A secondary amine-containing cationic lipid compound, compositions and uses thereof

Abstract

The application provides a kind of containing secondary amine cationic lipid compound and its composition and application, the application creatively proposes a single component containing secondary amine cationic lipid, to realize mRNA extrahepatic target delivery (such as spleen), enrich the kind of existing cationic lipid compound, simplify the preparation process of delivery system, play an important role in immunotherapy and application field.In addition to single component application, the secondary amine cationic lipid of the application can also be used to prepare lipid nanoparticles (LNPs) with auxiliary lipids, to efficiently deliver mRNA and other nucleic acid molecules in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to a secondary amine cationic lipid compound, its preparation method, its composition, and its application. Background Technology

[0002] Messenger ribonucleic acid (mRNA) is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and guides protein synthesis. By regulating the mRNA sequence, various proteins can be translated, thus mRNA therapy shows great promise in vaccine development, gene editing, and the treatment of genetic diseases. Compared to deoxyribonucleic acid (DNA) molecules, which only function in the cell nucleus, mRNA molecules can exert their effects in the cytoplasm, demonstrating numerous advantages in safety and efficiency. However, mRNA molecules themselves are unstable and easily degraded; they also carry a large negative charge, making them difficult for target cells to take up and exert their effects, requiring delivery vectors to enter the target tissues and cells. Developing efficient, safe, and targeted delivery systems has become a crucial issue and bottleneck in mRNA drug development, highlighting the urgent need for safe and effective mRNA delivery materials. Lipid nanoparticles (LNPs) are currently the most commonly used mRNA delivery carriers. LNP carriers typically consist of four components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids. Ionizable cationic lipids are the key component of LNPs and usually contain tertiary amine groups. However, LNPs are difficult to use for targeted extrahepatic mRNA delivery, and their four-component composition makes their preparation cumbersome. Therefore, continuous optimization of the types and structures of delivery carriers is needed to obtain targeted delivery materials that meet different requirements, thereby enriching the diversity and application range of mRNA carriers. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing lipid nanoparticles in extrahepatic targeted delivery and the complexity of their preparation processes. This invention provides a single-component cationic lipid containing secondary amines to achieve extrahepatic targeted delivery of mRNA (e.g., to the spleen), enriching the variety of existing cationic lipid compounds, simplifying the preparation process of delivery systems, and playing an important role in immunotherapy and related applications. In addition to single-component applications, the cationic lipid containing secondary amines of this invention can also be used in conjunction with auxiliary lipids to prepare LNPs for highly efficient in vivo mRNA delivery.

[0004] The specific technical solution adopted in this invention is as follows:

[0005] The secondary amine cationic lipid compound represented by general formula (I), or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, or solvates thereof, is a compound of general formula (I).

[0006]

[0007] Wherein, L1 and L2 are each independently one of C1-C20 alkyl or C1-C20 alkenyl groups, one or more Cs in the C1-C20 alkyl or C1-C20 alkenyl groups are optionally substituted with O or N, and the C1-C20 alkyl or C1-C20 alkenyl groups are unsubstituted or optionally substituted with one or more of the following substituents: C1-C17 alkyl or alkenyl, ester, carbonate, carbonyl, amide, ether, amino, hydrocarbon, olefin, acyl, alkoxy; one or more Cs in the C1-C17 alkyl or alkenyl groups are optionally substituted with O or N, and the C1-C17 alkyl or alkenyl groups are unsubstituted or optionally substituted with one or more of the following substituents: ester, carbonate, carbonyl, amide, ether, amino, hydrocarbon, olefin, acyl, alkoxy.

[0008] R1 and R2 are each independently a straight-chain or branched, saturated or unsaturated C1-C17 alkyl or alkenyl structure, wherein one or more Cs in the C1-C17 alkyl or alkenyl group are optionally substituted with O or N, and the C1-C17 alkyl or alkenyl structure is unsubstituted or optionally substituted with one or more of the following substituents: C1-C12 alkyl, ester, carbonate, carbonyl, amide, ether, amino, hydrocarbon, olefin, acyl, alkoxy.

[0009] Specifically, the C1-C20 alkyl or C1-C20 alkenyl group can be a C1-C20 branched or unbranched saturated or unsaturated hydrocarbon group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, etc. The alkyl or alkenyl group can be cyclic or acyclic. The alkyl or alkenyl group can be branched or unbranched. The alkyl or alkenyl group can also be substituted or unsubstituted. For example, the alkyl group can substitute for one or more groups, including but not limited to the optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silyl, thio-oxo, and mercapto groups described in this invention.

[0010] The terms “alkoxy” and “alkoxy group” used in this invention refer to an alkyl or cycloalkyl group of 1 to 30 carbon atoms bonded by an ether bond; that is, “alkoxy” can be defined as —OR1, where R1 is an alkyl or cycloalkyl group as defined above.

[0011] The terms "olefinic" and "alkenyl" as used in this invention refer to hydrocarbon groups of 2 to 30 carbon atoms, whose structural formula contains at least one carbon-carbon double bond. Asymmetric structures such as (R2R3)C=C (R4R5) include E and Z isomers. This can be presumed to indicate the presence of an asymmetric olefin in the structural formula of this invention, or it can be explicitly represented by the bond symbol C=C. The alkenyl group may be substituted with one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, and alkynyl groups as described in this invention.

[0012] The term "amino group" used in this invention is represented by the formula —NR6R7, wherein R6 and R7 can be independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkylamino, and ester.

[0013] The term "ester group" used in this invention is represented by the formula —OC(=O)R8 or —C(=O)OR8, wherein R8 can be an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, etc. as described in this invention.

[0014] The term "carbonate group" used in this invention is derived from the formula R9O-C(=O)-OH or R9O-C(=O)-OR10, wherein R9 and R10 can independently be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, etc. as described in this invention.

[0015] The term "carbonyl" used in this invention is derived from the formula —C(=O)—.

[0016] The term "amide group" used in this invention is derived from the formula —R11-CO-NH-R12, wherein R11 and R12 can be independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, etc., as described in this invention.

[0017] Specifically, the pharmaceutically acceptable salt refers to an acid addition salt or a base addition salt. The acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamido acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glucohepanoic acid, gluconic acid, glucuronic acid, and glutamic acid. Glutaric acid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthalene-1,5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid.

[0018] The base addition salt refers to a salt prepared by adding an inorganic or organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. The organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcohol, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0019] Furthermore, the secondary amine-containing cationic lipid compound specifically has one of the following structures:

[0020]

[0021] A composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a cationic lipid, the cationic lipid comprising a cationic lipid compound of the general formula (I), or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, or solvate thereof.

[0022] Furthermore, the composition comprises lipid nanoparticles (LNPs) formed from one or more of the cationic lipid compounds of general formula (I), phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0023] Furthermore, the therapeutic or preventative agent includes one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0024] Further, the nucleic acid molecule is RNA, mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator or shRNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

[0025] Furthermore, the composition also includes one or more of the excipients or diluents that are available for the pharmaceutical.

[0026] A method for preparing the secondary amine cationic lipid compound represented by general formula (I) specifically comprises:

[0027] Reactant 1 and reactant 2 were mixed uniformly and reacted at a gradient temperature of 25°C and 70°C to obtain a secondary amine-containing cationic lipid compound; wherein, reactant 1 is a compound with an amine core and a primary amine terminal, and has the following general formula:

[0028]

[0029] The general formula of reactant 2 is as follows:

[0030]

[0031] In the formula, R is one of C1-C18 alkyl or alkenyl groups;

[0032] Among them, the products of the primary amines in reactant 2 and reactant 1 still contain one or more secondary amine groups.

[0033] Furthermore, reactant 1 specifically has one of the following structures:

[0034]

[0035] Furthermore, reactant 2 specifically has one of the following structures:

[0036]

[0037] The use of a secondary amine cationic lipid compound of the general formula (I), or a pharmaceutically usable salt thereof, or the use of the composition thereof in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs.

[0038] The beneficial effects of this invention are:

[0039] This invention develops cationic lipids rich in secondary amine groups, which can be used alone to deliver mRNA (mRNA-encapsulated nanoparticles are denoted as CLNs), and exhibits excellent specificity in delivering mRNA to the spleen, demonstrating significant value in immunotherapy and applications. Furthermore, based on these secondary amine-containing cationic lipids, LNPs can also be constructed to achieve highly efficient in vivo mRNA delivery. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the design and preparation process of cationic lipid compounds containing secondary or tertiary amines.

[0041] Figure 2 This is the 1H NMR spectrum of the secondary amine cationic lipid compound TE-EP8-S obtained in Example 3;

[0042] Figure 3 The mRNA encapsulation efficiency of CLNs is determined by the presence of cationic lipid compounds containing secondary or tertiary amines.

[0043] Figure 4 This study analyzed the transfection efficiency of CLNs (Cat Lipid Compounds Containing Secondary or Tertiary Amines) in delivering mRNA to IGROV1 cells.

[0044] Figure 5 This is a graph showing the cytotoxicity analysis results of CLNs containing cationic lipid compounds containing secondary or tertiary amines.

[0045] Figure 6 The images show fluorescence images of endocytosis and endosome escape of Cy5 mRNA delivered by secondary or tertiary amine cationic lipid compounds as CLNs in IGROV1 cells. The scale bar in the images represents 20 μm.

[0046] Figure 7 The figure shows the results of a study on the efficiency of in vivo mRNA delivery by CLNs using cationic lipid compounds containing secondary or tertiary amines (0.25 mg / kg Fluc mRNA).

[0047] Figure 8 This is a graph showing the results of high-efficiency expression of mRNA (0.25 mg / kg FlucmRNA) in the spleen mediated by the secondary amine cationic lipid TE-EP8-S;

[0048] Figure 9 This is a comparison diagram of the mRNA delivery of cationic lipid compounds rich in secondary amines and cationic lipid compounds containing tertiary amines;

[0049] Figure 10The figure shows the in vivo mRNA delivery efficiency analysis results of LNPs based on TE-EP8-S. In the figure, A1: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 35 / 16 / 46.5 / 2.5; A2: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 46.3 / 9.4 / 42.7 / 1.6; A3: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 15 / 20 / 25 / 2.

[0050] Figure 11 This is a diagram showing the results of organ-targeting analysis of in vivo mRNA delivery of LNPs based on TE-EP8-S. In the diagram, A1: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 35 / 16 / 46.5 / 2.5; A2: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 46.3 / 9.4 / 42.7 / 1.6; A3: the molar ratio of TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 is 15 / 20 / 25 / 2. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The implementation conditions used in the embodiments can be further adjusted according to different specific application requirements; implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. In the specific embodiments of the present invention, the raw materials used are all commercially available.

[0052] Example 1: Synthesis of a novel cationic lipid An-RS / T

[0053] In this context, An represents different small molecule amine cores, R represents different hydrophobic molecules, S represents lipids rich in secondary amines, and T represents lipids containing tertiary amines.

[0054] The synthetic route for An-RS, a cationic lipid compound rich in secondary amines, is shown below:

[0055]

[0056] The synthetic route for the tertiary amine-rich cationic lipid compound An-RT is shown below:

[0057]

[0058] Based on the reaction principles of epoxy ring-opening or Michael addition, the reactants are placed in a closed space to allow the amino head and hydrophobic tail to react. For An-RS synthesis, the amine and hydrophobic reactant are stirred at 25°C for 2 days, with the primary amine reacting preferentially under mild conditions, followed by stirring at 70°C for 1 day to ensure complete reaction. For An-RT synthesis, the amine and hydrophobic reactant are stirred at 70°C for 3 days. All reactions are carried out in two proportions. When the molar ratio of primary amine in reactant 2 to primary amine in reactant 1 is 1:1, the reaction proceeds to secondary amine to obtain An-RS. An-RT is obtained when the primary amine in reactant 1 consumes two molecules of reactant 2 and the secondary amine consumes one molecule of reactant 2. Figure 1 For non-liquid raw materials, isopropanol (iPrOH) (epoxide) or dimethyl sulfoxide (DMSO) (acrylate) can be used for dissolution. The entire reaction process was monitored using thin-layer chromatography (TLC). After the reaction was completed, the remaining solvent was removed in a vacuum drying oven, and the crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH system with 0.1% triethylamine) to obtain the final product.

[0059] The following shows the molecular structures of some secondary amine cationic lipid compounds:

[0060]

[0061] Example 2: Synthesis of a novel secondary amine-containing cationic lipid compound TA-Ac10-S

[0062]

[0063] Tris(2-aminoethyl)amine (TA, 0.1 mmol, 1.0 eq) and decyl acrylate (Ac10, 0.3 mmol, 3.0 eq) were added to a dry reaction flask. The mixture was stirred at 500 rpm for 48 h at 25 °C, and then stirred at 70 °C for another 24 h. The reaction was monitored by TLC until the reaction was complete and the crude product was obtained. The product was finally purified by silica gel column chromatography. The determination was performed using a VarianINOVA 500 MHz NMR spectrometer at 25 °C with deuterated chloroform (CDCl3) as solvent. 1 H NMR spectroscopy. 1 H NMR(CDCl3,ppm)δ0.88(t,9H,-CH2CH2CH2CH3),1.22-1.36(m,42H,-OCH2CH2(CH2)7CH3),1.57-1.65(m,6H,-OCH2CH2( CH2)7CH3),1.84(s,12H,-NCH2CH2NH-),2.40-2.95(m,12H,-NHCH2CH2COO-),4.02-4.09(m,6H,-OCH2CH2(CH2)7CH3).

[0064] Example 3: Synthesis of a novel secondary amine cationic lipid compound TE-EP8-S

[0065]

[0066] Tetraethylenepentamine (TE, 0.2 mmol, 1.0 eq) and 1,2-epoxyoctane (EP8, 0.4 mmol, 2.0 eq) were added to a dry reaction flask. The mixture was stirred at 500 rpm for 48 h at 25 °C, and then stirred at 70 °C for another 24 h. The reaction was monitored by TLC until the reaction was complete and the crude product was obtained. The product was finally purified by silica gel column chromatography. Figure 2 The determination was performed using a VarianINOVA 500MHz NMR spectrometer at 25°C with deuterated chloroform (CDCl3) as the solvent. 1 H NMR spectroscopy. 1 H NMR(CDCl3,ppm)δ0.85-0.91(m,6H,-CH2CH2CH2CH3),1.23-1.48(m,20H,-CHOH(CH2)5CH3 ), 2.35-2.85(m,20H,-NCH2CH2NH-and-NHCH2COH-), 3.52-3.71(m,2H,-CHOH(CH2)5CH3).

[0067] Example 4: Preparation and characterization of a cationic lipid / mRNA nanoparticle (CLN) delivery system

[0068] Under aseptic conditions, a certain amount of cationic lipids were dissolved in an ethanol solution, and firefly luciferase mRNA was diluted in citrate buffer (pH 4, 10 mM). The ethanol solution of cationic lipids and the mRNA solution were rapidly mixed at a mass ratio of 20:1 and a volume ratio of 1:3. After incubation for 15 min, the mixture was diluted three times its original volume with 1×PBS buffer to obtain the CLNs delivery system for in vitro cellular mRNA delivery. The CLNs were dialyzed against 1×PBS before being used in in vivo experiments.

[0069] The mRNA encapsulation efficiency of CLNs was determined using the Quant-iT RiboGreen RNA Detection Kit in a 1×TE buffer system. Figure 3 As shown, cationic lipids rich in secondary amines can achieve an encapsulation efficiency of over 95%, while cationic lipids rich in tertiary amines have an encapsulation efficiency of around 50%.

[0070] Example 5: In vitro mRNA delivery and cytotoxicity evaluation

[0071] IGROV1 ovarian cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (PS). For in vitro screening, IGROV1 cells were cultured at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 cells / well in white 96-well plates, with three parallel wells per group. After incubation in a CO2 incubator (37°C) for 24 hours, and cell adhesion was achieved, the medium containing metabolic waste was discarded, and 100 μL of fresh medium containing 10% FBS was added. CLNs containing 25 ng of firefly luciferase mRNA (Fluc mRNA) were added to each well, with a cationic lipid / mRNA mass ratio of 20:1. After mixing, the cell culture plates were incubated for 24 hours. Luciferase expression was measured using a luciferase assay kit. Figure 4 The designed cationic lipids exhibited high efficiency in in vitro mRNA delivery, with secondary amine cationic lipids showing superior mRNA delivery efficiency compared to tertiary amine-rich cationic lipids. Some of these cationic liposomes even outperformed the LNPs (ALC-0315LNPs) used in currently FDA-approved mRNA COVID-19 vaccines. Cytotoxicity was measured using Alamar Blue reagent at a wavelength of Ex / Em = 530 / 590 nm. Results are as follows... Figure 5 As shown, most secondary amine cationic lipids exhibit low cytotoxicity, good safety, and cell survival rates exceeding 90%.

[0072] Example 6: Endosomal escape and cellular uptake assay

[0073] Confocal imaging was used to directly measure endosome escape and cellular uptake of CLNs. IGROV1 cells were cultured at 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well into Lab-Tek chamber slides. After 12 hours, the culture medium containing metabolic waste was discarded, and 1 mL of medium containing 10% FBS was added. CLNs were prepared by adding 500 ng Cy5-Fluc mRNA to each well, with a cationic lipid / mRNA mass ratio fixed at 20:1. After incubation at 37°C for 4 hours, lysosomes were stained with Lysotracker Green DND 26, incubated for 60 minutes, and then washed three times with PBS. The nuclei were then stained with Hoechst 33342 (1 / 100 dilution), incubated at 37°C for 5 minutes, and washed five times repeatedly with PBS. The slides were then imaged using a confocal microscope (FLUOVIEW FV3000). The results are shown below. Figure 6 As shown, cationic lipids rich in secondary amines can mediate efficient endosome escape and are superior to cationic lipids rich in tertiary amines in endocytosis.

[0074] Example 7: Evaluation of the efficiency of single-component cationic lipoprotein in vivo mRNA delivery

[0075] Female C57BL / 6 mice, aged 6 to 8 weeks and weighing 18-20g, were selected and injected via tail vein with 0.25 mg / kg of Fluc mRNA. -1 CLNs were detected. Six hours later, mice were anesthetized with isoflurane and intraperitoneally injected with 100 μL of D-luciferin potassium salt PBS solution (30 mg / mL). -1 Approximately 5 minutes later, the bioluminescence of the body was measured using a small animal in vivo imaging system (Perkin Elmer). The major organs were then isolated and imaged using the same method. Figure 7-9 As shown, the single-component cationic lipids of this invention can specifically deliver mRNA to the spleen, exhibiting good targeting specificity. The mRNA delivery efficiency of cationic lipids rich in secondary amines is superior to that of cationic lipids rich in tertiary amines. This spleen-targeting CLN (colony-forming nuclei) method has enormous potential for immunological applications.

[0076] Example 8: LNPs system constructed based on secondary amine cationic lipids for in vivo mRNA delivery.

[0077] Female C57BL / 6 mice, aged 6 to 8 weeks and weighing 18-20g, were selected and injected via tail vein with 0.25 mg / kg of Fluc mRNA. -1 LNPs were prepared using phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and PEGylated lipid 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000). Three molar ratios of cationic lipid TE-EP8-S / DOPE / cholesterol / DMG-PEG2000 were used: (A1) 35 / 16 / 46.5 / 2.5; (A2) 46.3 / 9.4 / 42.7 / 1.6; and (A3) 15 / 20 / 25 / 2. The components were dissolved in ethanol at their respective molar ratios, and mRNA was dissolved in citrate buffer (10 mM, pH 4). The two were rapidly mixed at a cationic lipid / mRNA mass ratio of 20:1 and an ethanol / water volume ratio of 1:3. The mixture was incubated for 15 min, and LNPs were formed after dialysis with PBS. Six hours after intravenous administration, the bioluminescence intensity of mouse bodies and isolated organs was measured using D-luciferin potassium salt substrate and a small animal in vivo imaging system. The results are as follows: Figure 10-11 As shown, secondary amine cationic lipids can not only deliver mRNA alone, but also synergistically construct LNPs with helper lipids, enabling efficient mRNA delivery in vivo.

Claims

1. A secondary amine cationic lipid compound or a pharmaceutically acceptable salt thereof, characterized in that, The secondary amine-containing cationic lipid compound specifically has one of the following structures: 。 2. A composition, characterized in that, The composition comprises a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a cationic lipid, said cationic lipid being one or more of the cationic lipid compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The composition according to claim 2, characterized in that, The composition comprises lipid nanoparticles formed from one or more of cationic lipid compounds, phospholipids, cholesterol, and polyethylene glycol-modified lipids.

4. The composition according to claim 2, characterized in that, The therapeutic or preventive agent includes one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

5. The composition according to claim 4, characterized in that, The nucleic acid molecule is RNA, DNA, or PNA.

6. The composition according to claim 5, characterized in that, The nucleic acid molecules specifically include mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitors, microRNA activators or shRNA, antisense nucleic acids, aptamers, ribozymes or immunostimulatory nucleic acids.

7. A method for preparing the secondary amine cationic lipid compound according to claim 1, characterized in that, Specifically: Reactant 1 and reactant 2 were mixed uniformly and reacted at a gradient temperature of 25 °C and 70 °C to obtain a secondary amine-containing cationic lipid compound; wherein, reactant 1 is a compound with an amine core and a primary amine terminal, and reactant 1 specifically has one of the following structures: ； Specifically, reactant 2 has one of the following structures: ； Among them, the products of the primary amines in reactant 2 and reactant 1 still contain one or more secondary amine groups.

8. The use of a secondary amine cationic lipid compound as described in claim 1, or a pharmaceutically acceptable salt thereof, or a composition as described in any one of claims 2 to 6, in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs.

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