LNP@si-NC with compounds I-1, II-1 and V-2 as the ionizable lipid components.

[0091] Figure 5 This demonstrates a basic LNP@mRNA with ALC-0315 as the ionizable lipid component. GFP The I-1 LNP@mRNA of the present invention, which uses compounds I-1, II-1, and V-2 as ionizable lipid components, and GFP II-1 LNP@mRNA GFP and V-2 LNP@mRNA GFP The expression of GFP protein in DC2.4 cells after in vitro transfection.

[0092] Figure 6 The ROS content of TGF-β-activated human hepatic stellate cells LX-2 after treatment with Basic LNP@si-NC (using ALC-0315 as the ionizable lipid component) and the I-1 LNP@si-NC and II-1 LNP@si-NC of the present invention (using compounds I-1 and II-1 as the ionizable lipid components) were shown.

[0093] Figure 7 The cell viability of mouse hepatocytes AML12 and human hepatic stellate cells LX-2 after treatment with hydrogen peroxide was shown by Basic LNP@si-NC with ALC-0315 as the ionizable lipid component and I-1 LNP@si-NC and II-1 LNP@si-NC of the present invention with compounds I-1 and II-1 as the ionizable lipid components.

[0094] Figure 8 The study presents a comparison of the in vivo anti-fibrotic abilities of Basic LNP@siHSP47 (with ALC-0315 as the ionizable lipid component) and the I-1 LNP@siHSP47 and II-1 LNP@siHSP47 of the present invention (with compounds I-1 and II-1 as the ionizable lipid components) against liver fibrosis in mice.

[0095] Figure 9 This demonstrates a basic LNP@mRNA with ALC-0315 as the ionizable lipid component. OVA And the I-1 LNP@mRNA of the present invention, which uses compound I-1 as an ionizable lipid component. OVA Comparison of tumor growth curves in mice after injection treatment.

[0096] Figure 10 This demonstrates a basic LNP@mRNA with ALC-0315 as the ionizable lipid component. OVA And the I-1 LNP@mRNA of the present invention, which uses compound I-1 as an ionizable lipid component. OVA Comparison of tumor growth inhibition rates in mice after injection treatment. Detailed Implementation

[0097] I. Definition In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are those widely used and standard practices in their respective fields. To better understand this disclosure, definitions and explanations of related terms are provided below.

[0098] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0099] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0100] The term "pharmaceutically acceptable salt" refers to the salt of the compounds in this application, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this application, with a specific substituent. When the compounds in this application contain relatively acidic functional groups, a base addition salt can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds in this application contain relatively basic functional groups, an acid addition salt can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds in this application contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0101] The pharmaceutically acceptable salts of this application can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both.

[0102] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemic, diastereomer, and enantiomers are all included within the scope of this disclosure.

[0103] In this disclosure, " "" refers to the position where the substituent is bonded.

[0104] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0105] When the lower and upper limits of a numerical range are disclosed, any value or subrange falling within that range is specifically disclosed. In particular, each numerical range of parameters disclosed herein (e.g., in the form of "about a to b", or equivalently "approximately a to b", or equivalently "about ab") should be understood to encompass every value and subrange therein. For example, "C..." 1-4 "This should be understood as encompassing any subrange and every point value, such as C." 2-4 C 3-4 C 1-2 C 1-3 C 1-4And so on, as well as C1, C2, C3, C4, etc. For example, "5-10 yuan" should be understood as covering any sub-range and each point value, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 6-7 yuan, 6-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

[0106] When any variable (e.g., R) n When a substituent appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by 1-5 Rs, the group can optionally be substituted by up to 5 Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0107] The terms "substituted" or "substituted" refer to the substitution of one or more hydrogen atoms on a particular atom or group by a substituent, provided that the valence state of the particular atom or group is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are substituted. Unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis. Substituents can be selected from one, two or more of the following: deuterium, halogen groups, cyano, nitro, -C(=O)R, -C(=O)OR', -OC(=O)R", imide, amide, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, etc., but not limited to these.

[0108] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0109] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0110] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, which have the indicated number of carbon atoms. For example, the term "C..." 1-10"Alkyl" refers to alkyl groups having 1 to 10 carbon atoms, including C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, and C1 alkyl. 10 Alkyl groups, examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. Alkyl groups may be optionally substituted or unsubstituted.

[0111] The term "alkylene" refers to a straight-chain or branched divalent saturated aliphatic hydrocarbon group to which the two groups (or segments) are attached can be connected to the same carbon atom or different carbon atoms. For example, the term "C" as used in this article... 1-10 "Alkylene" refers to an alkylene group having 1-10 carbon atoms (such as methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.). Alkylenes can be optionally substituted or unsubstituted.

[0112] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-30 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups can be optionally substituted or unsubstituted.

[0113] The term "alkenyl" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via two single bonds, including (but not limited to) vinylenes. For example, "C 2-10 "Alkenyl" refers to a divalent straight-chain or branched hydrocarbon group containing 2 to 10 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups can be optionally substituted or unsubstituted.

[0114] The term "alkynyl" refers to a monovalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments by a single bond. This includes (but is not limited to) ethynyl, propynyl, butynyl, and pentyynyl groups. For example, "C..." 2-30 "Alynyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond. The alkynyl group can be optionally substituted or unsubstituted.

[0115] The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic hydrocarbon group. For example, the term "C" as used in this invention...3-8 "Cycloalkyl" refers to a cycloalkyl group having 3 to 8 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Cycloalkyl groups can be optionally substituted or unsubstituted.

[0116] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4 Alkyl groups. If the valence requirements are met, the heterocyclic group can be connected to the rest of the molecule via any one ring atom. For example, the term "5-8 membered heterocyclic group" as used herein refers to a heterocyclic group having 5 to 8 ring atoms. Common heterocyclic groups include (but are not limited to) ethylene oxide, aziridine propane, aziridine butane, oxadiazine, tetrahydrofuranyl, dioxadiopentenyl, pyrrolyl, pyrrolidone, imidazoyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl. The heterocyclic groups in this invention may optionally be substituted with one or more substituents described herein.

[0117] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used in this invention... 6-10 "Aryl" refers to an aryl group having 6 to 10 carbon atoms. Common aryl groups include (but are not limited to) phenyl, naphthyl, anthraceneyl, phenanthryl, acenaphthene, azulel, fluorenyl, indene, pyrene, etc. The aryl group in this invention may optionally be substituted by one or more substituents described in this invention.

[0118] The term "heteroaryl" refers to an aromatic group consisting of a monocyclic or fused polycyclic ring with a conjugated π-electron system, the ring atom of which is composed of a carbon atom and at least one heteroatom selected from N, O, and S. If the valence requirement is met, the heteroaryl can be linked to the rest of the molecule through any one ring atom. For example, the term "5-10-membered heteroaryl" as used in this invention refers to a heteroaryl having 5 to 10 ring atoms. Common heteroaryl groups include (but are not limited to) thiophene, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl and their benzo[a] derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc. The heteroaryl group in this invention is optionally replaced by one or more substituents described in this invention (such as halogens, C...). 1-6Alkyl groups, etc., are substituted.

[0119] The term "pharmaceuticalally acceptable excipient" refers to an excipient administered co-administered with the above-described nucleic acid lipid nanoparticle composition, which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples include, but are not limited to: carriers, diluents, binders, absorbents, colorants, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0120] The tocopherols and tocotrienols mentioned in this article are all natural tocopherols and tocotrienols, and the amino acids mentioned in this article are all natural amino acids.

[0121] II. Specific Implementation Examples The present invention will be specifically described below through embodiments. These embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are within the scope of protection of the present invention.

[0122] All reagents and instruments used in the examples are commercially available conventional products. Unless otherwise specified, all conditions were performed under conventional conditions or conditions recommended by the manufacturer. The term "room temperature" as used in this invention refers to 20℃ ± 5℃. When used to modify a numerical value or range, the term "about" as used in this invention refers to the value or range and a range of errors acceptable to those skilled in the art, such as ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

[0123] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0124] The abbreviations used in this article have the following meanings:

[0125] Table 1. Abbreviation Meanings abbreviation meaning abbreviation meaning DMF N,N'-Dimethylformamide DMAP 4-Dimethylaminopyridine TEA Triethylamine DCM dichloromethane EDC 1-Ethyl-(3-dimethylaminopropyl)carbodiimide <![CDATA[SOCl2]]> sulfoxide NHS N-hydroxysuccinimide CDI N,N'-Carbonyldiimidazole

[0126] Example 1: Synthesis method of compound I-1

[0127] Compound 1 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DMF, and DMAP (0.1 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, compound 3 was precipitated with diethyl ether. Compound 4 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DMF, and DMAP (0.1 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, compound 5 was precipitated with diethyl ether. Compound 6 (1.0 eq) and compound 7 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 8. Compound 5 (1.0 eq) and compound 8 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 9. Compound 3 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.0 eq) and compound 9 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 10, i.e., compound I-1.

[0128] Example 2: Synthesis method of compound I-2

[0129] Compound 1 (1.0 eq) and compound 11 (1.0 eq) were dissolved in DMF, and DMAP (0.1 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, compound 12 was precipitated with diethyl ether. Compound 6 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.0 eq) and compound 13 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water to obtain compound 14. Compound 14 (1.0 eq) and compound 7 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 15. Compound 15 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 17. Compound 12 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, and then TEA (1.0 eq) and compound 17 (1.0 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 18, i.e., compound I-2.

[0130] Example 3: Synthesis method of compound II-1

[0131] Compound 19 (1.0 eq) and CDI (2.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, and then compound 20 (1.0 eq) was added. The mixture was stirred at room temperature for another 12 h. After the reaction was complete, compound 21 was obtained by column chromatography. Compound 21 (1.0 eq) and compound 22 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 23. Compound 23 (1.0 eq) and compound 24 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 25. Compound 25 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and separated by column chromatography to obtain compound 26, namely compound II-1.

[0132] Example 4: Synthesis method of compound II-2

[0133] Compound 19 (1.0 eq) and compound 27 (1.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. After the reaction was complete, excess solvent was removed under vacuum to obtain compound 28. Compound 28 (1.0 eq) and compound 22 (1.0 eq) were dissolved in DCM, and TEA (1.0 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was separated by column chromatography to obtain compound 29. Compound 29 (1.0 eq) and compound 30 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 31. Compound 31 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 32, i.e., compound II-2.

[0134] Example 5: Synthesis method of compound III-1

[0135] Compound 6 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.0 eq) and compound 33 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 34. Compound 34 (1.0 eq) and compound 35 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 36. Compound 36 (1.0 eq) and compound 37 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 38. Compound 3 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, and then TEA (1.0 eq) and compound 38 (1.0 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and separated by column chromatography to obtain compound 39, i.e., compound III-1.

[0136] Example 6: Synthesis method of compound III-2

[0137] Compound 6 (1.0 eq) and compound 40 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 41. Compound 41 (1.0 eq) and compound 35 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 42. Compound 42 (1.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 44. Compound 3 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.0 eq) and compound 44 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 45, which is compound III-2.

[0138] Example 7: Synthesis method of compound IV-1

[0139] Compound 1 (1.0 eq) and compound 46 (1.0 eq) were dissolved in DMF, and DMAP (0.1 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, compound 47 was precipitated with diethyl ether. Compound 19 (1.0 eq) and compound 48 (1.0 eq) were dissolved in DMF, and DMAP (0.1 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, compound 49 was precipitated with diethyl ether. Compound 49 (2.0 eq) and compound 50 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.2 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 51. Compound 51 (1.0 eq) and compound 47 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and separated by column chromatography to obtain compound 52, namely compound IV-1.

[0140] Example 8: Synthesis method of compound IV-2

[0141] Compounds 28 (1.0 eq) and 50 (1.0 eq) were dissolved in DCM, and TEA (1.0 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was separated by column chromatography to obtain compound 53. Compounds 53 (1.0 eq) and 54 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 55. Compounds 55 (1.0 eq) and 47 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 56, i.e., compound IV-2.

[0142] Example 9: Synthesis method of compound V-1

[0143] Compounds 28 (1.0 eq) and 57 (1.0 eq) were dissolved in DCM, and TEA (1.0 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was separated by column chromatography to obtain compound 58. Compound 59 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, and then TEA (1.0 eq) and compound 58 (1.0 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 60. Compound 3 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, and then TEA (1.0 eq) and compound 60 (1.0 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 61, i.e., compound V-1.

[0144] Example 10: Synthesis of compound V-2

[0145] Compound 6 (2.0 eq) was dissolved in DCM, and NHS (4.0 eq), EDC (4.0 eq), and DMAP (0.2 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (2.0 eq) and compound 57 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 62. Compound 47 (1.0 eq) was dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.0 eq) and compound 62 (1.0 eq). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 63, i.e., compound V-2.

[0146] Example 11: Synthesis method of compound VI-1

[0147] Compound 14 (1.0 eq) and compound 64 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 55. Compound 28 (1.0 eq) and compound 65 (1.0 eq) were dissolved in DCM, and TEA (1.0 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was separated by column chromatography to obtain compound 66. Compound 66 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 67, i.e., compound VI-1.

[0148] Example 12: Synthesis method of compound VI-2

[0149] Compounds 6 (1.0 eq) and 64 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 68. Compounds 24 (1.0 eq) and 68 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 69. Compounds 12 (1.0 eq) and 69 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and separated by column chromatography to obtain compound 70, i.e., compound VI-2.

[0150] Example 13: Preparation and characterization of basic LNPs carrying mRNA and LNPs of the present invention Ionizable lipid molecules (ALC-0315), DSPC, cholesterol, and polyethylene glycol lipids (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. Firefly luciferase mRNA was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The organic phase and aqueous phase solutions were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. Basic lipid nanoparticles encapsulating mRNA (represented as Basic LNP@mRNA) were obtained. Luc ); The compounds synthesized in Examples 1-12 (I-1 to VI-2), DSPC, cholesterol, and polyethylene glycol lipid (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. Firefly luciferase mRNA was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids (zinc chloride) was added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The lipid nanoparticles of the present invention encapsulating mRNA were obtained (named according to the compounds synthesized in Examples 1-12; for example, it can be represented as I-1 LNP@mRNA). Luc ).

[0151] The particle size distribution of the obtained basic lipid nanoparticles and the lipid nanoparticles of this invention was characterized using dynamic light scattering (DLS). Specifically, Basic LNP@mRNA... Luc And representative I-1 LNP@mRNA Luc II-1LNP@mRNA Luc and V-2 LNP@mRNA Luc The particle size distribution is as follows Figure 1 As shown.

[0152] DLS results (Table 2) show that Basic LNP@mRNA Luc Compared with the LNP@mRNA of the present invention prepared based on I-1 to VI-2 Luc In comparison, there was no significant difference in hydrated particle size, and both met the usable standards.

[0153] Basic LNP@mRNA was observed using transmission electron microscopy. Luc Ⅰ-1 LNP@mRNA Luc II-1 LNP@mRNA Luc and V-2 LNP@mRNA Luc Morphology of four types of lipid nanoparticles carrying mRNA. Electron micrographs show ( Figure 2 The four types of lipid nanoparticles carrying mRNA exhibited good morphology, all being nearly spherical, and with a particle size of approximately 100 nm.

[0154] LNP@mRNA Luc Encapsulation efficiency determination The resulting slightly white solution was dialyzed against a suitable volume of PBS for 4 hours. The filtrate was collected, and the mRNA content in the filtrate was determined using Nanodrop. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = mRNA 总量 -mRNA 滤液 / mRNA 总量 .

[0155] The results (Table 2) show that the LNP@mRNA prepared according to the present invention... Luc Both exhibited good mRNA encapsulation efficiency.

[0156] Table 2 LNP@mRNA Luc mRNA encapsulation efficiency <![CDATA[LNP@mRNA Luc ]]> Particle size (nm) mRNA encapsulation rate (%) <![CDATA[LNP@mRNA Luc ]]> Particle size (nm) mRNA encapsulation rate (%) Basic 99.6±5.1 90.4 Ⅳ-1 98.9±1.9 91.2 Ⅰ-1 100.8±2.2 95.9 Ⅳ-2 97.8±2.4 90.7 Ⅰ-2 97.9±4.3 94.5 Ⅴ-1 100.6±4.1 89.9 Ⅱ-1 101.2±4.7 90.3 Ⅴ-2 101.8±2.3 93.1 Ⅱ-2 100.1±3.3 89.4 Ⅵ-1 99.8±3.2 91.6 Ⅲ-1 103.6±2.1 88.8 Ⅵ-2 105.7±4.5 94.0 Ⅲ-2 101.6±3.6 90.9

[0157] Example 14: Preparation and characterization of basic LNPs loaded with siRNA and LNPs of the present invention Ionizable lipid molecules (ALC-0315), DSPC, cholesterol, and polyethylene glycol lipids (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The negative control siRNA (si-NC) was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier and siRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. Basic lipid nanoparticles encapsulating siRNA (denoted as Basic LNP@si-NC) were obtained. The compounds synthesized in Examples 1-12 (Ⅰ-1 to Ⅵ-2), DSPC, cholesterol, and polyethylene glycol lipid (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The negative control siRNA (si-NC) was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids of zinc chloride were added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and siRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The lipid nanoparticles of the present invention encapsulating siRNA were obtained (named according to the compounds synthesized in Examples 1-12; for example, it can be represented as Ⅰ-1 LNP@si-NC).

[0158] Dynamic light scattering (DLS) was used to characterize the particle size distribution of the obtained basic lipid nanoparticles and the lipid nanoparticles of this invention. The particle size distributions of Basic LNP@si-NC and representative I-1 LNP@si-NC, II-1 LNP@si-NC, and V-2 LNP@si-NC are shown below. Figure 3 As shown.

[0159] The DLS results (Table 3) show that the hydrated particle size of the Basic LNP@si-NC is not significantly different from that of the LNP@si-NC prepared based on I-1 to VI-2 of the present invention, and both meet the usable standards.

[0160] The morphology of four lipid nanoparticles carrying siRNA—Basic LNP@si-NC, I-1 LNP@si-NC, II-1 LNP@si-NC, and V-2 LNP@si-NC—was observed using transmission electron microscopy. The electron micrographs show (…). Figure 4 The four types of lipid nanoparticles carrying mRNA exhibited good morphology, all being nearly spherical, and with a particle size of approximately 100 nm.

[0161] Encapsulation efficiency determination of LNP@si-NC The resulting slightly white solution was dialyzed against a suitable volume of PBS for 4 hours. The filtrate was collected, and the siRNA content in the filtrate was determined using Nanodrop. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = siRNA 总量 -siRNA 滤液 / siRNA 总量 .

[0162] The results (Table 3) show that the LNP@si-NCs prepared according to the present invention all have good siRNA encapsulation efficiency.

[0163] Table 3. siRNA encapsulation efficiency of LNP@si-NC LNP@si-NC Particle size (nm) siRNA encapsulation efficiency (%) LNP@si-NC Particle size (nm) siRNA encapsulation efficiency (%) Basic 98.9±4.4 88.7 Ⅳ-1 100.8±4.7 88.7 Ⅰ-1 100.2±3.1 95.6 Ⅳ-2 102.5±2.8 91.7 Ⅰ-2 100.9±3.4 93.5 Ⅴ-1 100.4±3.2 90.5 Ⅱ-1 101.4±2.9 92.8 Ⅴ-2 99.8±4.3 92.5 Ⅱ-2 104.3±4.1 90.1 Ⅵ-1 99.3±3.4 90.4 Ⅲ-1 98.1±2.8 92.4 Ⅵ-2 103.1±3.9 92.1 Ⅲ-2 102.5±3.7 92.9

[0164] Example 15: LNP@mRNA of the present invention GFP In vitro transfection efficiency experiment Compounds I-1, II-1, V-2, polyethylene glycol lipid (ALC-0159), cholesterol, and DSPC synthesized in Examples 1, 3, and 10 were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was green fluorescent protein mRNA (GFP mRNA), dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids (zinc chloride) were added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The I-1 LNP@mRNA of the present invention, encapsulating mRNA, was obtained. GFP II-1 LNP@mRNA GFP and V-2 LNP@mRNA GFP Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. GFP .

[0165] DC2.4 cells were loaded at 5 × 10⁻⁶ 5 Cells were seeded at high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. After 24 hours of incubation, the medium was replaced with fresh medium. PBS and Basic LNP@mRNA were then added to the cells. GFP And the I-1 LNP@mRNA of the present invention GFP II-1 LNP@mRNA GFP and V-2 LNP@mRNA GFP (The mRNA dose per well was 1 μg). After incubating the cells for 24 hours, flow cytometry analysis was performed to detect the proportion of GFP-positive cells and compare the transfection efficiency of different LNP@mRNAs.

[0166] The results show that ( Figure 5 ), using Basic LNP@mRNA GFP After in vitro transfection, the proportion of GFP-positive cells was low, indicating that DC2.4 cells were transfected with Basic LNP@mRNA. GFP Transfection efficiency after GFP mRNA transfection is very limited. However, the I-1 LNP@mRNA method of this invention...GFP II-1 LNP@mRNA GFP and V-2 LNP@mRNA GFP After in vitro transfection with GFP mRNA, the proportion of GFP-positive cells significantly increased, demonstrating that the LNP@mRNA of this invention can significantly improve antigen presentation efficiency. *** P < 0.001 indicates extremely significant difference, and **** P < 0.0001 indicates highly significant difference.

[0167] Example 16: LNP@mRNA of the present invention Luc In vitro expression experiments The compounds synthesized in Examples 1-12 (I-1 to VI-2), polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA, firefly luciferase mRNA, was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids (zinc chloride) was added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The I-1 LNP@mRNA of this invention, encapsulating the mRNA, was obtained. Luc II-1 LNP@mRNA Luc and V-2 LNP@mRNA Luc Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. Luc .

[0168] DC2.4 cells were loaded at 5 × 10⁻⁶ 5 Cells were seeded at high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. After 24 hours of incubation, the medium was replaced with fresh medium. PBS and Basic LNP@mRNA were then added to the cells. Luc And the I-1 LNP@mRNA of the present invention Luc II-1 LNP@mRNA Luc and V-2 LNP@mRNA Luc(The mRNA dose per well was 1 μg). After incubating the cells for 24 hours, the cells were lysed, and the firefly luciferase detection kit (Luciferase Reporter Gene Assay Kit, Yeasen) was added. The mixture was thoroughly mixed, and the RLU (Relative Light Unit) was measured using the Luminescence mode of a multi-functional microplate reader to reflect the LNP@mRNA levels in each group. Luc The in vitro transfection efficiency.

[0169] The results (Table 4) show that Basic LNP@mRNA Luc In vitro transfection efficiency is low, while the LNP@mRNA of this invention... Luc In vitro transfection efficiency was significantly higher than that of Basic LNP@mRNA. Luc There has been a significant improvement, and the transfection effect is excellent.

[0170] Table 4 Basic LNP@mRNA Luc In vitro transfection efficiency - fluorescence intensity <![CDATA[LNP@mRNA Luc ]]> <![CDATA[Fluorescence intensity (RLU·ml -1 )]]> <![CDATA[LNP@mRNA Luc ]]> <![CDATA[Fluorescence intensity (RLU·ml -1 )]]> PBS 61 Ⅲ-2 89500 Basic 40000 Ⅳ-1 93500 Ⅰ-1 135000 Ⅳ-2 95700 Ⅰ-2 100000 Ⅴ-1 93300 Ⅱ-1 120000 Ⅴ-2 117000 Ⅱ-2 95050 Ⅵ-1 96730 Ⅲ-1 88360 Ⅵ-2 90240

[0171] Example 17: Antioxidant Function Experiment of LNP of the Present Invention Compounds I-1 and II-1 synthesized in Examples 1 and 3, polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The siRNA, a negative control siRNA (si-NC), was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids (zinc chloride) was added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. This yielded the I-1 LNP@si-NC and II-1 LNP@si-NC of the present invention, loaded with siRNA. Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@si-NC loaded with siRNA was obtained using the same method.

[0172] Human hepatic stellate cells LX-2 were used at a concentration of 8 × 10⁻⁶ 5Cells were seeded at high density in 6-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. After 24 hours of incubation, the medium was replaced with fresh medium. TGF-β (final concentration 10 ng / mL) was added to the cells, and incubation continued. After 24 hours, the medium was replaced with fresh medium, and PBS, Basic LNP@si-NC, and the I-1 LNP@si-NC and II-1 LNP@si-NC of this invention (siRNA dose of 1 μg per well) were added to the cells, respectively. After 24 hours of incubation, DCFH-DA was added for total reactive oxygen species (ROS) staining and measurement, followed by imaging with a laser confocal microscope to reflect the antioxidant status of each group of LNPs.

[0173] The results show that ( Figure 6 The group treated with Basic LNP@si-NC had higher ROS levels than other groups, demonstrating that Basic LNP@si-NC not only lacks antioxidant capacity but also exacerbates oxidative stress, further damaging the liver microenvironment. In contrast, the groups treated with the I-1 LNP@si-NC and II-1 LNP@si-NC of this invention showed significantly lower ROS levels, proving that the LNPs of this invention can significantly reduce ROS levels, alleviate oxidative stress, and thus remodel the liver microenvironment. **P<0.001 indicates a statistically significant difference, and ****P<0.0001 indicates a highly statistically significant difference.

[0174] Example 18: Experiment on the liver protective ability of LNP of the present invention Compounds I-1 and II-1 synthesized in Examples 1 and 3, polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The siRNA, a negative control siRNA (si-NC), was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids (zinc chloride) was added. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. This yielded the I-1 LNP@si-NC and II-1 LNP@si-NC of the present invention, loaded with siRNA. Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@si-NC loaded with siRNA was obtained using the same method.

[0175] Mouse hepatocytes AML-12 and human hepatic stellate cells LX-2 were used at a concentration of 8 × 10⁻⁶. 3Cells were seeded at various densities in 96-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. After 24 hours of incubation, the medium was replaced with fresh medium. Hydrogen peroxide (final concentration 600 μM) was added to the cells to simulate liver injury, and incubation continued. After 24 hours, the medium was replaced with fresh medium, and PBS, Basic LNP@si-NC, and the I-1 LNP@si-NC and II-1 LNP@si-NC of this invention (1 μg siRNA per well) were added to the cells, respectively. After 24 hours of incubation, CCK-8 reagent was added, and incubation continued at 37°C for 4 hours. The absorbance was then measured using a multi-mode microplate reader to calculate cell viability, thereby reflecting the liver-protective ability of each group of LNPs.

[0176] The results show that ( Figure 7 In the group treated with Basic LNP@si-NC, the cell viability of both hepatocytes and hepatic stellate cells was lower than that of other groups, demonstrating that Basic LNP@si-NC not only lacks liver protection capabilities but also exacerbates damage and further disrupts the liver microenvironment. In contrast, the groups treated with the I-1 LNP@si-NC and II-1 LNP@si-NC of this invention showed significantly increased cell viability, demonstrating that the LNPs of this invention can provide significant protection to a wide range of liver tissues, thereby reshaping the liver microenvironment. *** P<0.001 indicates a highly significant difference, and **** P<0.0001 indicates a very high degree of significance.

[0177] Example 19: In vivo antifibrotic ability test of LNP@siHSP47 of the present invention Compounds I-1 and II-1 synthesized in Examples 1 and 3, polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The siRNA, heat shock protein 47 siRNA (siHSP47), was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids, of zinc chloride was added. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. This yielded I-1 LNP@siHSP47 and II-1 LNP@siHSP47 of the present invention, loaded with HSP47 siRNA. Basic LNP@siHSP47 loaded with siRNA was obtained by using ALC-0315 instead of the compounds synthesized in the above examples, using the same method.

[0178] Balb / c mice (6-8 weeks old, male, weighing approximately 22-25g) were intraperitoneally injected with carbon tetrachloride twice weekly for 5 weeks to establish a liver fibrosis model. Week 1 was the first week of carbon tetrachloride injection. Starting from week 4, mice were intravenously injected with PBS, Basic LNP@siHSP47, and the I-1 LNP@siHSP47 and II-1 LNP@siHSP47 of this invention, twice weekly for 2 weeks. Each siRNA injection dose was 5 μg. After 5 weeks, the mice were sacrificed, and serum was collected for liver function testing. Liver samples were collected for hematoxylin-eosin staining and Sirius red staining to assess in vivo anti-fibrotic capacity.

[0179] The results show that ( Figure 8 In the group treated with Basic LNP@si-NC, liver function in mice did not show significant improvement, and hematoxylin-eosin and Sirius red staining revealed numerous pathological areas, demonstrating that Basic LNP@siHSP47 lacks the ability to remodel the liver microenvironment and has poor anti-fibrotic ability. However, in the groups treated with I-1 LNP@si-NC and II-1 LNP@si-NC of this invention, liver function significantly improved, and hematoxylin-eosin and Sirius red staining showed a significant reduction in fibrotic lesions, demonstrating that the LNP of this invention can remodel the liver microenvironment and possesses excellent anti-fibrotic ability. ** P < 0.001 indicates a statistically significant difference, *** P < 0.001 indicates a highly statistically significant difference, **** P < 0.0001 indicates a very high statistically significant difference, and ns indicates no statistically significant difference.

[0180] Example 20: LNP@mRNA of the present invention OVA Evaluation of antitumor performance Compound I-1 synthesized in Example 1, polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA, ovalbumin mRNA (OVA mRNA), was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids, of zinc chloride was added. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The I-1 LNP@mRNA of the present invention, encapsulating the mRNA, was obtained. OVA Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. OVA .

[0181] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. PBS and BasicLNP@mRNA were administered intramuscularly to the thigh twice, on days 9 and 15, respectively. OVA and I-1 LNP@mRNA OVA Each mRNA injection dose was 5 μg, and tumor volume changes in mice were recorded over 30 days.

[0182] The results show that ( Figure 9 ), accepts I-1 LNP@mRNA OVA The tumors in the treated mice grew slowly, and the treatment effect was significantly better than that of mice receiving Basic LNP@mRNA. OVA The LNP@mRNA of the present invention was demonstrated in treated mice. OVA It can significantly enhance the in vivo expression efficiency of mRNA and has excellent anti-tumor effects.

[0183] Example 21: LNP@mRNA with antioxidant properties OVA Tumor growth inhibition rate experiment Compound I-1 synthesized in Example 1, polyethylene glycol lipids (ALC-0159), cholesterol, and DSPC were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA, ovalbumin mRNA (OVA mRNA), was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0, and five times the molar amount of ionizable lipids, of zinc chloride was added. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol and excess zinc chloride were then removed by ultrafiltration. The I-1 LNP@mRNA of the present invention, encapsulating the mRNA, was obtained. OVA Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. OVA .

[0184] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. PBS and Basic LNP@mRNA were administered intramuscularly to the thigh twice, on days 9 and 15, respectively. OVA and I-1 LNP@mRNA OVAEach mRNA injection dose was 5 μg. The tumor volume of mice was measured on day 0 and day 30. The tumor growth inhibition rate was calculated according to the formula TGI (Tumor Growth Inhibition) = (1 - Tumor volume of treatment group / Tumor volume of control group) * 100%.

[0185] The results show that ( Figure 10 Basic LNP@mRNA OVA The tumor growth inhibition rate was approximately 54%, while the I-1LNP@mRNA of this invention... OVA The tumor growth inhibition rate reached approximately 86%, compared to Basic LNP@mRNA. OVA The significant improvement demonstrates the effectiveness of the LNP@mRNA of this invention. OVA It can enhance the in vivo expression efficiency of mRNA, thereby improving the anti-tumor effect. *** P<0.001 indicates a highly significant difference, and **** P<0.0001 indicates a very high degree of significance.

[0186] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise form disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments and various different options of this disclosure.

Claims

1. A compound of formula (1) or a pharmaceutically acceptable salt thereof, in, X is selected from N or CH; Indicates a single bond or a double bond; R a R b and R c Independently selected from H or methyl; L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2 - or combinations of these groups with amino acid residues; The amino acid residues are selected from divalent groups derived from twenty common amino acids, or combinations thereof; Each R 2 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10-membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is unsubstituted or substituted by one or more OH, NH2 or halogens; Each L a Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; L 4 L 5 and L 6 Independently selected from single bonds, C 1-20 Alkylene, C 2-10 Alkenyl group or -(OCH2CH2) n - The alkylene or alkenylene group is unsubstituted or substituted with one or more OH, NH2 or halogens; wherein n is an integer from 1 to 10; R 1 Selected from C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 The alkynyl group, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or is surrounded by one or more OH, NH2, halogen, or -OC groups. 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10 heteroaryl substitutions.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; Or, L 1 L 2 and L 3 Independently selected from single bonds or C 1-6 Alkylene, wherein the alkylene is unsubstituted or substituted with one or more OH groups; Or, L 1 L 2 and L 3 Independently selected from single bonds, -CH2-, -CH2CH2-, , , or .

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2 - or combinations of these groups with amino acid residues; the amino acid residues are selected from divalent groups derived from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine, or combinations thereof. Or, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NR 2 -、-O-、-S-、-OC(=O)NR 2 -、-NR 2 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)- 、 -C(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)O-、-C(=O)-L a -C(=O)NR 2 -、-NR 2 C(=O)NR 2 - or combinations of these groups with amino acid residues; the amino acid residues are selected from leucine, threonine, glutamic acid, phenylalanine, tryptophan, arginine, serine, histidine, alanine or glycine by removing H and / or OH to obtain divalent groups. Or, G 1 G 2 G 3 G 4 G 5 and G 6 Independently selected from single bonds, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -NHC(=O)-, -OC(=O)NH-, , , , , or ; Each R 2 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10-membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is unsubstituted or substituted by one or more OH, NH2 or halogens; Or, each R 2 Independently selected from H and C 1-6 Alkyl or C 2-6 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogen; Or, each R 2 H is independent; Each L a Independently selected from single bonds, C 1-10 Alkylene or C 2-10 The alkylene group or the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogens; Or, each L a Independently selected from single bonds or C 1-6 Alkylene, wherein the alkylene is unsubstituted or substituted by one or more OH, NH2 or halogens; Or, each L a Independently selected from single bonds, -CH2-, -CH2CH2-, , , or .

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, L 4 L 5 and L 6 Independently selected from single bonds, C 1-20 Alkylene, C 2-10 Alkenyl group or -(OCH2CH2) n - The alkylene or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens; Or, L 4 L 5 and L 6 Independently selected from single bonds, C 1-16 Alkylene or -(OCH2CH2) n - The alkylene group is unsubstituted or substituted with one or more OH, NH2 or halogens; Or, L 4 L 5 and L 6 Independently selected from single bonds, -CH2-, -CH2CH2-, , , , , , or ; n is an integer from 1 to 10; or, n is an integer from 1 to 6; or, n is an integer from 1 to 3.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, Selected from , , , , , , or .

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R 1 Selected from C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 The alkynyl group, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or is surrounded by one or more OH, NH2, halogen, or -OC groups. 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10 heteroaryl substitutions; Or, R 1 Selected from , , , , , , , C 8-20 Alkyl or C 8-20 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or converted by one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6 Alkyl substitution; Or, R 1 Selected from , , , , , , , , , , , , , , , , , , or .

7. The following compounds or their pharmaceutically acceptable salts: 。 8. A lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroidal lipid molecule, and an accessory lipid molecule, wherein the ionizable lipid molecule comprises a compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof; in, The polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide ALC-0159, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol PEG-DMG, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEG-disterol glycerol PEG-DSG, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearate, PEG-diacylglycerol amide PEG-DAG, PEG-dipalmitoylphosphatidylethanolamine PEG-DPPE, or PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA; The steroidal lipid molecules are selected from at least one of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, serotonol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; The auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline DSPC, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline DPPC, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine DOPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine DMPE, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)DOPG, oleoylphosphatidylcholine POPC, and 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE; The lipid carrier comprises, by molar percentage, 10%-70% ionizable lipid molecules, 5%-60% steroidal lipid molecules, 1%-60% accessory lipid molecules and 1%-30% polyethylene glycol lipid molecules.

9. A nucleic acid lipid nanoparticle composition comprising the lipid carrier according to claim 8 and nucleic acid; The nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, and circular RNA; The mass ratio of the lipid carrier to nucleic acid is 5:1-50:1; or the mass ratio of the lipid carrier to nucleic acid is 10:1-30:

1.

10. Use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, the lipid carrier of claim 8, or the nucleic acid lipid nanoparticle composition of claim 9 in the preparation of nucleic acid drugs or gene vaccines.

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