Spleen-targeted ionizable lipid compounds, compositions comprising same and uses

Abstract

The invention provides an ionizable lipid compound. The ionizable lipid compound can efficiently deliver biomacromolecules including nucleic acid drugs or nucleic acid vaccines in a spleen-targeted mode. The invention also relates to lipid nanoparticles comprising said ionizable lipid compound and an active molecule, as well as to a pharmaceutical composition comprising said lipid nanoparticles.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202311865464.4 (application date: 2023 / 12 / 29, invention name: spleen-targeted ionizable lipid compounds, compositions containing the same and uses thereof). Technical Field

[0002] The present invention provides an ionizable lipid compound, which can efficiently and spleen-targetedly deliver biological macromolecules including nucleic acid drugs or nucleic acid vaccines. The present invention also relates to lipid nanoparticles (lipid nanoparticles, LNPs) comprising the ionizable lipid compound and active molecules, and a pharmaceutical composition comprising the lipid nanoparticles. Background Art

[0003] Lipid nanoparticles (LNPs) can encapsulate nucleic acid drug molecules such as RNA and protect them from nuclease degradation. They are also highly stable and biocompatible, and are currently a drug delivery system that has attracted much attention in the development of nucleic acid drugs. LNPs are usually prepared by mixing four components: ionizable lipids, phospholipids, cholesterol, and pegylated lipids in a certain proportion. The drug delivery efficiency or tissue targeting of LNPs is affected by the proportion of its components and the chemical structure of ionizable lipids. After LNPs are administered intravenously, soluble apolipoprotein E (apoE) is usually absorbed onto the surface of LNPs during blood circulation, which in turn promotes the binding of LNPs to the low-density lipoprotein receptors (LDLr) highly expressed on the sinusoidal surface of hepatocytes, thereby enabling targeted liver delivery of drugs. The Chinese patent application CN202311261516.7 filed by the same applicant on September 27, 2023 describes ionizable lipid compounds with optimized hydrophobic carbon chain length and amine head. In this application, the inventors found that further reducing the length of the hydrophobic carbon chain actually increases the delivery efficiency in the liver.

[0004] Although the liver-targeted delivery technology based on LNPs has achieved rapid development, the problem of targeted and specific delivery to organs other than the liver needs to be solved urgently. The spleen is the largest secondary lymphoid organ in the human body, widely involved in various immune functions of the body, and has a large number of antigen-presenting cells and B / T lymphocytes, which can quickly induce stronger immune responses and can be used as a new target for the design of new vaccines. However, due to the influence of the human reticuloendothelial system, after intravenous administration or LNP carriers, most nanoparticles or drugs are absorbed by the liver, and the dose reaching the spleen is relatively small, which poses a challenge to nucleic acid drug delivery and vaccine effectiveness.

[0005] Therefore, the development of an LNP delivery system for spleen-targeted delivery, such as a technology for targeted delivery to antigen-presenting cells in the spleen (such as dendritic cells (DC)), is of great significance for the development of nucleic acid drugs and vaccines. Summary of the Invention

[0006] The inventors of the present invention have found that: LNPs containing ionizable lipid compounds with specific carbon chain lengths can highly selectively deliver bioactive molecules to the spleen (hereinafter sometimes also referred to as "spleen targeting"). For this reason, the inventors of the present invention have completed the present invention.

[0007] In a first aspect, the present invention provides a lipid compound of formula (I),

[0008]

[0009] wherein

[0010] R 2a and R 3a are each independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht;

[0011] t and s are each independently 0 or 1, and when t or s is zero, it means that this part is directly a single bond; provided that t and s are not both 0;

[0012] A1, A2, and A3 are each independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, a divalent heteroaromatic hydrocarbon group, or a combination of the above two;

[0013] Ht, each occurrence thereof, is independently -R1-X-R2-Y-R3-Z-R4,

[0014] wherein

[0015] R1, each occurrence thereof, is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or

[0016] a divalent heteroaromatic hydrocarbon group;

[0017] X, each occurrence thereof, is independently

[0018] wherein

[0019] m, n, p, q, and r are each independently 1-6;

[0020] W is O, S, or NR c ;

[0021] L1, L3, L5, L7, and L9, which are directly connected to R1 or R2 and are each independently a single bond, O, S, or

[0022] NR d ;

[0023] L2, L4, L6, L8, and L 10 are each independently a bond, O, S, or NR e ;

[0024] V is an aliphatic hydrocarbon group, OR f , SR g , or NR h R i ,

[0025] wherein R b , R c , R d , R e , R f , R g , R h , and R i are each independently hydrogen, hydroxy, oxyaliphatic radical, monovalent aliphatic hydrocarbon group, monovalent heteroaliphatic hydrocarbon group, monovalent aromatic hydrocarbon group, or monovalent heteroaromatic hydrocarbon group;

[0026] Y and Z are each independently S or O each time they appear;

[0027] R2 is each independently a single bond, divalent aliphatic hydrocarbon group, divalent heteroaliphatic hydrocarbon group, divalent aromatic hydrocarbon group, or divalent heteroaromatic hydrocarbon group each time it appears;

[0028] R3 is each independently a single bond, divalent aliphatic hydrocarbon group, divalent heteroaliphatic hydrocarbon group, divalent aromatic hydrocarbon group, or divalent heteroaromatic hydrocarbon group each time it appears;

[0029] R4 is each independently a hydrophobic group selected from -(CH2CH2O) y -C1-C2 alkyl, -(CH2CH2O) y -C2 alkenyl, or -(CH2CH2O) y -C2 alkynyl, where y is 0, 1, or 2.

[0030] In some embodiments, both t and s are 0; or t is 0 and s is 1; or t is 1 and s is 0.

[0031] In some embodiments, R1 is C 1-6 (e.g., C 1-4 ) divalent aliphatic hydrocarbon group or C 1-6 (e.g., C 1-4 ) divalent heteroaliphatic hydrocarbon group, preferably C 1-4 divalent alkyl or C 1-4 divalent heteroalkyl.

[0032] In some embodiments, X is where each variable is defined as in formula (I). In further embodiments, L1, L3, L5, L7, and L9 are connected to R1 and are each independently a single bond, O, S, or NH. In further embodiments, X is

[0033] where R d and R e are defined as in formula (I). In some embodiments, R d and R e are each independently H or a C 1-4 monovalent aliphatic hydrocarbon group, preferably H or a C 1-4 monovalent alkyl group.

[0034] In some embodiments, both Y and Z are S; or Y is S and Z is O; or Y is O and Z is S; or both Y and Z are O.

[0035] In some embodiments, R2 is each independently a single bond or a C 1-6 divalent aliphatic hydrocarbon group (e.g., a C 1-4 divalent aliphatic hydrocarbon group, preferably a C 1-4 divalent alkyl group, more preferably a C 1-2 divalent alkyl group).

[0036] In some embodiments, R3 is each independently a single bond or a C 1-6 divalent aliphatic hydrocarbon group (e.g., a C 1-4 divalent aliphatic hydrocarbon group, preferably a C 1-4 divalent alkyl group). In further embodiments, R3 is each independently a single bond or or methylene preferably

[0037] In some embodiments, R4 is each independently ethyl (C2H5), methyl (CH3), vinyl, ethynyl, -(CH2CH2O)-CH3, -(CH2CH2O)-C2H5, -(CH2CH2O)-CH═CH2, -(CH2CH2O)-C≡CH, -(CH2CH2O)2-CH3, -(CH2CH2O)2-C2H5, -(CH2CH2O)2-CH═CH2, or -(CH2CH2O)2-C≡CH.

[0038] In some embodiments, Ht is each independently wherein both Y and Z are S; or Y is S and Z is O; or Y is O and Z is S; or both Y and Z are O; R3 is each independently a single bond or or a methylene group R 44 is ethyl (C2H5), methyl (CH3), vinyl or ethynyl.

[0039] In the present invention, A1, A2 and A3 form the amine head of the lipid compound, and a series of lipid compounds are obtained by the addition reaction with the hydrophobic lipid tail. Since the amine head can be protonated, the lipid compound nanoparticles are overall positively charged, and thus can undergo electrostatic interaction with negatively charged mRNA, cell membrane and lysosomal membrane, so that the lipid compounds can effectively encapsulate and deliver mRNA. In some embodiments, the pKa value of the amine head is greater than 4, preferably greater than 6, more preferably greater than 8.

[0040] In some embodiments, t is 1, s is 0, A2 is a single bond, and A1 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in a further embodiment, A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (for example, a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group); in a further embodiment, A1 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or a C1-C6 alkyl group or methyl. In some embodiments, t is 1, s is 1, A1, A2 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in a further embodiment, A1, A2 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (for example, a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group); in a further embodiment, A1, A2 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a and R 3a are each independently hydrogen or a monovalent aliphatic hydrocarbon group, or are hydrogen or a C1-C6 alkyl group or methyl.

[0041] In some embodiments, is wherein R 2a and R 3a are each independently hydrogen or a monovalent aliphatic hydrocarbon group, or are hydrogen or a C1-C6 alkyl group or methyl.

[0042] In some embodiments, the lipid compound of formula (I) is

[0043]

[0044] In a second aspect, the present invention provides a method for preparing a lipid compound of formula (I) wherein R1 is ethylidene, the method comprising:

[0045] Mixing and reacting an acrylic compound of formula (II) CH2=CH-X-R2-Y-R3-Z-R4 (II) with a hydrophilic amine in a molar ratio, wherein each variable is as defined in formula (I), wherein the molar ratio of the hydrophilic amine to the acrylic ester compound of formula (II) is greater than 4, such as 4.3:1 or 4.5:1, to ensure complete reaction; then optionally purifying the product by column chromatography to obtain the desired lipid compound of formula (I);

[0046] wherein the variables R2, X, Y, R3, Z and R4 are as defined in formula (I). In some embodiments, X is

[0047] where each variable is as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH. In further embodiments, X is

[0048] where R d and R e are as defined in formula (I). In some embodiments, R d and R e are each independently H or C 1-4 a monovalent aliphatic hydrocarbon group, preferably H or C 1-4 a monovalent alkyl group.

[0049] In some embodiments, in method (a), the product is purified by column chromatography to obtain the desired lipid compound of formula (I), wherein the column chromatography purification method is well known in the art.

[0050] In some embodiments, in the present invention, A1, A2 and A3 and the connected N together form the amine head of the lipid compound, which undergoes an addition reaction with the hydrophobic lipid tail to obtain a series of lipid compounds. Since the amine head can be protonated, the lipid compound nanoparticles are overall positively charged, and thus can undergo electrostatic interaction with negatively charged mRNA and cell membranes, so that the lipid compounds can effectively encapsulate and deliver mRNA.

[0051] In some embodiments, the hydrophilic amine in the method is selected from

[0052]

[0053] In some embodiments, the ketal-containing acrylate CH2=CH-C(O)O-R2-Y-R3-Z-R4 is The remaining variables are as defined in formula (I).

[0054] In some embodiments, the reaction is an addition reaction, preferably a Michael addition reaction.

[0055] In some embodiments, the reaction is carried out at a temperature of 60 - 85 °C, for example at 70 °C for 24 to 96 hours or longer.

[0056] The ketal-containing acrylate of the present invention can be synthesized according to methods known in the art, such as the method disclosed in CN110101665 A. The ketal(thio)ketal-containing acrylate is an ester compound obtained by the esterification reaction of acrylic acid with a ketal(thio)ketal-containing alcohol compound.

[0057] Other lipid compounds of the present invention can be prepared using other suitable starting materials by the above synthetic routes and other routes known in the art. The methods listed above may include one or more additional steps to add or remove appropriate protecting groups to ultimately allow the synthesis of the lipid compounds. In addition, the individual synthetic steps can be carried out in an alternative order or sequence to obtain the desired materials. Synthetic chemical transformations and protecting group methods (protection and deprotection) useful for synthesizing suitable lipid compounds are known in the art.

[0058] In a third aspect, the present invention provides a pharmaceutical composition, which comprises a pharmaceutical carrier and lipid nanoparticle particles (LNP), wherein the lipid nanoparticle particles comprise the lipid compound of formula (I) of the present invention and a drug active molecule.

[0059] In some embodiments, the drug active molecule targets the spleen. In some embodiments, the drug active molecule is a nucleic acid, an antigen, a vaccine, an immunomodulator, or other active ingredient that targets the spleen, or a combination thereof. In some embodiments, the drug active molecule is mRNA. In a further embodiment, the mRNA can be Firefly luciferase mRNA that expresses luciferase.

[0060] In some embodiments, the lipid nanoparticles have a particle size distribution of about 50 to about 500 nm.

[0061] In some embodiments, the lipid compound binds to the drug active molecule via non-covalent interactions, covalent bonds, or both.

[0062] In some embodiments, the lipid nanoparticles further contain other lipids, such as phospholipids, cholesterol, and pegylated lipids, which are the lipids conventionally used to form LNPs. In the LNPs of the present invention, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is (about 15 to about 50):(about 38.5 to about 75):(about 10 to about 25):(about 0.5 to about 3). In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 50:about 38.5:about 10:about 1.5. In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 25:about 48.5:about 25:about 1.5. In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 15:about 74.5:about 10:about 0.5. In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 20:about 60:about 19.5:about 0.5. In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 25:about 65:about 9.5:about 0.5. In some embodiments, the molar ratio of lipid compound:cholesterol:phospholipid:pegylated lipid is about 30:about 60:about 9.5:about 0.5. In some embodiments, the N / P ratio of the lipid compound to the nucleic acid ranges from about 5:1 to about 20:1. In some embodiments, the N / P ratio of the lipid compound to the nucleic acid ranges from about 7:1 to about 15:1. In some embodiments, the N / P ratio of the lipid compound to the nucleic acid is about 6:1, about 7.5:1, or about 9:1.

[0063] In some embodiments, the pegylated lipid refers to a lipid modified with polyethylene glycol (PEG). The hydrophilic PEG stabilizes the LNPs, regulates the nanoparticle size, and increases the half-life of the nanoparticles by reducing non-specific interactions with macrophages. In some embodiments, the pegylated lipid is selected from: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, or any combination thereof. The molecular weight of the PEG-modified PEG is typically 350 - 5000 Da. In some embodiments, the pegylated lipid is selected from distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0064] In some embodiments, the phospholipids are selected from: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.In some embodiments, the phospholipids are selected from: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0065] In some embodiments, the lipid nanoparticles of the present invention are prepared by the following method: including separately dissolving the ionizable lipid compound, cholesterol, DOPE, and DSPE-PEG2000 of the present invention in absolute ethanol, and dissolving the pharmaceutically active molecule and 5 to 20 times its mass (the N / P ratio of the lipid compound to the nucleic acid ranges from about 5:1 to about 20:1) into 100 mM sodium acetate buffer solution (pH = about 5.2). The above ethanol solution and sodium acetate buffer solution are mixed and prepared on a microfluidic machine at a flow rate of 1:3. In a further embodiment, the pharmaceutically active molecule is mRNA.

[0066] The present invention also relates to a method for delivering an antigen to antigen-presenting cells in the spleen (preferably professional antigen-presenting cells, such as dendritic cells and / or macrophages) or expressing an antigen in antigen-presenting cells in the spleen (preferably professional antigen-presenting cells, such as dendritic cells and / or macrophages), the method comprising administering to a subject in need thereof a pharmaceutical composition of the present invention.

[0067] The present invention also relates to a method for inducing an immune response (preferably an immune response against cancer) in a subject, the method comprising administering to a subject in need thereof a pharmaceutical composition of the present invention.

[0068] The present invention also relates to a method for treating diseases caused by damaged or abnormal spleen, the method comprising administering to a subject in need thereof a pharmaceutical composition of the present invention. In some embodiments, the diseases caused by damaged or abnormal spleen include lymphoma, leukemia, and the like.

[0069] Definition

[0070] The term "aliphatic" refers to a saturated or unsaturated, straight-chain or branched-chain, acyclic, cyclic, or polycyclic hydrocarbon moiety. Examples include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl moieties.

[0071] The term "alkyl" includes a hydrocarbon group selected from straight-chain and branched-chain saturated hydrocarbon groups containing 1 to 30, such as 1 to 24, 1 to 18, such as 1 to 12, further such as 1 to 10, still further such as 1 to 8 or 1 to 6 or 1 to 4 carbon atoms. Examples of monovalent alkyl or alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, and triacontyl, etc. Examples of divalent alkyl, i.e., alkylene groups, include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, icosylene, and triacotylene, etc.

[0072] A monovalent group is a group formed by removing one hydrogen atom from the corresponding hydrocarbon moiety. A divalent group is a group formed by removing two hydrogen atoms from the corresponding hydrocarbon moiety.

[0073] The term "alkenyl" includes a hydrocarbon group selected from straight-chain and branched-chain hydrocarbon groups containing at least one C═C double bond and 2 to 30, such as 2 to 24, 2 to 18, such as 2 to 8, further such as 2 to 6 carbon atoms. Alkenyl, such as C 2-6 Examples of alkenyl groups include, but are not limited to, ethenyl / vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, but-1,3-dienyl, 2-methylbut-1,3-dienyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-1,3-dienyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, etc.

[0074] The term "alkynyl" includes a hydrocarbon group selected from straight-chain and branched-chain hydrocarbon groups containing at least one C≡C triple bond and 2 to 30, such as 2 to 24, 2 to 18, such as 2 to 8, further such as 2 to 6 carbon atoms. Alkynyl, such as C 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl, 2-butynyl, and 3-butynyl.

[0075] The term "cycloalkyl" includes a hydrocarbyl group selected from saturated cycloaliphatic hydrocarbyl groups containing monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged or spiro cycloalkyls. The cycloalkyl may contain 3 to 30, 3 to 12, such as 3 to 10, further such as 3 to 8, further such as 3 to 6, 3 to 5 or 3 to 4 carbon atoms. Further, by way of example, the cycloalkyl may be selected from monocyclic groups containing 3 to 12, such as 3 to 10, further such as 3 to 8, 3 to 6 carbon atoms. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. The term "cycloalkenyl" refers to a non-aromatic, cyclic hydrocarbyl moiety containing at least one double bond, such as cyclohexenyl, cyclohexenylene. The term "cycloalkynyl" refers to a non-aromatic, cyclic hydrocarbyl moiety containing at least one triple bond, cyclooctynyl and cyclooctynylene. Similarly, cycloalkylene, cycloalkenylene and cycloalkynylene are the corresponding divalent groups.

[0076] The term "heteroaliphatic" refers to an aliphatic moiety containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. The heteroaliphatic groups of the present invention include alkyl, alkenyl, or alkynyl groups containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge, and cycloalkyl, cycloalkenyl, or cycloalkynyl moieties containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. The cyclic heteroaliphatic groups include 3- to 7-membered monocyclic heteroaliphatic groups and 7- to 12-membered bicyclic heteroaliphatic groups, which contain 1-3 or more, for example, 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the cyclic heteroaliphatic group is a 3- to 7-membered monocyclic heteroaliphatic group containing 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. The cyclic heteroaliphatic groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, dithietanyl, dihydropyridine, tetrahydropyridine, thiomorpholine, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxathianyl, dioxepanyl, oxathiepanyl, oxazepanyl, dithiepanyl, thiazepanyl, diazepane, thiazinyl, oxazepine, diazepine, thiazepine, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrroline, indoline, dioxanyl, dioxolanyl, pyrazoline, pyrazolidinyl, dithianyl, dithiocyclopentyl, pyrimidinone, dioxo-thiomorpholinyl, azabicyclo[3.1.0]hexyl, azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, etc. A typical heteroaliphatic group is a heteroalkyl group, that is, an alkyl group containing at least one heteroatom of N, O, or S, such as a C 1-6 alkyl group containing one N atom, a C 1-6 alkyl group containing one O atom, or a C 1-6 alkyl group containing one S atom; or a C 1-4 heteroalkyl group containing one N atom, a C 1-4 heteroalkyl group containing one O atom, or a C 1-4 heteroalkyl group containing one S atom.

[0077] The term "oxyaliphatic" refers to -O-aliphatic hydrocarbon group. Examples of oxyaliphatic groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0078] The term "aryl" (aromatic hydrocarbon group) refers to an aromatic ring system of C6 monocyclic, C10 bicyclic, C14 tricyclic, C20 tetracyclic or C24 pentacyclic. Examples of aryl include phenyl, phenylene, naphthyl, naphthylene, anthryl, anthrylene, pyrenyl and pyrenylene.

[0079] The term "heteroaryl" (heteroaromatic hydrocarbon group) refers to a ring system of aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, 11- to 14-membered tricyclic and 15- to 20-membered tetracyclic having one or more heteroatoms (such as O, N, S or Se). Examples of heteroaryl include furyl, furylene, fluorenyl, fluorenylene, pyrrolyl, pyrrolylene, thienyl, thienylene, oxazolyl, oxazolylene, imidazolyl, imidazolylene, benzimidazolyl, benzimidazolylene, thiazolyl, thiazolylene, pyridyl, pyridylene, pyrimidinyl, pyrimidinylene, quinazolinyl, quinazolinylene, quinolinyl, quinolinylene, isoquinolinyl, isoquinolinylene, indolyl and indolylene.

[0080] Unless otherwise specified, the aliphatic hydrocarbon groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbon oxy groups, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynylene groups, cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenylene groups, cycloalkynyl groups, cycloalkynylene groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenylene groups, aryl groups and heteroaryl groups mentioned in this application include both substituted and unsubstituted moieties. Possible substituents on cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl and heteroaryl include, but are not limited to, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 20 cycloalkyl, C3-C 20 cycloalkenyl, C2-C 20 heterocycloalkyl, C3-C 20 heterocycloalkenyl, C1-C 10 alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C1-C 10 alkylamino, C2-C 20 dialkylamino, arylamino, diarylamino, C1-C 10 alkylsulfonylamino, arylsulfonylamino, C1-C 10 alkylimino, arylimino, C1-C 10Alkylsulfonylimino, arylsulfonylimino, hydroxy, halo, thio, C1-C10 alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amido, amidino, guanidine, ureido, thioureido, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxy, and carboxylic ester. On the other hand, possible substituents on aliphatic hydrocarbon groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbon oxy groups, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, and alkynylene groups include all of the substituents listed above, but C1-C 10 alkyl is excluded. Cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl, and heteroaryl may also be fused to each other.

[0081] The term "pharmaceutically active molecule" refers to any chemical substance intended for the medical diagnosis, cure, treatment, or prevention of disease. In some embodiments, the pharmaceutically active molecule targets the spleen. In some embodiments, the pharmaceutically active molecule is a nucleic acid, antigen, vaccine, immunomodulator, or other active ingredient, or a combination thereof, that targets the spleen. In some embodiments, the antigen is an antigenic peptide or protein derived from a pathogenic antigen, tumor antigen, allergic antigen, or autoimmune autoantigen. In some embodiments, the vaccine is a tumor, influenza, or rabies vaccine.

[0082] The term "RNA" or "nucleic acid" includes a nucleic acid molecule of ribonucleotide residues or a nucleic acid molecule with chemically modified bases. In some embodiments, the RNA comprises all or most of the ribonucleotide residues.

[0083] The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of β-D-ribofuranosyl.

[0084] RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from a naturally occurring RNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. In some embodiments, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA typically comprises a 5'-untranslated region (5'-UTR), a peptide-coding region, and a 3'-untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides. In some embodiments, the RNA is in vitro transcribed RNA (IVT RNA) and can be obtained by in vitro transcription of a suitable DNA template.

[0085] In some embodiments, the concentration of the RNA in the pharmaceutical composition comprising the LNP of the present application is from about 0.01 mg / mL to about 1 mg / mL, or from about 0.05 mg / mL to about 0.5 mg / mL. In some specific embodiments, the concentration of the RNA is about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL, or about 0.50 mg / mL.

[0086] In some embodiments, when administered systemically, the LNPs target the spleen or accumulate in the spleen. Preferably, when administered systemically, the LNPs deliver the RNA to antigen-presenting cells in the spleen, preferably professional antigen-presenting cells such as dendritic cells and / or macrophages. In some embodiments, the LNPs release the RNA at the target organ or tissue and / or enter the cells of the target organ or tissue. In some embodiments, the target organ or tissue is the targeted lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, and the cells at the target organ or tissue are antigen-presenting cells such as dendritic cells. In some embodiments, after systemic administration of the LNPs, RNA expression occurs in the antigen-presenting cells of the spleen and in professional antigen-presenting cells. In some embodiments, after systemic administration of the LNPs of the present invention, RNA expression occurs within the spleen. In some embodiments, when administered systemically, the LNPs do not target or substantially do not target the lung and / or liver or do not accumulate or substantially do not accumulate in the lung and / or liver. In some embodiments, after systemic administration of the LNPs of the present invention, RNA expression does not substantially occur in the lung and / or liver. In some embodiments, the amount of nanoparticles targeting the spleen or accumulating in the spleen is at least 5 times, preferably at least 8 times, preferably at least 10 times, preferably at least 20 times, preferably at least 50 times, preferably at least 100 times, preferably at least 1000 times or even higher than the amount targeting the lung or accumulating in the lung. In some embodiments, after systemic administration of the LNPs, the RNA expression in the spleen is at least 5 times, at least 8 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, preferably at least 1000 times or even higher than the amount of RNA expression in the liver.

[0087] The antigen encoded by the RNA contained in the LNPs of the present application is preferably a disease-related antigen, or it elicits an immune response against the disease-related antigen or the cells expressing the disease-related antigen.

[0088] The pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients. The pharmaceutical composition of the present invention may further comprise at least one adjuvant.

[0089] The pharmaceutical composition of the present application can be administered by conventional routes, such as by parenteral administration, including administration by injection or infusion. In some embodiments, the pharmaceutical composition of the present invention is administered parenterally, such as intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. The term "parenteral administration" refers to administration in a manner that does not pass through the digestive tract, such as intravenous injection or intramuscular injection. Systemic administration (also known as general administration) is a route of administration that includes enteral administration (i.e., administration involving absorption through the gastrointestinal tract) or parenteral administration. The pharmaceutical composition of the present invention can be formulated for systemic administration.

[0090] The pharmaceutical composition of the present invention can be used to induce an immune response, particularly an immune response against a disease-related antigen or cells expressing a disease-related antigen, such as an immune response against cancer. Thus, the pharmaceutical composition can be used for prophylactic and / or therapeutic treatment of diseases involving disease-related antigens or cells expressing disease-related antigens, such as cancer. In some embodiments, the disease-related antigen is a tumor antigen.

[0091] The terms "subject" or "patient" in this application include humans and mammals.

[0092] The term "therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, can improve a disease or symptom. "Prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, can prevent a disease or symptom. The amount of the therapeutic agent constituting the "therapeutically effective amount" or the amount of the prophylactic agent constituting the "prophylactically effective amount" varies with the therapeutic agent / prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient / subject to be treated / prevented. A person of ordinary skill in the art can routinely determine the therapeutically effective amount and the prophylactically effective amount based on their knowledge and this disclosure.

[0093] The term "pharmaceutically acceptable" in this application means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with the human or mammal for which it is used to prevent or treat a disease or disorder.

[0094] As used in this application, the term "treatment" refers to administering one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease, in order to cure, relieve, alleviate, improve or affect the disease or the symptoms of the disease. In the context of this application, unless specifically stated to the contrary, the term "treatment" may also include prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 Shows in vivo bioluminescence imaging of Firefly luciferase (Fluc) mRNA delivered by LNPs prepared from lipid compounds YX-30, YX-32, and YX-34 (dose administered: 150 μg / kg).

[0096] Figure 2 Shows in vivo and ex vivo tissue bioluminescence imaging of Firefly luciferase (Fluc) mRNA delivered by LNP prepared from lipid compound YX-32 (a) and the expression ratio of mRNA in spleen / whole body (b).

[0097] Figure 3 Shows the analysis of Luciferase intensity in different tissues of mice after delivery of Fluc mRNA by different YX-32 LNP formulations.

[0098] Figure 4 Shows the splenocyte uptake assay of YX-32 / Fluc mRNA nanoparticles. Detailed implementation mode

[0099] The present invention will be further described in detail below in conjunction with the specific implementation modes. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0100] In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged.

[0101] The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0102] In the following examples, cholesterol is a product of Macklin, with the product number C10006595, CAS: 57-88-5.

[0103] In the following examples, DOPE (dioleoyl phosphatidylethanolamine, CAS: 4004-05-1) and DMG-PEG 2000 (CAS: 147867-65-) are all products of AVT (Shanghai) Pharmaceutical Technology Co., Ltd.

[0104] In the following examples, Firefly luciferase mRNA is a product of Shanghai Zhaowei Technology Development Co., Ltd.

[0105] In the following examples, Balb / c mice are products of Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0106] Example 1: Synthesis of Ionizable Lipid Compounds

[0107] The cationizable lipid compound is synthesized by the Michael addition reaction of the corresponding hydrophilic amine compound and the acrylate TK2 containing a ketal.

[0108] The hydrophobic tail molecule TK2, namely 2-((2-((2-(ethyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate 2-((2-((2-(ethyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate

[0109] Synthesized by a method similar to Examples 1 and 2 of CN110101665A; specifically, 2,2'-(Propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) and the corresponding haloethane (such as bromoethane) are used as starting materials to prepare Then it reacts with an acyl halide (such as acryloyl chloride) to obtain TK2.

[0110] For TK2 1 The 1H NMR is as follows: 1 1H NMR (300 MHz, CDCl3) δ 6.41 (d, 1H), 6.12 (t, 1H), 5.83 (d, 1H), 4.32 (d, 2H), 3.59 (d, 2H), 3.46 (d, 2H), 2.92 (d, 2H), 2.82 (d, 2H), 1.61–1.38 (m, 10H), 0.89 (d, 3H).

[0111] YX-30 is synthesized from the hydrophilic amine compound N,N-bis(3-aminopropyl)methylamine and TK2. The hydrophilic amine compound N,N-bis(3-aminopropyl)methylamine and TK2 are mixed in a molar ratio of 4.3:1 and heated at 70 °C for 72 h. The crude product is purified on a silica gel column with dichloromethane / methanol as the eluent to obtain the cationizable lipid compound YX-30. The 1 1H NMR is as follows: 1 1H NMR (300 MHz, CDCl3) δ 4.22 (t, 8H), 3.59 - 3.50 (m, 16H), 2.86 - 2.77 (m, 24H), 2.45 (t, 12H), 2.28 (t, 4H), 2.18 (s, 3H), 1.60 (s, 32H), 1.20 (t, 12H).

[0112] YX-32 and YX-34 are respectively synthesized from the corresponding hydrophilic amine compounds 2,2'-diamino-N-methyldiethylamine and N,N'-bis(2-aminoethyl)-N,N'-dimethyl-1,3-propanediamine and TK2 according to the above method.

[0113] YX-32: 1 1H NMR (300 MHz, CDCl3) δ 4.22 (t, 8H), 3.60 (t, 8H), 3.52 (t, 8H), 2.88 - 2.77 (m, 24H), 2.47 (t, 8H), 1.60 (s, 24H), 1.25 - 1.18 (m, 20H), 1.10 (t, 12H)

[0114] YX-34: 11H NMR (300 MHz, CDCl3) δ 4.22 (t, 8H), 3.58 (t, 8H), 3.44 (t, 8H), 2.86 - 2.77 (m, 24H), 2.55 - 2.46 (m, 16H), 2.23 (s, 3H), 1.60 - 1.38 (m, 26H), 1.20 (t, 12H).

[0115]

[0116] Example 2: Encapsulation of Firefly luciferase mRNA, Preparation of Lipid Nanoparticles and Delivery Experiment

[0117] To achieve the delivery of spleen - targeted mRNA at the animal level, the inventors prepared mRNA / lipid nanoparticles, and the specific method is as follows: The cationizable lipid compounds (YX - 30, YX - 32, or YX - 34) prepared in Example 1, cholesterol, DOPE, and DMG - PEG2000 were respectively formulated into ethanol solutions with a concentration of 10 mg / mL. Taking YX - 32 as an example, when preparing the mRNA / LNP complex once, 1.1 mg of the lipid compound YX - 30 / YX - 32 / YX - 34 solution was measured and mixed with cholesterol, DOPE, and DMG - PEG2000 in a molar ratio of 50:38.5:10:1.5, and the total volume of the solution was adjusted to 450 μL by adding ethanol. 150 μg of Firefly luciferase mRNA was added to the sodium acetate buffer solution (50 mM, pH = 5.2), and the total volume of the solution was 1350 μL. The cationizable lipid compound solution and the mRNA solution were mixed by introducing them into a microfluidic machine (speed: 0.2 mL / min for the ethanol solution, 0.6 mL / min for the sodium acetate buffer solution) to prepare lipid nanoparticles encapsulating mRNA, which were dialyzed with PBS as the buffer for 3 hours, and the obtained sample was directly used for animal experiments. Among them, the ratio of the cationizable lipid compound YX - 30 / YX - 32 / YX - 34 to mRNA was calculated according to the molar ratio (N / P) of the nitrogen atoms in YX - 30 / YX - 32 / YX - 34 to the phosphorus atoms in the mRNA phosphate backbone. In this example, N / P was 7.5.

[0118] For the lipid nanoparticles encapsulating mRNA, the concentration of RNA can be measured using QUANT - IT TMRNA assay (Invitrogen Corporation, Carlsbad, CA) was used to evaluate the encapsulation efficiency of the nanoparticle composition on RNA. The sample was diluted to a concentration of approximately 5 μg / mL in TE buffer (Solarbio, T1120, pH 8.0). 50 μL of the diluted sample was transferred to a polystyrene 96-well plate, and 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution (Solarbio, T8200) was added to the wells. The plate was incubated at a temperature of 37 °C for 10 minutes. The reagent was diluted 1:200 in TE buffer, and 100 μL of this solution was added to each well. The fluorescence intensity could be measured using a fluorescence plate reader (BIOTEK / Synergy H1) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, and the percentage of free RNA was determined by dividing the fluorescence intensity of the intact sample (without adding Triton X-100) by the fluorescence value of the disrupted sample (caused by adding Triton X-100).

[0119] The encapsulation efficiencies of three kinds of LNPs on RNA were determined to be respectively:

[0120] LNP containing YX-30: 81.9%;

[0121] LNP containing YX-32: 52.1%

[0122] LNP containing YX-34: 53.9%.

[0123] In addition, conventional methods in the art were adopted to test the average particle size, particle dispersion coefficient (PDI), and zeta potential of the LNPs:

[0124] The LNP containing YX-30 in this example was tested as follows: the average particle size was 188 nm, the PDI was 0.01, and the zeta potential was -4.0 mV;

[0125] The LNP containing YX-32 in this example was tested as follows: the average particle size was 195 nm, the PDI was 0.03, and the zeta potential was -6.0 mV; and

[0126] The LNP containing YX-34 in this example was tested as follows: the average particle size was 265 nm, the PDI was 0.08, and the zeta potential was -3.1 mV.

[0127] Female Balb / c mice at 6 - 8 weeks of age were injected via the tail vein with YX-30LNP, YX-32LNP, and YX-34LNP encapsulating 150 μg / kg Fluc mRNA. Six hours later, 200 μL of D-Lucifin (30% mass fraction) was injected via intraperitoneal administration, and the PerkinElmer IVIS Lumina III small animal in vivo optical imaging system was used to detect the luciferase expression and chemiluminescence intensity throughout the body.

[0128] In vivo imaging diagrams of LNP containing lipid compounds YX-30, YX-32, and YX-34 delivering Fluc mRNA are shown in Figure 1 in. Figure 1 It shows that luciferase mRNA delivered by the three LNPs is mainly expressed in the spleen. Among them, the LNP containing YX-32 has the best targeting property, and the LNP containing YX-30 has the highest delivery efficiency, but its targeting property is slightly worse than that of the LNP containing YX-32.

[0129] Example 3: Splenic Targeted Delivery of mRNA by Lipid Nanoparticles of the Present Invention

[0130] To verify the spleen-targeted delivery effect of the above Fluc mRNA / LNP formulation, female Balb / c mice at 6 - 8 weeks of age were injected via the tail vein with YX-32LNP encapsulating Fluc mRNA (mRNA dosing: 500 μg / kg; the sample was prepared according to the method described in Example 2). Six hours later, 200 μL of D-Lucifin (30% mass fraction) was injected via intraperitoneal administration, and the PerkinElmer IVIS Lumina III small animal in vivo optical imaging system was used to detect the luciferase expression and chemiluminescence intensity throughout the body. Then the mice were sacrificed and dissected to obtain tissues, and the PerkinElmer IVIS Lumina III was used to test the luciferase expression and chemiluminescence intensity of the treated heart, liver, spleen, lung, and kidney tissues.

[0131] The results are shown in Figure 2 in. Figure 2 The left figure in (a) shows the in vivo imaging diagram (i.e., the whole body), Figure 2 The right figure in (a) shows the tissue imaging diagrams of each organ (including five organs: heart, liver, spleen, lung, and kidney). Figure 2 (b) shows the luminescence intensity of mRNA in the whole body and spleen. The ratio of the expression of mRNA in the spleen to the expression of mRNA in the whole body is as high as 90%, which fully proves the spleen-targeted delivery of the LNP of the present invention.

[0132] Example 4: Effect of YX-32 LNP Formulations with Different Component Ratios on Targeted Delivery of mRNA

[0133] To further study the effect of YX-32 LNP formulations with different component ratios on spleen-targeted mRNA delivery, the inventors prepared YX-32 / Fluc mRNA formulations with different ratios of the four components and studied their expression in the spleen when delivering Luciferase mRNA. In view of this, 1.1 mg of YX-32 solution was measured and mixed with cholesterol, DOPE, and DMG-PEG2000 at different molar ratios (as shown in Table 1), and the total volume of the solution was made up to 450 μL by adding ethanol. The preparation method of YX-32 lipid nanoparticles encapsulating Fluc mRNA was carried out according to the method described in Example 2. Among them, the ratio of the lipid compound YX-32 to mRNA was calculated according to the molar ratio (N / P) of nitrogen atoms in YX-32 and phosphorus atoms in the mRNA phosphate backbone, where the YX-32-01 formulation was the LNP formulation prepared in Example 2.

[0134] Table 1 YX-32 LNP formulations for spleen-targeted mRNA delivery

[0135]

[0136] In the same manner as in Example 3, each YX-32 LNP formulation encapsulating 500 μg / kg Fluc mRNA was injected into 6-8-week-old female Balb / c mice via the tail vein. After 6 hours, the mice were sacrificed and tissues (heart, liver, spleen, lung, kidney) were dissected, placed in PBS, washed on the surface, and weighed. 100 mg of the tissue was added to 500 μL of the prepared tissue lysate (IP cell lysate containing 1 mM PMSF), and then transferred to ice for tissue homogenization (35000 rpm, 10 seconds each time, repeated three times, with a 10-second interval each time). The lysate was centrifuged at 4 °C for 15 minutes, and the supernatant was taken for standby. The protein concentration was quantified by the BCA method. 500 μg of the lysate from different tissues was taken, and the luciferase enzyme activity in the lysates of different tissues was detected using the luciferase activity system (Promega, E1501) kit, and the proportion of the luciferase enzyme activity in the lysates of the 5 different tissues was calculated. Figure 3 It shows that the expression level of luciferase in the spleen and the proportion of enzyme activity in the five tissues of heart, liver, spleen, lung, and kidney after delivering mRNA by LNP formulations with different components are as high as more than 65%, and even as high as 90% or higher.

[0137] Example 5: Preparation and cell uptake experiment of DiO-fluorescently labeled YX-32 / mRNA lipid nanoparticles

[0138] In order to further verify that the expression of luciferase after administration of YX-32 / mRNA lipid nanoparticles is related to the uptake of YX-32 / mRNA by spleen cells, the inventors prepared fluorescently labeled mRNA / lipid nanoparticles and studied their cellular uptake efficiency. The specific method is as follows: 13 microliters of DiO ethanol solution (10 mg / mL) were added to the YX-32 / Fluc mRNA lipid nanoparticles prepared in Example 1, and mixed in dark. Dialyze for 3 hours in dark with PBS as buffer, and the obtained sample was directly used for animal experiments. To further study the spleen cell uptake efficiency of YX-32 / Fluc mRNA, 500 μg / kg DiO-labeled YX-32 / Fluc mRNA LNP was injected into 6-8 week old female Balb / c mice by tail vein administration. After 6 hours, the mice were killed and spleen tissue was dissected and washed in PBS. Add spleen tissue to 250 μL digestion solution (DMEM cell culture medium containing 45U / uL collagenase l+25U / uLDNAse I+30U / uL hyaluronidasel) and mince with a sterile blade. Transfer the minced spleen and digestion solution to a 15mL tube and add 5-10mL digestion solution. Shake in a 37° constant temperature incubator for 1 hour. After taking out, pass through a 70um cell filter, rinse the tube and filter with PBS (containing 2% FBS) to make the total liquid volume up to 10mL. Centrifuge the above cell suspension at 4°C for 5 minutes (speed: 300G) and discard the supernatant. Then resuspend the cell pellet with 2mL red blood cell lysis solution, lyse on ice for 5 minutes, and shake evenly several times during the period to fully lyse the red blood cells. Add 4mL PBS (containing 2% FBS) to stop the red blood cell lysis. Centrifuge the above cell suspension at 4°C for 5 minutes (speed: 300G) and discard the supernatant. Resuspend the cell pellet with 1mL PBS (containing 2% FBS). After resuspending, filter with a 70um cell strainer, pipette and mix well, and use flow cytometry to detect and analyze the DiO positive ratio, that is, the cell uptake efficiency of YX-32 / Fluc mRNA LNP. The results showed that the efficiency of spleen cells taking up YX-32 / mRNA lipid nanoparticles was about 35% or more ( Figure 4 ).

[0139] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

[0140] In summary, the present invention includes but is not limited to the following items:

[0141] 1. A lipid compound of formula (I),

[0142]

[0143] wherein

[0144] R 2a and R 3a are each independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group or Ht;

[0145] t and s are each independently 0 or 1, and when t or s is 0, it means that this part is directly a single bond; provided that t and s are not both 0;

[0146] A1, A2 and A3 are each independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group or a combination of the two above;

[0147] Ht, each occurrence of which is independently -R1-X-R2-Y-R3-Z-R4,

[0148] wherein

[0149] R1, each occurrence of which is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group;

[0150] X, each occurrence of which is independently

[0151] wherein

[0152] m, n, p, q and r are each independently 1-6;

[0153] W is O, S or NR c ;

[0154] L1, L3, L5, L7 and L9, which are directly connected to R1 or R2 and are each independently a single bond, O, S

[0155] or NR d ;

[0156] L2, L4, L6, L8 and L 10 are each independently a bond, O, S or NR e ;

[0157] V is an aliphatic hydrocarbon group, OR f 、SR g or NR h R i ,

[0158] where R b 、Rc , R d , R e , R f , R g , R h and R i Each independently is hydrogen, hydroxy, aliphatic hydrocarbon oxy, monovalent aliphatic hydrocarbon, monovalent heteroaliphatic hydrocarbon, monovalent aromatic hydrocarbon or monovalent heteroaromatic hydrocarbon;

[0159] Y and Z are each independently S or O each time they appear;

[0160] R2 is each independently a single bond, divalent aliphatic hydrocarbon, divalent heteroaliphatic hydrocarbon, divalent aromatic hydrocarbon or divalent heteroaromatic hydrocarbon each time it appears;

[0161] R3 is each independently a single bond, divalent aliphatic hydrocarbon, divalent heteroaliphatic hydrocarbon, divalent aromatic hydrocarbon or divalent heteroaromatic hydrocarbon each time it appears;

[0162] R4 is each independently a hydrophobic group selected from -(CH2CH2O) y -C1-C2 alkyl, -

[0163] (CH2CH2O) y -C2 alkenyl or -(CH2CH2O) y -C2 alkynyl, where y is 0 or 1 or 2.

[0164] 2. The lipid compound according to item 1, wherein both t and s are 0; or t is 0 and s is 1; or t is 1 and s

[0165] is 0.

[0166] 3. The lipid compound according to item 1 or 2, wherein R1 is C 1-6 (e.g., C 1-4 ) divalent aliphatic hydrocarbon or C 1-6 (e.g., C 1-4 ) divalent heteroaliphatic hydrocarbon, preferably C 1-4 divalent alkyl or C 1-4 divalent heteroalkyl.

[0167] 4. The lipid compound according to any one of items 1-3, wherein X is

[0168]

[0169] wherein each variable is defined as in formula (I).

[0170] 5. The lipid compound of item 4, wherein L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH.

[0171] 6. The lipid compound of any one of items 1 - 3, wherein X is

[0172]

[0173] wherein R d and R e are as defined in formula (I).

[0174] 7. The lipid compound of item 6, wherein R d and R e are each independently H or C 1-4 a monovalent aliphatic hydrocarbon group, preferably H or C 1-4 a monovalent alkyl group.

[0175] 8. The lipid compound of item 1, wherein both Y and Z are S; or Y is S and Z is O; or Y

[0176] is O and Z is S; or both Y and Z are O.

[0177] 9. The lipid compound of any one of items 1 - 8, wherein R2 is each independently a single bond or C 1-6 a divalent aliphatic hydrocarbon group each time it appears.

[0178] 10. The lipid compound of item 9, wherein R2 is each independently C 1-4 a divalent aliphatic hydrocarbon group, preferably C 1-4 a divalent alkyl group, more preferably C 1-2 a divalent alkyl group.

[0179] 11. The lipid compound of any one of items 1 - 10, wherein R3 is each independently a single bond or C 1-6 a divalent aliphatic hydrocarbon group (e.g., C 1-4 a divalent aliphatic hydrocarbon group, preferably C 1-4 a divalent alkyl group) each time it appears.

[0180] 12. The lipid compound of item 11, wherein R3 is each independently a single bond or or methylene preferably

[0181] 13. The lipid compound of any one of items 1 - 12, wherein R4 is each independently ethyl

[0182] (C2H5), methyl (CH3), vinyl, ethynyl, -(CH2CH2O)-CH3, -(CH2CH2O)-C2H5, -

[0183] (CH2CH2O)-CH=CH2, -(CH2CH2O)-C≡CH, -(CH2CH2O)2-CH3, -(CH2CH2O)2-C2H5, -

[0184] (CH2CH2O)2-CH=CH2 or -(CH2CH2O)2-C≡CH.

[0185] 14. The lipid compound of item 1, wherein each Ht is independently at each occurrence

[0186] wherein both Y and Z are S; or Y is S and Z is O; or Y is O and Z is S; or both Y and Z are O; R3 is independently at each occurrence a single bond or or methylene R 44 is ethyl (C2H5), methyl (CH3), vinyl or ethynyl.

[0187] 15. The lipid compound of item 1, wherein

[0188] t is 1, s is 0, A2 is a single bond, and A1 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group;

[0189] t is 1, s is 0, A2 is a single bond, and A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group);

[0190] t is 1, s is 0, A2 is a single bond, and A1 and A3 are each independently -CH2CH2- or -

[0191] CH2CH2CH2-, and

[0192] R 2a is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or a C1-C6 alkyl group or methyl.

[0193] 16. The lipid compound of item 1, wherein

[0194] t is 1, s is 1, and A1, A2 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group;

[0195] t is 1, s is 1, and each of A1, A2, and A3 is independently a C1-C6 divalent aliphatic hydrocarbon group (for example, a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group);

[0196] t is 1, s is 1, and each of A1, A2, and A3 is independently -CH2CH2- or -CH2CH2CH2-, and

[0197] R 2a and R 3a are each independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or methyl.

[0198] 17. The lipid compound of item 1, wherein is

[0199]

[0200] wherein R 2a and R 3a are each independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or methyl.

[0201] 18. The lipid compound of item 1, wherein the lipid compound of formula (I) is

[0202]

[0203] 19. A pharmaceutical composition, the pharmaceutical composition comprising a pharmaceutical carrier and lipid nanoparticle particles (LNP), wherein the lipid nanoparticle particles comprise the lipid compound of any one of items 1-18 and a pharmaceutically active molecule.

[0204] 20. The pharmaceutical composition of item 19, wherein the pharmaceutically active molecule targets the spleen as an organ.

[0205] 21. The pharmaceutical composition of item 19, wherein the pharmaceutically active molecule is a nucleic acid, an antigen, a vaccine, an immunomodulator, or other active ingredient, or a combination thereof that targets the spleen as an organ.

[0206] 22. The pharmaceutical composition of item 19 or 20, wherein the LNP further comprises phospholipids, cholesterol, and polyethylene glycolated lipids.

[0207] 23. The pharmaceutical composition of any one of items 19-22, wherein the molar ratio of the lipid compound of any one of items 1-18:cholesterol:phospholipids:polyethylene glycolated lipids is (about 15 to about 50):(about 38.5 to about 75):(about 10 to about 25):(about 0.5 to about 3).

[0208] 24. The pharmaceutical composition according to any one of items 19 - 22, wherein the N / P ratio of the lipid compound according to any one of items 1 - 18 to the nucleic acid ranges from about 5:1 to about 20:1.

[0209] 25. A method for delivering an antigen to an antigen - presenting cell of the spleen or expressing an antigen in an antigen - presenting cell of the spleen, the method comprising administering to a subject in need thereof the pharmaceutical composition according to any one of items 19 - 24.

[0210] 26. The method of item 25, wherein the antigen - presenting cell is a professional antigen - presenting cell, preferably a dendritic cell and / or a macrophage.

[0211] 27. A method for treating a disease caused by damaged or abnormal spleen, the method comprising administering to a subject in need thereof the pharmaceutical composition according to any one of items 19 - 24.

[0212] 28. The method of item 27, wherein the disease caused by damaged or abnormal spleen includes lymphoma and leukemia.

[0213] 29. The method of item 27 or 28, wherein the pharmaceutical composition is administered systemically.

[0214] 30. The method according to any one of items 27 - 29, wherein the LNP targets the spleen or accumulates in the spleen.

[0215] 31. The method of item 29 or 30, wherein the LNP delivers RNA to an antigen - presenting cell of the spleen or to an antigen - presenting cell in the spleen.

[0216] 32. The method of item 31, wherein the antigen - presenting cell is a professional antigen - presenting cell, preferably in dendritic cells and / or macrophages.

[0217] 33. The method according to any one of items 29 or 30, wherein the LNP releases RNA at the target organ or target tissue and / or enters cells of the target organ or target tissue.

[0218] 34. The method of item 33, wherein the target organ or target tissue is the lymphatic system, preferably a secondary lymphoid organ, more preferably the spleen.

Claims

1. A lipid compound of formula (I), wherein R 2a and R 3a each independently is hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group or Ht; t and s are each independently 0 or 1, and when t or s is 0, it means that this part is directly a single bond; provided that t and s are not both 0; A1, A2 and A3 are each independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group or a combination of the two above; Ht is independently -R1-X-R2-Y-R3-Z-R4 each time it appears, wherein R1 is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group each time it appears; X, each independently at each occurrence, is wherein m, n, p, q and r are each independently 1-6; W is O, S or NR c ; L1, L3, L5, L7 and L9, which are directly connected to R1 or R2 and are each independently a single bond, O, S or NR d ; L2, L4, L6, L8 and L 10 each independently is a bond, O, S or NR e ; V is an aliphatic hydrocarbon group, OR f , SR g or NR h R i , wherein R b , R c , R d , R e , R f , R g , R h and R i are each independently hydrogen, hydroxy, aliphatic hydrocarbyloxy, monovalent aliphatic hydrocarbyl, monovalent heteroaliphatic hydrocarbyl, monovalent aromatic hydrocarbyl or monovalent heteroaromatic hydrocarbyl; Y and Z are each independently S or O each time they appear; R2 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group each time it appears; R3 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group each time it appears; Each R4 is independently a hydrophobic group each time it appears, and it is selected from -(CH2CH2O) y -C1-C2 alkyl, - (CH2CH2O) y -C2 alkenyl or -(CH2CH2O) y -C2 alkynyl, where y is 0 or 1 or 2.

2. The lipid compound of claim 1, wherein both t and s are 0; or t is 0 and s is 1; or t is 1 and s is 0.

3. The lipid compound according to claim 1 or 2, wherein R1 is C 1-6 (e.g., C 1-4 ) a divalent aliphatic hydrocarbon group or C 1-6 (e.g., C 1-4 ) a divalent heteroaliphatic hydrocarbon group, preferably C 1-4 a divalent alkyl group or C 1-4 a divalent heteroalkyl group.

4. The lipid compound according to any one of claims 1-3, wherein X is Each variable is defined as in formula (I).

5. The lipid compound of claim 4, wherein L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH.

6. The lipid compound according to any one of claims 1-3, wherein X is wherein R d and R e are as defined in formula (I).

7. The lipid compound according to claim 6, wherein R d and R e are each independently H or C 1-4 a monovalent aliphatic hydrocarbon group, preferably H or C 1-4 a monovalent alkyl group.

8. The lipid compound of claim 1, wherein both Y and Z are S; or Y is S and Z is O; or Y is O and Z is S; or both Y and Z are O.

9. The lipid compound according to any one of claims 1-8, wherein R2 is, independently in each occurrence, a single bond or a C 1-6 divalent aliphatic hydrocarbon group.

10. The lipid compound of claim 9, wherein each occurrence of R2 is independently a divalent aliphatic hydrocarbon group, preferably a divalent alkyl group, more preferably a divalent alkyl group. 1-4 a divalent aliphatic hydrocarbon group, preferably a 1-4 divalent alkyl group, more preferably a 1-2 divalent alkyl group.

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