CN114907243B - Ionizable lipid, composition and application thereof - Google Patents

Abstract

The invention discloses an ionizable lipid, a composition and application thereof. The invention belongs to the technical field of medicines, and provides a general formula shown in a formula (1) or pharmaceutically usable salt thereof, which mainly comprises long carbon chains, biodegradable ester bonds, bioreductive disulfide bonds and other structures; the ionizable lipids and corresponding compositions and nucleic acids of the invention can be prepared as lipid nanoparticles having a particle size of about 100 nm; the lipid nanoparticle has high lysosome escape capacity and transfection efficiency, can obviously silence protein kinase 3 (PKN 3) genes related to prostate cancer, and can inhibit invasion, metastasis and tumor growth of prostate cancer cells; the invention has important significance for enriching the variety of ionizable lipid, delivering gene medicine and preventing or treating gene related diseases.

Description

An ionizable lipid, its composition and application

Technical field

The invention belongs to the field of medical technology and relates to an ionizable lipid, its composition and application. Specifically, it relates to an ionizable lipid or its pharmaceutically acceptable salt, including its composition and application.

Background technique

With the continuous development of molecular biology technology, diseases caused by gene defects or gene mutations have received widespread attention and research. Research has found that many human diseases (such as diabetes, asthma, cancer, and mental illness, etc.) are closely related to gene defects or gene mutations. Therefore, it is an effective way to prevent, interfere with and treat diseases at the genetic level. Strategy. Gene therapy is a therapeutic technology that interferes with endogenous genes of cells at the nucleic acid level. There are three commonly used strategies: 1. For some preventive diseases, genes encoding antigens can be introduced into the body in advance to trigger the body's immunity. reaction; 2. For highly expressed abnormal genes, RNAi interference technology can be used to inhibit the function of abnormal endogenous genes; 3. The purpose of treating diseases is to introduce normal genes to replace missing or abnormally mutated genes.

Gene drugs cannot exist stably inside or outside the body and are easily degraded by nucleases in the air and cells. Therefore, it is necessary to develop suitable carriers to deliver them efficiently in the body. Currently, common genetic drug vectors include viral vectors and non-viral vectors. As nucleic acid drug delivery carriers, viruses have the ability to infect cells. In addition, they also have the advantages of easy modification, easy preparation, high transfection efficiency, and fast onset of effect. However, viral vectors have safety issues such as cytotoxicity, immunogenicity and carcinogenicity. In addition, the limited size of loaded DNA and high production costs are also important reasons that limit their clinical application. With the rapid development of medical polymer materials and nanotechnology, various non-viral vectors have emerged on the market, such as polyethylenimine, micelles, cationic lipids, etc. Cationic lipids mainly form nanoparticles with negatively charged nucleic acid drugs through electrostatic adsorption, which can protect nucleic acid drugs from degradation by nucleases and achieve stable delivery of nucleic acid drugs in the body. Compared with viral vectors, the immunogenicity, carcinogenicity, and limited size of loaded DNA of cationic lipids have been further solved, but problems such as cytotoxicity and biocompatibility still exist. Based on the above-mentioned problems of cationic lipids, there is an urgent need to develop a low-toxic or non-toxic, good biocompatibility, biodegradable lipid carrier and its composition for efficient delivery of gene drugs, nucleic acid vaccines, and small molecule drugs. , peptide or protein drugs to prevent or treat diseases.

Contents of the invention

Purpose of the invention: The purpose of the present invention is to provide an ionizable lipid for delivering gene drugs, nucleic acid vaccines, small molecule drugs, polypeptides or protein drugs, its pharmaceutically available salts, including its compositions and applications; The invented lipid has pH response and glutathione response capabilities, enriches the types of ionizable lipids, provides more options for drug delivery, and has important practical significance.

Technical solution: an ionizable lipid represented by general formula (1) of the present invention, or a pharmaceutically acceptable salt thereof.

Wherein, n is a positive integer from 6 to 22 or the branch chain may be optionally substituted.

Specifically, the "optionally substituted" means that it may be substituted or unsubstituted. For example, optionally substituted alkenyl includes substituted alkenyl and unsubstituted alkenyl.

Specifically, when the groups described are "substituted," they may be substituted by any suitable substituent or substituents.

More specifically, when the group is "substituted", it means that it is fully or partially substituted by one or more of hydroxyl, alkoxy, halogen, alkyl, alkenyl, and cycloalkyl.

Specifically, the "pharmaceutically acceptable salts thereof" refer to acid addition salts or base addition salts.

The acids include, but are not limited to, phosphoric acid, lactic acid, oxalic acid, formic acid, acetic acid, propionic acid, caproic acid, sulfuric acid, nitric acid, hydrochloric acid, oleic acid, mucic acid, nicotinic acid, fumaric acid, lauric acid, cinnamic acid, propylene glycol Acid, methanesulfonic acid, glycolic acid, pyruvic acid, malic acid, mandelic acid, salicylic acid, maleic acid, hippuric acid, isobutyric acid, tartaric acid, succinic acid, gentisic acid, galactonic acid, cyclic acid, 4-Aminosalicylic acid, 2-oxoglutaric acid, 1-hydroxy-2-naphthoic acid.

The base addition salt refers to a salt prepared by adding an inorganic base or an organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, manganese salts, aluminum salts, calcium salts, magnesium salts, iron salts, zinc salts, potassium salts, lithium salts, ammonium salts, copper salts, sodium salts, etc.;

The organic base includes but is not limited to ammonia, ethanolamine, diethylamine, dicyclohexylamine, trimethylamine, isopropylamine, choline, betaine, procaine, hydrazine aniline, 2-diethylaminoethanol, 2 -Dimethylaminoethanol, choline and caffeine.

Preferably, n is a positive integer of 10-16.

More preferably, the branched chain containing n is an unsubstituted linear alkyl group.

According to some preferred embodiments, the ionizable lipid is one or more of the following compounds:

The present invention provides a composition, which can be used as a carrier for a therapeutic or preventive agent. The carrier includes an ionizable lipid, and the ionizable lipid includes the formula (1). One or more of the ionizable lipids, or pharmaceutically acceptable salts thereof.

Specifically, the active ingredients are encapsulated in the carrier or adsorbed with the carrier.

Specifically, the therapeutic or preventive agent includes one or more of genetic drugs, nucleic acid vaccines, polypeptides or proteins.

Specifically, the active ingredients of the genetic medicine include but are not limited to single-stranded DNA, double-stranded DNA, siRNA, shRNA, miRNA, mRNA, dsRNA, tRNA, LNA, PNA and other forms of RNA molecules known in the art.

According to some specific embodiments, the therapeutic or preventive agent comprises at least one shRNA.

According to some specific embodiments, the therapeutic or prophylactic agent comprises at least one mRNA.

More specifically, the shRNA interferes with the expression of protein kinase 3 in tumors.

Specifically, the therapeutic and/or preventive agents are currently known drugs, such as antineoplastic agents, anticonvulsants, antifungal agents, antiinfectious agents, antiglaucoma agents, anesthetics, antibiotics/antibacterial agents, local anesthetics , antidepressants, hormone antagonists, immunomodulators, antiparasitics, hormones or imaging agents.

Preferably, the ionizable lipid also includes one or more other charged lipids.

More specifically, the charged lipids include, but are not limited to: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-di Methylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3 ]-Dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), N-[1-(2,3-dioleenyl Oxy)propyl]-N,N,N-trimethylammonium chloride.

(DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dimyristoleoyl- sn-glyceryl-3-ethylcholine phosphate (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl), (R)-5-Guanidinopentane-1,2-diyldiolein hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis (Oleyloxy)pentan-1-ammonium chloride DOTAPen).

Preferably, the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 60:1, more preferably 8:1 to 40:1, and more preferably 10:1 to 30:1.

Preferably, the carrier and drug combination form a lipid nanopreparation, and the average size of the lipid nanopreparation is 10 nm to 250 nm, preferably 30 nm to 200 nm, further preferably 50 nm to 150 nm, and more preferably 70 nm to 100 nm.

Preferably, the polydispersity index of the lipid nanoformulation is ≤0.40, further preferably ≤0.25, and more preferably ≤0.20.

According to some specific embodiments, the carrier further includes a structural lipid, and the molar ratio of the ionizable lipid to the structural lipid is 1 to 5:1, preferably 1 to 4:1, and more preferably 1 ~2:1.

Structural lipids can well stabilize the structure of the carrier. Specifically, the structural lipids include, but are not limited to, cholesterol, campesterol, stigmasterol, brassisterol, sitosterol, ergosterol, nonsterols, corticosteroids, ursolic acid, tomatine, and alpha-tocopherol. of one or more.

According to some specific embodiments, the carrier further includes a neutral lipid, and the molar ratio of the ionizable lipid to the neutral lipid is 1 to 10:1, more preferably 3 to 6:1.

Specifically, the neutral lipid is any lipid molecule, disclosed or undisclosed, that exists in an uncharged form or a neutral zwitterionic form within a selected pH value or range.

More specifically, the neutral lipids include, but are not limited to, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1 1,2-dioleoyl-sn-glycerol-3- Phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) , 2-(((2,3-bis(oleoyloxy)propyl))dimethylammonium phosphate)ethyl hydrogen (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), ceramide, sterols and their derivatives.

According to some embodiments, the carrier further includes a polymer conjugated lipid, and the molar ratio of the ionizable lipid to the polymer conjugated lipid is 10 to 100:1, more preferably 10 to 50: 1, and further preferably 25 to 35:1.

Polymer-conjugated lipids can improve the stability of lipids and reduce protein absorption of lipids; specifically, the polymer-conjugated lipids mainly include disclosed or undisclosed PEG-modified lipids; the polymerized Material-conjugated lipids include but are not limited to PEG-DMG, PEG-c-DOMG, PEG-DMPE, PEG-DPPC, PEG-DLPE, PEG-DSPE, Chol-PEG2000, and ceramide-PEG2000; preferably, the polymer The conjugated lipid is DMPE-PEG2000 or DMG-PEG2000.

According to some embodiments, the carrier further includes structural lipids, neutral lipids and polymer conjugated lipids, the ionizable ionic lipids, the structural lipids, the neutral lipids, and The molar ratio of the polymer-conjugated lipid is (15-60): (15-45): (1-20): (0.5-2). More preferably, (20-35): (20-35): (1-10): (0.5-1.5).

According to some specific embodiments, the composition further includes one or more excipients or diluents commonly used in pharmaceuticals.

The third aspect of the present invention is to provide an application of the ionizable lipid represented by the general formula (1) or the composition in the preparation of gene drugs, small molecule drugs, polypeptides or protein drugs.

Beneficial effects: Compared with the prior art, the characteristics of the present invention are: the present invention provides a brand-new ionizable lipid, which is non-toxic, biodegradable and bioreducible. Especially in glutathione-rich tumor environments, gene release can be accelerated. The ionizable lipid enriches the types of lipid compounds, provides more options for delivering gene drugs, nucleic acid vaccines, small molecule drugs, polypeptides and protein drugs, etc., and has important practical significance.

Description of the drawings

Figure 1 is a schematic diagram of the synthetic route of compound 1 in the present invention;

Figure 2 is the mass spectrum and nuclear magnetic spectrum of compound 1 in the present invention;

Figure 3 In the present invention (a) particle size diagram of lipid nanoparticles; (b) polydispersity coefficient (PDI) of lipid nanoparticles; (c) potential diagram of lipid nanoparticles; (d) lipid nanoparticles Picture of one negative control gene (shNC) of the lipid nanoparticles; (e) Schematic diagram of one of the lipid nanoparticles silencing protein kinase 3 (shPKN3-2459); (f) Another one of the lipid nanoparticles silencing protein kinase 3 (shPKN3) -3357) Schematic diagram;

Figure 4 is a schematic diagram of the lysosomal escape phenomenon of lipid nanoparticles in the present invention;

Figure 5 In the present invention (a) lipid nanoparticles transfect 293T cells; (b) lipid nanoparticles transfect PC-3 cells; (c) green fluorescence intensity diagram; (d) glutathione concentration of lipid nanoparticles Peptide response;

Figure 6 is a schematic diagram of the cytotoxicity test of lipid nanoparticles in the present invention;

Figure 7 is an immunoblotting detection chart in the present invention;

Figure 8 In the present invention, (a) the effect of lipid nanoparticles on the migration of PC-3 tumor cells; (b) the effect of lipid nanoparticles on the invasion of PC-3 tumor cells;

Figure 9 Schematic diagram of (a) lipid nanopreparation used in the treatment of mouse prostate cancer in the present invention; (b) tumor volume; (c) tumor in mice after treatment; (d) mouse body weight; (e) HE dyeing;

Figure 10 is the synthetic route of compound 2 in the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1, synthesis of compound 1:

Compound 1 was synthesized according to the route in Figure 1:

Take 6g of 3-mercaptopropane-1,2-diol and 4ml of 30% hydrogen peroxide in the reaction bottle, stir at room temperature for about 12h; use silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 20:1) Purify compound 1-1; after purification is completed, dissolve dodecanoic acid in DCM, then add dicyclohexylcarbodiimide (DCC) (1.2 equivalents) and stir for 60 minutes; add white compound 1-1 and catalyst N,N-dimethyl-4-aminopyridine (DMAP) (400mg) were added to the above reaction mixture, and further stirred at room temperature overnight; after removing DCM and DCU, the intermediate product was purified by silica gel column chromatography, and Dry on a rotary evaporator and confirm by 1H NMR; In a nitrogen atmosphere, dissolve 300 mg of compound 1-2 in DCM, then add 160 ml of SO ₂ Cl ₂ to the mixture, and then stir at room temperature for 1 h; incubate at 45°C at high temperature The mixture was dried under vacuum, and then 20 mL anhydrous DCM and 68 mg 2-(dimethylamino)ethane-1-thiol were added; after stirring overnight, the final compound 1 was obtained by purification and vacuum drying, and was analyzed by TOF-MS, 1HNMR and 13C NMR confirmation (Figure 2).

Example 2. Detection of particle size, polydispersity coefficient, potential and transmission electron microscope of lipid nanoparticle (LNP) preparation:

The ionizable lipids in Example 1 were dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution; 1 negative control gene and 2 silenced The shRNA of protein kinase 3 was dissolved in 10 to 50mM citrate buffer (pH=4), respectively, and was recorded as shNC, shPKN3-2459 and shPKN3-3357. The specific sequences are as follows:

shPKN3-2459 sense:5’-GGGACCUGAAGUUGGAUAACC-3’;

shPKN3-3357 sense:5’-GACUGGACUUGCUUUAUAUUA-3’;

shNC(negative control)sense:5'-UUCUCCGAACGUGUCACGU-3'

Mix the ethanol solution of lipids: the citric acid solution of genes at a volume ratio of 1:3, and dialyze for 24 hours to remove the ethanol; the three obtained samples are recorded as LNP-shNC, LNP-shPKN3-2459, and LNP-shPKN3 respectively. -3357; Finally, filter the lipid nanoparticles using a 220nm sterile filter;

Use a Malvern particle size analyzer (Malvern UK) to detect the particle size, polydispersity coefficient, and potential of the lipid nanoparticles. As shown in Figure 3, the particle size of the lipid nanoparticles is about 100 nm, the PDI is less than 0.3, and the potential is about 6 mV. ;

The surface morphology of lipid nanoparticles was observed by transmission electron microscopy (TEM, JEOL, Japan); the lipid nanoparticles prepared above were dropped on a 200-mesh carbon film copper grid; after natural air drying, 2% (w /v) Phosphotungstic acid was added dropwise to the above copper mesh for about 1-2 minutes; the treated sample was photographed at an accelerating voltage of 200kV; the transmission electron microscope image showed that the lipid nanoparticles were spherical and the size was the same as the above Malvern The results measured by the particle size analyzer are consistent.

Example 3. Lysosomal escape phenomenon mediated by compound 1:

In order to detect the pH responsiveness of compound 1 and the effectiveness of the delivery system, a lysosomal escape assay was performed; briefly, PC-3 cells were added to a confocal culture dish (14 mm), and after 20 h of culture, the heterogeneous Fluorescein thiocyanate-labeled lipid nanoformulation was added to the confocal culture dish, and then cultured at 37°C for different times (2h, 4h, 6h); cells were washed with PBS (pH=7.2), and then treated with lysozyme The body red dye Lyso-Tracker Red was incubated for 20 minutes to clearly visualize lysosomes; after washing 3 times with PBS (pH=7.2), intracellular lysosomes were observed using a confocal laser scanning microscope (CLSM, Olympus, Japan). Escape behavior; In addition, the genes without using carriers were used as a control; Figure 4 shows that the number of lipid nanoparticles escaping from lysosomes is positively correlated with the passage of time, but on the experimental group without using carriers, the experimental results showed that naked The shRNA can be rapidly degraded by lysosomes, and no green fluorescence is observed; therefore, compound 1 has pH responsiveness in acidic lysosomes (pH=4) and can be protonated smoothly, triggering the proton sponge effect and helping genes escape smoothly. Exit lysosomes.

Example 4. Cell transfection and glutathione response experiments:

293T cells or PC-3 cells (2 × ¹⁰ cells/well) were seeded in a 6-well plate and cultured in DMEM medium until reaching 80% confluence; then, the cells were washed three times with PBS and the medium was replaced. into Opti-MEM serum-free medium, add LNP-shPKN3-2459 or LNP-shPKN3-3357 for incubation; in addition, according to the instructions, mix equimolar doses of shPKN3-2459 or shPKN3-3357 with lipo2000 as a positive control; Figure 5 It shows that the lipid nanoparticle preparation mediated by Compound 1 can effectively transfect normal cells and cancer cells, and the transfection effect is better than that of commercially available lipid lipo2000, which has good application prospects;

In order to study the glutathione responsiveness of compound 1, different concentrations of glutathione (2mM, 4mM and 6mM) were added to the complex of lipid nanoparticles, and then gel electrophoresis was performed; the steps were as follows: the lipids were The mass nanoformulation (5ul) and 6× Loading buffer (1ul) were gently mixed and added to the agarose gel (0.8% w/v); the gel was run in TAE buffer solution at a constant voltage of 110V for 40 minutes, using coagulation Images were obtained with a gel recording system (GelDoc XR, Bio-Rad, USA); Figure 5 shows that compound 1-mediated lipid nanoparticles can rapidly release genes in a glutathione environment, and as the glutathione concentration increases , the more genes are released.

Example 5:

293T cells were seeded in a 96-well plate (2000 cells/well) and cultured overnight; after the cells adhered, different concentrations of lipo 2000-shPKN3 and LNP-shPKN3 lipid nanoparticles were added to the 96-well plate; through After 24 hours of incubation, Cell Counting Kit-8 (CCK-8, APExBIO, USA) was used to evaluate cytotoxicity; the OD value at 450 nm was measured by a microplate reader; the results showed that the cytotoxicity of the lipid nanoformulation was very small, compared with Under the circumstances, the toxicity of commercially available lipid lipo2000 is much greater than that of self-synthesized lipid (Figure 6).

Example 6, protein immunoblotting experiment:

Western blot experiments were performed to detect the interference efficiency of genes on protein kinase 3 (PKN3); the preliminary in vitro cell culture and transfection tests were as described in Example 4; after transfection, cells were collected, lysed with lysis buffer, and then centrifuged for 20 minutes; according to the manufacturer's instructions, use the BCA kit to measure the protein concentration; after the gel preparation is completed, add the processed sample (25 μg per well) into the pores, electrophoresis for 2 hours, and carefully transfer the gel to the polymer on a vinylidene fluoride (PVDF) membrane; after cleaning with deionized water, the membrane was blocked with TBST and skim milk for 2 hours; in addition, the treated PVDF membrane was incubated with specific primary antibodies at 4°C overnight; subsequently, horseradish was The oxidase-labeled secondary antibody was added to the membrane and incubated at room temperature for 2 hours; after washing, it was incubated with ECL reagent for 2 minutes and observed through the gel imaging system; the results in Figure 7 show that the PKN3-2459 gene can efficiently interfere Protein kinase 3 reduces the expression of protein kinase 3 in tumor cells, which also proves the effectiveness of the vector.

Example 7. Effect of lipid nanoparticles on PC-3 tumor cell migration and invasion:

Plate PC-3 cells (2 × 10 cells/well) in a ⁶ -well plate until these cells reach 95% confluence; use a 1 ml pipette tip to make scratches in the 6-well plate, then wash the cells with PBS to remove exfoliated cells; then, add LNP-shRNA complex (LNP-shNC, LNP-shPKN3-2459, LNP-shPKN3-3357) and Opti-MEM I medium. After culturing for a period of time, record scratches under an inverted microscope. , and used Image J for subsequent analysis; Figure 8a results show that the cell migration rates of the control group and LNP-shNC group were 65.11% and 64.53%, respectively, while the cell migration rates of the LNP-shPKN3-2459 group and LNP-shPKN3-3357 group 28.16% and 41.39% respectively, indicating that shPKN3-2459 can effectively prevent the migration of PC-3 cells with blood and lymph nodes;

Invasion test to evaluate the effect of lipid nanoparticle formulation on the invasion ability of PC-3 cells; PC-3 cells were transfected with lipid nanoparticle formulation for 24 hours and then transferred to transwell containing Matrigel; 10% fetal bovine serum containing 500uLDMEM culture medium was added to the bottom chamber, and then the cells were placed in an incubator and cultured at 37°C for 24 hours; the PC-3 cells on the bottom surface of the membrane were washed three times with PBS (pH 7.2) to remove the matrix gel and residual cells. ; After the cells were fixed, they were stained with crystal violet for 30 minutes and observed with a fluorescence microscope; Figure 8b results show that compound 1-mediated LNP carrier can successfully deliver shPKN3-2459 and shPKN3-3357, which effectively inhibits PC-3 cells in vitro of invasion.

Example 8. Use of lipid nanopreparations for the treatment of prostate cancer in mice:

Female BALB/c nude mice (4-6 weeks old); ^after the tumor volume reached 100 mm, all PC-3 tumor-bearing mice were randomly divided into 4 groups (n=5), namely saline group and LNP-shNC. group, LNP-shPKN3-2459 group and LNP-shPKN3-3357 group; a given dose (1mg/kg shRNA) was intravenously injected every other day, and the tumor volume and body weight of nude mice were recorded at the same time; after 16 days, PC-3 tumor-bearing mice were sacrificed Mice, H&E staining analysis was performed to evaluate the safety of treatment; Figure 9 shows that the saline group and LNP-shNC group had no anti-tumor activity, while the LNP-shPKN3-2459 group and LNP-shPKN3-3357 group significantly inhibited tumor growth; treatment During this period, no congestion, inflammation, edema, or other reactions were observed in H&E staining in each group, further proving the effectiveness and safety of the Compound 1-mediated delivery system.

Example 9, synthesis of compound 2:

Take 6g of 3-mercaptopropane-1,2-diol and 4ml of 30% hydrogen peroxide in the reaction bottle, stir at room temperature for about 12h; use silica gel column chromatography (DCM:MeOH=20:1) to purify compound 1- 1; After purification is completed, dissolve linoleic acid in DCM, then add DCC (1.2 equivalents) and stir for 60 minutes; add white compound 1-1 and high-performance catalyst DMAP (400mg) to the above reaction mixture, at room temperature Further stir overnight under Add 160ml SO2Cl2 to the mixture, then stir at room temperature for 1h; dry the mixture under high vacuum at 45°C, then add 20mL anhydrous DCM and 68mg 2-(dimethylamino)ethane-1-thiol; after stirring overnight, The final compound 2 was obtained through purification and vacuum drying, weighing 120 mg, and the molecular ion peak measured by mass spectrometry was 737.21.

Example 10. Detection of mRNA-encapsulated lipid nanoparticles, their particle size, polydispersity coefficient, potential and transmission electron microscope:

According to the steps of Example 2, the ionizable lipid (Compound 2 of the Example) was dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution; The mRNA expressing the protein of the new coronavirus receptor binding domain (RBD) is dissolved in 10 to 50mM citrate buffer (pH=4), according to the volume of ethanol solution of lipid: citric acid solution of gene = 1:3 Mix the mixture and dialyze for 24 hours to remove ethanol; record the obtained sample as LNP-mRNA; finally, use a 220nm sterile filter to filter the lipid nanoparticles;

Use a Malvern particle size analyzer (Malvern UK) to detect the particle size, polydispersity coefficient, and potential of the lipid nanoparticles; the detection results show that the particle size of the lipid nanoparticles is about 80 nm, the PDI is 0.15, and the potential is about 10 mV; passed The surface morphology of the lipid nanoparticles was observed with a transmission electron microscope (TEM, JEOL, Japan). The experimental results showed that the lipid nanoparticles encapsulating the mRNA were spherical.

Example 11. Elisa method to detect RBD protein expressed in HeLa cells:

The transfection procedure is as shown in Example 4. HeLa cells (2×10 ⁴ cells/well) are seeded in a 6-well plate, and the cells are cultured using DMEM medium until reaching 80% confluence; then, the cells are washed 3 times with PBS. , change the medium to Opti-MEM serum-free medium, add LNP-mRNA system, and culture for 24 hours. After 24 hours, take 100ul of cell supernatant and detect the sample according to the operating method of the Elisa kit. The mRNA without LNP was used as a control group. The experimental results show that the OD450 value measured in the mRNA control group without LNP was 0.05, while the OD450 value measured in the LNP-mRNA experimental group was 2.64, proving that the ionizable lipid-mediated LNP delivery system can successfully deliver mRNA and express RBD protein.

Example 12. Elisa method to detect IgG antibodies in mice:

Ten female BALB/c mice (6-8 weeks old) were taken, and 5 mice were injected with physiological saline as a control group; 5 mice were injected with LNP-mRNA (10ug/mouse); two weeks later, the mice were harvested Blood, centrifuge at 5000r/min for 15min, take the supernatant, and detect IgG antibodies in mice; the detection method is as described in the instructions in the Elisa kit;

Experimental results: The OD450 of the mouse serum in the control group was 0.10 after being diluted 1000 times, and the OD450 of the mouse serum in the experimental group was 0.82 after being diluted 1000 times. The experimental results were positive, indicating that after injection of LNP-mRNA, a large amount of LNP-mRNA was produced in the mice. of IgG antibodies.

The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above-mentioned embodiments. All technical solutions that fall under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims (8)

1. An ionizable lipid, or a pharmaceutically acceptable salt thereof, characterized in that,

the ionizable lipid is shown as a general formula (1),

wherein n is a positive integer of 6-22.

2. A composition comprising a therapeutic agent, a prophylactic agent, and a carrier for delivering the therapeutic agent or prophylactic agent,

wherein the therapeutic or prophylactic agent is siRNA, shRNA, miRNA and one or more of an mRNA nucleic acid molecule, polypeptide or protein;

the carrier comprises an ionizable lipid,

the ionizable lipid is one or more of ionizable lipid shown in a general formula (1) or pharmaceutically usable salt thereof;

the mass ratio of the carrier to the therapeutic agent or the prophylactic agent is as follows: 1-100:1.

3. A composition according to claim 2, wherein,

the composition is lipid nano particles, and the average particle size of the lipid nano particles is 10 nm-1000 nm; the lipid nanoparticle has a polydispersity index of less than 0.5.

4. A composition according to claim 2, wherein,

the carrier also comprises structural lipid, wherein the structural lipid is one or more of cholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterol, corticosteroid, ursolic acid, lycorine and alpha-tocopherol;

the molar ratio of the ionizable lipid to the structural lipid is 1-10:1.

5. A composition according to claim 2, wherein,

the carrier also comprises neutral lipid, wherein the neutral lipid is one or more of ceramide, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and derivatives thereof;

the molar ratio of the ionizable lipid to the neutral lipid is 1:2-20:1.

6. A composition according to claim 2, wherein,

the carrier also comprises polymer conjugated lipid, wherein the polymer conjugated lipid is one or more of polyethylene glycol modified phosphatidylethanolamine, PEG modified ceramide, PEG modified diacylglycerol, PEG modified phosphatidic acid, PEG modified dialkyl amine and PEG modified dialkyl glycerol;

the molar ratio of the ionizable lipid to the polymer conjugated lipid is: 10-200:1.

7. A composition according to claim 4,5 or 6, characterized in that,

the molar ratio of the ionizable lipid, the structural lipid, the neutral lipid and the polymer conjugated lipid is as follows: (15-60): (15-45): (1-20): (0.5-2).

8. Use of an ionizable lipid of general formula (1) according to claim 1, or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 2 to 7, for the preparation of a genetic drug, a small molecule drug, a polypeptide or a protein drug.