Method for quantitative detection of nucleic acids in nucleic acid-lipid nanoparticle complexes

Abstract

The present application provides a method for quantitatively detecting nucleic acids in a nucleic acid lipid nanoparticle complex, wherein sodium dodecyl sulfate is used as a demulsifier. According to the method for quantitatively detecting nucleic acids in a nucleic acid lipid nanoparticle complex of the present application, the effective nucleic acid concentration and encapsulation rate thereof can be accurately and quantitatively determined.

Description

Technical Field

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

[0002] During the use of LNP preparations containing nucleic acids, it is necessary to confirm the effective drug encapsulation concentration. Therefore, it is important to accurately determine the actual drug concentration and encapsulation rate of LNP preparations.

[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, a certain concentration of Triton-100 was added to break the emulsion, so that all the nucleic acids inside the LNP preparation were released, and the total amount of nucleic acids in the solution was measured to obtain c LNP-RNA全部核酸 Finally, the encapsulation efficiency is calculated by a formula (as shown in Formula 1) to accurately determine the nucleic acid concentration and encapsulation efficiency of the LNP preparation. For example, the encapsulation efficiency and mRNA concentration of mRNA-LNP are determined using RiboGreen.

[0004]

[0005] 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.

[0006] Therefore, it is desirable to provide a LNP nucleic acid preparation that can fully lyse the permanent cationic lipids so as to accurately and quantitatively determine the effective nucleic acid concentration and encapsulation efficiency thereof.

[0007] Prior art literature

[0008] Non-patent literature 1Pharmaceutics 2022,14,2086

[0009] Patent Document 2WO 2022 / 204286 Al Summary of the invention

[0010] Non-patent document 1 discloses the use of 1% (v / v) Triton-100 to lyse LNPs containing DOTAP-encapsulated siRNA, and the use of a Ribogreen kit to detect its encapsulation efficiency, but does not explain whether this method can be used to accurately detect the content of siRNA. In addition, the article only conducts a qualitative analysis of LNP-siRNA by using agarose gel electrophoresis.

[0011] Patent Document 2 discloses the use of Triton-100 and Ribogreen kit to detect the encapsulation efficiency of LNP containing cationic lipid (SORT lipid)-encapsulated nucleic acid, wherein the nucleic acid includes mRNA, sgRNA, and Cas9 mRNA. There is no disclosure or detailed description of the encapsulation efficiency detection and quantitative detection of siRNA in Patent Document 1.

[0012] In existing studies, the encapsulation efficiency of LNPs containing cationic lipid-encapsulated siRNA is usually measured by demulsification using Triton-100 and detection of the encapsulation efficiency using a Ribogreen kit. However, under this method, there is no public and clear description for the quantitative detection of siRNA encapsulated in LNPs.

[0013] 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 perform quantitative detection of nucleic acid content and encapsulation efficiency detection.

[0014] The 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.

[0015] In a preferred embodiment, the nucleic acid lipid nanoparticle complex comprises a permanent cationic lipid, wherein the pKa of the permanent cationic lipid is 9 or more, and preferably, the pKa of the permanent cationic lipid is 14 or more.

[0016] The permanent cationic lipid comprises one or more selected from phospholipids, sugars, proteins, amino acids, vitamins and SORT lipids.

[0017] 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).

[0018] 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.

[0019] 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, and more preferably, the nucleic acid is siRNA.

[0020] 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%.

[0021] 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 of demulsification using sodium lauryl sulfate is 10 minutes to 12 hours, and further can be 15 minutes to 12 hours.

[0022] Preferably, the demulsification time is 10 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours or 12 hours.

[0023] In a preferred embodiment, the temperature for demulsification using sodium dodecyl 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.

[0024] In a preferred embodiment, the method for quantitative detection of nucleic acid in nucleic acid lipid nanoparticle complexes comprises the following steps:

[0025] S1: Detection of free nucleic acid concentration in nucleic acid lipid nanoparticle complexes;

[0026] S2: Demulsification of nucleic acid-lipid nanoparticle complexes using sodium dodecyl sulfate as a demulsifier;

[0027] S3: Detect total nucleic acid concentration.

[0028] Furthermore, the method further comprises:

[0029] S4: Calculate the encapsulation efficiency based on the free nucleic acid concentration and the total nucleic acid concentration.

[0030] 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 to obtain the complex.

[0031] In a preferred embodiment, the nucleic acid lipid nanoparticle complex is prepared by the following method, which comprises the following steps:

[0032] S1: dissolving an ionizable cationic lipid, a permanent cationic lipid, a structural lipid, and a polymer-conjugated lipid in an organic solvent to obtain a lipid solution. Optionally, this step further includes dissolving an auxiliary lipid in an organic solvent;

[0033] S2: dissolving the nucleic acid in a buffer to obtain a nucleic acid stock solution;

[0034] S3: The lipid solution and the nucleic acid stock solution are fully mixed to obtain a mixed solution, and the organic solvent in the mixed solution is replaced with a solvent buffer to obtain a nucleic acid lipid nanoparticle complex.

[0035] In one embodiment, the solvent buffer solution is a phosphate buffer, and may further be Dulbecco's phosphate buffered saline (DPBS).

[0036] In a preferred embodiment, a MicroRNA detection kit is used to quantitatively detect the nucleic acid in the nucleic acid-lipid nanoparticle complex.

[0037] 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; the formula and preparation method thereof can be obtained by those skilled in the art according to the prior art.

[0038] In order to more clearly describe the technical solution of the present application, we provide a nucleic acid lipid nanoparticle complex, which comprises:

[0039] (a) nucleic acids;

[0040] (b) an ionizable cationic lipid which accounts for 10 mol% to 80 mol% of the total lipids present in the particle;

[0041] (c) structural lipids, which account for 3 mol% to 80 mol% of the total lipids present in the particle;

[0042] (d) a helper lipid that accounts for 0 mol% to 20 mol% of the total lipids present in the particle;

[0043] (e) a permanent cationic lipid which accounts for 15 mol% to 80 mol% of the total lipids present in the particle;

[0044] (f) polymer-conjugated lipids, which account for 0.1 mol% to 7.5 mol% of the total lipids present in the particle.

[0045] In one embodiment, the ionizable cationic lipid accounts for 20 mol% to 55 mol% of the total lipids present in the particle, for example, it may be 23.8 mol% or 24 mol%.

[0046] 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%.

[0047] 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%.

[0048] 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%.

[0049] In one embodiment, the polymer-conjugated lipid accounts for 0.25 mol% to 2 mol% of the total lipids present in the particle, and may further be 0.9 mol%, 1 mol% or 1.5 mol%.

[0050] 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.

[0051] 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.

[0052] 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), 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, saponasterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol and campesterol, more preferably cholesterol and / or β-sitosterol.

[0053] In a preferred embodiment, the auxiliary lipid is selected from phospholipids of different structural types, and representative auxiliary lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.

[0054] Preferably, the auxiliary 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 -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 -phosphoserine (DMPS); 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA); 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); and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC); 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). ;

[0055] In this specification, permanent cationic lipids refer to lipid molecules that have a positive charge and whose charge does not change with changes in solution pH.

[0056] In a preferred embodiment, the permanent cationic lipid comprises one or more selected from phospholipids, carbohydrates, proteins, amino acids, vitamins and SORT lipids.

[0057] The permanent cationic lipid is selected from dimethyl dioctadecyl ammonium bromide (DDAB), 1,2-dimyristoyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane methyl sulfate, 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 Methyldi(dodecyl)ammonium bromide, N-(a-trimethylammonioacetyl)-di(dodecyl)-D-glutamine hydrochloride, N-(a-trimethylammonioacetyl)-O,O'-bis-(1H,1H,2H,2H-perfluorodecyl)-L-glutamine hydrochloride, O,O'-di(dodecanoyl)-N-(a-trimethylammonioacetyl)diethanolamine hydrochloride, methylallyldi(dodecyl)ammonium bromide, N-{p-(w-trimethylammoniobutyloxy)-benzoyl}-di(dodecyl)-L-glutamine hydrochloride, 9-(w-trimethylammoniobutyl)-3,6-bis(dodecanoyl)carbazole bromide, dimethyldi(octadecyl) ammonium hydrochloride, Nw-trimethylammonium decanoyl-di(hexadecyl)-D-glutamine bromide, N-{p-(w-trimethylammonium hexyloxy)-benzoyl}-di(tetradecyl)-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"-tri(dodecanoyl)-N-(w-trimethyl-ammonium decanoyl)-tri(hydroxymethyl)aminomethane bromide (TC-1-12), 1,2-dimethylaminodecanoyl ... 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-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride.

[0058] 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).

[0059] The saccharides are cationic saccharide polymers and copolymers. Cationic polysaccharides suitable for use in the present application include those based on 5 or 6 carbon sugars and water-soluble derivatives such as those derived 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 bonds. The monomers can be arranged in straight or branched geometrical arrangements.

[0060] Suitable non-limiting examples of the 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.

[0061] The protein is obtained by coupling ethylenediamine with the protein using a coupling agent. Preferably, the coupling agent is N,N'-carbonyldiimidazole, 4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimide ester or a mixture thereof. Preferably, the coupling agent is N,N'-carbonyldiimidazole. Preferably, the protein is silk protein.

[0062] The amino acids include at least one selected from arginine, lysine or ornithine.

[0063] The vitamins include at least one selected from vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, and inositol.

[0064] The SORT lipid may be any one disclosed in CN112996519A and US11229609B, and its structure may be as shown in the following formula (I).

[0065]

[0066] Where R 1 and R 2 are each independently an alkyl group (C8 -C 24 ), alkenyl (C 8 -C 24 ) or a substituted form of any group; R 3 , R 3 ' and R 3 "Each independently is an alkyl (C≤6) or a substituted alkyl (C≤6); X is a monovalent anion. It can also be other lipid molecules with permanent positive groups in the art.

[0067] In a preferred embodiment, the polymer-conjugated lipid is a PEGylated lipid.

[0068] 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;

[0069] Preferably, the PEGylated lipid comprises a PEG moiety with a molecular weight of 1000Da to 20kDa, preferably a PEG moiety of about 1000Da to about 5000Da;

[0070] 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-PEG2000 amine, 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).

[0071] 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);

[0072] Or, heptane-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);

[0073] 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);

[0074] Or, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (MC3), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), cholesterol (CHO), dimyristoylglycerol-polyethylene glycol (DMG-PEG).

[0075] In one embodiment, the dimyristoylglycerol-polyethylene glycol is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000 or DMG-PEG2K).

[0076] The present application can achieve the following excellent technical effects: the demulsification effect of SDS is significantly better than that of Triton-100 and other commonly used demulsifiers in the art. 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

[0077] Figure 1 The results of using a MicroRNA kit to detect the content of all siRNA in the LNP-siRNA stock solution containing permanent cationic lipids in Example 1 after demulsification with different surfactants for different times;

[0078] Figure 2It 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 Effect Example 2;

[0079] Figure 3 The results are as follows: in Example 3, different formulations and concentrations were used with a demulsification concentration of 0.25 wt% SDS and after demulsification for different times, MicroRNA was used to detect the content and encapsulation rate of all siRNA, wherein 1X represents one-fold concentration and 2X represents two-fold concentration. DETAILED DESCRIPTION

[0080] I. Definitions

[0081] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0082] As used herein and unless otherwise specified, 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.

[0083] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can 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.

[0084] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0085] The term "demulsifier" 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 compounds that separate a solution containing nucleic acid lipid nanoparticle complexes.

[0086] The term "nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and their hybrid molecules. 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). Nucleic acids 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 (Poly A), and a miRNA binding site.

[0087] The term "siRNA" refers to a double-stranded RNA (duplex RNA) that can induce RNAi (RNA interference) by the cutting of a 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-stranded inside the single-stranded RNA. In addition, siRNA can be composed of nucleotides introduced with various functional groups for purposes such as increasing blood stability or weakening immune response.

[0088] 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.

[0089] The term "helper lipid" refers to a lipid molecule that is uncharged under specific pH conditions, such as physiological pH conditions.

[0090] The term "structural lipid" refers to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentane pyrophenanthrene carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coprostanol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

[0091] The term "polymer conjugated lipid" refers to a molecule containing 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 products of lipid coupling with uncharged, hydrophilic, steric barrier moieties, can also be used.

[0092] The specific names of the abbreviations used in this manual are as follows:

[0093] Lipid 5: Octanoic acid heptane-9-yl 8-(2-hydroxyethyl)(8-nonyloxy)-8-oxooctyl)amino ester;

[0094]

[0095] BHEM or BHEM-Cholesterol: N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide;

[0096] CHO or Cholesterol: Cholesterol

[0097] DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine;

[0098] DOTAP: (2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride;

[0099] DMG-PEG2K: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000;

[0100] MC3: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA);

[0101] DPBS (Dulbecco's Phosphate-Buffered Saline): Dulbecco's phosphate buffered saline.

[0102] The SDS demulsification concentration described in the present application is the mass percentage concentration of SDS in the mixture obtained by mixing the configured SDS solution with the nucleic acid lipid nanoparticle complex solution (LNP-nucleic acid stock solution) or the nucleic acid stock solution.

[0103] 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 quantitative detection of nucleic acids in SiRNA lipid nanoparticles prepared by different sequences using the method provided in this application has the same effect. Therefore, only one of the sequences prepared CD31 lipid nanoparticles is provided in this application 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 prescriptions 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.

[0104] II. Embodiment

[0105] 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.

[0106] Before further describing the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are described. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0107] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products, which are obtained by purchasing commercially available products.

[0108] Preparation Example 1: Preparation of MC3-BHEM-CD31 preparation

[0109] The different components in LNP were mixed evenly in ethanol in the following proportions: 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, and the theoretical concentration was 162.1ug / mL. The lipid mixed solution and the siRNA stock solution were mixed through the chip at a flow rate ratio of 1:3 (1.25:3.75mL / min). The mixed solution was diluted 10 times into 1×DPBS, and then concentrated and purified using a 100K ultrafiltration membrane to obtain a MC3-BHEM-CD31 preparation (LNP-siRNA stock solution), wherein the theoretical concentration of siRNA was 162.1ug / mL.

[0110] Preparation Example 2: Preparation of Lipid5-DOTAP-CD31 Preparation

[0111] In Preparation Example 1, the formulation formula was replaced with Lipid5 / DOTAP / CHO / DMG-PEG2K=24 / 50 / 25 / 1, N / P=3. Otherwise, the same procedure as Preparation Example 1 was performed to obtain a Lipid5-DOTAP-CD31 formulation uniformly dispersed in 1×DPBS, and the theoretical concentration of siRNA in LNP-siRNA was 162.1 ug / mL.

[0112] Preparation Example 3: Preparation of MC3-BHEM-1X (or MC3-BHEM-2X) formulation

[0113] The different components in LNP were mixed in ethanol in the following ratios: MC3 / BHEM / CHO / DOPE / DMG-PEG2K=23.8 / 50 / 17.8 / 7.5 / 0.9, N / P=3 to prepare a lipid solution. The pH 4 sodium citrate buffer was used to prepare siRNA (CD31) at one-fold concentration (1X) to 162.1ug / mL (or two-fold concentration (2X) to 326.2ug / mL), and the two solutions were mixed through the chip at a flow rate ratio of 1:3 (1.25:3.75mL / min), and the mixture was diluted 10 times into 1×DPBS solution, and then concentrated and purified using a 100K ultrafiltration membrane to obtain MC3-BHEM-1X (or MC3-BHEM-2X) preparation (LNP-siRNA stock solution) uniformly dispersed in 1×DPBS, wherein the theoretical concentrations of siRNA were 162.1ug / mL (1X) and 326.2ug / mL (2X), respectively.

[0114] Preparation Example 4: Preparation of MC3-1X preparation

[0115] 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 in the same manner as Preparation Example 3, wherein the theoretical concentration of siRNA was 162.1 ug / mL.

[0116] Preparation Example 5: Preparation of MC3-DOTAP-1X Preparation

[0117] 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 concentration of siRNA was 162.1 ug / mL.

[0118] Effect Example 1: Comparison of Demulsification Effects of Different Surfactants

[0119] Detection Methods

[0120] Sodium dodecyl sulfate (SDS) was prepared into aqueous solutions with different concentrations, 0.5wt% and 1wt%, respectively. The LNP-siRNA stock solution was directly diluted in MicroRNA buffer and the siRNA was analyzed using a 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 solution (20uL) obtained in Preparation Example 1 and Preparation Example 2 was incubated (demulsified) at 37°C for 15 min or 12 hours with 20uL 0.5wt% SDS, 1wt% SDS or 0.5wt% Triton-100 solution (the demulsification concentrations of SDS and Triton-100 were 0.25%, 0.5% and 0.25%, respectively), and the content of siRNA in LNP-siRNA was detected after incubation, i.e., c LNP-RNA全部核酸 The demulsification effects of different demulsifiers on LNP-siRNA containing permanent cationic lipids are shown in the attached Figure 1 The encapsulation efficiency EE% of LNP-siRNA can be calculated by formula (1).

[0121] Test results

[0122] As attached Figure 1As shown, the Triton-100 solution with a demulsification concentration of 0.25wt% did not show good demulsification ability for LNP-siRNA preparations containing permanent cationic lipids (DOTAP, BHEM), but under the action of SDS with a demulsification concentration of 0.25wt% or 0.5wt%, LNP-siRNA containing BHEM and DOTAP showed a complete demulsification effect.

[0123] Effect Example 2: Effect of Sodium Dodecyl Sulfate (SDS) Concentration on the Accuracy of Detection Results

[0124] Preparation of reagents

[0125] Sodium dodecyl sulfate (SDS) was prepared into aqueous solutions of different concentrations, namely 0.5wt%, 1wt%, 2wt%, and 5wt% SDS solutions. siRNA (CD31) was diluted with 25mM sodium citrate buffer to obtain a siRNA stock solution with a theoretical concentration of 169.56ug / mL (determined by Nanodrop).

[0126] Detection Methods

[0127] 20uL of siRNA stock solution was mixed with 20uL of SDS solution with concentrations of 0wt%, 0.5wt%, 1wt%, 2wt%, and 5wt% (the demulsification concentrations of the SDS solution were 0%, 0.25wt%, 0.5wt%, 1wt%, and 2.5wt%, respectively), and incubated at 37°C for 15 minutes. The concentration of siRNA was then detected and calculated using a MicroRNA kit.

[0128] Test results

[0129] As attached Figure 2 As shown, the inventor 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 the MicroRNA kit was used for detection, wherein the single siRNA solution was used as a control, that is, the SDS solution concentration was 0%. The results showed that the siRNA stock solution was calibrated to a concentration of 169.56ug / mL by Nanodrop, and was detected as 172.63ug / mL using the MicroRNA kit, indicating that the MicroRNA kit can accurately and quantitatively detect the concentration of siRNA. In addition, as the demulsification concentration of the SDS solution increased (0.25wt%, 0.5wt%, 1wt%, 2.5wt%), the concentrations of the siRNA detected were 177.36ug / mL, 179.31ug / mL, 208.87ug / mL and 310.46ug / mL, respectively.

[0130] Effect Example 3: Effect of Demulsification Time on Demulsification Effect

[0131] Detection Methods

[0132] 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 detected, i.e., 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 solution (20 uL) prepared in Preparation Examples 3 to 5 was 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, and the amount of all siRNA in the LNP-siRNA was detected after incubation, i.e., c LNP-RNA全部核酸 , as attached Figure 3 As shown; the encapsulation efficiency EE% of LNP-siRNA was calculated by formula (1).

[0133] Test results

[0134] As attached Figure 3 As shown, the detected siRNA content c of LNP-siRNA without permanent cationic lipid (prepared in Example 4) decreases with the demulsification time (3 hours, 1 hour, 15 minutes). LNP-RNA全部核酸 The encapsulation efficiency of LNP-siRNA containing DOTAP and BHEM was 94.80, 93.86, and 91.98% respectively. The demulsification effect of LNP-siRNA containing DOTAP and BHEM was the same as that of demulsification for 3 hours after demulsification for 15 minutes. Compared with the data of 190.3ug / mL detected by siRNA stock solution under the same detection conditions of the MicroRNA kit, LNP-siRNA containing BHEM showed complete demulsification effect at 15 minutes, 1 hour, and 3 hours of demulsification. LNP-RNA全部核酸 The encapsulation efficiency and siRNA content of the LNP-siRNA containing BHEM with a theoretical double concentration (2X) obtained in Example 3 were further tested. LNP-RNA全部核酸They were 394.95, 405.47, and 409.63 ug / mL, respectively, and the encapsulation rates were 99.58%, 99.60%, and 99.59%, respectively. This proved that when the SDS demulsification concentration was 0.25%, it had similar demulsification abilities within 15 minutes, 1 hour, and 3 hours, and all exhibited complete demulsification effects.

[0135] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex, wherein: Sodium dodecyl sulfate is used as a demulsifier; the demulsification concentration of the sodium dodecyl sulfate is 0.25wt%-1wt%; the nucleic acid lipid nanoparticle complex contains a permanent cationic lipid, which has a positive charge or a pKa of 9 or more; the permanent cationic lipid is a cholesterol-based cationic lipid or a SORT lipid.

2. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1, wherein: The permanent cationic lipid is selected from one or more of 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).

3. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1 or 2, wherein: The number of nucleic acid base pairs is less than 1000.

4. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 3, wherein: The number of nucleic acid base pairs is 500 or less.

5. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1, 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 and triplex-forming oligonucleotide.

6. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1, wherein: 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% or 1wt%.

7. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1 or 2, wherein: The time for demulsification using sodium lauryl sulfate ranges from 10 minutes to 12 hours.

8. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1 or 2, wherein: The temperature for demulsification using sodium lauryl sulfate is 25±2°C to 37±2°C.

9. The method for quantitative detection of nucleic acid in a nucleic acid-lipid nanoparticle complex according to claim 1 or 2, comprising the following steps: S1: Detection of free nucleic acid concentration in nucleic acid lipid nanoparticle complexes; S2: Using sodium dodecyl sulfate as a demulsifier to demulsify the nucleic acid-lipid nanoparticle complex; S3: Detect total nucleic acid concentration; S4: Calculate the encapsulation efficiency based on the free nucleic acid concentration and the total nucleic acid concentration. The formula for calculating the encapsulation efficiency is: .

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