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WO-2026092599-A1 - NUCLEIC ACID DELIVERY CARRIER, COMPOSITION, AND PREPARATION METHOD THEREFOR

WO2026092599A1WO 2026092599 A1WO2026092599 A1WO 2026092599A1WO-2026092599-A1

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.

Inventors

  • QI, Jianping
  • WEI, Yuning
  • YANG, JINLONG

Assignees

  • 复旦大学

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241030

Claims (10)

  1. A nucleic acid delivery vector, characterized in that: the nucleic acid delivery vector comprises at least the following components: (a) a eutectic solvent; (b) lipid molecules.
  2. The nucleic acid delivery vector according to claim 1 is 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 according to claim 1, wherein the lipid molecule is 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, 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: [-2000](PEG2k-DMPE), 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), and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159).
  4. The nucleic acid delivery vector according to claim 1, wherein 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 a nucleic acid delivery vector according to any one of claims 1-4, characterized in that: it includes mixing lipid molecules and 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 it comprises the nucleic acid delivery carrier 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 carrier 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 it comprises: a nucleic acid delivery carrier as described in any one of claims 1-4, lipid nanoparticles as described in 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; The drugs mentioned 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.

Description

A nucleic acid delivery vector, a composition and a method for preparing the same Cross-reference of related applications 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 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 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. 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 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. In a first aspect, the present invention provides a nucleic acid delivery vector, the nucleic acid delivery vector comprising at least the following components: (a) a eutectic solvent; and (b) lipid molecules. 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. 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%. 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 2