Method for analyzing size of exosome

Abstract

The invention discloses a method for analyzing the size of an exosome. According to the invention, the SV-AUC is used for detecting the exosome for the first time, and it is found that impurity components in the exosome can be detected by using the method for detecting the exosome; in addition, the distribution difference of the settlement coefficients of exosomes from different sources can be distinguished by using the method. The invention provides a rapid, accurate and high-sensitivity exosome analysis method for the field, and the method has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of detection and analysis technology. Specifically, the present invention relates to a method for analyzing the size of exosomes. Background Art

[0002] Exosomes refer to vesicles (30-150nm) secreted by various organisms, wrapped in lipid membranes, and containing various biological molecules such as nucleic acids and proteins. As a new form of intercellular communication, exosomes have received widespread attention from the scientific research and pharmaceutical industry, and many studies and market applications have emerged that use the inherent characteristics and advantages of exosomes for clinical diagnosis and drug development. With the continuous deepening of research, scientific exosome evaluation and analysis methods are needed. The current detection methods based on the concentration and particle size distribution of exosome particles cannot accurately identify other impurities contained in the solution, such as free proteins, nucleic acids, etc. Therefore, the development of a rapid, accurate, and highly sensitive exosome analysis method has important application value.

[0003] Analytical Ultracentrifugation (AUC) technology is widely used in the characterization of biomacromolecules, heterogeneity analysis, aggregate formation and intermolecular interaction research due to its precision and accuracy. It is mainly divided into two experimental methods: sedimentation velocity (SV) and sedimentation equilibrium (SE). SV-AUC has a breakthrough application in the characterization of viral vectors (such as lentiviral vectors). It can analyze the particle size distribution, purity and particle impurities of viral particles with high resolution, providing important support for the production and quality control of viral vectors. However, the application of AUC technology in exosome analysis has not yet been studied. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for analyzing exosomes based on SV-AUC.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for analyzing exosomes.

[0006] Furthermore, the method comprises: detecting the exosome detection sample using an analytical ultracentrifugation-sedimentation rate method.

[0007] Furthermore, the method also includes the step of separating and purifying the detection object to obtain an exosome detection sample.

[0008] Furthermore, the method can detect impurity components in exosomes.

[0009] Furthermore, the method can distinguish exosomes from different sources.

[0010] Furthermore, before detection, the exosome detection sample was formed into OD 280 It is a solution of 0.6-1.2.

[0011] Furthermore, the analytical ultracentrifugation-sedimentation rate method uses an analytical ultracentrifuge, and its relevant parameters are set as follows: temperature, 4°C~40°C; rotation speed, 5000-8000rpm.

[0012] Preferably, the rotor used in the detection process is AN-60Ti.

[0013] Furthermore, the data measured by the analytical ultracentrifuge are processed by running the ls-g*(s) model using SEDFIT17 software, and whether the exosomes contain impurities and / or the distribution of the exosome sedimentation coefficient are analyzed based on the data processing results.

[0014] In the present invention, the term "exosome" is a vesicle that is secreted from a cell to the outside of the cell, or has a membrane structure composed of a lipid bilayer present in the cell. The diameter of the exosome is about 30-150nm, and the exosome is released from the cell when the multivesicular body fuses with the cell membrane, or is directly released from the cell membrane. It is known that exosomes play a role in transporting biomolecular proteins, bioactive lipids and RNA (miRNA) in cells to achieve their functional role in mediating coagulation, intercellular communication and cellular immunity.

[0015] The quality of separation and extraction of exosomes determines the stability and accuracy of subsequent experiments. Methods for extraction / purification of exosomes include: ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation, immunoaffinity separation (immunomagnetic beads, microfluidics), etc. The quality of exosomes obtained by different extraction methods varies, and some may contain impurities. However, the current detection methods based on the concentration and particle size distribution of exosome particles cannot accurately identify other impurities contained in the solution. In this application, the inventors applied SV-AUC technology to exosome analysis for the first time and found that SV-AUC can detect impurity components in exosomes and distinguish exosomes from different sources compared to existing technologies for detecting exosomes.

[0016] In the present invention, the sedimentation rate is a time-dependent technique, which records the real-time concentration change data over time under high-speed centrifugation, and uses ultraviolet and interferometric detection systems to monitor the sedimentation process in real time, thereby analyzing and determining the size distribution and purity of exosomes.

[0017] In the present invention, the exosome detection sample is added to an analytical ultracentrifuge for detection. The analytical ultracentrifuge is composed of a power system and an optical detection system.

[0018] The power system described above may include a driving system, a vacuum system, a control system, a protection system, a rotor and a sample cell.

[0019] In some embodiments of the present invention, the rotor has multiple models, which can be divided into AN-60Ti with 4 holes and AN-50Ti with 8 holes. During the detection process, the rotor with the corresponding number of holes can be selected according to the number of samples and the speed requirement. In the present invention, AN-60Ti with 4 holes is preferred. The material of the rotor is generally titanium alloy.

[0020] In addition, the sample pool is composed of a center piece, a window, a gasket, a screw and a shell; the material of the center piece is generally aluminum alloy and resin, and the resin center piece is preferably selected in the present invention; the material of the window is selected from sapphire or quartz, and more preferably, the material of the window is sapphire.

[0021] Furthermore, the optical detection system includes any one of an ultraviolet / visible light absorption detector, an interference light detector and a fluorescence detector.

[0022] The UV / visible light absorption detector can record the absorbance of the sample at different positions in the rotor radial direction during the centrifugation process to complete the detection when the sample absorbs within a certain wavelength. The interference light detector can detect the change in optical path length due to the different refractive indices of the reference solution and the sample when light passes through the reference solution and the sample, and the point of equal optical path length on the collector is displaced. The displacement distance of the interference fringes is proportional to the solute concentration. By monitoring the displacement of the interference fringes, the change of the solute concentration in the rotor radial direction over time can be obtained.

[0023] In some preferred embodiments of the present invention, a UV / visible light absorption detector is selected to detect the sample. Specifically, after the test sample is loaded, ultraviolet light is used for detection and data is recorded.

[0024] Furthermore, the sample loading volume is 350-400 μL, preferably 380 μL. After loading, the sample is detected at 4°C-40°C, 5000-8000 rpm, using a wavelength of 250-300 nm; preferably, after loading, the sample is detected at 20°C, 7000 rpm, using a wavelength of 280 nm and 260 nm, and a data is collected every 230 seconds.

[0025] In addition, since the test sample may contain a buffer component, in order to ensure the accuracy of the results, a control group with the same buffer component (such as PBS) solution as a sample is set to eliminate the interference of the PBS solution as a solvent on the results. The pH of the PBS solution is 7.0±0.5, and the sample volume is 350~400μL, more preferably 400μL.

[0026] The data obtained through the above test were processed by ls-g*(s) model using SEDFIT software to obtain the sedimentation coefficient distribution diagram of the test sample.

[0027] In SEDFIT, the ls-g*(s) model is an important model in sedimentation rate analysis, which is used to describe the sedimentation behavior of the test sample in the solution. Among them, ls (least square) is a procedure based on direct least squares boundary modeling, while the g*(s) model is used to describe the sedimentation distribution of a group of non-diffusive species. It provides information about different species in the sample by analyzing the sedimentation velocity distribution of species during the sedimentation process. According to the experimental data and the characteristics of the sample, appropriate parameters and models can be selected to obtain the most accurate fitting results.

[0028] In a specific embodiment of the present invention, the relevant parameters of the ls-g*(s) model are: Resolution, 400; s min, -500 / 0; s max, 1500 / 3000; check Baseline, Fit RI Noise, Fit Time Independent Noise, Meniscus, Bottom; Confidence level, 0.68.

[0029] The second aspect of the present invention provides application of the method described in the first aspect of the present invention in exosome quality control.

[0030] A third aspect of the present invention provides a system for analyzing exosomes.

[0031] Further, the system comprises: Determination module: Detect exosome detection samples by analyzing ultracentrifugation-sedimentation rate method; Data processing module: processes the data obtained by the measurement module.

[0032] Furthermore, the processing includes running the ls-g*(s) model through SEDFIT17 software for processing.

[0033] Furthermore, the system also includes a parameter setting module for setting parameters of the analytical ultracentrifuge.

[0034] A fourth aspect of the present invention provides a computer device and a computer-readable storage medium.

[0035] Furthermore, the device includes a memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method described in the first aspect of the present invention is implemented.

[0036] Furthermore, the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method described in the first aspect of the present invention is implemented.

[0037] In the present invention, the processor and the memory can be set separately or integrated together. For example, the memory can include random access memory, flash memory, read-only memory, programmable read-only memory, non-volatile memory or register, etc. The processor can be a central processing unit (CPU), etc. Or a graphic processing unit (GPU) The memory can store executable instructions. The processor can execute the executable instructions stored in the memory to implement the various processes described herein.

[0038] It can be understood that the memory in this embodiment can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. Among them, the non-volatile memory can be a ROM (Read-Only Memory), a PROM (Programmable ROM), an EPROM (Erasable PROM), an EEPROM (Electrically EPROM) or a flash memory. The volatile memory can be a RAM (Random Access Memory), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as SRAM (Static RAM), DRAM (Dynamic RAM), SDRAM (Synchronous DRAM), DDRSDRAM (Double Data Rate SDRAM), ESDRAM (Enhanced SDRAM), SLDRAM (Synchlink DRAM), and DRRAM (Direct Rambus RAM). The memories described herein are intended to include, but are not limited to, these and any other suitable types of memories.

[0039] In some embodiments, the memory stores the following elements, upgrade packages, executable units or data structures, or a subset thereof, or an extended set thereof: an operating system and an application program.

[0040] The operating system includes various system programs, such as a framework layer, a core library layer, a driver layer, etc., which are used to implement various basic services and process hardware-based tasks. The application program includes various application programs, which are used to implement various application services. The program for implementing the method of the embodiment of the present invention can be included in the application program.

[0041] In an embodiment of the present invention, the processor calls a program or instruction stored in a memory, specifically, a program or instruction stored in an application program, and the processor is used to execute the method steps provided in the first aspect.

[0042] In the present invention, the computer-readable storage medium may include but is not limited to various known and unknown types of non-volatile memories.

[0043] Advantages and beneficial effects of the present invention: The present invention uses SV-AUC to detect exosomes for the first time, and finds that the method can detect impurities in exosomes; in addition, the method can also distinguish exosomes from different sources. The present invention provides a rapid, accurate, and highly sensitive exosome analysis method for the art, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Shows TEM experimental results; among them, a is the Milk-UC TEM result, b is the Milk-AA-UC TEM result, c is the Milk-EXODUS TEM result, and d is the Urine-UC TEM result; Figure 2 Display DLS experimental results; a is Milk-UC DLS result, b is Milk-AA-UC DLS result, c is Milk-EXODUS DLS result, d is Urine-UC DLS result; Figure 3 Display NTA test results; a is Milk-UC NTA result, b is Milk-AA-UC NTA result, c is Milk-EXODUS NTA result, d is Urine-UC NTA result; Figure 4 Shows the Nanocoulter results; a is the Milk-UC Nanocoulter result, b is the Milk-AA-UC Nanocoulter result, c is the Milk-EXODUS Nanocoulter result, and d is the Urine-UC Nanocoulter result; Figure 5 The SV-AUC results are shown; among them, a is the Milk-UC SV-AUC result, b is the Milk-AA-UC SV-AUC result, c is the Milk-EXODUS SV-AUC result, and d is the Urine-UC SV-AUC result. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0046] The instruments and reagents used in the embodiments of the present invention are as follows: (1) Instruments: Preparative ultracentrifuge: XPN-100 (Beckman Coulter) Fully automatic exosome purification instrument EXODUS: EXODUS H-600 (Shenzhen Huixin Biomedical Technology Co., Ltd.) Analytical ultracentrifuge: Optima AUC (Beckman Coulter) Transmission electron microscope TEM: FEI Tecnai Spirit TEM D1266 (FEI) Dynamic light scattering DLS: WDPN-08 (Wyatt Technology) NanoSight (Malvern) Nanocoulter: resun-G03 (Shenzhen Ruixin Intelligent Manufacturing Technology Co., Ltd.) (2) Reagents: 1× phosphate buffer (1× PBS instant granules, Qingke Biotechnology, catalog number: 1GP20701, 1L / Pouch) Acetic acid (General-reagent, Product No.: P1718453) Example 1 Exosome isolation and purification 1. Milk exosome ultracentrifuge purification (Milk-UC) (1) 50 mL of fresh milk, 4°C, 15 min, take the supernatant; (2) Pour the supernatant into a new centrifuge tube, centrifuge at 3000g, 4°C, 15 min, take out the supernatant, and repeat once; (3) Pour the supernatant into a new centrifuge tube, centrifuge at 21500g, 4°C, 30 min, take out the supernatant, and repeat once; (4) Pour the supernatant into a new centrifuge tube, centrifuge at 100,000 g, 4 °C, 74 min, and take the supernatant; (5) Pour the supernatant into a new centrifuge tube, centrifuge at 100,000 g, 4°C, 74 min, take the precipitate, and resuspend it in PBS to obtain UC-EV.

[0047] 2. Milk exosome ultracentrifuge-acetic acid precipitation purification (Milk-AA-UC) (1) 50 mL of fresh milk, 4°C, 15 min, take the supernatant; (2) Pour the supernatant into a new centrifuge tube, centrifuge at 3000g, 4°C, 15 min, take out the supernatant, and repeat once; (3) Pour the supernatant into a new centrifuge tube, centrifuge at 21500g, 4°C, 30 min, and take the supernatant; (4) Pour the supernatant into a new centrifuge tube, add acetic acid to the supernatant to adjust the pH to 3.8, incubate at 21500g, 4°C, 30min to precipitate casein, and take the supernatant; (5) Pour the supernatant into a new centrifuge tube, centrifuge at 100,000 g, 4°C, 74 min, take the precipitate, and resuspend it in PBS to obtain UC-AA-EV.

[0048] 3. Milk exosomes EXODUS fully automatic exosome extraction system purification (Milk-EXODUS) (1) Centrifuge 50 mL of fresh milk at 4°C, 3000 g for 15 min to remove cell debris, and filter the supernatant through 3-4 layers of gauze; use a 1 mL pipette tip to block the fat layer at the mouth of the tube; (2) Add rennet in proportion (e.g. 50 mL colostrum: 0.01 rennet), shake and mix, and place in a 37°C water bath for 1 hour, shaking every 10-15 minutes; (3) Centrifuge at 4°C, 10,000 g for 30 min to remove casein and obtain whey; if the whey is yellowish in color, add proteinase K in proportion and incubate at 37°C for 5 min; (4) Dilute the sample 5-fold, filter it with a 0.22 μm filter to remove apoptotic bodies and microvesicles, and collect the sample in a new centrifuge tube; (5) Get on the computer.

[0049] 4. Urine exosomes ultracentrifuge purification (Urine-UC) (1) Centrifuge 50 mL of fresh urine at 3000 g for 20 min and collect the supernatant; (2) Centrifuge at 17000g for 30 min and take the supernatant; (3) Centrifuge the supernatant at 200,000 g for 2 h, take the precipitate, and resuspend it in 65 mL of PBS; (4) Resuspend the product and centrifuge at 200,000 g for 2 h; (5) Resuspend in 100 μL PBS to obtain EVs.

[0050] Example 2 SV-AUC Analysis 1. Sample dilution The exosome samples isolated above were diluted to OD 280 Approximately 1.

[0051] 2. Analytical Ultracentrifugation Experiment 1. Assemble and add samples to the sample pool: Place the window and center piece along the positioning strip on the sample pool shell, and place them vertically into the sample pool shell in the order of "window assembly-center piece-window assembly". Place red gaskets on both sides of the aluminum alloy center piece, and then put in the threaded ring gasket and threaded ring and tighten them fully with the help of a torque wrench. Place the sample pool flat on the sample tray and add samples using a special long pipette tip. The left side is the control well, and 400μL of buffer is added; the right side is the sample well. After the entire sample addition process is completed, the sample pool is axially symmetrically balanced, and the weight difference after balancing is controlled within 0.5g. Place the sample pool and the special balance piece in the rotor, and pay attention to the alignment of the rotor scale line and the sample pool scale line.

[0052] 2. Program setting and operation: Create a new experimental method on the equipment operating software, and select the corresponding rotor according to the speed and flux requirements to conduct the analytical ultracentrifugal sedimentation velocity experiment. The specific experimental method parameters are shown in Table 1.

[0053] Table 1 Experimental procedure settings

[0054]

[0055] 3. Data analysis: Open SEDFIT17 software, import the experimental data of the target sample, select the ls-g*(s) model, click Parameter, refer to Table 2 to enter the relevant parameters, click RUN and FIT to calculate and fit the data.

[0056] Table 2 Data processing parameter settings

[0057]

[0058] 3. Experimental Results The exosomes purified by ultracentrifuge and EXODUS exosome purification instrument were identified and characterized. The purified exosomes were identified by TEM, DLS, NTA, nanocoulter, and SV-AUC. We identified the morphological state of the exosomes through TEM results ( Figure 1 ), proving that exosomes were obtained by both purification methods.

[0059] Through the DLS results, we can see that using UC ( Figure 2 a) UC-AA ( Figure 2 b) and EXODUS ( Figure 2 c) The particle size of purified milk is too large, which is inconsistent with the exosome size of 30-150nm, and it is impossible to distinguish exosomes from different treatment methods and different sources, such as urine exosomes ( Figure 2 d) The differences in the exosomes obtained indicate that DLS is clearly insufficient in the characterization of exosomes.

[0060] At present, the most commonly used method for exosome detection is NTA detection. Through the NTA results, we can see that the overall size of exosomes is larger than the actual size. In different purification methods, we can see that after AA-UC ( Figure 3 b) or EXODUS ( Figure 3 The size distribution of milk exosomes in c) was significantly better than that in UC ( Figure 3 This indicates that NTA has the ability to distinguish changes in exosome size.

[0061] From the results of Nano Coulter, we can see that the average particle size of all results is most consistent with the size of exosomes, but the distribution of all results cannot distinguish between different treatment methods and different sources such as urine exosomes ( Figure 4 d) Differences in exosomes obtained.

[0062] Through the AUC results, we can see that the distribution of exosomes purified by all methods (OD260 / 280>1) is between 100-1000S, and the exosomes purified by UC ( Figure 5 a), through the comparison of dual wavelength detection at 260nm and 280nm, we can see that the UC purified milk exosomes have an obvious protein peak near 0S (OD260 / 280<1), which indicates that there are impurity proteins in the UC purified exosomes, while the UC-AA ( Figure 5 c) or EXODUS ( Figure 5 c) In the purification process, no protein peaks were found in the AUC results after casein was precipitated with acetic acid and treated with proteinase K, indicating that AUC can detect impurities in exosomes, which cannot be identified by DLS and NTA methods. Therefore, AUC can be used to optimize the purification method of exosomes. We used the sedimentation coefficient distribution of milk exosomes ( Figure 5 Comparison of the sedimentation coefficient distribution of urine exosomes with that of urine exosomes revealed that urine exosomes ( Figure 5 d) is more uniform than that of milk exosomes, which is consistent with the exosome morphology in TEM. In addition, we can see that milk exosomes are also distributed in negative sedimentation coefficients, which indicates that the density of various components in the exosome system is also different, and AUC analysis in this regard also has obvious potential.

[0063] It is obvious to those skilled in the art that the embodiments of the present invention are not limited to the details of the exemplary embodiments described above, and that the embodiments of the present invention can be implemented in other specific forms without departing from the spirit or essential features of the embodiments of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the embodiments of the present invention is defined by the appended claims rather than the above description, and therefore it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the embodiments of the present invention.

[0064] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for analyzing exosomes, characterized in that: The method comprises: using an analytical ultracentrifugation-sedimentation rate method to detect an exosome detection sample.

2. The method according to claim 1, characterized in that The method also includes the step of separating and purifying the detection object to obtain an exosome detection sample.

3. The method according to claim 1, characterized in that The method can detect impurity components in exosomes; Preferably, the method can distinguish exosomes from different sources.

4. The method according to claim 1, characterized in that: Before detection, the exosome detection samples were formed into OD 280 It is a solution of 0.6-1.

2.

5. The method according to claim 1, characterized in that: The analytical ultracentrifugation-sedimentation rate method uses an analytical ultracentrifuge, and its relevant parameters are set as follows: temperature, 4°C to 40°C; speed, 5000-8000rpm; Preferably, the rotor used in the detection process is AN-60Ti.

6. The method according to any one of claims 1 to 5, characterized in that: The data measured by the analytical ultracentrifuge are processed by running the ls-g*(s) model using SEDFIT17 software, and whether the exosomes contain impurities and / or the distribution of the exosome sedimentation coefficient are analyzed based on the data processing results.

7. Use of the method according to any one of claims 1 to 6 in exosome quality control.

8. A system for analyzing exosomes, characterized in that: The system comprises: Determination module: Detect exosome detection samples by analyzing ultracentrifugation-sedimentation rate method; Data processing module: processes the data obtained by the measurement module; Preferably, the processing comprises running the ls-g*(s) model through SEDFIT17 software for processing.

9. The system according to claim 8, characterized in that The system also includes a parameter setting module for setting parameters of the analytical ultracentrifuge.

10. A computer device and a computer readable storage medium, characterized in that: The device comprises a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method according to any one of claims 1 to 6 is implemented; The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method according to any one of claims 1 to 6 is implemented.

Images