Method for measuring one or more indexes of non-viral nucleic acid vector

Abstract

A method for measuring drug content indexes of a non-viral nucleic acid vector composition, comprising performing labeling by enabling a permeable nucleic acid dye to be in contact with a non-viral nucleic acid vector (for example, an mRNA-LNP). The drug content indexes are calculated by measuring, at a single-particle level, fluorescent light generated by free nucleic acids of a nucleic acid vector, and fluorescent light generated by externally adsorbed nucleic acids and internally entrapped nucleic acids of the non-viral nucleic acid vector. The drug content indexes include a drug loading capacity, entrapment efficiency and the like. The method optionally further comprises: treating a sample with a nuclease before or after performing labeling by using the permeable nucleic acid dye, and when performing treatment with the nuclease, measuring the fluorescence condition of the non-viral nucleic acid vector composition; or enabling the permeable nucleic acid dye to be in contact with the sample, and then measuring the fluorescence condition of the non-viral nucleic acid vector composition.

Description

Method for detecting one or more indicators of non-viral nucleic acid carriers TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology technology, and relates to a method for detecting one or more indicators such as drug content indicators of non-viral nucleic acid carriers. Specifically, it relates to a method for detecting drug loading and encapsulation efficiency of non-viral nucleic acid carriers such as liposome nanoparticles containing nucleic acids. BACKGROUND

[0002] Gene therapy is the ultimate treatment technology for diseases related to gene abnormalities. The emergence of nucleic acid drugs with various mechanisms brings more possibilities for gene therapy. However, nucleic acid molecules have poor in vivo stability and are difficult to efficiently enter target cells, often requiring the assistance of carriers to deliver them to specific tissues and target cells in the body. Therefore, the development of safe and efficient nucleic acid delivery systems is an important part of gene therapy.

[0003] Compared with viral vectors, non-viral nucleic acid carrier preparations have higher safety and have broad application prospects in the field of gene therapy. The "Guidelines for Microcapsule, Microsphere and Liposome Preparations" in Appendix IX E of Chinese Pharmacopoeia 2005 Edition (Part II) emphasizes that the drug loading rate and encapsulation efficiency of microcapsule, microsphere and liposome preparations must be detected.

[0004] Non-viral nucleic acid carrier preparations belong to microcapsule, microsphere and liposome preparations regulated by Chinese Pharmacopoeia. According to the requirements of Chinese Pharmacopoeia, characterization data of drug loading and encapsulation efficiency must be provided. Non-viral nucleic acid carriers have small particle size and strong individual heterogeneity, and non-viral nucleic acid carrier preparations (so-called microcapsule, microsphere and liposome preparations) have different drug loading rates and encapsulation efficiencies between different products.

[0005] There is still a need in the field of gene therapy biotechnology to develop a method for determining the drug content indicators such as encapsulation efficiency and drug loading of non-viral nucleic acid carrier preparations. SUMMARY

[0006] In the traditional method, the detection of drug loading rate, encapsulation efficiency and the like of non-viral nucleic acid carriers has the following problems: a centralized average method is used. In addition to the above problems, the inventors have unexpectedly found that the prior art has the following other problems: nucleic acids are adhered to the surface of non-viral nucleic acid carriers, which leads to overestimation of the effective drug content, and such drugs belonging to the type of externally adsorbed nucleic acids are ineffective and unsafe when entering the human body. This technical problem has not been reported in the prior art, and the inventors have unexpectedly found this technical problem and proposed the following technical solution.

[0007] The present application provides a method for detecting one or more indicators of non-viral nucleic acid carrier in the form of nanoparticles, which can be quantitative indicators such as drug content. As the non-viral nucleic acid carrier, for example, a lipid nanoparticle (LNP) encapsulating a nucleic acid of interest such as mRNA can be listed.

[0008] The present application provides a method for detecting a drug content indicator of a non-viral nucleic acid carrier composition, which comprises labeling the non-viral nucleic acid carrier (e.g. mRNA-LNP) composition by contacting with a permeable nucleic acid dye, measuring the fluorescence of free nucleic acid, the fluorescence of externally adsorbed nucleic acid and the fluorescence of internally encapsulated nucleic acid of the non-viral nucleic acid carrier at the single particle level, and calculating the drug content indicator. The drug content indicator includes drug loading, encapsulation efficiency, and other indicators such as surface nucleic acid signal positive rate, external adsorbed nucleic acid copy number distribution, etc. The method optionally further comprises: treating the sample with a nuclease before or after labeling with a permeable nucleic acid dye, measuring the fluorescence of the non-viral nucleic acid carrier composition after nuclease treatment; or contacting the sample with a non-permeable nucleic acid dye, and measuring the fluorescence of the non-viral nucleic acid carrier composition after contacting.

[0009] The method of the present application has the advantages of high efficiency, accuracy, easy operation, small sample size, and good repeatability.

[0010] In a first aspect of the present application, a method for detecting nanoparticle characterization indicators or parameters of a non-viral nucleic acid carrier composition is provided.

[0011] Herein, unless otherwise specified, nucleic acid signal positive refers to the fluorescence detection result of nucleic acid after being combined with a nucleic acid dye, and the same applies hereinafter.

[0012] The method comprises:

[0013] Step O: contacting the composition with a permeable dye, and detecting the fluorescence of free nucleic acid and the fluorescence intensity of nucleic acid signal positive non-viral nucleic acid carrier of the obtained mixture at the single particle level by flow nanometer particle detection technology.

[0014] Optionally, the method further comprises step a or step b, and the drug loading and encapsulation efficiency of the composition are calculated according to the fluorescence intensity of each component measured in step O and step a or b, wherein,

[0015] Step a: treating the composition before or after contacting with a permeable dye with a nuclease to degrade free nucleic acid and externally adsorbed nucleic acid;

[0016] Step b: independently contacting the composition with a non-permeable nucleic acid dye.

[0017] The inventors found that when there is extremely small amount of nucleic acid adhered to the surface of the non-viral nucleic acid carrier, the method according to step O can achieve accurate detection of the drug loading and encapsulation efficiency. Preferably, when the nucleic acid signal positive rate calculated according to step O is ≤30% and the average copy number of the externally adsorbed nucleic acid is ≤5, the method does not include steps a and b, and the drug loading and encapsulation efficiency of the composition are calculated according to the fluorescence intensity of each component measured according to step O.

[0018] In a second aspect of the present application, a method for detecting the surface nucleic acid signal positive rate and the copy number distribution of externally adsorbed nucleic acid of a non-viral nucleic acid carrier composition is provided, which comprises the steps of detecting the nucleic acid signal positive rate of the non-viral nucleic acid carrier composition and the average copy number of the externally adsorbed nucleic acid.

[0019] In a third aspect of the present application, a flow particle detection device used in the methods of the first and second aspects is provided. The flow particle detection device is a particle analysis detection device capable of achieving directional flow of sample flow, and comprises a directional fluid system and a particle analysis detection device.

[0020] The directional fluid system comprises a sample loading unit and a flow unit.

[0021] The particle analysis detection device comprises an optical system and a particle detector.

[0022] The particle detector comprises a photoelectric sensor and a signal conditioning circuit with band-pass filtering high-frequency noise function.

[0023] The sample enters the flow particle detection device through the sample loading unit and forms a liquid flow beam in the flow unit. The liquid flow beam is irradiated by the light beam of the optical system, forming multiple directional scattered light. Part of the light in the light beam is absorbed by the sample and emits fluorescence, which is received by the photoelectric sensor in the particle detector. These fluorescence signals are then converted into electrical signals by the signal conditioning circuit with band-pass filtering high-frequency noise function. Through further statistical analysis of the electrical signals, detection and analysis data are obtained.

[0024] In a fourth aspect of the present application, the use of a nuclease in the detection of characterization parameters of a non-viral nucleic acid carrier composition is provided.

[0025] In a fifth aspect of the present application, the use of a permeable nucleic acid dye in the detection of characterization parameters of a non-viral nucleic acid carrier composition is provided.

[0026] In a sixth aspect of the present application, a method for evaluating whether a non-viral nucleic acid carrier product meets the release standard of the encapsulation efficiency specified in the Chinese Pharmacopoeia is provided, wherein the release standard requires that the encapsulation efficiency of the non-viral nucleic acid carrier product should not be less than 80%.

[0027] The method can be used as a method for detecting the content of the encapsulated nucleic acid in the LNP drug, or a method for quantitatively analyzing the content of the encapsulated nucleic acid in a sample containing a non-viral nucleic acid carrier.

[0028] Advantages of the present application

[0029] The present application has the advantage of providing a method for detecting one or more indicators of nanoparticles of a non-viral nucleic acid carrier composition with high precision. Compared with existing methods, the method has the advantages of shorter time, high efficiency, accuracy, easy operation, small sample size, and better repeatability.

[0030] The present application incorporates external adsorbed nucleic acid into the calculation, so that the detection result more restores the true drug loading and encapsulation efficiency of the non-viral nucleic acid carrier, and avoids the interference of nucleic acid adhering to the surface of the non-viral nucleic acid carrier which cannot be used as a drug to affect the determination result of the drug loading and encapsulation efficiency.

[0031] The present application provides a nuclease and a permeable nucleic acid dye and a non-permeable nucleic acid dye, which are particularly suitable for the above-mentioned method.

[0032] The method of the present application is suitable for the determination of flow nanometer particle parameters, quality control, etc. of microparticle preparations such as LNP products encapsulating nucleic acids of interest, such as mRNA vaccines.

[0033] The technical solution provided by the present application can be used for the research and development or quality control of non-viral nucleic acid carrier products, greatly saving the time and sample size of the detection method in quality control. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a graph showing the particle concentration and particle size distribution of mRNA-lipid nanoparticles determined in Example 1.

[0035] Figure 2 is a two-dimensional scatter plot showing the scattering light signal and fluorescence signal of mRNA-lipid nanoparticles labeled with a first nucleic acid dye in Example 1.

[0036] Figure 3 is a two-dimensional scatter plot of the scattering light signal and fluorescence signal of the sample after nuclease treatment in Example 1.

[0037] Figure 4 is a regression curve of the peak area and concentration of the mRNA vaccine standard determined by HPLC method in Comparative Example 1, and the concentration detection result (C0) of the detected mRNA vaccine product is shown in the figure.

[0038] Figure 5 is an elution curve of the mRNA vaccine product detected by HPLC method in Comparative Example 1 and the concentration detection result (C1).

[0039] Figure 6 is a two-dimensional scatter plot of the scattered light signal and the fluorescent signal of the test sample solution 3 after the first nucleic acid dye labeling in Example 2.

[0040] Figure 7 is a two-dimensional scatter plot of the scattered light signal and the fluorescent signal of the test sample solution 4 after the first nucleic acid dye labeling in Example 2. DETAILED DESCRIPTION

[0041] The present application will be further clarified by the following examples which are intended to be exemplary of the present application.

[0042] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations. Unless specifically stated otherwise, the present disclosure is not to be construed as using terms or phrases such as "example," "for example," "exemplary," or "illustrative" to identify ideas or concepts that are preferred or important. The terms "example" and "exemplary" are used to provide examples of embodiments of the present disclosure. Unless specifically stated otherwise, embodiments described herein are not to be construed as preferred or advantageous over other embodiments.

[0043] Furthermore, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0044] Terminology

[0045] "Microcapsule" refers to a microcapsule in which a solid or liquid drug is encapsulated by a carrier excipient. In general, a microcapsule refers to a particle having a particle size of 1 to 250 μm, a sub-microcapsule refers to a particle having a particle size of 0.1 to 1 μm, and a nanocapsule refers to a particle having a particle size of 10 to 100 nm.

[0046] "Microsphere" refers to a microspherical entity in which a drug is dissolved or dispersed in a carrier excipient. In general, a microsphere refers to a particle having a particle size of 1 to 250 μm, a sub-microsphere refers to a particle having a particle size of 0.1 to 1 μm, and a nanosphere refers to a particle having a particle size of 10 to 100 nm.

[0047] "Liposome" refers to a microvesicle in which a drug is encapsulated by a lipid bilayer.

[0048] "Nanoparticle" refers to a solid particle having a particle size of less than 500 nm, in which a drug is dispersed by a nanofication technique together with a carrier excipient. A nanoparticle composed of only drug molecules is referred to as a nanocrystal or a nanodrug, and a nanoparticle formed using a lipid material as a drug carrier is referred to as a lipid nanoparticle.

[0049] As the "non-viral nucleic acid carrier", a lipid nanoparticle in which a nucleic acid molecule is encapsulated as a drug can be exemplified. In the present specification, "lipid nanoparticle" is used with the same meaning as "lipid nanoparticle". The "non-viral nucleic acid carrier" includes a non-viral nucleic acid carrier to which a nucleic acid is adsorbed on the outside, a non-viral nucleic acid carrier in which a nucleic acid is encapsulated in the inside, a non-viral nucleic acid carrier to which a nucleic acid is adsorbed on the surface and encapsulated in the inside, and a non-viral nucleic acid carrier without a nucleic acid.

[0050] The term "surface-adsorbed nucleic acid" is used with the same meaning as "externally adsorbed nucleic acid".

[0051] The "non-viral nucleic acid carrier composition" means a non-viral nucleic acid carrier externally adsorbed with a nucleic acid, a non-viral nucleic acid carrier internally encapsulated with a nucleic acid, a non-viral nucleic acid carrier externally adsorbed and internally encapsulated with a nucleic acid, a non-viral nucleic acid carrier without a nucleic acid, a free nucleic acid, and a complex with a solvent.

[0052] The "lipid nanoparticle-encapsulated nucleic acid drug" means a composition in which a nucleic acid is encapsulated in a lipid nanoparticle and can be used as a drug.

[0053] The "nucleic acid signal-positive non-viral nucleic acid carrier" means a non-viral nucleic acid carrier sample that is labeled with a nucleic acid dye (such as a permeable nucleic acid dye or a non-permeable nucleic acid dye) and shows a significant fluorescence intensity during the measurement in a flow particle group.

[0054] The "nucleic acid signal-negative non-viral nucleic acid carrier" means a non-viral nucleic acid carrier sample that is not labeled with a nucleic acid dye (such as a permeable nucleic acid dye or a non-permeable nucleic acid dye) and does not show a significant fluorescence intensity during the measurement in a flow particle group.

[0055] In the present application, the nucleic acid signal-positive non-viral nucleic acid carrier has a stronger fluorescence signal than the nucleic acid signal-negative non-viral nucleic acid carrier, and there is a relatively clear boundary between the two. For example, when the threshold is set to N=3 and Wmin=0.2 ms, the fluorescence signal is considered positive.

[0056] In the present application, the non-viral nucleic acid carrier has a certain particle size compared to the free nucleic acid, and thus the non-viral nucleic acid carrier has a stronger scattered light intensity than the free nucleic acid in the scattered light channel, and there is a relatively clear boundary between the two. The signal with a significant scattered light intensity is a scattered light positive signal.

[0057] The type of the "nucleic acid molecule" (in this document, the encapsulated nucleic acid molecule is sometimes also referred to as "test nucleic acid") is not particularly limited and includes, but is not limited to, DNA, mRNA, siRNA, ASO (antisense oligonucleotides), circular RNA, double-stranded DNA plasmid, etc.

[0058] The size of the encapsulated nucleic acid molecule or test nucleic acid is not particularly limited and can be, for example, greater than 200-300 bp / nt, for example, 200-10000 bp / nt, 400-7000 bp / nt.

[0059] In one embodiment, the non-viral nucleic acid vector is an mRNA-LNP or an LNP encapsulating a double-stranded DNA plasmid. Herein, "mRNA-LNP" is used with the same meaning as "mRNA vaccine" "mRNA-lipid nanoparticle".

[0060] The mRNA vaccine can include an mRNA molecule encoding a target protein (such as an antigenic peptide) from a virus such as a herpes simplex virus, a poxvirus, a rabies virus, an influenza virus, an adenovirus, an enterovirus, a rotavirus, a novel coronavirus, a dengue virus, a human syncytial virus, a monkeypox virus, a porcine circovirus, etc., can include an mRNA molecule encoding one or more immunostimulatory molecules, one or more pathogenic antigens, and can include a non-coding region of a virus, including a major histocompatibility complex (MHC) binding peptide, etc. The mRNA can be with or without nucleotide modification.

[0061] Typical LNP is generally a stable nanoparticle assembled from phospholipids, cationic lipids, sterol lipids, and PEGylated lipids in a certain ratio.

[0062] As a lipid component of LNP, those recognized as being able to be used can be listed, such as SM-102 cationic lipid, ALC-0315, DLin-MC3-DMA, DODAP, DSPC, cholesterol, ALC-0159, DMG-PEG 2000, etc.

[0063] As the zeta potential of LNP, the range recognized as being able to be allowed can be listed.

[0064] The particle size range of LNP detected by the method of the present application is not particularly limited and is adjusted according to the use scenario. It can be listed as: less than 800 nm, less than 500 nm, preferably less than 400 nm, more preferably less than 150 nm.

[0065] "Drug loading" refers to the weight percentage of the drug contained in the microparticle preparation.

[0066] "Drug loading" = weight of drug contained in microspheres / total weight of microspheres x 100%

[0067] "Encapsulation efficiency" = amount of drug encapsulated in the system / total amount of encapsulated and unencapsulated drug in the system x 100%.

[0068] In the present invention, the term "nucleic acid dye" refers to a dye having a fluorescent excitation wavelength capable of binding to all nucleic acid molecules. In a free state, the nucleic acid dye has almost no fluorescence, but once bound to nucleic acid, the fluorescence is greatly enhanced, and the fluorescence signal intensity is related to the number and length of the nucleic acid. The fluorescence signal intensity is positively correlated with the length of the nucleic acid fragment. When the length of the nucleic acid of interest is constant, the fluorescence signal intensity is uniform, and the number of fluorescence signal events is linearly related to the number of nucleic acids.

[0069] In the present invention, after treatment with a nuclease, free nucleic acids and nucleic acids adsorbed outside the nanoparticles are degraded, and the fluorescent dye originally bound thereto does not show fluorescence.

[0070] The nucleic acid dye independently includes at least one selected from a permeable nucleic acid dye, a permeable cyanine dye, an intercalating dye, or a DNA double helix minor groove binding dye, respectively.

[0071] As the nucleic acid dye, Acridine Orange, Actinomycin D, 7-AAD (7-aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodid, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroxyethidium (hydroethidine), Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride, POPO-1 Iodid, POPO-3 Iodide, Propidium Monoazide Bromide (PMA), SYTO-9, SYTO-11, SYTO-12, SYTO-13, SYTO-14, SYTO-15, SYTO-16, SYTO-17, SYTO-18, SYTO-19, SYTO-20, SYTO-21, SYTO-22, SYTO-23, SYTO-24, SYTO-25, SYTO-26, SYTO-27, SYTO-28, SYTO-29, SYTO-30, SYTO-31, SYTO-32, SYTO-33, SYTO-34, SYTO-35, SYTO-36, SYTO-37, SYTO-38, SYTO-39, SYTO-40, SYTO-41, SYTO-42, SYTO-43at least one of Ethidium Bromide, Hoechst 33258, Hoechst 33342, Hoechst 34580, Hoechst 34580 Trihydrate, Hoechst 34580 Trihydrate-FluoroPure Grade, LDS 751, NeuroTrace Blue Fluorescent Nissl Stain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615 Red Fluorescent Nissl Stain, NeuroTrace Deep-Red Fluorescent Nissl Stain, POPO-1 Iodide, POPO-3 Iodide, PO-PRO-1 Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe DNA Gel Stain, SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, TO-PRO-1 Iodide, TO-PRO-3 Iodide, TOTO-1 Iodide, TOTO-3 Iodide, YO-PRO-1 Iodide, YO-PRO-3 Iodide, YOYO-1 Iodide, YOYO-3 Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain.

[0072] In the present application, the term "permeable nucleic acid dye" means a nucleic acid dye that can permeate through the surface of a non-viral nucleic acid carrier into the inside without using other reagents or methods for enhancing the surface permeability of the non-viral nucleic acid carrier. That is, the so-called permeability refers to the permeability with respect to the inside and outside of the nucleic acid carrier.

[0073] As the permeable nucleic acid dye (or the first nucleic acid dye), at least one selected from the group consisting of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide can be exemplified.

[0074] In some embodiments, the permeable nucleic acid dye is used at a final concentration of 0.001 to 10 μM. In some embodiments, the permeable nucleic acid dye is used at a final concentration of 0.001 μM, 0.002 μM, 0.1 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, or 10 μM. Preferably, 0.5 to 2 μM.

[0075] In some embodiments, the permeable nucleic acid dye is selected from the group consisting of SYTO 9, SYTO 13, SYTO 16, SYTO 24. In some embodiments, the permeable nucleic acid dye is selected from the group consisting of SYTO 9 and SYTO 16, preferably SYTO 9.

[0076] In the present application, the term "non-permeable nucleic acid dye" means a nucleic acid dye that cannot permeate through the surface of a non-viral nucleic acid carrier into the inside without using other agents and methods for enhancing the surface permeability of a non-viral nucleic acid carrier.

[0077] As the non-permeable nucleic acid dye (or the second nucleic acid dye), at least one selected from the group consisting of SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), RiboGreen can be exemplified.

[0078] In some embodiments, the non-permeant nucleic acid dye is at a final concentration of 0.001-10 μΜ when labeled. In some embodiments, the non-permeant nucleic acid dye is at a final concentration of 0.001 μΜ, 0.002 μΜ, 0.1 μΜ, 0.5 μΜ, 1.0 μΜ, 1.5 μΜ, 2.0 μΜ, 2.5 μΜ, 3.0 μΜ, 3.5 μΜ, 4.0 μΜ, 5.0 μΜ, 6.0 μΜ, 7.0 μΜ, 8.0 μΜ, 9.0 μΜ, or 10 μΜ, preferably 0.5-2 μΜ when labeled.

[0079] In some embodiments, the permeant nucleic acid dye is one selected from the group consisting of SYTOX Green, RiboGreen, Pico Green, more preferably selected from the group consisting of SYTOX Green, RiboGreen. In some embodiments, the non-permeant nucleic acid dye is SYTOX Green.

[0080] As the nuclease, there can be mentioned: recombinant DNase I; DNase, RNase-free; DNase I; Deoxyribonuclease I; Exonuclease I; Exonuclease III; S1 nuclease; dsDNase; RNase A; RNase A (DNase and protease free), and the like.

[0081] In some embodiments, the nuclease can be at least one selected from the group consisting of recombinant DNase I (RNase-free), Dnase, RNase-free, DNase I (RNase-free), S1 nuclease, dsDNase, RNase A, RNase A (DNase and protease free), and combinations thereof, wherein preferably at least one selected from the group consisting of recombinant DNase I (RNase-free), RNase A, RNase A (DNase and protease free) is contained.

[0082] As specific examples of the nuclease, there can be mentioned, but not limited to, recombinant DNase I (RNase-free) (TKR-2270A, Takara Bio), DNase, RNase-free (EN0523, Thermo), DNase I (RNase-Free) (M0303S, NEB), Deoxyribonuclease I (DNase, Bovine Pancreas) (S10073-250mg, Sigma-Aldrich), Exonuclease I (2170A, Takara Bio), Exonuclease III (2650A, Takara Bio), S1 nuclease (EN0321, Thermo), 1X dsDNase (ENO771, Thermo), RNase A (ST576, Bioworld), RNase A (DNase-free and Protease-free) (EN0531, Thermo).

[0083] Statistical method

[0084] Note that, as the method of the present application, although a method using the median value of the fluorescence intensity for the calculation is mentioned, it is not limited thereto. As long as it is a mathematical method capable of calculating the one or more indexes using the fluorescence generated by the free nucleic acid of the nucleic acid carrier, the fluorescence generated by the externally adsorbed nucleic acid, and the internally encapsulated nucleic acid, other statistical methods not using the median value of the fluorescence intensity can be used, and these methods are also included in the scope of the method of the present application.

[0085] The summation symbol ∑ involved in the mathematical formula of the present application indicates the summation of a series of numbers, where

[0086] A part: upper limit of summation, B part: lower limit of summation, C part: content of summation.

[0087] For example, in the present application, when referring to all mean where i takes values from 1 to n1, that is, the sum of each term from θ1 to θ n1 .

[0088] The term "flow particle detection device" of the present application includes a device based on the detection of dispersed samples in a fluid, including flow cytometry devices, nanoflow, and also microfluidic devices, and even simple devices of microplates based on microscopes.

[0089] In some embodiments, when the sample is detected by the flow particle detection device, the sample is diluted to a certain concentration before use.

[0090] In some embodiments, the certain concentration is 1 x 10 5-1 x 10 12 In some embodiments, the dilution is performed using a buffer.

[0091] In some embodiments, the buffer is a PBS buffer.

[0092] In some embodiments, the PBS buffer is an aqueous solution of 100-150 mM NaCl, 2-4 mM KCl, 5-10 mM Na2HPO4 and 1-3 mM KH2PO4 at pH 7.2-7.6. In some embodiments, the PBS buffer is an aqueous solution of 100-140 mM NaCl, 2-3 mM KCl, 7-9 mM Na2HPO4 and 1-2 mM KH2PO4 at pH 7.2-7.6.

[0093] In some embodiments, the PBS buffer is an aqueous solution of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 at pH 7.2-7.6.

[0094] In some embodiments, the PBS buffer is an aqueous solution of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 at pH 7.4.

[0095] In some embodiments, the sample injection pressure for each sample can be the same or different when the samples are detected using a flow particle detection apparatus.

[0096] In some embodiments, the sample injection pressure is independently selected from 0.1-10.0 kPa, preferably, the sample injection pressure can be independently selected from 0.5-2.0 kPa. In some embodiments, the sample injection pressure is independently selected from 0.1 kPa, 0.5 kPa, 1.0 kPa, 1.5 kPa, 2.0 kPa, 3.0 kPa, 4.0 kPa, 5.0 kPa, 6.0 kPa, 7.0 kPa, 8.0 kPa or 10.0 kPa.

[0097] In some embodiments, the sample injection time for each sample can be the same or different when the samples are detected using a flow particle detection apparatus.

[0098] In some embodiments, the injection time is independently selected from 0.1 min to 10 min. In some embodiments, the injection time is independently selected from 0.1 min, 0.5 min, 1.0 min, 1.5 min, 2.0 min, 3.0 min, 4.0 min, 5.0 min, 6.0 min, 7.0 min, 8.0 min, 9.0 min, or 10 min.

[0099] In some embodiments, the method described above comprises taking pictures, counting and signal intensity values of the particles with specific optical features.

[0100] In the present application, "room temperature" means ambient temperature, which can be 4°C to 40°C, can be 20°C to 30°C, in some embodiments, 22°C to 28°C, in some embodiments, 24°C to 26°C, and in some embodiments, 25°C.

[0101] The incubation temperature of the lipid nanoparticle encapsulating the nucleic acid with the nucleic acid dye can be 20-40°C, can be room temperature to 37°C, and can be 25°C.

[0102] "Gating" or "setting a gate" means to define a range or an area in a flow particle distribution map, and to analyze the particles in the range or the area one by one according to single parameter or multiple parameters. The shape of the gate includes linear gate, cross gate, rectangular gate, circular gate, polygonal gate, arbitrary shape gate, and four-quadrant gate, etc.

[0103] In the above of the present application, all the numbers disclosed herein are approximate. Based on the numbers disclosed, each number can vary by ±10% or a reasonable variation as understood by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0104] The term "plurality" means 2 or more.

[0105] The term "optional", "optionally", or "may" means that the subsequent event or circumstance can or can not occur. For example, "optional surfactant" means that surfactant can or can not be present.

[0106] The term "final concentration" means the concentration of the ingredient in the system at the start of the reaction or labeling. The term "and / or" should be understood to mean either one of the options or a combination of any two or more of the options.

[0107] In the specific embodiment 1, the method of the present application comprises:

[0108] Step (1a): taking a sample of a non-viral nucleic acid carrier-containing composition to be tested, labeling with a nucleic acid dye that specifically binds to nucleic acid, as Mixture 1; preferably, the nucleic acid dye is a permeable nucleic acid dye.

[0109] As the non-viral nucleic acid carrier composition, a lipid nanoparticle encapsulating a nucleic acid molecule as a drug can be exemplified. The nucleic acid molecule as a drug can be selected from DNA, mRNA, siRNA, ASO (antisense oligonucleotides), circular RNA, double-stranded DNA plasmid, etc., and is preferably mRNA or a double-stranded DNA plasmid. When the nucleic acid molecule as a drug is mRNA, the non-viral nucleic acid carrier is an mRNA vaccine (or mRNA-LNP).

[0110] The non-viral nucleic acid carrier can include a non-viral nucleic acid carrier externally adsorbing a nucleic acid, a non-viral nucleic acid carrier internally encapsulating a nucleic acid, a non-viral nucleic acid carrier externally adsorbing and internally encapsulating a nucleic acid, and a non-viral nucleic acid carrier empty.

[0111] The non-viral nucleic acid carrier composition represents a complex including a non-viral nucleic acid carrier externally adsorbing a nucleic acid, a non-viral nucleic acid carrier internally encapsulating a nucleic acid, a non-viral nucleic acid carrier externally adsorbing and internally encapsulating a nucleic acid, a non-viral nucleic acid carrier empty, free nucleic acid, and a solvent.

[0112] As the solvent, those commonly used in the art can be used, for example, a buffer as an aqueous phase. As the buffer, Tris buffer, PBS buffer, etc. can be exemplified. The pH of the buffer can be exemplified as pH 7-8, for example, pH 7.2-7.6, and preferably pH 7.4.

[0113] Compared to free nucleic acid, the non-viral nucleic acid carrier has a certain particle size. In detection, in the scattered light channel, the non-viral nucleic acid carrier has a stronger scattered light intensity than the free nucleic acid, and the two have a relatively obvious boundary, and the signal with a clear scattered light intensity is a scattered light positive signal.

[0114] The scattered light channel is, for example, an SSC channel.

[0115] As the sample of the non-viral nucleic acid carrier-containing composition, it can be diluted to a certain concentration before use. The certain concentration can be a non-viral nucleic acid carrier particle concentration of 1 x 10 5 -1 x 10 12 In some embodiments, the dilution is performed with a buffer.

[0116] As the buffer, Tris buffer, PBS buffer, etc. can be exemplified. The pH of the buffer can be exemplified: pH 7-8, for example, pH 7.2-7.6.

[0117] The PBS buffer can be exemplified as an aqueous solution containing NaCl, KCl, Na2HPO4, and KH2PO4. The PBS buffer is, for example, an aqueous solution of 100 mM-150 mM NaCl, 2 mM-4 mM KCl, 5 mM-10 mM Na2HPO4, and 1 mM-3 mM KH2PO4; an aqueous solution of 100 mM-140 mM NaCl, 2 mM-3 mM KCl, 7 mM-9 mM Na2HPO4, and 1 mM-2 mM KH2PO4 at pH 7.2-7.6. Preferably, the PBS buffer is an aqueous solution of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4 at pH 7.2-7.6. The PBS buffer can have a pH of pH 7.2-7.6, preferably pH 7.4.

[0118] The nucleic acid dye independently includes at least one selected from the group consisting of a permeant nucleic acid dye, a permeant cyanine dye, an intercalating dye, or a DNA double helix minor groove binding dye, respectively.

[0119] As the nucleic acid dye, Acridine Orange, Actinomycin D, 7-AAD (7-aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodid, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroxyethidium (hydroethidine), Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride, POPO-1 Iodid, POPO-3 Iodide, Propidium Monoazide Bromide (PMA), SYBR® Gold, SYTO-9, SYTO-11, SYTO-12, SYTO-13, SYTO-14, SYTO-15, SYTO-16, SYTO-17, SYTO-18, SYTO-19, SYTO-20, SYTO-21, SYTO-22, SYTO-23, SYTO-24, SYTO-25, SYTO-26, SYTO-27, SYTO-28, SYTO-29, SYTO-30, SYTO-31, SYTO-32, SYTO-33, SYTO-34, SYTO-35, SYTO-36, SYTO-37, SYTO-38, SYTO-39, SYTO-40, SYTO-41, SYTO-42at least one of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, TO-PRO-1 Iodide, TO-PRO-3 Iodide, TOTO-1 Iodide, TOTO-3 Iodide, YO-PRO-1 Iodid, YO-PRO-3 Iodide, YOYO-1 Iodide, YOYO-3 Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, ethidium bromide.

[0120] The permeable nucleic acid dye is a nucleic acid dye that can permeate through the surface of the non-viral nucleic acid carrier into the inside without using other agents, methods for enhancing the surface permeability of the non-viral nucleic acid carrier. That is, the so-called permeability refers to the permeability with respect to the inside and outside of the nucleic acid carrier.

[0121] As the first nucleic acid dye (or permeant nucleic acid dye), at least one selected from the group consisting of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide can be exemplified.

[0122] In some embodiments, the permeant nucleic acid dye is selected from the group consisting of SYTO 9, SYTO 13, SYTO 16, SYTO 24. In some embodiments, the permeant nucleic acid dye is selected from the group consisting of SYTO 9 and SYTO 16, preferably SYTO 9.

[0123] The final concentration of the permeant nucleic acid dye at the time of labeling can be exemplified as 0.001-10 μM. The final concentration of the permeant nucleic acid dye at the time of labeling is, for example, 0.001 μM, 0.002 μM, 0.1 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, or 10 μM.

[0124] As the labeling, mixing the nucleic acid dye with the composition or the sample containing the composition or the dilution of the sample and incubating can be exemplified. As the time of incubation, there is no particular limitation, and 5 minutes-20 minutes, preferably 20 minutes can be exemplified. As the temperature of incubation, there is no particular limitation, and 20-40°C, preferably room temperature-37°C can be exemplified.

[0125] Optionally, after the incubation, further dilution can be performed on the liquid containing the non-viral nucleic acid carrier composition to be tested: for example, dilution using a buffer such as PBS buffer, for example, it can be diluted by about 100-1000 times.

[0126] Step (2a): using a flow particle detection device, detecting the fluorescence intensity θ of each of the free nucleic acids in the sample volume V1 of the mixture 1 at the sample injection pressure and the sample injection time, i.e., obtaining the fluorescence intensity data θ1-θ n1, and the fluorescence intensity γ of each non-viral nucleic acid carrier in the mixture 1 before the nuclease treatment, i.e., the fluorescence intensity data γ1~γ n2 of the non-viral nucleic acid carrier in the mixture 1, and then calculating the median value M1 of the fluorescence intensity θ1~θ n1 of the free nucleic acid, wherein n1 is the number of the free nucleic acid in the injection volume V1 of the mixture 1 under the certain injection pressure and the certain injection time, and n2 is the number of the non-viral nucleic acid carrier in the injection volume V1 of the mixture 1 under the certain injection pressure and the certain injection time.

[0127] The flow particle detection device can be a particle analysis detection device capable of achieving directional flow of a sample stream. The flow particle detection device can include a sample loading unit and a flow unit, and a particle analysis detection device. The particle analysis detection device can include an optical system and a particle detector, and the particle detector can include a photosensor and a signal conditioning circuit with a band-pass filter high-frequency noise function.

[0128] The flow particle detection device can be a flow nano detector. The flow nano detector can be a Flow NanoAnalyzer (manufactured by Xiamen Fu Li Biotechnology Co., Ltd.).

[0129] As for the certain injection pressure, in the present method and the following method, the certain injection pressure of each mixture can be the same or different, and can be independently selected from 0.1 kPa to 10.0 kPa, preferably 0.5 kPa to 2.0 kPa, and more preferably 1 kPa.

[0130] As for the certain injection time, in the present method and the following method, the certain injection time of each mixture can be the same or different, and can be independently selected from 0.1 min to 10.0 min, and preferably 2 min.

[0131] As for the injection volume, in the present method and the following method, the injection volume of each mixture can be the same or different.

[0132] The fluorescence intensity (e.g., represented by θ, γ, or Z) can be detected under conditions such as a laser detector 488 nm + 638 nm; single laser channel detection Laser: 10 / 50 mW, 488 nm. It should be noted that the wavelength of the detection laser detector can be adjusted according to the type of nucleic acid dye used. The fluorescence intensity can be obtained by the FITC channel of the device. The FITC fluorescence channel is a nucleic acid dye excitation signal, which is used to represent the mRNA-lipid nanoparticle signal and the free nucleic acid signal.

[0133] The number of the free nucleic acids and the number of non-viral nucleic acid carrier particles, for example, the number of non-viral nucleic acid carrier particles positive for nucleic acid signal and negative for nucleic acid signal, can be obtained by flow particle detection equipment, for example, under the conditions of laser detector 488nm+638nm; single laser channel detection Laser: 20 / 50mW, 488nm; scattered light attenuation: 0.2%; sample pressure: 1kpa; signal type: Large signal, using the scattered light channel, and the same below.

[0134] Step (3a): taking mixture 1, adding a nuclease to degrade the free nucleic acids and the nucleic acids adhered to the surface of the non-viral nucleic acid carrier composition, to obtain mixture 2-A; or taking the sample containing the non-viral nucleic acid carrier composition to be tested, adding a nuclease to degrade the free nucleic acids and the nucleic acids adhered to the surface of the non-viral nucleic acid carrier composition, and then labeling with the nucleic acid dye to obtain mixture 2-B.

[0135] Here, since the nuclease cannot penetrate the non-viral nucleic acid carrier, in mixture 2-A or mixture 2-B, the non-viral nucleic acid carrier internal nucleic acid in the non-viral nucleic acid carrier is not degraded, and the same below.

[0136] Since the nucleic acid dye needs to show fluorescence after binding or intercalating with the surface structure of the nucleic acid molecule, when using the nuclease treatment, since the free nucleic acids and the nucleic acids adhered to the surface of the non-viral nucleic acid carrier composition are degraded, even if the fragments of the free nucleic acids and the nucleic acids adhered to the surface of the non-viral nucleic acid carrier composition exist in the system with the nucleic acid dye, no fluorescence will be generated, and thus no background fluorescence is formed.

[0137] The nuclease can be at least one selected from the group consisting of recombinant DNase I (RNase-free), Dnase, RNase-free, DNase I (RNase-free), S1 nuclease, dsDNase, RNase A, RNase A (without DNase and protease), and combinations thereof, wherein it is preferred to comprise at least one selected from the group consisting of recombinant DNase I (RNase-free), RNase A, RNase A (without DNase and protease), or a combination of DNase I, S1 nuclease, 1X dsDNase.

[0138] As specific examples of the nuclease, the following can be mentioned but are not limited to: recombinant DNase I (RNase-free) (TKR-2270A, Takara Bio); DNase, RNase-free (EN0523, Thermo); DNase I (RNase-Free) (M0303S, NEB); Deoxyribonuclease I (DNase, Bovine Pancreas) (S10073-250mg, Sigma-Aldrich); Exonuclease I (2170A, Takara Bio); Exonuclease III (2650A, Takara Bio); S1 nuclease (EN0321, Thermo); 1X dsDNase (ENO771, Thermo); RNase A (ST576, Bioworld); RNase A (DNase-free and Protease-free) (EN0531, Thermo).

[0139] After the addition of the nuclease, the mixture is incubated with the nuclease. The time for which the nuclease is incubated with the composition is not particularly limited and can be mentioned as 30 to 60 minutes, preferably 60 minutes. The temperature for which the nuclease is incubated with the composition is not particularly limited and can be mentioned as 20 to 40°C, preferably room temperature 37°C.

[0140] As the final concentration of the nuclease, 0.05 to 60 U / μl can be mentioned.

[0141] In step (3a), the labeling of the nucleic acid dye can be performed after the addition of the nuclease or before the addition of the nuclease, and the method and materials for the labeling can be the same as those described in step (1a). As a method for performing the labeling after the addition of the nuclease, the nucleic acid dye can be supplemented to the system after the enzymatic reaction, and the permeable nucleic acid dye is preferably supplemented.

[0142] Step (4a): The mixture 2-A or the mixture 2-B obtained in step (3a) is subjected to flow particle detection equipment under a certain injection pressure for a certain injection time to obtain the fluorescence intensity γ' of each non-viral nucleic acid vector particle having a nucleic acid signal positive after nuclease treatment in the injection volume V1 of the mixture 2-A or the mixture 2-B under a certain injection pressure for a certain injection time, i.e., to obtain the fluorescence intensity data γ1' ~ γ n3 ' of the non-viral nucleic acid vector particles having a nucleic acid signal positive after nuclease treatment, where n3 is the number of non-viral nucleic acid vector particles having a nucleic acid signal positive after nuclease treatment in the injection volume V1 of the mixture 2-A or the mixture 2-B under a certain injection pressure for a certain injection time.

[0143] The number of non-viral nucleic acid vector particles can be obtained in the same method and conditions as in step (1a).

[0144] Step (5a): Data analysis, calculation of drug loading and / or encapsulation efficiency.

[0145] The drug loading of the non-viral nucleic acid carrier composition refers to the weight percentage of the drug contained in the microparticle preparation. For example, drug loading = weight of drug contained in the microspheres / total weight of the microspheres x 100%.

[0146] The encapsulation efficiency of the non-viral nucleic acid carrier composition refers to the amount of encapsulated drug in the system / total amount of encapsulated and unencapsulated drug in the system x 100%.

[0147] In the present method, the formula for calculating the encapsulation efficiency is:

[0148] The formula for calculating the drug loading is:

[0149] where M is the relative molecular mass of the nucleic acid to be measured, NA is the Avogadro constant, V3 is the total volume of the sample containing the non-viral nucleic acid carrier composition to be measured, and d is the total weight of the sample containing the non-viral nucleic acid carrier composition to be measured after removing the solvent.

[0150] In the mathematical formula of the present application, when the summation symbol ∑ is involved, it means summing a series of numbers, where

[0151] Part A: upper limit of summation, part B: lower limit of summation, part C: content of summation.

[0152] For example, in the present application, when referring to means where i starts from 1 and takes values up to n1, i.e., the sum of each term from θ1 to θ n1 .

[0153] It should be noted that, as a method of the present application, although a method using the median value of fluorescence intensity for calculation is mentioned, it is not limited thereto. As long as a mathematical method that can utilize the fluorescence produced by free nucleic acid, externally adsorbed nucleic acid and internally encapsulated nucleic acid of the nucleic acid carrier, and calculate one or more indicators, other statistical methods for normalization that do not use the median value of fluorescence intensity can be used, and these methods are also included within the scope of the method of the present application.

[0154] In the specific embodiment 2, the method of the present application comprises:

[0155] Step (1b): Take the sample containing the non-viral nucleic acid carrier composition to be measured, and label it with a permeable nucleic acid dye that can specifically bind to nucleic acid, as mixture i.

[0156] As the non-viral nucleic acid carrier composition, the non-viral nucleic acid carrier, the solvent, the dilution condition, the use concentration, and the like, the same ones as in Embodiment 1 can be listed.

[0157] As the nucleic acid dye, the permeable nucleic acid dye, the labeling concentration, and the labeling method, the dilution condition, and the like, the same ones as in Step 1a) of Embodiment 1 can be listed.

[0158] Step (2b): A sample of the non-viral nucleic acid carrier composition to be tested is labeled with a non-permeable nucleic acid dye that specifically binds to nucleic acid, as a mixture ii.

[0159] As the non-viral nucleic acid carrier, the non-viral nucleic acid carrier composition, the sample of the non-viral nucleic acid carrier composition to be tested of Step (2b), the same ones as used in Step (1b) are used.

[0160] In Step (2b), the concentration of the non-permeable nucleic acid dye, the incubation time, and the temperature at the time of labeling with the non-permeable nucleic acid dye can be adjusted as appropriate and can be different from those at the time of labeling with the permeable nucleic acid dye in Step (1b).

[0161] The non-permeable nucleic acid dye (or the second nucleic acid dye) means a nucleic acid dye that cannot permeate through the surface of the non-viral nucleic acid carrier into the inside without using other reagents and methods that enhance the surface permeability of the non-viral nucleic acid carrier.

[0162] As the non-permeable nucleic acid dye, at least one selected from the group consisting of SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), and RiboGreen can be listed. Preferably, the non-permeable nucleic acid dye is one selected from the group consisting of SYTOX Green, Ribo Green, and Pico Green, and more preferably, it is selected from the group consisting of SYTOX Green and Ribo Green. In some embodiments, the non-permeable nucleic acid dye is SYTOX Green.

[0163] The final concentration when labeled with the non-permeant nucleic acid dye can be 0.001 to 10 μM. The final concentration when labeled with the non-permeant nucleic acid dye can be 0.001 μM, 0.002 μM, 0.1 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, or 10 μM.

[0164] As the labeling, mixing the non-permeant nucleic acid dye with the composition or the composition-containing sample or the dilution of the sample and incubating can be exemplified. As the time of incubation, there is no particular limitation, and 5 minutes to 20 minutes, preferably 20 minutes, can be exemplified. As the temperature of incubation, there is no particular limitation, and 20 to 40°C, preferably room temperature to 37°C, can be exemplified.

[0165] Optionally, after the incubation, the liquid containing the non-viral nucleic acid vector composition to be tested can be further diluted: for example, diluted by about 100 to 1000 times using a buffer such as a PBS buffer.

[0166] Step (3b): The mixture i is detected at a certain injection pressure for a certain injection time using a flow particle detection apparatus, and the fluorescence intensity θ' of each free nucleic acid and the fluorescence intensity γ" of each non-viral nucleic acid vector having a positive nucleic acid signal after permeant nucleic acid dye treatment in the injection volume V2 of the mixture i at a certain injection pressure for a certain injection time are obtained, i.e., the fluorescence intensity data θ1' to θ n4 ' of the free nucleic acid and the fluorescence intensity data γ1" to γ n5 " of the non-viral nucleic acid vector having a positive nucleic acid signal after permeant nucleic acid dye treatment are obtained, and the median value M2 of the fluorescence intensity θ1' to θ n4 ' of the free nucleic acid is calculated and recorded, wherein n4 is the number of free nucleic acids in the injection volume V2 of the mixture i at a certain injection pressure for a certain injection time, and n5 is the number of particles of the non-viral nucleic acid vector having a positive nucleic acid signal after permeant nucleic acid dye treatment in the injection volume V2 of the mixture i at a certain injection pressure for a certain injection time.

[0167] The mixture ii is detected at a certain injection pressure for a certain injection time using a flow particle detection apparatus, and the fluorescence intensity θ" of each free nucleic acid and the fluorescence intensity γ" of each non-viral nucleic acid vector having a positive nucleic acid signal after non-permeant nucleic acid dye treatment are obtained, i.e., the fluorescence intensity data θ1" to θ n6 " of the free nucleic acid and the fluorescence intensity data γ1"' to γ n7" and calculating the median value M3 of the fluorescence intensity θ1" to θ n6 " and calculating the median value M3 of the fluorescence intensity θ1" to θ

[0168] The flow particle detection apparatus, the sample injection pressure, the sample injection time, the sample injection volume, the measurement conditions of the fluorescence intensity, the channel, and the like can be the same as those in Embodiment 1.

[0169] As the fluorescence detection conditions after labeling with the non-permeable nucleic acid dye, for example, detection can be performed using the FITC channel under the conditions of laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW, 488nm; scattering light attenuation: 10%; sample injection pressure: 1kpa; signal type: small signal. Preferably, the solution of the sample containing the non-viral nucleic acid carrier composition is detected under the same sample injection pressure and the same sample injection time as in step (3b).

[0170] Note that, as the sample injection volumes of the mixture i and the mixture ii, although the use of the same volumes is exemplified, different sample injection volumes can be used, followed by a conventional mathematical normalization process.

[0171] Step (4b): data analysis, calculation of the drug loading and / or the encapsulation efficiency.

[0172] As the drug loading of the non-viral nucleic acid carrier composition, the weight percentage of the drug contained in the microparticle preparation is meant. For example, drug loading = weight of the drug contained in the microspheres / total weight of the microspheres x 100%.

[0173] As the encapsulation efficiency of the non-viral nucleic acid carrier composition, the amount of the drug encapsulated in the system / total amount of the encapsulated and unencapsulated drugs in the system x 100% is meant.

[0174] In the present method, the formula for calculating the encapsulation efficiency is:

[0175] Alternatively, the formula for calculating the encapsulation efficiency is:

[0176] The formula for calculating the drug loading is:

[0177] wherein M is the relative molecular mass of the nucleic acid to be measured, NA is the Avogadro constant, V3 is the total volume of the sample containing the non-viral nucleic acid carrier composition to be measured, and d is the total weight of the sample containing the non-viral nucleic acid carrier composition after removal of the solvent.

[0178] In some embodiments, the drug loading amount is calculated by the following formula:

[0179] wherein M is the relative molecular mass of the nucleic acid to be measured, NA is the Avogadro constant, V3 is the total volume of the sample of the non-viral nucleic acid carrier composition to be measured, and d is the total weight of the sample of the non-viral nucleic acid carrier composition after removal of the solvent.

[0180] The explanation of the summation symbol ∑ in the mathematical formula of the present application is the same as in Embodiment 1.

[0181] Note that, as the method of the present application, although a method using the median value of the fluorescence intensity for calculation is exemplified, the present application is not limited thereto. As long as a mathematical method capable of calculating the one or more indexes using the fluorescence generated by the free nucleic acid of the nucleic acid carrier, the externally adsorbed nucleic acid, and the internally encapsulated nucleic acid is used, other statistical methods for normalization that do not use the median value of the fluorescence intensity can be used, and these methods are also included in the scope of the method of the present application.

[0182] In Embodiment 3, the method of the present application is:

[0183] By the detection, when the nucleic acid signal-positive rate of the non-viral nucleic acid carrier of the non-viral nucleic acid carrier composition is ≤30% and the average copy number of the externally adsorbed nucleic acid is ≤5, the method includes the following steps (B) and / or (A).

[0184] The nucleic acid signal-positive rate of the non-viral nucleic acid carrier refers to the percentage of the number of particles of the non-viral nucleic acid carrier that are nucleic acid signal-positive with respect to the total number of particles of the non-viral nucleic acid carrier. The average copy number of the externally adsorbed nucleic acid refers to the copy number of the nucleic acid externally adsorbed to each non-viral nucleic acid carrier that is nucleic acid signal-positive, averaged with respect to the number of particles of the non-viral nucleic acid carrier that is nucleic acid signal-positive.

[0185] (B) Detection of the drug loading amount and / or the encapsulation efficiency of the non-viral nucleic acid carrier composition, which includes:

[0186] Step (1c): Take the sample of the non-viral nucleic acid carrier composition to be measured, and label it with a permeable nucleic acid dye that specifically binds to nucleic acid, as mixture iii.

[0187] The same as in Embodiment 1 can be exemplified for the non-viral nucleic acid carrier composition, the non-viral nucleic acid carrier, the solvent, the dilution, the use concentration, and the like.

[0188] The nucleic acid dye independently includes at least one selected from the group consisting of a permeable nucleic acid dye, a permeable cyanine dye, an intercalating dye, or a DNA double helix minor groove binding dye.

[0189] As the nucleic acid dye, the permeable nucleic acid dye, the labeling concentration and the labeling method, the dilution condition, etc., the same as those in Step 1a) of Embodiment 1 can be cited.

[0190] Step (2c): The mixture iii is detected at a certain injection pressure for a certain injection time using a flow particle detection apparatus to obtain the fluorescence intensity θ' of each free nucleic acid and the fluorescence intensity γ" of each non-viral nucleic acid vector having a positive nucleic acid signal after permeable nucleic acid dye treatment in the injection volume V2 of the mixture iii at the certain injection pressure for the certain injection time, i.e., the fluorescence intensity data θ1' ~ θ n4 ' of the free nucleic acid and the fluorescence intensity data γ1" ~ γ n5 " of the non-viral nucleic acid vector having a positive nucleic acid signal after permeable nucleic acid dye treatment, and the median value M2 of the fluorescence intensity θ1' ~ θ n4 ' of the free nucleic acid is calculated and recorded, wherein n4 is the number of free nucleic acids in the injection volume V2 of the mixture iii at the certain injection pressure for the certain injection time, and n5 is the number of particles of the non-viral nucleic acid vector having a positive nucleic acid signal after permeable nucleic acid dye treatment in the injection volume V2 of the mixture iii at the certain injection pressure for the certain injection time.

[0191] The flow particle detection apparatus, the injection pressure, the injection time, the injection volume, the measurement conditions of the fluorescence intensity, the channel, etc., can cite the same as those in Embodiment 1.

[0192] Step (3c): Data analysis to calculate the drug loading and / or the encapsulation efficiency.

[0193] As the drug loading of the non-viral nucleic acid vector composition, the weight percentage of the drug contained in the microparticle preparation is meant. For example, drug loading = weight of the drug contained in the microsphere / total weight of the microsphere x 100%.

[0194] As the encapsulation efficiency of the non-viral nucleic acid vector composition, the amount of the drug encapsulated in the system / the total amount of the encapsulated and unencapsulated drugs in the system x 100% is meant.

[0195] In the present method, the formula for calculating the encapsulation efficiency is:

[0196] or

[0197] or

[0198] The detection method can further include, before step (B), detecting the nucleic acid signal positive rate of the non-viral nucleic acid vector and the average number of externally adsorbed nucleic acids.

[0199] The nucleic acid signal positive rate of the non-viral nucleic acid vector refers to the percentage of the number of non-viral nucleic acid vector particles that are nucleic acid signal positive with respect to the total number of non-viral nucleic acid vector particles. The average number of externally adsorbed nucleic acids refers to the average number of copies of nucleic acids externally adsorbed per non-viral nucleic acid vector that is nucleic acid signal positive.

[0200] In some embodiments, the detection of the nucleic acid signal positive rate of the non-viral nucleic acid vector and the average number of externally adsorbed nucleic acids includes:

[0201] (A) detecting the nucleic acid signal positive rate of the non-viral nucleic acid vector and the average number of externally adsorbed nucleic acids:

[0202] Step (lk): Taking a sample of the non-viral nucleic acid vector-containing composition to be tested, and labeling it with a nucleic acid dye that specifically binds to nucleic acids, as Mixture 7; preferably, the nucleic acid dye is a non-permeable nucleic acid dye.

[0203] As the non-viral nucleic acid vector composition, the non-viral nucleic acid vector, the solvent, the dilution conditions, the use concentration, and the like, the same as those in Embodiment 1 can be cited.

[0204] As the nucleic acid dye, the permeable nucleic acid dye, the labeling concentration and method, the dilution conditions, and the like, the same as those in Step la) of Embodiment 1 can be cited.

[0205] Step (2k): Using a flow particle detection device to detect the fluorescence intensity Z” of each free nucleic acid, the fluorescence intensity Z”’ of each non-viral nucleic acid vector that is nucleic acid signal positive after treatment with a non-permeable nucleic acid dye, and the number n10 of non-viral nucleic acid vector particles that are nucleic acid signal negative, in the sample volume V6 of Mixture 7 taken at a certain sample pressure and for a certain sample time, i.e., to obtain the fluorescence intensity data Z1” ~ Z n8 ” of free nucleic acids and the fluorescence intensity data Z1”’ ~ Z n9 ”’ of non-viral nucleic acid vectors that are nucleic acid signal positive after treatment with a non-permeable nucleic acid dye, and to calculate the median value M5 of the recorded fluorescence intensity Z1” ~ Z n8 ” of free nucleic acids, where n8 is the number of free nucleic acids in the sample volume V6 of Mixture 7 taken at a certain sample pressure and for a certain sample time, and n9 is the number of non-viral nucleic acid vectors that are nucleic acid signal positive after treatment with a non-permeable nucleic acid dye in the sample volume V6 of Mixture 7 taken at a certain sample pressure and for a certain sample time.

[0206] The flow particle detection device, sample introduction pressure, sample introduction time, sample introduction volume, measurement conditions of the fluorescence intensity, channel, and the like can be exemplified as those in Embodiment 1.

[0207] Step (3k): calculating the nucleic acid signal positive rate and the average copy number of externally adsorbed nucleic acid of the non-viral nucleic acid carrier;

[0208] The method for calculating the nucleic acid signal positive rate of the non-viral nucleic acid carrier is:

[0209] The method for calculating the average copy number of externally adsorbed nucleic acid is:

[0210] The explanation of the summation symbol ∑ in the mathematical formula of the present application is the same as in Embodiment 1.

[0211] It should be noted that, as the method of the present embodiment, other statistical methods for normalization that do not use the median of the fluorescence intensity can be used, and these methods are also included in the scope of the method of the present application.

[0212] In one embodiment, when it is determined that the nucleic acid signal positive rate of the non-viral nucleic acid carrier to be tested is ≤30% and the average copy number of externally adsorbed nucleic acid is ≤5, or some of the samples of the batch already meet the condition that the nucleic acid signal positive rate of the non-viral nucleic acid carrier is ≤30% and the average copy number of externally adsorbed nucleic acid is ≤5, the drug loading and / or encapsulation efficiency of the non-viral nucleic acid carrier composition can be detected according to the step (B).

[0213] The formula for calculating the encapsulation efficiency is:

[0214] The formula for calculating the drug loading is:

[0215] wherein M is the relative molecular mass of the nucleic acid to be tested, NA is the Avogadro constant, V3 is the total volume of the sample containing the non-viral nucleic acid carrier composition to be tested, and d is the total weight of the sample containing the non-viral nucleic acid carrier composition after removal of the solvent.

[0216] It should be noted that, as the method of the present embodiment, other statistical methods for normalization that do not use the median of the fluorescence intensity can be used, and these methods are also included in the scope of the method of the present application.

[0217] The present application includes the following.

[0218] Item 1. A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid carrier composition,

[0219] The method comprises:

[0220] labeling with a first nucleic acid dye in combination with the non-viral nucleic acid carrier composition to be tested to obtain a mixture 1;

[0221] detecting the mixture 1 of the volume V1 by a flow particle detection device to obtain the fluorescence intensity of each of the free nucleic acids in the mixture 1 of the volume V1, i.e. the fluorescence intensity of the free nucleic acids θ1~θn1, and the fluorescence intensity of each of the non-viral nucleic acid carriers with positive nucleic acid signal in the mixture 1 of the volume V1, i.e. the fluorescence intensity of the non-viral nucleic acid carriers with positive nucleic acid signal γ1~γn2; n1 n2 wherein n1 is the number of the free nucleic acids in the mixture 1 of the volume V1, and n2 is the number of the particles of the non-viral nucleic acid carriers with positive nucleic acid signal in the mixture 1 of the volume V1;

[0222] calculating the drug loading and / or the encapsulation efficiency of the non-viral nucleic acid carrier composition according to the fluorescence intensity data,

[0223] wherein the first nucleic acid dye is a permeable nucleic acid dye.

[0224] Item 2. The method according to item 1, further comprising the following steps:

[0225] taking the mixture 1 and adding a nuclease to obtain a mixture 2-A, or taking the sample containing the non-viral nucleic acid carrier composition to be tested and adding a nuclease to obtain a mixture 2-B, and then labeling with the first nucleic acid dye;

[0226] detecting the mixture 2-A or the mixture 2-B of the volume V1 by a flow particle detection device to obtain the fluorescence intensity of each of the non-viral nucleic acid carriers with positive nucleic acid signal in the mixture 2-A or the mixture 2-B of the volume V1, i.e. the fluorescence intensity of the non-viral nucleic acid carriers with positive nucleic acid signal γ’1~γ’n3; n3 wherein n3 is the number of the particles of the non-viral nucleic acid carriers with positive nucleic acid signal in the mixture 2-A or the mixture 2-B of the volume V1;

[0227] calculating the drug loading and / or the encapsulation efficiency of the non-viral nucleic acid carrier composition according to the fluorescence intensity data,

[0228] wherein the encapsulation efficiency is calculated according to formula 1

[0229] wherein the i in the formula above represents i in the formula above represents i from 1 to n1, i.e. θ1 to θn1, and i in the formula above represents i from 1 to n2, i.e. γ1 to γn2. n1 The same applies to the following.

[0230] the drug loading is calculated according to formula 2​​

[0231] M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample of the non-viral nucleic acid carrier composition to be detected, and d is the total weight of the sample of the non-viral nucleic acid carrier composition after removing the solvent.

[0232] M1 is the median value of the fluorescence intensity θ1~θ n1 of the free nucleic acid.

[0233] Item 3. The method according to item 1, further comprising:

[0234] detecting the nucleic acid signal positive rate and the average copy number of the externally adsorbed nucleic acid of the nucleic acid carrier,

[0235] when the nucleic acid signal positive rate of the nucleic acid carrier is ≤ 30% and the average copy number of the externally adsorbed nucleic acid is ≤ 5, the encapsulation efficiency is calculated according to formula 11:

[0236] The formula for calculating the drug loading amount is:

[0237] M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample of the non-viral nucleic acid carrier composition to be detected, and d is the total weight of the sample of the non-viral nucleic acid carrier composition after removing the solvent.

[0238] M1 is the median value of the fluorescence intensity θ1~θ n1 of the free nucleic acid.

[0239] 4. The method according to item 3, wherein the step of detecting the nucleic acid signal positive rate and the average copy number of the externally adsorbed nucleic acid of the nucleic acid carrier comprises:

[0240] The non-viral nucleic acid carrier composition to be detected is labeled by contacting with the first nucleic acid dye to obtain mixture 7;

[0241] A volume V6 of mixture 7 is injected into the flow particle detection device, and the fluorescence intensity Z” of each free nucleic acid in the injection volume V6 of mixture 7, i.e. the fluorescence intensity data Z1”~Z n8 ” of the free nucleic acid, and the fluorescence intensity Z”’ of each nucleic acid signal positive non-viral nucleic acid carrier in the injection volume V6 of mixture 7, i.e. the fluorescence intensity data Z1”’~Z n9 ”’ of the nucleic acid signal positive non-viral nucleic acid carrier are obtained.

[0242] n10 is the number of particles of non-viral nucleic acid carriers with nucleic acid signal negative in the mixture 7 of the sample volume V6, n8 is the number of free nucleic acids in the mixture 7 of the sample volume V6, and n9 is the number of particles of non-viral nucleic acid carriers with nucleic acid signal positive in the mixture 7;

[0243] calculating the nucleic acid signal positive rate of the non-viral nucleic acid carriers and the average copy number of surface-adsorbed nucleic acids;

[0244] The nucleic acid signal positive rate of the non-viral nucleic acid carriers is calculated according to formula 8:

[0245] The average copy number of surface-adsorbed nucleic acids is calculated according to formula 9:

[0246] M5 is the fluorescence intensity Z1”-Z n8 of the free nucleic acids.

[0247] 5. A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid carrier composition,

[0248] The method comprises:

[0249] labeling the non-viral nucleic acid carrier composition to be detected with a first nucleic acid dye to obtain a mixture i;

[0250] feeding the mixture i of the sample volume V2 into a flow particle detection device for detection to obtain the fluorescence intensity θ’ of each free nucleic acid in the mixture i of the sample volume V2, i.e. the fluorescence intensity θ1’-θ n4 of the free nucleic acids, and the fluorescence intensity γ” of each non-viral nucleic acid carrier with nucleic acid signal positive in the mixture i of the sample volume V2, i.e. the fluorescence intensity γ1”-γ n5 ” of the non-viral nucleic acid carriers with nucleic acid signal positive; wherein n4 is the number of free nucleic acids in the mixture i of the sample volume V2, and n5 is the number of particles of non-viral nucleic acid carriers with nucleic acid signal positive in the mixture i of the sample volume V2;

[0251] labeling the non-viral nucleic acid carrier composition to be detected with a second nucleic acid dye to obtain a mixture ii;

[0252] feeding the mixture ii of the sample volume V2 into a flow particle detection device for detection to obtain the fluorescence intensity θ” of each free nucleic acid in the mixture ii of the sample volume V2, i.e. the fluorescence intensity θ”-θ n6 ” of the free nucleic acids, and the fluorescence intensity γ” of each non-viral nucleic acid carrier with nucleic acid signal positive after the second nucleic acid dye treatment in the mixture i of the sample volume V2, i.e. the fluorescence intensity data γ1”’-γn7 wherein n6 is the number of free nucleic acids in the mixture ii of the injection volume V2, and n7 is the number of non-viral nucleic acid vector particles with positive nucleic acid signal in the mixture ii of the injection volume V2;

[0253] calculating the drug loading and / or the encapsulation efficiency of the non-viral nucleic acid vector composition according to the fluorescence intensity data,

[0254] wherein the first nucleic acid dye is a permeant nucleic acid dye, and the second nucleic acid dye is a non-permeant nucleic acid dye,

[0255] The formula for calculating the encapsulation efficiency is

[0256] or

[0257] and / or

[0258] The formula for calculating the drug loading is:

[0259] or

[0260] wherein M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample to be tested containing the non-viral nucleic acid vector composition, d is the total weight of the sample containing the non-viral nucleic acid vector composition after removing the solvent;

[0261] M2 is the median value of the fluorescence intensity θ1’~θ n4 ’ of the free nucleic acids, and M3 is the median value of the fluorescence intensity θ1”~θ n6 ” of the free nucleic acids.

[0262] 6. The method according to any one of items 1-5, wherein the first nucleic acid dye is selected from at least one of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide.

[0263] 7. The method according to item 6, wherein the first nucleic acid dye is selected from the group consisting of SYTO 9, SYTO 13, SYTO 16, SYTO 24, preferably from SYTO 9, SYTO 16.

[0264] 8. The detection method according to any one of items 5-7, wherein the second nucleic acid dye is selected from at least one of SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), RiboGreen, preferably from one of SYTOX Green, Ribo Green, Pico Green, more preferably from SYTOX Green, Ribo Green.

[0265] 9. The detection method according to item 2, wherein the nuclease is one or more selected from the group consisting of recombinant DNase I (RNase-free), Dnase, RNase-free, DNase I (RNase-free), S1 nuclease, dsDNase, RNase A, RNase A (DNase and protease free); preferably the nuclease comprises at least one selected from the group consisting of recombinant DNase I (RNase-free), RNase A, RNase A (DNase and protease free), or is a combination selected from DNase I, S1 nuclease, 1X dsDNase.

[0266] 10. The detection method according to any one of items 1-9, wherein the nucleic acid species to be detected encapsulated in the non-viral nucleic acid vector composition is selected from the group consisting of DNA, mRNA, siRNA, ASO (antisense oligonucleotide), circular RNA, double-stranded DNA plasmid,

[0267] wherein the size of the nucleic acid to be detected is greater than 200-300 bp / nt, preferably 200-10000 bp / nt, more preferably 400-7000 bp / nt.

[0268] 11. The detection method according to any one of items 1-10, wherein the non-viral nucleic acid vector is an mRNA-LNP.

[0269] 12. The detection method according to item 11, wherein the mRNA-LNP comprises one or more molecules selected from the group consisting of:

[0270] mRNA molecules encoding target proteins (such as antigen peptides) from a virus selected from the group consisting of herpes simplex virus, poxvirus, rabies virus, influenza virus, adenovirus, enterovirus, rotavirus, SARS-CoV-2, dengue virus, human syncytial virus, monkeypox virus, porcine circovirus; mRNA molecules encoding one or more immunostimulatory molecules, one or more pathogenic antigens; non-coding regions of a virus; (major histocompatibility complex) MHC binding peptides;

[0271] Optionally, the mRNA is modified with nucleoside modifications or unmodified.

[0272] 13. The detection method according to any one of items 1-12, wherein the first nucleic acid dye is labeled at a final concentration of 0.001 mM, 0.002 mM, 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM or 10 mM, preferably at a final concentration of 0.001-10 mM, more preferably 1 mM.

[0273] 14. The detection method according to any one of items 1-13, wherein the first nucleic acid dye is incubated with the composition for a time period of 5-20 minutes, preferably 20 minutes.

[0274] 15. The detection method according to any one of items 1-14, wherein the first nucleic acid dye is incubated with the composition at a temperature of 20-40 °C, preferably 37 °C.

[0275] 16. The detection method according to item 2 or 9, wherein the nuclease is incubated with the composition at a final concentration of 0.05-60 U / µl.

[0276] 17. The detection method according to item 2 or 9, wherein the nuclease is incubated with the composition for a time period of 30-60 minutes, preferably 60 minutes.

[0277] 18. The detection method according to item 2 or 9, wherein the nuclease is incubated with the composition at a temperature of 20-40 °C, preferably 37 °C.

[0278] 19. The method according to any one of items 1-18, comprising taking pictures, counting and signal intensity value measurements of the sample, measuring images of particles having specific optical characteristics.

[0279] 20. The method according to any one of items 1-19, wherein the sample injection pressure of the mixture is independently selected from 0.1 kPa-10.0 kPa, preferably 0.5 kPa-2.0 kPa, respectively.

[0280] 21. The method of any one of claims 1-20, wherein the injection time of the mixture is independently selected from the group consisting of 0.1 min to 10.0 min.

[0281] 22. The method of any one of claims 1-21, wherein the non-viral nucleic acid vector composition comprises: non-viral nucleic acid vectors adsorbing nucleic acid externally, non-viral nucleic acid vectors encapsulating nucleic acid internally, non-viral nucleic acid vectors adsorbing nucleic acid externally and encapsulating nucleic acid internally, empty non-viral nucleic acid vectors, free nucleic acid, solvent.

[0282] 23. The method of any one of claims 1-22, wherein the flow particle detection device is a particle analysis detection device capable of achieving directional flow of sample stream, the flow particle detection device comprises a directional fluid system and a particle analysis detection device, wherein

[0283] the directional fluid system consists of a sample loading unit and a flow unit;

[0284] the particle analysis detection device comprises an optical system and a particle detector;

[0285] the particle detector consists of a photosensor and a signal conditioning circuit with a band-pass filter high-frequency noise function.

[0286] Item 1.1. A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid vector composition, comprising:

[0287] Step (1a): taking a sample containing the non-viral nucleic acid vector composition to be tested, labeling it with a nucleic acid dye that can specifically bind to nucleic acid, as mixture 1;

[0288] Preferably, the nucleic acid dye is a permeable nucleic acid dye;

[0289] Step (2a): using a flow particle detection device to detect the fluorescence intensity of each free nucleic acid in the injection volume V1 of mixture 1 at a certain injection pressure and injection time, i.e. to obtain the fluorescence intensity data θ1~θ n1 of free nucleic acid, and the fluorescence intensity of each non-viral nucleic acid vector that is positive for nucleic acid signal before nuclease treatment in the injection volume V1 of mixture 1 at a certain injection pressure and injection time, i.e. to obtain the fluorescence intensity data γ1~γ n2 of non-viral nucleic acid vectors; and then calculating the median value M1 of the fluorescence intensity θ1~θ n1 of free nucleic acid, wherein n1 is the number of free nucleic acids in the injection volume V1 of mixture 1 at a certain injection pressure and injection time, and n2 is the number of non-viral nucleic acid vectors in the injection volume V1 of mixture 1 at a certain injection pressure and injection time;

[0290] Step (3a): taking mixture 1, adding nuclease to degrade free nucleic acid and nucleic acid adhered to the surface of non-viral nucleic acid carrier composition, to obtain mixture 2-A; or taking sample containing non-viral nucleic acid carrier composition to be tested, adding nuclease to degrade free nucleic acid and nucleic acid adhered to the surface of non-viral nucleic acid carrier composition, and then labeling with the nucleic acid dye to obtain mixture 2-B;

[0291] Step (4a): using the flow particle detection device to detect mixture 2-A or mixture 2-B obtained in step (3a) at a certain sample injection pressure for a certain sample injection time, to obtain the fluorescence intensity γ' of each non-viral nucleic acid carrier particle with nucleic acid signal positive after nuclease treatment in the sample volume V1 of mixture 2-A or mixture 2-B at a certain sample injection pressure for a certain sample injection time, i.e. to obtain the fluorescence intensity data γ1' ~ γ n3 ' of non-viral nucleic acid carrier particles with nucleic acid signal positive after nuclease treatment, wherein n3 is the number of non-viral nucleic acid carrier particles with nucleic acid signal positive after nuclease treatment in the sample volume V1 of mixture 2-A or mixture 2-B at a certain sample injection pressure for a certain sample injection time.

[0292] Step (5a): data analysis to calculate drug loading and / or encapsulation efficiency;

[0293] Wherein, the certain sample injection pressure used in step (2a) for detecting mixture 1 is the same as the certain sample injection pressure used in step (4a) for detecting mixture 2-A or mixture 2-B; the certain sample injection time used in step (2a) for detecting mixture 1 is the same as the certain sample injection time used in step (4a) for detecting mixture 2-A or mixture 2-B.

[0294] Item 1.2. A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid carrier composition, comprising:

[0295] Step (1b): taking sample containing non-viral nucleic acid carrier composition to be tested, labeling with permeable nucleic acid dye, as mixture i, which can specifically bind to nucleic acid;

[0296] Step (2b): taking sample containing non-viral nucleic acid carrier composition to be tested, labeling with non-permeable nucleic acid dye, as mixture ii, which can specifically bind to nucleic acid;

[0297] Step (3b): using the flow particle detection device to detect mixture i at a certain sample injection pressure for a certain sample injection time, to obtain the fluorescence intensity θ' of each free nucleic acid and the fluorescence intensity γ" of each non-viral nucleic acid carrier with nucleic acid signal positive after permeable nucleic acid dye treatment in the sample volume V2 of mixture i at a certain sample injection pressure for a certain sample injection time, i.e. to obtain the fluorescence intensity data θ1' ~ θ n4' and the fluorescence intensity data of non-viral nucleic acid carriers with positive nucleic acid signals after treatment with permeable nucleic acid dyes γ1"~γ n5 ", and calculate and record the fluorescence intensity of free nucleic acid θ1'~θ n4 ', wherein n4 is the number of free nucleic acids in the mixture i with a certain injection pressure and a certain injection time, and n5 is the number of non-viral nucleic acid carrier particles with positive nucleic acid signals after treatment with a permeable nucleic acid dye in the mixture i with a certain injection pressure and a certain injection time; a flow particle detection device is used to detect the mixture ii with a certain injection pressure and a certain injection time, and the fluorescence intensity θ" of each free nucleic acid in the mixture ii with a certain injection pressure and a certain injection time and the fluorescence intensity γ" of each non-viral nucleic acid carrier with positive nucleic acid signals after treatment with a non-permeable nucleic acid dye are obtained, that is, the fluorescence intensity data θ1" to θ n6 " and fluorescence intensity data of non-viral nucleic acid carriers with positive nucleic acid signals after treatment with non-permeable nucleic acid dyes γ1"'~γ n7 ”', and calculate and record the fluorescence intensity of free nucleic acid θ1”~θ n6 "The median value M3, where n6 is the number of free nucleic acids in the injection volume V2 mixture ii at a certain injection pressure and injection time, and n7 is the number of non-viral nucleic acid carriers with positive nucleic acid signals after treatment with a non-permeable nucleic acid dye in the injection volume V2 mixture ii at a certain injection pressure and injection time;

[0298] Step (4b): data analysis, calculation of drug loading and / or encapsulation efficiency;

[0299] In which, the certain injection pressure used in step (3b) when detecting mixture i is the same as the certain injection pressure used in detecting mixture ii; the certain injection time used in step (3b) when detecting mixture i is the same as the certain injection time used when detecting mixture ii.

[0300] Item 1.3. A method for detecting the drug loading capacity and / or encapsulation efficiency of a non-viral nucleic acid vector composition, wherein the non-viral nucleic acid vector composition is labeled with a non-permeable nucleic acid dye, and the nucleic acid signal positivity rate of the non-viral nucleic acid vector is ≤30% and the average copy number of surface-adsorbed nucleic acid is ≤5, and the detection method is performed comprising the following steps:

[0301] (B) Detection of drug loading and / or encapsulation efficiency of the non-viral nucleic acid vector composition, comprising:

[0302] Step (1c): taking a sample containing the non-viral nucleic acid vector composition to be tested and labeling it with a permeable nucleic acid dye as mixture iii, wherein the permeable nucleic acid dye can specifically bind to nucleic acid;

[0303] Step (2c): detecting the fluorescence intensity of each free nucleic acid and the fluorescence intensity of each non-viral nucleic acid carrier with positive nucleic acid signal after permeable nucleic acid dye treatment in the injection volume V2 of mixture iii at a certain injection pressure and injection time by using a flow particle detection device, i.e. obtaining the fluorescence intensity data θ1'~θ n4 ' of free nucleic acids and the fluorescence intensity data γ1"~γ n5 " of non-viral nucleic acid carriers with positive nucleic acid signal after permeable nucleic acid dye treatment, and calculating and recording the median value M2 of the fluorescence intensity θ1'~θ n4 ' of free nucleic acids, wherein n4 is the number of free nucleic acids in the injection volume V2 of mixture iii at a certain injection pressure and injection time, and n5 is the number of particles of non-viral nucleic acid carriers with positive nucleic acid signal after permeable nucleic acid dye treatment in the injection volume V2 of mixture iii at a certain injection pressure and injection time;

[0304] Step (3c): data analysis to calculate the drug loading and / or encapsulation efficiency;

[0305] Optionally, the detection method further comprises detecting the positive rate of nucleic acid signal and the average copy number of surface-adsorbed nucleic acids of non-viral nucleic acid carriers before step (B).

[0306] Optionally, the detection of the positive rate of nucleic acid signal and the average copy number of surface-adsorbed nucleic acids of non-viral nucleic acid carriers comprises:

[0307] (A) detecting the positive rate of nucleic acid signal and the average copy number of surface-adsorbed nucleic acids of non-viral nucleic acid carriers, which comprises:

[0308] Step (1k): taking a sample of the composition containing non-viral nucleic acid carriers to be tested, and labeling with a nucleic acid dye that can specifically bind to nucleic acids, as mixture 7; preferably, the nucleic acid dye is a non-permeable nucleic acid dye;

[0309] Step (2k): detecting and recording the fluorescence intensity Z1"~Z n8 " of each free nucleic acid, the fluorescence intensity Z1"'~Z n9 "' of each non-viral nucleic acid carrier with positive nucleic acid signal after non-permeable nucleic acid dye treatment, and the number n10 of non-viral nucleic acid carrier particles with negative nucleic acid signal in the injection volume V6 of mixture 7 at a certain injection pressure and injection time by using a flow particle detection device, i.e. obtaining the fluorescence intensity data Z1"~Z n8"The median value M5, where n8 is the number of free nucleic acids in the injection volume V6 mixture 7 at a certain injection pressure and injection time, and n9 is the number of non-viral nucleic acid carriers with positive nucleic acid signals after treatment with a non-permeable nucleic acid dye in the injection volume V6 mixture 7 at a certain injection pressure and injection time;

[0310] Step (3k) calculates the nucleic acid signal positive rate of the non-viral nucleic acid vector and the average copy number of the surface-adsorbed nucleic acid;

[0311] When the nucleic acid signal positive rate of the non-viral nucleic acid vector is ≤30% and the average copy number of the surface adsorbed nucleic acid is ≤5, the drug loading capacity and / or encapsulation efficiency of the non-viral nucleic acid vector composition is tested according to step (B).

[0312] Item 1.4. A method for detecting the empty shell ratio, nucleic acid copy number distribution, and / or nucleic acid copy number and particle size distribution of a non-viral nucleic acid vector composition, comprising:

[0313] Step (1d): taking a sample containing the non-viral nucleic acid vector composition to be tested and labeling it with a nucleic acid dye as mixture 3, wherein the nucleic acid dye can specifically bind to nucleic acid; preferably, the nucleic acid dye is a permeable nucleic acid dye;

[0314] Step (2d): Use a flow particle detection device to detect the number of non-viral nucleic acid carrier particles P1 with positive nucleic acid signals in the injection volume V4 mixture 3 at a certain injection pressure and a certain injection time, and the fluorescence intensity Z1 to Z1 of each non-viral nucleic acid carrier with positive nucleic acid signals. n13 , the number of non-viral nucleic acid carrier particles with negative nucleic acid signals P2 and the fluorescence intensity of free nucleic acid Z1'~Z n14 ', calculate the fluorescence intensity of free nucleic acid Z1'~Z n14 'Median value M4;

[0315] Step (3d): Prepare particle size standard solutions of particle size standards of different particle sizes, use a flow particle detection device to detect the particle size standard solution, record the scattered light channel detection data of the particle size standard solution, and record the particle size S1 to S2 of each non-viral nucleic acid carrier with a positive nucleic acid signal. n13 ;

[0316] Step (4d): data analysis, calculation of the empty shell rate, plotting of the nucleic acid copy number distribution graph and / or plotting of the nucleic acid copy number and particle size distribution graph.

[0317] Item 1.5. According to the detection method according to any one of Items 1.1, 1.3 or 1.4, the nucleic acid dye includes at least one selected from permeable nucleic acid dyes, cyanine dyes, intercalating dyes or DNA double helix minor groove binding dyes.

[0318] Clause 1.6. The detection method according to any of clauses 1.1, 1.3 or 1.4, said nucleic acid dye is selected from a cyanine dye, a non-permeant dye, a permeant dye, an intercalating dye or a DNA double helix minor groove binder dye; preferably, said nucleic acid dye is a permeant nucleic acid dye; and / or

[0319] the nucleic acid dye is selected from the group consisting of Acridine Orange, Actinomycin D, 7-AAD (7-aminoactinomycin D), ACMA (9-Amino-6-Chloro-2- Methoxyacridine), BOBO-1 Iodid, BOBO-3 Iodide, DAPI (4',6-Diamidino-2- Phenylindole, Dihydrochloride), Dihydroxyethidium (hydroethidine), Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride, Hoechst 34580, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341, Trihydrochloride, Hoechst 33342, Trihydrochloride, Hoechst 33341at least one of Ethidium Bromide, Ethidium Bromide, Trihydrate, Trihydrate-FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace Blue Fluorescent Nissl Stain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615 Red Fluorescent Nissl Stain, NeuroTrace Deep-Red Fluorescent Nissl Stain, POPO-1 Iodide, POPO-3 Iodide, PO-PRO-1 Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe DNA Gel Stain, SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, TO-PRO-1 Iodide, TO-PRO-3 Iodide, TOTO-1 Iodide, TOTO-3 Iodide, YO-PRO-1 Iodid, YO-PRO-3 Iodide, YOYO-1 Iodide, YOYO-3 Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, and / or,

[0320] the permeant nucleic acid dye is selected from at least one of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide; and / or

[0321] the permeant nucleic acid dye is SYTO 9.

[0322] Item 1.7. The detection method according to any one of items 1.1-1.6, wherein the step (4a), step (3b), step (2c), step (2d) or step (2k) comprises taking images of the particles having the specific optical characteristics, counting and signal intensity value measurement of the particles in the sample using a flow-particle detection device; and / or

[0323] the certain injection pressure is independently selected from 0.1 kPa to 10.0 kPa, preferably 0.5 kPa to 2.0 kPa; and / or

[0324] the certain injection time is independently selected from 0.1 min to 10.0 min.

[0325] Item 1.8. The detection method according to item 1.2 or 1.3, wherein the permeant nucleic acid dye is selected from at least one of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide; and / or

[0326] The non-permeable nucleic acid dye is selected from at least one of SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), RiboGreen.

[0327] Item 1.9. The detection method according to item 1.1, wherein the calculation formula of the encapsulation efficiency is:

[0328] and / or,

[0329] The calculation formula of the drug loading amount is:

[0330] wherein M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample to be detected containing the non-viral nucleic acid carrier composition, and d is the total weight of the sample containing the non-viral nucleic acid carrier composition after removing the solvent.

[0331] Item 1.10. The detection method according to item 1.2, wherein the calculation formula of the encapsulation efficiency is:

[0332] or,

[0333] and / or,

[0334] The calculation formula of the drug loading amount is:

[0335] or,

[0336] wherein M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample to be detected containing the non-viral nucleic acid carrier composition, and d is the total weight of the sample containing the non-viral nucleic acid carrier composition after removing the solvent.

[0337] Item 1.11. The detection method according to item 1.3, wherein the calculation formula of the encapsulation efficiency is:

[0338] and / or,

[0339] The calculation formula of the drug loading amount is:

[0340] Wherein, M is the relative molecular mass of the target nucleic acid, NA is the Avogadro constant, V3 is the total volume of the sample containing the non-viral nucleic acid carrier composition to be detected, d is the total weight of the sample containing the non-viral nucleic acid carrier composition after removing the solvent;

[0341] and / or,

[0342] The calculation method of the nucleic acid signal positive rate of the non-viral nucleic acid carrier is:

[0343] and / or,

[0344] The calculation method of the average number of surface-adsorbed nucleic acid copies is:

[0345] Item 1.12. The detection method according to item 1.4, wherein the calculation formula of the empty shell rate is:

[0346] and / or,

[0347] The drawing method of the nucleic acid copy number distribution map is:

[0348] Step (1e): divide the fluorescence intensity Z1-Z n13 of each nucleic acid signal positive non-viral nucleic acid carrier by M4, respectively, to obtain the nucleic acid copy number C1-C n13 of each nucleic acid signal positive non-viral nucleic acid carrier.

[0349] Step (2e): draw a statistical histogram of C1-C n13 and the corresponding number of events, i.e. a nucleic acid copy number distribution map.

[0350] and / or,

[0351] The drawing method of the nucleic acid copy number and particle size distribution map is:

[0352] Step (1f): divide the fluorescence intensity Z1-Z n13 of each nucleic acid signal positive non-viral nucleic acid carrier by M4, respectively, to obtain the nucleic acid copy number C1-C n13 of each nucleic acid signal positive non-viral nucleic acid carrier.

[0353] Step (2f): draw a two-dimensional scatter plot of C1-C n13 and S1-S n13 one-to-one correspondence, i.e. a nucleic acid copy number and particle size distribution map.

[0354] Item 1.13. The method according to any one of items 1.1-1.12, wherein the flow particle detection device is a particle analysis detection device capable of achieving directional flow of the sample stream; and / or

[0355] The directional fluid system is composed of a sample loading unit and a flow unit; and / or

[0356] The particle analysis detection device comprises an optical system and a particle detector; and / or

[0357] The particle detector is composed of a photosensor and a signal conditioning circuit with a limited band filter high frequency noise function.

[0358] Embodiments

[0359] In order for those skilled in the art to better understand the technical solutions of the present application, some non-limiting embodiments are further disclosed below to further illustrate the present application. The reagents used in the present application can be purchased from the market or can be prepared by the methods described in the present application. In order for those skilled in the art to better understand the technical solutions of the present application, some non-limiting embodiments are further disclosed below to further illustrate the present application.

[0360] The reagents used in the present application can be purchased from the market or can be prepared by the methods described in the present application.

[0361] In the present application, mRNA-lipid nanoparticles or lipid nanoparticles encapsulating nucleic acid drugs both belong to non-viral nucleic acid carriers.

[0362] Abbreviations

[0363] DSPC: distearoylphosphatidylcholine

[0364] DMG: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

[0365] SM-102 cationic lipid (SM102), CAS No.: 2089251-47-6.

[0366] Example 1 Determination of the encapsulation efficiency and drug loading of Luciferase mRNA-lipid nanoparticles

[0367] 1) Source of specimens

[0368] The Luciferase mRNA-lipid nanoparticles used in this example are in accordance with the already marketed Moderna mRNA-1273 vaccine (mRNA-1273 The formulation of Example 1) is prepared, wherein the ratio of SM-102 cationic lipid: DMG-PEG200: DSPC: cholesterol is 50:10:38.5:1.5, the nitrogen-phosphorus ratio of the formulation is 6:1, Tris buffer with pH 7-8 is used as the aqueous phase, 2750 nt of Luciferase mRNA standard (500 ng / μl) is used as the nucleic acid for encapsulation, and the encapsulated Luciferase mRNA lipid nanoparticles are prepared by using the PNI (Positioning Navigation Intelligence) ignite microfluidic chip according to a flow rate ratio of 3:1 (L:R) and an inlet flow rate of 12 ml / min.

[0369] 2) Reagents and instruments used

[0370] 2.1) Reagents:

[0371] SYTO 9 and SYTO 16 are purchased from Invitrogen;

[0372] RNase-free DNase I is purchased from Takara;

[0373] S1 nuclease and 1×dsDNase are purchased from ThermoFisher;

[0374] PBS buffer: an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 with pH 7.4.

[0375] 2.2) Instruments: Flow NanoAnalyzer is purchased from NanoFCM Inc; Alliance 2695 / 2487 high-performance liquid chromatography system, ODS C18 column is purchased from Waters, USA, Sephadex G-50 column is purchased from Shimadzu, Japan.

[0376] 1) Determination of the total concentration of encapsulated Luciferase mRNA lipid nanoparticles (including empty particles)

[0377] The encapsulated Luciferase mRNA lipid nanoparticles are diluted 100 times with PBS buffer as sample solution. The fluorescence microsphere standard solution with calibrated concentration and the sample solution are detected by Flow NanoAnalyzer, and the respective particle numbers of the fluorescence microsphere standard solution and the sample solution under the same sample pressure (1 kpa) and the same sample time (2 min) are recorded.

[0378] The detection parameters of Flow NanoAnalyzer were as follows: laser detector 488nm+638nm; single laser channel detection Laser: 20 / 50mW, 488nm; scattering light attenuation: 0.2%; sample pressure: 1kpa; signal type: Large signal, and scattering light channel was used.

[0379] The total concentration of encapsulated Luciferase mRNA lipid nanoparticles (including empty particles) (A0, pieces / mL) was calculated by dilution factor and the concentration of fluorescent microsphere standard solution.

[0380] The total concentration of encapsulated Luciferase mRNA lipid nanoparticles (pieces / mL) = concentration of fluorescent microsphere standard (pieces / mL) x number of particles in sample solution x dilution factor of encapsulated Luciferase mRNA lipid nanoparticles ÷ number of particles of fluorescent microsphere standard.

[0381] Results

[0382] As shown in Figure 1, the total concentration of encapsulated Luciferase mRNA lipid nanoparticles (including empty particles) was calculated to be 3.4x10 8 pieces / mL.

[0383] 2) Determination of the fluorescence intensity of free nucleic acids in encapsulated Luciferase mRNA lipid nanoparticles and encapsulated Luciferase mRNA lipid nanoparticles (without empty particles)

[0384] 100 μl of encapsulated Luciferase mRNA lipid nanoparticles were incubated with permeable nucleic acid dye SYTO 9 at a final concentration of 1 μM at 37°C for 20 minutes, and then diluted 100-fold with PBS buffer to serve as test sample solution 1.

[0385] Using FITC and SSC dual channels, test sample solution 1 was detected by Flow NanoAnalyzer under the following conditions: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW, 488nm; scattering light attenuation: 10%; sample pressure: 1kpa; signal type: small signal, under the same sample pressure and the same sample time as step 1) of Example 1.

[0386] FITC fluorescence, the excitation signal of the nucleic acid dye, was used to characterize the signal from Luciferase mRNA-encapsulated lipid nanoparticles and the free nucleic acid (mRNA) signal. A two-dimensional scatter plot was constructed with the scattered light signal from the SSC channel as the abscissa and the fluorescence signal from the FTTC channel as the ordinate. A gate was set. Quadrant 1 (upper left quadrant) of the two-dimensional scatter plot represented free nucleic acid, quadrant 2 (upper right quadrant) represented nucleic acid-positive Luciferase mRNA-encapsulated lipid nanoparticles, and quadrant 4 (lower right quadrant) represented empty lipid nanoparticles. The positivity rate for each quadrant was recorded, as shown in Figure 2.

[0387] Record the fluorescence intensity θ of each free nucleic acid in the test solution 1 at the same injection pressure and injection time as in step 1 of Example 1 (i.e., obtain θ1, θ2, .....θ n1 ) and the fluorescence intensity γ of each nucleic acid signal-positive Luciferase mRNA encapsulated lipid nanoparticle (excluding empty particles) (ie, γ1, γ2, ....γ n2 ), and calculate the fluorescence intensity of free nucleic acid θ1, θ2, .....θ n1 The median value M1 is as follows; wherein n1 is the number of free nucleic acids in the test solution 1 detected under the above injection pressure and injection time, and n2 is the number of particles of Luciferase mRNA encapsulated lipid nanoparticles (non-viral nucleic acid carriers) with positive nucleic acid signals in the test solution 1 detected under the above injection pressure and injection time.

[0388] 3) Determination of the mean fluorescence intensity of Luciferase mRNA-encapsulated lipid nanoparticles (excluding empty particles) after removal of externally adsorbed nucleic acids

[0389] Incubate 100 μl of Luciferase mRNA-encapsulated lipid nanoparticles (same as in Example 1, step 2) with 0.2 U / μl RNase-free DNase I, 2 U / μl S1 nuclease, and 1× dsDNase at 37°C for 30 minutes. Add SYTO 9 (dissolved in PBS) to a final concentration of 1 μM to the reaction system and incubate at 37°C for 20 minutes.

[0390] After diluting with PBS buffer to a concentration of encapsulated Luciferase mRNA lipid nanoparticles consistent with that of the test solution 1 in step 2) of Example 1, the enzymatically hydrolyzed test solution 2 was used as the test solution 2. The enzymatically hydrolyzed test solution 2 was detected by Flow NanoAnalyzer under the conditions of laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW, 488nm; scattered light attenuation: 10%; injection pressure: 1kPa; signal type: small signal, using FITC and SSC dual channels, at the same injection pressure and the same injection time as in step 1) of Example 1.

[0391] FITC fluorescence is the nucleic acid dye excitation signal, used to characterize the signal from Luciferase mRNA-encapsulated lipid nanoparticles and the signal from free nucleic acid. A two-dimensional scatter plot was constructed with the scattered light signal from the SSC channel as the horizontal axis and the fluorescence signal from the FTTC channel as the vertical axis. A gate was set. Quadrant 1 (upper left quadrant) of the two-dimensional scatter plot represented free nucleic acid, quadrant 2 (upper right quadrant) represented nucleic acid-positive Luciferase mRNA-encapsulated lipid nanoparticles, and quadrant 4 (lower right quadrant) represented empty lipid nanoparticles. The positivity rate for each quadrant was recorded. The empty liposome content was 10.4%, as shown in Figure 3. The "positivity rate" for a given quadrant refers to the percentage of positive events in that quadrant relative to the total number of events.

[0392] Record the fluorescence intensity γ' (i.e., γ1', γ2', .... γ) of each nucleic acid signal-positive Luciferase mRNA-encapsulated lipid nanoparticle (excluding empty particles) in the test solution 2 after enzymatic hydrolysis at the same injection pressure and the same injection time as in step 2) of Example 1. n3 '); wherein n3 is the number of particles encapsulating Luciferase mRNA lipid nanoparticles (non-viral nucleic acid carriers) that are positive for nucleic acid signal after the enzymatic treatment of the test solution 2 obtained under the above injection pressure and injection time.

[0393] 4) Determination of the encapsulation efficiency and drug loading of Luciferase mRNA-encapsulated lipid nanoparticles

[0394] Calculate the encapsulation efficiency and drug loading according to the formula:

[0395] Wherein, M is the relative molecular mass of the nucleic acid to be tested, NA is the Avogadro constant, V3 is the total volume of the Luciferase mRNA lipid nanoparticles to be tested (mL), d is the total weight of the Luciferase mRNA lipid nanoparticles to be tested after removing the solvent (g); M1 is the median value of the fluorescence intensity of the free nucleic acid in step 2), V1 is the volume of the test sample solution 2 after enzyme digestion (used in the same meaning as "nuclease treatment") detected within the sampling pressure and sampling time (mL).

[0396] Results

[0397] The encapsulation efficiency of the sample was calculated to be 2.86x10 6 / (2.95x10 6 +0.715x10 6 ) = 78.03%, and the median value of the fluorescence intensity of the free nucleic acid M1 was 938.7.

[0398] The total volume of the Luciferase mRNA lipid nanoparticles to be tested V3 was 1000ml, the relative molecular mass of the mRNA vaccine M was 487500g / mol, the Avogadro constant NA was 6.02x10 23 / mol, the total weight of the lipid nanoparticles d was 52x10 -6 g, and a total of 0.1nL (V1 = 1x10 -7 ml) of sample was measured within the same sampling pressure and sampling time during the fluorescence data collection process using the Flow NanoAnalyzer.

[0399] Discussion: Compared with the encapsulation efficiency measured by the dextran gel method used in Comparative Example 1, the method provided by the present application can accurately detect the encapsulation efficiency and drug loading of the Luciferase mRNA lipid nanoparticle non-viral nucleic acid vector, avoiding overestimation of the results.

[0400] Encapsulation efficiency measured by dextran gel filtration-HPLC method in Comparative Example 1

[0401] Preparation of standard curve

[0402] Precise suction concentration of 500 ng / μl of 2750 bp mRNA encapsulated Luciferase mRNA lipid nanoparticles 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL, 1.6 mL were placed in 10 mL volumetric flask, diluted with methanol to the mark, prepared concentration of 5 ng / μl, 10 ng / μl, 20 ng / μl, 40 ng / μl, 80 ng / μl series of standard solution, according to the chromatographic conditions: column ODS C18 column (150 mm x 3.9 mm, 5 μm), mobile phase is methanol: acetonitrile: triethylamine-phosphate buffer (24:20:50), detection wavelength 265 nm, flow rate 1.0 mL / min, column temperature 30 °C, injection volume 20 μl respectively, record the chromatographic peak area, with peak area concentration regression, the regression equation, the results shown in Figure 4.

[0403] Encapsulation rate detection: take 0.5 mL of encapsulated Luciferase mRNA lipid nanoparticles in 10 mL volumetric flask, methanol constant volume, using the HPLC chromatographic conditions in the "preparation of standard curve" under the comparative example 1 to determine its content, calculate the concentration Co.

[0404] Another 0.5 mL of encapsulated Luciferase mRNA lipid nanoparticles solution sample on Sephadex G-50, eluted with water, the flow rate control is 3 mL / min; take 10 mL eluent, 5 mL in 10 mL volumetric flask, methanol constant volume, using the HPLC chromatographic conditions in the "preparation of standard curve" under the comparative example 1 to determine its content, the results shown in Figure 5, calculate the concentration C1.

[0405] The encapsulation rate was calculated as follows. Encapsulation rate (%) = (lipid nanoparticle drug content after column separation / total drug amount) x 100% Equation 14;

[0406] That is, the encapsulation rate (%) = (C1*40 / C0*20) x 100%.

[0407] Results

[0408] The concentration Co of the encapsulated Luciferase mRNA lipid nanoparticles after passing through the Sephadex gel column was measured to be 16.43 ng / μl, and the concentration C1 of the encapsulated Luciferase mRNA lipid nanoparticle product was 6.67 ng / μl. The encapsulation rate measured by the Sephadex gel method was calculated to be 81.2%.

[0409] Discussion

[0410] It can be seen that the encapsulation efficiency of the Luciferase mRNA lipid nanoparticles obtained by using the dextran gel filtration-HPLC determination method is higher than the encapsulation efficiency of 78.03% determined by the method of Example 1, which is increased by 3.17%.

[0411] By using the determination method of the present application to determine the encapsulation efficiency of mRNA-LNP, the external adsorbed nucleic acid can be avoided to be counted into the total amount of encapsulated nucleic acid, so as to more accurately reflect the actual encapsulation efficiency of the nucleic acid drug.

[0412] Example 2 Determination of the encapsulation efficiency of eGFP mRNA lipid nanoparticle sample, the nucleic acid signal positive rate of eGFP mRNA lipid nanoparticle, and the average copy number of surface adsorbed nucleic acid

[0413] 1) Source of specimen

[0414] According to the formula of the Pfizer-BioNTech mRNA vaccine which has been publicly disclosed , the ratio of ALC-0315 cationic lipid:ALC-0159:DSPC:cholesterol is 46.3:9.4:42.7:1.6, the formula nitrogen-phosphorus ratio is 3:1, PBS buffer with pH 7-8 is used as the water phase, and 1226 nt eGFP mRNA standard (500 ng / μl) is used as the nucleic acid for encapsulation. The preparation process uses PNI ignite microfluidic chip, and the preparation is carried out according to the flow rate ratio of 3:1 (L:R) and the inlet flow rate of 12 ml / min.

[0415] Abbreviations

[0416] ALC-0315 cationic lipid, CAS No. 2036272-55-4. ALC-0159, CAS No. 1849616-42-7.

[0417] 2) Reagents and instruments used

[0418] 2.1) Reagents: SYTO 16, SYTOX Green, Triton X-100 purchased from Invitrogen Company; Quant-iT TM RNA Assay Kit purchased from Thermo Fisher Company;

[0419] PBS buffer: pH 7.4 aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0420] 2.2) Instrument: Flow NanoAnalyzer purchased from NanoFCM Inc. Specter MAX M2 Multifunctional Microplate Reader purchased from Molecular Devices, USA.

[0421] 3) Encapsulation efficiency determination of encapsulated eGFP mRNA lipid nanoparticles with permeant nucleic acid dye

[0422] 3.1) Labeling free nucleic acids and encapsulated eGFP mRNA lipid nanoparticles with adsorbed external nucleic acids with permeant nucleic acid dye

[0423] 300 μΐ of encapsulated eGFP mRNA lipid nanoparticles were incubated with 1 μΜ of permeant nucleic acid dye SYTO16 at 37 °C for 20 min, and then diluted 100 times with PBS buffer as test solution 3.

[0424] The test solution 3 was detected by Flow NanoAnalyzer with FITC and SSC dual channels under the conditions of laser detector 488 nm+638 nm; single laser channel detection Laser: 10 / 50 mW, 488 nm; scattered light attenuation: 10%; sample pressure: 1 kpa; signal type: small signal, and the sample time was 2 min.

[0425] FITC fluorescence was nucleic acid dye excitation signal, which was used to characterize the signal of encapsulated eGFP mRNA lipid nanoparticles and free nucleic acids. The two-dimensional scatter plot was made with the scattered light signal of SSC channel as the abscissa and the fluorescence signal of FITC channel as the ordinate, and the circle gate was set. One quadrant (upper left quadrant) of the two-dimensional scatter plot represented free nucleic acids, two quadrants (upper right quadrant) represented encapsulated eGFP mRNA lipid nanoparticles with nucleic acid signal positive, and four quadrants (lower right quadrant) represented empty lipid nanoparticles. The positive rate data of each quadrant were recorded. The results are shown in Figure 6.

[0426] The fluorescence intensity θ' of each free nucleic acid of test solution 3 (i.e. θ1', θ2',.... θn' were obtained) and the fluorescence intensity γ" of encapsulated eGFP mRNA lipid nanoparticles with nucleic acid signal positive in test solution 3 (i.e. γ1", γ2",.... γn" were obtained) were recorded, and the fluorescence intensity of free nucleic acids θ1', θ2',.... θn' was calculated and recorded. n4 n5 n4 ​​n4; wherein n4 is the number of free nucleic acids in the test sample solution 3 detected within the above-mentioned injection pressure and injection time, n5 is the number of encapsulated eGFP mRNA lipid nanoparticles (non-viral nucleic acid carrier) having nucleic acid signal positive after permeable nucleic acid dye treatment in the test sample solution 3 detected within the above-mentioned injection pressure and injection time.

[0427] 3.2) Labeling free nucleic acids and externally adsorbed nucleic acids of encapsulated eGFP mRNA lipid nanoparticles with non-permeable nucleic acid dye

[0428] 300 μl of encapsulated eGFP mRNA lipid nanoparticles were incubated with non-permeable nucleic acid dye SYTOX Green at a final concentration of 2 μM at 37°C for 20 minutes. Then diluted 100 times with PBS buffer as test sample solution 4.

[0429] The test sample solution 4 was detected within the same injection pressure and the same injection time as in step 3.1) of Example 2 by Flow NanoAnalyzer using FITC and SSC dual channels under the conditions of laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50 mW, 488nm; scattered light attenuation: 10%; injection pressure: 1 kpa; signal type: small signal.

[0430] FITC fluorescence is nucleic acid dye excitation signal, which is used to characterize the signal of encapsulated eGFP mRNA lipid nanoparticles and the signal of free nucleic acids. Scatter light signal of SSC channel is taken as abscissa, and fluorescence signal of FITC channel is taken as ordinate to make two-dimensional scatter plot, and to set circle gate. One quadrant (upper left quadrant) of the two-dimensional scatter plot composed of the above signals represents free nucleic acids, two quadrants (upper right quadrant) represent encapsulated eGFP mRNA lipid nanoparticles having nucleic acid signal positive, and four quadrants (lower right quadrant) represent empty lipid nanoparticles, and the positive rate data of each quadrant is recorded. The results are shown in Figure 7.

[0431] The fluorescence intensity θ” (i.e. θ1”, θ2”, … θn” are obtained) of each free nucleic acid, the fluorescence intensity γ”’ (i.e. γ1”’, γ2”’, … γn”’ are obtained) of each encapsulated eGFP mRNA lipid nanoparticles having nucleic acid signal positive, and the number n10 of non-viral nucleic acid carrier particles having nucleic acid signal negative in the test sample solution 4 detected within the same injection pressure and the same injection time as in step 3.1) of Example 2 were recorded. n6 n7 n6 ​​the median value M3 of the number of free nucleic acids in the sample solution 4 detected during the injection pressure and injection time, n7 is the number of encapsulated eGFP mRNA lipid nanoparticles (non-viral nucleic acid carriers) that are positive for nucleic acid signals after being treated with a non-permeable nucleic acid dye in the sample solution 4 detected during the injection pressure and injection time.

[0432] 3.3) Determination of the encapsulation efficiency of the encapsulated eGFP mRNA lipid nanoparticles

[0433] The encapsulation efficiencies 2, 3, 4 were calculated according to the following formula:

[0434] or

[0435] The nucleic acid signal positivity rate of the non-viral nucleic acid carriers was calculated according to the following formula:

[0436] The average copy number of the externally adsorbed nucleic acids was calculated according to the following formula:

[0437] Results

[0438] It was calculated that the encapsulation efficiency 2, the encapsulation efficiency 3 of the sample was 65.45%, the encapsulation efficiency 4 was 65.99%, which was consistent with the results of Comparative Example 2; the nucleic acid signal positivity rate of the encapsulated eGFP mRNA lipid nanoparticles was 12.7%; and the average copy number of the externally adsorbed nucleic acids was 0.857. It can be seen that when the nucleic acid signal positivity rate of the nucleic acid carriers is ≤30% and the average copy number of the externally adsorbed nucleic acids is ≤5, formula 3, formula 7 and formula 8 can be used to calculate the encapsulation efficiency in the method of the present application.

[0439] Comparative Example 2 Quant-iT TM RiboGreen method for determining the encapsulation efficiency 4 of the encapsulated eGFP mRNA lipid nanoparticles

[0440] From Quant-iT TM RNA Assay Kit kit (Thermo Fisher), take out 1 ml of RiboGreen dye that has been dissolved in ultradry DMSO, and equilibrate the solution to room temperature before use. According to the number of sample wells, dilute the RiboGreen dye with PBS buffer according to a ratio of 1:200 to prepare the working solution, and store it in an aluminum foil or dark place to avoid light.

[0441] 4.2) RiboGreen standard curve drawing

[0442] Quant-iT TM The 16S and 23S RNA standards with a concentration of 100 pg / mL are provided in the RNA Assay Kit, which are diluted 50 times with PBS buffer to form a working solution of 2 pg / mL. The working solution of 2 pg / mL is gradient diluted to 1000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, and 0 ng / mL.

[0443] Take 100 mΐ of each concentration of standard respectively, and add to the 96-well plate, 3 repeats for each sample. Add 100 mΐ of RiboGreen working solution to the sample wells respectively, mix well, and incubate at room temperature for 2-5 minutes in the dark.

[0444] According to the instrument operation manual, open the Specter MAX M2 multifunctional microplate reader, place the 96-well plate, and detect the fluorescence value of the microplate sample. The fluorescence value of each sample well minus the reagent background fluorescence value of the 0 ng / mL sample well is the corrected fluorescence value corresponding to the corresponding RNA concentration, and a standard curve is made.

[0445] 4.3) Pretreatment of eGFP mRNA lipid nanoparticles

[0446] Take 100 mΐ of eGFP mRNA lipid nanoparticles and 100 mΐ of empty lipid nanoparticles (prepared in the same way as eGFP mRNA lipid nanoparticles, but without nucleic acid) respectively, and mix with 20 mΐ of 10% (v / v) Triton X-100, and ultrasonic for 5 min for demulsification, and shake well. Take 100 mΐ of eGFP mRNA lipid nanoparticles and 100 mΐ of empty lipid nanoparticles respectively, and mix with 20 mΐ of PBS buffer, and shake well. Prepare 4 sample mixtures.

[0447] 4.4) Sample analysis of mRNA-lipid nanoparticles

[0448] Dilute the above 4 sample mixtures with PBS buffer to the appropriate proportion (diluted to a lipid nanoparticle concentration of 1 x 10 5 -1 x 10 12 / ml), and obtain respective dilution solutions, and take 100 mΐ of the above dilution solutions and mix with RiboGreen fluorescent dye in equal volume. Mix well, and incubate at room temperature for 2-5 minutes in the dark. Then add to the 96-well plate.

[0449] The fluorescence intensity of the sample was detected using a Specter MAX M2 multifunctional microplate reader. The corrected sample fluorescence value was obtained by subtracting the background fluorescence value of the empty lipid nanoparticle reagent from the mRNA-lipid nanoparticle sample detection value. The corrected sample fluorescence value was substituted into the standard curve obtained in step 4.2) of Example 2 to obtain the nucleic acid concentration V1 of the eGFP mRNA lipid nanoparticle before demulsification and the nucleic acid concentration V2 of the eGFP mRNA lipid nanoparticle after demulsification.

[0450] 4.5) Calculation of the encapsulation efficiency of eGFP mRNA lipid nanoparticles

[0451] The results are shown in Table 1.

[0452] Table 1 Nucleic acid concentration and fluorescence intensity (n = 3) Y = 6347.7X (R 2 = 0.9965)

[0453] Results

[0454] The encapsulation efficiency of eGFP mRNA lipid nanoparticles was measured using the Quant-iT TM RiboGreen method was 64.82%.

[0455] Conclusion: The Quant-iT TM RiboGreen method is also a method for detecting nucleic acid concentration using a fluorescent dye, and the difference from the method of the present application is that: in principle, the Quant-iT TM RiboGreen method still belongs to the centralized average method of measuring the total value of multiple particles, while the present method can measure each lipid nanoparticle and accurately grasp the nucleic acid content at the single particle level; in structure, the Quant-iT TM RiboGreen method uses Triton X-100 to destroy the structure of the lipid particle, while the method of the present application does not destroy the lipid structure of the lipid particle whether or not a nuclease is used.

[0456] The results of the encapsulation efficiency measured by the method of Example 2 and the Quant-iT TM RiboGreen method are basically consistent. The experimental results prove that the method of the present application can be used to measure the encapsulation efficiency of non-viral nucleic acid carrier compositions. The method of the present application has high accuracy and can avoid overestimation of the encapsulation efficiency. In addition, the method provided by the present application is simple to operate, short in time consumption, low in cost, high in efficiency, and has excellent technical effects.

[0457] Example 3: Detection of the encapsulation efficiency of eGFP mRNA lipid nanoparticles using different permeable dyes

[0458] 1) Specimen source

[0459] Prepared according to the formula of the publicly listed Moderna mRNA-1273 vaccine, with a SM-102 cationic lipid: DMG-PEG200: DSPC: Cholesterol ratio of 50: 10: 38.5: 1.5, a formula nitrogen-phosphorus ratio of 6: 1, using Tris buffer with a pH of 7-8 as the aqueous phase, and a 1226 nt eGFP mRNA standard (500 ng / μl) as the nucleic acid for encapsulation, using PNI ignite microfluidic chips for preparation, according to a flow rate ratio of 3: 1 (L: R), with a feed flow rate of 12 ml / min.

[0460] 2) Reagents and instruments used

[0461] 2.1) Reagents:

[0462] SYTO 16, SYTOX Green, Triton X-100 were purchased from Invitrogen company;

[0463] SYTO 9 (Thermo), SYTO 13 (Thermo), SYTO 24 (Thermo) (all are permeable nucleic acid dyes)

[0464] PBS buffer: pH 7.4 aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0465] 2.2) Instruments: Flow NanoAnalyzer purchased from NanoFCM Inc.

[0466] 3) Operation of permeable nucleic acid dye determination of the encapsulation rate of eGFP mRNA lipid nanoparticles

[0467] The difference from Example 2 is that the eGFP mRNA lipid nanoparticles are used as the sample to be tested, and SYTO9, SYTO13, SYTO16, SYTO24 (permeable nucleic acid dyes) are used instead of SYTO16 in Example 2 for determination, and the calculation of the results uses the encapsulation rate 2 calculation formula in Example 2, and the rest of the operations are the same as in Example 2. The results of the determination of the encapsulation rate of eGFP mRNA lipid nanoparticles using different nucleic acid dyes are shown in Table 2. (n = 3) indicates that the experiment was repeated 3 times.

[0468] Table 2: Encapsulation rate of eGFP mRNA lipid nanoparticles determined using different dye labeling (n = 3)

[0469] Conclusion: The method of the present application can use different permeable dyes, and can accurately detect the encapsulation efficiency of non-viral nucleic acid vectors.

[0470] Example 4 Determination of the encapsulation efficiency of nucleic acid drugs encapsulated by lipid nanoparticles with different weight ratios of lipids to oligonucleotides

[0471] The weight ratio of lipids to oligonucleotides will affect the encapsulation efficiency of lipid nanoparticles. The inventors performed preparation experiments with different weight ratios of lipids to oligonucleotides, and detected the encapsulation efficiency of encapsulated particles with different weight ratios of lipids to oligonucleotides.

[0472] 1) Source of specimens

[0473] The formulation nitrogen to phosphorus ratio (abbreviated as NP ratio) was adjusted to 4:1, 8:1, 12:1, 16:1, and 20:1 to prepare nucleic acid encapsulated lipid nanoparticles NP4, NP8, NP12, NP16, and NP20, respectively, using the lipid nanoparticle synthesis method provided in Example 1.

[0474] 2) Reagents and instruments used

[0475] 2.1) Reagents: SYTO 16, SYTOX Green, Triton X-100 were purchased from Invitrogen; Quant-iTTM RNA Assay Kit was purchased from Thermo Fisher;

[0476] PBS buffer: an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4 at pH 7.4.

[0477] 2.2) Instruments: Flow NanoAnalyzer was purchased from NanoFCM Inc. Specter MAX M2 multifunctional microplate reader was purchased from Molecular Devices, USA.

[0478] 3) Operation for permeable nucleic acid dye determination of lipid nanoparticle encapsulation efficiency

[0479] The difference from Example 2 is that nucleic acid encapsulated lipid nanoparticles with different NP ratios (NP4, NP8, NP12, NP16, and NP20) are used as the sample to be tested, and the encapsulation efficiency 2 calculation formula in Example 2 is used for result calculation, and the rest of the operation is the same as in Example 2. The encapsulation efficiency of nucleic acid encapsulated lipid nanoparticles with different NP ratios: NP4, NP8, NP12, NP16, and NP20 is calculated respectively, and the results are shown in Table 3 under the column of “Method of the present application”.​

[0480] Comparative Example 3 Quant-iT TM RiboGreen method for determining the encapsulation efficiency of lipid nanoparticles with different weight ratios of lipids to oligonucleotides

[0481] The encapsulation efficiency was calculated according to the determination operation of 4.1) to 4.5) of Comparative Example 2, with the difference being that

[0482] In the pretreatment of 4.3), 100 μl of lipid nanoparticles encapsulating nucleic acids with different NP ratios (NP4 (4: 1), NP8 (8: 1), NP12 (12: 1), NP16 (16: 1), NP20 (20: 1)) and 100 μl of empty lipid nanoparticles were taken respectively, mixed with 20 μl of 10% (v / v) Triton X-100, and emulsified by ultrasonic for 5 min, and fully shaken and mixed. Six sample mixtures were prepared.

[0483] 100 μl of lipid nanoparticles encapsulating nucleic acids with different NP ratios (NP4, NP8, NP12, NP16, NP20) and 100 μl of empty lipid nanoparticles were taken respectively, mixed with 20 μl of PBS buffer, and fully shaken and mixed. Six sample mixtures were prepared.

[0484] In 4.4), the above 12 sample mixtures were treated.

[0485] The encapsulation efficiency was calculated using the encapsulation efficiency calculation formula in Comparative Example 2, and the rest of the operation was the same as that in Comparative Example 2.

[0486] The encapsulation efficiency of lipid nanoparticles encapsulating nucleic acids with different NP ratios was determined using the method of the present application and Quant-iT TM The encapsulation efficiency determined by the RiboGreen method is shown in Table 3.

[0487] Results

[0488] Table 3 Results of determining the encapsulation efficiency of lipid nanoparticles encapsulating nucleic acids using the RiBoGreen Kit and the method of the present application (n = 3)

[0489] Conclusion: For lipid nanoparticles encapsulating nucleic acid drugs with different weight ratios of lipids to oligonucleotides, the encapsulation efficiency determined by Quant-iT TM RiboGreen method is higher than that determined by the method of the present application. Compared with the Quant-iT TM RiboGreen method, the results determined by the method of the present application are consistent. And it can be known that the method provided by the present application can accurately detect the encapsulation efficiency of lipid nanoparticles encapsulating nucleic acids prepared with different NP ratios.

[0490] Example 5 Effect of different nucleic acid dyes on the determination of the encapsulation efficiency of mRNA-Cas9 lipid nanoparticle

[0491] 1) Sample source

[0492] Prepared according to the formula of the publicly disclosed Moderna mRNA-1273 vaccine, with a SM-102 cationic lipid: DMG-PEG200: DSPC: cholesterol ratio of 50: 10: 38.5: 1.5, a formula nitrogen-phosphorus ratio of 6: 1, using Tris buffer with a pH of 7-8 as the aqueous phase, and a 4100 nt mRNA-Cas9 standard (500 ng / μl) as the nucleic acid for encapsulation. The preparation process used a PNI ignite microfluidic chip, with a flow rate ratio of 3: 1 (L: R) and an inlet flow rate of 12 ml / min.

[0493] 2) Reagents and instruments used

[0494] 2.1) Reagents:

[0495] SYTO 9, Triton X-100 purchased from Invitrogen company;

[0496] RiboGreen (Thermo Fisher), Pico Green (Thermo) (both non-permeable nucleic acid dyes)

[0497] PBS buffer: pH 7.4 aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0498] 2.2) Instruments: Flow NanoAnalyzer purchased from NanoFCM Inc.

[0499] 3) Operation of non-permeable nucleic acid dye on the determination of the encapsulation efficiency of eGFP mRNA lipid nanoparticle

[0500] The difference from Example 2 is that mRNA-Cas9 lipid nanoparticle is used as the sample to be tested, and RiboGreen and Pico Green (both non-permeable nucleic acid dyes) are used instead of SYTOX Green in Example 2 for determination. The result is calculated using the encapsulation efficiency 2 calculation formula in Example 2, and the rest of the operation is the same as Example 2. The results of different nucleic acid dyes on the determination of the encapsulation efficiency of mRNA-Cas9 lipid nanoparticle are shown in Table 4.

[0501] Results

[0502] Table 4 Encapsulation efficiency results determined using different dyes to label the mRNA-Cas9 lipid nanoparticles

[0503] Discussion

[0504] Using the methods provided by the present application, the encapsulation efficiency of non-viral nucleic acid vectors can be detected using different nucleic acid dyes. SYTOX Green, Ribo Green, and Pico Green can all be used as non-permeable nucleic acid dyes in the methods of the present application.

[0505] Example 6 Effect of different nucleases on the determination of the encapsulation efficiency of the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticles

[0506] 1) Source of specimens

[0507] Prepared according to the formula of the already marketed Moderna mRNA-1273 vaccine, with a ratio of SM-102 cationic lipid: DMG-PEG200: DSPC: cholesterol of 50: 10: 38.5: 1.5, a formula nitrogen-phosphorus ratio of 6: 1, using Tris buffer with a pH of 7-8 as the aqueous phase, and a 6173 bp pECMV-3xFLAG-CD9 plasmid (500 ng / μl) as the nucleic acid for encapsulation, the preparation process used a PNI ignite microfluidic chip, according to a flow rate ratio of 3: 1 (L: R), with an inlet flow rate of 12 ml / min.

[0508] 2) Reagents and instruments used

[0509] 2.1) Reagents: SYTO 9 was purchased from Invitrogen company;

[0510] Recombinant DNase I (RNase-free) (TKR-2270, Takara) was purchased from Takara company

[0511] DNase, RNase-free (EN0523, Thermo), RNase A (10 mg / mL, DNase and protease-free) (EN0531, Thermo) were purchased from Thermo company

[0512] DNase I (RNase-free) (M0303S, NEB) was purchased from NEB company

[0513] PBS buffer: pH 7.4 aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0514] 1) Determination of fluorescence intensity of free DNA and pECMV-3×FLAG-CD9 plasmid-encapsulated lipid nanoparticles

[0515] 100 μl of pECMV-3×FLAG-CD9 plasmid lipid nanoparticles encapsulated was incubated with SYTO 9 (a permeable nucleic acid dye) at a final concentration of 1 μM at 37° C. for 20 minutes, and then diluted 100-fold with PBS buffer as the test solution 1. The sample solution 1 was detected by Flow NanoAnalyzer using FITC and SSC dual channels at the same injection pressure and injection time as in step 1) of Example 1 under the following conditions: laser detector 488 nm + 638 nm; single laser channel detection Laser: 10 / 50 mW, 488 nm; scattered light attenuation: 10%; injection pressure: 1 kPa; signal type: small signal. FITC fluorescence is the nucleic acid dye excitation signal, which is used to characterize the encapsulated pECMV-3×FLAG-CD9 plasmid lipid nanoparticle signal and the free nucleic acid (DNA) signal. A two-dimensional scatter plot was drawn with the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate, and a gate was set. The first quadrant (upper left quadrant) of the two-dimensional scatter plot composed of the above signals represented free nucleic acid, the second quadrant (upper right quadrant) represented the encapsulated pECMV-3×FLAG-CD9 plasmid lipid nanoparticles with positive nucleic acid signal, and the fourth quadrant (lower right quadrant) represented empty lipid nanoparticles. The positive rate data of each quadrant was recorded.

[0516] Record the fluorescence intensity θ of each free nucleic acid in the test solution 1 at the same injection pressure and injection time as in step 1 of Example 1 (i.e., obtain θ1, θ2, .....θ n1 ) and the fluorescence intensity γ of each pECMV-3×FLAG-CD9 plasmid lipid nanoparticle (excluding empty lipid nanoparticles) with a positive nucleic acid signal (i.e., γ1, γ2, ....γ n2 ), and calculate the fluorescence intensity of free nucleic acid θ1, θ2, .....θ n1 wherein n1 is the number of free nucleic acids in the test solution 1 detected under the above injection pressure and injection time, and n2 is the number of encapsulated pECMV-3×FLAG-CD9 plasmid lipid nanoparticles (non-viral nucleic acid vectors) with positive nucleic acid signals in the test solution 1 detected under the above injection pressure and injection time.

[0517] 2) Determination of the mean fluorescence intensity of pECMV-3×FLAG-CD9 plasmid-encapsulated lipid nanoparticles (excluding empty lipid nanoparticles) after removal of surface-adsorbed nucleic acids

[0518] The 100 μl of encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticles (same as step 2 of Example 1) was aliquoted into 4 tubes, 300 μl in each tube, and each tube was incubated with 2 U / μl of recombinant DNase I (RNase-free) (TKR-2270, Takara), DNase, RNase-free (EN0523, Thermo), DNase I (RNase-free) (M0303S, NEB), RNase A (10 mg / mL, DNase and protease free) (EN0531, Thermo) at 37°C for 30 minutes, respectively.

[0519] In the above reaction system, 1 μM of SYTO 9 (dissolved in PBS solution) was added to the final concentration, and incubated at 37°C for 20 minutes. After dilution with PBS buffer to the same concentration of the test solution 1 of step 2) of Example 1, the test solution 2, test solution 3, test solution 4, and test solution 5 after enzymolysis were prepared.

[0520] The test solution 2, test solution 3, test solution 4, and test solution 5 after enzymolysis were detected by Flow NanoAnalyzer under the conditions of laser detector 488 nm+638 nm; single laser channel detection Laser: 10 / 50 mW, 488 nm; scattering light attenuation: 10%; sample injection pressure: 1 kpa; signal type: small signal, using FITC and SSC dual channels, under the same sample injection pressure and the same sample injection time as step 1) of Example 1.

[0521] The FITC fluorescence is the nucleic acid dye excitation signal, which is used to characterize the signal of the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticles and the free nucleic acid (DNA) signal. The two-dimensional scatter plot was drawn with the scattering light signal of the SSC channel as the abscissa and the fluorescence signal of the FITC channel as the ordinate. The circle gate was set, and the quadrant (upper left quadrant) of the two-dimensional scatter plot composed of the above signals represented free nucleic acid, the second quadrant (upper right quadrant) represented encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticles with positive nucleic acid signal, and the fourth quadrant (lower right quadrant) represented empty lipid nanoparticles. The positive rate data of each quadrant was recorded.

[0522] The fluorescence intensity of each of the test sample solution 2, test sample solution 3, test sample solution 4, and test sample solution 5 after enzymolysis under the same injection pressure and the same injection time as in step 2) of Example 1, which has a nucleic acid signal positive for the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticle (excluding empty lipid nanoparticles) is recorded as γ' (i.e., γ1', γ2',... γn3'); wherein n3 is the number of nucleic acid enzyme-treated particles with a nucleic acid signal positive for the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticle (non-viral nucleic acid vector) in the test sample solution 2, test sample solution 3, test sample solution 4, and test sample solution 5 obtained under the above injection pressure and injection time.

[0523] 4) Determination of the encapsulation efficiency and drug loading of the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticle

[0524] According to the formula:

[0525] The results are shown in Table 5.

[0526] Table 5. Encapsulation efficiency of the encapsulated pECMV-3xFLAG-CD9 plasmid lipid nanoparticle determined using different nucleic acid enzymes

[0527] Discussion

[0528] Using the method provided by the present application, the encapsulation efficiency of non-viral nucleic acid vectors can be detected using different nucleic acid enzymes. Recombinant DNase I (RNase-free), DNase (RNase-free), DNase I (RNase-free), and RNase A (free of DNase and protease) can be used as nucleic acid enzymes for the method of the present application, or one or a combination of several of them can be used.

[0529] The purpose of this example is to explore the types of nucleic acid enzymes that can be used in the method of the present application. For the convenience of parallel comparison, the concentration of all nucleic acid enzymes in the above experiment is set to the same condition, and does not represent that the encapsulation efficiency determination result is the best performance of the enzyme for determining the encapsulation efficiency of the sample.

[0530] It should be understood that the digestion efficiency of nucleic acids in the sample can be improved by exploring the optimal concentration of the nucleic acid enzyme and other processing conditions, and those skilled in the art can explore the optimal digestion conditions of different nucleic acid enzymes for the sample to better determine the encapsulation efficiency, or combine different types of nucleic acid enzymes.

[0531] Example 7: Effect of different nucleic acid enzymes on the determination of the encapsulation efficiency of the encapsulated lL22 mRNA lipid nanoparticle

[0532] 1) Source of the specimen

[0533] It is prepared according to the formula of the publicly available Moderna mRNA-1273 vaccine, in which the ratio of SM-102 cationic lipid: DMG-PEG200: DSPC: cholesterol is 50:10:38.5:1.5, the nitrogen-phosphorus ratio is 6:1, and Tris buffer with a pH of 7-8 is used as the aqueous phase. The 540nt lL22 mRNA standard (500ng / μl) is used as the nucleic acid encapsulation. The preparation process uses PNI's ignite microfluidic chip, with a flow rate ratio of 3:1 (L:R) and an inlet flow rate of 12ml / min.

[0534] 2) Reagents and instruments used

[0535] 2.1) Reagents:

[0536] SYTO 9 was purchased from Invitrogen;

[0537] Recombinant DNase I (RNase-free) (TKR-2270, Takara) was purchased from Takara

[0538] RNase A (10 mg / mL, without DNase and protease) (EN0531, Thermo) was purchased from Thermo

[0539] RNase A was purchased from Biyuntian Pharmaceutical Co., Ltd.

[0540] PBS buffer: an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 at pH 7.4.

[0541] 1) Determination of Fluorescence Intensity of Free mRNA and 1L22 mRNA-Encapsulated Lipid Nanoparticles

[0542] 100 μl of 1L22 mRNA-encapsulated lipid nanoparticles was incubated with SYTO 9 (a permeable nucleic acid dye) at a final concentration of 1 μM at 37°C for 20 minutes and then diluted 100-fold with PBS buffer to serve as test solution 1. Test solution 1 was detected on a Flow NanoAnalyzer using a dual FITC and SSC channel at the same injection pressure and time as in step 1) of Example 1, under the following conditions: laser detector 488 nm + 638 nm; single laser channel detection: Laser: 10 / 50 mW, 488 nm; scattered light attenuation: 10%; injection pressure: 1 kPa; signal type: small signal.

[0543] FITC fluorescence, the excitation signal of the nucleic acid dye, was used to characterize the signal from lipid nanoparticles encapsulating 1L22 mRNA and the free nucleic acid (mRNA) signal. A two-dimensional scatter plot was constructed with the scattered light signal from the SSC channel as the abscissa and the fluorescence signal from the FTTC channel as the ordinate. A gate was set. Quadrant 1 (upper left quadrant) of the two-dimensional scatter plot represented free nucleic acid, quadrant 2 (upper right quadrant) represented lipid nanoparticles encapsulating 1L22 mRNA with a positive nucleic acid signal, and quadrant 4 (lower right quadrant) represented empty lipid nanoparticles. The positive rate data for each quadrant was recorded.

[0544] Record the fluorescence intensity θ of each free nucleic acid in the test solution 1 at the same injection pressure and injection time as in step 1 of Example 1 (i.e., obtain θ1, θ2, .....θ n1 ) and the fluorescence intensity γ of each lipid nanoparticle encapsulating 1L22 mRNA with a positive nucleic acid signal (excluding empty lipid nanoparticles) (ie, γ1, γ2, .... γ n2 ), and calculate the fluorescence intensity of free nucleic acid θ1, θ2, .....θ n1 wherein n1 is the number of free nucleic acids in the test solution 1 detected under the above injection pressure and injection time, and n2 is the number of encapsulated 1L22 mRNA lipid nanoparticles (non-viral nucleic acid carriers) with positive nucleic acid signals in the test solution 1 detected under the above injection pressure and injection time.

[0545] 2) Determination of the mean fluorescence intensity of 1L22 mRNA-encapsulated lipid nanoparticles (excluding empty lipid nanoparticles) after removal of surface-adsorbed nucleic acids

[0546] 100 μl of 1L22 mRNA-encapsulated lipid nanoparticles (same as in step 2 of Example 1) was divided into four tubes, each containing 300 μl. Each tube was incubated with 2 U / μl of recombinant DNase I (RNase-free) (TKR-2270, Takara), RNase A (10 mg / mL, DNase- and protease-free) (EN0531, Thermo), and RNase A (ST576, Beyotime) at 37°C for 30 min.

[0547] In the above reaction system, SYTO 9 (dissolved in PBS solution) was added at a final concentration of 1 μM, incubated at 37° C. for 20 minutes, and then diluted with PBS buffer to a concentration of lipid nanoparticles encapsulating 1L22 mRNA consistent with that of test solution 1 in step 2) of Example 1, and used as test solution 2, test solution 3, and test solution 4 after enzymatic hydrolysis.

[0548] The FITC and SSC dual channels were used to detect the test sample solution 2, test sample solution 3, and test sample solution 4 after enzymolysis under the same sample pressure and the same sample time as in step 1) of Example 1 by the Flow NanoAnalyzer under the conditions of laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW, 488nm; scattered light attenuation: 10%; sample pressure: 1kpa; signal type: small signal.

[0549] The FITC fluorescence is a nucleic acid dye excitation signal, which is used to characterize the encapsulated lL22 mRNA lipid nanoparticle signal and the free nucleic acid (mRNA) signal in the state. The two-dimensional scatter plot is drawn with the SSC channel scattered light signal as the abscissa and the FITC channel fluorescence signal as the ordinate, and the circle gate is set. One quadrant (upper left quadrant) of the two-dimensional scatter plot composed of the above signals represents free nucleic acid, two quadrants (upper right quadrant) represent encapsulated lL22 mRNA lipid nanoparticles with positive nucleic acid signal, and four quadrants (lower right quadrant) represent empty lipid nanoparticles, and the positive rate data of each quadrant is recorded.

[0550] The fluorescence intensity γ' (i.e. γ1', γ2',... γn3') of each encapsulated lL22 mRNA lipid nanoparticle (excluding empty lipid nanoparticles) with a positive nucleic acid signal in the test sample solution 2, test sample solution 3, and test sample solution 4 after enzymolysis under the same sample pressure and the same sample time as in step 2) of Example 1 is recorded. n3 Wherein n3 is the number of encapsulated lL22 mRNA lipid nanoparticles (non-viral nucleic acid carriers) with a positive nucleic acid signal after nuclease treatment in the test sample solution 2, test sample solution 3, and test sample solution 4 after enzymolysis under the above sample pressure and sample time.

[0551] 4) Determination of the encapsulation efficiency and drug loading of the encapsulated lL22 mRNA lipid nanoparticles

[0552] According to the formula:

[0553] The results are shown in Table 6.

[0554] Table 6 Determination of the encapsulation efficiency of the encapsulated lL22 mRNA lipid nanoparticles using different nucleases

[0555] Results

[0556] The results show that the non-viral nucleic acid vector encapsulation rate can be detected using different nucleases according to the method of the present application. Recombinant DNase I (RNase-free), RNase A (without DNase and protease) can be used as nucleases in the method of the present application, or one or several of them can be combined.

[0557] The purpose of this example is to explore the types of nucleases that can be used in the method of the present application. For the convenience of parallel comparison, the concentration of all nucleases in the above experiment is set to the same condition, which does not mean that the encapsulation rate determination result is the best performance of the enzyme determination encapsulation rate.

[0558] It should be understood that the digestion efficiency of nucleic acids in the sample can be improved by exploring the optimal concentration of nucleases and other processing conditions. Those skilled in the art can explore the optimal digestion conditions of different nucleases for the sample to better determine the encapsulation rate, or combine different types of nucleases to obtain good digestion effect.

[0559] The method of the present application has been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein within the content, spirit and scope of the present application to realize and apply the present application technology. Those skilled in the art can refer to the content herein to appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present application.

Claims

1. A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid carrier composition, The method comprises: labeling with a first nucleic acid dye in contact with the non-viral nucleic acid carrier composition to be tested to obtain mixture 1; The mixture 1 of the sample volume V1 is injected into the flow-type particle detection device for detection, and the fluorescence intensity θ of each free nucleic acid in the mixture 1 of the sample volume V1, i.e. the fluorescence intensity θ1~θ n1 of the free nucleic acid, and the fluorescence intensity γ of each non-viral nucleic acid vector positive for the nucleic acid signal in the mixture 1 of the sample volume V1, i.e. the fluorescence intensity γ1~γ n2 of the non-viral nucleic acid vector positive for the nucleic acid signal are obtained. Wherein, n1 is the number of free nucleic acids in the sample volume V1 of mixture 1, n2 is the number of non-viral nucleic acid carrier particles with positive nucleic acid signal in the sample volume V1 of mixture 1; According to the fluorescence intensity data, the drug loading and / or encapsulation efficiency of the non-viral nucleic acid carrier composition is calculated, Wherein, the first nucleic acid dye is a permeable nucleic acid dye. 2.The method of claim 1, further comprising the following steps: Take mixture 1, add nuclease for treatment to obtain mixture 2-A;Or take the sample containing non-viral nucleic acid carrier composition to be tested, add nuclease for treatment, and then label with the first nucleic acid dye to obtain mixture 2-B; The flow-type particle detection device is fed with V1 volume of mixture 2-A or mixture 2-B for detection, and the fluorescence intensity γ' of each nucleic acid signal positive non-viral nucleic acid carrier in V1 volume of mixture 2-A or mixture 2-B is obtained, i.e. the fluorescence intensity γ'1-γ' of the nucleic acid signal positive non-viral nucleic acid carrier n3 wherein n3 is the particle number of the nucleic acid signal positive non-viral nucleic acid carrier in V1 volume of mixture 2-A or mixture 2-B; According to the fluorescence intensity data, the drug loading and / or encapsulation efficiency of the non-viral nucleic acid carrier composition is calculated, wherein the encapsulation efficiency is calculated according to Formula 1 Drug loading was calculated according to Formula 2 Wherein, M is the relative molecular mass of the target nucleic acid, NA is Avogadro's constant, V3 is the total volume of the sample containing non-viral nucleic acid carrier composition to be tested, and d is the total weight of the sample containing non-viral nucleic acid carrier composition after removing the solvent; M1 is the median value of the fluorescence intensity of the free nucleic acids θ1-θ n1 of the median value. 3.The method of claim 1, further comprising: The steps of detecting the nucleic acid signal positive rate and the average copy number of the external adsorbed nucleic acid of the nucleic acid carrier, When the nucleic acid signal positivity rate of the nucleic acid carrier is ≤ 30% and the average copy number of the externally adsorbed nucleic acid is ≤ 5, the encapsulation efficiency is calculated according to Formula 11: The calculation formula of the drug loading amount is: Wherein, M is the relative molecular mass of the target nucleic acid, NA is Avogadro's constant, V3 is the total volume of the sample containing non-viral nucleic acid carrier composition to be tested, and d is the total weight of the sample containing non-viral nucleic acid carrier composition after removing the solvent; M1 is the median value of the fluorescence intensity of the free nucleic acids θ1-θ n1 of the median value.

4. The method of claim 3, wherein, The steps of detecting the nucleic acid signal positive rate and the average copy number of the external adsorbed nucleic acid of the nucleic acid carrier comprise: Take the non-viral nucleic acid carrier composition to be tested, label with the first nucleic acid dye in contact to obtain mixture 7; into the flow-through particle detection device, obtaining the fluorescence intensity Z" of each free nucleic acid in the sample volume V6 of the mixture 7, i.e. the fluorescence intensity data Z1" ~ Z n8 " of each non-viral nucleic acid vector positive for the nucleic acid signal in the sample volume V6 of the mixture 7, i.e. the fluorescence intensity data Z1"" ~ Z n9 "" of each non-viral nucleic acid vector positive for the nucleic acid signal in the sample volume V6 of the mixture 7, i.e. the fluorescence intensity data Z1"" ~ Z Wherein, n10 is the number of non-viral nucleic acid carrier particles with negative nucleic acid signal in the sample volume V6 of mixture 7, n8 is the number of free nucleic acids in the sample volume V6 of mixture 7, and n9 is the number of non-viral nucleic acid carrier particles with positive nucleic acid signal in the sample volume V6 of mixture 7; The nucleic acid signal positive rate and the average copy number of the surface adsorbed nucleic acid of the non-viral nucleic acid carrier are calculated. wherein the nucleic acid signal positive rate of the non-viral nucleic acid vector is calculated according to formula 8: The average copy number of surface-adsorbed nucleic acids is calculated according to equation 9: wherein M5 is the median value of the fluorescence intensity of the free nucleic acids Z1" to Z n8 the median value of the fluorescence intensity of the free nucleic acids Z1" to Z 5.A method for detecting the drug loading and / or encapsulation efficiency of a non-viral nucleic acid carrier composition, The method comprises: labeling with a first nucleic acid dye in contact with the non-viral nucleic acid carrier composition to be tested to obtain mixture i; The mixture i of the volume V2 is injected into the flow-type particle detection device for detection, and the fluorescence intensity θ' of each free nucleic acid in the mixture i of the volume V2, i.e. the fluorescence intensity θ1' ~ θ n4 of the free nucleic acid, and the fluorescence intensity γ' of each nucleic acid signal positive non-viral nucleic acid vector in the mixture i of the volume V2, i.e. the fluorescence intensity γ1' ~ γ n5 of the nucleic acid signal positive non-viral nucleic acid vector; wherein n4 is the number of free nucleic acids in the mixture i in the mixture i of the volume V2, and n5 is the number of particles of the nucleic acid signal positive non-viral nucleic acid vector in the mixture i in the mixture i of the volume V2. labeling with a second nucleic acid dye in contact with the non-viral nucleic acid carrier composition to be tested to obtain mixture ii; The flow-through particle detection device is fed with the mixture ii of volume V2 for detection, and the fluorescence intensity θ" of each free nucleic acid in the mixture ii of volume V2, i.e. the fluorescence intensity data θ1" ~ θ n6 of the free nucleic acid, and the fluorescence intensity γ" of each non-viral nucleic acid vector with positive nucleic acid signal after nucleic acid dye treatment in the mixture i of volume V2, i.e. the fluorescence intensity data γ1" ~ γ n7 of the non-viral nucleic acid vector with positive nucleic acid signal after nucleic acid dye treatment, wherein n6 is the number of free nucleic acids in the mixture ii of volume V2, and n7 is the number of particles of the non-viral nucleic acid vector with positive nucleic acid signal in the mixture ii of volume V2. According to the fluorescence intensity data, the drug loading and / or encapsulation efficiency of the non-viral nucleic acid carrier composition is calculated, Wherein, the first nucleic acid dye is a permeable nucleic acid dye, and the second nucleic acid dye is a non-permeable nucleic acid dye, The formula for calculating the encapsulation efficiency is or And / or The calculation formula of the drug loading amount is: or Wherein, M is the relative molecular mass of the target nucleic acid, NA is Avogadro's constant, V3 is the total volume of the sample containing non-viral nucleic acid carrier composition to be tested, and d is the total weight of the sample containing non-viral nucleic acid carrier composition after removing the solvent. M2 is the median value of the fluorescence intensity of the free nucleic acids θ1' ~ θ n4 M3 is the median value of the fluorescence intensity of the free nucleic acids θ1" ~ θ n6 ".

6. The method of any one of claims 1-5, wherein, The first nucleic acid dye is selected from at least one of SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, TO-PRO-3 Iodide.

7. The method of claim 6, wherein, The first nucleic acid dye is selected from at least one of SYTO 9, SYTO 13, SYTO 16, SYTO 24, preferably from SYTO 9, SYTO 16.

8. The method of any one of claims 5-7, wherein, The second nucleic acid dye is selected from at least one of SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), RiboGreen, preferably from one of SYTOX Green, RiboGreen, Pico Green, more preferably from SYTOX Green, RiboGreen.

9. The method of claim 2, wherein, The nuclease is one or more selected from the group consisting of recombinant DNase I (RNase-free), Dnase, RNase-free, DNase I (RNase-free), S1 nuclease, dsDNase, RNase A, RNase A (DNase and protease free); preferably the nuclease comprises at least one selected from the group consisting of recombinant DNase I (RNase-free), RNase A, RNase A (DNase and protease free), or is a combination selected from DNase I, S1 nuclease, 1X dsDNase.

10. The method according to any one of claims 1 to 9, wherein the first nucleic acid dye is labeled at a final concentration of 0.001 to 10 μΜ, preferably at a final concentration of 0.001 μΜ, 0.002 μΜ, 0.1 μΜ, 0.5 μΜ, 1.0 μΜ, 1.5 μΜ, 2.0 μΜ, 2.5 μΜ, 3.0 μΜ, 3.5 μΜ, 4.0 μΜ, 5.0 μΜ, 6.0 μΜ, 7.0 μΜ, 8.0 μΜ, 9.0 μΜ or 10 μΜ, further preferably at a final concentration of 0.5 to 2 μΜ, more preferably at a final concentration of 1 μΜ.

11. The method of any one of claims 1-10, wherein the non-viral nucleic acid vector composition comprises a non-viral nucleic acid vector with externally adsorbed nucleic acid, a non-viral nucleic acid vector with internally encapsulated nucleic acid, a non-viral nucleic acid vector with both externally adsorbed and internally encapsulated nucleic acid, a non-viral nucleic acid vector with no nucleic acid, free nucleic acid, a complex of two or more of the foregoing.

12. The method of any one of claims 1-11, wherein the flow particle detection apparatus is an apparatus that can achieve directional flow of sample stream, the flow particle detection apparatus comprising a directional fluid system and a particle analysis detection apparatus; the directional fluid system is composed of a sample loading unit and a flow unit; the particle analysis detection apparatus comprises an optical system and a particle detector; the particle detector is composed of a photosensor and a signal conditioning circuit with band-pass filter high frequency noise function. wherein ​ ​ ​

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