Nucleic acid delivery carrier, composition, and preparation method therefor

Abstract

Provided are a nucleic acid delivery carrier, a composition, and a preparation method therefor. The nucleic acid delivery carrier at least comprises the following components: (a) a deep eutectic solvent; and (b) a lipid molecule. The composition comprises the nucleic acid delivery carrier and a nucleic acid drug. The provided composition significantly improves drug stability, increases drug delivery efficiency and cellular uptake rate, and achieves effective endosomal escape, thereby improving the therapeutic effect of the drug.

Description

A nucleic acid delivery vector, a composition and a method for preparing the same

[0001] Cross-references to related applications

[0002] This application claims priority to an earlier application filed on October 30, 2024, with patent application number 202411531847.2 and entitled "A Nucleic Acid Delivery Vector, Composition and Preparation Method Thereof", which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of nucleic acid delivery technology, and in particular to a nucleic acid delivery vector, composition and preparation method thereof. Background Technology

[0004] Eutectic solvents (DES) are eutectic mixtures composed of two or more components, primarily formed through hydrogen bonds between the components, with melting points lower than those of their constituent parts. Eutectic solvents mainly consist of two parts: hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs). The different hydrogen bond forces between different eutectic solvents determine their different properties and structures. Compared to traditional organic solvents and ionic liquids, DES are primarily prepared from some naturally occurring substances with good biocompatibility (such as acids, sugars, alcohols, and amines), thus offering lower costs, biodegradability, and high biocompatibility. Furthermore, DES exhibit chemical customizability, good solubility in a variety of substances, and chemical and thermal stability. Their preparation process is very simple, involving methods such as simple stirring, grinding, evaporation, and ultrasonication.

[0005] In recent years, nucleic acid drugs have seen rapid development in antiviral, genetic disease treatment, immunotherapy, tumor treatment, and vaccine development fields, including plasmid DNA (pDNA), messenger RNA (mRNA), antisense nucleotides (ASO), small interfering RNA (siRNA), aptamers, microRNA (miRNA), and the CRISPR / CAS9 system. However, nucleic acid drugs are unstable and easily degraded by nucleases in vivo. Furthermore, due to their size and charge, they are difficult for cells to take up and escape from the body, thus failing to exert their original efficacy. Therefore, nucleic acid therapy requires viral or non-viral drug delivery systems. Viral vectors pose certain safety concerns, leading to increasing attention on non-viral vector delivery systems primarily composed of biocompatible materials. Among these, lipid nanoparticles (LNPs) have been successfully applied clinically due to their advantages such as biocompatibility, low immunogenicity, good stability, tunable structure and composition, and low toxicity, achieving breakthrough results, particularly in COVID-19 vaccines. Lipid nanoparticles (LNPs) can be prepared using various methods, including liposome extrusion, thin-film hydration, solvent injection, solvent evaporation, high-pressure homogenization, supercritical fluid extraction, microemulsion, and microfluidics. Among these, microfluidic technology is currently the most mature and widely used method for preparing LNPs carrying nucleic acid drugs. However, microfluidic technology requires specialized chips, which are costly to purchase, difficult to maintain, and have limited production scale. Therefore, developing a low-cost, easily producible nucleic acid drug delivery carrier to supplement the shortcomings of existing microfluidic technologies is particularly important. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nucleic acid delivery carrier, composition, and preparation method thereof. This nucleic acid drug delivery carrier is not only low-cost, simple to prepare, easy to scale up for production, and convenient for storing and transporting nucleic acids and lipids, but also has high delivery efficiency, achieving effective endosome escape and improving the bioavailability of delivered molecules within cells, thereby enhancing therapeutic efficacy.

[0007] In a first aspect, the present invention provides a nucleic acid delivery vector comprising at least the following components: (a) a eutectic solvent; and (b) lipid molecules.

[0008] In one embodiment of the present invention, the mass ratio of the eutectic solvent to the lipid molecules is (0.25-5000):1, preferably (1-1000):1.

[0009] In one embodiment of the present invention, the content of the lipid molecules is 0.02% to 80% (mass percentage), preferably 0.1% to 50%, for example 5%, 10%, 20%, 30%, 40%, and 50%.

[0010] In one embodiment of the present invention, the lipid molecule is selected from one or more of the following: ionizable lipids, cationic lipids and zwitterionic lipids (e.g., DOTMA, Dlin-MC3-DMA, ALC-0315, SM-102, DOTAP, DODAB), accessory lipids (e.g., DSPC, DOPE, HSPC, DOPC, DPPC, DSPE, DOPA, DOPG), cholesterol and its derivatives (e.g., DC-cholesterol, sodium cholesterol sulfate), sterol lipids, fatty acid esters, mannosylated lipids, PEGylated lipids (e.g., DMG-PEG2000, DMPE-PEG 2000, DSPE-PEG2000, ALC-0159, DPPE-PEG 2000).

[0011] In one embodiment of the present invention, the lipid molecule comprises ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids, wherein the molar ratio of the ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids is (20-100):(5-20):(20-50):(0.5-5); preferably, the molar ratio is (30-60):(5-15):(35-45):(0.5-3). For example, in one embodiment of the present invention, the molar ratio of the ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids is 50:10:38.5:1.5. In another embodiment of the present invention, the molar ratio of the ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids is 46.3:9.4:42.7:1.6.

[0012] In one embodiment of the present invention, the ionizable lipid is selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(dimethylamino)butyric acid (6,9,28,31-tetraen-19-yl)heptadecyl alcohol ester (DLin-MC3-DMA), and 1,2-dioleoyl-3-dimethylammonium propane. (DODAP), (2,3-dioleoyl-propyl)-trimethylammonium chloride phospholipid (DOTAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (DOBAQ), YSK05, 4-(((2,3-bis(oleoyloxy)prop-1-ammonium)benzoic acid (DOBAT), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (D OBAQ), 3-((2,3-bis(oleoyloxy)propyl)(methyl)amino)propionic acid (DOPAT), N-(2-carboxypropyl)-N,N-dimethyl-2,3-bis-(oleoyloxy)-prop-1-ammonium (DOMPAQ), N-(carboxymethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (DOAAQ), Alny-100, 3-(dimethylamino)propyl(12Z, 15Z)-3-[(9Z The ionizable lipid is one or more of the following: [(2-hydroxyethyl)(6-oxo-6-decoxyhexyl)amino]octanoic acid (heptadecanoic acid-9-yl) ester or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate). In some embodiments, the ionizable lipid may be SM-102. In some embodiments, the ionizable lipid may be ALC-0315. In some embodiments, the ionizable lipid may be DOTAP.

[0013] In one embodiment of the present invention, the auxiliary lipids include phospholipids. Preferably, the phospholipids include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilaurylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2- One or more of the following lipids are selected: arachidoyl-sn-glycerol-3-phosphate choline (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycerol-3-phosphate choline (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylcholine, distearyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), and lysophosphatidylethanolamine. In some embodiments, the cofactor lipid may be dioleoyl phosphatidylethanolamine (DOPE). In some embodiments, the cofactor lipid may be distearyl phosphatidylcholine (DSPC).

[0014] In one embodiment of the present invention, the cholesterol and its derivatives are selected from one or more of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and phytosterols, preferably from one or more of cholesterol and cholesterol esters. In some embodiments, cholesterol and its derivatives may be cholesterol. In some embodiments, cholesterol and its derivatives may be 3β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride.

[0015] In one embodiment of the present invention, the PEG lipids include PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, and PEG-distearylglyceramide, PEG-cholesterol (1-[8′-(cholesterol-5-ene-3-) [β]-oxy]formamido-3′,6′-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMPE) or 1,2-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000 One or more of the following: PEG2k-DMG, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSPE), 1,2-distearyl-sn-glycerol, methoxy polyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSA), and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159). In some embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In some embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be ALC-0159.

[0016] In one embodiment of the present invention, the eutectic solvent is selected from at least one of ionic liquids, ionic eutectic solvents, and nonionic eutectic solvents. Preferably, it is an ionic liquid or a nonionic eutectic solvent, such as a sugar-based eutectic solvent.

[0017] In one embodiment of the present invention, the eutectic solvent is stable at room temperature, clear and transparent, and is a liquid substance.

[0018] In one embodiment of the present invention, the eutectic solvent includes a binary, ternary, or multi-component system composed of hydrogen bond acceptors and hydrogen bond donors.

[0019] In one embodiment of the present invention, the hydrogen bond acceptor is selected from at least one of quaternary ammonium salts, amines, acids, alcohols, sugars, water, and urea; the hydrogen bond donor is selected from at least one of acids, alcohols, sugars, amines, and water.

[0020] In one embodiment of the present invention, the quaternary ammonium salt is selected from at least one of choline chloride, choline bicarbonate, choline hydroxide, phospholipids or glycerophospholipids, acetylcholine chloride, choline phosphate, and choline bitartrate; the amine is selected from at least one of betaine, ethanolamine, ethylene glycolamine, acetamide, methylamine, ethylamine, dimethylamine, ethylamine, trimethylamine, and aniline; and the sugar is selected from at least one of sucrose, glucose, fructose, maltose, sorbitol, mannose, rhamnose, trehalose, lactose, glyceraldehyde, starch, cellulose, xylose, chitosan, fucose, lipopolysaccharide, dextran, and cyclodextrin. The alcohols are selected from at least one of sorbitol, xylitol, mannitol, lactitol, maltitol, erythritol, glucosyl alcohol, methanol, ethanol, propylene glycol, glycerol, urea, ethylene glycol, butanol, pentanol, hexanol, arabinitol, tert-butanol, isopropanol, polyethylene glycol, benzyl alcohol, phenylethanol, phenylpropanol, and menthol; the acids are selected from at least one of citral, benzenesulfonic acid, geranilic acid, sorbic acid, citric acid, malic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, ascorbic acid, benzoic acid, salicylic acid, oleic acid, palmitic acid, butyric acid, acetic acid, glycolic acid, malonic acid, caprylic acid, capric acid, linoleic acid, linolenic acid, stearic acid, adipic acid, lauric acid, myristic acid, amino acids, levulinic acid, gluconic acid, and pyruvic acid.

[0021] In one embodiment of the present invention, the hydrogen bond acceptor or hydrogen bond donor is selected from at least one of alcohols, sugars, acids, and water. In another embodiment of the present invention, the hydrogen bond acceptor or hydrogen bond donor is selected from one or more of the following: fructose, glucose, ethylene glycol, glycerol, sorbitol, amino acids, cyclodextrin, lactic acid, malic acid, geraniic acid, sorbic acid, citric acid, tartaric acid, malonic acid, succinic acid, and water.

[0022] In one embodiment of the present invention, the eutectic solvent is selected from nonionic eutectic solvents, such as sugar eutectic solvents. Specifically, it includes, for example, a ternary system composed of sugars, alcohols, and water. Preferably, the sugars are selected from fructose and glucose; the alcohols are selected from ethylene glycol, glycerol, propylene glycol, and sorbitol. Another example is a ternary system composed of sugars, acids, and water. Preferably, the sugars are selected from fructose and glucose; the acids are selected from lactic acid, malonic acid, malic acid, tartaric acid, and citric acid.

[0023] In one embodiment of the present invention, the stoichiometric ratio of the ternary system (sugars, alcohols, and water, or sugars, acids, and water) is (1-30):(1-30):(1-15), for example, 30:1:1, 20:1:1, 1:20:1, 1:30:1, 1:1:5, 1:1:10; preferably, the stoichiometric ratio is (1-10):(1-10):(1-10), for example, 10:1:1, 5:1:1, 1:5:1, 1:10:1, 1:1:5, 1:1:10.

[0024] In one embodiment of the present invention, the ionic liquid is selected from choline-based ionic liquids, such as a mixture of choline hydroxide (Ch) and acids; preferably, it is an ionic liquid composed of choline hydroxide (Ch) and oleic acid, linoleic acid, phenylalanine (Phe), geraniic acid (Ger), citric acid, malonic acid (Mal), sorbic acid (Sorb), succinic acid (Suc), or malic acid (Mala). Specifically, it includes, but is not limited to, geraniol choline, malate choline, citrate choline, malonic acid choline, sorbate choline, oleic acid choline, and linoleic acid choline.

[0025] In a second aspect, the present invention provides a method for preparing a nucleic acid delivery vector, comprising mixing lipid molecules and a eutectic solvent to obtain a nucleic acid delivery vector.

[0026] In one embodiment of the present invention, the preparation method includes: (1) dissolving lipid molecules in a solvent to obtain a lipid molecule solution; (2) adding the lipid molecule solution to a eutectic solvent, mixing, and then removing the solvent to obtain a nucleic acid delivery vector.

[0027] In one embodiment of the present invention, the solvent in step (1) is an organic solvent; preferably at least one of methanol, ethanol, hexane, petroleum ether, acetone, chloroform, tetrahydrofuran, isopropanol, and acetonitrile.

[0028] In this invention, the purpose of adding a solvent is twofold: firstly, it effectively dissolves lipid molecules and the eutectic solvent, ensuring thorough and uniform mixing of lipid molecules within the eutectic solvent system; secondly, it allows for the quantitative determination of the stoichiometric ratio between different lipid molecules, facilitating practical operation. Furthermore, organic solvents can negatively impact the overall composition and exacerbate the instability of nucleic acid drugs; therefore, organic solvents must be removed.

[0029] In one embodiment of the present invention, the solvent removal method in step (2) is vacuum drying; preferably, the drying temperature is 20-100°C; the drying time is 1-48h; and the vacuum degree is 0-900mbar.

[0030] In one embodiment of the present invention, the mixing method in step (2) is ultrasound, vortex oscillation and manual blowing, preferably vortex oscillation; more preferably, the frequency of vortex oscillation is 100 to 4000 rpm and the time of vortex oscillation is 1s to 10min.

[0031] In this invention, the purpose of vortex oscillation is to bring the entire system into a uniformly dispersed state.

[0032] In a third aspect, the present invention provides a lipid nanoparticle comprising the above-mentioned nucleic acid delivery carrier and nucleic acid drug.

[0033] In one embodiment of the present invention, the nucleic acid drug is selected from one or more of the following: antisense nucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, antibody-nucleic acid conjugate (ARC), DNA drug (e.g., plasmid DNA), and CRISPR / CAS9 drug.

[0034] In one embodiment of the present invention, the mass ratio of lipid molecules to nucleic acid drugs is (1-1000):1; preferably, the mass ratio is (1-100):1.

[0035] In one embodiment of the present invention, the lipid nanoparticles further include a diluent or an excipient.

[0036] In one embodiment of the present invention, the diluent or excipient includes, but is not limited to, deionized water, ultrapure water, physiological saline, and buffers of different pH (phosphate buffer, Tris buffer, borate buffer, succinate buffer, HEPES buffer, citrate buffer or histidine buffer); preferably, a buffer with a pH of 3 to 7.

[0037] In one embodiment of the present invention, the lipid nanoparticles have a particle size < 500 nm and a PDI (polymer dispersibility index) < 0.5; preferably a particle size < 300 nm and a PDI < 0.3; more preferably a particle size < 200 nm and a PDI < 0.2.

[0038] In one embodiment of the present invention, the encapsulation efficiency of the lipid nanoparticles is ≥60%; preferably ≥90%; more preferably ≥95%; and most preferably ≥98%.

[0039] In a fourth aspect, the present invention provides a method for preparing lipid nanoparticles, comprising: adding a nucleic acid drug to the above-mentioned nucleic acid delivery carrier to obtain a composition; adding a diluent or excipient to the composition, mixing, and filtering to obtain the final product (i.e., a co-preparation method);

[0040] Alternatively, nucleic acid drugs can be added to diluents or excipients, then added to nucleic acid delivery carriers, mixed, and filtered to obtain the final product (i.e., the separate preparation method).

[0041] In one embodiment of the present invention, the mixing method includes, but is not limited to, water bath ultrasound, vortex oscillation, and manual blowing.

[0042] In one embodiment of the present invention, the power of the water bath ultrasound is 10-200W, the frequency is 30-150Hz, and the duration is 1s-1h; preferably, the power of the water bath ultrasound is 30-100W, the frequency is 30-70Hz, and the duration is 1-30min.

[0043] In one embodiment of the present invention, the frequency of the vortex oscillation is 100-5000 rpm and the time is 1 s-1 h; preferably, the frequency of the vortex oscillation is 1000-3000 rpm and the time is 1-30 min.

[0044] In one embodiment of the present invention, the number of times the manual blowing is 1 to 100 times / min and the time is 0.5 to 10 min; preferably, the number of times the manual blowing is 10 to 50 times / min and the time is 1 to 3 min.

[0045] In one embodiment of the present invention, the filtration is a conventional operation in the preparation method of lipid nanoparticles in the art, the purpose of which is to remove bacteria, solid particles, and lipid nanoparticles with excessively large particle size; preferably, it is a microporous filter membrane with a diameter of 0.22 to 0.45 μm.

[0046] In this invention, nucleic acid drugs have good solubility in water and eutectic solvents, so no additional solvent is needed to assist in the dissolution of nucleic acids into the eutectic solvent system. In addition, since the structure and physicochemical properties of nucleic acids are unstable, organic solvents and vacuum drying during the preparation of nucleic acid delivery carriers can affect the structure and physicochemical properties of nucleic acids. Therefore, nucleic acid drugs are added after the preparation of nucleic acid delivery carriers is completed.

[0047] In one embodiment of the present invention, the volume-to-mass ratio of the diluent or excipient to the composition is (1-300):1 (μL / mg); preferably (1-50):1.

[0048] In one embodiment of the present invention, the method for preparing lipid nanoparticles further includes the steps of replacing the buffer medium and concentrating the obtained lipid nanoparticles.

[0049] In one embodiment of the present invention, the replacement of the buffer medium is a conventional operation in the preparation method of lipid nanoparticles in the art. The purpose is to replace the acidic buffer solution of the lipid nanoparticles with a neutral buffer solution and concentrate the lipid nanoparticles. This is because ionizable lipids (e.g., SM-102, ALC-0315) will protonate under acidic conditions, which will cause cytotoxicity of the lipid nanoparticles and affect the delivery performance of the formulation. Preferably, the buffer is replaced by ultrafiltration or dialysis, and more preferably by ultrafiltration tubes with a capacity of 30-300 kDa.

[0050] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the above-described nucleic acid delivery carrier, lipid nanoparticles, pharmaceutically acceptable excipients and / or other pharmaceutically active molecules.

[0051] In one embodiment of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: sustained-release agents (e.g., chitosan), pH adjusters (e.g., disodium hydrogen phosphate, citric acid and its salts), surfactants (e.g., polysorbate 80, poloxamer), osmotic pressure maintainers (e.g., sodium chloride, mannitol), adjuvants (e.g., aluminum salts), preservatives (e.g., sodium benzoate, benzalkonium chloride), absorption delay agents (e.g., aluminum hydroxide), half-life extenders (e.g., polyethylene glycol), stabilizers (e.g., sucrose), adsorbents (e.g., activated carbon, silica, magnesium oxide), antimicrobial agents (e.g., parabens, phenoxyethanol, sodium benzoate), binders (e.g., hydroxypropyl methylcellulose), solubilizers (e.g., cyclodextrin), humectants (e.g., hyaluronic acid), and thickeners (e.g., carbomer, xanthan gum).

[0052] In one embodiment of the present invention, the other pharmaceutically active molecules include, but are not limited to, one or more of the following: antibodies, protein molecules, enzyme molecules, polypeptide molecules, small molecule compounds, such as insulin, glucagon, semaglutide, paclitaxel, doxorubicin, irinotecan, docetaxel, aspirin, nitroglycerin, warfarin, propranolol hydrochloride, felodipine, vitamins, and metronidazole.

[0053] In one embodiment of the present invention, the lipid nanoparticles and pharmaceutical composition can be any dosage form known in the medical and pharmaceutical fields. For example, the dosage form can be capsules, pills, solutions, emulsions, injections, lyophilized powders, gels, ointments, sprays, dispersions, suspensions, tablets, granules, or suppositories.

[0054] In one embodiment of the present invention, the administration route of the lipid nanoparticles and pharmaceutical composition includes, but is not limited to, gastrointestinal administration (e.g., oral administration) or non-gastrointestinal administration (e.g., skin administration, intravenous injection, local injection, sublingual administration, ocular administration, vaginal administration, and nasal administration).

[0055] In a sixth aspect, the present invention provides the use of the above-described nucleic acid delivery vector in the preparation of at least one of the following reagents:

[0056] 1) Loading, storing, and transporting drugs;

[0057] 2) In vivo drug delivery;

[0058] 3) Improve drug stability;

[0059] 4) Transfection reagent;

[0060] The drugs mentioned include nucleic acid drugs, lipid molecules, and other pharmaceutically active molecules.

[0061] In a seventh aspect, the present invention provides the use of the above-mentioned lipid nanoparticles and pharmaceutical compositions in the preparation of cosmetics, health products, beauty products, and care products; or in the preparation of drugs for treating diabetes, hypoglycemia, cancer, analgesia, inflammation, fever, angina pectoris, thrombotic diseases, hypertension, parasitic infections, antiviral drugs, and hereditary diseases.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] 1) This invention provides a eutectic solvent that can be used as a medium for loading, storing and transporting drugs (such as nucleic acid drugs) and lipid molecules;

[0064] 2) The nucleic acid delivery vector described in this invention can significantly improve the stability of drugs (such as nucleic acid drugs);

[0065] 3) The nucleic acid delivery vector described in this invention can improve drug delivery efficiency and cellular uptake rate, and achieve effective endosome escape, thereby improving the therapeutic effect of drugs (such as nucleic acid drugs).

[0066] 4) The nucleic acid delivery vector described in this invention is applicable to a variety of drug types and can encapsulate and transport various types of drugs, including small molecule drugs, macromolecule drugs (such as proteins, nucleic acids, peptides, antibodies, enzymes, etc.), hydrophilic and hydrophobic drugs, and even complex drugs. It can be used in a variety of therapeutic fields, such as cancer, infectious diseases, gene therapy, etc., providing more flexibility and choices for drug design and adapting to different treatment needs.

[0067] 5) This invention provides two methods for preparing ready-to-use lipid nanoparticles: a co-preparation method and a separate preparation method; wherein the separate preparation method can be applied to transfection reagents (e.g., commercially available transfection reagent Lipofectamine 2000).

[0068] 6) This invention provides a method for preparing lipid nanoparticles, which can significantly reduce the production cost of lipid nanoparticles (LNPs), expand the preparation scale, and facilitate industrial-scale production; at the same time, it can solve the problems of structural instability and short shelf life of lipid nanoparticles (LNPs) during storage; and different preparation methods can be applied to different application scenarios.

[0069] 7) This invention provides a method for preparing lipid nanoparticles, which can effectively control the size of lipid nanoparticles, making the lipid nanoparticle (LNP) particle size distribution narrower, the uniformity better, and the encapsulation efficiency higher, thereby improving the efficiency and targeting of the drug delivery system.

[0070] 8) Compared with traditional complex nanoparticle preparation processes, the lipid nanoparticle preparation method provided by this invention only requires simple mixing equipment or basic experimental equipment, without the need for expensive high-precision instruments, making it more suitable for large-scale industrial production.

[0071] 9) The preparation process of the lipid nanoparticles of the present invention requires fewer operation steps, shortens the operation time, and improves production efficiency.

[0072] 10) The present invention has significant advantages for clinical use, and the compositions described herein can be widely used in different fields (e.g., pharmacy, medicine, health products). Attached Figure Description

[0073] Figure 1: Infrared spectra of the eutectic solvent and raw materials fructose and glycerol in the [Fru][Gly][Water] ternary system prepared in this invention.

[0074] Figure 2: Physicochemical characterization of different ready-to-use lipid nanoparticle preparation methods. In the figure, A represents the particle size distribution of lipid nanoparticles with different nucleic acids; B represents the encapsulation efficiency of lipid nanoparticles with different nucleic acids.

[0075] Figure 3: Transfection of ready-to-use lipid nanoparticles expressing luciferase nucleic acid in vitro. A shows the transfection of different doses of pDNA in DC 2.4 and HEK 293 cells; B shows the transfection of different doses of mRNA in DC 2.4 and HEK 293 cells.

[0076] Figures 4-9: In vitro transfection of ready-to-use lipid nanoparticles prepared in Examples 5-10, in order; a: luciferase expression of ready-to-use lipid nanoparticles in DC 2.4 cells; b: luciferase expression of ready-to-use lipid nanoparticles in HEK 293 cells.

[0077] Figure 10: Flow cytometry analysis of the uptake of ready-to-use lipid nanoparticles by cells. A represents the uptake of DiD-lipid nanoparticles in DC2.4 and HEK293 cells; B represents the uptake of siRNA in DC2.4 and HEK293 cells.

[0078] Figure 11: Observation of endosome escape from ready-to-use lipid nanoparticles using laser confocal microscopy. In the figure, A shows the co-localization results of DiD-lipid nanoparticles in lysosomes of DC 2.4 and HEK 293 cells; B shows the co-localization results of siRNA in lysosomes of DC 2.4 and HEK 293 cells.

[0079] Figure 12: In vivo distribution of ready-to-use lipid nanoparticles and expression of firefly luciferase. A shows the in vivo distribution of ready-to-use lipid nanoparticles; B shows the in vivo expression of firefly luciferase. Detailed Implementation

[0080] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0081] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0082] Experimental instruments: The models and sources of the instruments used in the following specific embodiments are as follows:

[0083] Experimental cells: HEK-293 and DC2.4 (Shanghai Cell Bank, Chinese Academy of Sciences)

[0084] Laboratory animals: Male BALB / c mice, 4-6 weeks old, approximately 20g. Purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd. All animals were kept in an SPF-grade environment for one week for acclimatization. Animal care and use were approved by the Laboratory Animal Center. All experimental procedures were conducted in accordance with the regulations of the Laboratory Animal Welfare and Ethics Committee of the Institute of Animal Sciences.

[0085] Abbreviation list

[0086] Example 1: Preparation of a eutectic solvent for a ternary system of fructose-glycerol-water ([Fru][Gly][Water])

[0087] 1. Preparation method

[0088] 18.16 g (0.1 mol) of fructose, 9.29 g (0.1 mol) of glycerol, and 9 g (0.5 mol) of DEPC water were accurately weighed into a 50 mL round-bottom flask, placed on a magnetic stirrer, and a rotor of appropriate size was added. The temperature was adjusted to about 50 °C, the stirring speed was 3000 rpm, and after stirring for about 5 hours, a ternary eutectic solvent [Fru][Gly][Water] was obtained. It was clear and transparent in appearance, without obvious solid crystals, and was in a liquid state at room temperature. At the same time, after being placed at room temperature for at least 48 hours, the appearance of [Fru][Gly][Water] did not change, indicating that fructose, glycerol, and water formed a ternary eutectic solvent.

[0089] 2. FI-IR characterization

[0090] The FI-IR spectra of fructose, glycerol, and [Fru][Gly][Water] were studied separately. Comparison of the infrared spectra of these three substances indicates the presence of hydrogen bonding interactions in the eutectic solvent. As shown in Figure 1, approximately 3388 cm⁻¹ -1 and 3379cm -1 The bands at 3352 cm⁻¹ correspond to the hydroxyl groups of the single components fructose and glycerol, respectively. For the ternary system [Fru][Gly][Water], the band at 3352 cm⁻¹ corresponds to the hydroxyl groups of the single components fructose and glycerol, respectively. -1 The observation of broad and diffuse bands of hydroxyl groups indicates that abundant hydrogen bonds are formed in [Fru][Gly][Water].

[0091] Example 2: Preparation of ready-to-use lipid nanoparticles

[0092] 1. Prescription

[0093] Lipid nanoparticles were prepared according to the formulation shown in Table 1.

[0094] Table 1. Lipid Nanoparticle Formulation

[0095] 2. Preparation of lipid nanoparticles

[0096] 2.1 Co-preparation method

[0097] (1) Preparation of lipid solutions: Accurately weigh a certain mass of SM-102, DOPE, DC-CHO and PEG2000-DMG, dissolve them fully in anhydrous ethanol, and prepare the stock solutions of different lipids in the above prescription table 1. According to the chemical molar ratio of lipids, use a pipette to draw the corresponding volume of lipid stock solution in the above prescription table 1 into a 2mL EP tube for later use.

[0098] (2) Accurately weigh 68 mg of the ternary system eutectic solvent prepared in Example 1 into a 2 mL EP tube using an electronic balance. Eutectic solvent: lipid = 100:1 (mass ratio);

[0099] (3) Mix (1) lipid solution and (2) ternary eutectic solvent evenly by vortexing;

[0100] (4) Place the well-mixed solution from (3) into a vacuum drying oven at 25°C and 50 mbar for 8 hours; the resulting lipid-eutectic solvent can transport and store lipid molecules and can be used in transfection reagents (e.g., commercially available transfection reagent Lipofectamine 2000) to effectively transfect pDNA.

[0101] (5) Accurately weigh 33.75 μg of pDNA (plasmid DNA), add it to the lipid-eutectic solvent composition, vortex at 3000 rpm for 30 s to mix evenly, and obtain the composition; lipid:pDNA = 20:1 (mass ratio); this composition can transport and store pDNA and lipid molecules; when using, hydrate the composition to form complete lipid nanoparticles;

[0102] (6) Hydration composition: Add 1 mL of PBS buffer to the composition and sonicate in a water bath at 37 Hz and 100 W for 3 min; PBS: composition = 15:1 (v / m, μL / mg). Visual inspection of the resulting solution showed no obvious lipid precipitation.

[0103] (7) The hydrated composition was filtered using a 0.22 μm microporous membrane to remove lipid nanoparticles with excessive particle size and to sterilize it;

[0104] (8) Using a 100kDa ultrafiltration tube, centrifuge at 1500g for 15min to concentrate 0.3mL of lipid nanoparticles.

[0105] 2.2 Separate preparation method

[0106] The difference from the co-preparation method is that step (5) is omitted. In step (6), when hydrating the composition, the pDNA is first completely dissolved with PBS buffer, and then 1 mL of PBS buffer containing 33.75 μg of completely dissolved pDNA is added to the composition.

[0107] Example 3: Preparation of ready-to-use lipid nanoparticles

[0108] 1. Prescription

[0109] Prepare lipid nanoparticles according to the formulation shown in Table 2.

[0110] Table 2 Lipid Nanoparticle Formulation

[0111] 2. Preparation of lipid nanoparticles

[0112] 2.1 Co-preparation method

[0113] (1) Preparation of lipid solutions: Accurately weigh a certain mass of SM-102, DSPC, CHO-HP, and PEG2000-DMG, dissolve them fully in anhydrous ethanol, and prepare stock solutions of different lipids in the above prescription table 2. According to the chemical molar ratio of lipids, use a pipette to draw the corresponding volume of lipid stock solution in the above prescription table 2 into a 2mL EP tube for later use.

[0114] (2) Accurately weigh 62 mg of the ternary eutectic solvent prepared in Example 1 into a 2 mL EP tube using an electronic balance, i.e., eutectic solvent: lipid = 100:1 (mass ratio);

[0115] (3) Mix (1) lipid solution and (2) ternary eutectic solvent evenly by vortexing;

[0116] (4) Place the well-mixed solution from (3) into a vacuum drying oven at 25°C and 50 mbar for 8 hours; the resulting lipid-eutectic solvent can transport and store lipid molecules and can be used in transfection reagents (e.g., commercially available transfection reagent Lipofectamine 2000) to effectively transfect mRNA.

[0117] (5) Accurately weigh 31 μg of mRNA and add it to the lipid-eutectic solvent composition. Vortex at 3000 rpm for 30 seconds to mix evenly and obtain the composition. Lipid:mRNA = 20:1 (mass ratio). This composition can transport and store mRNA and lipid molecules. When used clinically or in practice, the composition can be hydrated to form complete lipid nanoparticles.

[0118] (6) Preparation of citrate buffer (pH=4): 5 mM sodium citrate, 5 mM citric acid and 150 mM sodium chloride were diluted with DEPC water and then filtered through a 0.22 μm microporous membrane for sterilization.

[0119] (7) Hydration of the composition: Add 1 mL of citrate buffer to the composition and vortex at 3000 rpm for 1 min; that is, citrate buffer: composition = 15:1 (v / m, μL / mg); the resulting solution showed no obvious lipid precipitation upon visual inspection.

[0120] (8) The hydrated composition was filtered using a 0.22 μm microporous membrane to remove lipid nanoparticles with excessive particle size and to sterilize it;

[0121] (9) Dilute with 2 mL of PBS buffer;

[0122] (10) Using a 100kDa ultrafiltration tube, centrifuge at 1500g for 15min / time, for a total of 3 ultrafiltration centrifugations. Each ultrafiltration centrifugation requires the addition of 2mL of PBS buffer. The purpose is to replace the acidic buffer of the lipid nanoparticles with a neutral buffer and finally concentrate the lipid nanoparticle solution to obtain 0.3mL of the final preparation.

[0123] 2.2 Separate preparation method

[0124] The difference from the co-preparation method is that step (5) is omitted. In step (7), when hydrating the composition, the mRNA is first completely dissolved with citrate buffer, and then 1 mL of citrate buffer that completely dissolves the mRNA (31 μg) is added to the composition.

[0125] Example 4: Preparation of ready-to-use lipid nanoparticles

[0126] 1. Prescription

[0127] Lipid nanoparticles were prepared according to the formulation shown in Table 3.

[0128] Table 3 Lipid Nanoparticle Formulation

[0129] 2. Preparation of lipid nanoparticles

[0130] 2.1 Co-preparation method

[0131] (1) Preparation of lipid solution: Accurately weigh a certain mass of ALC-0315, DSPC, CHO-HP and ALC-0159, dissolve them fully in anhydrous ethanol, and prepare the stock solutions of different lipids in the above prescription table 3. According to the chemical molar ratio of lipids, use a pipette to take 2 mL of the corresponding volume of lipid stock solution in the above prescription table 3 into an EP tube for later use.

[0132] (2) Accurately weigh 62 mg of the eutectic solvent of the ternary system prepared in Example 1 into a 2 mL EP tube using an electronic balance. Eutectic solvent: lipid = 100:1 (mass ratio);

[0133] (3) Mix (1) lipid solution and (2) ternary eutectic solvent evenly by vortexing;

[0134] (4) Place the well-mixed solution from (3) into a vacuum drying oven at 25°C and 50 mbar for 8 hours; the resulting lipid-eutectic solvent can transport and store lipid molecules and can be used in transfection reagents (e.g., commercially available transfection reagent Lipofectamine 2000) to effectively transfect siRNA.

[0135] (5) Accurately weigh 32.44 μg of siRNA and add it to the lipid-eutectic solvent composition. Vortex at 3000 rpm for 30 s to mix evenly and obtain the composition. Lipid:siRNA = 20:1 (mass ratio). This composition can transport and store siRNA and lipid molecules. When using, the composition is hydrated to form complete lipid nanoparticles.

[0136] (6) Preparation of citrate buffer (pH=4): 5 mM sodium citrate, 5 mM citric acid and 150 mM sodium chloride were diluted with DEPC water and then filtered through a 0.22 μm microporous membrane for sterilization.

[0137] (7) Hydration of the composition: Add 1 mL of citrate buffer to the composition and manually pipette the mixture 30 times / min for 1 min; citrate buffer: composition = 15:1 (v / m, μL / mg); the resulting solution showed no obvious lipid precipitation upon visual inspection.

[0138] (8) The hydrated composition was filtered using a 0.22 μm microporous membrane to remove lipid nanoparticles with excessive particle size and to sterilize it;

[0139] (9) Dilute with 2 mL of PBS buffer;

[0140] (10) Using a 100kDa ultrafiltration tube, centrifuge at 1500g for 15min / time, for a total of 3 ultrafiltration centrifugations. Each ultrafiltration centrifugation requires the addition of 2mL of PBS buffer. The purpose is to replace the acidic buffer of the lipid nanoparticles with a neutral buffer and finally concentrate the lipid nanoparticle solution to obtain 0.3mL of the final preparation.

[0141] 2.2 Separate preparation method

[0142] The difference from the co-preparation method is that step (5) is omitted. In step (7), when hydrating the composition, the siRNA is first completely dissolved with citrate buffer, and then 1 mL of citrate buffer (32.44 μg) of completely dissolved siRNA is added to the composition.

[0143] Example 5: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose lactic acid aqueous solution)

[0144] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose lactic acid water.

[0145] Example 6: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose malonic acid aqueous solution)

[0146] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose malonic acid water.

[0147] Example 7: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose-malic acid aqueous solution)

[0148] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose-malic acid water.

[0149] Example 8: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose tartaric acid aqueous solution)

[0150] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose tartaric acid water.

[0151] Example 9: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose citric acid aqueous solution)

[0152] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose-citric acid solution.

[0153] Example 10: Preparation of ready-to-use lipid nanoparticles based on a eutectic solvent (fructose propylene glycol water)

[0154] Similar to the co-preparation method in Example 2, the difference is that the eutectic solvent used in the ternary system is fructose propylene glycol water.

[0155] Example 11 Preparation of self-assembled lipid nanoparticles of siRNA-DOTAP-ILs (siRNA-DILs) based on ionic liquid (geraniol choline)

[0156] 1. Prepare lipid self-assembled nanoparticles according to the formulation in Table 4 below.

[0157] Table 4. Formulation of lipid self-assembled nanoparticles

[0158] 2. Preparation of lipid self-assembled nanoparticles

[0159] (1) Preparation of lipid self-assembled nanoparticle solution: Accurately weigh a certain mass of DOTAP and dissolve it fully in methanol. Add an appropriate amount of DEPC water to 32 μg of siRNA to prepare the 5 mM stock solution in Table 1 above. According to the content ratio, use a pipette to draw the corresponding volume of stock solution in Table 1 above into a 2 mL EP tube for later use.

[0160] (2) Accurately weigh 8 mg of geranium ionic liquid ([Cho][Ger]) into a 2 mL EP tube using an electronic balance. Add different amounts of DOTAP from the prescription and vacuum dry for 2 hours to remove methanol, obtaining DOTAP-ILs droplets. Accurately weigh 8 mg of geranium ionic liquid ([Cho][Ger]) into a 2 mL EP tube using an electronic balance. Add the prescribed amount of siRNA at a ratio of siRNA:DOTAP = 1:500 (w / w), freeze at -80°C for four hours, vacuum dry for 2 hours to remove methanol, and obtain siRNA-ILs droplets.

[0161] (3) Mix DOTAP-ILs droplets and siRNA-DILs droplets evenly by vortexing to obtain siRNA-DILs droplets.

[0162] (4) Add 1 mL of deionized water to the siRNA-DILs droplet that was mixed evenly in (3), vortex for 30 seconds to hydrate, and visual inspection of the resulting solution showed no obvious lipid precipitation.

[0163] Example 12 Preparation of self-assembled lipid nanoparticles based on ionic liquid (malate choline) siRNA-DOTAP-ILs (siRNA-DILs)

[0164] Similar to Example 11, except that the ionic liquid used is malic choline ([Cho][Malic]).

[0165] Example 13 Preparation of self-assembled lipid nanoparticles based on ionic liquid (citric acid choline) siRNA-DOTAP-ILs (siRNA-DILs)

[0166] Similar to Example 11, except that the ionic liquid used is choline citrate ([Cho][Cit]).

[0167] Example 14: Preparation of self-assembled lipid nanoparticles based on ionic liquid (malonidocholine) siRNA-DOTAP-ILs (siRNA-DILs)

[0168] Similar to Example 11, except that the ionic liquid used is a malonic acid ionic liquid ([Cho][Malo]).

[0169] Example 15: Preparation of self-assembled lipid nanoparticles based on ionic liquid (sorbate choline) siRNA-DOTAP-ILs (siRNA-DILs)

[0170] Similar to Example 11, except that the ionic liquid used is a sorbic acid ionic liquid ([Cho][Sorb]).

[0171] Example 16: Preparation of self-assembled lipid nanoparticles based on ionic liquid (oleic choline) siRNA-DOTAP-ILs (siRNA-DILs)

[0172] Similar to Example 11, except that the ionic liquid used is an oleic acid ionic liquid ([Cho][Ole]).

[0173] Example 17 Preparation of self-assembled lipid nanoparticles based on ionic liquid (linoleic acid choline) siRNA-DOTAP-ILs (siRNA-DILs)

[0174] Similar to Example 11, except that the ionic liquid used is a linoleic acid ionic liquid ([Cho][Lin]).

[0175] Example 18: Preparation of 1% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline).

[0176] 1. Prescription

[0177] Lipid self-assembled nanoparticles were prepared according to the formulations shown in Table 5.

[0178] Table 5. Formulation of lipid self-assembled nanoparticles

[0179] 2. Preparation method

[0180] (1) Preparation of lipid self-assembled nanoparticle solution: Accurately weigh a certain mass of DOTAP and dissolve it fully in methanol. Add an appropriate amount of DEPC water to 32 μg of siRNA to prepare the 5 mM stock solution in Table 1 above. According to the content ratio, use a pipette to draw the corresponding volume of stock solution in Table 1 above into a 2 mL EP tube for later use.

[0181] (2) Accurately weigh 8 mg of geranium ionic liquid ([Cho][Ger]) into a 2 mL EP tube using an electronic balance. Add 1.6 mg of DOTAP and 0.05 mg of PTX and vacuum dry for 2 hours to remove methanol, obtaining PTX-DOTAP-ILs droplets. Accurately weigh 8 mg of geranium ionic liquid ([Cho][Ger]) into a 2 mL EP tube using an electronic balance. Add 3.2 μg of siRNA and freeze at -80℃ for four hours. Vacuum dry for 2 hours to remove methanol, obtaining siRNA-ILs droplets.

[0182] (3) PTX-DOTAP-ILs droplets and siRNA-DILs droplets are vortexed and mixed evenly to obtain PTX-siRNA-DILs droplets.

[0183] (4) Add 1 mL of deionized water to the PTX-siRNA-DILs droplet that was mixed evenly in (3), vortex for 30 seconds to hydrate, and visual inspection of the resulting solution showed no obvious lipid precipitation.

[0184] Example 19: Preparation of 2.5% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline).

[0185] Similar to Example 18, except that the PTX was 0.125 mg.

[0186] Example 20: Preparation of 5% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline).

[0187] Similar to Example 18, except that the PTX was 0.25 mg.

[0188] Example 21 Preparation of 10% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline)

[0189] Similar to Example 18, except that the PTX was 0.5 mg.

[0190] Example 22 Preparation of 25% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline)

[0191] Similar to Example 18, except that the PTX was 1.25 mg.

[0192] Example 23 Preparation of 100% PTX-siRNA-DOTAP-ILs (PTX-siRNA-DILs) self-assembled lipid nanoparticles based on ionic liquid (geraniol choline)

[0193] Similar to Example 18, except that the PTX is 5 mg.

[0194] Experimental Example 1: Physicochemical Properties of Ready-to-Use Lipid Nanoparticles

[0195] 1. Particle size and potential determination

[0196] Appropriate amounts of the finished products from Examples 2, 3, and 4 were diluted with water and subjected to dynamic light scattering measurements. The measurement wavelength λ = 633 nm, the measurement angle was 173°, and the temperature was 25°C. Particle size was expressed as intensity data. Simultaneously, the potential of the hydrated lipid nanoparticles was measured using a nanoparticle size analyzer.

[0197] The specific experimental results are shown in Table 6; and the particle size distribution diagram is shown in Figure 2.

[0198] Table 6. Particle size and potential of different embodiments

[0199] As shown in Table 6 and Figure 2(A) above, the ready-to-use lipid nanoparticles of this invention are suitable for different nucleic acids. The lipid nanoparticles obtained by hydration have a uniform particle size distribution, and the particle size distribution diagrams all show a single peak, all less than 200 nm, and the PDI is less than 0.2. The zeta potential in Example 2 is positive because DC-CHO is a cationic lipid, while Examples 3 and 4 are both CHO-HP, so their zeta potentials are negative in the neutral PBS environment, but the negative charge is not very strong.

[0200] 2. Determination of encapsulation efficiency

[0201] The encapsulation efficiency of RNA and pDNA was determined according to the instructions for use of the Ribogreen and Picogreen kits. The results are shown in Figure 2(B).

[0202] The results show that pDNA, mRNA, and siRNA all exhibited very high encapsulation efficiency, approaching 100%. This indicates that lipid nanoparticles can effectively load these nucleic acid substances into the carrier and have good loading capacity for different types of nucleic acid substances.

[0203] The physicochemical properties of the ready-to-use lipid nanoparticles from Examples 5-11 were tested using the same method. The results are shown in Tables 7-15. It can be seen that these nanoparticles have a uniform particle size distribution, good charge stability, and exhibit high encapsulation efficiency.

[0204] Table 7 Physicochemical properties of ready-to-use lipid nanoparticles

[0205] Table 8 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquid (geraniol choline)

[0206] Table 9 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquids (malate choline)

[0207] Table 10 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquids (citric acid choline).

[0208] Table 11 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquid (malonidocholine).

[0209] Table 12 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquid (sorbate choline).

[0210] Table 13 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquids (oleic choline).

[0211] Table 14 Physicochemical properties of siRNA-DILs self-assembled lipid nanoparticles based on ionic liquid (linoleic acid choline).

[0212] Table 15 Physicochemical properties of 1-10% PTX-siRNA-DILs self-assembled lipid nanoparticles based on ionic liquid (geraniol choline).

[0213] Experimental Example 2: In vitro transfection of ready-to-use lipid nanoparticles

[0214] HEK-293 cells or DC 2.4 cells in logarithmic growth phase were digested with trypsin for a period of time, and then the trypsin was neutralized with complete culture medium to prepare a cell solution of a certain concentration. Cells were counted using a fully automated cell counter, and then the cells were counted at a rate of 1.5 x 10⁻⁶. 5Cells were seeded per well in a 24-well plate, with 500 μL of complete culture medium per well. The plates were incubated for 24 h in a cell culture incubator (37°C, 5% CO2). When the cell density in each well reached 70%, ready-to-use lipid nanoparticles prepared in Examples 2 and 3 (diluted with the corresponding complete culture medium) with different nucleic acid amounts (0.2 μg / mL, 0.5 μg / mL, 1 μg / mL) were added. Three dose groups were set up for each sample, and three replicates were set up for each dose. The plates were incubated for another 48 h in a cell culture incubator (24 h for mRNA incubation). Then, following the instructions of the Firefly Luciferase Reporter Gene Assay Kit (Beyotime) and the Bradford Protein Assay Kit (Detergent Compatible) (Beyotime), the bioluminescence intensity (RLU, Relative Luminescence Units) and total protein content in the cell lysate were measured and compared quantitatively using RLU / mg.

[0215] The specific experimental results are shown in Figure 3. Figure A shows the transfection of luciferase pDNA lipid nanoparticles (Example 2) in DC2.4 and HEK 293 cells, and Figure B shows the transfection of luciferase mRNA lipid nanoparticles (Example 3) in DC2.4 and HEK 293 cells. The data in Figure 3 show that the pDNA / mRNA lipid nanoparticles exhibit a dose-response effect in in vitro transfection.

[0216] The in vitro transfection of the ready-to-use lipid nanoparticles prepared in Examples 5-10 was detected using the same method, as shown in Figures 4-9. a) shows the luciferase expression of the ready-to-use lipid nanoparticles in DC 2.4 cells. b) shows the luciferase expression of the ready-to-use lipid nanoparticles in HEK 293 cells.

[0217] Experimental Example 3: Study on cellular uptake and endosome escape of ready-to-use lipid nanoparticles

[0218] Lipid nanoparticle transfection mainly involves the following processes: uptake into cells, escape from endosomes, and entry into the cytoplasm or nucleus to express corresponding proteins or regulate corresponding genes or proteins. Therefore, this invention also investigates the uptake and endosome escape of ready-to-use lipid nanoparticles.

[0219] To better explain the uptake and endosome escape of lipid nanoparticles, in this experiment, DiD probes were added to the lipid mixture at a ratio of 0.5% mol of all lipid components to label the lipid nanoparticles (Example 2); or Cy5-siRNA was used instead of ordinary non-fluorescent siRNA to label the nucleic acids inside the lipid nanoparticles (Example 4).

[0220] 1. Cell uptake assay

[0221] HEK-293 cells or DC 2.4 cells in the logarithmic growth phase were digested with trypsin for a period of time, then the trypsin was neutralized with complete culture medium to prepare a cell solution of a certain concentration. Cells were counted using an automated cell counter, and then the cells were counted at a rate of 5 x 10⁻⁶. 5 Cells were seeded per well in a 12-well plate, with 1 mL of complete culture medium per well. The plates were incubated for 24 h at 37°C with 5% CO2. When the cell density in each well reached 70% or higher, ready-to-use lipid nanoparticles were added, and the final nucleic acid concentration was 500 ng / mL per well, with three replicates. After another 6 h of incubation, the cells were digested with trypsin, washed twice with pre-cooled 1xPBS, and then analyzed by flow cytometry.

[0222] The results are shown in Figure 10. In A, lipid nanoparticles were labeled with DiD (Example 2), and in B, siRNA was labeled with Cy5 (Example 4). Flow cytometry analysis showed that the ready-to-use lipid nanoparticles of the present invention achieved an uptake rate of over 98% in DC 2.4 and HEK-293 cells. Furthermore, both lipid nanoparticles and nucleic acids demonstrated that the ready-to-use lipid nanoparticles of the present invention have high uptake efficiency at the cellular level.

[0223] 2. Internal escape test

[0224] HEK-293 cells or DC 2.4 cells in the logarithmic growth phase were digested with trypsin for a period of time, then the trypsin was neutralized with complete culture medium to prepare a cell solution of a certain concentration. Cells were counted using an automated cell counter, and then the cells were counted at a rate of 5 x 10⁻⁶. 5 Cells were seeded per confocal microsphere, with 1 mL of complete culture medium per well. Cells were incubated in a cell culture incubator (37°C, 5% CO2) for 24 h. When the cell density reached 70% or higher, ready-to-use lipid nanoparticles were added, bringing the final nucleic acid concentration to 500 ng / mL per well. After another 6 h of incubation, the culture medium was aspirated, and the cells were washed twice with pre-chilled 1×PBS. Then, lysosomal green fluorescent probes were added according to the manufacturer's instructions and incubated for 2 h. The cells were then washed twice with pre-chilled 1×PBS, and 500 μL of 4% paraformaldehyde was added, incubating for 15 min at room temperature. The cells were then washed twice with pre-chilled 1×PBS, and 500 μL of DAPI was added according to the manufacturer's instructions, incubating for 10 min at room temperature. The cells were then washed twice with pre-chilled 1×PBS. The cells were blotted dry as much as possible before analysis using laser confocal microscopy.

[0225] The results are shown in Figure 11. A represents the escape of DiD-labeled lipid nanoparticles (Example 2) from endosomes, and B represents the escape of Cy5-labeled siRNA (Example 4) from endosomes. Laser confocal microscopy analysis showed that the co-localization of lipid nanoparticles and nucleic acids in the lysosomes of DC 2.4 and HEK-293 was not very high.

[0226] As shown in Figure A, the signals of the red DiD-labeled lipid nanoparticles and the green LyoTracker-labeled endosomes / lysosomes do not completely overlap, indicating that the lipid nanoparticles partially escape from the endosomes. In Figure B, the red signal of the Cy5-labeled siRNA also separates from the green LyoTracker signal, indicating that the siRNA successfully escaped from the endosomes. This shows that both lipid nanoparticles and siRNA can escape from the endosomes. This indicates that the uptake of ready-to-use lipid nanoparticles mainly occurs via the endosome pathway. Furthermore, because lipid nanoparticles contain ionizable lipids, and the endosome environment is acidic, the ionizable lipids undergo protonation within the endosome, preventing nucleic acids from being phagocytosed by lysosomes and allowing them to reach the cytoplasm or nucleus.

[0227] Experimental Example 4: In vivo distribution and luciferase expression of ready-to-use lipid nanoparticles

[0228] To better illustrate the in vivo distribution of ready-to-use lipid nanoparticles in this validation example, a DiD probe (0.5% mol of all lipid components) was added to a lipid mixture solution, and Fluc-mRNA-ready-to-use lipid nanoparticles were prepared according to the protocol in Example 3. The day before the formal experiment, the hair on the abdomen and right leg of the mice was removed to avoid the influence of hair on the fluorescence values. Each mouse was injected with Fluc-mRNA-ready-to-use lipid nanoparticles into the right leg muscle at a dose of 0.25 g / kg. At different time points, the mice were intraperitoneally injected with D-fluorescein potassium salt at a dose of 150 mg / kg; then, the mice were anesthetized and placed on an imaging platform, with 2% isoflurane passing through the nasal cone. The in vivo distribution of the lipid nanoparticles was captured using IVIS Fluorescence mode with automatic exposure time, an imaging height of 1.5 cm, Ex = 640 nm, and Em = 680 nm. The in vivo expression of Fluc-mRNA was captured in the Bioluminescence mode of IVIS, with the excitation filter set to Block, the emission filter set to Open, the exposure time set to automatic, and the imaging height set to 1.5 cm.

[0229] The results are shown in Figure 12. Data in A shows that the ready-to-use lipid nanoparticles are mainly distributed at the injection site. Data in B shows that Fluc-mRNA expresses luciferase protein well in mice and also shows a certain amount of expression in the liver after a certain period of time, but the expression of luciferase protein is mainly at the injection site. This result verifies that the ready-to-use lipid nanoparticles described in this invention also have a certain transfection ability and nucleic acid delivery ability in vivo.

[0230] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nucleic acid delivery vector, characterized in that: The nucleic acid delivery vector comprises at least the following components: (a) an eutectic solvent; and (b) lipid molecules.

2. The nucleic acid delivery vector as described in claim 1, characterized in that: The mass ratio of the eutectic solvent to the lipid molecules is (0.25–5000):1, preferably (1–1000):1; Preferably, the content of the lipid molecules is 0.02% to 80% (mass percentage), more preferably 0.1% to 50%.

3. The nucleic acid delivery vector as described in claim 1, characterized in that: The lipid molecules are selected from one or more of the following: ionizable lipids, cationic lipids, zwitterionic lipids, accessory lipids, cholesterol and its derivatives, sterol lipids, fatty acid esters, mannosylated lipids, and PEGylated lipids. Preferably, the lipid molecule comprises ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids, wherein the molar ratio of the ionizable lipids, cofactor lipids, cholesterol and its derivatives, and PEGylated lipids is (20-100):(5-20):(20-50):(0.5-5); more preferably, the molar ratio is (30-60):(5-15):(35-45):(0.5-3). Preferably, the ionizable lipid is selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(dimethylamino)butyric acid (6,9,28,31-tetraen-19-yl)heptadecyl alcohol ester (DLin-MC3-DMA), and 1,2-dioleoyl-3-dimethylammonium propane (DODA). P), (2,3-dioleoyl-propyl)-trimethylammonium chloride phospholipid (DOTAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (DOBAQ), YSK05, 4-(((2,3-bis(oleoyloxy)prop-1-ammonium)benzoic acid (DOBAT), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (DOBAQ) Q), 3-((2,3-bis(oleoyloxy)propyl)(methyl)amino)propionic acid (DOPAT), N-(2-carboxypropyl)-N,N-dimethyl-2,3-bis-(oleoyloxy)-prop-1-ammonium (DOMPAQ), N-(carboxymethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)-prop-1-ammonium (DOAAQ), Alny-100, 3-(dimethylamino)propyl(12Z,15Z)-3-[(9Z, [12Z)-octadec-9,12-dien-1-yl]-tetradec-12,15-dien ester (DMAP-BLP) and derivatives of ionizable amino lipids, SM-102, ALC-0315, MC3, 8-[(2-hydroxyethyl)(6-oxo-6-decoxyhexyl)amino]octanoic acid (heptadecanoic acid 9-yl) ester or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ester; Preferably, the auxiliary lipid comprises phospholipids; more preferably, the phospholipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilaurylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), and 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC). One or more of the following: 1,2-diarachido-sn-glycerol-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyl oleyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylcholine, distearate phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleyl phosphatidylethanolamine (POPE), and lysophosphatidylethanolamine; Preferably, the cholesterol and its derivatives are selected from one or more of cholesterol, cholesterol esters, sterol hormones, sterol vitamins and phytosterols; Preferably, the PEG lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE), PEG-dilaurylglycerylamide, PEG-dimyristoylglycerylamide, PEG-dipalmitoylglycerylamide and PEG-distearylglycerylamide, PEG-cholesterol (1-[8′-(cholest-5-en-3[β]-oxy)formamido-3′,6′-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine N-[methoxy(polyethylene glycol)] The following are one or more of the following: [PEG2k-DMPE] or 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (PEG2k-DMG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearyl-sn-glycerol, methoxy polyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159).

4. The nucleic acid delivery vector as described in claim 1, characterized in that: The eutectic solvent is selected from at least one of ionic liquids, ionic eutectic solvents, and nonionic eutectic solvents; Preferably, the eutectic solvent is stable at room temperature, clear and transparent, and is a liquid substance; Preferably, the eutectic solvent comprises a binary, ternary, or multi-component system composed of hydrogen bond acceptors and hydrogen bond donors; Preferably, the hydrogen bond acceptor is selected from at least one of quaternary ammonium salts, amines, acids, alcohols, sugars, water, and urea; the hydrogen bond donor is selected from at least one of acids, alcohols, sugars, amines, and water; more preferably, the hydrogen bond acceptor or hydrogen bond donor is selected from at least one of alcohols, sugars, acids, and water; even more preferably, the hydrogen bond acceptor or hydrogen bond donor is selected from one or more of the following: fructose, glucose, ethylene glycol, glycerol, sorbitol, amino acids, cyclodextrin, lactic acid, malic acid, geranilic acid, sorbic acid, citric acid, tartaric acid, malonic acid, succinic acid, and water; Preferably, the eutectic solvent is selected from nonionic eutectic solvents, more preferably from sugar eutectic solvents, and even more preferably from ternary systems composed of sugars, alcohols, and water; or from ternary systems composed of sugars, acids, and water. Preferably, the sugars are selected from fructose and glucose; the acids are selected from lactic acid, malonic acid, malic acid, tartaric acid, and citric acid; and the alcohols are selected from ethylene glycol, glycerol, propylene glycol, and sorbitol. Preferably, the stoichiometric ratio of the ternary system is (1-30):(1-30):(1-15), more preferably, the stoichiometric ratio is (1-10):(1-10):(1-10); Preferably, the eutectic solvent is selected from ionic liquids, which are selected from choline-based ionic liquids, such as a mixture of choline hydroxide (Ch) and acids; more preferably, it is an ionic liquid composed of choline hydroxide (Ch) and oleic acid, linoleic acid, phenylalanine (Phe), geranic acid (Ger), citric acid, malonic acid (Mal), sorbic acid (Sorb), succinic acid (Suc), or malic acid (Mala).

5. A method for preparing the nucleic acid delivery vector according to any one of claims 1-4, characterized in that: This includes mixing lipid molecules with a eutectic solvent to obtain a nucleic acid delivery vector; Preferably, the preparation method includes: (1) dissolving lipid molecules in a solvent to obtain a lipid molecule solution; (2) adding the lipid molecule solution to a eutectic solvent, mixing, and then removing the solvent to obtain a nucleic acid delivery vector; Preferably, the solvent in step (1) is an organic solvent; preferably at least one of methanol, ethanol, hexane, petroleum ether, acetone, chloroform, tetrahydrofuran, isopropanol, and acetonitrile. Preferably, the solvent removal method in step (2) is vacuum drying; preferably, the drying temperature is 20-100℃; the drying time is 1-48h; and the vacuum degree is 0-900mbar. Preferably, the mixing method in step (2) is ultrasound, vortex oscillation, or manual blowing, with vortex oscillation being the most preferred; more preferably, the frequency of vortex oscillation is 100 to 4000 rpm, and the duration of vortex oscillation is 1 to 10 min.

6. A lipid nanoparticle, characterized in that: Includes the nucleic acid delivery vector and nucleic acid drug as described in any one of claims 1-4; Preferably, the nucleic acid drug is selected from one or more of the following: antisense nucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, antibody-nucleic acid conjugate (ARC), DNA drug and CRISPR / CAS9 drug; Preferably, the mass ratio of lipid molecules to nucleic acid drugs is (1-1000):1; more preferably, the mass ratio is (1-100):

1. Preferably, the lipid nanoparticles further include a diluent or excipient; Preferably, the diluent or excipient includes, but is not limited to, deionized water, ultrapure water, physiological saline, and buffers of different pH values; preferably, a buffer solution with a pH of 3 to 7. Preferably, the lipid nanoparticles have a particle size < 500 nm and a PDI (polymer dispersibility index) < 0.5; more preferably, a particle size < 300 nm and a PDI < 0.3; and even more preferably, a particle size < 200 nm and a PDI < 0.

2. Preferably, the encapsulation efficiency of the lipid nanoparticles is ≥60%; more preferably ≥90%; further preferably ≥95%; and most preferably ≥98%.

7. A method for preparing the lipid nanoparticles according to claim 6, characterized in that: A nucleic acid drug is added to the nucleic acid delivery vector to obtain a composition; a diluent or excipient is added to the composition, mixed, and filtered to obtain the final product. Alternatively, nucleic acid drugs can be added to diluents or excipients, then added to nucleic acid delivery vectors, mixed, and filtered to obtain the final product. Preferably, the volume-to-mass ratio of the diluent or excipient to the composition is (1-300):1 (μL / mg); more preferably (1-50):

1. Preferably, the mixing method includes, but is not limited to, water bath ultrasound, vortex oscillation, and manual blowing; Preferably, the power of the water bath ultrasound is 10-200W, the frequency is 30-150Hz, and the duration is 1s-1h; more preferably, the power of the water bath ultrasound is 30-100W, the frequency is 30-70Hz, and the duration is 1-30min. Preferably, the frequency of the vortex oscillation is 100–5000 rpm and the duration is 1 s–1 h; more preferably, the frequency of the vortex oscillation is 1000–3000 rpm and the duration is 1–30 min. Preferably, the number of manual blows is 1 to 100 times / min, and the time is 0.5 to 10 minutes; more preferably, the number of manual blows is 10 to 50 times / min, and the time is 1 to 3 minutes. Preferably, the preparation method further includes the steps of replacing the buffer medium and concentrating the obtained lipid nanoparticles.

8. A pharmaceutical composition, characterized in that: Includes the nucleic acid delivery carrier according to any one of claims 1-4, the lipid nanoparticles according to claim 6, pharmaceutically acceptable excipients and / or other pharmaceutically active molecules; Preferably, the other pharmaceutically active molecules include, but are not limited to, one or more of the following: antibodies, protein molecules, enzyme molecules, polypeptide molecules, small molecule compounds, and more preferably insulin, glucagon, semaglutide, paclitaxel, doxorubicin, irinotecan, docetaxel, aspirin, nitroglycerin, warfarin, propranolol hydrochloride, felodipine, vitamins, and metronidazole.

9. The use of the nucleic acid delivery vector according to any one of claims 1-4 in the preparation of at least one of the following reagents: 1) Loading, storing, and transporting drugs; 2) In vivo drug delivery; 3) Improve drug stability; 4) Transfection reagent; in, The drugs include nucleic acid drugs, lipid molecules, and other pharmaceutically active molecules.

10. The use of the lipid nanoparticles of claim 6 and the pharmaceutical composition of claim 8 in the preparation of cosmetics, health products, beauty products, and nursing products; or in the preparation of drugs for treating diabetes, hypoglycemia, cancer, analgesia, inflammation, fever, angina pectoris, thrombotic diseases, hypertension, parasitic infections, antiviral drugs, and hereditary diseases.

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