Lipid compounds and lipid nanoparticles for delivery

Abstract

This application discloses a compound with the structural formula shown in Formula (I), as well as pharmaceutically acceptable salts or stereoisomers thereof. This application also discloses a nanoparticle composition comprising the aforementioned compound, its pharmaceutically acceptable salts or stereoisomers thereof. The nanoparticles of this application can efficiently deliver drugs and vaccines into cells, achieving therapeutic or preventive purposes of drugs and vaccines. #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 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 lies in whether the therapeutic drugs can be safely and effectively delivered into the target cells through the vector 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 efficient delivery systems to achieve target gene transfection and therapeutic purposes, viral vectors contain immunogenic viral proteins, have limited target gene loading capacity and are expensive. As a result, lipid nanoparticles (LNPs) as non-viral vectors have received widespread attention due to their advantages such as good in vitro stability, in vivo degradation, 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 demonstrated to be effective delivery vehicles for bioactive substances such as small molecule drugs, proteins, and nucleic acids into and / or within cells. LNPs, small vesicles formed from one or more lipid components, can effectively encapsulate and deliver a variety of nucleic acid molecules, from DNA and RNA to chromosomes and even cells. Their defined construction scheme and ease of modification with targeting ligands facilitate large-scale production.

[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 efficiency, 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 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), 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 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(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, Y is (C=O)O, and Z is O(C=O), or In one embodiment, 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 all (C=O)O, or X is NH(C=O), Y and Z are all 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 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, 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.

[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 the compound of 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 claim 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-cholesteryldimethylsuccinate-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-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0062] 18. The nanoparticle composition of claim 16, wherein the phospholipid is DOPE.

[0063] 19. The nanoparticle composition of claim 16, wherein the phospholipid is DSPC.

[0064] 20. The nanoparticle composition of 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. The nanoparticle composition of any one of claim 20, wherein the structural lipid is cholesterol.

[0067] 23. The nanoparticle composition of any one of items 15-22, wherein the lipid component further comprises a PEG lipid.

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

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

[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 a mammalian cell, the method comprising administering to a subject the nanoparticle composition of any one of items 15-29 or the pharmaceutical composition of item 31, wherein the administration comprises contacting the cell with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or prophylactic agent to the cell.

[0078] 33. The method of 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. The method of 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 polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition of any one of items 15-29 or the pharmaceutical composition of item 31 to deliver a therapeutic and / or prophylactic agent to the cell, wherein the therapeutic and / or prophylactic agent is mRNA encoding the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

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

[0083] 38. The method of 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 pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0085] 40. A method of 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 aberrant protein or polypeptide activity.

[0087] 42. The method of claim 40 or 41, wherein the disease or condition is selected from an infectious disease, cancer and proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

[0088] 43. The method of 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 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 the nanoparticle composition of any one of items 15-29 or the pharmaceutical composition of 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. The method of any one of items 45-47, wherein the mammal is pretreated 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 to prepare lipid nanoparticles. Nanoparticle compositions containing the compounds provided herein can achieve the encapsulation and delivery of therapeutic / prophylactic agents, safely deliver the therapeutic / prophylactic agents to the targeted location, achieve high expression, and exert the effects of the therapeutic / prophylactic agents.

[0097] The lipid nanoparticles prepared in this application have a small average particle size, high encapsulation efficiency, 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 solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0099] Figure 1 Shown are the hEPO expression levels of 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 are the luciferase fluorescence intensities of different LNP formulations in the spleen 6 hours after intramuscular injection. DETAILED DESCRIPTION

[0102] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0103] Terms and Definitions

[0104] As used herein, the term "alkyl" refers to a group comprising 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 to 12 carbon atoms. Unless otherwise indicated, the alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0105] Unless otherwise indicated, the alkyl group may be optionally substituted. The optional substituents may be selected from, but are not limited to, a halogen atom (e.g., chloro, bromo, fluoro, or iodo), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl group, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g., -C(O)H), a carbonyl (e.g., -C(O)R, or represented by C=O), an acyl halide (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy group (e.g., -OR), an acetal, a phosphate, a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfite (e.g., -S(O)R), a thiophene ... In some embodiments, the substituents include, but are not limited to, alkyl, alkyl, alkylene ... 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 intended to include all isomers and isotopes of the described structure. "Isotopes" refer to atoms having the same atomic number but differing in mass due to the number of neutrons in their nuclei. 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 by combining 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. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. 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 amounts of the nanoparticle composition. 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 can include administering to the subject a nanoparticle composition comprising the therapeutic and / or prophylactic agent (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell can involve contacting one or more cells with the nanoparticle composition.

[0109] As used herein, term " enhanced delivery " refers to that by nanoparticle more (for example, 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 drug is delivered to target target tissue (for example, mammal liver) and control nanoparticle is delivered to target tissue (for example MC3, KC2 or DLinDMA) and / or the level of preventive drug is compared.Can be by the amount of protein produced in tissue and the weight of described tissue compared, by the therapeutic and / or preventive amount and the weight of tissue in tissue, by the protein amount produced in tissue and the amount of total protein in tissue compared, or by the amount of treatment and / or preventive agent in tissue and the total treatment and / or preventive agent in described tissue compared.Should be understood that nanoparticle does not need to determine in treated experimenter to the enhanced delivery of target tissue, but can determine in such as animal model (for example, rat model) surrogate. 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) of therapeutic and / or prophylactic drugs to a target tissue of interest (e.g., mammalian liver) by nanoparticles as compared to non-target tissues. The level of delivery of nanoparticles to a specific tissue can be measured by comparing the weight of protein produced in the tissue with the weight of the tissue, comparing the therapeutic and / or prophylactic amount in the tissue with the weight of the tissue, comparing the weight of protein produced in the tissue with the total protein weight in the tissue, or comparing the therapeutic and / or prophylactic amount in the tissue with the total therapeutic and / or prophylactic 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 and / 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 can be 97%. As used herein, "encapsulation" can 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, therefore, may 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). This 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 comprises one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids, such as phospholipids.

[0118] As used herein, a "linker" is a portion connecting two parts, for example, the connection between two nucleosides in a cap. A linker 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 modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. "Modified" substances may also be referred to herein as "engineered" substances. Such substances can be modified or engineered chemically, structurally, or functionally. For example, a modified nucleobase species can 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 typically on the order of microns or less and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle composition can be a liposome having a lipid bilayer with 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 currently receiving treatment, is about to receive treatment, or is being cared for by a trained professional for a particular condition.

[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 humans 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, the structural formula of the compound represents certain isomers for convenience, 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 salts may be pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified 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, dodecylsulfate, 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. Typically, 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. Typically, 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. Phospholipid can comprise one or more multiple (such as double or triple bonds) bonds (such as one or more unsaturated bonds). Specific phospholipid can promote fusion with membrane. For example, cationic phospholipid can interact with one or more negatively charged phospholipids of membrane (such as cell membrane or intracellular membrane). The fusion of phospholipid and membrane can allow one or more elements containing lipid composition to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to the cell.

[0131] As used herein, the "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. A smaller value indicates a 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 can be produced naturally (eg, isolated or purified) or synthetically.

[0133] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, the RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. The RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, the RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of the mRNA inside a mammalian cell, may produce the encoded polypeptide. The RNA may be selected from a non-limiting group including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (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 a 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 a composition according to the present application can be administered, e.g., for experimental, diagnostic, prophylactic 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 cells 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 tissue types of interest to which 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 may be the kidney, lung, spleen, vascular endothelium in the kidney (e.g., intracoronary or intrafemoral) or in a blood vessel (e.g., by intratumoral injection). "Non-target tissue" refers to any one or more tissue types to which expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In specific applications, non-target tissues may include the liver and spleen.

[0141] The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as "active agents" or "active ingredients." Such agents 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 (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered 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, resolving, 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, the 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 A straight-chain alkyl group, R1 is selected from a C10-C11 straight-chain alkyl group, and R1 is selected from a C7-C9 straight-chain alkyl group. For example, R1 is a C1 alkyl group, R1 is a C2 alkyl group, R1 is a C3 straight-chain alkyl group, R1 is a C4 straight-chain alkyl group, R1 is a straight-chain C5 alkyl group, R1 is a C6 straight-chain alkyl group, R1 is a C7 straight-chain alkyl group, R1 is a C8 straight-chain alkyl group, R1 is a C9 straight-chain alkyl group, R1 is a C10 straight-chain alkyl group, and R1 is a C11 straight-chain alkyl group. In a further preferred embodiment, R1 is a C10 straight-chain alkyl group or a C11 straight-chain alkyl group.

[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 A straight-chain alkyl group, R2 is selected from a C10-C11 straight-chain alkyl group, and R2 is selected from a C7-C9 straight-chain alkyl group. For example, R2 is a C1 alkyl group, R2 is a C2 alkyl group, R2 is a C3 straight-chain alkyl group, R2 is a C4 straight-chain alkyl group, R2 is a straight-chain C5 alkyl group, R2 is a C6 straight-chain alkyl group, R2 is a C7 straight-chain alkyl group, R2 is a C8 straight-chain alkyl group, R2 is a C9 straight-chain alkyl group, R2 is a C10 straight-chain alkyl group, and R2 is a C11 straight-chain alkyl group. In a further preferred embodiment, R2 is a C8 straight-chain alkyl group.

[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 A straight-chain alkyl group, R3 is selected from a C10-C11 straight-chain alkyl group, and R3 is selected from a C7-C9 straight-chain alkyl group. For example, R3 is a C1 alkyl group, R3 is a C2 alkyl group, R3 is a C3 straight-chain alkyl group, R3 is a C4 straight-chain alkyl group, R3 is a straight-chain C5 alkyl group, R3 is a C6 straight-chain alkyl group, R3 is a C7 straight-chain alkyl group, R3 is a C8 straight-chain alkyl group, R3 is a C9 straight-chain alkyl group, R3 is a C10 straight-chain alkyl group, and R3 is a C11 straight-chain alkyl group. In a further preferred embodiment, R3 is a C8 straight-chain alkyl group.

[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 an even 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 an even 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 an even 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 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 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 all (C=O)O, or X is (C=O)NH, Y and Z are all 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 all (C=O)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 can include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes.

[0167] The nanoparticle compositions described herein comprise a lipid component comprising at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition can include one or more of compounds 1-13. The nanoparticle composition can 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 can 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 can be selected from the non-limiting group of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid can 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.Structural lipids can be selected from, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, stigmasterol and mixtures thereof, but are not limited thereto. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (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-cholesteryldimethylsuccinate-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-phosphate-(1-glycerol) 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 lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 35 mol%-65 mol%, the content of the sum of phospholipid and cholesterol is 35 mol%-65 mol%, and the content of PEG lipid is 0.5 mol%-5 mol%.

[0193] The nanoparticle composition may contain one or more therapeutic and / or prophylactic agents, wherein the therapeutic and / or prophylactic 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 condition in a mammal in need thereof, the method comprising administering to the mammal and / or contacting the mammalian cells with a composition comprising therapeutic and / or prophylactic nanoparticles.

[0195] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any target polypeptide, including any naturally occurring 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), a phospholipid (eg, an unsaturated lipid such as DOPE or DSPC), a PEG lipid, and a structured lipid.

[0197] The nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle compositions. 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 60 nm to 130 nm.

[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 therapeutic and / or preventative specific delivery to mammalian organs, the method comprising administering any one of the nanoparticle compositions described above to a mammal, wherein the administration comprises contacting a mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or preventative to the organ. Therapeutic and / or preventative, for example, a protein, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, or a nucleic acid (such as RNA, for example mRNA) can be delivered to a cell or organ. In the case where therapeutic and / or preventative is mRNA, when a cell contacts the nanoparticle composition, translatable mRNA can be translated in the cell to produce a target polypeptide. However, substantially non-translatable mRNAs also can be delivered to the cell. Substantially non-translatable mRNA can be used as a vaccine and / or can isolate the translation component of a cell to reduce the expression of other species in the cell.

[0201] In some embodiments, the nanoparticle composition can be targeted to a specific type or class 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 class of cells, organs, or systems or groups thereof 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 the nanoparticle composition is administered to a mammal. In some embodiments, the target tissue is selected from the group consisting of 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, to a solution of compound A (100.00 g, 512.67 mmol) in dichloromethane (500 ml) were added compound B (116.36 g, 563.94 mmol) and DMAP (3.13 g, 25.63 mmol). The mixture was stirred at room temperature until uniform, and DCC (116.36 g, 563.94 mmol) was added portionwise. 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 mixture was filtered, and the filter cake was rinsed with dichloromethane (500 ml x 2). The filtrate was concentrated and purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-5%) to obtain compound C (170.68 g, 95.30%).

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

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

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

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

[0212] To a solution of compound J (1.00 g, 1.47 mmol) in methanol (30 mL) was added NaBH4 (0.22 g, 5.88 mmol) in batches and stirred at room temperature for 2 h. After completion of the reaction monitored by TLC, hydrochloric acid was added to quench the reaction. The solvent was distilled off and the mixture was washed with ethyl acetate (30 mL) and purified water (20 mL). The organic phase was then washed with 20 mL of brine. The organic phase was distilled and wet-loaded onto a column. 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). The mixture was stirred at room temperature for 24 hours and monitored by TLC. After the reaction was complete, purified water was added to the reaction solution with stirring. The layers were separated, and the filtrate was concentrated and eluted with a silica gel column (MeOH:DCM = 1:150, 1:120, 1:90, 1:70) using a gradient elution method to afford compound 1 (86 mg, 37.6% yield). 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, to a solution of compound K (0.30 g, 0.44 mmol) in dichloromethane (30 ml) were added M (0.11 g, 0.66 mmol), DCC (0.27 g, 1.32 mmol), and DMAP (54 mg, 0.44 mmol), and the mixture was stirred at room temperature for 5 hours. The mixture was concentrated and eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 100:1, 80:1, 60:1, 40:1) with gradient elution to obtain compound N (0.28 g, yield 77.8%).

[0219] To compound N (0.28 g, 0.34 mmol) were added a tetrahydrofuran solution of dimethylamine (2 mL, 4 mmol) and triethylamine (0.45 g, 4.42 mmol), and the mixture was stirred at room temperature overnight. Dichloromethane (30 ml) and purified water (20 mL) were added for washing. The organic phase was distilled and eluted with a silica gel column chromatography (MeOH:DCM = 1:90, 1:60, 1:40, 1:20) in a gradient elution to give compound 2 (0.21 g, yield 77.8%). 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, but 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), stirred at room temperature for 1 hour, concentrated to obtain P, which was set aside; Q (0.20 g, 0.29 mmol) and triethylamine (0.33 g, 3.30 mmol) were dissolved in 10 mL of dichloromethane solution and added dropwise to the above solution, stirred at room temperature overnight, washed with purified water (20 mL), and the organic phase was distilled and eluted with silica gel column chromatography (petroleum ether: ethyl acetate = 100:1, 80:1, 40:1, 20:1) to obtain compound R (0.18 g, yield 19.4%).

[0232] To compound R (0.18 g, 0.21 mmol) were added a tetrahydrofuran solution of dimethylamine (2 mL, 4 mmol) and triethylamine (1.5 mL). The mixture was stirred at room temperature overnight, washed with dichloromethane (50 mL) and purified water (30 mL). The organic phase was distilled and eluted with a silica gel column chromatography (MeOH:DCM = 1:30, 1:20) using a gradient elution method to give 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), and the mixture was stirred at room temperature for 5 hours. The mixture was washed with brine (20 mL), and the organic phase was distilled. The mixture was eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 40:1, 30:1, 20:1) with a gradient elution to obtain compound T (0.21 g, yield 84.0%).

[0238] To compound T (0.21 g, 0.24 mmol) were 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 eluted with a silica gel column chromatography (MeOH:DCM = 1:40, 1:30, 1:20) in a gradient elution to give 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, but 5-bromovaleric acid is used as the 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 the 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, except that 6-bromohexanol, undecanoic acid and N,N-dimethylglycine are used as raw materials instead of 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 solution of compound K (100 mg, 0.15 mmol) in DCM (5 ml), and the mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After completion of the reaction, the solvent was removed by concentration, the reaction solution was diluted with water, and extracted with ethyl acetate (30 ml x 3). 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] To a solution of compound V (183 mg, 0.22 mmol) in DCM (5 ml) were added compound W (109 mg, 1.10 mmol), DMAP (11 mg, 0.09 mmol), and DIPEA (83 mg, 0.64 mmol), and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed three times with 1 M NaOH. The organic layer was dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to afford 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 was the same as compound 10, except that N,N-dimethylethanolamine was used as the raw material instead of 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, to a solution of compound K (1.0 g, 1.47 mmol) in DCM (10 ml) at 0°C, methylsulfonic anhydride (383 mg, 2.20 mmol) and triethylamine (300 mg, 2.93 mmol) were added and stirred at room temperature for 12 h. The solvent was concentrated and removed, and the reaction mixture was diluted with water and extracted with ethyl acetate (30 ml x 3). This afforded 1.8 g of crude compound X, which was then dissolved in ultra-dry DMF and NaN3 (600 mg, 1.18 mmol) was added. The mixture was heated under reflux at 70°C for 4 h. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate (30 ml x 3). The organic layer was dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to afford compound Y (1.2 g, 71.73%).

[0266] Under argon protection, palladium on 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 purified by silica gel column chromatography (dichloromethane: methanol = 15:1) to give compound Z (420 mg, 43.65%).

[0267] To a solution of compound Z (100 mg, 0.15 mmol) in dichloromethane (50 ml) were added compound L (35 mg, 0.23 mmol), EDC (57 mg, 0.30 mmol), and DMAP (8 mg, 0.06 mmol). The mixture was stirred at room temperature for 7 hours and the reaction was monitored by TLC. After completion of the reaction, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to afford 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 the 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] Test Example 1 Lipid Nanoparticle (LNP) Encapsulation

[0277] The mRNA stock solution was dispersed in 20 mM acetic acid (pH 5.0) to a final concentration of 200 μg / mL (aqueous phase). A lipid mixture (oil phase) was prepared by mixing the Example compound: cholesterol: DSPC: DMG-PEG2000 at a molar ratio of 50:38.5:10:1.5. The mRNA and lipid mixture were mixed by controlling the flow rates of the aqueous and oil phases using a T-flow system to obtain LNP-encapsulated mRNA. The encapsulated LNPs were diluted with buffer and then concentrated by ultrafiltration. The buffer was then replaced, ultimately concentrating the LNPs to a concentration of 150 μg / mL. The pH of the LNPs was adjusted to approximately 7-8. Finally, the total and free mRNA content in the LNPs was determined using a Ribogreen assay kit and 10% Triton as a demulsifier, and the LNP encapsulation efficiency was calculated. The final LNP product was diluted with the diluent and added to a 1 ml particle size cell. The LNPs were then placed on a Malvern ZetaSizer instrument for particle size measurement. The results are shown in Table 1.

[0278] Particle size, PDI, and encapsulation efficiency are all important quality attributes of lipid nanoparticles. As shown in the table below, all tested compounds exhibited good encapsulation efficiency, a particle size suitable for mRNA delivery, and a narrow PDI. Compounds 4, 7, 8, and 9 exhibited precipitation during preparation, resulting in poor drugability and were not further studied. The precipitation of compound 9 further hindered characterization of the corresponding LNPs.

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

[0280] Compound number Encapsulation efficiency / % 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. hEPO mRNA was encapsulated into the formulations of Compound 1, Compound 2, Compound 3, Compound 5, and Compound 6. The LNP formulation preparation method and mRNA encapsulation method were 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. Blood was collected from the eye sockets at 6 hours and serum was separated.

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

[0286] 5. The hEPO protein content was 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 clearly 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 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. Fluorescence expression intensity detection: 10 minutes before the test, inject D-luciferin sodium salt (dose: 150 mg / kg) into the abdominal cavity of each mouse. Then, anesthetize the mouse with isoflurane and place it in the IVIS instrument, and select bioluminescence for detection.

[0293] 5. Use fluorescence intensity results to determine the delivery effect of the compound.

[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 the 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 show that compounds 2 and 5 have excellent mRNA delivery performance, and both have obvious spleen-targeted delivery advantages.

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

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, in, The compound of formula (I) is selected from Compound 2 Compound 5 2. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The lipid nanoparticle composition of claim 2, wherein the lipid component further comprises phospholipids, structural lipids and / or PEG lipids; The phospholipids are selected from one or more of the following compounds: Dilauroyl lecithin, Dimyristoylphosphatidylcholine, Dioleoyl lecithin, Dipalmitoylphosphatidylcholine, Distearoylphosphatidylcholine, Dioleoylphosphatidylcholine, Palmitoyloleoylphosphatidylcholine, 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine, l-hexadecyl-sn-glycero-3-phosphocholine, 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diaryl acyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine, 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-phosphate-(1-glycerol) sodium salt or sphingomyelin, wherein the structural lipid is selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol; and / or 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.

4. The lipid nanoparticle composition according to claim 3, wherein the phospholipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine or distearoylphosphatidylcholine.

5. The lipid nanoparticle composition of claim 3, wherein the structural lipid is cholesterol.

6. The lipid nanoparticle composition according to claim 3, wherein the PEG lipid is DMG-PEG2000.

7. The lipid nanoparticle composition according to any one of claims 2 to 6, further comprising a therapeutic and / or prophylactic agent, wherein the therapeutic and / or prophylactic agent is a nucleic acid.

8. The lipid nanoparticle composition according to claim 7, wherein the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

9. A pharmaceutical composition comprising the lipid nanoparticle composition according to any one of claims 2 to 8 and a pharmaceutically acceptable carrier.

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