Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application

Abstract

The invention provides a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof, the cationic lipid compound has a structure as shown in formula (I): # imgabs0 #, x and y are respectively selected from positive integers between 0 and 2, and x + y is greater than or equal to 1; r1 is selected from H and substituted or unsubstituted C1-C10 alkyl groups; r1 and R2 are respectively selected from straight chain or branched chain C1-C30 saturated or unsaturated alkyl groups; r1, R2, R3 are respectively selected from H and substituted or unsubstituted C1-C20 alkyl groups; each L1 is selected from a straight chain or branched chain substituted or unsubstituted C0-C30 saturated or unsaturated alkylene group; l2 represents a linking group for linking R2 and an N atom; wherein when the substituted or unsubstituted groups have substituent groups, the substituent groups are selected from one or a combination of more of C1-C10 alkyl groups, C3-C7 cycloalkyl groups, hydroxyl groups, ester groups, amino groups, acyl groups, ether groups, carboxyl groups and sulfydryl groups.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a cationic lipid compound, a preparation method thereof, a composition containing the same, and applications thereof. Background Art

[0002] Nucleic acid drugs have broad application prospects in the treatment of major infectious diseases, tumors and other diseases, but are restricted by the lack of effective delivery systems. Delivery systems based on cationic lipid molecules have been widely used in clinical practice. For example, mRNA therapy has broad application prospects in the medical field, including protein replacement, vaccine development, and gene editing. Despite this, how to efficiently deliver mRNA to target cells remains a major challenge to its clinical application. Lipid nanoparticles (LNPs) have become the preferred carrier for mRNA therapy due to their excellent delivery efficiency, especially in the development of COVID-19 and RSV mRNA vaccines, and have been used in multiple clinical trials.

[0003] With the success of mRNA vaccines, people have a deeper understanding of the potential of mRNA therapy, which has further stimulated the demand for more efficient delivery technology. This demand is intended to support the continuous expansion of mRNA therapy to cover a wider range of therapeutic areas. In the LNP mRNA delivery system, ionizable cationic lipids are the core components, which are usually composed of three basic parts: an amine-containing head, a hydrophobic tail, and a connector connecting the two parts. Studies have shown that slight changes in lipid structure can have a significant impact on the delivery effect.

[0004] Therefore, in order to improve the delivery efficiency of nucleic acid drugs and broaden their therapeutic applications, it is particularly important to develop new ionizable lipids with different structures. This can not only improve the therapeutic effect, but also unlock new uses of nucleic acid drug therapy in other medical fields. Summary of the invention

[0005] In view of this, the present invention provides a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof, wherein the cationic lipid compound has a structure shown in formula (I):

[0006]

[0007] Wherein, x and y are each selected from positive integers between 0 and 2, and x+y≥1;

[0008] R1 is selected from H, substituted or unsubstituted C1-C10 alkyl;

[0009] R2 is each selected from a linear or branched C1-C30 saturated or unsaturated hydrocarbon group;

[0010] R3 is independently selected from H, substituted or unsubstituted C1-C20 alkyl, wherein R3 is connected to any of the connectable positions 1-6;

[0011] L1 is independently selected from a linear or branched substituted or unsubstituted C0-C30 saturated or unsaturated alkylene group;

[0012] L2 represents a linking group for connecting R2 and the N atom;

[0013] Wherein, when the substituted or unsubstituted group has a substituent, the substituent is selected from one or more combinations of C1-C10 alkyl, C3-C7 cycloalkyl, hydroxyl, ester, amino, acyl, ether, carboxyl and thiol.

[0014] As another aspect of the present invention, there is provided a method for preparing a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof, comprising the following steps:

[0015] The compound represented by formula (1) is reacted with a reducing agent and carbon dioxide to obtain an intermediate represented by formula (2);

[0016]

[0017] The intermediate represented by formula (2) is reacted with the compound represented by formula (3) to obtain the intermediate represented by formula (4);

[0018]

[0019] The intermediate represented by formula (4) is reacted with Acn or EPm to obtain a cationic lipid compound represented by formula (III-1) or formula (III-2);

[0020]

[0021] or

[0022]

[0023] In yet another aspect of the present invention, a composition is provided, comprising the cationic lipid compound as described above or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

[0024] In another aspect of the present invention, there is provided a use of the cationic lipid compound as described above or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer or the composition as described above in the preparation of a delivery drug for nucleic acids, polypeptides or proteins.

[0025] The cationic lipid compound shown in the above formula (I-1) provided by the present invention introduces a bicyclic aromatic hydrocarbon group into the amine core, which enhances hydrophobicity and has a higher affinity for RNA, and has a suitable particle size and zeta potential, as well as a strong binding ability with nucleic acid drugs and endosomal escape efficiency. At the same time, compared with DLin-MC3-DMA LNPs in the prior art, it shows a higher delivery efficiency of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and advantages of the present disclosure will become more apparent through the following description of the embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0027] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate represented by formula (1-4) prepared in Example 1 of the present invention;

[0028] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the cationic lipid compound CPA4-Ac8-1 prepared in Example 1 of the present invention;

[0029] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate represented by formula (2-4) prepared in Example 1 of the present invention;

[0030] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the cationic lipid compound CPA6-EP12 prepared in Example 1 of the present invention;

[0031] Figure 5 These are the results of characterizing the physicochemical properties of LNPs according to the examples of the present invention, including: (A) particle size of typical PIL LNPs; (B) Zeta potential of typical PIL LNPs; (C) lipid fusion efficiency between PIL LNPs and in vivo mimics; (D) lipid fusion efficiency of representative CPA4-Ac8-1 LNPs and CPA6-EP12 LNPs at different time intervals.

[0032] Figure 6 This is a diagram of the binding efficiency of LNPs and mRNA in an embodiment of the present invention;

[0033] Figure 7 The results of in vitro delivery of mRNA by 198 kinds of PIL LNPs in Example 1 of the present invention are shown; (A) Heat map of delivery of Fluc mRNA containing luciferase by PIL LNPs in IGROV1 cells; (B) Comparison of relative hit rates of PIL lipids prepared by Acn and EPm; (C) Relative hit rates of PIL lipids with different amine core L1 structures; (D) Relative hit rates of PILs prepared by Acn with different R2; (E) Relative hit rates of PILs prepared by EPm with different R2;

[0034] Figure 8 This is a representative cell viability graph of PIL LNPs in IGROV1 cells according to an embodiment of the present invention;

[0035] Fig. 9 This is a comparison chart of the mRNA delivery efficiency of PIL LNPs prepared in the embodiment of the present invention and SM-102 in comparative example 1 in cells in vitro;

[0036] Fig.10 The results of in vivo mRNA delivery of 44 PIL LNPs in the examples of the present invention are shown in Figure 1; (A) the results of in vivo luminescence imaging of mRNA delivery of 44 PIL LNPs IV-administered in C57BL / 6 mice; (B) the results of the average (ave) radiant brightness of the quantitative expression of Fluc proteins of PIL lipids with different amine core L1 structures; (C) the mRNA delivery efficiency of CPA4-Ac8-1 LNPs in different organs; (D) the comparison of mRNA translation of CPA6-EP12 LNPs in different organs;

[0037] Fig.11 This is a comparison diagram of the mRNA delivery results of CPA6-EP12 and four-tailed PIL in vivo in the present invention;

[0038] Fig.12 Comparison diagram of mRNA delivery in liposomes of Example 4 of the present invention. (A) Chemical structure diagram of CPA4-Ac8-1 and CPA6-EP12 lipids; (B) Comparison diagram of in vivo imaging of CPA4-Ac8-1 LNPs and CPA6-EP12 LNPs after intravenous administration; (C) Comparison diagram of in vivo imaging of CPA4-Ac8-1 LNPs, CPA6-EP12 LNPs, SM-102 and ALC-0315 after intramuscular administration. DETAILED DESCRIPTION

[0039] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings.

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. However, the present invention is not limited to the following embodiments.

[0041] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The terms "including", "comprising", etc. used herein indicate the presence of features, steps, operations and / or components, but do not exclude the presence or addition of one or more other features, steps, operations or components. All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art unless otherwise defined. It should be noted that the terms used herein should be interpreted as having meanings consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0042] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the common meanings understood by technicians in the field to which the present invention belongs. If the full text involves descriptions such as "first", "second", etc., the descriptions such as "first", "second", etc. are only used to distinguish similar objects, and cannot be understood as indicating or implying their relative importance, order of precedence, or implicitly indicating the number of technical features indicated. It should be understood that the data described by "first", "second", etc. can be interchangeable under appropriate circumstances.

[0043] In the present invention, the term "pharmaceutically acceptable" refers to compounds, substances, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0044] In the present invention, the term "solvate" refers to a complex formed by combining a compound of formula (I-1) or formula (I-2) or a pharmaceutically acceptable salt thereof and a solvent (e.g., ethanol or water). The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water. Further, the compound of formula (I-1) or formula (I-2) or a pharmaceutically acceptable salt thereof can be separated in the form of a solvate, and any solvate is included in the scope of protection of the present invention.

[0045] In the present invention, the term "pharmaceutically acceptable salt" refers to a relatively non-toxic organic acid or inorganic acid addition salt of a compound of formula (I-1) or formula (I-2). Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, etc., organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, cinnamic acid, picric acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, itaconic acid, naphthalene disulfonic acid, malic acid, adipic acid, alginic acid, maleic acid, D-gluconic acid, aspartic acid, etc.

[0046] In the present invention, the term "stereoisomer" includes geometric isomers, diastereomers and enantiomers. Therefore, the compounds claimed in the present invention also include racemic mixtures, single stereoisomers and optically active mixtures. It is understood that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. Racemates can be chirally resolved by chromatographic resolution or chemical resolution.

[0047] In the present invention, the term "substituted or unsubstituted" group may be substituted with one substituent or multiple substituents. When there are multiple substituents, they may be selected from different substituents. When the same expressions are involved in the present invention, they all have the same meaning, and the selection range of substituents is as shown above and will not be repeated one by one.

[0048] In the present invention, "each independently" means that when there are multiple subjects, they may be the same or different from each other.

[0049] In the present invention, the expression of chemical elements, unless otherwise specified, generally includes the concept of isotopes with the same chemical properties. For example, the expression "hydrogen (H)" also includes isotopes with the same chemical properties. 1 H (hydrogen or H), 2 The concept of H (deuterium or D); carbon (C) includes 12 C. 13 C, etc., no further details.

[0050] In the present invention, the expression of Ca to Cb represents that the number of carbon atoms in the group is a to b. Unless otherwise specified, the number of carbon atoms generally does not include the number of carbon atoms in the substituent.

[0051] In the present invention, the term "alkyl" may include a branched or straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. For example, examples of C1 to C20 alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. C0 can be understood as a direct connection without any intermediate groups.

[0052] In the present invention, the term "straight or branched saturated or unsaturated hydrocarbon group" may include straight or branched saturated aliphatic monovalent hydrocarbon groups or unsaturated aliphatic monovalent hydrocarbon groups having a specified number of carbon atoms. For example, C3-C30 straight or branched saturated hydrocarbon groups include: propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, isooctyl, nonyl, decyl, undecyl, dodecyl, etc. The C3-C30 straight or branched unsaturated hydrocarbon group may be one in which one or more single bonds in the C3-C30 straight or branched saturated hydrocarbon group are replaced with double bonds, etc.

[0053] In the present disclosure, the term "saturated or unsaturated alkylene" may include a saturated aliphatic divalent hydrocarbon group or an unsaturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. For example, C1-C20 straight-chain or branched saturated alkylene groups include methylene, ethylene, propylene, pentylene, etc. C1-C20 unsaturated alkylene groups may be C1-C20 saturated alkylene groups in which one or more single bonds are replaced with double bonds or triple bonds.

[0054] In the present invention, CPA3-Ac10-1 represents PIL cationic lipid, and CPA3-Ac10-1LNPs represents lipid nanoparticles (LNPs) using CPA3-Ac10-1 as cationic lipid.

[0055] In the process of developing more efficient ionizable lipids (PILs) to optimize mRNA delivery systems, researchers have extensively customized the hydrophobic tails and connectors of lipids. These customizations are intended to enhance the delivery performance of lipids, including:

[0056] Optimization of the hydrophobic tail: The design of the tail was refined to improve the hydrophobicity of the lipid by adjusting the length of the alkyl chain, increasing the number of alkyl groups, introducing unsaturated bonds, changing the branching pattern, and embedding stimuli-responsive groups.

[0057] Connector design: The connector part is also carefully designed to contain degradable esters, carbonates, amides, hydroxyls and stimulus-responsive elements to enhance the stability and safety of mRNA and achieve specific organ targeting.

[0058] However, the amine cores of most PILs are still mainly derived from commercially available small amine molecules, which limits the functional diversity of PILs. Therefore, the delivery function of PILs may be further improved by introducing functional groups into the amine core. By introducing bicyclic aromatic hydrocarbon groups with strong RNA affinity and hydrophobicity into the amine core, the electrostatic interaction can be synergistically used to enhance the connection between PILs and RNA, thereby improving the overall delivery efficiency of PILs.

[0059] According to an embodiment of one aspect of the present invention, a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof is provided, wherein the cationic lipid compound has a structure shown in formula (I):

[0060]

[0061] Preferably, it has the structure shown in formula (I-1):

[0062]

[0063] More preferably, the cationic lipid compound has a structure shown in formula (I-2):

[0064]

[0065] wherein x and y are each selected from a positive integer between 0 and 2, and x+y≥1, for example, x=0, y=1; x=1, y=1; x=2, y=1; x=2, y=2; R1 is selected from H, substituted or unsubstituted C1-C10 alkyl, preferably H, C1-C6 alkyl, more preferably H, methyl or ethyl; R2 is each selected from a linear or branched C1-C30 saturated or unsaturated hydrocarbon group, preferably a C2-C18 saturated or unsaturated hydrocarbon group, more preferably a C4-C14 saturated or unsaturated hydrocarbon group, further preferably a C8-C14 saturated or unsaturated hydrocarbon group; R3 is each selected from H, substituted or unsubstituted C1-C20 alkyl, wherein R3 and any one of 1 to 6 may be connected Position connection; L1 is each selected from a linear or branched substituted or unsubstituted C0~C30 saturated or unsaturated alkylene group, which can be C0, that is, N atoms and N atoms are directly connected to form a bond, and can be a linear or branched C1~C20 alkyl group, a linear or branched C2~C20 olefin group, preferably a C1~C12 linear or branched saturated or unsaturated hydrocarbon group, and more preferably a C3~C7 linear or branched saturated or unsaturated hydrocarbon group; L2 represents a connecting group for connecting R2 and the N atom; wherein, when the substituted or unsubstituted group has a substituent, the substituent is selected from a combination of one or more of C1~C10 alkyl, C3~C7 cycloalkyl, hydroxyl, ester group, amino group, acyl group, ether group, carboxyl group, and thiol group.

[0066] According to an embodiment of the present invention, by introducing a bicyclic aromatic hydrocarbon group into the amine core, the hydrophobicity and affinity for RNA can be enhanced at the same time. The affinity of the bicyclic aromatic hydrocarbons for RNA and the electrostatic interaction between the secondary amine and tertiary amine groups and RNA will synergistically enhance the transfer function. At the same time, the length of the three hydrophobic tails connected by R2 and the length of L1 in the amine core affect the balance between hydrophilicity and hydrophobicity and mediate the delivery effect on RNA.

[0067] According to an embodiment of the present invention, L2 represents a linking group for connecting R2 and the N atom, preferably selected from H, a substituted or unsubstituted C1-C30 alkylene group, or a substituted or unsubstituted C2-C30 alkylene ester group;

[0068] More preferably, L2 is selected from one of the following formula (II-1) or formula (II-2):

[0069] Wherein, in formula (II-1), position 1 is connected to the N atom, and position 4 is connected to R2;

[0070] Among them, in formula (II-2), position 1 is connected to the N atom, and position 2 is connected to R2.

[0071] According to an embodiment of the present invention, in the compound represented by formula (I-1) or formula (I-2), R2 is selected from a C1 to C30 straight-chain or branched alkyl, alkene or alkyne group, and further R2 is preferably any of the following structures:

[0072]

[0073] According to an embodiment of the present invention, in the compound represented by formula (I-1) or formula (I-2), L1 is independently selected from a C0-C30 straight-chain or branched alkyl, alkene or alkyne group, and further L1 is preferably any of the following structures:

[0074]

[0075] According to an embodiment of the present invention, the above types of L1 and R2 can affect the delivery efficiency of PILs to RNA and the therapeutic effect of RNA by regulating the amphiphilic balance of the hydrophilicity of the amine core and the hydrophobic tail. Further, different L1 and R2 can specifically mediate mRNA delivery in the liver or in the liver and spleen at the same time.

[0076] According to an embodiment of the present invention, the above-mentioned compound of the present invention is preferably selected from any of the following structures, but these compounds are only representative:

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] According to another aspect of the present invention, there is provided a method for preparing the cationic lipid compound or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer as described above, comprising the following steps S101 to S105.

[0106] In step S101, the compound represented by formula (1) is reacted with a reducing agent and carbon dioxide to obtain an intermediate represented by formula (2);

[0107]

[0108] In step S102, the intermediate represented by formula (2) is reacted with the compound represented by formula (3) to obtain the intermediate represented by formula (4);

[0109]

[0110] In step S103, the intermediate represented by formula (4) is reacted with Acn or EPm to obtain a cationic lipid compound represented by formula (III-1) or formula (III-2);

[0111]

[0112] or

[0113]

[0114]

[0115] According to the embodiments of the present invention, the preparation method has mild conditions and is easy to operate, and can effectively prepare the cationic lipid compound.

[0116] According to an embodiment of the present invention, in step S101, the method may specifically include: adding a first solvent to 4,16-dibromo[2.2]paracyclic aromatic hydrocarbon of formula (1) under the protection of an inert gas atmosphere, cooling the mixture, slowly adding a strong base and a strong nucleophilic agent to the reaction system, heating the mixture for a period of time, cooling the mixture, introducing carbon dioxide gas for a period of time, and heating the mixture to room temperature. The reaction product is extracted, precipitated, and washed to obtain an intermediate of formula (2). The inert gas atmosphere may be, for example, argon, helium, etc.; the first solvent may be, for example, anhydrous tetrahydrofuran, anhydrous dimethylformamide (DMF), anhydrous dimethyl sulfoxide (DMSO), or anhydrous ether, etc.; the strong base and the strong nucleophilic agent may be, for example, n-butyl lithium hexane, n-hexyl lithium, or sec-butyl lithium, etc.

[0117] According to an embodiment of the present invention, in step S102, the method may specifically include: adding a second solvent, an organic base catalyst and a condensing agent to the intermediate of formula (2) for condensation reaction at room temperature, and then slowly adding the mixture to the compound of formula (3) dissolved in a third solvent to obtain the intermediate of formula (4). The second solvent and the third solvent may be at least one of dichloromethane, chloroform, ethyl acetate or n-hexane; the organic base catalyst may be, for example, N,N-diisopropylethylamine (DIEA), triethylamine or piperidine; and the condensing agent may be, for example, BOP, EDC, DCC or HATU.

[0118] In some embodiments of the present invention, in step S102, the progress of the reaction can also be monitored by high performance liquid chromatography (HPLC) to determine whether the intermediate represented by formula (2) disappears completely.

[0119] According to an embodiment of the present invention, in step S103, it can specifically include: subjecting the intermediate shown in formula (4) to Michael addition reaction or epoxy ring-opening reaction with Acn or EPm and an alkali reagent in a fourth solvent and a fifth solvent, filtering and purifying the crude product after the reaction to obtain a cationic lipid compound shown in formula (III-1) or formula (III-2). The alkali reagent is used to help deprotonate Acn or EPm, for example, sodium bicarbonate, potassium carbonate or triethylamine; the fourth solvent is used to provide an inert environment, for example, dichloromethane, chloroform, ethyl acetate or n-hexane; the fifth solvent is used to adjust the solvent system of the reaction and as a co-solvent to improve solubility or reaction selectivity, for example, it can be isopropanol, ethanol or n-butanol. The purification process can be carried out by silica gel column chromatography, and thin layer chromatography (TLC), gas chromatography (GC) or liquid chromatography (HPLC) can also be used to monitor the progress of the reaction.

[0120] According to an embodiment of the present invention, Acn includes at least one of the following structures:

[0121]

[0122]

[0123] According to an embodiment of the present invention, EPm includes at least one of the following structures:

[0124]

[0125] According to another embodiment of the present invention, there is provided a composition comprising the cationic lipid compound as described above or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

[0126] According to an embodiment of the present invention, the above composition may further include a preventive agent and / or a therapeutic agent.

[0127] According to an embodiment of the present invention, the preventive agent or therapeutic agent may be at least one of a nucleic acid, a polypeptide, a protein or a protein-like compound.

[0128] According to an embodiment of the present invention, the preventive agent includes a cationic lipid compound, a stabilizer for stabilizing the structure of the composition, and a filler for filling the structure of the composition.

[0129] According to an embodiment of the present invention, the molar ratio of the cationic lipid compound to the stabilizer and filler in the preventive agent includes 1:2, 1:3, 1:4, 1:5, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0130] According to an embodiment of the present invention, the stabilizer comprises PEGylated lipids, and the filler comprises neutral phospholipids and / or steroidal lipids.

[0131] The nucleic acid includes but is not limited to single-stranded DNA, double-stranded DNA and RNA. The RNA includes but is not limited to small interfering RNA (siRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), messenger RNA (mRNA), small hairpin RNA, asymmetric interfering RNA and mixtures thereof.

[0132] Also, proteins or protein-like compounds include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), interferon, and hepatitis B surface antigen.

[0133] According to an embodiment of the present invention, phospholipids are suitable for improving the stability of lipid nanoparticles, regulating the fluidity of lipid nanoparticles, and may also affect target specificity. The neutral phospholipids may be, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1,2-behenoyl-phosphatidylcholine (DLPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-myristoylphosphatidylcholine (SMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DPE), and 1,2-dioleoylphosphatidylcholine (DOPE). MPE), dilauroyl phosphatidylethanolamine (DLPE), distearoyl phosphatidic acid sodium salt (DSPA), dipalmitoyl phosphatidic acid sodium salt (DPPA), dimyristoyl phosphatidic acid sodium salt (DMPA), dioleoyl phosphatidic acid sodium salt (DOPA), dilauroyl phosphatidic acid sodium salt (DLPA), distearoyl phosphatidyl glycerol sodium salt (DSPG), dipalmitoyl phosphatidyl glycerol sodium salt (DPPG), dimyristoyl phosphatidyl glycerol sodium salt (DMPG), dioleoyl phosphatidyl glycerol sodium salt (DOPG), dilauroyl phosphatidyl glycerol sodium salt (DLPG), distearoyl phosphatidyl serine sodium (DSPS), dipalmitoyl phosphatidyl serine sodium (DPPS), dimyristoyl phosphatidyl serine sodium (DMPS), dioleoyl phosphatidyl serine sodium salt (DOPS), dilauroyl phosphatidyl serine sodium salt (DLPS), etc.

[0134] According to an embodiment of the present invention, the steroidal lipid is selected from one or more of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginasterol, sitostanol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, and deoxycholic acid.

[0135] According to an embodiment of the present invention, the steroidal lipid is selected from cholesterol, which is a structural lipid. It adjusts the fluidity of lipid nanoparticles by filling the gaps between phospholipids and enhances the stability of lipid nanoparticles.

[0136] According to embodiments of the present invention, PEGylated lipid (i.e. PEGylated lipid), can affect the size, stability, in vivo distribution and transfection efficiency of lipid nanoparticle delivery system, in the preparation and storage process of lipid nanoparticle delivery system, PEGylated lipid can drive self-assembly and prevent particle aggregation. PEGylated lipid can for example be distearoyl phosphatidylethanolamine polyethylene glycol (DSPE-PEG), dimyristoyl glycerol-rac-methoxy polyethylene glycol 2000 (DMG-PEG2000), methoxy polyethylene glycol ditetradecyl acetamide (ALC-0159), polyethylene glycol-diacylglycerol amide (PEG-DAG), polyethylene glycol-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), polyethylene glycol-disterol glycerol (PEG-DSG), polyethylene glycol-diacylglycerol amide (PEG-DAG) etc.

[0137] According to an embodiment of the present invention, the molar ratios of the cationic lipid compound to the neutral phospholipid, the steroidal lipid and the pegylated lipid in the above composition include 10:15:20:0.5, 10:15:20:1.0, 10:15:20:2.0, 10:15:20:3.0, 10:20:25:0.5, 10:20:25:1.0, 10:20:25:2.0, 10:20:25:3.0, 10:20:25:0.5, 10:20:25:1.0, 10:20:25:2.0, 10:20:25:3.0, 10:20:25:0.5, 10:20:25:1.0 0:25:1.0, 15:20:25:2.0, 15:20:25:3.0, 15:25:30:0.5, 15:25:30:1.0, 15:25:30:2.0, 15:25:30:3.0, 20:25:30:0.5, 20:25:30:1.0, 20:25:30:2.0, 20:25:30:3.0, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0138] According to an embodiment of the present invention, the above composition may further include pharmaceutically acceptable excipients or auxiliary ingredients, including but not limited to: binders, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations. The pharmaceutical preparation may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically).

[0139] According to an embodiment of the present invention, the above-mentioned composition can be formulated into several dosage forms according to the mode of administration, such as oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which can be used immediately by adding injection water before injection), etc.

[0140] According to an embodiment of the present invention, the above composition can be administered at a therapeutically effective dose, which can vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient. For example, a nucleic acid at a dose of about 0.001 mg / Kg to about 10 mg / Kg can be administered to a subject, which can be a mammal (such as a human) or the like.

[0141] According to another embodiment of the present invention, there is also provided the use of the cationic lipid compound as described above or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer or the composition as described above in the preparation of a delivery drug for nucleic acids, polypeptides or proteins.

[0142] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. The test materials and reagents used in the following embodiments, unless otherwise specified, can be obtained from commercial sources. If no specific techniques or conditions are specified in the embodiments, they are all conventional methods and can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0143] Example 1 Synthesis of Cationic Lipid Compound CPA4-Ac8-1

[0144] The synthetic route is as follows:

[0145]

[0146] Step S101, add 1 equivalent of 4,16-dibromo[2.2]paracyclophane (1-1) to a dry round-bottom flask, then add anhydrous tetrahydrofuran, i.e., the first solvent, under argon protection, and cool the mixture to -70°C to -80°C. After that, slowly add 2.5M 6 equivalents of n-butyl lithium hexane, i.e., a solution of a strong base and a strong nucleophile, to the system, and gradually heat the system to -20°C to -30°C for 1 hour. Subsequently, cool the system to -40°C to -50°C, continue to pass carbon dioxide gas into the system for about 1 hour, then allow the system to warm to room temperature and stir for 4 hours.

[0147] After the reaction is completed, the solvent is removed by rotary evaporation, 1M sodium hydroxide aqueous solution is added, and then methyl tert-butyl ether is added for extraction. The aqueous phase is washed 3 times with methyl tert-butyl ether, and then 6M hydrochloric acid is added to adjust the pH to 2-3, and a white solid precipitates out. After filtration, the precipitate is washed twice with clean water and dried at 50°C to obtain the intermediate represented by formula (1-2) with a yield of 50-60%.

[0148] Step S102, in a round-bottomed flask, add equimolar intermediates shown in formula (1-2) and dichloromethane, i.e., the second solvent, and add 3 equivalents of DIEA, i.e., an organic base catalyst, and 3 equivalents of BOP, i.e., a condensing agent, at room temperature. The mixture is stirred at room temperature for 1 hour. Afterwards, the reaction mixture is slowly added to another flask containing a dichloromethane solution of 5 equivalents of the compound of formula (1-3), i.e., the third solvent, and stirred at room temperature overnight to obtain a crude mixture of the intermediate shown in formula (1-4). The reaction is monitored by high performance liquid chromatography (HPLC) until the intermediate shown in formula (1-2) disappears completely, and then the crude mixture can be directly used in the next step without any treatment.

[0149] Purification step: Add di-tert-butyl dicarbonate to the obtained crude intermediate mixture of formula (1-4) at room temperature in an amount of 20 molar equivalents, and keep the reaction at room temperature for 4 hours. After the reaction is completed, remove the solvent by rotary evaporation, and then purify by column chromatography, and the eluent used is a mixed solvent system of dichloromethane and methanol, and the ratio gradually changes from 100:1 to 10:1. Through the above two-step reaction process, the total yield of the intermediate represented by formula (1-4) is expected to be between 65% and 75%.

[0150] The purified intermediate of formula (1-4) is added to a mixed solution of 4 molar hydrochloric acid and 1,4-dioxane, and then the mixed system is continuously stirred at room temperature until overnight. After the reaction is completed, the solvent is removed by rotary evaporation technology, and then further purified by high performance liquid chromatography (HPLC) technology. In this way, the intermediate of product formula (1-4) can be obtained with a yield of up to 80%.

[0151] In addition, the amino protection and deprotection steps via Boc group pairs should be taken during the purification process as shown below:

[0152]

[0153] Step S103, the purified intermediate represented by formula (1-4) is added to a proportional vial with Ac8 and sodium bicarbonate at a molar ratio of 1:3:4.5. Then, Michael addition reaction is carried out with 0.5 mL of dichloromethane, and the reaction is monitored by TLC until Ac8 completely disappears to obtain a crude product. The crude product after the reaction is filtered and purified, and the cationic lipid compound CPA4-Ac8-1 is obtained by filtering and removing the solvent under vacuum.

[0154] The intermediate represented by formula (1-4) and the cationic lipid compound CPA4-Ac8-1 were characterized by nuclear magnetic resonance (NMR) and mass spectrometry, respectively, and the results were as follows: Figure 1 and Figure 2shown.

[0155] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate represented by formula (1-4) prepared in Example 1 of the present invention; Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the cationic lipid compound CPA4-Ac8-1 prepared in Example 1 of the present invention.

[0156] exist Figure 1 In the formula (1-4), the intermediate 1 H NMR spectrum (D2O), in addition, the molecular weight calculated by the structural formula is 436.288, and [M+H] + (ESI-MS): 437.20, all of which indicate that the intermediate represented by formula (1-4) was successfully prepared.

[0157] exist Figure 2 In the figure, the cationic lipid compound CPA4-Ac8-1 is represented by 1 H NMR spectrum (CDCl3), the characterization results are as follows:

[0158] 1H NMR(CDCl3,ppm)δ6.90(ddd,2H),6.69(dd,2H),6.41(dd,2H),6.10(t,1H),5.87(t,1 H),3.90-4.03(m,5H),3.58-3.66(m,2H),3.35-3.47(m,3H),3.23-3.32(m,2H),2.87 -3.02(m,5H),2.72-2.80(m,5H),2.60(t,2H),2.45(t,2H),2.41(t,4H),1.65-1.70( m,3H),1.49-1.62(m,7H),1.36-1.38(m,2H),1.29-1.36(m,8H),1.23-1.27(m,20H).

[0159] In addition, the molecular weight calculated by the structural formula is 988.72, and the [M+H] + The m / z value of the ion is 989.74, which indicates that the cationic lipid compound CPA4-Ac8-1 was successfully prepared.

[0160] Example 2 Synthesis of cationic lipid compound CPA6-EP12

[0161] The synthetic route is as follows:

[0162]

[0163] Step S101, add 1 equivalent of 4,16-dibromo[2.2]paracyclophane (2-1) to a dry round-bottom flask, then add anhydrous tetrahydrofuran, i.e., the first solvent, under argon protection, and cool the mixture to -70°C to -80°C. After that, slowly add 2.5M 6 equivalents of n-butyl lithium hexane, i.e., a solution of a strong base and a strong nucleophile, to the system, and gradually heat the system to -20°C to -30°C for 1 hour. Subsequently, cool the system to -40°C to -50°C, continue to pass carbon dioxide gas into the system for about 1 hour, then allow the system to warm to room temperature and stir for 4 hours.

[0164] After the reaction is completed, the solvent is removed by rotary evaporation, 1M sodium hydroxide aqueous solution is added, and then methyl tert-butyl ether is added for extraction. The aqueous phase is washed 3 times with methyl tert-butyl ether, and then 6M hydrochloric acid is added to adjust the pH to 2-3, and a white solid precipitates out. After filtration, the precipitate is washed twice with clean water and dried at 50°C to obtain the intermediate shown in formula (2-2) with a yield of 50-60%.

[0165] Step S102, in a round-bottom flask, add equimolar intermediates shown in formula (2-2) and dichloromethane, i.e., the second solvent, and add 3 equivalents of DIEA, i.e., an organic base catalyst, and 3 equivalents of BOP, i.e., a condensing agent, at room temperature. The mixture is stirred at room temperature for 1 hour. Afterwards, the reaction mixture is slowly added to another flask containing a dichloromethane solution of 5 equivalents of the compound of formula (2-3), i.e., the third solvent, and stirred at room temperature overnight to obtain a crude mixture of the intermediate shown in formula (2-4). The reaction is monitored by high performance liquid chromatography (HPLC) until the intermediate shown in formula (2-2) disappears completely, and then the crude mixture can be directly used in the next step without any treatment.

[0166] Purification step: Add di-tert-butyl dicarbonate to the obtained crude intermediate mixture of formula (2-4) at room temperature in an amount of 20 molar equivalents, and keep the reaction at room temperature for 4 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and then column chromatography is used for purification. The eluent used is a mixed solvent system of dichloromethane and methanol, and the ratio gradually changes from 100:1 to 10:1. Through the above two-step reaction process, the total yield of the intermediate represented by formula (2-4) is expected to be between 65% and 75%.

[0167] The purified intermediate of formula (2-4) is added to a mixed solution of 4 molar hydrochloric acid and 1,4-dioxane, and then the mixed system is continuously stirred at room temperature until overnight. After the reaction is completed, the solvent is removed by rotary evaporation technology, and then further purified by high performance liquid chromatography (HPLC) technology. In this way, the intermediate of product formula (2-4) can be obtained with a yield of up to 80%.

[0168] In addition, the amino protection and deprotection steps via Boc group pairs should be taken during the purification process as shown below:

[0169]

[0170] Step S103, the purified intermediate shown in formula (2-4) is added to a proportional vial with EP12 and sodium bicarbonate in a molar ratio of 1:3:4.5. Then, Michael addition reaction is carried out with 0.5 mL of dichloromethane, and the reaction is monitored by TLC until EP12 completely disappears to obtain a crude product. The crude product after the reaction is filtered and purified, and the cationic lipid compound CPA6-EP12 is obtained by filtering and removing the solvent under vacuum.

[0171] The intermediate shown in formula (2-4) and the cationic lipid compound CPA6-EP12 were characterized by nuclear magnetic resonance (NMR) and mass spectrometry, respectively, and the results were as follows: Figure 3 and Figure 4 shown.

[0172] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate represented by formula (2-4) prepared in Example 1 of the present invention; Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the cationic lipid compound CPA6-EP12 prepared in Example 1 of the present invention.

[0173] exist Figure 3 In the formula (2-4), the intermediate 1 H NMR spectrum (D2O), in addition, the molecular weight calculated by the structural formula is 492.35, and [M+H] + (ESI-MS): 493.20, all of which indicate that the intermediate represented by formula (2-4) was successfully prepared.

[0174] exist Figure 4 In the figure, the cationic lipid compound CPA6-EP12 is represented by 1 H NMR spectrum (CDCl3), the characterization results are as follows:

[0175] 1H NMR(CDCl3,ppm)δ6.81-6.88(m,2H),6.66-6.72(m,2H),6.38-6.44(m,2H),6.04-6.16( m,1H),5.85-6.02(m,1H),3.91-3.99(m,1H),3.76-3.90(m,2H),3.54-3.63(m,2H),3.29 -3.45(m,4H),3.16-3.26(m,2H),2.87-3.04(m,7H),2.70-2.86(m,4H),2.61(s,3H),1.7 3-1.81(m,1H),1.51-1.65(m,6H),1.34-1.49(m,15H),1.16-1.32(m,48H),0.87(t,9H).

[0176] In addition, the molecular weight calculated by the structural formula is 1044.89, and the [M+H] + The m / z value of the ion is 1045.91, which indicates that the cationic lipid compound CPA6-EP12 was successfully prepared.

[0177] Example 3 Preparation method and physicochemical properties of lipid nanoparticles (LNPs) prepared from PIL

[0178] Lipid Nanoparticles (LNPs) Prepared by PIL:

[0179] The PIL lipid, DOPE, cholesterol and DMG-PEG 2000 prepared by the preparation method of the present invention are mixed in an ethanol solution at a molar ratio of 15:20:25:2; then the mRNA is diluted in a citric acid buffer (pH 4.4, 10mM) under sterile conditions; wherein the mass ratio of PIL:mRNA is fixed at 10:1 for preparation. The mRNA aqueous solution and the ethanol solution are mixed at a volume ratio of 3:1 and incubated at room temperature for 15 minutes to obtain LNPs.

[0180] LNPs including the following PILs: CPA3-Ac10-1, CPA4-Ac8-1, CPA6-Ac8-1, CPA4-EP12, CPA5-EP12, CPA6-EP12 and CPA7-EP12 were prepared and characterized for their physicochemical properties.

[0181] FRET probe detection method: FRET probes NBD-PE and Rho-PE were formulated in a liposome environment that simulates endosomes. When liposomes fuse, due to the FRET effect, the fluorescence intensity of NBD decreases due to energy transfer to rhodamine. As the distance between the two probes increases, the fluorescence signal of NBD increases accordingly. By detecting the change in fluorescence intensity, the membrane fusion and endosomal escape can be evaluated. DOPC:DOPS:DOPE:Rho-PE:BD-PE were mixed in chloroform at a molar ratio of 25:25:48:1:1, the solvent was removed by rotary evaporation, and then dried under vacuum for 2 hours to obtain a lipid membrane. After that, 1× PBS buffer (pH 7.4) was added and ultrasonicated for 30 minutes to obtain anionic liposomes that simulated endosomal concentrations with a total lipid concentration of 1mM.

[0182] PIL LNPs were prepared using the above method at a total lipid concentration of 1 mM. 100 μL of 1× PBS buffer (pH 5.5), 10 μL of PIL LNPs, and 1 μL of simulated endosomal liposomes were added to the wells of a black 96-well plate. After incubation at 37°C for 30 minutes, the fluorescence intensity (F) was measured on a multifunctional microplate reader at a wavelength of Ex / Em=465 / 520 nm. The simulated endosomal liposomes in 1× PBS were set as negative controls (F min The liposomes containing the FRET probe and Triton X-100 (2 wt.%) were set as the positive control (F max Lipid fusion rate (%) was calculated by (FF min ) / (F max -F min )×100% calculation.

[0183] The particle size and Zeta potential of PIL LNPs were detected by using the dynamic light scattering instrument Zetasizer Nano-ZS; the in vivo escape characteristics of LNPs were detected by fluorescence resonance energy transfer (FRET), and the degree of membrane fusion and endosomal rupture was determined by detecting the change in the fluorescence signal of the FRET probe after mixing with the in vivo mimics. The results are shown in Figure 5 shown.

[0184] The mRNA binding efficiency was measured by Quant-iT RiboGreen RNA Quantification Kit, and the results were shown in Figure 6 shown.

[0185] Figure 5The figures are the characterization results of the physicochemical properties of LNPs in the examples of the present invention, (A) particle size of typical PIL LNPs (mean ± standard deviation, n = 3); (B) Zeta potential of typical PIL LNPs (mean ± standard deviation, n = 3); (C) lipid fusion efficiency between PIL LNPs and in vivo mimics (mean ± standard deviation, n = 3); (D) lipid fusion efficiency of representative CPA4-Ac8-1 LNPs and CPA6-EP12 LNPs at different time intervals (mean ± standard deviation, n = 3).

[0186] Figure 6 This is a graph showing the binding efficiency of LNPs and mRNA in an example of the present invention (mean ± standard deviation, n = 3).

[0187] according to Figure 5 (A) Figure 5 (B) and Figure 6 It can be seen that the particle size of the multiple PIL LNPs prepared by the present invention ranges from 100 nanometers to 150 nanometers, which is suitable for further in vivo application; unlike the cationic polymer-mRNA polymers that usually exhibit high zeta potential, the PIL LNPs prepared by the present invention are usually negatively charged, and the negatively charged property helps to avoid adsorption and aggregation with proteins in the blood, so it has better stability in serum. Moreover, although the PIL LNPs have less positive charge than the cationic polymer-mRNA polymers, they effectively encapsulate the negatively charged mRNA, and the binding rate with the mRNA will not be affected by the negative charge of the prepared PIL LNPs.

[0188] according to Figure 5 (C) and Figure 5 (D) It can be seen that fluorescence resonance energy transfer (FRET) analysis was used to detect the endosomal escape properties of LNPs. The PIL LNPs were mixed with in vivo mimics and the fluorescence signal changes of the FRET probe were detected to determine the degree of membrane fusion and endosomal rupture. Representative LNPs mediated efficient endosomal rupture, indicating that they have great potential in promoting endosomal escape. These physicochemical properties highlight the application prospects of PIL LNPs in efficient mRNA delivery.

[0189] Example 4 Lipid Nanoparticles (LNPs) Prepared from PIL to Deliver mRNA in Vitro

[0190] The 198 PILs prepared by the preparation method of the present invention were used to prepare 198 PIL LNPs respectively by the same method as in Example 3.

[0191] General method for in vitro delivery of mRNA detection: Ovarian cancer cells IGROV1 were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution in an environment of 37°C and 5% carbon dioxide.

[0192] IGROV1 cells were plated at 1.5 × 10 4 The cells were inoculated into a white 96-well plate at a density of 1.50 μg / mL. Each sample was cultured three times. After 24 hours of culture, the culture medium containing metabolic waste was discarded and 150 μl of new culture medium containing 10% FBS was added to each well. Then 25 μl of the PIL LNPs mixed preparation was added to each well, using 25 ng of mRNA per well. The cell culture plate was cultured in the incubator for another 24 hours. TM II Firefly Luciferase Assay Kit was used to measure luciferase expression, and Alamar Blue Cell Viability Assay Kit was used to determine cell viability.

[0193] The results of 198 PIL LNPs testing are as follows Figure 7 shown.

[0194] Figure 7 The figures are the results of in vitro mRNA delivery of 198 PIL LNPs in Example 1 of the present invention; (A) Heat map of PIL LNPs delivering Fluc mRNA containing luciferase in IGROV1 cells (25ng mRNA, n=3, unit is relative light RLU); (B) Comparison of relative hit rates of PIL lipids prepared by Acn and EPm; (C) Relative hit rates of PIL lipids with different amine core L1 structures; (D) Relative hit rates of PILs prepared by Acn with different R2s; (E) Relative hit rates of PILs prepared by EPm with different R2s.

[0195] according to Figure 7 It can be seen that all 198 PIL LNPs showed delivery effects in in vitro mRNA delivery, especially when the L1 structure was A3-A7, which can be understood as the medium alkyl length of the amine core is better than the amine core with shorter and longer carbon chain lengths. The R2 structure was more efficient when selected from C8-C14, which can be understood as the tail chain of the PIL showed better mRNA delivery performance when it had a medium tail chain length of about 10 carbon atoms and had shorter and longer tail chain lengths.

[0196] Figure 8 The representative cell viability graph of PIL LNPs in IGROV1 cells of the present invention example (mean ± standard deviation, n = 3).

[0197] according to Figure 8It can be seen that the various PIL LNPs prepared by the present invention still show high activity in IGROV1 cells after delivering mRNA. Therefore, the PIL LNPs prepared by the present invention have high safety and negligible toxicity.

[0198] Comparative Example 1: In vitro delivery of mRNA by SM-102LNPS

[0199] The same experimental method and test method as in Example 4 were used to detect the in vitro mRNA delivery efficiency of LNPsSM-102 used in the mRNA vaccine, and the results were as follows: Fig. 9 shown.

[0200] Fig. 9 This is a comparison chart of the mRNA delivery efficiency of PIL LNPs prepared in the present invention and SM-102 in comparative example 1 in vitro. Statistical significance was calculated by one-way ANOVA and Dunnett's multiple comparison test: *P<0.05.

[0201] according to Fig. 9 It can be seen that the in vitro mRNA delivery efficiency of the PIL LNPs prepared by the present invention is much higher than that of the LNPs SM-102 used in the COVID-19 mRNA vaccine.

[0202] Example 5 In vivo delivery of mRNA by lipid nanoparticles (LNPs) prepared from PIL

[0203] All mouse experiments complied with ethical requirements.

[0204] The prepared LNPs were diluted with 1× PBS buffer and then injected intravenously (IV) into 6-8 week old C57BL / 6 mice at a Fluc mRNA dose of 0.25 mg / kg 1 hour later. After 6 hours, the mice were anesthetized under isoflurane anesthesia and then intraperitoneally injected with 100 μL of D-luciferin potassium solution (GoldBio, 30 mg / mL). After 3-5 minutes, bioluminescence was measured using a live imaging system (PerkinElmer). Afterwards, the mice were sacrificed, and the main organs were isolated and imaged using the same method. The results are shown in Figure 2. Fig.10 shown.

[0205] Fig.10These are the results of in vivo mRNA delivery of 44 PIL LNPs in the examples of the present invention; (A) the results of in vivo luminescence imaging of mRNA delivery in C57BL / 6 mice by IV administration of 44 PIL LNPs; (B) the results of the average (ave) radiation brightness of the quantitative expression of Fluc protein of PIL lipids with different amine core L1 structures; (C) the mRNA delivery efficiency of CPA4-Ac8-1 LNPs in different organs (mean ± standard deviation, n = 3); (D) the comparison of mRNA translation of CPA6-EP12LNPs in different organs (mean ± standard deviation, n = 3).

[0206] according to Fig.10 It can be seen that all 44 PIL LNPs showed good in vivo delivery effects, wherein preferably, the amine core L1 structure played a key role in the final delivery efficiency, the alkyl length affected the expression of mRNA, and the carbon chain was too short or too long, which would reduce the translation of mRNA. The amine core L1 structure with a preferred length of 3 to 7 carbon atoms was conducive to the delivery of mRNA in vivo, and different R2 structures, i.e., tail chains with different lengths, had different delivery efficiencies, and preferably the length of 8 to 14 carbon atoms was superior to other lengths. At the same time, the PIL LNPs prepared by the present invention mediated targeted mRNA expression in the liver, and compared with other organs (such as heart, lung, spleen and kidney), the efficiency was higher and had certain organ selectivity.

[0207] Example 6 In vivo delivery of mRNA by PIL with four tail strands

[0208] The same method as in Example 5 was used to detect CPA6-EP12 and a structure similar to CPA6-EP12 but having four hydrophobic tail chains as shown in the following formula (V) for in vivo delivery mRNA detection. The results are as follows Fig.11 shown.

[0209] Formula (V):

[0210]

[0211] Fig.11 This is a comparison diagram of the mRNA delivery results of CPA6-EP12 and four-stranded PIL in vivo according to the present invention.

[0212] according to Fig.11 It can be seen that the PIL with three tail chains CPA6-EP12, i.e., three R2 groups, and the PIL with four tail chains in the embodiment of the present invention can both deliver mRNA in vivo, but the efficiency of the three tail chains is better than that of the four tail chain PIL lipid.

[0213] Comparative Example 3 DLin-MC3-DMA (MC3) and ALC-0315 in vivo delivery of mRNA DLin-MC3-DMA (MC3) is a lipid for delivery of Onpattro (siRNA drug), and its structure is shown in the following formula (VI-1):

[0214]

[0215] The same detection method as in Example 5 was used to test the mRNA delivery results in vivo. Fig.12 shown.

[0216] ALC-0315 is a common LNP small molecule available on the market, and its structure is shown in the following formula (VI-2):

[0217]

[0218] Fig.12 Comparison diagram of mRNA delivery in liposomes of Example 4 of the present invention. (A) Chemical structure diagram of CPA4-Ac8-1 and CPA6-EP12 lipids; (B) Comparison diagram of in vivo imaging of CPA4-Ac8-1 LNPs and CPA6-EP12 LNPs after intravenous administration; (C) Comparison diagram of in vivo imaging of CPA4-Ac8-1 LNPs, CPA6-EP12 LNPs, SM-102 and ALC-0315 after intramuscular administration.

[0219] according to Fig.12 It can be seen that the mRNA delivery efficiency of CPA4-Ac8-1 LNPs and CPA6-EP12 LNPs prepared by the present invention after in vivo administration is higher than that of commercially available LNPs DLin-MC3-DMA and ALC-0315, showing higher mRNA delivery efficiency in vivo.

Claims

1. A cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof, characterized in that: The cationic lipid compound has a structure shown in formula (I): Wherein, x and y are each selected from positive integers between 0 and 2, and x+y≥1; R1 is selected from H, substituted or unsubstituted C1-C10 alkyl; R2 is each selected from a linear or branched C1-C30 saturated or unsaturated hydrocarbon group; R3 is independently selected from H, substituted or unsubstituted C1-C20 alkyl, wherein R3 is connected to any of the connectable positions 1-6; L1 is independently selected from a linear or branched substituted or unsubstituted C0-C30 saturated or unsaturated alkylene group; L2 represents a linking group for connecting R2 and the N atom; Wherein, when the substituted or unsubstituted group has a substituent, the substituent is selected from one or more combinations of C1-C10 alkyl, C3-C7 cycloalkyl, hydroxyl, ester, amino, acyl, ether, carboxyl and thiol.

2. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, characterized in that: The cationic lipid compound has a structure shown in formula (I-1):

3. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1 or 2, characterized in that: The cationic lipid compound has a structure shown in formula (I-2): The structure shown in formula (I-2):

4. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, 2 or 3, characterized in that: L2 is selected from H, substituted or unsubstituted C1-C30 alkylene, or substituted or unsubstituted C2-C30 alkylene ester; Preferably, L2 is selected from one of the following formula (II-1) or formula (II-2): Wherein, in formula (II-1), position 1 is connected to the N atom, and position 4 is connected to R2; Among them, in formula (II-2), position 1 is connected to the N atom, and position 2 is connected to R2.

5. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, 2 or 3, characterized in that: R1 is selected from H, C1-C6 alkyl; More preferably, it is selected from H, methyl and ethyl.

6. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, 2 or 3, characterized in that: L1 is selected from C1-C12 linear or branched saturated or unsaturated hydrocarbon groups, preferably C3-C7 linear or branched saturated or unsaturated hydrocarbon groups.

7. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to any one of claims 1, 2, 3 and 4, characterized in that: R2 is selected from any of the following structures:

8. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, 2 or 3, characterized in that: L1 is selected from any of the following structures:

9. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof according to claim 1, 2 or 3, characterized in that: The cationic lipid compound is selected from the following compounds:

10. A method for preparing a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof as claimed in any one of claims 1 to 9, characterized in that: The following steps are involved: The compound represented by formula (1) is reacted with a reducing agent and carbon dioxide to obtain an intermediate represented by formula (2); The compound represented by formula (2) is reacted with the compound represented by formula (3) to obtain the intermediate represented by formula (4); The intermediate represented by formula (4) is reacted with Acn or EPm to obtain a cationic lipid compound represented by formula (III-1) or formula (III-2); or 11. The preparation method according to claim 10, wherein: Acn includes at least one of the following structures: EPm includes at least one of the following structures:

12. A composition, characterized in that The composition comprises the cationic lipid compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

13. The composition according to claim 12, characterized in that The composition may also include a prophylactic and / or therapeutic agent.

14. The composition according to claim 13, characterized in that The preventive or therapeutic agent is a nucleic acid, a polypeptide, a protein or a protein-like compound.

15. The composition according to claim 12, characterized in that The preventive agent includes the cationic lipid compound, a stabilizer for stabilizing the structure of the composition, and a filler for filling the structure of the composition.

16. The composition according to claim 15, characterized in that The molar ratio of the cationic lipid compound to the stabilizer and filler in the preventive agent is 1:(2-5).

17. The composition according to claim 15, characterized in that The stabilizer includes PEGylated lipids, and the filler includes neutral phospholipids and / or steroidal lipids.

18. The composition according to claim 14, characterized in that: The nucleic acid is selected from one or more of single-stranded DNA, double-stranded DNA, small interfering RNA, micro RNA, Dicer-substrate RNA, messenger RNA, small hairpin RNA, and asymmetric interfering RNA; The protein or protein-like compound is selected from one or more of gentamicin, amikacin, insulin, erythropoietin, granulocyte colony stimulating factor, interferon, and hepatitis B surface antigen.

19. The composition according to claim 17, characterized in that: The neutral phospholipid is selected from diarachidoylphosphatidylcholine, 1,2-behenoyl-phosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, 1-stearoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, One or more of dilauroylphosphatidylethanolamine, distearoylphosphatidic acid sodium salt, dipalmitoylphosphatidic acid sodium salt, dimyristoylphosphatidic acid sodium salt, dioleoylphosphatidic acid sodium salt, dilauroylphosphatidic acid sodium salt, distearoylphosphatidylglycerol sodium salt, dipalmitoylphosphatidylglycerol sodium salt, dimyristoylphosphatidylglycerol sodium salt, dioleoylphosphatidylglycerol sodium salt, dilauroylphosphatidylglycerol sodium salt, distearoylphosphatidylserine sodium salt, dipalmitoylphosphatidylserine sodium salt, dimyristoylphosphatidylserine sodium salt, dioleoylphosphatidylserine sodium salt, dilauroylphosphatidylserine sodium salt; The steroidal lipid is selected from one or more of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, and deoxycholic acid; The PEGylated lipid is selected from one or more of distearoylphosphatidylethanolamine polyethylene glycol, dimyristoylglycerol-rac-methoxy polyethylene glycol 2000, methoxy polyethylene glycol ditetradecyl acetamide, polyethylene glycol-diacylglyceramide, polyethylene glycol-dipalmitoylphosphatidylethanolamine, polyethylene glycol-disterol glycerol, and polyethylene glycol-diacylglyceramide.

20. The composition according to claim 17, characterized in that The molar ratio of the cationic lipid compound to the neutral phospholipid, steroidal lipid and pegylated lipid is (10-20):(15-25):(20-30):(0.5-3.0), preferably 15:20:25:

2.

21. Use of the cationic lipid compound or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer as claimed in any one of claims 1 to 9 or the composition as claimed in any one of claims 12 to 20 in the preparation of a nucleic acid, polypeptide or protein delivery drug.

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