Spleen-targeted ionizable lipid compounds, compositions containing the same and uses thereof

Abstract

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 comprising the ionizable lipid compound and active molecules, and a pharmaceutical composition comprising the lipid nanoparticles.

Description

Technical Field

[0001] 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

[0002] 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.

[0003] 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.

[0004] Therefore, the development of LNP delivery systems for spleen-targeted delivery, such as targeted delivery to spleen antigen-presenting cells (e.g., dendritic cells (DCs)), is of great significance for the development of nucleic acid drugs and vaccines. Summary of the invention

[0005] The inventors of the present invention have discovered that LNPs containing ionizable lipid compounds of specific carbon chain lengths can deliver pharmaceutically active molecules to the spleen with high selectivity (hereinafter sometimes referred to as "spleen targeting"). To this end, the inventors of the present invention have completed the present invention.

[0006] In a first aspect, the present invention provides a lipid compound of formula (I),

[0007]

[0008] in

[0009] R 2a and R 3a Each is independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group or Ht;

[0010] t and s are each independently 0 or 1, and when t or s is zero, it indicates that the part is directly a single bond; provided that t and s are not 0 at the same time;

[0011] A 1 , A 2 and A 3 Each 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 or a combination of the above;

[0012] Ht is independently -R at each occurrence 1 -XR 2 -YR 3 -ZR 4 ,

[0013] in

[0014] R 1 is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or

[0015] Divalent heteroaromatic hydrocarbon group;

[0016] X is independently

[0017] in

[0018] m, n, p, q and r are each independently 1-6;

[0019] W is O, S or NR c ;

[0020] L 1 , L 3 , L 5 , L 7 and L9 , which is directly related to R 1 or R 2 connected and each independently is a single bond, O, S or

[0021] NR d ;

[0022] L 2 , L 4 , L 6 , L 8 and L 10 are each independently a bond, O, S or NR e ;

[0023] V is an aliphatic hydrocarbon group, OR f , SR g or NR h R i ,

[0024] Where R b , R c , R d , R e , R f , R g , R h and R i are each independently hydrogen, hydroxyl, an oxyaliphatic radical, a monovalent aliphatic radical, a monovalent heteroaliphatic radical, a monovalent aromatic radical or a monovalent heteroaromatic radical;

[0025] Y and Z are each independently S or O at each occurrence;

[0026] R 2 is independently at each occurrence a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group;

[0027] R 3 is independently at each occurrence a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group;

[0028] R 4 Each occurrence is independently a hydrophobic group selected from -(CH 2 CH 2 O) y -C 1 -C 2 Alkyl, -(CH 2 CH 2 O) y -C 2 Alkenyl or -(CH 2 CH 2 O)y -C 2 Alkynyl, wherein y is 0 or 1 or 2.

[0029] In some embodiments, t and s are both 0; or t is 0 and s is 1; or t is 1 and s is 0.

[0030] In some embodiments, R 1 C 1-6 (For example, C 1-4 ) a divalent aliphatic hydrocarbon group or C 1-6 (For example, C 1-4 ) a divalent heteroaliphatic hydrocarbon group, preferably C 1-4 Divalent alkyl or C 1-4 A divalent heteroalkyl group.

[0031] In some embodiments, X is wherein the variables are as defined in formula (I). In a further embodiment, L 1 , L 3 , L 5 , L 7 and L 9 With R 1 are connected and are each independently a single bond, O, S or NH. In a further embodiment, X is

[0032] Where R d and R e As defined in formula (I). In some embodiments, R d and R e Each independently is H or C 1-4 A monovalent aliphatic hydrocarbon group, preferably H or C 1-4 A monovalent alkyl group.

[0033] In some embodiments, Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O.

[0034] In some embodiments, R 2 Each occurrence is 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, more preferably C 1-2 divalent alkyl).

[0035] In some embodiments, R 3 Each occurrence is independently a single bond or C 1-6 A divalent aliphatic hydrocarbon group (e.g., C1-4 A divalent aliphatic hydrocarbon group, preferably C 1-4 In a further embodiment, R 3 Each occurrence is independently a single bond or or methylene Preferably

[0036] In some embodiments, R 4 Each occurrence is independently ethyl (C 2 H 5 ), methyl (CH 3 ), vinyl, ethynyl, -(CH 2 CH 2 O)-CH 3 、-(CH 2 CH 2 O)-C 2 H 5 、-(CH 2 CH 2 O)-CH=CH 2 、-(CH 2 CH 2 O)-C≡CH, -(CH 2 CH 2 O) 2 -CH 3 、-(CH 2 CH 2 O) 2 -C 2 H 5 、-(CH 2 CH 2 O) 2 -CH=CH 2 or -(CH 2 CH 2 O) 2 -C≡CH.

[0037] In some embodiments, Ht is independently at each occurrence wherein Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O; R 3 Each occurrence is independently a single bond or or methylene R 44 Ethyl (C 2 H 5 ), methyl (CH 3 ), vinyl or ethynyl.

[0038] In the present invention, A1 , A 2 and A 3 The amine head of the lipid compound is reacted with the hydrophobic lipid tail to obtain a series of lipid compounds. Since the amine head can be protonated, the lipid compound nanoparticles are positively charged as a whole, and thus can interact electrostatically with negatively charged mRNA and cell membranes and lysosomal membranes, so that the lipid compound can effectively encapsulate and deliver mRNA. In some embodiments, the pKa value of the amine head is greater than 4, preferably greater than 6, and more preferably greater than 8.

[0039] In some embodiments, t is 1, s is 0, A 2 is a single bond, and A 1 and A 3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in a further embodiment, A 1 and A 3 Each is independent of C 1 -C 6 A divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 A divalent aliphatic hydrocarbon group, preferably C 1 -C 4 divalent alkyl); in a further embodiment, A 1 and A 3 Each is independent of -CH 2 CH 2 -or-CH 2 CH 2 CH 2 -, and R 2a is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or C 1 -C 6 In some embodiments, t is 1, s is 1, A 1 , A 2 and A 3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in a further embodiment, A 1 , A 2 and A 3 Each is independent of C 1 -C 6 A divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 A divalent aliphatic hydrocarbon group, preferably C 1 -C 4 divalent alkyl); in a further embodiment, A 1 , A 2 and A 3 Each is independent of -CH 2 CH2 -or-CH 2 CH 2 CH 2 -, and R 2a and R 3a Each independently is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or C 1 -C 6 Alkyl or methyl.

[0040] In some embodiments, for Where R 2a and R 3a Each independently is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or C 1 -C 6 Alkyl or methyl.

[0041] In some embodiments, the lipid compound of formula (I) is

[0042]

[0043] In a second aspect, the present invention provides a method for preparing R 1 A method for a lipid compound of formula (I) which is ethylene, the method comprising:

[0044] The acrylic compound of formula (II) CH 2 =CH-XR 2 -YR 3 -ZR 4 (II) reacting with a hydrophilic amine in a molar ratio, wherein the variables are defined as in formula (I), wherein the molar ratio of the hydrophilic amine to the acrylate compound of formula (II) is greater than 4, for example, 4.3:1 or 4.5:1, to ensure that the reaction is fully completed; and then optionally purifying the product by column chromatography to obtain the desired lipid compound of formula (I);

[0045] The variable R 2 , X, Y, R 3 , Z and R 4 As defined in formula (I). In some embodiments, X is

[0046] wherein the variables are as defined in formula (I). In a further embodiment, L 1 , L 3 , L 5 , L 7 and L 9 With R 1 are connected and are each independently a single bond, O, S or NH. In a further embodiment, X is

[0047] Where R d and R e As defined in formula (I). In some embodiments, R d and R e Each independently is H or C 1-4 A monovalent aliphatic hydrocarbon group, preferably H or C 1-4 A monovalent alkyl group.

[0048] 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.

[0049] In some embodiments, in the present invention, A 1 , A 2 and A 3 The amine head and the N to which it is connected together constitute the amine head of the lipid compound, which reacts with the hydrophobic lipid tail to obtain a series of lipid compounds. Since the amine head can be protonated, the lipid compound nanoparticles are positively charged as a whole, and can thus undergo electrostatic interactions with negatively charged mRNA and cell membranes, thereby enabling the lipid compound to effectively encapsulate and deliver mRNA.

[0050] In some embodiments, the hydrophilic amine in the method is selected from

[0051]

[0052] In some embodiments, the ketal-containing acrylate CH 2 =CH-C(O)OR 2 -YR 3 -ZR 4 for The remaining variables are as defined in formula (I).

[0053] In some embodiments, the reaction is an addition reaction, preferably a Michael addition reaction.

[0054] In some embodiments, the reaction is carried out at a temperature of 60-85°C, such as at 70°C, for 24 to 96 hours or longer.

[0055] The acrylate containing ketal of the present invention can be synthesized according to methods known in the art, such as the method disclosed in CN110101665 A. The acrylate containing ketal (thio) is an ester compound obtained by esterification reaction of acrylic acid and alcohol compound containing ketal (thio) ester.

[0056] Other lipid compounds of the present invention can be prepared by other suitable starting materials through the above-mentioned synthetic routes and other routes known in the art. The method listed above can include one or more additional steps to add or remove appropriate protecting groups to ultimately allow the lipid compound to be synthesized. In addition, each synthesis step can be carried out in an alternative order or sequence to obtain the required material. Synthetic chemical transformations and protecting group methods (protection and deprotection) that can be used to synthesize applicable lipid compounds are known in the art.

[0057] In a third aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutical carrier and lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise a lipid compound of formula (I) of the present invention and a pharmaceutically active molecule.

[0058] 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 pharmaceutically active molecule is mRNA. In further embodiments, the mRNA may be Firefly luciferase mRNA that expresses luciferase.

[0059] In some embodiments, the lipid nanoparticles have a particle size distribution of about 50 to about 500 nm.

[0060] In some embodiments, the lipid compound is bound to the pharmaceutically active molecule via non-covalent interactions, covalent bonds, or both.

[0061] In some embodiments, the lipid nanoparticles further contain other lipids, such as phospholipids, cholesterol and pegylated lipids, i.e., 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 lipid compound to nucleic acid ranges from about 5: 1 to about 20: 1. In some embodiments, the N / P ratio of lipid compound to nucleic acid ranges from about 7: 1 to about 15: 1. In some embodiments, the N / P ratio of lipid compound to nucleic acid ranges from about 6: 1, about 7.5: 1, or about 9: 1.

[0062] In some embodiments, the PEGylated lipid refers to a lipid modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNP, adjusts the size of nanoparticles, and increases the half-life of nanoparticles by reducing nonspecific 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 PEG-modified PEG has a molecular weight of typically 350-5000Da. 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 ditetradecyl acetamide (ALC-0159).

[0063] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine 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 phospholipid is selected from the group consisting of: 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0064] In some embodiments, the lipid nanoparticles of the present invention are prepared by the following method: comprising dissolving the ionizable lipid compound of the present invention, cholesterol, DOPE and DSPE-PEG2000 in anhydrous ethanol, respectively, dissolving the active pharmaceutical molecule and 5-20 times the mass thereof (the N / P ratio of the lipid compound to the nucleic acid ranges from about 5:1 to about 20:1) in a 100 mM sodium acetate buffer solution (pH = about 5.2). The above ethanol solution and the sodium acetate buffer solution are mixed and prepared on a microfluidic machine at a flow rate of 1:3. In a further embodiment, the active pharmaceutical molecule is mRNA.

[0065] The present invention also relates to a method for delivering an antigen to antigen-presenting cells of the spleen (preferably professional antigen-presenting cells, such as dendritic cells and / or macrophages) or expressing an antigen in antigen-presenting cells of the spleen (preferably professional antigen-presenting cells, such as dendritic cells and / or macrophages), the method comprising administering the pharmaceutical composition of the present invention to a subject having the same.

[0066] 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 the subject a pharmaceutical composition of the present invention.

[0067] The present invention also relates to a method for treating diseases caused by spleen damage or abnormality, the method comprising administering the pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the diseases caused by spleen damage or abnormality include lymphoma and leukemia, etc.

[0068] definition

[0069] The term "aliphatic" refers to a saturated or unsaturated, linear or branched, acyclic, cyclic or polycyclic hydrocarbon moiety. Examples include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl moieties.

[0070] The term "alkyl" includes hydrocarbon groups selected from straight and branched 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, 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, triacontyl, and the like. Examples of divalent alkyl groups, 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, triacotylene, and the like.

[0071] A monovalent group is a group formed by removing one hydrogen atom from the corresponding hydrocarbon part. A divalent group is a group formed by removing two hydrogen atoms from the corresponding hydrocarbon part.

[0072] The term "alkenyl" includes hydrocarbon groups selected from straight and branched 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. 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, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-1,3-dienyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, and the like.

[0073] The term "alkynyl" includes hydrocarbon groups selected from straight and branched 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. 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.

[0074] The term "cycloalkyl" includes a saturated cycloalkyl selected from monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged or spiral cycloalkyl.Cycloalkyl can include 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, for example, cycloalkyl can be selected from a monocyclic group including 3 to 12, such as 3 to 10, further such as 3 to 8, 3 to 6 carbon atoms.The example of monocyclic cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.The term "cycloalkenyl" refers to a non-aromatic, cyclic hydrocarbon moiety containing at least one double bond, such as cyclohexenyl and cyclohexenylene. The term "cycloalkynyl" refers to non-aromatic, cyclic hydrocarbon moieties containing at least one triple bond, cyclooctynyl and cyclooctynylene. Similarly, cycloalkylene, cycloalkenylene and cycloalkynylene are corresponding divalent radicals.

[0075] 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 moieties of the present invention include alkyl, alkenyl or alkynyl moieties 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. Cyclic heteroaliphatic moieties include 3-7 membered monocyclic heteroaliphatic moieties and 7-12 membered bicyclic heteroaliphatic moieties containing 1-3 or more, for example 1-3 heterocyclic rings selected from oxygen, nitrogen and sulfur. In some embodiments, the cyclic heteroaliphatic hydrocarbon group is a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1-3 heterocyclic rings selected from oxygen, nitrogen and sulfur. Cyclic heteroaliphatic hydrocarbon groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, oxiranyl, aziridine, thioranyl, azetidinyl, oxetanyl, thiol, dithiol, dihydropyridine, tetrahydropyridine, thiomorpholine, homopiperazinyl, homopiperidinyl, azepanyl, oxetanyl, thiol, oxathiol, dioxetanyl, oxathiol, azacycloheptane, dithiol, azathiol, cycloheptane, dioxetanyl ... , diazepanyl, azathiocanyl, oxazepine, diazepine, thiazepine, dihydrothiophenyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolinyl, indolyl, dioxane, dioxolane, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinyl, dioxo-thiomorpholinyl, azabicyclo[3.1.0]hexyl, azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexyl, etc. Typical heteroaliphatic hydrocarbon groups are heteroalkyl groups, i.e., alkyl groups containing at least one heteroatom of N, O or S, such as C containing one N atom. 1-6 Alkyl, C containing one O atom 1-6 Alkyl, or C containing one S atom 1-6 Alkyl; or C containing one nitrogen atom 1-4 Heteroalkyl, C containing one O atom 1-4 Heteroalkyl, or C containing one S atom 1-4 Heteroalkyl.

[0076] The term "oxyaliphatic" refers to an -O-aliphatic hydrocarbon group. Examples of oxyaliphatic groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.

[0077] The term "aryl" refers to a C6 monocyclic, C10 bicyclic, C14 tricyclic, C20 tetracyclic or C24 pentacyclic aromatic ring system. Examples of aryl include phenyl, phenylene, naphthyl, naphthylene, anthracenyl, anthrylene, pyrenyl and pyrenylene.

[0078] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, 11-14 membered tricyclic and 15-20 membered tetracyclic ring system having one or more heteroatoms (such as O, N, S or Se). Examples of heteroaryl include furanyl, furanylene, fluorenyl, fluorenylene, pyrrolyl, pyrrolylene, thienyl, thienylene, oxazolyl, oxazolylene, imidazolyl, imidazolylene, benzimidazolylene, benzimidazolylene, thiazolyl, thiazolylene, pyridinyl, pyridinylene, pyrimidinyl, pyrimidinylene, quinazolinyl, quinazolinylene, quinolyl, quinolylene, isoquinolyl, isoquinolylene, indolyl and indolylene.

[0079] Unless otherwise indicated, the aliphatic hydrocarbon groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbon group oxy groups, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynyl groups, cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenyl groups, cycloalkynyl groups, cycloalkynyl groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenyl groups, aryl groups and heteroaryl groups mentioned in this application include both substituted and unsubstituted parts. Possible substituents on cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenyl groups, cycloalkynyl groups, cycloalkynyl groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenyl groups, aryl groups and heteroaryl groups include, but are not limited to, C 1 -C 10 Alkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, C 3 -C 20 Cycloalkyl, C 3 -C 20 Cycloalkenyl, C 2 -C 20 Heterocycloalkyl, C 3 -C 20 Heterocycloalkenyl, C 1 -C 10 Alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C 1 -C 10 Alkylamino, C 2 -C 20 Dialkylamino, arylamino, diarylamino, C 1 -C 10 Alkylsulfonylamino, arylsulfonylamino, C 1 -C10 Alkyl imino, aryl imino, C 1 -C 10 alkylsulfonyl imino, arylsulfonyl imino, hydroxy, halo, thio, C1-C10 alkylthio, arylthio, C 1 -C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amide, amidino, guanidine, urea, thiourea, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylate. On the other hand, possible substituents on aliphatic hydrocarbon, heteroaliphatic hydrocarbon, aliphatic hydrocarbonoxy, alkyl, alkylene, alkenyl, alkenylene, alkynyl, and alkynylene include all of the substituents listed above, but C10 1 -C 10 The cycloalkyl group, cycloalkylene group, cycloalkenyl group, cycloalkenylene group, heterocycloalkyl group, heterocycloalkylene group, heterocycloalkenyl group, heterocycloalkenylene group, aryl group and heteroaryl group may be condensed with each other.

[0080] The term "pharmaceutically active molecule" refers to any chemical substance intended for medical diagnosis, cure, treatment or prevention of a 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, a tumor antigen, an allergic antigen, or an autoimmune autoantigen. In some embodiments, the vaccine is a tumor, influenza, or rabies vaccine.

[0081] The term "RNA" or "nucleic acid" includes nucleic acid molecules of ribonucleotide residues or nucleic acid molecules whose bases are chemically modified. In some embodiments, the RNA comprises all or most ribonucleotide residues.

[0082] The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group.

[0083] 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 is different from naturally occurring RNA by adding, deleting, replacing, and / or changing one or more nucleotides. In some embodiments, RNA is a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA generally comprises a 5'-untranslated region (5'-UTR), a peptide coding region, and a 3'-untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid comprising deoxyribonucleotides. In some embodiments, RNA is in vitro transcribed RNA (IVT RNA), and can be obtained by in vitro transcription of a suitable DNA template.

[0084] In some embodiments, the concentration of RNA in the pharmaceutical composition comprising LNP of the present application is about 0.01 mg / mL to about 1 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL. In some 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 In some embodiments, the present invention relates to an intravenous in vitro medicated pharmaceutical composition or the like. In some embodiments, the intravenous in vitro medicated pharmaceutical composition or the like is preferably a 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.

[0085] In some embodiments, when systemic administration is performed, the LNP targets the spleen, or accumulates in the spleen. Preferably, when systemic administration is performed, the LNP delivers RNA to the antigen presenting cells of the spleen, preferably professional antigen presenting cells, such as dendritic cells and / or macrophages. In some embodiments, the LNP releases RNA and / or enters the cells of the target organ or target tissue at the target organ or target tissue. In some embodiments, the target organ or target tissue is a targeted lymphatic system, particularly a secondary lymphoid organ, more specifically the spleen, and the cells at the target organ or target tissue are antigen presenting cells, such as dendritic cells. In some embodiments, after systemic administration of the LNP, RNA expression occurs in the antigen presenting cells of the spleen, in professional antigen presenting cells. In some embodiments, after systemic administration of the LNP of the present invention, RNA expression occurs in the spleen. In some embodiments, when systemic administration is performed, the LNP does not target or substantially does not target the lungs and / or liver, or does not or substantially does not accumulate in the lungs and / or liver. In some embodiments, after systemic administration of the LNP of the present invention, RNA expression does not occur substantially in the lungs and / or liver. In some embodiments, the amount of nanoparticles targeted to the spleen or accumulated in the spleen is at least 5 times the amount targeted to the lungs or accumulated in the lungs, 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. In some embodiments, after systemic administration of the LNP, 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.

[0086] The antigen encoded by the RNA contained in the LNP described herein is preferably a disease-associated antigen, or it triggers an immune response against the disease-associated antigen or a cell expressing the disease-associated antigen.

[0087] 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.

[0088] 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 intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration or intramuscular administration. The term "parenteral administration" refers to administration in a manner not passing through the digestive tract, such as intravenous injection or intramuscular injection. Systemic administration (also referred to as systemic administration) is a route of administration, including enteral administration (that is, administration involving absorption by the gastrointestinal tract) or parenteral administration. The pharmaceutical composition of the present invention can be formulated for systemic administration.

[0089] The pharmaceutical composition of the present invention can be used to induce an immune response, particularly a disease-associated antigen or a cellular immune response expressing a disease-associated antigen, such as an immune response against cancer. Therefore, the pharmaceutical composition can be used for preventive and / or therapeutic treatment of diseases involving disease-associated antigens or cells expressing disease-associated antigens, such as cancer. In some embodiments, the disease-associated antigen is a tumor antigen.

[0090] The term "subject" or "patient" in this application includes humans and mammals.

[0091] The term "therapeutically effective amount" is an amount of a therapeutic agent that ameliorates a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "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 a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present disclosure.

[0092] The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients constituting the formulation and / or with humans or mammals for the prevention or treatment of a disease or condition.

[0093] The term "treatment" used in this application refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, mitigate, improve or affect the disease or the symptoms of the disease. In the context of this application, unless otherwise specifically stated, the term "treatment" may also include prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 In vivo bioluminescence imaging of LNPs prepared from lipid compounds YX-30, YX-32, and YX-34 delivering Fireflyluciferase (Fluc) mRNA (administered dose: 150 μg / kg).

[0095] Figure 2 Shown are in vivo and anatomical tissue bioluminescence imaging of Firefly luciferase (Fluc) mRNA delivered by LNPs prepared from lipid compound YX-32 (a) and the mRNA expression ratio in the spleen / whole body (b).

[0096] Figure 3 Shown is the analysis of luciferase intensity in different tissues of mice after delivery of Fluc mRNA by different YX-32LNP formulations.

[0097] Figure 4 Shown is the spleen cell uptake analysis of YX-32 / Fluc mRNA nanoparticles. DETAILED DESCRIPTION

[0098] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0099] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0100] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0101] In the following examples, cholesterol is a product of MacLean, with the product number C10006595, CAS: 57-88-5.

[0102] In the following examples, DOPE (dioleoylphosphatidylethanolamine, CAS: 4004-05-1) and DMG-PEG 2000 (CAS: 147867-65-) are all products of Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0103] In the following examples, Firefly luciferase mRNA is a product of Shanghai Zhaowei Technology Development Co., Ltd.

[0104] In the following examples, Balb / c mice were produced by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0105] Example 1: Synthesis of ionizable lipid compounds

[0106] The cationizable lipid compounds were synthesized from the corresponding hydrophilic amine compounds and ketal-containing acrylate TK2 via Michael addition reaction.

[0107] Hydrophobic tail molecule TK2, 2-((2-((2-(ethyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate Synthesized by a method similar to CN110101665A Examples 1 and 2; specifically, 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) and the corresponding halogenated ethane (e.g. ethyl bromide) are used as starting materials to prepare

[0108] It is then reacted with an acryloyl halide (eg acryloyl chloride) to give TK2.

[0109] TK2 1 The H NMR is shown below: 1 H NMR (300 MHz, CDCl 3 )δ6.41(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).

[0110] YX-30 is synthesized from a hydrophilic amine compound N,N-bis(3-aminopropyl)methylamine and TK2, wherein the hydrophilic amine compound N,N-bis(3-aminopropyl)methylamine and TK2 are mixed at a molar ratio of 4.3:1, heated at 70°C for 72 hours, and the crude product is purified on a silica gel column using dichloromethane / methanol as an eluent to obtain a cationizable lipid compound YX-30. 1 The H NMR is shown below: 1 HNMR (300MHz, CDCl 3 )δ4.22(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).

[0111] YX-32 and YX-34 were synthesized from the corresponding hydrophilic amine compounds 2,2'-diamino-N-methyldiethylamine and N,N'-bis(2-aminoethyl)-N,N'-dimethyl-1,3-propylenediamine and TK2 according to the above method.

[0112] YX-32: 1 H NMR (300 MHz, CDCl 3 )δ4.22(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)

[0113] YX-34: 1 H NMR (300 MHz, CDCl 3 )δ4.22(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).

[0114]

[0115] Example 2: Encapsulation of Fireflyluciferase mRNA, Preparation of Lipid Nanoparticles and Delivery Experiment

[0116] In order to achieve animal-level spleen-targeted mRNA delivery, the inventors prepared mRNA / lipid nanoparticles, and the specific method is as follows: the cationizable lipid compound (YX-30, YX-32 or YX-34), cholesterol, DOPE, and DMG-PEG2000 prepared in Example 1 are respectively configured into 10 mg / mL ethanol solutions. Taking YX-32 as an example, when preparing mRNA / LNP complexes once, the lipid compound YX-30 / YX-32 / YX-34 solution (1.1 mg) is 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 is 450 microliters by adding ethanol. Take 150 micrograms of Firefly luciferase mRNA and add it to a sodium acetate buffer solution (50mM, pH=5.2), and the total volume of the solution is 1350 microliters. The cationizable lipid compound solution and the mRNA solution were mixed by introducing into a microfluidic machine (speed: 0.2 mL / min ethanol solution, sodium acetate buffer solution 0.6 mL / min), and lipid nanoparticles encapsulating mRNA were prepared, which were dialyzed for 3 hours with PBS as a buffer, and the obtained samples were 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 nitrogen atoms in YX-30 / YX-32 / YX-34 to phosphorus atoms in the mRNA phosphate backbone, and N / P in this embodiment was 7.5.

[0117] For lipid nanoparticles encapsulating mRNA, the RNA concentration can be measured using QUANT-IT TMRNA assay (Invitrogen Corporation, Carlsbad, CA) was used to evaluate the encapsulation efficiency of the nanoparticle composition for RNA. The sample was diluted to a concentration of approximately 5 μg / mL in TE buffer (Solarbio / Solaibao, 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 / Solaibao, T8200) was added to the well. 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 can 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 can be determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0118] The encapsulation efficiency of the three LNPs for RNA was determined to be:

[0119] LNP containing YX-30: 81.9%;

[0120] LNP containing YX-32: 52.1%

[0121] LNP containing YX-34: 53.9%.

[0122] In addition, the average particle size, particle dispersion index (PDI) and zeta potential of LNP were tested using conventional methods in the art:

[0123] The LNPs containing YX-30 in this example were tested as follows: average particle size 188 nm, PDI 0.01, and zeta potential -4.0 mV;

[0124] The LNPs comprising YX-32 in this example were tested as follows: average particle size 195 nm, PDI 0.03, and zeta potential -6.0 mV; and

[0125] The LNPs comprising YX-34 in this example were tested as follows: average particle size 265 nm, PDI of 0.08, and zeta potential of -3.1 mV.

[0126] YX-30LNP, YX-32LNP and YX-34LNP encapsulated with 150 μg / kg Fluc mRNA were injected into 6-8 week old female Balb / c mice via tail vein administration. Six hours later, 200 μL of D-Lucifin (30% by mass) was injected intraperitoneally, and the whole body luciferase expression and chemiluminescence intensity were detected using the PerkinElmer IVIS Lumina III small animal in vivo optical imaging system.

[0127] In vivo imaging of Fluc mRNA delivered by LNPs containing lipid compounds YX-30, YX-32, and YX-34 is shown in Figure 1 middle. Figure 1 The results showed that Luciferase mRNA delivered by the three LNPs were mainly expressed in the spleen, among which the LNP containing YX-32 had the best targeting, and the LNP containing YX-30 had the highest delivery efficiency, but its targeting was slightly worse than that of the LNP containing YX-32.

[0128] Example 3: Spleen-targeted delivery of mRNA by lipid nanoparticles of the present invention

[0129] To verify the spleen-targeted delivery effect of the above-mentioned Fluc mRNA / LNP preparation, YX-32LNP encapsulating Fluc mRNA was injected into 6-8 week-old female Balb / c mice by tail vein administration (mRNA dosage: 500 μg / kg; the sample was prepared according to the method described in Example 2), and 200 microliters of D-Lucifin (mass fraction 30%) were injected by intraperitoneal administration 6 hours later, and the whole body luciferase expression and chemiluminescence intensity were detected by PerkinElmer IVIS Lumina III test mouse in vivo optical imaging system. Then the mice were killed and the tissues were dissected and the luciferase expression and chemiluminescence intensity of the treated heart, liver, spleen, lung and kidney tissues were tested by PerkinElmer IVIS Lumina III.

[0130] The results are shown in Figure 2 middle. Figure 2 The left image of (a) shows the in vivo imaging (i.e., whole body). Figure 2 The right figure of (a) shows the tissue imaging diagram 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, wherein the expression ratio of mRNA in spleen tumor to mRNA in the whole body is as high as 90%, which fully proves the targeted delivery of LNP of the present invention to the spleen.

[0131] Example 4: Effects of YX-32LNP formulations containing different component ratios on mRNA targeted delivery

[0132] To further study the effect of YX-32LNP preparations containing different component ratios on spleen-targeted mRNA delivery, the inventors prepared YX-32 / Fluc mRNA preparations containing different four component ratios, and studied the expression of Luciferase mRNA in the spleen when it was delivered. In view of this, YX-32 solution (1.1 mg) was measured and mixed with cholesterol, DOPE, and DMG-PEG2000 in different molar ratios (as shown in Table 1), and the total volume of the solution was 450 microliters 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 lipid compound YX-32 to mRNA is calculated according to the molar ratio (N / P) of nitrogen atoms in YX-32 and phosphorus atoms in mRNA phosphate backbone, wherein the YX-32-01 preparation is the LNP preparation prepared in Example 2.

[0133] Table 1 YX-32 LNP formulations that can be used for spleen-targeted mRNA delivery

[0134]

[0135] In the same manner as in Example 3, each YX-32LNP preparation encapsulating 500 μg / kg Fluc mRNA was injected into 6-8 week old female Balb / c mice by tail vein administration. After 6 hours, the mice were killed and the tissues (heart, liver, spleen, lung, kidney) were dissected and placed in PBS to clean the surface and weighed. Take 100 mg of tissue and add 500 μL of prepared tissue lysate (IP cell lysate containing 1 mM PMSF) and transfer to crushed ice for tissue homogenization (35,000 rpm, 10 seconds each time, repeated three times, 10 seconds each time). The lysate was centrifuged at 4 ° C for 15 minutes, and the supernatant was taken for standby use, and its protein concentration was quantified by BCA method. Take 500 μg of lysate of different tissues, use luciferase activity system (Promega, E1501) kit to detect luciferase enzyme activity in different tissue lysates, and calculate the proportion of luciferase enzyme activity of 5 different tissue lysates. Figure 3 It was shown that after the LNP preparations with different components delivered mRNA, the expression level of luciferase in the spleen and the enzyme activity in the five tissues of heart, liver, spleen, lung and kidney accounted for more than 65%, and even up to 90% or higher.

[0136] Example 5: Preparation of DiO fluorescently labeled YX-32 / mRNA lipid nanoparticles and cellular uptake experiment

[0137] 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 hyaluronidase l), and mince with a sterile blade. Transfer the minced spleen and digestion solution to a 15mL tube, add 5-10mL digestion solution, and shake in a 37° constant temperature incubator for 1 hour. After taking it out, pass it 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 it on ice for 5 minutes, and shake it 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, and 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 ).

[0138] 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.

Claims

1. A lipid compound, wherein the lipid compound is 2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise the lipid compound of claim 1 and a pharmaceutically active molecule.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically active molecule targets the spleen.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutically active molecule is a nucleic acid, antigen, vaccine, immunomodulator, or other active ingredient, or a combination thereof, that targets the spleen.

5. The pharmaceutical composition of claim 2 or 3, wherein the LNP further comprises phospholipids, cholesterol and pegylated lipids.

6. The pharmaceutical composition of claim 5, wherein the molar ratio of the lipid compound of claim 1: cholesterol: phospholipid: PEGylated lipid is (15 to 50): (38.5 to 75): (10 to 25): (0.5 to 3).

7. The pharmaceutical composition of claim 4, wherein the pharmaceutically active molecule is a nucleic acid that targets the spleen, and the N / P ratio of the lipid compound of claim 1 to the nucleic acid ranges from 5:1 to 20:

1.

8. Use of the pharmaceutical composition according to any one of claims 2 to 7 for the preparation of a medicament for delivering an antigen to antigen-presenting cells of the spleen or expressing an antigen in antigen-presenting cells of the spleen.

9. The use according to claim 8, wherein the antigen presenting cells are professional antigen presenting cells.

10. The use according to claim 8, wherein the antigen presenting cells are dendritic cells and / or macrophages.

11. Use of the pharmaceutical composition according to any one of claims 2 to 7 in the preparation of a medicament for treating diseases caused by spleen damage or abnormality.

12. The use according to claim 11, wherein the diseases caused by the spleen damage or abnormality include lymphoma and leukemia.

13. The use according to claim 11 or 12, wherein the pharmaceutical composition is administered systemically.

14. The use of claim 11 or 12, wherein the LNP is targeted to the spleen, or accumulates in the spleen.

15. The use of claim 13, wherein the LNP delivers the RNA to antigen presenting cells of the spleen or to antigen presenting cells in the spleen.

16. The use according to claim 15, wherein the antigen presenting cells are professional antigen presenting cells.

17. The use according to claim 15, wherein the antigen presenting cells are dendritic cells and / or macrophages.

18. The use of claim 13, wherein the LNP releases RNA at the target organ or target tissue and / or enters the cells of the target organ or target tissue.

19. The use according to claim 18, wherein the target organ or target tissue is the spleen.

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