Lipid nanoparticle for delivering load

Abstract

Provided are a lipid nanoparticle for delivering a load, a composition comprising same, and use of the lipid nanoparticle and the composition in the preparation of a drug or vaccine for treating a disease, wherein the load is selected from any one or more of a small molecule compound, a polypeptide, a protein, and a nucleic acid molecule.

Description

Lipid nanoparticles for delivering payloads Technical Field

[0001] This application relates to the field of biomedicine, and more specifically to lipid nanoparticles for delivering payloads, compositions comprising the same, and the use of said lipid nanoparticles and compositions in the preparation of medicaments or vaccines for treating diseases, wherein said payload is selected from any one or more small molecule compounds, peptides, proteins, antibodies, or nucleic acid molecules. Background Technology

[0002] Gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate for gene defects or abnormalities within the cells, thereby achieving therapeutic goals. Over the past few decades, research into using gene therapy to treat clinical diseases has received increasing attention, especially in recent years with the FDA approval of siRNA-related drugs and mRNA vaccines for clinical treatment, further advancing research and investment in the field of gene therapy.

[0003] Nucleic acids are readily degraded in vivo by nucleases, and their negative charge makes them difficult to cross cell membranes and enter cells. Lipid nanoparticles (LNPs), as a nucleic acid delivery material, offer advantages such as simple preparation, good biodegradability, non-immunogenicity, and good safety, making them one of the most important nucleic acid delivery systems currently available. The main components of LNPs include ionizable lipid molecules, cholesterol, neutral lipids, and polymeric lipids (such as PEG lipids). Ionizable lipids are the core component of the LNP structure, and their molecular structure plays a crucial role in the overall delivery efficiency, targeting, and formulation stability of the lipid nanoparticles. The latter three components determine the structure and stability of the LNP.

[0004] In recent years, the rapid development of nucleic acid technology and lipid nanoparticle (LNP) delivery systems has made nucleic acid therapy a promising application for the prevention and treatment of various diseases, with a very broad range of applications, including vaccines, protein replacement therapy, tumor immunotherapy, cell reprogramming, and gene editing.

[0005] Building upon this, there has been an urgent need in the field for drugs and vaccines targeting tumors, infectious diseases, immune diseases, genetic diseases, and other diseases, as well as lipid nanoparticles for delivering such drugs and vaccines. However, despite extensive research on the targeted delivery of LNPs to different organs, it remains inconclusive which LNPs are best suited for delivering nucleic acid molecules or active pharmaceutical ingredients to specific organs or to organs in certain mammals, such as the liver. This invention explores this area and provides a feasible solution. Summary of the Invention

[0006] The inventors discovered that when using LNPs to deliver nucleic acid drugs to the liver, LNPs with excellent liver targeting in mice showed significantly reduced liver targeting in primates, while expression increased in the spleen, leading to a significant decrease in the liver / spleen ratio. To address this issue, the inventors, after further research, surprisingly discovered that by providing a specific LNP, it was possible to achieve higher expression in the liver and lower expression in the spleen of primates, thus enabling the specific expression of nucleic acid molecules in specific tissues or organs.

[0007] Therefore, the first aspect of this application provides a lipid nanoparticle comprising the following components in the molar percentage of the total lipids in the particle:

[0008] Ionizable lipids: about 25 mol% to about 75 mol%, preferably about 30 mol% to about 70 mol%, more preferably about 40 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, more preferably about 50 mol%;

[0009] Structural lipids: about 5 mol% to about 60 mol%, preferably about 10 mol% to about 60 mol%, more preferably about 20 mol% to about 50 mol%, more preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, more preferably about 37 mol%;

[0010] Neutral lipids: about 5 mol% to about 30 mol%, preferably about 5 mol% to about 25 mol%, more preferably about 5 mol% to about 20 mol%, more preferably about 8 mol% to about 20 mol%, more preferably about 8 mol% to about 15 mol%, more preferably about 10 mol%;

[0011] Polymer lipids: about 1.5 mol% to about 6 mol%, preferably about 1.5 mol% to about 5 mol%, more preferably about 2 mol% to about 4.5 mol%, more preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, more preferably about 3 mol%.

[0012] In some embodiments, this application provides lipid nanoparticles as described above, wherein the ionizable lipid is a compound of formula (II) or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof.

[0013] in,

[0014] a = 1, 2, 3, 4, 5 or 6;

[0015] b = 4, 5, 6, 7, 8, 9 or 10;

[0016] c = 1, 2, 3, 4, 5 or 6;

[0017] d = 0, 1, 2, 3 or 4;

[0018] c+d = 3, 4, 5, 6, 7, 8 or 9;

[0019] M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O )S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -SS-, and -S(O) 0-2 -;

[0020] R1 and R2 are independently selected from C 4-25 Alkyl, C 4-25 alkenyl and C 4-25 Alkyne group, which is optionally surrounded by one or more R groups 1s Substitution, and one or more methylene units therein are optionally and independently substituted with -NR'-;

[0021] R 1s Independently selected from H and C 1-20 Alkyl, -L c -OR c -L c -SR c and -L c -NR c R' c ;

[0022] R and R' are each independently selected from H and C. 1-20 alkyl;

[0023] L c Independently selected from chemical bonds and C 1-20 Alkylene;

[0024] R c and R' c Independently selected from H and C 1-20 Alkyl, C 3-14 cycloalkyl groups and 3 to 14-membered heterocyclic groups;

[0025] R4 and R5 are independently selected from C 1-8 Alkyl groups, which are optionally composed of one or more R groups 4s replace;

[0026] Or R4, R5, together with the carbon atoms they are attached to, form C. 3-14 Cycloalkyl or 3 to 14-membered heterocyclic group, optionally surrounded by one or more R 4s replace;

[0027] R 4s Independently selected from H, halogen, cyano, C 1-8 Alkyl, C 1-8 Halogenated alkyl groups, -L d -OR d -L d -SR d and -L d -NR d R' d ;

[0028] L d Independently selected from chemical bonds and C 1-8 Alkylene;

[0029] R d and R' d Independently selected from H and C 1-8 Alkyl, C 3-14 Cycloalkyl and 3 to 14-membered heterocyclic groups.

[0030] In some embodiments, this application provides lipid nanoparticles as described above, wherein M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably wherein M1 is -OC(O)- and M2 is -C(O)O-.

[0031] In some embodiments, this application provides lipid nanoparticles as described above, wherein R4 and R5 are independently C 1- 8-alkyl, preferably C 1-3 Alkyl, more preferably methyl; or

[0032] R4 and R5, together with the carbon atoms they are attached to, form C. 3-8 Cycloalkyl or 3 to 8-membered heterocyclic group, optionally surrounded by one or more R 4s Replace, where R 4s As defined in compound (II) above.

[0033] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 and R2 are independently selected from C 4-22 Alkyl groups, which are optionally composed of one or more R groups 1s Replace, where R 1s As defined in compound (II) above; preferably, R1 is C 8-12 Straight-chain alkyl with R2 being C12-22 Branched alkyl; more preferably, R1 is C 9-11 Straight-chain alkyl with R2 being C 15- 22 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 15-20 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 16 C 17 C 18 C 19 Or C 20 Branched alkyl groups.

[0034] In some embodiments, this application provides lipid nanoparticles as described above, wherein a is an integer from 1 to 4, such as 1, 2, 3 or 4; b is an integer from 5 to 8, such as 5, 6, 7 or 8; c is an integer from 2 to 6, such as 2, 3, 4, 5 or 6; and d is an integer from 1 to 4, such as 1, 2, 3 or 4.

[0035] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) has the structure of formula (II-1):

[0036] R1, R2, a, b, c, and d are defined as above.

[0037] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) has the structure of formula (II-2):

[0038] R1, R2, a, b, c, and d are defined as above.

[0039] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is nonyl or decyl, and R2 is selected from one of the following structures:

[0040] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) is selected from the following compounds or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers:

[0041] In some embodiments, this application provides lipid nanoparticles as described above, wherein the structural lipid is a steroid selected from one or more of the following: cholesterol, sitosterol, coccosterol, lycopene, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, and campesterol; preferably, the structural lipid is selected from cholesterol and / or β-sitosterol; more preferably, the structural lipid is cholesterol.

[0042] In some embodiments, this application provides lipid nanoparticles as described above, wherein the neutral lipid is a phospholipid selected from phosphatidylcholine and / or phosphatidylethanolamine; preferably, the phospholipid is selected from one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMP). C), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonium)ethyl hydrogen phosphate (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and dipalmitoylphosphatidylethanolamine (DPPE) and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol)sodium salt (DOPG), sphingomyelin and mixtures thereof, more preferably DSPC and / or DOPE.

[0043] In some embodiments, this application provides lipid nanoparticles as described above, wherein the polymeric lipid is a polyethylene glycol-modified lipid;

[0044] Preferably, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

[0045] Preferably, the PEGylated lipid contains a PEG portion of 1000 Da to 20 kDa, and more preferably contains a PEG portion of about 1000 Da to about 5000 Da;

[0046] Preferably, the PEGylated lipid is selected from one or more of the following: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000, DSPE-PEG2000-Mannose, DSPE-PEG2000 The amine, DSPE-PEG3350, DSPE-PEG3500, DMG-PEG3500, DPPE-PEG3500, DSPE-PEG4000, DMG-PEG4000, Ceramide-PEG5000, DSPE-PEG5000, DMG-PEG5000 and ALC-0159, preferably ALC-0159, DMG-PEG2000, DSPE-PEG2000, DMG-PEG3500, DMG-PEG4000 and / or DMG-PEG5000, more preferably DMG-PEG2000.

[0047] In some embodiments, this application provides lipid nanoparticles as described above, comprising the following molar percentages of components:

[0048] Ionizable lipids: about 40 mol% to about 60 mol%, preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, even more preferably about 50 mol%;

[0049] Structural lipids: about 20 mol% to about 50 mol%, preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, even more preferably about 37 mol%, or even more preferably about 37.5 mol%;

[0050] Neutral lipids: about 5 mol% to about 20 mol%, preferably about 8 mol% to about 15 mol%, more preferably about 8 mol% to about 12 mol%, even more preferably about 10 mol%; and

[0051] Polymer lipids: about 1.5 mol% to about 5 mol%, preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, more preferably about 2.2 mol% to about 3.2 mol%, more preferably about 2.5 mol% to about 3 mol%, more preferably about 2.5 mol%, or more preferably about 3 mol%.

[0052] In a preferred embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0053] Ionizable lipids: approximately 50 mol%;

[0054] Structural lipids: approximately 37 mol%;

[0055] Neutral lipids: approximately 10 mol%; and

[0056] Polymer lipids: approximately 3 mol%.

[0057] In a preferred embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0058] Ionizable lipids: approximately 50 mol%;

[0059] Structural lipids: approximately 37.5 mol%;

[0060] Neutral lipids: approximately 10 mol%; and

[0061] Polymer lipids: approximately 2.5 mol%.

[0062] In some embodiments, this application provides lipid nanoparticles as described above, which further comprise at least one load selected from therapeutic agents, preventive agents, or diagnostic agents;

[0063] Preferably, the therapeutic agent, preventive agent, or diagnostic agent is selected from any one or more of the following: small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules;

[0064] More preferably, the small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules are small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules used for the prevention, treatment, or diagnosis of the following diseases: tumor diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, or autoimmune diseases; or liver diseases or liver-related diseases, preferably malignant tumors of the liver.

[0065] In some embodiments, this application provides lipid nanoparticles as described above, wherein the nucleic acid molecule is selected from antisense oligonucleotides (ASO), RNA, or DNA;

[0066] Preferably, the RNA is selected from one or more of the following: interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozymes; more preferably, the RNA is selected from one or more of the following: messenger RNA (mRNA), modified mRNA (mmRNA), circular RNA (circRNA), siRNA, gRNA, and self-replicating RNA (SrRNA), preferably mRNA or modified mRNA, siRNA, or gRNA, more preferably modified mRNA; or

[0067] Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and more preferably from one or more of plasmid DNA (pDNA), microcircular DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multicopy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); more preferably, the DNA is selected from at least one of plasmid DNA (pDNA) and microcircular DNA (mcDNA).

[0068] In some embodiments, this application provides lipid nanoparticles as described above, wherein the N:P molar ratio of N atoms in the ionizable lipid to P atoms in the loaded molecule is (1-15):1, preferably (2-12):1, and more preferably (2-9):1;

[0069] Preferably, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the loaded molecule is (2-15):1, more preferably (2-12):1, more preferably (2-10):1, more preferably (2-8):1, and even more preferably (2-6):1;

[0070] Preferably, the N:P molar ratio of the N atom in the ionizable lipid to the P atom in the loaded molecule is (1-12):1, more preferably (4-11):1, more preferably (4-10):1, and even more preferably (4-9):1.

[0071] In some embodiments, this application provides lipid nanoparticles as described above, wherein the particle size of the lipid nanoparticles is 30-500 nm, such as 30-300 nm, 30-275 nm, 30-250 nm, 30-225 nm, 30-200 nm, 30-175 nm, 30-150 nm, 30-120 nm, 30-100 nm, 40-300 nm, 40-275 nm, 40-250 nm, 40-225 nm, 40-200 nm, 40-175 nm, 40-150 nm, 40-120 nm or 40-100 nm, preferably 40-80 nm, more preferably 50-70 nm.

[0072] On the other hand, this application provides a method for preparing lipid nanoparticles as described above, comprising: mixing the lipid components in the lipid nanoparticles and then mixing them with a loading device to obtain lipid nanoparticles.

[0073] In another aspect, this application provides a composition comprising the lipid nanoparticles as described above.

[0074] On the other hand, this application provides a pharmaceutical composition comprising the lipid nanoparticles as described above and optionally pharmaceutically acceptable excipients, such as carriers, adjuvants, or mediators.

[0075] On the other hand, this application provides an immunogenic composition comprising lipid nanoparticles as described above and optionally pharmaceutically acceptable excipients, such as carriers, adjuvants, or mediators.

[0076] On the other hand, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above for the delivery of a load, preferably for the use of specifically delivering a load to the liver.

[0077] On the other hand, this application provides a method for delivering a payload to a subject, comprising administering to the subject the lipid nanoparticles or composition or pharmaceutical composition or immunogenic composition as described above; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection.

[0078] On the other hand, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above in the preparation of products for gene editing, protein replacement and / or supplementation, and gene interference.

[0079] On the other hand, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above in the preparation of a medicament for delivery of a payload.

[0080] On the other hand, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above in the preparation of medicaments or vaccines for the prevention, treatment or diagnosis of diseases.

[0081] On the other hand, this application provides a method for preventing, treating, or diagnosing a disease in a subject, comprising administering to the subject an effective amount of the lipid nanoparticles or composition or pharmaceutical composition or immunogenic composition as described above; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection.

[0082] In some embodiments of the uses and methods described above, the diseases include tumor diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, or autoimmune diseases.

[0083] In some embodiments of the uses and methods described above, the disease includes liver disease or liver-related disease; preferably, the liver disease or liver-related disease is a malignant tumor of the liver.

[0084] In some embodiments of the uses and methods described above, the subject is a mammal or human, preferably a primate such as a monkey, ape, orangutan, chimpanzee, or gorilla, such as a cynomolgus monkey or rhesus monkey, and more preferably a human.

[0085] On the other hand, this application provides a kit containing the lipid nanoparticles or composition, pharmaceutical composition or immunogenic composition as described above, and optionally other drugs or vaccines.

[0086] In some embodiments, the other drugs or vaccines are selected from one or more immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, or one or more antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies that target tumor antigens, or one or more drugs used to prevent or treat infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections.

[0087] In some embodiments, the viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, or fungi treated by the drug for the prevention or treatment of infectious diseases are selected from the Catalogue of Human Infectious Pathogens (2023 Edition) published on the website of the National Health Commission of the People's Republic of China in 2023, the entire contents of which are incorporated herein by reference.

[0088] In some implementations, the drug used to prevent or treat viral infection is an anti-SARS-CoV-2 drug.

[0089] In some implementations, the anti-SARS-CoV-2 drug may be any directly acting anti-SARS-CoV-2 drug, including but not limited to remdesivir (Gilead), REGN-COV2 (Regeneron), LY-CoV555 (Lilly), MK-4482 / EIDD-2801 (Merck / Ridgeback Bio); or immunomodulators, including but not limited to CD24Fc (Oncoimmune), T-COVIDTM (Altimmune), itutuzumab (Equillium Inc); or vaccines, including but not limited to AdCOVIDTM (Altimmune), BNT162b1 / 2 (Pfizer / BioNTech), mRNA-1273 (Moderna), AZD1222 / ChAdOx1 (AstraZeneca / Oxford Univ), Ad5-vectored COVID-19 vaccine (CanSino Biologics), CoronaVac (Sinovac), NVX-CoV2373 (Novavax).

[0090] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions, immunogenic compositions, or methods of this application may be used in combination with immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, one or more antibodies targeting tumor-specific antigens, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies.

[0091] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions of this application may also be combined with radiotherapy, for example, including the administration of ionizing radiation to a subject earlier, during, and / or later than the administration of the nucleic acid molecules, lipid nanoparticles (LNPs) or compositions, pharmaceutical compositions or immunogenic compositions of this application.

[0092] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions of this application may be used in combination with drugs for the prevention or treatment of diseases caused by infections of viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes or fungi, wherein the viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes or fungi are selected from the "Catalogue of Human Infectious Pathogenic Microorganisms" (2023 Edition) published on the website of the National Health Commission of the People's Republic of China in 2023, the entire contents of which are incorporated herein by reference.

[0093] GPC3 (Glypican-3) is a cell surface glycoprotein primarily involved in cell proliferation, differentiation, migration, and apoptosis. GPC3 is expressed in the liver and kidneys of fetuses, but is almost entirely absent in healthy human tissues. However, it is highly expressed in 70-80% of hepatocellular carcinoma (HCC) tissues, and also expressed in small amounts in other tumors such as ovarian clear cell carcinoma, melanoma, and lung squamous cell carcinoma. Although the function of GPC3 is not fully understood, numerous studies have shown that it is closely related to the occurrence, metastasis, and prognosis of HCC. Therefore, GPC3 has become a novel target for the diagnosis and immunotherapy of HCC.

[0094] In some embodiments, the lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application can be combined with immunotherapies targeting tumor antigens such as GPC3 protein. Immunotherapies targeting tumor antigens such as GPC3 protein mainly include monoclonal antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies.

[0095] The actual dose level of the active ingredient in the pharmaceutical or immunogenic compositions of this application may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response to a specific subject, composition, and route of administration, without toxicity to the subject. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this application, route of administration, time of administration, excretion rate of the specific compound applied, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the subject being treated, and similar factors known in the medical field.

[0096] An "effective amount" of the nucleic acid molecules, lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumors, an "effective amount" of the nucleic acid molecules, lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system for predicting the efficacy of treatment against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by tests known to those skilled in the art. An effective amount of the lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application is capable of reducing tumor size or otherwise alleviating symptoms in subjects, such as preventing and / or treating metastasis or recurrence. Those skilled in the art can determine this amount based on factors such as the subject's body size, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0097] The lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the term "parenteral administration" refers to a mode of administration other than gastrointestinal and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0098] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions of this application are administered parenterally, such as by intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection.

[0099] definition

[0100] Chemical definition

[0101] The definitions of specific functional groups and chemical terms are described in more detail below.

[0102] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 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 alkyl.

[0103] The term "alkyl" refers to the straight-chain or branched saturated hydrocarbon moiety. "C 1-25 Alkyl", C 1-22 Alkyl or C 1-20 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 25, 1 to 22, or 1 to 20 carbon atoms. In some embodiments, the straight-chain or branched C... 4-25 Alkyl, C 4-22 Alkyl, C 4-20 Alkyl, C 8-20 Alkyl, C 6-14 Alkyl, C 7-12 Alkyl, C 8-12 Alkyl, C 12-20 Alkyl, C 12-22 Alkyl, C 15-22 Alkyl, C 15-20 Alkyl, C 16-20 Alkyl, C 4-10 Alkyl, C 7-11 Alkyl, C 8-11 Alkyl, C 8-10 Alkyl, C 9-10 Alkyl, C 9-11 Alkyl, C 8-9 Alkyl, C 4-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C9 alkyl, C 2-8 Alkyl, C 5-8 Alkyl, C 7-8 Alkyl, C 4-6 Alkyl, C 1-20 Alkyl, C 1-14 Alkyl, C 2-14 Alkyl, C 1-13 Alkyl, C 1- 12 Alkyl, C1-10 Alkyl, C 1-9 Alkyl, C 1-8 Alkyl, C 1-7 Alkyl, C 2-7 Alkyl, C 1-6 Alkyl, C 2-6 Alkyl, C 1-5 Alkyl, C5 alkyl, C 1-4 Alkyl, C 2-4 Alkyl, C 1-3 Alkyl, C 2-3 Alkyl, C 1-2 Alkyl groups and Me are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2). In some embodiments, the alkyl group is preferably a straight-chain alkyl group.

[0104] The term "alkenyl" refers to a straight-chain or branched hydrocarbon moiety having one or more carbon-carbon double bonds. 4-25 "alkenyl", "C" 4-22 "Alkenyl" or "C" 4-20 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 4 to 25, 4 to 22, or 4 to 20 carbon atoms and at least one carbon-carbon double bond. In some embodiments, the straight-chain or branched C... 4-22 alkenyl, C 4-20 alkenyl, C 2-13 alkenyl, C 4- 14 alkenyl, C 6-14 alkenyl, C 4-10 alkenyl, C 2-10 alkenyl, C 2-9 alkenyl, C 2-6 alkenyl and C 2-4 Alkenyl groups are preferred. C 2-6Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0105] The term "alkynyl" refers to a straight-chain or branched hydrocarbon moiety having one or more carbon-carbon triple bonds. 4-25 "Alkyne", "C" 4-22 "Alkyne" or "C" 4-20 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 4 to 25, 4 to 22, or 4 to 20 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, the straight-chain or branched C... 4-22 alkynyl group, C 4-20 alkynyl group, C 2-13 alkynyl group, C 4-14 alkynyl group, C 6-14 alkynyl group, C 4-10 alkynyl group, C 2-10 alkynyl group, C 2-9 alkynyl group, C 2-6 alkynyl group and C 2-4 The alkynyl group is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0106] The term "alkylene" refers to a divalent group formed by removing one hydrogen atom from an alkyl group; it can be substituted or unsubstituted. "C" 1-20 "Alkylene" refers to the removal of C 1-20 The alkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. In some embodiments, C 4-20 Alkylene, C 6-14 Alkylene, C 7-12 Alkylene, C 8-12 Alkylene, C4-12 Alkylene, C 4-10 Alkylene, C 7-11 Alkylene, C 8-11 Alkylene, C 8-10 Alkylene, C 9-10 Alkylene, C 8-9 Alkylene, C 4-9 Alkylene, C 6-9 Alkylene, C 7-9 Alkylene, C9 alkylene, C 2-8 Alkylene, C 5-8 Alkylene, C 7-8 Alkylene, C 4-6 Alkylene, C 1-20 Alkylene, C 1-14 Alkylene, C 2-14 Alkylene, C 1-13 Alkylene, C 1-12 Alkylene, C 1-10 Alkylene, C 1- 9-alkylene, C 1-8 Alkylene, C 1-7 Alkylene, C 2-7 Alkylene, C 1-6 Alkylene, C 2-6 Alkylene, C 1-5 Alkylene, C5 alkylene, C 1-4 Alkylene, C 2-4 Alkylene, C 1-3 Alkylene, C 2-3 Alkylene, C 1-2 Alkylenes and methylene groups are preferred. Unsubstituted alkylenes include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2-), and so on. Exemplary substituted alkylenes, for example, those substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylidenes (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0107] “C 0-6 "alkylene" refers to chemical bonds and the aforementioned "C" 1-6 Alkylene", "C" 0-4 "alkylene" refers to chemical bonds and the aforementioned "C" 1-4 Alkylene".

[0108] The term "the total length of variables A and B is x carbon atoms" means that the sum of the number of carbon atoms in the main chain of the group represented by variable A and the number of carbon atoms in the main chain of the group represented by variable B is x.

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

[0110] Therefore, "C" 1-10 "Halogenated alkyl" refers to the above "C 1-10 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-8 Haloalkyl, C 1-6 Haloalkyl, C 1-4 Haloalkyl, C 1-3 Haloalkyl groups are particularly preferred, and C4 groups are more preferred. 1-2 Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halide group can be substituted at any available junction, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0111] “C 3-14"Cycloalkyl" or "3- to 14-membered cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 14 ring carbon atoms and zero heteroatoms, optionally containing 1, 2, or 3 double or triple bonds. In some embodiments, 3- to 10-membered cycloalkyl, 5- to 10-membered cycloalkyl, 3- to 8-membered cycloalkyl, 3- to 7-membered cycloalkyl, and 3- to 6-membered cycloalkyl are particularly preferred, more preferably 5- to 7-membered cycloalkyl, 4- to 6-membered cycloalkyl, 3- to 5-membered cycloalkyl, 3- to 4-membered cycloalkyl, and 5- to 6-membered cycloalkyl, more preferably 5-membered cycloalkyl, more preferably 6-membered cycloalkyl, and more preferably cyclopropyl. Cycloalkyl also includes a cyclic system in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the cycloalkyl ring, and in such cases, the number of carbons continues to indicate the number of carbons in the cycloalkyl system. Cycloalkyl also includes a cyclic system in which the aforementioned cycloalkyl ring, Substituents on any non-adjacent carbon atoms are linked together to form a bridged ring, forming a polycyclic alkane sharing two or more carbon atoms. Cycloalkyl groups also include the aforementioned cycloalkyl rings, where substituents on the same carbon atom are linked together to form a ring, forming a polycyclic alkane sharing one carbon atom. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0112] “C 3-14 "Cycloalkylene" refers to the alkylene oxide that has had C removed. 3-14 The cycloalkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. In some embodiments, C 3-10 Cycloalkylene, C 3-7 Cycloalkylene, C 3-6 Cycloalkylene, C 3-5 Cycloalkylene and C 3-4 Cycloalkylene compounds are particularly preferred.

[0113] "3-14 membered heterocyclic group" or "3 to 14 membered heterocyclic group" refers to a saturated or unsaturated group having a 3 to 14 membered non-aromatic ring system with a cyclic carbon atom and 1 to 5 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, and optionally contains 1, 2, or 3 double or triple bonds. In heterocyclic groups containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. In some embodiments, a 3- to 10-membered heterocyclic group is preferred, which is a 3- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms; in some embodiments, a 5- to 10-membered heterocyclic group is preferred, which is a 5- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms; in some embodiments, a 3- to 8-membered heterocyclic group is preferred, which is a 3- to 8-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; in some embodiments, a 3- to 7-membered heterocyclic group is preferred, which is a 3- to 7-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; preferably, a 5- to 7-membered heterocyclic group is preferred, which is a 3- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms. A 5- to 7-membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; preferably a 3- to 6-membered heterocyclic group having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; preferably a 4- to 6-membered heterocyclic group having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; more preferably a 5- to 6-membered heterocyclic group having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; preferably a 5-membered heterocyclic group having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; preferably a 6-membered heterocyclic group having a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include ring systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl groups, wherein the linking point is on the heterocyclic ring; or ring systems in which the aforementioned heterocyclic ring is fused with one or more aryl or heteroaryl groups, wherein the linking point is on the heterocyclic ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Heterocyclic groups also include the aforementioned heterocyclic ring in which substituents on any non-adjacent carbon or nitrogen atom are linked to form a bridged ring, together forming a polycyclic heteroalkane sharing two or more carbon or nitrogen atoms. Heterocyclic groups also include the aforementioned heterocyclic ring in which substituents on the same carbon atom are linked to form a ring, together forming a polycyclic heteroalkane sharing one carbon atom. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirropropyl, oxetane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirrobutyl, oxetane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione.Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: pyrazolyl, dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinoneyl, etc. Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. Heterocyclic groups also include those that share one or two atoms with a cycloalkyl, heterocyclic, aryl, or heteroaryl group to form a bridged or spirocyclic ring, wherein the shared atom may be a carbon or nitrogen atom, provided that the valence allows. Heterocyclic groups also include those that can be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0114] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0115] "5-14 membered heteroaryl" or "5 to 14 membered heteroaryl" refers to a 4n+2 aromatic ring system of a 5-14 membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms (e.g., having 6, 10, or 14 shared π electrons arranged in a ring), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryls containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryls also include ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryls are preferred, which are 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring systems having a ring carbon atom and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 4n+2 aromatic ring systems of 5-6 membered monocyclic or bicyclic rings having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridinyl or pyridoneyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirmonoheptatrienyl, oxadiazinyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazoleyl, benzotriazolyl, benzothiopheneyl, isobenzothiopheneyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxolinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0116] "Optionally replaced by..." means that it can be replaced by a specified substituent or not replaced.

[0117] The divalent groups formed by removing one hydrogen atom from the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups defined above are collectively referred to as "subunits". Cyclic groups such as cycloalkyl, heterocyclic, aryl, and heteroaryl are collectively referred to as "cyclogroups".

[0118] The alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups defined in this article are optional substituted groups.

[0119] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa -C(=O)N(R) bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa -NR bb CO2R aa -NR bb C(=O)N(R bb )2、-C(=NR bb )R aa -C(=NR) bb OR aa -OC(=NR) bb )R aa -OC(=NR) bb OR aa -C(=NR) bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa-NR bb SO2R aa -SO2N(R) bb )2、-SO2R aa -SO2OR aa -OSO2R aa -S(=O)R aa -OS(=O)R aa 、-Si(R aa 3. -OSi(R) aa 3. -C(=S)N(R) bb )2、-C(=O)SR aa -C(=S)SR aa -SC(=S)SR aa -SC(=O)SR aa -OC(=O)SR aa -SC(=O)OR aa -SC(=O)R aa -P(=O)2R aa -OP(=O)2R aa -P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0120] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R)bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0121] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0122] R bb Each is independently selected from: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0123] Rcc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0124] R dd Each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2Ree -S(=O)R ee 、-Si(R ee 3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. Alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently marked by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can combine to form =O or =S;

[0125] R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0126] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R ff The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0127] R gg Each of these is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C) 1- 6-alkyl)2、-N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - -NH(C 1-6 Alkyl)2 + X - -NH2(C 1-6 alkyl) + X --NH3 + X - -N(OC) 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6 Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6 Alkyl)2、-OC(=O)NH(C 1-6 Alkyl), -NHC(=O)(C 1-6 alkyl), -N(C) 1-6 Alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 Alkyl), -NHC(=O)N(C 1-6 Alkyl)2、-NHC(=O)NH(C 1-6 Alkyl groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl), -OC (=NH)(C 1-6 Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2、-SO2NH(C 1-6 Alkyl groups, -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1- 6-alkyl, -Si(C) 1-6 Alkyl)3、-OSi(C 1-6 Alkyl)3, -C(=S)N(C 1-6 Alkyl)2、C(=S)NH(C1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC (=S)SC 1-6 Alkyl group, -P(=O)2(C 1-6 Alkyl), -P(=O)(C 1-6 Alkyl)2、-OP(=O)(C 1-6 Alkyl)2、-OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C7 cycloalkyl, C6-C 10 aryl, 3-7 membered heterocyclic, 5-10 membered heteroaryl; or two geminal Rs gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0128] Exemplary substituents on the nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. ccThe groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0129] "Bifurcation carbon atom" can be understood as the carbon atom marked with "*" in the following structure:

[0130] The term "branched carbon atom of a branched alkyl group separated from M2 by XX carbon atoms" refers to the sum of the number of carbon atoms (including N atoms replaced by -NR'-) between the branched carbon atom and M2. Other cases follow the same logic, for example:

[0131] In this compound, the branched carbon atom is separated from M2 by one carbon atom.

[0132] "Nucleic acid" or "nucleic acid molecule" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and their hybrid molecules. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASO). Nucleic acids can be further chemically modified, with modifications selected from one or a combination of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methylcytosine. mRNA molecules contain protein-coding regions and may further contain expression regulatory sequences, typical of which include, but are not limited to, a 5' cap (5'cap), a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), a polyadenylated nucleotide sequence (PolyA), and a miRNA binding site.

[0133] "Ionizable lipids" refers to lipids that are ionizable, allowing them to exist in a positively charged or neutral form depending on pH. In some embodiments, ionizable lipids are aminolipids.

[0134] "Neutral lipids" refer to lipid molecules that are uncharged under specific pH conditions, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), monomethylphosphatidylethanolamine, and dimethylphosphatidylethanolamine. Methylphosphatidylethanolamine, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine or 1,2-dioleoyl-sn-glycero-3-phosphate-L-serine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylglycerol (DMPG), distearylphosphatidylglycerol (DSPG), rutinoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphate ethanolamine (DLPE), 1,2-diphydanoyl-sn-glycero-3-phosphate ethanolamine (DPHyPE), lecithin, sphingomyelin, and any two or more mixtures thereof.

[0135] "Structural lipids" refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentane and polyhydrophenanthrene carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coccosterol, rock saponin, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol.

[0136] "Polymer lipids" refer to molecules containing both polymer and lipid moieties. In some embodiments, polymer lipids are polyethylene glycol (PEG)-modified lipids. Other lipids that can reduce aggregation, such as products of lipid coupling with compounds having uncharged, hydrophilic, or sterically barrier moieties, may also be used.

[0137] "Lipid nanoparticles" refer to particles containing lipid components and having a nanoscale size.

[0138] "Biodegradable groups" refer to functional groups containing biodegradable bonds, such as esters, disulfide bonds, and amides. Biodegradation can affect the process of clearing compounds from the body. The orientation of the biodegradable groups in this application is from the head to the tail of the ionizable lipid molecule.

[0139] "Pharmaceutically acceptable excipients" refer to non-toxic carriers, adjuvants, or mediators that do not impair the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants, or mediators that can be used in the compositions of this invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acid metaglycerides, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin, etc.

[0140] Other definitions

[0141] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0142] As used herein, the terms “comprising,” “having,” and “including” are open-ended, meaning that all elements, components, steps, etc., that are expressly stated thereafter are included in the scheme, but do not exclude other elements, components, steps, etc. that are not expressly stated.

[0143] As used herein, the term “about” means approximately, roughly, roughly, or generally. When the term “about” is used with a numerical range, it modifies the range by extending the boundaries to be above and below the stated numerical value, typically including all values ​​within the range of +20%, preferably +10%, more preferably +5% of the stated value. For example, a value of about 100 means any value in the range of 80-120, preferably any value in the range of 90-110, more preferably any value in the range of 95-105.

[0144] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0145] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this application to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0146] When the term “comprising” is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activities described in this application.

[0147] Sequence identity between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the online alignment tool EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm with default parameters. Sequence identity can be along the full length of a given sequence.

[0148] The terms “mRNA” or “messenger RNA” are used interchangeably in this article and refer to single-stranded ribonucleic acid molecules that carry genetic information and guide protein synthesis within cells. mRNA typically includes a 5' UTR, a protein-coding sequence (CDS), and a 3' UTR. mRNA may also include a poly(A) sequence at the 3' end and a 5' cap. mRNA is generally linear; however, the term can also encompass covalently closed circular RNA molecules.

[0149] As used herein, the "5'UTR" generally refers to the sequence from the 5' end of an mRNA molecule to the translation initiation codon, which recruits the ribosome complex and initiates mRNA translation. The 5'UTR regulates post-transcriptional modifications, translation initiation complex formation, and stability by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The term "5'UTR" also encompasses the ribosome entry site (IRES) sequence, particularly for circular RNA molecules.

[0150] As used in this article, "3'UTR" refers to the sequence between the stop codon of the polypeptide coding sequence in mRNA and the poly(A) sequence. The 3'UTR can regulate mRNA translation through interactions with mRNA-binding proteins, miRNAs, and other mechanisms. The sequence and structural features of the 3'UTR can affect mRNA stability, ribosome scanning and translation, and the formation of the termination complex, thereby influencing protein expression levels.

[0151] Poly(A) sequences typically contain multiple adenine nucleotides. The addition of a poly(A) sequence contributes to mRNA stability and transport, prevents its degradation, and plays an important role in post-transcriptional modifications. A poly(A) sequence can be a continuous chain of pure adenine nucleotides, or it can be a variant containing non-adenine nucleotides, as long as its function is equivalent to the conventional poly(A) sequence, providing similar biological functions as the natural poly(A) sequence, such as affecting mRNA stability, translation efficiency, or ribosome binding. Known poly(A) sequences include the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants may differ in nucleotide composition but are functionally considered equivalent to the conventional poly(A) sequence.

[0152] As used in this article, the "5' cap" for RNA includes the 5' cap structure present on native mRNA and its analogues. The 5' cap structure on native mRNA refers to the methylated guanosine monophosphate linked to the 5' terminal nucleotide of RNA via pyrophosphate, forming a 5',5'-triphosphate linkage. There are generally three types of 5' caps (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as Cap0, Cap1, and Cap2, respectively. Cap0 indicates that the ribose of the terminal nucleotide is unmethylated, Cap1 indicates that the ribose of the terminal nucleotide is methylated, and Cap2 indicates that the ribose of both terminal nucleotides are methylated.

[0153] Methods for capping mRNA molecules are known in the art. The 5' cap structure of the mRNA molecule can be added via an enzymatic reaction after the mRNA molecule has been obtained through chemical synthesis or in vitro transcription (e.g., using a commercially available kit containing a vaccinia capping enzyme and a 2'-O-methyltransferase for the mRNA cap structure). However, capped mRNA can also be produced by directly incorporating a capped nucleotide analog as the first nucleotide into the transcript during in vitro transcription.

[0154] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), bispecific antibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0155] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain, containing an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), the CDR being part of the antigen-binding site domain.

[0156] As used herein, “hypervariant region,” “HV,” “complementarity-determining region,” and “CDR” and “antibody CDR” are interchangeably used to refer to one of the multiple portions within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3. The three CDRs in the variable region are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbers (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J.Mol.Biol. 196: 901-917).

[0157] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0158] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously and specifically bind to two antigens or epitopes. "BiTE" (Bispecific T-cell Engager) refers to a bispecific antibody that connects the antigen-binding region of an antibody targeting a specific antigen on a target cell to the antigen-binding region of an antibody targeting CD3 via a linker. It acts as a "bridge" between T cells and target cells, mediating the T cell's targeted recognition and specific killing of the target cell. The target cell is, for example, a tumor cell.

[0159] As used herein, "vector" refers to a segment of DNA extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has already been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in the organism. A vector may contain, for example, an origin of replication, a selection marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). This term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, plasmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc.

[0160] As used herein, the term "pharmaceutically acceptable salt" refers to those organic or inorganic acid or base addition salts, amino acid addition salts of the compounds of this application, which are suitable for contact with patient tissues to the extent of reliable medical judgment without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of this application.

[0161] Pharmaceutically acceptable base addition salts are those formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include sodium, potassium, magnesium, and calcium. Suitable amines include N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine.

[0162] The base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the required base in a conventional manner to form a salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and then separating the free acid. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents; however, for the purposes of this application, the salts are equivalent to their respective free acids.

[0163] Salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, and iodides prepared from inorganic acids, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthate, methanesulfonate, gluconate, lactobionate, laurylsulfonate, and hydroxyethanesulfonate. Salts can also be prepared from organic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, alkyl diacids, aromatic acids, and aliphatic and aromatic sulfonic acids. Representative salts include acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, naphthates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, and methanesulfonates. Pharmaceutically acceptable salts may include alkali metal and alkaline earth metal-based cations, such as sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. It also covers salts of amino acids, such as arginine salts, gluconates, galacturons, etc. (see, for example, Berge S. et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977; 66: 1-19, which is incorporated herein by reference).

[0164] The term "subject" or "subject" includes, but is not limited to: humans (i.e., men or women of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, such as mammals, such as primates (e.g., monkeys, apes, orangutans, chimpanzees, or gorillas, e.g., cynomolgus monkeys or rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "subject," "patient," and "subject" are used interchangeably herein.

[0165] The terms “disease,” “disorder,” and “symptom” are used interchangeably in this article.

[0166] In the context of disease, the terms “prevention,” “avoidance,” and their various grammatical variations refer to reducing the risk of acquiring or developing a disease or condition, for example, preventing the occurrence or development of at least one clinical symptom of the disease in subjects who have not yet been exposed to the causative agent or in subjects who are susceptible to the disease before its onset.

[0167] Unless otherwise stated, the term “treatment” as used herein includes effects that occur when a subject has a specific disease, disorder, or condition, which reduce the severity of the disease, disorder, or condition, or delay or slow the development of the disease, disorder, or condition (“therapeutic treatment”), and also includes effects that occur before a subject begins to have a specific disease, disorder, or condition (“preventive treatment”).

[0168] As used herein, the term "vaccine" refers to a composition that can provide active acquired immunity and / or therapeutic effects (e.g., treatment) against a specific disease or pathogen. Vaccines typically contain one or more substances that can induce an immune response in an individual against a pathogen or disease (i.e., the target pathogen or disease). The immunogenic composition stimulates the body's immune system to recognize the substance as a threat or indication of the presence of the target pathogen or disease, thereby inducing immune memory so that the immune system can more easily recognize and eliminate any pathogen upon subsequent exposure. Vaccines can be prophylactic (e.g., preventing or mitigating the effects of any natural or pathogenic future infection or the anticipated effects of cancer in susceptible individuals) or therapeutic (e.g., treating cancer in an individual who has been diagnosed with cancer). Administration of a vaccine is called vaccination. In some instances, the vaccine composition can provide an individual with nucleic acids, such as mRNA encoding antigenic molecules (e.g., peptides). The nucleic acids delivered into the individual via the vaccine composition can be expressed as antigenic molecules and enable the individual to acquire immunity against said antigenic molecules. In the case of vaccination against infectious diseases, the vaccine composition may provide mRNA encoding an antigenic molecule associated with a specific pathogen, such as one or more peptides known to be expressed in pathogens (e.g., pathogenic bacteria or viruses). In the case of viral vaccines, the vaccine composition may provide mRNA encoding certain viral peptides that are characteristic of viruses for which they seek immunity, such as peptides that are substantially proprietary or highly expressed on the surface of the virus (e.g., capsid proteins). After vaccination with the viral vaccine composition, an individual may develop immunity against the viral peptides, specifically killing cells that express them. In the case of tumor vaccines, tumor-associated antigens, such as tumor-associated proteins or peptides, may be introduced into the patient in the form of mRNA encoding them to overcome tumor-induced immunosuppression or enhance immunogenicity, thereby activating the patient's own immune system, inducing cellular and humoral immune responses, and ultimately achieving the goal of controlling or eliminating the tumor.

[0169] The term "nucleic acid vaccine" used in this article, also known as a gene vaccine, involves directly introducing genetic material (DNA or RNA) that determines specific antigens of a pathogen into human cells, allowing the human cells to produce these antigens themselves and stimulating the body to produce an immune response to the antigen, thereby providing the recipient with corresponding immune protection. It may also contain adjuvants.

[0170] As used herein, the term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when administered in conjunction with, or before, during, or after the administration of the antigen to a subject. This includes, but is not limited to: nucleic acid adjuvants (such as nucleic acid carriers), plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), and emulsion adjuvants (such as Freund's adjuvant).

[0171] The term "virus," used in its general sense in the field of biology, refers to a non-cellular microorganism that parasitizes within cells and multiplies by replication. It includes a viral genome (e.g., DNA, RNA, whether single-stranded or double-stranded), a protein protective capsid (e.g., capsid proteins), and related proteins. It includes both non-enveloped and enveloped viruses. In the case of enveloped viruses (e.g., herpesviruses), the envelope includes lipids and optional host cell membrane components, and / or viral proteins.

[0172] The term “viral infection” or “viral disease” refers to an illness or condition caused by a virus, including symptomatic and asymptomatic infections. Non-limiting examples of viral infections include hepatitis virus diseases (e.g., hepatitis A, B, C, D, and E), herpesvirus infections (e.g., HSV-1, HSV-2, and shingles), flavivirus infections, Zika virus infections, cytomegalovirus infections, respiratory virus infections (e.g., adenovirus infections, influenza, severe acute respiratory syndrome), coronavirus infections (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, COVID-19, and MERS), gastrointestinal virus infections (e.g., norovirus infections, rotavirus infections, and astrovirus infections), exanthematous virus infections (e.g., measles, shingles, smallpox, and rubella), viral hemorrhagic diseases (e.g., Ebola virus, Lassa fever, dengue fever, and yellow fever), nervous system virus infections (e.g., West Nile virus infections, poliomyelitis, viral meningitis, viral encephalitis, Japanese encephalitis, and rabies), and human papillomavirus infections.

[0173] SARS-CoV-2 belongs to the beta-coronavirus family, which includes two other zoonotic viruses that have caused severe disease outbreaks since the 21st century: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The term "SARS-CoV" refers to the SARS coronavirus. The term "SARS-CoV" includes any coronavirus, such as SARS-CoV-2, SARS-CoV-1, and MERS-CoV.

[0174] The term "anti-SARS-CoV-2 drug" refers to drugs used to treat and / or prevent suspected / confirmed SARS-CoV-2 infection. These can be any direct-acting anti-SARS-CoV-2 drug, including but not limited to remdesivir (Gilead), REGN-COV2 (Regeneron), LY-CoV555 (Lilly), MK-4482 / EIDD-2801 (Merck / Ridgeback Bio); or immunomodulators, including but not limited to CD24Fc (Oncoimmune), T-COVIDTM (Altimmune), itutuzumab (Equillium Inc); or vaccines, including but not limited to AdCOVIDTM (Altimmune), BNT162b1 / 2 (Pfizer / BioNTech), mRNA-1273 (Moderna), AZD1222 / ChAdOx1 (AstraZeneca / Oxford Univ), Ad5-vectored COVID-19 vaccine (CanSino Biologics Inc.). Biologics), CoronaVac (Sinovac), NVX-CoV2373 (Novavax).

[0175] The term "hereditary disease" refers to diseases caused by alterations in genetic material or controlled by pathogenic genes. These diseases are often congenital, but can also develop later in life. Some hereditary diseases are entirely determined by genetic factors and develop symptoms some time after birth, sometimes taking years, decades, or even longer to show obvious symptoms. Other hereditary diseases require the combined effects of genetic and environmental factors and are not entirely determined by genetic factors. Hereditary diseases include chromosomal disorders, chromosomal syndromes, single-gene diseases, and polygenic diseases. They are usually caused by chromosomal or gene abnormalities and can potentially be inherited by offspring. Examples include Down syndrome (trisomy 21), gonadal dysgenesis, achondroplasia, polydactyly, colonic polyps, albinism, phenylketonuria, alkaptonuria, congenital deafness, high myopia, thalassemia, hemophilia, essential hypertension, juvenile diabetes, rheumatoid arthritis, epilepsy, congenital heart disease, spina bifida, anencephaly, cleft lip, cleft palate, clubfoot, asthma, depression, schizophrenia, Alzheimer's disease, chronic progressive chorea, and cancer.

[0176] The term "infectious disease" refers to a disease caused by infection with viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, or fungi, wherein the viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, or fungi are selected from the "Catalogue of Human Infectious Pathogens" (2023 Edition) published on the website of the National Health Commission of the People's Republic of China in 2023, the entire contents of which are incorporated herein by reference.

[0177] Generally, the "effective amount" of a pharmaceutical composition refers to the amount sufficient to elicit a target biological response. As will be understood by those skilled in the art, the effective amount of the pharmaceutical composition of this application can vary depending on factors such as the biological target, the pharmacokinetics of the pharmaceutical composition, the disease being treated, the administration method, and the age, health status, and symptoms of the subject. Effective amounts include therapeutic effective amounts and prophylactic effective amounts.

[0178] Unless otherwise stated, the “therapeuticly effective amount” of a pharmaceutical composition as used herein is an amount sufficient to provide therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. Therapeuticly effective amount of a pharmaceutical composition refers to the amount of a therapeutic agent, used alone or in combination with other therapies, that provides therapeutic benefit in the treatment of a disease, disorder, or condition. The term “therapeuticly effective amount” may include amounts that improve overall treatment, reduce or prevent symptoms or causes of a disease or condition, or enhance the therapeutic effects of other therapeutic agents.

[0179] Unless otherwise stated, the “preventive effective amount” of a pharmaceutical composition as used herein is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount sufficient to prevent recurrence of a disease, disorder, or condition. The preventive effective amount of a pharmaceutical composition refers to the amount of the therapeutic agent, used alone or in combination with other agents, that provides preventive benefit in the prevention of a disease, disorder, or condition. The term “preventive effective amount” may include amounts that improve overall prevention or enhance the preventive effect of other preventive agents.

[0180] The terms "combination," "in combination," or "in conjunction," and related terms, refer to the simultaneous or sequential administration of the lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application with other therapeutic agents. For example, the pharmaceutical compositions of this application may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or simultaneously with other therapeutic agents in a single unit dosage form.

[0181] Invention Details

[0182] This application provides, on the one hand, lipid nanoparticles for delivering nucleic acid molecules, comprising the following components in the following molar percentages of the total lipids in the particles:

[0183] Ionizable lipids: about 25 mol% to about 75 mol%, preferably about 30 mol% to about 70 mol%, more preferably about 40 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, more preferably about 50 mol%;

[0184] Structural lipids: about 5 mol% to about 60 mol%, preferably about 10 mol% to about 60 mol%, more preferably about 20 mol% to about 50 mol%, more preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, more preferably about 37 mol%, or more preferably about 37.5 mol%.

[0185] Neutral lipids: about 5 mol% to about 30 mol%, preferably about 5 mol% to about 25 mol%, more preferably about 5 mol% to about 20 mol%, more preferably about 8 mol% to about 20 mol%, more preferably about 8 mol% to about 15 mol%, more preferably about 10 mol%;

[0186] Polymer lipids: about 1.5 mol% to about 6 mol%, preferably about 1.5 mol% to about 5 mol%, more preferably about 2 mol% to about 4.5 mol%, more preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, more preferably about 2.2 mol% to about 3.2 mol%, more preferably about 2.5 mol% to about 3 mol%, more preferably about 2.5 mol%, or more preferably about 3 mol%.

[0187] In some embodiments, this application provides lipid nanoparticles as described above, wherein the ionizable lipid is a compound of formula (II), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof.

[0188] in,

[0189] a = 1, 2, 3, 4, 5 or 6;

[0190] b = 4, 5, 6, 7, 8, 9 or 10;

[0191] c = 1, 2, 3, 4, 5 or 6;

[0192] d = 0, 1, 2, 3 or 4;

[0193] c+d = 3, 4, 5, 6, 7, 8 or 9;

[0194] M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O )S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -SS-, and -S(O) 0-2 -;

[0195] R1 and R2 are independently selected from C 4-25 Alkyl, C 4-25 alkenyl and C 4-25 Alkyne group, which is optionally surrounded by one or more R groups 1s Substitution, and one or more methylene units therein are optionally and independently substituted with -NR'-;

[0196] R 1s Independently selected from H and C 1-20 Alkyl, -L c -OR c -Lc -SR c and -L c -NR c R' c ;

[0197] R and R' are each independently selected from H and C. 1-20 alkyl;

[0198] L c Independently selected from chemical bonds and C 1-20 Alkylene;

[0199] R c and R' c Independently selected from H and C 1-20 Alkyl, C 3-14 cycloalkyl groups and 3 to 14-membered heterocyclic groups;

[0200] R4 and R5 are independently selected from C 1-8 Alkyl groups, which are optionally composed of one or more R groups 4s replace;

[0201] Or R4, R5, together with the carbon atoms they are attached to, form C. 3-14 Cycloalkyl or 3 to 14-membered heterocyclic group, optionally surrounded by one or more R 4s replace;

[0202] R 4s Independently selected from H, halogen, cyano, C 1-8 Alkyl, C 1-8 Halogenated alkyl groups, -L d -OR d -L d -SR d and -L d -NR d R' d ;

[0203] L d Independently selected from chemical bonds and C 1-8 Alkylene;

[0204] R d and R' d Independently selected from H and C 1-8 Alkyl, C 3-14 Cycloalkyl and 3 to 14-membered heterocyclic groups.

[0205] In some specific implementation schemes, R1 and R2 are independently selected from C. 4-22 Alkyl, C 4-22 alkenyl and C 4-22The alkynyl group, wherein one or more methylene units selected from the alkyl, alkenyl, and alkynyl groups are optionally and independently replaced by -NR'-, wherein each R' is independently selected from H and C. 1-20 alkyl.

[0206] In some specific implementation schemes, R1 and R2 are independently selected from C. 4-20 Alkyl, C 4-20 alkenyl and C 4-20 The alkynyl group, wherein one or more methylene units selected from the alkyl, alkenyl, and alkynyl groups are optionally and independently replaced by -NR'-, wherein each R' is independently selected from H and C. 1-20 Alkyl group. In some specific embodiments, R1 and R2 are independently selected from the following groups: -(CH2)5CH3, -(CH2)6CH3, -(CH2)7CH3, -(CH2)8CH3, -(CH2)9CH3, -(CH2) 10 CH3, -(CH2) 11 CH3, -CH2-C≡C-(CH2)5CH3, -CH2-C≡C-(CH2)6CH3, -(CH2)2-C≡C-(CH2)5CH3, -(CH2)4-C≡C-(CH2)3CH3, -CH2-CH=CH-( CH2)5CH3, -CH2-CH=CH-(CH2)6CH3, -(CH2)2-CH=CH-(CH2)5CH3, -(CH2)4-CH=CH-(CH2)3CH3, -(CH2)5-CH=CH-CH2CH3,

[0207] In some embodiments, this application provides lipid nanoparticles as described above, wherein M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably wherein M1 is -OC(O)- and M2 is -C(O)O-.

[0208] In some embodiments, this application provides lipid nanoparticles as described above, wherein R4 and R5 are independently C 1-8 Alkyl group. In some embodiments, this application provides lipid nanoparticles as described above, wherein R4 and R5 are independently C4 and R5. 1-3 Alkyl groups, such as methyl, ethyl, or propyl. In some embodiments, this application provides lipid nanoparticles as described above, wherein R4 and R5 are independently methyl.

[0209] In some embodiments, this application provides lipid nanoparticles as described above, wherein R4, R5, together with the carbon atoms to which they are attached, form C 3-8Cycloalkyl or 3 to 8-membered heterocyclic group, optionally surrounded by one or more R 4s Replace, where R 4s As defined in compound (II) above.

[0210] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 and R2 are independently selected from C 4-22 Alkyl groups, which are optionally composed of one or more R groups 1s Replace, where R 1s As defined above.

[0211] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is C 6-14 Straight-chain alkyl; preferably, R1 is C 8-12 Straight-chain alkyl; more preferably, R1 is C 9-11 Straight-chain alkyl groups.

[0212] In some embodiments, this application provides lipid nanoparticles as described above, wherein R2 is C 12-25 Branched alkyl group; preferably, R2 is C 12-22 Branched alkyl group; preferably, R2 is C 12-20 Branched alkyl group; more preferably, R2 is C 15-20 Branched alkyl groups; or,

[0213] R2 is C 6-14 Straight-chain alkyl, preferably C 8-12 Straight-chain alkyl group, and it is bounded by 1 R 1s replace;

[0214] R 1s C 1-10 Alkyl; preferably, R 1s C 6-10 Alkyl group; preferably selected from C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl or C 10 alkyl.

[0215] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is C 8-12 Straight-chain alkyl groups.

[0216] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is a C8 linear alkyl group, a C9 linear alkyl group, or a C4 linear alkyl group. 10 Straight-chain alkyl or C 11 Straight-chain alkyl, preferably C9 straight-chain alkyl, or preferably C 10 Straight-chain alkyl groups.

[0217] In some embodiments, this application provides lipid nanoparticles as described above, wherein R2 is C12-22 Branched alkyl groups, preferably C 15-22 Branched alkyl groups.

[0218] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is C 8-12 Straight-chain alkyl with R2 being C 12-22 Branched alkyl groups, more preferably, R1 is C 9-11 Straight-chain alkyl with R2 being C 15-22 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 15-20 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 16 C 17 C 18 C 19 Or C 20 Branched alkyl groups.

[0219] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is a C9 straight-chain alkyl group and R2 is a C... 16 Branched alkyl groups.

[0220] In some embodiments, this application provides lipid nanoparticles as described above, wherein R2 is C 12-22 Branched alkyl group, wherein the branched carbon atoms of the branched alkyl group are spaced 0-6 carbon atoms apart from M2; preferably 1-5 carbon atoms apart, for example -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2- or -CH2CH2CH2CH2CH2-; more preferably 1-3 carbon atoms apart, for example -CH2-, -CH2CH2- or -CH2CH2CH2-.

[0221] In some embodiments, this application provides lipid nanoparticles as described above, wherein a is an integer from 1 to 4, such as 1, 2, 3 or 4; b is an integer from 5 to 8, such as 5, 6, 7 or 8; c is an integer from 2 to 6, such as 2, 3, 4, 5 or 6; and d is an integer from 1 to 4, such as 1, 2, 3 or 4.

[0222] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) has the following structure:

[0223] R1, R2, a, b, c, and d are defined as above.

[0224] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) has the following structure:

[0225] R1, R2, a, b, c, and d are defined as above.

[0226] In some embodiments, this application provides lipid nanoparticles as described above, wherein R1 is nonyl or decyl, and R2 is selected from one of the following structures:

[0227] In some embodiments, this application provides lipid nanoparticles as described above, wherein the compound of formula (II) is selected from the following compounds or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers:

[0228] In some embodiments, the ionizable lipids comprise about 25 mol% to about 75 mol% of the total lipids, preferably about 30 mol% to about 70 mol%, more preferably about 40 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, and more preferably about 50 mol%.

[0229] In some embodiments, the ionizable lipids comprise about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, or about 25 mol% to about 50 mol%; in some embodiments, the ionizable lipids comprise about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, or about 30 mol% to about 55 mol%; in some embodiments, the ionizable lipids comprise about [missing information - likely a percentage] of the total lipids. The molar percentage content is about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, or about 35 mol% to about 55 mol%; in some embodiments, the molar percentage content of the ionizable lipids in the total lipids is about 30 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 40 mol% to about 65 mol%, about 40 mol% to about 60 mol%, about 45 mol% to about 60 mol%, or about 45 mol% to about 55 mol%, more preferably about 50 mol%.In some embodiments, the molar percentage of the ionizable lipids in the total lipids is about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 40.5 mol%, about 41 mol%, about 41.5 mol%, about 42 mol%, about 42.5 mol%, about 43 mol%, about 43.5 mol%, about 44 mol%, about 44.5 mol%, about 45 mol%, about 45.5 mol%, about 46 mol%, about 46.5 mol%, about 47 mol%, about 47.5 mol%, about 48 mol%, about 48.5 mol%, about 49 mol%. Approximately 49.5 mol%, approximately 50 mol%, approximately 50.5 mol%, approximately 51 mol%, approximately 51.5 mol%, approximately 52 mol%, approximately 52.5 mol%, approximately 53 mol%, approximately 53.5 mol%, approximately 54 mol%, approximately 54.5 mol%, approximately 55 mol%, approximately 55.5 mol%, approximately 56 mol%, approximately 56.5 mol%, approximately 57 mol%, approximately 57.5 mol%, approximately 58 mol%, approximately 58.5 mol%, approximately 59 mol%, approximately 59.5 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, approximately 65 mol%, approximately 66 mol%, approximately 67 mol%, approximately 68 mol%, approximately 69 mol%, approximately 70 mol%, approximately 71 mol%, approximately 72 mol%, approximately 73 mol%, approximately 74 mol%, or approximately 75 mol%.

[0230] In some embodiments, the structural lipid is a steroid or an analogue thereof.

[0231] In some embodiments, the steroid or its analogue has the following tetracyclic skeleton structure.

[0232] This includes naturally occurring or synthetic steroids and their analogues, including but not limited to: alfalfa sterol, β-sitosterol, campesterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, demosterol, cholesterol, cholesterol, cholestenone, 3p-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (D C-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cyclolutenol, 22-ketocholesterol, 20-hydroxycholesterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, luminesterol, citopalcitol, calcipotriol, fecal prostaglandin, cholecalciferol, lupeol, ergocalciferol, 22-dihydrocalciferidol, tomatine, ursolic acid, chenodeoxycholic acid, yeast sterol, diosgenin, etc. In some embodiments, the structural lipid is selected from one or more of cholesterol, β-sitosterol, coccosterol, rock saponin, rapeseed sterol, ergosterol, tomatine, ursolic acid, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol.

[0233] In some embodiments, the steroid is selected from one or more of the following: cholesterol, sitosterol, coccosterol, lycopene, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, and campesterol; preferably, the structural lipid is selected from cholesterol and / or β-sitosterol; more preferably, the structural lipid is cholesterol.

[0234] In some embodiments, the structural lipids comprise about 5 mol% to about 60 mol% of the total lipids, preferably about 10 mol% to about 60 mol%, more preferably about 20 mol% to about 50 mol%, more preferably about 30 mol% to about 50 mol%, more preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, and more preferably about 37 mol%.

[0235] In some embodiments, the structural lipids constitute about 5 mol% to about 55 mol%, about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, or about 5 mol% to about 40 mol%; in some embodiments, the structural lipids constitute about 10 mol% to about 60 mol%, about 10 mol% to about 55 mol%, about 10 mol% to about 50 mol%, or about 10 mol% to about 40 mol%; in some embodiments, the structural lipids constitute about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 20 mol% to about 45 mol%, or about 20 mol% to about 40 mol%; in some... In some embodiments, the structural lipids comprise about 15 mol% to about 50 mol%, about 15 mol% to about 45 mol%, or about 15 mol% to about 40 mol%, preferably about 25 mol% to about 40 mol%, more preferably about 37 mol%. In some embodiments, the structural lipids comprise about 10 mol% to about 60 mol%, about 15 mol% to about 55 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol%, preferably about 25 mol% to about 50 mol%, preferably about 30 mol% to about 50 mol%, more preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, more preferably about 37 mol%, more preferably about 37.5 mol%.

[0236] In some embodiments, the structural lipids constitute about 5 mol% of the total lipids, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 32.5 mol%, about 35 mol%, about 35.5 mol%, about 36 mol%, about 36.5 mol%, about 37 mol%, about 37.5 mol%, about 38 ... 1 mol%, approximately 38.5 mol%, approximately 39 mol%, approximately 39.5 mol%, approximately 40 mol%, approximately 40.5 mol%, approximately 41 mol%, approximately 41.5 mol%, approximately 42 mol%, approximately 42.5 mol%, approximately 43 mol%, approximately 43.5 mol%, approximately 44 mol%, approximately 44.5 mol%, approximately 45 mol%, approximately 45.5 mol%, approximately 46 mol%, approximately 46.5 mol%, approximately 47 mol%, approximately 47.5 mol%, approximately 48 mol%, approximately 48.5 mol%, approximately 49 mol%, approximately 49.5 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, or approximately 60 mol%.

[0237] In some embodiments, the neutral lipid is a phospholipid selected from phosphatidylcholine and / or phosphatidylethanolamine.

[0238] Preferably, the phospholipid is selected from one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycerol- 3-Phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonium)ethyl hydrogen phosphate (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), and dipalmitoyl phosphatidylethanolamine (DPPE) and 1,2-dioleoyl Sodium 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol)sodium salt (DOPG), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, 18-1-trans-PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine or 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS) Sphingomyelin (SM), dimyristoyl phosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), rutinoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (DPHyPE), lecithin, sphingomyelin, and mixtures thereof, preferably DSPC and / or DOPE.

[0239] In some embodiments, the neutral lipids constitute about 5 mol% to about 30 mol% of the total lipids, preferably about 5 mol% to about 25 mol%, more preferably about 5 mol% to about 20 mol%, more preferably about 8 mol% to about 20 mol%, more preferably about 8 mol% to about 15 mol%, and more preferably about 10 mol%.

[0240] In some embodiments, the neutral lipids constitute about 5 mol% to about 15 mol% of the total lipids. In some embodiments, the neutral lipids constitute about 8 mol% to about 30 mol%, about 8 mol% to about 25 mol%, about 8 mol% to about 20 mol%, about 8 mol% to about 15 mol%, or about 8 mol% to about 12 mol%. In some embodiments, the neutral lipids constitute about 10 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 20 mol%, or about 10 mol% to about 15 mol%.

[0241] In some embodiments, the molar percentage of neutral lipids in total lipids is about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 8 mol%, about 8.5 mol%, about 9 mol%, about 9.5 mol%, about 10 mol%, about 10.5 mol%, about 11 mol%, about 11.5 mol%, about 12 mol%, about 12.5 mol%, about 13 mol%, about 13.5 mol%, about 14 mol%. Approximately 14.5 mol%, approximately 15 mol%, approximately 15.5 mol%, approximately 16 mol%, approximately 16.5 mol%, approximately 17 mol%, approximately 17.5 mol%, approximately 18 mol%, approximately 18.5 mol%, approximately 19 mol%, approximately 19.5 mol%, approximately 20 mol%, approximately 21 mol%, approximately 22 mol%, approximately 23 mol%, approximately 24 mol%, approximately 25 mol%, approximately 26 mol%, approximately 27 mol%, approximately 28 mol%, approximately 29 mol%, or approximately 30 mol%.

[0242] In some embodiments, the polymeric lipid is a polyethylene glycol-modified lipid.

[0243] In some embodiments, the PEG-modified lipids (PEG lipids) of this invention are lipids covalently or non-covalently linked to one or more polyethylene glycol (PEG) chains.

[0244] Preferably, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

[0245] Preferably, the PEGylated lipid contains a PEG portion of 1000 Da to 20 kDa, and more preferably contains a PEG portion of about 1000 Da to about 5000 Da.

[0246] In some embodiments, the polyethylene glycol-modified lipids of the present invention include polyethylene glycol-modified diacylglycerol (PEG-DAG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), polyethylene glycol-modified succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-bis(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (salt) (PEG-S-DMG), polyethylene glycol-modified ceramide (PEG-cer), or polyethylene glycol-modified diekoxypropylamine. Carbamates (e.g., ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)carbamate or 2,3-di(tetradecoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate), PEG-dialkoxypropylcarbamate, N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine sodium, PEG-dilauroyloxypropyl, PEG-dimyristoyloxypropyl, PEG-dipalmitoyloxypropyl PEG-disteayloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol-PEG (DMG-PEG), disteayl-rac-glycerol-PEG (DSG-PEG), PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-disteaylglycerol, PEG-dilauroylglycerol amide, PEG-dimyristoylglycerol amide, PEG-dipalmitoylglycerol amide, PEG-disteaylglycerol amide, (1-[8'-(cholesterol-5-ene- [3β-oxy]formamido-3',6'-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-bistetradecoxybenzyl-ω-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH).

[0247] Preferably, the PEGylated lipid is selected from one or more of the following: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000, DSPE-PEG2000-Mannose, DSPE-PEG2000 The amine, DSPE-PEG3350, DSPE-PEG3500, DMG-PEG3500, DPPE-PEG3500, DSPE-PEG4000, DMG-PEG4000, Ceramide-PEG5000, DSPE-PEG5000, DMG-PEG5000 and ALC-0159, preferably ALC-0159, DMG-PEG2000, DSPE-PEG2000, DMG-PEG3500, DMG-PEG4000 and / or DMG-PEG5000, more preferably DMG-PEG2000.

[0248] In some embodiments, the polyethylene glycol-modified lipids comprise about 1.5 mol% to about 6 mol% of the total lipids, preferably about 1.5 mol% to about 5 mol%, more preferably about 2 mol% to about 4.5 mol%, more preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, and more preferably about 3 mol%.

[0249] In some embodiments, the PEGylated lipids comprise about 1.5 mol% to about 5 mol%, about 1.5 mol% to about 4.5 mol%, about 1.5 mol% to about 4 mol%, about 1.5 mol% to about 3.5 mol%, or about 1.5 mol% to about 3 mol%; in some embodiments, the PEGylated lipids comprise about 2 mol% to about 6 mol%, about 2 mol% to about 5.5 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4.5 mol%, about 2 mol% to about 4 mol%, about 2 mol% to about 3.5 mol%, or about 2 mol% to about 3 mol%; in some embodiments, the PEGylated lipids comprise about 2 mol% of the total lipids. 2 mol% to about 6 mol, about 2.2 mol% to about 5.5 mol%, about 2.2 mol% to about 5 mol%, about 2.2 mol% to about 4.5 mol%, about 2.2 mol% to about 4 mol%, about 2.2 mol% to about 3.8 mol%, about 2.2 mol% to about 3.5 mol%, about 2.2 mol% to about 3.2 mol%, about 2.2 mol% to about 3 mol%; in some embodiments, the PEGylated lipids constitute about 2.5 mol% to about 6 mol%, about 2.5 mol% to about 5.5 mol%, about 2.5 mol% to about 5 mol%, about 2.5 mol% to about 4.5 mol%, about 2.5 mol% to about 4 mol%, or about 2.5 mol% to about 3.5 mol%.

[0250] In some embodiments, the PEGylated lipids comprise about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, and about 3. 6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, approximately 5 mol%, approximately 5.1 mol%, approximately 5.2 mol%, approximately 5.3 mol%, approximately 5.4 mol%, approximately 5.5 mol%, approximately 5.6 mol%, approximately 5.7 mol%, approximately 5.8 mol%, approximately 5.9 mol%, or approximately 6 mol%.

[0251] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0252] Ionizable lipids: approximately 25 mol% - approximately 75 mol%;

[0253] Structural lipids: approximately 5 mol% to approximately 60 mol%;

[0254] Neutral lipids: approximately 5 mol% to approximately 30 mol%;

[0255] Polymer lipids: approximately 1.5 mol% to approximately 6 mol%.

[0256] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0257] Ionizable lipids: approximately 30 mol% - approximately 70 mol%;

[0258] Structural lipids: approximately 10 mol% to approximately 60 mol%;

[0259] Neutral lipids: approximately 5 mol% to approximately 25 mol%;

[0260] Polymer lipids: approximately 1.5 mol% to approximately 5 mol%.

[0261] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0262] Ionizable lipids: approximately 40 mol% - approximately 60 mol%;

[0263] Structural lipids: approximately 20 mol% to approximately 50 mol%;

[0264] Neutral lipids: approximately 5 mol% to approximately 20 mol%;

[0265] Polymer lipids: approximately 2 mol% - approximately 4.5 mol%.

[0266] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0267] Ionizable lipids: approximately 40 mol% - approximately 55 mol%;

[0268] Structural lipids: approximately 30 mol% to approximately 45 mol%;

[0269] Neutral lipids: approximately 8 mol% to approximately 20 mol%;

[0270] Polymer lipids: approximately 2 mol% - approximately 4 mol%.

[0271] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0272] Ionizable lipids: approximately 40 mol% - approximately 55 mol%;

[0273] Structural lipids: approximately 30 mol% to approximately 45 mol%;

[0274] Neutral lipids: approximately 8 mol% to approximately 20 mol%;

[0275] Polymer lipids: approximately 2 mol% - approximately 3.5 mol%.

[0276] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0277] Ionizable lipids: approximately 45 mol% - approximately 55 mol%;

[0278] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0279] Neutral lipids: approximately 8 mol% to approximately 20 mol%;

[0280] Polymer lipids: approximately 2.5 mol% - approximately 3.5 mol%.

[0281] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0282] Ionizable lipids: approximately 45 mol% - approximately 55 mol%;

[0283] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0284] Neutral lipids: approximately 8 mol% - approximately 15 mol%;

[0285] Polymer lipids: approximately 2.5 mol% - approximately 3.5 mol%.

[0286] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0287] Ionizable lipids: approximately 45 mol% - approximately 55 mol%;

[0288] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0289] Neutral lipids: approximately 8 mol% - approximately 15 mol%;

[0290] Polymer lipids: approximately 2.2 mol% - approximately 3.2 mol%.

[0291] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0292] Ionizable lipids: approximately 45 mol% - approximately 55 mol%;

[0293] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0294] Neutral lipids: approximately 8 mol% - approximately 15 mol%;

[0295] Polymer lipids: approximately 2.5 mol% - approximately 3 mol%.

[0296] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0297] Ionizable lipids: approximately 50 mol%;

[0298] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0299] Neutral lipids: approximately 10 mol%;

[0300] Polymer lipids: approximately 2.2 mol% - approximately 3.2 mol%.

[0301] In one specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0302] Ionizable lipids: approximately 50 mol%;

[0303] Structural lipids: approximately 35 mol% - approximately 40 mol%;

[0304] Neutral lipids: approximately 10 mol%;

[0305] Polymer lipids: approximately 2.5 mol% - approximately 3 mol%.

[0306] In a more specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0307] Ionizable lipids: approximately 50 mol%;

[0308] Structural lipids: approximately 37 mol%;

[0309] Neutral lipids: approximately 10 mol%;

[0310] Polymer lipids: approximately 3 mol%.

[0311] In a more specific embodiment, the lipid nanoparticles comprise the following components in molar percentage:

[0312] Ionizable lipids: approximately 50 mol%;

[0313] Structural lipids: approximately 37.5 mol%;

[0314] Neutral lipids: approximately 10 mol%;

[0315] Polymer lipids: approximately 2.5 mol%.

[0316] In some embodiments, the lipid nanoparticles contain about 25-75 mol% of the ionizable lipid compound provided in this application, about 5-60 mol% of cholesterol, about 5-30 mol% of DSPC, and about 1.5-6 mol% of DMG-PEG2000.

[0317] In some embodiments, the lipid nanoparticles contain about 30-70 mol% of the ionizable lipid compound provided in this application, about 10-60 mol% of cholesterol, about 5-25 mol% of DSPC, and about 1.5-5 mol% of DMG-PEG2000.

[0318] In some embodiments, the lipid nanoparticles contain about 40-60 mol% of the ionizable lipid compound provided in this application, about 20-50 mol% of cholesterol, about 5-20 mol% of DSPC, and about 2-4.5 mol% of DMG-PEG2000.

[0319] In some embodiments, the lipid nanoparticles contain about 40-55 mol% of the ionizable lipid compound provided in this application, about 30-45 mol% of cholesterol, about 8-20 mol% of DSPC, and about 2-4 mol% of DMG-PEG2000.

[0320] In some embodiments, the lipid nanoparticles contain about 45-55 mol% of the ionizable lipid compound provided in this application, about 35-40 mol% of cholesterol, about 8-15 mol% of DSPC, and about 2.5-3.5 mol% of DMG-PEG2000.

[0321] In some embodiments, the lipid nanoparticles contain about 45-55 mol% of the ionizable lipid compound provided in this application, about 35-40 mol% of cholesterol, about 8-15 mol% of DSPC, and about 2.2-3.2 mol% of DMG-PEG2000.

[0322] In some embodiments, the lipid nanoparticles contain about 45-55 mol% of the ionizable lipid compound provided in this application, about 35-40 mol% of cholesterol, about 8-15 mol% of DSPC, and about 2.5-3 mol% of DMG-PEG2000.

[0323] In some embodiments, the lipid nanoparticles contain about 50 mol% of the ionizable lipid compound provided in this application, about 35-40 mol% of cholesterol, about 10 mol% of DSPC, and about 2.2-3.2 mol% of DMG-PEG2000. In some embodiments, the lipid nanoparticles contain about 50 mol% of the ionizable lipid compound provided in this application, about 35-40 mol% of cholesterol, about 10 mol% of DSPC, and about 2.5-3 mol% of DMG-PEG2000. In some embodiments, the lipid nanoparticles contain about 50 mol% of the ionizable lipid compound provided in this application, about 37 mol% of cholesterol, about 10 mol% of DSPC, and about 3 mol% of DMG-PEG2000.

[0324] In some embodiments, the lipid nanoparticles contain about 50 mol% of the ionizable lipid compound provided in this application, about 37.5 mol% of cholesterol, about 10 mol% of DSPC, and about 2.5 mol% of DMG-PEG2000. In some embodiments, the ionizable lipid compound provided in this application described in the above embodiments is MTS001. In some embodiments, the ionizable lipid compound provided in this application described in the above embodiments is MTS003.

[0325] In some embodiments, the lipid nanoparticles described above further comprise at least one load selected from therapeutic agents, preventative agents, or diagnostic agents.

[0326] In some embodiments, the therapeutic agent, preventative agent, or diagnostic agent is selected from any one or more of the following: small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules.

[0327] In some embodiments, the small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules are small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules used to prevent, treat, or diagnose the following diseases: tumor diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, or autoimmune diseases; or liver diseases or liver-related diseases, preferably malignant tumors of the liver.

[0328] In some embodiments, the therapeutic, preventative, or diagnostic agent is a small molecule compound, peptide, protein, antibody, or nucleic acid molecule used for gene therapy.

[0329] In some embodiments, the therapeutic, preventative, or diagnostic agent is a small molecule compound, peptide, protein, antibody, or nucleic acid molecule used for gene editing, protein replacement and / or supplementation, or gene interference.

[0330] In some embodiments, the nucleic acid molecule is selected from antisense oligonucleotides (ASO), RNA, or DNA;

[0331] Preferably, the RNA is selected from one or more of the following: interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozymes; more preferably, the RNA is selected from one or more of the following: messenger RNA (mRNA), modified mRNA (mmRNA), circular RNA (circRNA), siRNA, gRNA, and self-replicating RNA (SrRNA), preferably mRNA or modified mRNA, siRNA, or gRNA, more preferably modified mRNA; or

[0332] Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and more preferably from one or more of plasmid DNA (pDNA), microcircular DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multicopy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); more preferably, the DNA is selected from at least one of plasmid DNA (pDNA) and microcircular DNA (mcDNA).

[0333] In some embodiments, the therapeutic, preventive, or diagnostic agent is selected from therapeutic or preventive vaccines. In some embodiments, the therapeutic or preventive vaccine is a nucleic acid molecule. In some embodiments, the therapeutic or preventive vaccine is a nucleic acid molecule for the prevention, treatment, or diagnosis of the following diseases: oncological diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, or autoimmune diseases; or liver diseases or liver-related diseases, preferably malignant tumors of the liver.

[0334] In some implementations, the tumor disease is selected from solid tumors and hematologic cancers, such as lung cancer, prostate cancer, colorectal cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, cervical cancer, ovarian cancer, thyroid cancer, pancreatic cancer, skin cancer, central nervous system tumors, epithelial cancer, squamous cell carcinoma, oral cancer, bile duct cancer, head and neck cancer, leiomyosarcoma, osteosarcoma, colon cancer, melanoma, leukemia, lymphoma, multiple myeloma, etc.

[0335] In some embodiments, the tumor disease is selected from the following: acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, connective tissue proliferative small round cell tumor, diffuse large B-cell lymphoma, adrenocortical carcinoma, HIV-related cancer, anal cancer, rectal cancer, appendiceal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, malignant fibrous histiocytoma, breast cancer, bronchial tumor, Burkitt lymphoma, spinal cord tumor, carcinoid tumor, embryonal tumor of the central nervous system, central nervous system lymphoma, cervical cancer, chordoma, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, Cezari syndrome, endometrial cancer, endothelioma, ependymoma, esophageal cancer, Ewing sarcoma. Germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, Waldenström macroglobulinemia, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, plasmacytoma, nasal cavity carcinoma, paranasal sinus carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, intermediately differentiated pineal medullary tumors, pineal cell tumors, supratentorial primitive neuroectodermal tumors, pituitary tumors, pleural pulmonary blastoma, prostate cancer, renal and pelvic cancer, ureteral cancer, retinoblastoma, and rhabdomyosarcoma.

[0336] In some implementations, the parasitic disease is a worm infection, such as trematode disease, tapeworm disease, and nematode disease.

[0337] In some implementations, the parasitic disease is a protozoan infection, such as flagellation disease, amoebiasis, ciliate disease, and spore disease.

[0338] In some embodiments, the infectious disease is a disease caused by infection with a virus, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, or fungi, wherein the virus, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, or fungi are selected from the "Catalogue of Human Infectious Pathogens" (2023 Edition) published on the website of the National Health Commission of the People's Republic of China in 2023, the entire contents of which are incorporated herein by reference.

[0339] In some implementations, the infectious disease is a viral infection, such as diseases caused by novel coronavirus infection, influenza virus infection, adenovirus infection, herpes zoster virus infection, hepatitis B virus infection, hepatitis C virus infection, hepatitis A virus infection, rotavirus infection, rabies virus infection, HIV infection, herpes simplex virus infection, rubella virus infection, cytomegalovirus infection, etc.

[0340] In some implementations, the disease caused by the novel coronavirus infection is a disease caused by SARS-CoV-2.

[0341] In some implementations, the infectious disease is a disease caused by bacterial infection, such as systemic or localized infections caused by pathogenic or opportunistic pathogens, such as infections caused by hemolytic streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Neisseria meningitidis, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Klebsiella spp., Serratia spp., Proteus spp., Acinetobacter spp., Neisseria spp., Yersinia pestis, or Haemophilus influenzae, such as purulent tonsillitis, pneumonia, bronchitis, rhinitis, sinusitis, skin or soft tissue infections, osteomyelitis, meningitis, colitis, gastritis, diarrhea, urethritis, vaginitis, sepsis, or endocarditis, etc.

[0342] In some implementations, the infectious disease is a disease caused by a fungal infection, such as fungal vaginitis, athlete's foot, etc.

[0343] In some implementations, the infectious disease is a disease caused by a parasitic infection, such as a disease caused by roundworms or malaria.

[0344] In some implementations, the infectious diseases include central nervous system infections such as purulent meningitis, tuberculous meningitis, encephalitis, etc.; skin infections such as pustules, rashes, etc.; lung infections such as pneumonia; liver infections such as hepatitis; and intestinal infections such as enteritis.

[0345] In some implementations, the allergic diseases include respiratory allergies, skin allergies, eye allergies, digestive allergies, etc.

[0346] In some implementations, the allergic diseases include allergic rhinitis, allergic bronchitis, asthma, dermatitis, eczema, drug eruption, papular urticaria, contact dermatitis, chronic urticaria, allergic conjunctivitis, etc.

[0347] In some implementations, the immune diseases include immunodeficiency, hyperimmune function, immunoproliferative diseases, and allergic reactions.

[0348] In some implementations, the immune diseases include mononucleosis syndrome, urticaria, asthma, allergic rhinitis, systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, vasculitis, autoimmune liver diseases (e.g., primary biliary cirrhosis, autoimmune hepatitis), rheumatic immune diseases (e.g., spondyloarthritis, degenerative diseases, metabolic and endocrine-related rheumatic diseases), and other autoimmune diseases (e.g., chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Graves' disease, Hashimoto's thyroiditis, polymyositis, glomerulonephritis, progressive systemic sclerosis).

[0349] In some embodiments, this application provides lipid nanoparticles as described above, wherein the nucleic acid molecule is selected from antisense oligonucleotides (ASO), RNA, or DNA.

[0350] In some embodiments, the RNA is selected from one or more of the following: interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long noncoding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozymes.

[0351] In some embodiments, the RNA is selected from at least one of the following: messenger RNA (mRNA), modified mRNA (mmRNA), circular RNA (circRNA), siRNA, gRNA, and self-replicating RNA (SrRNA).

[0352] In some implementations, the RNA is selected from mRNA or modified mRNA.

[0353] In some more specific implementations, the RNA is modified mRNA.

[0354] In some embodiments, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).

[0355] In some embodiments, the DNA is selected from one or more of plasmid DNA (pDNA), microcircular DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multicopy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA).

[0356] In some more specific embodiments, the DNA is selected from at least one of the following: plasmid DNA (pDNA) and microcircular DNA (mcDNA).

[0357] In some embodiments, the nucleic acid molecule is an mRNA molecule. In some embodiments, the mRNA molecule also contains a 5' UTR and / or a 3' UTR.

[0358] Various available 5'UTRs and / or 3'UTRs are known in the art. Those skilled in the art can determine the 5'UTR and / or 3'UTR applicable to this application.

[0359] In some implementations, the mRNA molecule also includes a poly(A) sequence.

[0360] In some embodiments of this application, the poly(A) sequence comprises approximately 20 to approximately 500 (e.g., consecutive) adenine nucleotides (A), for example, approximately 25, approximately 50, approximately 100, approximately 150, approximately 175, approximately 200, approximately 300, approximately 400, or approximately 500 (e.g., consecutive) adenine nucleotides (A). In some preferred embodiments, the poly(A) sequence comprises 120 (e.g., consecutive) adenine nucleotides (A). The addition of the poly(A) sequence contributes to the stability and transport of mRNA, prevents its degradation, and plays an important role in post-transcriptional modification. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but it can also be a variant containing non-adenine nucleotides, as long as its function is equivalent to that of a conventional poly(A) sequence, i.e., it can provide biological functions similar to those of the natural poly(A) sequence, such as affecting mRNA stability, translation efficiency, or ribosome binding. Known poly(A) sequences include the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants may differ in nucleotide composition but are considered functionally equivalent to the conventional poly(A) sequence.

[0361] In some embodiments, the mRNA molecule further includes a 5' cap structure. In some embodiments of this application, the 5' cap structure is a Cap1 cap structure.

[0362] In some embodiments, the mRNA molecule of this application may also contain at least one nucleotide modification. The at least one nucleotide modification includes, but is not limited to, cytidine modification, uridine modification, or adenosine modification. In some embodiments, the at least one nucleoside modification includes, but is not limited to, 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5molU).

[0363] In some embodiments, the mRNA is chemically synthesized. In some embodiments, the mRNA molecule is obtained through in vitro transcription.

[0364] Furthermore, the mRNA molecules involved in this application possess a certain degree of stability, tolerating a certain degree of insertion of additional sequences (such as microRNA binding sites) without affecting their translation ability or the stability of the mRNA molecules. In some embodiments, the additional sequences (such as microRNA binding sites) may be inserted into the 3'UTR. The microRNA binding site includes a full-length microRNA inverse complementary sequence (exemplary length may be 19-25 nt) or an inverse complementary sequence of its seed sequence (exemplary length may be 7-8 nt).

[0365] On the other hand, this application provides a nucleic acid vector containing the coding sequence of a nucleic acid molecule, such as an mRNA molecule, of this application. In some embodiments, the nucleic acid vector is used to generate a nucleic acid molecule, such as an mRNA molecule, of this application.

[0366] As used herein, a "vector" refers to a segment of DNA extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has already been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in the organism. A vector may contain, for example, an origin of replication, a selection marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, granules, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc.

[0367] In some embodiments, the nucleic acid vector further comprises an RNA polymerase promoter sequence operatively linked to the coding sequence of the nucleic acid molecule, such as an mRNA molecule. The operatively linked promoter allows for in vivo and / or in vitro transcription of the RNA molecule. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0368] In some implementations, the nucleic acid vector is a plasmid vector.

[0369] In some embodiments, the nucleic acid vector includes a restriction endonuclease site, such as an IIS-type restriction endonuclease site, on the 3' flanking of the coding sequence of the nucleic acid molecule, such as mRNA. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, and SapI. The restriction endonuclease site can be used to linearize the nucleic acid vector for in vitro transcription.

[0370] Methods for obtaining mRNA molecules from nucleic acid vectors through in vitro transcription are known in the art, for example, in vitro transcription can be performed using commercially available kits.

[0371] In some embodiments, this application provides lipid nanoparticles as described above, wherein the N:P molar ratio of N atoms in the ionizable lipid to P atoms in the loaded molecule is (1-15):1, preferably (2-12):1, and more preferably (2-9):1;

[0372] Preferably, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the loaded molecule is (2-15):1, more preferably (2-12):1, more preferably (2-10):1, more preferably (2-8):1, and even more preferably (2-6):1;

[0373] Preferably, the N:P molar ratio of the N atom in the ionizable lipid to the P atom in the loaded molecule is (1-12):1, more preferably (4-11):1, more preferably (4-10):1, and even more preferably (4-9):1.

[0374] In some embodiments, this application provides lipid nanoparticles as described above, wherein the particle size of the lipid nanoparticles is 30-500 nm, such as 30-300 nm, 30-275 nm, 30-250 nm, 30-225 nm, 30-200 nm, 30-175 nm, 30-150 nm, 30-120 nm, 30-100 nm, 40-300 nm, 40-275 nm, 40-250 nm, 40-225 nm, 40-200 nm, 40-175 nm, 40-150 nm, 40-120 nm or 40-100 nm, preferably 40-80 nm, more preferably 50-70 nm.

[0375] In some embodiments, the lipid nanoparticles have a particle size of about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, or about 180 nm.

[0376] In another aspect, this application provides a method for preparing lipid nanoparticles as described above, comprising: mixing the lipid components in the lipid nanoparticles and then mixing them with a loading device to obtain lipid nanoparticles.

[0377] In another aspect, this application provides a composition comprising the lipid nanoparticles as described above.

[0378] In another aspect, this application provides pharmaceutical compositions comprising lipid nanoparticles as described above and optionally pharmaceutically acceptable excipients, such as carriers, adjuvants, or mediators.

[0379] In another aspect, this application provides an immunogenic composition comprising the lipid nanoparticles as described above and optionally a pharmaceutically acceptable carrier.

[0380] In another aspect, this application provides the use of the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions as described above for the delivery of payloads.

[0381] In another aspect, this application provides a method for delivering a payload to a subject, comprising administering to the subject the lipid nanoparticles or composition or pharmaceutical composition or immunogenic composition as described above; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection.

[0382] In another aspect, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above in the preparation of products for gene editing, protein replacement and / or supplementation, and gene interference.

[0383] In another aspect, this application provides the use of the lipid nanoparticles or compositions or pharmaceutical compositions or immunogenic compositions as described above in the preparation of a medicament for delivery of a payload.

[0384] In another aspect, this application provides the use of the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions as described above in the preparation of medicaments or vaccines for the prevention, treatment or diagnosis of diseases.

[0385] In another aspect, this application provides a method for preventing, treating, or diagnosing a disease in a subject, comprising administering to the subject an effective amount of the lipid nanoparticles or composition, pharmaceutical composition, or immunogenic composition as described above; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection, or intraperitoneal injection.

[0386] In some embodiments of the uses or methods described above, the diseases include tumor diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, parasitic diseases, or autoimmune diseases.

[0387] In some embodiments of the uses and methods described above, the disease includes liver disease or liver-related disease; preferably, the liver disease or liver-related disease is a malignant tumor of the liver.

[0388] In some embodiments of the uses and methods described above, the subject is a mammal or human, preferably a primate such as a monkey, ape, orangutan, chimpanzee, or gorilla, such as a cynomolgus monkey or rhesus monkey, and more preferably a human.

[0389] In some embodiments, the lipid nanoparticles or composition, pharmaceutical composition or immunogenic composition are administered by injection, such as intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection, preferably intramuscular injection.

[0390] Those skilled in the art will understand that the actual dose level of the active ingredient in the pharmaceutical or immunogenic compositions of this application may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response to a particular subject, composition, and route of administration, without toxicity to the subject. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this application, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the subject receiving treatment, and similar factors known in the medical field.

[0391] The lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intrabursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0392] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions, immunogenic compositions, or methods of this application may be used in combination with one or more drugs or therapies selected from: immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, or one or more antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T-cell (CAR-T) therapy, and bispecific antibodies targeting tumor antigens, or one or more drugs for the prevention or treatment of infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections, or radiotherapy.

[0393] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions, immunogenic compositions, or methods of this application may be used in combination with one or more drugs for the prevention or treatment of infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections. In some embodiments, the viruses, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetal, or fungi are selected from the "Catalogue of Human Infectious Pathogens" (2023 Edition) published on the website of the National Health Commission of the People's Republic of China in 2023, the entire contents of which are incorporated herein by reference. In some embodiments, the drug for the prevention or treatment of viral infections is an anti-SARS-CoV-2 drug. In some implementations, the anti-SARS-CoV-2 drug may be any directly acting anti-SARS-CoV-2 drug, including but not limited to remdesivir (Gilead), REGN-COV2 (Regeneron), LY-CoV555 (Lilly), MK-4482 / EIDD-2801 (Merck / Ridgeback Bio); or immunomodulators, including but not limited to CD24Fc (Oncoimmune), T-COVIDTM (Altimmune), itutuzumab (Equillium Inc); or vaccines, including but not limited to AdCOVIDTM (Altimmune), BNT162b1 / 2 (Pfizer / BioNTech), mRNA-1273 (Moderna), AZD1222 / ChAdOx1 (AstraZeneca / Oxford Univ), Ad5-vectored COVID-19 vaccine (CanSino Biologics), CoronaVac (Sinovac), NVX-CoV2373 (Novavax).

[0394] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions, immunogenic compositions, or methods of this application may be used in combination with immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, one or more antibodies targeting tumor-specific antigens, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies.

[0395] In some embodiments, the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions of this application may be combined with radiotherapy, for example, including the administration of ionizing radiation to a subject earlier than, during, and / or later than the administration of the lipid nanoparticles or compositions, pharmaceutical compositions or immunogenic compositions of this application.

[0396] In some embodiments, the lipid nanoparticles, compositions, pharmaceutical compositions, or immunogenic compositions of this application can be combined with immunotherapies targeting tumor antigens such as GPC3 protein. Immunotherapies targeting tumor antigens such as GPC3 protein mainly include monoclonal antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies.

[0397] In another aspect, this application also provides a kit containing the lipid nanoparticles or composition, pharmaceutical composition, or immunogenic composition of this application, and optionally one or more drugs or therapies selected from: immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, or one or more antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies targeting tumor antigens, or one or more drugs for the prevention or treatment of infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections.

[0398] In some embodiments, this application provides a kit containing the lipid nanoparticles or composition, pharmaceutical composition, or immunogenic composition of this application, and one or more drugs for the prevention or treatment of infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsial, spirochetal, or fungal infections. In some embodiments, the drug for the prevention or treatment of viral infections is an anti-SARS-CoV-2 drug as described above.

[0399] There are no particular limitations on immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, and antibodies, antibody-drug conjugates, or tumor vaccines that can be used in combination with the lipid nanoparticles or compositions, pharmaceutical compositions, or immunogenic compositions of this application. Examples of the immunomodulators, chemotherapeutic agents, immune checkpoint inhibitors, and antibodies, antibody-drug conjugates, or tumor vaccines targeting tumor antigens include, but are not limited to: ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enoxabin, capecitabine, carmoflurane, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, deoxyfluorouracil, nerabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzosen, docetaxel, paclitaxel, vinorelbine, vincristine, vindesin, actinomycin D, arubicin, amrubicin, idarubicin, epirubicin, and fentostatin. Daunorubicin, Doxorubicin, Pirarubicin, Bleomycin, Pepromycin, Mitomycin C, Mitoxantrone, Oxaliplatin, Carboplatin, Cisplatin, Nedaplatin, Anastrozole, Exemestane, Ethinylestradiol, Chlormadinone, Goserelin, Tamoxifen, Dexamethasone, Bicalutamide, Toremifene, Flutamide, Prednisolone, S-estradiol, Mitotan, Methyltestosterone, Leuprorelin, Letrozole, Methylamphetamine Progesterone, tiimomab, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarbitine, trastuzumab, retinoic acid, perumumab, bevacizumab, bortezomib, lapatinib, atezolizumab, pembrolizumab, tislelizumab, camrelizumab, sugemalimab, and nivolumab, etc.

[0400] The lipid nanoparticles or compositions, pharmaceutical compositions, or immunogenic compositions of this application, along with the chemotherapeutic agents, immune checkpoint inhibitors, or antibodies, antibody-drug conjugates, or tumor vaccines targeting tumor antigens, or one or more of the drugs used to prevent or treat infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections, can be administered together or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0401] The dosage of the lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application, and the chemotherapeutic agents, immune checkpoint inhibitors, or antibodies, antibody-drug conjugates, or tumor vaccines targeting tumor antigens, or one or more of the drugs used to prevent or treat infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections, in the combination of this application, is not particularly limited. As mentioned above, the dosage of the lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application can be determined by referring to the dosage when the antibody is used alone. The chemotherapeutic agents, immune checkpoint inhibitors, or antibodies, antibody-drug conjugates, or tumor vaccines targeting tumor antigens, or one or more of the drugs used to prevent or treat infectious diseases such as viral, bacterial, actinomycete, chlamydia, mycoplasma, rickettsia, spirochetal, or fungal infections can be used according to the dosage specified for each drug or the dosage can be reduced (taking into account the combined effect with the lipid nanoparticles, pharmaceutical compositions, or immunogenic compositions of this application). Attached Figure Description

[0402] Figure 1 shows the in vivo luminescence of lipid nanoparticles LNP-3 and LNP-6 in six mice from two groups at a dose of 0.25 mpk.

[0403] Figure 2 shows: (A) fluorescence expression in different isolated organs of mice after LNP-3 administration, from left to right: heart, liver, spleen, lung, kidney, and brain; and (B) fluorescence expression in different isolated organs of mice after LNP-6 administration, from left to right: heart, liver, spleen, lung, kidney, and brain.

[0404] Figure 3 shows the fluorescence expression of LNP-4 in isolated liver and spleen of cynomolgus monkeys after administration.

[0405] Figure 4 shows the fluorescence expression of LNP-5 in different isolated organs of cynomolgus monkeys after administration.

[0406] Figure 5 shows the expression of LNP-2 in different organs of mice. All three mice (n=3) are shown in the figure. From left to right, the organs are heart, liver, spleen, lung, kidney and brain. Detailed Implementation

[0407] Example

[0408] To make the technical solution of this application clearer and more explicit, the following embodiments are provided for further detailed explanation. These embodiments are only used to illustrate specific implementation methods of this application, enabling those skilled in the art to understand it, but do not imply limitation of the scope of protection of this application. In the specific embodiments of this application, technical means or methods not specifically described are conventional technical means or methods in the art. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0409] Abbreviation Table

[0410] Example 1:

[0411] Preparation of MTS001

[0412] Under an inert nitrogen atmosphere, sodium hydride (2.0 g, 50.64 mmol, 1.6 eq.) was added to a tetrahydrofuran (50.0 mL) solution of nonanoic acid (5.0 g, 31.65 mmol, 1.0 eq.), and the resulting mixture was stirred at 0 °C for 10 min. Diisopropylaminolithium (2 mol / L, 28.50 mL, 56.97 mmol, 1.8 eq.) was added to the mixture at 0 °C. The resulting mixture was stirred at 0 °C for 10 min, and 1-iodoheptane (10.7 g, 47.48 mmol, 1.5 eq.) was added. The mixture was stirred at 40 °C for 12 h, the reaction was quenched with saturated ammonium chloride solution (10.0 mL), diluted with 500 mL of water and extracted with 3 x 500 mL of dichloromethane. The combined organic layers were washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate (3:1), to give 5 g (62%) 1-1, which was a yellow oily substance; 1 H NMR(300MHz, CDCl3)δ:0.85-0.89(m,6H),1.25-1.32(m,19H),1.41-1.67-2.27(m,4H),2.29-2.33(m,1H),10.29(s,1H);

[0413] At 0 °C, a borane-tetrahydrofuran complex solution (1 mol / L, 23.4 mL, 23.43 mmol, 3.0 eq.) was added to a stirred solution of 1-1 (2.0 g, 7.81 mmol, 1.0 eq.) in 5.0 mL of tetrahydrofuran. The resulting solution was heated to 75 °C and stirred for 3 hours. The mixture was cooled to room temperature, and the reaction was quenched with methanol (10.0 mL). The mixture was diluted with 100 mL of water and extracted with 3 × 200 mL of dichloromethane. The organic layers were combined, washed with 3 × 200 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate (20:1), to give 1.6 g (85%) of 1-2 as a colorless oil.

[0414] Under an inert nitrogen atmosphere, 8-bromooctanoic acid (1.11 g, 4.96 mmol, 1.2 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.20 mmol, 1.5 eq.), and 4-dimethylaminopyridine (756.0 mg, 6.20 mmol, 1.5 mol) were added to a solution of 1-2 (1.0 g, 4.13 mmol, 1.0 eq.) in 10.0 mL of dichloromethane. The resulting mixture was stirred at 25 °C for 6 hours, diluted with 100 mL of water, extracted with 3 x 300 mL of dichloromethane, and the organic layers were combined. The mixture was washed with 3 x 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate (50:1), to give 1 g (56%) of 1-3 as a yellow oil.

[0415] At 0 °C, lithium diisopropylamino (43.0 mL, 86.00 mmol, 2.0 eq.) was added to a stirred solution of methyl isobutyrate (4.4 g, 43.0 mmol, 1.0 eq.) in tetrahydrofuran (100.0 mL). The resulting mixture was stirred at 0 °C for 30 minutes. 1,5-Dibromo-pentane (20.0 g, 86.0 mmol, 1.0 eq.) was added to the above solution at 0 °C, and the resulting mixture was stirred at 25 °C for 5 hours. The reaction was quenched with saturated ammonium chloride solution (1.0 mL), diluted with 300 mL of water, extracted with 3 x 500 mL dichloromethane, and the combined organic layers were washed with 3 x 500 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with petroleum ether / ethyl acetate (50:1) to give 10 g (46%) 1-4, which was a light yellow oil.

[0416] At 0°C, a borane-tetrahydrofuran complex solution (100.0 mL) was added to a stirred solution of 1-4 (10.0 g, 40.0 mmol, 1.0 eq.) in 20.0 mL of tetrahydrofuran. The resulting solution was stirred at 75°C for 3 hours. The mixture was cooled to 25°C, diluted with 100 mL of water, and extracted with 3 x 500 mL of dichloromethane. The organic layers were combined, washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 8 g (90%) of 1-5 was obtained as a colorless oil. The preparation process was repeated to obtain a sufficient quantity of product.

[0417] At 0 °C, triethylamine (13.5 g, 134.0 mmol, 3.0 eq.) and decanoyl chloride (11.0 g, 58.0 mmol, 1.3 eq.) were added to a stirred solution of 1-5 (10.0 g, 44.00 mmol, 1.0 eq.) in 100.0 mL of dichloromethane. The resulting mixture was stirred at 25 °C for 3 hours, diluted with 100 mL of water, and extracted with 3 x 300 mL of dichloromethane. The organic layers were combined, washed with 3 x 300 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (10:1). 10 g (60%) of 1-6 was obtained as a colorless oil.

[0418] Under an inert nitrogen atmosphere, potassium carbonate (1.5 g, 11.1 mmol, 3.0 eq.) and 1-6 (1.4 g, 3.70 mmol, 1.0 eq.) were added to a stirred solution of ethanolamine (2.3 g, 37.2 mmol, 10.0 eq.) in acetonitrile (15.0 mL). The resulting solution was stirred at 70 °C for 3 hours. The mixture was cooled to 25 °C, diluted with 10 mL of water, extracted with 3 x 50 mL of dichloromethane, and the organic layers were combined. The mixture was washed with 3 x 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with dichloromethane / methanol (10:1), to give 1 g (76%) of 1-7 as a colorless oil. 1 H NMR(300MHz,CD3Cl)δ:0.85-0.89(m,9H),1.20-1.26(m,21H),1.59-1.62(m,4H) ),2.29-2.34(m,2H),2.73-2.76(m,2H),2.87-2.91(m,2H),3.72-3.77(m,3H);

[0419] Under a nitrogen-inert atmosphere, potassium carbonate (161.5 mg, 1.17 mmol, 3.0 eq.), sodium iodide (141.5 mg, 0.95 mmol, 2.5 eq.), and 1-7 (135.0 mg, 0.39 mmol, 1.0 eq.) were added to a solution of 1-3 (202.8 mg, 0.45 mmol, 1.2 eq.) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at 70 °C for 6 h. The mixture was cooled to 25 °C, diluted with 10 mL of water, extracted with 3 x 50 mL dichloromethane, and the organic layers were combined. The mixture was washed with 3 x 50 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography: Xselect CSH F-phenyl OBD column 19x 250 mm, 5 μm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 20 mL / min; gradient: from 75% B to 95% B in 9 minutes, yielding 83 mg (30%) MTS001 as a yellow oily substance.

[0420] 1 H NMR(300MHz, CDCl3)δ:0.80-0.90(m,15H),1.13-1.72(m,62H),2.22-2.27(m,4H),2.64-2.79(m,4H),3.71(s,2H),3.88-3.90(m,2H); MS m / z[M+H] + (ESI): 724.80.

[0421] Preparation of MTS003

[0422] 3-4 tert-butyl cyclopropylformate (1.5 g, 10.55 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (15.0 mL). Under nitrogen protection at -60 °C, LDA (2 mol / L, 10.5 mL, 21.10 mmol, 2.0 eq.) was added, and the reaction was stirred for 30 min. Then, 1-bromo-5-chloropentane (2.35 g, 12.66 mmol, 1.2 eq.) was added, and the reaction mixture was heated to room temperature and stirred for another 3 hours. After the reaction was complete, the mixture was quenched with saturated ammonium chloride solution (10.0 mL), diluted with water (500 mL), extracted with dichloromethane (300 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride aqueous solution (300 mL x 3) and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation to obtain a crude product. Purification by silica gel column chromatography yielded 1.3 g of a yellow oily compound 3-5.

[0423] Compound 3-5 (1.3 g, 5.27 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (15.0 mL) solution. Borane tetrahydrofuran complex (1 mol / L, 15.81 mL, 15.81 mmol, 3.0 eq.) was added at 0 °C under nitrogen protection. The mixture was stirred at 75 °C for 3 hours. After the reaction was complete, it was quenched with methanol (10.0 mL), diluted with water (100 mL), extracted with dichloromethane (200 mL x 3), and the organic phases were combined. The organic phases were washed with saturated sodium chloride aqueous solution (200 mL x 3), collected, and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation to obtain the crude product. Purification by silica gel column chromatography yielded 0.58 g of yellow oily compound 3-6.

[0424] Compound 3-6 (0.58 g, 3.28 mmol, 1.0 eq.) was dissolved in dichloromethane (10.0 mL), and triethylamine (993.8 mg, 9.84 mmol, 3.0 eq.) and decanoyl chloride (0.75 g, 3.94 mmol, 1.2 eq.) were added sequentially under nitrogen protection. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was diluted with water (50 mL), extracted with dichloromethane (100 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride aqueous solution (100 mL x 3), and the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to give 0.6 g of yellow oily compound 3-7.

[0425] Compounds 3-7 (0.6 g, 1.81 mmol, 1.0 eq.) and ethanolamine (1.11 g, 18.13 mmol, 10 eq.) were added to an acetonitrile (2 mL) solution. Under nitrogen protection, potassium carbonate (749.3 mg, 5.43 mmol, 3.0 eq.) and sodium iodide (678.8 mg, 4.53 mmol, 2.5 eq.) were added. The reaction was stirred at 50 °C for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with 10 mL of water, and extracted with dichloromethane (50 mL x 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL x 3), collected, and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation to obtain the crude product. Purification by silica gel column chromatography yielded 0.32 g of a yellow oily compound 3-8.

[0426] Compounds 1-3 (151.6 mg, 0.34 mmol, 1.2 eq.) and 3-8 (100 mg, 0.28 mmol, 1.0 eq.) were added to a 2 mL DMF solution. Under nitrogen protection, potassium carbonate (115.9 mg, 0.84 mmol, 3.0 eq.) and sodium iodide (105.0 mg, 0.70 mmol, 2.5 eq.) were added. The reaction was carried out at 70 °C with stirring for 6 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with 10 mL of water, and extracted with dichloromethane (50 mL x 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL x 3), collected, and dried over anhydrous sodium sulfate. The organic phase was filtered and the organic solvent was removed by rotary evaporation to obtain the crude product. The compound MTS003, a yellow oil, was purified by high performance liquid chromatography (column: Xselect CSH F-Phenyl OBD column 19x250 mm, 5 μm; phase A: acetonitrile / water (10 mM ammonium bicarbonate + 0.05% ammonia), phase B: isopropanol / acetonitrile; flow rate: 20 mL / min; gradient: 75% B to 95% B, 9 min) to obtain 88 mg of the compound MTS003.

[0427] 1 H NMR(300MHz, CDCl3)δ:0.44-0.48(m,4H),0.86-0.90(m,9H),1.27-1.46(m,52H),1.58-1.68(m,6H),2.27-2.32 (m,4H),2.34-2.48(m,4H),2.60-2.64(m,2H),3.55(t,J=5.1Hz,2H),3.90(s,2H),3.96(d,J=5.7Hz,2H); ESI-MS m / z:722.70[M+H] + .

[0428] Other ionizable lipid compounds can be prepared similarly using the method described in Example 1, and their physical data are as follows.

[0429] Example 2: Preparation of lipid nanoparticles

[0430] Materials used for assembling lipid nanoparticles include: (1) ionizable lipid compounds: ionizable lipids provided in this application, or Lipid5, SM102, and MC3 (purchased from AVT) as control groups; (2) structural lipids: such as cholesterol (purchased from Sigma-Aldrich); (3) phospholipids: such as DSPC, i.e., 1,2-distearyl-SN-glycerol-3-phosphocholine (purchased from AVT); (4) polyethylene glycol-modified lipid compounds: such as DMG-PEG2000, i.e., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (purchased from AVT); (5) nucleic acid molecules: such as Luciferase mRNA, siRNA, CRISPR Cas9 mRNA, etc. The sequences of several nucleic acid molecules used in the examples are known in the art and can be purchased from commercial sources. The names and structural formulas of the lipid nanoparticle assembly materials are detailed in Table 1.

[0431] Table 1

[0432] Preparation method of lipid nanoparticles: (1) Ionizable lipid compounds, cholesterol, phospholipids and polyethylene glycol-modified lipids are dissolved and mixed in ethanol in proportion (see specific examples below for formulation); (2) mRNA active ingredient is dissolved in 25mM sodium citrate solution (pH=4); (3) The organic phase containing the lipid mixture and the aqueous phase containing the mRNA are mixed at a flow rate ratio of 1:1 to 1:4 using an automated high-throughput microfluidic system, with a total mixing flow rate of 10mL / min to 20mL / min; (4) The prepared lipid nanoparticles (N / P ratio of 6) are diluted with phosphate buffer solution and ultrafiltered to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 100kDa (purchased from Millipore); (5) The obtained nanoparticles are sterilized by filtration through a 0.22μm sterile filter membrane and then stored at low temperature in a sealed glass bottle.

[0433] Lipid nanoparticles can be prepared using microfluidic mixing systems, but are not limited to this method; other methods include T-type mixers and ethanol injection.

[0434] Example 3: Characterization of the physical properties of lipid nanoparticles

[0435] The particle size and particle size distribution index (PDI) of the prepared lipid nanoparticles were measured using a Malvern Nanometer particle size potentiometer (purchased from Malvern Instruments Ltd.). The degree of RNA encapsulation by the lipid nanoparticles was characterized by the encapsulation efficiency (%), which reflects the degree of binding between the lipid nanoparticles and the RNA fragments. This coefficient was obtained from Quant-it... TM The RiboGreen RNA Assay (purchased from Invitrogen) method was used for measurement. Lipid nanoparticle samples were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and a portion of the sample solution was added to 0.5% Triton X-100 and incubated at 37°C for 30 minutes. Immediately after the reaction, the fluorescence values ​​were read using a Varioskan LUX multi-functional microplate reader (purchased from Thermofisher) at an absorption wavelength of 485 nm and an emission wavelength of 528 nm to obtain the encapsulation rate.

[0436] Example 4: Delivery effect of lipid nanoparticles in mice

[0437] The delivery efficiency of lipid nanoparticles loaded with luciferase mRNA (commercially purchased, Trilink, L-7202) (specific formulation shown in Table 2, N:P = 6) in mice was evaluated. The experimental mice were SPF-grade C57BL / 6 mice, female, 6-8 weeks old, weighing 18-22g, n=3, purchased from Beijing Spefol Biotechnology Co., Ltd. All animals underwent acclimatization for more than 7 days before the experiment, with free access to food and water, 12 / 12h light / dark cycles, an indoor temperature of 20-26℃, and a humidity of 40-70%. Mice were randomly assigned to groups. The prepared lipid nanoparticles loaded with luciferase mRNA were administered intravenously to mice at a single dose of 0.25 mg / kg mRNA. Six hours after administration, in vivo bioluminescence detection was performed using a small animal in vivo imaging system (IVIS LUMINA III, purchased from PerkinElmer). The specific operational steps for the detection are as follows: A 20 mg / mL D-fluorescein solution was prepared using physiological saline, and the substrate was administered to each mouse via intraperitoneal injection. Ten minutes after administration, the mice were anesthetized in an anesthesia box with 2.5% isoflurane. The anesthetized mice were placed in an IVIS for fluorescence imaging, and data were collected and analyzed from areas of concentrated fluorescence distribution. The physical property characterization data and the average total photon count in vivo 6 hours after administration are shown in Table 3. After imaging, the mice were removed, euthanized by cervical dislocation, and their organs were dissected. The removed mouse organs were placed in an IVIS for imaging again, and data were collected and analyzed from areas of concentrated fluorescence distribution. The in vivo delivery efficiency of the lipid nanoparticle carrier is expressed as the average of the fluorescence intensity and total photon count of different animals within the same test group. Higher values ​​of fluorescence intensity and total photon count indicate higher in vivo delivery efficiency of the lipid nanoparticles for the mRNA fragment. AVE_Total Flux represents the average Total Flux of 3 mice.

[0438] Table 2

[0439] Table 3

[0440] Example 5: Delivery effect of lipid nanoparticles in non-human primates (NHP)

[0441] The delivery efficiency of lipid nanoparticles loaded with luciferase mRNA (commercially purchased, Trilink, L-7202) (specific formulation shown in Table 4, N / P = 6) in cynomolgus monkeys was investigated. The experimental cynomolgus monkeys were ordinary females, 3-5 years old, weighing 2-6 kg, n = 1, purchased from Shanghai Medicilon Biopharmaceutical Co., Ltd. All animals underwent acclimatization for more than 5 days prior to the experiment, with free access to food and water, 12 / 12h light / dark alternation, an indoor temperature of 18-26℃, and a humidity of 40-70%. The prepared lipid nanoparticles loaded with luciferase mRNA were administered intravenously to the cynomolgus monkeys at a single dose of 1.5 mg / kg mRNA. Six hours after administration, in vitro bioluminescence detection of the cynomolgus monkey organs was performed using a small animal in vivo imaging system. The specific operational steps for the detection are as follows: A 30 mg / mL D-fluorescein solution (substrate) was prepared using physiological saline. The substrate was administered to the cynomolgus monkeys via intraperitoneal injection at a dose of 37.5 mg / kg. Ten to five minutes after substrate administration, the cynomolgus monkeys were euthanized by intramuscular or intravenous injection of salbutamol and xylazine hydrochloride. Heart, liver, spleen, lungs, kidneys, brain, spinal cord (cervical, thoracic, and lumbar regions), and ovarian tissues were collected. A 0.15 mg / mL substrate solution was then prepared, and the organs were immersed in the substrate for 1 minute before being placed in an IVIS (IVIS LUMINA III, purchased from PerkinElmer) for fluorescence imaging. Data was collected and analyzed from areas of concentrated fluorescence distribution.

[0442] The specific formulation of the lipid nanoparticles is shown in Table 4, and the characterization data is shown in Table 5. The in vivo delivery efficiency of the lipid nanoparticle carrier is expressed as the average of the fluorescence intensity and total photon count of different animals within the same test group. The higher the value of the total bioluminescence photon count, the higher the expression level of luciferase. The expression of LNP-4 in various organs (in vitro) of cynomolgus monkeys 6 hours after administration is shown in Table 6 (corresponding to Figure 3). The higher the values ​​of fluorescence intensity and total photon count, the higher the in vivo delivery efficiency of the lipid nanoparticles for the mRNA fragment in that organ (liver, lung, or spleen), and the better the targeting of the corresponding organ or tissue. The expression of LNP-3 and LNP-6 in mice is shown in Table 7. The ratio of the total photon count of LNP-4 and LNP-5 in the liver bioluminescence of NHP (cynomolgus monkey) to that in the spleen bioluminescence, and the ratio of the total photon count of LNP-2 and LNP-6 in the in vitro liver bioluminescence of mice to that in the in vitro spleen bioluminescence are shown in Table 8. Avg Radiance represents the average photon count (or average radiance).

[0443] Table 4

[0444] Table 5

[0445] Table 6

[0446] Table 7: Expression in different organs (ex-vivo) of mice (dose administered as 0.25 mpk mRNA).

[0447] The experimental results of LNP-3 in Example 4 and LNP-6 in Example 5 are shown in Figures 1 and 2. The results show that, under the same LNP formulation ratio, the ionizable lipid MTS001 is more effective than Lipid5 in delivering the payload in mice and has better targeting to the liver. Combined with the data in Table 7, this further demonstrates that the delivery effect of the ionizable lipid MTS001 is superior to that of Lipid5.

[0448] Table 8

[0449] As shown in Table 8, increasing the proportion of polymeric lipids, such as polyethylene glycol-modified lipids, in LNPs significantly increases liver-specific targeted delivery in both mice and NHP (e.g., 19x represents the factor of 19 in liver / spleen (ex-vivo) fluorescence intensity).

[0450] Example 6: Delivery effect of lipid nanoparticles prepared from the ionizable lipid compounds of this application in mice.

[0451] The preparation of lipid nanoparticles was carried out according to Example 2, the characterization of the physical properties of lipid nanoparticles was carried out according to Example 3, and the animal experimental methods were carried out according to Example 4. The specific formulation (molar percentage) of the lipid nanoparticles was as follows: ionizable lipid compounds (MTS002-MTS031): cholesterol: DSPC: DMG-PEG2000 = 50: 38.5: 10: 1.5, N / P = 6, loaded with luciferase mRNA (Luc mRNA, purchased from Trilink, L-7202). The ionizable lipid compounds used as controls were MC3 and SM102, whose LNP formulations were the same as those of MTS002-MTS031. The lipid nanoparticles loaded with luciferase mRNA prepared above were injected intravenously into mice at a single dose of 0.5 mg / kg mRNA. Six hours after administration, in vivo bioluminescence detection was performed on the mice using a small animal in vivo imaging system (IVIS LUMINAIII, purchased from PerkinElmer) (data acquisition and analysis of the areas of concentrated fluorescence distribution). The in vivo delivery efficiency of lipid nanoparticles is expressed as the average fluorescence intensity and total photon count of the same test group of animals, as shown in Table 9.

[0452] Table 9

[0453] Example 7: Delivery effect of lipid nanoparticles prepared using the ionizable lipid compounds of this application in mice.

[0454] The preparation of lipid nanoparticles was carried out according to Example 2, the physical properties of lipid nanoparticles were characterized according to Example 3, and the animal experimental methods were carried out according to Example 4. The experimental animals were rats, with 3 rats in each group. The drug was administered via tail vein injection at a dose of 0.5 mpk based on mRNA. The specific formulation (molar percentage) of the lipid nanoparticles was as follows: ionizable lipid compound (MTS001 or MTS003): cholesterol (CHO): DSPC: DMG-PEG2000 = 50:37.5:10:2.5 or 50:38.5:10:1.5, N / P = 6, and luciferase mRNA (purchased from Trilink, L-7202) was loaded. For the LNP designations below, for example, MTS001-2.5PEG indicates that the ionizable lipid used to prepare the LNP is MTS001, and the PEG lipid used in the formulation accounts for 2.5 mol% of the total lipids; MTS003-1.5PEG indicates that the ionizable lipid used to prepare the LNP is MTS003, and the PEG lipid used in the formulation accounts for 1.5 mol% of the total lipids (the dosage is 0.25 mpk based on mRNA), and so on. The physical characterization data of the LNPs and the average total flux data of the isolated liver are shown in Table 10.

[0455] Table 10

[0456] As shown in Table 10, by using the ionizable lipid compounds and LNP formulation of the present invention, increasing the proportion of polymer lipids, such as polyethylene glycol-modified lipids, in the LNP can significantly increase the liver-specific targeted delivery of the loaded nucleic acid molecules.

[0457] Example 8: Effect of lipid nanoparticles prepared using the ionizable lipid compounds of this application on gene editing.

[0458] Experiment 8-1: This experiment selected two LNP formulations, LNP-1 and LNP-2. First, the liver delivery efficiency of the two formulations was compared using the known lipid LP01. The LNP formulation with superior liver delivery efficiency was selected. Then, the ionizable lipid was replaced with MTS003 to investigate the liver delivery effect of the ionizable lipid provided by this invention under the preferred formulation. The preparation of the lipid nanoparticles was as described in Example 2, encapsulating luciferase mRNA (purchased from Trilink, L-7202). The physical properties of the lipid nanoparticles were characterized as described in Example 3. The animal experimental method was as described in Example 4. C57 mice, n=3, were administered the drug via tail vein injection at a dose of 0.5 mpk (mRNA). IVIS imaging was used to collect the average total photon count (Total Flux) of the liver in each group of animals. The specific formulation, physical characterization data, and liver delivery effect data of the LNPs in mice are shown in Table 11.

[0459] Table 11

[0460] Experiment 8-2: Gene Editing Experiment

[0461] The gene-editing effect of lipid nanoparticles (specific formulation shown in Table 12) loaded with Cas9 mRNA and sgRNA targeting the mouse TTR gene (sgRNA sequence WO2022271780A1, SEQ ID NO:35) in mice was evaluated. The experimental mice were SPF-grade C57BL / 6 mice (purchased from Beijing Spefol Biotechnology Co., Ltd.), female, 6-8 weeks old, weighing 18-22g. All animals were acclimatized for more than 7 days before the experiment, with free access to food and water, 12 / 12h light / dark cycles, an indoor temperature of 20-26℃, and a humidity of 40-70%. Mice were randomly divided into 8 groups, with 2 mice in each group. The prepared lipid nanoparticles loaded with Cas9 mRNA and sgRNA (mass 2:1) were injected intravenously into mice at single doses of 0.6, 0.3, 0.15, and 0.075 mpk (Cas9 mRNA + sgRNA). Seven days after drug administration, mice were euthanized by cervical dislocation, and their livers were dissected and genomic DNA was extracted. The DNA was then amplified by PCR using specific primer pairs (upstream primer: 5'-TCAGCAGGTTTGGAGTCAGC-3'; downstream primer: 5'-CAGACCCAATGCGCTAAAGC-3'). The product was then sequenced by Sanger sequencing, and the sequencing map was analyzed using TIDE software to calculate the gene editing efficiency.

[0462] Table 12

[0463] Table 13

[0464] The gene editing efficiency results in mouse liver are shown in Table 13. The results indicate that, at the same dosage, the lipid nanoparticles MTS003-1 prepared with MTS003 significantly improved the TTR gene editing efficiency of Cas9 mRNA / sgRNA in mouse liver compared to the lipid nanoparticles LP01-1 prepared with LP01. This demonstrates that the gene editing effect of MTS003-1 is significantly superior to that of the existing technology LP01-1.

[0465] Under similar experimental conditions, the ionizable lipids MTS001, MTS002, MTS004 to MTS031 of the present invention also achieved good gene editing efficiency in the LNP formulation provided by the present invention, with TTR gene editing efficiency in mouse liver ranging from about 10% to about 70%, especially from about 20% to about 70%.

[0466] The structural formula for LP01 (commercial purchase):

[0467] While the invention has been fully described through its embodiments, it is worth noting that various changes and modifications to the embodiments will be apparent to those skilled in the art, and such changes and modifications should be included within the scope of the appended claims.

[0468] The sequence involved in this application

Claims

1. A lipid nanoparticle comprising the following components in molar percentage of the total lipids in the particle: Ionizable lipids: about 25 mol% to about 75 mol%, preferably about 30 mol% to about 70 mol%, more preferably about 40 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, more preferably about 50 mol%; Structural lipids: about 5 mol% to about 60 mol%, preferably about 10 mol% to about 60 mol%, more preferably about 20 mol% to about 50 mol%, more preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, more preferably about 37 mol%; Neutral lipids: about 5 mol% to about 30 mol%, preferably about 5 mol% to about 25 mol%, more preferably about 5 mol% to about 20 mol%, more preferably about 8 mol% to about 20 mol%, more preferably about 8 mol% to about 15 mol%, more preferably about 10 mol%; Polymer lipids: about 1.5 mol% to about 6 mol%, preferably about 1.5 mol% to about 5 mol%, more preferably about 2 mol% to about 4.5 mol%, more preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, more preferably about 3 mol%.

2. The lipid nanoparticles of claim 1, wherein the ionizable lipid is a compound of formula (II) or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof. in, a = 1, 2, 3, 4, 5 or 6; b = 4, 5, 6, 7, 8, 9 or 10; c = 1, 2, 3, 4, 5 or 6; d = 0, 1, 2, 3 or 4; c+d = 3, 4, 5, 6, 7, 8 or 9; M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O)S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -S-S- and -S(O) 0-2 -; R1 and R2 are independently selected from C 4-25 Alkyl, C 4-25 alkenyl and C 4-25 Alkyne group, which is optionally surrounded by one or more R groups 1s Substitution, and one or more methylene units therein are optionally and independently substituted with -NR'-; R 1s Independently selected from H and C 1-20 Alkyl, -L c -OR c -L c -SR c and -L c -NR c R' c ; R and R' are each independently selected from H and C. 1-20 alkyl; L c Independently selected from chemical bonds and C 1-20 Alkylene; R c and R' c Independently selected from H and C 1-20 Alkyl, C 3-14 cycloalkyl groups and 3 to 14-membered heterocyclic groups; R4 and R5 are independently selected from C 1-8 Alkyl groups, which are optionally composed of one or more R groups 4s replace; Or R4, R5, together with the carbon atoms they are attached to, form C. 3-14 Cycloalkyl or 3 to 14-membered heterocyclic group, optionally surrounded by one or more R 4s replace; R 4s Independently selected from H, halogen, cyano, C 1-8 Alkyl, C 1-8 Halogenated alkyl groups, -L d -OR d -L d -SR d and -L d -NR d R' d ; L d Independently selected from chemical bonds and C 1-8 Alkylene; R d and R' d Independently selected from H and C 1-8 Alkyl, C 3-14 Cycloalkyl and 3 to 14-membered heterocyclic groups.

3. The lipid nanoparticles of claim 2, wherein M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably wherein M1 is -OC(O)- and M2 is -C(O)O-.

4. The lipid nanoparticles according to any one of claims 2-3, wherein R4 and R5 are independently C 1-8 Alkyl group, preferably C 1-3 Alkyl, more preferably methyl; or R4 and R5, together with the carbon atoms they are attached to, form C. 3-8 Cycloalkyl or 3 to 8-membered heterocyclic group, optionally surrounded by one or more R 4s Replace, where R 4s As defined in claim 2.

5. The lipid nanoparticles according to any one of claims 2-4, wherein R1 and R2 are independently selected from C 4-22 Alkyl groups, which are optionally composed of one or more R groups 1s Replace, where R 1s As defined in claim 2; preferably, R1 is C 8-12 Straight-chain alkyl with R2 being C 12-22 Branched alkyl; more preferably, R1 is C 9-11 Straight-chain alkyl with R2 being C 15-22 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 15-20 Branched alkyl; more preferably, R1 is a C9 straight-chain alkyl and R2 is a C9 branched alkyl. 16 C 17 C 18 C 19 Or C 20 Branched alkyl groups.

6. The lipid nanoparticles according to any one of claims 2-5, wherein a is an integer from 1 to 4, such as 1, 2, 3 or 4; b is an integer from 5 to 8, such as 5, 6, 7 or 8; c is an integer from 2 to 6, such as 2, 3, 4, 5 or 6; and d is an integer from 1 to 4, such as 1, 2, 3 or 4.

7. The lipid nanoparticles according to any one of claims 2-6, wherein the compound of formula (II) has the structure of formula (II-1) or (II-2): R1, R2, a, b, c, and d are defined as in any one of claims 2-6.

8. The lipid nanoparticles according to any one of claims 2-7, wherein R1 is nonyl or decyl, and R2 is selected from one of the following structures:

9. The lipid nanoparticles according to any one of claims 2-8, wherein the compound of formula (II) is selected from the following compounds or their pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers:

10. The lipid nanoparticles according to any one of claims 1-9, wherein the structural lipid is a steroid or an analogue thereof, the steroid or analogue thereof being selected from one or more of the following: cholesterol, sitosterol, coccosterol, lycopene, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, and campesterol; preferably, the structural lipid is selected from cholesterol and / or β-sitosterol; more preferably, the structural lipid is cholesterol.

11. The lipid nanoparticles according to any one of claims 1-10, wherein the neutral lipid is a phospholipid, the phospholipid being selected from phosphatidylcholine and / or phosphatidylethanolamine; preferably, the phospholipid is selected from one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC). 1-Palmyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonium)ethyl hydrogen phosphate (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), 1-palmyl-2-oleoylphosphatidylethanolamine (POPE) and dipalmitoylphosphatidylethanolamine (DPPE) and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol)sodium salt (DOPG), sphingomyelin and mixtures thereof, more preferably DSPC and / or DOPE.

12. The lipid nanoparticles according to any one of claims 1-11, wherein the polymeric lipid is a polyethylene glycol-modified lipid; Preferably, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; Preferably, the PEGylated lipid contains a PEG portion of 1000 Da to 20 kDa, and more preferably contains a PEG portion of about 1000 Da to about 5000 Da; Preferably, the PEGylated lipid is selected from one or more of the following: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000, DSPE-PEG2000-Mannose, DSPE-PEG2000 The amine, DSPE-PEG3350, DSPE-PEG3500, DMG-PEG3500, DPPE-PEG3500, DSPE-PEG4000, DMG-PEG4000, Ceramide-PEG5000, DSPE-PEG5000, DMG-PEG5000 and ALC-0159, preferably ALC-0159, DMG-PEG2000, DSPE-PEG2000, DMG-PEG3500, DMG-PEG4000 and / or DMG-PEG5000, more preferably DMG-PEG2000.

13. The lipid nanoparticles according to any one of claims 1-12, comprising the following components in molar percentage: Ionizable lipids: about 40 mol% to about 60 mol%, preferably about 40 mol% to about 55 mol%, more preferably about 45 mol% to about 55 mol%, more preferably about 50 mol%; Structural lipids: about 20 mol% to about 50 mol%, preferably about 30 mol% to about 45 mol%, more preferably about 35 mol% to about 40 mol%, even more preferably about 37 mol%, or even more preferably about 37.5 mol%; Neutral lipids: about 5 mol% to about 20 mol%, preferably about 8 mol% to about 15 mol%, more preferably about 8 mol% to about 12 mol%, even more preferably about 10 mol%; and Polymer lipids: about 1.5 mol% to about 5 mol%, preferably about 2 mol% to about 4 mol%, more preferably about 2.5 mol% to about 3.5 mol%, more preferably about 2.2 mol% to about 3.2 mol%, more preferably about 2.5 mol% to about 3 mol%, more preferably about 2.5 mol%, or more preferably about 3 mol%; Preferably, the lipid nanoparticles comprise the following components in molar percentage: Ionizable lipids: approximately 50 mol%; Structural lipids: approximately 37 mol%; Neutral lipids: approximately 10 mol%; and Polymer lipids: Approximately 3 mol%; Preferably, the lipid nanoparticles comprise the following components in molar percentage: Ionizable lipids: approximately 50 mol%; Structural lipids: approximately 37.5 mol%; Neutral lipids: approximately 10 mol%; and Polymer lipids: approximately 2.5 mol%.

14. The lipid nanoparticles according to any one of claims 1-13, further comprising at least one load selected from therapeutic agents, preventive agents, or diagnostic agents; Preferably, the therapeutic agent, preventive agent, or diagnostic agent is selected from any one or more of the following: small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules; More preferably, the small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules are small molecule compounds, peptides, proteins, antibodies, and nucleic acid molecules used for the prevention, treatment, or diagnosis of the following diseases: tumor diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases, and other diseases such as cancer, viral infections, or autoimmune diseases; or liver diseases or liver-related diseases, preferably malignant tumors of the liver.

15. The lipid nanoparticles of claim 14, wherein the nucleic acid molecule is selected from antisense oligonucleotides (ASO), RNA, or DNA; Preferably, the RNA is selected from one or more of the following: interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozymes; more preferably, the RNA is selected from one or more of the following: messenger RNA (mRNA), modified mRNA (mmRNA), circular RNA (circRNA), siRNA, gRNA, and self-replicating RNA (SrRNA), preferably mRNA or modified mRNA, siRNA, or gRNA, more preferably modified mRNA; or Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and more preferably from one or more of plasmid DNA (pDNA), microcircular DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multicopy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); more preferably, the DNA is selected from at least one of plasmid DNA (pDNA) and microcircular DNA (mcDNA).

16. The lipid nanoparticles according to any one of claims 1-15, wherein the N:P molar ratio of N atoms in the ionizable lipid to P atoms in the loaded molecule is (1-15):1, preferably (2-12):1, and more preferably (2-9):1; Preferably, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the loaded molecule is (2-15):1, more preferably (2-12):1, more preferably (2-10):1, more preferably (2-8):1, and even more preferably (2-6):1; Preferably, the N:P molar ratio of the N atom in the ionizable lipid to the P atom in the loaded molecule is (1-12):1, more preferably (4-11):1, more preferably (4-10):1, and even more preferably (4-9):

1.

17. The lipid nanoparticles according to any one of claims 1-16, wherein the particle size of the lipid nanoparticles is 30-500 nm, for example 30-300 nm, 30-275 nm, 30-250 nm, 30-225 nm, 30-200 nm, 30-175 nm, 30-150 nm, 30-120 nm, 30-100 nm, 40-300 nm, 40-275 nm, 40-250 nm, 40-225 nm, 40-200 nm, 40-175 nm, 40-150 nm, 40-120 nm or 40-100 nm, preferably 40-80 nm, more preferably 50-70 nm.

18. A method for preparing the lipid nanoparticles according to any one of claims 1-17, comprising: The lipid components in the lipid nanoparticles are mixed and then mixed with a load to obtain lipid nanoparticles.

19. A composition comprising the lipid nanoparticles according to any one of claims 1-17.

20. A pharmaceutical composition comprising any one of the lipid nanoparticles of claims 1-17 and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant, or mediator.

21. An immunogenic composition comprising lipid nanoparticles according to any one of claims 1-17 and optionally pharmaceutically acceptable excipients, such as carriers, adjuvants, or mediators.

22. Use of the lipid nanoparticles of any one of claims 1-17, or the composition of claim 19, or the pharmaceutical composition of claim 20, or the immunogenic composition of claim 21, for the delivery of a payload, preferably for the specific delivery of a payload to the liver.

23. A method of delivering a payload to a subject, comprising administering to the subject any of the lipid nanoparticles of claims 1-17 or the composition of claim 19 or the pharmaceutical composition of claim 20 or the immunogenic composition of claim 21; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection or intraperitoneal injection.

24. Use of the lipid nanoparticles of any one of claims 1-17, the composition of claim 19, the pharmaceutical composition of claim 20, or the immunogenic composition of claim 21 in the preparation of products for gene editing, protein replacement and / or supplementation, or gene interference.

25. Use of the lipid nanoparticles of any one of claims 1-17, the composition of claim 19, the pharmaceutical composition of claim 20, or the immunogenic composition of claim 21 in the preparation of a medicament for delivery of a payload.

26. Use of the lipid nanoparticles of any one of claims 1-17, the composition of claim 19, the pharmaceutical composition of claim 20, or the immunogenic composition of claim 21 in the preparation of a medicament or vaccine for the prevention, treatment, or diagnosis of a disease.

27. A method for preventing, treating, or diagnosing a disease in a subject, comprising administering to the subject an effective amount of any of claims 1-17, or the composition of claim 19, or the pharmaceutical composition of claim 20, or the immunogenic composition of claim 21; preferably, the administration is performed via parenteral administration, such as by intramuscular injection, intravenous injection, arterial injection, or intraperitoneal injection.

28. The use or method according to any one of claims 22-27, wherein the disease includes tumor diseases, parasitic diseases, infectious diseases, allergic diseases, immune diseases, genetic diseases and other diseases such as cancer, viral infections or autoimmune diseases.

29. The use or method according to any one of claims 22-27, wherein the disease includes liver disease or liver-related disease; preferably, the liver disease or liver-related disease is a malignant tumor of the liver.

30. The use or method according to any one of claims 22-29, wherein the subject is a mammal or human, preferably a primate, such as a monkey, ape, orangutan, chimpanzee or gorilla, such as a cynomolgus monkey or rhesus monkey, more preferably a human.

31. A kit comprising any one of 1-17 lipid nanoparticles or the composition of claim 19 or the pharmaceutical composition of claim 20 or the immunogenic composition of claim 21, and optionally other pharmaceuticals or vaccines.

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