Liposome detection method

Abstract

The invention discloses a detection method of lipidosome. The present invention provides a method for detecting lipidosome, the method comprising: a dyeing step in which a nucleic acid-loaded lipidosome sample to be detected is dyed using a fluorescent nucleic acid dyeing agent, and an analysis step in which the dyed lipidosome sample is analyzed using a nanoflow analyzer, wherein the analysis comprises at least one of particle size determination of the liposome, particle counting, nucleic acid entrapment rate, and nucleic acid copy number determination in the liposome. The detection method of the lipidosome provided by the invention has intuitiveness and high efficiency, can rapidly and accurately perform comprehensive qualitative and quantitative analysis on the nucleic acid loaded nano-lipidosome, and covers simultaneous characterization of the particle size, particle concentration and nucleic acid encapsulation efficiency of the nucleic acid loaded nano-lipidosome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological product quality detection and analysis, and relates to a liposome detection method. BACKGROUND

[0002] In the field of drug delivery, nanoliposomes show great application potential. However, the standardization, sensitivity and non-destructive analysis technology of its quality detection method still face challenges. In order to comprehensively and accurately evaluate the safety of nanoliposomes, the current detection means must consider data and information from various aspects such as in vitro experiments, animal model experiments and clinical monitoring. At the same time, great attention should also be paid to the whole process quality evaluation during the preparation of nanoliposomes. Particle size, particle concentration and nucleic acid encapsulation rate are key indicators for evaluating quality, therefore, it is crucial to establish a standardized detection method. At present, there are limitations and deficiencies in the technical methods for detecting the particle size and nucleic acid encapsulation rate of liposomes. Future research should focus on developing more accurate and efficient evaluation techniques to effectively monitor and control the quality of liposomes, minimize their potential risks in clinical application, and improve their safety and reliability in clinical use.

[0003] As the core carrier of drug delivery system, nanoliposomes show significant advantages in clinical application, but their safety issues still need to be focused on. The safety problem of nanoliposomes in clinical application is mainly due to the insufficient and effective encapsulation of active ingredients. Incomplete encapsulation will lead to the leakage of free PEG, cationic lipids and other components. The leaked substances do not achieve effective nucleic acid delivery and precise release in cells, but also cause a series of side effects and potential risks, including changes in the physical properties of nanoliposomes (such as particle size, charge distribution) and their behavior in the body (such as being captured by the reticuloendothelial system, long-term accumulation). For example, allergic reactions such as skin rash, shortness of breath, anaphylactic shock, blood toxicity reactions such as thrombocytopenia, hemolysis, coagulation abnormalities, organ toxicity reactions such as elevated liver enzymes, abnormal kidney function, etc. Therefore, the encapsulation rate of nanoliposomes is one of the important factors for its quality control.

[0004] In mRNA vaccines, conventional characterization methods of liposomes have many limitations, which affect the accuracy, efficiency and subsequent clinical application of the research. Particle size and dispersion (PDI) detection is usually detected by dynamic light scattering (DLS), but this method has low accuracy for polydisperse samples (PDI > 0.3) and is difficult to distinguish between liposomes and impurity particles. Electron microscopy (TEM / SEM) detection technology has problems such as possible structural deformation caused by sample preparation (such as drying) and insufficient statistical representativeness. Cryo-TEM is an important tool for studying the structure of liposomes, but it requires expensive cryogenic equipment and maintenance, and has high technical requirements for operators. Statistical analysis of liposome size and morphology requires a large number of images, and manual processing is inefficient; traditional methods for measuring encapsulation efficiency, such as ultracentrifugation, dialysis or gel chromatography, can damage the structure of liposomes and cause drug leakage. New technologies such as HPLC or fluorescent labeling have high costs and low sensitivity for certain drugs (such as lipid-soluble molecules). Although nucleic acid detection kits are widely used and have high sensitivity and specificity, they also have disadvantages: they may not detect very low concentrations of samples (such as certain cytokines or early infection markers), especially when the sample is not properly pretreated; there is a risk of false positives due to cross-reactions and interference; the operation is complex and prone to human error; sample processing has limitations (dilution or concentration of certain samples increases the complexity of the operation); the dynamic linear range is limited (dilution of high-concentration samples may introduce errors, standard curve fitting errors are larger in the low-concentration region, and the control of color development time affects signal intensity); reagents and equipment are dependent and batch differences (different batches of antibodies or enzyme conjugates have different activities, which affect the consistency of the results, and the use of equipment such as a microplate reader may result in reading errors due to improper calibration or inadequate maintenance).

[0005] Flow cytometry provides a rapid and accurate quantitative analysis method at the cellular and molecular level with its high sensitivity and high throughput characteristics. The core of this technology is to use laser to irradiate single cells and obtain biological information of cells by detecting scattered light and fluorescent signals. Due to its single-particle analysis capability, flow cytometry is an ideal tool for studying the heterogeneity of extracellular vesicles. However, the flow cytometers currently available on the market are not optimized for extracellular vesicles as small as 100 nanometers in diameter, so further technical improvements are needed when detecting and characterizing these small extracellular vesicles.

[0006] Although nano-flow analyzers such as CytoFLEX nano nano-flow analyzer have been developed, which can detect and characterize microexosomes as small as 40 nanometers in diameter, there is currently no method for detecting nanoliposomes using such nano-flow analyzers, and it is unknown whether they can effectively detect nanoliposomes.

[0007] In addition, it is unknown how to select appropriate dyes from the numerous dyes in the field to cooperate with nano-flow analyzers for nanoliposome detection.

[0008] Therefore, there is still a need to develop new detection methods for liposomes, especially nanoliposomes. SUMMARY

[0009] Problems to be solved by the invention

[0010] Based on the above-mentioned defects existing in the prior art, the purpose of the present application is to provide a detection method for liposomes, in particular a detection method for rapid qualitative and quantitative analysis of nucleic acid-loaded nanoliposomes based on nanoflow detection technology.

[0011] Further, the purpose of the present application is to solve the problem of qualitative and quantitative analysis of nucleic acid-loaded nanoliposomes, and to provide a method for simultaneously detecting multiple indicators of particle size detection, particle counting, and nucleic acid encapsulation rate of nanoliposomes.

[0012] Solution to the problem

[0013] [1]. A detection method for liposomes, wherein the method comprises:

[0014] a staining step, wherein a fluorescent nucleic acid stain is used to stain a nucleic acid-loaded liposome sample to be tested, and

[0015] an analysis step, wherein the stained liposome sample is analyzed by a nanoflow analyzer.

[0016] The analysis includes at least one of particle size determination, particle counting, nucleic acid encapsulation rate, and nucleic acid copy number determination in liposomes.

[0017] [2]. The detection method according to [1], wherein the liposomes are nanoliposomes with a diameter ranging from 40 to 1000 nm; and / or

[0018] The nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0019] [3]. The detection method according to [1] or [2], wherein the nucleic acid-loaded liposome sample to be tested is a dispersion of nucleic acid-loaded liposomes to be tested,

[0020] Preferably, the nucleic acid-loaded liposomes to be tested are dispersed in water,

[0021] More preferably, the concentration of nucleic acid-loaded liposomes to be tested in the liposome sample is 0.1 to 5 x 10 5 particles / μL.

[0022] [4]. The detection method according to any one of [1] to [3], wherein the fluorescent nucleic acid stain is SYTO TM 9 green fluorescent nucleic acid stain;

[0023] Preferably, the concentration of the fluorescent nucleic acid stain is 0.05-0.5 mM.

[0024] [5]. The detection method according to any one of [1] to [4], wherein the volume ratio of the liposome sample to the fluorescent nucleic acid stain is 50-150:1, preferably 70-130:1, more preferably 80-120:1, and further preferably 90-110:1.

[0025] [6]. The detection method according to any one of [1] to [5], wherein, in the staining step, the incubation is at a temperature of 33-40°C, and / or the incubation time is 1-30 min.

[0026] [7]. The detection method according to any one of [1] to [6], wherein, in the particle size determination, the standard particle size microspheres are detected under the same test conditions as the liposome sample by using the nano-flow cytometer, the signal position of the standard particle size microspheres in the side scattering light channel for characterizing particle size is determined, and the particle size of the liposome sample is determined.

[0027] [8]. The detection method according to any one of [1] to [7], wherein, in the nucleic acid encapsulation rate determination, the nucleic acid-loaded liposome sample to be detected is detected in the side scattering light channel for characterizing particle size, the position of the background noise population is determined according to particle size, the position of the nucleic acid-loaded liposome to be detected is determined, and a gate containing the nucleic acid-loaded liposome to be detected is delimited, and the gate containing the nucleic acid-loaded liposome to be detected is analyzed in the fluorescence detection channel for detecting the fluorescent nucleic acid stain, to obtain the nucleic acid encapsulation rate.

[0028] [9]. The detection method according to any one of [1] to [8], wherein, in the determination of the nucleic acid copy number in the liposome, a two-dimensional scatter plot is established according to the side scattering light for characterizing particle size and the fluorescence detection channel for detecting the fluorescent nucleic acid stain, the position of the free nucleic acid dyed by the fluorescent nucleic acid stain, i.e. the P1 gate, is determined, and the position of the liposome particle dyed by the fluorescent nucleic acid stain, i.e. the P3 gate, is determined; the average nucleic acid copy number in the liposome is obtained by calculating the ratio of the average fluorescence intensity of the particles in the P3 gate in the fluorescence detection channel to the average fluorescence intensity of the particles in the P1 gate in the fluorescence detection channel.

[0029]

[10] . The detection method according to any one of [1] to [9], wherein, in addition to the nucleic acid-loaded liposome sample to be detected, the following controls are included;

[0030] Control for the medium for dispersing the liposome to be tested for loading nucleic acid;

[0031] Control for staining the medium using a fluorescent nucleic acid stain;

[0032] Control for the sample of the liposome to be tested for loading nucleic acid, which is not stained using a fluorescent nucleic acid stain;

[0033] Control for the sample of the liposome to be tested for empty loading nucleic acid, which is stained using a fluorescent nucleic acid stain.

[0034] Effects of the invention

[0035] The liposome detection method provided by the present application has intuitiveness and high efficiency, can rapidly and accurately comprehensively qualitatively and quantitatively analyze the nucleic acid-loaded nanoliposome, and covers the simultaneous characterization of the particle size, particle concentration and nucleic acid encapsulation rate of the nanoliposome. The liposome detection method performs well in terms of accuracy and precision, has high sensitivity, research flexibility, and multiple advantages such as convenience and comprehensiveness of detection. It is not only suitable for full-process quality evaluation of the nucleic acid-loaded nanoliposome preparation process, but also can provide solid data support for formulating scientific and reasonable quality standards, and is helpful for more effectively controlling and evaluating the quality of biological products, thereby significantly improving the safety and reliability of the nanoliposome in clinical medication.

[0036] The dye used in the liposome detection method provided by the present application can specifically bind to the nucleic acid component in the liposome and emit a fluorescence signal, and in combination with the advanced nano-flow analysis detection technology, the technology can accurately characterize and quantify the nanoscale liposome at the single particle level, thereby comprehensively and deeply analyzing the quality of the liposome. Through this comprehensive method, not only can the sensitivity of the detection be significantly improved to ensure that even a small amount of liposome can be accurately captured and recognized, but also the effect of non-destructive analysis can be realized, that is, the structure and function of the liposome will not be damaged during the analysis process, ensuring the reliability of the analysis result and the integrity of the liposome.

[0037] The liposome detection method provided by the present application can accurately quantitatively analyze the encapsulation rate of the nanoliposome particle, cover various conditions such as free nucleic acid and empty liposome, and can simultaneously characterize and research the particle size and particle number. The detection method is suitable for comprehensive quality evaluation of the nanoliposome preparation process, provides data support for formulating relevant quality standards, is helpful for effectively controlling and evaluating the quality of biological products, and further improves the safety and reliability of the liposome in clinical application. In addition, the detection method of the present application has high sensitivity and can realize non-destructive analysis. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Signal position of standard particle size microspheres in VSSC1 channel.

[0039] Figure 2 Deionized water as sample top sample.

[0040] Figure 3 Syto9 dye and deionized water as sample top sample.

[0041] Figure 4 Syto9 dye and TE buffer as sample top sample.

[0042] Figure 5 Liposome nanoparticle without Syto9 dye staining as sample top sample.

[0043] Figure 6 Empty liposome nanoparticle plus Syto9 staining as sample top sample.

[0044] Figure 7 Deoxyribonucleic acid (DNA) nanoliposome sample loaded with Syto9 dye as sample top sample.

[0045] Figure 8 Ribonucleic acid (RNA) nanoliposome sample loaded with Syto9 dye as sample top sample.

[0046] Figure 9 Method for detecting the number of encapsulated nucleic acids in liposome nanoparticles, whereinA in Figure 9 is an LNP blank sample (LNP not stained with Syto9);B in Figure 9 is an LNP sample + Syto9 stained sample;C in Figure 9 is an LNP sample + Syto9 stained sample + nuclease treatment.

[0047] Figure 10 Comparison of the accuracy of the sample detection results of the present application and the detection results of conventional detection methods, whereinA in Figure 10 is the particle size and PDI measured by Malvern 3000 laser particle size analyzer;B in Figure 10 is a Cryo-TEM cryogenic transmission electron microscope image;C in Figure 10 is the detection result of the method of the present application.

[0048] Figure 11 Comparison chart of different fluorescent dyes. DETAILED DESCRIPTION

[0049] ​​​​​​Various illustrative embodiments, features and aspects of the present application are described below. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0050] In addition, for the purpose of convenience and brevity, detailed descriptions of well-known functions and structures incorporated in the present application can be omitted. It will be appreciated that those skilled in the art will be able to devise various modes of implementing the application without the application departing from the spirit and scope thereof, and that the omissions therefore should not be regarded as a disadvantage of the application.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The meaning of "a", "an", and "the" includes plural references. The meaning of "in" includes "in" and "on." The use of "including", "comprising", "having" and "with" should be interpreted as specifically reciting the presence of the stated features or components and any associated steps, but not the exclusion of others. The use of "including" and "comprising" should not be interpreted as implying that any other features are optional, optional, essential, or preferred.

[0052] In this specification, the meaning expressed by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0053] In this specification, the terms "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like, refer to the specific elements (e.g., features, structures, properties, and / or characteristics) described in relation to that embodiment, which are included in at least one embodiment described herein, and can or can not be present in other embodiments. In addition, it should be understood that the described elements can be combined in various embodiments in any suitable manner.

[0054] In this specification, the numerical range expressed by "numerical value A to numerical value B" means a range including the end point values A and B. Detailed description of the invention

[0056] In some aspects of the present application, a method for detecting a liposome is provided, which is a detection method for rapid qualitative and quantitative analysis of nucleic acid-loaded nanoliposomes based on nanoflow detection technology.

[0057] Specifically, a method for detecting a liposome is provided, the method comprising:

[0058] a staining step in which a nucleic acid-loaded liposome sample to be measured is stained using a fluorescent nucleic acid stain, and an analysis step in which the stained liposome sample is analyzed using a nanoflow analyzer.

[0059] The analysis includes at least one of particle size measurement, particle counting, and nucleic acid encapsulation rate measurement of the liposome.

[0060] The detection method provided by the application can qualitatively and quantitatively analyze liposomes, especially nanoliposomes, and the quantitative analysis method of single particle of the nanoliposome can evaluate the encapsulation of the nanoliposome, including free nucleic acid, empty loading of the liposome, and can also simultaneously characterize and study the particle size, the particle number and the nucleic acid encapsulation rate. The method is suitable for quality evaluation of the whole process of preparation of the nanoliposome, can provide data support for formulating quality standards, is helpful for controlling and evaluating the product quality, and can improve the safety and reliability of the liposome in clinical medication.

[0061] (dyeing step)

[0062] In the dyeing step, a fluorescent nucleic acid dye is used to dye the nucleic acid-loaded liposome sample to be detected.

[0063] Liposome

[0064] In the application, the "liposome" is an artificially prepared spherical vesicle with a closed bilayer membrane structure. The membrane is mainly composed of amphiphilic phospholipid (with both hydrophilic head and hydrophobic tail) molecules arranged into a bilayer structure similar to a cell membrane.

[0065] In some embodiments, the liposome is a nanoliposome. In the application, the nanoliposome is a liposome with a size in the nanometer range (usually with a diameter range of about 40-1000 nm).

[0066] In some preferred embodiments, the method of the application can detect liposomes with a diameter of less than 200 nm. In some preferred embodiments, the method of the application can detect liposomes with a diameter of less than 100 nm. In some preferred embodiments, the method of the application can detect liposomes with a diameter of less than 80 nm. In some preferred embodiments, the method of the application can detect liposomes with a diameter of less than 50 nm.

[0067] In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0068] In some embodiments, the nucleic acid-loaded liposome sample to be detected is a dispersion (suspension) of the nucleic acid-loaded liposome to be detected. In some embodiments, the nucleic acid-loaded liposome to be detected is dispersed in water. In some embodiments, the concentration of the nucleic acid-loaded liposome to be detected in the liposome sample is 0.1-5 x 10 5 particles / μL, preferably 0.5-5 x 10 5 particles / μL, more preferably 0.5-3 x 10 5 particles / μL, further preferably 0.5-2 x 10 5 particles / μL, for example 0.5 x 10 5particles / μL, 1.5 x 10 5 particles / μL, 2 x 10 5 particles / μL, 2 x 10 5 particles / μL.

[0069] fluorescent nucleic acid stain

[0070] In some embodiments, the fluorescent nucleic acid stain is a green fluorescent nucleic acid stain. The stain has the ability to penetrate the cell membrane and enter the interior of the cell, and is capable of binding to nucleic acids (including RNA and DNA) in the cell, thereby producing green fluorescence.

[0071] In some embodiments, the fluorescent nucleic acid stain is SYTO TM 9 green fluorescent nucleic acid stain.

[0072] In some embodiments, the concentration of the fluorescent nucleic acid stain is 0.05-0.5 mM, preferably 0.1-0.5 mM, more preferably 0.1-0.3 mM, for example 0.1 mM, 0.2 mM, 0.3 mM.

[0073] In the step of staining, the volume ratio of the liposome sample to the fluorescent nucleic acid stain is 50-150:1, preferably 70-130:1, more preferably 80-120:1, further preferably 90-110:1, for example: 90:1, 100:1, 110:1.

[0074] In the step of staining, the incubation is at a temperature of 33-40°C, preferably at a temperature of 35-38°C, for example 36°C, 37°C, 38°C.

[0075] In the step of staining, the incubation time is 1-30 min, preferably 2-25 min, more preferably 5-20 min, for example 5 min, 10 min, 15 min, 20 min.

[0076] (analyzing step)

[0077] In the analyzing step, the stained liposome sample is analyzed by a nano-flow cytometer.

[0078] In the analyzing step, the speed of detection by the nano-flow cytometer is less than 8000 particles / s, preferably less than 7000 particles / s, more preferably less than 5000 particles / s.

[0079] In some specific embodiments, the nano-flow cytometer is a CytoFLEX nano flow cytometer.

[0080] In some exemplary embodiments, the analyzing step specifically comprises the following steps:

[0081] 1) The prepared liposome sample is placed in the sample tube of the nano-flow cytometer. Under the action of gas pressure, the liposome particles in the liposome sample are introduced into the flow chamber. At the same time, the sheath liquid is ejected from the sheath liquid tube under high pressure to form a high-speed sheath flow, which wraps the liposome particles to ensure that the particles pass through the detection area of the instrument in a single column;

[0082] 2) During the laser irradiation process, the laser beam is vertically projected on the liposome particles labeled by the fluorescent dye, so as to excite the generation of fluorescence signal and scattering light signal;

[0083] 3) In the signal detection stage, the fluorescence signal and the scattering light signal are transmitted to the detector via the optical filter. The detector captures these light signals and converts them into electrical signals, which are then transmitted to the computer for further processing and analysis to obtain the relevant characteristic data of the nanoliposome.

[0084] Particle size determination

[0085] In some embodiments, in the step of analyzing, the particle size of the liposome is determined.

[0086] In some specific embodiments, the nano-flow analyzer is used to detect standard particle size microspheres under the same test conditions as the liposome sample, determine the signal intensity interval, determine the signal position of the standard particle size microspheres in the side scattering light channel, and then determine the particle size of the liposome sample.

[0087] In some specific embodiments, the detection of standard particle size microspheres includes microspheres with diameters of 53 nm, 73 nm, 96 nm and 120 nm.

[0088] In some exemplary embodiments, the microspheres are silica microspheres.

[0089] In the method provided by the present application, liposome particles with a particle size as low as 40 nm can be accurately distinguished.

[0090] Nucleic acid encapsulation efficiency and copy number of nucleic acid in liposomes

[0091] In some embodiments, in the step of analyzing, the nucleic acid encapsulation efficiency of the liposome is determined.

[0092] In some specific embodiments, the position of the background noise population is determined by the particle size, and the position of the liposome is set.

[0093] In some embodiments, in the nucleic acid encapsulation rate assay, the nucleic acid loaded liposome sample to be tested is detected in a side scatter light channel for characterizing particle size, the position of background noise population is determined according to particle size, the position of the nucleic acid loaded liposome to be tested is determined, and a gate containing the nucleic acid loaded liposome to be tested is delimited, the gate containing the nucleic acid loaded liposome to be tested is analyzed in a fluorescence detection channel for detecting the fluorescence of the fluorescent nucleic acid stain, and the nucleic acid encapsulation rate is obtained.

[0094] In some embodiments, in the analyzing step, the position of the background noise population is determined according to particle size, and the position of the LNP gate is delimited to the right thereof, the single LNP gate analyzes single LNP particles in the LNP gate, and the Syto9+ gate positive signal indicates that the LNP particles in the single LNP gate encapsulate nucleic acids, i.e., the LNP nucleic acid encapsulation rate.

[0095] In some embodiments, in the analyzing step, the number of nucleic acid copies in the liposome is determined.

[0096] In some specific embodiments, in the nucleic acid copy number determination in the liposome, a two-dimensional scatter plot is established according to the side scatter light for characterizing particle size and the fluorescence detection channel for detecting the fluorescence of the fluorescent nucleic acid stain, the position of free nucleic acid stained by the fluorescent nucleic acid stain, i.e., the P1 gate, is determined, the position of the liposome particle stained by the fluorescent nucleic acid stain, i.e., the P3 gate, is determined, and the average number of nucleic acid copies in the liposome is obtained by calculating the ratio of the average fluorescence intensity of particles in the P3 gate in the fluorescence detection channel to the average fluorescence intensity of particles in the P1 gate in the fluorescence detection channel.

[0097] In some more specific embodiments, a two-dimensional scatter plot is established according to the side scatter light VSSC1 (for characterizing particle size) and the B531-H fluorescence detection channel for detecting the fluorescence of the fluorescent nucleic acid stain, the position of free nucleic acid stained by the fluorescent nucleic acid stain, i.e., the P1 gate, is determined, the position of the liposome particle stained by the fluorescent nucleic acid stain, i.e., the P3 gate, is determined, and the average number of nucleic acid copies in the liposome is obtained by calculating the ratio of the average fluorescence intensity of particles in the P3 gate in the B531-H channel to the average fluorescence intensity of particles in the P1 gate in the B531-H channel.

[0098] In the present application, the method provided by the present application utilizes nano-flow detection technology, irradiates a single cell with a laser, detects the scattering light and fluorescence signal thereof, separates and identifies different small particle subpopulations according to the SSC scattering light ratio of different channels, easily detects, characterizes and distinguishes nanoparticles with a particle size as low as 40 nm, and can determine the particle concentration, and the positive signal proportion of the sample represents the proportion of effective nucleic acid encapsulation, as confirmed by Examples 2 and 3, etc.

[0099] (Control)

[0100] In some embodiments, in addition to the sample of the nucleic acid-loaded liposome to be tested, the following controls are included:

[0101] a control for the medium used to disperse the nucleic acid-loaded liposome to be tested, such as a deionized water control;

[0102] a control in which the medium is stained using a fluorescent nucleic acid stain;

[0103] a control of the sample of the nucleic acid-loaded liposome to be tested that is not stained using a fluorescent nucleic acid stain;

[0104] a control of the sample of the empty nucleic acid-loaded liposome to be tested that is stained using a fluorescent nucleic acid stain.

[0105] The method for detecting liposomes provided by the present application utilizes nanoflow detection technology, irradiates a single liposome with a laser, detects the scattered light and fluorescence signal thereof, and can separate and identify different small particle subpopulations according to the SSC scattered light ratio of different channels, so that nanoparticles with a particle size as low as 40 nm can be easily detected, characterized and distinguished. The quantitative analysis method for single nanoparticles can not only effectively evaluate the encapsulation of liposomes, including free nucleic acids, empty packages or unevenly encapsulated nucleic acid proportions, but also can simultaneously characterize the particle size, particle number and nucleic acid encapsulation rate. It is not only suitable for quality evaluation of the whole process of nanoliposome preparation, but also can provide data support for formulating scientific and reasonable quality standards, which is helpful for more effective control and evaluation of product quality, and thus significantly improves the safety and reliability of liposomes in clinical medication.

[0106] The fluorescence staining technology used in the detection method provided by the present application not only has simple operation, but also can significantly shorten the experimental operation steps and time. Compared with the traditional detection method, this innovative fluorescence staining technology undoubtedly provides a more efficient and convenient solution for the research of nanoliposomes.

[0107] Examples

[0108] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0109] The unloaded (empty), deoxyribonucleic acid (DNA)-loaded nanoliposome or ribonucleic acid (RNA)-loaded nanoliposome samples used in the following examples were prepared by the applicant; the SYTO TM9 green fluorescent nucleic acid stain (in this specification, also referred to as Syto9 dye for short), SYTO TM 13, SYTO TM 9, SYTO TM RNA was purchased from Invitrogen TM , concentration: 5 mM; TE buffer was purchased from Merck, HPLC grade water was purchased from Alfa Aesar, deionized water was purchased from Merck, 20 nm filter membrane was purchased from cytiva; 5 mL syringe was purchased from beyotime, 53 ± 2 nm, 73 ± 2 nm, 96 ± 3 nm and 120 ± 3 nm silica nanoparticles were purchased from NanoFCM TM , item number: S23M-SEV. Nano flow cytometer: CytoFLEX nano flow cytometer was provided by Beckman Coulter Biotechnology (Suzhou) Co., Ltd., Malvern 3000 laser particle size analyzer was purchased from Shanghai Sibaiji Instrument System Co., Ltd., Quant-iTTM OliGreenTm ssDNA and Quant-iTTM OliGreenTm ssRNA quantification kit were purchased from Thermo Fisher Scientific, Cryo-TEM cryogenic transmission electron microscope was tested by Suzhou Qingyunruijing Biotechnology Co., Ltd.

[0110] The operation steps for precise detection of nanoliposome single particle suspension using a nano flow cytometer are as follows:

[0111] (1) The prepared nanoliposome particle suspension was placed in the sample tube of the nano flow cytometer. Under the action of gas pressure, the nanoliposome particles were introduced into the flow chamber. At the same time, the sheath liquid was ejected from the sheath liquid tube under high pressure environment, forming a high-speed sheath flow, which wrapped the nanoliposome particles to ensure that the particles passed through the detection area of the instrument in single column;

[0112] (2) During the laser irradiation process, the laser beam was vertically projected on the nanoliposome particles labeled with fluorescent dye, thereby exciting the generation of fluorescence signal and scattering light signal;

[0113] (3) In the signal detection stage, the fluorescence signal and the scattering light signal were transmitted to the detector via the optical filter. The detector captured these light signals and converted them into electrical signals, which were then transmitted to the computer for further processing and analysis to obtain the relevant characteristic data of the nanoliposome;

[0114] For example, using a nano-flow cytometer to analyze the nucleic acid encapsulation rate in the nano-liposome (LNP) particles, the position of the background noise population is determined by particle size, and the position of single LNP (Single LNP gate) is set based on the position of the background noise population. Among them, the Single LNP gate is used to analyze single LNP particles in the LNP gate, and the Syto9+ gate is used to identify LNP particles containing nucleic acids in the Single LNP gate, and a two-dimensional scatter plot is established according to the side scatter light channel (characterizing particle size) and the fluorescence detection channel detecting the fluorescent dye (characterizing the fluorescent dye staining of LNP particles).

[0115] Example 1: Sensitivity and high-precision detection of the method of the present invention

[0116] (1) Reagent preparation:

[0117] ① TE buffer is filtered through a 20 nm filter membrane

[0118] Pull out the 5ml syringe piston, directly pour into 1xTE buffer, insert the syringe piston and discharge the air, install the 20nm filter membrane, press the piston along the flow tube nozzle to filter PBS, first filter out 1-2mL TE to rinse the flow tube wall, then pour out and discard, continue to filter PBS into the rinsed flow tube, and seal the film tightly for use. All TE in the following refers to filtered TE.

[0119] ② Deionized water is filtered through a 20 nm filter membrane

[0120] The steps are the same as ①. All deionized water in the following refers to filtered.

[0121] (2) Nano-liposome initial concentration verification: gradient dilution with deionized water, determine the optimal concentration of nano-liposome sample after dilution as 10 5 events / μL, too high concentration, multiple particles will be detected at the same time, causing false positives, samples at this optimized concentration can be detected on the machine at less than 5000 events / s;

[0122] (3) Nano-liposome sample staining: first dilute 5mM Syto9 dye to a concentration of 0.2mM with deionized water, add 10 5 events / μL nano-liposome to 0.2mM Syto9 dye according to the volume ratio of 100:1, incubate at 37°C for 15min, and directly detect on the machine;

[0123] (4) Standard particle size microspheres detection: Take the optimized concentration of standard particle size microspheres: 53 nm, 73 nm, 96 nm and 120 nm silica microspheres in a volume ratio of 1:1000 with deionized water, run under the same test conditions as the sample, determine the signal intensity interval, and determine the signal position of the standard particle size microspheres in the side scatter light channel (VSSC1 channel), such as Figure 1 ;

[0124] (5) To verify that the results are true positives, the present application sets up the following several groups of control experiments:

[0125] Deionized water control, i.e. deionized water as a sample;

[0126] Syto9 dye and deionized water staining control: 0.2 mM Syto9 dye solution: deionized water mixed in a volume ratio of 1:100, incubated at 37°C water bath for 15 min to obtain Syto9 dye and deionized water staining control;

[0127] Syto9 dye and 1x TE buffer staining control: 0.2 mM Syto9 dye solution: 1x TE buffer added in a volume ratio of 1:100, incubated at 37°C water bath for 15 min to obtain Syto9 dye and 1x TE buffer staining control;

[0128] No Syto9 dye staining of nucleic acid-loaded liposome nanoparticles control: 10 5 events / μL of LNP particles were not stained;

[0129] Empty nucleic acid-loaded liposome nanoparticles with Syto9 staining control: 0.2 mM Syto9 dye solution: 10 5 events / μL of empty LNP particles were added in a volume ratio of 1:100, incubated at 37°C water bath for 15 min to obtain.

[0130] Specifically:

[0131] Deionized water control sample detection: deionized water as a sample was loaded, after loading, according to the LNP gate position, the nanoliposome sample detected in the above step (2) was analyzed again, the background noise population position was determined according to the particle size, and the Single LNP gate position was circled on the right side of the background noise population (as shown in Figure 2 Single LNP gate analyzed single LNP particles in the LNP gate, and Syto9+ gate represented LNP particles in the Single LNP gate that encapsulated mRNA nucleic acid; the aggregation of the dye was determined, the positive proportion in the Syto9+ gate was relatively low, only about 0.86%, which met the expectation, as shown in Figure 2shown.

[0132] Syto9 dye and deionized water staining control sample loading detection: Syto9 dye and deionized water were loaded as samples, 0.2 mM Syto9 dye solution: deionized water was added at a volume ratio of = 1:100, incubated at 37°C water bath for 15 min, directly loaded into the machine for detection, to determine the aggregation of the dye, the positive proportion in the Syto9+ gate was low, only about 6%, as shown in Figure 3 .

[0133] Syto9 dye and 1x TE buffer staining control sample loading detection: Syto9 dye and TE buffer were loaded as samples, 0.2 mM Syto9 dye solution: 1x TE buffer was added at a volume ratio of = 1:100, incubated at 37°C water bath for 15 min, directly loaded into the machine for detection, to determine the aggregation of the dye, compared with Syto9 dye and deionized water staining control, the positive proportion in the Syto9+ gate of Syto9 dye and TE buffer staining control was higher, so it was determined that deionized water was the buffer for suspended LNP, as shown in Figure 4 .

[0134] Control sample loading detection of liposome nanoparticles loaded with nucleic acid without Syto9 dye staining: 10 5 events / μL of LNP particles were directly loaded as samples without staining, and it was observed that the positive proportion in the Syto9+ gate was 0.3%, which met the expectation, as shown in Figure 5 .

[0135] Control sample loading detection of empty liposome nanoparticles with Syto9 staining: 0.2 mM Syto9 dye solution: 10 5 events / μL of empty LNP particles were added at a volume ratio of = 1:100, incubated at 37°C water bath for 15 min, directly loaded into the machine for detection, and it was observed that the positive proportion in the Syto9+ gate was less than 5%, which met the expectation, as shown in Figure 6 .

[0136] Particle size calibration: The particle size is determined based on the nano-flow detection technology, and the appropriate characterization calibration microspheres matching the calibration method are used for calibration. The FCMpass software can infer the particle size to obtain the particle size distribution graph, and the data is tested by polystyrene microspheres with different particle sizes, which is then used for fitting analysis of the particle size of the previous detection sample; 144nm CytoFLEX nano QC microspheres (Lot: 2692902), 500nm Peak 8 single-peak fluorescent microspheres (Lot: BAQ03), 500nm 8Peaks multi-peak fluorescent microspheres (Lot: BAQ05), Nano VISLow microspheres, which are quality control microspheres, are machine quality control. The manufacturing process of polystyrene latex microspheres is reliable, consistent and meets the traceability requirements of NIST, and is the preferred tool for this task. Based on this calibration method, the nano-flow detection technology can obtain accurate and reliable particle size data reports of LNP samples, so that the data of different experiments can be compared and correlated.

[0137] Example 2: Detection of fluorescence signal of Syto9 dye labeling of deoxyribonucleic acid (DNA) nanoliposome sample

[0138] (1) Loading deoxyribonucleic acid (DNA) nanoliposome sample initial concentration verification: The deoxyribonucleic acid (DNA) nanoliposome loaded with deoxyribonucleic acid (DNA) was gradiently diluted with deionized water, and the optimized concentration of the deoxyribonucleic acid (DNA) nanoliposome sample after dilution was 10 5 events / μL, which was detected by the machine, and the concentration detection was less than 5000 / s;

[0139] (2) Detection of standard particle size microspheres: The optimized concentration of 53nm, 73nm, 96nm and 120nm silica microspheres was 1:1000 by volume ratio, which was run under the same test conditions as the sample to determine the signal intensity interval and the signal position of the standard particle size microspheres in the side scatter light channel (VSSC1 channel);

[0140] (3) Deoxyribonucleic acid (DNA) nanoliposome sample staining: 10 5 events / μL of nanoliposome was added with 0.2mM Syto9 dye solution at a volume ratio of 100:1, and then incubated at 37°C for 15min, and then directly detected by the machine;

[0141] (4) Deoxyribonucleic acid (DNA) nanoliposome sample detection results: The diameter is in the range of 53-120nm, the peak signal is near 73nm, the positive signal ratio in the sample Syto9+ gate is 91.44%, i.e. the encapsulation efficiency is 91.44%; The sample concentration after dilution is about 1.86x10 5 events / μL, such as Figure 7The NIST traceable known standard concentration microspheres were used for testing to verify the accuracy of the data, and the calibrated results showed that the particle size was 73 nm, which was consistent with the detection results.

[0142] Example 3: Detection of fluorescence signal of Syto9 dye labeling of ribonucleic acid (RNA) nanoliposome sample

[0143] (1) Initial concentration verification of loaded ribonucleic acid (RNA) nanoliposome samples: The loaded ribonucleic acid (RNA) nanoliposomes were gradient diluted with deionized water, and it was determined that the optimized multiple after dilution of the nanoliposome sample was 10 5 events / μL, and the concentration detection was less than 5000 / s;

[0144] (2) Detection of standard particle size microspheres: The optimized concentration of 53 nm, 73 nm, 96 nm and 120 nm silica microspheres was 1:1000 by volume ratio, and was run under the same test conditions as the sample to determine the signal intensity interval and the signal position of the standard particle size microspheres in the side scatter light channel (VSSC1 channel);

[0145] (3) Staining of loaded ribonucleic acid (RNA) nanoliposome samples: 10 5 events / μL of nanoliposomes were added with 0.2 mM Syto9 dye solution at a volume ratio of 100:1, and then incubated at 37°C for 15 min, and then directly detected on the machine;

[0146] (4) Detection of loaded ribonucleic acid (RNA) nanoliposome samples: The peak signal was near 73 nm within the range of 53-120 nm in diameter, the positive signal ratio in the Syto9+ gate of the sample was 90.92%, i.e. the encapsulation rate was 90.92%; the sample concentration after dilution was about 9.24 x 10 4 events / μL, and the concentration detection was less than 5000 / s; Figure 8 The NIST traceable known standard concentration microspheres were used for testing to verify the accuracy of the data, and the calibrated results showed that the particle size was 73 nm, which was consistent with the detection results.

[0147] Example 4: Calculation of the number of encapsulated nucleic acids in nucleic acid-based liposome particles

[0148] (1) Loaded nucleic acid liposome nanoparticles without staining and direct loading: The concentration of LNP particles was 10 5 events / μL: After adding deionized water at a volume ratio of 100:1, incubation at 37°C for 15 min, direct detection on the machine; it can be seen that there are basically no particles in the P1 gate, and only liposome particles exist on the right side of VSSC1-H;

[0149] (2) Loaded nucleic acid liposome sample + Syto9 stained sample: 10 5events / μL nano-liposome and 0.2 mM Syto9 staining solution were added in a volume ratio of 100:1, incubated at 37°C for 15 min, and directly detected on the machine; after machine testing, obvious particles were observed in the P1 gate, which were free nucleic acids, and particles in the P3 gate were liposome particles encapsulating nucleic acids, as shown in Figure 9 ;

[0150] (3) Liposome sample + Syto9 staining sample + nuclease treatment: 10 5 events / μL nano-liposome and 0.2 mM Syto9 staining solution were added in a volume ratio of 100:1, incubated at 37°C for 15 min, and directly detected on the machine; after machine testing, obvious particles were observed in the P1 gate, which were free nucleic acids, and particles in the P3 gate were liposome particles encapsulating nucleic acids, as shown in

[0151] (4) Calculation of the number of nucleic acids encapsulated in the liposome particles: for the nucleic acid-loaded liposome sample + Syto9 staining sample, the average fluorescence intensity of the particles in the P3 gate / B531-H channel / the average fluorescence intensity of the particles in the P1 gate / B531-H channel was calculated, and the number of nucleic acid copies in the liposome (the average number of copies in a single liposome) was obtained.

[0152] (5) A two-dimensional scatter plot was established according to the side scatter channel VSSC (characterizing particle size) and the fluorescence detection channel B531-H (characterizing the Syto9 staining of liposome particles). The position of free nucleic acids stained by fluorescent dye (P1 gate), and the position of liposome particles stained by fluorescent dye (P3 gate) were detected. Free nucleic acid groups (P1) and nucleic acid-loaded liposomes (P3) were detected; after RNAase digestion, the free RNA group disappeared, proving that P1 was the real free nucleic acid.

[0153] For the nucleic acid-loaded liposome sample + Syto9 staining sample, the average fluorescence intensity of the P3 gate / the average fluorescence intensity of the P1 gate was calculated, and the number of nucleic acid copies in the liposome was obtained.

[0154] CytoFLEX nano can successfully detect free nucleic acids, realize nucleic acid localization and nucleic acid copy number detection.

[0155] Example 5: Comparison of the accuracy of the detection results of the detection method of the present invention and the conventional detection method

[0156] (1) The prepared nucleic acid-loaded liposome sample with a concentration of 10 5 events / μL nucleic acid-loaded liposome sample and Syto9 staining solution (concentration 0.2 mM) were added in a volume ratio of 100:1, incubated at 37°C for 15 min, and directly detected on the machine;

[0157] (2) The particle size, particle concentration, and nucleic acid encapsulation rate results obtained by the detection method provided by the application are compared with the particle size measured by a Malvern 3000 laser particle size analyzer, the liposome size and morphology measured by Cryo-TEM cryogenic transmission electron microscopy, and the nucleic acid encapsulation rate measured by Quant-iTTM OliGreenTm ssDNA and Quant-iTTM OliGreenTm ssRNA quantitative reagent kits.

[0158] When the detection method of the application detects a sample, the obtained results show that the diameter of the sample ranges from 53 nm to 120 nm, and the peak signal appears at about 73 nm. The calculated particle size is 73.8 nm. In addition, the nucleic acid encapsulation rate of the sample shows that the proportion of positive signals is as high as 91.44%, and after dilution treatment, the concentration of the sample is about 1.86 x 10 5 6 / μL. At the same time, the nucleic acid detection kit is used for detection, and the obtained nucleic acid positive encapsulation rate is 66%. By using the Malvern 3000 laser particle size analyzer, the obtained particle size data is 120 nm, and the polydispersity index (PDI) is 0.05. Further, the sample is observed by using the Cryo-TEM cryogenic transmission electron microscopy technology, and the particle size is counted, and the results show that the average particle size is stable at about 73 nm, and the overall particles show a regular and uniform distribution state, as shown in Figure 10 .

[0159] Through comprehensive analysis of the experimental results, it can be found that the Malvern 3000 laser particle size analyzer has certain limitations in measuring the particle size and its dispersity (PDI), especially it cannot effectively distinguish liposomes from impurity particles, which is particularly prominent in practical application. Similarly, electron microscopes (including transmission electron microscopes TEM and scanning electron microscopes SEM) also have the problem of insufficient statistical representativeness in detection technology, and it is difficult to fully reflect the true situation of the sample. Although the nucleic acid detection kit is widely used in encapsulation rate determination and has the advantages of high sensitivity and high specificity, it still faces the challenge of insufficient detection sensitivity when facing extremely low concentration samples.

[0160] In comparison, the detection method of the present application is more simple and easy to operate, greatly reducing the experimental difficulty of small particle detection. Based on the optimization design of the light path and flow path, the method can easily realize the accurate distinction of silica microspheres with a particle size in the range of 53-120 nm, and the accuracy of the absolute count of the volume method is as high as 95% or more. For the samples in this experiment, the method can clearly detect the positive groups of Syto9 and fluorescent dyes, which fully proves the excellent performance of the detection method in terms of fluorescence sensitivity and accuracy, and provides strong technical support for related research and application.

[0161] Example 6: Comparison of different fluorescent dyes

[0162] The dyeing effects of different dyes are compared, and SYTO TM 13, SYTO TM 9, and SYTO TM RNA are selected for equi-proportion dyeing of liposomes:

[0163] The same 10 5 events / μL of nanoliposome samples are respectively added with 0.2mM of SYTO TM 13, SYTO TM 9, and SYTO TM RNA at a ratio of 100:1, and then incubated at 37°C for 15min, and directly detected by machine; it is found that the fluorescence signal of SYTO TM 13 is 80.96, the fluorescence signal of SYTO TM 9 is 56.26, and the fluorescence signal of SYTO TM RNA is 48.40, and the results are shown in the following figure:

[0164] The same sample, different dyes, different results. Fluorescence signal: SYTO TM 13>SYTO TM 9>SYTO TM RNA. SYTO TM 13 shows significant difference in sample concentration compared with other samples, SYTO TM 9, and SYTO TM RNA have appropriate fluorescence signals, but SYTO TM RNA has high selectivity for RNA, and has strong fluorescence signal when combined with RNA, and only produces weak fluorescence signal when combined with DNA. SYTO TM 9 is more stable, and it is determined through experiments that the ratio of 100:1 of the mixed dyeing concentration is the best dyeing concentration, and too high dyeing concentration is easy to cause false positive.

[0165] The liposome detection method provided by the application is a quantitative analysis method for single nano-liposome particles, successfully solves the technical route complexity, large operation difficulty, long time consumption and many other shortcomings in the process of conventional particle size detection, particle counting and qualitative analysis of encapsulation rate.

[0166] In the detection method provided by the application, the types of reagents used are extremely simple, only including SYTO TM 9 green fluorescent nucleic acid staining agent. This reagent is not only easy to purchase on the market, but also has a very simple staining method, without complex pretreatment steps, and can realize efficient staining of nano-liposomes. In the detection method provided by the application, the experimental steps are greatly compressed and simplified. Specifically, only 10 5 events / μL of LNP is mixed with a staining solution with a concentration of 0.2mM at a volume ratio of 100:1, and then incubated in a 37℃ water bath for 15 minutes, and then directly detected on the machine. This method is not only convenient and efficient, but also accurate and reproducible, and has the characteristics of high throughput, and can quickly and accurately detect a large number of liposome samples.

[0167] The detection method provided by the application involves simple operation of the analyzer, is easy to use, reduces the experimental difficulty of small particle detection, and based on the optimization of light path and flow path, can easily meet the particle size range of 53nm-120nm of silica microspheres, and has an absolute volume method count with a precision of more than 95%.

[0168] The detection method provided by the application can also realize simultaneous quantitative detection of particle size detection, particle counting and nucleic acid encapsulation rate. This multi-index synchronous detection capability effectively avoids the shortcomings of traditional qualitative analysis method in single detection, and provides more comprehensive and in-depth data support for researchers.

[0169] The detection method provided by the application is a quantitative analysis method for single nano-liposome particles, which can effectively evaluate the encapsulation of liposomes, including free nucleic acid, empty package or uneven proportion of nucleic acid wrapping, and can also characterize and research the particle size, particle number and nucleic acid encapsulation rate. It is not only suitable for quality evaluation of the whole process of nano-liposome preparation, but also can provide data support for formulating scientific and reasonable quality standards, which is helpful for more effective control and evaluation of product quality, and thus significantly improves the safety and reliability of liposomes in clinical drug use.

[0170] The detection method provided by the application greatly compresses the experimental steps, simplifies the experimental operation, has the advantages of convenient operation, intuitive detection, high efficiency, accuracy and precision, high throughput, high sensitivity, research flexibility, convenient use and comprehensive detection, and the like.

[0171] It should be noted that although the technical solutions of the application are described with specific examples, those skilled in the art can understand that the application should not be limited thereto.

[0172] The above has described various embodiments of the application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles, practical applications or technical improvements in the market of the embodiments, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting a liposome, wherein, The method comprises: a staining step, wherein the nucleic acid-loaded liposome sample to be tested is stained using a fluorescent nucleic acid stain, and an analysis step, wherein the stained liposome sample is analyzed using a nano-flow analyzer; wherein the analysis comprises at least one of liposome particle size determination, particle counting, nucleic acid encapsulation efficiency, and nucleic acid copy number determination in liposomes.

2. The detection method according to claim 1, wherein, The liposome is a nanoliposome, with a diameter ranging from 40 to 1000 nm; and / or The nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

3. The detection method according to claim 1 or 2, wherein, The nucleic acid-loaded liposome sample to be tested is a dispersion of nucleic acid-loaded liposomes to be tested, Preferably, the nucleic acid-loaded liposome to be tested is dispersed in water, More preferably, the concentration of the nucleic acid-loaded liposomes to be measured in the liposome sample is 0.1-5 x 10 5 particles / μL.

4. The assay of any one of claims 1-3, wherein, The fluorescent nucleic acid stain is SYTO TM 9 green fluorescent nucleic acid stain; Preferably, the concentration of the fluorescent nucleic acid stain is 0.05 to 0.5 mM.

5. The assay of any one of claims 1-4, wherein, The volume ratio of the liposome sample to the fluorescent nucleic acid stain is 50 to 150:1, preferably 70 to 130:1, more preferably 80 to 120:1, and further preferably 90 to 110:

1.

6. The assay of any one of claims 1-5, wherein, In the staining step, incubation is carried out at a temperature of 33 to 40°C, and / or the incubation time is 1 to 30 min.

7. The assay of any one of claims 1-6, wherein, In the particle size determination, standard particle size microspheres are detected under the same test conditions as the liposome sample using the nano-flow analyzer, the signal position of the standard particle size microspheres in the side scatter light channel characterizing particle size is determined, and the particle size of the liposome sample is determined.

8. The assay of any one of claims 1-7, wherein, In the nucleic acid encapsulation efficiency determination, the nucleic acid-loaded liposome sample to be tested is detected in the side scatter light channel characterizing particle size, the position of the background noise population is determined according to particle size, the position of the nucleic acid-loaded liposome to be tested is determined, a gate containing the nucleic acid-loaded liposome to be tested is drawn, and the gate containing the nucleic acid-loaded liposome to be tested is analyzed in the fluorescence detection channel detecting the fluorescent nucleic acid stain, to obtain the nucleic acid encapsulation efficiency.

9. The assay of any one of claims 1-8, wherein, In the nucleic acid copy number determination in liposomes, a two-dimensional scatter plot is established according to the side scatter light characterizing particle size and the fluorescence detection channel detecting the fluorescent nucleic acid stain, the position of free nucleic acid stained by the fluorescent nucleic acid stain, i.e. the P1 gate, is determined, and the position of the liposome particle stained by the fluorescent nucleic acid stain, i.e. the P3 gate, is determined; the average nucleic acid copy number in the liposome is obtained by calculating the ratio of the average fluorescence intensity of the particles in the P3 gate in the fluorescence detection channel to the average fluorescence intensity of the particles in the P1 gate in the fluorescence detection channel.

10. The assay of any one of claims 1-9, wherein, In addition to the nucleic acid-loaded liposome sample to be tested, the following controls are included: a control for the medium in which the nucleic acid-loaded liposome to be tested is dispersed; a control in which the medium is stained using the fluorescent nucleic acid stain; a control of the nucleic acid-loaded liposome sample to be tested without staining using the fluorescent nucleic acid stain; a control of the nucleic acid-loaded liposome sample to be tested stained using the fluorescent nucleic acid stain. a control of the empty nucleic acid-loaded liposome sample to be tested stained using the fluorescent nucleic acid stain.

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