Quantitative measurement method for nucleic acid in nucleic acid-lipid nanoparticle complex

Abstract

A quantitative measurement method for a nucleic acid in a nucleic acid-lipid nanoparticle complex, sodium lauryl sulfate serving as a demulsifier. Said quantitative measurement method can accurately and quantitatively determine the effective nucleic acid concentration and the encapsulation rate.

Description

Quantitative detection method for nucleic acids in nucleic acid-lipid nanoparticle complexes Technical Field

[0001] The present application relates to the technical field of nucleic acid detection, and in particular to a method for quantitative detection of nucleic acids in nucleic acid-lipid nanoparticle complexes. Background Art

[0002] During the use of nucleic acid-encapsulated LNP formulations, it is necessary to confirm the effective drug entrapment concentration. Therefore, it is important to accurately determine the actual drug entrapment concentration and entrapment efficiency of the LNP formulation.

[0003] Generally, the method for determining the nucleic acid content in LNP preparations containing nucleic acids is to first detect the free nucleic acid concentration c in the solution before demulsification. LNP中游离的核酸 Then, by adding a certain concentration of Triton-100 to break the emulsion, all nucleic acids inside the LNP preparation are released. The total amount of nucleic acids in the solution is measured to obtain the total nucleic acid of cLNP-RNA. Finally, the encapsulation efficiency is calculated by the formula (as shown in Formula 1), thereby accurately determining the concentration of nucleic acids entrapped inside the LNP preparation and its encapsulation efficiency. For example, RiboGreen is used to determine the encapsulation efficiency of mRNA-LNP and its mRNA concentration.

[0004] With the widespread application of permanent cationic lipids or compounds in LNP nucleic acid preparations, the inventors encountered the problem of insufficient demulsification when using Triton-100 to demulsify and detect the total nucleic acid concentration, resulting in a large difference between the detected concentration and the theoretical value.

[0005] Therefore, it is desirable to provide an LNP nucleic acid preparation that can fully lyse the permanent cationic lipids to accurately and quantitatively determine the effective nucleic acid concentration and encapsulation efficiency thereof.

[0006] Prior art literature

[0007] Non-patent literature 1Pharmaceutics 2022, 14, 2086

[0008] Patent Document 2WO 2022 / 204286A1 Summary of the Invention

[0009] Non-patent document 1 discloses the use of 1% (v / v) Triton-100 to cleave LNPs containing DOTAP-encapsulated siRNA and the use of a Ribogreen kit to detect the encapsulation efficiency. However, it does not specify whether this method can be used to accurately detect the siRNA content. In addition, the article only performs a qualitative analysis of LNP-siRNA by using agarose gel electrophoresis.

[0010] Patent Document 2 discloses the use of Triton-100 and a Ribogreen kit to detect the encapsulation efficiency of LNPs containing cationic lipids (SORT lipids) encapsulating nucleic acids, including mRNA, sgRNA, and Cas9 mRNA. Patent Document 1 does not disclose or provide detailed descriptions of siRNA encapsulation efficiency and quantitative detection.

[0011] In existing studies, the encapsulation efficiency of LNPs containing cationic lipid-encapsulated siRNA is usually determined by using Triton-100 for demulsification and a Ribogreen kit for detection of the encapsulation efficiency. However, under this method, there is no public and clear description of the quantitative detection of LNP-encapsulated siRNA.

[0012] The present invention uses sodium dodecyl sulfate (hereinafter sometimes referred to as "SDS") to demulsify LNPs encapsulating nucleic acids, and uses methods and kits for nucleic acid content detection in the art, such as a MicroRNA kit, to quantitatively detect the nucleic acid content and the encapsulation efficiency.

[0013] A first aspect of the present application provides a method for quantitatively detecting nucleic acids in nucleic acid-lipid nanoparticle complexes, using sodium dodecyl sulfate as a demulsifier.

[0014] In a preferred embodiment, the nucleic acid-lipid nanoparticle complex comprises a permanent cationic lipid, and the pKa of the permanent cationic lipid is 9 or greater, preferably, the pKa of the permanent cationic lipid is 14 or greater.

[0015] The permanent cationic lipid comprises one or more selected from phospholipids, sugars, proteins, amino acids, vitamins and SORT lipids.

[0016] In a preferred embodiment, the permanent cationic lipid is one or more selected from N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA).

[0017] In a preferred embodiment, the number of nucleic acid base pairs is 1000 or less, preferably, the number of nucleic acid base pairs is 500 or less.

[0018] Preferably, the nucleic acid is selected from one or more of siRNA, mRNA, tRNA, rRNA, cDNA, miRNA, ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, triplex forming oligonucleotide (TFO), and gene. More preferably, the nucleic acid is siRNA.

[0019] In a preferred embodiment, the demulsification concentration of the sodium lauryl sulfate is 0.20 wt%-3 wt%, preferably 0.25 wt%-2.5 wt%, more preferably 0.25%-1 wt%.

[0020] In a preferred embodiment, the demulsification concentration of the sodium lauryl sulfate is 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.5wt%, 2wt% or 2.5wt%. In a preferred embodiment, the time range for demulsification using sodium lauryl sulfate is 10 minutes to 12 hours, and further can be 15 minutes to 12 hours.

[0021] Preferably, the demulsification time is 10 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours or 12 hours.

[0022] In a preferred embodiment, the temperature for demulsification using sodium lauryl sulfate is 25±2°C to 37±2°C, preferably, the temperature for demulsification is 30±2°C to 37±2°C, preferably, the temperature for demulsification is 25±2°C, 26±2°C, 27±2°C, 28±2°C, 29±2°C, 30±2°C, 31±2°C, 32±2°C, 33±2°C, 34±2°C, 35±2°C, 36±2°C, 37±2°C.

[0023] In a preferred embodiment, the method for quantitative detection of nucleic acids in nucleic acid-lipid nanoparticle complexes comprises the following steps:

[0024] S1: Detection of free nucleic acid concentration in nucleic acid-lipid nanoparticle complexes;

[0025] S2: Sodium dodecyl sulfate was used as a demulsifier to demulsify the nucleic acid-lipid nanoparticle complex;

[0026] S3: Detect total nucleic acid concentration.

[0027] Furthermore, the method further comprises:

[0028] S4: Calculate the encapsulation efficiency based on the free nucleic acid concentration and the total nucleic acid concentration.

[0029] In one embodiment, the nucleic acid-lipid nanoparticle complex is prepared by the following method: mixing lipids according to a formula amount, and then mixing with a load.

[0030] In a preferred embodiment, the nucleic acid-lipid nanoparticle complex is prepared by the following method, which comprises the following steps:

[0031] S1: dissolving an ionizable cationic lipid, a permanent cationic lipid, a structured lipid, and a polymer-conjugated lipid in an organic solvent to prepare a lipid solution. Optionally, this step further includes dissolving an auxiliary lipid in the organic solvent;

[0032] S2: dissolving the nucleic acid in a buffer solution to prepare a nucleic acid stock solution;

[0033] S3: The lipid solution and the nucleic acid stock solution are thoroughly mixed to obtain a mixed solution, and the organic solvent in the mixed solution is replaced with a solvent buffer to prepare a nucleic acid-lipid nanoparticle complex.

[0034] In one embodiment, the solvent buffer solution is a phosphate buffer, and may further be Dulbecco's phosphate buffered saline (DPBS).

[0035] In a preferred embodiment, a MicroRNA detection kit is used to quantitatively detect the nucleic acid in the nucleic acid-lipid nanoparticle complex.

[0036] In a preferred embodiment, the nucleic acid-lipid nanoparticle complex includes but is not limited to nucleic acids, ionizable cationic lipids, structural lipids, auxiliary lipids, permanent cationic lipids and polymer-conjugated lipids; its formula and preparation method can be obtained by those skilled in the art based on existing technology.

[0037] In order to more clearly describe the technical solution of the present application, we provide a nucleic acid lipid nanoparticle complex comprising:

[0038] (a) nucleic acids;

[0039] (b) an ionizable cationic lipid that constitutes 10 mol% to 80 mol% of the total lipids present in the particle;

[0040] (c) structural lipids, which constitute 3 mol% to 80 mol% of the total lipids present in the particle;

[0041] (d) a helper lipid comprising 0 mol% to 20 mol% of the total lipids present in the particle;

[0042] (e) a permanent cationic lipid comprising 15 mol% to 80 mol% of the total lipids present in the particle;

[0043] (f) polymer-conjugated lipids, which constitute 0.1 mol% to 7.5 mol% of the total lipids present in the particle.

[0044] In one embodiment, the ionizable cationic lipid accounts for 20 mol% to 55 mol% of the total lipids present in the particle, for example, 23.8 mol% or 24 mol%.

[0045] In one embodiment, the structural lipids account for 10 mol% to 60 mol% of the total lipids present in the particle, for example, 17.8 mol%, 25 mol% or 38.5 mol%.

[0046] In one embodiment, the helper lipid accounts for 0 mol% to 10 mol% of the total lipids present in the particle, for example, it may be 0 mol% or 7.5 mol%.

[0047] In one embodiment, the permanent cationic lipid accounts for 20 mol% to 50 mol% of the total lipids present in the particle, for example, it may be 50 mol%.

[0048] In one embodiment, the polymer-conjugated lipid accounts for 0.25 mol% to 2 mol% of the total lipids present in the particle, and further can be 0.9 mol%, 1 mol% or 1.5 mol%.

[0049] In one embodiment, the nucleic acid lipid nanoparticle complex N / P is 1-20, preferably 2-10, more preferably 3, 4, 5, 6, 7 or 8.

[0050] In one embodiment, the ionizable cationic lipid (also referred to as a cationic lipid in some literature) can be selected from molecules disclosed in the prior art in the art, such as WO2013086354, WO2011153493, CN115850104A, or any one or more of the ionizable cationic lipids disclosed in Chinese applications CN202310744585.7, CN2023107445013.0, and CN202311255442.6.

[0051] In a preferred embodiment, the ionizable cationic lipids include 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102) and [(4-hydroxybutyl) )azepine diyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); preferably one or more selected from 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA) and / or [(4-hydroxybutyl)azepine diyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester (SM-102), heptane-9-yl 8-(2-hydroxyethyl) (8-nonyloxy)-8-oxooctyl) amino ester (Lipid5), and 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester (MC3). In a preferred embodiment, the structured lipids include one or more of cholesterol and its derivatives, preferably one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol and campesterol, more preferably cholesterol and / or β-sitosterol.

[0052] In a preferred embodiment, the helper lipid is selected from phospholipids of different structural types, and representative helper lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.

[0053] Preferably, the helper lipid comprises a member selected from the group consisting of hydrogenated soybean phosphatidylcholine (HSPC); 1,2-didecanoyl-sn-glyceryl-3-phosphocholine (DDPC); 1,2-dioleoyl-sn-glyceryl-3-phosphate (DEPA); 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DEPC); 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DEPE); 1,2-dioleoyl-phosphatidylglycerol (DEPG); 1,2-dilinoleoyl-sn-glyceryl-3-phosphocholine (DLOPC); 1,2-dilauroyl-sn-glyceryl-3-phosphate (DLPA); 1,2-dilauroyl-sn-glyceryl-3 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-Dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG); 1,2-Dilauroyl-sn-glycero-3-phosphoserine (DLPS); 1,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA); 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-Dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG); 1,2-Dimyristoyl-sn-glycero-3 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-Dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG); 1,2-Dioleoyl-sn-glycero-3-phosphoserine (DOPS); 1,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) ; 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG); 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS); 1,2-distearoyl-sn-glycero-3-phosphate (DSPA); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG); 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS); 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC);1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC); 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphatidylglycerol (POPG); 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC); 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC); 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC).

[0054] In this specification, permanent cationic lipids refer to lipid molecules that have a positive charge and do not change their charge with changes in solution pH.

[0055] In a preferred embodiment, the permanent cationic lipid comprises one or more selected from phospholipids, carbohydrates, proteins, amino acids, vitamins and SORT lipids.

[0056] The permanent cationic lipid is selected from dimethyldioctadecyl ammonium bromide (DDAB), 1,2-dimyristoyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane methylsulfate, 1,2-dipalmitoyl-3-trimethylammonium propane, 1,2-distearoyl-3-trimethylammonium propane, N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dimyristoyloxypropyl dimethylhydroxyethylammonium bromide (DMRIE), dioleoyloxypropyl dimethylhydroxyethylammonium bromide (DORIE), di Methyldidodecylammonium bromide, N-(a-trimethylammonioacetyl)-didodecyl-D-glutamine hydrochloride, N-(a-trimethylammonioacetyl)-O,O'-bis-(1H,1H,2H,2H-perfluorodecyl)-L-glutamine hydrochloride, O,O'-didodecanoyl-N-(a-trimethylammonioacetyl)diethanolamine hydrochloride, methylallyldidodecylammonium bromide, N-{p-(w-trimethylammoniobutyloxy)-benzoyl}-didodecyl-L-glutamine hydrochloride, 9-(w-trimethylammoniobutyl)-3,6-bis(dodecanoyl)carbazole bromide, dimethyldioctadecyl Ammonium hydrochloride, Nw-trimethylammonium decanoyl-dihexadecyl-D-glutamine bromide, N-{p-(w-trimethylammonium hexyloxy)-benzoyl}-ditetradecyl-L-glutamine bromide, p-(w-trimethylammonium decyloxy)-ρ'-octyloxyazobenzene bromide (MC-1-0810), p-{w-(b-hydroxyethyl)dimethyl-ammonium-decyloxy}-ρ'-octyloxyazobenzene bromide (MC-3-0810), O,O',O"-tridodecanoyl-N-(w-trimethyl-ammonium decanoyl)-tris(hydroxymethyl)aminomethane bromide (TC-1-12), 1,2-dimethylaminodecanoyl ...MC-1-0810), 1,2-dimethylaminodecanoyl-tris(hydroxymethyl)aminomethane bromide (TC-1-12), 1,2-dimethylaminodecanoyl-tris(hydroxymethyl)aminomethane bromide (MC-3-0810), 1,2-dimethylaminodecanoyl-tris(hydroxymethyl)aminomethane bromide (TC-1-12), 1,2-dimethylaminodecanoyl-tris(hydroxymethyl)aminomethane bromide (TC-1-12), 1,2-dimethylamino One or more of cinnamic-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-glycero-3-ethylphosphocholine, 1,2-distearoyl-glycero-3-ethylphosphocholine, 1,2-dioleoyl-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-ethylphosphocholine, N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), and N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride.

[0057] Preferably, the permanent cationic lipid is one or more selected from N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA).

[0058] The carbohydrates are cationic carbohydrate polymers and copolymers. Cationic polysaccharides suitable for use in this application include those based on 5- or 6-carbon sugars and water-soluble derivatives thereof, such as those derivatized with ethylene oxide. These polymers can be linked together by any of several arrangements, such as 1,4-α, 1,4-β, 1,3-α, 1,3-β, and 1,6 linkages. The monomers can be arranged in a linear or branched geometry.

[0059] Suitable non-limiting examples of cationic polysaccharides include those based on cellulose and hydroxyalkylcellulose; starch and hydroxyalkyl starch; polymers based on arabinose monomers; polymers derived from xylose monomers; polymers derived from fucose; polymers derived from fructose monomers; polymers based on acid-containing sugar monomers such as galacturonic acid and glucuronic acid; polymers based on amine sugar monomers such as galactosamine and glucosamine, especially acetylglucosamine; polymers based on 5- or 6-membered ring polyol monomers; polymers based on galactose monomers; polymers based on mannose monomers and polymers based on galactomannan monomers.

[0060] The protein is formed by coupling ethylenediamine to the protein using a coupling agent. Preferably, the coupling agent is N,N'-carbonyldiimidazole, 4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimidyl ester, or a mixture thereof. Preferably, the coupling agent is N,N'-carbonyldiimidazole. Preferably, the protein is silk protein.

[0061] The amino acids include at least one selected from arginine, lysine or ornithine.

[0062] The vitamins include at least one selected from vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, and inositol.

[0063] The SORT lipid may be any one disclosed in CN112996519A or US11229609B, and its structure may be as shown in the following formula (I).

[0064] wherein R1 and R2 are each independently an alkyl group (C8-C 24 ), alkenyl (C8-C 24) or a substituted form of any group; R3, R3' and R3" are each independently an alkyl group (C≤6) or a substituted alkyl group (C≤6); X is a monovalent anion. It can also be other lipid molecules with permanent positive groups in the art.

[0065] In a preferred embodiment, the polymer-conjugated lipid is a PEGylated lipid.

[0066] In a preferred embodiment, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

[0067] Preferably, the PEGylated lipid comprises a PEG moiety with a molecular weight of 1000 Da to 20 kDa, preferably a PEG moiety of about 1000 Da to about 5000 Da;

[0068] Preferably, the PEGylated lipid is selected from DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, Ceramide-PEG2000, DPPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, DSPE-PEG5000, DSPE-PEG2000amine, ALC-0159, dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) , dimyristoylphosphatidylethanolamine-polyethylene glycol 2000 (DMPE-PEG2000)), 2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DSG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol 2000 (DSPE-PEG2000), 1,2-dioleoyl-rac-glycerol (DOG-PEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethylene glycol 2000 (DOPE-PEG2000).

[0069] In one embodiment, the complex comprises: nucleic acid, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (MC3), N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), cholesterol (CHO), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoylglycerol-polyethylene glycol (DMG-PEG);

[0070] Or, heptadecyl-9-yl 8-(2-hydroxyethyl)(8-nonyloxy)-8-oxooctyl)amino octanoate (Lipid5), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), cholesterol (CHO), dimyristoylglycerol-polyethylene glycol (DMG-PEG);

[0071] or, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (MC3), N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), cholesterol (CHO), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoylglycerol-polyethylene glycol (DMG-PEG);

[0072] Or, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (MC3), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), cholesterol (CHO), dimyristoylglycerol-polyethylene glycol (DMG-PEG).

[0073] In one embodiment, the dimyristoylglycerol-polyethylene glycol is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000 or DMG-PEG2K).

[0074] The present application can achieve the following excellent technical effects: the demulsification effect of SDS is significantly better than Triton-100 and other commonly used demulsifiers in this field. SDS can completely demulsify the nucleic acid-lipid nanoparticle complex, and has a better demulsification effect on the nucleic acid-lipid nanoparticle complex containing permanent cationic lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 is the result of detecting the content of all siRNA using a MicroRNA kit after demulsification of the LNP-siRNA solution containing permanent cationic lipids in Example 1 at different surfactants and different times;

[0076] Figure 2 is the result of quantitative detection using a MicroRNA kit after incubation of SDS solutions with different demulsification concentrations (2.5wt%, 1wt%, 0.5wt%, 0.25wt%, 0) with CD31-siRNA stock solution in Example 2;

[0077] Figure 3 shows the content and encapsulation efficiency of all siRNA detected by MicroRNA after demulsification for different times using 0.25 wt% SDS at different concentrations and different formulations in Example 3, where 1X represents one-fold concentration and 2X represents two-fold concentration. DETAILED DESCRIPTION

[0078] I. Definition

[0079] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0080] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0081] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0082] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0083] The term "emulsifier" refers to one or more compounds that reduce the interfacial tension between at least two liquids in an emulsion and can cause the emulsion to separate into at least two liquid phases, such as a compound that separates a solution containing nucleic acid-lipid nanoparticle complexes.

[0084] The term "nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and hybrid molecules thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASOs). The nucleic acid may be further chemically modified, and the chemical modification is selected from one or a combination of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methylcytosine. The mRNA molecule contains a protein coding region and may further contain an expression regulatory sequence. Typical expression regulatory sequences include, but are not limited to, a 5' cap (5' cap), a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a polyadenylic acid sequence (PolyA), and a miRNA binding site.

[0085] The term "siRNA" refers to a double-stranded RNA (duplex RNA) that can induce RNAi (RNA interference) by cutting of specific mRNA, or a single-stranded RNA with a double-stranded form inside a single-stranded RNA. It consists of a sense RNA chain with a sequence homologous to the target gene mRNA and an antisense RNA chain with a sequence complementary thereto. The length of the siRNA can be about 15 to 60, specifically about 15 to 50, about 15 to 40, about 15 to 30, about 15 to 25, about 16 to 25, about 19 to 25, about 20 to 25 or about 20 to 23 nucleotides. The siRNA length refers to the number of nucleotides on one side of the double-stranded RNA, i.e., the number of base pairs, and in the case of single-stranded RNA, refers to the length of the double chain inside the single-stranded RNA. In addition, siRNA can be composed of nucleotides having various functional groups introduced for purposes such as increasing blood stability or weakening immune response.

[0086] The term "cationic lipid" refers to a lipid molecule that is capable of being positively charged under physiological pH conditions. In some embodiments, the cationic lipid is an amino lipid.

[0087] The term "helper lipid" refers to a lipid molecule that is uncharged under specific pH conditions, such as physiological pH.

[0088] The term "structured lipid" refers to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentanylphenyl carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coproposterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol.

[0089] The term "polymer-conjugated lipid" refers to a molecule comprising a polymer portion and a lipid portion. In some embodiments, the polymer-lipid is a polyethylene glycol (PEG) lipid. Other lipids that can reduce aggregation, such as compounds having uncharged, hydrophilic, steric barrier moieties coupled to lipids, can also be used.

[0090] The specific names of the abbreviations used in this manual are as follows:

[0091] Lipid 5: heptadecyl-9-yl 8-(2-hydroxyethyl)(8-nonyloxy)-8-oxooctyl)amino octanoate;

[0092] BHEM or BHEM-Cholesterol: N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide;

[0093] CHO or Cholesterol: cholesterol;

[0094] DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine;

[0095] DOTAP: (2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride;

[0096] DMG-PEG2K: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000;

[0097] MC3: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA);

[0098] DPBS (Dulbecco's Phosphate-Buffered Saline): Dulbecco's phosphate buffered saline.

[0099] The SDS demulsification concentration described in this application is the mass percentage concentration of SDS in the mixture obtained by mixing the prepared SDS solution with the nucleic acid lipid nanoparticle complex solution (LNP-nucleic acid stock solution) or the nucleic acid stock solution.

[0100] For the Anti-CD31-SiRNA sequence, see Reference 3 (Lung Endothelium Targeted Nanoparticles based on a pH-Sensitive Lipid and the GALA Peptide Enable Robust Gene Silencing and the Regression of Metastatic Lung Cancer, Mahmoud M.Abd Elwakil et al., Advanced functional materials, 2019) Supporting Information Table (S6). Many sequences are disclosed in the prior art. The method provided in this application has the same effect on the quantitative detection of nucleic acids in siRNA lipid nanoparticles prepared with different sequences. Therefore, this application only provides CD31 lipid nanoparticles prepared with one of the sequences to illustrate the technical solution of this application. Different LNP formulations containing permanent cations can be demulsified using the SDS solution described in the present invention and achieve the effect of complete demulsification. Only some of the formulations are listed below to illustrate the technical effects of the present invention. Those skilled in the art can also select corresponding kits or content detection methods for nucleic acid content determination according to different nucleic acids.

[0101] II. Embodiment

[0102] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0103] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0104] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

[0105] Preparation Example 1: Preparation of MC3-BHEM-CD31 preparation

[0106] The different components in LNP were mixed evenly in ethanol in the following ratio: MC3 / BHEM / CHO / DOPE / DMG-PEG2K=23.8 / 50 / 17.8 / 7.5 / 0.9, N / P=3 to obtain a lipid mixed solution. At the same time, siRNA stock solution (CD31) was prepared with 25mM pH4 sodium citrate buffer with a theoretical concentration of 162.1ug / mL. The lipid mixed solution and siRNA stock solution were mixed through the chip at a flow rate ratio of 1:3 (1.25:3.75mL / min). The mixture was diluted 10 times into 1×DPBS and then concentrated and purified using a 100K ultrafiltration membrane to obtain the MC3-BHEM-CD31 preparation (LNP-siRNA stock solution), wherein the theoretical siRNA concentration was 162.1ug / mL.

[0107] Preparation Example 2: Preparation of Lipid5-DOTAP-CD31 Preparation

[0108] In Preparation Example 1, the preparation formula was replaced with Lipid5 / DOTAP / CHO / DMG-PEG2K=24 / 50 / 25 / 1, N / P=3. Otherwise, the same procedure as in Preparation Example 1 was followed to obtain a Lipid5-DOTAP-CD31 preparation uniformly dispersed in 1×DPBS. The theoretical concentration of siRNA in the LNP-siRNA was 162.1 ug / mL.

[0109] Preparation Example 3: Preparation of MC3-BHEM-1X (or MC3-BHEM-2X) formulation

[0110] The different components of LNP were mixed in ethanol in the following ratio: MC3 / BHEM / CHO / DOPE / DMG-PEG2K=23.8 / 50 / 17.8 / 7.5 / 0.9, N / P=3 to prepare lipid solution. siRNA (CD31) was prepared in pH 4 sodium citrate buffer at a concentration of one times (1X) to 162.1 ug / mL (or at a concentration of two times (2X) to 326.2 ug / mL). The two solutions were mixed through the chip at a flow rate ratio of 1:3 (1.25:3.75 mL / min). The mixture was diluted 10-fold into 1X DPBS solution and then concentrated and purified using a 100K ultrafiltration membrane to obtain MC3-BHEM-1X (or MC3-BHEM-2X) formulation (LNP-siRNA stock solution) uniformly dispersed in 1X DPBS. The theoretical concentrations of siRNA were 162.1 ug / mL (1X) and 326.2 ug / mL (2X), respectively.

[0111] Preparation Example 4: Preparation of MC3-1X Preparation

[0112] The formulation in Preparation Example 3 was replaced with MC3 / CHO / DSPC / DMG-PEG2K=50 / 38.5 / 10 / 1.5, N / P=3, and the MC3-1X formulation was prepared according to the same method as Preparation Example 3, wherein the theoretical siRNA concentration was 162.1 ug / mL.

[0113] Preparation Example 5: Preparation of MC3-DOTAP-1X formulation

[0114] The formulation in Preparation Example 3 was replaced with MC3 / DOTAP / CHO / DMG-PEG2K=24 / 50 / 25 / 1, N / P=3, and the MC3-DOTAP-1X formulation was prepared in the same manner as in Preparation Example 1, wherein the theoretical siRNA concentration was 162.1 ug / mL.

[0115] Effect Example 1: Comparison of Demulsification Effects of Different Surfactants

[0116] Detection method

[0117] Sodium dodecyl sulfate (SDS) was prepared into aqueous solutions with different concentrations, namely 0.5wt% and 1wt%. The LNP-siRNA stock solution was directly diluted in MicroRNA buffer and the microRNA kit (Quant-iT TM The amount of free siRNA in the LNP-siRNA solution was determined by using MicroRNA Assay Kit (Cat.Q32882). LNP中游离的核酸 The LNP-siRNA stock solutions (20 μL) obtained in Preparation Examples 1 and 2 were incubated (demulsified) at 37° C. for 15 min or 12 h with 20 μL of 0.5 wt% SDS, 1 wt% SDS, or 0.5 wt% Triton-100 solution, respectively (the demulsification concentrations of SDS and Triton-100 were 0.25%, 0.5%, and 0.25%, respectively). After incubation, the siRNA content in the LNP-siRNA, i.e., the total nucleic acid content of the cLNP-RNA, was measured. The demulsification effects of different demulsifiers on LNP-siRNA containing permanent cationic lipids are shown in FIG1 . The LNP-siRNA encapsulation efficiency (EE%) was calculated using formula (1).

[0118] Test results

[0119] As shown in Figure 1, a Triton-100 solution with a demulsification concentration of 0.25 wt% did not show good demulsification ability for LNP-siRNA formulations containing permanent cationic lipids (DOTAP, BHEM). However, under the action of SDS with a demulsification concentration of 0.25 wt% or 0.5 wt%, LNP-siRNA containing BHEM and DOTAP showed a complete demulsification effect.

[0120] Effect Example 2: Effect of Sodium Dodecyl Sulfate (SDS) Concentration on the Accuracy of Detection Results

[0121] Preparation of reagents

[0122] Sodium dodecyl sulfate (SDS) was prepared into aqueous solutions of varying concentrations: 0.5 wt%, 1 wt%, 2 wt%, and 5 wt%. siRNA (CD31) was diluted with 25 mM sodium citrate buffer to produce a theoretical siRNA stock solution of 169.56 μg / mL (as measured by Nanodrop).

[0123] Detection method

[0124] 20 μL of siRNA stock solution was mixed with 20 μL of SDS solution with concentrations of 0 wt%, 0.5 wt%, 1 wt%, 2 wt%, and 5 wt%, respectively (the demulsification concentrations of the SDS solution were 0%, 0.25 wt%, 0.5 wt%, 1 wt%, and 2.5 wt%, respectively), and incubated at 37°C for 15 minutes. The siRNA concentration was then detected and calculated using a MicroRNA kit.

[0125] Test results

[0126] As shown in Figure 2, the inventors explored the effect of different concentrations of SDS solution on the concentration detection of siRNA stock solution. During the experiment, different concentrations of SDS solution were added to the siRNA stock solution and detected using a MicroRNA test kit. The siRNA solution alone was used as a control, i.e., the SDS solution concentration was 0%. The results showed that the siRNA stock solution was calibrated to a concentration of 169.56 ug / mL by Nanodrop and 172.63 ug / mL using the MicroRNA test kit, indicating that the MicroRNA test kit can accurately and quantitatively detect the concentration of siRNA. In addition, as the demulsification concentration of the SDS solution increased (0.25 wt%, 0.5 wt%, 1 wt%, 2.5 wt%), the concentrations of the siRNA detected were 177.36 ug / mL, 179.31 ug / mL, 208.87 ug / mL, and 310.46 ug / mL, respectively.

[0127] Effect Example 3: Influence of Demulsification Time on Demulsification Effect

[0128] Detection method

[0129] The siRNA in the LNP-siRNA stock solution was quantitatively detected using a MicroRNA kit. The LNP-siRNA stock solution was directly diluted in MicroRNA buffer and the amount of free siRNA in the LNP-siRNA solution was determined, i.e., the amount of c LNP中游离的核酸 ; The free nucleic acid concentration in the LNP-siRNA-1X stock solution prepared in Preparation Example 3 was 1.59 ug / mL, and the free nucleic acid concentration in the LNP-siRNA-2X stock solution was 1.63 ug / mL; the free nucleic acid concentration in the LNP-siRNA stock solution prepared in Preparation Example 4 was 10.85 ug / mL; the LNP-siRNA stock solutions (20 uL) prepared in Preparation Examples 3 to 5 were incubated with 20 uL of 0.5 wt% SDS solution (demulsification concentration was 0.25%) at 37° C. for 15 minutes, 1 hour, and 3 hours. After incubation, the amount of total siRNA in the LNP-siRNA, i.e., total nucleic acid of cLNP-RNA, was detected, as shown in FIG3 ; and the LNP-siRNA encapsulation efficiency EE% was calculated by formula (1).

[0130] Test results

[0131] As shown in Figure 3, the LNP-siRNA (prepared in Example 4) without permanent cationic lipids showed increased siRNA content (cLNP-RNA total nucleic acid) of 176.28 μg / mL, 149.18 μg / mL, and 114.27 μg / mL, respectively, with encapsulation efficiencies of 94.80, 93.86, and 91.98%, as the demulsification time decreased (3 hours, 1 hour, and 15 minutes). LNP-siRNA containing DOTAP and BHEM, however, achieved the same demulsification effect after 15 minutes of demulsification as after 3 hours of demulsification. Compared to the siRNA stock solution measured at 190.3 μg / mL under the same detection conditions as the microRNA kit, LNP-siRNA containing BHEM demonstrated complete demulsification, with cLNP-RNA total nucleic acid content of 194.62, 191.10, and 185.59 μg / mL, respectively, at 15 minutes, 1 hour, and 3 hours of demulsification, with encapsulation efficiencies of 99.16%, 99.14%, and 99.11%, respectively. Further testing of the encapsulation efficiency and siRNA content of the LNP-siRNA containing BHEM, which was theoretically prepared in Example 3 at twice the concentration (2X), showed that the total nucleic acid content of the cLNP-RNA was 394.95, 405.47, and 409.63 μg / mL, respectively, and the encapsulation efficiency was 99.58%, 99.60%, and 99.59%, respectively. This demonstrates that when the SDS demulsification concentration is 0.25%, the demulsification ability is similar within 15 minutes, 1 hour, and 3 hours, and all exhibit complete demulsification effects.

[0132] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for quantitatively detecting nucleic acid in a nucleic acid-lipid nanoparticle complex, wherein, Sodium dodecyl sulfate is used as a demulsifier.

2. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 1, wherein, The nucleic acid-lipid nanoparticle complex contains a permanent cationic lipid, and the permanent cationic lipid is positively charged or has a pKa of 9 or more.

3. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 2, wherein, The permanent cationic lipid contains one or more selected from phospholipids, cholesterol-based cationic lipids, saccharides, proteins, amino acids, vitamins, and SORT lipids.

4. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 2, wherein, The permanent cationic lipid is one or more selected from N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethyl ammonium bromide (BHEM-Chol), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA).

5. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 1 or 2, wherein, The number of nucleic acid base pairs is 1000 or less, preferably 500 or less.

6. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 5, wherein, The nucleic acid is selected from one or more of siRNA, mRNA, tRNA, rRNA, cDNA, miRNA, ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, triple helix-forming oligonucleotide, and gene.

7. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to any one of claims 1 to 6, wherein, The demulsification concentration of the sodium dodecyl sulfate is 0.20 wt% - 3 wt%, preferably 0.25 wt% - 2.5 wt%, more preferably 0.25 wt% - 1 wt%.

8. The method for quantitative detection of nucleic acid in the nucleic acid-lipid nanoparticle complex according to claim 7, wherein the demulsification concentration of the sodium dodecyl sulfate is 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.5 wt%, 2 wt%, or 2.5 wt%.

9. The method for quantitatively detecting nucleic acid in the nucleic acid-lipid nanoparticle complex according to any one of claims 1 to 8, wherein, The time range for demulsification using sodium dodecyl sulfate is 10 minutes to 12 hours; Preferably, the temperature for demulsification using sodium dodecyl sulfate is 25 ± 2°C to 37 ± 2°C.

10. The method for quantitative detection of nucleic acid in the nucleic acid-lipid nanoparticle complex according to any one of claims 1 to 9, which comprises the following steps: S1: Detect the concentration of free nucleic acid in the nucleic acid-lipid nanoparticle complex; S2: Use sodium dodecyl sulfate as a demulsifier to demulsify the nucleic acid-lipid nanoparticle complex; S3: Detect the total nucleic acid concentration; Further, the method further comprises: S4: Calculate the encapsulation efficiency according to the concentration of free nucleic acid and the total nucleic acid concentration.

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