Antigen presenting cell targeting lipids for delivery of nucleic acids and compositions and uses thereof

Abstract

The present disclosure provides a class of antigen presenting cell targeting lipids and compositions comprising the same. Specifically, the invention relates to a nanoparticle containing the lipid, which is used for delivering nucleic acid drugs, can improve the transfection efficiency of antigen presenting cells such as macrophages of the nucleic acid drugs and reduce the transfection efficiency of the nucleic acid drugs on other non-target cells, and has important significance on development and application of the nucleic acid drugs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority and benefits of the following Chinese patent applications filed with the State Intellectual Property Office of China: Chinese Patent Application No. 202311444250.X filed on November 01, 2023, and Chinese Patent Application No. 202311545292.2 filed on November 17, 2023. The entire text of the above patent applications is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of lipid delivery vectors, and specifically to a class of antigen presenting cell targeting lipid compounds, which can form lipid particles carrying nucleic acid drugs with other lipid components, thereby achieving extracellular to intracellular delivery of nucleic acids in vitro and in vivo. Specifically, the present invention relates to antigen presenting cell targeting lipids for delivering nucleic acids, and compositions and uses thereof. Background Art

[0004] Nucleic acid drugs replace, compensate, block or correct specific genes by introducing exogenous genes into target cells or tissues to achieve the purpose of treating and preventing diseases. Nucleic acid drugs show therapeutic potential in viral vaccines, protein replacement therapy, tumor immunity, cell therapy and gene editing. In order to achieve the above therapeutic effects, nucleic acid drugs must first enter the target cells and be able to produce enough target proteins. However, the development of safe and effective delivery nucleic acid vectors has become the key to the targeted delivery of nucleic acid drugs.

[0005] Researchers have developed a series of delivery systems for mRNA delivery, including lipid-based delivery systems and polymer-based delivery systems. Among them, lipid-based lipid nanoparticles (LNPs) have become the most promising mRNA delivery system due to their good biosafety and delivery efficiency. The approved mRNA new crown vaccines all use lipid nanoparticles to deliver mRNA antigens. Systemic mRNA vaccination by intravenous injection is currently believed to be the best route of administration for tumor vaccines, as it may promote CD8+T cell responses and circumvent the side effects of mRNA-inherent innate immunity. However, when administered systemically, more than 90% of the LNPs will be delivered to the liver, an effect that greatly reduces the effectiveness and safety of mRNA tumor vaccines. Charge-dependent organ-specific accumulation has been shown to enhance non-liver accumulation of mRNA, but the initial priming of vaccine-derived antigens by tumor-killing cells such as CD8+ T cells depends on antigen processing and presentation by antigen presenting cells (APCs). Currently, there is still a lack of effective targeted delivery systems for antigen presenting cells (APCs). Therefore, APCs-targeted expression of mRNA in vivo can minimize side effects and improve efficacy, which is considered to be the key point of the next generation of LNPs. Summary of the invention

[0006] The purpose of the present disclosure is to provide a new type of antigen presenting cell targeted lipid compound. The antigen presenting cell targeted lipid compound can form lipid particles carrying nucleic acid drugs with other lipid components, thereby achieving antigen presenting cell targeted delivery of nucleic acids in vitro and in vivo.

[0007] The present disclosure provides a compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in,

[0010] W is selected from a bond, O, NH or S;

[0011] Z is selected from a bond, O, NH or S;

[0012] R 1 Selected from C1-C 20 Alkyl, the C1-C 20 The alkyl group is optionally substituted with deuterium, OH, halogen;

[0013] R 2 Selected from C2-C 20 Alkenyl or C2-C 20Alkynyl, the C2-C 20 Alkenyl or C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen;

[0014] R 3 Selected from the following groups:

[0015] a)H, C1-C 20 Alkyl or C2-C 20 alkenyl;

[0016] or,

[0017] b)

[0018] wherein X is selected from a bond, O, NH or S;

[0019] Y is selected from a bond, O, NH or S;

[0020] R 4 Selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen;

[0021] R 5 Selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen;

[0022] R 6 Selected from H, C1-C 10 Alkyl or C2-C 10 alkenyl;

[0023] n is selected from 0, 1 or 2;

[0024] m is selected from 0, 1 or 2.

[0025] In some embodiments, W is selected from a bond or O.

[0026] In some embodiments, W is a bond.

[0027] In some embodiments, Z is selected from a bond or O.

[0028] In some embodiments, Z is a bond.

[0029] In some embodiments, R 1 Selected from C6-C 20 Alkyl, the C6-C 20 The alkyl group is optionally substituted with deuterium, OH, halogen.

[0030] In some embodiments, R 1 Selected from unsubstituted C6-C 20 alkyl.

[0031] In some embodiments, R 1 Select from unsubstituted C 10 -C 20 alkyl.

[0032] In some embodiments, R 1 Select from unsubstituted C 17 -C 20 alkyl.

[0033] In some embodiments, R 1 Selected from the following groups:

[0034]

[0035]

[0036] In some embodiments, R 2 Selected from C2-C 20 Alkenyl, the C2-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0037] In some embodiments, R 2 Selected from C6-C 20 Alkenyl, the C6-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0038] In some embodiments, R 2 Selected from unsubstituted C6-C 20 Alkenyl.

[0039] In some embodiments, R 2 Select from unsubstituted C 10 -C 20 Alkenyl.

[0040] In some embodiments, R 2 Select from unsubstituted C 17 -C 20 Alkenyl.

[0041] In some embodiments, R 2Selected from p is selected from 4, 5, 6, 7, 8, 9 or 10; q is selected from 1, 2 or 3; k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0042] In some embodiments, R 2 Selected from p is selected from 4, 5, 6, 7, 8, 9 or 10; k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0043] In some embodiments, R 2 Selected from p is selected from 6, 7, 8, 9 or 10; k is selected from 4, 5 or 6.

[0044] In some embodiments, R 2 Selected from the following groups:

[0045]

[0046]

[0047] In some embodiments, R 3 Selected from H or C1-C 10 alkyl.

[0048] In some embodiments, R 3 For H.

[0049] In some embodiments, R 3 Selected from the following groups:

[0050]

[0051] Among them, X, Y, R 4 , R 5 , R 6 , n, and m are as defined above.

[0052] In some embodiments, X is selected from a bond.

[0053] In some embodiments, Y is selected from a bond.

[0054] In some embodiments, R 4 Selected from C1-C 20 Alkyl or C2-C 20 Alkenyl, the C1-C 20 Alkyl or C2-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0055] In some embodiments, R 4 Selected from C6-C 20 Alkyl or C6-C 20Alkenyl, the C6-C 20 Alkyl or C6-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0056] In some embodiments, R 4 Selected from p is selected from 4, 5, 6, 7, 8, 9 or 10; q is selected from 1, 2 or 3; k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0057] In some embodiments, R 5 Selected from C1-C 20 Alkyl or C2-C 20 Alkenyl, the C1-C 20 Alkyl or C2-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0058] In some embodiments, R 5 Selected from C6-C 20 Alkyl or C6-C 20 Alkenyl, the C6-C 20 Alkyl or C6-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen.

[0059] In some embodiments, R 5 Selected from

[0060] p is selected from 4, 5, 6, 7, 8, 9 or 10; q is selected from 1, 2 or 3; k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0061] In some embodiments, p is selected from 5, 6, 7, 8, 9, or 10. In some embodiments, k is selected from 1, 2, 3, 4, 5, or 6. In some embodiments, R 4 , R 5 Independently selected from the following groups:

[0062]

[0063]

[0064] In some embodiments, R 6 Selected from H.

[0065] In some embodiments, n is selected from 1.

[0066] In some embodiments, m is selected from 1.

[0067] In some embodiments, the present disclosure provides the following compounds, or stereoisomers or pharmaceutically acceptable salts thereof:

[0068]

[0069]

[0070]

[0071] Another object of the present disclosure is to provide a composition, which comprises the compound represented by the above formula (I), or its stereoisomers or pharmaceutically acceptable salts thereof.

[0072] In some embodiments, the composition further comprises a nucleic acid.

[0073] In some embodiments, the composition further comprises one or more of a cationic lipid, a neutral helper lipid, cholesterol, and a PEG-modified lipid. Preferably, two or more of the cationic lipids are included.

[0074] In some embodiments, the composition comprises:

[0075] a) cationic lipid; b) neutral auxiliary lipid; c) cholesterol; d) PEG-modified lipid; and e) a compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0076] In some embodiments, in the composition, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0077] a) Cationic lipid 45% to 50%;

[0078] b) Neutral auxiliary lipids 5% to 10%;

[0079] c) Cholesterol 35% to 48%;

[0080] d) PEG-modified lipids 0-3%;

[0081] and e) 1% to 10% of the compound represented by formula (I) as described above, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0082] In some embodiments, in the composition, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0083] a) Cationic lipid 45% to 50%;

[0084] b) Neutral auxiliary lipids 5% to 10%;

[0085] c) Cholesterol 35% to 48%;

[0086] d) PEG-modified lipids 0-3%;

[0087] and e) 2% to 10% of the compound represented by formula (I) as described above, or its stereoisomer, or its pharmaceutically acceptable salt.

[0088] Another object of the present disclosure is to provide a nanoparticle, wherein the nanoparticle comprises the compound represented by the above formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments, the nanoparticles further include one or more of cationic lipids, neutral auxiliary lipids, cholesterol and PEG-modified lipids. Preferably, two or more of the cationic lipids are included.

[0090] In some embodiments, the nanoparticles include:

[0091] a) cationic lipids; b) neutral helper lipids; c) cholesterol; d) PEG-modified lipids; and

[0092] e) A compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0093] In some embodiments, the nanoparticles include:

[0094] a) cationic lipids; b) neutral helper lipids; c) cholesterol; d) PEG-modified lipids; and

[0095] e) a compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof;

[0096] and f) APCs targeting lipids.

[0097] In some embodiments, the APCs targeting lipid is selected from lipids that activate integrin receptors.

[0098] In some embodiments, the integrin receptor activating lipid is selected from RGD-modified lipids.

[0099] In some embodiments, the RGD-modified lipid is selected from RGD-modified DSPE.

[0100] In some embodiments, the RGD-modified lipid is selected from RGD-DSPE, DSPE-PEG-RGD or DSPE-PEG-iRGD. Preferably, the RGD-modified lipid is selected from RGD-DSPE.

[0101] In some embodiments, the APCs targeting lipid is selected from lipids that bind to S1P receptors.

[0102] In some embodiments, the lipid that binds to the S1P receptor is selected from any one of sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod, or Ceramide. Preferably, the lipid that binds to the S1P receptor is selected from S1P.

[0103] In some embodiments, the APCs targeting lipid is selected from lipids that bind to the G2A receptor.

[0104] In some embodiments, the lipid binding to the G2A receptor is selected from any one of lysophosphatidylcholine (Lyso PC), 9-HODE or 13-HODE. Preferably, the lipid binding to the G2A receptor is selected from Lyso PC.

[0105] In some embodiments, the APCs targeting lipid is selected from any one of RGD-modified DSPE, sphingosine 1-phosphate (S1P) or lysophosphatidylcholine (Lyso PC).

[0106] In some embodiments, the nanoparticles further comprise phosphatidylserine (PS).

[0107] In some embodiments, the cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, any one or more. Preferably, the cationic lipid is selected from DLin-MC3-DMA, SM-102 or ALC-0315, and more preferably, the cationic lipid is selected from SM-102.

[0108] In some embodiments, the neutral helper lipid is selected from any one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE). Preferably, the neutral helper lipid is selected from DSPC.

[0109] In some embodiments, the PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), DMG-PEG2000 (i.e., PEG2000-DMG), DMG-PEG5000, DSPE-PEG5000, and DSPE-PEG2000. Preferably, the PEG-modified lipid is selected from DMG-PEG2000.

[0110] In some embodiments, in the nanoparticles, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0111] a) Cationic lipid 45% to 50%;

[0112] b) Neutral auxiliary lipids 5% to 10%;

[0113] c) Cholesterol 35% to 48%;

[0114] d) PEG-modified lipids 0-3%;

[0115] and e) 1% to 10% of the compound represented by formula (I) as described above, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0116] In some embodiments, in the nanoparticles, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0117] a) Cationic lipid 45%-50%; b) Neutral auxiliary lipid 5%-10%;

[0118] c) Cholesterol 35% to 48%; d) PEG-modified lipid 0 to 3%;

[0119] and e) 2% to 10% of the compound represented by formula (I) as described above, or its stereoisomer, or its pharmaceutically acceptable salt.

[0120] In some embodiments, in the nanoparticles, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0121] a) Cationic lipid 45% to 50%;

[0122] b) Neutral auxiliary lipids 5% to 10%;

[0123] c) Cholesterol 35% to 48%;

[0124] d) PEG-modified lipids 0-3%;

[0125] e) 1% to 10% of the compound represented by formula (I) as described above, or its stereoisomer, or its pharmaceutically acceptable salt;

[0126] and f) APCs targeting lipid 0.1% to 10%.

[0127] In some embodiments, in the nanoparticles, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0128] a) Cationic lipid 45% to 50%;

[0129] b) Neutral auxiliary lipids 5% to 10%;

[0130] c) Cholesterol 35% to 48%;

[0131] d) PEG-modified lipids 0-3%;

[0132] e) 2% to 10% of the compound represented by formula (I) as described above, or its stereoisomer, or its pharmaceutically acceptable salt;

[0133] and f) APCs targeting lipid 0.1% to 10%.

[0134] In some embodiments, in the nanoparticles, based on the total molar amount of lipid as 100%, the molar ratio of each lipid component is:

[0135] a) Cationic lipid 45% to 50%;

[0136] b) Neutral auxiliary lipids 5% to 10%;

[0137] c) Cholesterol 35% to 48%;

[0138] d) PEG-modified lipids 0-3%;

[0139] e) the compound represented by formula (I) as described above, or its stereoisomer, or a pharmaceutically acceptable 1% to 10% thereof;

[0140] f) APCs targeting lipid 0.1% to 10%;

[0141] g) PS 0.5~5%.

[0142] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the cationic lipid is 45% to 48%.

[0143] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the cationic lipid is 45%, 46%, 47%, 48%, 49%, 50%, or any range selectable therein, including but not limited to 45% to 47% or 45% to 49%.

[0144] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 6% to 10%.

[0145] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 8% to 10%.

[0146] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 6%, 7%, 8%, 9%, 10%, or any range selectable therein, including but not limited to 7% to 10% or 7% to 9%.

[0147] In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 3% based on the total molar amount of the lipid as 100%. In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 2% based on the total molar amount of the lipid as 100%.

[0148] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the PEG-modified lipid is 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.5%, 3%, or any range selectable therein, including but not limited to 1.2% to 2.0% or 1.2% to 1.6%.

[0149] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of cholesterol is 35% to 42%.

[0150] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of cholesterol is 36% to 40%.

[0151] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of cholesterol is 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, or any range selectable therein, including but not limited to 36% to 38%, or 36% to 39%.

[0152] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I), or its stereoisomer, or its pharmaceutically acceptable salt is 1% to 5%.

[0153] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I), or its stereoisomer, or its pharmaceutically acceptable salt is 1%, 2%, 3%, 4%, 5%, or any selectable range therein, including but not limited to 1% to 3% or 1% to 4% or 1% to 5% or 2% to 5%.

[0154] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I), or its stereoisomer, or its pharmaceutically acceptable salt is 4% to 8%.

[0155] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I), or its stereoisomer, or its pharmaceutically acceptable salt is 4% to 6%.

[0156] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the APCs targeting lipid is 0.2% to 5%.

[0157] In some embodiments, based on the total molar amount of the lipid as 100%, the molar ratio of the APCs targeting lipid is 0.2% to 4%.

[0158] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the APCs targeting lipid is 0.4% to 3.5%.

[0159] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of APCs targeting lipid is 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.5%, 3.0%, 3.5%, or any range selectable therein, including but not limited to 1% to 2% or 0.4% to 2.5%.

[0160] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of PS is 0.5% to 4%.

[0161] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of PS is 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, or any range selectable therein, including but not limited to 1% to 2% or 1% to 3%.

[0162] In some embodiments, the nucleic acid is selected from RNA or DNA. Preferably, the nucleic acid is selected from RNA.

[0163] In some embodiments, the RNA includes one or more of non-self-replicating RNA (mRNA), self-replicating RNA (repRNA), trans-replicating RNA (taRNA), circular RNA (circRNA), micro RNA (miRNA), small (short) interfering (siRNA), lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

[0164] In some embodiments, DNA includes linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotide, antisense oligonucleotide.

[0165] In some embodiments, the nanoparticles can be in liquid or solid formulations.

[0166] In some embodiments, the nanoparticles are suitable for administration by injection.

[0167] In some embodiments, the nanoparticles are suitable for administration by inhalation.

[0168] Another object of the present disclosure is to provide a compound represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof, or a use of the above-mentioned nanoparticles in the preparation of nucleic acid drugs.

[0169] In another aspect, the present disclosure provides a method for targeted delivery of nucleic acids, the method comprising administering a therapeutically effective amount of the composition or nanoparticles to a subject in need thereof, the composition or nanoparticles comprising the compound represented by formula (I) described above, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0170] In another aspect, the present disclosure provides a method of regulating gene expression comprising delivering a nucleic acid to a cell, the method comprising: contacting the cell with a composition or nanoparticle described herein under conditions sufficient to cause uptake of the nucleic acid into the cell.

[0171] In some embodiments, the cells are contacted in vitro or ex vivo.

[0172] In some embodiments, the cells are contacted in vivo.

[0173] In some embodiments, modulation of gene expression is sufficient to treat or prevent a disease or disorder.

[0174] Any embodiment of any aspect of the present disclosure can be combined with other embodiments without contradiction. In addition, in any embodiment of any aspect of the present disclosure, any technical feature can be applied to the technical feature in other embodiments without contradiction.

[0175] Compared with the prior art, the present invention has the following beneficial effects:

[0176] The antigen presenting cell targeting lipid disclosed in the present invention can be combined with one or more of other components such as cationized lipids, neutral lipids, cholesterol and PEG lipids to form lipid particles for nucleic acid delivery. The lipid particles prepared by adding the antigen presenting cell targeting lipid disclosed in the present invention can improve the transfection efficiency of nucleic acid drugs in antigen presenting cells such as macrophages, reduce the transfection efficiency of other non-target cells, and especially improve the spleen-targeted delivery effect.

[0177] Definitions and explanations of terms

[0178] Unless otherwise indicated, the terms used in this disclosure have the following meanings, and the groups and term definitions recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in embodiments, etc., can be arbitrarily combined and combined with each other. A specific term should not be considered as uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. Scientific and technical terms related to this disclosure should have the meanings understood by ordinary technicians in the art. When a trade name appears in this article, it is intended to refer to its corresponding commodity or its active ingredient.

[0179] The term "nucleic acid" as used herein includes any compound and / or substance comprising a nucleotide polymer in its broadest sense. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides disclosed herein include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) or their hybrids.

[0180] The term "mRNA" as used herein refers to messenger ribonucleic acid. mRNA can be naturally or non-naturally occurring or synthetic. For example, mRNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or joints. mRNA can include a cap structure, a 5' transcription leader, a 5' untranslated region, a start codon, an open reading frame, a stop codon, a chain terminating nucleoside, a stem loop, a hairpin, polyadenylic acid (polyA), a polyadenylation signal and / or one or more cis-regulatory elements. mRNA can have a nucleotide sequence encoding a polypeptide. Translation of mRNA, such as in vivo translation of mRNA in mammalian cells, can produce a polypeptide. Traditionally, the basic components of a natural mRNA molecule include at least one coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap and a polyA sequence.

[0181] The term "untranslated region" or "UTR" as used herein refers to the portion of the mRNA upstream of the start codon and downstream of the stop codon that is not translated and is therefore referred to as the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed with the coding region and are therefore exonic when present in the mature mRNA.

[0182] As used herein, the term "5' untranslated region, 5'UTR" generally refers to a part of mRNA that is located 5' (i.e., "upstream") of the open reading frame and is not translated into protein. 5'UTR is generally understood to be a specific segment of messenger RNA (mRNA) that is located at the 5' end of the open reading frame of the mRNA. Typically, 5'UTR starts at the transcription start site and terminates at one nucleotide before the start codon of the open reading frame. Preferably, the 5'UTR has a length of more than 20, 30, 40 or 50 nucleotides. 5'UTR may include elements for controlling gene expression, also referred to as regulatory elements. The regulatory element may be, for example, a ribosome binding site. 5'UTR may be post-transcriptionally modified, for example, by adding a 5'-cap modification. The 5'UTR of mRNA is not translated into an amino acid sequence. 5'UTR sequences are generally encoded by genes that are transcribed into individual mRNAs during gene expression. The genomic sequence is first transcribed into pre-mature mRNA, which includes optional introns. Pre-mature mRNA is then further processed into mature mRNA during maturation. The maturation process includes the following steps: 5' capping, splicing of pre-mature mRNA to remove optional introns and 3' terminal modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 5'UTR corresponds to the mature mRNA sequence located between the start codon and, for example, the 5'-cap. Preferably, the 5'UTR corresponds to the nucleotide located at the 3' side of the 5' cap, more preferably from the 3' side nucleotide immediately adjacent to the 5' cap, to the nucleotide located at the 5' side of the start codon of the protein coding region, preferably to the sequence extending to the nucleotide immediately adjacent to the 5' side of the start codon of the protein coding region. The nucleotide immediately adjacent to the 3' side of the mature mRNA 5' cap typically corresponds to the transcription start site. The term "corresponding to" means that the 5'UTR sequence can be an RNA sequence in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence.

[0183] As used herein, the term "3' untranslated region, 3'UTR" generally refers to a part of mRNA that is located 3' (i.e., "downstream") of the open reading frame and is not translated into protein. Typically, 3'UTR is a part of the mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylic acid sequence of the mRNA. In the context of the present disclosure, the term 3'UTR may also include elements that are not encoded in the template, from which the RNA is transcribed, but added after transcription during the maturation process, such as polyadenylic acid sequences. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is typically encoded by a gene that is transcribed into a respective mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA comprising an optional intron. The pre-mature mRNA is then further processed into a mature mRNA during the maturation process. The maturation process comprises the following steps: 5' capping, splicing of pre-mature mRNA to remove optional introns and 3' terminal modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 3'-UTR corresponds to the polyadenylic acid sequence located between the 3' end of the protein coding region stop codon, preferably immediately following the 3' end of the protein coding region stop codon and the mRNA. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence. Preferably, the 3'UTR has a length of more than 20, 30, 40 or 50 nucleotides.

[0184] The term "RNA" as used herein includes, but is not limited to, non-self-replicating RNA (mRNA), self-replicating RNA (repRNA), trans-replicating RNA (taRNA), circular RNA (circRNA), micro RNA (miRNA), small (short) interfering RNA (siRNA), lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

[0185] As used herein, the term "deoxyribonucleic acid (DNA)" may be naturally occurring or non-naturally occurring, including but not limited to single-stranded DNA, double-stranded DNA, circular DNA. The DNA may comprise one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. The DNA in the nanoparticle may comprise any useful modification or alteration, such as modification or alteration of nucleobases, sugars, or internucleoside linkages (e.g., to phosphate linkages, to phosphodiester linkages, to phosphodiester backbones).

[0186] In some embodiments, DNA includes, but is not limited to, linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotides, antisense oligonucleotides. As used herein, the term "nucleic acid drug" or "nucleic acid-based drug" means a drug used to prevent or treat a specific disease, condition or disorder.

[0187] As used herein, a "nanoparticle" is a particle comprising one or more lipids. Nanoparticles are typically on the order of microns or smaller in size.

[0188] The term "cationic lipid" as used herein is ionizable, and the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated and are uncharged at pH above 8, 9, 10, 11 or 12. The ionizable cationic group can contain one or more protonatable amines that can form cationic groups at physiological pH. Other examples of cationic lipids include, but are not limited to, N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl ...methyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB ammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315.

[0189] The CAS number of the structural compound of DLin-MC3-DMA is 1224606-06-7, and the structural formula is as follows:

[0190]

[0191] The CAS number of the structural compound of SM-102 is 2089251-47-6, and the structural formula is as follows:

[0192]

[0193] The structural compound of ALC-0315 has a CAS number of 2036272-55-4 and a structural formula as follows:

[0194]

[0195] Other examples of the term "neutral lipid" as used herein include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), or phosphatidylethanolamine (DLPE).

[0196] Other examples of the term "PEG-modified lipid" as used herein include, but are not limited to, methoxypolyethylene glycol ditetradecylacetamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, DSPE-PEG5000, DSPE-PEG2000.

[0197] In some aspects, the APCs targeting lipid is selected from lipids that bind to the G2A receptor. The lipid binds to the G2A receptor, thereby inducing the tropism of macrophages, improving the uptake and transfection of nucleic acids in antigen-presenting cells, improving the expression efficiency of antigen proteins, and enhancing the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0198] In some embodiments, the lipids that bind to the G2A receptor include lysophosphatidylcholine (Lyso PC), 9-HODE, 13-HODE.

[0199] Lysophosphatidylcholine (Lyso PC) includes, but is not limited to, 16:0Lyso PC, 18:0Lyso PC, 20:0Lyso PC, and 24:0Lyso PC.

[0200] 9-HODE, CAS: 98524-19-7, structural formula is as follows:

[0201]

[0202] 13-HODE, CAS: 5204-88-6, has the following structural formula:

[0203]

[0204] In some aspects, the APCs targeting lipid is selected from lipids that activate integrin receptors. By adding the above lipids, integrin receptors can be activated, and the bridging molecules can bind to PS, thereby improving the uptake and transfection of nucleic acids in antigen presenting cells, improving the expression efficiency of antigen proteins, and enhancing the phagocytosis of apoptotic cells by antigen presenting cells, thereby improving the immune effect.

[0205] In some embodiments, the integrin receptor activating lipid comprises an RGD-modified lipid.

[0206] In some embodiments, the RGD-modified lipid is selected from RGD-modified DSPE.

[0207] In some embodiments, the RGD-modified lipid is selected from RGD-DSPE, DSPE-PEG-RGD, or DSPE-PEG-iRGD.

[0208] Wherein, RGD-DSPE refers to polypeptide modified distearoyl phosphatidylethanolamine. Wherein, RGD refers to Arg-Gly-Asp. The RGD-DSPE, CAS number: 2260795-79-5, has the following structural formula:

[0209]

[0210] DSPE-PEG-RGD refers to phospholipid-polyethylene glycol-RGD, wherein RGD refers to Arg-Gly-Asp. DSPE-PEG-RGD includes but is not limited to DSPE-PEG2000-RGD, DSPE-PEG3400-RGD, and DSPE-PEG5000-RGD.

[0211] DSPE-PEG-iRGD refers to phospholipid-polyethylene glycol-iRGD, wherein the amino acid composition of iRGD is: c(CRGDKGPDC) or c(CRGDRGPDC). DSPE-PEG-iRGD includes but is not limited to DSPE-PEG2000-iRGD and DSPE-PEG5000-iRGD.

[0212] In some aspects, the APCs targeting lipid is selected from lipids that bind to S1P receptors. The lipids bind to S1P receptors, induce the tropism of macrophages, improve the uptake and transfection of nucleic acids in antigen-presenting cells, improve the expression efficiency of antigen proteins, and enhance the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0213] In some embodiments, the S1P receptor binding lipid includes sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod, and Ceramide.

[0214] Sphingosine 1-phosphate (S1P) includes but is not limited to Sphingosine-1-phosphate (d17:0), Sphingosine-1-phosphate (d18:1), and Sphingosine-1-phosphate (d20:0).

[0215] Sphingosine (D-Sphingosine), CAS: 123-78-4, has the following structure:

[0216]

[0217] Fingolimod, CAS: 162359-56-0, has the following structure:

[0218]

[0219] Ozanimod, CAS: 1306760-87-1, has the following structure:

[0220]

[0221] Ceramide, CAS: 100403-19-8, has the following structure:

[0222]

[0223] In some embodiments, the lipid nanoparticle further comprises phosphatidylserine (PS).

[0224] In some embodiments, phosphatidylserine (PS) includes but is not limited to 10:0PS, 12:0PS, 17:0PS, 18:0PS.

[0225] The phosphatidylserine (PS) includes but is not limited to the following structural compounds, and the structural formula is as follows:

[0226]

[0227] In this article Indicates the connection site.

[0228] The graphic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, the wedge and dashed wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0229] The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer usually produce a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; while in phenols, the enol form predominates. The present disclosure includes all tautomeric forms of the compounds.

[0230] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0231] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E-type and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and their mixtures. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of these isomers and their mixtures involved in all substituents are also included within the definition of the compounds of the present invention. The compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0232] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo will not occur on an aromatic group.

[0233] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes the occurrence of the event or situation and the non-occurrence of the event or situation. For example, ethyl is "optionally" substituted by halogen, meaning that ethyl can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2 etc.) or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3 etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any substitution or substitution pattern that may not exist and / or cannot be synthesized in space will not be introduced.

[0234] When any variable (such as R a , R b ) occurs more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by 2 R b is replaced, then each R b All have independent options.

[0235] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0236] In this article, C m -C n It means having an integer number of carbon atoms in the range of mn. For example, "C1-C 10 ” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0237] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C 20 The term "alkyl" is understood to mean a straight or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3- dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C6 alkyl" may be understood to mean an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, specific examples of which include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc. The term "C1-C3 alkyl" may be understood to mean a straight or branched saturated alkyl group having 1, 2 or 3 carbon atoms. The "C1-C 20 The alkyl group may contain C6-C 18 Alkyl" or "C1-C 10 "C1-C6 alkyl" or "C1-C3 alkyl" and the like.

[0238] The term "alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. 20"Alkenyl" is understood to mean a linear or branched unsaturated hydrocarbon radical containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, "C2-C 20 "Alkenyl" is preferably "C2-C 10 "C2-C6 alkenyl" is further preferred. It will be understood that when the alkenyl contains more than one double bond, the double bonds may be separated or conjugated with each other. Specific examples of the alkenyl include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl or (Z)-1-methylprop-1-enyl, etc.

[0239] The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. 20 “Alkynyl” is understood to mean a linear or branched, unsaturated hydrocarbon radical containing one or more triple bonds and having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. “C2-C 20 "Alkynyl" is preferably "C2-C 10 Alkynyl". "C2-C 10 Examples of "alkynyl" include, but are not limited to, ethynyl (-C≡CH), propynyl (-C≡CCH 3、 -CH2C≡CH), but-1-ynyl, but-2-ynyl or but-3-ynyl. "C2-C 10 The "alkynyl group" may include "C2-C3 alkynyl group". Examples of "C2-C3 alkynyl group" include ethynyl (-C≡CH), prop-1-ynyl (-C≡CCH3), prop-2-ynyl (-CH2C≡CH).

[0240] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

[0241] The term "hydroxy" refers to an -OH group.

[0242] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H.3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0243] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically-labeled reagent for a non-isotopically-labeled reagent.

[0244] The term "therapeutically effective amount" means

[0245] An amount of a compound of the present disclosure that (i) treats a particular disease, condition or disorder, (ii) alleviates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition or disorder as described herein.

[0246] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on his or her knowledge and this disclosure.

[0247] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0248] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable acid addition or base addition salts, including salts formed between a compound and an inorganic acid or an organic acid, and salts formed between a compound and an inorganic base or an organic base.

[0249] The term "composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0250] The term "comprise" or "comprises" and its English variations such as comprises or comprising are to be construed in an open, non-exclusive sense, ie, "including but not limited to".

[0251] Typical routes of administration of the pharmaceutical compositions of the present disclosure include, but are not limited to, inhalation, intraperitoneal, mucosal, intramuscular, subcutaneous, intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0252] Figure 1 It is a diagram showing the in vitro macrophage transfection effect of the LNP formulation in Example 2 and the LNP formulation in the control group;

[0253] Figure 2 This is a diagram showing the in vivo spleen transfection effect of the LNP formulation in Example 2 and the LNP formulation in the control group;

[0254] Figure 3 This is a diagram showing the in vitro macrophage transfection effects of different LNP formulations in Example 3;

[0255] Figure 4 This is a diagram showing the in vitro macrophage transfection effects of different LNP formulations in Example 4;

[0256] Figure 5 This is a diagram showing the in vivo spleen transfection effects of different LNP prescriptions in Example 4 and the control group LNP prescription. DETAILED DESCRIPTION

[0257] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0258] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0259] GFP-luciferase mRNA (5'UTR-LUC-P2A-GFP-3'UTR-polyA) was homemade, and the sequence is as follows:

[0260]

[0261] The compounds and English abbreviations involved in the examples are described as follows:

[0262] SM-102 lipid, CAS number: 2089251-47-6, the structural formula is as follows:

[0263]

[0264] DMG-PEG2000, CAS No.: 160743-62-4, the structural formula is as follows:

[0265]

[0266] DSPC, CAS No.: 816-94-4, structural formula is as follows:

[0267]

[0268] Cholesterol: CAS No.: 57-88-5

[0269] Abbreviations:

[0270] THF: tetrahydrofuran; TBS: tert-butyldimethylsilyl; i-Pr2NEt: N,N-diisopropylethylamine; PE: petroleum ether; EA: ethyl acetate; DCC: dicyclohexylcarbodiimide; DMAP: 4-dimethylaminopyridine; octadecanoic acid: octadecanoic acid; heptane: n-heptane; DCM: dichloromethane; BF3·Et2O: boron trifluoride etherate; DMF: dimethylformamide; BnBr: benzyl bromide; AcOH: acetic acid; Ac2O: acetic anhydride; Et3N or TEA: triethylamine; DCI: 4,5-dicyanoimidazole; MeCN: acetonitrile; t-BuOOH: tert-butylperoxide; MeOH: methanol; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0271] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 The solvents for NMR measurement are deuterated dimethyl sulfoxide, methanol, chloroform, etc., and the internal standard is tetramethylsilane (TMS).

[0272] Synthesis of intermediate 4a:

[0273]

[0274] in,

[0275]

[0276] Step 1: Synthesis of intermediate 2a

[0277] (E)-10-octadecenoic acid (3.82 g, 13.54 mmol) and Jacobsen catalyst (80.14 mg, 132.74 μmol) were dissolved in tetrahydrofuran (5 mL), stirred in air for 20 minutes, and then dried by rotation. N, N-diisopropylethylamine (1.72 g, 13.27 mmol, 2.31 mL) was added, and after stirring for 10 minutes, compound 1a (2.5 g, 13.27 mmol) was added, and the mixture was stirred at room temperature for one day, and then detected by TLC. The mixture was dried by rotation, and purified by column chromatography (eluent: PE:EA=20:1-10:1) to obtain intermediate 2a (2.5 g, yield 40%).

[0278] 1 H NMR (400MHz, DMSO-d6) δ5.37–5.30(m,2H),4.96(d,J=5.1Hz,1H),4.03(dd,J=11.1,4.4Hz,1H),3.93(dd,J=11.1,5.7Hz,1H),3.71–3.62( m,1H),3.58–3.45(m,2H),2.32–2.23(m,2H),2.01–1.89(m,4H),1.56–1.46(m,2H),1.33–1.20(m,20H),0.89–0.84(m,12H),0.29(s,6H).

[0279] Step 2: Synthesis of intermediate 3a

[0280] Octadecanoic acid (2a, 1.18 g, 4.15 mmol) and intermediate 2a (1.7 g, 3.61 mmol), 4-dimethylaminopyridine (22.06 mg, 180.54 μmol) were dissolved in n-heptane (4 mL), dicyclohexylcarbodiimide (894.03 mg, 4.33 mmol) was added at 0°C, stirred at room temperature overnight, new spots were detected by TLC, the mixture was spin-dried, and purified by column chromatography (eluent: PE:EA=20:1-10:1) to obtain intermediate 3a (2.5 g, yield 93%).

[0281] 1H NMR(400MHz,Chloroform-d)δ5.41–5.26(m,2H),5.08–4.99(m,1H),4.29(dd,J=11.9,3.7Hz,1H),4.11(dd,J=11.9,6.3Hz,1H ),3.70–3.64(m,2H),2.31–2.21(m,4H),2.03–1.85(m,4H),1.59–1.53(m,4H),1.34–1.13(m,48H),0.83(s,15H),0.20(s,6H).

[0282] Step 3: Synthesis of intermediate 4a

[0283] Intermediate 3a (1.2 g, 1.63 mmol) was dissolved in dichloromethane (15 mL), and BF3-Et2O (231.01 mg, 1.63 mmol) was added at 0°C, and the reaction was continued for 10 minutes. TLC was performed, and the reaction solution was diluted with DCM (40 mL) and quenched with chilled phosphate buffer (8 mL, 1 M, pH = 7). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate 4a (970 mg, yield 95%).

[0284] 1 H NMR(400MHz,Chloroform-d)δ5.44–5.31(m,2H),4.45–4.04(m,4H),3.93(s,1H),2.40–2.29( m,4H),2.08–1.90(m,4H),1.66–1.56(m,4H),1.27(d,J=12.5Hz,48H),0.88(t,J=6.7Hz,6H).

[0285] Synthesis of intermediate 5a:

[0286]

[0287] Step 1: Synthesis of intermediate 11a

[0288] Compound 10a (2 g, 11.10 mmol) was dissolved in dimethylformamide (10 mL), and NaH (3.55 g, 88.81 mmol, 60% content) was added in batches at 0°C, and stirred at room temperature for one hour. Benzyl bromide (17.09 g, 99.91 mmol) was slowly added, and the reaction was allowed to proceed overnight. TLC detection was performed, and ice water (50 mL) was slowly added at 0°C to quench, and the mixture was extracted with ethyl acetate (3*50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (eluent: PE:EA=2:1-1:1) to obtain intermediate 11a (5 g, yield 71%).

[0289] 1 H NMR (400MHz, DMSO-d6) δ7.40–7.20(m,25H),4.91–4.50(m,11H),3.85–3.44(m,4H),3.41–3.28(m,2H).

[0290] Step 2: Synthesis of intermediate 5a

[0291] Dissolve ZnCl2 (5.40 g, 39.63 mmol) in acetic acid: acetic anhydride (12 mL, 1:5 (v / v)). Add intermediate 11a (5 g, 7.93 mmol) dissolved in acetic acid: acetic anhydride (6 mL, 1:5 (v / v)) at 0°C under nitrogen protection, warm to room temperature, stir for 4 hours, pour the mixture into 20 mL of ice water, filter the solid, spin dry and dissolve in methanol, add NaOH (634.10 mg, 15.85 mmol) dissolved in 5 mL of water, react for 5 hours. Add water (100 mL), extract with ethyl acetate (3*100 mL), wash with saturated brine, dry with anhydrous sodium sulfate, filter, spin dry, and purify by column chromatography (eluent PE:EA=1:1-1:2) to obtain intermediate 5a (2.6 g, yield 60%).

[0292] 1 H NMR (400MHz, DMSO-d6) δ7.42–7.05(m,20H),4.92–4.57(m,9H),3.77–3.42(m,4H),3.38–3.35(m,1H),3.33–3.30(m,1H).

[0293] Example 1 [1-[[Hydroxy-[(3,4,5,6-tetrahydroxytetrahydropyran-2-yl)methoxy]phosphoryl]oxymethyl]-2-[(E)-octadec-10-enoyl]oxyethyl]octadecanoate (Compound 1, referred to as Lipid N)

[0294]

[0295] The synthesis method is as follows:

[0296]

[0297] in,

[0298]

[0299] Step 1: Synthesis of intermediate 1-2

[0300] Dissolve intermediate 5a (2g, 3.70mmol) and triethylamine (1.50g, 14.80mmol, 2.06mL) in dichloromethane (40mL), add intermediate 1-1 (1.53g, 6.47mmol) dropwise at -60°C for more than 5 minutes, and after the addition is complete, transfer to room temperature and stir for 30 minutes, and detect by TLC. Add 0.3mL of triethylamine and diatomaceous earth to mix the sample, spin dry, and purify by column chromatography (eluent PE:EA=4:1~3:1 (containing 2% triethylamine)) to obtain intermediate 1-2 (2.5g, yield 91%).

[0301] 1 H NMR (400MHz, DMSO-d6) δ7.40–7.23(m,20H),4.89–4.59(m,9H),3.95–3.45(m,9H),3.40–3.35(m,1H),2.79–2.71(m,2H),1.14(s,12H).

[0302] Step 2: Synthesis of intermediate 1-3

[0303] Intermediate 4a (546.59 mg, 877.36 μmol) and intermediate 1-2 (500 mg, 674.89 μmol) were dissolved in dichloromethane (4 mL), 4,5-dicyanoimidazole (1M in MeCN, 2.02 mL) was added, and the mixture was stirred at room temperature for 1.5 hours. After cooling to 0°C, tert-butyl peroxide (217.22 mg, 1.69 mmol, 20.25 μL, content 70%) was added, and the mixture was stirred at 0°C for 30 minutes. After TLC detection, after the reaction was completed, the mixture was diluted with dichloromethane (20 mL), stirred with 5% NaHCO3 aqueous solution (10 mL) for 5 minutes, extracted with dichloromethane three times, washed with HCl (10 mL, 1 M) and saturated brine, dried by rotation, and purified by column chromatography (eluent: DCM: MeOH = 50: 1-10: 1) to obtain intermediate 1-3 (800 mg, yield 92%).

[0304] 1H NMR (400MHz, DMSO-d6) δ7.54–7.04(m,20H),5.38–5.31(m,2H),4.99–4.54(m,10H),4.34–4.16(m,6H),3.73–3.62(m,2H),3.55– 3.44(m,2H),2.95–2.88(m,2H),2.30–2.22(m,4H),1.97–1.87(m,4H),1.55–1.45(m,4H),1.35–1.19(m,48H),0.88–0.83(m,6H).

[0305] Step 3: Synthesis of intermediate 1-4

[0306] Intermediate 1-3 (700 mg, 547.44 μmol) was dissolved in dichloromethane (6 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (250.02 mg, 1.64 mmol, 245.12 μL) was added, stirred at room temperature for 10 minutes, and detected by TLC. 0.1 mL of acetic acid was added, the mixture was spin-dried, and purified by column chromatography (eluent: DCM:MeOH=15:1-8:1) to obtain intermediate 1-4 (500 mg, yield 74%).

[0307] 1 H NMR(400MHz, Methanol-d4)δ7.47–7.07(m,20H),5.41–5.31(m,2H),5.02–4.92(m,2H),4.90–4.53(m,8H),4.46–3.89(m, 6H),3.64–3.51(m,4H),2.29–2.20(m,4H),2.00–1.91(m,4H),1.58–1.50(m,4H),1.40–1.26(m,48H),0.94–0.88(m,6H).

[0308] Step 4: Synthesis of Compound 1

[0309] Intermediate 1-4 (150 mg, 122.39 μmol) and DDQ (277.83 mg, 1.22 mmol) were dissolved in carbon tetrachloride (2 mL) and dichloromethane (2 mL), and stirred at 80°C overnight under nitrogen protection. TLC detection, spin-dried, purified by column chromatography (eluent PE:EA=1:1), and then purified by column chromatography (eluent DCM:MeOH=10:1-4:1) to obtain compound 1 (referred to as Lipid N) (65 mg, yield 61%).

[0310] HR-MS:887.5625[M+Na] +.

[0311] 1 H NMR(400MHz,Methanol-d4)δ5.43–5.24(m,2H),4.50–4.36(m,2H),4.29–4.05(m,4H),3.90 –3.70(m,4H),2.51–1.80(m,8H),1.64–1.50(m,4H),1.44–1.04(m,48H),0.94–0.83(m,6H).

[0312] Other compounds except the compound synthesized in Example 1 can be synthesized by referring to the synthesis route and source materials of Example 1.

[0313] Example 2 Preparation of lipid nanoparticles

[0314] Different lipid nanoparticles were prepared by adding different molar ratios of antigen presenting cell targeting lipids into lipid nanoparticles. The specific formulations are shown in Table 1.

[0315] Table 1 Formulation of lipid nanoparticles

[0316]

[0317]

[0318] 2.1 Preparation process:

[0319] (1) According to Table 1, SM102, DSPC, DMG-PEG2000, cholesterol and Lipid N (lipid prepared in Example 1, compound 1) were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, GFP-luciferase mRNA (SEQ ID NO: 1) was added to the citric acid buffer to obtain an aqueous phase solution containing mRNA with a volume of three times that of ethanol.

[0320] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the resulting mixed solution was diluted with PBS buffer (pH = 7.4), and ultrafiltration was concentrated to a final mRNA concentration of 0.2 mg / mL to obtain the final lipid nanoparticle solution, which was stored in a -80°C refrigerator.

[0321] 2.2 LNP physical and chemical characterization

[0322] The LNP particle size, polydispersity index (PDI), and zeta potential (Zeta) obtained by dilution of aqueous solution were measured by Malvern particle size analyzer, and each sample was tested three times. The specific results are shown in Table 2 below.

[0323] Table 2 Physical and chemical characterization results of lipid nanoparticles

[0324] prescription Particle size (nm) PDI Zeta(mV) Prescription 1 95.39 0.12 11.57 Prescription 2 93.79 0.14 9.77 Prescription 3 98.32 0.17 6.38 Prescription 4 101.17 0.25 5.66 Prescription 5 132.33 0.35 3.09 Comparative Prescription A 96.57 0.05 15.11

[0325] As can be seen from Table 2, compared with the control group, the addition of Lipid N reduced the potential and reduced nonspecific transfection of non-target cells, thereby reducing toxicity and off-target effects.

[0326] 2.3 In vitro cell level study on the macrophage transfection effect of APCs-targeted lipid-modified LNP

[0327] On day 0, THP-1 cells in good culture state were mixed with 100 nM PMA (phorbol 12-myristate 13-acetate, Stemcell, cat: 74044) in RPMI1640 (Gibco, 61870127) medium and cultured at 2×10 4 cells / well were plated in a white opaque 96-well plate to induce THP-1 into macrophages; on the third day, the expression of CD14 was determined by flow cytometry to verify the successful induction. First, the control prescription A and prescriptions 1-3 were mixed with Opti-MEM (Gbico, 2492867) and β2-GPI (Probio, PB-BTA-02) at 0.25 μg / mL, respectively, so that the final concentration of β2-GPI in the culture medium was 1 for the mass ratio of β-GPI to lipid in LNP, and incubated at 37°C for 2 hours. 10 minutes before the end of the incubation, the THP-1 cells induced with PMA (phorbol12-myristate 13-acetate, Stemcell, cat: 74044) for 3 days were replaced with new RPMI1640 culture medium. After the incubation, the mixed sample after incubation was added to the 96-well plate, and 2 replicate wells were set. On the 4th day, prepare the luciferin substrate, take 1mL of luciferin bioluminescent substrate (D-luciferin), mix it evenly with 9mL PBS, and prepare it into working solution. Add 30μL / well of luciferin substrate to the cells transfected with LNPs sample, and react at room temperature for 10 minutes in the dark. After the reaction is completed, use the microplate reader to read the results, set the detection mode to chemiluminescence, and set the detection light source to Luminescence. Put the 96-well plate into the microplate reader to read directly, export the data for result analysis, and the results Figure 1 shown.

[0328] The results showed that after adding Lipid N lipid, due to the decrease in potential, the transfection rate of prescriptions 1-3 was lower than that of the control prescription A. As the proportion of Lipid N increased, the potential decreased, and the transfection rate was further reduced. After adding β2-GPI to the protein corona of LNP in the simulated systemic circulation, the transfection rate of LNP with Lipid N was significantly improved. LNPs with higher potential can non-specifically bind to negatively charged cell membranes, resulting in higher non-specific transfection. Therefore, lower potential can reduce non-specific cell transfection of LNP. The specific transfection level of macrophages in vivo simulated by β2-GPI protein corona is higher because macrophages in vivo specifically phagocytose through β2-GPI. After adding β2-GPI, the phagocytic process in vivo is simulated. By comparing the increase in phagocytic efficiency after adding β2-GPI, the efficiency of specific delivery of different LNPs to macrophages in vivo can be reflected. Therefore, Lipid N-modified LNPs can simulate antigen presenting cells (APCs) through biomimetic strategies to increase the transfection of LNPs in APCs-rich tissues such as the spleen.

[0329] 2.4 In vivo animal study on the APCs targeting ability of LNPs modified with APCs targeting lipids

[0330] The animal model was female BALB / c mice of 8 weeks (SHANGHAI SLAC); after tail vein injection of LNP sample (formulation 1) and control formulation A, spleens were taken for in vitro imaging at 3h, 6h, 48h and 72h, and the fluorescence intensity was measured and the area under the fluorescence intensity-time curve (3h-72h) was calculated. The results are shown in Figure 2 shown.

[0331] The results showed that the Lipid N prescription (prescription 1) disclosed herein significantly increased the intensity and AUC of luciferase mRNA in the spleen enriched with APCs compared to the control prescription A, and had a significantly improved transfection effect in the spleen.

[0332] Example 3 Preparation of Lipid N-containing targeted lipid composition nanoparticles

[0333] Different lipid nanoparticles were prepared by adding different molar ratios of the antigen presenting cell targeting lipid composition to the lipid nanoparticles. The specific prescriptions are shown in Table 3.

[0334] Table 3 Prescription of targeted lipid nanoparticles

[0335]

[0336]

[0337]

[0338] 3.1 Process steps:

[0339] (1) According to Table 3, SM102, DSPC, DMG-PEG2000, cholesterol, Lipid N and lyso PC (or RGD-DSPE or S1P) were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, GFP-luciferase mRNA was added to the citric acid buffer to obtain an aqueous phase solution containing mRNA with a volume of three times that of ethanol.

[0340] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the resulting mixed solution was diluted with PBS and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0341] 3.2 LNP physicochemical characterization

[0342] The LNP particle size, polydispersity index (PDI), and zeta potential (Zeta) obtained by dilution of aqueous solution were measured by Malvern particle size analyzer, and each sample was tested three times. The specific results are shown in Table 4 below.

[0343] Table 4 Physical and chemical characterization results of lipid nanoparticles

[0344] prescription <![CDATA[Particle size ( nm ) > PDI Zeta(mV) Prescription 6 110.47 0.18 11.23 Prescription 7 105.60 0.14 5.04 Prescription 8 108.47 0.17 5.05 Prescription 9 106.07 0.15 7.79 Prescription 10 108.27 0.18 6.83 Prescription 11 112.57 0.21 6.02 Prescription 12 102.73 0.10 11.09 Prescription 13 103.37 0.14 9.29 Prescription 14 112.90 0.14 8.09

[0345] 3.3 In vitro cell level study on macrophage transfection effect of Lipid N modified LNP

[0346] On day 0, THP-1 cells in good culture state were mixed with 100 nM PMA (phorbol 12-myristate 13-acetate, Stemcell, cat: 74044) in RPMI1640 (Gibco, 61870127) medium and cultured at 2×10 4Cells / well were plated in a white opaque 96-well plate to induce THP-1 into macrophages; on the third day, the expression of CD14 was measured by flow cytometry to verify the successful induction. First, prescription 1 and prescription 12-14 were mixed with Opti-MEM (Gbico, 2492867) and β2-GPI (Probio, PB-BTA-02) at 0.25 μg / mL, respectively, so that the final concentration of β2-GPI in the culture medium was 1 for the mass ratio of β-GPI to lipid in LNP, and incubated at 37°C for 2 hours. 10 minutes before the end of the incubation, the THP-1 cells induced with PMA (phorbol12-myristate 13-acetate, Stemcell, cat: 74044) for 3 days were replaced with new RPMI1640 culture medium, and the mixed sample was added to a 96-well plate, and 2 replicate wells were set. On the 4th day, prepare the luciferin substrate, take 1mL of luciferin bioluminescent substrate (D-luciferin), mix it evenly with 9mL PBS, and prepare it into working solution. Add 30μL / well of luciferin substrate to the cells transfected with LNPs sample, and react at room temperature for 10 minutes in the dark. After the reaction is completed, use the microplate reader to read the results, set the detection mode to chemiluminescence, and set the detection light source to Luminescence. Put the 96-well plate into the microplate reader to read directly, export the data for result analysis, and the results Figure 3 shown.

[0347] The results showed that after adding S1P (or Lyso PC or RGD-DSPE) to LNPs with Lipid N, the zeta potential decreased, but the level of macrophages induced by LNP transfection of THP-1 increased with the increase in the modification level of S1P (or Lyso PC or RGD-DSPE). It is expected that the transfection of APCs-rich tissues such as the spleen by LNPs in vivo can be increased.

[0348] Example 4 Preparation of Lipid N-containing targeted lipid composition nanoparticles

[0349] Different lipid nanoparticles were prepared by adding different molar ratios of the antigen presenting cell targeting lipid composition to the lipid nanoparticles. The specific formula is shown in Table 5.

[0350] Table 5 Prescription of targeted lipid nanoparticles

[0351]

[0352]

[0353]

[0354] 4.1 Preparation process

[0355] (1) According to Table 5, SM102, DSPC, DMG-PEG2000, cholesterol, Lipid N, PS and lysoPC (or S1P, or RGD-DSPE) were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, GFP-luciferase mRNA was added to the citric acid buffer to obtain an aqueous phase solution containing mRNA with a volume of three times that of ethanol.

[0356] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the obtained mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0357] 4.2 Physicochemical characterization of LNPs

[0358] The determination method is the same as 3.2 of Example 3. The results are shown in Table 6.

[0359] Table 6 Physical and chemical characterization results of targeted lipid nanoparticles

[0360] prescription Particle size (nm) PDI Zeta(mV) Prescription 15 101.83 0.12 18.58 Prescription 16 101.27 0.10 16.47 Prescription 17 103.07 0.13 14.28 Prescription 18 114.60 0.13 6.05 Prescription 19 109.83 0.13 6.69 Prescription 20 112.03 0.21 6.06 Prescription 21 111.30 0.10 11.63 Prescription 22 106.97 0.12 11.28 Prescription 23 112.47 0.10 10.46

[0361] The results showed that the potential of LNP modified with Lyso PC, PS and Lipid N decreased with the increase of the modified Lyso PC ratio, and the physical and chemical properties of LNP were good. The particle size of the prepared LNP was below 150nm, and PDI was <0.20. The physical and chemical properties of LNP modified with RGD-DSPE, PS and Lipid N were good. The particle size of LNP modified with RGD-DSPE, PS and Lipid N was good. The particle size of LNP modified with RGD-DSPE, PS and Lipid N was good. The potential of LNP modified with S1P, PS and Lipid N decreased with the increase of S1P modification ratio, and the physical and chemical properties of LNP were good. The particle size of LNP modified with RGD-DSPE, PS and Lipid N was good.

[0362] 4.3 In vitro cell level study on macrophage transfection effect of Lipid N modified LNP

[0363] The in vitro macrophage transfection effects of LNP prescriptions 1, 21, 22, and 23 were determined using the same method as in Example 3, 3.3. Figure 4 shown.

[0364] The results showed that after adding LipidN to PS-containing LNPs, the level of macrophages induced by LNP transfection of THP-1 was further increased after further adding S1P (or Lyso PC, or RGD-DSPE), and the level of macrophages induced by LNP transfection of THP-1 was increased with the increase of the modification level of S1P (or Lyso PC, or RGD-DSPE). It is expected that the transfection of APCs-rich tissues such as spleen by LNPs in vivo can be increased.

[0365] 4.4 In vivo animal study on the APCs targeting ability of LNPs modified with APCs targeting lipids

[0366] The animal model was: female, BALB / c mice, 8 weeks (SHANGHAI SLAC); the spleens were taken out for in vitro imaging at 3h, 6h and 72h after tail vein injection of prescription 1, prescription 23, control prescription A and positive control prescription 1. The fluorescence intensity was measured and the area under the fluorescence intensity-time curve (3h-72h) was calculated. The results are shown in Figure 5 shown.

[0367] The results showed that the positive control prescription 1 containing PS increased the distribution ratio of the spleen compared with the control prescription A without PS, but due to the overall reduced transfection ability caused by the potential, its AUC was not significantly different from the control prescription A. The prescription 1 containing Lipid N or the combination prescription 23 containing Lipid N, S1P and PS had a significantly increased spleen ratio compared with the control prescription A without PS and the positive control prescription 1 containing 5% PS, and the AUC increased three times. In general, the prescription containing LipidN and the combination prescription containing S1P, Lipid N and PS have significantly improved spleen / APCs targeting effects, especially in the long-term (72 hours) delivery of spleen targeting.

[0368] RGD-DSPE or lysophosphatidylcholine (Lyso PC) are similar to S1P in that they actively target antigen-presenting cells from different pathways. When combined with Lipid N and PS in a certain ratio to modify LNP, they can also better mimic apoptotic cells under physiological conditions, thereby enhancing antigen presentation and increasing its targeting effect. It is expected that it can also increase the transfection of LNPs in APCs-enriched tissues such as the spleen in vivo.

[0369] Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in, W is selected from a bond, O, NH or S; Z is selected from a bond, O, NH or S; R 1 Selected from C1-C 20 Alkyl, the C1-C 20 The alkyl group is optionally substituted with deuterium, OH, halogen; R 2 Selected from C2-C 20 Alkenyl or C2-C 20 Alkynyl, the C2-C 20 Alkenyl or C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen; R 3 Selected from the following groups: a)H, C1-C 20 Alkyl or C2-C 20 alkenyl; or, b) wherein X is selected from a bond, O, NH or S; Y is selected from a bond, O, NH or S; R 4 Selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen; R 5 Selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl is optionally substituted with deuterium, OH, halogen; R 6 Selected from H, C1-C 10 Alkyl or C2-C 10 alkenyl; n is selected from 0, 1 or 2; m is selected from 0, 1 or 2.

2. The compound represented by formula (I) according to claim 1, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: W is selected from a bond or O; Z is selected from a bond or O.

3. The compound of formula (I) according to any one of claims 1 to 2, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 2 Selected from C2-C 20 Alkenyl, the C2-C 20 Alkenyl is optionally substituted with deuterium, OH, halogen; Or, R 2 Selected from p is selected from 4, 5, 6, 7, 8, 9 or 10; q is selected from 1, 2 or 3; k is selected from 0, 1, 2, 3, 4, 5 or 6; Or, R 2 Selected from p is selected from 4, 5, 6, 7, 8, 9 or 10; k is selected from 0, 1, 2, 3, 4, 5 or 6; Or, R 2 Selected from the following groups:

4. The compound of formula (I) according to any one of claims 1 to 3, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from C6-C 20 Alkyl, the C6-C 20 The alkyl group is optionally substituted with deuterium, OH, halogen.

5. The compound of formula (I) according to any one of claims 1 to 4, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 3 Selected from H or C1-C 10 alkyl; Or, R 3 For H.

6. The following compound, or its stereoisomer or pharmaceutically acceptable salt, 7. A composition, wherein The composition comprises the compound of formula (I) as described in any one of claims 1 to 6, or its stereoisomer or a pharmaceutically acceptable salt thereof.

8. A nanoparticle, wherein The nanoparticles include the compound of formula (I) as described in any one of claims 1 to 6, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

9. The nanoparticle according to claim 8, wherein The nanoparticles include: a) cationic lipids; b) neutral helper lipids; c) cholesterol; d) PEG-modified lipids; and e) A compound of formula (I) according to any one of claims 1 to 6, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

10. The nanoparticle according to claim 9, wherein The cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOT AP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, any one or more thereof; The neutral auxiliary lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPP) C), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), Any one or more of dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE); The PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, DSPE-PEG2000, and DSPE-PEG5000.

11. The nanoparticle according to claim 8 or 9, wherein In the nanoparticles, the molar ratio of each lipid component is as follows, based on the total molar amount of lipids as 100%: a) Cationic lipid 45% to 50%; b) Neutral auxiliary lipids 5% to 10%; c) Cholesterol 35% to 48%; d) PEG-modified lipids 0-3%; e) 1-10% of the compound of formula (I) according to any one of claims 1 to 6, or its stereoisomer, or its pharmaceutically acceptable salt.

12. The nanoparticle according to any one of claims 8 to 11, wherein The nanoparticles include: a) cationic lipids; b) neutral helper lipids; c) cholesterol; d) PEG-modified lipids; and e) a compound of formula (I) according to any one of claims 1 to 6, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and f) APCs targeting lipids.

13. The nanoparticle according to claim 12, wherein The APCs targeting lipid is selected from lipids that activate integrin receptors; preferably, the lipid that activates integrin receptors is selected from lipids modified with RGD; preferably, the RGD-modified lipid is selected from RGD-modified DSPE; more preferably, the RGD-modified lipid is selected from any one of RGD-DSPE, DSPE-PEG-RGD or DSPE-PEG-iRGD. Alternatively, the APCs targeting lipid is selected from lipids that bind to S1P receptors; preferably, the lipid that binds to S1P receptors is selected from any one of sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod or Ceramide. Alternatively, the APCs targeting lipid is selected from lipids that bind to the G2A receptor; preferably, the lipid that binds to the G2A receptor is selected from any one of lysophosphatidylcholine (Lyso PC), 9-HODE or 13-HODE.

14. The nanoparticle according to any one of claims 12 to 13, wherein The targeted lipid nanoparticles further include phosphatidylserine (PS).

15. The nanoparticle according to any one of claims 8 to 14, wherein The nanoparticles also include nucleic acids.

16. The nanoparticle according to claim 15, wherein The nucleic acid is selected from RNA or DNA.

17. The nanoparticle according to claim 16, wherein The RNA includes non-self-replicating RNA One or more of mRNA, self-replicating RNA (repRNA), anti-replicating RNA (taRNA), circular RNA (circRNA), microRNA (miRNA), siRNA, lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

18. The nanoparticle according to claim 16, wherein The DNA includes linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotide, and antisense oligonucleotide.

19. Use of the compound of formula (I) according to any one of claims 1 to 6, or its stereoisomer or pharmaceutically acceptable salt, or the composition according to claim 7, or the nanoparticles according to any one of claims 8 to 18 in the preparation of nucleic acid drugs.

Images