Lipid compounds and lipid nanoparticles for delivery

Abstract

The invention discloses a compound, a structural formula of the compound is shown as a formula (I), pharmaceutically acceptable salt or stereoisomer of the compound, and further discloses a nanoparticle composition containing the compound or the pharmaceutically acceptable salt or stereoisomer of the compound. The treatment or prevention purposes of medicines and vaccines are achieved. # imgabs0 #

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to lipid compounds and lipid nanoparticle compositions for delivering effective ingredients such as drugs and vaccines. Background Art

[0002] The effective and targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids is an ongoing medical challenge. The key to the success of gene therapy is whether the therapeutic drugs can be safely and effectively delivered into the target cells through the carrier in vivo. Due to the relative instability of nucleic acids and the low cell permeability of such substances, the delivery of nucleic acids to cells becomes difficult. Therefore, it is necessary to develop methods and compositions to promote the delivery of therapeutic and / or preventive drugs such as nucleic acids to cells. Gene therapy vectors are divided into viral vectors and non-viral vectors. Although viral vectors are used as efficient delivery systems to achieve target gene transfection and treatment purposes, viral vectors contain immunogenic viral proteins, limited target gene loading capacity and high prices. As a non-viral carrier, lipid nanoparticles (LNPs) have received widespread attention due to their good in vitro stability, degradability in vivo, safety and reliability, and are widely used in gene therapy research for congenital and acquired genetic defects.

[0003] Lipid-containing nanoparticles or lipid nanoparticles, liposomes and lipid complexes have been shown to be effective transport carriers for bioactive substances such as small molecule drugs, proteins and nucleic acids entering and / or inside cells. LNPs refer to small vesicles formed by one or more lipid components, which can effectively compress and deliver various nucleic acid molecules, from DNA, RNA to chromosomes and even cells; LNPs are conducive to large-scale production due to their defined construction scheme and easy modification of targeting ligands.

[0004] LNPs generally include one or more cationic lipids and / or amino (ionizable) lipids, phospholipids containing polyunsaturated lipids, structural lipids (such as sterols) and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Summary of the invention

[0005] Based on this, the present application discloses a lipid compound and a lipid nanoparticle composition comprising the compound, wherein the lipid has the advantages of high encapsulation rate, high expression, spleen targeting, etc.

[0006] Specifically, this application adopts the following technical solutions

[0007] 1. A compound of formula (I), or a pharmaceutically acceptable salt or a stereoisomer thereof,

[0008]

[0009] Wherein R1 is a C1-C12 alkyl group; R2 and R3 are each independently a C1-C11 alkyl group; R4 and R5 are each independently a C1-C3 alkyl group;

[0010] X is (C=O)O, O(C=O), O(C=O)O, (C=O)NH or NH(C=O), and Y and Z are each independently (C=O)O or O(C=O);

[0011] o Selected from 1, 2, 3, 4 or 5;

[0012] p is selected from 3, 4, 5, 6, 7 or 8;

[0013] q is selected from 3, 4, 5, 6, 7 or 8.

[0014] 2. The compound according to item 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is a C7-C12 alkyl group, preferably a C10 alkyl group or a C11 alkyl group.

[0015] 3. The compound according to item 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is a C7-C12 straight-chain alkyl group, preferably a C10 straight-chain alkyl group or a C11 straight-chain alkyl group.

[0016] 4. The compound according to any one of items 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R2 is a C6-C11 alkyl group, preferably a C7-C9 alkyl group, and more preferably a C8 alkyl group.

[0017] 5. The compound according to any one of items 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R2 is a C6-C11 straight-chain alkyl group, preferably a C7-C9 straight-chain alkyl group, and more preferably a C8 straight-chain alkyl group.

[0018] 6. The compound according to any one of items 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R3 is a C6-C11 alkyl group, preferably a C7-C9 alkyl group, and more preferably a C8 alkyl group.

[0019] 7. The compound according to any one of items 1 to 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R3 is a C6-C11 straight-chain alkyl group, preferably a C7-C9 straight-chain alkyl group, and more preferably a C8 straight-chain alkyl group.

[0020] 8. The compound according to any one of items 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R4 is methyl, ethyl, propyl or isopropyl, preferably methyl.

[0021] 9. The compound according to any one of items 1 to 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R5 is methyl, ethyl, propyl or isopropyl, preferably methyl.

[0022] 10. The compound according to any one of items 1 to 9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein X, Y, and Z are all (C=O)O, or X, Y are all (C=O)O and Z is O(C=O), or X is (C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)O, Y and Z are all O(C=O), or X is O(C=O), Y and Z are all (C=O)O, or X is O(C=O), Y is (C=O)O, and Z is O(C=O), or X, Y are all O(C=O), and Z is (C=O)O, or X, Y, and Z are all O(C=O), or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all O(C=O), or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all O(C=O), or X is is O(C=O)O, Y is (C=O)O, and Z is O(C=O), or X is O(C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)NH, and both Y and Z are (C=O)O, or X is (C=O)NH, and both Y and Z are O(C=O), or X is (C=O)NH, and both Y and Z are O(C=O), or X is (C=O)NH, and Y is (C=O)O, and Z is O(C=O), or Preferably, X is (C=O)NH, Y is O(C=O), and Z is (C=O)O, or X is NH(C=O), and Y and Z are both (C=O)O, or X is NH(C=O), and Y and Z are both O(C=O), or X is NH(C=O), and Y and Z are both O(C=O), or X is NH(C=O), and Y is (C=O)O, and Z is O(C=O), or X is NH(C=O), and Y is O(C=O), and Z is (C=O)O. Preferably, X, Y, and Z are all (C=O)O, or X is (C=O)O, and Y is O(C=O), and Z is (C=O)O, or X is (C=O)NH, and Y and Z are all O(C=O), or X is O(C=O)O, and Y and Z are all (C=O)O.

[0023] 11. The compound according to any one of items 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein o is selected from 2, 3 or 4, preferably 3 or 4.

[0024] 12. The compound according to any one of items 1 to 11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein p is selected from 5, 6 or 7, preferably 5 or 6.

[0025] 13. The compound according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein q is selected from 6, 7 or 8, preferably 7.

[0026] 14. The compound according to item 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the compound of (I) is selected from

[0027] Compound 1

[0028] Compound 2

[0029] Compound 3

[0030] Compound 4

[0031] Compound 5

[0032] Compound 6

[0033] Compound 7

[0034] Compound 8

[0035] Compound 9

[0036] Compound 10

[0037] Compound 11

[0038] Compound 12 Compound 13

[0039] 15. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises a compound as described in any one of items 1-14.

[0040] 16. A lipid nanoparticle composition according to claim 15, wherein the lipid component further comprises a phospholipid.

[0041] 17. The lipid nanoparticle composition according to item 16, wherein the phospholipid is selected from one or more of the following compounds:

[0042] Dilauroyl phosphatidylcholine (DLPC),

[0043] Dimyristoylphosphatidylcholine (DMPC),

[0044] Dioleoylphosphatidylcholine (DOPC),

[0045] Dipalmitoylphosphatidylcholine (DPPC),

[0046] Distearoylphosphatidylcholine (DSPC),

[0047] Dioleoylphosphatidylcholine (DUPC),

[0048] Palmitoyloleoylphosphatidylcholine (POPC),

[0049] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0050] 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0051] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0052] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0053] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0054] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0055] 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE),

[0056] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0057] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0058] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0059] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0060] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0061] 1,2-Diol-sn-glycero-3-phospho-(1-glycero) sodium salt (DOPG) or sphingomyelin.

[0062] 18. A nanoparticle composition according to claim 16, wherein the phospholipid is DOPE.

[0063] 19. A nanoparticle composition according to claim 16, wherein the phospholipid is DSPC.

[0064] 20. A nanoparticle composition according to any one of items 15-19, wherein the lipid component further comprises a structural lipid.

[0065] 21. The nanoparticle composition of claim 20, wherein the structural lipid is selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol.

[0066] 22. A nanoparticle composition according to any one of claim 20, wherein the structural lipid is cholesterol.

[0067] 23. A nanoparticle composition according to any one of items 15-22, wherein the lipid component further comprises PEG lipids.

[0068] 24. A nanoparticle composition according to any one of claim 23, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol.

[0069] 25. A nanoparticle composition according to any one of items 15-24, wherein the lipid component further comprises cationic and / or ionizable lipids.

[0070] 26. The nanoparticle composition according to any one of items 15-25, further comprising a therapeutic and / or prophylactic agent selected from a vaccine or a compound capable of eliciting an immune response, a nucleic acid,

[0071] Preferably, the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

[0072] 27. A nanoparticle composition according to any one of items 15-26, wherein the encapsulation efficiency of the therapeutic agent and / or prophylactic agent is ≥50%; or ≥80%; or ≥90%.

[0073] 28. A nanoparticle composition according to any one of items 15-27, wherein the average particle size of the nanoparticle composition is 60nm-130nm.

[0074] 29. The nanoparticle composition of any one of items 15-28, wherein the nanoparticle composition has a dispersibility index of 0.04-0.20.

[0075] 30. Use of a compound according to any one of items 1 to 14 in the preparation of a lipid nanoparticle composition.

[0076] 31. A pharmaceutical composition comprising the nanoparticle composition of any one of items 15-29 and a pharmaceutically acceptable carrier.

[0077] 32. A method for delivering a therapeutic and / or prophylactic agent to mammalian cells, the method comprising administering to a subject the nanoparticle composition described in any one of items 15-29 or the pharmaceutical composition described in item 31, wherein the administration comprises contacting the cells with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or prophylactic agent to the cells.

[0078] 33. A method according to claim 32, wherein the mammalian cell is in a mammal.

[0079] 34. The method of item 32 or 33, wherein the mammal is a human.

[0080] 35. A method according to any one of items 32-34, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0081] 36. A method for producing a target polypeptide in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition described in any one of items 15-29 or the pharmaceutical composition described in item 31 to deliver a therapeutic and / or prophylactic agent to the cell, wherein the therapeutic and / or prophylactic agent is mRNA, which encodes the target polypeptide, such that the mRNA can be translated in the cell to produce the target polypeptide.

[0082] 37. The method of claim 36, wherein the mammalian cell is in a mammal.

[0083] 38. A method according to any one of items 36 or 37, wherein the mammalian cell is human.

[0084] 39. The method of any one of items 36-38, wherein the nanoparticle composition or the pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0085] 40. A method for treating a disease or condition in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of items 15-29 or the pharmaceutical composition of item 31.

[0086] 41. A method according to claim 40, wherein the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0087] 42. A method according to item 40 or 41, wherein the disease or condition is selected from an infectious disease, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases or metabolic diseases.

[0088] 43. A method according to any one of items 40-42, wherein the mammal is a human.

[0089] 44. The method of any one of items 40-43, wherein the nanoparticle composition or the pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0090] 45. A method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising administering to the mammal a nanoparticle composition described in any one of items 15-29 or a pharmaceutical composition described in item 31, wherein the administration comprises contacting the mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the organ.

[0091] 46. ​​The method of claim 45, wherein the mammal is a human.

[0092] 47. The method of item 45 or 46, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0093] 48. A method according to any one of items 45-47, wherein the mammal is pre-treated 24 hours or less prior to the contacting or administering step.

[0094] 49. The method of any one of items 45-48, wherein the mammal is pretreated about one hour prior to the contacting or administering step.

[0095] Effects of the Invention

[0096] The compounds of the present application can be used for the preparation of lipid nanoparticles. The nanoparticle compositions containing the compounds provided in the present application can achieve the encapsulation and delivery of therapeutic / prophylactic agents, safely deliver the therapeutic / prophylactic agents to the targeted locations, achieve high expression, and exert the effects of the therapeutic / prophylactic agents.

[0097] The lipid nanoparticles prepared in the present application have a small average particle size, a high encapsulation rate, a high expression, and spleen targeting characteristics, and have broad application prospects in the field of drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0099] Figure 1 Shown are the hEPO expression levels exhibited by different LNP formulations 6 hours after intramuscular injection.

[0100] Figure 2 Shown are the luciferase fluorescence intensities of different LNP formulations 6 hours after intramuscular injection.

[0101] Figure 3 Shown is the luciferase fluorescence intensity displayed in the spleen 6 hours after intramuscular injection of different LNP formulations. DETAILED DESCRIPTION

[0102] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0103] Terms and Definitions

[0104] As used herein, the term "alkyl" refers to a group containing one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms), which is optionally substituted. The term "C1-C12 alkyl" refers to an optionally substituted straight or branched saturated hydrocarbon comprising 1-12 carbon atoms. Unless otherwise specified, the alkyl described herein refers to unsubstituted and substituted alkyls.

[0105] Unless otherwise indicated, the alkyl group may be optionally substituted. The optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro or iodo), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxyl, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride and iodide), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals, phosphates, thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfites (e.g., -S(O)R), In some embodiments, the substituents include, but are not limited to, -S(O)OH), sulfonic acid (e.g., -S(O)OH), thiol (e.g., -C(S)H), sulfate, sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR2 or -N(R)C(O)R), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, -NRH or -NH2), carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH or -OC(O)NH2), sulfonamide, alkyl, alkenyl and cyclic (e.g., carbocyclyl or heterocyclyl). In any of the foregoing, R is alkyl or alkenyl as defined herein. In some embodiments, the substituents themselves may be further substituted with one, two, three, four, five or six substituents, e.g., as defined herein. For example, a C6-11 alkyl group can be further substituted with 6, 7, 8, 9, 10, or 11 substituents as described herein.

[0106] As used herein, the term "compound" is meant to include all isomers and isotopes of the described structure. "Isotopes" refer to atoms with the same atomic number, but with different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts or complexes of the present application can be prepared by conventional methods in combination with solvents or water molecules to form sols and hydrates.

[0107] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. The method of contacting a cell with an external entity in vivo and in vitro is well known in the biological field. For example, a nanoparticle composition can be contacted with a mammalian cell placed in a mammal by a variety of routes of administration (e.g., intravenous, intramuscular, intradermal and subcutaneous), and can involve a variety of nanoparticle compositions. In addition, the nanoparticle composition can contact more than one mammalian cell.

[0108] As used herein, the term "delivery" refers to providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may include administering to the subject a nanoparticle composition comprising a therapeutic and / or prophylactic agent (e.g., by intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0109] As used herein, the term "enhanced delivery" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) therapeutic and / or preventive drugs to target tissue (e.g., mammal liver) by nanoparticles compared to the level of therapeutic and / or preventive drugs delivered to target tissue (e.g., MC3, KC2 or DLinDMA) by control nanoparticles. Can be by comparing the amount of protein produced in tissue with the weight of the tissue, the therapeutic and / or preventive amount in tissue with the weight of tissue, the amount of protein produced in tissue with the amount of total protein in tissue, or the amount of treatment and / or preventive agent in tissue with the total treatment and / or preventive agent in the tissue. It should be understood that the enhanced delivery of nanoparticles to target tissue does not need to be determined in the treated subject, but can be determined in a substitute such as an animal model (e.g., rat model). In some embodiments, nanoparticle compositions including a compound of Formula (I) have substantially the same level of delivery enhancement regardless of the route of administration.

[0110] As used herein, the term "specific delivery" or "specific transport" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) therapeutic and / or preventive drugs to a target tissue (e.g., mammalian liver) by nanoparticles than to a non-target tissue. The level of delivery of nanoparticles to a specific tissue can be measured by comparing the weight of protein produced in a tissue with the weight of the tissue, comparing the therapeutic and / or preventive amount in a tissue with the weight of the tissue, comparing the protein weight produced in a tissue with the total protein weight in the tissue, or comparing the therapeutic and / or preventive amount in a tissue with the total therapeutic and / or preventive amount in the tissue.

[0111] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition, relative to the total amount of therapeutic or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of the therapeutic and / or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency may be 97%. As used herein, "encapsulation" may refer to complete, substantial or partial encapsulation, enclosure, surrounding or encapsulation.

[0112] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0113] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism).

[0114] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant or microorganism, or a cell or tissue thereof).

[0115] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0116] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may contain one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers). The present application encompasses any and all isomers of the compounds described herein. Enantiomeric and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0117] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, PEGylated lipids, structural lipids or other lipids, such as phospholipids.

[0118] As used herein, a "linker" is a portion that connects two parts, for example, the connection between two nucleosides in a cap. A connector can include one or more groups, including but not limited to a phosphate group (e.g., phosphate, borophosphate, thiophosphate, selenophosphate, and phosphate), an alkyl, an amide, or a glycerol. For example, two nucleosides of a cap analog can be connected at their 5' positions by a triphosphate group or by a chain comprising two phosphate moieties and a boric acid phosphate moiety.

[0119] As used herein, "administration method" may include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering the composition to the subject. Any administration method can be selected to target delivery (e.g., specific delivery) to a specific area or system of the body.

[0120] As used herein, "modified" refers to non-natural. For example, RNA can be a modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides or linkers. A "modified" substance may also be referred to as an "engineered" substance herein. The substance may be modified or engineered chemically, structurally or functionally. For example, a modified nucleobase species may include one or more non-naturally occurring substitutions.

[0121] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. The particle size of the nanoparticle composition is generally in the order of microns or less, and may include a lipid bilayer. The nanoparticle composition includes lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle composition may be a liposome having a lipid bilayer having a diameter of 500 nm or less.

[0122] As used herein, "naturally occurring" means occurring in nature without human assistance.

[0123] As used herein, "patient" refers to a subject who may seek or need treatment, is in need of treatment, is receiving treatment, is about to receive treatment, or is being cared for by a trained professional for a particular disease.

[0124] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0125] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0126] In the present application, for convenience, the structural formula of the compound represents certain isomers, but the present application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers and the like.

[0127] The nanoparticle compositions of the present application may also include salts of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glucoheptonate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,

[0128] Oleate, oxalate, palmitate, palmitate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, etc.

[0129] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present application include, for example, conventional non-toxic salts of the parent compound formed by non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an alkaline or acidic part by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0130] As used herein, "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Specific phospholipids may promote fusion with membranes. For example, cationic phospholipids may interact with one or more negatively charged phospholipids of membranes (e.g., cell membranes or intracellular membranes). The fusion of phospholipids with membranes may allow one or more elements containing lipid compositions to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to cells.

[0131] As used herein, "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. Smaller values ​​indicate narrower particle size distribution.

[0132] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which may be produced naturally (eg, isolated or purified) or synthetically.

[0133] As used herein, "RNA" refers to ribonucleic acid that can be naturally or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA can include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence and / or a polyadenylation signal. RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, RNA can be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, can produce the encoded polypeptide. RNA can be selected from a non-limiting group including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), mRNA and mixtures thereof.

[0134] As used herein, a "single unit dose" is a dose of any therapeutic agent that is administered in one dose / at one time / through a single route / at a single point of contact, ie, a single time.

[0135] As used herein, a "split dose" is the division of a single unit dose or total daily dose into two or more doses.

[0136] As used herein, the "total daily dose" is the amount given or prescribed over a 24-hour period. It may be administered in a single unit dose.

[0137] As used herein, "particle size" or "average particle size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0138] As used herein, the term "subject" or "patient" refers to any organism to which the composition according to the present application can be administered, e.g., for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates and humans) and / or plants.

[0139] As used herein, "target cell" refers to any one or more cells of interest. The cell can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0140] As used herein, "target tissue" refers to any one or more target tissue types, where therapeutic and / or prophylactic delivery will result in a desired biological and / or pharmacological effect. Examples of target tissues include specific tissues, organs, and systems or groups thereof. In specific applications, the target tissue can be the kidney, lung, spleen, vascular endothelium in the kidney (e.g., intracoronary or intrafemoral) or blood vessels (e.g., by intratumoral injection). "Non-target tissue" refers to any one or more tissue types, where expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In specific applications, non-target tissues can include the liver and spleen.

[0141] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active agents" or "active ingredients". Such substances include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins and nucleic acids.

[0142] As used herein, the term "therapeutically effective amount" refers to an amount of an agent to be delivered (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) that is sufficient when administered to a subject suffering from or susceptible to an infection, disease, disorder and / or condition to treat, ameliorate the symptoms of, diagnose, prevent and / or delay the onset of the infection, disease, disorder and / or condition.

[0143] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can be performed, for example, in vitro, ex vivo, or in vivo.

[0144] As used herein, the term "treat" refers to partially or completely alleviating, relieving, ameliorating, remitting, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment may be performed on subjects who do not exhibit the disease, disorder, and / or condition and / or on subjects who only exhibit early signs of the disease, disorder, and / or condition in order to reduce the risk of developing a pathological condition associated with the disease, disorder, and / or condition.

[0145] The present application discloses a compound of formula (I), or a salt thereof or an isomer thereof,

[0146]

[0147] Wherein R1 is a C1-C12 alkyl group; R2 and R3 are each independently a C1-C11 alkyl group; R4 and R5 are each independently a C1-C3 alkyl group;

[0148] X is (C=O)O, O(C=O), O(C=O)O, (C=O)NH or NH(C=O), and Y and Z are each independently (C=O)O or O(C=O);

[0149] o Selected from 1, 2, 3, 4 or 5;

[0150] p is selected from 3, 4, 5, 6, 7 or 8;

[0151] q is selected from 3, 4, 5, 6, 7 or 8.

[0152] In a preferred embodiment, R1 is selected from C1-C11 alkyl, for example, R1 is selected from C2-C11 alkyl, R1 is selected from C3-C11 alkyl, R1 is selected from C4-C11 alkyl, R1 is selected from C5-C11 alkyl, R1 is selected from C6-C11 alkyl, R1 is selected from C7-C11 alkyl, R1 is selected from C8-C11 alkyl, R1 is selected from C9-C11 alkyl, R1 is selected from C10-C11 alkyl, for example, R1 is C1 alkyl, R1 is C2 alkyl, R1 is C3 alkyl, R1 is C4 alkyl, R1 is C5 alkyl, R1 is C6 alkyl, R1 is C7 alkyl, R1 is C8 alkyl, R1 is C9 alkyl, R1 is C10 alkyl, R1 is C11 alkyl. In a further preferred embodiment, R1 is C10 alkyl or C11 alkyl.

[0153] In a preferred embodiment, R1 is selected from C1-C11 straight chain alkyl, for example, R1 is selected from C2-C11 straight chain alkyl, R1 is selected from C3-C11 straight chain alkyl, R1 is selected from C4-C11 straight chain alkyl, R1 is selected from C5-C11 straight chain alkyl, R1 is selected from C6-C11 straight chain alkyl, R1 is selected from C7-C11 straight chain alkyl, R1 is selected from C8-C11 straight chain alkyl, R1 is selected from C9-C11 Straight chain alkyl, R1 is selected from C10-C11 straight chain alkyl, R1 is selected from C7-C9 straight chain alkyl, for example, R1 is C1 alkyl, R1 is C2 alkyl, R1 is C3 straight chain alkyl, R1 is C4 straight chain alkyl, R1 is straight chain C5 alkyl, R1 is C6 straight chain alkyl, R1 is C7 straight chain alkyl, R1 is C8 straight chain alkyl, R1 is C9 straight chain alkyl, R1 is C10 straight chain alkyl, R1 is C11 straight chain alkyl. In a further preferred embodiment, R1 is C10 straight chain alkyl or C11 straight chain alkyl.

[0154] In a preferred embodiment, in the compounds of the present invention, R2 is selected from C1-C11 alkyl, for example, R2 is selected from C2-C11 alkyl, R2 is selected from C3-C11 alkyl, R2 is selected from C4-C11 alkyl, R2 is selected from C5-C11 alkyl, R2 is selected from C6-C11 alkyl, R2 is selected from C7-C11 alkyl, R2 is selected from C8-C11 alkyl, R2 is selected from C9-C11 alkyl, R2 is selected from C10-C11 alkyl, R2 is selected from C7-C9 alkyl, for example, R2 is C1 alkyl, R2 is C2 alkyl, R2 is C3 alkyl, R2 is C4 alkyl, R2 is C5 alkyl, R2 is C6 alkyl, R2 is C7 alkyl, R2 is C8 alkyl, R2 is C9 alkyl, R2 is C10 alkyl, R2 is C11 alkyl. In a further preferred embodiment, R2 is C8 alkyl.

[0155] In a preferred embodiment, R2 is selected from C1-C11 straight chain alkyl, for example, R2 is selected from C2-C11 straight chain alkyl, R2 is selected from C3-C11 straight chain alkyl, R2 is selected from C4-C11 straight chain alkyl, R2 is selected from C5-C11 straight chain alkyl, R2 is selected from C6-C11 straight chain alkyl, R2 is selected from C7-C11 straight chain alkyl, R2 is selected from C8-C11 straight chain alkyl, R2 is selected from C9-C11 Straight chain alkyl, R2 is selected from C10-C11 straight chain alkyl, R2 is selected from C7-C9 straight chain alkyl, for example, R2 is C1 alkyl, R2 is C2 alkyl, R2 is C3 straight chain alkyl, R2 is C4 straight chain alkyl, R2 is straight chain C5 alkyl, R2 is C6 straight chain alkyl, R2 is C7 straight chain alkyl, R2 is C8 straight chain alkyl, R2 is C9 straight chain alkyl, R2 is C10 straight chain alkyl, R2 is C11 straight chain alkyl. In a further preferred embodiment, R2 is C8 straight chain alkyl.

[0156] In a preferred embodiment, in the compounds of the present invention, R3 is selected from C1-C11 alkyl, for example, R3 is selected from C2-C11 alkyl, R3 is selected from C3-C11 alkyl, R3 is selected from C4-C11 alkyl, R3 is selected from C5-C11 alkyl, R3 is selected from C6-C11 alkyl, R3 is selected from C7-C11 alkyl, R3 is selected from C8-C11 alkyl, R3 is selected from C9-C11 alkyl, R3 is selected from C10-C11 alkyl, R3 is selected from C7-C9 alkyl, for example, R3 is C1 alkyl, R3 is C2 alkyl, R3 is C3 alkyl, R3 is C4 alkyl, R3 is C5 alkyl, R3 is C6 alkyl, R3 is C7 alkyl, R3 is C8 alkyl, R3 is C9 alkyl, R3 is C10 alkyl, R3 is C11 alkyl. In a further preferred embodiment, R3 is C8 alkyl.

[0157] In a preferred embodiment, R3 is selected from C1-C11 straight chain alkyl, for example, R3 is selected from C2-C11 straight chain alkyl, R3 is selected from C3-C11 straight chain alkyl, R3 is selected from C4-C11 straight chain alkyl, R3 is selected from C5-C11 straight chain alkyl, R3 is selected from C6-C11 straight chain alkyl, R3 is selected from C7-C11 straight chain alkyl, R3 is selected from C8-C11 straight chain alkyl, R3 is selected from C9-C11 Straight chain alkyl, R3 is selected from C10-C11 straight chain alkyl, R3 is selected from C7-C9 straight chain alkyl, for example, R3 is C1 alkyl, R3 is C2 alkyl, R3 is C3 straight chain alkyl, R3 is C4 straight chain alkyl, R3 is straight chain C5 alkyl, R3 is C6 straight chain alkyl, R3 is C7 straight chain alkyl, R3 is C8 straight chain alkyl, R3 is C9 straight chain alkyl, R3 is C10 straight chain alkyl, R3 is C11 straight chain alkyl. In a further preferred embodiment, R3 is C8 straight chain alkyl.

[0158] In a preferred embodiment, in the compounds of the present invention, R4 is selected from C1-C3 alkyl, for example, R4 is selected from methyl, ethyl, propyl or isopropyl. In a further preferred embodiment, R4 is methyl.

[0159] In a preferred embodiment, in the compounds of the present invention, R5 is selected from C1-C3 alkyl, for example, R5 is selected from methyl, ethyl, propyl or isopropyl. In a further preferred embodiment, R5 is methyl.

[0160] In a preferred embodiment, o is selected from 1, 2, 3, 4 or 5. In a further preferred embodiment, o is 2, 3 or 4. In a further preferred embodiment, o is 3 or 4.

[0161] In a preferred embodiment, p is selected from 3, 4, 5, 6, 7 or 8. In a further preferred embodiment, p is 5, 6 or 7. In a further preferred embodiment, p is 5 or 6.

[0162] In a preferred embodiment, q is selected from 3, 4, 5, 6, 7 or 8. In a further preferred embodiment, q is 6, 7 or 8. In a further preferred embodiment, q is 7.

[0163] In a preferred embodiment, X, Y, and Z are all (C=O)O, or X, Y are all (C=O)O and Z is O(C=O), or X is (C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)O, Y and Z are all O(C=O), or X is O(C=O), Y and Z are all (C=O)O, or X is O(C=O), Y is (C=O)O, and Z is O(C=O), or X, Y are all O(C=O), and Z is (C=O)O, or X, Y, and Z are all O(C=O), or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all O(C=O), or X is O(C=O)O, Y and Z are all O(C=O), or X is O(C=O)O, Y is (C=O)O, and Z is )O, Z is O(C=O), or X is O(C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)NH, Y and Z are both (C=O)O, or X is (C=O)NH, Y and Z are both O(C=O), or X is (C=O)NH, Y is (C=O)O, and Z is O(C=O), or X is (C=O)NH, Y is (C=O)O, and Z is O(C=O), or X is (C=O)NH, Y is O(C=O), and Z is (C=O)O, or X is NH(C=O), Y and Z are both (C=O)O, or X is NH(C=O), Y and Z are both O(C=O), or X is NH(C=O), Y is (C=O)O, and Z is O(C=O), or X is NH(C=O), Y is (C=O)O, and Z is (C=O)O. In a further preferred embodiment, X, Y and Z are all (C=O)O, or X is (C=O)O, Y is O(C=O) and Z is (C=O)O, or X is (C=O)NH, Y and Z are all O(C=O), or X is O(C=O)O, Y and Z are all (C=O)O.

[0164] The present application further provides a nanoparticle composition comprising a lipid component, wherein the lipid component comprises a compound of formula (I) provided herein.

[0165] In some embodiments, the nanoparticle composition has an average particle size of 60 nm to 130 nm.

[0166] The nanoparticle compositions may include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes.

[0167] The nanoparticle composition described herein comprises a lipid component, the lipid component comprising at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition may include one or more of compounds 1-13. The nanoparticle composition may also include a variety of other components. For example, in addition to the compound according to formula (I), the lipid component of the nanoparticle composition may also include one or more other lipids.

[0168] The lipid component of the nanoparticle composition may include one or more PEG or PEG-modified lipids. Such substances may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from the non-limiting group of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids, preferably DMG-PEG2000.

[0169] The lipid component of the nanoparticle composition can include one or more structural lipids. The structural lipid can be selected from, but not limited to, cholesterol, coprostanol, sitosterol, ergosterol, stigmasterol and mixtures thereof, but not limited thereto. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone), or a combination thereof.

[0170] The lipid component of the nanoparticle composition may include one or more phospholipids. The phospholipids used in the nanoparticle composition and method may be selected from

[0171] Dilauroyl phosphatidylcholine (DLPC),

[0172] Dimyristoylphosphatidylcholine (DMPC),

[0173] Dioleoylphosphatidylcholine (DOPC),

[0174] Dipalmitoylphosphatidylcholine (DPPC),

[0175] Distearoylphosphatidylcholine (DSPC),

[0176] Dioleoylphosphatidylcholine (DUPC),

[0177] Palmitoyloleoylphosphatidylcholine (POPC),

[0178] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0179] 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0180] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0181] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0182] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0183] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0184] 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE),

[0185] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0186] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0187] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0188] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0189] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0190] 1,2-Diol-sn-glycero-3-phospho-(1-glycero) sodium salt (DOPG) or sphingomyelin.

[0191] In some embodiments, the nanoparticle composition comprises DSPC.In some embodiments, the nanoparticle composition comprises DOPE.In some embodiments, the nanoparticle composition comprises DSPC and DOPE.

[0192] In some embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 35 mol%-65 mol%, the content of the sum of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%.

[0193] The nanoparticle composition may contain one or more therapeutic agents and / or preventive agents, and the therapeutic agents and / or preventive agents are selected from vaccines or compounds capable of inducing an immune response, nucleic acids, preferably the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

[0194] The present application provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing a polypeptide of interest in mammalian cells, and treating a disease or disorder in a mammal in need thereof, the method comprising administering to the mammal and / or contacting the mammalian cells with a therapeutic and / or prophylactic nanoparticle composition.

[0195] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can have any size and can have any secondary structure or activity. In some embodiments, when expressed in a cell, the polypeptide encoded by the mRNA can have a therapeutic effect.

[0196] The lipid component of the nanoparticle composition may include, for example, a compound according to formula (I), phospholipids (eg, unsaturated lipids such as DOPE or DSPC), PEG lipids and structured lipids.

[0197] The nanoparticle composition can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size.

[0198] The average particle size of the nanoparticle composition is 60nm-130nm.

[0199] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index generally indicates a narrow particle size distribution.

[0200] The application further provides a method for delivering therapeutic and / or preventive agents specifically to mammalian organs, the method comprising administering any of the aforementioned nanoparticle compositions to a mammal, the administration comprising contacting a mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or preventive agent to the organ. Therapeutic and / or preventive agents, for example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents or nucleic acids (e.g., RNA, e.g., mRNA) can be delivered to cells or organs. In the case of therapeutic and / or preventive being mRNA, when cells are contacted with the nanoparticle composition, translatable mRNA can be translated in cells to produce target polypeptides. However, substantially untranslatable mRNAs can also be delivered to cells. Substantially untranslatable mRNA can be used as a vaccine and / or can isolate the translation components of cells to reduce the expression of other species in the cell.

[0201] In some embodiments, the nanoparticle composition can target a specific type or category of cells (e.g., cells of a specific organ or system thereof). For example, a nanoparticle composition comprising a target treatment and / or prevention can be specifically delivered to the liver, kidney, spleen, gastrointestinal tract, femur or lung of a mammal. Specific delivery to a specific category of cells, organs or their systems or groups means that a higher proportion of the nanoparticle composition comprising a therapeutic and / or preventive agent, including treatment and / or prevention, is delivered to the target destination (e.g., tissue) relative to other destinations, for example, when administering a nanoparticle composition to a mammal. In some embodiments, the target tissue is selected from the liver, kidney, lung, spleen, femur, gastrointestinal tract, eye tissue (e.g., by intraocular, subretinal or intravitreal injection), vascular endothelium in a blood vessel (e.g., intracoronary or intrafemoral) or kidney, and tumor tissue (e.g., by intratumoral injection).

[0202] Example

[0203] Example 1 Synthesis of Compound 1

[0204] Compound 1

[0205] The synthetic route is as follows:

[0206]

[0207] Specifically, compound B (116.36 g, 563.94 mmol) and DMAP (3.13 g, 25.63 mmol) were added to a solution of compound A (100.00 g, 512.67 mmol) in dichloromethane (500 ml), stirred evenly at room temperature, and DCC (116.36 g, 563.94 mmol) was added in batches. After the addition, the mixture was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered, and the filter cake was rinsed with dichloromethane (500 ml x 2). The filtrate was concentrated and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0-5%) to obtain compound C (170.68 g, 95.30%).

[0208] Compound E (126.19 g, 493.03 mmol) and DMAP (3.04 g, 22.41 mmol) were added to a solution of compound D (100.04 g, 448.21 mmol) in dichloromethane (400 ml), stirred at room temperature, and a solution of DCC (102.63 g, 493.03 mmol) in dichloromethane (100 ml) was slowly added dropwise. After the addition, the mixture was stirred at room temperature for 3 hours and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered and the filter cake was rinsed with dichloromethane (500 ml x 2). The filtrate was concentrated and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0-5%) to obtain compound F (160.43 g, 77.53%).

[0209] Compound G (25.38 g, 129.99 mmol), NBu4I (3.20 g, 8.67 mmol) and K2CO3 (17.97 g, 129.99 mmol) were added to a DMF (120 ml) solution of compound F (20.00 g, 43.33 mmol), and the mixture was stirred at room temperature for 20 hours. The reaction was monitored by TLC. After the reaction was complete, water (200 ml) and ethyl acetate (200 ml) were added to the reaction solution, and the mixture was allowed to stand for separation. The aqueous phase was extracted with ethyl acetate (100 ml), and the organic phases were combined, washed with saturated brine (200 ml), and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and chromatographed on a silica gel column (ethyl acetate: petroleum ether = 0-5%) to obtain compound H (22.03 g, yield 88.28%).

[0210] Compound C (9.10 g, 26.05 mmol), NBu4I (12.83 g, 34.73 mmol) and Cs2CO3 (16.97 g, 52.09 mmol) were added to a DMF (50 ml) solution of compound H (10.00 g, 17.36 mmol), and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After the reaction was complete, water (100 ml) and ethyl acetate (100 ml) were added to the reaction solution, and the mixture was allowed to stand for separation. The aqueous phase was extracted with ethyl acetate (50 ml), and the organic phases were combined, washed with saturated brine (100 ml), and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and chromatographed on a silica gel column (ethyl acetate: petroleum ether = 0-5%) to obtain compound I (15.19 g, yield 98.71%).

[0211] Concentrated hydrochloric acid (60 ml) was added to a solution of compound I (13.15 g, 15.57 mmol) in dichloromethane (100 ml), and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the organic layer was separated, and the organic layer was washed with saturated sodium bicarbonate solution (100 ml), washed with saturated brine (100 ml), dried over anhydrous sodium sulfate, concentrated, and chromatographed on a silica gel column (ethyl acetate: petroleum ether = 0-5%) to obtain compound J (10.09 g, 95.40%).

[0212] NaBH4 (0.22 g, 5.88 mmol) was added in batches to methanol (30 mL) of compound J (1.00 g, 1.47 mmol), and the mixture was stirred at room temperature for 2 h. After the reaction was completed under TLC monitoring, hydrochloric acid was added to quench the reaction. The solvent was distilled off, and ethyl acetate (30 mL) and purified water (20 mL) were added for washing. The organic phase was then washed with 20 mL of brine. The organic phase was distilled and wet-column loaded. Gradient elution was performed on a silica gel column (petroleum ether: ethyl acetate = 40:1, 30:1, 20:1, 10:1) to give compound K (0.72 g, yield 72.0%).

[0213] To a solution of compound K (0.20 g, 0.29 mmol) in dichloromethane (30 ml) were added L (0.14 g, 0.88 mmol), EDCI (0.23 g, 1.17 mmol), DMAP (36 mg, 0.29 mmol) and DIEA (0.30 g, 2.35 mmol), stirred at room temperature for 24 hours, and the reaction was monitored by TLC. After the reaction was complete, purified water was added to the reaction solution and stirred, the liquids were separated, the filtrate was concentrated, and the compound 1 (86 mg, yield 37.6%) was obtained by gradient elution on a silica gel column (MeOH: DCM = 1: 150, 1: 120, 1: 90, 1: 70); 1H NMR (400MHz, CDCl3) δ4.95–4.89(m,2H),4.11–4.08(t,J=8.0Hz,2H),2.69–2.66(t,J=8.0Hz,2H),2.54–2.50(t,2H) ,2.34–2.30(m,10H),1.67–1.53(m,14H),1.36–1.30(m,52H),0.94–0.90(t,J=8.0Hz,9H);MS-ESI(m / z):780.8(M+H) + .

[0214] Example 2 Synthesis of Compound 2

[0215] Compound 2

[0216] The synthetic route is as follows:

[0217]

[0218] Specifically, M (0.11 g, 0.66 mmol), DCC (0.27 g, 1.32 mmol), and DMAP (54 mg, 0.44 mmol) were added to a solution of compound K (0.30 g, 0.44 mmol) in dichloromethane (30 ml), and the mixture was stirred at room temperature for 5 hours, concentrated, and gradient eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, 80: 1, 60: 1, 40: 1) to obtain compound N (0.28 g, yield 77.8%).

[0219] To compound N (0.28 g, 0.34 mmol), a tetrahydrofuran solution of dimethylamine (2 mL, 4 mmol) and triethylamine (0.45 g, 4.42 mmol) were added, and the mixture was stirred at room temperature overnight. Dichloromethane (30 ml) and purified water (20 mL) were added for washing, and the organic phase was distilled. Compound 2 (0.21 g, yield 77.8%) was obtained by gradient elution with silica gel column chromatography (MeOH:DCM=1:90, 1:60, 1:40, 1:20); 1 H NMR (400MHz, CDCl3) δ4.93–4.87(m,2H),4.11–4.07(t,J=8.0Hz,2H),2.47–2.29(m,14H),1.92–1.85( m,2H),1.69–1.55(m,14H),1.38–1.30(m,52H),0.94–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):794.6(M+H) + .

[0220] Example 3 Synthesis of Compound 3

[0221] Compound 3

[0222] The preparation method is the same as compound 1, and compound 3 can be prepared by using 6-bromohexanol and undecanoic acid as raw materials instead of compound A and compound B; 1 H NMR (400MHz, CDCl3) δ4.96–4.89(m,2H),4.10–4.08(t,J=8.0Hz,2H),2.69–2.65(t,J=8.0Hz,2H),2.57–2.50(t,J=8.0Hz ,2H),2.37–2.30(m,10H),1.67–1.55(m,14H),1.35–1.30(m,52H),0.94–0.90(t,J=8.0Hz,9H);MS-ESI(m / z):780.5(M+H) + .

[0223] Example 4 Synthesis of Compound 4

[0224] Compound 4

[0225] The synthetic route is as follows:

[0226] The preparation method is the same as compound 2, and compound 4 can be prepared by using 6-bromohexanol and undecanoic acid as raw materials instead of compound A and compound B; 1 H NMR (400MHz, CDCl3) δ4.94–4.87(m,2H),4.13–4.07(t,J=8.0Hz,2H),2.47–2.30(m,14H),1.92–1.84( m,2H),1.67–1.55(m,14H),1.38–1.30(m,52H),0.94–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):794.9(M+H) + .

[0227] Example 5 Synthesis of Compound 5

[0228] Compound 5

[0229] The synthetic route is as follows:

[0230]

[0231] Specifically, to a solution of compound O (0.20 g, 1.10 mmol) in dichloromethane (20 mL) was added thionyl chloride (0.39 g, 3.30 mmol), the mixture was stirred at room temperature for 1 hour, and concentrated to obtain P for standby use; Q (0.20 g, 0.29 mmol) and triethylamine (0.33 g, 3.30 mmol) were dissolved in 10 mL of dichloromethane solution, and the mixture was dropped into the above solution, stirred at room temperature overnight, purified water (20 mL) was added for washing, the organic phase was distilled, and compound R (0.18 g, yield 19.4%) was obtained by gradient elution on a silica gel column (petroleum ether: ethyl acetate = 100:1, 80:1, 40:1, 20:1).

[0232] To compound R (0.18 g, 0.21 mmol), a tetrahydrofuran solution of dimethylamine (2 mL, 4 mmol) and triethylamine (1.5 ml) were added, and the mixture was stirred at room temperature overnight. Dichloromethane (50 ml) and purified water (30 mL) were added for washing. The organic phase was distilled and gradient eluted by silica gel column chromatography (MeOH: DCM = 1:30, 1:20) to obtain compound 5 (90 mg, yield 52.3%). 1 HNMR(400MHz, CDCl3)δ4.93–4.86(m,2H),4.15–4.07(t,J=8.0Hz,2H),2.45–2.30(m,14H),1.94–1.84( m,2H),1.67–1.55(m,16H),1.38–1.31(m,52H),0.93–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):808.7(M+H) + .

[0233] Example 6 Synthesis of Compound 6

[0234] Compound 6

[0235] The synthetic route is as follows:

[0236]

[0237] Specifically, S (0.39 g, 3.30 mmol) was added to a solution of compound Q (0.20 g, 0.29 mmol) in dichloromethane (30 ml), stirred at room temperature for 5 hours, washed with brine (20 mL), and the organic phase was distilled. Compound T (0.21 g, yield 84.0%) was obtained by gradient elution with silica gel column chromatography (petroleum ether: ethyl acetate = 40:1, 30:1, 20:1).

[0238] To compound T (0.21 g, 0.24 mmol) was added a tetrahydrofuran solution of dimethylamine (2 mL, 4 mmol) and triethylamine (0.5 mL), and the mixture was stirred at room temperature for 96 h. Dichloromethane (40 mL) and purified water (40 mL) were added for washing. The organic phase was distilled and gradient eluted by silica gel column chromatography (MeOH:DCM=1:40, 1:30, 1:20) to obtain compound 6 (50 mg, yield 25.0%). 1 H NMR(400MHz, CDCl3) δ4.94–4.86(m,2H),4.17–4.07(t,J=8.0Hz,2H),2.46–2.30(m,14H),1.94–1.84( m,2H),1.67–1.53(m,16H),1.38–1.30(m,54H),0.93–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):822.8(M+H) + .

[0239] Example 7 Synthesis of Compound 7

[0240] Compound 7

[0241] The synthetic route is as follows:

[0242] The preparation method is the same as compound 2, using 5-bromovaleric acid as a raw material instead of bromobutyric acid to obtain compound 7 (0.16 g, 64.0%); 1 H NMR(400MHz, CDCl3) δ4.94–4.86(m,2H),4.17–4.07(t,J=8.0Hz,2H),2.46–2.30(m,14H),1.93–1.84( m,2H),1.68–1.55(m,16H),1.39–1.31(m,52H),0.94–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):808.5(M+H) + .

[0243] Example 8 Synthesis of Compound 8

[0244] Compound 8

[0245] The synthetic route is as follows:

[0246] The preparation method is the same as compound 6, and compound K is used as a raw material instead of compound Q to obtain compound 8 (0.11 g, 43.7%); 1H NMR(400MHz, CDCl3) δ4.95–4.86(m,2H),4.17–4.05(t,J=8.0Hz,2H),2.44–2.30(m,14H),1.93–1.84( m,2H),1.65–1.53(m,16H),1.38–1.32(m,54H),0.94–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):822.7(M+H) + .

[0247] Example 9 Synthesis of Compound 9

[0248] Compound 9

[0249] The synthetic route is as follows:

[0250] The preparation method is the same as compound 1, using 6-bromohexanol, undecanoic acid and N,N-dimethylglycine as raw materials to replace compound A, compound B and compound L to obtain compound 9 (0.11 g, 55.0%); 1 H NMR (400MHz, CDCl3) δ4.95–4.89(m,2H),4.12–4.08(t,J=8.0Hz,2H),2.70–2.65(t,J=8.0Hz,2H),2.55–2.50(t,J=8.0Hz ,2H),2.37–2.30(m,8H),1.67–1.54(m,14H),1.34–1.30(m,52H),0.93–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):766.7(M+H) + .

[0251] Example 10 Synthesis of Compound 10

[0252] Compound 10

[0253] The synthetic route is as follows:

[0254]

[0255] Specifically, compound U (46 mg, 0.23 mmol) and pyridine (22 mg) were added to a DCM (5 ml) solution of compound K (100 mg, 0.15 mmol), and the mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was removed by concentration, and the reaction solution was diluted with water, extracted with ethyl acetate (30 ml × 3), and the organic layer was dried over anhydrous magnesium sulfate, concentrated, and chromatographed on a silica gel column (petroleum ether: ethyl acetate = 5:1) to obtain compound V.

[0256] Compound W (109 mg, 1.10 mmol), DMAP (11 mg, 0.09 mmol) and DIPEA (83 mg, 0.64 mmol) were added to a DCM (5 ml) solution of compound V (183 mg, 0.22 mmol) and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed three times with 1M NaOH aqueous solution, and the organic layer was dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column chromatography (dichloromethane: methanol = 10: 1) to obtain compound 10 (34 mg, 19.44%).

[0257] 1 H NMR(500MHz,Chloroform-d)δ4.91–4.80(m,1H),4.71–4.60(m,1H),4.17(t,J=7.5Hz,2H),4.04(t,J=7.3Hz,2H),2.39(d,J=7.9Hz,2H),2. 26(d,J=13.0Hz,10H),1.85(q,J=7.3Hz,2H),1.65–1.45(m,14H),1.27(d,J=19.2Hz,52H),0.86(t,J=7.9Hz,9H).MS-ESI(m / z):810.6(M+H) + .

[0258] Example 11 Synthesis of Compound 11

[0259] Compound 11

[0260] The preparation method is the same as compound 10, except that N,N-dimethylethanolamine is used as a raw material to replace compound W to obtain compound 11 (11 mg, 6.57%). 1 H NMR(500MHz,Chloroform-d)δ4.89–4.82(m,1H),4.70–4.59(m,1H),4.23(t,J=5.8Hz,2H),4.04(t,J=6.7Hz,2H),2.65(t,J =6.0Hz,2H),2.37–2.18(m,10H),1.65–1.45(m,14H),1.34–1.23(m,52H),0.87(t,J=6.8Hz,9H).MS-ESI(m / z):796.6(M+H) + .

[0261] Example 12 Synthesis of Compound 12

[0262] Compound 12

[0263] The synthetic route is as follows:

[0264]

[0265] Specifically, at 0°C, methanesulfonic anhydride (383 mg, 2.20 mmol) and triethylamine (300 mg, 2.93 mmol) were added to a DCM (10 ml) solution of compound K (1.0 g, 1.47 mmol) and stirred at room temperature for 12 h. The solvent was concentrated to remove the solvent, water was added to dilute the reaction solution, and ethyl acetate was extracted (30 ml×3) to obtain 1.8 g of crude compound X, which was dissolved in ultra-dry DMF, and NaN3 (600 mg, 1.18 mmol) was added. The mixture was heated to reflux at 70°C for 4 h. After the reaction was complete, water was added to quench the mixture, and ethyl acetate was extracted (30 ml×3). The organic layer was dried over anhydrous magnesium sulfate, concentrated, and chromatographed on a silica gel column (petroleum ether: ethyl acetate = 15:1) to obtain compound Y (1.2 g, 71.73%).

[0266] Under argon protection, palladium carbon was added to a solution of compound Y (1 g, 1.42 mmol) in ethyl acetate (10 ml), and hydrogen was added for reduction at room temperature for 12 h. After the reaction was complete, the mixture was filtered, concentrated, and chromatographed on a silica gel column (dichloromethane: methanol = 15: 1) to give compound Z (420 mg, 43.65%).

[0267] Compound L (35 mg, 0.23 mmol), EDC (57 mg, 0.30 mmol) and DMAP (8 mg, 0.06 mmol) were added to a solution of compound Z (100 mg, 0.15 mmol) in dichloromethane (50 ml), and the mixture was stirred at room temperature for 7 hours. The reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to the reaction solution for dilution, and the layers were separated. The organic layer was dried by adding anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and subjected to silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain compound 12 (53 mg, 45.373%). 1 H NMR(500MHz,Chloroform-d)δ4.88–4.82(m,1H),4.04(t,J=6.8Hz,2H),3.86(q,J=7.0Hz,1H),2.70(d,J=7.3Hz,2H),2.44(t,J=6.2Hz,2H),2.3 7(s,6H),2.27(q,J=7.2Hz,4H),1.60(t,J=7.1Hz,6H),1.53–1.42(m,6H),1.34–1.22(m,54H),0.87(t,J=6.8Hz,9H).MS-ESI(m / z):779.6(M+H) + .

[0268] Example 13 Synthesis of Compound 13

[0269] Compound 13

[0270] The synthetic route is as follows:

[0271] The preparation method is the same as compound 12, but using 4-(dimethylamino)butyric acid instead of compound L as a raw material to obtain oily compound 13. 1 H NMR(500MHz,Chloroform-d)δ4.89–4.81(m,1H),4.04(t,J=6.8Hz,2H),3.86(q,J=6.2Hz,1H),2.99(t,J=6.7Hz,2H),2.75(s,6H),2.46(t,J=6.5Hz,2H), 2.27(q,J=7.5Hz,4H),2.10(q,J=6.9Hz,2H),1.64–1.56(m,6H),1.53–1.44( m,6H),1.34–1.21(m,54H),0.87(t,J=6.6Hz,9H).MS-ESI(m / z):793.7(M+H) + .

[0272] Example 14 Synthesis of Compound LP-C27

[0273] Compound LP-C27

[0274] The preparation method is the same as compound 1, using 6-bromohexanol, undecanoic acid and 4-(dimethylamino)butyric acid as raw materials instead of compound A, compound B and compound L as raw materials to obtain oily compound LP-C27. 1 H NMR (400MHz, CDCl3) δ4.95–4.87(m,2H),4.11–4.08(t,J=8.0Hz,2H),2.38–2.30(m,8H),2.29(s,6H),1.88– 1.80(m,2H),1.69–1.52(m,14H),1.37–1.30(m,52H),0.94–0.90(t,J=8.0Hz,9H); MS-ESI(m / z):794.9(M+H) + .

[0275] Test example

[0276] Experimental Example 1 Lipid Nanoparticle (LNP) Encapsulation

[0277] The mRNA stock solution was dispersed in a 20mM acetic acid solution (pH5.0) to a final concentration of 200μg / mL (aqueous phase). The mixture was mixed into a lipid mixture (oil phase) according to the molar ratio of Example Compound: Cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5. The flow rates of the aqueous phase and the oil phase were controlled to mix the mRNA with the lipid mixture by T mixing to obtain the mRNA encapsulated in LNP. The encapsulated LNP was diluted with buffer, then concentrated by ultrafiltration, and the buffer was replaced. Finally, the LNP was concentrated to 150μg / mL of mRNA, and the pH of the LNP was adjusted to about 7-8. Finally, the total content and free content of mRNA in the LNP were detected using a Ribogreen kit and 10% Triton as a demulsifier, and the encapsulation rate of the LNP was calculated. The final LNP product was diluted with a diluent, added to 1ml of the particle size pool, and placed on a Malvern ZetaSizer instrument to detect the particle size of the LNP. The results are shown in Table 1.

[0278] Particle size, PDI and encapsulation efficiency are important quality attributes of lipid nanoparticles. As shown in the table below, the test compounds all have good encapsulation efficiency, particle size suitable for mRNA delivery and narrow PDI. Compounds 4, 7, 8 and 9 had precipitation during the preparation process and poor drugability, so no follow-up studies were conducted. The precipitation of compound 9 made it impossible to characterize the corresponding LNPs.

[0279] Table 1: LNP characterization data of example compounds

[0280] Compound No. Encapsulation rate / % Particle size / nm PDI Compound 1 91.94 58.98 0.1342 Compound 2 97.94 66.15 0.05831 Compound 3 95.29 53.25 0.1265 Compound 4 96.89 79.60 0.1022 Compound 5 97.4 67.4 0.05389 Compound 6 95.7 70.97 0.09461 Compound 7 89.6 103.4 0.1526 Compound 8 93.2 103.1 0.1984 Compound LP-C27 96.28 70.38 0.101

[0281] Test Example 2 Test of human erythropoietin (hEPO) delivery effect

[0282] 1. Encapsulate hEPO mRNA into the formulations of Compound 1, Compound 2, Compound 3, Compound 5, and Compound 6. The LNP formulation preparation method and mRNA encapsulation method are as described in Experimental Example 1;

[0283] 2. hEPO protein was expressed by intramuscular injection of 5 μg mRNA into 5 Balb / c mice.

[0284] 3. Collect blood from the eye sockets at 6 hours and separate the serum.

[0285] 4. The corresponding absorbance was detected by ELISA kit (Biyuntian, #PE230), and the hEPO protein content in unit volume of serum was calculated by absorbance.

[0286] 5. The hEPO protein content is used as the criterion for judging the delivery effect.

[0287] The hEPO experiment is a common method to test the expression of mRNA in vivo by hEPO expression level. Figure 1 The results show that 6 hours after intramuscular injection, compound 1 and compound 3 basically do not express hEPO, while compound 6, compound 2, and compound 5 can all obviously express hEPO, showing a trend of increasing in sequence, with the expression of compound 5 being the most obvious.

[0288] Test Example 3: Test of luciferase delivery effect

[0289] 1. Encapsulate the mRNA expressing Luciferase into the LNP formulations of compound LP-C27, compound 2, and compound 5. The LNP formulation preparation method and the mRNA encapsulation method are as described in Experimental Example 1;

[0290] 2. The loaded LNP formulation was injected intramuscularly into Balb / c mice at a dose of 5 μg / mouse, with 5 mice in each group.

[0291] 3. Detect the Luciferase fluorescence expression intensity of each mouse at 6 hours.

[0292] 4. Detection of fluorescence expression intensity: D-luciferin sodium salt (dose: 150 mg / kg) was injected intraperitoneally into each mouse 10 minutes before the test. The mice were then anesthetized with isoflurane and placed in an IVIS instrument, and bioluminescence was selected for detection.

[0293] 5. Determine the delivery effect of the compound based on the fluorescence intensity results.

[0294] Luciferase assay is the main method to detect mRNA expression in vivo by fluorescence intensity. Figure 2 The results show that 6 hours after intramuscular injection, both compounds 2 and 5 can express fluorescent protein and produce fluorescence. The expression of compound 5 is significantly better than that of the control compound LP-C27, while compound 2 is comparable to LP-C27.

[0295] Figure 3 The expression of each group in spleen, among which compound 5 and compound 2 were significantly higher than the control group compound LP-C27, especially compound 5.

[0296] The above results indicate that compounds 2 and 5 have excellent mRNA delivery performance, and both have obvious advantages in spleen targeted delivery.

[0297] Although the embodiments of the present application are described above, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the protection of the present application.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R1 is a C7-C12 alkyl group; R2 and R3 are each independently a C6-C11 alkyl group; R4 and R5 are each independently a C1-C3 alkyl group; X is (C=O)O, O(C=O), O(C=O)O, (C=O)NH or NH(C=O), and Y and Z are each independently (C=O)O or O(C=O); o Selected from 1, 2, 3, 4 or 5; p is selected from 3, 4, 5, 6, 7 or 8; q is selected from 3, 4, 5, 6, 7 or 8.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein o is selected from 2, 3 or 4, preferably 3 or 4.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein p is selected from 5, 6 or 7, preferably 5 or 6. 4 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein q is selected from 6, 7 or 8, preferably 7.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is a C7-C12 straight-chain alkyl group, preferably a C10 straight-chain alkyl group or a C11 straight-chain alkyl group; R2 and R3 are each independently a C7-C9 straight-chain alkyl group, preferably a C8 straight-chain alkyl group; R4 and R5 are each independently a methyl group, an ethyl group, a propyl group or an isopropyl group, preferably a methyl group.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein X, Y, and Z are all (C=O)O, or X, Y are all (C=O)O and Z is O(C=O), or X is (C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)O, Y and Z are all O(C=O), or X is O(C=O), Y and Z are all (C=O)O, or X is O(C=O), Y is (C=O)O, and Z is O(C=O), or X, Y are all O(C=O), and Z is (C=O)O, or X, Y, and Z are all O(C=O), or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all (C=O)O, or X is O(C=O)O, Y and Z are all O(C=O), or X is O(C=O)O, Y is (C=O)O, and Z is O(C=O), or X is O(C=O)O, Y is O(C=O), and Z is (C=O)O, or X is (C=O)NH, and both Y and Z are (C=O)O, or X is (C=O)NH, and both Y and Z are O(C=O), or X is (C=O)NH, and both Y and Z are O(C=O), or X is (C=O)NH, Y is (C=O)O, and Z is O(C=O), or X is (C=O)NH, Y is O(C=O), and Z is (C=O)O, or X is NH(C=O), and both Y and Z are (C=O)O, or X is NH(C=O), and both Y and Z are O(C=O), or X is NH(C=O), Y is (C=O)O, and Z is O(C=O), or X is NH(C=O), Y is (C=O)O, and Z is (C=O)O. Preferably, X, Y and Z are all (C═O)O, or X is (C═O)O, Y is O(C═O) and Z is (C═O)O, or X is (C═O)NH, Y and Z are all O(C═O), or X is O(C═O)O, Y and Z are all (C═O)O.

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: The compound (I) is selected from Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 8. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

9. The lipid nanoparticle composition of claim 8, wherein the lipid component further comprises phospholipids, structural lipids and / or PEG lipids; The phospholipids are preferably selected from one or more of the following compounds: Dilauroyl phosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DUPC), Palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diaryl acyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-glycero-3-phosphoethanolamine, 1,2-Diaryl-sn-glycero-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-diol-sn-glycero-3-phospho-(1-glycero) sodium salt (DOPG) or sphingomyelin, For example, the phospholipid is DOPE or DSPC; The structural lipid is preferably selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol; For example, the structured lipid is cholesterol; and / or The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol, preferably DMG-PEG2000.

10. The nanoparticle composition according to any one of claims 8 to 9, further comprising a therapeutic agent and / or a prophylactic agent, wherein the therapeutic agent and / or the prophylactic agent is selected from a vaccine or a compound capable of eliciting an immune response, a nucleic acid, Preferably, the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

11. A pharmaceutical composition comprising the nanoparticle composition according to any one of claims 8 to 10 and a pharmaceutically acceptable carrier.

12. A method for delivering a therapeutic and / or prophylactic agent to a mammalian cell, the method comprising administering to a subject the nanoparticle composition of any one of claims 8-10 or the pharmaceutical composition of claim 11, wherein the administration comprises contacting a cell with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or prophylactic agent to the cell.

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