Lipid Compounds and Lipid Nanoparticles for Delivery

Abstract

The present application discloses a compound, the structural formula of which is shown in formula (I), as well as its salts and isomers. The present application also discloses a nanoparticle composition comprising the above compound or its salt or its isomer. The nanoparticles of the present application can efficiently deliver drugs and vaccines into cells to achieve the therapeutic or prophylactic purposes of the drugs and vaccines.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically to lipid compounds and lipid nanoparticle compositions for the delivery of active 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 lies in whether therapeutic drugs can be safely and effectively delivered into target cells through a 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 has become difficult. Therefore, methods and compositions need to be developed to facilitate the delivery of therapeutic and / or prophylactic 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 for achieving the transfection of target genes and therapeutic purposes, due to problems such as the presence of immunogenic viral proteins, limited loading capacity of target genes, and high cost, lipid nanoparticles (LNPs), as non-viral vectors, have received extensive attention due to their good in vitro stability, biodegradability 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 proven to be effective transport carriers for bioactive substances such as small molecule drugs, proteins, and nucleic acids entering cells and / or intracellularly. 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 schemes 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, lipids containing amines, which can be easily protonated. Various such lipid-containing nanoparticle compositions have been demonstrated in the prior art. For example, patent document WO2017049245A2 discloses a compound and composition for intracellular delivery of a therapeutic agent. Summary of the Invention

[0005] Based on this, this application discloses a lipid compound and a lipid nanoparticle composition containing the compound, and the lipid has advantages such as high encapsulation efficiency and high expression.

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

[0007] 1. A compound of formula (I), or a salt or an isomer thereof,

[0008]

[0009] wherein R1 and R2 are independently C1-C6 alkyl, R3 and R4 are independently C1-C12 straight-chain alkyl, and R5 and R6 are independently selected from hydrogen or C1-C12 straight-chain alkyl;

[0010] X is selected from (C=0)O or O(C=0), and Y and Z are independently selected from (C=0)O or O(C=0);

[0011] m, n, o, and p are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0012] 2. The compound according to item 1, wherein R1 and R2 are each independently C1-C3 alkyl, preferably methyl.

[0013] 3. The compound according to item 1 or 2, wherein R3 and R4 are each independently C5-C9 straight-chain alkyl, preferably C6-C8 straight-chain alkyl.

[0014] 4. The compound according to any one of items 1-3, wherein R5 is hydrogen and R6 is C8-C11 straight-chain alkane.

[0015] 5. The compound according to any one of items 1-3, wherein R5 and R6 are each independently C5-C9 straight-chain alkyl, preferably C6-C8 straight-chain alkyl.

[0016] 6. The compound according to any one of items 1-5, wherein X is (C=O)O, Y is (C=O)O, Z is (C=O)O, or X is (C=O)O, Y is O(C=O), Z is (C=O)O, or X is (C=O)O, Y is (C=O)O, Z is O(C=O), or X is (C=O)O, Y is O(C=O), Z is O(C=O), or X is O(C=O), Y is (C=O)O, Z is (C=O)O, or X is O(C=O), Y is O(C=O), Z is (C=O)O, or X is O(C=O), Y is (C=O)O, Z is O(C=O), or X is O(C=O), Y is O(C=O), Z is O(C=O).

[0017] 7. The compound according to any one of items 1-6, wherein m is selected from 2, 3, 4, 5, 6, preferably 2, 3, 4.

[0018] 8. The compound according to any one of items 1 - 7, wherein n is selected from 2, 3, 4, 5, 6, preferably 2, 3, 4.

[0019] 9. The compound according to any one of items 1 - 8, wherein o is selected from 4, 5, 6, 7, 8, 9, preferably 5, 6, 7, 8.

[0020] 10. The compound according to any one of items 1 - 9, wherein p is selected from 4, 5, 6, 7, 8, 9, preferably 5, 6, 7, 8.

[0021] 11. The compound according to item 1, wherein the compound of (I) is selected from

[0022] Compound 1

[0023] Compound 2

[0024] Compound 3

[0025] Compound 4

[0026] Compound 5

[0027] Compound 6

[0028] Compound 7 Compound 8

[0029] 12. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises the compound according to any one of items 1 - 11.

[0030] 13. The lipid nanoparticle composition according to item 12, wherein the lipid component further comprises a phospholipid.

[0031] 14. The lipid nanoparticle composition according to item 13, wherein the phospholipid is selected from one or more of the following compounds:

[0032] Dilauroyl phosphatidylcholine (DLPC),

[0033] Dimyristoyl phosphatidylcholine (DMPC),

[0034] Dioleoyl phosphatidylcholine (DOPC),

[0035] Dipalmitoyl phosphatidylcholine (DPPC),

[0036] Distearoyl phosphatidylcholine (DSPC),

[0037] Dioleoyl phosphatidylcholine (DUPC),

[0038] Palmitoyl oleoyl phosphatidylcholine (POPC),

[0039] 1,2 - Di - O - octadecyl - sn - glycero - 3 - phosphocholine (18:0 Diether PC),

[0040] 1 - Oleoyl - 2 - cholesteryl dimethyl succinate - sn - glycero - 3 - phosphocholine (OChemsPC),

[0041] 1 - Hexadecyl - sn - glycero - 3 - phosphocholine (C16 Lyso PC),

[0042] 1,2 - Divinyl - sn - glycero - 3 - phosphocholine,

[0043] 1,2 - Diaroyl - sn - glycero - 3 - phosphocholine,

[0044] 1,2 - Dioleoyl - SN - glycero - 3 - phosphoethanolamine (DOPE),

[0045] 1,2 - Dihydroxystannyl - sn - glycero - 3 - phosphoethanolamine (ME 16.0PE),

[0046] 1,2 - Distearoyl - sn - glycero - 3 - phosphoethanolamine,

[0047] 1,2 - Divinylol - sn - glycero - 3 - phosphoethanolamine,

[0048] 1,2 - Divinyl - sn - glycero - 3 - phosphoethanolamine,

[0049] 1,2 - Diaryl - sn - glycero - 3 - phosphoethanolamine,

[0050] 1,2 - Dithiohexadecanoic acid - sn - glycero - 3 - phosphoethanolamine,

[0051] 1,2 - Dihydroxy - sn - glycero - 3 - phospho - (1 - glycerol) sodium salt (DOPG) or sphingomyelin.

[0052] 15. The nanoparticle composition according to item 13, wherein the phospholipid is DOPE.

[0053] 16. The nanoparticle composition according to item 13, wherein the phospholipid is DSPC.

[0054] 17. The nanoparticle composition according to any one of items 12 - 16, wherein the lipid component further comprises a structural lipid.

[0055] 18. The nanoparticle composition according to item 17, wherein the structural lipid is selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, and stigmasterol.

[0056] 19. The nanoparticle composition according to any one of item 17, wherein the structural lipid is cholesterol.

[0057] 20. The nanoparticle composition according to any one of items 12 - 19, wherein the lipid component further comprises a PEG lipid.

[0058] 21. The nanoparticle composition according to any one of item 20, 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.

[0059] 22. The nanoparticle composition according to any one of items 12 - 21, wherein the lipid component further comprises a cationic and / or ionizable lipid.

[0060] 23. The nanoparticle composition according to any one of items 12 - 22, further comprising a therapeutic agent and / or a prophylactic agent, wherein the therapeutic agent and / or prophylactic agent is selected from vaccines or compounds capable of eliciting an immune response, nucleic acids,

[0061] Preferably, the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, or mRNA.

[0062] 24. The nanoparticle composition according to any one of items 12 - 23, wherein the encapsulation efficiency of the therapeutic agent and / or prophylactic agent is ≥50%; or ≥80%; or ≥90%.

[0063] 25. The nanoparticle composition according to any one of items 12 - 24, wherein the average particle size of the nanoparticle composition is 50 nm - 110 nm.

[0064] 26. The nanoparticle composition according to any one of items 12 - 24, wherein the dispersity index of the nanoparticle composition is 0.04 - 0.20.

[0065] 27. Use of the compound according to any one of items 1 - 11 in the preparation of a lipid nanoparticle composition.

[0066] 28. A pharmaceutical composition comprising the nanoparticle composition according to any one of items 12 - 26 and a pharmaceutically acceptable carrier.

[0067] 29. A method of delivering a therapeutic agent and / or a prophylactic agent to mammalian cells, the method comprising administering to a subject the nanoparticle composition according to any one of items 12-26 or the pharmaceutical composition according to item 28, the administration comprising contacting the cells with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic agent and / or the prophylactic agent to the cells.

[0068] 30. The method according to item 29, wherein the mammalian cells are in a mammal.

[0069] 31. The method according to item 29 or 30, wherein the mammal is a human.

[0070] 32. The method according to any one of items 29-31, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0071] 33. A method of producing a polypeptide of interest in mammalian cells, the method comprising contacting the cells with the nanoparticle composition according to any one of items 12-26 or the pharmaceutical composition according to item 28 to deliver a therapeutic agent and / or a prophylactic agent to the cells, wherein the therapeutic agent and / or the prophylactic agent is mRNA that encodes the polypeptide of interest, whereby the mRNA is capable of being translated in the cells to produce the polypeptide of interest.

[0072] 34. The method according to item 33, wherein the mammalian cells are in a mammal.

[0073] 35. The method according to any one of items 33 or 34, wherein the mammalian cells are human.

[0074] 36. The method according to any one of items 33-35, wherein the nanoparticle composition or the pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0075] 37. A method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition according to any one of items 12-26 or the pharmaceutical composition according to item 31.

[0076] 38. The method according to item 37, wherein the disease or disorder is characterized by a dysfunctional or abnormal protein or polypeptide activity.

[0077] 39. The method according to item 37 or 38, wherein the disease or disorder is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases or metabolic diseases.

[0078] 40. The method according to any one of items 37 - 39, wherein the mammal is a human.

[0079] 41. The method according to any one of items 37 - 40, wherein the nanoparticle composition or the pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0080] 42. A method for specifically delivering a therapeutic agent and / or a prophylactic agent to an organ of a mammal, the method comprising administering to the mammal the nanoparticle composition according to any one of items 12 - 26 or the pharmaceutical composition according to item 28, the administration comprising contacting the organ of the mammal with the nanoparticle composition, thereby delivering the therapeutic agent and / or the prophylactic agent to the organ.

[0081] 43. The method according to item 42, wherein the mammal is a human.

[0082] 44. The method according to item 42 or 43, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0083] 45. The method according to any one of items 42 - 44, wherein the mammal is pretreated 24 hours or less before the contacting or administration step.

[0084] 46. The method according to any one of items 42 - 45, wherein the mammal is pretreated approximately one hour before the contacting or administration step.

[0085] Advantages of the Invention

[0086] The compounds of the present application can be used for the preparation of lipid nanoparticles. The nanoparticle composition containing the compounds provided by the present application can achieve the encapsulation and delivery of a therapeutic agent / prophylactic agent, safely deliver the therapeutic agent / prophylactic agent to the target position, achieve high expression, and exert the effect of the therapeutic agent / prophylactic agent.

[0087] The lipid nanoparticles prepared by the present application have a small average particle size, a high encapsulation rate, and high expression, and have broad application prospects in the field of drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art.

[0089] Figure 1 Shows the Luciferase fluorescence intensity shown by different LNP formulations 6 hours after intravenous injection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0090] The following describes exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered merely exemplary. Therefore, those of ordinary skill in the art should recognize 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, descriptions of well-known functions and structures are omitted in the following description for clarity and conciseness.

[0091] Terms and Definitions

[0092] 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 a straight-chain or branched-chain saturated hydrocarbon containing 1-12 carbon atoms, optionally substituted. Unless otherwise specified, the alkyl groups described herein refer to unsubstituted and substituted alkyl groups.

[0093] As used herein, the term "carbocyclic" or "carbocyclic group" refers to an optionally substituted monocyclic or polycyclic system including one or more carbon atom rings. The rings can be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty-membered rings. The term "5-7 membered ring" refers to a carbocyclic ring including a monocyclic, fused-ring or spiro-ring having 5-7 carbon atoms. The carbocyclic ring can include one or more carbon-carbon double bonds or triple bonds and can be non-aromatic or aromatic (e.g., cycloalkyl or aryl). Examples of carbocyclic rings include cyclopentyl, cyclohexyl, phenyl, naphthyl and 1,2-dihydronaphthyl.

[0094] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring and may or may not include any double or triple bonds. Unless otherwise specified, the carbocyclic rings described herein refer to unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocyclic rings.

[0095] As used herein, the term "heterocycle" or "heterocyclic group" refers to an optionally substituted monocyclic or polycyclic system comprising one or more rings, wherein at least one ring comprises at least one heteroatom, such as one, two, three, four, five heteroatoms, and the heteroatoms can be, for example, nitrogen, oxygen or sulfur atoms. The ring can be a three-, four-, five-, six-, seven-, eight-, nine-, ten-, eleven-, twelve-, thirteen- or fourteen-membered ring. The heterocycle can comprise one or more double bonds or triple bonds, and can be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, thienyl, pyrrolyl, pyrrolidinyl, furyl, thienyl, phenyl.

[0096] As used herein, the term "heterocycloalkyl" refers to a non-aromatic heterocycle and can include or can not include any double bonds or triple bonds. Unless otherwise specified, the heterocycles described herein refer to unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

[0097] As used herein, "aryl" includes an optionally substituted carbocyclic group comprising one or more aromatic rings. Examples of aryl include phenyl and naphthyl.

[0098] As used herein, "heteroaryl" refers to an optionally substituted heterocyclic group comprising one or more aromatic rings. Examples of heteroaryl include pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl and thiazolyl. Both aryl and heteroaryl can be selectively substituted. Unless otherwise specified, the aryl or heteroaryl described herein refers to unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl.

[0099] Unless otherwise indicated, the alkyl group may be optionally substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chlorine, bromine, fluorine or iodine groups), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxyl group, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyl groups (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, where 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), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thioesters (e.g., -C(S)H), sulfates, sulfonyl groups (e.g., -S(O)2-), amides (e.g., -C(O)NR2 or -N(R)C(O)R), azide groups (e.g., -N3), nitro groups (e.g., -NO2), cyano groups (e.g., -CN), isocyano groups (e.g., -NC), acyloxy groups (e.g., -OC(O)R), amino groups (e.g., -NR2, -NRH or -NH2), carbamoyl groups (e.g., -OC(O)NR2, -OC(O)NRH or -OC(O)NH2), sulfonamides, alkyl groups, alkenyl groups and cyclic groups (e.g., carbocyclic groups or heterocyclic groups). In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted with, for example, one, two, three, four, five or six substituents as defined herein. For example, a C1-6 alkyl group may be further substituted with 1, 2, 3, 4, 5 or 6 substituents as described herein.

[0100] As used herein, the term "compound" refers to all isomers and isotopes including the described structures. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the atomic 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 combining with solvents or water molecules by conventional methods to form sols and hydrates.

[0101] As used herein, the term "contact" means 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 ex vivo are well known in the biological field. For example, a nanoparticle composition can be contacted with mammalian cells placed in a mammal by a variety of administration routes (e.g., intravenous, intramuscular, intradermal and subcutaneous), and can involve various amounts of the nanoparticle composition. In addition, the nanoparticle composition can contact more than one mammalian cell.

[0102] 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.

[0103] As used herein, the term "enhanced delivery" refers to delivering a greater amount (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) of a therapeutic and / or prophylactic agent to a target tissue (e.g., mammalian liver) via nanoparticles compared to the level of delivery of the therapeutic and / or prophylactic agent to the target tissue (e.g., MC3, KC2, or DLinDMA) by control nanoparticles. It can be by comparing the amount of protein produced in the tissue with the weight of the tissue, comparing the amount of therapeutic and / or prophylactic in the tissue with the weight of the tissue, comparing the amount of protein produced within the tissue with the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue with the total amount of therapeutic and / or prophylactic agent in the tissue. It should be understood that enhanced delivery of nanoparticles to the target tissue need not be determined in the subject being treated, but can be determined in an alternative such as an animal model (e.g., a rat model). In certain embodiments, when the nanoparticle composition comprises a compound of formula (I), (IA), (IB), (IC), (ID), regardless of the route of administration, it has a substantially the same level of enhanced delivery.

[0104] As used herein, the term "specific delivery" or "specific transport" refers to delivering a greater amount (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) of a therapeutic and / or prophylactic agent to a target tissue (such as the mammalian liver) via nanoparticles compared to non-target tissues. The level of transport of nanoparticles to a specific tissue can be measured by comparing the weight of the protein produced in the tissue with the weight of the tissue, comparing the amount of therapeutic and / or prophylactic in the tissue with the weight of the tissue, comparing the weight of the protein produced in the tissue with the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue with the total amount of therapeutic and / or prophylactic agent in the tissue.

[0105] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the nanoparticle composition, relative to the total amount of therapeutic or prophylactic agent used in preparing 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 the 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 envelopment.

[0106] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or the post-translational modification of the polypeptide or protein.

[0107] 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 cell culture, in a Petri dish, etc., rather than events that occur within a living organism (e.g., an animal, a plant, or a microorganism).

[0108] As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, or a microorganism or its cells or tissues).

[0109] As used herein, the term "ex vivo" refers to events that occur outside of a living organism (e.g., an animal, a plant, or a microorganism or its cells or tissues). Ex vivo events can occur in an environment with minimal alteration from the natural (e.g., in vivo) environment.

[0110] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds and thus may exist in the form of 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. Mixtures of enantiomers and stereoisomers of a compound and methods for separating them into their component enantiomers or stereoisomers are well known.

[0111] As used herein, "lipid component" is a component of a nanoparticle composition that comprises one or more lipids. For example, the lipid component may include one or more cationic / ionizable lipids, PEGylated lipids, structural lipids, or other lipids such as phospholipids.

[0112] As used herein, a "linker" is a moiety that joins two portions, for example, the linkage between two nucleosides in a cap. The linker can include one or more groups including, but not limited to, phosphate groups (e.g., phosphate, borophosphate, thiophosphate, selenophosphate, and phosphonate), alkyl groups, amides, or glycerol. For example, two nucleosides of a cap analog can be linked at their 5' positions by a triphosphate group or by a chain including two phosphate moieties and a borophosphate moiety.

[0113] As used herein, "route of administration" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. Any one of the routes of administration can be selected to target delivery (e.g., specific delivery) to a particular region or system of the body.

[0114] 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. A "modified" substance can also be referred to herein as a "modified" substance. The substance can be modified or altered chemically, structurally, or functionally. For example, modified nucleobase species can include one or more non-naturally occurring substitutions.

[0115] 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 smaller and can 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.

[0116] As used herein, "naturally occurring" refers to occurring in nature without human assistance.

[0117] As used herein, a "patient" is a subject who may seek or require treatment, who needs treatment, who is receiving treatment, who will receive treatment, or who is the subject of care by a trained professional for a particular disease.

[0118] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety.

[0119] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0120] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) and that is substantially non-toxic and non-inflammatory to the patient. Excipients can include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorants, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate ( dibasic), calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0121] In this application, for convenience, the structural formulas of the compounds represent certain isomers, but this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like. It should be understood that not all isomers may have the same activity level. Additionally, the compounds represented by the structural formulas of the compounds described in this application may exhibit crystal polymorphism. Note that any crystal form, mixture of crystal forms, or its anhydride or hydrate is included within the scope of this application.

[0122] The terms "crystal polymorph", "polymorph", or "crystal form" refer to crystal structures in which a compound (or its salt or solvate) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can cause one crystal form to predominate. Crystal polymorphs of a compound can be prepared by crystallization under different conditions.

[0123] The nanoparticle composition of the present application may further comprise 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 existing acid or base moiety is modified by converting it to its salt form (e.g., by reacting the 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; base 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, glucuronate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate salts, etc.

[0124] 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 compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid or base forms 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 both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0125] As used herein, "phospholipid" is a lipid that includes a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. The phospholipid may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Specific phospholipids can promote fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of a membrane (e.g., cell membrane or intracellular membrane). The fusion of the phospholipid with the membrane can allow one or more elements of the lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more elements to cells.

[0126] 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 narrow particle size distribution.

[0127] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues that are typically linked by peptide bonds and can be produced naturally (e.g., isolated or purified) or synthetically.

[0128] 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, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, RNA can be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, e.g., in vivo translation of an mRNA inside a mammalian cell, can produce the encoded polypeptide. RNA can be selected from the 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.

[0129] As used herein, a "single unit dose" is the dose of any therapeutic agent administered as one dose / one time / one route / one point of contact, i.e., a single administration.

[0130] As used herein, a "divided dose" is the splitting of a single unit dose or the total daily dose into two or more doses.

[0131] As used herein, the "total daily dose" is the amount given or prescribed within 24 hours. It can be administered as a single unit dose.

[0132] As used herein, in the context of a nanoparticle composition, the "particle size" or "average particle size" refers to the average diameter of the nanoparticle composition.

[0133] As used herein, the terms "subject" or "patient" refer to any living 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 such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0134] As used herein, a "target cell" refers to any one or more cells of interest. 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.

[0135] As used herein, "target tissue" refers to any one or more target tissue types in 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 their systems or groups. In a particular application, the target tissue can be the kidney (e.g., intrarenal or femoral), the lung, the spleen, the vascular endothelium, or the kidney within a blood vessel (e.g., by intratumoral injection). "Non-target tissue" refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In a particular application, non-target tissues can include the liver and the spleen.

[0136] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or causes a desired biological and / or pharmacological effect when administered to a subject. A therapeutic agent is also referred to as an "active agent" or "active ingredient". Such substances include, but are not limited to, cytotoxins, radioisotopes, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0137] As used herein, the term "therapeutically effective amount" refers to the amount of a reagent to be delivered (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, 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 its symptoms, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0138] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can be carried out, for example, in vitro, ex vivo, or in vivo.

[0139] As used herein, the term "treatment" refers to partial or complete remission, alleviation, improvement, mitigation, delay of its onset, inhibition of its progression, reduction of its severity, and / or reduction of the incidence of one or more of its symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment can be carried out on a subject who does not exhibit a disease, disorder, and / or condition and / or on a subject who exhibits only early signs of a disease, disorder, and / or condition in order to reduce the risk associated with the pathological development of the disease, disorder, and / or condition.

[0140] This application discloses a compound of formula (I), or a salt or an isomer thereof,

[0141]

[0142] wherein R1 and R2 are independently C1-C6 alkyl, R3 and R4 are independently C1-C12 straight-chain alkyl, and R5 and R6 are independently selected from hydrogen or C1-C12 straight-chain alkyl;

[0143] X is selected from (C=0)O, O(C=0) or N, and Y and Z are independently selected from (C=0)O or O(C=0);

[0144] m, n, o, p are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0145] In a preferred embodiment, R1 is selected from C1-C6 straight-chain alkyl groups. For example, R1 is selected from C1-C5 straight-chain alkyl groups, R1 is selected from C1-C4 straight-chain alkyl groups, R1 is selected from C1-C3 straight-chain alkyl groups. 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 straight-chain C6 alkyl. In a further preferred embodiment, R1 is methyl or ethyl. In an even more preferred embodiment, R1 is methyl.

[0146] In a preferred embodiment, R2 is selected from C1-C6 straight-chain alkyl groups. For example, R2 is selected from C1-C5 straight-chain alkyl groups, R2 is selected from C1-C4 straight-chain alkyl groups, R2 is selected from C1-C3 straight-chain alkyl groups. 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 straight-chain C6 alkyl. In a further preferred embodiment, R2 is methyl or ethyl. In an even more preferred embodiment, R2 is methyl.

[0147] In a preferred embodiment, R3 is selected from C1-C12 straight-chain alkyl groups. For example, R3 is selected from C2-C12 straight-chain alkyl groups, R3 is selected from C3-C12 straight-chain alkyl groups, R3 is selected from C4-C12 straight-chain alkyl groups, R3 is selected from C5-C12 straight-chain alkyl groups, R3 is selected from C6-C12 straight-chain alkyl groups, R3 is selected from C6-C8 straight-chain alkyl groups, R3 is selected from C7-C12 straight-chain alkyl groups, R3 is selected from C8-C12 straight-chain alkyl groups, R3 is selected from C9-C12 straight-chain alkyl groups, R3 is selected from C10-C12 straight-chain alkyl groups, R3 is selected from C11-C12 straight-chain alkyl groups. 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, R3 is C12 straight-chain alkyl. In a further preferred embodiment, R3 is C8 straight-chain alkyl or C6 straight-chain alkyl.

[0148] In a preferred embodiment, R4 is selected from C1-C12 straight-chain alkyl groups. For example, R4 is selected from C2-C12 straight-chain alkyl groups, R4 is selected from C3-C12 straight-chain alkyl groups, R4 is selected from C4-C12 straight-chain alkyl groups, R4 is selected from C5-C12 straight-chain alkyl groups, R4 is selected from C6-C12 straight-chain alkyl groups, R4 is selected from C6-C8 straight-chain alkyl groups, R4 is selected from C7-C12 straight-chain alkyl groups, R4 is selected from C8-C12 straight-chain alkyl groups, R4 is selected from C9-C12 straight-chain alkyl groups, R4 is selected from C10-C12 straight-chain alkyl groups, R4 is selected from C11-C12 straight-chain alkyl groups. For example, R4 is a C1 alkyl group, R4 is a C2 alkyl group, R4 is a C3 straight-chain alkyl group, R4 is a C4 straight-chain alkyl group, R4 is a straight-chain C5 alkyl group, R4 is a C6 straight-chain alkyl group, R4 is a C7 straight-chain alkyl group, R4 is a C8 straight-chain alkyl group, R4 is a C9 straight-chain alkyl group, R4 is a C10 straight-chain alkyl group, R4 is a C11 straight-chain alkyl group, R4 is a C12 straight-chain alkyl group. In a further preferred embodiment, R4 is a C8 straight-chain alkyl group or a C6 straight-chain alkyl group.

[0149] In a preferred embodiment, R5 is hydrogen, and at the same time, R6 is selected from C1-C12 straight-chain alkyl groups. For example, R6 is selected from C2-C12 straight-chain alkyl groups, R6 is selected from C3-C12 straight-chain alkyl groups, R6 is selected from C4-C12 straight-chain alkyl groups, R6 is selected from C5-C12 straight-chain alkyl groups, R6 is selected from C6-C12 straight-chain alkyl groups, R6 is selected from C6-C8 straight-chain alkyl groups, R6 is selected from C7-C12 straight-chain alkyl groups, R6 is selected from C8-C12 straight-chain alkyl groups, R6 is selected from C9-C12 straight-chain alkyl groups, R6 is selected from C10-C12 straight-chain alkyl groups, R6 is selected from C11-C12 straight-chain alkyl groups. For example, R6 is a C1 alkyl group, R6 is a C2 alkyl group, R6 is a C3 straight-chain alkyl group, R6 is a C4 straight-chain alkyl group, R6 is a straight-chain C5 alkyl group, R6 is a C6 straight-chain alkyl group, R6 is a C7 straight-chain alkyl group, R6 is a C8 straight-chain alkyl group, R6 is a C9 straight-chain alkyl group, R6 is a C10 straight-chain alkyl group, R6 is a C11 straight-chain alkyl group, R6 is a C12 straight-chain alkyl group. In a further preferred embodiment, R5 is hydrogen and R6 is a C9-C11 straight-chain alkyl group. In an even more preferred embodiment, R5 is hydrogen and R6 is a C9 straight-chain alkyl group or a C10 straight-chain alkyl group or a C11 straight-chain alkyl group.

[0150] In a preferred embodiment, R5 and R6 are each independently selected from C1-C12 linear alkyl groups. For example, they are selected from C2-C12 linear alkyl groups, C3-C12 linear alkyl groups, C4-C12 linear alkyl groups, C5-C12 linear alkyl groups, C6-C12 linear alkyl groups, C6-C8 linear alkyl groups, C7-C12 linear alkyl groups, C8-C12 linear alkyl groups, C9-C12 linear alkyl groups, C10-C12 linear alkyl groups, C11-C12 linear alkyl groups. For example, R5 and R6 are independently a C1 alkyl group, a C2 alkyl group, a C3 linear alkyl group, a C4 linear alkyl group, a linear C5 alkyl group, a C6 linear alkyl group, a C7 linear alkyl group, a C8 linear alkyl group, a C9 linear alkyl group, a C10 linear alkyl group, a C11 linear alkyl group, a C12 linear alkyl group. In a further preferred embodiment, R5 is a C6 linear alkyl group and R8 is a C8 linear alkyl group or R8 is a C6 linear alkyl group and R5 is a C8 linear alkyl group.

[0151] In a preferred embodiment, m is 2, 3, 4, 5, 6. In a further preferred embodiment, m is 2, 3, 4.

[0152] In a preferred embodiment, n is 2, 3, 4, 5, 6. In a further preferred embodiment, n is 2, 3, 4.

[0153] In a preferred embodiment, o is 4, 5, 6, 7, 8, 9. In a further preferred embodiment, o is 5, 6, 7, 8.

[0154] In a preferred embodiment, p is 4, 5, 6, 7, 8, 9. In a further preferred embodiment, p is 5, 6, 7, 8.

[0155] In a preferred embodiment, both o and p are 6.

[0156] In a preferred embodiment, m is 2 and n is 4.

[0157] In a preferred embodiment, m is 2 and n is 2.

[0158] In a preferred embodiment, X is (C=O)O, Y is (C=O)O, Z is (C=O)O, or X is (C=O)O, Y is O(C=O), Z is (C=O)O, or X is (C=O)O, Y is (C=O)O, Z is O(C=O), or X is (C=O)O, Y is O(C=O), Z is O(C=O), or X is O(C=O), Y is (C=O)O, Z is (C=O)O, or X is O(C=O), Y is O(C=O), Z is (C=O)O, or X is O(C=O), Y is (C=O)O, Z is O(C=O), or X is O(C=O), Y is O(C=O), Z is O(C=O). In a further preferred embodiment, X is (C=O)O, Y is O(C=O), Z is O(C=O), or X is (C=O)O, Y is (C=O)O, Z is (C=O)O. In an even more preferred embodiment, X is (C=O)O, Y is O(C=O), Z is O(C=O).

[0159] In a preferred embodiment, the compound of formula (I) is compound 1

[0160]

[0161] In a preferred embodiment, the compound of formula (I) is compound 2

[0162]

[0163] In a preferred embodiment, the compound of formula (I) is compound 3

[0164]

[0165] In a preferred embodiment, the compound of formula (I) is compound 4

[0166]

[0167] In a preferred embodiment, the compound of formula (I) is compound 5

[0168]

[0169] In a preferred embodiment, the compound of formula (I) is compound 6

[0170]

[0171] In a preferred embodiment, the compound of formula (I) is compound 7

[0172]

[0173] In a preferred embodiment, the compound of formula (I) is compound 8

[0174]

[0175] The present application further provides a nanoparticle composition comprising a lipid component, the lipid component comprising a compound of formula (I) provided by the present application.

[0176] In some embodiments, the average particle size of the nanoparticle composition is 60 nm - 140 nm.

[0177] The nanoparticle composition may include, for example, lipid nanoparticles (LNP), liposomes, lipid vesicles, and lipid complexes.

[0178] The nanoparticle composition described in the present application 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 - 8. The nanoparticle composition may also comprise a variety of other components. For example, in addition to the compound according to formula (I), the lipid component of the nanoparticle composition may further include one or more other lipids.

[0179] 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 consisting 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, the PEG lipid may be PEG - c - DOMG, PEG - DMG, PEG - DLPE, PEG - DMPE, PEG - DPPC, or PEG - DSPE lipid.

[0180] The lipid component of the nanoparticle composition may include one or more structural lipids. The structural lipids may 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 corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0181] The lipid component of the nanoparticle composition may include one or more phospholipids, and the phospholipids for the nanoparticle composition and method may be selected from

[0182] dilauroyl phosphatidylcholine (DLPC),

[0183] Dipalmitoyl phosphatidylcholine (DMPC),

[0184] Dioleoyl phosphatidylcholine (DOPC),

[0185] Dipalmitoyl phosphatidylcholine (DPPC),

[0186] Distearoyl phosphatidylcholine (DSPC),

[0187] Dioleoyl phosphatidylcholine (DUPC),

[0188] Palmitoyl oleoyl phosphatidylcholine (POPC),

[0189] 1,2 - Di - O - octadecyl - sn - glycero - 3 - phosphocholine (18:0 Diether PC),

[0190] 1 - Oleoyl - 2 - cholesteryl dimethyl succinate - sn - glycero - 3 - phosphocholine (OChemsPC),

[0191] 1 - Hexadecyl - sn - glycero - 3 - phosphocholine (C16 Lyso PC),

[0192] 1,2 - Divinyl - sn - glycero - 3 - phosphocholine,

[0193] 1,2 - Diaroyl - sn - glycero - 3 - phosphocholine,

[0194] 1,2 - Dioleoyl - SN - glycero - 3 - phosphoethanolamine (DOPE),

[0195] 1,2 - Dihydroxystearoyl - sn - glycero - 3 - phosphoethanolamine (ME 16.0PE),

[0196] 1,2 - Distearoyl - sn - glycero - 3 - phosphoethanolamine,

[0197] 1,2 - Divinylol - sn - glycero - 3 - phosphoethanolamine,

[0198] 1,2 - Divinyl - sn - glycero - 3 - phosphoethanolamine,

[0199] 1,2 - Diaryl - sn - glycero - 3 - phosphoethanolamine,

[0200] 1,2 - Dithiohexadecanoic acid - sn - glycero - 3 - phosphoethanolamine,

[0201] 1,2 - Dihydroxy - sn - glycero - 3 - phosphate - (1 - glycerol) sodium salt (DOPG) or sphingomyelin.

[0202] 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.

[0203] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a PEG lipid, wherein the content of the ionizable lipid is 35 mol% - 65 mol%, the combined content of the phospholipid and cholesterol is 35 mol% - 65 mol%, and the content of the PEG lipid is 0.5 mol% - 5 mol%.

[0204] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a PEG lipid. In the LNP, the content of the ionizable lipid is 40 mol% - 50 mol%, the content of the phospholipid is 10 mol% - 15 mol%, the content of cholesterol is 35 mol% - 45 mol%, and the content of the PEG lipid is 1.5 mol% - 2.5 mol%.

[0205] The nanoparticle composition may comprise one or more therapeutic and / or prophylactic agents selected from vaccines or compounds capable of eliciting 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.

[0206] The present application provides methods for delivering a therapeutic and / or prophylactic agent 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 methods comprising administering to a mammal and / or contacting mammalian cells with a therapeutic and / or prophylactic nanoparticle composition.

[0207] In certain embodiments, the therapeutic and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, 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, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0208] The lipid component of the nanoparticle composition can include, for example, a compound according to formula (I), a phospholipid (such as an unsaturated lipid, such as DOPE or DSPC), a PEG lipid, and a structural lipid.

[0209] The nanoparticle composition can be characterized by a variety of methods. For example, microscopes (e.g., transmission electron microscope or scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the ζ potential. Dynamic light scattering can also be used to determine the particle size.

[0210] The average particle size of the nanoparticle composition is 60 nm - 140 nm.

[0211] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to represent the uniformity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index generally indicates a narrow particle size distribution.

[0212] This application further provides a method for specifically delivering a therapeutic agent and / or a prophylactic agent to a mammalian organ, the method comprising administering to the mammal a nanoparticle composition according to any one of the foregoing, the administration comprising contacting the mammalian organ with the nanoparticle composition, thereby delivering the therapeutic agent and / or the prophylactic agent to the organ. Therapeutic agents and / or prophylactic agents, e.g., 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 where the therapeutic and / or prophylactic is mRNA, when the cell contacts the nanoparticle composition, the translatable mRNA can be translated in the cell to produce the target polypeptide. However, substantially non-translatable mRNAs can also be delivered to cells. Substantially non-translatable mRNAs can be used as vaccines and / or can sequester the translation components of the cell to reduce the expression of other species in the cell.

[0213] In some embodiments, the nanoparticle composition can target a specific type or class of cells (e.g., cells of its specific organ or system). For example, a nanoparticle composition containing a target therapy and / or prophylaxis 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 their systems or groups means that a higher proportion of the nanoparticle composition comprising the therapeutic and / or prophylactic agent is delivered to the target destination (e.g., tissue) relative to other destinations, e.g., when the nanoparticle composition is administered 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 blood vessels (e.g., intracoronary or intr femoral), or kidney, and tumor tissue (e.g., by intratumoral injection).

[0214] Examples

[0215] Example 1 Synthesis of Compound 1

[0216] Compound 1

[0217] The synthetic route is as follows:

[0218]

[0219] Specifically, to a solution of Compound A1 (10.53 g, 46.80 mmol) in dichloromethane (50 ml) was added Compound B1 (10.0 g, 38.99 mmol), EDCI (9.7 g, 50.68 mmol) and DMAP (1.92 g, 15.66 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with an equal volume of saturated sodium bicarbonate solution, separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) gave Compound C1 (16.0 g, 88.00%).

[0220] To a solution of Compound D1 (9.6 g, 49.0 mmol) in dichloromethane (60 ml) was added Compound E1 (7.0 g, 40.8 mmol), EDCI (10.2 g, 53.1 mmol) and DMAP (2.0 g, 16.4 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with an equal volume of saturated sodium bicarbonate solution, separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) gave Compound F1 (12.20 g, 86.15%).

[0221] To a solution of Compound F1 (10.0 g, 28.70 mmol) in DMF (50 ml) was added Compound G1 (13.72 g, 70.28 mmol), NBu4I (13.0 g, 35.14 mmol) and K2CO3 (14.57 g, 105.43 mmol). The mixture was stirred at room temperature for 12 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with an equal volume of water, extracted with ethyl acetate (50 ml × 3), concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) gave Compound H1 (11.0 g, yield 82.7%).

[0222] To a solution of compound H1 (11.0 g, 23.70 mmol) in DMF (100 ml) was added compound C1 (16.0 g, 35.60 mmol), NBu4I (13.10 g, 35.6 mmol) and Cs2CO3 (15.4 g, 47.4 mmol). The mixture was stirred at room temperature for 15 h, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of water was added to the reaction mixture for dilution, and the mixture was extracted with ethyl acetate (100 ml × 3). The extract was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to give compound I1 (17.6 g, yield 88.10%).

[0223] To a solution of compound I1 (17.6 g, 20.86 mmol) in dichloromethane (100 ml) was added concentrated hydrochloric acid (60 ml). The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction was complete, the layers were separated. The organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound J1 (7.50 g, 53.00%).

[0224] To a solution of compound J1 (5 g, 7.36 mmol) in toluene (25 ml) were added compound K1 (2.9 g, 22.10 mmol) and pyridinium p-toluenesulfonate (1.9 g, 7.36 mmol). The mixture was heated under reflux for 20 h using a Dean-Stark apparatus, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, an equal volume of water was added for dilution, and the mixture was extracted with ethyl acetate (50 ml × 3). The extract was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 40:1) to give compound L1 (1.9 g, yield 32.54%).

[0225] At 0 °C, to a solution of compound L1 (1.9 g, 2.4 mmol) in super dry dichloromethane (10 ml) were added methanesulfonic anhydride (0.59 g, 3.4 mmol) and anhydrous triethylamine (0.7 ml). The mixture was stirred for 12 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was allowed to warm to room temperature, an equal volume of water was added for dilution, and the layers were separated. The organic layer was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 40:1) to give compound M1 (1.4 g, yield 67.31%).

[0226] Triethylamine (0.12 g, 1.15 mmol) and compound N1 (0.12 g, 1.15 mmol) were added to a solution of compound M1 (0.20 g, 0.23 mmol) in DMF (10 ml). The mixture was heated under reflux at 80 °C for 20 h, and the reaction was monitored by TLC. After the reaction was complete, it was cooled to room temperature. An equal volume of water was added to the reaction solution for dilution, and then extracted with ethyl acetate (50 ml × 3). The extract was concentrated, and silica gel column chromatography (dichloromethane:methanol = 40:1) was performed to obtain compound 1 (121 mg, yield 59.31%). 1H NMR (500 MHz, CDCl3) δ 4.90 - 4.71 (m, 1H), 4.17 - 3.90 (m, 4H), 3.49 - 3.37 (m, 1H), 2.36 - 2.24 (m, 10H), 2.20 (d, J = 6.6 Hz, 9H), 1.67 - 1.54 (m, 12H), 1.52 - 1.42 (m, 7H), 1.41 - 1.19 (m, 55H), 0.86 (t, J = 6.9 Hz, 9H); MS-ESI (m / z): 893.8 (M + H)+.

[0227] Synthesis of Compound 2 in Example 2

[0228] Compound 2

[0229] The synthetic route is as follows:

[0230]

[0231] Specifically, compound L1 (50 mg, 0.30 mmol), EDCI (62 mg, 0.33 mmol) and DMAP (13 mg, 0.10 mmol) were added to a solution of compound M2 (0.20 g, 0.25 mmol) in dichloromethane (10 ml). The mixture was stirred at room temperature for 7 h, and 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 then liquid separation was performed. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 30:1) was performed to obtain compound 2 (107 mg, 47.35%). 1H NMR (500 MHz, CDCl3) δ 4.89 - 4.81 (m, 1H), 4.11 - 3.99 (m, 6H), 3.43 (t, 1H), 2.41 - 2.33 (m, 4H), 2.32 - 2.24 (m, 10H), 1.87 - 1.80 (m, 2H), 1.69 - 1.54 (m, 12H), 1.49 (d, J = 6.2 Hz, 6H), 1.39 - 1.19 (m, 54H), 0.87 (t, J = 6.9 Hz, 9H); MS-ESI (m / z): 908.9 (M + H)+.

[0232] Synthesis of Compound 3 in Example 3

[0233] Compound 3

[0234] The synthesis route is as follows:

[0235]

[0236] Specifically, to a solution of Compound A3 (10.00 g, 55.23 mmol) in dichloromethane (100 ml) was added Compound B3 (15.6 g, 60.72 mmol), EDCI (12.72 g, 66.30 mmol) and DMAP (2.8 g, 22.1 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with an equal volume of saturated sodium bicarbonate solution, separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) was used to obtain Compound C3 (18 g, 78.26%).

[0237] To a solution of Compound C3 (10 g, 21.60 mmol) in DMF (100 ml) was added Compound G1 (1.4 g, 7.26 mmol), NBu4I (3.9 g, 10.56 mmol) and K2CO3 (7.10 g, 21.79 mmol). The mixture was stirred at room temperature for 12 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with an equal volume of water, extracted with ethyl acetate (100 ml × 3), concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 40:1) was used to obtain Compound I3 (5.00 g, yield 79.67%).

[0238] To a solution of Compound I3 (5.00 g, 5.73 mmol) in dichloromethane (100 ml) was added concentrated hydrochloric acid (60 ml). The mixture was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, it was separated, the organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) was used to obtain Compound J3 (3.75 g, 84.27%).

[0239] To a toluene (25 ml) solution of compound J3 (1.10 g, 1.56 mmol) was added compound K1 (0.63 g, 4.71 mmol) and pyridinium 4-methylbenzenesulfonate (0.59 g, 2.30 mmol). With a Dean-Stark apparatus, the mixture was heated under reflux for 20 h, and the reaction was monitored by TLC. After the reaction was complete, it was cooled to room temperature. An equal volume of water was added to the reaction solution for dilution, and then extracted with ethyl acetate (50 ml × 3). After concentration, silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) gave compound L3 (1.3 g, yield 78.12%).

[0240] To a dichloromethane (5 ml) solution of compound L3 (0.20 g, 0.24 mmol) was added compound M3 (0.08 g, 0.49 mmol), EDCI (0.09 g, 0.48 mmol), and DMAP (0.02 g, 0.13 mmol). The reaction was monitored by TLC. After the reaction was complete, it was concentrated, and silica gel column chromatography (dichloromethane:methanol = 20:1) gave compound 3 (109 mg, yield 48.88%). 1H NMR (600 MHz, Chloroform-d) δ 4.28 - 4.23 (m, 1H), 4.18 - 4.11 (m, 2H), 4.09 - 4.03 (m, 5H), 3.50 (td, J = 7.8, 2.6 Hz, 1H), 2.63 (t, J = 7.2 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.33 - 2.27 (m, 3H), 2.25 (s, 6H), 1.97 - 1.91 (m, 1H), 1.90 - 1.82 (m, 1H), 1.65 - 1.54 (m, 12H), 1.46 - 1.20 (m, 50H), 0.88 (t, J = 7.1 Hz, 9H); MS-ESI (m / z): 922.8 (M + H)+.

[0241] Example 4 Synthesis of Compound 4

[0242] Compound 4

[0243] The preparation method was the same as that of compound 3. Using 5-(dimethylamino)pentanoic acid instead of compound M3 as the raw material, an oily compound 4 was obtained. 1H NMR (600 MHz, Chloroform-d) δ 4.11 - 3.94 (m, 8H), 3.48 - 3.38 (m, 1H), 2.50 (t, J = 7.5 Hz, 2H), 2.36 - 2.26 (m, 4H), 1.69 - 1.54 (m, 18H), 1.54 - 1.38 (m, 7H), 1.38 - 1.19 (m, 54H), 0.87 (t, J = 7.1 Hz, 12H). MS-ESI (m / z): 951.1 (M + H)+.

[0244] Synthesis of Compound 5 in Example 5

[0245] Compound 5

[0246] The synthetic route is as follows:

[0247]

[0248] Specifically, to a solution of Compound A3 (10.0 g, 55.21 mmol) in dichloromethane (50 ml) was added Compound B3 (15.6 g, 60.72 mmol), EDCI (12.7 g, 66.31 mmol) and DMAP (2.82 g, 22.10 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with an equal volume of saturated sodium bicarbonate solution, and the layers were separated. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) gave Compound C3 (18.0 g, 78.26%).

[0249] To a solution of Compound D4 (1.0 g, 71.70 mmol) in dichloromethane (60 ml) was added Compound E4 (16 g, 86.00 mmol), EDCI (17.8 g, 93.00 mmol) and DMAP (3.5 g, 26.68 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with an equal volume of saturated sodium bicarbonate solution, and the layers were separated. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) gave Compound F4 (23.0 g, 85.28%).

[0250] To a solution of Compound F4 (7.0 g, 18.60 mmol) in DMF (50 ml) was added Compound G1 (7.3 g, 37.28 mmol), NBu4I (6.8 g, 18.61 mmol) and K2CO3 (5.12 g, 37.21 mmol). The mixture was stirred at room temperature for 12 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with an equal volume of water, extracted with ethyl acetate (50 ml × 3), concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) gave Compound H4 (4.4 g, yield 48.35%).

[0251] To a solution of compound H4 (4.4 g, 8.91 mmol) in DMF (100 ml) was added compound C3 (5.6 g, 13.31 mmol), NBu4I (3.3 g, 8.92 mmol) and Cs2CO3 (5.8 g, 17.80 mmol). The mixture was stirred at room temperature for 15 h and monitored by TLC. After the reaction was complete, an equal volume of water was added to the reaction solution for dilution, and the mixture was extracted with ethyl acetate (100 ml×3). After concentration, silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) was carried out to obtain compound I4 (4.5 g, yield 60.97%).

[0252] To a solution of compound I4 (4.5 g, 5.42 mmol) in dichloromethane (100 ml) was added concentrated hydrochloric acid (60 ml). The mixture was stirred at room temperature for 3 h and monitored by TLC. After the reaction was complete, the layers were separated. The organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) was carried out to obtain compound J4 (3.10 g, 86.11%).

[0253] To a solution of compound J4 (2 g, 3.01 mmol) in toluene (25 ml) were added compound K1 (1.2 g, 8.94 mmol) and pyridinium 4-methylbenzenesulfonate (1.1 g, 4.36 mmol). The reaction was carried out in a Dean-Stark apparatus under reflux for 20 h and monitored by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature, an equal volume of water was added for dilution, and the mixture was extracted with ethyl acetate (50 ml×3). After concentration, silica gel column chromatography (dichloromethane:methanol = 40:1) was carried out to obtain compound L4 (1.06 g, yield 45.30%).

[0254] To a solution of compound L4 (0.20 g, 0.26 mmol) in dichloromethane (10 ml) were added compound M4 (93 mg, 0.52 mmol), EDC (108 mg, 0.52 mmol) and DMAP (16 mg, 0.13 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to the reaction mixture for dilution. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 30:1) gave compound 5 (179 mg, 75.53%). 1H NMR (600 MHz, CDCl3) δ 4.09 - 4.00 (m, 8H), 3.44 (t, J = 5.3 Hz, 1H), 2.45 (t, J = 7.5 Hz, 2H), 2.37 - 2.25 (m, 11H), 1.68 - 1.54 (m, 18H), 1.53 - 1.51 (m, 1H), 1.49 - 1.39 (m, 3H), 1.38 - 1.21 (m, 52H), 0.87 (t, J = 7.1 Hz, 9H); MS-ESI (m / z): 909.1 (M + H)+.

[0255] Example 6 Synthesis of Compound 6

[0256] Compound 6

[0257] The preparation method was the same as that of compound 5. Using 1,2,4-butanetriol and 4-(dimethylamino)butyric acid instead of compounds K1 and M4 as raw materials, an oily compound 6 was obtained. 1H NMR (500 MHz, Chloroform-d) δ 4.22 (dt, J = 11.6, 6.0 Hz, 1H), 4.12 (dt, J = 12.7, 6.5 Hz, 2H), 4.05 (td, J = 6.6, 4.6 Hz, 4H), 3.50 (t, J = 7.7 Hz, 1H), 2.95 (s, 1H), 2.88 (s, 1H), 2.41 - 2.32 (m, 4H), 2.32 - 2.25 (m, 8H), 2.21 (t, J = 7.6 Hz, 1H), 2.04 - 1.78 (m, 4H), 1.65 - 1.51 (m, 11H), 1.46 - 1.37 (m, 3H), 1.36 - 1.19 (m, 52H), 0.87 (t, J = 5.8 Hz, 9H); MS-ESI (m / z): 894.8 (M + H)+.

[0258] Example 7 Synthesis of Compound 7

[0259] Compound 7

[0260] The preparation method is the same as that of Compound 5. Using 1,2,4-butanetriol and 3-(dimethylamino)propionic acid instead of Compound K1 and M4 as raw materials, the oily Compound 7 can be obtained. 1H NMR (600 MHz, CDCl3) δ 4.27 - 4.23 (m, 5.7 Hz, 1H), 4.18 - 4.11 (m, 2H), 4.09 - 4.03 (m, 5H), 3.50 (t, 1H), 2.63 (t, J = 7.2 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.32 - 2.22 (m, 9H), 1.97 - 1.91 (m, 1H), 1.89 - 1.82 (m, 1H), 1.64 - 1.54 (m, 12H), 1.45 - 1.40 (m, 2H), 1.36 - 1.22 (m, 46H), 0.88 (t, 9H). MS-ESI (m / z): 825.0 (M + H)+.

[0261] Synthesis of Compound 8 in Example 8

[0262] Compound 8

[0263] The preparation method is the same as that of Compound 5. Using 1,2,4-butanetriol instead of Compound K1 as the raw material, the oily Compound 8 can be obtained. 1H NMR (600 MHz, CDCl3) δ 4.25 - 4.18 (m, 1H), 4.15 - 4.07 (m, 2H), 4.07 - 4.01 (m, 5H), 3.49 (td, J = 7.8, 2.6 Hz, 1H), 2.45 (t, J = 7.5 Hz, 2H), 2.39 - 2.24 (m, 11H), 1.96 - 1.89 (m, 1H), 1.88 - 1.80 (m, 1H), 1.68 - 1.53 (m, 16H), 1.45 - 1.38 (m, 2H), 1.36 - 1.20 (m, 46H), 0.87 (t, J = 7.0 Hz, 9H). MS-ESI (m / z): 853.0 (M + H)+.

[0264] Test Example

[0265] Test Example 1 Lipid Nanoparticle (LNP) Encapsulation

[0266] Disperse the mRNA stock solution in 20 mM acetic acid solution (pH 5.3) to a final concentration of 200 μg / mL (aqueous phase). Mix the lipid components according to the molar ratio of the compound in the example: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5 to form a lipid mixture (oil phase). Control the flow rates of the aqueous and oil phases and mix the mRNA with the lipid mixture by T-mixing to obtain the mRNA encapsulated in LNP. Dilute the encapsulated LNP with buffer, then concentrate it by ultrafiltration and replace the diluent. Finally, concentrate the LNP to a mRNA concentration of 100 μg / mL, and adjust the pH of the LNP to around 7.0 - 8.5. Finally, use the Ribogreen kit and 10% OTG as a demulsifier to detect the total and free mRNA content in the LNP, and calculate the encapsulation efficiency of the LNP. Dilute the final LNP product with the diluent, add 1 ml to the particle size cell, and measure the particle size of the LNP on a Malvern ZetaSizer instrument. The results are shown in Table 1.

[0267] Particle size, PDI, and encapsulation efficiency are all important quality attributes of lipid nanoparticles. As can be seen from the following table, the tested compounds all have good encapsulation efficiency, particle sizes suitable for mRNA delivery, and narrow PDI.

[0268] Table 1: LNP characterization data of the compounds in the examples

[0269]

[0270]

[0271] Test Example 2: Testing of the delivery effect

[0272] 1. Encapsulate Luciferase mRNA into the LNP formulations of Compounds 1 - 8. The preparation method of the LNP formulation and the encapsulation method of mRNA are as described in Test Example 1.

[0273] 2. Inject the encapsulated LNP formulation into Balb / c mice via intravenous injection at a dose of 1 mg / kg intravenously, with 5 mice in each group.

[0274] 3. Detect the Luciferase fluorescence expression intensity in each mouse at 6 hours.

[0275] 4. Detection of fluorescence expression intensity: Inject sodium D-luciferin (dose: 150 mg / kg) into the peritoneal cavity of each mouse 10 minutes before detection. Then anesthetize the mouse with isoflurane and place it in the IVIS instrument to select bioluminescence for detection.

[0276] 5. Judge the delivery effect of the compound based on the fluorescence intensity results.

[0277] The luciferase experiment is the main method for detecting the in vivo expression of mRNA by fluorescence intensity. According to Figure 1 the results, after 6 hours of intravenous injection, among compounds 1-8, except for compound 1, the rest can express fluorescent proteins and generate fluorescence, and the effects of compounds 3 and 7 are the most significant.

[0278] Although the above combinations have been described for the embodiments of the present application, the present application is not limited to the above specific embodiments and application fields. The above specific embodiments are merely illustrative and guiding, rather than restrictive. Those of ordinary skill in the art can also make many forms under the inspiration of this specification and without departing from the scope protected by the claims of the present application, and these all fall within the scope of protection of the present application.

Claims

1. A compound, or a salt thereof, wherein The compound is selected from Compound 3 Compound 7 2. A lipid nanoparticle composition comprising a lipid component, wherein the lipid comprises the compound of claim 1.

3. A pharmaceutical composition comprising the lipid nanoparticle composition according to claim 2 and a pharmaceutically acceptable carrier.

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