Method for detecting index of non-viral nucleic acid vector

Abstract

The invention provides a method for detecting an indicator of a non-viral nucleic acid vector composition. The method comprises the following steps: mixing a permeable nucleic acid dye with a sample containing a non-viral nucleic acid vector composition for marking; detecting the mixture by adopting flow type particle detection equipment, and recording the fluorescence intensity and the particle number of the single-particle-level non-viral nucleic acid vector with positive nucleic acid signals, the particle number of the non-viral nucleic acid vector with negative nucleic acid signals, the fluorescence intensity of free nucleic acid, the particle size of the non-viral nucleic acid vector with positive nucleic acid signals and the like; and calculating to obtain indexes such as empty shell rate, nucleic acid copy number distribution, nucleic acid copy number-particle size distribution and the like of the non-viral nucleic acid vector composition.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology and relates to a method for detecting characterization indexes of a non-viral nucleic acid carrier composition. Specifically, it relates to methods for detecting the empty shell rate, nucleic acid copy number distribution, nucleic acid copy number-particle size distribution, etc. of a non-viral nucleic acid carrier composition. Background Art

[0002] Gene therapy is the ultimate treatment technology for gene abnormality-related diseases, and the emergence of nucleic acid drugs with various different mechanisms has brought more possibilities for gene therapy. However, nucleic acid molecules have problems such as poor in vivo stability and difficulty in efficiently entering target cells, and often need the assistance of a carrier for delivery to specific tissues and target cells in the body. Therefore, the development of a safe and efficient nucleic acid delivery system has become an important link in gene therapy.

[0003] Non-viral nucleic acid carriers are tiny in particle size and have strong inter-individual heterogeneity. As non-viral nucleic acid carriers, cationic lipids, lipid nanoparticles, cationic polymers, inorganic nanoparticles, extracellular vesicles (exosomes), nucleic acid conjugates, etc. can be enumerated. For any type of non-viral nucleic acid carrier, determining the ratio of microparticles (such as lipid nanoparticles) that effectively load the target molecule is an important characterization means.

[0004] The empty shell rate is one of the important indexes for evaluating the quality of a non-viral nucleic acid carrier microparticle preparation. For example, in mRNA-LNP preparations, blank LNPs (empty shell LNPs) that do not contain mRNA or other payloads widely exist. In preparations with some standard LNP lipid compositions, the proportion of empty shell LNPs can reach 40%-80%. These blank LNPs not only waste the preparation materials, but may also have a negative impact on the overall performance of the LNP preparation. Therefore, it is necessary to monitor their proportion. The correlation between the distribution of the copy number of the target nucleic acid and the particle size in the microparticle preparation can reflect the uniformity and quality of the preparation.

[0005] The field of gene therapy biotechnology still needs to develop a method for determining the characterization indexes of non-viral nucleic acid carrier preparations. Summary of the Invention

[0006] As non-viral nucleic acid carriers, cationic lipids, lipid nanoparticles, cationic polymers, inorganic nanoparticles, extracellular vesicles (exosomes), nucleic acid conjugates, etc. can be enumerated. In the composition of non-viral nucleic acid carriers, empty particles do not contain the target molecule (target nucleic acid). These empty particles may be truly empty, or they may contain some small fragments of the target nucleic acid, such as fragments of the target-encapsulated mRNA. Non-viral nucleic acid carriers containing intact target nucleic acids have the highest therapeutic performance. The empty particles do not contain therapeutically useful target nucleic acids and do not contribute to the efficacy of the product. On the contrary, they may contain nucleic acid fragments that cause immunogenicity.

[0007] For example, since the particle size distribution, shape, and density of the blank lipid nanoparticles and those loaded with the target RNA are very similar, even using an extremely expensive cryo-transmission electron microscope (the test fee for one sample may be several hundred euros), it is still impossible to confirm whether the nanoparticles have successfully loaded the target RNA.

[0008] The present invention provides a method for detecting one or more indicators of a non-viral nucleic acid carrier in the form of nanoparticles, and the indicators include characterization parameters such as the empty shell rate, nucleic acid copy number distribution, nucleic acid copy number-particle size distribution, etc. of the non-viral nucleic acid carrier. As the non-viral nucleic acid carrier, for example, lipid nanoparticles (LNP) encapsulating a target nucleic acid such as mRNA can be cited.

[0009] The method of the present invention contacts a nucleic acid dye (such as a permeable nucleic acid dye) with a sample, generates fluorescence through the binding of the nucleic acid dye to the nucleic acid, and uses flow cytometry nanoparticle detection technology to detect the labeled mixture, thereby determining whether there are empty shell particles that do not contain the target nucleic acid, the relationship between the copy number of the target nucleic acid and the particle size distribution, and calculating the characterization parameters of the non-viral nucleic acid carrier, such as the empty shell rate, nucleic acid copy number distribution, nucleic acid copy number-particle size distribution, etc. in the sample.

[0010] The detection includes detecting the fluorescence intensity, particle number, and particle size of the particles with positive nucleic acid signals in the mixture, the particle number of the particles with negative nucleic acid signals, the fluorescence intensity of free nucleic acid, and the particle size of the particles with positive labels.

[0011] The method of the present invention has advantages such as high efficiency, accuracy, easy operation, small sample consumption, and good repeatability.

[0012] In the first aspect of the present invention, a method for detecting the empty shell rate of a non-viral nucleic acid carrier composition is provided. The method includes:

[0013] Contacting a nucleic acid dye with a sample containing a non-viral nucleic acid carrier composition;

[0014] Detecting the test solution after mixing to obtain the fluorescence intensity, particle number of the non-viral nucleic acid carriers with positive nucleic acid signals at the single-particle level, the particle number of the non-viral nucleic acid carriers with negative nucleic acid signals at the single-particle level, and the fluorescence intensity of free nucleic acid at the single-particle level;

[0015] Obtaining the particle size of the non-viral nucleic acid carriers with positive nucleic acid signals at the single-particle level;

[0016] Calculating the empty shell rate based on the above result data, drawing a nucleic acid copy number distribution diagram, and drawing a nucleic acid copy number-particle size distribution diagram,

[0017] Among them, preferably, the nucleic acid dye is a non-permeable nucleic acid dye.

[0018] In a second aspect of the present invention, there is provided the use of the above method in the research and development or quality control of non-viral nucleic acid carrier products, for evaluating whether the non-viral nucleic acid carrier products have good uniformity, have a qualified degree of uniformity, and / or whether it is necessary to improve the preparation indexes through process optimization.

[0019] In a third aspect of the present invention, there is provided a nucleic acid dye particularly suitable for the above method, and the use conditions.

[0020] Use

[0021] The method described in the present invention can be used as a method for detecting the empty shell rate, nucleic acid copy number distribution, nucleic acid copy number-particle size distribution in drugs such as cationic lipids, lipid nanoparticles, cationic polymers, inorganic nanoparticles, extracellular vesicles (exosomes), nucleic acid conjugates, etc., or a method for quantitatively analyzing the ratio of particles encapsulating nucleic acids to empty particles in a sample containing non-viral nucleic acid carriers. The method can be used to evaluate whether the non-viral nucleic acid carrier products have good uniformity, have a qualified degree of uniformity, and / or whether it is necessary to improve the preparation indexes through process optimization.

[0022] Advantages of the present invention

[0023] The empty shell rate is one of the important indexes for evaluating the preparation quality of particulate preparations. Understanding the empty shell rate can help producers evaluate the stability and reliability of the preparation process, so as to ensure the quality and consistency of particulate preparation products. The method of the present invention is applicable to the determination of flow cytometry nanoparticle parameters, quality control, etc. of particulate preparations such as mRNA-LNP products, such as mRNA vaccines. The method of the present invention can be used for the research and development or quality control of non-viral nucleic acid carrier products, greatly saving the time and sample amount used in the detection method for quality control.

[0024] The correlation between the distribution of copy number and particle size can reflect the uniformity and quality of the preparation. The method of the present invention is beneficial to the research and development or quality control of non-viral nucleic acid carrier products. For example: if the encapsulation rate of a certain non-viral nucleic acid carrier product reaches 80% required by the pharmacopoeia, it can theoretically be released for clinical treatment as a medicament. However, if the empty shell situation of the product is detected and it is found that its empty shell rate is relatively high, and / or the distribution of its copy number has no correlation or poor correlation with the particle size and is randomly distributed in a disorderly manner, it is considered that the non-viral nucleic acid carrier needs process optimization.

[0025] Through the method of the present invention, it can be judged whether the non-viral nucleic acid carrier has good uniformity, has a qualified degree of uniformity or whether it is necessary to improve its encapsulation rate through process optimization. Thus, it can promote the improvement of the process, make the particulate preparation products more uniform, and make the expected clinical treatment effect of the non-viral nucleic acid carrier more stable.

[0026] The advantages of the present invention are that, compared with the existing methods for detecting the empty shell rate, nucleic acid copy number distribution, and nucleic acid copy number - particle size distribution, the method has the advantages of shorter time consumption, higher efficiency, accuracy, easy operation, less sample consumption, and better repeatability. Description of the Drawings

[0027] Figure 1 It is the concentration and particle size distribution diagram of the lipid nanoparticle - encapsulated mRNA vaccine sample solution in Example 1.

[0028] Figure 2 It is the two - dimensional scatter plot of the scattered light signal and fluorescence signal of the test solution 1 in Example 1.

[0029] Figure 3 It is the two - dimensional scatter plot of the scattered light signal and fluorescence signal of the test solution 2 after enzymatic digestion in Example 1.

[0030] Figure 4 It is the regression curve of the peak area and concentration of the lipid nanoparticle - encapsulated mRNA vaccine standard product by HPLC determination in Comparative Example 1 and the detection result diagram of the lipid nanoparticle - encapsulated mRNA vaccine product (C0 = 16.43 ng / μl).

[0031] Figure 5 It is the elution curve diagram of the lipid nanoparticle - encapsulated mRNA vaccine product in Comparative Example 1.

[0032] Figure 6 It is the two - dimensional scatter plot of the scattered light signal and fluorescence signal of the test solution 3 after treatment with a permeable nucleic acid dye in Example 2.

[0033] Figure 7 It is the two - dimensional scatter plot of the scattered light signal and fluorescence signal of the test solution 4 after treatment with a non - permeable nucleic acid dye in Example 2.

[0034] Figure 8 It is the two - dimensional scatter plot of the encapsulated mRNA - mChery lipid nanoparticles in the SSC and FITC fluorescence channels in Example 3.

[0035] Figure 9 It is the scatter plot (left) and nucleic acid copy number distribution histogram (right) of the encapsulated mRNA - mChery lipid nanoparticles in the SSC and FITC fluorescence channels in Example 3.

[0036] Figure 10 It is the copy number - particle size distribution scatter plot of the encapsulated mRNA - mChery lipid nanoparticles in Example 3.

[0037] Figure 11 It is the copy number - particle size distribution diagram of the lipid nanoparticles of lipid nanoparticle formulation A in Example 4.

[0038] Figure 12 It is the copy number - particle size distribution diagram of the lipid nanoparticles of formulation B of the lipid nanoparticles in Example 4.

[0039] Figure 13 It is the event frequency - copy number curve diagram of the lipid nanoparticles of formulation A and B of the lipid nanoparticles in Example 4. Detailed implementation manners

[0040] The present application will be further described in detail below with reference to the accompanying drawings and examples. Through these descriptions, the features and advantages of the present application will become clearer and more definite.

[0041] The special term "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior to or better than other embodiments. Although various aspects of the embodiments are shown in the drawings, the drawings do not have to be drawn to scale unless otherwise specified.

[0042] In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0043] Term description

[0044] "Microcapsule" refers to a tiny capsule in which a solid or liquid drug is encapsulated by carrier excipients. Generally, those with a particle size between 1 and 250 μm are called microcapsules, those with a particle size between 0.1 and 1 μm are called sub - microcapsules, and those with a particle size between 10 and 100 nm are called nanocapsules.

[0045] "Microsphere" refers to a tiny spherical entity in which a drug is dissolved or dispersed in carrier excipients. Generally, those with a particle size between 1 and 250 μm are called microspheres, those with a particle size between 0.1 and 1 μm are called sub - microspheres, and those with a particle size between 10 and 100 nm are called nanospheres.

[0046] "Liposome" refers to a tiny vesicle in which a drug is encapsulated by a lipid bilayer.

[0047] "Nanoparticle" refers to solid particles with a particle size < 500 nm formed by nanosizing a drug or a combination of a drug and carrier excipients. Nanoparticles composed only of drug molecules are called nanocrystals or nano - drugs, and nanoparticles formed with lipid materials as drug carriers are called lipid nanoparticles.

[0048] As non - viral nucleic acid carriers, cationic lipids, lipid nanoparticles, cationic polymers, inorganic nanoparticles, extracellular vesicles (exosomes), nucleic acid conjugates, etc. can be cited. As an example, lipid nanoparticles encapsulating nucleic acid molecules as drugs can be cited. Herein, "lipid nanoparticle" and "lipid nanoparticle" are used with the same meaning.

[0049] "Non-viral nucleic acid carriers" include non-viral nucleic acid carriers that adsorb nucleic acids externally, non-viral nucleic acid carriers that encapsulate nucleic acids internally, non-viral nucleic acid carriers that adsorb nucleic acids on the surface and encapsulate nucleic acids internally, and empty non-viral nucleic acid carriers.

[0050] "Non-viral nucleic acid carrier composition" refers to a complex composed of non-viral nucleic acid carriers that adsorb nucleic acids externally, non-viral nucleic acid carriers that encapsulate nucleic acids internally, non-viral nucleic acid carriers that adsorb nucleic acids on the surface and encapsulate nucleic acids internally, empty non-viral nucleic acid carriers, free nucleic acids, and solvents.

[0051] "mRNA-lipid nanoparticles" or "nucleic acid drugs" "lipid nanoparticle-encapsulated nucleic acid drugs" mean that mRNA is encapsulated in lipid nanoparticles and can be used as a drug.

[0052] "Non-viral nucleic acid carriers with positive nucleic acid signals" refer to the set of event signals of non-viral nucleic acid carriers that are labeled by nucleic acid dyes (such as permeable nucleic acid dyes or non-permeable nucleic acid dyes, etc.) and show obvious fluorescence intensity during the measurement of non-viral nucleic acid carrier samples in a flow cytometry particle population.

[0053] "Non-viral nucleic acid carriers with negative nucleic acid signals" refer to the set of event signals of non-viral nucleic acid carriers that are not labeled by nucleic acid dyes (such as permeable nucleic acid dyes or non-permeable nucleic acid dyes, etc.) and do not show obvious fluorescence intensity during the measurement of non-viral nucleic acid carrier samples in a flow cytometry particle population.

[0054] In the present invention, compared with non-viral nucleic acid carriers with negative nucleic acid signals, the non-viral nucleic acid carriers with positive nucleic acid signals of the present invention have stronger fluorescence signals, and there is a relatively obvious 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.

[0055] In a scatter plot, the "positive rate" of a certain quadrant refers to the percentage of positive events in the total number of events in that quadrant.

[0056] In the present invention, compared with free nucleic acids, the non-viral nucleic acid carriers have a certain particle size. Therefore, in the scattered light channel, the non-viral nucleic acid carriers have stronger scattered light intensity than free nucleic acids, and there is a relatively obvious boundary between the two. The signal with obvious scattered light intensity is the scattered light positive signal.

[0057] The "nucleic acid molecule" includes but is not limited to DNA, mRNA, siRNA, ASO (antisense oligonucleotides), circular RNA, etc. In this article, for the sake of simplicity, the encapsulated nucleic acid molecule is sometimes also referred to as the "target nucleic acid".

[0058] There is no particular limitation on the size of the encapsulated nucleic acid molecule or target nucleic acid, which can be, for example, greater than 200 - 300 bp / nt, such as 200 - 10000 bp / nt, 400 - 7000 bp / nt.

[0059] In one embodiment, the non-viral nucleic acid carrier is mRNA-LNP. Herein, "mRNA-LNP" is used with the same meaning as "mRNA vaccine" and "mRNA-lipid nanoparticle".

[0060] The mRNA vaccine can contain mRNA molecules encoding target proteins (such as antigenic peptides) from viruses such as herpes simplex virus, poxvirus, rabies virus, influenza virus, adenovirus, enterovirus, rotavirus, SARS-CoV-2, dengue virus, human respiratory syncytial virus, monkeypox virus, porcine circovirus, etc., or can contain mRNA molecules encoding one or more immune-stimulating molecules, one or more pathogenic antigens, or can contain non-coding regions of the virus, including (major histocompatibility complex) MHC-binding peptides, etc. The mRNA can be nucleoside-modified or unmodified.

[0061] Typical LNPs are usually stable nanoparticles assembled from phospholipids, cationic lipids, sterol lipids, and PEGylated lipids in a certain ratio.

[0062] As the lipid components of the LNP, those that are generally recognized as usable can be listed. As the Zeta potential of the LNP, the generally recognized allowable range can be listed.

[0063] There is no special limitation on the particle size range of the LNP detected by the method of the present invention, which is adjusted according to the usage scenario. It can be listed as: less than 800 nm, less than 500 nm, preferably less than 400 nm, and more preferably less than 150 nm.

[0064] The "empty shell rate" refers to the proportion of empty shell particles that do not contain the target nucleic acid in the non-viral nucleic acid carrier sample.

[0065] The "nucleic acid copy number distribution" reflects the average number of target nucleic acid molecules in each particle of the lipid nanoparticle and the distribution of these numbers among different particles. The nucleic acid copy number distribution can be represented, for example, by a scatter plot.

[0066] The "nucleic acid copy number - particle size distribution" reflects the relationship between the above nucleic acid copy number distribution and the particle size of the particles in the non-viral nucleic acid carrier composition. The nucleic acid copy number distribution can be represented, for example, by a scatter plot.

[0067] In the present invention, the term "nucleic acid dye" refers to all dyes with a fluorescence excitation wavelength that can bind to the minor groove region of the nucleic acid helix. In the free state, the nucleic acid dye has almost no fluorescence, but once it binds to nucleic acid, the fluorescence is greatly enhanced. The fluorescence signal intensity is positively correlated with the length of the nucleic acid fragment. When the length of the target nucleic acid is constant, the fluorescence signal intensity is uniform, and the number of fluorescence signal events is linearly related to the number of nucleic acids.

[0068] The nucleic acid dyes each independently include at least one selected from the group consisting of permeable nucleic acid dyes, permeable cyanine dyes, intercalating dyes, or DNA minor groove binding dyes.

[0069] Examples of the nucleic acid dye include: Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroethidium (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,At least one of 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, 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 RNA Select, 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 ethidium bromide.,

[0070] In the present invention, the term "permeable nucleic acid dye" refers to a nucleic acid dye that can penetrate through the surface of a non-viral nucleic acid carrier and enter the interior without using other reagents or methods to enhance the surface permeability of the non-viral nucleic acid carrier. That is, the so-called permeability refers to the permeability between the inside and outside of the nucleic acid carrier.,

[0071] Examples of the permeable nucleic acid dye (or nucleic acid dye 1) include at least one selected from SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO 24, SYTOBC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1 Iodide, TOTO-3 Iodide, TO-PRO-1 Iodide, and TO-PRO-3 Iodide.

[0072] In some embodiments, the final concentration during labeling with the permeable nucleic acid dye is 0.001 - 10 μM. In some embodiments, the final concentration during labeling with the permeable nucleic acid dye is 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.

[0073] In some embodiments, the permeable nucleic acid dye is selected from SYTO 9, SYTO 13, SYTO 16, and SYTO 24. In some embodiments, the permeable nucleic acid dye is selected from SYTO 9 and SYTO 16, preferably SYTO 16.

[0074] In some embodiments, when detecting a sample using a flow particle detection device, the sample is diluted to a certain concentration before use.

[0075] In some embodiments, the certain concentration is such that the non-viral nucleic acid carrier particle concentration is 1×10 5 -1×10 12 particles / ml. In some embodiments, the dilution is performed using a buffer.

[0076] In some embodiments, the buffer is PBS buffer.

[0077] In some embodiments, the PBS buffer is an aqueous solution containing 100 mM - 150 mM NaCl, 2 mM - 4 mM KCl, 5 mM - 10 mM Na2HPO4 and 1 mM - 3 mM KH2PO4 with a pH of 7.2 - 7.6. In some embodiments, the PBS buffer is an aqueous solution containing 100 mM - 140 mM NaCl, 2 mM - 3 mM KCl, 7 mM - 9 mM Na2HPO4 and 1 mM - 2 mM KH2PO4 with a pH of 7.2 - 7.6.

[0078] In some embodiments, the PBS buffer is an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 with a pH of 7.2 - 7.6.

[0079] In some embodiments, the PBS buffer is an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 with a pH of 7.4.

[0080] In some embodiments, when using a flow particle detection device to detect samples, the injection pressures of the samples can be the same or different.

[0081] In some embodiments, the injection pressures are independently selected from 0.1 kPa - 10.0 kPa. Preferably, the injection pressures can be independently selected from 0.5 kPa - 2.0 kPa. In some embodiments, the injection pressures are 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.

[0082] In some embodiments, when using a flow particle detection device to detect samples, the injection times of the samples can be the same or different.

[0083] In some embodiments, the injection times are independently selected from 0.1 min - 10 min. In some embodiments, the injection times are 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.

[0084] In some embodiments, the flow particle detection device used in any of the above methods. The flow particle detection device is a particle analysis detection device capable of realizing the directional flow of the sample stream, wherein

[0085] The flow cytometry particle detection device includes a directed fluid system and a particle analysis and detection device;

[0086] Among them, the directed fluid system consists of a sampling unit and a flow unit;

[0087] The particle analysis and detection device includes an optical system and a particle detector;

[0088] The particle detector is composed of a photoelectric sensor and a signal conditioning circuit with a function of limited-band filtering of high-frequency noise.

[0089] In some embodiments, the above method includes using a flow cytometry particle detection device to photograph, count, and measure the signal intensity value of a particle image with specific optical characteristics for a sample.

[0090] In the present invention, "room temperature" refers to the ambient temperature, which can be 10°C - 40°C, can be 20°C - 30°C; in some embodiments, it is 22°C - 28°C; in some embodiments, it is 24°C - 26°C; in some embodiments, it is 25°C.

[0091] As the incubation temperature of lipid nanoparticles encapsulating nucleic acids with a nucleic acid dye, it can be 20°C - 40°C, can be room temperature - 37°C, can be 25°C.

[0092] "Gating" or "setting a gate" means delimiting a range or an area in a flow cytometry particle distribution diagram and performing single-parameter or multi-parameter analysis on the particle groups therein one by one. The shapes of the gates include linear gates, cross gates, rectangular gates, circular gates, polygonal gates, arbitrarily shaped gates, and quadrant gates, etc.

[0093] In the foregoing of the present invention, whether or not words such as "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the numerical value of each quantitative word may have a difference of less than ±10% or a reasonable difference considered by those skilled in the art, such as a difference of ±1%, ±2%, ±3%, ±4%, or ±5%.

[0094] The term "a variety of" means a quantity of 2 or more.

[0095] The terms "optional", "optionally", or "optionally" mean that the subsequent described event or situation may but does not necessarily occur. For example, "optional surfactant" means that the surfactant may or may not be present.

[0096] The term "final concentration" represents the concentration of this component in the system at the start of the reaction or labeling. The term "and / or" should be understood to mean any one of the alternatives or any combination of any two or more of the alternatives.

[0097] The present invention includes the following content.

[0098] One embodiment of the present invention is a method for detecting the empty shell rate, nucleic acid copy number distribution, and / or nucleic acid copy number - particle size distribution of a non-viral nucleic acid carrier composition, which includes:

[0099] Step (1d): Take a sample containing the non-viral nucleic acid carrier composition to be tested, label it with a nucleic acid dye to obtain mixture 3; preferably, the nucleic acid dye is a permeable nucleic acid dye.

[0100] The non-viral nucleic acid carrier may include a non-viral nucleic acid carrier that externally adsorbs nucleic acid, a non-viral nucleic acid carrier that internally encapsulates nucleic acid, a non-viral nucleic acid carrier that externally adsorbs and internally encapsulates nucleic acid, and an empty non-viral nucleic acid carrier.

[0101] The non-viral nucleic acid carrier composition refers to a complex of a non-viral nucleic acid carrier that externally adsorbs nucleic acid, a non-viral nucleic acid carrier that internally encapsulates nucleic acid, a non-viral nucleic acid carrier that externally adsorbs and internally encapsulates nucleic acid, an empty non-viral nucleic acid carrier, free nucleic acid, and a solvent.

[0102] As the solvent, those commonly used in the art can be used, for example, a buffer solution is used as the aqueous phase. As the buffer solution, Tris buffer solution, PBS buffer solution, etc. can be cited. The pH of the buffer solution can be cited as: pH 7 - 8, for example, pH 7.2 - 7.6, and preferably pH 7.4.

[0103] Compared with free nucleic acid, the non-viral nucleic acid carrier has a certain particle size. During detection, in the scattered light channel, the non-viral nucleic acid carrier has a greater scattered light intensity than free nucleic acid, and there is a relatively obvious boundary between the two. The signal with an obvious scattered light intensity is the scattered light positive signal.

[0104] The scattered light channel is, for example, the SSC (Side Scatter) channel.

[0105] As the sample containing the non-viral nucleic acid carrier composition, it can be diluted to a certain concentration before use. The certain concentration can be that the non-viral nucleic acid carrier particle concentration is 1×10 5 -1×10 12 per ml. In some embodiments, the dilution is carried out using a buffer solution.

[0106] As the buffer solution, Tris buffer solution, PBS buffer solution, etc. can be cited. The pH of the buffer solution can be cited as: pH 7 - 8, for example, pH 7.2 - 7.6.

[0107] The PBS buffer solution may be exemplified by an aqueous solution containing NaCl, KCl, Na2HPO4 and KH2PO4. The PBS buffer solution is, for example, an aqueous solution containing 100 mM - 150 mM NaCl, 2 mM - 4 mM KCl, 5 mM - 10 mM Na2HPO4 and 1 mM - 3 mM KH2PO4; an aqueous solution containing 100 mM - 140 mM NaCl, 2 mM - 3 mM KCl, 7 mM - 9 mM Na2HPO4 and 1 mM - 2 mM KH2PO4 with a pH of 7.2 - 7.6. Preferably, the PBS buffer solution is an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4 with a pH of 7.2 - 7.6. The pH of the PBS buffer solution may be 7.2 - 7.6, preferably pH 7.4.

[0108] The nucleic acid dyes each independently include at least one selected from permeable nucleic acid dyes, permeable cyanine dyes, intercalating dyes or DNA minor groove binding dyes.

[0109] Examples of the nucleic acid dye include: Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), Dihydroethidium (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,At least one of Trihydrate, Trihydrate-FluoroPure Grade, Hoechst 34580, LDS751, 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, 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 ethidium bromide.,

[0110] The permeable nucleic acid dye is a nucleic acid dye that can penetrate through the surface of a non-viral nucleic acid carrier and enter the interior without using other reagents or methods to enhance the surface permeability of the non-viral nucleic acid carrier. That is, the so-called permeability refers to the permeability between the inside and outside of the nucleic acid carrier.,

[0111] Examples of the permeable nucleic acid dyes include at least one selected from 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, SYTO82, 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, and TO-PRO-3 Iodide.

[0112] In some embodiments, the permeable nucleic acid dye is selected from SYTO 9, SYTO 13, SYTO 16, and SYTO 24. In some embodiments, the permeable nucleic acid dye is selected from SYTO 9 and SYTO 16, preferably SYTO 16.

[0113] Examples of the labeling include mixing and incubating the nucleic acid dye with the composition, the sample containing the composition, or the dilution of the sample. There is no particular limitation on the incubation time, and examples include 5 minutes to 20 minutes, preferably 20 minutes. There is no particular limitation on the incubation temperature, and it can be, for example, 20 - 40°C, preferably room temperature - 37°C.

[0114] Optionally, after incubation, the liquid containing the composition of the non-viral nucleic acid carrier to be measured can be further diluted: for example, diluted using a buffer such as PBS buffer, and for example, it can be diluted about 100 - 1000 times.

[0115] The final concentration of the permeable nucleic acid dye during labeling is 0.001 - 10 μM. In some embodiments, the final concentration of the permeable nucleic acid dye during labeling is 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. The final concentration is preferably 0.5 - 2 μM.

[0116] The incubation time of the permeable nucleic acid dye with the sample is 5 - 20 minutes, preferably 20 minutes.

[0117] The incubation temperature of the permeable nucleic acid dye with the sample is 20 - 40°C, preferably 37°C.

[0118] Step (2d): Use a flow particle detection device to detect mixture 3, and obtain the number P1 of non-viral nucleic acid carrier particles with positive nucleic acid signals, the fluorescence intensities Z1 to Z of each non-viral nucleic acid carrier with positive nucleic acid signals n13 , the number P2 of non-viral nucleic acid carrier particles with negative nucleic acid signals, and the free nucleic acid fluorescence intensities Z1' to Z n14 '.

[0119] M4 is the median value of the free nucleic acid fluorescence intensities Z1' to Z n14 '; V4 is the injection volume of mixture 3.

[0120] The flow particle detection device is a particle analysis and detection device that can achieve the directional flow of the sample stream. The flow particle detection device may include a sampling unit, a flow unit, and a particle analysis and detection device. The particle analysis and detection device may include an optical system and a particle detector, and the particle detector includes a photoelectric sensor and a signal conditioning circuit with a function of limiting and filtering high-frequency noise.

[0121] The flow particle detection device may be a flow nano-detector. The flow nano-detector may be FlowNanoAnalyzer (manufactured by Xiamen FlowBio Technology Co., Ltd.).

[0122] The injection pressure can be selected from 0.1 kPa - 10.0 kPa, preferably 0.5 kPa - 2.0 kPa, and more preferably 1 kPa.

[0123] The injection time can be selected from 0.1 min - 10.0 min, preferably 2 min.

[0124] Regarding the injection volume, in this method and the following methods, as the injection volume of each mixture, it can be the same or different.

[0125] The fluorescence intensity (for example, represented by Z) can be detected under the conditions of, for example, a laser detector at 488 nm + 638 nm; single laser channel detection, Laser: 10 / 50 mW, 488 nm. It should be noted that as the wavelength of the detection laser detector, it can be adjusted according to the type of nucleic acid dye used. The fluorescence intensity can be obtained through the FITC channel of the device. The FITC fluorescence channel is the excitation signal of the nucleic acid dye and is used to characterize the mRNA-lipid nanoparticle signal and the free nucleic acid signal.

[0126] Regarding the number of the free nucleic acids and the number of non-viral nucleic acid carrier particles, for example, the number of particles of non-viral nucleic acid carriers that are positive and negative for nucleic acid signals can be obtained using a flow particle detection device under conditions such as a laser detector of 488 nm + 638 nm; single laser channel detection, Laser: 20 / 50 mW, 488 nm; scattered light attenuation: 0.2%; injection pressure: 1 kPa; signal type: Large signal, and the same hereinafter.

[0127] Step (3d): Prepare size standard solutions of size standards with different particle sizes, detect the size standard solutions using a flow particle detection device, record the detection data of the scattered light channel of the size standard solutions, and calculate the particle size S1 to S of each non-viral nucleic acid carrier with a positive nucleic acid signal n13 。

[0128] The flow particle detection device used in step (3d) can be the same device used in step (2d). The scattered light channel can be, for example, the SSC channel.

[0129] The particle size can be detected under conditions such as setting the detection parameters to: laser detector 488 nm + 638 nm; single laser channel detection, Laser: 10 / 50 mW 488; scattered light attenuation: 10%; injection pressure: 1 kPa; signal type: small signal, using the scattered light channel; and fixing the Sampling pressure at 1.0 kPa, but not limited to this.

[0130] As the range of different particle sizes of the size standard solution, it can be adjusted according to the properties of the predicted non-viral nucleic acid carrier to be tested. The size standard solution can, for example, contain size standards with particle sizes selected from 68 ± 2 nm, 91 ± 3 nm, 113 ± 3 nm, and 155 ± 3 nm. As such size standards, commercially available microsphere particle size standard substances can be used. The size standards can be non-fluorescent.

[0131] The calculation can be performed by generating a fitting curve based on the Median value and Events value of the display peak of the size standard (standard solution). The particle size of each non-viral nucleic acid carrier with a positive nucleic acid signal can be obtained by substituting the data of the non-viral nucleic acid carrier sample (lipid nanoparticle product) tested under the same detection parameters into the fitting curve.

[0132] Step (4d): Data analysis, calculate the empty shell rate, draw a nucleic acid copy number distribution map, and / or draw a nucleic acid copy number - particle size distribution map.

[0133] The calculation formula for the empty shell rate is:

[0134]

[0135] The empty shell rate refers to the proportion of empty shell particles that do not contain the target nucleic acid in the non-viral nucleic acid carrier sample. The non-inclusion includes the cases of "completely not containing" and "containing incomplete target nucleic acid or fragments of the target nucleic acid".

[0136] As a method for drawing the nucleic acid copy number distribution map, for example, it may include the following steps:

[0137] Step (1e): Divide the fluorescence intensity Z1-Z of each non-viral nucleic acid carrier with positive nucleic acid signal n13 by M4 respectively to obtain the nucleic acid copy numbers C1-C of each non-viral nucleic acid carrier with positive nucleic acid signal n13 ;

[0138] Step (2e): Draw a statistical histogram of C1-C n13 and the corresponding number of events, which is the nucleic acid copy number distribution map of the non-viral nucleic acid carrier composition.

[0139] As the statistical histogram, for example, the abscissa is the corresponding number of events and the ordinate is the nucleic acid copy number.

[0140] As a method for drawing the nucleic acid copy number-particle size distribution map, for example, it may include the following steps:

[0141] Step (1f): Divide the fluorescence intensity Z1-Z of each non-viral nucleic acid carrier with positive nucleic acid signal n13 by M4 respectively to obtain the nucleic acid copy numbers C1-C of each non-viral nucleic acid carrier with positive nucleic acid signal n13 ;

[0142] Step (2f): Draw a two-dimensional scatter plot of the one-to-one correspondence between C1-C n13 and S1-S n13 which is the nucleic acid copy number-particle size distribution map.

[0143] As the two-dimensional scatter plot, for example, the abscissa is the particle size and the ordinate is the nucleic acid copy number.

[0144] The present invention includes the following contents.

[0145] Item 1. A method for detecting the empty shell rate, nucleic acid copy number distribution and / or nucleic acid copy number-particle size distribution of a non-viral nucleic acid carrier composition, which includes:

[0146] Step (1d): Take a sample of the non-viral nucleic acid carrier composition to be tested, label it with a nucleic acid dye to obtain a mixture 3, and the nucleic acid dye can specifically bind to nucleic acid; preferably, the nucleic acid dye is a permeable nucleic acid dye;

[0147] Step (2d): Use a flow particle detection device to detect the number P1 of non-viral nucleic acid carrier particles with positive nucleic acid signals, and the fluorescence intensities Z1-Z of each non-viral nucleic acid carrier with positive nucleic acid signals in the injection volume V4 of the mixture 3 at a certain injection pressure and a certain injection time. n13 , the number P2 of non-viral nucleic acid carrier particles with negative nucleic acid signals, and the fluorescence intensities Z1'-Z of free nucleic acids n14 ', and calculate the median value M4 of the fluorescence intensities Z1'-Z of free nucleic acids n14 '.

[0148] Step (3d): Prepare particle size standard solutions of different particle sizes, use a flow particle detection device to detect the particle size standard solutions, record the detection data of the scattered light channels of the particle size standard solutions, and record the particle size sizes S1-S of each non-viral nucleic acid carrier with positive nucleic acid signals n13 ;

[0149] Step (4d): Data analysis, calculate the empty shell rate, draw a nucleic acid copy number distribution map and / or draw a nucleic acid copy number - particle size distribution map.

[0150] Item 2. According to the detection method described in Item 1, the nucleic acid dye includes at least one selected from permeable nucleic acid dyes, cyanine dyes, intercalating dyes, or DNA minor groove binding dyes.

[0151] Item 3. According to the detection method described in Item 1 or 2, the nucleic acid dye is a permeable nucleic acid dye; and / or

[0152] The nucleic acid dyes are selected from 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), dihydroethidium (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,At least one of Trihydrate, Trihydrate - FluoroPure Grade, Hoechst 34580, LDS751, 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, 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, ethidium bromide; and / or,

[0153] The permeable 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, SYTO83, 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

[0154] The permeable nucleic acid dye is SYTO 9.

[0155] Item 4. The detection method according to any one of Items 1-3, the calculation formula of the empty shell rate is:

[0156] and / or

[0157] The method for drawing the nucleic acid copy number distribution map is:

[0158] Step (1e): Divide the fluorescence intensity Z1-Z of each non-viral nucleic acid carrier with positive nucleic acid signal n13 by M4 respectively to obtain the nucleic acid copy numbers C1-C of each non-viral nucleic acid carrier with positive nucleic acid signal n13 ;

[0159] Step (2e): Draw a statistical histogram of C1-C n13 and the corresponding number of events, namely the nucleic acid copy number distribution map; and / or

[0160] The method for drawing the nucleic acid copy number-particle size distribution map is:

[0161] Step (1f): Divide the fluorescence intensity Z1-Z of each non-viral nucleic acid carrier with positive nucleic acid signal n13 by M4 respectively to obtain the nucleic acid copy numbers C1-C of each non-viral nucleic acid carrier with positive nucleic acid signal n13 ;

[0162] Step (2f): Draw a two-dimensional scatter plot of the one-to-one correspondence between C1-C n13 and S1-S n13 to obtain the nucleic acid copy number-particle size distribution map.

[0163] Example

[0164] To enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below for further detailed description of the present invention. All the reagents used in the present invention can be purchased from the market or prepared by the methods described in the present invention.

[0165] In the present invention, mRNA-lipid nanoparticles or mRNA-lipid nanoparticles both belong to non-viral nucleic acid carriers.

[0166] 1) Source of specimens

[0167] Prepared according to the formula of the commercially available Moderna mRNA-1273 vaccine (mRNA-1273 ), in which the ratio of SM-102 cationic lipid: DMG-PEG200: DSPC: cholesterol is 50:10:38.5:1.5, the nitrogen-phosphorus ratio of the formula is 6:1, Tris buffer with a pH of 7-8 is used as the aqueous phase, and the 2750nt Luciferase mRNA standard (500 ng / μL) is used as the nucleic acid for encapsulation. The encapsulation of Luciferase mRNA lipid nanoparticles was prepared using the ignite microfluidic chip of PNI Corporation according to the flow rate ratio of 3:1 (L:R) and the inlet flow rate of 12 ml / min.

[0168] SM-102 cationic lipid (cationic·lipid·SM102), CAS·No.: 2089251-47-6.

[0169] 2) Reagents and instruments used

[0170] 2.1) Reagents:

[0171] SYTO 9 and SYTO 16 were purchased from Invitrogen;

[0172] RNase-free DNase I was purchased from Takara;

[0173] S1 nuclease and 1×dsDNase were purchased from ThermoFisher;

[0174] 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.

[0175] 2.2) Instruments: Flow NanoAnalyzer was purchased from NanoFCM Inc; Alliance 2695 / 2487 high-performance liquid chromatography system and ODS C18 column were purchased from Waters, USA, and Sephadex G-50 dextran gel column was purchased from Shimadzu, Japan.

[0176] Comparative Example 1: Determination of encapsulation efficiency by Sephadex gel filtration-HPLC method

[0177] Drawing of standard curve: Accurately pipette 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL, and 1.6 mL of 2750 bp encapsulated Luciferase mRNA lipid nanoparticles with a concentration of 500 ng / μL into 10 mL volumetric flasks respectively, dilute to the mark with methanol, and prepare a series of standard solutions with concentrations of 5 ng / μL, 10 ng / μL, 20 ng / μL, 40 ng / μL, and 80 ng / μL. According to the chromatographic conditions: chromatographic column ODS C18 column (150 mm × 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, inject samples respectively, record the chromatographic peak area, regress the peak area against the concentration, obtain the regression equation, and the results are as Figure 4 shown.

[0178] Detection of encapsulation efficiency: Take 0.5 mL of the product of encapsulated Luciferase mRNA lipid nanoparticles in a 10 mL volumetric flask, make up the volume with methanol, and determine its content under the HPLC chromatographic conditions in the "Drawing of standard curve" item of Comparative Example 1, and calculate the concentration C0. Take another 0.5 mL of the solution sample of the product of encapsulated Luciferase mRNA lipid nanoparticles onto Sephadex G-50 dextran gel, elute with water, and control the flow rate at 3 mL / min; collect 10 mL of the eluate, take 5 mL into a 10 mL volumetric flask, make up the volume with methanol, and determine its content under the HPLC chromatographic conditions in the "Drawing of standard curve" item of Comparative Example 1 (the results are as Figure 5 shown), calculate the concentration C1, and calculate the encapsulation efficiency according to the following formula: Encapsulation efficiency (%) = (drug content of lipid nanoparticles separated by the column / total drug amount) × 100%, that is, Encapsulation efficiency (%) = (C 1* 40 / C 0* 20) × 100%.

[0179] Results: The concentration C0 of the product of encapsulated Luciferase mRNA lipid nanoparticles after passing through the Sephadex gel column was measured to be 16.43 ng / μL, the concentration C1 of the product of encapsulated Luciferase mRNA lipid nanoparticles was 6.67 ng / μL, and the encapsulation efficiency was 81.2%.

[0180] Example 1: Determination of the encapsulation efficiency and drug loading of the Luciferase mRNA-encapsulated lipid nanoparticle product

[0181] 1) Determination of the total concentration of the Luciferase mRNA-encapsulated lipid nanoparticle product and empty lipid nanoparticles

[0182] Dilute the Luciferase mRNA-encapsulated lipid nanoparticles 100-fold with PBS buffer as the sample solution. Detect the fluorescence microsphere standard solution with a calibrated concentration and the sample solution by Flow NanoAnalyzer, and record the number of particles of the fluorescence microsphere standard solution and the sample solution respectively under the same injection pressure (1 kPa) and the same injection time (2 min). The detection parameters of Flow NanoAnalyzer are: laser detector 488 nm + 638 nm; single laser channel detection Laser: 20 / 50 mW, 488 nm; scattered light attenuation: 0.2%; injection pressure: 1 kPa; signal type: Large signal. Use the scattered light channel under these conditions.

[0183] Calculate the total concentration (A0, particles / mL) of the Luciferase mRNA-encapsulated lipid nanoparticles and empty lipid nanoparticles through the dilution factor and the concentration of the fluorescence microsphere standard solution.

[0184] Total concentration of Luciferase mRNA-encapsulated lipid nanoparticles and empty lipid nanoparticles (particles / mL) = Concentration of fluorescence microsphere standard solution (particles / mL) × Number of particles in the sample solution × Dilution factor of Luciferase mRNA-encapsulated lipid nanoparticles diluted to the sample solution ÷ Number of particles in the fluorescence microsphere standard solution.

[0185] Result: As Figure 1 shown, the total concentration of the mRNA vaccine product encapsulated in lipid nanoparticles and empty lipid nanoparticles is 3.4×10 8 particles / mL.

[0186] 2) Determination of the fluorescence intensity of free mRNA vaccine and Luciferase mRNA-encapsulated lipid nanoparticles (excluding empty lipid nanoparticles) in the Luciferase mRNA-encapsulated lipid nanoparticles

[0187] 100 μL of luciferase mRNA-encapsulated lipid nanoparticles were 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 obtain test sample solution 1. Using a Flow NanoAnalyzer, under the conditions of laser detectors 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, and using the FITC and SSC dual channels, test sample solution 1 was detected within the same injection pressure and injection time as in step 1) of Example 1. The FITC fluorescence was the excitation signal of the nucleic acid dye, used to characterize the signal of luciferase mRNA-encapsulated lipid nanoparticles and the signal of free nucleic acids (mRNA vaccine). Taking the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate, a two-dimensional scatter plot was created, and a gate was set. In the two-dimensional scatter plot composed of the above signals, the first quadrant (upper left quadrant) represents free nucleic acids, the second quadrant (upper right quadrant) represents luciferase mRNA-encapsulated lipid nanoparticles with positive nucleic acid signals, and the fourth quadrant (lower right quadrant) represents empty lipid nanoparticles, and the positive rate data of each quadrant were recorded (see Figure 2 ).

[0188] Record the fluorescence intensity θ of each free nucleic acid in test sample solution 1 within 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 luciferase mRNA-encapsulated lipid nanoparticle with positive nucleic acid signal (excluding empty lipid nanoparticles) (i.e., obtain γ1, γ2,....γ n2 ), and calculate the median value M1 of the fluorescence intensities θ1, θ2,.....θ n1 ; where n1 is the number of free nucleic acids in test sample solution 1 detected under the above injection pressure and injection time, and n2 is the number of luciferase mRNA-encapsulated lipid nanoparticles with positive nucleic acid signals (non-viral nucleic acid carriers) in test sample solution 1 detected under the above injection pressure and injection time.

[0189] 3) Determination of the average fluorescence intensity of luciferase mRNA-encapsulated lipid nanoparticles (excluding empty lipid nanoparticles) after removing surface-adsorbed nucleic acids

[0190] Incubate 100 μL of luciferase mRNA-encapsulated lipid nanoparticles with 0.2 U / μL of RNase-free DNase I, 2 U / μL of S1 nuclease, and 1×dsDNase at 37 °C for 30 minutes. In the above reaction system, add SYTO 9 (dissolved in PBS solution) with a final concentration of 1 μM, incubate at 37 °C for 20 minutes, and then dilute with PBS buffer to the same concentration of luciferase mRNA-encapsulated lipid nanoparticles as the test solution 1 in step 2) of Example 1 to obtain the test solution 2 after enzymatic digestion. Under the conditions of 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 of Flow NanoAnalyzer, use the FITC and SSC dual channels to detect the test solution 2 after enzymatic digestion within the same injection pressure and the same injection time as in step 1) of Example 1. The FITC fluorescence is the excitation signal of the nucleic acid dye, which is used to characterize the signal of luciferase mRNA-encapsulated lipid nanoparticles and the signal of free nucleic acid (mRNA).

[0191] Using the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate, create a two-dimensional scatter plot, set the gating. In the two-dimensional scatter plot composed of the above signals, the first quadrant (upper left quadrant) represents free nucleic acid, the second quadrant (upper right quadrant) represents luciferase mRNA-encapsulated lipid nanoparticles with positive nucleic acid signals, and the fourth quadrant (lower right quadrant) represents empty lipid nanoparticles, and record the positive rate data of each quadrant (see Figure 3 ).

[0192] Record the fluorescence intensity γ' of each luciferase mRNA-encapsulated lipid nanoparticle (excluding empty lipid nanoparticles) with a positive nucleic acid signal in the test solution 2 after enzymatic digestion within the same injection pressure and the same injection time as in step 2) of Example 1 (i.e., obtain γ1', γ2',....γ n3 '); where n3 is the number of luciferase mRNA-encapsulated lipid nanoparticles (non-viral nucleic acid carriers) with positive nucleic acid signals after nuclease treatment in the test solution 2 detected under the above injection pressure and injection time.

[0193] 4) Determination of the encapsulation efficiency and drug loading of luciferase mRNA-encapsulated lipid nanoparticles

[0194] Calculate according to the formula:

[0195]

[0196] Wherein, M is the relative molecular mass of the target nucleic acid, NA is Avogadro's constant, V3 is the total volume (mL) of the lipid nanoparticles encapsulating Luciferase mRNA to be tested, d is the total weight (g) of the lipid nanoparticles encapsulating Luciferase mRNA to be tested after removing the solvent; M1 is the median fluorescence intensity of the free nucleic acid in step 2), and V1 is the volume (mL) of the test sample solution 2 after enzymatic hydrolysis detected within the injection pressure and injection time.

[0197] Result: The encapsulation efficiency is 2.86×10 6 / (2.95×10 6 +0.715×10 6 ) = 78.03%, and the median fluorescence intensity of the free nucleic acid M1 = 938.7; the drug loading is 51.7%.

[0198] The total volume V3 of the lipid nanoparticles encapsulating Luciferase mRNA to be tested is 1000 ml. The relative molecular mass M of the known mRNA vaccine is 487500 g / mol, Avogadro's constant NA is 6.02×10 23 per mol, the total weight d of the lipid nanoparticles is 52×10 -6 g. During the process of collecting fluorescence data by Flow NanoAnalyzer, within the same injection pressure and the same injection time, a total of 0.1 nL (V1 = 1×10 -7 ml) of the sample was measured.

[0199] Conclusion: The method provided by the present invention can more accurately detect the encapsulation efficiency and drug loading of non-viral nucleic acid carriers, avoiding overestimation of the results.

[0200] Example 2: Determination of the encapsulation efficiency of eGFP mRNA lipid nanoparticles, the positive rate of nucleic acid signal of eGFP mRNA lipid nanoparticles, and the average copy number of surface-adsorbed nucleic acid

[0201] 1) Source of specimens

[0202] According to the commercially available Pfizer-BioNTech mRNA vaccine (mRNA-1273 ) were prepared according to the formula, in which the ratio of ALC-0315 cationic lipid: ALC-0159;: DSPC: cholesterol was 46.3:9.4:42.7:1.6, the nitrogen-phosphorus ratio of the formula was 3:1, PBS buffer with a pH of 7-8 was used as the aqueous phase, and a 1226-nt eGFP mRNA standard product (500 ng / μL) was used as the nucleic acid for encapsulation. The preparation process used the ignite microfluidic chip of PNI Company and was prepared according to the flow rate ratio of 3:1 (L:R) and an inlet flow rate of 12 ml / min.

[0203] 2) Reagents and instruments used

[0204] 2.1) Reagents: SYTO 16, SYTOX Green, and Triton X-100 were purchased from Invitrogen; Quant-iT TM RNAAssay Kit was purchased from Thermo Fisher;

[0205] PBS buffer: An aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4 with a pH of 7.4.

[0206] 2.2) Instruments: Flow NanoAnalyzer was purchased from NanoFCM Inc. Specter MAX M2 multi-functional microplate reader was purchased from Molecular Devices, USA.

[0207] 3) Operation for determining the encapsulation efficiency of permeable nucleic acid dyes for eGFP mRNA-encapsulated lipid nanoparticles

[0208] 3.1) Determination of the fluorescence intensity of free nucleic acids in eGFP mRNA-encapsulated lipid nanoparticles and the fluorescence intensity of eGFP mRNA-encapsulated lipid nanoparticles containing surface-adsorbed nucleic acids using permeable nucleic acid dyes

[0209] Incubate 300 μL of eGFP mRNA-encapsulated lipid nanoparticles with SYTO16 (a permeable nucleic acid dye) at a final concentration of 1 μM at 37 °C for 20 minutes, and then dilute it 100-fold with PBS buffer to obtain Test Sample Solution 3. Using FlowNanoAnalyzer, under the conditions of laser detectors 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, use the FITC and SSC dual channels to detect Test Sample Solution 3. The injection time is 2 min. The FITC fluorescence is the excitation signal of the nucleic acid dye, which is used to characterize the signal of eGFP mRNA-encapsulated lipid nanoparticles and the free nucleic acid signal. Taking the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate, a two-dimensional scatter plot is plotted. Set the gating. In the first quadrant (upper left quadrant) of the two-dimensional scatter plot composed of the above signals represents free nucleic acid, the second quadrant (upper right quadrant) represents eGFP mRNA-encapsulated lipid nanoparticles with positive nucleic acid signals, and the fourth quadrant (lower right quadrant) represents empty lipid nanoparticles; and record the positive rate data of each quadrant (see Figure 6 ).

[0210] Record the fluorescence intensity θ' of each free nucleic acid in Test Sample Solution 3 (i.e., obtain θ1', θ2',.....θ n4 ') and the fluorescence intensity γ'' of eGFP mRNA-encapsulated lipid nanoparticles with positive nucleic acid signals in Test Sample Solution 3 (i.e., obtain γ1'', γ2'',....γ n5 "), and calculate and record the median value M2 of the free nucleic acid fluorescence intensities θ1', θ2',.....θ n4 '; where n4 is the number of free nucleic acids in Test Sample Solution 3 detected under the above injection pressure and injection time, and n5 is the number of eGFP mRNA-encapsulated lipid nanoparticles (non-viral nucleic acid carriers) with positive nucleic acid signals after treatment with permeable nucleic acid dyes in Test Sample Solution 3 detected under the above injection pressure and injection time.

[0211] 3.2) Determination of the fluorescence intensity of free nucleic acids and the fluorescence intensity of eGFP mRNA-encapsulated lipid nanoparticles containing surface-adsorbed nucleic acids in eGFP mRNA-encapsulated lipid nanoparticles using non-permeable nucleic acid dyes

[0212] Incubate 300 μL of encapsulated eGFP mRNA lipid nanoparticles with SYTOX Green (a non-permeable nucleic acid dye) at a final concentration of 2 μM at 37 °C for 20 minutes, and then dilute 100-fold with PBS buffer to obtain the test sample solution 4. Using a Flow NanoAnalyzer, under the conditions of a laser detector at 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, use the FITC and SSC dual channels to detect the test sample solution 4 under the same injection pressure and the same injection time as in step 3.1) of Example 2. The FITC fluorescence is the excitation signal of the nucleic acid dye, which is used to characterize the signal of encapsulated eGFP mRNA lipid nanoparticles and the free nucleic acid signal.

[0213] Using the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate, create a two-dimensional scatter plot, set the gating. In the two-dimensional scatter plot composed of the above signals, the first quadrant (upper left quadrant) represents free nucleic acid, the second quadrant (upper right quadrant) represents encapsulated eGFP mRNA lipid nanoparticles with positive nucleic acid signals, and the fourth quadrant (lower right quadrant) represents empty lipid nanoparticles, and record the positive rate data of each quadrant (see Figure 7 ).

[0214] Record the fluorescence intensity θ” of each free nucleic acid in the test sample solution 4 detected under the same injection pressure and the same injection time as in step 3.1) of Example 2 (i.e., obtain θ1”, θ2”, ….θ n6 ”), the fluorescence intensity γ”’ of each encapsulated eGFP mRNA lipid nanoparticle with a positive nucleic acid signal (i.e., obtain γ1”’, γ2”’,....γ n7 ”’) and the number n10 of non-viral nucleic acid carrier particles with negative nucleic acid signals; and calculate and record the median value M3 of the free nucleic acid fluorescence intensities θ1”~θ n6 ”. Where n6 is the number of free nucleic acids in the test sample solution 4 detected under the above injection pressure and injection time, and n7 is the number of encapsulated eGFP mRNA lipid nanoparticles (non-viral nucleic acid carriers) with positive nucleic acid signals after treatment with the non-permeable nucleic acid dye in the test sample solution 4 detected under the above injection pressure and injection time.

[0215] 3.3) Determination of the encapsulation rate of encapsulated eGFP mRNA lipid nanoparticles

[0216] Calculate according to the formula:

[0217]

[0218] Or

[0219]

[0220] or

[0221]

[0222] Calculate the positive rate of nucleic acid signal of the non-viral nucleic acid vector according to the following formula:

[0223]

[0224] Calculate the average copy number of the surface-adsorbed nucleic acid according to the following formula:

[0225]

[0226] Results: Encapsulation rate 2 and encapsulation rate 3 were 65.45%, and encapsulation rate 4 was 65.99%; the positive rate of nucleic acid signal of the non-viral nucleic acid vector was 12.7%; the average copy number of the surface-adsorbed nucleic acid was 0.857.

[0227] 4)Quant-iT TM Operation for determining the encapsulation rate of eGFP mRNA-loaded lipid nanoparticles by Quant-iT

[0228] 4.1) Preparation of RiboGreen working solution

[0229] Take out 1 ml of RiboGreen dye already dissolved in ultradry DMSO from the Quant-iT TM RNAAssay Kit kit, and equilibrate the solution to room temperature before use. According to the number of sample wells, dilute the RiboGreen dye with PBS buffer at a ratio of 1:200 to prepare the working solution, and wrap it with aluminum foil or store it in the dark to avoid light.

[0230] 4.2) Plotting of the RiboGreen standard curve

[0231] Quant-iT TM When taking out the RNA Assay Kit, 16S and 23S RNA standards with a concentration of 100 μg / mL are provided. Dilute them 50-fold with PBS buffer to form a working solution of 2 μg / mL. Gradiently dilute the 2 μg / mL working solution 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, 0 ng / mL. Take 100 μl of each concentration of the standard and add it to a 96-well plate, with 3 replicates for each sample. Add 100 μL of the RiboGreen working solution to each sample well, mix well, and incubate in the dark at room temperature for 2 - 5 minutes.

[0232] Open the Specter MAX M2 multimode microplate reader according to the instrument operation guide, place the 96-well plate in it, and detect and read the fluorescence values of the microplate samples. Subtract the reagent background fluorescence value of the 0 ng / mL sample well from the fluorescence value of each sample well, and the corrected fluorescence value corresponds to the corresponding RNA concentration to make a standard curve.

[0233] 4.3) Pretreatment of eGFP mRNA-encapsulated lipid nanoparticles

[0234] Take 100 μL of eGFP mRNA-encapsulated lipid nanoparticles and 100 μL of empty lipid nanoparticles respectively, mix them with 20 μL of 10% (v / v) Triton X-100 respectively, and perform demulsification by ultrasonic treatment for 5 min, and shake well. Take 100 μL of eGFP mRNA-encapsulated lipid nanoparticles and 100 μL of empty lipid nanoparticles respectively, and mix them with 20 μL of PBS buffer respectively, and shake well. Prepare 4 sample mixtures.

[0235] 4.4) Sample analysis of eGFP mRNA-encapsulated lipid nanoparticles

[0236] Dilute the above 4 sample mixtures with PBS buffer to an appropriate ratio (dilute to a lipid nanoparticle concentration of 1×10 5 -1×10 12(in units of / ml) to obtain respective diluted solutions. Then, take 100 μL of each of the above diluted solutions and mix them with an equal volume of RiboGreen fluorescent dye. Mix well and incubate in the dark at room temperature for 2 - 5 minutes. Then add them to a 96-well plate. Use a Specter MAXM2 multi-functional microplate reader to detect the fluorescence intensity of the samples. Subtract the background fluorescence value of the empty lipid nanoparticle reagent from the detected value of the encapsulated eGFP mRNA lipid nanoparticle sample to obtain the corrected sample fluorescence value. Substitute the corrected sample fluorescence value into the standard curve obtained in step 4.2) of Example 2 to obtain the nucleic acid concentration V1 of the encapsulated eGFP mRNA lipid nanoparticle before demulsification and the nucleic acid concentration V2 of the encapsulated eGFP mRNA lipid nanoparticle after demulsification.

[0237] 4.5) Calculation of the encapsulation efficiency of the encapsulated eGFP mRNA lipid nanoparticles

[0238]

[0239] Results: The detection results are shown in Table 1. After calculation, the encapsulation efficiency of the encapsulated eGFP mRNA lipid nanoparticles is 64.82%.

[0240] Table 1 Nucleic acid concentration and fluorescence intensity (n = 3) Y = 6347.7X (R 2 = 0.9965)

[0241]

[0242] Conclusion: Compared with the gold standard Quant-iT TM RiboGreen method, the detection results of the method of Example 2 are basically the same, indicating that the method of the present invention can be used to determine the drug loading and encapsulation efficiency of non-viral nucleic acid carrier compositions, with high accuracy, and can avoid overestimation of the drug loading and encapsulation efficiency. In addition, the method provided by the present invention is simple to operate, time-consuming, low-cost, and highly efficient, and has excellent technical effects.

[0243] Example 3: Determination of the empty shell rate, copy number distribution, and copy number - particle size distribution of the encapsulated mRNA-mChery lipid nanoparticles

[0244] 1) Source of specimens

[0245] Prepared according to the formula of the commercially 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 of the formula is 6:1, Tris buffer with a pH of 7-8 is used as the aqueous phase, and the 711-nt mRNA-mChery (cherry fluorescent protein) standard (Beijing Tsingke, 500 ng / μL) is used as the nucleic acid for encapsulation. The preparation process uses the PNI Company's ignite microfluidic chip and is prepared at a flow rate ratio of 3:1 (L:R) and an inlet flow rate of 12 ml / min.

[0246] Abbreviation

[0247] DSPC: Distearoylphosphatidylcholine

[0248] DMG: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

[0249] SM-102 cationic lipid (cationic lipid SM102), CAS No.: 2089251-47-6.

[0250] 2) Reagents and instruments used

[0251] 2.1) Reagents: SYTO 16 was purchased from Invitrogen;

[0252] 2.2) Instruments: Flow NanoAnalyzer was purchased from NanoFCM Inc.

[0253] 3) Determination of the empty shell rate of encapsulated mRNA-mChery lipid nanoparticles by permeable nucleic acid dyes

[0254] 3.1) Determination of the empty shell rate of encapsulated mRNA-mChery lipid nanoparticles after nucleic acid dye labeling: The FlowNanoAnalyzer was used to detect the scattered light and FITC fluorescence of the lipid nanoparticles after nucleic acid dye labeling.

[0255] 300 μL of encapsulated mRNA-mChery lipid nanoparticles was incubated with SYTO16 (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 obtain the test solution 5; the test solution 5 was detected by FlowNanoAnalyzer.

[0256] Detection parameters of Flow NanoAnalyzer: Laser detectors 488nm + 638nm; Single laser channel detection Laser: 10 / 50mW 488; Scattered light attenuation: 10%; Injection pressure: 1kpa; Signal type: small signal. Under these conditions, the test solution 5 was detected using the SSC scattered light channel and the FITC fluorescence channel.

[0257] The FITC fluorescence is the excitation signal of the SYTO16 dye, which is used to characterize the nucleic acid in the lipid nanoparticles. SSC is the scattered light channel, which is used to receive the scattered light signal and has nothing to do with whether the nucleic acid is labeled with a fluorescent dye.

[0258] 3.2) According to the detection results of the test solution 5 in the SSC and FITC fluorescence channels, a scatter plot of the detection results was plotted with the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FITC channel as the ordinate, as shown in Figure 8 .

[0259] A two-quadrant gate was set for the obtained scatter plot. Specifically, the intensity of the stained nucleic acid signal is relatively high, and the positive signal is significantly distinguishable from the background. With FITC as the Y-axis, two subpopulations distributed up and down can be clearly seen on the scatter plot, and the positive subpopulation can be easily circled using the gating tool.

[0260] Among them, the particle population with a positive SSC scattered light signal and a positive SYTO16 dye signal in the figure was used as the encapsulated mRNA-mChery lipid nanoparticles containing nucleic acid, and the number of particles P1 of the encapsulated mRNA-mChery lipid nanoparticles containing nucleic acid was obtained; the particle population with a positive SSC scattered light signal and a negative SYTO16 dye signal was used as the empty-shell lipid nanoparticles, and the number of particles P2 of the empty-shell lipid nanoparticles was obtained.

[0261] The empty-shell rate was calculated according to the following formula:

[0262]

[0263] Result

[0264] After calculation, the empty-shell rate of the encapsulated mRNA-mChery lipid nanoparticles was 40.9%. The empty-shell rate of the encapsulated mRNA-mChery lipid nanoparticles, the positive rates of P1 and P2, etc. are shown in Figure 8 . The method of the present invention can be used to evaluate the nucleic acid loading of non-viral nucleic acid carrier formulations and obtain the proportion of empty particles in the total number of particles.

[0265] 4) Determination of the copy number distribution and copy number-diameter distribution of encapsulated mRNA-mChery lipid nanoparticles

[0266] 4.1) Incubate 300 μL of encapsulated mRNA-mChery lipid nanoparticles with SYTO16 (a permeable nucleic acid dye) at a final concentration of 1 μM at 37 °C for 20 minutes, and then dilute 100-fold with PBS buffer to obtain Test Sample Solution 3; detect Test Sample Solution 3 using FlowNanoAnalyzer.

[0267] Flow NanoAnalyzer detection parameters: Laser detectors 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. Under these conditions, use the dual channels of the SSC scattered light channel and FITC fluorescence, and the injection time is 2 min.

[0268] 4.2) The FITC fluorescence is the excitation signal of the nucleic acid dye, used to characterize the signal of encapsulated mRNA-mChery lipid nanoparticles and the free nucleic acid signal. Set the gate with the scattered light signal of the SSC channel as the abscissa and the fluorescence signal of the FTTC channel as the ordinate.

[0269] In the two-dimensional scatter plot composed of the above signals, the first quadrant (upper left quadrant) represents free nucleic acid, the second quadrant (upper right quadrant) represents encapsulated mRNA-mChery lipid nanoparticles, and the fourth quadrant (lower right quadrant) represents empty lipid nanoparticles.

[0270] Record the positive rate data of each quadrant (the above operation is the same as step 3.1 of Example 2). The "positive rate" of a certain quadrant refers to the percentage of positive events in the total number of events in that quadrant.

[0271] 4.3) Record the fluorescence intensities Z1’~Z n14 ’ of each free nucleic acid in Test Sample Solution 5 and the fluorescence intensities Z1~Z n13 of the encapsulated mRNA-mChery lipid nanoparticles with positive nucleic acid signals in Test Sample Solution 5;

[0272] And calculate and record the median value M4 of the fluorescence intensities Z1’~Z n14 ’;;

[0273] Divide the fluorescence intensities Z1~Z n13 of each non-viral nucleic acid carrier with positive nucleic acid signals by M4 to obtain the nucleic acid copy numbers C1~C n13 of each encapsulated mRNA-mChery lipid nanoparticle with positive nucleic acid signals, and plot C1~C n13The statistical histogram corresponding to the number of events, i.e., the nucleic acid copy number distribution map. The scatter plot and nucleic acid copy number distribution map of the encapsulated mRNA-mChery lipid nanoparticles are shown in Figure 9 .

[0274] Results

[0275] After calculation, on average, each encapsulated mRNA-mChery lipid nanoparticle encapsulates 1.4 copies of the nucleic acid drug. From Figure 9 it can be seen that the copy number distribution of the encapsulated mRNA-mChery lipid nanoparticles in this example is concentrated, and the proportion of empty particles is low.

[0276] 4.4) Detection of the copy number - particle size distribution of encapsulated mRNA-mChery lipid nanoparticles

[0277] Source: Encapsulated mRNA-mChery lipid nanoparticles (same as above)

[0278] Set the detection parameters as: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; injection pressure: 1kpa; signal type: small signal, use the scattered light channel under this condition; fix the Sampling pressure at 1.0 kPa.

[0279] Under this condition, the data of the particle size standard mixture of 68 ± 2nm, 91 ± 3nm, 113 ± 3nm, and 155 ± 3nm were tested, and the Median value and Events value of the peaks shown by the particle size standard were used to generate a fitting curve.

[0280] For the data of the concentrated solution of the encapsulated mRNA-mChery lipid nanoparticles tested under the same detection parameters after dilution by 10 times, the gating tool was used to define the target particle size distribution range. Record the number of particles within the gate, the percentage of the number of particles within the gate, and the median value, average value, and standard deviation of the particle size within the gate, and combine the fitting curve to convert the single-particle size value of each encapsulated mRNA-mChery lipid nanoparticle with a positive nucleic acid signal, and use the single-particle size of each encapsulated mRNA-mChery lipid nanoparticle with a positive nucleic acid signal as the abscissa of the scatter plot. The nucleic acid copy numbers C1 to C of each encapsulated mRNA-mChery lipid nanoparticle with a positive nucleic acid signal n13 , are used as the ordinate of the copy number - particle size distribution scatter plot.

[0281] Make a scatter plot of the above abscissa and ordinate data, that is, the copy number - particle size distribution map of the encapsulated mRNA-mChery lipid nanoparticles, and the figure is shown in Figure 10 .

[0282] Result

[0283] After calculation, the empty shell rate of the mRNA-mChery-encapsulated lipid nanoparticles is 40.9% ( Figure 8 ); the copy number distribution of the mRNA-mChery-encapsulated lipid nanoparticles ( Figure 9 ), with an average of 1.4 copies of nucleic acid drug encapsulated in each mRNA-mChery-encapsulated lipid nanoparticle; the copy number-size distribution of the mRNA-mChery-encapsulated lipid nanoparticles is as shown in Figure 10 .

[0284] Copy number-size distribution of lipid nanoparticles in Example 4

[0285] To illustrate the use of the method of the present invention, specifically, to illustrate the relationship between the copy number-size distribution of lipid nanoparticles and the judgment of whether the lipid nanoparticle preparation process should be improved. The inventors measured two examples of lipid nanoparticle formulations A and B with relatively large and typical differences in the copy number-size distribution of lipid nanoparticles, and the measurement method was the same as in Examples 2 and 3.

[0286] The copy number-size distribution diagram of the lipid nanoparticles of lipid nanoparticle formulation A is shown in Figure 11 , and the copy number-size distribution diagram of the lipid nanoparticles of lipid nanoparticle formulation B is shown in Figure 12 , and the event frequency-copy number curves of the lipid nanoparticles of the two formulations are combined and shown in Figure 13 .

[0287] Discussion

[0288] In Figure 11 , it can be seen that the group of scattered points above the horizontal line are fluorescent-positive lipid nanoparticles containing the target nucleic acid, and the group of scattered points below the horizontal line are empty nanoparticles. The proportion of P1 is 68.6%, and the proportion of P2 is 31.4%.

[0289] In Figure 12 , it can be seen that the group of scattered points above the horizontal line are fluorescent-positive lipid nanoparticles containing the target nucleic acid, and the group of scattered points below the horizontal line are empty nanoparticles. The proportion of P1 is 86.7%, and the proportion of P2 is 13.3%.

[0290] In Figure 13 , it can be seen that the peak of lipid nanoparticle formulation A is higher and the peak shape is sharper. On the other hand, the peak of lipid nanoparticle formulation B is shorter and the peak shape is flatter.

[0291] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all such similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention.

Claims

1. Method for detecting the empty shell rate, nucleic acid copy number distribution and / or nucleic acid copy number - particle size distribution of a non-viral nucleic acid carrier composition, comprising: Taking a sample containing the non-viral nucleic acid carrier composition to be tested, labeling it with a nucleic acid dye to obtain a mixture; Use a flow cytometry particle detection device to detect and record the number P1 of non-viral nucleic acid carrier particles with positive nucleic acid signals in the mixture, the fluorescence intensities Z1 to Z of each non-viral nucleic acid carrier with positive nucleic acid signals n13 , the number P2 of non-viral nucleic acid carrier particles with negative nucleic acid signals, and the free nucleic acid fluorescence intensities Z1' to Z n14 '; Record the particle size S1 to S of each non-viral nucleic acid carrier with positive nucleic acid signal n13 ; Calculating the empty shell rate, plotting the nucleic acid copy number distribution map and / or the nucleic acid copy number - particle size distribution map.

2. The method according to claim 1, wherein The calculation formula for the empty shell rate is: Wherein, P1 is the number of non-viral nucleic acid carrier particles with positive nucleic acid signal, and P2 is the number of non-viral nucleic acid carrier particles with negative nucleic acid signal.

3. The method according to claim 1, wherein, The method for plotting the nucleic acid copy number distribution map is: Divide the fluorescence intensities Z1 to Z of each non-viral nucleic acid vector with positive nucleic acid signals n13 by M4 respectively to obtain the nucleic acid copy numbers C1 to C of each non-viral nucleic acid vector with positive nucleic acid signals n13 , where M4 is the median value of the fluorescence intensities Z1' to Z n14 '; Draw C1 to C n13 and the statistical histogram corresponding to the number of events, that is, the nucleic acid copy number distribution map.

4. The method according to claim 1, wherein The method for plotting the nucleic acid copy number - particle size distribution map is: For each non-viral nucleic acid vector with positive nucleic acid signal, divide the fluorescence intensities Z1 to Z n13 by M4 respectively to obtain the nucleic acid copy numbers C1 to C of each non-viral nucleic acid vector with positive nucleic acid signal n13 , where M4 is the median value of the fluorescence intensities Z1' to Z n14 '; Plot C1 to C n13 in one-to-one correspondence with S1 to S n13 to obtain a two-dimensional scatter plot, namely the nucleic acid copy number - particle size distribution plot.

5. The method according to any one of claims 1-4, the method comprising: Preparing a particle size standard solution of particle size standards with different particle sizes, detecting the particle size standard solution by a flow particle detection device, and recording the detection data of the scattered light channel of the particle size standard solution.

6. The method according to any one of claims 1-5, wherein, The nucleic acid dye is at least one selected from permeable nucleic acid dyes, cyanine dyes, intercalating dyes or DNA minor groove binding dyes.

7. The method according to any one of claims 1-6, wherein The nucleic acid dye is selected from Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroethidium (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,At least one of 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, 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, SYTO17, 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., 8. The method according to claim 7, wherein The nucleic acid dye is a permeable nucleic acid dye, and the permeable nucleic acid dye is at least one selected from 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, SYTO85, SYTO 17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, TOTO-1Iodide, TOTO-3Iodide, TO-PRO-1Iodide, TO-PRO-3Iodide; preferably, the permeable nucleic acid dye is selected from SYTO 9 and SYTO 16.

9. The method according to any one of claims 1-8, wherein The injection pressure range of the mixture is 0.1 kPa - 10.0 kPa, preferably 0.5 kPa - 2.0 kPa, more preferably 1 kPa.

10. The method according to any one of claims 1-9, wherein, The injection time range of the mixture is 0.1 min - 10.0 min, preferably 2 min.

11. The method according to any one of claims 1-10, wherein, The detection includes using a flow particle detection device to photograph, count and measure the signal intensity value of a sample containing a non-viral nucleic acid carrier composition, and measure the particle image with specific optical characteristics.

12. The method according to any one of claims 1-11, the non-viral nucleic acid carrier composition is a complex composed of two or more of non-viral nucleic acid carriers with surface-adsorbed nucleic acid, non-viral nucleic acid carriers with internally encapsulated nucleic acid, non-viral nucleic acid carriers with surface-adsorbed and internally encapsulated nucleic acid, empty non-viral nucleic acid carriers, free nucleic acid, solvents, etc.

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