CN114907243B

CN114907243B - Ionizable lipid, composition and application thereof

Info

Publication number: CN114907243B
Application number: CN202210187286.3A
Authority: CN
Inventors: 李新松; 王吉; 张延好; 刘超; 邢寒磊; 董硕; 查文慧
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2023-10-27
Anticipated expiration: 2042-02-28
Also published as: CN114907243A

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

Ionizable lipid, composition and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to an ionizable lipid, a composition and application thereof, and in particular relates to an ionizable lipid or a pharmaceutically usable salt thereof, and a composition and application thereof.

Background

With the continuous development of molecular biology techniques, diseases caused by gene defects or gene mutations have been widely paid attention to and studied. It has been found that various diseases (such as diabetes, asthma, cancer, psychosis, etc.) of human beings are closely related to gene defects or gene mutations, and thus prevention, interference and treatment of diseases at the gene level are effective strategies. Gene therapy is a therapeutic technique for interfering with endogenous genes of cells at the nucleic acid level, and the common strategies include three kinds of strategies, namely, for partial preventive diseases, the genes encoding antigens can be introduced into the body in advance so as to induce immune response of the human body; 2. for highly expressed abnormal genes, RNAi interference techniques can be used to suppress the function of abnormal endogenous genes; 3. the purpose of treating diseases is achieved by introducing normal genes to replace the deleted or abnormally mutated genes.

The gene medicine can not exist stably in vitro and in vivo and is easy to be degraded by air and nuclease in cells, so that a proper carrier needs to be developed to be delivered in vivo with high efficiency. Vectors for gene drugs that are currently common include viral vectors and non-viral vectors. The virus serving as a nucleic acid drug delivery carrier has the instinct of infecting cells, and has the advantages of easy transformation, easy preparation, high transfection efficiency, quick effect and the like. However, viral vectors have safety problems such as cytotoxicity, immunogenicity and carcinogenicity, and besides, the size of the loaded DNA is limited, and the high production cost is an important reason for limiting the clinical application of the viral vectors. With the rapid development of medical polymer materials and nanotechnology, various non-viral vectors such as polyethylenimine, micelles, cationic lipids and the like are emerging on the market. The cationic lipid forms nano particles with the nucleic acid medicine with negative charge mainly through electrostatic adsorption, and can protect the nucleic acid medicine from degradation of nuclease, so that stable delivery of the nucleic acid medicine in vivo is realized. Compared with virus vectors, the problems of immunogenicity, carcinogenicity, limited size of loaded DNA and the like of cationic lipids are further solved, but the problems of cytotoxicity, biocompatibility and the like still exist. Based on the problems of the cationic lipid, development of a lipid carrier with low toxicity or no toxicity, good biocompatibility and biodegradability and a composition thereof for high-efficiency delivery of gene drugs, nucleic acid vaccines, small molecule drugs, polypeptides or protein drugs is urgently needed to achieve the purpose of preventing or treating diseases.

Disclosure of Invention

The invention aims to: the invention aims at providing an ionizable lipid for delivering gene drugs, nucleic acid vaccines, small molecule drugs, polypeptides or protein drugs, pharmaceutically usable salts thereof, compositions comprising the same and applications thereof; the lipid provided by the invention has pH response and glutathione response capabilities, enriches the variety of ionizable lipids, provides more choices for drug delivery, and has important practical significance.

The technical scheme is as follows: the invention relates to an ionizable lipid shown in a general formula (1) or a pharmaceutically usable salt thereof.

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

In particular, the term "optionally substituted" refers to alkenyl groups that may or may not be substituted, e.g., optionally substituted alkenyl groups include substituted alkenyl groups and unsubstituted alkenyl groups.

In particular, where the groups are "substituted," they may be substituted with any suitable substituent or substituents.

More specifically, when said group is "substituted" it means substituted in whole or in part by one or more of hydroxy, alkoxy, halogen, alkyl, alkenyl, cycloalkyl.

In particular, the "pharmaceutically acceptable salts thereof" refers 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, malonic acid, methanesulfonic acid, glycolic acid, pyruvic acid, malic acid, mandelic acid, salicylic acid, maleic acid, isobutyric acid, tartaric acid, succinic acid, gentisic acid, galactonic acid, cyclic amic acid, 4-aminosalicylic acid, 2-oxoglutarate, 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, and the like;

the organic bases include, but are not limited to, ammonia, ethanolamine, diethylamine, dicyclohexylamine, trimethylamine, isopropylamine, choline, betaine, procaine, hydrazinaniline, 2-diethylaminoethanol, 2-dimethylaminoethanol, choline, and caffeine.

Preferably, n is a positive integer from 10 to 16.

More preferably, the branched chain comprising 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 therapeutic or prophylactic agents, the carrier comprising an ionizable lipid comprising one or more of the ionizable lipids represented by the general formula (1), or pharmaceutically acceptable salts thereof.

Specifically, the active ingredient is encapsulated in or adsorbed to a carrier.

In particular, the therapeutic or prophylactic agent comprises one or more of a genetic drug, a nucleic acid vaccine, a polypeptide or a protein.

Specifically, the active ingredients of the gene 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 prophylactic agent comprises at least one shRNA.

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

More specifically, the shRNA is interfering with the expression of intratumoral protein kinase 3.

In particular, the therapeutic and/or prophylactic agent is a currently known drug, such as an antineoplastic agent, anticonvulsant, antifungal agent, anti-infective agent, anti-glaucoma agent, anesthetic, antibiotic/antibacterial agent, local anesthetic, antidepressant, hormone antagonist, immunomodulator, antiparasitic, hormone or imaging agent.

Preferably, the ionizable lipid further comprises one or more other charged lipids.

More specifically, the charged lipids include, but are not limited to: 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 2-dioleyloxy-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2-dioleoyloxy-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DODMA), N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride.

(DOTMA), N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethyl ammonium chloride (DOTAP), 1, 2-dimyristoyl oleoyl-sn-glycero-3-ethylcholine phosphate (MOEPC), (R) -5- (dimethylamino) pentane-1, 2-diyl dioleate hydrochloride (DODAPEN-Cl), (R) -5-guanidinopentane-1, 2-diyl dioleate hydrochloride (DOPen-G), and (R) -N, N, N-trimethyl-4, 5-bis (oleoyloxy) pentane-1-ammonium chloride DOTAPEN.

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

Preferably, the carrier forms a lipid nanopreparation with the pharmaceutical composition, the average size of the lipid nanopreparation is 10nm to 250nm, preferably 30nm to 200nm, more preferably 50nm to 150nm, more preferably 70nm to 100nm.

Preferably, the lipid nanoformulations have a polydispersity index of 0.40 or less, more preferably 0.25 or less, and even more preferably 0.20 or less.

According to some embodiments, the carrier further comprises a structural lipid, the molar ratio of the ionizable lipid to the structural lipid being from 1 to 5:1, preferably from 1 to 4:1, more preferably from 1 to 2:1.

Structural lipids can stabilize the structure of the carrier well. In particular, the structural lipids include, but are not limited to, one or more of cholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterols, corticosteroids, ursolic acid, lycorine, alpha-tocopherol.

According to some embodiments, the carrier further comprises neutral lipids, the molar ratio of the ionizable lipid to the neutral lipids being from 1 to 10:1, further preferably from 3 to 6:1.

In particular, the neutral lipid is any lipid molecule that is disclosed or not disclosed as being in an uncharged form or a neutral zwitterionic form at a selected pH or range.

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

According to some embodiments, the carrier further comprises a polymer conjugated lipid, the molar ratio of the ionizable lipid to the polymer conjugated lipid being 10-100:1, more preferably 10-50:1, still more preferably 25-35:1.

The polymer conjugated lipid can improve the stability of the lipid and reduce the protein absorption of the lipid; specifically, the polymer conjugated lipid mainly comprises a disclosed or unpublished PEG modified lipid; the polymer conjugated lipids include, but are not limited to, PEG-DMG, PEG-c-DOMG, PEG-DMPE, PEG-DPPC, PEG-DLPE, PEG-DSPE, chol-PEG2000, ceramide-PEG 2000; preferably, the polymer conjugated lipid is DMPE-PEG2000 or DMG-PEG2000.

According to some embodiments, the carrier further comprises a structural lipid, a neutral lipid, and a polymer conjugated lipid, the molar ratio of the ionizable ionic lipid, the structural lipid, the neutral lipid, and the polymer conjugated lipid being (15-60): (15-45): (1-20): (0.5-2). Further preferably (20 to 35): (20-35): (1-10): (0.5-1.5).

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

The third aspect of the invention provides an application of the ionizable lipid shown in the general formula (1) or the composition in preparing gene medicines, small molecule medicines, polypeptides or protein medicines.

The beneficial effects are that: compared with the prior art, the invention has the characteristics that: the invention provides a brand new ionizable lipid, which has no toxicity, biodegradability and biodegradability. Especially in glutathione-rich tumor environments, the release of genes can be accelerated. The ionizable lipid enriches the types of lipid compounds, provides more choices for delivering gene drugs, nucleic acid vaccines, micromolecular drugs, polypeptides, protein drugs and the like, and has important practical significance.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of Compound 1 of the present invention;

FIG. 2 is a mass spectrum and a nuclear magnetic resonance diagram of compound 1 in the present invention;

FIG. 3 is a graph showing particle diameters of (a) lipid nanoparticles of the present invention; (b) the polydispersity index (PDI) of the lipid nanoparticle; (c) potential map of lipid nanoparticle; (d) 1 negative control gene (shNC) map of lipid nanoparticle; (e) 1 of the lipid nanoparticles represented a schematic representation of a silent protein kinase 3 (shPKN 3-2459); (f) The lipid nanoparticle has another 1 silencing protein kinase 3 (shPKN 3-3357) schematic diagram;

FIG. 4 is a schematic diagram showing lysosomal escape of lipid nanoparticles according to the present invention;

FIG. 5 (a) lipid nanoparticle transfection 293T cells in the present invention; (b) transfecting the PC-3 cells with the lipid nanoparticle; (c) a green fluorescence intensity map; (d) a glutathione response of the lipid nanoparticle;

FIG. 6 is a schematic diagram of a cytotoxicity test of lipid nanoparticles of the present invention;

FIG. 7 is a diagram of immunoblotting detection in the present invention;

FIG. 8 shows the effect of (a) lipid nanoparticles on PC-3 tumor cell migration in the present invention; (b) Influence of lipid nanoparticles on PC-3 tumor cell invasion;

FIG. 9 is a schematic representation of (a) lipid nanoformulations of the invention for use in the treatment of prostate cancer in mice; (b) tumor volume; (c) tumor in mice after treatment; (d) mouse body weight; (e) HE staining;

FIG. 10 shows the synthetic route of Compound 2 of the present invention.

Detailed Description

The invention is further described below with reference to the drawings and examples.

Example 1, synthesis of compound 1:

compound 1 was synthesized according to the scheme of fig. 1:

6g of 3-mercaptopropane-1, 2-diol and 4ml of 30% hydrogen peroxide are taken in a reaction flask and stirred at room temperature for about 12 hours; compound 1-1 was purified using silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) =20:1); after purification was completed, dodecanoic acid was dissolved in DCM, dicyclohexylcarbodiimide (DCC) (1.2 eq) was then added and stirred for 60 minutes; white compound 1-1 and catalyst N, N-dimethyl-4-aminopyridine (DMAP) (400 mg) were added to the above reaction mixture and further stirred at room temperature overnight; after removal of DCM and DCU, the mixture was purified by silica gel column chromatographyThe intermediate was purified, dried on a rotary evaporator and confirmed by 1H NMR; 300mg of Compound 1-2 were dissolved in DCM under nitrogen and 160ml of SO was then added to the mixture ₂ Cl ₂ Then stirring for 1h at room temperature; the mixture was dried under high vacuum at 45 ℃ and then 20mL of anhydrous DCM and 68mg of 2- (dimethylamino) ethane-1-thiol were added; after stirring overnight, final compound 1 was obtained by purification, vacuum drying and confirmed by TOF-MS, 1HNMR and 13C NMR (fig. 2).

Example 2 detection of Lipid Nanoparticle (LNP) formulation particle size, polydispersity, potential and transmission electron microscopy:

the ionizable lipids of example 1 were combined with DSPC, cholesterol and DMG-PEG2000 at 50:10:38.5:1.5 mol ratio is dissolved in ethanol to prepare ethanol lipid solution; 1 negative control gene and 2 shRNAs of silent protein kinase 3 were dissolved in 10 to 50mM citrate buffer (pH=4), denoted shNC, shPKN3-2459 and shPKN3-3357, respectively, and specific sequences were as follows:

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

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

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

according to the ethanol solution of lipid: gene citric acid solution = 1:3, mixing the materials according to the volume ratio, and dialyzing the mixture for 24 hours to remove ethanol; the three samples obtained are respectively marked as LNP-shNC, LNP-shPKN3-2459 and LNP-shPKN3-3357; finally, the lipid nanoparticles were filtered using a 220nm sterile filter;

detecting the particle size, polydisperse coefficient, and potential of the lipid nanoparticles using a Malvern particle sizer (Malvern UK); as shown in fig. 3, the lipid nanoparticle has a particle size of about 100nm, PDI less than 0.3, and a potential of about 6mV;

observing the surface morphology of the lipid nanoparticles by transmission electron microscopy (TEM, JEOL, japan); dropping the lipid nano particles prepared above on a 200-mesh carbon film copper grid; after naturally air-drying, 2% (w/v) phosphotungstic acid is dripped into the copper mesh for about 1-2 minutes; shooting the treated sample under an acceleration voltage of 200 kV; the transmission electron microscopy image showed that the lipid nanoparticle was spherical in shape and the size was consistent with the results measured by the above-described malvern particle sizer.

Example 3, compound 1 mediated lysosomal escape phenomenon:

to test the pH response capacity 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 dish (14 mm), after 20h incubation, fluorescein isothiocyanate-labeled lipid nanoformulations were added to the confocal dish, and then incubated at 37℃for different times (2 h, 4h, 6 h); cells were washed with PBS (ph=7.2) and then incubated with lysosomal Red dye Lyso-Tracker Red for 20 minutes to clearly color lysosomes; after washing 3 times with PBS (ph=7.2), the intracellular lysosome escape behavior was observed using a confocal laser scanning microscope (CLSM, olympus, japan); in addition, a gene without using a vector was used as a control; FIG. 4 shows that the number of lipid nanoparticles escaping lysosomes correlated positively with the passage of time, but on the experimental group without vector, experimental results indicated that naked shRNA was able to be rapidly degraded by lysosomes, with no green fluorescence observed; therefore, compound 1 has pH response capability in acid lysosomes (ph=4), can be successfully protonated, induces proton sponge effect, and helps genes to successfully escape from lysosomes.

Example 4, cell transfection and glutathione response experiments:

293T cells or PC-3 cells (2X 10) ⁴ Cells/well) were seeded in 6-well plates and cells were cultured using DMEM medium until 80% confluence was achieved; then, the cells were washed 3 times with PBS, the medium was changed to Opti-MEM serum-free medium, and LNP-shPKN3-2459 or LNP-shPKN3-3357 was added for incubation; furthermore, according to the instructions, equimolar doses of shPKN3-2459 or shPKN3-3357 were mixed with lipo2000 as positive controls; FIG. 5 shows that the lipid nanoparticle preparation mediated by the compound 1 can effectively transfect normal cells and cancer cells, and the transfection effect is better than that of the commercially available lipid lipo2000, so that the lipid nanoparticle preparation has good application prospect;

to investigate the glutathione-responsive ability of compound 1, different concentrations of glutathione (2 mM, 4mM and 6 mM) were added to the complex of lipid nanoparticles, followed by gel electrophoresis assay; the method comprises the following steps: the lipid nanofabricated (5 ul) and 6×loading buffer (1 ul) were gently mixed and added to the agarose gel (0.8% w/v); the gel was run in TAE buffer for 40 minutes at a constant voltage of 110V, and images were obtained using 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 that as glutathione concentration increases, the more gene is released.

Example 5:

293T cells were seeded in 96-well plates (2000 cells/well) and cultured overnight; after cell attachment, lipo 2000-shPKN3 and LNP-shPKN3 lipid nanoparticles of different concentrations were added to 96-well plates; cytotoxicity was assessed by 24 hour incubation using Cell Counting Kit-8 (CCK-8, apexbio, usa); measuring an OD value at 450nm by an enzyme label instrument; the results indicate that the cytotoxicity of the lipid nanoformulations is small, compared to the toxicity of the commercially available lipid lipo2000, which is much greater than that of the autonomously synthesized lipids (fig. 6).

Example 6 western blot experiments:

detecting the interference efficiency of a gene on protein kinase 3 (PKN 3) by a western blot experiment; the early in vitro cell culture and transfection assays were as described in example 4; after transfection, cells were collected, lysed with lysis buffer, and then centrifuged for 20 min; protein concentration was measured using BCA kit according to manufacturer's instructions; after the gel preparation was completed, the treated sample (25 μg per well) was added to the pores and the gel was carefully transferred to a polyvinylidene fluoride (PVDF) membrane by 2 hours electrophoresis; after deionized water cleaning, sealing the membrane for 2 hours by using TBST and skimmed milk; in addition, the treated PVDF membrane was incubated with a specific primary antibody overnight at 4 ℃; subsequently, horseradish peroxidase-labeled secondary antibodies were added to the membrane and incubated for 2 hours at room temperature; after washing, incubation with ECL reagent for 2 minutes and observation was performed by gel imaging system; the results in FIG. 7 show that PKN3-2459 gene can interfere with protein kinase 3 effectively, reduce the expression of protein kinase 3 in tumor cells, and demonstrate the effectiveness of the vector from the side.

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

PC-3 cells (2X 10) ⁴ Cells/well) were seeded in 6-well plates until the cells reached 95% confluence; scratches were made in 6-well plates using a 1ml pipette tip, and then the cells were washed with PBS to remove shed cells; subsequently, LNP-shRNA complexes (LNP-shNC, LNP-shPKN3-2459, LNP-shPKN 3-3357) and Opti-MEM I medium were added, and after a period of incubation, scratches were recorded under an inverted microscope and subjected to subsequent analysis using Image J; the results in FIG. 8a show that the cell mobilities of the control and LNP-shNC groups are 65.11% and 64.53%, respectively, and the cell mobilities of the LNP-shPKN3-2459 and LNP-shPKN3-3357 groups are 28.16% and 41.39%, respectively, indicating that shPKN3-2459 is effective in preventing migration of PC-3 cells with blood and lymph nodes;

invasive tests to assess the effect of lipid nanoparticle formulations on PC-3 cell invasive capacity; PC-3 cells were transfected with lipid nanoparticle formulations for 24 hours and then transferred to a transwell containing Matrigel; 500uLDMEM medium containing 10% fetal bovine serum was added to the bottom chamber, and then the cells were placed in an incubator and cultured at 37℃for 24 hours; washing the PC-3 cells on the bottom surface of the membrane three times with PBS (pH 7.2) to remove matrix gel and residual cells; after the cells are fixed, staining with crystal violet for 30 minutes, and observing with a fluorescence microscope; the results in FIG. 8b demonstrate that compound 1 mediated LNP vectors can successfully deliver shPKN3-2459 and shPKN3-3357, which are effective in inhibiting PC-3 cell invasion in vitro.

Example 8 lipid nanoformulations for treatment of prostate cancer in mice:

female BALB/c nude mice (4-6 weeks old); tumor volume up to 100mm ³ Afterwards, all PC-3 tumor-bearing mice were randomly divided into 4 groups (n=5), namely, physiological saline group, LNP-shNC group, LNP-shPKN3-2459 group and LNP-shPKN3-3357 group; every other day a given dose (1 mg/kg shRNA) was intravenously injected while tumor volume and body weight of nude mice were recorded; after 16 days, PC-3 tumor-bearing mice were sacrificed for H&E staining analysis to assess safety of treatment; drawing of the figure9 shows that the physiological saline group and the LNP-shNC group have no anti-tumor activity, and the LNP-shPKN3-2459 group and the LNP-shPKN3-3357 group obviously inhibit tumor growth; h of each group during treatment&No congestion, inflammation, edema, and other reactions were observed with E staining, further demonstrating the efficacy and safety of compound 1-mediated delivery systems.

Example 9 Synthesis of Compound 2:

6g of 3-mercaptopropane-1, 2-diol and 4ml of 30% hydrogen peroxide are taken in a reaction flask and stirred at room temperature for about 12 hours; compound 1-1 was purified using silica gel column chromatography (DCM: meoh=20:1); after purification was completed, linoleic acid was dissolved in DCM, DCC (1.2 eq) was then added and stirred for 60 min; white compound 1-1 and high performance catalyst DMAP (400 mg) were added to the above reaction mixture, and stirred further overnight at room temperature; after removal of DCM and DCU, the intermediate was purified by silica gel column chromatography, dried on a rotary evaporator and confirmed by 1H NMR; 220mg of Compound 2-2 was dissolved in DCM under nitrogen, then 160ml of SO2Cl2 was added to the mixture, and then stirred at room temperature for 1h; the mixture was dried under high vacuum at 45 ℃ and then 20mL of anhydrous DCM and 68mg of 2- (dimethylamino) ethane-1-thiol were added; after stirring overnight, the final compound 2 was obtained by purification, vacuum drying, weighing 120mg and measuring molecular ion peak 737.21 by mass spectrometry.

Example 10 detection of coated mRNA lipid nanoparticles, and their particle size, polydispersity, potential and transmission electron microscopy:

the procedure of example 2 was followed, with the ionizable lipid (compound 2 of example) and DSPC, cholesterol and DMG-PEG2000 at 50:10:38.5:1.5 mol ratio is dissolved in ethanol to prepare ethanol lipid solution; mRNA expressing the novel coronavirus Receptor Binding Domain (RBD) protein was solubilized in 10 to 50mM citrate buffer (ph=4) as an ethanol solution of lipids: gene citric acid solution = 1:3, mixing the materials according to the volume ratio, and dialyzing the mixture for 24 hours to remove ethanol; the resulting samples were designated LNP-mRNA; finally, the lipid nanoparticles were filtered using a 220nm sterile filter;

detecting the particle size, polydisperse coefficient, and potential of the lipid nanoparticles using a Malvern particle sizer (Malvern UK); the detection result shows that the particle size of the lipid nanoparticle is about 80nm, the PDI is 0.15, and the potential is about 10mV; the surface morphology of the lipid nanoparticles was observed by transmission electron microscopy (TEM, JEOL, japan), and experimental results showed that the lipid nanoparticles encapsulating mRNA were spherical.

Example 11, elisa assay for RBD protein expressed in HeLa cells:

transfection procedure HeLa cells (2X 10) were transfected as described in example 4 ⁴ Cells/well) were seeded in 6-well plates and cells were cultured using DMEM medium until 80% confluence was achieved; cells were then washed 3 times with PBS, medium was changed to Opti-MEM serum-free medium, LNP-mRNA system was added and incubated for 24 hours. After 24 hours, 100ul of cell supernatant was taken and samples were assayed according to the procedure of the Elisa kit. LNP-free mRNA was used as a control. Experimental results show that the OD450 value measured by the mRNA control group without LNP is 0.05, and the OD450 value measured by the LNP-mRNA experimental group is 2.64, which proves that the ionic lipid mediated LNP delivery system can successfully deliver mRNA and express RBD protein.

Example 12 Elisa method for detection of IgG antibodies in mice:

taking 10 female BALB/c mice (6-8 weeks old), and injecting physiological saline into 5 mice as a control group; 5 mice were injected with LNP-mRNA (10 ug/mouse); after two weeks, the mice were bled, centrifuged at 5000r/min for 15min, the supernatant was taken, and IgG antibodies in the mice were detected; the detection method is described in the specification of the Elisa kit;

experimental results: the OD450 of the control group after 1000 times dilution is 0.10, and the OD450 of the experimental group after 1000 times dilution is 0.82; the experimental result was positive, indicating that a large amount of IgG antibodies were produced in mice after LNP-mRNA injection.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.

Claims

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.