CN113461577A

CN113461577A - Amino lipid and application thereof

Info

Publication number: CN113461577A
Application number: CN202111017566.1A
Authority: CN
Inventors: 查高峰; 彭星华; 夏恒
Original assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Current assignee: SHENZHEN HONGXIN BIOTECHNOLOGY CO Ltd
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-10-01
Anticipated expiration: 2041-09-01
Also published as: CN113461577B; WO2023029928A1

Abstract

The invention relates to the technical field of medical chemistry, in particular to amino lipid and application thereof. In vitro, in vivo delivery studies, an excellent ability to deliver nucleic acids into cells was demonstrated. The amino lipid compound has two sulfonamides, and the introduction of the group obviously enhances the stability of the lipid nanoparticles and improves the in vivo circulation time, thereby improving the in vivo delivery efficiency. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements on instruments and equipment and simple operation.

Description

Amino lipid and application thereof

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to amino lipid and application thereof.

Background

The gene medicine is to introduce exogenous normal gene into target cell to correct or compensate diseases caused by gene defect and abnormality so as to achieve the aim of treatment; or the gene is expressed to generate corresponding antigen, thereby inducing memory immune response. During the past two decades, gene medicine has been developed in many disease treatment fields from the clinical point of view, and has no alternative advantages for diseases caused by gene abnormality and difficult to solve so far in the medical field, such as tumor and the like. Common genetic drugs are plasmid DNA (pDNA), antisense oligonucleic acid (antisense ODN), small interfering rna (sirna), and messenger rna (mrna).

However, when a foreign gene is introduced into the body, it is degraded by nuclease in the body and is degraded into small-molecule nucleotides before entering target cells, thereby losing therapeutic effects. Therefore, the key to achieving gene therapy is an efficient, safe gene delivery system. Gene vectors undergo a number of complex processes in the delivery of genes: the gene substance is released by the carrier through blood circulation to reach target cells, cellular uptake, escape of endosomes, intracellular movement. The major obstacles are mainly extracellular obstacles to the complex blood environment and intracellular obstacles to lysosomal enzyme degradation. Therefore, the search for a good gene vector to enable the target gene to reach the target point and exert the utility is a problem to be solved urgently by gene vector researchers.

Currently, there are two main categories of gene delivery vector systems: one is a viral vector system and the other is a non-viral vector system. The virus vector is a natural vector resource, the virus genome has simple structure, high transfection efficiency and strong target cell specificity, but the use of the virus vector is limited by the defects of poor targeting property, low carrying capacity, immunogenicity and the like. Thus, diverse, non-immunogenic and easily controlled production non-viral vector systems have attracted attention in recent years and find application in many therapeutic areas. The non-viral vector systems commonly used are mainly cationic lipid (cationic lipids) vectors.

Cationic lipids have three important structural regions, a positively charged hydrophilic polar head gene, a linker chain in the middle responsible for linking the two ends, polar and non-polar, and a hydrophobic lipid chain. The polar head containing amine group plays a role in combining liposome and RNA, and the liposome/RNA complex and cell membrane mutually, influences the charging condition of lipid and plays a main role in the escape process of lysosome. The linker chain determines the chemical and biological stability of the cationic liposome, particularly the cytotoxicity resulting therefrom. The hydrophobic region may be in the form of a carbon chain or a variety of structures such as steroids, and the length, saturation and specific type of the carbon chain will influence the lipid behavior, which will both provide sufficient mobility to the lipid bilayer and promote lipid fusion of the cationic liposome in vivo.

The cationic liposome and the negatively charged group form a liposome/gene complex through electrostatic interaction. The complex is positively charged due to the excess of cationic liposome, and the positively charged liposome/gene complex is adsorbed on the negatively charged cell surface due to electrostatic interaction. And then enters the cell by fusion with the cell membrane or endocytosis of the cell. The main feature of cationic lipids for gene therapy is charge-influenced membrane fusion during endosome escape. However, the excessive positive charge of the cationic lipid/gene complex and the difficult degradation of part of the cationic lipid also contribute to cytotoxicity. Thus lower transfection efficiency and cytotoxicity are major drawbacks limiting the use of cationic lipids. At present, cationic lipid is used as a gene vector, which is the most widely used non-viral vector at present due to the characteristics of simple structure, simple and convenient operation, high biological safety and the like, but the problems of low transfection efficiency and cytotoxicity caused by positive charge still need to be solved, so the ionizable cationic lipid is designed to solve the problems so as to achieve better gene therapy effect.

Disclosure of Invention

Aiming at the technical problems of low transfection efficiency of cationic liposome, cytotoxicity caused by positive charge and the like in the prior art, the invention provides amino lipid and application thereof.

The purpose of the invention is realized by the following technical scheme:

an amino lipid, the structure of which is shown in formula (I):

wherein L is C₁-C₂₄Alkylene radical, C₁-C₂₄Alkenylene radical, C₃-C₈Cycloalkylene radical, C₃-C₈Cycloalkenylene;

R¹is H, OR⁵、CN、-C (=O) OR⁴、-OC(=O) R⁴、- C (=O) N R⁴R⁵、-NR⁵ C (=O) R⁴Or N R⁴R⁵；

R²、R³、R⁴And R⁵Are identical or different from each other and are each independently selected from H, C₁-C₂₄Alkyl radical, C₂-C₂₄Alkenyl radical, C₂-C₂₄An alkynyl group; said C is₁-C₂₄Alkyl radical, C₂-C₂₄Alkenyl radical, C₂-C₂₄Alkynyl may optionally be substituted by C₁-C₆Hydrocarbyl substitution;

or R²And R³Are connected to form a 4-10 membered heterocyclic ring, wherein the heterocyclic ring contains 1-6 heteroatoms selected from nitrogen, sulfur or oxygen.

Preferably, said R is²Is selected from C₆-C₂₄Alkyl radical, C₆-C₂₄Alkenyl radical, C₆-C₂₄An alkynyl group; the quilt C₆-C₂₄Alkyl radical, C₆-C₂₄Alkenyl radical, C₆-C₂₄Alkynyl may optionally be substituted by C₁-C₆Hydrocarbyl substitution.

Preferably, as NH₂As positions of free radicals, L and R¹Are linked to form NH₂-L-R¹One selected from a group consisting of a1, a2, A3, A4, A5, A6, a7, A8, a9, a10, a11, a12, a13, a14, a15, a16, a17, a18, a19, a20, a21, a22, a23, a24, a25, a26, a27, a28, a29, a30, a31, a32, a33, a34, a35, a36, a37, a38, a39, a 40.

In the compounds of the formula (I) L and R¹After attachment, the N atom is attached. NH as described above₂-L-R¹Middle NH₂The position of substitution is the radical position attached to the compound of formula (I).

Preferably, R²、R³Form R with the adjacent N atom²R³-NH, wherein H is the position of a radical; r²R³-NH is selected from one of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23.

In the compounds of the formula (I) R²、R³To the same N atom which is in turn linked to the S atom of the sulfonyl group. R is as defined above²R³The H atom in NH is the position of the bond to the S atom in formula (I).

The preparation method of the amino lipid comprises the following steps:

s1 Compound NH₂-L-R¹Stirring and reacting with vinyl sulfonyl fluoride in a solvent;

s2 adding R into the reaction system in the step S1²R³NH, and heating and reacting in the presence of alkali.

The reaction scheme is as follows:

preferably, the method comprises the steps of:

(1) a first reaction between vinyl sulfonyl fluoride (ESF) and a compound represented by R1-L-NH2 at a temperature of-20 ℃ to 40 ℃ to give a first intermediate;

(2) reacting the first intermediate with HNR in the presence of a base as an acid-binding agent with or without isolation of the first intermediate²R³And (3) carrying out a second reaction on the amine under the heating condition to obtain the amino lipid compound shown in the formula I. Preferably, the heating temperature in the step S2 is 50 to 120 ℃. The base used in the above preparation method is an organic base or an inorganic base, such as: triethylamine, DIPEA, pyridine, DMAP, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc

The use of said amino lipids and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof for the preparation of a medicament for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering with RNA.

Preferably, the above-mentioned use is for the manufacture of a medicament for the treatment of cancer or genetic diseases.

Preferably, the application is the application in preparing medicaments for treating lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymph cancer, blood cancer or prostate cancer, and the genetic diseases are one or more of hemophilia, thalassemia and gaucher's disease.

Preferably, the above-mentioned use is in the manufacture of a medicament for the treatment of cancer, allergy, toxicity and pathogen infection.

Preferably, the above-mentioned use is use in the preparation of a medicament for nucleic acid transfer.

Preferably, the nucleic acids are RNA, messenger RNA (mrna), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rrna), micro RNA (mirna), transfer RNA (trna), small inhibitory RNA (sirna), and small nuclear RNA (snrna).

Compared with the prior art, the invention has the following technical effects:

according to the amino lipid compound disclosed by the invention, an amino head group and a hydrophobic chain are constructed into amino lipid through a bifunctional electrophilic reagent of vinyl sulfonyl fluoride (ESF), the click chemistry reaction characteristic of ESF is fully utilized, the reaction condition is mild in the process of constructing the amino lipid, protection and deprotection are not required, and the atom economy is high. In vitro, in vivo delivery studies, an excellent ability to deliver nucleic acids into cells was demonstrated. The amino lipid compound has two sulfonamides, and the introduction of the group obviously enhances the stability of the lipid nanoparticles and improves the in vivo circulation time, thereby improving the in vivo delivery efficiency. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements on instruments and equipment and simple operation.

Drawings

Figure 1 is a ratio graph of differentiation into cell populations presenting OVA antigen following stimulation of BMDCs by representative amino lipid compound delivery of OVA mRNA in example 10;

FIG. 2 is a graph of the proportion of a representative amino lipid compound delivery OVA mRNA in example 10 differentiated into a mature DC cell population following stimulation of BMDCs;

FIG. 3 is a graph of body fluid antibody titers generated by OVA mRNA delivered by subcutaneous administration of representative amino lipid compounds of example 12;

FIG. 4 survival profiles of tumor-bearing mice following intramuscular injection of OVA mRNA vaccines in example 13.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The term "optionally substituted" as used herein means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or substituted with one or more, e.g., one, two, three or four, substituents. When an atom or group is substituted with a plurality of substituents, the plurality of substituents may be the same or different.

Abbreviations herein:

DNA deoxyribonucleic acid

RNA ribonucleic acid

DOPE dioleoyl phosphatidylethanolamine

DSPC distearoyl phosphatidylcholine

PEG2000-DMG (1- (monomethoxypolyethylene glycol) -2, 3 dimyristoyl glycerol

kD kilodalton

PBS phosphate buffer solution.

Example 1 parallel Synthesis and characterization of A1Ny series amino lipid Compound library

To a 250mL reaction flask were added n-butylamine (25.3 mg, 0.25 mmol), vinylsulfonylfluoride (55 mg, 0.5 mmol) and anhydrous tetrahydrofuran (2.5 mL) in this order, and the mixture was stirred at room temperature for 5min to obtain a Step I reaction solution (2.5 mL, 0.1M).

Each Step I reaction was transferred separately to 22 1.5 mL EP tubes (0.1 mL, 0.01 mmol each) using a pipette, and a solution of diamine in THF (0.12 mL, 0.024 mmol, 0.2M), a solution of DIPEA in THF (0.2 mL, 0.04 mmol, 0.2M) were added to each corresponding EP tube and reacted in a heated shaking reactor (Thermo-Shaker) at 78 ℃ for 1 h with TLC detection of no Step I starting material. After the reaction is finished, the solvent in the reaction tube is volatilized at normal temperature, and 23 amido lipid compounds A1Ny are obtained. Mass spectrometry was performed and the results are shown in Table 1 below.

Table 1: MW/z values of A1Ny series Aminoamino Liposome library

Example 22, 2' - (nonyl Azadiyl) bis (N-undecylethane-1-sulfonamide)

Into a 250mL reaction flask were added n-nonanamine (1.43 g, 10 mmol), vinylsulfonyl fluoride (2.2 g, 20 mmol) and 80 mL of anhydrous tetrahydrofuran in this order, and the mixture was stirred at room temperature for 5min, followed by addition of n-undecanamine (13.7 g, 80 mmol) and diisopropylethylamine (20.6 g, 160 mmol) and heating to 75 ℃ for 4 h. After concentration, purification using flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) gave compound A4N4 (5.06 g, 76%).¹H NMR (400 MHz, DMSO-d ₆)：δ 3.62 (m, 4H), 3.03 (m, 2H), 2.94 (m, 4H), 2.89 (m, 4H), 1.54-1.23 (m, 50H), 0.89 (m, 9H). ESI-MS calculated for C₃₅H₇₆N₃O₄S₂ ⁺ [M+H]⁺ 667.1, found 667.3。

Example 32, 2' - (Hexadecyl-azadialkyl) bis (N-nonylethane-1-sulfonamide)

6-aminoundecane (1.71 g, 10 mmol), vinylsulfonyl fluoride (2.2 g, 20 mmol) and anhydrous tetrahydrofuran (80 mL) were sequentially charged into a 250mL reaction flask, and the mixture was stirred at room temperature for reaction for 5min, followed by addition of n-nonanamine (11.4 g, 80 mmol) and diisopropylethylamine (20.6 g, 160 mmol), and the mixture was warmed to 75 ℃ for reaction for 4 h. After concentration, purification using flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) gave compound a12N2 (5.06 g, 76%).¹H NMR (400 MHz, DMSO-d ₆)：δ 3.62 (m, 4H), 3.03 (m, 2H), 2.94 (m, 4H), 2.89 (m, 4H), 1.54-1.23 (m, 50H), 0.89 (m, 9H). ESI-MS calculated for C₃₅H₇₆N₃O₄S₂ ⁺ [M+H]⁺667.1, found 667.3。

Example 42, 2' - (nonyl Azadiyl) bis (N-undecylethane-1-sulfonamide)

Ethanolamine (0.61 g, 10 mmol), vinyl sulfonyl fluoride (2.2 g, 20 mmol) and anhydrous tetrahydrofuran (80 mL) were added sequentially to a 250mL reaction flask, and stirred at room temperature for 5min, followed by addition of n-pentadecylmethylamine (14.5 g, 60 mmol) and diisopropylethylamine (20.6 g, 160 mmol) and warmed to 75 ℃ for 4 h. After concentration, purification using flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) gave compound a14N13 (5.06 g, 76%).¹H NMR (400 MHz, DMSO-d ₆)：δ 3.62 (m, 4H), 2.49 (m, 1H), 2.94 (m, 4H), 2.89 (m, 4H), 1.54-1.23 (m, 44H), 0.89 (m, 12H). ESI-MS calculated for C₃₃H₇₂N₃O₄S₂ ⁺ [M+H]⁺ 667.1, found 667.3。

Example 52, 2' - (nonyl Azadiyl) bis (N-undecylethane-1-sulfonamide)

4-Aminotetrahydropyran (1.01 g, 10 mmol), vinylsulfonyl fluoride (2.2 g, 20 mmol) and anhydrous tetrahydrofuran (80 mL) were sequentially added to a 250mL reaction flask, and the mixture was stirred at room temperature for 5min, followed by addition of dioxane amine (14.8 g, 80 mmol) and diisopropylethylamine (20.6 g, 160 mmol), and the mixture was heated to 75 ℃ for 4 h. After concentration, purification using flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) gave compound a21N16 (4.43 g, 68%).¹H NMR (400 MHz, DMSO-d ₆)：δ 3.69-3.61 (m, 4H), 3.52 (m, 4H), 2.94 (m, 4H), 2.72 (m, 1H), 2.39 (m, 8H), 1.78-1.52 (m, 4H), 1.39-1.23 (m, 32H), 0.89 (m, 12H). ESI-MS calculated for C₃₃H₇₀N₃O₅S₂ ⁺ [M+H]⁺ 652.5, found 652.9。

Example 62, 2' - (nonyl Azadiyl) bis (N-undecylethane-1-sulfonamide)

N, N-dimethylpropylenediamine (204 mg, 2 mmol), vinylsulfonylfluoride (440 mg, 4 mmol) and anhydrous tetrahydrofuran (40 mL) were sequentially added to a 250mL reaction flask, and the mixture was stirred at room temperature for reaction for 5min, followed by addition of methyldialenyloctadecylamine (3.36 g, 12 mmol) and diisopropylethylamine (4.12 g, 32 mmol), and the mixture was heated to 75 ℃ for reaction for 4 h. After concentration, purification using flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) gave compound a32N23 (1.41 g, 84%).¹H NMR (400 MHz, DMSO-d ₆)：δ 5.42-5.28 (m, 8H), 3.52 (m, 4H), 2.94 (m, 4H), 2.89 (s, 6H), 2.80 (m, 4H), 2.39-2.35 (m, 8H), 2.16 (m, 8H), 2.14 (m, 6H), 1.54-1.23 (m, 38H), 0.89 (m, 6H). ESI-MS calculated for C₄₇H₉₃N₄O₄S₂ ⁺ [M+H]⁺841.7, found 841.9。

Example 7 in vitro evaluation of amino lipid Compounds as mRNA vectors

Cell line: HeLa cell line (ATCC)

Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

Screening form: 96-well plate cell transfection

Detection (readout): percentage of GFP fluorescent cells relative to total cells (total cells were determined using the nuclear dye Hoechst-see figure 2). Lipofectamine2000 (Invitrogen) was used as a positive control according to the manufacturer's instructions.

The method comprises the following steps: samples were loaded using an 8-channel pipette. The contents shown are single wells of a 96 well plate.

1. The molar ratio of the amino lipid compound described in example 1 to Dioleoylphosphatidylethanolamine (DOPE), cholesterol, PEG2000-DMG was 45: 10: 42.5: 2.5, mixing and dissolving in absolute ethyl alcohol; EGFP mRNA (TriLink) was dissolved in a sodium acetate solution (50 mM, pH = 4.0), the above mixed lipid solution was taken out using a line gun, added to the EGFP-mRNA solution, and thoroughly mixed at a ratio of an ethanol solution to a sodium acetate solution (50 mM, pH = 4.0) of 1:3 to prepare a lipid nanoparticle solution. The mass ratio of aminolipid compound to green fluorescent protein mRNA (EGFP mRNA) was about 8:1, using 100 ng of mRNA per well.

2. After incubation of the lipid nanoparticle solution at room temperature for 30min, 90. mu.L of freshly resuspended HeLa cells (3-5X 10)⁴Cells) and mixed with a pipette. Transfer 100 μ L of cell + lipid nanoparticles to separate wells of a 96-well culture plate and place at 37 ℃ with 5% CO₂In an incubator.

3. 20 to 24 hours after initial transfection of cells, Hoechst33258 (Invitrogen) was added to the cells at a final concentration of 0.2. mu.g/ml and incubated at 37 ℃ for 15min in the dark. The cells were then washed 1 time with PBS solution and cultured for 20 to 24 hours with additional medium.

4. The cells were placed in a high throughput confocal microscope (Molecular Devices ImageXpress), 4 image fields of the cells were captured from each well, and for each sample, 3 laser wavelength images were captured: bright field images of cells, Hoechst stained images showing total nuclei and GFP images showing successful transfection with plasmid DNA and expression of GFP. And respectively counting the cells of the obtained Hoechst staining image and the GFP image by using MetaXpress software, and dividing the number of the cells expressing GFP by the total number of cell nuclei to obtain the absolute cell transfection efficiency. The absolute transfection efficiency was calculated as follows:

。

as a result: the transfection efficiency of the partial compound library for eGFP-mRNA of HeLa cells is shown in Table 2.

Table 2: transfection efficiency of partial compound library on eGFP-mRNA of HeLa cells

Example 8: transfection of lipid nanoparticles prepared with amino-lipid compounds on BMDC primary cells

The preparation method comprises the following steps: the same as in example 7.

Animal preparation: female C57BL/6 mice of 6 weeks old are selected, the weight is about 20 g, the breeding environment is a SPF-grade breeding room, and animal experiments are carried out strictly according to the guidelines of the national health institution and the ethical requirements of animals.

Cell acquisition: removing neck and socket of C57BL/6 mouse, killing, soaking in 75% alcohol for 5min for sterilization, deplaning to obtain thigh tibia, removing attached muscle to expose bone, blowing bone marrow out of tibia with 1ml syringe with PBS, blowing the bone marrow out, filtering out impurities with 50um filter screen, adding erythrocyte lysate into the filtrate, standing for 5min, centrifuging for 100g and 5min to remove supernatant, resuspending the obtained cells in 1640 culture medium (containing 10% fetal calf serum, 20ng/ml GMCSF, 10ng/ml IL 4), inoculating in 6-well plate,inoculating into culture medium with density of 100000 cells/ml, standing at 37 deg.C with 5% CO₂In the cell culture box, half liquid change is carried out once every 2 days, suspension cells and loosely attached cells are collected on the seventh day, and the suspension cells and the loosely attached cells are inoculated to a 96-hole full-white enzyme standard plate, the inoculation density is 10000 cells per hole, and the volume of a culture medium is 100 ul.

Cell transfection: and adding lipid nanoparticles wrapping luciferase mRNA into a 96-well full-white-enzyme standard plate paved with primary cells, and controlling the addition amount of the lipid nanoparticles wrapping the luciferase mRNA in each well to be 3 ug. Then placed at 37 ℃ with 5% CO₂The luciferase mRNA was expressed sufficiently in an incubator at a concentration of 12 hours.

And (3) detecting the transfection efficiency: 10ul of 10mg/ml potassium D-luciferin salt is added to each well of a 96-well total-white-enzyme standard plate, and immediately placed in a microplate reader to detect the luminescence intensity. The expression intensity of representative amino lipid compounds on BMDCs to transfect Fluc mRNA is shown in table 3. DLin-MC3 served as a control, and many of the amino lipids were similar in expression intensity to MC3 and were significantly better than the positive control.

Table 3: expression intensity of transfection of representative amino lipid compounds on BMDCs

Example 9 evaluation of luciferase mRNA in vivo delivery Performance of lipid nanoparticles prepared from amine-based lipid Compounds

1. Preparation of lipid nanoparticles

Preparation method 1:

mixing the amino lipid compound of the present invention with DOPE, cholesterol, (1- (monomethoxypolyethylene glycol) -2, 3 dimyristoyl glycerol (PEG 2000-DMG) at a molar ratio of 45: 10: 42.5: 2.5 and dissolving in absolute ethanol so that the molar concentration of the amino lipid compound is 0.001 to 0.01 mmol/L, using a micro-syringe pump, mixing the resulting ethanol solution and a sodium acetate solution (50 mM, pH = 4.0) in which Fluc-mRNA (TriLink) is dissolved in a volume ratio of 1:3 in a micro flow channel chip to prepare a crude solution of lipid nanoparticles, which is then dialyzed with a dialysis cassette (Fisher, MWCO 20,000) at 1 XPBS at a controlled temperature of 4 ℃ for 6 hours, and filtered through a 0.22 μm micro-pore filter before use, the mass ratio of the amino lipid compound to the luciferase mRNA (P) is about 10: 1 Administered to the subject animal by subcutaneous administration.

Characterization of lipid nanoparticles:

characterization of particle size: the particle size and PDI of the prepared lipid nanoparticles were determined by Nano-ZSZEN3600 (Malvern). Particle size measurements were taken at 40 uL of LNP solution, cycled three times for 30s each.

And (3) detecting the encapsulation efficiency: using qubits^®The RNA HS Assay kit detects the LNP RNA concentration. The theoretical RNA concentration is the total RNA amount dosed divided by the total volume of the final solution.

。

Table 4: characterization data for LNP prepared with representative amino lipid compounds using formulation method 1

	Amino lipid numbering	Z-Average (d.nm)	PDI	Encapsulation efficiency
						1	A4N4	85	0.06	98.7%
2	A3N10	80	0.02	99.0%
					3	A12N4	85	0.03	99.0%
4	A12N9	105	0.02	98.5%
					5	A14N13	90	0.06	97.2%
6	A15N22	123	0.10	99.6%
					7	A14N16	122	0.04	99.0%
8	A26N16	118	0.03	98.3%
					9	A21N16	90	0.02	98.8%
10	A32N23	116	0.05	99.3%
					11	A34N22	98	0.04	99.2%
12	A35N21	100	0.03	99.1%
					13	DLin-MC3	120	0.08	98.4%

Preparation method 2:

the preparation method is the same as preparation method 1 except that the amino lipid compound, DSPC, cholesterol and PEG2000-DMG are used in a molar ratio of 50: 10: 38.5: 1.5. The resulting Lipid Nanoparticle (LNP) solution is administered to the subject animal by tail vein and intramuscular injection.

Table 5: characterization data for LNP prepared with representative amino lipid compounds using formulation method 2

	Amino lipid numbering	Z-Average (d.nm)	PDI	Encapsulation efficiency
						1	A4N4	127	0.04	98.6%
2	A3N10	95	0.05	98.3%
					3	A12N4	96	0.04	98.2%
4	A12N9	92	0.03	98.5%
					5	A14N13	120	0.05	98.9%
6	A15N22	95	0.03	98.3%
					7	A14N16	130	0.08	99.2%
8	A26N16	90	0.07	98.4%
					9	A21N16	115	0.04	99.0%
10	A32N23	125	0.05	99.5%
					11	A34N22	88	0.04	99.0%
12	A35N21	110	0.06	99.3%
					13	DLin-MC3	120	0.07	99.0%

2. Animal experiments

Animal preparation: female C57BL/6 mice 6 weeks old, weighing approximately 20 g, were selected and housed in SPF-rated housing. Animal experiments were performed strictly according to the guidelines of the national health authorities and the ethical requirements of animals.

In vivo delivery: 9C 57BL/6 mice were randomly selected per group and injected with lipid nanoparticle solutions using three administration modes of subcutaneous, intramuscular, and caudal vein injection (3 mice per administration mode) at a dose of 0.5 mg/kg mRNA, respectively. After 12 hours, 200. mu.L of 10mg/mL potassium D-luciferin was injected into each mouse via the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded by photographing. The expression intensities of Fluc mRNA delivered by 3 administration modes for representative amino lipid compounds are shown in tables 6-8. DLin-MC3 served as a control.

Table 6: expression intensity of Fluc mRNA delivered by subcutaneous administration of representative amino lipid compounds

	Amino lipid numbering	Intensity of fluorescence
			1	A4N4	2.7E+06
2	A3N10	1.9E+07
			3	A12N4	7.6E+06
4	A12N9	1.4E+07
			5	A14N13	3.7E+08
6	A15N22	4.8E+08
			7	A14N16	3.1E+07
8	A26N16	1.1E+07
			9	A21N16	8.2E+06
10	A32N23	9.7E+06
			11	A34N22	1.9E+07
12	A35N21	6.8E+06
			13	DLin-MC3	3.1E+06

Table 7: expression intensity of Fluc mRNA delivered by intramuscular administration of representative amino lipid compounds

	Amino lipid numbering	Intensity of fluorescence
			1	A4N4	4.1E+06
2	A3N10	2.7E+06
			3	A12N4	1.2E+07
4	A12N9	4.3E+07
			5	A14N13	2.8E+07
6	A15N22	2.7E+07
			7	A14N16	4.7E+06
8	A26N16	4.7E+06
			9	A21N16	8.2E+06
10	A32N23	4.2E+06
			11	A34N22	9.1E+06
12	A35N21	1.8E+06
			13	DLin-MC3	8.5E+06

Table 8: expression intensity of Fluc mRNA delivered by tail vein administration of representative amino lipid compounds

	Amino lipid numbering	Intensity of fluorescence
			1	A4N4	4.6E+06
2	A3N10	5.2E+06
			3	A12N4	3.6E+06
4	A12N9	5.1E+07
			5	A14N13	7.2E+06
6	A15N22	5.1E+07
			7	A14N16	5.2E+06
8	A26N16	2.1E+06
			9	A21N16	6.5E+06
10	A32N23	3.9E+06
			11	A34N22	2.1E+06
12	A35N21	8.2E+06
			13	DLin-MC3	2.7E+07

Example 10: immunity evaluation of lipid nanoparticles prepared from amino lipid compounds on BMDC primary cells

The preparation method comprises the following steps: the mol ratio of the amino lipidic compound to DOPE, cholesterol and PEG2000-DMG is 45: 10: 42.5: 2.5 in the absolute ethyl alcohol. Ovalbumin mrna (ova mrna) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Two micro syringe pumps were used, the ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled to be 1:3, preparing a crude solution of lipid nanoparticles in a micro-flow channel chip, dialyzing with a dialysis cartridge (Fisher, MWCO 20,000) at 1 XPBS and a controlled temperature of 4 ℃ for 6h, and filtering with a 0.22 μm microporous membrane before use. The mass ratio of aminolipid compound to ovalbumin mRNA (OVA mRNA) was about 8: 1.

Cell acquisition: the same as in example 8.

Activation of immune cells: add 1ug ovalbumin mRNA lipid to 12-well plates per wellNanoparticles, incubated at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24 hours. The cells were blown down with PBS solution and centrifuged (100 g, 5 min) three times with PBS washes, followed by incubation for 30min with CD11c-APC antibody and SIINFEKL-H-2Kb-PE antibody, CD11c-APC antibody and MHC-II-PE antibody, followed by centrifugation (100 g, 5 min) once with PBS washes to remove unbound antibody, and then detected with a flow cytometer (Beckmann cytoflex LX). Wherein CD11 is a marker for BMDCs, CD11c-APC antibody is used for labeling of DC populations, SIINFEKL-H-2Kb-PE antibody is used for labeling of OVA antigen presenting cell populations in cell populations, and MHC-II-PE antibody is used for labeling of mature DC cell populations. The results are shown in fig. 1 and 2, the abilities of A14N13 and A15N12 to stimulate BMDC to mature and present OVA antigen are equivalent to those of MC3, and the immune activation effects of A32N22, A34N22 and A35N20 are obviously better than those of the MC3 control group.

Example 11: evaluation of luciferase mRNA in vivo delivery Performance of lipid nanoparticles prepared from amino lipid Compound

The preparation method comprises the following steps: the mol ratio of the amino lipidic compound to DOPE, cholesterol and PEG2000-DMG is 45: 10: 42.5: 2.5 in the absolute ethyl alcohol. Luciferase mrna (fluc mrna) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Two micro syringe pumps were used, the ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled to be 1:3, preparing a crude solution of lipid nanoparticles in a micro-flow channel chip, dialyzing with a dialysis cartridge (Fisher, MWCO 20,000) at 1 XPBS and a controlled temperature of 4 ℃ for 6h, and filtering with a 0.22 μm microporous membrane before use. The mass ratio of aminolipid compound to luciferase mrna (fluc mrna) was about 8: 1.

In vivo delivery: 3 mice were randomly selected per group and injected subcutaneously with lipid nanoparticles at a dose of 0.5 mg/kg. After 6 hours, 200. mu.L of 10mg/mL potassium D-luciferin was injected into each mouse via the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded by photographing. Representative amino lipid compounds the expression intensity of Fluc mRNA delivered by the three modes of administration is shown in tables 9-10, DLin-MC3 as a control. The plurality of amino lipids expressed with similar intensity to dilin-MC 3 and were significantly better than the positive control.

Table 9: characterization data for LNP prepared with representative amino lipid compounds using formulation method 2

	Amino lipid numbering	Z-Average (d.nm)	PDI	Encapsulation efficiency
						1	A4N4	117.8	0.05	98.9%
2	A3N10	110.9	0.06	98.6%
					3	A12N4	109.8	0.04	98.6%
4	A12N9	140.3	0.04	98.9%
					5	A14N13	133.9	0.06	98.5%
6	A15N22	113.2	0.05	98.8%
					7	A14N16	131.2	0.07	99.2%
8	A26N16	127.5	0.08	98.5%
					9	A21N16	124.7	0.06	99.0%
10	A32N23	134.1	0.04	99.3%
					11	A34N22	115.6	0.08	99.2%
12	A35N21	136.8	0.05	99.0%
					13	DLin-MC3	125.4	0.08	98.6%

Table 10: expression intensity of representative amino lipid compounds administered intramuscularly to deliver Fluc mRNA

Numbering	Amino lipid numbering	Intensity of fluorescence
			1	A4N4	2.8E+06
2	A3N10	1.8E+06
			3	A12N4	2.1E+05
4	A12N9	4.5E+06
			5	A14N13	5.1E+06
6	A15N22	2.8E+06
			7	A14N16	8.1E+06
8	A26N16	7.8E+06
			9	A21N16	6.1E+05
10	A32N23	4.1E+06
			11	A34N22	1.2E+07
12	A35N21	6.4E+06
			13	DLin-MC3	4.2E+06

Example 12: in vivo delivery of ovalbumin mRNA and evaluation of immunological properties of lipid nanoparticles prepared from amino lipid compounds

In vivo delivery: 3 mice were randomly selected per group and injected subcutaneously with lipid nanoparticles (Day 0) at a dose of 0.5 mg/kg. After 7 days, the same amount was used for another boost (Day 7). Tail vein bleeds were taken on day 21 for serological analysis, with DLin-MC3 as a control.

Enzyme-linked immunosorbent assay (ELISA): flat bottom 96 well plates (Nunc) were pre-plated in 50 mM carbonate buffer at 0.5 μ g protein per well (pH 9.6) with OVA protein concentration overnight at 4 ℃ and then blocked with 5% glycine. Sera from immunized animals were removed from 10 using PBS-0.05% Tween (PBS-T, pH 7.4)^-2Diluting to 10^-6Added to the wells and incubated at 37 ℃ for 1 hour. Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG was purified in PBS-T-1% BSA at 1: a dilution of 10,000 was labeled. After addition of the HRP substrate, absorbance at 450 nm was measured in an optical density ELISA plate reader (Bio-Rad) at one wavelength. As shown in fig. 3, a14N13 was comparable to the IgG antibody titer produced by MC3, while the IgG antibody titers of a32N22, a34N22, a35N20 were significantly better than the MC3 control.

Example 13: evaluation of in vivo Immunity and tumor treatment Effect of lipid nanoparticles prepared from amino lipid Compound

In vivo delivery: will be provided withB16-OVA melanoma cells (1.5X 10)⁵) Mice of 4-6 weeks of age were injected subcutaneously on the right side. When the tumor size is less than 50 mm³Vaccination was started (approximately

day

4 or 5 after tumor vaccination). Animals were immunized by intramuscular injection of an LNP preparation containing 15 μ g OVA-mRNA. Tumor growth was measured 3 times per week using digital caliper, and the formula was 0.5 x length x width. When the tumor volume reaches 2,000 mm³Mice were euthanized at time. Tumor inhibition was compared to mice carrying freshly inoculated tumors. The median survival time of the group without vaccine injection is 29 weeks, and after vaccine injection, the corresponding median survival times are respectively as follows: week 38 (MC 3), week 49 (a 32N 22), and week 52 (a 35N 20), as shown in fig. 4.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. An amino lipid, characterized in that it has the structure shown in formula (I):

R¹is H, OR⁵、CN、-C (=O) OR⁴、-OC (=O) R⁴、- C (=O) N R⁴R⁵、-NR⁵ C (=O) R⁴Or N R⁴R⁵；

R²、R³ _、R⁴And R⁵Are identical or different from each other and are each independently selected from H, C₁-C₂₄Alkyl radical, C₂-C₂₄Alkenyl radical, C₂-C₂₄An alkynyl group; said C is₁-C₂₄Alkyl radical, C₂-C₂₄Alkenyl radical, C₂-C₂₄Alkynyl may optionally be substituted by C₁-C₆Hydrocarbyl substitution;

2. The amino lipid of claim 1, wherein R is²Is selected from C₆-C₂₄Alkyl radical, C₆-C₂₄Alkenyl radical, C₆-C₂₄An alkynyl group; the quilt C₆-C₂₄Alkyl radical, C₆-C₂₄Alkenyl radical, C₆-C₂₄Alkynyl may optionally be substituted by C₁-C₆Hydrocarbyl substitution.

3. Amino lipid according to claim 1 or 2, characterized in that it is constituted by NH₂As positions of free radicals, L and R¹Are linked to form NH₂-L-R¹One selected from a1, a2, A3, A4, A5, A6, a7, A8, a9, a10, a11, a12, a13, a14, a15, a16, a17, a18, a19, a20, a21, a22, a23, a24, a25, a26, a27, a28, a29, a30, a31, a32, a33, a34, a35, a36, a37, a38, a39, a 40:

。

4. amino lipid according to claim 1 or 2, characterized in that R²、R³Form R with the adjacent N atom²R³-NH, wherein H is the position of a radical; r²R³-NH is selected from one of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23:

。

5. a process for the preparation of an amino lipid according to any of claims 1 to 4, comprising the steps of:

6. Use of an amino lipid according to any one of claims 1 to 4 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA.

7. Use according to claim 6, for the preparation of a medicament for the treatment of cancer or genetic diseases.

8. The use according to claim 7, in the manufacture of a medicament for the treatment of lung, stomach, liver, oesophagus, colon, pancreas, brain, lymph, blood or prostate cancer.

9. Use according to claim 7, for the preparation of a medicament for the treatment of cancer, allergy, toxicity and pathogen infections.

10. Use according to claim 6, for the preparation of a medicament for nucleic acid transfer.