Peg lipid and use thereof

Abstract

The present disclosure relates to a PEG lipid and the use thereof. The PEG lipid can be used for preparing a lipid nanoparticle that delivers an active ingredient. The present disclosure further relates to a pharmaceutical composition and a lipid nanoparticle comprising the PEG lipid, as well as the use thereof in preparing a pharmaceutical.

Description

PEG lipids and their applications Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a PEG lipid and an application thereof. Background Art

[0002] Nucleic acid drugs work by targeting RNA or DNA, and include mRNA, antisense oligonucleotides (ASOs), microRNA (miRNA), small interfering RNA (siRNA), and aptamers. Research has shown that nucleic acid drugs have enormous therapeutic potential in areas such as cancer treatment, infectious disease prevention, and genetic disease treatment.

[0003] To avoid degradation by nucleases in the blood, nucleic acid drugs require delivery vehicles to deliver them into the body for efficacy. Common delivery vehicles for nucleic acid drugs include lipid nanoparticles (LNPs). Lipid nanoparticles generally contain cationic lipids, phospholipids, helper lipids, and PEG lipids. PEG lipids can both drive self-assembly and prevent particle aggregation, making the preparation of lipid nanoparticles easier. It is generally believed that PEG lipids can prolong the stability and residence time of lipid nanoparticles in the blood circulation. However, recent studies have found that repeated injections of lipid nanoparticles containing PEG lipids can trigger an immune response. This immune response leads to a decrease in the in vivo circulation time of lipid nanoparticles and an increase in their accumulation in the liver and spleen. This phenomenon is known as "accelerated blood clearance" (ABC). The accelerated blood clearance of PEG lipids poses significant challenges to their development and clinical application. Summary of the Invention

[0004] In a first aspect, the present disclosure provides a PEG lipid as shown in formula (I):

[0005] or a salt thereof, or a stereoisomer thereof, wherein

[0006] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0007] Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond,

[0008] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0009] Q1 is -C(=O)-, -OC(=O)- or a bond,

[0010] Q2 is -C(=O)- or a bond,

[0011] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0012] L1 is C1~C 20 an alkylene group or bond,

[0013] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0014] R2 is C4~C 24 Hydrocarbon group,

[0015] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0016] L2 is a C1-C4 alkylene group,

[0017] L3 is a C1-C4 alkylene group,

[0018] R3 is H or a C1-C3 hydrocarbon group,

[0019] n is any integer from 30 to 60.

[0020] In a second aspect, the present disclosure also provides a PEG lipid as shown in formula (IX):

[0021] or a salt thereof, or a stereoisomer thereof, wherein

[0022] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0023] Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond,

[0024] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0025] Q1 is -C(=O)-, -OC(=O)- or a bond,

[0026] Q2 is -C(=O)- or a bond,

[0027] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0028] L1 is C1~C 20an alkylene group or bond,

[0029] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0030] R2 is C4~C 24 Hydrocarbon group,

[0031] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0032] L2 is a C1-C4 alkylene group,

[0033] L3 is a C1-C4 alkylene group,

[0034] R3 is H or a C1-C3 hydrocarbon group,

[0035] n is any integer from 30 to 60,

[0036] L1, A1 and R2 form a four-membered ring, a five-membered ring or a six-membered ring.

[0037] In some embodiments, L1 in formula (IX) is C1 to C 20 Hydrocarbylene.

[0038] In a third aspect, the present disclosure further provides a PEG lipid as shown in formula (X):

[0039] or a salt thereof, or a stereoisomer thereof, wherein

[0040] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0041] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0042] Q1 is -C(=O)-, -OC(=O)- or a bond,

[0043] Q2 is -C(=O)- or a bond,

[0044] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0045] L1 is C1~C 20 an alkylene group or bond,

[0046] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0047] R2 is C4~C 24 Hydrocarbon group,

[0048] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0049] L2 is a C1-C4 alkylene group,

[0050] L3 is a C1-C4 alkylene group,

[0051] R3 is H or a C1-C3 hydrocarbon group,

[0052] n is any integer from 30 to 60,

[0053] Z2 forms a four-membered ring, a five-membered ring or a six-membered ring with Q3, L1, A1 and R2.

[0054] In some embodiments, in Formula (X), Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2-, or a bond, and Z2 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-.

[0055] In some embodiments, R1 is H,

[0056] In some embodiments, R1 is H,

[0057] In some embodiments, the structure of the PEG lipid is shown in Formula (II):

[0058] wherein R1 is not H and is not unsubstituted methyl.

[0059] In some embodiments, R1 is HO-Z1-Q1-, A1 is a bond, and Z1 and Q1 are not bonds at the same time; preferably, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-. In some such embodiments, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-, and Q1 is -C(=O)-, -OC(=O)-, or a bond. In preferred embodiments, L2 is C1-C3 alkylene, L3 is C1-C3 alkylene, and R3 is H or C1-C3 alkyl. In more preferred embodiments, R1 is

[0060] In some embodiments, R1 is H-Z1-Q1-, wherein when Q1 is a bond, Z1 is not a bond and is not an unsubstituted methylene. In preferred embodiments, R1 is H-Z1-Q1-, Z1 is -L2-, Q1 is -C(=O)-, and A1 is a bond. In such embodiments, L2 is a C1-C3 alkylene group. In preferred embodiments, R1 is

[0061] In some embodiments, the PEG lipid is represented by formula (II-1):

[0062] wherein A1 is -OC(=O)-, -OC(=O)O-, -O- or -S-.

[0063] In some embodiments, the PEG lipid is represented by formula (II-1-1):

[0064] In some embodiments, the PEG lipid has a structure as shown in Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII):

[0065] In some embodiments, the PEG lipid has the structure:

[0066] In some embodiments, the total number of carbon atoms in L1 and R2 is 12-25.

[0067] In some embodiments, the total number of carbon atoms in L1 and R2 is 16-22.

[0068] In some embodiments, L1 is a C1-C8 alkylene group or a bond.

[0069] In some embodiments, L1 is C1-C8 alkylene, C2-C8 alkenylene, or C2-C8 alkynylene.

[0070] In some embodiments, R2 is C7 to C 22 Hydrocarbon group.

[0071] In some embodiments, R2 is C7 to C 22 Alkyl, C7~C 22 Alkenyl or C7~C 22 Alkynyl.

[0072] In some embodiments, L2 is a C1-C3 alkylene group.

[0073] In some embodiments, L2 is C1-C3 alkylene, C2-C3 alkenylene, or C2-C3 alkynylene.

[0074] In some embodiments, L3 is a C1-C2 alkylene group.

[0075] In some embodiments, L3 is C1-C2 alkylene, C2 alkenylene, or C2 alkynylene.

[0076] In some embodiments, R3 is H or C1-C3 alkyl.

[0077] In some embodiments, n is any integer from 30 to 55.

[0078] In some embodiments, n is any integer from 35 to 50.

[0079] In some embodiments, n is any integer from 40 to 50.

[0080] In some embodiments, the PEG lipid is:

[0081] In another aspect, the present disclosure provides use of the PEG lipid according to any one of the above embodiments in preparing lipid nanoparticles.

[0082] In another aspect, the present disclosure provides a lipid nanoparticle comprising the PEG lipid of any one of the above embodiments.

[0083] In some embodiments, the lipid nanoparticle further comprises an active ingredient. In some embodiments, the active ingredient is a pharmaceutical active ingredient. In some embodiments, the active ingredient is a nucleic acid. In some embodiments, the pharmaceutical active ingredient is a nucleic acid.

[0084] In some embodiments, the lipid nanoparticles further comprise one or more of a cationic lipid, a helper lipid, a structural lipid, and a polymeric lipid.

[0085] In another aspect, the present disclosure provides a composition comprising the lipid nanoparticle of any one of the above embodiments, and a biologically acceptable carrier.

[0086] In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticles according to any one of the above embodiments, and a pharmaceutically acceptable carrier.

[0087] In another aspect, the present disclosure provides use of the lipid nanoparticles according to any of the above embodiments or the pharmaceutical composition according to any of the above embodiments in the preparation of a medicament.

[0088] In some embodiments, the medicament is for gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA.

[0089] In some embodiments, the lipid particles are used as carriers for transferring or delivering active ingredients. In some embodiments, the active ingredient is a pharmaceutical active ingredient. In some embodiments, the pharmaceutical active ingredient is a nucleic acid.

[0090] In some embodiments, the medicament is for the treatment and / or prevention of a disease.

[0091] In some embodiments, the medicament is used to treat and / or prevent one or more of the following diseases: rare diseases, cancer, infectious diseases, autoimmune diseases, metabolic diseases, neurological diseases, cardiovascular diseases, transplant rejection, inflammatory response, genetic diseases and musculoskeletal diseases.

[0092] In some embodiments, the rare disease comprises one or more of osteogenesis imperfecta, Wilson's disease, spinal muscular atrophy, Huntington's disease, Rett syndrome, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Friedreich's ataxia, methylmalonic acidemia, cystic fibrosis, glycogen storage disease 1a, glycogen storage disease III, Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, propionic acidemia, phenylketonuria, hemophilia A, hemophilia B, beta-thalassemia, Lafora disease, Dravet syndrome, Alexander disease, Leber congenital amaurosis, myelodysplastic syndrome, and CBS-deficiency homocystinuria.

[0093] In some embodiments, the cancer comprises one or more of the following: hematologic malignancies, lung cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, brain cancer, prostate cancer, gallbladder cancer, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, bladder cancer, and melanoma.

[0094] In some embodiments, the infectious disease includes diseases caused by one or more infections selected from the group consisting of viruses, fungi, and bacteria.

[0095] In some embodiments, the autoimmune disease comprises one or more of: acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, celiac disease, type 1 diabetes mellitus, and diffuse toxic goiter.

[0096] In some embodiments, the genetic disease comprises one or more of the following: hemophilia, thalassemia, and Gaucher disease.

[0097] In some embodiments, the neurological disease comprises one or more of the following: amyotrophic lateral sclerosis, Alzheimer's disease, and glioma.

[0098] In some embodiments, the medicament is a vaccine.

[0099] In some embodiments, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA) and Cas9mRNA.

[0100] In another aspect, the present disclosure provides the use of the lipid nanoparticles described above in preparing a carrier for transferring or delivering an active ingredient. In some embodiments, the active ingredient is a pharmaceutical active ingredient, such as a nucleic acid. In other embodiments, the active ingredient is a transfection reagent or a detection reagent.

[0101] In some embodiments, the nucleic acid includes at least one of RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 shows the results of the hEPO concentration test in the serum of rats after multiple administration. "001" in Figure 1 represents lipid nanoparticles containing the PEG lipid numbered 001 in Table 1, and so on.

[0103] Figure 2 shows the results of detecting anti-PEG IgG antibodies in the serum of rats after multiple administration. "001" in Figure 2 represents lipid nanoparticles containing the PEG lipid numbered 001 in Table 1, and so on.

[0104] Figure 3 shows the results of detecting IL-6 in the serum of rats after administration. "001" in Figure 3 represents lipid nanoparticles containing the PEG lipid numbered 001 in Table 1, and so on.

[0105] Figure 4 shows the results of detecting the cytokine IFN-γ in the serum of rats after administration. "001" in Figure 4 represents lipid nanoparticles comprising the PEG lipid numbered 001 in Table 1, and so on.

[0106] Detailed Description of the Invention

[0107] 1. Definition

[0108] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0109] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0110] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0111] As used herein, unless the context indicates otherwise, the singular expressions "a," "an," "the," and similar references used in the context of describing the invention (particularly in the context of the claims) should be interpreted to cover both the singular and the plural. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The terms "at least one" or "one or more" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0112] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10. For another example, "C1-C6 alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, C 1～6 、C 1～5 、C 1～4 、C 1～3 、C 1～2 、C 2～6 、C 2～5 、C 2～4 、C 2～3 、C 3～6 、C 3～5 、C 3～4 、C 4～6 、C 4～5 and C 5～6 This principle also applies to ranges where only one value is used as a minimum or maximum value.

[0113] As used herein, the terms "and / or," "any combination thereof," and their grammatical equivalents are used interchangeably. These terms may expressly refer to any combination. For example, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may refer to "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C."

[0114] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0115] As used herein, the term "hydrocarbyl" means the group remaining after an aliphatic hydrocarbon loses one hydrogen atom, including straight-chain or branched, saturated or unsaturated hydrocarbon groups. Hydrocarbyl groups include, but are not limited to, alkyl, alkenyl, and alkynyl groups. In some embodiments, the hydrocarbon group has 1 to 24 carbon atoms (C1 to C2). 24 hydrocarbon radicals), for example, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms (C1, C2, C3, ... C 21 、C 22 、C 23 or C 24 Examples of hydrocarbon groups include, but are not limited to, C1 to C 24 Hydrocarbon, C1~C 22 Hydrocarbon, C1~C 20 Hydrocarbon, C1~C18 Hydrocarbon, C1~C 16 Hydrocarbon, C1~C 12 Hydrocarbon, C1~C 10 Hydrocarbon group, C1-C8 hydrocarbon group, C1-C7 hydrocarbon group, C1-C6 hydrocarbon group, C1-C4 hydrocarbon group, C1-C3 hydrocarbon group, C1-C2 hydrocarbon group, C2-C8 hydrocarbon group, C2-C4 hydrocarbon group, C4-C8 hydrocarbon group, C4-C9 hydrocarbon group, C5-C8 hydrocarbon group, C3 hydrocarbon group, C4 hydrocarbon group, C5 hydrocarbon group, C6 hydrocarbon group, C7 hydrocarbon group, C8 hydrocarbon group, C9 hydrocarbon group, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon, C 21 Hydrocarbon and C 22 Hydrocarbyl. Unless otherwise expressly stated in this specification, a hydrocarbyl group is optionally substituted, and the substituents are defined below regarding "optionally substituted." In certain embodiments, the hydrocarbyl group has zero branches (i.e., a straight chain), one branch, two branches, or more branches.

[0116] As used herein, the term "alkylene" refers to a divalent group remaining after the above-mentioned alkyl group further loses one hydrogen atom. Unless otherwise specified in the specification, the alkylene group may also be optionally substituted.

[0117] As used herein, the term "alkyl" is a linear or branched saturated monovalent hydrocarbon group. In some embodiments, the alkyl group has 1 to 24 carbon atoms (C1 to C2). 24 alkyl), for example having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms (C1, C2, C3, ... C 21 、C 22 、C 23 or C 24 Examples of alkyl groups include, but are not limited to, C1 to C 24 Alkyl, C1~C 22 Alkyl, C1~C 20 Alkyl, C1~C 18 Alkyl, C1~C 16 Alkyl, C1~C 12 Alkyl, C1~C 10Alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C2-C4 alkyl, C4-C8 alkyl, C4-C9 alkyl, C5-C8 alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl and tridec-7-yl. Unless otherwise expressly stated in the specification, an alkyl group may be optionally substituted.

[0118] As used herein, the term "alkylene" refers to a divalent group remaining after the above-mentioned alkyl group further loses one hydrogen atom. Unless otherwise specifically stated in the specification, the alkylene group may also be optionally substituted.

[0119] As used herein, the term "alkenyl" is a linear or branched monovalent hydrocarbon group containing one or more double bonds (C=C). In some embodiments, the alkenyl group has 2 to 24 carbon atoms (C2 to C 24 alkenyl), for example, having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms (C2, C3, C4, ... C 21 、C 22 、C 23 or C 24 Alkenyl) and has 1, 2, 3, 4 or more double bonds. Alkenyl includes but is not limited to C2 to C 24 Alkenyl, C2~C 22 Alkenyl, C2~C 20 Alkenyl, C2~C 18 Alkenyl, C2~C 16 Alkenyl, C2~C 12 Alkenyl, C2~C 10 Alkenyl, C2~C8 alkenyl, C2~C7 alkenyl, C2~C6 alkenyl, C2~C4 alkenyl, C2~C3 alkenyl, C4~C8 alkenyl, C4~C9 alkenyl, C5~C8 alkenyl, it has 1,2,3,4 or more double bonds.Some more specific examples include but are not limited to vinyl, propenyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hept-1-enyl, hept-2-enyl, hept-3-enyl, oct-1-enyl, oct-2-enyl, oct-3-enyl, non-1-enyl, non-2-enyl and non-3-enyl.In some embodiments, the alkenyl has 1 double bond.Unless otherwise clearly stated in this specification, alkenyl is optionally substituted.

[0120] As used herein, the term "alkenylene" refers to a divalent group remaining after the above alkenyl group further loses one hydrogen atom. Unless otherwise specifically stated in the specification, the alkenylene group may also be optionally substituted.

[0121] As used herein, the term "alkynyl" is a linear or branched monovalent hydrocarbon radical containing one or more triple bonds (C≡C). In some embodiments, the alkynyl group has 2 to 24 carbon atoms (C2 to C 24 alkynyl), for example, having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms (C2, C3, C4, ... C 21 、C 22 、C 23 or C 24 Alkynyl) and has 1, 2, 3, 4 or more triple bonds. Alkynyl includes but is not limited to C2 to C 24 Alkynyl, C2~C 22 Alkynyl, C2~C 20 Alkynyl, C2~C 18 Alkynyl, C2~C 16 Alkynyl, C2~C 12 Alkynyl, C2~C 10 Alkynyl, C2~C8 alkynyl, C2~C7 alkynyl, C2~C6 alkynyl, C2~C4 alkynyl, C2~C3 alkynyl, C4~C8 alkynyl, C4~C9 alkynyl, C5~C8 alkynyl, it has 1,2,3,4 or more triple bonds.Some more specific examples include but are not limited to ethynyl, propynyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hept-1-ynyl, hept-2-ynyl, hept-3-ynyl, oct-1-ynyl, oct-2-ynyl, oct-3-ynyl, non-1-ynyl, non-2-ynyl and non-3-ynyl.In some embodiments, the alkynyl has 1 triple bond.Unless otherwise clearly stated in this specification, alkynyl is optionally substituted.

[0122] As used herein, the term "alkynylene" refers to a divalent group remaining after the above-mentioned alkynyl group further loses one hydrogen atom. Unless otherwise specifically stated in the specification, the alkynylene group may also be optionally substituted.

[0123] As used herein, the term "optionally substituted" means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or independently substituted with one or more (e.g., 1, 2, 3, or 4) substituents. The substituents may be independently selected from, but are not limited to, halogen (e.g., chlorine, bromine, fluorine, or iodine), carboxylic acid (e.g., -C(=O)OH), oxygen (e.g., =O), sulfur (e.g., =S), hydroxyl (e.g., -OH), ester (e.g., -C(=O)ORiii or -OC(=O)Riii), aldehyde (e.g., -C(=O)H), carbonyl (e.g., -C(=O)Riii, or represented by C=O), acyl halide (e.g., -C(=O)X, wherein X is selected from bromine, fluorine, chlorine, or iodine), carbonate (e.g., -OC(=O)ORiii), alkoxy (e.g., -ORiii), acetal (e.g., -C(ORiii)2Riii, wherein each ORiii is the same or different alkoxy), phosphate (e.g., P(=O)4 3- ), sulfhydryl (e.g. -SH), sulfoxide (e.g. -S(=O)Riii), sulfinic acid (e.g. -S(=O)OH), sulfonic acid (e.g. -S(=O)2OH), thialdehyde (e.g. -C(=S)H), sulfate (e.g. S(=O)4 2-), sulfonyl (e.g., -S(=O)2Riii), sulfinyl (e.g., -S(=O)Riii), amide (e.g., -C(=O)N(Riii)2 or -N(Riii)C(=O)Riii), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(=O)Riii), amino (e.g., -N(Riii)2, -N(Riii)H or -NH2), carbamoyl (e.g., -OC(=O)N(Riii)2, -OC(=O)N(Riii) i)H or -OC(=O)NH2), sulfonamide (e.g., -S(=O)2N(Riii)2, -S(=O)2N(Riii)H, -S(=O)2NH2, -N(Riii)S(=O)2Riii, -N(H)S(=O)2Riii, -N(Riii)S(=O)2H or -N(H)S(=O)2H), alkyl, alkenyl, alkynyl, cycloalkyl (e.g., cycloalkyl, cycloalkenyl or cycloalkynyl), heterocycloalkyl (e.g., heterocycloalkyl containing one or more heteroatoms selected from S, N and O, or containing one or more heteroatoms selected from S, N and O) heterocycloalkenyl), aryl (e.g., phenyl or fused ring), heteroaryl (e.g., 8- to 10-membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur), -C(=O)SRiii, -C(=N-CN)N(Riii)2, -C(=NO-CH3)N(Riii)2, -C(=N-SO2-NH2)N(Riii)2, -C(=CH-NO2)N(Riii)2, -OC(=O)N(Riii)2, -CHN(Riii)N(Riii)2, -C(=O)N(Riii)ORiii, -N (Riii)2C(=O)ORiii、-OP(=O)(ORiii)2、-P(=O)(ORiii)2、-N(ORiii)C(=O)Riii、-N(ORiii)S(=O)2Riii、-N(ORiii)C(=O)ORiii、-N(ORiii)C(=O)N(Riii)2、-N(ORiii)C(=S)N(Riii)2、-N(ORiii)C(NRiii)N(Riii)2、-N(ORiii)C(CHRiii)N(Riii)2。 In any of the foregoing, Riii is hydrogen as defined herein, or alkyl, or alkenyl, or alkynyl, or heteroalkyl, or heteroalkenyl, or heteroalkynyl. In some embodiments, Riii is hydrogen as defined herein, or C1~C 12 Alkyl, or C2~C 12 Alkenyl, or C2~C 12 Alkynyl, or C2~C 12 Heteroalkyl, or C3~C12 Heteroalkenyl, or C3~C 12 In certain embodiments, the substituents themselves may be further substituted, for example, with one or more substituents as defined herein. For example, a C1-C6 alkyl substituent may be further substituted with one or more substituents as described herein.

[0124] As used herein, the term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0125] As used herein, "pharmaceutically acceptable salts" refer to acid addition salts or base addition salts of the disclosed compounds that retain the biological effectiveness and properties of the disclosed compounds and, generally, are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0126] Pharmaceutically acceptable acid addition salts can be formed with inorganic and / or organic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, and phosphoric acid; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactose. Diacid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid and undecylenic acid, etc.

[0127] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and / or organic bases and the compounds of the present disclosure. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. For example, inorganic salts include ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Examples of organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0128] As used herein, the term "isomer" means different compounds with the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space. "Atropisomers" are stereoisomers resulting from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is found and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0129] In certain embodiments, "stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof.

[0130] As used herein, the term "nucleic acid" generally refers to a polymer comprising deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer comprising ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof. In addition, nucleic acids herein also include derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of the nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In addition, herein, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0131] As used herein, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, digesting a circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in the cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In an alternative embodiment, the DNA plasmid is a medium copy or high copy plasmid. In another alternative embodiment, the DNA plasmid is a high copy plasmid. Examples of such high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.

[0132] As used herein, the term "vaccine" is typically understood to mean a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0133] As used herein, the term "treatment" or the like is used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. Therefore, the treatment of the present application may relate to the treatment of the state of a certain disease, but may also relate to prophylactic treatment for preventing a disease or its symptoms in whole or in part. Preferably in some embodiments, the term "treatment" is understood to be therapeutic in terms of partially or completely curing a disease and / or the adverse effects and / or symptoms owing to the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, such as to prevent infection and / or the onset of a disease.

[0134] As used herein, the term "administer" refers to providing or administering a medicament to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, disorder, condition, or symptom thereof, the administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom. When used to prevent a disease, disorder, condition, or symptom, the administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.

[0135] As used herein, the terms "subject" and "patient" can be used interchangeably. In certain embodiments, the subject is a mammal, such as a human, non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), domesticated animal (including dog and cat and livestock (e.g., horse, cattle, pig, sheep, and goat)), or other mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0136] As used herein, the term "cationic lipid" refers to a lipid that becomes positively charged when the pH is lowered below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. At pH values ​​below the pKa, the positively charged lipid is able to bind to negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid.

[0137] As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory agency (e.g., the State Food and Drug Administration (CFDA), the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopeia).

[0138] Herein, some elements of the application will be described, and these elements are listed together with specific embodiments, but it should be understood that they can be combined in any way and in any number to produce other embodiments. The examples and preferred embodiments of different descriptions should not be interpreted as limiting the application to only the embodiments clearly described. This specification should be understood as supporting and including embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any arrangement and combination of all description elements in this application should be considered as disclosed by the specification of the application.

[0139] 2. PEG lipids

[0140] The present disclosure provides a PEG lipid as shown in formula (I):

[0141] or a salt thereof, or a stereoisomer thereof, wherein

[0142] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0143] Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond,

[0144] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0145] Q1 is -C(=O)-, -OC(=O)- or a bond,

[0146] Q2 is -C(=O)- or a bond,

[0147] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0148] L1 is C1~C 20 an alkylene group or bond,

[0149] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0150] R2 is C4~C 24 Hydrocarbon group,

[0151] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0152] L2 is a C1-C4 alkylene group,

[0153] L3 is a C1-C4 alkylene group,

[0154] R3 is H or a C1-C3 hydrocarbon group,

[0155] n is any integer from 30 to 60.

[0156] The present disclosure also provides another PEG lipid as shown in formula (IX):

[0157] or a salt thereof, or a stereoisomer thereof, wherein

[0158] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0159] Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond,

[0160] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0161] Q1 is C(=O)-, -OC(=O)- or a bond,

[0162] Q2 is C(=O)- or a bond,

[0163] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0164] L1 is C1~C 20 an alkylene group or bond,

[0165] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0166] R2 is C4~C 24 Hydrocarbon group,

[0167] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0168] L2 is a C1-C4 alkylene group,

[0169] L3 is a C1-C4 alkylene group,

[0170] R3 is H or a C1-C3 hydrocarbon group,

[0171] n is any integer from 30 to 60,

[0172] L1, A1 and R2 form a four-membered ring, a five-membered ring or a six-membered ring.

[0173] In some embodiments, L1 in formula (IX) is C1 to C 20 Hydrocarbylene.

[0174] In addition, the present disclosure also provides a PEG lipid as shown in formula (X):

[0175] or a salt thereof, or a stereoisomer thereof, wherein

[0176] R1 is H, HO-Z1-Q1- or H-Z1-Q1-,

[0177] Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2- or a bond,

[0178] Q1 is C(=O)-, -OC(=O)- or a bond,

[0179] Q2 is C(=O)- or a bond,

[0180] Q3 is -C(=O)O-, -OC(=O)-, -O-, -S- or a bond,

[0181] L1 is C1~C 20 an alkylene group or bond,

[0182] A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0183] R2 is C4~C 24 Hydrocarbon group,

[0184] The total number of carbon atoms in L1 and R2 is 10 to 35.

[0185] L2 is a C1-C4 alkylene group,

[0186] L3 is a C1-C4 alkylene group,

[0187] R3 is H or a C1-C3 hydrocarbon group,

[0188] n is any integer from 30 to 60,

[0189] Z2 forms a four-membered ring, a five-membered ring or a six-membered ring with Q3, L1, A1 and R2.

[0190] In some embodiments, in Formula (X), Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2-, or a bond, and Z2 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-.

[0191] In some embodiments, the PEG lipid of formula (I) has a structure as shown in formula (II):

[0192] wherein A1, L1, R1, R2 and n are as defined in formula (I), and R1 is not H and is not unsubstituted methyl.

[0193] In some embodiments, the PEG lipid of formula (I) has a structure as shown in formula (III):

[0194] wherein A1, L1, R1, R2 and n are as defined in formula (I).

[0195] In some embodiments, the PEG lipid of formula (I) has a structure as shown in formula (IV):

[0196] wherein A1, L1, R1, R2 and n are as defined in formula (I).

[0197] In some embodiments, the PEG lipid of formula (I) has a structure as shown in formula (V):

[0198] wherein A1, L1, R1, R2 and n are as defined in formula (I).

[0199] In some embodiments, the PEG lipid of formula (I) has the structure shown in formula (VI):

[0200] wherein A1, L1, R1, R2 and n are as defined in formula (I).

[0201] In some embodiments, the PEG lipid of formula (I) has the structure shown in formula (VII):

[0202] wherein A1, L1, R1, R2, Q2, Z2, Q3 and n are as defined in formula (I).

[0203] PEG lipids as shown in Formula (A)-(I), (IA), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), when applicable, include one or more of the following features.

[0204] In some embodiments, the salt is a pharmaceutically acceptable salt.

[0205] In some embodiments, R1 is H or a C1-C9 hydrocarbon group.

[0206] In some embodiments, R1 is H, C1-C9 alkyl, C2-C9 alkenyl, or C2-C9 alkynyl.

[0207] In some embodiments, R1 is not H and is not unsubstituted methyl.

[0208] In some embodiments, R1 is a substituted methyl group or a C2-C9 hydrocarbon group.

[0209] In some embodiments, R1 is a substituted methyl group or a C2-C6 hydrocarbon group.

[0210] In some embodiments, R1 is substituted methyl, C2-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0211] In some embodiments, R1 is a substituted hydrocarbon group. In some embodiments, R1 is a substituted C1-C9 hydrocarbon group. In some embodiments, R1 is a substituted C1-C6 hydrocarbon group.

[0212] In some embodiments, R1 is HO-Z1-Q1-, wherein Z1 and Q1 are as defined in formula (I).

[0213] In some embodiments, R1 is H-Z1-Q1-, wherein Z1 and Q1 are as defined in formula (I), and when Q1 is a bond, Z1 is not a bond and is not unsubstituted methylene.

[0214] In some embodiments, R1 is HO-Z1-Q1-, wherein Z1 and Q1 are as defined in formula (I), and Z1 and Q1 are not simultaneously a bond.

[0215] In some embodiments, R1 is HO-Z1-Q1-, wherein Q1 is as defined in formula (I), and Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-.

[0216] In some embodiments, R1 is HO-Z1-Q1-, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3- or -L2-, and Q1 is -C(=O)-, -OC(=O)- or a bond. In some preferred embodiments, L2 is C1-C3 alkylene, L3 is C1-C3 alkylene, and R3 is H or C1-C3 alkyl. Preferably, L2 and L3 are each a linear C1-C3 alkylene, more preferably a C1 alkylene or a C2 alkylene. In a more preferred embodiment, R1 is

[0217] In some embodiments, R1 is H-Z1-Q1-, Z1 is -L2-, and Q1 is -C(=O)-. In some preferred embodiments, L2 is a C1-C3 alkylene group. In some preferred embodiments, R1 is

[0218] In some embodiments, R1 is H, wherein Q1, L2, L3 and R3 are as defined in formula (I).

[0219] In some embodiments, R1 is H,

[0220] In some embodiments, R1 is

[0221] In some embodiments, R1 is

[0222] In some embodiments, R1 is H or HO-Z1-Q1-. In some embodiments, R1 is H or

[0223] In some embodiments, R1 is HO-Z1-Q1-, Z1 is -L2-, -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, or -L2-CH(R3)-L3-, and Q1 is -OC(=O)-.

[0224] In some embodiments, R1 is HO-Z1-Q1-, wherein Z1 is -L2-, Q1 is -OC(=O)-, and L2 is as defined in formula (I).

[0225] In some embodiments, R1 is HO-Z1-Q1-, wherein Z1 is -L2-, Q1 is -OC(=O)-, and L2 is C1-C3 alkylene.

[0226] In some embodiments, R1 is HO-Z1-Q1-, wherein Z1 is -L2-, Q1 is -OC(=O)-, and L2 is C1-C3 alkylene or C2-C3 alkenylene.

[0227] In some embodiments, R1 is H.

[0228] In some embodiments, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, or a bond.

[0229] In some embodiments, A1 is -OC(=O)-, -OC(=O)O-, -O-, or -S-.

[0230] In some embodiments, A1 is -C(=O)O-, -OC(=O)-, -O-, or a bond.

[0231] In some embodiments, A1 is -O- or a bond.

[0232] In some embodiments, A1 is a bond.

[0233] In some embodiments, A1 is -C(=O)O-.

[0234] In some embodiments, A1 is -OC(=O)-.

[0235] In some embodiments, Q2 is a bond or -C(=O)-.

[0236] In some embodiments, Q2 is a bond.

[0237] In some embodiments, Q3 is a bond.

[0238] In some embodiments, Q2 and Q3 are a bond.

[0239] In some embodiments, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-.

[0240] In some embodiments, Z2 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, or -L2-CH(R3)-L3-.

[0241] In some embodiments, L2 is independently at each occurrence a C1-C3 alkylene group, L3 is independently at each occurrence a C1-C3 alkylene group, and R3 is independently at each occurrence H or a C1-C3 alkyl group.

[0242] In some embodiments, L2 is independently C1-C3 hydrocarbylene at each occurrence, preferably C1-C3 alkylene, more preferably C1 alkylene or C2 alkylene.

[0243] In some embodiments, L3 is independently C1-C3 hydrocarbylene at each occurrence, preferably C1-C3 alkylene, more preferably C1 alkylene or C2 alkylene.

[0244] In some embodiments, R3 at each occurrence is independently H or C1-C3 alkyl.

[0245] In some embodiments, the PEG lipid of formula (II) has a structure as shown in formula (II-1):

[0246] wherein A1 is -OC(=O)-, -OC(=O)O-, -O- or -S-, and L1, R2 and n are as defined in formula (II).

[0247] In some embodiments, the PEG lipid of formula (II) has the structure shown below:

[0248] wherein L1, R2 and n are as defined in formula (II).

[0249] In some embodiments, in the PEG lipid represented by formula (II), R1 is not H and is not unsubstituted methyl, and A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-.

[0250] In some embodiments, in the PEG lipid represented by formula (II), R1 is HO-Z1-Q1-, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-, wherein Z1 and Q1 are as defined in formula (I), and Z1 and Q1 are not bonds at the same time.

[0251] In some embodiments, in the PEG lipid represented by formula (II), R1 is H-Z1-Q1-, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-, wherein Z1 and Q1 are as defined in formula (I), and when Q1 is a bond, Z1 is not a bond or Z1 is a substituted methylene.

[0252] In some embodiments, in the PEG lipid represented by formula (II), R1 is H-Z1-Q1-, Q1 is -C(=O)-, Z1 is -L2-, and A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-.

[0253] In some embodiments, in the PEG lipid of formula (II), A1 is a bond, and R1 is not H and is not unsubstituted methyl.

[0254] In some embodiments, in the PEG lipid of formula (II), R1 is HO-Z1-Q1-, A1 is a bond, and Z1 and Q1 are not simultaneously bonds. In some such embodiments, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-. In more preferred embodiments, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2-, and Q1 is -C(=O)-, -OC(=O)-, or a bond. In more preferred embodiments, L2 is a C1-C3 alkylene group, L3 is a C1-C3 alkylene group, and R3 is H or a C1-C3 alkyl group. In a further more preferred embodiment, L2 and L3 are each C1-C3 alkylene, more preferably linear C1-C3 alkylene, further more preferably C1 alkylene or C2 alkylene. In an even more preferred embodiment, R1 is

[0255] In some embodiments, in the PEG lipid of formula (II), R1 is H-Z1-Q1-, Z1 is -L2-, Q1 is -C(=O)-, and A1 is a bond. In some such embodiments, L2 is a C1-C3 alkylene group, preferably a C1-C3 alkylene group, more preferably a C1 alkylene group or a C2 alkylene group. In a more preferred embodiment, R1 is

[0256] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0257] wherein Z1, Q1, L1, R2 and n are as defined in formula (II), and Z1 and Q1 are not bonds at the same time.

[0258] In some embodiments, Z1 and Q1 of the PEG lipid represented by formula (II-2) do not contain -C(=O)- at the same time.

[0259] In some embodiments, Z1 of the PEG lipid represented by formula (II-2) is -L2-C(=O)-L3-.

[0260] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0261] wherein Z1, Q1, L1, R2 and n are as defined in formula (II), and when Q1 is a bond, Z1 is not a bond and is not an unsubstituted methylene group.

[0262] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0263] wherein L1, L2, L3, Q1, R2 and n are as defined in formula (II).

[0264] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0265] wherein L1, L2, Q1, R2 and n are as defined in formula (II).

[0266] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0267] wherein L1, L2, L3, Q1, R2, R3 and n are as defined in formula (II).

[0268] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0269] wherein L1, L2, L3, Q1, R2, R3 and n are as defined in formula (II).

[0270] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0271] wherein L1, L2, Q1, R2 and n are as defined in formula (II).

[0272] In some embodiments, Q1 of the PEG lipid represented by formula (II-2-1) is -C(=O)-.

[0273] In some embodiments, in the PEG lipid represented by formula (II-2-1), when Q1 is -C(=O)-, L3 is a C1 alkylene group or a C4 alkylene group.

[0274] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0275] wherein L1, L2, L3, R2 and n are as defined in formula (II).

[0276] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0277] wherein L1, L2, R2 and n are as defined in formula (II).

[0278] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0279] wherein L1, L2, R2 and n are as defined for formula (II).

[0280] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0281] wherein L1, L2, L3, R2, R3 and n are as defined for formula (II).

[0282] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0283] wherein L1, L2, L3, R2, R3 and n are as defined in formula (II).

[0284] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0285] wherein L1, L2, L3, R2, R3 and n are as defined in formula (II).

[0286] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0287] wherein L1, L2, L3, R2, R3 and n are as defined in formula (II).

[0288] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0289] wherein L1, L2, R2 and n are as defined in formula (II).

[0290] In some embodiments, the PEG lipid of formula (II) has the following structure:

[0291] in,

[0292] R2 and L1 are as defined in formula (II),

[0293] (i) A1 is a bond, and R2 and n satisfy the following conditions: (1) the total number of carbon atoms in L1 and R2 is 5 to 12, 10 to 12, 14 to 16, 18, or 20 to 32; and (2) n is any integer from 36 to 54; or

[0294] (ii) A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-.

[0295] In some embodiments, in the PEG lipid represented by formula (II-4), A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-, R2 and n are as defined in formula (I), and satisfy one or more of the following conditions: (1) R2 is C4 to C 17 Hydrocarbon or C 19 ~C 24 and (2) the total number of carbon atoms in the main chain of L1 and the total number of carbon atoms in R2 is 5 to 19 or 21 to 32.

[0296] In some embodiments, in the PEG lipid shown in formula (III), R1 is H or HO-Z1-Q1-, wherein Z1 and Q1 are as defined in formula (I), and Z1 and Q1 are not bonds at the same time, and A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or a bond.

[0297] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0298] wherein R1, L1, R2 and n are as defined in formula (III).

[0299] In some embodiments, in the PEG lipid represented by formula (III), R1 is H or HO-Z1-Q1-, wherein Z1 is -L2-, Q1 is -OC(=O)-, and A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or a bond.

[0300] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0301] wherein L1, R2 and n are as defined in formula (III).

[0302] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0303] wherein L1, R2 and n are as defined in formula (III).

[0304] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0305] wherein L1, R2 and n are as defined in formula (III).

[0306] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0307] wherein L1, R2 and n are as defined in formula (III).

[0308] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0309] wherein L1, R2 and n are as defined in formula (III).

[0310] In some embodiments, the PEG lipid of formula (III) has the following structure:

[0311] wherein L1, L2, R2 and n are as defined in formula (III).

[0312] In some embodiments, in the PEG lipid represented by Formula (IV), Formula (V) or Formula (VI), R1 is H and A1 is a bond, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O- or -S-.

[0313] In some embodiments, in the PEG lipid represented by Formula (IV), Formula (V) or Formula (VI), R1 is HO-Z1-Q1- or H-Z1-Q1-, and A1 is a bond, -C(=O)O-, -OC(=O)- or -O-.

[0314] In some embodiments, the PEG lipid of formula (IV) has the following structure:

[0315] wherein L1, R2 and n are as defined in formula (IV).

[0316] In some embodiments, the PEG lipid of formula (V) has the following structure:

[0317] wherein R2 and n are as defined in formula (V).

[0318] In some embodiments, the PEG lipid of formula (VI) has the following structure:

[0319] wherein R2 and n are as defined in formula (VI).

[0320] The PEG lipids of formula (VII), when applicable, include one or more of the following features.

[0321] In some embodiments, R1 is HO-Z1-Q1- or H-Z1-Q1-, and A1 is a bond, -C(=O)O-, -OC(=O)-, or -O-.

[0322] In some embodiments, R1 is HO-Z1-Q1- or H-Z1-Q1-, and Q2 and Q3 are a bond.

[0323] In some embodiments, R1 is HO-Z1-Q1- or H-Z1-Q1-, Q2 and Q3 are a bond, and A1 is -OC(=O)-.

[0324] In some embodiments, R1 is HO-Z1-Q1- or H-Z1-Q1-, Q2 and Q3 are a bond, A1 is -OC(=O)-, and Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, or -L2-CH(R3)-L3-.

[0325] In some embodiments, R1 is HO-Z1-Q1- or H-Z1-Q1-, Q2 and Q3 are a bond, A1 is -OC(=O)-, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, or -L2-CH(R3)-L3-, and L1 is a bond or C1 alkylene.

[0326] In some embodiments, R1 is H, and A1 is a bond, -C(=O)O-, -OC(=O)-, -OC(=O)O-, or -O-.

[0327] In some embodiments, Q2 and Q3 are a bond, R1 is H, and Z2 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, or -L2-CH(R3)-L3-.

[0328] In some embodiments, the PEG lipid of formula (VII) has the following structure:

[0329] wherein L1, L2, L3, R2, R3 and n are as defined in formula (VII).

[0330] In some embodiments, the PEG lipid of formula (VII) has the following structure:

[0331] wherein L1, L2, L3, R2, R3 and n are as defined in formula (VII).

[0332] In some embodiments, the PEG lipid of formula (VII) has the following structure:

[0333] wherein L1, L2, L3, R2 and n are as defined in formula (VII).

[0334] In some embodiments, in the PEG lipid of formula (I), Y is a bond, i.e., the PEG lipid of formula (I) has the following structure:

[0335] wherein R1, A1, L1, R2 and n are as defined in formula (I), and

[0336] When R1 is H and A1 is -O-, L1 and R2 satisfy at least one of the following conditions: (1) the total number of carbon atoms is not 16, (2) at least one of L1 and R2 has a branched chain, and (3) L1 is a C3-C8 alkylene group or a bond.

[0337] In some embodiments, A1 of the PEG lipid represented by formula (VIII) is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -S-, or a bond.

[0338] Formula (I), formula (II), formula (II-1), formula (II-1-1), formula (II-2), formula (II-2-1), formula (II-2-1-a), formula (II-2-2), formula (II-2-2-a), formula (II-2-2-b), formula (II-2-3), formula (II-2-3-a), formula (II-2-3-b), formula (II-2-3-c), formula (II-2-4), formula (II-2-4-a), formula (II-3), formula (II-3-1), formula (II-3-1-a), formula ( The PEG lipids represented by formula (II-4), formula (III), formula (III-A), formula (III-1), formula (III-2), formula (III-3), formula (III-4), formula (III-5), formula (III-6), formula (IV), formula (IV-1), formula (V), formula (V-1), formula (VI), formula (VI-1), formula (VII), formula (VII-1), formula (VII-2), formula (VII-3), formula (VIII), formula (IX) or formula (X), when applicable, include one or more of the following features.

[0339] In some embodiments, the total number of carbon atoms of L1 and R2 is 12 to 25. For example, the total number of carbon atoms of L1 and R2 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0340] In some embodiments, the total number of carbon atoms in L1 and R2 is 11-22.

[0341] In some embodiments, the total number of carbon atoms in L1 and R2 is 14-24.

[0342] In some embodiments, the total number of carbon atoms in L1 and R2 is 16-22.

[0343] In some embodiments, the total number of carbon atoms in L1 and R2 is 15-20.

[0344] In some embodiments, the total number of carbon atoms in L1 and R2 is 16-20.

[0345] In some embodiments, the total number of carbon atoms in L1 and R2 is 10-18.

[0346] In some embodiments, the total number of carbon atoms of L1 and R2 is 14 to 16. In some embodiments, L1 is a C1 to C8 alkylene group or a bond.

[0347] In some embodiments, L1 is C1 to C 20 Alkylene, C2~C 20 Alkenylene or C2~C 20 Alkynylidene.

[0348] In some embodiments, L1 is C1-C8 alkylene, C2-C8 alkenylene, or C2-C8 alkynylene.

[0349] In some embodiments, L1 is substituted or unsubstituted C1-C 20 Alkylene, such as substituted or unsubstituted C1-C 15 alkylene, C1-C8 alkylene, C4-C8 alkylene, C1-C4 alkylene or C1-C2 alkylene.

[0350] In some embodiments, L1 is substituted or unsubstituted C1-C 20 Alkylene, such as substituted or unsubstituted C1-C 15 C1~C8 alkylene, C4~C8 alkylene, C1~C4 alkylene or C1~C2 alkylene.

[0351] In some embodiments, L1 is a linear or branched C1-C 20 Alkylene, such as straight-chain or branched C1-C 15 alkylene, C1-C8 alkylene, C4-C8 alkylene, C1-C4 alkylene or C1-C2 alkylene.

[0352] In some embodiments, L1 is a linear or branched C1-C 20 Alkylene, such as linear or branched C1-C 15 Alkylene, C1~C8 alkylene, C4~C8 alkylene, C1~C4 alkylene or C1~C2 alkylene.

[0353] In some embodiments, L1 is a linear C1-C 20 Alkylene, such as straight chain C1-C 15 Alkylene, linear C1-C8 alkylene, linear C4-C8 alkylene, linear C1-C4 alkylene or linear C1-C2 alkylene.

[0354] In some embodiments, L1 is a bond.

[0355] In some embodiments, R2 is C4 to C 24 Alkyl, C4~C 24 Alkenyl or C4~C 24 Alkynyl.

[0356] In some embodiments, R2 is substituted or unsubstituted C4-C 24 Hydrocarbon groups, such as substituted or unsubstituted C7-C 22 Hydrocarbon, C 11 ~C 22 Hydrocarbon, C4~C20 Hydrocarbon, C8~C 20 Hydrocarbon, C 15 ~C 20 Hydrocarbon, C 12 ~C 18 Hydrocarbon, C 10 ~C 16 Hydrocarbon, C4~C 15 Hydrocarbon, C8~C 15 Hydrocarbon or C5~C 11 Hydrocarbon group.

[0357] In some embodiments, R2 is substituted or unsubstituted C4-C 24 Alkyl, such as substituted or unsubstituted C7-C 22 Alkyl, C 11 ~C 22 Alkyl, C4~C 20 Alkyl, C8~C 20 Alkyl, C 15 ~C 20 Alkyl, C 12 ~C 18 Alkyl, C 10 ~C 16 Alkyl, C4~C 15 Alkyl, C8~C 15 Alkyl or C5~C 11 alkyl.

[0358] In some embodiments, R2 is a linear or branched C4 to C 24 Hydrocarbon groups, such as linear or branched C7-C 22 Hydrocarbon, C 11 ~C 22 Hydrocarbon, C4~C 20 Hydrocarbon, C8~C 20 Hydrocarbon, C 15 ~C 20 Hydrocarbon, C 12 ~C 18 Hydrocarbon, C 10 ~C 16 Hydrocarbon, C4~C 15 Hydrocarbon, C8~C 15 Hydrocarbon or C5~C 11 Hydrocarbon group.

[0359] In some embodiments, R2 is a linear or branched C4 to C 24 Alkyl, such as linear or branched C7-C 22 Alkyl, C 11 ~C 22 Alkyl, C4~C 20 Alkyl, C8~C 20Alkyl, C 15 ~C 20 Alkyl, C 12 ~C 18 Alkyl, C 10 ~C 16 Alkyl, C4~C 15 Alkyl, C8~C 15 Alkyl or C5~C 11 alkyl.

[0360] In some embodiments, R2 is a linear C4 to C 24 Alkyl, such as straight chain C7~C 22 Alkyl, straight chain C 11 ~C 22 Alkyl, straight chain C4~C 20 Alkyl, straight chain C8~C 20 Alkyl, straight chain C 15 ~C 20 Alkyl, straight chain C 12 ~C 18 Alkyl, straight chain C 10 ~C 16 Alkyl, straight chain C4~C 15 Alkyl, straight chain C8~C 15 Alkyl or straight chain C5~C 11 alkyl.

[0361] In some embodiments, R2 is C7 to C 22 Hydrocarbon group.

[0362] In some embodiments, R2 is C7 to C 22 Alkyl, C7~C 22 Alkenyl or C7~C 22 Alkynyl.

[0363] In some embodiments, L1 is a linear C1-C8 alkylene group, and R2 is a linear C7-C 22 alkyl.

[0364] In some embodiments, -L1-R2 is a linear C 16 ~C 22 Hydrocarbon group.

[0365] In some embodiments, -L1-R2 is C 16 ~C 22 alkyl.

[0366] In some embodiments, -L1-R2 is a linear C 16 ~C 22 alkyl.

[0367] In some embodiments, -L1-R2 is C 16 ~C 18 Hydrocarbon group.

[0368] In some embodiments, -L1-R2 is a linear C 16 ~C 18 Hydrocarbon group.

[0369] In some embodiments, -L1-R2 is C 16 ~C 18 alkyl.

[0370] In some embodiments, -L1-R2 is a linear C 16 ~C 18 alkyl.

[0371] In some embodiments, R3 is H, C1-C3 alkyl, C2-C3 alkenyl, or C2-C3 alkynyl.

[0372] In some embodiments, R3 is H or C1-C3 alkyl.

[0373] In some embodiments, R3 is C1-C3 alkyl, C2-C3 alkenyl, or C2-C3 alkynyl.

[0374] In some embodiments, R3 is a C1-C2 hydrocarbon group.

[0375] In some embodiments, R3 is a C1 hydrocarbon group or a C2 hydrocarbon group.

[0376] In some embodiments, R3 is C1 alkyl or C2 alkyl.

[0377] In some embodiments, R3 is H.

[0378] In some embodiments, R3 is C1 alkyl.

[0379] In some embodiments, R3 is C2 alkyl.

[0380] In some embodiments, n is any integer from 30 to 55, for example, n is any integer from 30 to 50, 34 to 50, 34 to 55, 36 to 55, 40 to 55, 44 to 55, 48 to 55, 50 to 55, 36 to 54, 40 to 54, 44 to 54, 48 to 54, or 50 to 54.

[0381] In some embodiments, n is any integer from 35 to 55.

[0382] In some embodiments, n is any integer from 35 to 50.

[0383] In some embodiments, n is any integer from 40 to 50.

[0384] In some embodiments, n is 45.

[0385] In some embodiments, L2 is a C1-C3 alkylene group.

[0386] In some embodiments, L2 is C1-C3 alkylene, C2-C3 alkenylene, or C2-C3 alkynylene.

[0387] In some embodiments, L2 is C1-C3 alkylene.

[0388] In some embodiments, L2 is a linear C1-C3 alkylene group.

[0389] In some embodiments, L2 is C1 alkylene or C2 alkylene.

[0390] In some embodiments, L3 is a C1-C2 alkylene group.

[0391] In some embodiments, L3 is C1-C2 alkylene, C2 alkenylene, or C2 alkynylene.

[0392] In some embodiments, L3 is C1-C2 alkylene.

[0393] In some embodiments, the PEG lipid of formula (I) has the structure shown in Table 1 below:

[0394] [Corrected 31.12.2024 in accordance with Rule 26] Table 1

[0395] 3. Lipid Nanoparticles, Pharmaceutical Compositions, Drugs, and Their Applications

[0396] The present disclosure also provides a lipid nanoparticle comprising the PEG lipid of any one of the above embodiments.

[0397] In some embodiments, the lipid nanoparticles are particles having a nanometer scale (eg, 1 nm to 1000 nm).

[0398] In some embodiments, the average diameter of the lipid nanoparticles is 20nm to 800nm, 20nm to 500nm, 20nm to 400nm, 20nm to 300nm, 20nm to 200nm, 20nm to 100nm, 30nm to 700nm, 30nm to 500nm, 30nm to 300nm, 30nm to 200nm, 30nm to 100nm, 40nm to 800nm, 40nm to 600nm, 40nm to 500nm, 40nm to 600nm, 300nm, 40nm~200nm, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of the lipid nanoparticles is 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm, 231 nm, 236 nm, 241 nm, 246 nm or 249 nm. Herein, the average diameter of lipid nanoparticles can be expressed as the z-average value determined by dynamic light scattering.

[0399] In some embodiments, the lipid nanoparticles include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nonlamellar lipid nanoparticles. In an optional specific example, the lipid nanoparticles are cationic lipid nanoparticles.

[0400] In some embodiments, the lipid nanoparticles further contain one or more of the following substances: cationic lipids, helper lipids, and structural lipids.

[0401] In some embodiments, the cationic lipid comprises a compound represented by the following formula (XI), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0402] in,

[0403] R 1 Select from the group consisting of: C5~C 30 Alkyl, C5~C 20 alkenyl, -R*Y1R", -Y1R", and -R"'M'R';

[0404] R 2 and R 3 Independently selected from the group consisting of: H, C1-C 14 Alkyl, C2~C 14 alkenyl, -R*Y1R", -Y1R" and -R*OR", or R 2 and R 3 Together with the atoms to which they are attached, they form a heterocyclic or carbocyclic ring;

[0405] R4 is selected from the group consisting of: hydrogen, C3-C6 carbocycle, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2 and unsubstituted C1-C6 alkyl, wherein Q is selected from carbocyclic, heterocyclic, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O) R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, N(R)R8, -N(R)S(O)2R8, -O(CH2) nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N( R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9) R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, each o is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5;

[0406] each R5 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl, and H;

[0407] Each R6 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl and H;

[0408] M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O), -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl groups and heteroaryl groups, wherein M" is a bond, C1-C 13 Alkylene or C 2- ~C 13 alkenylene;

[0409] R7 is selected from C 1-3 a group consisting of an alkyl group, a C2-C3 alkenyl group, and H;

[0410] R8 chooses from C 3-6 a group consisting of carbocyclic and heterocyclic rings;

[0411] R9 is selected from the group consisting of H, CN, NO2, C1-C6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-C6 alkenyl, C3-C6 carbocycle and heterocycle;

[0412] R 10 selected from the group consisting of H, C1-C3 alkyl and C2-C3 alkenyl;

[0413] Each R is independently selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, (CH2) q OR** and H,

[0414] and each q is independently selected from 1, 2, and 3;

[0415] Each R' is independently selected from the group consisting of: C1 to C 18 Alkyl, C2~C 18 alkenyl, -R*Y1R", -Y1R", H and And R 11 Choose from C1 to C 12 Alkylene and C2~C 12 The group consisting of alkenylene, R 12 and R 13 Each independently selected from C1 to C 12 Alkyl and C2~C 12 a group consisting of alkenyl groups;

[0416] Each R" is independently selected from C3 to C 15 Alkyl and C3~C 15 a group consisting of alkenyl groups;

[0417] Each R'' is independently selected from C3 to C 15 Alkylene and C3~C 15 a group consisting of alkenylene;

[0418] Each R* is independently selected from the group consisting of: 12 Alkylene and C2~C 12 a group consisting of alkenylene;

[0419] Each R** is independently selected from the group consisting of: 12 Alkyl and C2~C 12 a group consisting of alkenyl groups;

[0420] Each Y1 is independently a C3-C6 carbocyclic ring;

[0421] each X is independently selected from the group consisting of: F, Cl, Br, and I; and

[0422] m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and wherein when R4 is -(CH2) n Q, -(CH2) n When CHQR, -CHQR or -CQ(R)2, then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R)2; or (ii) when n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl.

[0423] In an optional specific example, the cationic lipid is a compound represented by (XI), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0424] Among them, R 1 ~R3 , R4 to R7, M and m are as defined above in formula (XI).

[0425] In an optional specific example, the cationic lipid is a compound represented by formula (XI-1), its N-oxide, its salt or its isomer:

[0426] In some embodiments, the cationic lipid comprises a compound represented by the following formula (XII), an N-oxide, a salt thereof, or an isomer thereof:

[0427] in,

[0428] R 1 、R 2 、R 3 , R5, R6, M and R7 are as defined above for formula (XI),

[0429] R N is H or C1-C3 alkyl;

[0430] X a and X b each independently O or S;

[0431] R 14 Selected from H, halogen, -OH, R b 、-N(R b )2, -CN, -N3, -C(O)OH, -C(O)OR b 、-OC(O)R b 、-OR b 、-SR b 、-S(O)R b 、-S(O)OR b 、-S(O)2OR b 、-NO2、-S(O)2N(R b )2、-N(R b )S(O)2R b 、-NH(CH2) t1 N(R b )2、-NH(CH2) p1 O(CH2) q1 N(R b )2、-NH(CH2) s1 OR b 、-N((CH2) s1 OR b )2、-N(R b )-carbocyclic ring, -N(R b )-heterocyclic, -N(R b )-aryl, -N(Rb )-heteroaryl, -N(R b )(CH2) t1 -Carbocyclic ring, -N(R b )(CH2) t1 -heterocycle, -N(R b )(CH2) t1- Aryl, -N(R b )(CH2) t1 - the group consisting of heteroaryl, carbocycle, heterocycle, aryl and heteroaryl;

[0432] Each R b independently selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, and H;

[0433] u is 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0434] w is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0435] r is 0 or 1;

[0436] t 1 is 1, 2, 3, 4, or 5;

[0437] p 1 is 1, 2, 5, 4, or 5;

[0438] q 1 is 1, 2, 5, 4, or 5; and

[0439] s 1 1, 2, 3, 4, or 5.

[0440] In an optional specific example, the cationic lipid is a compound represented by formula (XII), its N-oxide, its salt or its isomer:

[0441] Among them, R 1 ~R 3 、R5~R7、R 14 、X a 、X b 、R N , M, u, w and r are as defined above in formula (XII).

[0442] In some embodiments, the cationic lipid comprises a compound represented by the following formula (XIII), an N-oxide, a salt thereof, or an isomer thereof:

[0443] in:

[0444] L 1 or L 2One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or bond;

[0445] G 1 and G 2 are each independently unsubstituted C1 to C 12 Alkylene or C2~C 12 alkenylene;

[0446] G 3 C1~C 24 Alkylene, C2~C 24 Alkenylene, C3~C8 cycloalkylene, C3~C8 cycloalkenylene;

[0447] R a H or C1~C 12 hydrocarbon group;

[0448] R 15 and R 16 Each independently is C6~C 24 Alkyl or C6~C 24 alkenyl;

[0449] R 17 H, OR 18 、CN、-C(=O)OR 19 、-OC(=O)R 19 or –NR 18 C(=O)R 19 ;

[0450] R19 C1~C 12 hydrocarbon group;

[0451] R 18 is H or a C1-C6 hydrocarbon group; and

[0452] x is 0, 1, or 2.

[0453] In an optional specific example, the cationic lipid is a compound represented by formula (XIII), its N-oxide, its salt or its isomer:

[0454] Where: R 15 ~R 17 , G 1 ~G 3 、L 1 ~L 2 As defined above for formula (XIII).

[0455] In some embodiments, the cationic lipid comprises a compound represented by the following formula (XIV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0456] in:

[0457] L 3 and L 4 The same or different, each independently C1 to C 12 Alkylene, C2~C 12 Alkenylene or C2~C 12 Alkyne; in some embodiments L 3 and L 4 The same or different, each independently C3~C 10 Alkylene, C3~C 10 Alkenylene or C3~C 10 Alkyne; in some embodiments, L 3 and L 4 The same or different, each independently C3~C 10 Alkylene; in some embodiments, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group;

[0458] G 4 and G 5 are the same or different and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; in some embodiments, G 4 and G 5are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, or -O-; in some embodiments, G 4 and G 5 the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-;

[0459] R 18 and R 19 The same or different, each independently C5~C 27 Alkyl or C5~C 27 alkenyl; in some embodiments, R 18 and R 19 The same or different, each independently C8~C 20 Alkyl or C8~C 20 alkenyl; in some embodiments, R 18 and R 19 The same or different, each independently C9 to C 17 Alkyl or C9~C 18 alkenyl; in some embodiments, R 18 and R 19 the same or different, each independently

[0460] R 20 is halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; in some embodiments, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido;

[0461] z is 1, 2 or 3.

[0462] In some embodiments, the cationic lipid is a compound represented by formula (XIV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0463] Where: R18 ~R 20 , G 4 ~G 5 、L 3 ~L 4 and z are as defined above for formula (XIV).

[0464] In some embodiments, the cationic lipid comprises the following compound (XIV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0465] In some embodiments, the cationic lipid comprises the following compound, or a pharmaceutically acceptable salt thereof:

[0466] In some embodiments, the cationic lipids of the lipid nanoparticles include one or more of the following: ALC-0315 (CAS No. 2036272-55-4), SM-102 (CAS No. 2089251-47-6), and

[0467] In some embodiments, the helper lipids of the lipid nanoparticles include phospholipids. Phospholipids are typically semi-synthetic, but can also be naturally derived or chemically modified. In an alternative specific example, the helper lipids of the lipid nanoparticles are phospholipids. In some embodiments, the phospholipids of the lipid nanoparticles include one or more of the following: DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the helper lipid of the lipid nanoparticles is selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticles is DSPC and / or DOPE.

[0468] In some embodiments, the structural lipids of the lipid nanoparticles include sterols. In an alternative specific example, the structural lipids of the lipid nanoparticles are sterols. In some embodiments, the sterols of the lipid nanoparticles include one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipids of the lipid nanoparticles include at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids. In some embodiments, the structural lipid of the lipid nanoparticles is cholesterol. In an alternative specific example, the structural lipid of the lipid nanoparticles is high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0469] In some embodiments, the lipid nanoparticles contain cationic lipids, helper lipids, structural lipids and the PEG lipids of any of the above embodiments. In some embodiments, the lipid nanoparticles comprise the following amount (molar percentage) of cationic lipids based on the total amount of cationic lipids, helper lipids, structural lipids and PEG lipids: about 25% to 75%, for example, about 25% to 28%, 28% to 32%, 32% to 35%, 35% to 40%, 40% to 42%, 42% to 45%, 45% to 46.3%, 46.3% to 48%, 48% to 49.5%, 49.5% to 50%, 50% to 55%, 55% to 60%, 60% to 65% or 65% to 75%.

[0470] In some embodiments, the lipid nanoparticles comprise the following amount (molar percentage) of helper lipids, based on the total amount of cationic lipids, helper lipids, structural lipids and PEG lipids: about 5% to 45%, for example, about 5% to 9%, 9% to 9.4%, 9.4% to 10%, 10% to 10.5%, 10.5% to 11%, 11% to 15%, 15% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 33.5%, 33.5% to 37%, 37% to 40%, 40% to 42% or 42% to 45%.

[0471] In some embodiments, the lipid nanoparticles comprise the following amounts (molar percentages) of structural lipids, based on the total amount of cationic lipids, helper lipids, structural lipids and PEG lipids: about 0% to 50%, for example, about 0% to 10%, 10% to 15.5%, 15.5% to 18.5%, 18.5% to 22.5%, 22.5% to 23.5%, 23.5% to 28.5%, 28.5% to 33.5%, 33.5% to 35%, 35%-36.5%, 36.5%-38%, 38%-38.5%, 38.5%-39%, 39%-39.5%, 39.5%-40.5%, 40.5%-41.5%, 41.5%-42.5%, 42.5%-42.7%, 42.7%-43%, 43%-43.5%, 43.5%-45%, 45%-46.5%, 46.5%-48.5% or 46.5%-50%.

[0472] In some embodiments, the lipid nanoparticles comprise the following amount (molar percentage) of PEG lipids, based on the total amount of cationic lipids, helper lipids, structural lipids and PEG lipids: about 0.5% to 5%, for example, about 0.5% to 1%, 1% to 1.5%, 1.5% to 1.6%, 1.6% to 2%, 2% to 2.5%, 2.5% to 3%, 3% to 3.5%, 3.5% to 4%, 4% to 4.5% or 4.5% to 5%.

[0473] In some embodiments as described above, in the lipid nanoparticles, the molar ratio of cationic lipid:helper lipid:structural lipid:PEG lipid is about 45:10:42.5:2.5, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 35:16:46.5:2.5, 35:25:36.5:3.5, 28:33.5:35:3.5, 32:37:40.5:0.5, 35:40:22.5:2.5, 40:42:15.5:2.5, 40:20:38.5:1.5, 5:15:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:3 In some such embodiments, the helper lipid is DOPE and the structural lipid is CHO-HP.

[0474] In other embodiments as described above, in the lipid nanoparticles, the molar ratio of cationic lipid:helper lipid:structural lipid:PEG lipid is about 50:10:38.5:1.5, 50:9:38:3, 49.5:10:39:1.5, 48:10:40.5:1.5, 46.3:9.4:42.7:1.6, 45:9:43:3, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 45:1 5:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:33. In some such embodiments, the helper lipid is DSPC and the structural lipid is CHO-HP.

[0475] In some embodiments, the lipid nanoparticles further comprise an active ingredient.

[0476] In some embodiments, the active ingredient is a pharmaceutical active ingredient.

[0477] In some embodiments, the active ingredient is a nucleic acid, such as DNA, RNA. In some embodiments, the pharmaceutical active ingredient is a nucleic acid, such as DNA, RNA. In some embodiments, the pharmaceutical active ingredient is one or more of the following: RNA and DNA. In some embodiments, RNA is one or more of the following: mRNA, antisense oligonucleotides, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA) and Cas9 mRNA. In some embodiments, the pharmaceutical active ingredient is mRNA. In some embodiments, the pharmaceutical active ingredient is a plasmid.

[0478] In some embodiments, the nucleic acid used as a pharmaceutical active ingredient encodes a polypeptide and / or protein of interest. A polypeptide or protein of interest refers to a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect, whose function in or near a cell is necessary or beneficial. For example, a protein whose deficiency or defective form leads to a disease, and whose provision can modulate or prevent the disease, or a protein whose presence in or near a cell is beneficial to the body. The polypeptide or protein may comprise the entire protein or a functional variant thereof.

[0479] In some embodiments, the polypeptide and / or protein expressed by the nucleic acid encoding the polypeptide and / or protein of interest comprises or is one or more of: (a) an antigen; (b) a therapeutic protein or polypeptide, fragment, fragment or variant thereof; and (c) other polypeptides or proteins.

[0480] The PEG lipids of formula (I) or lipid nanoparticles (LNPs) containing the PEG lipids of formula (I) avoid immune responses against LNPs in a subject.

[0481] In some embodiments, repeated administration (e.g., 2, 3, 4, or more administrations) of a PEG lipid as shown in formula (I) or LNPs containing a PEG lipid as shown in formula (I) does not induce an immune response against the LNPs in the subject.

[0482] In some embodiments, repeated administration (e.g., 2, 3, 4, or more times) of a PEG lipid as shown in formula (I) or LNPs containing a PEG lipid as shown in formula (I) does not induce the production of IgM or IgG bound to the LNPs. Not inducing the production of IgM or IgG bound to the LNPs means not inducing natural IgM or IgG bound to the LNPs, or inducing low levels of IgM or IgG, but not sufficient to cause ABC.

[0483] The PEG lipids of formula (I) or LNPs containing the PEG lipids of formula (I) reduce the immune response of a subject against the LNPs.

[0484] In some embodiments, repeated administration (e.g., 2, 3, 4, or more administrations) of LNPs containing a PEG lipid of formula (I) induces less IgM or IgG, e.g., at least a 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction, compared to control LNPs (e.g., LNPs whose PEG lipid is DMG-PEG, where DMG refers to 1,2-dimyristoyl-sn-glycerol and DMG-PEG is a polyethylene glycol derivative of DMG).

[0485] The PEG lipid represented by formula (I) or LNPs containing the PEG lipid represented by formula (I) avoids the occurrence of accelerated blood clearance (ABC).

[0486] In some embodiments, repeated administration of the PEG lipid of formula (I) or LNPs containing the PEG lipid of formula (I) also does not result in accelerated blood clearance (ABC).

[0487] The PEG lipid of formula (I) or LNPs containing the PEG lipid of formula (I) reduces the extent of accelerated blood clearance (ABC).

[0488] In some embodiments, the clearance of the active ingredient is less when the active ingredient is delivered for the second or more times using LNPs containing a PEG lipid as shown in formula (I) compared to control LNPs (e.g., LNPs whose PEG lipid is DMG-PEG), for example, at least 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% reduction.

[0489] In some embodiments, the clearance of the active ingredient is less when the active ingredient is delivered for the second or more times using LNPs containing a PEG lipid as shown in formula (I) compared to control LNPs (e.g., LNPs whose PEG lipid is DMG-PEG), for example, at least 1% to 5%, 10% to 100%, 10% to 50%, 20% to 100%, 20% to 50%, 30% to 100%, 30% to 50%, 40% to 100%, 40% to 80%, 50% to 90% or 50% to 100%.

[0490] In addition, the present disclosure further provides a composition comprising the lipid nanoparticles of any of the above embodiments and a biologically acceptable carrier. The term "biologically acceptable carrier" as used herein refers to carriers that have no significant stimulatory effect on natural or artificial organisms (including humans, animals, plants, or tissues or cells in vivo or in vitro thereof; unicellular organisms such as bacteria and fungi; and artificial cells) and do not impair the activity and performance of the active ingredient in the lipid nanoparticles.

[0491] In addition, the present disclosure also provides a pharmaceutical composition comprising the lipid nanoparticles according to any of the above embodiments and a pharmaceutically acceptable carrier.

[0492] The above-mentioned pharmaceutical composition comprises the above-mentioned lipid nanoparticles and has its corresponding advantages.

[0493] In addition, the present disclosure also provides a use of the lipid nanoparticles according to any of the above embodiments or the pharmaceutical composition according to any of the above embodiments in preparing a drug.

[0494] In some embodiments, the above-mentioned medicament is used for the treatment and / or prevention of a disease.

[0495] In some embodiments, the above-mentioned medicament is used for gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.

[0496] In some embodiments, the lipid particles described above are used as carriers for transferring or delivering active ingredients. In some embodiments, the active ingredient is a pharmaceutical active ingredient, such as a nucleic acid.

[0497] In some embodiments, the above-mentioned drug is used to treat a disease corresponding to the above-mentioned polypeptide or protein of interest.

[0498] In some embodiments, the above-mentioned drugs are used to treat and / or prevent one or more of the following diseases: rare diseases, cancer, infectious diseases, autoimmune diseases, metabolic diseases, neurological diseases, cardiovascular diseases, transplant rejection, inflammatory response, genetic diseases and musculoskeletal diseases.

[0499] In some embodiments, the rare diseases include one or more of the following: osteogenesis imperfecta, Wilson disease, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome, amyotrophic lateral sclerosis (ALS), Duchenne type muscular dystrophy, Friedrich's ataxia, methylmalonic acidemia (MMA), cystic fibrosis (CF), glycogen storage disease 1a (GSD1a), glycogen storage disease III (GSDIII), Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, The following rare diseases are included in this article: 1) OTCD, 2) propionic acidemia (PA), 2) phenylketonuria (PKU), 3) hemophilia A, 4) hemophilia B, β-thalassemia, 5) Lafora disease, 6) Dravet syndrome (DS), 7) Alexander disease, 8) Leber's congenital amaurosis (LCA), 9) myelodysplastic syndrome (MDS), and 10) homocystinuria due to CBS deficiency. In addition, the rare diseases listed at www.orpha.net / consor / cgi-bin / Disease_Search_List.php and rarediseases.info.nih.gov / diseases are also included in this article.

[0500] In some embodiments, the cancers include one or more of the following: hematological malignancies, lung cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, brain cancer, prostate cancer, gallbladder cancer, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, bladder cancer, and melanoma;

[0501] In some embodiments, the infectious diseases include diseases caused by one or more infections selected from the group consisting of viruses, fungi, and bacteria.

[0502] In some embodiments, the autoimmune diseases include one or more of the following: acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, celiac disease, type 1 diabetes mellitus, and diffuse toxic goiter.

[0503] In some embodiments, the genetic diseases include one or more of the following: hemophilia, thalassemia, and Gaucher disease.

[0504] In some embodiments, the neurological diseases include one or more of the following: amyotrophic lateral sclerosis, Alzheimer's disease, and glioma.

[0505] In some embodiments, the above-mentioned drug is a vaccine.

[0506] In some embodiments, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA and DNA. In an alternative embodiment, the DNA is a plasmid. In an alternative embodiment, the RNA is one or more of the following: mRNA, antisense oligonucleotide, rRNA, miRNA, tRNA, siRNA, snRNA, shRNA, sgRNA, and Cas9 mRNA.

[0507] In addition, the present disclosure provides the use of the lipid nanoparticles of any of the above embodiments in preparing a carrier for transferring or delivering an active ingredient. In some embodiments, the active ingredient is a pharmaceutical active ingredient, such as a nucleic acid.

[0508] In addition, the present disclosure also provides a method for delivering the above-mentioned active ingredient to mammalian cells, the method comprising: administering to a mammal a lipid nanoparticle comprising an active ingredient according to any of the above-mentioned embodiments, wherein the administering comprises contacting the mammalian cells with the lipid nanoparticles, thereby delivering the active ingredient to the mammalian cells. In some embodiments, the active ingredient is a pharmaceutical active ingredient, such as a nucleic acid.

[0509] In some embodiments, the nucleic acid includes at least one of RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA.

[0510] In other embodiments, the active ingredient is a transfection reagent or a detection reagent.

[0511] In addition, the present disclosure also provides a drug comprising the PEG lipid according to any of the above embodiments, the lipid nanoparticle according to any of the above embodiments, or the pharmaceutical composition according to any of the above embodiments.

[0512] In some embodiments, the diseases or conditions that can be prevented or treated with the medicament are as described above.

[0513] In addition, the present disclosure also provides a drug prepared from the pharmaceutical composition according to any of the above embodiments.

[0514] In some embodiments, the diseases or conditions that can be prevented or treated with the medicament are as described above.

[0515] In addition, the present disclosure also provides a method for preventing or treating a disease or condition, comprising the step of administering the lipid nanoparticle, pharmaceutical composition or drug of the present invention to a subject in need thereof.

[0516] In some embodiments, the disease or condition to be prevented or treated is as described above.

[0517] The above-mentioned method for preventing or treating a disease has corresponding advantages due to the use of the above-mentioned lipid nanoparticles, pharmaceutical composition or drug.

[0518] Additionally, the present disclosure provides a method for reducing accelerated blood clearance (ABC) in a treated subject, comprising delivering a pharmaceutically active ingredient using lipid nanoparticles (LNPs) comprising the PEG lipid of any of the above embodiments.

[0519] In addition, the present disclosure also provides a method for delivering lipid nanoparticles (LNPs) encapsulating a pharmaceutically active ingredient to a subject without affecting subsequent doses of LNPs by promoting an immune response that accelerates blood clearance (ABC), the method comprising:

[0520] Providing LNPs encapsulating a pharmaceutically active ingredient, comprising the PEG lipid of any one of the above embodiments;

[0521] administering a first dose of LNPs to the subject, wherein the first dose of LNPs does not induce an immune response that promotes ABC upon administration of a second dose of LNPs; and

[0522] A second dose of LNPs is administered to the subject, wherein the subject does not have an ABC response to the second dose of LNPs. Example

[0523] In order to make the purpose and technical solutions of the present disclosure clearer, the following detailed description is given in conjunction with specific examples. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other implementation methods obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the manufacturer.

[0524] In the examples, "Hep" refers to n-heptane, "EA" refers to ethyl acetate, "DMF" refers to N,N-dimethylformamide, "TEA" refers to triethylamine, "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, "DMAP" refers to 4-dimethylaminopyridine, and "DCM" refers to dichloromethane.

[0525] Example 1 Synthesis of Compound 001

[0526] The first step is to synthesize compound 001-A1

[0527] Experimental process

[0528] To a 250 mL single-necked flask, add 1-hexadecanol (10.0 g, 41.25 mmol) and dissolve in DMF (100 mL). The mixture was cooled to below 10°C and slowly added with NaH (4.95 g, 123.75 mmol) in portions. After addition, the mixture was stirred for 30 minutes. Bromoacetic acid (6.88 g, 49.5 mmol) was slowly added to the reaction mixture. After addition, the mixture was stirred for 30 minutes. The mixture was then allowed to react at room temperature (25°C) for 15 hours. Water (200 mL) was added to the reaction mixture, and the pH was adjusted to 6.5-7.5 with dilute hydrochloric acid. The mixture was extracted twice with EA (200 mL). The EA phases were combined and washed with saturated brine (200 mL). The combined EA phases were collected and evaporated under reduced pressure. The concentrate was purified by silica gel column chromatography using Hep:EA = 3:1 as the eluent to afford compound 001-A1 as a white solid (4.5 g, 36.3% yield).

[0529] Step 2: Synthesis of Compound 001

[0530] Experimental process

[0531] To a 250 mL single-necked flask, add PEG2000 (19.96 g, 9.98 mmol) and DCM (100 mL). Dissolve the mixture by sonication. EDCI (1.91 g, 9.98 mmol), TEA (1.05 g, 9.98 mmol), and DMAP (0.24 g, 2.0 mmol) were then added. After stirring for 5 min, compound 001-A1 (2 g, 6.66 mmol) was added and allowed to react at room temperature for 15 h. Water (200 mL) was added to the reaction mixture, stirred for 10 min, and the lower organic phase was collected. The aqueous phase was extracted once with DCM (100 mL). The combined DCM phases were washed once with saturated brine (100 mL), and the DCM phase was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography using Hep:EA=15:1 as eluent. The product was then purified by reverse phase column chromatography using EtOH:H2O gradient elution (20% to 70%) to give compound 001 (800 mg) with a purity of 99.3% and a yield of 5.3%.

[0532] 1 H NMR(400MHz, CDCl3)δ4.31(m,2H),4.10(s,2H),3.76-3.57(m,184H),3.52(t ,J=6.7Hz,2H),1.67-1.55(m,2H),1.40-1.20(m,26H),0.88(t,J=6.8Hz,3H).

[0533] LC-MS (ESI): Mn=2277.

[0534] Example 2 Synthesis of Compound 003

[0535] The first step is to synthesize compound 003-A1

[0536] Experimental process

[0537] To a 250 mL single-necked flask, 1-octadecylamine (5 g, 18.55 mmol) was added and dissolved in DCM (100 mL). Saturated aqueous sodium bicarbonate (50 mL) was then added and the temperature was lowered to below 10°C. Triphosgene (2.2 g, 7.42 mmol) was added and allowed to react for 1 h. The organic phase was separated and collected, and the aqueous phase was extracted once with DCM (100 mL). The DCM phases were combined and washed once with saturated brine (100 mL). The DCM phase was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography using Hep:EA = 5:1 as the eluent to afford 4.64 g of compound 003-A1 as a solid, in an 84.6% yield.

[0538] Step 2: Synthesis of Compound 003

[0539] Experimental process

[0540] To a 250 mL single-necked flask, PEG2000 (30.46 g, 15.23 mmol) was added and dissolved in DMF (60 mL). Cuprous iodide (0.39 g, 2.03 mmol) was then added, followed by compound 003-A1 (3 g, 10.15 mmol). The mixture was allowed to react at room temperature for 3 h. The solid was removed by filtration, and water (200 mL) was added to the filtrate. The mixture was extracted twice with EA (100 mL). Ammonia was added dropwise with stirring to remove copper ions until the EA phase became colorless. The EA phases were combined, washed once with saturated brine (100 mL), and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography using a 5:1 ratio of Hep:EA. The resulting product was then purified by reverse-phase column chromatography using a 20% to 70% gradient of EtOH:H₂O to afford compound 003 (1.4 g) with a purity of 98.9% and a yield of 6.2%.

[0541] 1 H NMR (400MHz, CDCl3) δ4.25-4.16(m,2H),3.86-3.78(m,1H),3.77-3.53(m,184H) ,3.15(dd,J=13.4,6.8Hz,2H),1.47(m,2H),1.25(m,30H),0.88(t,J=6.8Hz,3H).

[0542] LC-MS (ESI): Mn=2230.

[0543] Example 3 Synthesis of Compound 006

[0544] The first step is to synthesize compound 006-A1

[0545] Experimental process

[0546] To a 100 mL single-necked flask, 1-hexadecanol (5 g, 20.62 mmol) was added and dissolved in DCM (50 mL). Pyridine (2.45 g, 30.39 mmol) and DMAP (0.76 g, 6.19 mmol) were then added and stirred at room temperature. p-Nitrophenyl chloroformate (4.99 g, 24.74 mmol) was then slowly added portionwise and stirred at room temperature for 3 h. Water (100 mL) was added, stirred, and the organic phase was collected. The aqueous phase was extracted with DCM (100 mL). The combined DCM phases were washed with saturated brine (100 mL) and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography using Hep:EA = 20:1 as the eluent to afford compound 006-A1 as a solid (8.1 g, 96.9% yield).

[0547] Step 2: Synthesis of Compound 006

[0548] Experimental process

[0549] PEG2000 (49.16 g, 24.58 mmol) was added to a 500 mL single-necked flask and dissolved in DCM (150 mL). Pyridine (4.9 g, 61.45 mmol) and DMAP (0.45 g, 3.69 mmol) were then added, followed by compound 006-A1 (5 g, 12.29 mmol). The mixture was allowed to react at room temperature for 3 days. Water (200 mL) was added to the reaction mixture, stirred, and the organic phase was separated and collected. The aqueous phase was extracted with DCM (100 mL). The combined organic phases were washed once with saturated brine (100 mL), collected, and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography using Hep:EA (15:1) as the eluent. The resulting product was then purified by reverse phase column chromatography using an EtOH:H2O gradient elution (20% to 70%) to afford compound 006 (9.3 g) with a purity of 99.1% and a yield of 31.7%.

[0550] 1 H NMR (600MHz, CDCl3) δ4.27-4.23(t,J=4.8Hz,2H),4.13-4.06(t,J=6.8Hz,2H),3.62(m, 184H),1.66-1.59(m,2H),1.36-1.29(m,2H),1.29-1.19(m,24H),0.85(t,J=6.8Hz,3H).

[0551] LC-MS (ESI): Mn=2156.

[0552] Example 4 Synthesis of Compound 002

[0553] Compound 002-A1 was synthesized by referring to the first step of Example 3, and then compound 002 was synthesized by using compound 002-A1 and referring to the second step of Example 3, wherein compound 002-A1 (2 g, 4.59 mmol) and PEG2000 (13.8 g, 6.89 mmol) were used to obtain compound 002 (1.67 g) with a purity of 98.3% and a yield of 15.6%.

[0554] 1 H NMR (400MHz, CDCl3) δ4.27(m,2H),4.12(t,J=6.8Hz,2H),3.65(d,J=7.7Hz,184H),1.70–1.59(m,2H),1.38-1.21(m,30H),0.88(t,J=6.8Hz,3H).

[0555] LC-MS (ESI): Mn = 2240

[0556] Example 5 Synthesis of Compound 004

[0557] Compound 004-A1 was synthesized by referring to the first step of Example 3, and then compound 004 was synthesized by using compound 004-A1 and referring to the second step of Example 3, wherein compound 004-A1 (1.5 g, 3.34 mmol) and PEG2000 (10.0 g, 5.00 mmol) were used to obtain compound 004 (2.3 g) with a purity of 98.8% and a yield of 30.14%.

[0558] 1 H NMR (400MHz, CDCl3) δ4.31-4.24(m,2H),4.12(t,J=6.8Hz,2H),3.65(m,184H),1.67-1.60(m,2H),1.25(m,32H),0.88(t,J=6.8Hz,3H).

[0559] LC-MS (ESI): Mn=2287.

[0560] Example 6 Synthesis of Compound 005

[0561] Compound 005-A1 was synthesized by referring to the first step of Example 3, and then compound 005 was synthesized by using compound 005-A1 and referring to the second step of Example 3, wherein compound 005-A1 (1.2 g, 2.59 mmol) and PEG2000 (7.76 g, 3.88 mmol) were used to obtain compound 005 (2.12 g) with a purity of 98.1% and a yield of 36.49%.

[0562] 1 H NMR (400MHz, CDCl3) δ4.27(m,2H),4.12(t,J=6.8Hz,2H),3.65(m,184H),1.63(m,2H),1.28(m,34H),0.88(t,J=6.8Hz,3H).

[0563] LC-MS (ESI): Mn=2243.

[0564] Example 7 Synthesis of Compound 013

[0565] Compound 013-A1 was synthesized by referring to the first step of Example 3, and then compound 013 was synthesized by using compound 013-A1 and referring to the second step of Example 3, wherein compound 013-A1 (1.6 g, 3.35 mmol) and PEG2000 (10.05 g, 5.02 mmol) were used to obtain compound 013 (2.9 g) with a purity of 98.5% and a yield of 39.7%.

[0566] 1 H NMR (400MHz, CDCl3) δ4.314.25(m,2H),4.12(t,J=6.8Hz,2H),3.65(m,184H),1.63(m,2H),1.25(m,36H),0.88(t,J=6.8Hz,3H).

[0567] LC-MS (ESI): Mn=2180.

[0568] Example 8 Synthesis of Compound 014

[0569] Compound 014-A1 was synthesized by referring to the first step of Example 3, and then compound 014-A1 was used to synthesize compound 014 by referring to the second step of Example 3, wherein compound 014-A1 (2 g, 4.07 mmol) and PEG2000 (12.2 g, 6.11 mmol) were used to obtain compound 014 (2.58 g) with a purity of 99.2% and a yield of 29.5%.

[0570] 1 H NMR (600MHz, CDCl3) δ4.26-4.19 (m, 2H), 4.15-4.09 (t, J = 6.8Hz, 2H), 3.77-3 .42(m,184H),1.65-1.55(m,2H),1.34-1.15(m,38H),0.83(t,J=6.8Hz,3H).

[0571] LC-MS (ESI): Mn=2150.

[0572] Example 9 Synthesis of Compound 015

[0573] Compound 015-A1 was synthesized by referring to the first step of Example 3, and then compound 015 was synthesized by using compound 015-A1 and referring to the second step of Example 3, wherein compound 015-A1 (2.1 g, 5.98 mmol) and PEG2000 (17.94 g, 8.96 mmol) were used to obtain compound 015 (2.68 g) with a purity of 98.2% and a yield of 20.1%.

[0574] 1 H NMR (600MHz, CDCl3) δ4.33-4.26(m,2H),4.14(t,J=6.8Hz,2H),3.87-3.58(m,184H),1.72-1.62(m,2H),1.41-1.24(m,18H),0.90(t,J=6.8Hz,3H).

[0575] LC-MS (ESI): Mn=2230.

[0576] Example 10 Synthesis of Compound 016

[0577] Compound 016-A1 was synthesized by referring to the first step of Example 3, and then compound 016 was synthesized by using compound 016-A1 and referring to the second step of Example 3, wherein compound 016-A1 (2.3 g, 6.06 mmol) and PEG2000 (18.2 g, 9.09 mmol) were used to obtain compound 016 (3.0 g) with a purity of 98.9% and a yield of 22.1%.

[0578] 1 H NMR (600MHz, CDCl3) δ4.31-4.26(m,2H),4.14(t,J=6.8Hz,2H),3.76-3.57(m,184H),1.72-1.64(m,2H),1.41-1.24(m,22H),0.90(t,J=6.8Hz,3H).

[0579] LC-MS (ESI): Mn=2240.

[0580] Example 11 Synthesis of Compound 007

[0581] Compound 007-A1 was synthesized by referring to the first step of Example 3, and then compound 007 was synthesized using compound 007-A1 and referring to the second step of Example 3, wherein compound 007-A1 (1.5 g, 3.56 mmol) and PEG2000 (28.48 g, 14.24 mmol) were used to obtain compound 007 (2.61 g) with a purity of 99.1% and a yield of 33.2%.

[0582] 1 H NMR (600MHz, CDCl3) δ4.29-4.23(t,J=4.8Hz,2H),4.13-4.07(t,J=6.8Hz,2H),3.62(m, 184H),1.65-1.59(m,2H),1.35-1.29(m,2H),1.29-1.19(m,28H),0.87(t,J=6.8Hz,3H).

[0583] LC-MS (ESI): Mn=2211.

[0584] Experimental Example 1. Preparation of lipid nanoparticles encapsulating human erythropoietin mRNA (hEPO mRNA)

[0585] (1) Ingredients:

[0586] A specified amount of hEPO mRNA stock solution, 0.2 M sodium acetate buffer, and DEPC water were added to a container and mixed to prepare an aqueous phase.

[0587] Cationic lipid (XI-1), helper lipid (DSPC), structural lipid (cholesterol), and the disclosed PEG lipid or control PEG lipid (DMG-PEG2000) were dissolved in anhydrous ethanol to prepare respective solutions at concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 25 mg / mL, respectively. The molar ratio of cationic lipid (XI-1): DSPC: CHO-HP: PEG lipid was 48:10:40.5:1.5. The four solutions were pipetted separately and mixed to prepare the alcohol phase.

[0588] (2) Encapsulation: The aqueous phase and alcohol phase were drawn into the aqueous phase and alcohol phase syringes at a flow rate of 9 mL / min and 3 mL / min, respectively, using a microfluidic preparation apparatus (MPE-L2). Encapsulation was performed at a flow rate of aqueous phase:alcohol phase = 12 mL / min:4 mL / min to obtain lipid nanoparticles encapsulating mRNA (mRNA-LNP).

[0589] (3) Dialysis: The product from step (2) was placed in a dialysis bag and placed in a Tris buffer-8% (m / v) sucrose solution for displacement to remove residual ethanol, unassembled lipids, and other components. Dialysis was performed at room temperature in the dark under magnetic stirring for 2 hours (dialysis solution was changed every hour).

[0590] (4) The product of step (3) is sterilized by passing it through a 0.22 μm microporous filter membrane, and then packaged to prepare a plurality of lipid nanoparticle preparations (LNP preparations) encapsulating hEPO mRNA, wherein the PEG lipid contained in each LNP preparation is different, the concentration of hEPO mRNA in each LNP preparation is 0.2 μg / μL, the molar ratio of hEPO mRNA:Lipid (cationic lipid (XI-1)) is 1:4, the particle size is 80 nm to 130 nm, and the encapsulation efficiency is greater than 85%.

[0591] Test Example 2. Detection of hEPO Concentration in Rat Serum after Multiple Administration

[0592] Animal Preparation: Sprague-Dawley rats, 5-7 weeks old, half male and half female, were housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with national health agency guidelines and animal ethics requirements.

[0593] In vivo delivery: Before injection of the test LNP formulation, gently invert the LNP formulation repeatedly to thoroughly mix the formulation sample. Use a 2 mL syringe to draw up the corresponding amount of LNP formulation and inject the LNP formulation via tail vein (IV) injection. Four to six rats, half male and half female, are injected with the corresponding LNP formulation encapsulating human erythropoietin mRNA (hEPO mRNA) prepared in Test Example 1 at a dose of 0.5 mg / kg mRNA. Dosing is continued once weekly for 4 to 6 times.

[0594] Whole blood was collected from rats 3 hours, 8 hours, and 24 hours after the first injection of the LNP formulation, and 8 hours after the second, third, fourth, fifth, and sixth injections of the LNP formulation. Serum was obtained by collecting whole blood from the rats and placing it in a tube without an anticoagulant. The blood was allowed to coagulate naturally at room temperature for 30 to 60 minutes. After the blood coagulated, it was centrifuged at 3500 rpm for 10 minutes to obtain the supernatant, which was the serum.

[0595] The specific method for detecting hEPO concentration in serum is as follows:

[0596] 1. Solution Preparation

[0597] 0.05% PBST Wash Solution: Measure 250 mL of 20× PBS, add water to 5 L, add 2.5 mL of Tween 20, mix well, and set aside; 3% BSA Blocking Solution: Weigh 3.0 g of BSA powder using an electronic balance, add 0.05% PBST to 100 mL, mix well, and set aside; 1% BSA Diluent: Weigh 1.0 g of BSA powder using an electronic balance, add 0.05% PBST to 100 mL, mix well, and set aside; Standard Curve Reagents: 1) Stock Solution Dilution: Take 2 μL of the stock solution (Cat. No.: EPO-H4214, Manufacturer: Acro) (initial concentration: 0.6 mg / mL) and add it to 998 μL of diluent, mix well, and the standard concentration is now 1.2 μg / mL; 2) Standard Curve Dilution: Prepare a 3-fold serial dilution.

[0598] 2.ELISA test:

[0599] (1) Coating: Dilute the coating antibody (catalog number: ab272358, manufacturer: Abcam) to a concentration of 1 μg / mL with PBS, 100 μL / well, and coat overnight at 4°C; (2) Washing: Wash the plate with 0.05% PBST solution, 250 μL / well, and wash three times; (3) Blocking: Block the plate with 0.05% PBST solution containing 3% BSA, 250 μL / well, and incubate at 37°C for 1 hour; (4) Washing: Wash the plate with 0. 0.05% PBST washing solution, 250 μL / well, wash 3 times; (5) Incubation of samples to be tested: add diluted standard curve and test sample according to the plate layout, 100 μL / well, incubate at 37℃ for 2h; (6) Washing plate: Use 0.05% PBST washing solution to wash the plate, 250 μL / well, wash 3 times; (7) Incubation of detector antibody: Use 1% BSA diluent to prepare 0.2 μg / mL detection antibody (biotin-labeled ), 100 μL / well, incubate at 37°C for 1 hour; (Cat. No.: ab272359, Manufacturer: Abcam); (8) Washing: Use 0.05% PBST washing solution to wash the plate, 250 μL / well, wash 3 times; (9) SA-HRP incubation: Use diluent 1:5000 dilution of detection secondary antibody (Cat. No.: 21126, Manufacturer: Invitrogen), 100 μL / well, incubate at 37°C for 45 minutes; (10) Washing: Use 0.05% PBST diluent to wash the plate, 250 μL / well, wash 3 times; (11) Color development: Add TMB (Cat. No.: PR1200, Manufacturer: Solarbio) for color development, 100 μL / well, incubate at 37°C for 5 min; (12) Stopping: Add stop solution, 50 μL / well; (13) Reading: Microplate reader OD450 and OD630; (14) Data analysis: OD450-OD630.

[0600] The results of serum hEPO concentration detection are shown in Figure 1. As can be seen from Figure 1, when multiple doses are administered, the serum hEPO concentration of rats administered with the LNP formulation containing DMG-PEG2000 gradually decreases with the increase in the number of doses, suggesting that the LNP formulation containing DMG-PEG2000 exhibits accelerated blood clearance (ABC). When multiple doses are administered, the serum hEPO concentration of rats administered with the LNP formulation containing the PEG lipids of the present disclosure does not gradually decrease with the increase in the number of doses, indicating that the LNP formulation containing the PEG lipids of the present disclosure can significantly reduce the accelerated blood clearance (ABC) phenomenon.

[0601] Test Example 3. Detection of anti-PEG IgG in the serum of rats after multiple administration

[0602] Serum acquisition: From the rats in Experimental Example 2, whole blood was collected 96 h or 120 h after each administration, and serum was obtained. The method for obtaining serum was as shown in Experimental Example 2.

[0603] The specific method for serum Anti-PEG IgG detection is as follows:

[0604] 1. Solution Preparation

[0605] 0.05% CHAPS wash solution: Take 200 mL of 20× PBS, add water to make up to 4 L, add 2 g of CHAPS, mix well, and set aside;

[0606] 2.3% BSA blocking solution: weigh 9.0 g BSA powder, add 0.05% PBS-CHAPS to 300 mL, mix well and set aside; 3.1% BSA diluent: weigh 1.0 g BSA powder, add 0.05% PBS-CHAPS to 100 mL, mix well and set aside;

[0607] 2.ELISA test

[0608] (1) Coating reagent: 0.1M sodium carbonate-sodium bicarbonate solution (pH = 9.51) dilute the coating reagent (PEG corresponding to the coating) to 10μg / mL, 100μL / well, 4℃ overnight; (2) Washing: Use 0.05% CHAPS washing solution (Product No.: ST1146-5g Manufacturer: Biyuntian) to wash the plate, 250μL / well, wash 3 times; (3) Blocking: Use 0.05% CHAPS washing solution with 3% BSA, 250 μL / well, incubate at 37℃ for 1.5h; (4) Washing: Wash the plate with 0.05% CHAPS washing solution, 250 μL / well, wash 3 times; (5) Incubate the sample to be tested: Add the diluted test sample according to the plate layout, 100 μL / well, incubate at 37℃ for 2h; (6) Washing: Wash the plate with 0.05% CHAPS washing solution, 250 μL / well, wash 3 times; (7) Rat-HRP incubation: 1:4000 1% BSA in Detection secondary antibody (catalog number: 62-9520 manufacturer: Invitrogen) diluted with 0.05% CHAPS washing solution, 100 μL / well, incubated at 37°C for 1 hour; (8) Washing: Wash the plate with 0.05% PBST diluent, 250 μL / well, wash three times; (9) Color development: Add TMB color development, 100 μL / well, incubate at 37°C for 10 minutes; (10) Stop: Add stop solution, 50 μL / well; (11) Read the plate: OD450 and OD630 on a microplate reader; (12) Data analysis: OD450-OD630.

[0609] The results of anti-PEG IgG detection in the serum 96 hours after the third administration are shown in Figure 2. As can be seen from Figure 2, compared with rats administered with the LNP formulation containing DMG-PEG2000, the anti-PEG IgG antibody titer in the serum of rats administered with the LNP formulation containing the PEG lipid of the present invention was extremely low, especially the LNP formulation containing 001, the LNP formulation containing 002, the LNP formulation containing 006, and the LNP formulation containing 007, the anti-PEG IgG antibody detection results in the serum of rats were basically negative, which suggests that LNPs containing the PEG lipid of the present invention can significantly reduce the production of anti-PEG IgG antibodies or do not produce anti-PEG IgG antibodies.

[0610] Test Example 4 Detection of cytokines IL-6 and IFN-γ in the serum of rats after administration

[0611] Serum collection: In the rats of Experimental Example 2, whole blood was collected 5 hours after the first administration to obtain serum using a kit (LEGENDplex TM The Rat Inflammation Panel (13-plex) w / VbP V02 (BIOLEGEND, cat# 741396) was used to detect the levels of IL-6 and IFN-γ in serum. The specific steps included:

[0612] 1. Prepare 1× Wash Buffer, standards, and beads according to the instructions provided in the kit.

[0613] 2. Sample addition and incubation: Add the standard and 4-fold diluted rat serum sample to the V-bottom plate, then add beads to each well and shake the V-bottom plate at 500 rpm on a microplate shaker. Incubate at room temperature in the dark for 2 h; after incubation, wash the plate with 1× Wash Buffer.

[0614] 3. Add detection antibodies: Add 25 μL of detection antibodies to each well of the V-bottom plate, shake the V-bottom plate at 500 rpm on a microplate shaker, and incubate at room temperature in the dark for 1 hour.

[0615] 4. After incubation, add 25 μL of SA-PE directly to each well, shake the V-bottom plate on a microplate shaker at 500 rpm, and incubate at room temperature in the dark for 30 minutes.

[0616] 5. After incubation, wash the plate once, then add 200 μL 1× Wash Buffer to resuspend and detect on a flow cytometer.

[0617] The results are shown in Figures 3 and 4. The LNP formulations prepared with PEG lipids of different structures induced significant differences in cytokine responses. When the linking groups connecting the polyethylene glycol moiety and the alkyl tail chain in the PEG lipids were different, the LNP formulations prepared with the corresponding PEG lipids induced significant differences in the levels of IL-6 or IFN-γ. For example, the LNP formulation prepared with PEG lipid 006 induced significantly lower levels of IFN-γ than the LNP formulation prepared with PEG lipid 001.

Claims

1. A PEG lipid as shown in formula (I): or a salt thereof, or a stereoisomer thereof, wherein R1 is H, HO-Z1-Q1-, or H-Z1-Q1- Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2-, or a bond Q1 is -C(=O)-, -OC(=O)-, or a bond Q2 is -C(=O)- or a bond Q3 is -C(=O)O-, -OC(=O)-, -O-, -S-, or a bond L1 is C1-C 20 a divalent hydrocarbon radical or a bond, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond R2 is a C4-C 24 hydrocarbyl group, The total number of carbon atoms of L1 and R2 is 10 to 35 L2 is a C1-C4 alkylene group L3 is a C1-C4 alkylene group R3 is H or a C1-C3 hydrocarbon group n is any integer from 30 to 60 2. PEG lipids as shown in formula (IX): or a salt thereof, or a stereoisomer thereof, wherein R1 is H, HO-Z1-Q1-, or H-Z1-Q1- Y is -C(=O)-, -C(=O)O-, -C(=O)NH-, -Q2-Z2-Q3-, or a bond Z1 and Z2 are each independently -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2-, or a bond Q1 is -C(=O)-, -OC(=O)-, or a bond Q2 is -C(=O)- or a bond Q3 is -C(=O)O-, -OC(=O)-, -O-, -S-, or a bond L1 is C1-C 20 alkylene group, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond R2 is a C4-C 24 hydrocarbyl group, The total number of carbon atoms of L1 and R2 is 10 to 35 L2 is a C1-C4 alkylene group L3 is a C1-C4 alkylene group R3 is H or a C1-C3 hydrocarbon group n is any integer from 30 to 60 L1 forms a four-membered, five-membered, or six-membered ring with A1 and R2 3. PEG lipids as shown in formula (X): or a salt thereof, or a stereoisomer thereof, wherein R1 is H, HO-Z1-Q1-, or H-Z1-Q1- Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, -L2-, or a bond Z2 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3-, or -L2- Q1 is -C(=O)-, -OC(=O)-, or a bond Q2 is -C(=O)- or a bond Q3 is -C(=O)O-, -OC(=O)-, -O-, -S-, or a bond L1 is C1 to C 20 an alkylene group or a bond, A1 is -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond R2 is a C4-C 24 hydrocarbyl group, The total number of carbon atoms of L1 and R2 is 10 to 35 L2 is a C1-C4 alkylene group L3 is a C1-C4 alkylene group R3 is H or a C1-C3 hydrocarbon group n is any integer from 30 to 60 Z2 forms a four-membered, five-membered or six-membered ring with Q3, L1, A1 and R2.

4. The PEG lipid according to any one of claims 1 to 3, wherein R1 is H, Preferably, R1 is H, 5. The PEG lipid according to claim 1 or 4, wherein the structure of the PEG lipid is as shown in formula (II): Among them, R1 is not H and is not an unsubstituted methyl group.

6. The PEG lipid according to claim 5, wherein (1) R1 is HO-Z1-Q1-, A1 is a bond, and Z1 and Q1 are not both bonds at the same time; Preferably, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3- or -L2-; More preferably, Z1 is -L2-CH(OR3)-L3-, -L2-C(=O)-L3-, -L2-CH(R3)-L3- or -L2-, and Q1 is -C(=O)-, -OC(=O)- or a bond, wherein L2 is preferably a C1-C3 alkylene group, L3 is preferably a C1-C3 alkylene group, and R3 is preferably H or a C1-C3 alkyl group; Preferably, R1 is Or (2) R1 is H-Z1-Q1-, wherein when Q1 is a bond, Z1 is not a bond and is not an unsubstituted methylene group; Preferably, R1 is H-Z1-Q1-, Z1 is -L2-, Q1 is -C(=O)-, and A1 is a bond, wherein L2 is preferably a C1-C3 alkylene group, more preferably a C1-C3 alkylidene group, More preferably, R1 is 7. The PEG lipid according to claim 1, wherein the PEG lipid is represented by formula (II-1): wherein A1 is -OC(=O)-, -OC(=O)O-, -O- or -S-; Preferably, the PEG lipid is represented by the formula (II-1-1):

8. The PEG lipid according to claim 1 or 4, wherein the PEG lipid has a structure represented by formula (III), formula (IV), formula (V), formula (VI) or formula (VII):

9. The PEG lipid according to any one of claims 1 and 4 to 8, wherein the PEG lipid has the following structure: wherein in the formula (II-2-3), formula (II-2-4), formula (VII-1) or formula (VII-2), R3 is preferably H or a C1-C3 alkyl group, more preferably H, a C1 alkyl group or a C2 alkyl group.

10. The PEG lipid according to claim 1 or 4, wherein the PEG lipid has the following structure:

11. The PEG lipid according to claim 10, wherein the PEG lipid has the following structure:

12. The PEG lipid according to any one of claims 1 to 11, wherein the total number of carbon atoms of L1 and R2 is 12 to 25.

13. The PEG lipid according to claim 12, wherein the total number of carbon atoms of L1 and R2 is 16 to 22.

14. The PEG lipid according to any one of claims 1 to 13, wherein L1 is a C1-C8 alkylene group or a bond.

15. The PEG lipid according to claim 14, wherein L1 is a C1-C8 alkylidene group, a C2-C8 alkenylene group or a C2-C8 alkynylene group.

16. The PEG lipid according to any one of claims 1 to 15, wherein R2 is a C7-C 22 hydrocarbyl group.

17. The PEG lipid according to claim 16, wherein R2 is a C7-C 22 alkyl group, a C7-C 22 alkenyl group or a C7-C 22 alkynyl group.

18. The PEG lipid according to any one of claims 1 to 17, wherein L1 is a linear C1-C8 alkylene group and R2 is a linear C7-C 22 alkyl group.

19. The PEG lipid according to any one of claims 10 to 18, wherein -L1-R2 is C 16 to C 22 alkyl.

20. The PEG lipid according to claim 19, wherein -L1-R2 is a straight-chain C 16 ~C 22 alkyl group.

21. The PEG lipid according to claim 19, wherein -L1-R2 is C 16 to C 18 alkyl.

22. The PEG lipid according to claim 21, wherein -L1-R2 is a straight-chain C 16 ~C 18 alkyl group.

23. The PEG lipid according to any one of claims 1 to 6, 8 to 10, 12 to 22, wherein L2 is a C1-C3 alkylene group.

24. The PEG lipid according to claim 23, wherein L2 is a C1-C3 alkylidene group, a C2-C3 alkenylene group or a C2-C3 alkynylene group.

25. The PEG lipid according to claim 24, wherein L2 is a straight-chain C1-C3 alkylidene group, preferably a C1 alkylidene group or a C2 alkylidene group.

26. The PEG lipid according to any one of claims 1 to 6, 8 to 10, 12 to 26, wherein L3 is a C1-C2 alkylene group.

27. The PEG lipid according to claim 26, wherein L3 is a C1-C2 alkylidene group, a C2 alkenylene group or a C2 alkynylene group.

28. The PEG lipid according to any one of claims 1 to 27, wherein n is any integer from 30 to 55.

29. The PEG lipid according to claim 28, wherein n is any integer from 35 to 50.

30. The PEG lipid according to claim 29, wherein n is any integer from 40 to 50.

31. The PEG lipid according to claim 30, wherein n is 45.

32. The PEG lipid according to claim 1, wherein the PEG lipid is:

33. Use of the PEG lipid according to any one of claims 1 to 32 in the preparation of lipid nanoparticles.

34. A lipid nanoparticle comprising the PEG lipid according to any one of claims 1 to 32.

35. The lipid nanoparticle according to claim 34, wherein the lipid nanoparticle further comprises an active ingredient; wherein the active ingredient is preferably a pharmaceutically active ingredient, more preferably a nucleic acid; Preferably, the lipid nanoparticle further comprises one or more of a cationic lipid, a co-lipid, a structural lipid, and a polymeric lipid.

36. A pharmaceutical composition comprising the lipid nanoparticle according to claim 34 or 35, and a pharmaceutically acceptable carrier.

37. Use of the lipid nanoparticle according to claim 34 or 35 or the pharmaceutical composition according to claim 36 in the preparation of a drug; Preferably, the drug is used for gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or therapy by interfering RNA; Preferably, the lipid particle is used as a carrier for transferring or delivering a pharmaceutically active ingredient; more preferably, the pharmaceutically active ingredient is a nucleic acid; Preferably, the drug is used for the treatment and / or prevention of diseases; Preferably, the drug is used for the treatment and / or prevention of one or more of the following diseases: rare diseases, cancers, infectious diseases, autoimmune diseases, metabolic diseases, neurological diseases, cardiovascular diseases, transplant rejection reactions, inflammatory reactions, genetic diseases, and musculoskeletal diseases; Preferably, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA.

38. Use of the lipid nanoparticle according to claim 34 or 35 in the preparation of a carrier for transferring or delivering an active ingredient; Preferably, the active ingredient is a pharmaceutically active ingredient; Preferably, the pharmaceutically active ingredient is a nucleic acid; Preferably, the nucleic acid comprises at least one of the following: RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA.

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