Compound, liposome and use thereof

Abstract

The present invention provides a compound, a liposome, a method for preparing a liposome, a drug carrier, a drug composition, and the use of the drug composition in treating or preventing hyperlipidemia and related diseases. The compound has a structure shown in formula (I), and can be used as a liposome and a drug carrier, can load a negatively charged active pharmaceutical ingredient, and deliver the active pharmaceutical ingredient to the cell and effectively escape from the endosome to the cytoplasm, which is beneficial for the loaded active pharmaceutical ingredient to exert its efficacy, and has the advantages of low toxicity and high stability.

Description

[0001] Priority information

[0002] This application claims priority to the PCT patent application with an application date of May 20, 2021 and international application number PCT / CN2021 / 094937, and incorporates its entire contents into this application by reference. Technical Field

[0003] The present invention belongs to the field of biomedicine, and specifically relates to compounds, liposomes, drug carriers and their uses, methods for preparing liposomes, pharmaceutical compositions and uses. Background Art

[0004] Nucleic acid molecules, such as siRNA and mRNA, are very promising drug molecules that need to enter cells to exert their effects. However, nucleic acid molecules themselves are large molecular weight, highly negatively charged, and easily degradable compounds that cannot enter cells by themselves. Therefore, a suitable delivery vector is needed to carry them into cells or tissues in the body.

[0005] Liposomes have been proven to be an effective nucleic acid carrier, and the siRNA drug Onpattro encapsulated by liposomes has been launched on the market. With the progress of research on nucleic acid drugs, especially siRNA drugs and mRNA drugs, there is a continuous demand for research on nucleic acid drug carriers, including research on liposomes targeting different cell types and tissue types, better protection of nucleic acids from enzyme degradation, or better release of liposomes in cells, and liposomes with lower toxicity and easy biodegradation.

[0006] Therefore, it is necessary to develop a liposome with high stability, low toxicity and easy biodegradability. Summary of the invention

[0007] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0008] To this end, in the first aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (I),

[0009]

[0010] Wherein, X, Y, R1, R2, and R3 have the same definitions as those described in the present invention.

[0011] According to an embodiment of the present invention, each X and Y is independently selected from -CH2- or -CO-; X and Y are not simultaneously -CH2- or -CO-, wherein each X is the same or different, and each Y is the same or different; R2 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl or an optionally substituted C1-C20 alkynyl; R3 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl or an optionally substituted C1-C20 alkynyl, wherein the substituent groups are independently selected from H, F, Cl, Br, CN, NO2; R1 is selected from wherein n is an integer selected from 0 to 10, and Z is selected from R4 is selected from an optionally substituted C4-C20 alkyl, an optionally substituted C4-C20 alkenyl or an optionally substituted C4-C20 alkynyl, wherein the substituents are each independently selected from H, F, Cl, Br, CN, NO2.

[0012] According to an embodiment of the present invention, R2 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl, an optionally substituted C1-C20 alkynyl or R1, and R3 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl, an optionally substituted C1-C20 alkynyl or R1, wherein the substituted groups are each independently selected from H, F, Cl, Br, CN, and NO2.

[0013] According to an embodiment of the present invention, R2 is selected from H, CH3- or R1; R3 is selected from H or R1; R1 is selected from wherein n is an integer selected from 1 to 5, and Z is selected from R4 is selected from C4-C20 alkyl, C4-C20 alkenyl or C4-C20 alkynyl, wherein the C4-C20 alkenyl contains 1-3 double bonds, and the C4-C20 alkynyl contains 1-3 triple bonds.

[0014] According to an embodiment of the present invention, R4 is selected from C5-C15 alkyl and C5-C15 alkenyl, wherein the C5-C15 alkenyl contains 1-3 double bonds.

[0015] According to an embodiment of the present invention, R1, R2 and R3 are the same; R4 is selected from C10-C15 alkyl and C10-C15 alkenyl, wherein the C10-C15 alkenyl contains 1-3 double bonds.

[0016] According to an embodiment of the present invention, the compound has one of the following structures:

[0017]

[0018] According to an embodiment of the present invention, the compound skeleton comprises a backbone structure of 4 amine groups and a plurality of branched alkyl or substituted alkyl structures. On the backbone chain, each amine group is connected by an alkyl or acyl group, wherein at least two acyl groups are present, which reduces the alkalinity of the compound, reduces the chemical stability effect of the ester group contained in the branched chain by the alkalinity of the alkylamine group, and reduces the positive charge of the compound, which is beneficial to reduce the positive charge of the liposome, and at the same time, the amide bond enhances the metabolizability in vivo, avoiding the accumulation of the compound in vivo. In addition, the inventors have found through a large number of experiments that if the backbone structure contains four amide groups, that is, there is no alkylamino group in the backbone structure, it will cause its electrical properties to be greatly reduced, so that its ability to bind to nucleic acids is weakened, and the nucleic acid load capacity is deteriorated.

[0019] In the second aspect of the present invention, the present invention proposes the use of the above compound in preparing liposomes. According to an embodiment of the present invention, the above compound is an amine-containing lipid compound, has ionizable properties, and can be used to prepare liposomes.

[0020] According to an embodiment of the present invention, the above-mentioned compound can be used to prepare liposomes in the form of a polymer; the above-mentioned compound can be covalently linked with other substances to prepare liposomes; the above-mentioned compound can react chemically with other substances to prepare liposomes. According to an embodiment of the present invention, the specific method of preparing liposomes with the above-mentioned compound is not limited. The use of the above-mentioned compound to prepare liposomes, and the liposomes containing all or part of the structure of the above-mentioned compound are regarded as the use of the present invention.

[0021] In the third aspect of the present invention, the present invention provides a liposome. The liposome according to the embodiment of the present invention is made of the compound provided in the first aspect of the present invention, and the liposome can encapsulate the added substance to be loaded through the flow effect of the lipid bilayer, and the encapsulation effect is good, so that the loaded substance can effectively enter the cell and escape from the endosome to the cytoplasm.

[0022] According to an embodiment of the present invention, the above compound can form liposomes by self-assembly with hydrophobic lipids and / or amphiphilic lipids.

[0023] According to an embodiment of the present invention, the liposome further includes hydrophobic lipids, amphiphilic lipids, a buffer agent and / or an organic solvent, the amphiphilic lipids include at least one selected from neutral lipids, cationic lipids and PEG-lipids, and the hydrophobic lipids include sterols.

[0024] According to an embodiment of the present invention, the neutral lipid includes at least one selected from the following: distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EPC).

[0025] According to an embodiment of the present invention, the cationic lipid is selected from at least one of the following: trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), didodecyl dimethyl ammonium bromide (DDAB), trifluoroacetic acid dimethyl-2,3-dioleyloxypropyl-2-(2-spermine formamido)ethylammonium (DOSPA), trimethyl dodecyl ammonium bromide (DTAB), trimethyl tetradecyl ammonium bromide (TTAB), trimethyl hexadecyl ammonium bromide (CTA B), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2,3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2,3- Dihexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE), N-(2-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), lipid poly-L-lysine (LPLL), stearyl ammonium (SA).

[0026] According to an embodiment of the present invention, the PEG-lipid includes at least one selected from the following: dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), distearoylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol-PEG (DMG-PEG) and dimethacrylate-PEG (DMA-PEG).

[0027] According to an embodiment of the present invention, the sterol is cholesterol.

[0028] According to an embodiment of the present invention, the buffer reagent is an acidic buffer reagent.

[0029] According to an embodiment of the present invention, the buffer reagent includes at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tris(hydroxymethylaminomethane)-hydrochloric acid, potassium dihydrogen phosphate-sodium hydroxide, boric acid-borax, glycine-hydrochloric acid, phthalic acid-hydrochloric acid, potassium hydrogen phthalate and sodium dihydrogen phosphate-citric acid.

[0030] According to an embodiment of the present invention, the organic solvent includes at least one selected from methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, ethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.

[0031] According to an embodiment of the present invention, the amount of the compound is 3-80% mol / mol based on the liposome.

[0032] According to an embodiment of the present invention, the amount of the compound is 20-80% mol / mol based on the liposome.

[0033] According to an embodiment of the present invention, the molar ratio of the compound, the neutral lipid, the sterol and the PEG-lipid is (20-80): (5-50): (10-60): (0.2-20).

[0034] According to an embodiment of the present invention, the molar ratio of the compound, the neutral lipid, the sterol and the PEG-lipid is (40-61): (8-20): (30-60): (0.5-5).

[0035] According to an embodiment of the present invention, the molar ratio of the compound, sterol and PEG-lipid is (20-80):(5-50):(10-60).

[0036] According to an embodiment of the present invention, the molar ratio of the compound, sterol and PEG-lipid is (40-70): (5-20): (20-40).

[0037] According to an embodiment of the present invention, the amount of the compound is 3-50% mol / mol based on the liposome.

[0038] According to an embodiment of the present invention, the molar ratio of the compound, the neutral lipid, the sterol, the PEG-lipid and the cationic lipid is (3-50): (1-20): (2-50): (0.01-10): (5-92).

[0039] According to an embodiment of the present invention, the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-41): (1-12): (2-45): (0.05-2): (10-90).

[0040] According to an embodiment of the present invention, the volume of the buffer reagent is 50-90% v / v based on the liposome.

[0041] According to an embodiment of the present invention, the volume of the buffer reagent is 75% v / v based on the liposome.

[0042] According to the embodiments of the present invention, the liposomes with the above ratio are used, which has high transfection efficiency, good encapsulation effect and strong drug presentation ability.

[0043] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.2-20); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0044] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, sterol and PEG-lipid is (20-80):(5-50):(10-60); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0045] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 3-50% mol / mol; the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-50):(1-20):(2-50):(0.01-10):(5-92); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0046] In a fourth aspect of the present invention, the present invention provides a method for preparing the above liposome. According to an embodiment of the present invention, the method comprises: mixing a predetermined amount of the compound provided in the first aspect of the present invention with an organic solvent to obtain the liposome. According to an embodiment of the present invention, dissolving the compound in an organic solvent facilitates the formation of the liposome.

[0047] According to an embodiment of the present invention, the method further comprises: dissolving a predetermined amount of lipid in the above-mentioned organic solution and mixing it with a buffer reagent to obtain the liposome. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80): (5-50): (10-60): (0.2-20); based on the liposome, the volume of the buffer reagent is 75% v / v. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, sterol and PEG-lipid is (20-80): (5-50): (10-60); based on the liposome, the volume of the buffer reagent is 75% v / v. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 4-50% mol / mol; the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-50): (1-20): (2-50): (0.01-10): (5-92); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0048] In the fifth aspect of the present invention, the present invention provides a drug carrier, including the compound provided in the first aspect of the present invention or the liposome provided in the third aspect of the present invention. According to an embodiment of the present invention, the compound is an ionizable amine-containing lipid, and the drug carrier is an ionizable carrier, which can carry negatively charged or hydrophobic drugs, and deliver the drugs into cells and effectively escape from the endosomes to the cytoplasm, which is beneficial to the efficacy of the loaded drugs.

[0049] In the sixth aspect of the present invention, the present invention proposes the use of the compound proposed in the first aspect of the present invention, the liposome proposed in the third aspect of the present invention or the drug carrier proposed in the fifth aspect of the present invention in the preparation of drugs.

[0050] In the seventh aspect of the present invention, the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising a carrier and a pharmaceutically active ingredient, wherein the carrier comprises the pharmaceutical carrier provided in the fifth aspect of the present invention. According to an embodiment of the present invention, the pharmaceutical carrier is an ionizable carrier, which can carry a negatively charged or hydrophobic pharmaceutically active ingredient, and deliver the pharmaceutically active ingredient into the cell and effectively escape from the endosome to the cytoplasm, which is beneficial for the loaded pharmaceutically active ingredient to exert its efficacy.

[0051] According to an embodiment of the present invention, the active pharmaceutical ingredient includes at least one selected from the following: a DNA molecule, an RNA molecule, a protein, a polypeptide and a small molecule drug.

[0052] According to an embodiment of the present invention, the active pharmaceutical ingredient is negatively charged or hydrophobic. According to an embodiment of the present invention, the drug carrier contains ionizable amine-containing lipids, which are positively charged at a specific pH and can be combined with the negatively charged active pharmaceutical ingredient. In addition, the active pharmaceutical ingredient can be encapsulated by the flow of the lipid bilayer and delivered to the cell.

[0053] According to an embodiment of the present invention, the active pharmaceutical ingredient contains nucleic acid. According to an embodiment of the present invention, the active pharmaceutical ingredient contains DNA, RNA, nucleic acid-protein complex, nucleic acid-lipid complex, nucleic acid-nuclide complex, etc., and the type of nucleic acid is not particularly limited.

[0054] According to an embodiment of the present invention, the mass ratio of the carrier to the active pharmaceutical ingredient is (5-50):1; preferably, the mass ratio of the carrier to the active pharmaceutical ingredient is (5-40):1.

[0055] According to an embodiment of the present invention, the mass ratio of the carrier to the active ingredient of the medicine is (8-20):1.

[0056] According to an embodiment of the present invention, the mass ratio of the carrier to the active pharmaceutical ingredient is 15:1.

[0057] In the eighth aspect of the present invention, the present invention provides a transfection complex, characterized in that the transfection complex comprises the above-mentioned compound or the above-mentioned liposome.

[0058] According to an embodiment of the present invention, the transfection complex comprises at least one bioactive agent.

[0059] According to an embodiment of the present invention, the bioactive agent includes at least one selected from the following: a DNA molecule, an RNA molecule, a protein, a polypeptide and a drug.

[0060] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0062] Figure 1 is the gel retardation test result according to an embodiment of the present invention;

[0063] Figure 2 is the stability result of the preparation of siA4-13 according to the example of the present invention at 4°C;

[0064] Figure 3is the quantitative data of the distribution of Cy-siFLA4-13 in C57BL / 6 mice according to an embodiment of the present invention;

[0065] Figure 4 It is based on the changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels at different time points after animals receive different concentrations of siA4-13 preparations according to the examples of the present invention, wherein Figure a shows the changes in ApoB mRNA expression at different time points after animals receive 1 mg / kg or 3 mg / kg siA4-13 preparations, and Figure b shows the changes in CHO, TG, LDL-c, and HDL-c levels;

[0066] Figure 5 is the change of serum biochemical indexes according to the embodiment of the present invention;

[0067] Figure 6 The lipid-lowering effect and ANGPTL3 mRNA expression inhibition ability of siANA4-13 in db / db mice according to the examples of the present invention;

[0068] Figure 7 The lipid deposition in the liver of animals in different treatment groups according to the embodiments of the present invention;

[0069] Figure 8 The lipid-lowering effect and lipid changes of siApoCA4-13 treatment according to the embodiment of the present invention and within 4 weeks of drug withdrawal;

[0070] Fig. 9 Results of luciferase mRNA expression on cells mediated by liposomes with different formulations according to the embodiments of the present invention;

[0071] Fig.10 The expression results of luciferase mRNA in mice mediated by mLuA4-13 according to the embodiment of the present invention;

[0072] Fig.11 The expression of mLuA4-13 and mLuDA4-1 to mLuDA4-9 in mouse lungs, livers and kidneys according to the embodiments of the present invention;

[0073] Fig.12 Comparison of the cell killing ability of siGA4-1 to siGA4-9 obtained by encapsulating GOx proteins in different proportions with the same amount of GOx alone according to the example of the present invention;

[0074] Fig.13 is the chemical structure of lipid E;

[0075] Fig.14 The stability test results of lipid A4 and lipid E ​​at 40 degrees Celsius according to the embodiments of the present invention;

[0076] Fig.15 The figure is a comparison of the efficacy of A4-13 and Lipofactamine 2000 in C57 mice according to an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0077] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0078] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0079] Certain embodiments of the present invention are now described in detail, examples of which are illustrated by the accompanying structural formula and chemical formula. The present invention is intended to cover all substitutions, modifications and equivalent technical solutions, which are all included in the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described in the present invention can be used to practice the present invention. The present invention is by no means limited to the methods and materials described in the present invention. In the event that one or more of the combined documents, patents and similar materials are different from or contradictory to the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0080] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0081] Unless otherwise noted, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. Unless otherwise noted, all patent publications cited in the entire disclosure of the present invention are incorporated herein by reference in their entirety.

[0082] The following definitions shall apply to the present invention unless otherwise indicated. For the purposes of the present invention, chemical elements are defined according to the Periodic Table of the Elements, CAS version and Handbook of Chemicals, 75, th Ed, 1994. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, and all references are incorporated herein by reference.

[0083] In this article, "liposome" or "lipid nanoparticle (LNP)" refers to a drug delivery system that uses a biocompatible lipid material as a carrier to dissolve or encapsulate drugs or other biologically active substances in the lipid core or adsorb or attach them to the surface of the nanoparticle.

[0084] The present invention also includes isotopically labeled compounds of the present invention which are identical to those described herein except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Exemplary isotopes that may also be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H. 3 H. 13 C. 14 C. 15 N. 16 O. 17 O. 31 P. 32 P. 36 S. 18 F and 37 Cl.

[0085] Compounds of the invention containing the aforementioned isotopes and / or other isotopes of other atoms and pharmaceutically acceptable salts of the compounds are included within the scope of the invention. Isotopically labeled compounds of the invention, for example radioactive isotopes, such as 3 H and 14 C is incorporated into the compounds of the present invention for drug and / or substrate tissue distribution analysis. Due to the ease of preparation and detection, tritiated, i.e. 3 H, and carbon-14, i.e. 14 C, isotopes are particularly preferred. In addition, heavy isotopes, such as deuterium, 2H substitution may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0086] The stereochemical definitions and conventions used herein are generally in accordance with SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof such as racemic mixtures, are also included within the scope of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to the chiral center (or multiple chiral centers) in the molecule. The prefixes d and l or (+) and (-) are the signs used to designate the rotation of plane polarized light caused by a compound, where (-) or l indicates that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. With respect to a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and a mixture of such isomers is often referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0087] Depending on the choice of starting materials and processes, the compounds of the invention may exist in the form of one of the possible isomers or a mixture thereof, for example as pure optical isomers, or as isomer mixtures, such as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)-isomers may be prepared using chiral synthons or chiral agents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis- or trans- configuration.

[0088] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

[0089] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomers, diastereomeric atropisomers, and geometric (or conformational)) forms of such structures; for example, R and S configurations at various asymmetric centers, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric isomer (or conformational) mixtures of the compounds of the invention are within the scope of the invention.

[0090] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be mutually converted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be reached. For example, proton tautomers (protontautomers) (also known as prototropic tautomers) include mutual conversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include mutual conversions by the reorganization of some bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one tautomers. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is the interconversion of pyridine-4-ol and pyridine-4 (1H)-one tautomers. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0091] As used herein, "nitrogen oxide" means that when a compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide. Special examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms of nitrogen-containing heterocyclic rings. The corresponding amines can be treated with oxidizing agents such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) to form N-oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), wherein, for example, in an inert solvent such as dichloromethane, an amine compound is reacted with meta-chloroperbenzoic acid (MCPBA).

[0092] The "solvate" of the present invention refers to an association formed by one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol. The term "hydrate" refers to an association formed when the solvent molecule is water.

[0093] The "pharmaceutically acceptable salt" used in the present invention refers to the organic salt and inorganic salt of the compound of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts formed by non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates) formed by reaction with amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates), or other methods described in books and literature, such as ion exchange methods, to obtain these salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. The present invention also contemplates quaternary ammonium salts formed by compounds of any group containing N. Water-soluble or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter-balancing ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates and aromatic sulfonates.

[0094] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the invention may exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)-configuration. Substituents on atoms with unsaturated double bonds may exist in cis-(Z)- or trans-(E)-forms, if possible.

[0095] Therefore, as described herein, the compounds of the present invention may exist in the form of one or a mixture of possible isomers, rotamers, atropisomers, tautomers, for example, in the form of substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0096] Any resulting mixtures of isomers can be separated on the basis of the physicochemical differences of the components into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0097] Any racemate of the final product or intermediate obtained can be separated into optical antipodes by known methods by methods familiar to those skilled in the art, such as by separation of the diastereoisomeric salts obtained thereof. The racemic product can also be separated by chiral chromatography, such as high pressure liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers can be prepared by asymmetric synthesis (e.g., Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0098] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as the general formula compounds above, or as the specific examples, subclasses, and classes of compounds included in the embodiments. It should be understood that the term "optionally substituted" can be used interchangeably with the term "substituted or unsubstituted". The term "optionally", "optional" or "optionally" means that the event or situation described below may but may not occur, and the description includes situations in which the event or situation occurs, as well as situations in which the event or situation does not occur. In general, the term "optionally", whether or not it is preceded by the term "substituted", means that one or more hydrogen atoms in the given structure are replaced by a specific substituent. Unless otherwise indicated, an optional substituent group can be substituted at each substitutable position of the group. When more than one position in the given structural formula can be substituted with one or more substituents selected from a specific group, the substituents can be substituted at each position in the same or different manner. The substituents described herein can be, but are not limited to, F, Cl, Br, CN, N3, OH, NH2, NO2, oxo (=O), etc.

[0099] The term "N3" refers to an azide structure. This group can be linked to other groups, for example, to a methyl group to form methyl azide (MeN3), or to a phenyl group to form phenyl azide (PhN3).

[0100] In addition, it should be noted that, unless explicitly stated otherwise, the description methods used in the present invention, "each... is independently" and "... are each independently" and "... are independently" can be interchanged and should be understood in a broad sense, which can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0101] In various parts of this specification, the substituents of the disclosed compounds of the present invention are disclosed according to group types or ranges. It is particularly pointed out that the present invention includes each independent secondary combination of the individual members of these group types and ranges. For example, the term "C1-6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.

[0102] In various parts of the present invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood as a linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents an alkylene group or an arylene group, respectively, that is linked.

[0103] The term "alkyl" or "alkyl group" used in the present invention refers to a saturated straight or branched monovalent hydrocarbon radical containing 1 to 20 carbon atoms. Unless otherwise specified, the alkyl group contains 1 to 20 carbon atoms, in some embodiments, the alkyl group contains 1 to 10 carbon atoms, in other embodiments, the alkyl group contains 1 to 9 carbon atoms; in other embodiments, the alkyl group contains 1 to 8 carbon atoms, in other embodiments, the alkyl group contains 1 to 6 carbon atoms, in other embodiments, the alkyl group contains 1 to 4 carbon atoms, in other embodiments, the alkyl group contains 1 to 3 carbon atoms, in other embodiments, the alkyl group contains 10 to 20 carbon atoms, in other embodiments, the alkyl group contains 10 to 18 carbon atoms, in other embodiments, the alkyl group contains 10 to 16 carbon atoms, in other embodiments, the alkyl group contains 10 to 14 carbon atoms, in other embodiments, the alkyl group contains 10 to 13 carbon atoms.

[0104] Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like, wherein the alkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.

[0105] As used herein, the term "alkyl" and its prefix "alkane" include both straight and branched saturated carbon chains.

[0106] The term "alkenyl" refers to a straight or branched monovalent hydrocarbon group of 2-20 carbon atoms, or 2-18 carbon atoms, or 2-16 carbon atoms, or 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position CC is an sp2 double bond unsaturated state, wherein the alkenyl group can be independently unsubstituted or substituted with one or more substituents described in the present invention, including groups with "cis", "trans" or "Z" "E" orientation, wherein specific examples include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2) and the like.

[0107] The term "alkynyl" refers to a straight or branched monovalent hydrocarbon group of 2-20 carbon atoms, or 2-18 carbon atoms, or 2-16 carbon atoms, or 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position CC is in an sp triple bond unsaturated state, wherein the alkynyl group may be independently unsubstituted or substituted with one or more substituents described in the present invention, specific examples include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3) and the like.

[0108] The term “comprise” or “include” is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

[0109] As described in the present invention, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, adhesive, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or a combination thereof, which are known to those skilled in the art (such as Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical composition is covered.

[0110] Compound

[0111] In the first aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (I),

[0112]

[0113] Wherein, X, Y, R1, R2, and R3 have the same definitions as those described in the present invention.

[0114] In some embodiments, each X and Y are each independently selected from -CH2- or -CO-; X and Y are not simultaneously -CH2- or -CO-, wherein each X is the same or different, and each Y is the same or different; R2 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl, or an optionally substituted C1-C20 alkynyl; R3 is selected from H, an optionally substituted C1-C20 alkyl, an optionally substituted C1-C20 alkenyl, or an optionally substituted C1-C20 alkynyl, wherein the substituents are each independently selected from H, F, Cl, Br, CN, NO2; R1 is selected from wherein n is an integer selected from 0 to 10, and Z is selected from R4 is selected from an optionally substituted C4-C20 alkyl, an optionally substituted C4-C20 alkenyl or an optionally substituted C4-C20 alkynyl, wherein the substituents are each independently selected from H, F, Cl, Br, CN, NO2.

[0115] In some embodiments, R2 is selected from H, CH3- or R1; R3 is selected from H or R1; R1 is selected from wherein n is an integer selected from 1 to 5, and Z is selected from R4 is selected from C4-C20 alkyl, C4-C20 alkenyl or C4-C20 alkynyl, wherein the C4-C20 alkenyl contains 1-3 double bonds, and the C4-C20 alkynyl contains 1-3 triple bonds.

[0116] In some embodiments, R4 is selected from C5-C15 alkyl, C5-C15 alkenyl, wherein the C5-C15 alkenyl contains 1-3 double bonds.

[0117] In some embodiments, R1, R2 and R3 are the same; R4 is selected from C10-C15 alkyl, C10-C15 alkenyl, wherein the C10-C15 alkenyl contains 1-3 double bonds.

[0118] Furthermore, the compound has one of the following structures:

[0119]

[0120]

[0121] The compound described in the present invention refers to a compound having a hydrophilic head and a hydrophobic tail on a macromolecular chain. Wherein, the hydrophilic head can be N1, N4-bis(3-aminopropyl)succinamide or 3,3'-(butane-1,4-diylbis(methylazanediyl))dipropanamide or 3,3'-(butane-1,4-diylbis(methylazanediyl))dipropanamide. The hydrophobic tail can be a straight carbon chain with double bonds and / or ester bonds, and the total chain length can be 12, 14, 16, or 18 atoms. The compound of the present invention can be formed by one of the hydrophilic heads and one or more of the hydrophobic tails in a variety of ratios such as 1:1, 1:2, or 1:3.

[0122] In some embodiments, the hydrophobic tail has at least one of the following chemical structures:

[0123]

[0124] Among them, the positions of double bonds and ester bonds in Formula 1 can be changed, based on the convenient availability of raw materials, and the total chain length is set to 16 atoms or 18 atoms; the position of the ester bond in Formula 2 can be changed, based on the convenient availability of raw materials, and the total chain length is set to 14, 16 or 18 atoms; the chain length in Formula 3 can be changed, and can be 12, 14, 16, or 18 (among which 12 and 14 atoms are more preferred).

[0125] In some embodiments, the salts are pharmaceutically acceptable salts.The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0126] The compounds of the present invention also include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts and can be used as intermediates for preparing and / or purifying the compounds of the present invention and / or for separating the enantiomers of the compounds of the present invention.

[0127] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorophylline, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalactonate, propionate, stearate, succinate, sulfosalicylate, tartrate, toluenesulfonate and trifluoroacetate.

[0128] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0129] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, and the like.

[0130] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0131] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0132] Organic bases from which salts can be derived include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include, for example, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0133] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, the basic or acidic part by conventional chemical methods. In general, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (such as Na, Ca, Mg or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. Such reactions are usually carried out in water or an organic solvent or a mixture of the two. Generally, in appropriate cases, it is necessary to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. For example, "Remington's Pharmaceutical Sciences", 20th edition, Mack Publishing Company, Easton, Pa., (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) can be found in some other suitable salt lists.

[0134] Moreover, the compounds of the present invention, including their salts, may also be obtained in the form of their hydrates, or include other solvents for their crystallization. The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the present invention is intended to include solvated and unsolvated forms.

[0135] Any structural formula given herein is also intended to represent unlabeled forms of these compounds as well as isotopically labeled forms. Isotopically labeled compounds have structures depicted by the general formula given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Exemplary isotopes that can be introduced into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 36 S. 37 Cl or 125 I.

[0136] On the other hand, the compounds of the present invention include compounds defined in the present invention which are labeled with various isotopes, for example, wherein a radioactive isotope is present, such as 3 H. 14C and 18 Those compounds of F, or in which non-radioactive isotopes are present, such as 2 H and 13 C. This type of isotope-labeled compound can be used for metabolic studies (using 14 C), reaction kinetics studies (using e.g. 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution determination, or may be used in radiation therapy of the patient. 18 F-labeled compounds are particularly ideal for PET or SPECT studies. Isotope-labeled compounds of formula (I) can be prepared by conventional techniques familiar to those skilled in the art or as described in the examples and preparations herein by replacing the unlabeled reagent originally used with an appropriate isotope-labeled reagent.

[0137] In addition, heavier isotopes, especially deuterium (i.e. 2 H or D) can provide certain therapeutic advantages, which are brought about by higher metabolic stability. For example, increased half-life in vivo or reduced dosage requirements or improved therapeutic index. It should be understood that deuterium in this context is regarded as a substituent of the compound of formula (I). The concentration of such heavier isotopes, especially deuterium, can be defined by isotopic enrichment factors. The term "isotopic enrichment factor" used in the present invention refers to the ratio between the isotopic abundance and the natural abundance of a specified isotope. Where a substituent of a compound of the invention is designated as deuterium, the compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). Pharmaceutically acceptable solvates according to the invention include those wherein the solvent of crystallization may be isotopically substituted, for example D2O, acetone-d6, or DMSO-d6.

[0138] Liposome and preparation method

[0139] The present invention provides a liposome. The liposome according to the embodiment of the present invention is made of the compound proposed in the first aspect of the present invention, and the liposome can encapsulate the added loaded substance through the flow effect of the lipid bilayer, and has a good encapsulation effect, so that the loaded substance can effectively enter the cell and escape from the endosome to the cytoplasm.

[0140] The above compounds can form liposomes by self-assembly with hydrophobic lipids.

[0141] In some embodiments, the liposome further comprises a hydrophobic lipid, an amphiphilic lipid, a buffering agent and / or an organic solvent, wherein the amphiphilic lipid comprises at least one selected from a neutral lipid, a cationic lipid and a PEG-lipid, and the hydrophobic lipid comprises a sterol.

[0142] In some embodiments, the neutral lipid comprises at least one selected from the group consisting of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylcholine, diethyl pyrocarbonate, dimyristoylphosphatidylcholine, and egg yolk lecithin.

[0143] In some embodiments, the cationic lipid is selected from at least one of the following: trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium chloride (, didodecyldimethylammonium bromide, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-hydroxybutyl-2,3-dioleyloxypropylammonium bromide, dimethyl-5-hydroxybutyl-2,3-dioleyloxypropylammonium bromide, Hydroxypentyl-2,3-dioleyloxypropylammonium, dimethyl-2-hydroxyethyl-2,3-dihexadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide, N-(2-sperminecarbonyl)-N',N'-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl] cholesterol, lipid poly-L-lysine, stearammonium.

[0144] In some embodiments, the PEG-lipid comprises at least one selected from the group consisting of dipalmitoylphosphatidylethanolamine-PEG, distearoylphosphatidylethanolamine-PEG, dimyristylglycerol-PEG, and dimethacrylate-PEG.

[0145] In some embodiments, the sterol is cholesterol.

[0146] In some embodiments, the buffering agent is an acidic buffering agent.

[0147] In some embodiments, the buffer reagent includes at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tris(hydroxymethylaminomethane)-hydrochloric acid, potassium dihydrogen phosphate-sodium hydroxide, boric acid-borax, glycine-hydrochloric acid, phthalic acid-hydrochloric acid, potassium hydrogen phthalate and sodium dihydrogen phosphate-citric acid.

[0148] In some embodiments, the organic solvent includes at least one selected from methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, ethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.

[0149] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 3 to 80% mol / mol. Further, the amount of the compound is 3% mol / mol, 4% mol / mol, 5% mol / mol, 6% mol / mol, 7% mol / mol, 8% mol / mol, 9% mol / mol, 10% mol / mol, 11% mol / mol, 12% mol / mol, 13% mol / mol, 14% mol / mol, 15% mol / mol, 16% mol / mol, 17% mol / mol, 18% mol / mol, 19% mol / mol, 20% mol / mol, 21% mol / mol, 22% mol / mol, 23% mol / mol, 24% mol / mol, 25% mol / mol, 26% mol / mol, 27% mol / mol, 28% mol / mol, 29% mol / mol, 30% mol / mol, 31% mol / mol, 32% mol / mol, 33% mol / mol, 34% mol / mol, 35% mol / mol, 36% mol / mol, 37% mol / mol, 38% mol / mol, 39% mol / mol, 40% mol / mol, 41% mol / mol, 42% mol / mol, 43% mol / mol, 44% mol / mol, 45% mol / mol, 46% mol / mol, 47% mol / mol, 48% mol / mol, 49% mol / mol, 50% mol / mol, 51% mol / mol, 52% mol / mol, 53% mol / mol, 54% mol / mol, 55% mol / mol, 56% mol / mol, 57% mol / mol, 58% mol / mol, 59% mol / mol, 60% mol / mol, 61% mol / mol, 62% mol / mol, 63% mol / mol, 64% mol / mol, 65% mol / mol, 66% mol / mol, 67% mol / mol, 68% mol / mol, 69% mol / mol, 70% mol / mol, 71% mol / mol, 72% mol / mol, 73% mol / mol, 74% mol / mol, 75% mol / mol, 76% mol / mol, 77% mol / mol, 78% mol / mol, 79% mol / mol, 80% mol / mol.

[0150] In some embodiments, the amount of the compound is 20-80% mol / mol based on the liposome, and further, the amount of the compound is 20% mol / mol, 21% mol / mol, 22% mol / mol, 23% mol / mol, 24% mol / mol, 25% mol / mol, 26% mol / mol, 27% mol / mol, 28% mol / mol, 29% mol / mol, 30% mol / mol, 31% mol / mol, 32% mol / mol, 33% mol / mol, 34% mol / mol, 35% mol / mol, 36% mol / mol, 37% mol / mol, 38% mol / mol, 39% mol / mol, 40% mol / mol, 41% mol / mol, 42% mol / mol, 43% mol / mol, 44% mol / mol, 45% mol / mol, 46% mol / mol, 47% mol / mol, mol / mol, 48% mol / mol, 49% mol / mol, 50% mol / mol, 51% mol / mol, 52% mol / mol, 53% mol / mol, 54% mol / mol, 55% mol / mol, 56% mol / mol, 57% mol / mol, 58% mol / mol, 59% mol / mol, 60% mol / mol, 61% mol / mol, 62% mol / mol, 63% mol / mol, 64% mol / mol, 65% mol / mol, 66% mol / mol, 67% mol / mol, 68% mol / mol, 69% mol / mol, 70% mol / mol, 71% mol / mol, 72% mol / mol, 73% mol / mol, 74% mol / mol, 75% mol / mol, 76% mol / mol, 77% mol / mol, 78% mol / mol, 79% mol / mol, 80% mol / mol.

[0151] In some embodiments, the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.2-20).

[0152] According to an embodiment of the present invention, the molar ratio of the compound, the neutral lipid, the sterol and the PEG-lipid is (40-61): (8-20): (30-60): (0.5-5).

[0153] According to an embodiment of the present invention, the molar ratio of the compound, sterol and PEG-lipid is (20-80):(5-50):(10-60).

[0154] According to an embodiment of the present invention, the molar ratio of the compound, sterol and PEG-lipid is (40-70): (5-20): (20-40).

[0155] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 3-50% mol / mol, further, the amount of the compound is 3% mol / mol, 4% mol / mol, 5% mol / mol, 6% mol / mol, 7% mol / mol, 8% mol / mol, 9% mol / mol, 10% mol / mol, 11% mol / mol, 12% mol / mol, 13% mol / mol, 14% mol / mol, 15% mol / mol, 16% mol / mol, 17% mol / mol, 18% mol / mol, 19% mol / mol, 20% mol / mol, 21% mol / mol, 22% mol / mol, 23% mol / mol, 24% mol / mol, 25% mol / mol, 26% mol / mol, 27% mol / mol, 28% mol / mol, 29% mol / mol, 30% mol / mol, 31% mol / mol, 32% mol / mol, 33% mol / mol, 34% mol / mol, 35% mol / mol, 36% mol / mol, 37% mol / mol, 38% mol / mol, 39% mol / mol, 40% mol / mol, 41% mol / mol, 42% mol / mol, 43% mol / mol, 44% mol / mol, 45% mol / mol, 46% mol / mol, 47% mol / mol, 48% mol / mol, 49% mol / mol, 50% mol / mol.

[0156] According to an embodiment of the present invention, the molar ratio of the compound, the neutral lipid, the sterol, the PEG-lipid and the cationic lipid is (3-50): (1-20): (2-50): (0.01-10): (5-92).

[0157] According to an embodiment of the present invention, the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-41): (1-12): (2-45): (0.05-2): (10-90).

[0158] In some embodiments, the volume of the buffer reagent is 50-90% v / v based on the liposome. Further, the volume of the buffer reagent is 50% v / v, 51% v / v, 52% v / v, 53% v / v, 54% v / v, 55% v / v, 56% v / v, 57% v / v, 58% v / v, 59% v / v, 60% v / v, 61% v / v, 62% v / v, 63% v / v, 64% v / v, 65% v / v, 66% v / v, 67% v / v, 68% v / v, 69% v / v, 70% v / v, 71% v / v, 72% v / v, 73% v / v, 74% v / v, 75% v / v, 76% v / v, 77% v / v, 78% v / v, 79% v / v, 80% v / v, 81% v / v, 82% v / v, 83% v / v, 84% v / v, 85% v / v, 86% v / v, 87% v / v, 88% v / v, 89% v / v, 90% v / v.

[0159] In some embodiments, the volume of the buffering agent is 75% v / v based on the liposomes.

[0160] According to the embodiments of the present invention, the liposomes with the above ratio are used, which has high transfection efficiency, good encapsulation effect and strong drug presentation ability.

[0161] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.2-20); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0162] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, sterol and PEG-lipid is (20-80):(5-50):(10-60); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0163] According to an embodiment of the present invention, based on the liposome, the amount of the compound is 3-50% mol / mol; the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-50):(1-20):(2-50):(0.01-10):(5-92); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0164] In some embodiments, the above compounds can also form liposomes with phospholipid derivatives, including but not limited to distearoylphosphatidylcholine, distearoylphosphatidylethanolamine, and dioleoylphosphatidylethanolamine.

[0165] In a specific embodiment, the liposome comprises the above-mentioned compound, and further, the liposome further comprises a neutral lipid and a lipid capable of reducing aggregation of liposome particles.

[0166] In a specific embodiment, the liposomes are mainly composed of: (i) at least one lipid of the present invention; (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, DLPC, DEPC, DMPC, EPC, DOTAP, etc.; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as DPPE-PEG, DSPE-PEG, PEG-DMG or PEG-DMA, in a molar ratio of about 20-80% amine-containing lipid: 5-50% neutral lipid: 10-60% sterol: 0.2-20% PEG-lipid.

[0167] In another aspect of the present invention, the present invention provides a method for preparing the above liposomes. According to an embodiment of the present invention, the method comprises: mixing a predetermined amount of the compound provided in the first aspect of the present invention with an organic solvent to obtain the liposomes.

[0168] In some embodiments, the concentration of the above compound in the organic solution is 10-30 mg / mL, further, the concentration of the above compound in the organic solution is 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL.

[0169] In some embodiments, the method further comprises: dissolving a predetermined amount of hydrophobic lipids and amphiphilic lipids in the above-mentioned organic solution and mixing with a buffer reagent to obtain the liposome. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the mass ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80): (5-50): (10-60): (0.2-20), preferably, the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (40-61): (8-20): (30-60): (0.5-5); based on the liposome, the volume of the buffer reagent is 75% v / v. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 4-50% mol / mol; the molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-50): (1-20): (2-50): (0.01-10): (5-92); based on the liposome, the volume of the buffer reagent is 75% v / v. According to an embodiment of the present invention, based on the liposome, the amount of the compound is 20-80% mol / mol; the molar ratio of the compound, sterol and PEG-lipid is (20-80): (5-50): (10-60); based on the liposome, the volume of the buffer reagent is 75% v / v.

[0170] In some embodiments, the amount of the compound is 20% mol / mol, 21% mol / mol, 22% mol / mol, 23% mol / mol, 24% mol / mol, 25% mol / mol, 26% mol / mol, 27% mol / mol, 28% mol / mol, 29% mol / mol, 30% mol / mol, 31% mol / mol, 32% mol / mol, 33% mol / mol, 34% mol / mol, 35% mol / mol, 36% mol / mol, 37% mol / mol, 38% mol / mol, 39% mol / mol, 40% mol / mol, 41% mol / mol, 42% mol / mol, 43% mol / mol, 44% mol / mol, 45% mol / mol, 46% mol / mol, 47% mol / mol, 48% mol / mol, 49% mol / mol, 50% mol / mol, 51% mol / mol, 52% mol / mol, 53% mol / mol, 54% mol / mol, 55% mol / mol, 56% mol / mol, 57% mol / mol, 58% mol / mol, 59% mol / mol, 60% mol / mol, 61% mol / mol, 62% mol / mol, 63% mol / mol, 64% mol / mol, 44% mol / mol, 45% mol / mol, 46% mol / mol, 47% mol / mol, 48% mol / mol, 49% mol / mol, 50% mol / mol, 51% mol / mol, 52% mol / mol, 53% mol / mol, 54% mol / mol, 55% mol / mol, 56% mol / mol, 57% mol / mol, 58% mol / mol, 59% mol / mol, 60% mol / mol, 61% mol / mol, 62% mol / mol, 63% mol / mol, 64% mol / mol, 65% mol / mol, 66% mol / mol, 67% mol / mol, 68% mol / mol, 69% mol / mol, 70% mol / mol, 71% mol / mol, 72% mol / mol, 73% mol / mol, 74% mol / mol, 75% mol / mol, 76% mol / mol, 77% mol / mol, 78% mol / mol, 79% mol / mol, 80% mol / mol.

[0171] Drug carrier, drug composition, transfection complex

[0172] In another aspect of the present invention, the present invention provides a drug carrier, comprising the above-mentioned compound or liposome. According to an embodiment of the present invention, the compound is an ionizable amine-containing lipid, and the drug carrier is an ionizable carrier, which can carry negatively charged or hydrophobic drugs, and deliver the drugs into cells and effectively escape from the endosomes to the cytoplasm, which is beneficial to the efficacy of the loaded drugs.

[0173] In another aspect of the present invention, the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising a carrier and a pharmaceutically active ingredient, wherein the carrier comprises the pharmaceutical carrier provided in the fifth aspect of the present invention. According to an embodiment of the present invention, the pharmaceutical carrier is an ionizable carrier, which can carry a negatively charged or hydrophobic pharmaceutically active ingredient, and deliver the pharmaceutically active ingredient into the cell and effectively escape from the endosome to the cytoplasm, which is beneficial for the loaded pharmaceutically active ingredient to exert its efficacy.

[0174] In some embodiments, the active pharmaceutical ingredient comprises at least one selected from the group consisting of DNA molecules, RNA molecules, proteins, polypeptides, and hydrophobic drugs. According to an embodiment of the present invention, the drug carrier contains a long carbon chain and can carry a hydrophobic substance, for example, a hydrophobic small molecule drug.

[0175] Furthermore, the drug carrier of the present invention can also load proteins and polypeptides. The replication of the proteins and polypeptides is not particularly limited and can be peptide chains, proteins themselves, derivatives of the above substances, or complexes with other substances, for example, Cas9 protein or its partial domain peptides, proteins loaded with nuclides, antibodies and other substances.

[0176] Furthermore, the drug carrier of the present invention can also load nucleic acid substances, such as antisense nucleic acid.

[0177] In some embodiments, the active pharmaceutical ingredient is negatively charged. According to an embodiment of the present invention, the drug carrier contains ionizable amine-containing lipids, which are positively charged at a specific pH and can bind to the negatively charged active pharmaceutical ingredient. In addition, the active pharmaceutical ingredient can be encapsulated by the flow of the lipid bilayer and delivered to the cell.

[0178] In some embodiments, the active pharmaceutical ingredient contains nucleic acid. According to an embodiment of the present invention, the active pharmaceutical ingredient contains DNA, RNA, nucleic acid-protein complex, nucleic acid-lipid complex, nucleic acid-nuclide complex, etc., and the type of nucleic acid is not particularly limited. As a specific example of the nucleic acid, it can be siRNA, mRNA, tRNA, rRNA, cDNA, miRNA (microRNA), ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, triple-stranded oligonucleotide (Triplex FormingOligonucleotide, TFO), gene, etc. Among them, the vector of the present invention is particularly effective in transporting siRNA into cells. The nucleic acid applicable to the vector of the present invention can be nucleic acid from humans, animals, plants, bacteria, viruses, etc., and can also be nucleic acid prepared by chemical synthesis. Furthermore, the above-mentioned nucleic acid can be any one of single-stranded, double-stranded, and triple-stranded, and there is no special limitation on its molecular weight. In addition, in the present invention, the nucleic acid can be a nucleic acid modified by chemical, enzyme or peptide. In the present invention, the nucleic acid can be used alone or in combination of two or more. In a preferred embodiment, the nucleic acid delivery vector composition of the present invention preferably delivers small interfering nucleic acid (siRNA) or its analogs.

[0179] In some embodiments, the mass ratio of the carrier to the pharmaceutically active ingredient is (5-50):1; preferably, the mass ratio of the carrier to the pharmaceutically active ingredient is (5-40):1.

[0180] In some embodiments, the mass ratio of the carrier to the pharmaceutically active ingredient is (8-20):1.

[0181] In some embodiments, the mass ratio of the carrier to the active pharmaceutical ingredient is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1.

[0182] In some embodiments, the mass ratio of the carrier to the pharmaceutically active ingredient is 15:1.

[0183] In another aspect of the present invention, the present invention provides a transfection complex. According to an embodiment of the present invention, the transfection complex comprises the above compound or the above liposome.

[0184] In some embodiments, the helper lipids suitable for preparing and forming the transfection complexes disclosed herein may include, but are not limited to, cholesterol, cholesterol derivatives, or any of neutral or cationic lipids known to enable or facilitate the introduction of exogenous bioactive molecules into the interior of cells or tissues. In certain embodiments, more than one helper lipid may be used in the formulation of the transfection complexes described herein.

[0185] In some embodiments, the transfection complex comprises at least one bioactive agent. The transfection complex according to an embodiment of the present invention is suitable for delivering one or more bioactive agents to cells, tissues or organs in vitro or in vivo.

[0186] In some embodiments, the transfection complex may include one or more bioactive agents to be delivered to a cell or to a target tissue in vitro or in vivo. Suitable bioactive agents may include any molecule that is capable of forming a transfection complex with a transfection reagent as described herein and that causes a biological response when delivered to one or more cells or to a tissue in vivo or in vitro. The bioactive agent used in the embodiments described herein may be a cationic, neutral, or anionic agent.

[0187] In some embodiments, the bioactive agent includes at least one selected from the following: DNA molecules, RNA molecules, proteins, polypeptides and drugs. According to embodiments of the present invention, exemplary bioactive agents suitable for preparing transfection complexes may include, but are not limited to: nucleic acids (including, but not limited to single-stranded or double-stranded linear or circular DNA molecules (including cDNA molecules), single-stranded or double-stranded RNA molecules, small interfering RNA (siRNA) molecules, small hairpin RNA (shRNA) molecules, microRNA (miRNA) molecules, oligonucleotides, antisense oligonucleotides, sense oligonucleotides), polypeptides, antibodies, oligopeptides, therapeutic peptides or protein molecules, peptide nucleic acids (PNA), cationic, anionic or neutral organic molecules or drugs, and pharmaceutically acceptable salts thereof.

[0188] In some embodiments, the transfection complex described herein may optionally include one or more fusion peptides or cell penetrating peptides. Fusion peptides or cell penetrating peptides are any peptide macromolecules that can promote the fusion of lipid-containing complexes with cell membranes (plasma membranes or endosomal membranes). A variety of fusion peptides or cell penetrating peptides are known in the art, and within the skill level of practitioners, suitable fusion peptides or cell penetrating peptides and their conditions of use in the present invention can be identified without excessive experimentation.

[0189] In some embodiments, the transfection complexes described herein may optionally include one or more transfection adjuvants or targeting moieties. The targeting moiety may be a peptide, a modified peptide, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single-stranded or double-stranded nucleic acid molecule, a modified single-stranded or double-stranded nucleic acid molecule, a peptide or nucleic acid aptamer, a modified peptide or nucleic acid aptamer, an organic molecule, a polysaccharide, or any other molecule that is capable of targeting the transfection complex to a specific tissue or cell type for targeted delivery of a bioactive agent thereto.

[0190] In some embodiments, the transfection complexes provided herein can be stable for up to 1 year, and can be contacted with cells or tissues to be transfected, or administered to animals or humans immediately or soon after formation, or can be optionally stored for a period of time before contacting with cells or tissues or administering to animals or humans. The transfection complexes are stable and can be stored at room temperature or at a temperature greater than freezing until about room temperature for the following time period, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year. It should be understood that the formulations described herein may include one or more stabilizers, preservatives, buffers, etc., which assist in the long-term stability and storage of biologically active formulations, as will be readily understood by skilled practitioners in the fields of biology and pharmacy, and can be achieved without excessive experimentation. It should also be understood that the storage period can be any of these time periods, such as 31 minutes to 1 hour, or 1 hour to 24 hours.

[0191] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0192] Detection and calculation methods:

[0193] 1. Nuclear magnetic resonance spectrometer detection to determine the structure of ammonium-containing lipids

[0194] The nuclear magnetic resonance spectrometer used is a Bruker 400M nuclear magnetic resonance spectrometer. The specific calculation method is referenced to the literature, Boulmedais F., Frisch B., Etienne O., Lavalle Ph., Picart C., Ogier J., Voegel JC., Schaaf P., Eggles C. Polyelectrolyte multilayer films with pegylatedpolypeptides as a new type of anti-microbial protection for biomaterials, Biomaterials, 2004, 25, 2003-2011.

[0195] 2. Gel retardation experiment, used to prove the loading of nucleic acid in the preparation

[0196] Anti-apolipoprotein B siRNA (siApoB) was dissolved in DEPC-treated water and freshly assembled with LNPs of different mass ratios (w / w). Thereafter, it was mixed with 6× loading buffer (Baoriyi Biotechnology (Beijing) Co., Ltd.), and the preparation containing equal amounts of siRNA was added to a 1% (w / w) agarose gel containing 0.01% gel stain (w / w) (Beijing Quanshijin Biological). Electrophoresis was performed at 90v for 30min in 1×TAE electrophoresis buffer. The results were recorded at a UV wavelength of 320nm using a gel imaging system (Shanghai Tianneng Technology Co., Ltd.). The siApoB sequence is shown in Table 1:

[0197] Table 1:

[0198]

[0199] 3. Determination of particle size and surface potential to preliminarily determine whether the lipid formula is usable

[0200] The preparation obtained after LNP encapsulation of siRNA was first diluted 10 times before measuring the particle size and surface potential. The instrument used for particle size and surface potential measurement was Zetasizer 3000HS laser particle size analyzer from Malven Company, USA, with a measurement temperature of 25°C, an angle of 90°, and an incident light wavelength of 677nm.

[0201] 4. Determination of siRNA concentration in the preparation

[0202] The RiboGreen method was used to determine the concentration of siRNA. The test preparation was diluted 20 times and 100 μl was added to a 96-well plate to detect the fluorescence of siRNA outside the preparation. Another 15 μl of the diluted test preparation was used to destroy the lipid structure with 50 μl of a 2% Triton-X-100 (w / w) mixture to completely release the internal siRNA, and then 35 μl of 1×TE buffer was added to the well to make up the volume, which was used to detect the fluorescence of all siRNA in the preparation. In addition, a standard curve was prepared using siRNA standards, 1×TE, and 2% Triton-X-100 to calculate the concentration of siRNA in the test samples. After all the samples to be tested were added, incubated at room temperature for 30 minutes. Then, 100 μl / well of RiboGreen working solution was added in a dark place and fluorescence detection was immediately performed using an enzyme reader. The detection conditions are shown in Table 2:

[0203] Table 2:

[0204] parameter set up Detection Methods Fluorescence type Endpoint / kinetics Type of related components Monochromator Excitation light 480 / 17.0 Emitted light 520 / 17.0 Optical element position top Gain 60

[0205] 5. Transmission electron microscopy (TEM) experiment, used to detect the particle morphology of the preparation

[0206] The transmission electron microscope used was a Dutch JEM-100CX II transmission electron microscope. First, the copper mesh was immersed in the sample solution to be tested, and then stained with a 0.1 wt% phosphotungstic acid solution. After about 3 minutes, the excess liquid was filtered off with filter paper and dried at room temperature. Then the morphology of the particles in the system was observed and photographed with a transmission electron microscope.

[0207] 6. In vitro transfection experiment

[0208] To test the siRNA delivery efficiency of candidate preparations, human liver cancer cell line HepG2 was seeded in 12-well plates with DMEM complete medium containing 10% fetal bovine serum at a seeding density of 1×10 5 cells / well, 1 mL of culture medium per well, cultured overnight at 37°C.

[0209] On the second day, the cell culture medium in the 12-well plate was aspirated and discarded, and 1 mL of fresh DMEM complete medium containing 10% (v / v) fetal bovine serum was added to each well to be transfected. The Mock group was not further treated, and the corresponding preparation was added to the sample wells to be tested. The sample volume was calculated based on the siRNA concentration in the LNP previously determined by the RiboGreen method, so that the final concentration of siApoB was 50 nM. The cells were cultured at 37°C for 4 hours, and 1 mL of DMEM complete medium containing 10% fetal bovine serum was added to each well, and cultured at 37°C overnight.

[0210] Afterwards, the relative expression of ApoB mRNA in the sample to be tested was detected by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR). The specific steps are as follows: after culturing the transfected cells for 24 hours, use RNA isolator (Nanjing Novogene Biotechnology Co., Ltd., catalog number R401-01-AA), chloroform, and isopropanol to extract the total RNA in the cells; take 1 μg of total RNA and reverse transcribe it according to the method of reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AT311-03) to obtain cDNA. Real-time fluorescence quantitative PCR mix (Shanghai Yishen Biotechnology Co., Ltd., catalog number 11201ES08) was used to detect the expression of the target gene ApoB and the internal reference gene glyceraldehyde phosphate dehydrogenase (GAPDH) using cDNA as a template according to the steps in the instruction manual, where the expression of ApoB needs to refer to GAPDH. The PCR primers used to amplify ApoB and GAPDH as an internal reference gene are shown in Table 3.

[0211] Table 3:

[0212]

[0213] 7. Cytotoxicity assay

[0214] The cytotoxicity of the test substance was detected by MTT method (tetrazolium salt colorimetric method), and the specific method is as follows:

[0215] (1) 1.25×10 4 HepG2 cells / well, 100 μl of culture medium per well, incubated overnight at 37°C in the presence of 5% CO2.

[0216] (2) After 24 hours, a corresponding amount of siFLA4-13 encapsulating insect-resistant luciferase siRNA (siFirefly) was added to each well, so that the siRNA content in the well ranged from 1 nM to 200 nM. At the same time, control wells (cells, PBS with a pH value of 7.4 and the same volume of liquid as the preparation, and culture medium) were set up and incubated at 5% CO2 and 37°C for 48 hours.

[0217] (3) Add 20 μl of MTT solution (5 mg / mL) to each well and continue incubation at 5% CO2 and 37°C for 4 hours.

[0218] (4) Carefully discard the supernatant culture medium in each well, add 150 μl of dimethyl sulfoxide to each well, and shake on a low-speed shaker for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at 540 nm using a continuous spectrum multifunctional microplate reader (TECAN Infinite 200).

[0219] (5) Calculate the relative cell viability of each sample well according to the following formula:

[0220]

[0221] The siFirefly sequence is shown in Table 4:

[0222] Table 4

[0223]

[0224]

[0225] 8. Subcellular localization, used to determine whether nucleic acids have entered cells

[0226] On the day before the test, 2 × 10 5 HepG2 cells will be seeded in 35mm culture dishes and cultured overnight at 5% CO2 and 37℃. The next day, Cy-siFLA4-13 or Cy-siFLLipo was prepared by encapsulating Cy5 group at the 3' end of siFirefly sense chain with A4-13 or Lipofectamine 2000 (Invitrogen), and added to the corresponding culture dishes and cultured for 4 hours to allow cells to endocytose the transfection reagent. After the specified time, all the culture medium containing the transfection reagent was discarded and the cells on the culture dishes were washed three times with 1×PBS. After that, 1mL of staining working solution (Lysotracker diluted 1:3000 (v / v) with 1×PBS, and Hoechst 33342 0.1mg / ml) was added to the culture dishes and stained for 15 minutes at 37℃ in the dark. After staining, a laser confocal microscope (LSM 700, Zeiss) was used to observe the distribution of fluorescent signals in cells.

[0227] 9. Cellular uptake, used to determine the efficiency of nucleic acid entering cells

[0228] HepG2 cells were seeded in 12-well plates at a concentration of 2 × 10 5 cells / mL, volume 1mL / well. After 24 hours, Cy-siFLA4-13 was added to the wells to a final concentration of 50nM, and cultured for 4 hours at 5% CO2 and 37°C. Thereafter, all the culture medium containing the transfection reagent was discarded, the cells were digested with trypsin and complete culture medium to terminate the digestion, and the liquid was collected into a centrifuge tube. After centrifugation at 800 rpm for 5 minutes, the cells were resuspended with 1×PBS and the cells were rinsed three times. Thereafter, the endocytosis efficiency of HepG2 cells to the preparation was detected by flow cytometry (FACSverse, BD). The data were analyzed using FlowJo7.6.

[0229] 10. In vivo distribution experiment, used to determine the metabolism and distribution characteristics of drugs in the body

[0230] The experiment used 6-8 week old C57BL / 6 mice as experimental subjects, which were divided into two groups and given 1×PBS or Cy-siFLA4-13 through the tail vein, where the amount of Cy5-siFirefly was 2mg / kg. The distribution of Cy5 signals in mice will be detected by the small animal in vivo imaging system FX Pro (Kodak) at different time points after administration. In addition, after the completion of in vivo imaging at 0.5 hours, 4 hours and 24 hours after administration, 1-2 animals in each group will be euthanized and the heart, liver, spleen, lung, kidney, stomach, intestine, submandibular gland and thymus will be separated for organ imaging. The results were analyzed using Carestream software, and the line graph was generated by GraphpadPrism 8.

[0231] In another experiment, 6-8 week old C57BL / 6 mice were given 1×PBS or mLuA4-13 formulations through the tail vein, with the amount of mRNA being 20 μg / animal. At 6 and 9 hours after administration, the animals were euthanized and the heart, liver, spleen, lung, kidney, stomach, intestine, submandibular gland, and thymus were isolated for organ imaging. The results were analyzed using livingimage software.

[0232] In another experiment, 6-8 week old C57BL / 6 mice were administered 1×PBS, mLuA4-13, or mLuDA4-1 to mLuDA4-9 formulations by intrapulmonary spray, with the amount of mRNA being 4 μg / animal. The animals were euthanized 6 hours after administration, and the heart, liver, spleen, and lungs were isolated for organ imaging. The results were analyzed using livingimage software.

[0233] 11. Dose dependence experiment

[0234] Thirty-two 6-8 week old C57BL / 6 mice were divided into 8 groups and received 1×PBS or 0.01mg / kg to 1mg / kg siA4-13. After 42 hours, the animals were fasted for 6 hours and euthanized. Serum and liver tissue samples were collected for analysis of serum triglyceride (TG), total cholesterol (CHO), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and ApoB mRNA expression changes in liver tissue. The PCR primers used to amplify mouse ApoB and GAPDH as an internal reference gene are shown in Table 5.

[0235] Table 5:

[0236]

[0237] 12. Duration of action experiment

[0238] 96 6-8 week old C57BL / 6 mice were divided into three groups and received 1×PBS or 1mg / kg or 3mg / kg siA4-13, respectively. On days 7, 14, 21, 28, 35, 42, and 49 after administration, 4 animals in each group were fasted for 6 hours and euthanized, and serum and liver tissue samples were collected for analysis of changes in serum TG, CHO, LDL-c, HDL-c, and ApoB mRNA expression in liver tissue. The duration of action curve was generated by Graphpad Prism 8.

[0239] 13. Drug efficacy test

[0240] The experiment was divided into two parts. In the first part, leptin receptor knockout db / db mice (C57BLKS / JGpt-Leprem2Cd / Gpt) were used as experimental subjects, and non-gene knockout mice (m / m) of the same genetic background were used as controls. This part of the experiment was designed to detect the lipid-lowering effect of the preparation siANA4-13 encapsulated with anti-angiopoietin-like protein 3 siRNA (siANGPTL3) and the expression changes of target genes. In the second part, humanized apolipoprotein C3 (ApoC3) transgenic mice (hApoC3, Tg(APOC3)3707Bres) were used as experimental subjects, and C57BL / 6 mice were used as controls. This part of the experiment was designed to detect the lipid-lowering effect of the preparation siApoCA4-13 encapsulated with siApoC3 (siApoC3 is siRNA targeting ApoC3 mRNA) and the lipid recovery of animals after drug withdrawal.

[0241] In the first part, db / db mice will be injected with 1×PBS or 0.5mg / kg siFLA4-13 or 0.5mg / kg siANA4-13 or 0.25mg / kg siANA4-13 through the tail vein, respectively. The test will be conducted 4 times, once a week. On the third day after each administration, all animals will be fasted for 6 hours and blood will be collected through the orbit for analysis of changes in TG and CHO. On the second day after the last administration, all animals will be fasted for 6 hours and then sacrificed, and blood and liver tissue samples will be collected to further determine changes in blood lipids and the expression of ANGPTL3 mRNA in liver tissue. The liver of one animal in each group was collected for Oil Red O staining to observe changes in lipid deposition in the liver.

[0242] In the second part, hApoC3 mice will be injected with 1×PBS or 0.5mg / kg siFLA4-13 or 0.5mg / kg siApoCA4-13 or 0.25mg / kg siApoCA4-13 through the tail vein. The experiment is planned to be administered 5 times, with one week interval between each administration, during which changes in blood lipids will be detected. After the last administration, all animals will still undergo blood lipid testing once a week for a total of 4 times. Thereafter, the animals will be euthanized to collect blood for blood lipid testing. The sequences of siANGPTL3 and siApoC3 involved in the experiment, as well as the PCR primers used to amplify mouse ANGPTL3 and human ApoC3 are shown in Tables 6 and 7, respectively:

[0243] Table 6:

[0244]

[0245] Table 7:

[0246]

[0247] 14. In vitro mRNA expression test

[0248] HepG2 cells were seeded in 12-well plates at a concentration of 2 × 10 5 cells / mL, with a volume of 1mL / well. After 24 hours, different LNPs encapsulating luciferase mRNA were added to the wells, so that the amount of mRNA added to each well was 400ng, and cultured for 6 hours under 5% CO2 and 37°C. When the predetermined time is reached, the cells are lysed with passive lysis buffer and the cell lysate is collected. Thereafter, 10μl / sample of cell lysis buffer is added to the 96-well plate, and 50μl of luciferase substrate is added to each well. Immediately after completion, the amount of fluorescence emitted by the sample is detected using an ELISA reader.

[0249] 15. Comparative test of gene inhibition by siRNA and CRISPR / Cas system

[0250] HepG2-luc cells were seeded in 6-well plates 24 hours before transfection at a concentration of 2×10 5cells / mL, with a volume of 1mL / well. The next day, LNPs encapsulated with siRNA against polo-like kinase gene 1 (siPLK1) and plasmids (pCPLK1) simultaneously expressing Cas protein and PLK1 sgRNA were added to the corresponding wells, so that the amount of siRNA and plasmid was 1600ng. The Mock group was not further treated. The cells were cultured at 5% CO2 and 37°C for 4 hours. Thereafter, 1mL of fresh culture medium was added to each well, and further culture was continued for 20 hours. After the specified time, the relative expression of PLK1 mRNA in the test sample was detected by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR). The nucleic acid and primer sequences of PLK1 siRNA are shown in Tables 8 and 9, respectively.

[0251] Table 8

[0252]

[0253] Table 9

[0254]

[0255] 16. Detection of protein concentration in LNP by BCA method

[0256] First, 2% Triton-X-100 was used to destroy the structure of LNP to release the encapsulated GOx. After that, 50 μl of the treated sample was added to a 96-well plate. At the same time, 50 μl of protein standards of different concentrations were added to make a standard curve. 200 μl of BCA working solution was added to each well and incubated at 37°C for 30 minutes. Finally, the absorbance at 562 nm was detected using an ELISA reader. The concentration of the protein in the sample to be tested was calculated by drawing a standard curve.

[0257] Preparation Example 1 Synthesis of amine-containing lipid compound A4

[0258] Synthesis of compound A2:

[0259]

[0260] The reaction was carried out in a three-necked flask containing tert-butyl (3-aminopropyl) carbamate (1.01 g, 5.7 mmol), 1,2-epoxydodecane (2.33 g, 12.6 mmol) and 12.00 mL of ethanol, and refluxed with nitrogen at 50 °C. The reaction was monitored by thin layer chromatography (TLC) and stopped after the reaction was completed. The crude product was concentrated under reduced pressure and purified by silica gel column separation using dichloromethane: methanol = 30:1 (v / v) as the developing solvent. The product A2 (1.61 g, yield 78.1%) was obtained. 1H NMR (400 MHz, CDCl3) δ (ppm) 0.88 (t, J = 13.68 Hz, 6H), 1.26-1.44 (m, 45H), 1.67 (m, 2H), 2.38-2.70 (m, 6H), 3.18 (m, 2H), 3.70 (m, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm) 156.22, 79.39, 69.58, 62.90, 52.70, 38.58, 38.53, 35.13, 34.98, 31.92, 29.62, 29.35, 28.44, 25.65, 22.70, 14.13; high resolution mass spectrometry (EI+ mode) scanned compound A2 (molecular weight 543.5023), and the scanning result was 543.5108.

[0261] Synthesis of compound A3:

[0262]

[0263] In a three-necked flask containing compound A2 (1.04 g, 1.8 mmol) and 20.00 mL of dichloromethane, hydrochloric acid was slowly added while stirring. The reaction was carried out at room temperature for 16 hours, and then the reaction was stopped and detected by TLC plate. The pH of the reaction mixture was adjusted to 7-8 with 1M NaOH aqueous solution, and then extracted with water and ethyl acetate for 3 times. Concentrated under reduced pressure to obtain 398.0 mg of product A3 with a yield of 49.9%. 1H NMR(400MHz, CDCl3)δ(ppm)0.88(t,J=13.08Hz,6H),1.26(m,32H),1.43(m,4H),1.96(m,J=1 4.5Hz,2H),2.60(m,J=20.9Hz,4H),3.01(m,2H),3.25(m,2H),3.80(m,2H); 13C NMR (100MHz, CDCl3) δ (ppm) 70.41, 63.21, 58.29, 40.79, 36.15, 31.94, 29.91, 29.69, 29.39, 23.90, 22.70, 14.11; high resolution mass spectrometry (EI+ mode) scanned compound A3 (molecular weight 443.4498), and the scanning result was 443.4576.

[0264] Synthesis of compound A4:

[0265]

[0266] A3 (2.2 g, 4.9 mmol) and succinic acid (263.3 mg, 2.2 mmol) were dissolved in 20.00 mL of dichloromethane and placed in a three-necked flask. Then, catalyst 1-hydroxybenzotriazole (753.3 mg, 5.6 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.13 g, 5.6 mmol) were added. Under the protection of nitrogen, the reaction was allowed to proceed for 12 hours. The reaction was stopped after detection by thin layer chromatography. The crude product was washed twice with water, twice with saturated brine, and twice with the organic phase. 0.1 g of product A4 was obtained with a yield of 3.3%. 1H NMR (400MHz, CDCl3) δ (ppm) 0.88 13C NMR (100MHz, CDCl3) δ (ppm) 173.26, 67.14, 59.69, 51.69, 35.70, 34.47, 34.21, 33.21, 30.95, 30.52, 29.30, 28.72, 28.42, 21.70, 13.11; high resolution mass spectrometry (EI+ mode) scanned compound (molecular weight 967.9051), the scan result was 967.9137.

[0267] Example 1

[0268] The lipid A4 obtained in Preparation Example 1 was dissolved in anhydrous ethanol with DSPC, cholesterol, and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials were prepared, the four raw materials were mixed according to the mass ratio of A4: DSPC: cholesterol: DMG-PEG = 66.8: 10.9: 20.0: 2.2 and all of them were sucked into an insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer was prepared, and all of them were also sucked into the insulin syringe for use. After that, the insulin syringes containing the organic phase mixture and the buffer were placed in the same centrifuge tube and all the liquid was quickly released. The centrifuge tube was gently shaken to prepare lipid nanoparticles A4-1.

[0269] Test Example 1

[0270] This test example aims to detect the ability of the lipid nanoparticles obtained in Example 1 to encapsulate siRNA through a gel retardation experiment, and at the same time explore the optimal mass ratio of lipid nanoparticles to siRNA. The test results are as follows Figure 1 As shown. The results show that when the mass ratio of LNP to siRNA is lower than 5:1, a large amount of siRNA still leaks, indicating that the low mass ratio is not suitable for the delivery of siRNA. When the mass ratio is greater than or equal to 5:1, siRNA is efficiently encapsulated by LNP, and when the mass ratio is 15:1, siRNA is almost completely encapsulated. This shows that when the mass ratio of LNP to siRNA is 5:1-30:1, it is the suitable mass ratio range of LNP to siRNA, and a mass ratio greater than or equal to 15:1 may achieve efficient delivery of siRNA. Therefore, in the subsequent preparation examples, this mass ratio is selected to prepare LNP.

[0271] Example 2

[0272] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 58.5: 14.3: 23.4: 3.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-2.

[0273] Example 3

[0274] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 50.2: 16.4: 25.1: 8.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-3.

[0275] Example 4

[0276] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 42.8: 17.5: 25.7: 14.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-4.

[0277] Example 5

[0278] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 61.8: 8.6: 21.0: 8.6 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-5.

[0279] Example 6

[0280] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 57.4: 12.0: 14.6: 16.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-6.

[0281] Example 7

[0282] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 55.0: 15.3: 28.1: 1.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-7.

[0283] Example 8

[0284] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 54.7: 19.0: 23.3: 3.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-8.

[0285] Example 9

[0286] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 57.7: 7.0: 21.3: 14.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-9.

[0287] Example 10

[0288] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 57.2: 10.4: 25.4: 7.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-10.

[0289] Embodiment 11

[0290] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 66.1: 16.0: 14.7: 3.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-11.

[0291] Example 12

[0292] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 61.6: 18.6: 18.2: 1.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-12.

[0293] Embodiment 13

[0294] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 64.8: 6.9: 25.5: 2.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-13.

[0295] Embodiment 14

[0296] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 66.2: 10.6: 21.7: 1.4 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-14.

[0297] Embodiment 15

[0298] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 59.1: 12.7: 15.5: 12.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-15.

[0299] Example 16

[0300] The four dissolved raw materials were mixed in a mass ratio of A4: DSPC: cholesterol: DMG-PEG = 63.6: 17.0: 12.5: 6.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-16.

[0301] In the above embodiment, A4, DSPC, cholesterol, and DMG-PEG were set to 4 levels, as shown in Table 10:

[0302] Table 10:

[0303] A4 DSPC cholesterol DMG-PEG 40 8 30 0.5 47 12 37 1 54 16 44 2.5 61 20 51 5

[0304] The ratios of Examples 1-16 were designed using orthogonal design software (Orthogonal Design Assistant ii V3.1). The molar ratios and mass ratios of the 16 LNPs with different ratios are shown in Table 11:

[0305] Table 11:

[0306]

[0307] Examples 17-32

[0308] The concentration of siApoB was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl siApoB solution with an ethanol content of 25%.

[0309] The 16 lipid nanoparticles of different proportions obtained in Examples 1-16 were mixed with the siApoB solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoB to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the 16 lipid nanoparticle / siApoB complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0310] Test Example 2

[0311] This test example is intended to detect the particle size and polydispersity index (PDI) of 16 lipid nanoparticles without siRNA encapsulated obtained in Examples 1-16. The results are shown in Table 12. The preparation of the present invention can meet the requirements of being an excellent delivery carrier.

[0312] Table 12:

[0313] serial number size(nm) PDI A4-1 106.75±0.21 0.173±0.057 A4-2 146.90±0.99 0.547±0.374 A4-3 120.45±0.21 0.112±0.002 A4-4 171.45±1.63 0.179±0.033 A4-5 191.70±0.14 0.141±0.009 A4-6 203.85±3.18 0.254±0.013 A4-7 201.95±0.49 0.263±0.018 A4-8 200.70±0.99 0.274±0.010 A4-9 155.80±0.28 0.268±0.006 A4-10 187.20±0.71 0.248±0.001 A4-11 129.90±0.71 0.142±0.013 A4-12 106.34±0.49 0.124±0.028 A4-13 97.16±0.37 0.133±0.008 A4-14 92.25±0.32 0.130±0.007 A4-15 137.10±0.99 0.143±0.004 A4-16 120.50±0.42 0.278±0.011

[0314] Test Example 3

[0315] This test example is intended to detect the particle size (size), polydispersity index (PDI), encapsulation efficiency (EE) and intra-particle siRNA concentration (conc) of the 16 siApoB-encapsulated preparations obtained in Examples 17-32. The results are shown in Table 13. All formulations can efficiently encapsulate siRNA.

[0316] Table 13:

[0317]

[0318]

[0319] Test Example 4

[0320] In vitro transfection experiments were performed on the 16 preparations obtained in Examples 17-32. The results of real-time fluorescence quantitative PCR are shown in Table 14. All formulations can deliver the encapsulated siRNA into cells and achieve gene inhibition, among which siA4-13 has the best performance.

[0321] Table 14:

[0322] serial number active serial number active siA4-1 0.413±0.171 siA4-9 0.605±0.167 siA4-2 0.802±0.059 siA4-10 0.622±0.062 siA4-3 0.820±0.033 siA4-11 0.488±0.067 siA4-4 1.396±0.521 siA4-12 0.761±0.032 siA4-5 0.582±0.028 siA4-13 0.370±0.073 siA4-6 0.679±0.267 siA4-14 0.396±0.056 siA4-7 0.619±0.099 siA4-15 0.624±0.149 siA4-8 0.769±0.303 siA4-16 0.543±0.291

[0323] Test Example 5

[0324] The 16 preparations obtained in Examples 17-32 were verified for activity at the animal level. The experiment was conducted on C57BL / 6 mice, and 68 female mice were divided into 17 groups, which were injected with 1×PBS or a preparation at a dose of 0.5 mg / kg through the tail vein. 42 hours after administration, the animals will be fasted for 6 hours and euthanized. Liver tissue samples were collected for analysis of the expression changes of ApoB mRNA relative to animals receiving 1×PBS. ApoB mRNA expression was detected by real-time fluorescence quantitative PCR. The analysis results of the activity test were generated by Graphpad Prism 8 and are shown in Table 15. All preparations can achieve gene inhibition at the animal level, among which siA4-13 has the best performance.

[0325] Table 15:

[0326] serial number active serial number active siA4-1 0.263±0.112 siA4-9 0.628±0.200 siA4-2 0.500±0.198 siA4-10 0.295±0.086 siA4-3 0.540±0.196 siA4-11 0.228±0.068 siA4-4 0.850±0.213 siA4-12 0.433±0.101 siA4-5 0.364±0.186 siA4-13 0.133±0.057 siA4-6 0.690±0.421 siA4-14 0.213±0.094 siA4-7 0.531±0.320 siA4-15 0.509±0.133 siA4-8 0.840±0.315 siA4-16 0.424±0.154

[0327] Embodiment 33

[0328] The concentration of siFirefly was adjusted to 1000 ng / μl with DEPC-treated water and mixed with an equal volume of 50% ethanol solution to prepare a siFirefly solution containing 500 ng / μl and having an ethanol concentration of 25%.

[0329] The lipid nanoparticles numbered A4-13 obtained in Example 13 were mixed with the siFirefly solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow the siFirefly to be wrapped by the lipid nanoparticles as much as possible. Thereafter, the complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035), and dialyzed in 1×PBS for 3 hours to obtain siFLA4-13.

[0330] Test Example 6

[0331] This test example is intended to test the cytotoxicity of the siFLA4-13 preparation obtained in Example 33. The test results are shown in Table 16:

[0332] Table 16:

[0333] content Relative Vitality content Relative Vitality 1nM 0.98±0.06 50nM 0.96±0.02 5nM 0.99±0.02 100nM 0.97±0.06 10nM 0.98±0.05 200nM 0.94±0.04

[0334] Test Example 7

[0335] This test example is to test the stability of the preparation numbered siA4-13 obtained in Example 29 at 4°C. Specifically, after the preparation is prepared, the particle size of the preparation is detected by a laser particle size analyzer. After the operation is completed, it is placed at 4°C for storage. Thereafter, the particle size of the preparation is detected again on the 3rd, 7th, and 21st days. The test results are as follows: Figure 2 As shown, the preparation was proven to be stable at 4°C for at least 3 weeks.

[0336] Embodiment 34

[0337] The concentration of Cy5-siFirefly was adjusted to 1000 ng / μl with DEPC-treated water and mixed with an equal volume of 50% ethanol solution to prepare a 25% ethanol solution containing 500 ng / μl Cy5-siFirefly.

[0338] The lipid nanoparticles numbered A4-13 obtained in Example 13 were mixed with the Cy5-siFirefly solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow the Cy5-siFirefly to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035), and dialyzed in 1×PBS for 3 hours to obtain Cy-siFLA4-13. The above process must be protected from light throughout.

[0339] Test Example 8

[0340] This test example is intended to detect the metabolic characteristics of Cy-siFLA4-13 obtained in Example 34 in the liver of C57BL / 6 mice. Figure 3 As shown, the signal of Cy-siFLA4-13 in mouse liver is significantly higher than that of Cy5-siFirefly which is not coated by A4-13, indicating that A4-13 can significantly increase the accumulation of siRNA in the liver.

[0341] Test Example 9

[0342] This test example is intended to test the dose-dependent behavior of the siA4-13 preparation obtained in Example 29 in C57BL / 6 mice. The changes in ApoB mRNA expression and the changes in total cholesterol (CHO), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels are shown in Tables 17 and 18, respectively. After calculation, the half effective dose of the preparation is about 0.18 mg / kg.

[0343] Table 17:

[0344] dose mRNA relative expression 1mg / kg 0.19±0.08 0.5mg / kg 0.27±0.06 0.25mg / kg 0.38±0.15 0.1mg / kg 0.56±0.23 0.05mg / kg 0.94±0.29 0.025mg / kg 0.92±0.23 0.01mg / kg 0.84±0.24

[0345] Table 18:

[0346] dose TG CHO HDL-c LDL-c PBS 1.68±0.14 2.79±0.19 2.30±0.12 0.68±0.05 1mg / kg 0.86±0.21 1.01±0.26 0.89±0.19 0.17±0.05 0.5mg / kg 1.06±0.24 1.33±0.14 1.23±0.11 0.20±0.05 0.25mg / kg 1.11±0.21 1.73±0.25 1.58±0.19 0.31±0.09 0.1mg / kg 1.20±0.26 2.09±0.53 1.81±0.46 0.42±0.10 0.05mg / kg 1.17±0.28 2.06±0.63 1.69±0.56 0.58±0.14 0.025mg / kg 1.21±0.16 2.62±0.20 2.19±0.11 0.75±0.10 0.01mg / kg 1.47±0.19 2.57±0.16 2.09±0.14 0.64±0.08

[0347] Embodiment 35

[0348] The preparation obtained in Example 33 was concentrated using an ultrafiltration tube with a pore size of 100Kd. After the siRNA concentration was determined by the RiboGreen method, the concentration of siRNA in the preparation was adjusted to 300 ng / μl (corresponding to a dosing concentration of 3 mg / kg) and 100 ng / μl (corresponding to a dosing concentration of 1 mg / kg), respectively.

[0349] Test Example 10

[0350] This test example is designed to test the changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels at different time points after a single injection of the siA4-13 preparation obtained in Example 35 in C57BL / 6 mice. Figure 4 As shown, after 4 weeks of administration, ApoB mRNA, TG and LDL-c levels were still lower than those in the control group, supporting administration once every 4 weeks.

[0351] Embodiment 36

[0352] The preparation obtained in Example 34 was concentrated using an ultrafiltration tube with a pore size of 100Kd. After the siRNA concentration was determined by the RiboGreen method, the concentration of siRNA in the preparation was adjusted to 300 ng / μl (corresponding to a dosing concentration of 3 mg / kg) and 100 ng / μl (corresponding to a dosing concentration of 1 mg / kg), respectively.

[0353] Test Example 11

[0354] This test example is intended to test the changes in the main serum biochemical indicators of CD1 mice after receiving the preparation obtained in Example 36, and to determine the safety of the preparation. The results are as follows: Figure 5 As shown, the test experiments showed that the toxicity of the preparation met the requirements.

[0355] Embodiment 37

[0356] The concentration of siANGPTL3 was adjusted to 1000 ng / μl with DEPC-treated water and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl siANGPTL3 solution with an ethanol concentration of 25%.

[0357] The lipid nanoparticles numbered A4-13 obtained in Example 13 were mixed with the siANGPTL3 solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siANGPTL3 to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours to obtain siANA4-13. Thereafter, the concentration of siANA-13 was adjusted to 50 ng / μl to obtain siANG-H (corresponding to a dosing concentration of 0.5 mg / kg) and 25 ng / μl to obtain siANG-L (corresponding to a dosing concentration of 0.25 mg / kg).

[0358] Test Example 12

[0359] This test example is intended to test the lipid-lowering effect and ANGPTL3 mRNA expression inhibition ability of siANA4-H and siANG-L obtained in Example 36 in db / db mice. The changes in TG and CHO in mice after treatment are as follows: Figure 6 As shown, this experiment proves that the blood lipids of the model animals can be significantly reduced to the level of normal animals after treatment.

[0360] The target gene expression comparison between mice treated with siANG-H and siANG-L and mice treated with 0.5 mg / kg siNC is shown in Table 19, indicating that Figure 6 The lipid-lowering effect was caused by the inhibition of target genes.

[0361] Table 19:

[0362] Group Changes in mRNA expression siANG-H 0.03±0.02 siANG-L 0.15±0.06

[0363] Lipid deposition in the liver of animals in different treatment groups Figure 7 As shown, the results showed that the preparation had the ability to reverse lipid deposition in the liver.

[0364] Embodiment 38

[0365] The concentration of siApoC3 was adjusted to 1000 ng / μl with DEPC-treated water and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl siApoC3 solution with an ethanol concentration of 25%.

[0366] The lipid nanoparticles numbered A4-13 obtained in Example 13 were mixed with the siApoC3 solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoC3 to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035), and dialyzed in 1×PBS for 3 hours to obtain siApoCA4-13. Thereafter, the concentration of siApoCA-13 was adjusted to 50 ng / μl to obtain siApoC-H (corresponding to a dosing concentration of 0.5 mg / kg) and 25 ng / μl to obtain siApoC-L (corresponding to a dosing concentration of 0.25 mg / kg).

[0367] Test Example 13

[0368] This test example is intended to test the lipid-lowering effect and lipid changes of hApoC3 mice after treatment with siApoC-H and siApoC-L obtained in Example 37 and within 4 weeks of drug withdrawal. The changes in TG and CHO in mice after treatment are shown in the following table. Figure 8 As shown, the experiment showed that the blood lipids of the model animals were significantly reduced after treatment. When the drug was stopped, it took some time for the blood lipids to return to the level before treatment.

[0369] Preparation Example 2 Synthesis of amine-containing lipid compound A10

[0370] Synthesis of Compound A7

[0371]

[0372] Under the protection of Ar gas, 20 ml of anhydrous dichloromethane was added, and 2 g of trans-2-tridecene-1-ol and 1.71 g of 3-bromopropionyl chloride were added under stirring, and the mixture was stirred at room temperature for 2 h. After the reaction was completed by TLC detection, the dichloromethane was removed by concentration under reduced pressure, and then purified by silica gel column to obtain 2.7 g of a light yellow oil with a yield of 89%. 1H NMR (400 MHz, CDCl3) δ: 5.75-5.86 (1, m, CH=CH), 5.52-5.65 (1, m, CH=CH), 4.56-4.61 (2H, d OCH2), 3.56-3.62 (2H, t CH2), 2.89-2.97 (2H, tCH2), 2.16-2.97 (2H, m CH2), 1.22-1.45 (16H, m), 0.90 (3H, t, CH3) ppm.

[0373] Synthesis of Compound A8

[0374]

[0375] Under Ar protection, 10 ml of anhydrous DMF was added to the reaction flask, and 280 mg of N-Boc-1,3-diaminopropane hydrochloride and 721.4 mg of DIPEA were added under stirring. After stirring at room temperature for 2 hours, 1 g of compound A7 was added dropwise, and stirred at room temperature overnight. After the reaction was completed, DMF was removed by pressure concentration, and 163 mg of light yellow oil, namely compound A8, was obtained by silica gel column chromatography. The yield was 18%.

[0376] 1H NMR (400MHz, CDCl3) δ: 5.76-5.83 (2H, m, CH=CH), 5.54-5.62 (2, m, CH=CH), 4.53-4.57 (4H, d OCH2), 2.71-2.78 (4H, t CH2), 2.43-2.52 (6H, m CH2), 2.04-2.18 (4H, mCH2), 1.60-1.68 (2H, m), 1.43-1.50 (12H, m), 1.22-1.36 (32H, m), 0.84-0.90 (6H, m, CH3)ppm.

[0377] Synthesis of Compound A9

[0378]

[0379] Add 163 mg of compound A8 to 5 ml of 4M HCl ethyl acetate solution at room temperature and stir at room temperature to react. After 1 hour, the reaction was detected by TLC. No more starting material was found, indicating that the reaction was complete. The solvent was removed by concentrating under reduced pressure, and the solution was directly used for the next step reaction after concentrating with a vacuum pump for 5 minutes.

[0380] Synthesis of compound A10

[0381]

[0382] Under the protection of Ar gas, the product of the previous step was dissolved in 2ml of anhydrous dichloromethane, 45mg of triethylamine was added, and 14mg of succinyl chloride was added dropwise after stirring at room temperature for 30min. The reaction was stirred at room temperature for 1h, and the reaction was detected by TLC. The reaction solution was diluted with 10ml of dichloromethane and washed with 1M hydrochloric acid. The organic phase was collected and concentrated under reduced pressure and passed through a silica gel column. The mobile phase was DCM:MeOH=60:1 with 5‰ ammonia water (the product was not added with ammonia water and hung on the column) to obtain the product A10, 56mg of light yellow oil. The two-step yield was 37%.

[0383] 1H NMR (400MHz, CDCl3) δ: 6.85 (2H, s, NH), 5.77-5.83 (4H, m, CH=CH), 5.52-5.61 (4, m, CH=CH), 4.51-4.55 (8H, d OCH2), 3.20-3.26(4H, m), 2.71-2.77(8H, t CH2), 2.56(4H, s), 2.45-2.51(12H, m CH2), 2.07-2.15(8H, m CH2), 1.61-1.68(4H m,),1.21-1.33(64H,m),0.85-0.91(12H,m,CH3)ppm.

[0384] The theoretical value of MASS is 1239.88, and the measured value is M+1=1239.9.

[0385] Embodiment 39

[0386] The lipid A10 obtained in the preparation example was dissolved in anhydrous ethanol with DSPC, cholesterol, and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials are prepared, the four raw materials are mixed according to the mass ratio of A10: DSPC: cholesterol: DMG-PEG = 72.2: 8.1: 14.9: 4.9 and all of them are sucked into an insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer solution is prepared, and all of them are also sucked into the insulin syringe for use. After this, the insulin syringes containing the organic phase mixture and the buffer solution are placed in the same centrifuge tube and all the liquid is quickly released. The centrifuge tube is gently shaken to prepare LNP A10-1.

[0387] Embodiment 40

[0388] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 61.8: 12.1: 19.1: 7.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-2.

[0389] Embodiment 41

[0390] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 52.1: 14.6: 21.5: 11.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-3.

[0391] Embodiment 42

[0392] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 65.9: 5.9: 16.3: 11.9 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-4.

[0393] Embodiment 43

[0394] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 64.9: 10.2: 21.4: 3.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-5.

[0395] Embodiment 44

[0396] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 69.3: 15.6: 11.4: 3.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-6.

[0397] Embodiment 45

[0398] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 70.2: 5.3: 19.3: 5.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-7.

[0399] Embodiment 46

[0400] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 70.5: 9.2: 9.7: 10.6 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-8.

[0401] Embodiment 47

[0402] The four dissolved raw materials were mixed in a mass ratio of A10: DSPC: cholesterol: DMG-PEG = 69.5: 13.0: 14.3: 3.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP A10-9.

[0403] In the above examples, A10, DSPC, cholesterol, and DMG-PEG were set to 3 levels, as shown in Table 20:

[0404] Table 20:

[0405] A10 DSPC cholesterol DMG-PEG 40 8 30 1.5 50 14 45 2.5 60 20 60 5

[0406] The ratios of Examples 39-47 were designed using orthogonal design software (Orthogonal Design Assistant ii V3.1). The molar ratios and mass ratios of the nine LNPs with different ratios are shown in Table 21:

[0407] Table 21:

[0408]

[0409]

[0410] Examples 48-56

[0411] The 9 LNPs with different ratios obtained in Examples 39-47 were mixed with the siApoB solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoB to be encapsulated by the LNPs as much as possible. Thereafter, the 9 LNP / siApoB complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0412] Test Case 14

[0413] This test example aims to detect the particle size (size), polydispersity index (PDI), and surface potential (zeta) of the 9 LNPs without siApoB encapsulated obtained in Examples 39-47. The results are shown in Table 22. All preparations have the prerequisites to become excellent delivery carriers.

[0414] Table 22:

[0415] serial number size(nm) PDI zeta(mV) A10-1 86.54 0.190±0.004 33.65±5.30 A10-2 62.87±0.02 0.138±0.016 35.35±0.35 A10-3 62.58±0.03 0.123±0.027 34.10±0.28 A10-4 72.58±0.01 0.233±0.013 30.25±3.89 A10-5 72.32±0.01 0.172±0.013 32.90±1.56 A10-6 77.2 0.201±0.005 44.70±0.42 A10-7 64.41±0.01 0.100±0.010 46.65±1.06 A10-8 75.53±0.01 0.206±0.006 36.00±2.83 A10-9 79.68 0.218±0.004 39.78±2.21

[0416] Test Example 15

[0417] This test example aims to detect the particle size (size), polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE) and intraparticle siRNA concentration (conc) of the 9 siApoB-encapsulated preparations obtained in Examples 48-56. The results are shown in Table 23. All preparations have the prerequisites to become excellent delivery carriers.

[0418] Table 23:

[0419] serial number size(nm) PDI zeta(mV) EE(%) conc.(ng / μl) siA10-1 180.65±3.32 0.266±0.032 -6.05±1.76 90.0±1.1 151.1 siA10-2 136.40±1.41 0.181±0.040 -3.83±0.64 88.7±0.1 142.2±0.9 siA10-3 126.15±1.20 0.172±0.006 -3.06±0.44 86.3±0.1 134.3±0.4 siA10-4 149.75±2.19 0.163±0.027 -4.13±0.21 88.1±0.2 127.7±4.0 siA10-5 148.10±0.85 0.201±0.017 -3.88±0.13 93.1±0.4 131.6±0.2 siA10-6 153.05±0.49 0.240±0.011 -0.42±0.84 90.8±0.3 149.3±2.3 siA10-7 135.25±1.20 0.119±0.013 -5.22±0.42 87.2±0.5 120.8±2.7 siA10-8 149.05±0.92 0.188±0.014 0.09±0.19 81.3±0.8 152.1±0.5 siA10-9 164.65±2.62 0.190±0.009 -3.89±0.92 89.2±0.2 110.4±0.8

[0420] Test Example 16

[0421] The 9 preparations obtained in Examples 48-56 were subjected to in vitro transfection experiments, and the results of real-time fluorescence quantitative PCR are shown in Table 24. All preparations were able to achieve gene inhibition.

[0422] Table 24:

[0423] serial number mRNA relative expression siA10-1 0.82±0.17 siA10-2 0.89±0.24 siA10-3 0.59±0.18 siA10-4 0.70±0.32 siA10-5 0.84±0.20 siA10-6 0.85±0.04 siA10-7 0.67±0.04 siA10-8 0.60±0.10 siA10-9 0.67±0.06

[0424] Preparation Example 3 Synthesis of amine-containing lipid compound B8

[0425] Synthesis of compound B3

[0426]

[0427] 1.0 g of compound B1 (1.0 eq) was dissolved in 15 ml of anhydrous dichloromethane, 0.97 g (1.01 eq) of triethylamine was added under ice bath, and then 0.87 g (1.01 eq) of compound B2 was dropped into the system. The reaction was protected by argon. After 2 hours, TLC monitored the completion of the reaction of the raw materials. The crude product of compound B3 was directly used for the next step without treatment.

[0428] Synthesis of compound B4

[0429]

[0430] 1.92 g (2.1 eq) of triethylamine was injected into the reaction system of the previous step, and then 4.92 g of myristic acid chloride was injected into the system. The reaction was continued under the protection of argon in an ice bath. After 2 hours, the reaction of the raw material was completed by TLC monitoring. 1 M dilute hydrochloric acid was added to wash three times, and H2O was washed once. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and purified on a silica gel column (DCM / DCM:MeOH=500:1) to obtain 3.0 g of a white solid, namely compound B4. 1H NMR (400 MHz, CDCl3) δ: 6.58-5.63 (1H, m, CH=CH), 6.32-5.42 (1H, d, CH=CH), 5.68-5.73 (1, d, CH=CH), 4.18-4.30 (4H, dt NCH2), 3.64-3.74 (4H, t CH2), 2.30-2.38(4H, t CH2), 1.58-1.68(4H, m), 1.29-1.29(42H, m), 0.85-0.90(6H, m, CH3)ppm.

[0431] Synthesis of compound B5

[0432]

[0433] 1.0 g (1.0 eq) of butanediamine was dissolved in 30 ml of anhydrous dichloromethane. 5.2 g (2.1 eq) of Boc anhydride was added dropwise into the system under ice bath. The reaction was stirred for 6 h. After the reaction of the raw material was completed, 1 M dilute hydrochloric acid and saturated NaHCO3 were added in sequence for washing. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain 2.2 g of white solid powder, i.e., compound B5.

[0434] Synthesis of compound B6

[0435]

[0436] 1.0 g (1.0 eq) of compound B5 was dissolved in 20 ml of anhydrous dichloromethane. 0.55 g (3.0 eq) of NaH with a mass fraction of 60% was slowly added to the system under ice bath. After stirring for 15 min, 1.0 g (2.0 eq) of CH3I was slowly dripped into the system using a constant pressure dropping funnel. After reacting at low temperature for 1 hour, the mixture was moved to room temperature and reacted overnight. The reaction of the raw material was monitored the next day. 1 M dilute hydrochloric acid was added to quench the reaction. The DMF was removed by concentrating under reduced pressure, and then DCM was added to dissolve. The organic phase was extracted and washed with DCM / H2O. The organic phase was collected and concentrated under reduced pressure to remove the solvent. The organic phase was purified by silica gel column (PE; EA = 10:1) to obtain 3.0 g of white solid, namely compound B6. 1H NMR (400 MHz, CDCl3) δ: 3,24 (4H, m), 2.83 (6H, s, CH3), 1.46-1.51(4H, m), 1.38-1.48(18H, m)ppm.

[0437] Synthesis of compound B7

[0438]

[0439] 2.5 g (1.0 eq) of compound B6 was dissolved in 30 ml of 4 M HCl solution in EA, and the mixture was stirred at room temperature for 3 hours. The solvent was removed by concentration under reduced pressure to obtain 0.8 g of white solid powder, namely compound B7.

[0440] 1H NMR (400MHz, CDCl3) δ: 2.97 (4H, s), 2.57 (6H, s, CH3), 1.56-1.61 (4H, m)ppm

[0441] Synthesis of compound B8

[0442]

[0443] 130 mg (1.0 eq) of compound B7 was dissolved in 15 ml of isopropanol, and 142.6 mg (2.05 eq) of triethylamine was added. The reaction was stirred at room temperature for 30 min, and then 817.1 mg (2.05 eq) of compound B4 was added. The reaction was stirred at 90°C for 24 h. TLC showed that a small amount of raw material remained, so the reaction was stopped, and the solvent was removed by concentration under reduced pressure. DCM was added to dissolve the mixture, and then the mixture was washed three times with 1 M dilute hydrochloric acid and once with H2O. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The mixture was purified on a silica gel column to obtain 136 mg of compound B8 with a yield of 16%. 1H NMR (400 MHz, CDCl3) δ: 4.19-2.24 (8H, m), 3.58-3.68 (8H, m), 2.67-2.76 (4H, m), 2.54-2.62 (4H, m), 2.25-2.34 (8H, m), 2.24-2.25 (6H, m), 1.61-1.70 (8H, m), 1.43-1.50 (4H, m), 1.23-1.32 (80H, m), 0.76-0.89 (12H, t, CH3) ppm. MASS theoretical value = 1275.99, found value M+1 = 1276.0.

[0444] Embodiment 57

[0445] The lipid B8 obtained in Preparation Example 3 was dissolved in anhydrous ethanol with DSPC, cholesterol, and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials are prepared, the four raw materials are mixed according to the mass ratio of B8: DSPC: cholesterol: DMG-PEG = 70.1: 8.7: 15.9: 5.2 and all of them are sucked into the insulin syringe for standby use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer solution is prepared, and all of them are also sucked into the insulin syringe for standby use. After this, the insulin syringes containing the organic phase mixture and the buffer solution are placed in the same centrifuge tube and all the liquids are quickly released. The centrifuge tube is gently shaken to prepare LNP B8-1.

[0446] Embodiment 58

[0447] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 59.5: 12.9: 20.3: 7.4 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-2.

[0448] Embodiment 59

[0449] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 49.7: 15.4: 22.6: 12.4 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-3.

[0450] Embodiment 60

[0451] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 63.7: 6.3: 17.4: 12.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-4.

[0452] Embodiment 61

[0453] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 62.6: 10.9: 22.8: 3.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-5.

[0454] Embodiment 62

[0455] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 67.1: 16.6: 12.2: 4.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-6.

[0456] Embodiment 63

[0457] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 68.1: 5.6: 20.6: 5.6 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-7.

[0458] Embodiment 64

[0459] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 68.4: 9.9: 10.4: 11.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-8.

[0460] Embodiment 65

[0461] The four dissolved raw materials were mixed in a mass ratio of B8: DSPC: cholesterol: DMG-PEG = 67.4: 13.9: 15.3: 3.4 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP B8-9.

[0462] In the above examples, B8, DSPC, cholesterol, and DMG-PEG were set to 3 levels, as shown in Table 25:

[0463] Table 25:

[0464] B8 DSPC cholesterol DMG-PEG 40 8 30 1.5 50 14 45 2.5 60 20 60 5

[0465] The ratios of Examples 57-65 were designed using orthogonal design software (Orthogonal Design Assistant ii V3.1). The molar ratios and mass ratios of the 9 LNPs with different ratios are shown in Table 26:

[0466] Table 26:

[0467]

[0468] Examples 66-74

[0469] The 9 LNPs with different ratios obtained in Examples 57-65 were mixed with the siApoB solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoB to be encapsulated by the LNPs as much as possible. After that, the 9 LNP / siApoB complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0470] Test Example 17

[0471] This test example is intended to detect the particle size (size), polydispersity index (PDI), and surface potential (zeta) of the 9 LNPs without siApoB encapsulated obtained in Examples 57-65. The results are shown in Table 27. All preparations have good physical and chemical properties and have the potential to become excellent delivery vectors.

[0472] Table 27:

[0473]

[0474]

[0475] Test Example 18

[0476] This test example is intended to detect the particle size (size), polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE) and intraparticle siRNA concentration (conc) of the 9 siApoB-encapsulated preparations obtained in Examples 66-74. The results are shown in Table 28. All preparations have good physicochemical properties and have the potential to become excellent delivery vectors.

[0477] Table 28:

[0478] serial number size(nm) PDI zeta(mV) EE(%) conc.(ng / μl) siB8-1 184.05±0.78 0.172±0.021 -9.16±0.85 84.9±0.8 191.2±2.1 siB8-2 131.75±0.64 0.236±0.019 1.15±0.86 90.4±1.1 189.2±0.6 siB8-3 142.40±0.14 0.160±0.002 -3.08±0.04 93.2±0.8 200 siB8-4 99.19±0.38 0.191±0.001 2.13±0.23 91.1±0.3 200 siB8-5 213.30±1.98 0.332±0.046 2.33±0.15 91.3±0.3 185.1±3.7 siB8-6 212.50±2.83 0.325±0.011 6.93±0.25 89.2±0.8 200 siB8-7 186.90±0.42 0.213 2.28±0.31 92.6±1.1 193.8±0.5 siB8-8 119.70±0.57 0.179±0.041 4.53±0.93 85.8±1.4 200 siB8-9 210.75±7.85 0.309±0.033 1.91±0.44 87.2±1.0 174.4±10.6

[0479] Test Case 19

[0480] The 9 preparations obtained in Examples 66-74 were subjected to in vitro transfection experiments. The results of real-time fluorescence quantitative PCR are shown in Table 29. All preparations were able to achieve gene inhibition.

[0481] Table 29:

[0482] serial number mRNA relative expression siB8-1 0.74±0.01 siB8-2 0.96±0.07 siB8-3 0.70±0.05 siB8-4 0.80±0.12 siB8-5 0.84±0.14 siB8-6 0.49±0.04 siB8-7 0.63±0.02 siB8-8 0.66±0.05 siB8-9 0.84±0.02

[0483] Preparation Example 4 Synthesis of amine-containing lipid compound C3

[0484] Synthesis of compound C2

[0485]

[0486] Dissolve 200 mg (2.27 mmol) of 1,4-butanediamine in 10 ml of isopropanol, add 1.1 g (4.76 mmol) of compound C1, the system is colorless and transparent, heat to 70 ° C and stir, after 3 hours, white solid precipitates, continue to heat and stir for 5 hours, TLC detects that the raw material C1 is completely reacted, and the reaction is stopped. After removing isopropanol under reduced pressure, add 20 ml of n-hexane, stir at room temperature for 1 hour, and filter to obtain compound C2 (540 mg, yield 47%). 1H NMR (400 MHz, CDCl3) δ: 3.61 (2H, s), 2.58-2.65 (6H, m), 2.44-2.52 (2H, m), 1.47-1.59 (8H, m), 1.23-1.34 (40H, m), 0.73-0.88 (6H, t, CH3) ppm.

[0487] Synthesis of compound C3

[0488]

[0489] 130 mg (0.224 mmol) of compound B4 was dissolved in 5 ml of isopropanol, and then 57.49 mg (0.112 mmol) of compound C2 was added. After reacting at 90°C for 10 h, TLC detected that both raw materials B4 and C2 were completely reacted, and the reaction was stopped. The final product C3 (54 mg, yield 29%) was directly purified by silica gel column without post-treatment. 1H NMR (400 MHz, CDCl3) δ: 4.25 (8H, m), 3.56-3.67 (10H, m), 2.24-2.95 (24H, m), 1.44-1.62 (12H, m), 1.17-1.39 (124H, m), 0.76-0.91 (18H, t, CH3) ppm. Mass theoretical value = 1672.68, measured M+1 = 1673.6.

[0490] Embodiment 75

[0491] The lipid C3 obtained in Preparation Example 4 was dissolved in anhydrous ethanol with DSPC, cholesterol, and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials are prepared, the four raw materials are mixed according to the mass ratio of C3: DSPC: cholesterol: DMG-PEG = 75.5: 7.1: 13.1: 4.3 and all of them are sucked into the insulin syringe for standby use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer solution is prepared, and all of them are also sucked into the insulin syringe for standby use. After this, the insulin syringes containing the organic phase mixture and the buffer solution are placed in the same centrifuge tube and all the liquids are quickly released. The centrifuge tube is gently shaken to prepare LNP C3-1.

[0492] Embodiment 76

[0493] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 65.8: 10.9: 17.1: 6.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-2.

[0494] Embodiment 77

[0495] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 56.4: 13.3: 19.6: 10.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-3.

[0496] Embodiment 78

[0497] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 69.7: 5.3: 14.5: 10.6 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-4.

[0498] Embodiment 79

[0499] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 68.7: 9.1: 19.1: 3.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-5.

[0500] Embodiment 80

[0501] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 72.8: 13.8: 10.1: 3.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-6.

[0502] Embodiment 81

[0503] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 73.7: 4.6: 17.0: 4.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-7.

[0504] Embodiment 82

[0505] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 74.0: 8.2: 8.5: 9.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-8.

[0506] Embodiment 83

[0507] The four dissolved raw materials were mixed in a mass ratio of C3: DSPC: cholesterol: DMG-PEG = 73.1: 11.5: 12.7: 2.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP C3-9.

[0508] In the above examples, C3, DSPC, cholesterol, and DMG-PEG were set to 3 levels, as shown in Table 30:

[0509] Table 30:

[0510] C3 DSPC cholesterol DMG-PEG 40 8 30 1.5 50 14 45 2.5 60 20 60 5

[0511] The ratios of Examples 75-83 were designed using orthogonal design software (Orthogonal Design Assistant ii V3.1). The molar ratios and mass ratios of the 9 LNPs with different ratios are shown in Table 31:

[0512] Table 31:

[0513]

[0514] Examples 84-92

[0515] The 9 LNPs with different ratios obtained in Examples 75-83 were mixed with the siApoB solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoB to be encapsulated by the LNPs as much as possible. After that, the 9 LNP / siApoB complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0516] Test Example 20

[0517] This test example is intended to detect the particle size (size), polydispersity index (PDI), and surface potential (zeta) of the 9 LNPs without siApoB encapsulated obtained in Examples 75-83. The results are shown in Table 32. All preparations have good physical and chemical properties and have the potential to become excellent delivery vectors.

[0518] Table 32:

[0519]

[0520]

[0521] Test Example 21

[0522] This test example is intended to detect the particle size (size), polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE) and intraparticle siRNA concentration (conc) of the 9 siApoB-encapsulated preparations obtained in Examples 84-92. The results are shown in Table 33. All preparations have good physicochemical properties and have the potential to become excellent delivery vectors.

[0523] Table 33:

[0524] serial number size(nm) PDI zeta(mV) EE(%) conc.(ng / μl) siC3-1 197.10±0.14 0.353±0.026 -6.56±1.00 87.4±0.2 200 siC3-2 176.15±3.04 0.221±0.018 -3.82±0.01 85.3 174.2±2.0 siC3-3 112.85±0.78 0.212±0.009 1.59±0.60 83.1 200 siC3-4 152.30±0.57 0.249 -2.51±0.16 89.3±0.1 200 siC3-5 166.80±0.42 0.149±0.029 -1.95±1.47 81.4±2.6 124.8±0.8 siC3-6 302.65±5.44 0.406±0.016 -1.53±0.88 78.9±8.1 38.6±3.9 siC3-7 179.75±1.20 0.196±0.021 -2.10±0.40 91.4±2.7 81.0±1.1 siC3-8 142.25±2.33 0.187±0.022 -9.57±0.41 90.1±0.1 200 siC3-9 178.15±2.62 0.246±0.008 -0.55±0.12 78.4±5.0 46.8±1.1

[0525] Test Example 22

[0526] The 9 preparations obtained in Implementation 84-92 were subjected to in vitro transfection experiments, and the results of real-time fluorescence quantitative PCR are shown in Table 34. All preparations except siC3-2 were able to achieve gene inhibition.

[0527] Table 34:

[0528] serial number mRNA relative expression siC3-1 0.89±0.04 siC3-2 1.10±0.30 siC3-3 0.91±0.03 siC3-4 0.40±0.12 siC3-5 0.68±0.10 siC3-6 0.78±0.09 siC3-7 0.62±0.16 siC3-8 0.65±0.13 siC3-9 0.57±0.08

[0529] Embodiment 93

[0530] The lipid A4 obtained in Preparation Example 1 was dissolved in anhydrous ethanol with DOPE, cholesterol, and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials were prepared, the four raw materials were mixed according to the mass ratio of A4: DOPE: cholesterol: DMG-PEG = 45.0: 23.1: 30: 2.0 and all of them were sucked into the insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer was prepared, and all of them were also sucked into the insulin syringe for use. After that, the insulin syringes containing the organic phase mixture and the buffer were placed in the same centrifuge tube and all the liquid was quickly released. The centrifuge tube was gently shaken to prepare lipid nanoparticles mA4-1.

[0531] Embodiment 94

[0532] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=36.9:28.4:31.5:3.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-2.

[0533] Embodiment 95

[0534] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=31.3:32.1:32.5:4.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-3.

[0535] Embodiment 96

[0536] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=27.3:35.1:32.8:4.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-4.

[0537] Embodiment 97

[0538] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=47.1:18.1:30.1:4.6 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-5.

[0539] Embodiment 98

[0540] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=45.3:26.1:22.6:5.9 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-6.

[0541] Embodiment 99

[0542] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=37.0:28.5:33.3:1.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-7.

[0543] Embodiment 100

[0544] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=35.6:34.3:27.8:2.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-8.

[0545] Embodiment 101

[0546] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=49.1:15.1:30.6:5.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-9.

[0547] Embodiment 102

[0548] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=44.2:20.4:31.8:3.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-10.

[0549] Embodiment 103

[0550] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=48.4:29.8:19.3:2.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-11.

[0551] Embodiment 104

[0552] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=43.3:33.3:22.2:1.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-12.

[0553] Embodiment 105

[0554] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=52.6:13.5:31.6:2.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-13.

[0555] Embodiment 106

[0556] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=51.9:20.0:27.0:1.1 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-14.

[0557] Embodiment 107

[0558] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=49.3:25.3:21.0:4.3 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-15.

[0559] Embodiment 108

[0560] The four dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG=49.0:31.4:16.3:3.2 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mA4-16.

[0561] In the above embodiment, A4, DOPE, cholesterol, and DMG-PEG are set to 4 levels, as shown in Table 35:

[0562] Table 35:

[0563] A4 DOPE cholesterol DMG-PEG 15 10 25 0.25 20 15 32 0.5 25 20 39 0.75 30 25 45 1

[0564] The ratios of Examples 93-108 were designed using orthogonal design software (Orthogonal Design Assistant ii V3.1). The molar ratios and mass ratios of the 16 LNPs with different ratios are shown in Table 36:

[0565] Table 36:

[0566]

[0567] Examples 109-124

[0568] The concentration of luciferase mRNA (mLuc) was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl mLuc solution with an ethanol content of 25%.

[0569] The 16 lipid nanoparticles of different proportions obtained in Examples 93-108 were mixed with the mLuC solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow mLuC to be encapsulated by the lipid nanoparticles as much as possible. After that, the 16 LNP / mLuc complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1× PBS for 3 hours. The obtained mRNA-encapsulated preparations mLuA4-1–mLuA4-16 were obtained.

[0570] Test Example 23

[0571] This test example is intended to detect the in vitro mRNA expression efficiency of the 16 preparations obtained in Examples 109-124. The test results are as follows Fig. 9 The results showed that all 16 preparations could mediate the efficient expression of mRNA in cells, among which mLuA4-13 was the best.

[0572] Test Case 24

[0573] This test example is intended to detect the metabolic characteristics of mLuA4-13 obtained in Example 121 in C57BL / 6 mice. The average fluorescence signal intensity of each major organ 6 hours after the tail vein injection of mLuA4-13 is as follows: Fig.10 As shown, mA4-13 can mediate efficient expression of mRNA in vivo.

[0574] Embodiment 125

[0575] The concentration of the plasmid pCPLK1 expressing both PLK1 sgRNA and Cas9 protein was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl pCPLK1 solution with an ethanol content of 25%. The mA4-13 obtained in Example 105 was mixed with the pCPLK1 solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow pCPLK1 to be encapsulated by lipid nanoparticles as much as possible and obtain pCPA4-13. Thereafter, the pCPA4-13 complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0576] Embodiment 126

[0577] The concentration of PLK1 siRNA was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl siPLK1 solution with an ethanol content of 25%. A4-13 was mixed with siPLK1 solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siPLK1 to be encapsulated by lipid nanoparticles as much as possible and obtain siPLKA4-13. After this, the siPLKA4-13 complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours.

[0578] Test Case 25

[0579] This test example is intended to test the editing efficiency of the target gene of pCPA4-12 obtained in Example 125 at the cellular level, and compare the gene inhibition efficiency with that of siPLKA4-13 obtained in Example 126, and the results are shown in Table 37. The results show that the expression of the target gene PLK1 after cells were treated with pCPA4-12 was significantly higher than that of siPLKA4-13, but compared with the PBS control group, there was still a very significant difference, indicating that mA4-12 has a good prospect for gene editing applications.

[0580] Table 37:

[0581] Group mRNA relative expression PBS 1.00±0.02 siPLKA4-13 0.14±0.04 pCPA4-13 0.66±0.13

[0582] Embodiment 127

[0583] The lipid A4 obtained in Preparation Example 1 was dissolved in anhydrous ethanol with DOPE, cholesterol, DMG-PEG, and DOTAP (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. The five raw materials were mixed according to the mass ratio of A4: DOPE: cholesterol: DMG-PEG: DOTAP = 46.4: 12.6: 27.8: 2.0: 11.1 and all of them were sucked into an insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer solution was prepared, and all of them were also sucked into the insulin syringe for use. After that, the insulin syringes containing the organic phase mixture and the buffer solution were placed in the same centrifuge tube and all the liquid was quickly released. The centrifuge tube was gently shaken to prepare lipid nanoparticles mDA4-1.

[0584] Embodiment 128

[0585] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=40.8:11.1:24.5:1.8:21.9 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-2.

[0586] Embodiment 129

[0587] The five dissolved raw materials were mixed in a mass ratio of A4: DOPE: cholesterol: DMG-PEG: DOTAP = 35.2: 9.6: 21.1: 1.5: 32.5 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-3.

[0588] Embodiment 130

[0589] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=29.9:8.1:17.9:1.3:42.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-4.

[0590] Embodiment 131

[0591] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=24.6:6.7:14.8:1.1:52.9 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-5.

[0592] Embodiment 132

[0593] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=19.5:5.3:11.7:0.9:62.7 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into an insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-6.

[0594] Embodiment 133

[0595] The five dissolved raw materials were mixed in a mass ratio of A4: DOPE: cholesterol: DMG-PEG: DOTAP = 14.4: 3.9: 8.7: 0.6: 72.4 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-7.

[0596] Embodiment 134

[0597] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=9.5:2.6:5.7:0.4:81.8 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-8.

[0598] Embodiment 135

[0599] The five dissolved raw materials were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG:DOTAP=4.7:1.3:2.8:0.2:91.0 and sucked into an insulin syringe. Three volumes of citric acid / sodium citrate buffer were prepared and sucked into the insulin syringe and mixed with the organic phase, and gently shaken to prepare LNP mDA4-9.

[0600] Examples 136-144

[0601] The concentration of luciferase mRNA (mLuc) was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl mLuc solution with an ethanol content of 25%.

[0602] The 9 lipid nanoparticles of different proportions obtained in Examples 127-135 were mixed with the mLuC solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow mLuC to be encapsulated by the lipid nanoparticles as much as possible. After that, the 9 LNP / mLuc complexes were transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1× PBS for 3 hours. The obtained mRNA-encapsulated preparations mLuDA4-1–mLuDA4-9 were obtained.

[0603] Test Case 26

[0604] This test example is intended to test the ability of the LNP-mediated mRNA targeted delivery in mice. Fig.11The results show that the addition of DOTAP can change the distribution behavior of LNP in mice, among which mLuDA4-2 to mLuDA4-9 can achieve specific lung-targeted delivery of mRNA, and the delivery effect of mLuDA4-2 is the best. This formula overcomes the disadvantage that LNP can only effectively deliver drugs to the liver, realizes lung delivery, and expands the application range of LNP.

[0605] Examples 145-149

[0606] Glucose oxidase protein (GOx) was dissolved in DEPC water and the concentration of GOx was adjusted to 5000 μg / ml, 10000 μg / ml, 20000 μg / ml, 40000 μg / ml and 80000 μg / ml, respectively. The GOx solutions of different concentrations were mixed with equal volumes of A4-13 obtained in Example 13, respectively, so that the mass ratios of LNP to GOx were 1:1, 1:2, 1:4, 1:8 and 1:16, respectively. After that, the above-mentioned complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours to obtain the preparations GA4-1–GA4-5 encapsulating the GOx protein.

[0607] Test Case 27

[0608] This test case is designed to test the loading capacity of LNP on GOx at different mass ratios of LNP to GOx. This test case was completed by BCA method, and the results are shown in Table 38.

[0609] Table 38:

[0610] LNP:GOx(w / w) Actual GOx concentration (μg / ml) GOx theoretical concentration (μg / ml) Load factor 1：1 525 2500 0.21 1：2 675 5000 0.135 1：4 990 10000 0.099 1：8 1035 20000 0.052 1：16 1285 40000 0.032

[0611] The results showed that LNP can encapsulate GOx protein, and the process is not affected by the ratio of the two, but the loading efficiency of LNP on GOx is obviously affected by the ratio of the two.

[0612] Embodiment 150

[0613] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 156.25 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15:1 and the mass ratio of LNP to GOx 32:1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-1.

[0614] Embodiment 151

[0615] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 312.5 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 16: 1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-2.

[0616] Embodiment 152

[0617] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 625 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15:1 and the mass ratio of LNP to GOx 8:1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-3.

[0618] Embodiment 153

[0619] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 1250 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 4: 1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-4.

[0620] Embodiment 154

[0621] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 2500 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 2: 1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-5.

[0622] Embodiment 155

[0623] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 5000 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 1: 1. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-6.

[0624] Embodiment 156

[0625] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 10000 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 1: 2. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-7.

[0626] Embodiment 157

[0627] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 20,000 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 1: 4. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-8.

[0628] Embodiment 158

[0629] SiFirefly and glucose oxidase protein (GOx) were dissolved in DEPC water to a concentration of 500 ng / μl for siFirefly and 40,000 μg / ml for GOx. The above solution was mixed with A4-13 obtained in Example 13 to make the mass ratio of LNP to siRNA 15: 1 and the mass ratio of LNP to GOx 1: 8. After that, the above complex was incubated at 50°C for 20 minutes and then transferred to a dialysis bag with a molecular weight cutoff of 500,000 Daltons (Ruitaibio), and dialyzed in 1×PBS for 3 hours. The resulting preparation was siGA4-9.

[0630] Test Case 28

[0631] This test case is designed to test the ability of LNP to load protein and siRNA at the same time. This test case was completed by BCA method and RiboGreen method, and the results are shown in Table 39.

[0632] Table 39

[0633]

[0634] The results showed that LNP could load GOx protein and siRNA simultaneously.

[0635] Test Case 29

[0636] This test example is intended to test the lethality of GOx in the preparations obtained in Examples 150-158, that is, to detect whether LNP affects the function of the encapsulated GOx. This test was completed by the MTT method, and the results are as follows Fig.12 The results showed that LNP did not affect the function of GOx.

[0637] Embodiment 159

[0638] The four raw materials dissolved in anhydrous ethanol were mixed according to the mass ratio of A4: DSPC: cholesterol: DMG-PEG = 64.8: 6.9: 25.5: 2.8, and then the BET bromodomain inhibitor JQ1 was added to the mixture. JQ1 is a small molecule that can inhibit the biological activity of BRD4. After all the components were completely dissolved in ethanol, the mixture was sucked into an insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer, was prepared and also sucked into the insulin syringe for use. The insulin syringes containing the organic phase mixture and the buffer were placed in the same centrifuge tube and all the liquid was quickly released. At this time, the concentration of the small molecule JQ1 in the mixture was 1.85mg / ml. After that, the complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours. The resulting preparation is JQA4.

[0639] Test Case 29

[0640] This test case is designed to test the encapsulation efficiency of LNP for small molecule JQ1. Take 50μl JQA4 and incubate with 17μl 2% Triton-X-100 at room temperature for 30 minutes to destroy the lipid structure of LNP and release the encapsulated JQ1. The sample was frozen at -80℃ for 12 hours and then lyophilized, then dissolved in 100μl acetonitrile and detected by HPLC-MS. The peak areas of different concentrations of JQ1 (standard) and JQA4 are shown in Table 40. It was calculated that the encapsulation efficiency of LNP for JQ1 was 0.1095. It was proved that LNP can achieve the encapsulation of JQ1.

[0641] Table 40:

[0642] sample Retention time (minutes) Peak area (mAU.s) Concentration (mg / ml) Standard 1 17.93333333 48364.81209 0.5 Standard 2 17.9 26243.95475 0.25 Standard 3 17.93333333 14262.96759 0.125 Standard 4 17.93333333 7019.228105 0.0625 Standard 5 17.96666667 3727.460656 0.03125 Standard 6 17.96666667 2111.725313 0.015625 JQA4-1 17.933 20643.45596 0.202632659

[0643] Embodiment 160

[0644] The lipid A4 obtained in Preparation Example 1 was dissolved in anhydrous ethanol with cholesterol and DMG-PEG (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a concentration of 20 mg / ml. After the above materials were prepared, the three raw materials were mixed in a mass ratio of A4: cholesterol: DMG-PEG = 56.3: 11.1: 32.6 and all of them were sucked into an insulin syringe for use. In addition, three times the volume of the organic phase mixture, pH = 4.0, 50mM citric acid / sodium citrate buffer was prepared, and all of them were also sucked into the insulin syringe for use. After that, the insulin syringes containing the organic phase mixture and the buffer were placed in the same centrifuge tube and all the liquid was quickly released. The centrifuge tube was gently shaken to prepare lipid nanoparticles 3A4.

[0645] Embodiment 161

[0646] The concentration of siApoB was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl siApoB solution with an ethanol content of 25%.

[0647] The lipid nanoparticles obtained in Example 160 were mixed with the siApoB solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow siApoB to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the lipid nanoparticle / siApoB complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours to obtain si3A4.

[0648] Embodiment 162

[0649] The concentration of luciferase mRNA (mLuc) was adjusted to 1000 ng / μl and mixed with an equal volume of 50% ethanol solution to prepare a 500 ng / μl mLuc solution with an ethanol content of 25%.

[0650] The lipid nanoparticles obtained in Example 160 were mixed with the mLuc solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken and incubated at 50°C for 20 minutes to allow mLuc to be encapsulated by the lipid nanoparticles as much as possible. Thereafter, the lipid nanoparticle / mLuc complex was transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (spectrum, G235035) and dialyzed in 1×PBS for 3 hours to obtain m3A4.

[0651] Test Case 30

[0652] This test example is intended to test the particle size (size), polydispersity index (PDI), encapsulation efficiency (EE) and intra-particle siRNA concentration (conc) of the test samples obtained in Examples 160-162. The results are shown in Table 41, which proves that the preparation can efficiently encapsulate siRNA and mRNA.

[0653] Table 41

[0654] serial number size(nm) PDI EE(%) conc.(ng / μl) 3A4 123.12±0.03 0.257±0.080 / / si3A4 134.64±0.25 0.182±0.019 92％ 125.8 mA4-2 139.85±0.88 0.171±0.003 89％ 102.1

[0655] Test Case 31

[0656] This test example is intended to test the ability of si3A4 obtained in Example 161 to inhibit the expression of target mRNA at the cellular level. The results are shown in Table 42, which proves that at the cellular level, the performance of si3A4 is comparable to that of the commercial professional reagent Lipo.

[0657] Table 42

[0658] serial number active Lipo / siApoB 0.823±0.066 si3A4 0.853±0.221

[0659] Test Case 32

[0660] This test example is intended to test the mRNA delivery ability of m3A4 obtained in Example 162 at the cellular level. The results are shown in Table 43, which proves that at the cellular level, the performance of si3A4 is comparable to that of the commercial professional reagent Lipo.

[0661] Table 43

[0662] serial number Mean fluorescence signal intensity Lipo / siApoB 6730.2±234.0 m3A4 6624.9±365.2

[0663] Comparative Example 1

[0664] This comparative example is intended to compare the stability of lipid A4 obtained in Preparation Example 1 with the control lipid E. The chemical structure of lipid E ​​is as follows: Fig.13 shown.

[0665] Lipid A4 and lipid E ​​were dissolved in a solvent containing 90% anhydrous ethanol and 10% citrate (w / w) with a concentration of 0.05M and a pH of 4.0. 100 μl of the sample was immediately taken as the pre-incubation sample and immediately stored at -80°C to prevent sample degradation. This day is the 0th day of the experiment. Thereafter, the A4 and E test samples were placed at 40°C for continuous incubation, and 100 μl of the sample was taken on the 1st, 3rd, 5th and 7th days of the experiment and stored at -80°C. After all samples were obtained, the stability of the samples was tested by HPLC. The test results are as follows: Fig.14 The test conditions are as follows:

[0666] Table 44:

[0667] parameter set up Column Type Waters XBridge C8: 186006050 Flow rate 1ml / min Column temperature 50℃ High voltage limit 10000psi Sample temperature 10℃ Move Item A 0.05% TFA-Water Move Item A 0.05% TFA-33% ACN-67% IPA Testing time 16 minutes

[0668] Table 45:

[0669]

[0670] Comparative Example 2

[0671] Lipofectime 2000 (Lipo) is a commercial transfection reagent that can mediate efficient nucleic acid delivery at the cellular level, but it can hardly achieve effective delivery at the animal level. This experiment aims to compare the efficacy of siA4-13 in C57 mice (dosage is 0.5 mg / kg). The relative expression of Lipo / siApoB is 0.85, and the relative expression of siA4-13 is 0.27. The results are shown in Fig.15 The experimental results show that Lipo has low delivery efficiency in vivo, while A4-13 can achieve efficient siRNA delivery.

[0672] Comparative Example 3

[0673] This test case is designed to detect the cellular uptake efficiency of Cy-siFLA4-13. The test analysis results are shown in Table 46, indicating that the delivery efficiency of the preparation at the cellular level is comparable to that of commercial transfection reagents. Considering that Lipo cannot achieve efficient nucleic acid delivery at the animal level, A4-13 has obvious application potential advantages in the field of nucleic acid delivery.

[0674] Table 46:

[0675] Group Mean fluorescence signal intensity Positive rate (%) Mock 4.3±2.3 0.4±0.1 Lipo / Cy5-sifirefly 5370.5±313.2 91.9±3.9 Cy-siFLA4-13 4502.5±477.3 90.4±4.2

[0676] Comparative Example 4

[0677] This comparative example is intended to compare the endocytosis efficiency mediated by the complex obtained after Lipo encapsulated mRNA and mLuA4-13 at the cellular level. The test results are shown in Table 47, indicating that both Lipo and mLuA4-13 enable almost all cells to successfully endocytose luciferase mRNA, and Lipo achieves a higher average fluorescence signal intensity, that is, a single cell endocytoses more luciferase mRNA. However, considering that Lipo cannot achieve efficient nucleic acid delivery at the animal level, mA4-13 still has a broader range of applications.

[0678] Table 47:

[0679] Group Mean fluorescence signal intensity Positive rate (%) Mock 5.3±1.9 0.8±0.2 Lipo / mLuc 6262 98.85±0.07 mLuA4-13 4381 98.25±0.07

[0680] Comparative Example 5

[0681] This comparative example is intended to compare the delivery efficiency mediated at the cellular level by liposome E obtained by using lipid E ​​as the core lipid, and further incubated with siApoB to obtain siE, and siA4-13 obtained in Example 29. The results are shown in Table 48, indicating that siA4-13 can achieve efficient target gene inhibition at the cellular level, while siE can hardly achieve target gene inhibition at the cellular level.

[0682] Table 48:

[0683] Group mRNA relative expression iE 0.891±0.072 siA-13 0.402±0.066

[0684] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0685] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A compound or a stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound has one of the following structures:

2. A liposome, characterized in that: The liposome is made of the compound according to claim 1.

3. The liposome according to claim 2, characterized in that Further comprising at least one selected from the group consisting of: a hydrophobic lipid, an amphiphilic lipid; Wherein, the amphiphilic lipid comprises at least one selected from neutral lipids, cationic lipids and PEG-lipids, and the hydrophobic lipid is selected from sterols.

4. The liposome according to claim 3, characterized in that The neutral lipid includes at least one selected from the following: distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylcholine, diethyl pyrocarbonate, dimyristoylphosphatidylcholine and egg yolk lecithin.

5. The liposome according to claim 3, characterized in that The cationic lipid is selected from at least one of the following: trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium chloride, didodecyldimethylammonium bromide, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-bromide, dimethyl-2-hydroxyethyl ...2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-bromide, dimethyl-2- -Hydroxybutyl-2,3-dioleyloxypropylammonium, dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dixadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide, N-(2-spermine formyl)-N',N'-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, lipid poly-L-lysine, stearammonium.

6. The liposome according to claim 3, characterized in that The PEG-lipid comprises at least one selected from the group consisting of dipalmitoylphosphatidylethanolamine-PEG, distearoylphosphatidylethanolamine-PEG, dimyristylglycerol-PEG and dimethacrylate-PEG.

7. The liposome according to claim 3, characterized in that The sterol is cholesterol.

8. The liposome according to claim 3, characterized in that The amount of the compound is 3 to 80% mol / mol based on the liposome.

9. The liposome according to claim 3, characterized in that The amount of the compound is 20 to 80% mol / mol based on the liposome.

10. The liposome according to claim 3, characterized in that The molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.01-20).

11. The liposome according to claim 3, characterized in that The molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (40-61):(8-20):(30-60):(0.5-5).

12. The liposome according to claim 3, characterized in that The molar ratio of the compound, sterol and PEG-lipid is (20-80):(5-50):(10-60).

13. The liposome according to claim 3, characterized in that The molar ratio of the compound, sterol and PEG-lipid is (40-70):(5-20):(20-40).

14. The liposome according to claim 3, characterized in that The amount of the compound is 3 to 50% mol / mol based on the liposome.

15. The liposome according to claim 3, characterized in that The molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-50): (1-20): (2-50): (0.01-10): (5-92).

16. The liposome according to claim 3, characterized in that The molar ratio of the compound, neutral lipid, sterol, PEG-lipid and cationic lipid is (3-41): (1-12): (2-45): (0.05-2): (10-90).

17. A method for preparing the liposome according to any one of claims 2 to 16, characterized in that: include: A predetermined amount of the compound of claim 1 is mixed with an organic solvent to obtain the liposome.

18. The method according to claim 17, characterized in that Further including: A predetermined amount of a hydrophobic lipid, an amphiphilic lipid and / or a buffering agent is mixed with the solution obtained in claim 17 to obtain the liposome.

19. A drug carrier comprising the compound according to claim 1 or the liposome according to any one of claims 2 to 16.

20. Use of the compound of claim 1, the liposome of any one of claims 2 to 16, or the drug carrier of claim 19 in the preparation of a drug for targeted delivery to the lung.

21. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a carrier and a pharmaceutically active ingredient, and the carrier includes the pharmaceutical carrier according to claim 19.

22. The pharmaceutical composition according to claim 21, characterized in that The pharmaceutically active ingredient comprises at least one selected from the following: DNA molecules, RNA molecules, proteins, peptides and small molecule drugs.

23. The pharmaceutical composition according to claim 21, characterized in that The pharmaceutically active ingredient is negatively charged or hydrophobic.

24. The pharmaceutical composition according to claim 21, characterized in that The mass ratio of the carrier to the active ingredient of the medicine is (5-50):

1.

25. The pharmaceutical composition according to claim 21, characterized in that The mass ratio of the carrier to the active ingredient of the medicine is (5-40):

1.

26. The pharmaceutical composition according to claim 21, characterized in that The mass ratio of the carrier to the active ingredient of the medicine is (8-20):

1.

27. The pharmaceutical composition according to claim 21, characterized in that The mass ratio of the carrier to the active ingredient of the medicine is 15:

1.

28. The pharmaceutical composition according to claim 21, characterized in that The pharmaceutical active ingredient contains nucleic acid.

29. A transfection complex, characterized in that The transfection complex comprises the compound according to claim 1 or the liposome according to any one of claims 2 to 16.

30. The transfection complex according to claim 29, characterized in that The transfection complex comprises at least one biologically active agent.

31. The transfection complex according to claim 30, characterized in that The bioactive agent comprises at least one selected from the following: DNA molecules, RNA molecules, proteins, peptides and drugs.

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