Degradable liposome for active molecule delivery, and lipid nanoparticle thereof

Abstract

The present invention provides an ionizable lipid compound, having an optimized carbon chain length and an amine head for an active molecule, comprising but not limited to a nucleic acid, a protein, a small molecule drug, etc., and having increased delivery efficiency. The present invention also relates to a lipid nanoparticle (LNP) including the ionizable lipid compound and the active molecule, and a pharmaceutical composition including the lipid nanoparticle.

Description

A class of degradable liposomes and lipid nanoparticles for active molecule delivery Technical Field

[0001] The present invention provides an ionizable lipid compound having an optimized hydrophobic carbon chain length and an ionizable amine head structure, thereby being capable of efficiently delivering active molecules, including but not limited to nucleic acids, proteins, and small molecule drugs. The present invention also relates to lipid nanoparticles (LNPs) comprising the ionizable lipid compound and the active molecule, and pharmaceutical compositions comprising the lipid nanoparticles. Background Art

[0002] Biomacromolecules such as nucleic acids and proteins have enormous advantages and potential in regulating gene expression and preventing and treating malignant diseases. However, biomacromolecules are a type of charged molecules with strong hydrophilicity, making it difficult for them to independently cross the cell membrane and enter the cell interior. In addition, nucleic acid drugs such as RNA are easily degraded by nucleases, which limits their stability in the body and further restricts the drugability of RNA.

[0003] Lipid nanoparticles (LNPs) are typically composed of four components: cationic or ionizable lipids, phospholipids, cholesterol, and PEGylated lipids. LNPs are capable of encapsulating nucleic acid drug molecules such as RNA and protecting them from nuclease degradation. Their high stability and biocompatibility make them a promising drug delivery system with clinical applications.

[0004] The drug delivery efficiency of LNPs is influenced by their composition. The amine head of cationic or ionizable lipids can electrostatically interact with biomacromolecules to promote LNP binding and encapsulation, thereby increasing the efficiency of LNP uptake by cells. Studies have shown that the hydrophobic carbon chain length of cationic or ionizable lipids influences LNP stability, drug encapsulation, and delivery efficiency. For example, Min Qiu et al. (PNAS 2022, 119, e2116271119) reported that the delivery efficiency of LNP increases with the increase of the length of the lipid hydrophobic carbon chain. For example, lipid molecules containing a hydrophobic chain of 16 carbon atoms have the highest nucleic acid delivery efficiency than lipid molecules containing a hydrophobic chain of 12-14 carbon atoms; Chun Chan et al. (Curr Top Med Chem 2021, 21, 92–114) also reported that lipid molecules with a carbon chain length of 12 to 18 carbon atoms provide sufficient fluidity and rigidity for LNP; Shuai Liu et al. (Nature Mater, 2021, 20, 701–710) showed in Figure 2e of the document that the carbon chain length affects the delivery efficiency of nucleic acid drugs, and lipid molecules with carbon chain lengths of 10, 11 and 12 have higher delivery efficiencies.

[0005] In summary, the hydrophobic carbon chain length of cationic lipids or ionizable lipids affects the formation and drug delivery efficiency of LNPs. Therefore, a balance of these factors needs to be considered when designing LNPs to improve their delivery efficiency and stability in vivo.

[0006] Summary of the Invention

[0007] The present invention provides an ionizable lipid compound with an optimized hydrophobic carbon chain length and an amine head. Unlike prior art cationic lipids or ionizable lipid compounds containing carbon chains of 12 to 18 carbon atoms, the inventors discovered that for ionizable lipid compounds of this structure, further reducing the hydrophobic carbon chain length actually increases delivery efficiency. Consequently, the inventors completed the present invention.

[0008] In a first aspect, the present invention provides a liposome compound of formula (I),

[0009] in

[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 indicates that the portion is directly a single bond; provided that t and s are not 0 at the same time;

[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 or a divalent heteroaromatic hydrocarbon group, or a combination thereof;

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

[0014] in

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

[0021] L2, L4, L6, L8 and L 10 Each independently is a bond, O, S or NR e ;

[0022] V is an aliphatic hydrocarbon group, OR f SR g or NR h R i ,

[0023] 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 hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heteroaromatic hydrocarbon group;

[0024] Y and Z are each independently S or O at each occurrence;

[0025] R2 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;

[0026] R3, at each occurrence, 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;

[0027] R4 is independently a hydrophobic group at each occurrence selected from -(CH2CH2O) y -C3-C8 alkyl, -(CH2CH2O) y -C3-C8 alkenyl or -(CH2CH2O) y -C3-C8 alkynyl, wherein y is 0, 1 or 2.

[0028] In some embodiments, t and s are both 0; or t is 0 and s is 1; or t is 01 and s is 0.

[0029] In some embodiments, R1 is 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.

[0030] In some embodiments, X is wherein the variables are as defined in formula (I). In a further embodiment, L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH. In a further embodiment, X is 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 Monovalent aliphatic hydrocarbon group, preferably H or C 1-4 Monovalent alkyl group.

[0031] 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.

[0032] In some embodiments, R2 is independently a single bond at each occurrence or C 1-6 A divalent aliphatic hydrocarbon group (e.g., C 1-4 A divalent aliphatic hydrocarbon group, preferably C 1-4Divalent alkyl, more preferably C 1-2 divalent alkyl).

[0033] In some embodiments, each occurrence of R3 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 In a further embodiment, R3 is independently a single bond at each occurrence or or methylene Preferably

[0034] In some embodiments, R4 at each occurrence is independently C3-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, -(CH2CH2O)-C3-C8 alkyl, -(CH2CH2O)-C3-C8 alkenyl, -(CH2CH2O)-C3-C8 alkynyl, -(CH2CH2O)2-C3-C8 alkyl, -(CH2CH2O)2-C3-C8 alkenyl, or -(CH2CH2O)2-C3-C8 alkynyl. In some embodiments, R4 at each occurrence is independently C4-C8 alkyl, C4-C8 alkenyl, C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkenyl, -(CH2CH2O)2-C4-C8 alkynyl, -(CH2CH2O)2-C4-C8 alkyl, -(CH2CH2O)2-C4-C8 alkenyl, or -(CH2CH2O)2-C4-C8 alkynyl. In some embodiments, the alkyl group in the C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkyl or -(CH2CH2O)2-C4-C8 alkyl is a straight-chain alkyl group; the alkenyl group in the C4-C8 alkenyl, -(CH2CH2O)-C4-C8 alkenyl or -(CH2CH2O)2-C4-C8 alkenyl is a straight-chain alkenyl group; the alkynyl group in the C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkynyl or -(CH2CH2O)2-C4-C8 alkynyl is a straight-chain alkynyl group. In some embodiments, R4 at each occurrence is independently -(CH2CH2O)-(CH2)3CH3, -(CH2CH2O)2-(CH2)3CH3, -(CH2CH2O)-(CH2)4CH3, -(CH2CH2O)2-(CH2)4CH3, -(CH2CH2O)-(CH2)5CH3, -(CH2CH2O)2-(CH2)5CH3, -(CH2CH2O)-(CH2)6CH3, -(CH2CH2O)2-(CH2)6CH3, -(CH2CH2O)-(CH2)7CH3, or -(CH2CH2O)2-(CH2)7CH3.

[0035] 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; R3 is independently a single bond or or methylene R 44is C4-C8 alkyl, C4-C8 alkenyl or C4-C8 alkynyl; in some embodiments, R4 at each occurrence is independently -(CH2)3CH3, -(CH2CH2O)-(CH2)3CH3, -(CH2CH2O)-(CH2)4CH3, -(CH2CH2O)-(CH2)5CH3, -(CH2CH2O)-(CH2)6CH3 or -(CH2CH2O)-(CH2)7CH3.

[0036] In the present invention, A1, A2, and A3 constitute the amine head of the liposome, which reacts with the hydrophobic lipid tail to form a series of liposomes. Because the amine head can be protonated, the liposome-like nanoparticles are positively charged overall, enabling electrostatic interactions with negatively charged mRNA, cell membranes, and lysosomal membranes, thereby enabling the liposomes to 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.

[0037] 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 further embodiments, 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); in further embodiments, 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 a methyl group. 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 further embodiments, A1, A2 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); in further embodiments, A1, A2 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or a methyl group.

[0038] In some embodiments, for where R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or a methyl group.

[0039] In some embodiments, the liposome of formula (I) is

[0040] The liposomes of formula (I) of the present invention are biodegradable because they contain a ketal-responsive unit (especially a thioketal-responsive unit when both Y and Z are S) in the Ht group. Such liposomes can efficiently bind to mRNA and form nanostructures through assembly. After entering diseased cells, they selectively respond to and degrade the highly oxidative environment within the cells, thereby efficiently releasing mRNA and expressing responsive proteins.

[0041] In a second aspect, the present invention provides a method for preparing a liposome of formula (I) wherein R1 is ethylene, the method comprising:

[0042] reacting an acrylic acid compound of formula (II) CH2=CH-X-R2-Y-R3-Z-R4 (II) with a hydrophilic amine in a molar ratio, wherein the variables are as defined in formula (I), wherein the molar ratio of the hydrophilic amine to the acrylic acid 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 optionally purifying the product by column chromatography to obtain the desired liposomes of formula (I);

[0043] wherein the variables R2, X, Y, R3, Z and R4 are as defined in formula (I). In some embodiments, X is wherein the variables are as defined in formula (I). In a further embodiment, L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH. In a further embodiment, X is 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 Monovalent aliphatic hydrocarbon group, preferably H or C 1-4 Monovalent alkyl group.

[0044] In some embodiments, in method (a), the product is purified by column chromatography to obtain the desired liposomes of formula (I), wherein the column chromatography purification method is well known in the art.

[0045] In some embodiments, in the present invention, A1, A2 and A3 and their connected N together constitute the amine head of the liposome, which reacts with the hydrophobic lipid tail to obtain a series of liposomes. Since the amine head can be protonated, the liposome nanoparticles are positively charged as a whole, and can thus undergo electrostatic interactions with negatively charged mRNA and cell membranes and lysosomal membranes, thereby enabling the liposomes to effectively encapsulate and deliver mRNA.

[0046] In some embodiments, the hydrophilic amine in the method is selected from

[0047] 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).

[0048] In some embodiments, the reaction is an addition reaction, preferably a Michael addition reaction.

[0049] In some embodiments, the reaction is carried out at a temperature of 60-85°C, such as 70°C, for 24 to 96 hours or longer.

[0050] The ketal-containing acrylate of the present invention can be synthesized according to methods known in the art, such as the method disclosed in CN 110101665 A. The ketal-containing acrylate is an ester compound obtained by esterification of acrylic acid with an alcohol compound containing a ketal-containing acrylate.

[0051] Other liposomes 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 methods listed above may include one or more additional steps to add or remove appropriate protecting groups to ultimately allow the liposomes to be synthesized. In addition, each synthetic step may be performed in an alternative order or sequence to obtain the desired material. Synthetic chemical transformations and protecting group methods (protection and deprotection) that can be used to synthesize applicable liposomes are known in the art.

[0052] In a third aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise the liposomes of formula (I) of the present invention and a pharmaceutically active molecule.

[0053] In some embodiments, the pharmaceutically active molecule is a small molecule drug, a protein, a peptide, a nucleic acid, a carbohydrate, or a combination thereof. In further embodiments, the pharmaceutically active molecule is mRNA. In further embodiments, the mRNA may be Firefly luciferase mRNA that expresses luciferase.

[0054] In some embodiments, the lipid nanoparticles have a particle size distribution of 50 to 500 nm.

[0055] In some embodiments, the liposomes are associated with the pharmaceutically active molecule via non-covalent interactions, covalent bonds, or both.

[0056] In some embodiments, the lipid nanoparticles further contain other lipids selected from cholesterol, dioleoylphosphatidylethanolamine (DOPE, CAS: 4004-05-1) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000, CAS: 147867-65-0) or 1,2-dimyristoyl-RAC-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000, CAS: 160743-62-4) or a combination thereof. At the same time, replacing DOPE with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, CAS: 4235-95-4) or distearoylphosphatidylcholine (DSPC, CAS: 816-94-4) in the same proportion can achieve similar delivery effects. In a further embodiment, the lipid nanoparticles further contain cholesterol, dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-dimyristoyl-RAC-glycero-3-methoxypolyethylene glycol 2000. In the example of the present invention, the molar ratio of liposome: cholesterol: DOPE: DSPE-PEG2000 is 50:38.5:10:1.5.

[0057] In some embodiments, the lipid nanoparticles of the present invention are prepared by the following method: the ionizable lipid compound of the present invention, cholesterol, DOPE, and DSPE-PEG2000 are dissolved in anhydrous ethanol, and the active pharmaceutical molecule is dissolved in anhydrous ethanol at a mass ratio of 5-20 times that of the active pharmaceutical molecule (the N / P ratio of the liposome to the nucleic acid ranges from 5:1 to 20:1) in 100 mM sodium acetate buffer (pH 5.2). The ethanol solution and sodium acetate buffer are mixed at a flow rate of 1:3 on a microfluidic machine. In further embodiments, the active pharmaceutical molecule is mRNA.

[0058] In a fourth aspect, the present invention relates to a method for regulating gene expression comprising delivering a nucleic acid to a cell, the method comprising: contacting the cell with the pharmaceutical composition of the third aspect under conditions sufficient to cause the nucleic acid to be taken up into the cell. In some embodiments, the cell is contacted in vitro or ex vivo. In some embodiments, the cell is contacted in vivo. In some embodiments, the regulation of gene expression is sufficient to treat a disease or condition such as cancer.

[0059] The present invention also relates to a method for treating a disease or condition in a patient, comprising administering to a patient in need thereof a pharmaceutically effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition comprises a therapeutic nucleic acid or protein for the disease or condition.

[0060] definition

[0061] 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, and cycloalkynyl moieties.

[0062] The term "alkyl" includes hydrocarbon groups selected from straight 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, 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, 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.

[0063] A monovalent group is a group formed by removing one hydrogen atom from a corresponding hydrocarbon moiety, and a divalent group is a group formed by removing two hydrogen atoms from a corresponding hydrocarbon moiety.

[0064] 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, and further such as 2 to 6 carbon atoms. Examples of alkenyl groups, such as C2-6 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, and the like.

[0065] 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, and further such as 2 to 6 carbon atoms. Examples of alkynyl groups, such as C2-6 alkynyl groups, include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl, 2-butynyl, and 3-butynyl.

[0066] The term "cycloalkyl" includes alkyl groups selected from saturated cyclic hydrocarbon groups comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged, or spirocyclic alkyl groups. Cycloalkyl groups 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 groups can be selected from monocyclic groups comprising 3 to 12, such as 3 to 10, further such as 3 to 8, 3 to 6 carbon atoms. Examples of monocyclic cycloalkyl groups include 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.

[0067] 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. 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- to 7-membered monocyclic heteroaliphatic moieties and 7- to 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 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, thioethanethiol, azetidinyl, oxetanyl, thietanyl, dithietanyl, dihydropyridine, tetrahydropyridine, thiomorpholine, homopiperazinyl, homopiperidinyl, azepanyl, oxetanyl, thietanyl, oxathianyl, dioxetanyl, oxathianyl, azoxanyl, dithianyl, azothianyl, , diazepanyl, azathiolanyl, oxazepine, diazepine, thiazepine, dihydrothiophenyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolinyl, indolinyl, dioxanyl, dioxolane, pyrazolinyl, pyrazolidinyl, dithianyl, dithiacyclopentyl, 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, for example, 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.

[0068] 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.

[0069] The term "aryl" refers to a C6 monocyclic, C10 bicyclic, C14 tricyclic, C20 tetracyclic or C24 pentacyclic aromatic ring system. Examples of aryl groups include phenyl, phenylene, naphthyl, naphthylene, anthracenyl, anthrylene, pyrenyl and pyrenylene.

[0070] 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 (e.g., O, N, S, or Se). Examples of heteroaryl groups 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.

[0071] Unless otherwise indicated, the aliphatic hydrocarbon group, heteroaliphatic hydrocarbon group, aliphatic hydrocarbon group oxy, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl and heteroaryl groups mentioned in this application include both substituted and unsubstituted parts. Possible substituents on cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl and heteroaryl groups 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 Alkyl imino, aryl imino, C1-C 10Alkylsulfonylimino, arylsulfonylimino, hydroxy, halo, thio, C1-C10 alkylthio, arylthio, C1-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 groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbon oxy groups, alkyl, alkylene, alkenyl, alkenylene, alkynyl, and alkynylene groups include all of the substituents listed above, but C1-C10 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 fused with each other.

[0072] The term "pharmaceutically active molecule" refers to any chemical substance intended for medical diagnosis, cure, treatment or prevention of disease, including small molecule drugs and macromolecule drugs, wherein small molecule drugs are such as chemotherapeutic drugs, and macromolecule drugs are such as oligopeptides, oligosaccharides, oligonucleotides, peptides, proteins or nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1a shows whole-body bioluminescence imaging (IVIS) 6 hours after the delivery of Firefly luciferase (Fluc) mRNA by LNPs containing lipid compounds with different hydrophobic chain lengths. Mice were administered via the tail vein (dose: 150 μg / kg); Figure 1b shows the expression level and bioluminescence intensity of luciferase in liver tissue.

[0074] Figure 2a shows whole-body bioluminescence imaging (IVIS) 6 hours after administration of Firefly luciferase mRNA containing liposomes of different chain lengths to mice via intramuscular administration (dose: 250 μg / kg); Figure 1b shows the expression level and bioluminescence intensity of luciferase at the tissue level.

[0075] FIG3 shows the expression level and bioluminescence intensity of luciferase in liver tissue 6 hours after Firefly luciferase mRNA was delivered by YX27 lipid nanoparticles under different N / P conditions. Mice were administered via the tail vein (dose: 150 μg / kg).

[0076] Figure 4a shows whole-body bioluminescence imaging (IVIS) of YX27, SM102 and D-Lin-MC3-DMA (MC3) lipid nanoparticles 6 hours after delivery, and mice were administered via the tail vein (dose: 150 μg / kg); Figure 4b shows the expression level and bioluminescence intensity of luciferase in liver tissue.

[0077] FIG5 shows a curve showing the changes in the expression level of luciferase and bioluminescence intensity over time in liver tissues of Firefly luciferase mRNA delivered by YX27 lipid nanoparticles.

[0078] FIG6 shows the bioluminescence intensity of liver tissue 6 hours after Firefly luciferase mRNA was delivered by lipid nanoparticles with different molecular structures. DETAILED DESCRIPTION

[0079] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0080] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0081] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0082] In the following examples, cholesterol was a product of Maclean, with the product number C10006595, CAS: 57-88-5.

[0083] 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.

[0084] In the following examples, Firefly luciferase mRNA was a product of Shanghai Zhaowei Technology Development Co., Ltd.

[0085] In the following examples, Balb / c mice were produced by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0086] Example

[0087] Example 1: Synthesis of ionizable lipid compounds

[0088] The ionizable lipid compound is synthesized from a corresponding hydrophilic amine compound and a corresponding hydrophobic tail through a Michael addition reaction, wherein the hydrophobic tail is an acrylate containing a ketal, specifically an acrylate containing a thioketal.

[0089] The hydrophobic tail compound TK is synthesized or prepared as follows:

[0090] TK12 is 2-((2-((2-dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate That is, the compound of formula I-1 of Example 2 of CN110101665A, TK-12 1 H NMR: 1 H NMR (300MHz, CDCl3) δ6.18 (s, 1H), 5.97 (d, J = 10.2Hz, 1H), 4.26 (s, 2H), 3.51 (s, 4H), 3.34 ( s,6H),2.87(s,1H),2.74(s,1H),1.55(s,4H),1.50–1.39(m,1H),1.25(s,6H),0.86(s,1H).

[0091] TK2, TK4, TK6, TK8 and TK10 were synthesized by a method similar to that of Examples 1 and 2 of CN110101665A. 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) and the corresponding short-chain alkyl halides Halo-C u H 2u+1 As starting material, prepare Then react with acryloyl halide (such as acryloyl chloride) to obtain the corresponding TK2, TK4, TK6, TK8 and TK10, wherein

[0092] TK10, 2-((2-((2-(decyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK10 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ6.45(d,1H),6.13(m,1H),5.86(d,1H),4.31(t,2H),3.58( t,2H),3.43(t,2H),2.91(t,2H),2.82(t,2H),1.61-1.26(m,23H),0.87(t,3H).

[0093] TK8, 2-((2-((2-(octyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK8 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ6.40(d,1H),6.13(m,1H),5.86(d,1H),4.31(t,2H),3.58( t,2H),3.43(t,2H),2.91(t,2H),2.82(t,2H),1.61-1.27(m,17H),0.87(t,3H).

[0094] TK6, 2-((2-((2-(hexyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK-6 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ6.41(d,1H),6.12(m,1H),5.83(d,1H),4.32(t,2H),3.59(t, 2H),3.46(t,2H),2.92(t,2H),2.82(t,2H),1.61(m,8H),1.31(m,6H),0.88(t,3H).

[0095] TK4, 2-((2-((2-(butyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK4 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)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).

[0096] The synthesis of the ionizable lipid compound YX of the present invention can be found in CN110101665A and the like.

[0097] YX9, i.e., compound II-10 of Example 5 of CN110101665A, is synthesized from a hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine (CAS: 105-83-9) and TK12 in a manner similar to Example 5 of CN110101665A, except that the molar ratio of the hydrophilic amine compound to TK12 is 4.3:1. 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ4.21(t,8H),3.57(t,8H),3.42(t,8H),2.85-2.76(m,24H ),2.44(t,12H),2.29(s,4H),2.18(s,3H),1.59-1.25(m,114H),0.87(t,12H).

[0098] YX8 is synthesized from the hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine and TK10, wherein the hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine and TK10 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 with dichloromethane / methanol as the eluent to obtain the ionizable lipid compound YX8. 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ4.21(t,8H),3.57(t,8H),3.42(t,8H),2.85-2.76(m,24H ),2.44(t,12H),2.28(s,4H),2.19(s,3H),1.60-1.25(m,100H),0.87(t,12H).

[0099] YX17, YX27 and YX31 were synthesized by mixing the hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine with the corresponding hydrophobic tail compound at a molar ratio of 4.3:1.

[0100] YX17 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.21(t,8H),3.58(t,8H),3.43(t,8H),2.85-2.77(m,24 H),2.45(m,12H),2.28(s,4H),2.18(s,3H),1.60-1.28(m,83H),0.87(t,12);

[0101] YX27 1The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.44(t,8H),2.86-2.77(m,24H ),2.45(t,12H),2.28(t,4H),2.18(s,3H),1.60-1.38(m,45H),0.92(t,12H); and

[0102] YX31 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H),2 .45(t,12H),2.28(t,4H),2.18(s,3H),1.60(m,37H),1.29(s,25H),0.88(t,12H).

[0103] Ionizable lipid compounds YX28, YX35, YX29, and YX33 were synthesized by combining the hydrophilic amine compounds N-methyl-2,2-diaminodiethylamine (CAS No. 4097-88-5) or N,N'-bis(2-aminoethyl)-N,N'-dimethyl-1,3-propanediamine (CAS No. 152829-22-6) with the corresponding hydrophobic tail compounds using a similar method as described above.

[0104] YX28 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) 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,41H),0.91(t,12H);

[0105] YX29 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.44(t,8H),2.86-2.77(m,24H),2. 56(t,4H),2.46(t,12H),2.34(t,4H),2.22(s,6H),1.6-1.38(m,50H),0.92(t,12H);

[0106] YX331 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H),2.5 7(s,4H),2.46(t,12H),2.35(s,4H),2.23(s,6H),1.60-1.29(m,60H),0.88(t,12H); and

[0107] YX35 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H ),2.55-2.46(m,16H),2.23(s,3H),1.60(m,35H),1.29(m,24H),0.89(t,12H).

[0108] Example 2: Encapsulation of Firefly luciferase mRNA and Preparation of Lipid Nanoparticles

[0109] To achieve animal-level mRNA delivery, the inventors prepared mRNA / lipid nanoparticles using the following method: The ionizable lipid compounds (YX8, YX17, YX31, YX27, YX28, YX35, YX29, or YX33) prepared in Example 1, along with cholesterol, DOPE, and DMG-PEG2000, were each prepared in a 10 mg / mL ethanol solution. For a single mRNA / LNP complex preparation, YX27 solution (1.6 mg) was mixed with cholesterol, DOPE, and DMG-PEG2000 in a molar ratio of 50:38.5:10:1.5, and ethanol was added to a total volume of 450 μL. 150 μg of Firefly luciferase mRNA was then added to a 50 mM sodium acetate buffer solution (pH 5.2) for a total volume of 1350 μL. The ionizable lipid compound solution and the mRNA solution were mixed via a microfluidic system (at a rate of 0.2 mL / min for ethanol and 0.6 mL / min for sodium acetate buffer) to prepare mRNA-encapsulated lipid nanoparticles. The mRNA was then dialyzed against PBS for two hours, and the resulting sample was used directly in animal experiments. The ratio of the ionizable lipid compound YX27 to the mRNA was calculated based on the molar ratio of nitrogen atoms in YX27 to phosphorus atoms in the mRNA phosphate backbone (N / P). In this example, the N / P ratio was 7.5.

[0110] For lipid nanoparticles encapsulating mRNA, Quant-it TM The RiboGreen RNA Assay Kit (ThermoFisher Scientific, USA) was used to determine the concentration of free RNA. Based on this, a method for determining the RNA encapsulation efficiency of the nanoparticle composition was established. 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 (no Triton X-100 added) by the fluorescence value of the lysed sample (caused by the addition of Triton X-100).

[0111] The LNPs comprising YX27 in this example were tested as follows: average particle size 185 nm, PDI 0.148, and Zeta potential -7.87 mV.

[0112] Example 3: Animal delivery experiment of mRNA using lipid nanoparticles of the present invention

[0113] To verify the delivery efficacy of the Fluc mRNA / LNP formulation, 6-8 week-old female Balb / c mice were injected intravenously with YX27 LNPs encapsulating 150 μg / kg Fluc mRNA or intramuscularly with 250 μg / kg Fluc mRNA into their hind legs. Six hours later, 200 μL of D-Lucifin (30% by mass) were injected intraperitoneally. Luciferase expression and intensity were measured systemically, in the liver, and at the injection site using a PerkinElmer IVIS Lumina III in vivo optical imaging system. To investigate the effect of lipid hydrophobic chain length on mRNA delivery efficiency, Fluc mRNA-encapsulated LNPs were prepared using lipid compounds YX9, YX8, YX17, and YX31, following the YX27 protocol, and their luciferase expression efficiency in mice was investigated. The study found that lipid compounds with 4 and 6 carbon atoms in the hydrophobic carbon chain tail structure are significantly more efficient in delivering mRNA than other lipid molecules.

[0114] The results of in vivo Luciferase live imaging luminescence intensity after using different lipid compounds to deliver Fluc mRNA are shown in the following table and Figures 1 and 2:

[0115] Unit: p / s

[0116] Example 4: Effect of N / P ratio of YX27 formulation on mRNA delivery

[0117] To study the effect of the mixing ratio of ionizable lipids to mRNA (N / P ratio) in the LNP component on the mRNA delivery efficiency, YX27 and luciferase mRNA mixed samples at N / P ratios of 7.5 and 9 were prepared according to the operating procedures of Example 2, and the delivery of mRNA and the expression and intensity of luciferase in the whole body and liver and other parts were studied by tail vein administration (the delivery results are shown in Figure 3 and the table below).

[0118] Example 5: Comparison of the effects of YX27, SM102, and MC3 on mRNA delivery

[0119] Following the procedures of Example 2, LNPs containing either SM102 or MC3 as the cationic lipid and luciferase mRNA at an N / P ratio of 7.5 were prepared. Fluc mRNA was administered at doses of 25 μg / kg, 50 μg / kg, or 150 μg / kg, respectively. The mRNA delivery of the prepared LNPs was then investigated via tail vein administration to investigate luciferase expression and intensity throughout the body and in the liver. The delivery results are shown in Figure 4 and the table below.

[0120] Unit: p / s

[0121] As can be seen from the above table, the ionizable lipid YX27 of the present invention exhibits a better delivery effect than the commercial cationic lipid SM102 or MC3 at each mRNA dose.

[0122] Example 6: Time-dependent experiments on mRNA delivery and expression

[0123] To verify the relationship between luciferase expression efficiency and time after Fluc mRNA / LNP administration, 6-8 week old female Balb / c mice were injected with a YX27 solution encapsulating 150 μg / kg Fluc mRNA via the tail vein. 2 hours, 4 hours, 6 hours, 18 hours, and 24 hours after administration, 200 microliters of D-Lucifin (30% by mass) were injected intraperitoneally. Luciferase expression and intensity were measured systemically and in the liver using the PerkinElmer IVIS Lumina III Small Animal In Vivo Optical Imaging System. The delivery results are shown in Figure 5 and the table below. The imaging results show that in vivo expression peaks 6 hours after mRNA delivery.

[0124] Unit: p / s

[0125] Example 7 Evaluation of mRNA delivery efficiency of YX lipid compounds with different structures

[0126] LNPs containing SM102, YX27, YX28, YX29, YX33, YX31, or YX35, and luciferase mRNA at an N / P ratio of 7.5 were prepared according to the procedures of Example 2. The Fluc mRNA was administered at a dose of 150 μg / kg. The results of in vivo luciferase live imaging luminescence intensity after Fluc mRNA delivery using different lipid compounds are shown in Figure 6 and the table below. It can be seen that liposomes composed of different amine head groups, when containing 4 and 6 carbon atoms, can efficiently deliver mRNA, with an efficiency close to that of the commercially available ionizable lipid SM102.

[0127] Unit: p / s

[0128] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A liposome compound of formula (I), in 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; 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; 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 above; Ht is independently -R1-X-R2-Y-R3-Z-R4 at each occurrence, in R1, at each occurrence, is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group; X is independently in 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 are each independently a bond, O, S or NR e ; V is an aliphatic hydrocarbon group, OR f , SR g or NR h R i , Where R b , R c , R d , R e , R f , R g , R h and R i are each independently hydrogen, hydroxy, aliphatic hydrocarbon group, monovalent aliphatic hydrocarbon group, monovalent heteroaliphatic hydrocarbon group, monovalent aromatic hydrocarbon group or monovalent heteroaromatic hydrocarbon group; Y and Z are each independently S or O at each occurrence; R2, at each occurrence, 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; R3, at each occurrence, 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; R4 is independently a hydrophobic group at each occurrence selected from -(CH2CH2O) y -C3-C8 alkyl, -(CH2CH2O) y -C3-C8 alkenyl or -(CH2CH2O) y -C3-C8 alkynyl, wherein y is 0 or 1 or 2.

2. The liposomal compound of claim 1, wherein t and s are both 0; or t is 0 and s is 1; or t is 01 and s is 0.

3. The liposome compound of claim 1 or 2, wherein R1 is 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.

4. The liposome compound of any one of claims 1 to 3, wherein X is wherein the variables are as defined in formula (I).

5. The liposome 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 liposome compound of any one of claims 1 to 3, wherein X is Where R d and R e As defined in formula (I).

7. The liposome compound of claim 6, wherein 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.

8. The liposomal compound of any one of claims 1 to 7, 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.

9. The liposome compound of any one of claims 1 to 8, wherein R2 is independently a single bond or C 1-6 A divalent aliphatic hydrocarbon group.

10. The liposome compound of claim 9, wherein R2 is independently C at each occurrence 1-4 A divalent aliphatic hydrocarbon group, preferably C 1-4 A divalent alkyl group, more preferably C 1-2 Divalent alkyl group.

11. The liposome compound of any one of claims 1 to 10, wherein R3 at 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 divalent alkyl).

12. The liposome compound of claim 11, wherein R3 is independently a single bond or or methylene Preferably 13. The liposomal compound of any one of claims 1 to 12, wherein R4 is independently, at each occurrence, C3-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, -(CH2CH2O)-C3-C8 alkyl, -(CH2CH2O)-C3-C8 alkenyl, -(CH2CH2O)-C3-C8 alkynyl, -(CH2CH2O)2-C3-C8 alkyl, -(CH2CH2O)2-C3-C8 alkenyl or -(CH2CH2O)2-C3-C8 alkynyl; or R4 at each occurrence is independently C4-C8 alkyl, C4-C8 alkenyl, C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkenyl, -(CH2CH2O)2-C4-C8 alkynyl, -(CH2CH2O)2-C4-C8 alkyl, -(CH2CH2O)2-C4-C8 alkenyl or -(CH2CH2O)2-C4-C8 alkynyl.

14. The liposomal compound of claim 13, wherein the alkyl group in the C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkyl or -(CH2CH2O)2-C4-C8 alkyl is a straight-chain alkyl group; the alkenyl group in the C4-C8 alkenyl, -(CH2CH2O)-C4-C8 alkenyl or -(CH2CH2O)2-C4-C8 alkenyl is a straight-chain alkenyl group; the alkynyl group in the C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkynyl or -(CH2CH2O)2-C4-C8 alkynyl is a straight-chain alkynyl group.

15. The liposomal compound of claim 13, wherein R4 at each occurrence is independently -(CH2CH2O)-(CH2)3CH3, -(CH2CH2O)2-(CH2)3CH3, -(CH2CH2O)-(CH2)4CH3, -(CH2CH2O)2-(CH2)4CH3, -(CH2CH2O)-(CH2)5CH3, -(CH2CH2O)2-(CH2)5CH3, -(CH2CH2O)2-(CH2)5CH3, -(CH2CH2O)-(CH2)6CH3, -(CH2CH2O)2-(CH2)6CH3, -(CH2CH2O)-(CH2)7CH3 or -(CH2CH2O)2-(CH2)7CH3.

16. The liposomal compound of claim 1, wherein Ht is independently 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; R3 is independently a single bond or or methylene R 44 It is a C4-C8 alkyl, C4-C8 alkenyl or C4-C8 alkynyl.

17. The liposome compound of claim 16, wherein R 44 Each occurrence is independently -(CH2)3CH3, -(CH2)4CH3, -(CH2)5CH3, -(CH2)6CH3 or -(CH2)7CH3.

18. The liposomal compound of claim 1, wherein 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; or 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); or t is 1, s is 0, A2 is a single bond, A1 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a It is hydrogen or a monovalent aliphatic hydrocarbon group, or it is hydrogen or a C1-C6 alkyl group or a methyl group.

19. The liposomal compound of claim 1, wherein t is 1, s is 1, A1, A2 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; or t is 1, s is 1, A1, A2 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); or t is 1, s is 1, A1, A2 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or a methyl group.

20. The liposomal compound of claim 1, wherein for Where R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group.

21. The liposome compound of claim 1, wherein the liposome of formula (I) is 22. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and lipid nanoparticles, wherein the lipid nanoparticles comprise the liposome compound according to any one of claims 1 to 21 and a pharmaceutically active molecule.

23. A method of regulating gene expression comprising delivering a nucleic acid to a cell, the method comprising: The cell is contacted with the pharmaceutical composition of claim 22 under conditions sufficient to cause uptake of the nucleic acid into the cell.

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