Compounds, liposomes and uses thereof

Abstract

The present invention provides compounds, liposomes, methods for preparing liposomes, drug carriers, drug compositions and uses of the drug compositions for treating or preventing hyperlipidemia and its related diseases.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine, and specifically relates to compounds, liposomes, drug carriers and their uses, methods for preparing liposomes, drug compositions and uses. [Background technology]

[0002] Nucleic acid molecules such as siRNA and mRNA are highly potent drug molecules that must enter cells to function. However, nucleic acid molecules themselves are large, negatively charged, and easily degraded compounds, making them unable to enter cells by themselves. Therefore, they require suitable delivery vehicles to transfect cells or tissues.

[0003] Liposomes have already proven to be effective nucleic acid carriers, and the liposome-encapsulated siRNA drug Onpattro has been released. With the advancement of research into nucleic acid-based drugs, particularly siRNA drugs and mRNA drugs, there is a continuing demand for research into nucleic acid drug carriers, including liposomes that target different cell and tissue types, liposomes that better protect nucleic acids from enzymatic degradation or improve intracellular release, and liposomes that are less toxic and easier to biodegrade.

[0004] Therefore, there is a need to research and develop liposomes that are highly stable, less toxic, and easily biodegradable. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention solves, at least to some extent, one of the technical problems in the related art. [Means for solving the problem]

[0006] Therefore, in a first aspect, the present invention proposes a compound of formula (I) or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of a compound of formula (I), [ka] wherein X, Y, R1, R2, and R3 have the definitions as set forth in the present invention.

[0007] According to an embodiment of the present invention, each X and Y is independently selected from -CH2- or -CO-, provided that X and Y are not simultaneously -CH2- or -CO-, provided that each X is the same or different, each Y is the same or different, and R2 is H, optionally substituted C1-C 20 Alkyl groups, optionally substituted C1-C 20 Alkenyl group or optionally substituted C-C 20 alkynyl groups, R3 is H, optionally substituted C1-C 20 Alkyl groups, optionally substituted C1-C 20 Alkenyl group or optionally substituted C-C 20 alkynyl groups, wherein each substituent is independently selected from H, F, Cl, Br, CN, and NO2; and R1 is [ka] or [ka] where n is an integer selected from 0-10, and Z is [ka] or [ka] and R4 is an optionally substituted C4-C 20 Alkyl groups, optionally substituted C4-C 20 Alkenyl group or optionally substituted C4-C 20alkynyl groups, wherein each substituent is independently selected from H, F, Cl, Br, CN, and NO2.

[0008] According to an embodiment of the present invention, R2 is selected from H, CH3-, or R1, R3 is selected from H, or R1, and R1 is [ka] or [ka] where n is an integer selected from 1 to 5, and Z is [ka] or [ka] and R4 is C4-C 20 Alkyl groups, C4-C 20 Alkenyl group or C4-C 20 alkynyl groups, provided that the C4-C 20 The alkenyl group contains 1-3 double bonds, and the C4-C20 alkynyl group contains 1-3 triple bonds.

[0009] According to an embodiment of the present invention, R4 is C5-C 15 Alkyl groups, C5-C 15 alkenyl groups, provided that the C5-C 15 Alkenyl groups contain 1 to 3 double bonds.

[0010] According to an embodiment of the present invention, R1, R2 and R3 are the same, and R4 is C 10 -C 15 Alkyl group, C 10 -C 15 alkenyl groups, provided that the C 10 -C 15 Alkenyl groups contain 1 to 3 double bonds.

[0011] According to an embodiment of the present invention, the compound is [ka] It has one of the following structures.

[0012] According to an embodiment of the present invention, the compound skeleton comprises a backbone structure of four amino groups and a plurality of branched alkyl or substituted alkyl groups. In the backbone, each amino group is connected to an alkyl or acyl group, of which there are at least two acyl groups, thereby reducing the basicity of the compound, reducing the effect of the basicity of the alkylamine group on the chemical stability of the ester group contained in the branched chain, and reducing the electropositive charge of the compound, which is advantageous for reducing the electropositive charge of liposomes, while the amide bond enhances metabolism in the body and prevents the compound from accumulating in the body.

[0013] In a second aspect, the present invention proposes the use of the above compound in the preparation of 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.

[0014] According to an embodiment of the present invention, the compound can be used to prepare liposomes in the form of a polymer, can form a covalent bond with other substances to prepare liposomes, or can undergo a chemical reaction with other substances to prepare liposomes. According to an embodiment of the present invention, the specific method for preparing liposomes from the compound is not limited, but the use of the present invention can be achieved by using the compound to prepare liposomes, and the liposomes contain all or part of the structure of the compound.

[0015] In a third aspect of the present invention, the present invention provides liposomes. The liposomes according to the present invention are prepared from the compounds according to the first aspect of the present invention, and the liposomes can encapsulate the added substance to be loaded by the fluidity of the lipid bilayer, and have a good encapsulation effect, so that the substance can effectively enter the cell and escape from the encapsulated body into the cytoplasm.

[0016] According to an embodiment of the present invention, the compound is capable of self-assembling with hydrophobic lipids and / or amphiphilic lipids to form liposomes.

[0017] According to an embodiment of the present invention, the liposome further comprises a hydrophobic lipid, an amphipathic lipid, a buffering agent and / or an organic solvent, wherein the amphipathic lipid comprises at least one selected from a neutral lipid and a PEG-lipid, and the hydrophobic lipid comprises a sterol.

[0018] According to an embodiment of the present invention, the neutral lipid comprises at least one selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), dilaurylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristylphosphatidylcholine (DMPC), and egg yolk lecithin (EPC).

[0019] According to an embodiment of the present invention, the PEG-lipid comprises at least one selected from bispalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), bisstearylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol (DMG-PEG), and dimethacrylate (DMA-PEG).

[0020] According to an embodiment of the present invention, the sterol is cholesterol.

[0021] According to an embodiment of the present invention, the buffering agent is an acidic buffering agent.

[0022] According to an embodiment of the present invention, the buffer solution comprises at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trimethylolaminomethane-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.

[0023] According to an embodiment of the present invention, the organic solvent includes at least one selected from methanol, ethanol, isopropyl alcohol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl ethyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, and triethanolamine.

[0024] According to an embodiment of the present invention, the amount of said compound is 20-80% mol / mol based on said liposome.

[0025] According to an embodiment of the present invention, the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.2-20).

[0026] According to an embodiment of the present invention, the volume of the buffer reagent is 50-90% v / v based on the liposome.

[0027] According to an embodiment of the present invention, the volume of the buffer reagent is 75% v / v, based on the liposomes.

[0028] According to the embodiment of the present invention, the liposomes having the above composition ratio have high transfection efficiency, good encapsulation effect, and strong drug presentation ability.

[0029] In a fourth aspect, the present invention provides a method for preparing the liposomes. According to an embodiment of the present invention, the method comprises mixing a predetermined amount of the compound according to the first aspect of the present invention with an organic solvent to obtain the liposomes. According to an embodiment of the present invention, dissolving the compound in an organic solvent is advantageous for the formation of liposomes.

[0030] According to an embodiment of the present invention, the method further comprises dissolving a predetermined amount of lipid in the organic solution and then mixing it with a buffer reagent to obtain the liposomes. According to an embodiment of the present invention, based on the liposomes, 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), and the volume of the buffer reagent is 75% v / v based on the liposomes.

[0031] In a fifth aspect of the present invention, the present invention provides a drug carrier, which comprises the compound according to the first aspect of the present invention or the liposome according to 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 that can carry a negatively charged drug, deliver the drug into cells, and allow the drug to effectively escape from the encapsulation into the cytoplasm, which is advantageous for the drug to exert its efficacy.

[0032] In a sixth aspect of the present invention, the present invention proposes the use of a compound according to the first aspect of the present invention, a liposome according to the third aspect of the present invention or a drug carrier according to the fifth aspect of the present invention in the preparation of a drug.

[0033] In a seventh aspect of the present invention, the present invention provides a pharmaceutical composition, comprising a carrier and an active pharmaceutical ingredient, wherein the carrier comprises the drug carrier according to the fifth aspect of the present invention. According to an embodiment of the present invention, the drug carrier is an ionizable carrier, capable of carrying a negatively charged active pharmaceutical ingredient, and delivering the active pharmaceutical ingredient into cells so that the active pharmaceutical ingredient can effectively escape from the encapsulation body into the cytoplasm, which is advantageous for the exertion of the carried active pharmaceutical ingredient.

[0034] According to an embodiment of the present invention, the active pharmaceutical ingredient comprises at least one selected from a DNA molecule, an RNA molecule, a protein, and a small molecule drug.

[0035] According to an embodiment of the present invention, the active pharmaceutical ingredient is negatively charged. According to an embodiment of the present invention, the drug carrier comprises an ionizable amine-containing lipid, which is positively charged at a specific pH and can bind to the negatively charged active pharmaceutical ingredient, and the fluidity of the lipid bilayer can encapsulate the active pharmaceutical ingredient and deliver it to cells.

[0036] According to an embodiment of the present invention, the active pharmaceutical ingredient comprises a nucleic acid, which may include DNA, RNA, a nucleic acid-protein complex, a nucleic acid-lipid complex, a nucleic acid-nuclide complex, etc., and the type of the nucleic acid is not limited.

[0037] According to an embodiment of the present invention, the mass ratio of the carrier to the active pharmaceutical ingredient is (5-40):1.

[0038] According to an embodiment of the present invention, the mass ratio of the carrier to the active pharmaceutical ingredient is (8-20):1.

[0039] According to an embodiment of the present invention, the mass ratio of the carrier to the active pharmaceutical ingredient is 15:1.

[0040] In an eighth aspect, the present invention provides a transfection complex comprising the above-mentioned compound or the above-mentioned liposome.

[0041] According to an embodiment of the present invention, the transfection complex comprises at least one bioactive agent.

[0042] According to an embodiment of the present invention, the bioactive agent comprises at least one selected from a DNA molecule, an RNA molecule, a protein, and a drug.

[0043] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present invention. [Brief explanation of the drawings]

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] 10 shows the results of a gel block experiment according to an embodiment of the present invention. [Figure 2] 1 shows the stability results at 4° C. of a formulation of siA4-13 according to an example of the present invention. [Figure 3] 1 shows quantitative data on the distribution of Cy-siFLA4-13 in the body of a C57BL / 6 mouse according to an embodiment of the present invention. [Figure 4] Changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels after administration of different concentrations of siA4-13 formulations of an embodiment of the present invention to animals or at different time points, where Figure a shows the changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels at different time points after administration of 1 mg / kg or 3 mg / kg siA4-13 formulations to animals, and Figure b shows the changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels 48 hours after administration of siA4-13 formulations at doses ranging from 0.01 mg / kg to 1 mg / kg to animals. [Figure 5] 1 shows changes in serum biochemical indices according to an embodiment of the present invention. [Figure 6] 1 shows the lipid-lowering effect and ANGPTL3 mRNA expression suppression ability of siANA4-13 according to an embodiment of the present invention in db / db mice. [Figure 7] 1 shows the lipid deposition status in the liver of animals in different treatment groups according to an embodiment of the present invention. [Figure 8] 1 shows the lipid-lowering effect and blood lipid changes within 4 weeks after treatment with siApoCA4-13 according to an embodiment of the present invention and discontinuation of the drug. [Figure 9]1 shows the results of luciferase mRNA expression in cells mediated by different formulations of liposomes according to an embodiment of the present invention. [Figure 10] 1 shows the results of in vivo expression of luciferase mRNA mediated by liposomes according to an embodiment of the present invention in mice. [Figure 11] 1 is a chemical structure of lipid E according to an embodiment of the present invention. [Figure 12] 1 shows the stability test results of lipid A4 and lipid E at 40° C. according to an embodiment of the present invention. [Figure 13] 1 shows a comparison of the efficacy of Lipo and A4-13 in C57 mice according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0045] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings, examples of which are shown in the accompanying drawings. The following description of the preferred embodiments is for illustrative purposes only and is not intended to limit the present invention, but rather to illustrate the present invention.

[0046] The terms "first" and "second" are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the number of technical features. Thus, a feature qualified as "first" or "second" can expressly or imply the inclusion of at least one of the feature. In the description of the present invention, unless otherwise expressly and specifically limited, the concept of "plurality" is at least two, e.g., two or three.

[0047] Some embodiments of the present invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulae. The present invention is intended to cover all alternatives, modifications, and equivalent technical solutions, which are included within the scope of the present invention as defined by the claims. Those skilled in the art should recognize that many methods and materials similar or equivalent to the present invention can be used to practice the present invention. The present invention is not limited to the methods and materials described in the present invention. In the event that one or more of the references, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terms, application of terms, described techniques, etc.), the present application shall prevail.

[0048] Furthermore, it should be appreciated that certain features of the invention, which are, for clarity, described in the context of multiple separate embodiments, may also be provided in combination in a single example. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided alone or in any suitable subcombination.

[0049] Unless otherwise indicated, technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art of the present invention, and unless otherwise indicated, all patent publications cited in the disclosure of the entire contents of the present invention are incorporated by reference in their entirety into this disclosure.

[0050] The present invention applies the following definitions unless otherwise indicated. For the purposes of this invention, the chemical elements are defined according to the Periodic Table, CAS version, and the Chemical Handbook, 75, th Ed., 1994. General principles of organic chemistry are also referred to 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 the entire contents of this invention are incorporated by reference.

[0051] As used herein, "liposome" or "lipid nanoparticle (LNP)" refers to a drug-carrying system in which a drug or other bioactive substance is dissolved or encapsulated in the lipid core, or adsorbed or attached to the surface of the nanoparticle, using a biocompatible lipid material as a carrier.

[0052] The present invention further includes isotopically labeled compounds of the present invention, which are the same as the compounds described in the present invention except for the fact that one or more atoms are replaced with an atom whose atomic mass or mass number is different from the mass or mass number of the common atom in nature. Exemplary isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H, 3H, 13C, 14C, 15N, 16O, 17O, 31P, 32P, 36S, 18F, and 37Cl.

[0053] Compounds of the present invention and pharmaceutically acceptable salts of said compounds containing the aforementioned isotopes and / or other isotopes of other atoms are all within the scope of the present invention. Isotopically labeled compounds of the present invention, for example, radioactive isotopes such as H and C, can be incorporated into the compounds of the present invention for use in drug and / or substrate tissue distribution assays. Tritium-substituted, i.e., H, and carbon-14, i.e., C, isotopes are particularly preferred due to their ease of preparation and detectability. However, substitution with heavy isotopes such as deuterium, i.e., H, can offer several therapeutic advantages resulting from increased metabolic stability, such as increased half-life in the body or reduced dosage requirements, and therefore may be preferred in some cases.

[0054] The stereochemical definitions and conventions used herein generally follow those of S.P. 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 or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and transatropisomers, and mixtures thereof, such as racemic mixtures, are expected to be included within the scope of the present invention. Many organic compounds exist in optically active forms, i.e., have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to the chiral center(s) in the molecule. The prefixes d and l or (+) and (-) are symbols used to designate the rotation of plane-polarized light by a compound, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are similar except that they are mirror images of each other. Specific stereoisomers can also be referred to as enantiomers, and mixtures of such isomers are commonly referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when a chemical reaction or process lacks stereoselectivity or stereospecificity.

[0055] Depending on the selection of raw materials and methods, the compounds of the present disclosure may exist as one of the possible isomers or as a mixture thereof, e.g., a pure optical isomer, or as a mixture of isomers, e.g., racemates and diastereomeric mixtures, as determined by the number of asymmetric carbon atoms. Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations or resolved using conventional techniques. If the compound contains one double bond, the substituents may be in the E- or Z-configuration, and if the compound contains a disubstituted cycloalkyl, the cycloalkyl substituents may be in the cis- or trans-configuration.

[0056] The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including, but not limited to, diastereomers, enantiomers, transatropisomers, and geometric (or conformational) isomers, and mixtures thereof, such as racemic mixtures, are expected to be included within the scope of the present invention.

[0057] Unless otherwise indicated, structures depicted in the present invention represent all isomeric (e.g., enantiomeric, diastereomeric, transatropisomers, and geometric (or conformational)) forms encompassed by the structure, such as the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the invention.

[0058] The term "tautomer" or "tautomeric form" refers to structural isomers that can be interconverted by a low energy barrier, with different energies. When tautomerism is possible (e.g., in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton transfer, such as ketone-enol isomerization and imi-enamine isomerization. Valence bond tautomers include interconversions via recombination of some bond-forming electrons. A specific example of ketone-enol tautomerism is the tautomerism between pentane-2,4-dione and 4-hydroxypentyl-3-en-2-one. Another example of tautomerism is phenol-ketone tautomerism. One specific example of phenol-ketone tautomerism is the tautomerism between pyridin-4-ol and pyridin-4(1H)-ketone. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0059] 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. Specific examples of N-oxides are the N-oxides of tertiary amines or the N-oxides of nitrogen atoms in nitrogen-containing heterocycles. N-oxides are formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) (see Advanced Organic Chemistry, Wiley Interscience, 4th Edition, Jerry March, pages 1977). In particular, N-oxides can be prepared by the method of L.W. Ready (Syn. Comm. 1977, 7, 509-514), which involves reacting an amine compound with m-chloroperbenzoic acid (MCPBA) in an inert solvent such as methylene chloride.

[0060] The term "solvate" as used herein refers to an association formed between one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropyl alcohol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed between solvent molecules and water.

[0061] As used herein, the term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are known in the art, and are described, for example, in SM Berge et al., "Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19." Salts derived from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and perchlorate, which are formed by reaction with an amino group, and organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, and malonate, or these salts can be obtained by other methods described in the literature, such as ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutacate, glucoheptanoate, glyceryl phosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethylsulfonate, lactate, and the like. Salts obtained with appropriate bases include, but are not limited to, thiaminaldehyde, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, methylenebishydroxynaphthalenate, pectinate, persulfate, 3-phenylpropionate, picrate, tetradecanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. Salts obtained with appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-4 alkyl)4 salts. The present invention also contemplates any quaternary ammonium salts of compounds containing an N group. Water-soluble or oil-soluble or dispersible compounds can be obtained by quaternary ammonium formation. Alkali metal and alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.Pharmaceutically acceptable salts further include suitable non-toxic ammonium and quaternary ammonium salts, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates and aromatic sulfonates, and amine cations consisting of anti-equilibrium ions.

[0062] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present invention may be present in racemic or enantiomerically enriched form, such as the (R)-, (S)-, or (R,S)-configuration. In some 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. Where possible, substituents on atoms having unsaturated double bonds may be present in cis-(Z)- or trans-(E)-form.

[0063] Thus, as described herein, the compounds of the present invention can exist in one form or a mixture of possible isomers, spin isomers, hindrance isomers, tautomers, for example in the form of substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0064] Depending on the physicochemical differences of the components, the resulting mixture of isomers can be separated into pure or nearly pure geometric or optical isomers, diastereomers, racemates, etc. by chromatography and / or stepwise crystallization.

[0065] The racemates of the final product or intermediate obtained by known methods can be resolved into their optical enantiomers in a manner well known to those skilled in the art, for example by separating the diastereomeric salts obtained therefrom. Racemic products 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 Aube, 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., University of Notre Dame Press, Notre Dame, IN 1972).

[0066] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as the general formula compounds described above, or specific examples, subclasses, and compounds included in the examples. The term "optionally substituted" can be used interchangeably with the term "substituted or unsubstituted." The terms "optionally," "optional," or "optionally" mean that the event or circumstance described below may occur, but does not necessarily occur, and this description includes both cases where this event or circumstance occurs and cases where this event or circumstance does not occur. In general, the term "optionally," whether preceded by the term "substituted," indicates that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise specified, one optional substituent may be substituted at each substitutable position of the group. When multiple positions in a given structural formula can be substituted with one or more substituents selected from a specific group, the substituents, whether the same or different, may be substituted at each position. The substituents may be F, Cl, Br, CN, N, OH, NH, NO, oxo (=O), etc.

[0067] The term "N3" denotes an azido structure. Such a group may be connected to other groups, for example, to a methyl group to form an azido methane (MeN3) or to a phenyl group to form an azido benzene (PhN3).

[0068] Furthermore, unless otherwise specified, the descriptive methods used in the present invention "respectively...independently," "respectively...independently," and "independently..." are interchangeable and should be understood in a broad sense, and may mean that specific options represented by the same symbol in different groups do not affect each other, or that specific options represented by the same symbol in the same group do not affect each other.

[0069] In various parts of this specification, the substituents of the compounds disclosed in the present invention are disclosed according to group types or ranges. In particular, it is pointed out that the present invention is each independent subcombination of each member of these group types or ranges. For example, "C 1-6 The term "alkyl group" specifically refers to methyl, ethyl, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, and C6 alkyl groups, as disclosed independently.

[0070] In each section of the present invention, connecting substituents are described. When the structure clearly requires a linking group, the Kusch variable listed for that group should be understood to be the linking group. For example, when the structure requires a linking group and an "alkyl group" or "aryl group" is listed for the Kusch variable definition, the "alkyl group" or "aryl group" represents the connected alkylene group or arylene group, respectively.

[0071] As used herein, the term "alkyl" or "alkyl group" refers to a monovalent hydrocarbon group containing a saturated straight or branched chain of 1-20 carbon atoms. Unless otherwise specified, alkyl groups contain 1-20 carbon atoms, and in some embodiments, alkyl groups contain 1-10 carbon atoms, in other embodiments, alkyl groups contain 1-9 carbon atoms, in other embodiments, alkyl groups contain 1-8 carbon atoms, in other embodiments, alkyl groups contain 1-6 carbon atoms, in other embodiments, alkyl groups contain 1-4 carbon atoms, in other embodiments, alkyl groups contain 1-3 carbon atoms, in other embodiments, alkyl groups contain 10-20 carbon atoms, in other embodiments, alkyl groups contain 10-18 carbon atoms, in other embodiments, alkyl groups contain 10-16 carbon atoms, in other embodiments, alkyl groups contain 10-14 carbon atoms, and in other embodiments, alkyl groups contain 10-13 carbon atoms.

[0072] Examples of alkyl groups are 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 (-CH2CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH2CH3), 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( Examples of alkyl groups include, but are not limited to, 3-methyl-3-pentyl (-C(CH)(CHCH)), 2-methyl-3-pentyl (-CH(CHCH)CH(CH)), 2,3-dimethyl-2-butyl (-C(CH)CH(CH)), 3,3-dimethyl-2-butyl (-CH(CH)C(CH)), n-heptyl, n-octyl, and the like, wherein said alkyl groups may independently be unsubstituted or substituted with one or more substituents according to the present invention.

[0073] As used herein, the term "alkyl group" and its prefix "alkyl" are inclusive of both straight and branched saturated carbon chains.

[0074] The term "alkenyl group" refers to a straight or branched chain monovalent hydrocarbon radical 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 C-C is sp2 double bond unsaturated, and wherein alkenyl groups may be independently unsubstituted or substituted with one or more substituents according to the invention, including groups having "cis," "trans," or "Z" and "E" positions, where specific examples include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like.

[0075] The term "alkynyl group" refers to a straight or branched chain monovalent hydrocarbon radical 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 C-C contains sp triple bond unsaturation, and wherein the alkynyl group can be independently unsubstituted or substituted with one or more substituents according to the invention; specific examples include, but are not limited to, ethynyl (-C≡CH), propargyl (-CHC≡CH), 1-propynyl (-C≡C-CH), and the like.

[0076] The terms "comprise" and "include" are non-limiting expressions, ie, include the subject matter set forth in the present invention, but do not exclude the subject matter of other embodiments.

[0077] As used herein, 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, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, coloring agent, or combination thereof, which carriers are well known to those skilled in the art (e.g., as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, it is included for use in therapeutic or pharmaceutical compositions.

[0078] compound In a first aspect, the present invention provides a compound of formula (I) or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of a compound of formula (I), [ka] wherein X, Y, R1, R2, and R3 have the definitions as set forth in the present invention.

[0079] In some embodiments, each X and Y is independently selected from -CH2- or -CO-, provided that X and Y are not both -CH2- or -CO-, provided that each X is the same or different, each Y is the same or different, and R2 is H, optionally substituted C1-C 20 Alkyl groups, optionally substituted C1-C 20 Alkenyl group or optionally substituted C-C 20 alkynyl groups, R3 is H, optionally substituted C1-C 20 Alkyl groups, optionally substituted C1-C 20 Alkenyl group or optionally substituted C-C 20 alkynyl groups, wherein each substituent is independently selected from H, F, Cl, Br, CN, and NO2; and R1 is [ka] or [ka] where n is an integer selected from 0-10, and Z is [ka] or [ka] and R4 is an optionally substituted C4-C 20 Alkyl groups, optionally substituted C4-C 20 Alkenyl group or optionally substituted C4-C 20 alkynyl groups, wherein each substituent is independently selected from H, F, Cl, Br, CN, and NO2.

[0080] In some embodiments, R2 is H, CH 3- , or R1, R3 is selected from H, or R1, and R1 is [ka] or [ka] where n is an integer selected from 1 to 5, and Z is [ka] or [ka] and R4 is C4-C 20 Alkyl groups, C4-C 20 Alkenyl group or C4-C 20 alkynyl groups, provided that the C4-C 20 The alkenyl group contains 1-3 double bonds and the C4-C 20 Alkynyl groups contain 1 to 3 triple bonds.

[0081] In some embodiments, R4 is C5-C 15 Alkyl groups, C5-C 15 alkenyl groups, provided that the C5-C 15 Alkenyl groups contain 1 to 3 double bonds.

[0082] In some embodiments, R1, R2, and R3 are the same and R4 is C 10 -C 15 Alkyl group, C 10 -C 15 alkenyl groups, provided that the C 10 -C 15 Alkenyl groups contain 1 to 3 double bonds.

[0083] Furthermore, the compound is [ka] It has one of the following structures.

[0084] The compounds described in this invention are meant to have a hydrophilic head and a hydrophobic tail in one large molecular chain. However, the hydrophilic head may be N1,N4-bis(3-aminopropyl)succinamide, 3,3'-(butane-1,4-diylbis(methylazanediyl))dipropanamide, or 3,3'-(butane-1,4-diylbis((2-hydroxypropyl)azanediyl))dipropanamide. The hydrophobic tail may be a straight carbon chain with double bonds and / or ester bonds, and the total chain length may be 12, 14, 16, or 18 atoms. The compounds of the present invention may have one type of hydrophilic head and one or more types of hydrophobic tail, formed in multiple ratios such as 1:1, 1:2, or 1:3.

[0085] In some embodiments, the hydrophobic tail is [ka] and having at least one of the chemical structures However, the positions of the double bond and ester bond in Formula 1 may be varied depending on the availability of convenient raw materials, with the total chain length being 16 atoms or 18 atoms; the position of the ester bond in Formula 2 may be varied depending on the availability of convenient raw materials, with the total chain length being 14, 16, or 18 atoms; and the chain length in Formula 3 may be varied and may be 12, 14, 16, or 18 atoms (12 or 14 atoms being more preferred).

[0086] In some embodiments, the salt refers to a pharmaceutically acceptable salt. 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 being treated therewith.

[0087] The compounds of the present invention further include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts, and which may be used as intermediates for the preparation and / or purification of the compounds of the present invention and / or for the separation of the enantiomers of the compounds of the present invention.

[0088] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, hydrogensulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorotheophylline, citrate, ethylenedisulfonate, fumarate, glucoheptylsaccharate, gluconate, glucaldehyde acid salt, hippurate, hydrochloride / iodide, hydroxyethylsulfonate, lactate, lactaldehyde acid salt. , lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthalenesulfonate, nicotinate, nitrate, octadecylate, oleate, oxalate, palmitate, methylenebishydroxynaphthalene, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalactosate, propionate, stearate, succinate, sulfosalicylate, tartrate, toluenesulfonate and trifluoroacetate.

[0089] Inorganic acids from which salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0090] Organic acids from which salts can be derived include acetic acid, propionic acid, hydroxyacetic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, paratoluenesulfonic acid, sulfosalicylic acid, and the like.

[0091] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0092] Inorganic bases from which salts can be derived include ammonium salts and periodic Group I to XII metals. In some embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including ammonium, potassium, sodium, calcium, and magnesium salts.

[0093] Organic bases from which salts can be derived include primary amines, secondary amines, tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Some organic amines include, for example, isopropylamine, benzyl penicillin, choline salt, diethanolamine, diethylamine, lysine, glutamine, piperazine, and aminobutanetriol.

[0094] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometrically appropriate base (e.g., Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, etc.) or the free base form of these compounds with a stoichiometrically appropriate acid. Such reactions are usually carried out in water or an organic solvent, or a mixture of both. Generally, non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropyl alcohol, or acetonitrile, as appropriate, must be used. 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), provide information on several other suitable salts.

[0095] The compounds of the present invention, including salts thereof, may also be obtained in the form of their hydrates or contain other solvents for their crystallization. The compounds of the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water), and therefore the present invention is intended to encompass both solvated and unsolvated forms.

[0096] Any structural formulas given herein are intended to represent the unlabeled and isotopically labeled forms of these compounds. Isotopically labeled compounds have the structure represented by the general formula given herein, except that one or more atoms are replaced with atoms having a selected atomic mass or mass number. Exemplary isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 18F, 31P, 32P, 36S, 37Cl, or 125I.

[0097] In another embodiment, the compounds described herein include compounds defined herein that are labeled with various isotopes, for example, compounds containing radioactive isotopes such as H, C, and F, or compounds containing non-radioactive isotopes such as H and C. Such isotopically labeled compounds can be used in metabolic studies (using C), reaction kinetic studies (using H or H, for example), detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution measurements, or for patient radiotherapy. F-labeled compounds are particularly ideal for PET or SPECT studies. Instead of previously used non-labeled reagents, isotopically labeled compounds of formula (I) can be prepared using appropriate isotopically labeled reagents, as described in the Examples and preparation procedures of the present invention, or by conventional techniques known to those skilled in the art.

[0098] It should be noted that substitution with heavier isotopes, particularly deuterium (i.e., 2H or D), can offer several therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improved therapeutic index. In this context, it should be understood that deuterium is considered to be a substituent of the compound of formula (I). The concentration of such heavy isotopes, particularly deuterium, can be defined by an isotopic enrichment factor. As used herein, the term "isotopic enrichment factor" refers to the ratio between the isotopic abundance and the natural abundance of a specified isotope. When a substituent of a compound of the present invention is designated as deuterium, the compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium loading at each designated deuterium atom), at least 4000 (60% deuterium loading), at least 4500 (67.5% deuterium loading), at least 5000 (75% deuterium loading), at least 5500 (82.5% deuterium loading), at least 6000 (90% deuterium loading), at least 6333.3 (95% deuterium loading), at least 6466.7 (97% deuterium loading), at least 6600 (99% deuterium loading), or at least 6633.3 (99.5% deuterium loading). Pharmaceutically acceptable solvates of the present invention include solvates in which the crystalline solvent is isotopically substituted, such as DO, acetone-d6, or DMSO-d6.

[0099] Liposomes and preparation methods

[0100] In another aspect of the present invention, the present invention provides liposomes. The liposomes according to the present invention are prepared using the compounds according to the first aspect of the present invention, and the liposomes can encapsulate the added substance to be loaded by the fluidity of the lipid bilayer, and have a good encapsulation effect, allowing the substance to effectively enter cells and escape from the encapsulated body into the cytoplasm.

[0101] The above compound can form liposomes by self-assembly with hydrophobic lipids.

[0102] In some embodiments, the liposome further comprises a hydrophobic lipid, an amphipathic lipid, a buffering agent, and / or an organic solvent, wherein the amphipathic lipid comprises at least one selected from a neutral lipid and a PEG-lipid, and the hydrophobic lipid comprises a sterol.

[0103] In some embodiments, the neutral lipid comprises at least one selected from distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine, dioleoylphosphatidylethanolamine, dilaurylphosphatidylcholine, diethyl pyrocarbonate, dimyristylphosphatidylcholine, and egg yolk lecithin.

[0104] In some embodiments, the PEG-lipid comprises at least one selected from bispalmitoylphosphatidylethanolamine-PEG, bisstearylphosphatidylethanolamine-PEG, dimyristylglycerol, and dimethacrylate.

[0105] In some embodiments, the sterol is cholesterol.

[0106] In some embodiments, the buffering reagent is an acidic buffering reagent.

[0107] In some embodiments, the buffering reagent comprises at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trimethylolaminomethane-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.

[0108] In some embodiments, the organic solvent comprises at least one selected from methanol, ethanol, isopropyl alcohol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl ethyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, and triethanolamine.

[0109] 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 l, 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.

[0110] 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).

[0111] In some embodiments, the volume of the buffer reagent is 50-90% v / v based on the liposomes. Further, the volume of the buffer reagent may be 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, or any of the following: 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.

[0112] In some embodiments, the volume of the buffering reagent is 75% v / v based on the liposomes.

[0113] In some embodiments, the compounds may also form liposomes with phospholipid derivatives, including but not limited to distearoylphosphatidylcholine, bisstearylphosphatidylethanolamine, and dioleoylphosphatidylethanolamine.

[0114] In one specific embodiment, the liposome comprises the compound described above, and further comprises a neutral lipid and a lipid capable of reducing aggregation of the liposome particles.

[0115] In one specific embodiment, the liposomes are composed primarily of (i) at least one lipid of the present invention, (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, DLPC, DEPC, DMPC, and EPC, (iii) a sterol, e.g., cholesterol, and (iv) a PEG-lipid, e.g., DPPE-PEG, DSPE-PEG, PEG-DMG, or PEG-DMA, and about 20-80% of an amine-containing lipid. : 5-50% neutral lipids : 10-60% sterols :It consists of a mixture of 0.2-20% PEG-lipid molar ratio.

[0116] In another aspect, the present invention provides a method for preparing the above liposomes, which, according to an embodiment of the present invention, comprises mixing a predetermined amount of the compound according to the first aspect of the present invention with an organic solvent to obtain the liposomes.

[0117] In some embodiments, the concentration of the compound in the organic solution is 10-30 mg / mL, and further, the concentration of the 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, or 30 mg / mL.

[0118] In some embodiments, the method further comprises dissolving a predetermined amount of hydrophobic lipid and amphipathic lipid in the organic solution and then 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), and the volume of the buffer reagent is 75% v / v based on the liposome.

[0119] In some embodiments, the amount of the compound, based on the liposome, 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, 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 9%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.

[0120] Drug carriers, drug compositions, transfection complexes

[0121] In another aspect, 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 that can carry a negatively charged drug, deliver the drug into cells, and allow the drug to effectively escape from the encapsulation body into the cytoplasm, which is advantageous for the drug to exert its medicinal effect.

[0122] In a further aspect of the present invention, the present invention provides a pharmaceutical composition comprising a carrier and a pharmaceutical active ingredient, wherein the carrier comprises the pharmaceutical carrier according to the fifth aspect of the present invention. According to an embodiment of the present invention, the pharmaceutical carrier is an ionizable carrier, capable of carrying a negatively charged pharmaceutical active ingredient, and delivering the pharmaceutical active ingredient into cells so that it can effectively escape from the encapsulation body into the cytoplasm, which is advantageous for the pharmaceutical effect of the carried pharmaceutical active ingredient.

[0123] In some embodiments, the active pharmaceutical ingredient comprises at least one selected from a DNA molecule, an RNA molecule, a protein, and a hydrophobic drug. According to embodiments of the present invention, the drug carrier comprises a long carbon chain capable of carrying a hydrophobic substance, such as a hydrophobic small molecule drug.

[0124] Furthermore, the drug carrier of the present invention can also carry proteins or polypeptide-based substances, and the replica of the protein or polypeptide is not particularly limited and may be a peptide chain or the protein itself, or a derivative of the above substance or a complex of other substances, such as a Cas9 protein or a partial domain peptide segment thereof, a nuclide-containing carrier protein, an antibody, or the like.

[0125] Furthermore, the drug carrier of the present invention can also carry nucleic acid substances, such as antisense nucleic acids.

[0126] In some embodiments, the active pharmaceutical ingredient is negatively charged. According to embodiments of the present invention, the drug carrier comprises an ionizable amine-containing lipid, which is positively charged at a specific pH and can bind to the negatively charged active pharmaceutical ingredient, and the lipid bilayer fluidity allows the active pharmaceutical ingredient to be encapsulated and delivered to cells.

[0127] In some embodiments, the active pharmaceutical ingredient comprises a nucleic acid. According to embodiments of the present invention, the active pharmaceutical ingredient may comprise DNA, RNA, a nucleic acid-protein complex, a nucleic acid-lipid complex, a nucleic acid-nucleic acid complex, or the like, and the type of nucleic acid is not limited. Specific examples of the nucleic acid include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA (microRNA), ribozymes, antisense oligonucleosides, plasmid DNA, peptide nucleic acids, triplex-forming oligonucleosides (TFOs), and genes. The carrier of the present invention is particularly effective for delivering siRNA into cells. The nucleic acid used in the carrier of the present invention may be derived from humans, animals, plants, bacteria, viruses, or the like, or may be prepared by chemical synthesis. Furthermore, the nucleic acid may be single-stranded, double-stranded, or triple-stranded, and its molecular weight is not particularly limited. Furthermore, in the present invention, the nucleic acid may be modified with a chemical, enzyme, or peptide. In the present invention, one type of nucleic acid may be used alone, or two or more types may be used in appropriate combination. In a preferred embodiment, the nucleic acid transport carrier composition of the present invention preferably transports a low-interfering nucleic acid (siRNA) or its analog.

[0128] In some embodiments, the mass ratio of the carrier to the active pharmaceutical ingredient is (5-40:1).

[0129] In some embodiments, the mass ratio of the carrier to the active pharmaceutical ingredient is (8-20:1).

[0130] 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.

[0131] In some embodiments, the weight ratio of the carrier to the active pharmaceutical ingredient is 15:1.

[0132] In a further aspect, the invention proposes a transfection complex, which according to an embodiment of the invention comprises a compound as defined above or a liposome as defined above.

[0133] In some embodiments, co-lipids suitable for preparing and forming the transfection complexes disclosed herein may be, but are not limited to, any neutral or cationic lipid that provides or facilitates the introduction of cholesterol, cholesterol derivatives, or exogenous bioactive molecules into cells or tissues. In some embodiments, one or more co-lipids can be used in preparing the transfection complexes described herein.

[0134] In some embodiments, the transfection complex comprises at least one bioactive agent. Transfection complexes according to embodiments of the present invention are applicable to the delivery of one or more bioactive agents to cells, tissues, or organs in vitro or in vivo.

[0135] In some embodiments, the transfection complex includes one or more bioactive agents for delivery to a cell or target tissue in vitro or in vivo. Suitable bioactive agents can include any molecule that can form a transfection complex with a transfection reagent described herein and that elicits a biological response when delivered to one or more cells or tissues in vivo or in vitro. Bioactive agents used in the embodiments described herein can be cationic, neutral, or anionic.

[0136] In some embodiments, the bioactive agent comprises at least one selected from a DNA molecule, an RNA molecule, a protein, and a drug. Exemplary bioactive agents suitable for preparing transfection complexes, according to embodiments of the present invention, include, but are not limited to, nucleic acids (including, but not limited to, single- or double-stranded linear or circular DNA molecules (including cDNA molecules), single- or double-stranded RNA molecules, small interfering RNA (siRNA) molecules, short hairpin RNA (shRNA) molecules, microRNA (miRNA) molecules, oligoxylates, antisense oligoxylates, and sense oligoxylates), multiple skins, antibodies, oligoskins, therapeutic peptide or protein molecules, peptide nucleic acids (PNAs), cationic, anionic, or neutral organic molecules or drugs, and pharmaceutically acceptable salts thereof.

[0137] In some embodiments, the transfection complexes described herein can optionally include one or more fusion or cell-penetrating peptides. A fusion or cell-penetrating peptide is any peptide polymer that promotes fusion of a lipid-containing complex with a cell membrane (plasma membrane or intracellular membrane). Multiple fusion or cell-penetrating peptides are known in the art, and it is within the skill of one of ordinary skill in the art to identify fusion or cell-penetrating peptides and conditions for their use in the present invention without undue experimentation.

[0138] In some embodiments, the transfection complexes described herein can optionally include one or more transfection adjuvants or targeting moieties, which can be skin, modified skin, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single- or double-stranded nucleic acid molecule, a modified single- 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 capable of targeting the transfection complex to a particular tissue or cell type for targeted delivery of a bioactive agent thereto.

[0139] In some embodiments, the transfection complexes provided herein can be stable for up to one year and can be contacted with cells or tissues to be transfected, or can be administered to an animal or human immediately after formation or for a period of time, or can be stored for a period of time, optionally before contacting cells or tissues or administering to an animal or human. The transfection complexes are stable and can be stored at room temperature or above freezing to about room temperature for 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 can include one or more stabilizers, preservatives, buffers, etc., that aid in the long-term stability and storage of the bioactive formulations, as would be readily understood by one skilled in the biological and pharmaceutical arts, and can be accomplished without undue experimentation. It should further be understood that the storage period can be any of these periods, such as 31 minutes to 1 hour, or 1 hour to 24 hours. [Example]

[0140] The present invention will now be described with reference to specific examples, which are for illustrative purposes only and are not intended to limit the invention in any manner.

[0141] Detection and calculation methods: 1. Nuclear magnetic resonance spectroscopy detection for determining the structure of ammonium-containing lipids The nuclear magnetic resonance spectrometer used is a Bruker 400M nuclear magnetic resonance spectrometer. For specific calculation methods, please refer to the literature: Boulmedais F., Frisch B., Etienne O., Lavalle Ph., Picart C., Ogier J., Voegel JC., Schaaf P., Egles C. Polyelectrolyte multilayer films with pegylated polypeptides as a new type of antimicrobial protection for biomaterials, Biomaterials, 2004, 25, 2003-2011.

[0142] 2. Gel block experiment to prove the loading of nucleic acids in the formulation Anti-apolipoprotein B siRNA (siApoB) was dissolved in DEPC-treated water and freshly assembled with LNP at different mass ratios (w / w). Then, it was mixed with 6x loading buffer (Beijing Jinnan Biotechnology Co., Ltd.). The formulation containing the same amount of siRNA was loaded onto a 1% (w / w) agarose gel containing 0.01% gel stain (w / w) (Beijing Jinnan Biotechnology Co., Ltd.). Electrophoresis was performed for 30 min at 90V in 1x TAE electrophoresis buffer. The results were recorded using a gel imaging system (Shanghai Jinnan Biotechnology Co., Ltd.) under UV light at 320 nm. The siApoB sequence is shown in Table 1.

[0143] [Table 1]

[0144] 3. Measure particle size and surface potential to determine whether lipid formulations can be used. After encapsulating the siRNA into LNPs, the resulting formulation was diluted 10-fold before measuring particle size and surface potential. The instrument used to measure particle size and surface potential was a Zetasizer 3000HS laser particle sizer from Malven, USA, at a temperature of 25°C, an angle of 90°, and an incident light wavelength of 677 nm.

[0145] 4. Measurement of siRNA concentration in the formulation siRNA concentration was measured using the RiboGreen method. After diluting the test formulation 20-fold, 100 μL was added to a 96-well plate and used to detect the external siRNA fluorescence of the formulation. 15 μL of the diluted test formulation was taken separately and added to a 96-well plate with 50 μL of 2% Triton-X-100 (w / w) to disrupt the lipid structure and completely release the internal siRNA. 35 μL of 1x TE buffer was added to the well to make up the volume and used to detect the total siRNA fluorescence of the formulation. A calibration curve was created using siRNA standard samples, 1x TE, and 2% Triton-X-100, and used to calculate the siRNA concentration in the test samples. After loading all test samples, the plate was incubated at room temperature for 30 minutes. Next, 100 μL of RiboGreen working solution was added per well in the dark, and fluorescence detection using the enzyme marker was immediately performed. The detection conditions are shown in Table 2.

[0146] [Table 2]

[0147] 5. Transmission electron microscope (TEM) experiment to detect the particle morphology of the formulation The transmission electron microscope used is a Dutch JEM-100CX II transmission electron microscope. First, a copper mesh is immersed in the test sample solution, then stained with a 0.1% by weight solution of phosphotungstic acid. After about 3 minutes, the excess liquid is filtered through filter paper and allowed to dry at room temperature. Next, the system is observed under a transmission electron microscope to photograph the morphology of the particles in the system.

[0148] 6. In vitro transfection experiments For the detection of siRNA delivery efficiency of candidate formulations, human hepatoma cell line HepG2 was seeded into 12-well plates in DMEM complete medium containing 10% fetal bovine serum at an inoculation density of 1 × 10 5 cells / well in 1 mL medium per well and cultured overnight at 37°C.

[0149] The next 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 per well to all transfected wells. The mock group was not further treated. The corresponding formulation was added to the test sample wells, and the sample loading volume was calculated based on the siRNA concentration in the LNPs determined by RiboGreen analysis, resulting in a final siApoB concentration of 50 nM. The cells were incubated at 37°C for 4 hours, after which 1 mL of DMEM complete medium containing 10% fetal bovine serum was added per well, followed by overnight incubation at 37°C.

[0150] Real-time fluorescent quantitative PCR was then used to detect the relative expression of ApoB mRNA in the test samples. After culturing the transfected cells for 24 hours, total RNA was extracted from the cells using an RNA isolator (Nanjing Nuo Weizhan Biotechnology Co., Ltd., product number R401-01-AA), chloroform, and isopropyl alcohol. 1 μg of total RNA was then reverse-transcribed to obtain cDNA according to the instructions for a reverse transcription kit (Beijing Quanshi Jin Biotechnology Co., Ltd., product number AT311-03). A real-time fluorescent quantitative PCR mix (Shanghai Yisheng Biotechnology Co., Ltd., product number 11201ES08) was used to detect the expression of the target gene ApoB and the internal reference gene glyceraldehyde phosphate dehydrogenase (GAPDH) according to the instructions. ApoB expression was referenced to GAPDH. PCR primers for amplifying ApoB and the internal reference gene GAPDH are listed in Table 3.

[0151] [Table 3]

[0152] 7. Cytotoxicity detection The cytotoxicity of the polymer was detected using the MTT method (tetrazole salt colorimetric method), and the specific method is as follows. (1) 1.25 x 10 cells in a 96-well plate 4 HepG2 cells are seeded per well in a medium volume of 100 μL per well and incubated overnight at 37°C under 5% CO 2 conditions. (2) After 24 hours, add an appropriate amount of siFLA4-13 containing insect repellent luciferase siRNA (siFirefly) to each well to adjust the siRNA concentration in the well to 1 nM to 200 nM. At the same time, set up control wells (containing PBS at pH 7.4, culture medium, MTT, and dimethyl sulfoxide, with the same volume of liquid as the cells and formulation) and incubate at 5% CO2 and 37°C for 48 hours. (3) 20 μL of MTT solution (5 mg / mL) is added to each well, followed by incubation at 5% CO 2 and 37°C for 4 hours. (4) Carefully discard the supernatant and add 150 μL of dimethyl sulfoxide to each well. Shake in a slow oscillator for 10 minutes to dissolve the crystals thoroughly. Measure the absorbance of each well at 540 nm using a continuous spectroscopic multimode microplate reader (TECAN Infinite 200). (5) Calculate the relative vitality of each sample well cell by the following formula:

number

[0153] [Table 4]

[0154] 8. Subcellular localization used to determine whether nucleic acids have entered cells The day before the exam, 2 x 10 5HepG2 cells were seeded into 35 mm dishes and cultured overnight at 37°C under 5% CO2. The next day, LNP Cy-siFLA4-13 (SEQ ID NO. 7, 3'-terminally Cy5-linked) or Lipofectamine 2000 (Invitrogen) was added to the corresponding dishes and cultured for 4 hours to allow the cells to absorb the transfection reagent. After the designated time, the medium containing the transfection reagent was discarded and the cells on the dishes were washed three times with 1x PBS. Then, 1 mL of staining solution (1x PBS for Lysotracker, 1:3000 (v / v) diluted with 0.1 mg / ml Hoechst 33342) was added to the dishes and stained for 15 minutes at 37°C in the dark. After staining was complete, the intracellular distribution of the fluorescent signal was observed using a laser confocal microscope (LSM700, Zeiss).

[0155] 9. Cellular uptake to determine the efficiency of nucleic acid entry into cells HepG2 cells were seeded into 12-well plates the day before detection at a concentration of 2 × 10 5 The cells were incubated at 1 mL / mL per well. After 24 hours, Cy-siFLA4-13 was added to the wells to a final concentration of 50 nM and incubated at 37°C for 4 hours in 5% CO2. The medium containing the transfection reagent was then discarded, and the cells were digested with trypsin. The digestion was stopped with complete medium and the liquid was collected in a centrifuge tube. After centrifugation at 800 rpm for 5 minutes, the cells were resuspended in 1x PBS and washed three times. The efficiency of the formulation uptake by HepG2 cells was then measured using a flow cytometer (FACSverse, BD). Data were analyzed using FlowJo 7.6.

[0156] 10. Biodistribution experiments to determine the metabolism and distribution characteristics of drugs in the body The experiment involved 6-8 week-old C57BL / 6 mice. They were divided into two groups and administered 1x PBS or Cy-siFLA4-13 (2 mg / kg Cy5-siFirefly) via the tail vein. At different time points after administration, the distribution of Cy5 signals within the mice was detected using a small animal in vivo imaging system (FX Pro, Kodak). After in vivo imaging was completed at 0.5, 4, and 24 hours after administration, one or two animals from each group were euthanized and the heart, liver, spleen, lungs, kidneys, stomach, intestine, submandibular gland, and thymus were isolated for organ imaging. Analysis of the results was completed using Carestream software, and line graphs were generated using Graphpad Prism 8.

[0157] In other experiments, 6-8 week old C57BL / 6 mice were administered 1x PBS or mLuA4-12 formulation via the tail vein at a dose of 20 μg of mRNA per animal. Six and nine hours after administration, the animals were euthanized, and the heart, liver, spleen, lungs, kidneys, stomach, intestine, submandibular gland, and thymus were isolated for organ imaging. Analysis of the results was completed using Carestream software.

[0158] 11. Dose-dependence experiment Thirty-two 6-8 week old C57BL / 6 mice were divided into eight groups and received either 1x PBS or 0.01 mg / kg to 1 mg / kg siA4-13. After 42 hours, the animals were fasted for 6 hours and then euthanized. Serum and liver tissue samples were collected to analyze serum triglycerides (TG), total cholesterol (CHO), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and ApoB mRNA expression in liver tissue. PCR primers for amplifying mouse ApoB and GAPDH as an internal reference gene are listed in Table 5.

[0159] [Table 5]

[0160] 12. Duration of action experiment Ninety-six 6-8 week-old C57BL / 6 mice were divided into three groups and received 1x PBS or 1 or 3 mg / kg siA4-13, respectively. At 7, 14, 21, 28, 35, 42, and 49 days after administration, four animals from each group were fasted for 6 hours and then euthanized. Serum and liver tissue samples were collected for analysis of serum TG, CHO, LDL-c, HDL-c, and ApoB mRNA expression in liver tissue. Effect-time curves were generated using Graphpad Prism 8.

[0161] 13. Drug efficacy testing The experiment was divided into two parts. In the first part, leptin receptor knockout db / db mice (C57BLKS / JGpt- Leprem2Cd / Gpt) were used as subjects, and genetically identical null knockout mice (m / m) served as controls. This part of the experiment aimed to examine the lipid-lowering effect of siANA4-13, a formulation containing anti-angiogenin-like protein 3 siRNA (siANGPTL3), and changes in target gene expression. In the second part, humanized apolipoprotein C3 (ApoC3) transgenic mice (hApoC3, Tg(APOC3)3707Bres) were used as subjects, and C57BL / 6 mice served as controls. This part of the experiment aimed to examine the lipid-lowering effect of siApoCA4-13, a formulation containing siApoC3, and the recovery of blood lipids in the animals after drug discontinuation.

[0162] In the first part, db / db mice were injected via the tail vein with 1x PBS, 0.5 mg / kg siFLA4-13, 0.5 mg / kg siANA4-13, or 0.25 mg / kg siANA4-13. The test was conducted once a week for a total of five doses. On the third day after dosing, the animals were fasted for 6 hours and blood was collected via the orbit to analyze changes in TG and CHO. On the second day after the final dose, all animals were fasted for 6 hours and then sacrificed. Blood and liver samples were collected to further measure changes in blood lipids and ANGPTL3 mRNA expression in liver tissue. The liver from one animal per group was collected and stained with Oil Red O to observe changes in lipid deposition in the liver.

[0163] In the second part, hApoC3 mice were injected via the tail vein with 1x PBS, 0.5 mg / kg siFLA4-13, 0.5 mg / kg siApoCA4-13, or 0.25 mg / kg siApoCA4-13. The experiment was similarly planned with five doses, each administered weekly, during which blood lipid changes were monitored. After the final dose, all animals underwent blood lipid monitoring once a week for a total of four doses. The animals were then euthanized, and blood was collected for blood lipid monitoring. The sequences of siANGPTL3 and siApoC3 used in the experiment, as well as the PCR primers for amplifying mouse ANGPTL3 and human ApoC3, are listed in Tables 6 and 7, respectively.

[0164] [Table 6]

[0165] [Table 7]

[0166] 14. In vitro mRNA expression test HepG2 cells were seeded into 12-well plates the day before detection at a concentration of 2 × 10 5The cells / mL were added, with a volume of 1 mL per well. After 24 hours, mLuA4-12 containing luciferase mRNA was added to the wells, bringing the amount of mRNA added to each well to 400 ng. The wells were then incubated at 37°C in 5% CO2 for 6 hours. After the specified time, the cells were cleaved with a passive cleavage solution and the cleavage solution was collected. Then, 10 μL of the cleavage solution was added per sample to a 96-well plate, and 50 μL of luciferase substrate was added to each well. Upon completion, the amount of fluorescence from the samples was immediately detected using an enzyme marker.

[0167] 15. Comparative test of gene silencing between siRNA and CRISPR / Cas system Twenty-four hours before transfection, HepG2-luc cells were seeded into 6-well plates at a concentration of 2 × 10 5 The cells were cultured at 1 mL / mL per well. The next day, siPLK1, an siRNA containing anti-polo-like kinase gene 1 (PLK1), and siPLKA4-13 and pCPA4-12, plasmids (pCPLK1) co-expressing Cas protein and PLK1 sgRNA, were added to the corresponding wells, resulting in a total of 1600 ng of siRNA and plasmid. The mock group was not further treated. The cells were cultured at 37°C under 5% CO2 for 4 hours. Then, 1 mL of fresh medium was added to each well, and the culture was continued for an additional 20 hours. After the specified time, the relative expression of PLK1 mRNA in the test samples was detected using quantitative real-time fluorescent PCR. The nucleic acid and primer sequences for PLK1 siRNA are listed in Tables 8 and 9, respectively.

[0168] [Table 8]

[0169] [Table 9]

[0170] Preparation Example 1 Synthesis of amine-containing lipid compound A4

[0171] Synthesis of Compound A2 [ka] The reaction was carried out in a three-neck 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, refluxed with nitrogen at 50 °C. The reaction was monitored using thin-layer chromatography (TLC) and stopped upon completion. The crude product was concentrated under reduced pressure and then purified on a silica gel column using dichloromethane:methanol = 30:1 (v / v) as a eluent. Product A2 (1.61 g, 78.1% yield) was obtained. 1H NMR (400 MHz, CDCl3) δ (ppm) 0.88 (t, J = 13.68 Hz, 6 H), 1.26-1.44 (m, 45 H), 1.67 (m, 2 H), 2.38-2.70 (m, 6 H), 3.18 (m, 2 H), 3.70 (m, 2 H); 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; Compound A2 (molecular weight 543.5023) was scanned by high resolution mass spectrometry (EI+ mode), and the scan result was 543.5108.

[0172] Synthesis of compound A3 [ka] To a three-neck flask containing compound A2 (1.04 g, 1.8 mmol) and 20.00 mL of dichloromethane, slowly add hydrochloric acid while stirring. The reaction is allowed to proceed at room temperature for 16 hours, and the reaction is stopped after completion and detected by TLC. The pH of the reaction mixture is adjusted to 7-8 with 1 M aqueous NaOH, and the mixture is extracted three times with water and ethyl acetate. The mixture is concentrated under reduced pressure to obtain product A3 (398.0 mg, 49.9% yield). 1H NMR (400 MHz, CDCl3) δ (ppm) 0.88 (t, J = 13.08 Hz, 6 H), 1.26 (m, 32 H), 1.43 (m, 4 H), 1.96 (m, J = 14.5 Hz, 2 H), 2.60 (m, J=20.9 Hz, 4 13C NMR (100 MHz, 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; Compound A3 (molecular weight 443.4498) was scanned by high-resolution mass spectrometry (EI+ mode), and the scan result was 443.4576.

[0173] Synthesis of compound A4 [ka] 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-neck flask. Then, 1-hydroxybenzotriazole (753.3 mg, 5.6 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt (1.13 g, 5.6 mmol) were added as activators. The reaction was carried out under nitrogen gas protection for 12 hours. After detection by thin-layer chromatography, the reaction was stopped. The crude product was washed twice with water, twice with saturated salt, and twice with the organic phase. Product A4 (0.1 g, 3.3% yield) was obtained. 1H NMR (400 MHz, CDCl3) δ (ppm) 0.88 (t, J = 13.24 Hz, 12 H), 1.26-1.47 (m, 72 H), 1.93 (m, 4 H), 2.56-3.41.74 (m, 20 H), 3.89 (m, 4 H), 5.03 (m, 4 H), 8.04(m, 2 H); 13C NMR (100 MHz, 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; the compound (molecular weight 967.9051) was scanned by high-resolution mass spectrometry (EI+ mode), and the scan result was 967.9137.

[0174] Example 1 Lipid A4 obtained in Preparation Example 1, DSPC, cholesterol, and DMG-PEG (purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in absolute ethanol to a concentration of 20 mg / ml. A4 was completely soluble at room temperature and was a pale yellow, transparent liquid. DSPC, cholesterol, and DMG-PEG were then heated in a water bath at 50°C for several minutes until completely dissolved, resulting in a colorless, transparent liquid. After the above materials were prepared, the four ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 66.8:10.9:20.0:2.2, and the mixture was then drawn into an insulin syringe for use in preparation. A citric acid / sodium citrate buffer solution with a pH of 4.0 and a concentration of 10 nM was separately prepared at a volume three times the volume of the organic phase mixture, and similarly drawn into an insulin syringe for use in preparation. Then, 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 expelled. The centrifuge tube was gently shaken to prepare lipid nanoparticles A4-1.

[0175] Test example 1 This test example was conducted using gel block experiments to determine the siRNA encapsulation ability of the lipid nanoparticles obtained in Example 1 and to explore the optimal mass ratio of lipid nanoparticles to siRNA. The test results are shown in Figure 1. The results indicate that a mass ratio of LNP to siRNA lower than 5:1 results in significant siRNA leakage, explaining that low mass ratios are unsuitable for siRNA delivery. At a mass ratio of 5:1 or higher, siRNA is efficiently encapsulated in LNPs, and at a mass ratio of 15:1, siRNA is almost completely encapsulated. A mass ratio of 5:1-30:1 is an appropriate range for the LNP to siRNA mass ratio, and a mass ratio of 15:1 or higher can achieve efficient siRNA delivery. Therefore, in subsequent preparation examples, LNPs are prepared at this mass ratio.

[0176] Example 2 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 58.5:14.3:23.4:3.8 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-2.

[0177] Example 3 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 50.2:16.4:25.1:8.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-3.

[0178] Example 4 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 42.8:17.5:25.7:14.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare lipid nanoparticles A4-4.

[0179] Example 5 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 61.8:8.6:21.0:8.6 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-5.

[0180] Example 6 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 57.4:12.0:14.6:16.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-6.

[0181] Example 7 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 55.0:15.3:28.1:1.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was also prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare lipid nanoparticles A4-7.

[0182] Example 8 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 54.7:19.0:23.3:3.1 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-8.

[0183] Example 9 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 57.7:7.0:21.3:14.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-9.

[0184] Example 10 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 57.2:10.4:25.4:7.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was also prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare lipid nanoparticles A4-10.

[0185] Example 11 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 66.1:16.0:14.7:3.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-11.

[0186] Example 12 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 61.6:18.6:18.2:1.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-12.

[0187] Example 13 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 64.8:6.9:25.5:2.8 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-13.

[0188] Example 14 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 66.2:10.6:21.7:1.4 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-14.

[0189] Example 15 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 59.1:12.7:15.5:12.7 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare lipid nanoparticles A4-15.

[0190] Example 16 The four dissolved ingredients were mixed in a mass ratio of A4:DSPC:cholesterol:DMG-PEG = 63.6:17.1:21.5:6.8 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was also prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare lipid nanoparticles A4-16.

[0191] In the above example, A4, DSPC, cholesterol, and DMG-PEG were each set to four levels, as shown in Table 8.

[0192] [Table 10]

[0193] The blending ratios of Examples 1-16 were designed using orthogonal design software (Orthogonal Design Assistant II V3.1), and the molar ratios and mass ratios of the 16 LNPs with different blending ratios are shown in Table 9.

[0194] [Table 11]

[0195] Examples 17-32 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%.

[0196] The 16 lipid nanoparticles 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 then incubated at 50°C for 20 minutes to ensure that as much siApoB as possible was encapsulated in the lipid nanoparticles. The 16 lipid nanoparticle / siApoB complexes were then transferred to dialysis tubing (Spectrum, G235035) with a molecular weight cutoff of 100,000 daltons and dialyzed against 1x PBS for 3 hours.

[0197] Test example 2 The purpose of this test example was to detect the particle size and polydispersity index (PDI) of the 16 lipid nanoparticles that did not encapsulate siApoB obtained in Examples 1-16. The results are shown in Table 10, and most of the formulations meet the requirements for being excellent delivery carriers.

[0198] [Table 12]

[0199] Test example 3 This test example aimed to detect the particle size, polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE) and intra-particle siRNA concentration (conc) of the 16 siApoB-encapsulated formulations obtained in Examples 17-32. The results are shown in Table 11. All formulations can encapsulate siRNA efficiently.

[0200] [Table 13]

[0201] Test example 4 In vitro transfection experiments were conducted on the 16 formulations obtained in Examples 17-32, and the real-time fluorescent quantitative PCR results are shown in Table 12. All formulations were able to deliver the encapsulated siRNA into cells and achieve gene silencing, with siA4-13 showing the best performance.

[0202] [Table 14]

[0203] Test example 5 The 16 formulations obtained in Examples 17-32 were tested for activity at the animal level. The study involved C57BL / 6 mice. Sixty-eight female mice were divided into 17 groups and injected via the tail vein with either 1x PBS or 16 formulations at a dose of 0.5 mg / kg. Forty-two hours after administration, the animals were fasted for six hours and then euthanized. Liver tissue samples were collected to analyze changes in ApoB mRNA expression compared to animals receiving 1x PBS. ApoB mRNA expression was detected by real-time fluorescent quantitative PCR. The analysis results of the activity test were generated using Graphpad Prism 8 and are shown in Table 13. All formulations were able to achieve gene silencing at the animal level, with siA4-13 showing the best performance.

[0204] [Table 15]

[0205] Example 33 Adjust the concentration of siFirefly to 1000 ng / μL with DEPC-treated water and mix with an equal volume of 50% ethanol solution to prepare a 500 ng / μL siFirefly solution with an ethanol concentration of 25%.

[0206] The lipid nanoparticles (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 then incubated at 50°C for 20 minutes to ensure that as much siFirefly as possible was encapsulated in the lipid nanoparticles. The complex was then transferred to a dialysis tube (Spectrum, G235035) with a molecular weight cutoff of 100,000 Daltons, and dialyzed against 1x PBS for 3 hours to obtain siFLA4-13.

[0207] Test Example 6 This test example aims to test siFLA4-13, numbered as obtained in Example 33. The cytotoxicity and test results of the preparation are shown in Table 14.

[0208] [Table 16]

[0209] Test Example 7 This test example aims to test the stability of the formulation No. siA4-13 obtained in Example 29 at 4°C. Specifically, after preparation of the formulation, a laser granulometer is used to measure the particle size of the formulation. After the procedure is completed, the formulation is stored at 4°C. The particle size of the formulation is then measured again on days 3, 7, and 21. The results are shown in Figure 2, demonstrating that the formulation is stable at 4°C for at least 3 weeks.

[0210] Example 34 Adjust the concentration of Cy5-siFirefly to 1000 ng / μL with DEPC-treated water and mix with an equal volume of 50% ethanol solution to prepare a 500 ng / μL siFirefly solution with an ethanol concentration of 25%.

[0211] 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), shaken gently, and then incubated at 50°C for 20 minutes to ensure that as much Cy5-siFirefly as possible was encapsulated in the lipid nanoparticles. The complex was then transferred to a dialysis tube (Spectrum, G235035) with a molecular weight cutoff of 100,000 Daltons and dialyzed against 1x PBS for 3 hours to obtain Cy-siFLA4-13. The above process must be protected from light throughout.

[0212] Test Example 8 The purpose of this test example is to detect the metabolic characteristics of Cy-siFLA4-13 obtained in Example 34 in C57BL / 6 mice.

[0213] Test Example 9 This test example aims to test the dose-dependent activity of the formulation No. siA4-13 obtained in Example 29 in C57BL / 6 mice. The changes in ApoB mRNA expression and total cholesterol (CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels are shown in Tables 16 and 17, respectively. Calculations show that the half-effective dose of this formulation is approximately 0.18 mg / kg.

[0214] [Table 17]

[0215] [Table 18]

[0216] Test Example 10 This test example aims to test the changes in ApoB mRNA expression and CHO, TG, LDL-c, and HDL-c levels at different time points after C57BL / 6 mice received a single injection of the siA4-13 formulation obtained in Example 29. The results are shown in Figure 4, which shows that after 4 weeks of treatment, the ApoB mRNA, TG, and LDL-c levels were still lower than those in the control group, supporting the use of treatment once every 4 weeks.

[0217] Example 35 The preparation obtained in Example 33 was concentrated using an ultrafiltration tube with a pore size of 100 Kd, and the siRNA concentration was measured using the RiboGreen method. The siRNA concentrations in the preparation were then adjusted to 300 ng / μL (corresponding to a dosage concentration of 3 mg / kg) and 100 ng / μL (corresponding to a dosage concentration of 1 mg / kg), respectively.

[0218] Test Example 11 This test example aims to test the changes in major serum biochemical indicators after C57BL / 6 mice receive the preparation obtained in Example 34, thereby determining the safety of the preparation. The results are shown in Figure 5, and the test experiment shows that the toxicity of the preparation meets the requirements.

[0219] Example 36 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%.

[0220] The lipid nanoparticles (numbered A4-13) obtained in Example 13 and the siANGPTL3 solution were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much siANGPTL3 as possible was encapsulated in the lipid nanoparticles. The complex was then transferred to a dialysis tube (Spectrum, G235035) with a molecular weight cutoff of 100,000 Daltons and dialyzed against 1x PBS for 3 hours to obtain siANA4-13.

[0221] Test Example 12 This test example aimed to test the lipid-lowering effect and ANGPTL3 mRNA suppression ability of siANA4-13 obtained in Example 35 in db / db mice. The changes in TG and CHO of the mice after treatment are shown in Figure 6, demonstrating that the blood lipids of the model animals were significantly reduced to the levels of normal animals after treatment.

[0222] A comparison of target gene expression between mice treated with 0.5 mg / kg and 0.25 mg / kg siANGPT3 and mice treated with 0.5 mg / kg siNC is shown in Table 18, and the reduction in blood lipids in Figure 6 is due to the suppression of target genes.

[0223] [Table 19]

[0224] The lipid deposition status in the liver of animals from different treatment groups is shown in Figure 7 and the results show that the formulation has the ability to reverse lipid deposition in the liver.

[0225] Example 37 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 siANGPTL3 solution with an ethanol concentration of 25%.

[0226] The lipid nanoparticles No. A4-13 obtained in Example 13 and the siApoC3 solution were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much siApoC3 as possible was encapsulated in the lipid nanoparticles. The complex was then transferred to a dialysis tube with a molecular weight cutoff of 100,000 Daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours to obtain siApoCA4-13.

[0227] Test Example 13 This test example aims to test the lipid-lowering effect and changes in blood lipids in hApoC3 mice treated with siApoCA4-13 obtained in Example 34 and within 4 weeks of discontinuing the drug. The changes in TG and CHO in the treated mice are shown in Figure 8. This test demonstrates that blood lipids in the model animals were significantly reduced after treatment, and that blood lipids returned to pre-treatment levels after a certain period of time after discontinuing the drug.

[0228] Preparation Example 2 Synthesis of amine-containing lipid compound A10

[0229] Synthesis of Compound A7 [ka] Add 20 ml of anhydrous dichloromethane under Ar gas protection, add 2 g of trans-2-tridecen-1-ol and 1.71 g of 3-bromopropionyl chloride with stirring, and react at room temperature for 2 hours with stirring. After detecting the completion of the reaction by TLC, remove the dichloromethane by concentration under reduced pressure and then purify with a silica gel column to obtain a pale yellow oil (2.7 g, 89% yield). 1H NMR (400MHz, 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, t CH2), 2.16-2.97(2H, m CH2), 1.22-1.45(16H, m), 0.90(3H, t, CH3)ppm

[0230] Synthesis of compound A8 [ka] Under Ar gas protection, add 10 ml of anhydrous DMF to the reaction flask, add 280 mg of N-Boc-1,3-diaminopropane hydrochloride and 721.4 mg of DIPEA while stirring, and after stirring at room temperature for 2 hours, add 1 g of compound A7 dropwise and stir at room temperature overnight. After the reaction is complete, remove the DMF by pressure concentration, and obtain 163 mg of a pale yellow oil, compound A8, by silica gel column chromatography. Yield: 18%. 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, m CH2), 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

[0231] Synthesis of Compound A9 [ka] Add 163 mg of compound A8 to 5 ml of 4 M HCl ethyl acetate solution at room temperature and stir. After 1 hour, monitor the reaction by TLC. The reaction is complete until no raw material remains. Remove the solvent by concentrating under reduced pressure, and use the mixture in a vacuum pump for 5 minutes to directly proceed to the next step.

[0232] Synthesis of compound A10 [ka] Under Ar gas protection, dissolve the product from the previous step in 2 ml of anhydrous dichloromethane, add 45 mg of triethylamine, and stir at room temperature for 30 min. Then add 14 mg of succinyl chloride dropwise. Stir and react at room temperature for 1 h, and monitor the reaction by TLC. Add 10 ml of dichloromethane to the reaction mixture, dilute with 1 M hydrochloric acid, collect the organic phase, concentrate under reduced pressure, and pass through a silica gel column. Add 5% aqueous ammonia to the mobile phase (DCM:MeOH = 60:1) (if no aqueous ammonia is added, the product will be passed through the column) to obtain 56 mg of a pale yellow oil, product A10. The yield of the two steps is 37%. 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 Theoretical MASS value is 1239.88, and the actual measured value is M+1=1239.9.

[0233] Example 38 The lipid A10 obtained in Preparation Example 1, DSPC, cholesterol, and DMG-PEG (purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in absolute ethanol to a concentration of 20 mg / ml. A10 was completely soluble at room temperature and was a pale pink, transparent liquid. DSPC, cholesterol, and DMG-PEG were then heated in a water bath at 50°C for several minutes until completely dissolved, resulting in a colorless, transparent liquid. After the above materials were prepared, the four ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 72.2:8.1:14.9:4.9, and the mixture was then drawn into an insulin syringe for use in preparation. A citric acid / sodium citrate buffer solution with a pH of 4.0 and a concentration of 10 nM was separately prepared at a volume three times the volume of the organic phase mixture, and similarly drawn into an insulin syringe for use in preparation. Then, place the insulin syringes containing the organic phase mixture and the buffer solution in the same centrifuge tube, quickly expel all the liquid, and gently shake the centrifuge tube to prepare LNP A10-1.

[0234] Example 39 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 61.8:12.1:19.1:7.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-2.

[0235] Example 40 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 52.1:14.6:21.5:11.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-3.

[0236] Example 41 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 65.9:5.9:16.3:11.9 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-4.

[0237] Example 42 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 64.9:10.2:21.4:3.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-5.

[0238] Example 43 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 69.3:15.6:11.4:3.7 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-6.

[0239] Example 44 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 70.2:5.3:19.3:5.3 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-7.

[0240] Example 45 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 70.5:9.2:9.7:10.6 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-8.

[0241] Example 46 The four dissolved ingredients were mixed in a mass ratio of A10:DSPC:cholesterol:DMG-PEG = 69.5:13.0:14.3:3.1 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP A10-9.

[0242] In the above example, A10, DSPC, cholesterol, and DMG-PEG were each set to three levels, as shown in Table 19. [Table 20]

[0243] The blending ratios of Examples 38-46 were designed using orthogonal design software (Orthogonal Design Assistant II V3.1), and the molar ratios and mass ratios of the nine LNPs with different blending ratios are shown in Table 20.

[0244] [Table 21]

[0245] Examples 47-55 The nine LNP / siApoB solutions obtained in Examples 38-46 with different formulations were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much siApoB as possible was encapsulated in the LNP. The nine LNP / siApoB complexes were then transferred to dialysis tubing with a molecular weight cutoff of 100,000 daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours.

[0246] Test Example 14 This test example aimed to detect the particle size, polydispersity index (PDI), and surface potential (zeta) of the nine LNPs obtained in Examples 38-46 that did not encapsulate siApoB. The results are shown in Table 21, and all formulations are expected to be excellent delivery carriers.

[0247] [Table 22]

[0248] Test Example 15 This test example aimed to detect the particle size, polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE), and intraparticle siRNA concentration (conc) of the nine siApoB-encapsulated formulations obtained in Examples 47-55. The results are shown in Table 22, and all formulations are expected to be excellent delivery carriers.

[0249] [Table 23]

[0250] Test Example 16 The nine preparations obtained in Examples 47-55 were subjected to in vitro transfection experiments, and the results of real-time fluorescent quantitative PCR are shown in Table 23. All preparations can achieve gene suppression.

[0251] [Table 24]

[0252] Preparation Example 3 Synthesis of amine-containing lipid compound B8

[0253] Synthesis of Compound B3 [ka] 1.0 g (1.0 eq) of compound B1 was dissolved in 15 ml of anhydrous dichloromethane, and 0.97 g (1.01 eq) of triethylamine was added in an ice bath. Then, 0.87 g (1.01 eq) of compound B2 was added dropwise to the system and reacted under argon gas protection. After 2 hours, the reaction of the raw materials was monitored by TLC to confirm completion. The crude product of compound B3 obtained was used in the next step without further treatment.

[0254] Synthesis of compound B4 [ka] Inject 1.92g (2.1eq) of triethylamine into the reaction system from the previous step, then inject 4.92g of myristate chloride into the system, continue the reaction under argon gas protection in an ice bath, after 2 hours, monitor the completion of the reaction of the raw materials by TLC, add 1M diluted hydrochloric acid and wash three times, wash once with H2O, collect the organic phase, dry with anhydrous sodium sulfate, remove the solvent by concentration under reduced pressure, purify with silica gel column (DCM / DCM:MeOH=500:1) to obtain 3.0g of white solid compound B4. 1H NMR (400MHz, 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

[0255] Synthesis of Compound B5 [ka] Dissolve 1.0g (1.0eq) of butanediamine in 30ml of anhydrous dichloromethane, add 5.2g (2.1eq) of Boc anhydride dropwise to the system in an ice bath, stir and react for 6 hours, monitor the reaction completion of the raw materials by TLC, add 1M dilute hydrochloric acid and saturated NaHCO3 sequentially to wash, collect the organic phase, dry it with anhydrous sodium sulfate, and then remove the solvent by concentration under reduced pressure to obtain 2.2g of white solid powder, compound B5.

[0256] Synthesis of Compound B6 [ka] Dissolve 1.0g (1.0eq) of compound B5 in 20ml of anhydrous dichloromethane, add 0.55g (3.0eq) of 60% mass fraction NaH to the system slowly in an ice bath, stir and react for 15min, then add 1.0g (2.0eq) of CHI slowly using a constant pressure dropping funnel to the system, react at low temperature for 1 hour, then transfer to room temperature and react overnight, the next day, monitor the completion of the raw material, add 1M diluted hydrochloric acid to perform the quenching reaction, remove DMF by concentrating under reduced pressure, then add DCM to dissolve, extract and wash with DCM / H2O, collect the organic phase, remove the solvent by concentrating under reduced pressure, and purify with a silica gel column (PE; EA=10:1) to obtain 3.0g of white solid compound B6. 1H NMR (400MHz, CDCl3)δ: 3,24(4H, m), 2.83(6H, s, CH3), 1.46-1.51(4H, m), 1.38-1.48(18H, m)ppm

[0257] Synthesis of Compound B7 [ka] 2.5 g (1.0 eq) of compound B6 is dissolved in 30 ml of HCl in EA solution (4 M), stirred at room temperature for 3 hours, and the solvent is removed by vacuum concentration to obtain 0.8 g of white solid powder, compound B7. 1H NMR (400MHz, CDCl3)δ: 2.97 (4H, s), 2.57 (6H, s, CH3), 1.56-1.61 (4H, m) ppm

[0258] Synthesis of compound B8 [ka] Dissolve 130mg (1.0eq) of compound B7 in 15ml of isopropyl alcohol, add 142.6mg (2.05eq) of triethylamine, stir at room temperature for 30min, then add 817.1mg (2.05eq) of compound B4, stir at 90℃ for 24h, TLC shows that a small amount of raw material remains, stop the reaction, remove the solvent by concentrating under reduced pressure, add DCM to dissolve, then add 1M diluted hydrochloric acid to wash three times, wash once with H2O, collect the organic phase, dry with anhydrous sodium sulfate, remove the solvent by concentrating under reduced pressure, purify with silica gel column to obtain 136mg of compound B8, yield 16%. 1H NMR (400MHz, 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, actual value M+1 = 1276.0.

[0259] Example 56 Lipid B8 obtained in Preparation Example 2, DSPC, cholesterol, and DMG-PEG (purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in absolute ethanol to a concentration of 20 mg / ml. B8 required 5 minutes of incubation at 30°C before it could be completely dissolved, resulting in a colorless, transparent liquid. DSPC, cholesterol, and DMG-PEG were then placed in a water bath at 50°C for several minutes until they were completely dissolved, resulting in a colorless, transparent liquid. After the above materials were prepared, the four ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 70.1:8.7:15.9:5.2, and the mixture was then drawn into an insulin syringe for use in preparation. A 10 nM citric acid / sodium citrate buffer solution with a pH of 4.0 and a concentration of 3 times the volume of the organic phase mixture was prepared separately, and similarly drawn into an insulin syringe for use in preparation. Then, 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 expelled. The centrifuge tube was gently shaken to prepare LNP B8-1.

[0260] Example 57 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 59.5:12.9:20.3:7.4 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-2.

[0261] Example 58 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 49.7:15.4:22.6:12.4 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-3.

[0262] Example 59 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 63.7:6.3:17.4:12.7 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-4.

[0263] Example 60 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 62.6:10.9:22.8:3.7 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-5.

[0264] Example 61 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 67.1:16.6:12.2:4.0 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-6.

[0265] Example 62 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 68.1:5.6:20.6:5.6 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-7.

[0266] Example 63 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 68.4:9.9:10.4:11.3 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-8.

[0267] Example 64 The four dissolved ingredients were mixed in a mass ratio of B8:DSPC:cholesterol:DMG-PEG = 67.4:13.9:15.3:3.4 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP B8-9.

[0268] In the above example, B8, DSPC, cholesterol, and DMG-PEG were each set to three levels, as shown in Table 24.

[0269] [Table 25]

[0270] The blending ratios of Examples 56-64 were designed using orthogonal design software (Orthogonal Design Assistant II V3.1), and the molar ratios and mass ratios of the nine LNPs with different blending ratios are shown in Table 25.

[0271] [Table 26]

[0272] Examples 65-73 The nine LNP / siApoB solutions obtained in Examples 56-64 with different composition ratios were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much siApoB as possible was encapsulated in the LNP. The nine LNP / siApoB complexes were then transferred to dialysis tubing with a molecular weight cutoff of 100,000 daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours.

[0273] Test Example 17 This test example aimed to detect the particle size, polydispersity index (PDI), and surface potential (zeta) of the nine LNPs obtained in Examples 56-64 that did not encapsulate siApoB. The results are shown in Table 26. All formulations have good physicochemical properties and have the potential to be excellent delivery vehicles.

[0274] [Table 27]

[0275] Test Example 18 The purpose of this test example was to detect the particle size, polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE), and intraparticle siRNA concentration (conc) of the nine siApoB-encapsulated formulations obtained in Examples 65-73. The results are shown in Table 27. All formulations have good physicochemical properties and have the potential to be excellent delivery carriers.

[0276] [Table 28]

[0277] Test Example 19 The nine preparations obtained in Examples 65-73 were subjected to in vitro transfection experiments, and the results of real-time fluorescent quantitative PCR are shown in Table 28. All preparations can achieve gene suppression.

[0278] [Table 29]

[0279] Preparation Example 4 Synthesis of amine-containing lipid compound C3

[0280] Synthesis of compound C2 [ka] Dissolve 200 mg (2.27 mmol) of 1,4-butanediamine in 10 mL of isopropyl alcohol, add 1.1 g (4.76 mmol) of compound C1, and when the system is colorless and transparent, heat to 70 °C and stir. After 3 hours, a white solid precipitates. Continue heating and stirring for 5 hours. When TLC shows that raw material C1 has completely reacted, stop the reaction. After removing the isopropyl alcohol under reduced pressure, add 20 mL of n-hexane, stir at room temperature for 1 hour, and filter with suction to obtain compound C2 (540 mg, 47% yield). 1H NMR (400MHz, 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

[0281] Synthesis of compound C3 [ka] Dissolve 130 mg (0.224 mmol) of compound B4 in 5 mL of isopropyl alcohol, add 57.49 mg (0.112 mmol) of compound C2, and react at 90 °C for 10 h. After TLC showed complete reaction of both raw materials B4 and C2, the reaction was stopped. Purify the mixture directly on a silica gel column without further workup to obtain the final product C3 (54 mg, 29% yield). H 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. Theoretical mass value = 1672.68, actual measured value M+1 = 1673.6.

[0282] Example 74 The lipid C3 obtained in Preparation Example 3, DSPC, cholesterol, and DMG-PEG (purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in absolute ethanol to a concentration of 20 mg / ml. Complete dissolution of C3 required 5 minutes of sonication at 50°C, resulting in a colorless, transparent liquid. DSPC, cholesterol, and DMG-PEG were then placed in a water bath at 50°C for several minutes until completely dissolved, resulting in a colorless, transparent liquid. After completing the preparation of the above materials, the four ingredients were mixed in a mass ratio of C3:DSPC:cholesterol:DMG-PEG = 75.5:7.1:13.1:4.3, and the entire mixture was then drawn into an insulin syringe for use in preparation. A 10 nM citric acid / sodium citrate buffer solution with a pH of 4.0 and a concentration of 3 times the volume of the organic phase mixture was separately prepared, and similarly drawn into an insulin syringe for use in preparation. Then, 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 expelled. The centrifuge tube was gently shaken to prepare LNP C3-1.

[0283] Example 75 The four dissolved ingredients were mixed in a mass ratio of C3:DSPC:cholesterol:DMG-PEG = 65.8:10.9:17.1:6.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-2.

[0284] Example 76 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-3.

[0285] Example 77 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNPs C3-4.

[0286] Example 78 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-5.

[0287] Example 79 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-6.

[0288] Example 80 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-7.

[0289] Example 81 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-8.

[0290] Example 82 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 drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP C3-9.

[0291] In the above example, C3, DSPC, cholesterol, and DMG-PEG were each set to three levels, as shown in Table 29.

[0292] [Table 30]

[0293] The blending ratios of Examples 74-82 were designed using orthogonal design software (Orthogonal Design Assistant II V3.1), and the molar ratios and mass ratios of the nine LNPs with different blending ratios are shown in Table 30.

[0294] [Table 31]

[0295] Examples 83-91 The nine LNP / siApoB solutions obtained in Examples 74-82 with different composition ratios were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much siApoB as possible was encapsulated in the LNP. The nine LNP / siApoB complexes were then transferred to dialysis tubing with a molecular weight cutoff of 100,000 daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours.

[0296] Test Example 20 This test example aimed to detect the particle size, polydispersity index (PDI), and surface potential (zeta) of the nine LNPs obtained in Examples 74-82 that did not encapsulate siApoB. The results are shown in Table 31. All formulations have good physicochemical properties and have the potential to be excellent delivery vehicles.

[0297] [Table 32]

[0298] Test Example 21 The purpose of this test example was to detect the particle size, polydispersity index (PDI), surface potential (zeta), encapsulation efficiency (EE) and intra-particle siRNA concentration (conc) of the nine siApoB-encapsulated formulations obtained in Examples 83-91. The results are shown in Table 32. All formulations have good physicochemical properties and have the potential to be excellent delivery carriers.

[0299] [Table 33]

[0300] Test Example 22 The nine preparations obtained in Examples 83-91 were subjected to in vitro transfection experiments, and the results of real-time fluorescent quantitative PCR are shown in Table 33. All preparations were able to achieve gene suppression.

[0301] [Table 34]

[0302] Example 92 Lipid A4 obtained in Preparation Example 1, DOPE, cholesterol, and DMG-PEG (purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in absolute ethanol to a concentration of 20 mg / ml. A4 was completely soluble at room temperature and was a pale yellow, transparent liquid. DOPE, cholesterol, and DMG-PEG were heated in a water bath at 50°C for several minutes until completely dissolved, resulting in a colorless, transparent liquid. After the above materials were prepared, the four ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 45.0:23.1:30:2.0, and the mixture was then drawn into an insulin syringe for use in preparation. A citric acid / sodium citrate buffer solution with a pH of 4.0 and a concentration of 10 nM was separately prepared at a volume three times the volume of the organic phase mixture, and similarly drawn into an insulin syringe for use in preparation. Then, place the insulin syringes containing the organic phase mixture and the buffer solution in the same centrifuge tube, quickly expel all the liquid, and gently shake the centrifuge tube to prepare lipid nanoparticles mA4-1.

[0303] Example 93 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 36.9:28.4:31.5:3.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-2.

[0304] Example 94 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 31.3:32.1:32.5:4.1 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-3.

[0305] Example 95 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 27.3:35.1:32.8:4.8 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-4.

[0306] Example 96 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 47.1:18.1:30.1:4.6 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-5.

[0307] Example 97 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 45.3:26.1:22.6:5.9 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-6.

[0308] Example 98 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 37.1:28.5:33.3:1.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-7.

[0309] Example 99 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 35.6:34.3:27.8:2.3 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-8.

[0310] Example 100 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 49.1:15.1:30.6:5.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-9.

[0311] Example 101 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 44.2:20.4:31.8:3.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-10.

[0312] Example 102 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 48.4:29.8:19.3:2.5 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-11.

[0313] Example 103 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 43.3:33.3:22.2:1.1 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into an insulin syringe, mixed with the organic phase, and gently shaken to prepare LNP mA4-12.

[0314] Example 104 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 52.6:13.5:31.6:2.3 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-13.

[0315] Example 105 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 51.9:20.0:27.0:1.1 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-14.

[0316] Example 106 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 49.3:25.3:21.0:4.3 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was prepared and drawn into an insulin syringe. The solution was mixed with the organic phase and gently shaken to prepare LNP mA4-15.

[0317] Example 107 The four dissolved ingredients were mixed in a mass ratio of A4:DOPE:cholesterol:DMG-PEG = 49.0:31.4:16.3:3.2 and drawn into an insulin syringe. A three-fold volume of citric acid / sodium citrate buffer solution was then drawn into the insulin syringe, mixed with the organic phase, and gently shaken to prepare LNP mA4-16.

[0318] In the above example, A4, DOPE, cholesterol, and DMG-PEG were each set to four levels, as shown in Table 34.

[0319] [Table 35]

[0320] The blending ratios of Examples 92-107 were designed using orthogonal design software (Orthogonal Design Assistant II V3.1), and the molar ratios and mass ratios of 16 LNPs with different blending ratios are shown in Table 35.

[0321] [Table 36]

[0322] Examples 108-123 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%.

[0323] The 16 different lipid nanoparticles obtained in Examples 92-107 were mixed with mLuc solution at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), gently shaken, and then incubated at 50°C for 20 minutes to ensure that as much mLuc as possible was encapsulated in the lipid nanoparticles. The 16 LNP / mLuc complexes were then transferred to a dialysis tube (Spectrum, G235035) with a molecular weight cutoff of 100,000 Daltons and dialyzed against 1x PBS for 3 hours.

[0324] Test Example 23 This test example aimed to detect the in vitro mRNA expression efficiency of the 16 preparations obtained in Examples 108-123, and the test results are shown in Figure 9. The results showed that all 16 preparations could mediate efficient mRNA expression in cells, with mLuA4-12 being the most preferred.

[0325] Test Example 24 This test example aimed to detect the metabolic characteristics of mLuA4-12 obtained in Example 119 in C57BL / 6 mice. mLuA4-12 was injected via the tail vein, and 6 hours later, the luciferase expression levels in each major organ were shown in Figure 10, indicating that mLuA4-12 can mediate efficient intracellular expression of mRNA.

[0326] Example 124 The concentration of the plasmid pCPLK1, which co-expresses PLK1 sgRNA and Cas9 protein, was adjusted to 1000 ng / μL and mixed with an equal volume of 50% ethanol to prepare a 500 ng / μL pCPLK1 solution with 25% ethanol. The mA4-12 and pCPLK1 solutions obtained in Example 92 were mixed at a mass ratio of 15:1 (i.e., a volume ratio of 1.5:1), shaken gently, and incubated at 50°C for 20 minutes. The pCPLK1 was encapsulated in lipid nanoparticles to obtain pCPA4-12. The sgPLK1-12 complex was then transferred to a dialysis tube with a molecular weight cutoff of 100,000 daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours.

[0327] Example 125 The PLK1 siRNA concentration was adjusted to 1000 ng / μL and mixed with an equal volume of 50% ethanol to prepare a 500 ng / μL siPLK1 solution with 25% ethanol. The A4-13 and siPLK1 solutions were mixed 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. The siPLK1 was encapsulated in the lipid nanoparticles to obtain siPLKA4-13. The siPLKA4-13 complex was then transferred into dialysis tubing with a 100,000 dalton molecular weight cutoff (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours.

[0328] Example 126 sgRNA targeting PLK1 was dissolved in DEPC-treated water to a concentration of 1000 ng / μL and mixed with an equal volume of 50% ethanol to prepare a 500 ng / μL sgPLK1 solution with 25% ethanol. Cas9 protein was dissolved in DEPC-treated water, its concentration measured by the BCA method, and then mixed with an equal volume of sgPLK1. A4-13 and the above solution were then mixed at a mass ratio of 30:1, shaken gently, and incubated at 50°C for 20 minutes. The sgPLK1 and Cas9 proteins were encapsulated in lipid nanoparticles to obtain CasPLKA4-13. The CasPLKA4-13 complex was then transferred to a dialysis tube with a molecular weight cutoff of 100,000 daltons (Spectrum, G235035) and dialyzed against 1x PBS for 3 hours. HepG2 cells were used as test subjects. 24 hours after plating, cells were transfected with CasPLKA4-13, and siPLKA4-13 was used as a positive control. 24 hours after receiving the cells, qPCR was performed to detect the relative expression of PLK1.

[0329] Example 127 Azithromycin hydrochloride was dissolved in PBS until completely dissolved, and the concentration was measured. A4-13 was mixed with the above solution at a mass ratio of 15:1, gently shaken, and then incubated at room temperature for 20 minutes to form the DOXA4-13 complex. The DOXA4-13 complex was then transferred to a dialysis tube (Spectrum, G235035) with a molecular weight cutoff of 100,000 daltons and dialyzed against 1x PBS for 3 hours. HepG2 cells were used as test subjects. 24 hours after plating, the cells were transfected with DOXA4-13, and free DOX was used as a control. 24 hours later, the cells were received and the relative activity of the cells after different sample treatments was detected using the MTT assay.

[0330] Test Example 25 The purpose of this test example was to test the target gene editing efficiency of pCPA4-12 obtained in Example 124 at the cellular level and compare the gene silencing efficiency with siPLKA4-13 obtained in Example 125. The results are shown in Table 34. 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 there was a significant difference compared to the PBS control group, indicating that mA4-12 has good prospects for gene editing applications.

[0331] [Table 37]

[0332] Comparative Example 1 The purpose of this comparative example is to compare the stability of lipid A4 obtained in Preparation Example 1 with that of the control lipid E. The chemical structural formula of lipid E is shown in FIG.

[0333] Lipids A4 and E were dissolved in 90% absolute ethanol and 10% 0.05M citrate (w / w) at pH 4.0, respectively. 100 μL of the sample was immediately aspirated as a pre-incubation sample and immediately stored at -80°C to prevent sample degradation. This was day 0 of the experiment. The A4 and E test samples were then incubated at 40°C, and 100 μL of each sample was aspirated and stored at -80°C on days 1, 3, 5, and 7 of the experiment. After all samples were obtained, the sample stability was tested by HPLC under the following test conditions:

[0334] [Table 38]

[0335] [Table 39]

[0336] Comparative Example 2 Lipofectime 2000 (Lipo) is a commercially available transfection reagent that can mediate efficient nucleic acid delivery at the cellular level, but is unable to achieve effective delivery at the animal level. This experiment aimed to compare the efficacy of A4-13 in C57 mice (dosage: 0.5 mg / kg). The relative expression of Lipo / siApoB was 0.85, while the relative expression of A4-13 / siApoB was 0.27. The results are shown in Figure 12. These experimental results demonstrate that Lipo has low delivery efficiency in vivo, while A4-13 can achieve efficient siRNA delivery.

[0337] Comparative Example 3 This test example aims to detect the cellular uptake efficiency of Cy-siFLA4-13 obtained in Example 34. The test analysis results are shown in Table 35. The delivery efficiency of this formulation 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, the potential application advantages of A4-13 in the field of nucleic acid delivery are obvious.

[0338] [Table 40]

[0339] Comparative Example 4 This comparative example aims to compare the endocytosis mediated by the complex obtained after mRNA encapsulation with Lipo and mLuA4-12 obtained in Example 119 at the cellular level. The test results are shown in Table 36, which show that all cells successfully endocytose the luciferase mRNA with Lipo and mLuA4-12, and Lipo achieves a higher average fluorescent signal intensity, i.e., a larger amount of luciferase mRNA per single cell. However, considering that Lipo cannot achieve efficient nucleic acid delivery at the animal level, mLuA4-12 also has a wider range of applications.

[0340] [Table 41]

[0341] Comparative Example 5 This comparative example aims to compare the cellular delivery efficiency of liposome E, obtained using lipid E as the core lipid, and siE, obtained by further incubating with siApoB, with siA4-13 obtained in Example 29. The results are shown in Table 37, which indicate that siA4-13 can achieve efficient target gene suppression at the cellular level, while siE can hardly achieve target gene suppression at the cellular level.

[0342] [Table 42]

[0343] In the description herein, references such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In the description herein, the terms "exemplary" and "specific examples" do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, if not in conflict with each other, those skilled in the art may combine and combine features of different embodiments or examples described herein.

[0344] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention, and those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. A compound represented by any one of the following formulae (A4), (A10), (B8) and (C3): 【Chemistry 10】 or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of said compound.

2. 10. Use of the compound of claim 1 in the preparation of liposomes.

3. A liposome comprising the compound according to claim 1.

4. The liposome of claim 3, further comprising at least one selected from a hydrophobic lipid, an amphipathic lipid, a buffering agent, and an organic solvent, wherein the amphipathic lipid comprises at least one selected from a neutral lipid and a PEG-lipid, and the hydrophobic lipid is selected from a sterol.

5. The liposome described in claim 4, wherein the neutral lipid comprises at least one selected from distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine, dioleoylphosphatidylethanolamine, dilaurylphosphatidylcholine, diethyl pyrocarbonate, dimyristylphosphatidylcholine and egg yolk lecithin.

6. The liposome of claim 4, wherein the PEG-lipid comprises at least one selected from bispalmitoylphosphatidylethanolamine-PEG, bisstearylphosphatidylethanolamine-PEG, dimyristylglycerol, and dimethacrylate.

7. The liposome described in claim 4, wherein the sterol is cholesterol.

8. The liposome described in claim 4, wherein the buffer reagent is an acidic buffer reagent.

9. The liposome of claim 4, wherein the buffer reagent comprises at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trimethylolaminomethane-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.

10. The liposome of claim 4, characterized in that the organic solvent comprises at least one selected from methanol, ethanol, isopropyl alcohol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl ethyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, and triethanolamine.

11. The liposome according to claim 4, wherein the amount of the compound is 20 to 80% mol / mol based on the liposome.

12. The liposome of claim 4, wherein the molar ratio of the compound, neutral lipid, sterol and PEG-lipid is (20-80):(5-50):(10-60):(0.01-20).

13. The liposome described in claim 4, wherein the volume of the buffer reagent is 50 to 90% v / v based on the liposome.

14. The liposome described in claim 4, wherein the volume of the buffer reagent is 75% v / v based on the liposome.

15. 15. A method for preparing the liposome according to any one of claims 3 to 14, comprising the step of mixing a predetermined amount of the compound according to claim 1 with an organic solvent to obtain the liposome.

16. 16. The method of claim 15, further comprising the step of mixing a predetermined amount of a hydrophobic lipid, an amphipathic lipid and / or a buffer reagent with the solution obtained in claim 15 to obtain the liposomes.

17. A drug carrier comprising the compound according to claim 1 or the liposome according to any one of claims 3 to 14.

18. Use of the compound according to claim 1, the liposome according to any one of claims 3 to 14, or the drug carrier according to claim 17 in the preparation of a drug.

19. A pharmaceutical composition comprising a carrier and an active pharmaceutical ingredient, wherein the carrier comprises the pharmaceutical carrier described in claim 17.

20. The active pharmaceutical ingredient is 20. The pharmaceutical composition of claim 19, comprising at least one selected from a DNA molecule, an RNA molecule, a protein, a polypeptide, and a small molecule drug.

21. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical active ingredient is negatively charged.

22. 21. The pharmaceutical composition of claim 20, wherein the active pharmaceutical ingredient comprises a nucleic acid.

23. The pharmaceutical composition of claim 19, wherein the mass ratio of the carrier to the active pharmaceutical ingredient is (5-40):

1.

24. The pharmaceutical composition of claim 19, wherein the mass ratio of the carrier to the active pharmaceutical ingredient is (8-20):

1.

25. The pharmaceutical composition of claim 19, wherein the mass ratio of the carrier to the active pharmaceutical ingredient is 15:

1.

26. A transfection complex comprising the compound according to claim 1 or the liposome according to any one of claims 3 to 14.

27. 27. The transfection complex of claim 26, wherein the transfection complex comprises at least one bioactive agent.

28. The bioactive agent is 28. The transfection complex of claim 27, comprising at least one selected from a DNA molecule, an RNA molecule, a protein, and a drug.

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