Cationic lipid analogs, compositions and uses thereof

Abstract

The present invention belongs to the field of biomedicine technology, and discloses cationic lipid analogues, their compositions and uses. The present invention uses aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds as raw materials, synthesizes cationic lipid analogues by Ugi reaction, and the obtained cationic lipid analogues are combined with sterols, helper lipids and polyethylene glycol lipid derivatives to form lipid nanoparticle-like compositions, which can be used to deliver drugs including small molecule drugs, nucleic acid drugs such as mRNA, DNA, siRNA, protein / polypeptide drugs, and gene editing complexes such as mRNA / sgRNA, Cas9 / sgRNA, and can achieve mRNA expression in various organs in animal bodies through various administration methods, and can meet the needs of use such as mRNA nucleic acid therapy, nucleic acid vaccines and gene editing.

Description

[Technical field]

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to cationic lipid analogs, compositions and uses thereof. [Background technology]

[0002] Liposomes are bilayer vesicles made of lipidoids such as phospholipids, which are completely closed and can encapsulate two types of substances, water-soluble and lipid-soluble, and have the characteristics of high targeting efficiency, controlled sustained release, and safety and non-toxicity. In recent years, molecular drug delivery technology using liposomes and their analogues has matured, and there has been an increase in research showing the potential of various lipid nanoparticles in molecular drug therapy.

[0003] Liposomes are divided into cationic liposomes, neutral liposomes and anionic liposomes according to the nature of the charge. Here, cationic liposomes have the advantages of being naturally degradable, having no toxicity or immunogenicity, and being capable of repeated transfection. Cationic liposomes are usually used as gene vectors, which can recombine with DNA or RNA, protect them from inactivation and degradation by nucleases, and transport genes to specific sites, ensuring that the transfection process is simple, easy to carry out, and highly reproducible. A liposome delivery system with high gene transfer efficiency can properly express a specific gene in a specific organ in the body, thereby realizing therapeutic applications. Gene therapy applications developed by conventional technology include nucleic acid therapy, nucleic acid vaccines, protein replacement therapy, gene editing, etc. In particular, with the rapid development of nucleic acid vaccines in recent years, messenger RNA vaccines are considered important due to their ease of production, high efficiency and stability. Currently, many studies on cationic lipid particles have been reported, and commercialized cationic lipid materials such as the cationic phospholipid DOTAP and the ionizable cationic lipid DLin-MC3 can be selected. However, there are problems such as low delivery efficiency in the body, toxicity of cations to the living body, and poor targeting of internal delivery. Considering that subtle differences in the structure of cationic lipid analogues can lead to significant changes in their biological properties, it is important to develop novel cationic lipid analogue structures that have high in vivo transfection efficiency, low toxicity, and specific targeting. Summary of the Invention [Problem to be solved by the invention]

[0004] It is an object of the present invention to overcome the shortcomings of the prior art and to provide cationic lipid analogs, compositions and uses thereof. [Means for solving the problem]

[0005] In order to achieve the above objectives, the technical solutions adopted in the present invention are as follows: In a first aspect, the present invention produces a cationic lipid analog having a structure represented by any one of formulas (I) to (V) below. JPEG2025503982000002.jpg18397 [wherein m1 is independently at least one selected from the group consisting of a hydrogen atom, a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a substituted alkynyl group, a cycloalkyl group, a phenyl group, and a heteroatom-containing aromatic group; m2 is independently at least one selected from the group consisting of a linear alkyl group, a linear alkenyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing linear alkylene group, a heteroatom-containing branched alkyl group, a heteroatom-containing branched alkylene group, a heterocycle-containing alkyl group, a heterocycle-containing alkylene group, an ester group-containing alkyl group, and an ether group-containing alkyl group; m3 is independently at least one selected from the group consisting of a linear alkyl group, a linear alkenyl group, a cycloalkyl group, a linear alkyl group containing a heteroatom, a branched alkyl group containing a heteroatom, a branched alkylene group containing a heteroatom, an alkyl group containing a heterocycle, an alkyl group containing a heteroatom and an aromatic ring, an alkyl group containing a hydroxyl group, an alkyl group containing an ester group, an alkyl group containing an ether group, and an alkyl group containing a sulfonate group; m4 is independently at least one selected from the group consisting of a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing branched alkyl group, and a heterocycle-containing alkyl group. In the present invention, aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds are used as raw materials to synthesize cationic lipid analogues by Ugi reaction.Cationic lipid analogues have high in vivo transfection efficiency, low toxicity and specific targeting, and can achieve mRNA expression in various organs in animals through various administration methods, and can meet the needs of mRNA nucleic acid therapy, nucleic acid vaccines, gene editing and the like. JPEG2025503982000003.jpg224161JPEG2025503982000004.jpg235159JPEG2025503982000005.jpg240160In a second aspect, the present invention provides a method for producing a cationic lipid analogue, in which an aldehyde compound and an amine compound are added to an organic solution and reacted for 10 to 120 minutes, and then a carboxylic acid compound and an isonitrile compound are added in sequence, and the reaction is continued at 4 to 60°C for 6 to 72 hours, and the product is separated and purified to obtain a cationic lipid analogue. Preferably, the molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound and the isonitrile compound is (0.1-1.5):(0.1-1.5):(0.1-1.5):(0.1-1.5), and more preferably, the molar ratio is 1:(0.33-1):1:(0.33-1). The cationic lipid analogues of the present invention are produced under mild reaction conditions, have a simple synthetic process, and are highly stable. In a preferred embodiment of the method for producing a cationic lipid analogue according to the present invention, the isonitrile compound is one of a monofunctional isonitrile compound, a difunctional isonitrile compound, and a trifunctional isonitrile compound. Preferably, the isonitrile compound is any one of the following compounds I3 to I38. JPEG2025503982000006.jpg95135 In a preferred embodiment of the method for producing a cationic lipid analogue according to the present invention, the aldehyde compound is one of monofunctional aldehyde compounds. Preferably, the aldehyde compound is any one of the following compounds A1 to A35. JPEG2025503982000007.jpg8072 In a preferred embodiment of the method for producing a cationic lipid analogue according to the present invention, the amine compound is one of a monofunctional amine compound, a bifunctional amine compound, and a trifunctional amine compound. In a preferred embodiment of the method for producing a cationic lipid analogue according to the present invention, the amine compound is any one of the following compounds R1 to R53. JPEG2025503982000008.jpg115150 In a preferred embodiment of the method for producing a cationic lipid analogue according to the present invention, the carboxylic acid compound is any one of the following compounds C8 to C30 or S11 to S19. JPEG2025503982000009.jpg110144 In a third aspect, the present invention provides a composition of said cationic lipid analogue comprising said cationic lipid analogue and at least one of a sterol, a helper lipid, and a polyethylene glycol lipid derivative. In a preferred embodiment of the cationic lipid analogue composition according to the present invention, the sterol comprises at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives, and the helper lipid is a non-cationic lipid, more preferably the helper lipid is a neutral phospholipid, and even more preferably the helper lipid is at least one of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramide. As a preferred embodiment of the cationic lipid analogue composition according to the present invention, the molar ratio of the cationic lipid analogue, cholesterol, helper lipid and polyethylene glycol lipid derivative is (20-70):(20-50):(2-30):(0.1-20), and preferably, the molar ratio is (40-60):(25-45):(5-15):(0.5-5). In a fourth aspect, the present invention provides a drug-loaded nanoparticle comprising a composition of the cationic lipid analogue and a drug. In a preferred embodiment of the drug-loaded nanoparticles described in the present invention, the drug comprises at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide-based molecule, and a gene editing complex. Preferably, the nucleic acid molecule is at least one of messenger RNA (mRNA), transfer RNA (tRNA), dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA, and the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA. Preferably, the mass ratio of the cationic lipid analogue to the nucleic acid molecule is cationic lipid analogue:nucleic acid molecule=(2-50):1, and more preferably cationic lipid analogue:nucleic acid molecule=(5-20):1. In a fifth aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: (1) dissolving the cationic lipid analogue composition in an organic solution to obtain an organic phase; Step (2) of dissolving the nucleic acid molecule in a buffer solution to obtain an aqueous phase, in which the volume of the aqueous phase: the volume of the organic phase = (2 to 5): 1; (3) adding the aqueous phase to the organic phase quickly (or the organic phase to the aqueous phase quickly), mixing uniformly, and dialyzing to obtain drug-loaded nanoparticles; The present invention also provides a method for producing the drug-loaded nanoparticles, comprising the steps of: In a sixth aspect, the present invention provides the use of said cationic lipid analogues, compositions of said cationic lipid analogues, said drug-loaded nanoparticles in the manufacture, delivery or transport of molecular drugs or nucleic acid vaccines. Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds as raw materials to synthesize cationic lipid analogues by Ugi reaction. The cationic lipid analogues of the present invention are produced under mild reaction conditions, have a simple synthesis process and are stable. The synthesized cationic lipid analogues, sterols, helper lipids and polyethylene glycol lipid derivatives form lipid nanoparticle-like compositions, which can be used to deliver drugs including small molecule drugs, nucleic acid molecules such as mRNA, DNA and siRNA, protein / polypeptide molecules, and gene editing complexes such as mRNA / sgRNA and Cas9 / sgRNA. Various administration methods can achieve mRNA expression in various organs in the animal body, and meet the needs of use such as mRNA nucleic acid therapy, nucleic acid vaccines and gene editing. [Brief description of the drawings]

[0006] [Figure 1] 1 is a hydrogen nuclear magnetic resonance spectrum of A4I18R3C18-2 in Example 1. [Diagram 2] 1 is a mass spectrum of A4I18R3C18-2 in Example 1. [Diagram 3] 1 is a hydrogen nuclear magnetic resonance spectrum of A1I4R22C18-2 in Example 2. [Figure 4] 1 is a mass spectrum of A1I4R22C18-2 in Example 2. [Diagram 5] 1 is a hydrogen nuclear magnetic resonance spectrum of A1I5R22S15 in Example 3. [Figure 6] 1 is a mass spectrum of A1I5R22S15 in Example 3. [Figure 7] 1 is a hydrogen nuclear magnetic resonance spectrum of A1I32R46C18-2 in Example 4. [Figure 8] 1 is a mass spectrum of A1I32R46C18-2 in Example 4. [Figure 9]1 is a hydrogen nuclear magnetic resonance spectrum of A1I32R47C18-2 in Example 5. [Figure 10] 1 is a mass spectrum of A1I32R47C18-2 in Example 5. [Figure 11] FIG. 1 is a statistical diagram of antibody titers after immunization with OVA-mRNA drug-loaded nanoparticles prepared from the compound of Example 1. [Figure 12] FIG. 13 is a statistical diagram of antibody titers after immunization with OVA-mRNA drug-loaded nanoparticles prepared from the compound of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Those skilled in the art should understand that the specific examples described herein are for illustrating the present invention, and are not intended to limit the present invention. The test methods used in the examples are conventional unless otherwise specified, and the materials, reagents etc. used are commercially available unless otherwise specified.

[0008] Example 1: Cationic lipid analogues The cationic lipid analogue is synthesized according to the chemical reaction general formula (I), and the general formula of the structure of the product is as follows: JPEG2025503982000010.jpg3385The specific manufacturing method is as follows. At room temperature, 1.0 mmol of an aldehyde compound and 1.0 mmol of an amine compound were added to 0.5 mL of a methanol solution, and reacted at room temperature for 60 minutes. Then, 1.0 mmol of a carboxylic acid compound was added and reacted at room temperature for 60 minutes. Then, 1.0 mmol of an isonitrile compound was added and reacted at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by column chromatography, and a mixture of methanol and dichloromethane was used as the mobile phase. The general scheme for the synthesis of formula (I) is as follows: JPEG2025503982000011.jpg20147Here, the aldehyde compound was A1, A4, or A23, the isonitrile compound was I18, the amine compound was R1, R2, or R3, and the carboxylic acid compound was C18, C18-1, C18-2, C18-3, or C23. The following 45 cationic lipid analogs were synthesized from the above preferred compounds: A1I18R1C18, A1I18R1C18-1, A1I18R1C18-2, A1I18R1C18-3, A1I18R1C23, A1I18R2C1 8, A1I18R2C18-1, A1I18R2C18-2, A1I18R2C18-3, A1I18R2C23, A1I18R3C18, A1I18R3 C18-1, A1I18R3C18-2, A1I18R3C18-3, A1I18R3C23, A4I18R1C18, A4I18R1C18-1, A4I 18R1C18-2, A4I18R1C18-3, A4I18R1C23, A4I18R2C18, A4I18R2C18-1, A4I18R2C18-2, A4I18R2C18-3, A4I18R2C23, A4I18R3C18, A4I18R3C18-1, A4I18R3C18-2, A4I18R3C1 8-3, A4I18R3C23, A23I18R1C18, A23I18R1C18-1, A23I18R1C18-2, A23I18R1C18-3, A2 3I18R1C23, A23I18R2C18, A23I18R2C18-1, A23I18R2C18-2, A23I18R2C18-3, A23I18 R2C23, A23I18R3C18, A23I18R3C18-1, A23I18R3C18-2, A23I18R3C18-3, A23I18R3C23 In addition, the aldehyde compound was either A1, A4, or A23, the isonitrile compound was I18, the amine compound was either R5 or R12, and the carboxylic acid compound was either C18 or C18-2. The following 12 cationic lipid analogues were synthesized from the above preferred compounds: A1I18R5C18, A1I18R5C18-2, A1I18R12C18, A1I18R12C18-2, A4I18R5C18, A4I18R5C18-2, A4 I18R12C18, A4I18R12C18-2, A23I18R5C18, A23I18R5C18-2, A23I18R12C18, A23I18R12C18-2 In addition, the aldehyde compound was A4 or A5, the isonitrile compound was I10, I14, or I18, the amine compound was R4, R6, R7, R8, R9, R10, or R11, and the carboxylic acid compound was C18 or C18-2. The cationic lipid analogues synthesized from the above preferred compounds were the following 10 types: A5I18R2C18-2, A4I10R2C18-2, A4I14R2C18-2, A4I18R4C18-2, A4I18R6C18-2, A4I18R7C18-2, A4I18R8C18-2, A4I18R9C18-2, A4I18R10C18-2, A4I18R11C18-2 The hydrogen nuclear magnetic resonance spectrum and mass spectrum of A4I18R3C18-2 are shown in Figures 1 and 2.

[0009] Example 2: Cationic lipid analogues The cationic lipid analogue is synthesized according to the chemical reaction general formula (II), and the general formula of the product structure is as follows: JPEG2025503982000012.jpg25111The specific manufacturing method is as follows. At room temperature, 1.0 mmol of an aldehyde compound and 1.0 mmol of an amine compound were added to 0.5 mL of a methanol solution, and reacted at room temperature for 60 minutes. Then, 1.0 mmol of a carboxylic acid compound was added and reacted at room temperature for 60 minutes. Then, 0.5 mmol of an isonitrile compound was added and reacted at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by column chromatography, and a mixture of methanol and dichloromethane was used as the mobile phase. The general scheme for the synthesis of formula (II) is as follows: JPEG2025503982000013.jpg15140Here, the aldehyde compound was any of A1, A4, A21, and A23, the isonitrile compound was I4, the amine compound was any of R2, R13, R21, R22, and R23, and the carboxylic acid compound was any of C18, C18-1, and C18-2. The following 60 cationic lipid analogues were synthesized using the above compounds: A1I4R2C18, A1I4R2C18-1, A1I4R2C18-2, A1I4R13C18, A1I4R13C18-1, A1I4R13C18-2, A1I4R21C18, A1I4R21C18-1 , A1I4R21C18-2, A1I4R22C18, A1I4R22C18-1, A1I4R22C18-2, A1I4R23C18, A1I4R23C18-1, A1I4R23C18-2, A4I4R2C 18, A4I4R2C18-1, A4I4R2C18-2, A4I4R13C18, A4I4R13C18-1, A4I4R13C18-2, A4I4R21C18, A4I4R21C18-1, A4I4R2 1C18-2, A4I4R22C18, A4I4R22C18-1, A4I4R22C18-2, A4I4R23C18, A4I4R23C18-1, A4I4R23C18-2, A21I4R2C18, A21 I4R2C18-1, A21I4R2C18-2, A21I4R13C18, A21I4R13C18-1, A21I4R13C18-2, A21I4R21C18, A21I4R21C18-1, A21I4 R21C18-2, A21I4R22C18, A21I4R22C18-1, A21I4R22C18-2, A21I4R23C18, A21I4R23C18-1, A21I4R23C18-2, A23I4R 2C18, A23I4R2C18-1, A23I4R2C18-2, A23I4R13C18, A23I4R13C18-1, A23I4R13C18-2, A23I4R21C18, A23I4R21C18- 1, A23I4R21C18-2, A23I4R22C18, A23I4R22C18-1, A23I4R22C18-2, A23I4R23C18, A23I4R23C18-1, A23I4R23C18-2 In addition, the aldehyde compound was A1, the isonitrile compound was I4, the amine compound was any of R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R42, R43, and R48, and the carboxylic acid compound was C18-2. The following 13 types of cationic lipid analogues were synthesized using the above compounds. A1I4R27C18-2, A1I4R28C18-2, A1I4R29C18-2, A1I4R30C18-2, A1I4R31C18-2, A1I4R32C18-2, A1I4R3 3C18-2, A1I4R34C18-2, A1I4R35C18-2, A1I4R36C18-2, A1I4R42C18-2, A1I4R43C18-2, A1I4R48C18-2 In addition, the aldehyde compound was any of A1, the isonitrile compound was I4, the amine compound was R22, and the carboxylic acid compound was any of C12, C14, C16, S11, S13, S15, S17, and S19. The following eight cationic lipid analogues were synthesized using the above compounds: A1I4R22C12, A1I4R22C14, A1I4R22C16, A1I4R22S11, A1I4R22S13, A1I4R22S15, A1I4R22S17, A1I4R22S19 The hydrogen nuclear magnetic resonance spectrum and mass spectrum of A1I4R22C18-2 are shown in Figures 3 and 4.

[0010] Example 3: Cationic lipid analogs The cationic lipid analogue is synthesized according to the chemical reaction general formula (III), and the general formula of the product structure is as follows: JPEG2025503982000014.jpg6694The specific manufacturing method is as follows. At room temperature, 1.0 mmol of an aldehyde compound and 1.0 mmol of an amine compound were added to 0.5 mL of a methanol solution, and reacted at room temperature for 60 minutes. Then, 1.0 mmol of a carboxylic acid compound was added and reacted at room temperature for 60 minutes. Then, 0.33 mmol of an isonitrile compound was added and reacted at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by column chromatography, and a mixture of methanol and dichloromethane was used as the mobile phase. The general scheme for the synthesis of formula (III) is as follows: JPEG2025503982000015.jpg43124Here, the aldehyde compound is A1, the isonitrile compound is I5, the amine compound is R2, R13, R21, R22, R23, R26, R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R37, R38, R39, R40, R41 or R50, and the carboxylic acid compound is C8, C10, C12, C14, C16, C18, C18-2, C21, S11, S13, S15, S17 or S19. The following 29 types of cationic lipid analogues were synthesized using the above compounds: A1I5R22C8, A1I5R22C10, A1I5R22C12, A1I5R22C14, A1I5R22C16, A1I5R22C18, A1I5R22C18-2, A 1I5R22S11, A1I5R22S13, A1I5R22S15, A1I5R22S17, A1I5R22S19, A1I5R26S15, A1I5R27S15, A1I5 R28S15, A1I5R29S15, A1I5R30S15, A1I5R31S15, A1I5R32S15, A1I5R33S15, A1I5R34S15, A1I5R35 S15, A1I5R36S15, A1I5R37S15, A1I5R38S15, A1I5R39S15, A1I5R40S15, A1I5R41S15, A1I5R50C21 The hydrogen nuclear magnetic resonance spectrum and mass spectrum of A1I5R22S15 are shown in FIG. 5 and FIG.

[0011] Example 4: Cationic lipid analogs The cationic lipid analogue is synthesized according to the chemical reaction general formula (IV), and the general formula of the structure of the product is as follows: JPEG2025503982000016.jpg5267The specific manufacturing method is as follows. At room temperature, 1.0 mmol of an aldehyde compound and 0.5 mmol of an amine compound were added to 0.5 mL of a methanol solution, and reacted at room temperature for 60 minutes. Then, 1.0 mmol of a carboxylic acid compound was added and reacted at room temperature for 60 minutes. Then, 1.0 mmol of an isonitrile compound was added and reacted at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by column chromatography, and a mixture of methanol and dichloromethane was used as the mobile phase. The general scheme for the synthesis of formula (IV) is as follows: JPEG2025503982000017.jpg30106Here, the aldehyde compound was A1, the isonitrile compound was I10, I12, I31 or I32, the amine compound was R46 or R51, and the carboxylic acid compound was C8 or C18-2. The following four types of cationic lipid analogues were synthesized using the above compounds: A1I12R51C8, A1I10R51C18-2, A1I31R46C18-2, A1I32R46C18-2 The hydrogen nuclear magnetic resonance spectrum and mass spectrum of A1I32R46C18-2 are shown in FIG. 7 and FIG.

[0012] Example 5: Cationic lipid analogs Cationic lipid analogues are synthesized according to the general chemical reaction formula (V), and the general formula of the product structure is as follows: JPEG2025503982000018.jpg6782The specific manufacturing method is as follows. At room temperature, 1.0 mmol of an aldehyde compound and 0.33 mmol of an amine compound were added to 0.5 mL of methanol solution, and reacted at room temperature for 60 minutes. Then, 1.0 mmol of a carboxylic acid compound was added and reacted at room temperature for 60 minutes. Then, 1.0 mmol of an isonitrile compound was added and reacted at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by column chromatography, and a mixture of methanol and dichloromethane was used as the mobile phase. The general scheme for the synthesis of formula (V) is as follows: JPEG2025503982000019.jpg39116Here, the aldehyde compound was A1, the isonitrile compound was I12, I31 or I32, the amine compound was R47, and the carboxylic acid compound was C21 or C18-2. The cationic lipid analogues synthesized using the above compounds were of three types: A1I12R47C21, A1I31R47C18-2, A1I32R47C18-2 The hydrogen nuclear magnetic resonance spectrum and mass spectrum of A1I32R47C18-2 are shown in FIG. 9 and FIG.

[0013] Example 6: Drug-loaded nanoparticles The drug-loaded nanoparticles contained cationic lipid analogues, cholesterol, distearoylphosphatidylcholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). Here, the molar ratio of cationic lipid analogue:cholesterol:DSPC:DMG-PEG was 50:38.5:10:1.5. In the drug-loaded nanoparticles, the mass ratio of the cationic lipid analogue to the nucleic acid drug such as mRNA or siRNA was cationic lipid analogue:nucleic acid drug=11:1. The manufacturing method of the drug-loaded nanoparticles carrying mRNA is taken as an example. The cationic lipid analogue, cholesterol, DSPC, and DMG-PEG were dissolved in an ethanol solution and mixed in the above molar ratio to obtain an organic phase. The corresponding mass ratio of the mRNA nucleic acid drug was dissolved in a sodium acetate buffer solution (25 mM, pH = 3.0) so that the aqueous phase volume: organic phase volume = 3:1 to obtain an aqueous phase. The aqueous phase was quickly added to the organic phase, mixed uniformly, and allowed to stand for 10 minutes, then placed in a 1000 Da dialysis bag and dialyzed in 1x PBS solution at 4°C for 2 hours to obtain nanoparticles for direct injection. Table 1. Identification data of lipid nanoparticles produced in Example 6 JPEG2025503982000020.jpg35169

[0014] Example 7: Drug-loaded nanoparticles The drug-loaded nanoparticles contained cationic lipid analogues, β-sitosterol, dioleylphosphatidylethanolamine (DOPE), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). Here, the molar ratio was cationic lipid analogue:β-sitosterol:DOPE:DMG-PEG=35:25:5:2. In the drug-loaded nanoparticles, the mass ratio of the cationic lipid analogue to the nucleic acid drug such as mRNA or siRNA was cationic lipid analogue:nucleic acid drug=5:1.

[0015] Example 8: Drug-loaded nanoparticles The drug-loaded nanoparticles contained a cationic lipid analog, stigmasterol, ceramide phosphatidylcholine, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). Here, the molar ratio of cationic lipid analogue:stigmasterol:ceramide phosphatidylcholine:DMG-PEG was 55:40:12:2.5. In the drug-loaded nanoparticles, the mass ratio of the cationic lipid analogue to the nucleic acid drug such as mRNA or siRNA was cationic lipid analogue:nucleic acid drug=15:1.

[0016] Example 9: Drug-loaded nanoparticles The drug-loaded nanoparticles contained cationic lipid analogues, cholesteryl succinate (a cholesterol derivative), sphingomyelin, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). Here, the molar ratio of cationic lipid analogue:cholesteryl succinate:sphingomyelin:DMG-PEG was 60:45:15:3.5. In the drug-loaded nanoparticles, the mass ratio of the cationic lipid analogue to the nucleic acid drug such as mRNA or siRNA was cationic lipid analogue:nucleic acid drug=18:1.

[0017] Example 10: Measurement of expression levels in animals when mRNA is delivered intravenously Drug-loaded nanoparticles were produced according to the method of Example 6 using the cationic lipid analogs of Examples 1 to 5, with Luciferase-mRNA as the model mRNA. The specific operation method is as follows: 200 μL of nanoparticles containing 5 μg of Luciferase-mRNA or the drug-loaded nanoparticles prepared above were injected into the tail vein of 6-8 week-old C57BL / 6 mice. Six hours later, 200 μL of 1x PBS solution containing 3 mg of D-Luciferin potassium salt was injected into the abdominal cavity of the mice. After 10 minutes, the mRNA expression status in the body was evaluated by bioluminescence imaging. The expression status of drug-loaded nanoparticles in the body is shown in Tables 2 to 6.

[0018] Table 2. Expression status in vivo after intravenous injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 1 JPEG2025503982000021.jpg252164JPEG2025503982000022.jpg248164JPEG2025503982000023.jpg253164 JPEG2025503982000024.jpg251164JPEG2025503982000025.jpg244164JPEG2025503982000026.jpg153164

[0019] Table 3. Expression status in vivo after intravenous injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 2 JPEG2025503982000027.jpg246169JPEG2025503982000028.jpg245169JPEG20255039820 00029.jpg253169JPEG2025503982000030.jpg253169JPEG2025503982000031.jpg100169

[0020] Table 4. Expression status in vivo after intravenous injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 3 JPEG2025503982000032.jpg213166JPEG2025503982000033.jpg233166JPEG20255039820 00034.jpg248166JPEG2025503982000035.jpg245166JPEG2025503982000036.jpg235166 JPEG2025503982000037.jpg236166JPEG2025503982000038.jpg231166JPEG20255039820 00039.jpg230166JPEG2025503982000040.jpg253166JPEG2025503982000041.jpg162166

[0021] Table 5. Expression status in vivo after intravenous injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 4 JPEG2025503982000042.jpg75169

[0022] Table 6. Expression status in vivo after intravenous injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 5 JPEG2025503982000043.jpg230170

[0023] Example 11: Measurement of expression levels in animals when mRNA is delivered intramuscularly Drug-loaded nanoparticles were produced according to the method of Example 6 using the cationic lipid analogs of Examples 1 to 5, with Luciferase-mRNA as the model mRNA. The specific operation method is as follows: 100 μL of nanoparticles containing 5 μg of Luciferase-mRNA or the drug-loaded nanoparticles prepared above were injected into the thigh muscle of 6-8 week-old C57BL / 6 mice. Six hours later, 200 μL of 1x PBS solution containing 3 mg of D-luciferin potassium salt was injected into the abdominal cavity of the mice. After 10 minutes, the mRNA expression status in the body was evaluated by bioluminescence imaging. Table 7. Expression in vivo after intramuscular injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 1 JPEG2025503982000044.jpg230168JPEG2025503982000045.jpg240168JPEG2025503982000046.jpg143168Table 8. Expression in vivo after intramuscular injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 2 JPEG2025503982000047.jpg232170JPEG2025503982000048.jpg240170JPEG2025503982000049.jpg57170Table 9. Expression in vivo after intramuscular injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 3 JPEG2025503982000050.jpg241160JPEG2025503982000051.jpg232160JPEG2025503982000052.jpg230160JPEG2025503982000053.jpg222160JPEG2025503982000054.jpg224160JPEG2025503982000055.jpg228160JPEG2025503982000056.jpg75160Table 10. Expression in vivo when mRNA drug-loaded nanoparticles prepared from the compound of Example 4 are injected intramuscularly JPEG2025503982000057.jpg77169Table 11. Expression in vivo after intramuscular injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 5 JPEG2025503982000058.jpg232169

[0024] Example 12: Measurement of expression levels in animals when mRNA is delivered in vivo by intraperitoneal injection Drug-loaded nanoparticles were prepared according to the method of Example 6 using the cationic lipid analog A1I4R22C18-2 of Example 2, with Luciferase-mRNA as the model mRNA. The specific operation method is as follows: 200 μL of nanoparticles containing 5 μg of Luciferase-mRNA or the drug-loaded nanoparticles prepared above were injected into the abdominal cavity of 6-8 week-old C57BL / 6 mice. Six hours later, 200 μL of 1x PBS solution containing 3 mg of D-luciferin potassium salt was injected into the abdominal cavity of the mice. After 10 minutes, the mRNA expression status in the body was evaluated by bioluminescence imaging. Table 12. Expression in vivo after intraperitoneal injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 2 JPEG2025503982000059.jpg26169

[0025] Example 13: Measurement of expression levels in animals when mRNA is delivered in vivo by tracheal injection Drug-loaded nanoparticles were prepared according to the method of Example 6 using the cationic lipid analog A1I4R22C18-2 of Example 2, with Luciferase-mRNA as the model mRNA. The specific operation method is as follows: 100 μL of nanoparticles containing 5 μg of Luciferase-mRNA or the drug-loaded nanoparticles prepared above were injected into the trachea of ​​6-8 week-old C57BL / 6 mice. Six hours later, 200 μL of 1x PBS solution containing 3 mg of D-luciferin potassium salt was injected into the abdominal cavity of the mice. After 10 minutes, the mRNA expression status in the body was evaluated by bioluminescence imaging. Table 13. Expression in vivo after tracheal injection of mRNA drug-loaded nanoparticles prepared from the compound of Example 2 JPEG2025503982000060.jpg25169

[0026] Example 14: Evaluation of immunological efficacy of in vivo delivery of mRNA in animal studies Drug-loaded nanoparticles were prepared according to the method of Example 6 using OVA-mRNA as a model mRNA and the cationic lipid analogues of Examples 1 and 2. The specific operation method is as follows: C57BL / 6 mice aged 6 to 8 weeks were selected and the experiment was carried out in an SPF animal room. Five mice were selected for each group, and 100 μL of nanoparticles containing 5 μg of OVA-mRNA or the drug-loaded nanoparticles prepared above was injected intramuscularly or intraperitoneally into the thigh of each mouse. After another boost with the same dose, blood was collected and antibody titer analysis was performed by enzyme-linked immunosorbent assay (ELISA). Taking the cationic lipid analogue in Example 1 as an example, nanoparticles containing 5 μg of OVA-mRNA or the drug-loaded nanoparticles prepared above were intramuscularly injected on days 0 and 14, respectively, and blood was collected on day 21 for antibody titer analysis. The results are shown in Figure 11. Taking the cationic lipid analog A1I4R22C18-2 in Example 2 as an example, nanoparticles containing 5 μg of OVA-mRNA or the drug-loaded nanoparticles prepared above were injected intramuscularly or intraperitoneally on days 0 and 7, respectively, and then blood was collected on day 14 for antibody titer analysis. The results are shown in Figure 12.

[0027] Example 15: Measurement of the in vivo delivery level of siRNA by intravenous injection in animal experiments Drug-loaded nanoparticles were prepared according to the method of Example 6 using the cationic lipid analogs A4I18R2C18-2 and A1I4R22C18-2 of Example 1 with Cy5-siRNA as the model siRNA. The specific operation method is as follows: 200 μL of nanoparticles containing 10 μg of Cy5-siRNA or the drug-loaded nanoparticles prepared above were intravenously injected into the thigh region of 6-8 week-old C57BL / 6 mice, and 1 hour later, the delivery status of siRNA in the body was evaluated by biofluorescence imaging. Table 14. Delivery status in the body when siRNA drug-loaded nanoparticles prepared from the compound of Example 1 were intravenously injected JPEG2025503982000061.jpg70169Table 15. Delivery status in the body when siRNA drug-loaded nanoparticles prepared from the compound of Example 2 were intravenously injected. JPEG2025503982000062.jpg57169

[0028] It should be noted that the above examples are merely for illustrating the technical solution of the present invention, and are not intended to limit the scope of the present invention. In addition, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood that those skilled in the art can make modifications or equivalent substitutions to the technical solution of the present invention without departing from the substance and scope of the technical solution of the present invention.

Claims

1. A cationic lipid analogue having a structure represented by any one of the following formulas (I) to (V): [In the formula, m １ is independently at least one selected from the group consisting of a hydrogen atom, a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a substituted alkynyl group, a cycloalkyl group, a phenyl group, and a heteroatom-containing aromatic group; m ２ is independently at least one selected from the group consisting of a linear alkyl group, a linear alkenyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing linear alkylene group, a heteroatom-containing branched alkyl group, a heteroatom-containing branched alkylene group, a heterocycle-containing alkyl group, a heterocycle-containing alkylene group, an ester group-containing alkyl group, and an ether group-containing alkyl group; m ３ is independently at least one selected from the group consisting of a linear alkyl group, a linear alkenyl group, a cycloalkyl group, a linear alkyl group containing a heteroatom, a branched alkyl group containing a heteroatom, a branched alkylene group containing a heteroatom, an alkyl group containing a heterocycle, an alkyl group containing a heteroatom and an aromatic ring, an alkyl group containing a hydroxyl group, an alkyl group containing an ester group, an alkyl group containing an ether group, and an alkyl group containing a sulfonate group; m ４ is independently at least one selected from the group consisting of a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing branched alkyl group, and a heterocycle-containing alkyl group.

2.

3. A cationic lipid analogue as described in claim 2.

4. The method for producing the cationic lipid analogue according to any one of claims 1 to 3, characterized in that the aldehyde compound and the amine compound are added to an organic solution, reacted for 10 to 120 minutes, then the carboxylic acid compound and the isonitrile compound are added in sequence, and reacted at 4 to 60°C for 6 to 72 hours, and the product is separated and purified to obtain the cationic lipid analogue.

5. The method for producing a cationic lipid analogue according to claim 4, characterized in that the molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound and the isonitrile compound is (0.1-1.5):(0.1-1.5):(0.1-1.5):(0.1-1.5).

6. The method for producing a cationic lipid analogue according to claim 4, characterized in that the isonitrile compound is one of a monofunctional isonitrile compound, a difunctional isonitrile compound, and a trifunctional isonitrile compound.

7. The method for producing a cationic lipid analogue according to claim 5, wherein the isonitrile compound is any one of the following compounds I3 to I38.

8. The method for producing a cationic lipid analogue according to claim 4, characterized in that the aldehyde compound is one of monofunctional aldehyde compounds.

9. The method for producing a cationic lipid analogue according to claim 8, wherein the aldehyde compound is any one of the following compounds A1 to A35.

10. The method for producing a cationic lipid analogue according to claim 4, characterized in that the amine compound is one of a monofunctional amine compound, a bifunctional amine compound, and a trifunctional amine compound.

11. The method for producing a cationic lipid analogue according to claim 10, wherein the amine compound is any one of the following compounds R1 to R53.

12. The method for producing a cationic lipid analogue according to claim 4, wherein the carboxylic acid compound is any one of the following compounds C8 to C30 or S11 to S19.

13. A composition of cationic lipid analogues according to any one of claims 1 to 3, comprising the cationic lipid analogues and at least one of a sterol, a helper lipid, and a polyethylene glycol lipid derivative.

14. 14. The composition of cationic lipid analogs of claim 13, wherein the sterol comprises at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives, and the helper lipid is a neutral phospholipid.

15. The cationic lipid analogue composition according to claim 13, characterized in that the molar ratio of the cationic lipid analogue, cholesterol, helper lipid and polyethylene glycol lipid derivative is (20-70):(20-50):(2-30):(0.1-20).

16. A drug-loaded nanoparticle comprising the cationic lipid analogue composition of claim 13 and a drug.

17. The drug-loaded nanoparticle of claim 16, characterized in that the drug comprises at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide-based molecule, and a gene editing complex.

18. The drug-loaded nanoparticle of claim 17, characterized in that the nucleic acid molecule is at least one of messenger RNA, transfer RNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA, and the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

19. The drug-loaded nanoparticle of claim 17, wherein the mass ratio of the cationic lipid analogue to the nucleic acid molecule is cationic lipid analogue:nucleic acid molecule=(2-50):

1.

20. (1) dissolving the cationic lipid analogue composition in an organic solution to obtain an organic phase; Step (2) dissolving the nucleic acid molecule in a buffer to obtain an aqueous phase, wherein the volume of the aqueous phase: the volume of the organic phase = (2-5):1; (3) rapidly and uniformly mixing the aqueous phase and the organic phase and dialyzing the mixture to obtain drug-loaded nanoparticles; The method for producing drug-loaded nanoparticles according to claim 19, comprising the steps of:

21. Use of a cationic lipid analogue according to any one of claims 1 to 3, a composition of a cationic lipid analogue according to any one of claims 13 to 15, or a drug-loaded nanoparticle according to any one of claims 16 to 19 in the manufacture, delivery or transportation of a molecular drug or a nucleic acid vaccine.

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