Exosome absolute purity evaluation method

Abstract

The invention discloses an exosome absolute purity evaluation method. The invention relates to a method for quantitatively detecting exosome content based on gel permeation chromatography (GPC). The relative purity is determined by measuring the ratio of the particle number to the protein amount of the exosome, then the protein content of the exosome is further detected through a gel permeation chromatography (GPC) method, and the purity of the exosome can be perfectly evaluated in a product mode by combining the two modes. The purity of the exosome can be completely evaluated according to the result of the product of the ratio of the high particle number to the high protein amount and the high chromatographic purity, and the method is scientific, objective and accurate. The technical problems that an existing exosome protein content evaluation process is complex, operation is tedious, accuracy is low and repeatability is poor are solved, and the method has the beneficial effects that detection efficiency and detection accuracy are improved.

Description

Technical Field

[0001] The invention relates to a method for evaluating the absolute purity of exosomes and belongs to the field of biotechnology. Background Art

[0002] Exosomes are a type of extracellular vesicles (EVs), with a size of 30-150nm. They are released into the extracellular space in the form of exocytosis after the fusion of multivesicular bodies (MVB) in the cell with the cell membrane. Exosomes are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, urine, semen, breast milk, etc. Exosomes have many functional effects, so they also have high utilization value. Exosomes are widely involved in intercellular material transport and information transmission, regulating cell physiological activities; and exosomes have the functions of antigen presentation, immune escape, inducing normal cell transformation, promoting tumor occurrence and metastasis, etc. In addition, exosomes also have important applications in drug delivery and can be used as natural nanoparticles for drug delivery. There are currently many methods for characterizing exosomes. In 2014 and 2018, the International Society for Extracellular Vesicles (MISEV2018) proposed guidelines for basic characterization requirements that exosomes (including research and exosome preparation) should meet, including protein concentration, surface markers, Zeta potential, particle number, particle size, morphology and purity, etc. Among them, the exosome protein concentration can be measured more accurately using the BCA method; the expression of exosome surface marker proteins is identified by Western Blotting (WB), which can be qualitatively detected, and the exosome particle size and particle number concentration can be detected more accurately using nanoparticle tracking technology (NTA) or nanoflow cytometry (NanoFCM); Zeta potential can be accurately measured by Brookhaven's potentiostat, and exosome morphology is identified by transmission electron microscopy (TEM) to observe and detect the morphology of exosomes in an intuitive way; However, there is currently no clear method for the detection of the more critical exosome purity, and there is no relevant exosome pure product or standard product that can be used for purity identification, and sometimes the purity of exosomes is not even reflected. However, in practical applications, the purity of exosomes is actually a crucial parameter. Because there are many impurities in the exosomes secreted by cells, including cell fragments, miscellaneous proteins, liposomes, microvesicles and other large particle impurities, which are not conducive to the application of exosomes. For example, in the process of drug loading, the purity of exosomes needs to be high, otherwise it will interfere with the final result. Therefore, this parameter has a great impact on medical research. Therefore, a relatively fast, accurate, reproducible, objective and accurate method for evaluating the purity of exosomes is very necessary.

[0003] One of the existing related technologies is to dye the exosome sample with a fluorescent membrane dye, and then detect the dyed exosome sample by flow cytometry to obtain an exosome-related scatter plot, and obtain the purity by comparing the successfully stained particles with the total particles; another method is to simply use HPLC to detect the purity of exosomes, using the absorption of exosomes under ultraviolet wavelengths, and detecting the absorbance value by chromatography, so as to calculate the purity using the obtained exosome area and the total area.

[0004] Although there are some methods for detecting the purity of exosomes, these methods have great limitations and drawbacks in the actual detection and evaluation of the purity of exosomes. They are not accurate and reliable enough. The detection of the purity of exosomes by simply staining with dyes is likely to stain impurities or insufficient staining, which affects the detection of the purity of exosomes. In addition, the detection time of staining is long, and the pretreatment and dyes will destroy the structure of exosomes. The use of two-dimensional liquid chromatography and the need to use two chromatographic columns at the same time requires very high hardware requirements, high cost, and complex operation. Two different chromatographic methods need to be used at the same time, especially ion exchange chromatography. Slight changes in the concentration gradient of the solution have a greater impact on the test results. The operation is complex and prone to errors, the results are easily interfered with, and the repeatability of the test method is poor. The simple chromatographic purity detection method is single and cannot accurately detect the purity of exosomes. It is easily interfered by impurities, liposomes, fragments of similar size, and protein aggregates, resulting in a situation where the purity is too high. The purity of exosomes is determined using q-PCR technology. This process has high requirements and requires professional equipment, personnel and a harsh environment. It is prone to contamination and the operation is complex and error-prone, resulting in biased results. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for evaluating the absolute purity of exosomes.

[0006] The steps of the technical solution adopted by the present invention are as follows:

[0007] The total number of particles contained in the sample is detected by a particle number detector to obtain the total number of particles result B.

[0008] The protein concentration of the sample was detected by BCA protein quantification method, and the protein concentration result A was obtained.

[0009] The chromatographic ratio of the sample was detected by the GPC chromatographic method, and the chromatographic ratio result C was obtained.

[0010] Through the above steps, the absolute purity of exosomes is B÷A×C.

[0011] Preferably, the number of particles B in the exosome sample is detected by a nanoparticle tracking instrument.

[0012] Preferably, the protein concentration A is detected by the BCA protein quantification method, and the exosomes need to be mixed first.

[0013] Preferably, the relative purity of the exosomes is B÷A.

[0014] Preferably, the detection steps have no order of precedence and can be performed either before or after.

[0015] Preferably, the GPC detection method is adopted, and the chromatographic column is SRT-C SEC-500, 5 μm, 4.6x300mm.

[0016] The screened GPC columns use chemical bonding technology and are completely covered with a hydrophilic coating on the surface. Therefore, they not only have excellent stability, but also have very little non-specific adsorption effect on biological samples such as proteins.

[0017] Preferably, the column temperature in the chromatographic system is 20-40°C, the flow rate is 0.1-0.4 mL / min, the sample chamber temperature is 8-12°C, the injection volume is 10-50 μL, and the ultraviolet detection wavelength is 280 nm.

[0018] Preferably, the buffer solution in the mobile phase used is a buffer such as phosphate, acetate, HEPES, Tris, etc., and the concentration used is 10-100 mmol / L.

[0019] Preferably, sodium chloride solution is added to the mobile phase at a concentration of 180-220 mmol / L.

[0020] The concentration of the sodium chloride solution is beneficial to promoting the separation of samples, improving the resolution of chromatographic analysis, reducing the adsorption of samples, and increasing the separation and analysis effect.

[0021] The advantages and positive effects of the present invention are: the purity of the exosome sample is analyzed by detecting the sample protein concentration, the number of particles and the chromatographic ratio, and the size and type are considered respectively, so as to fully exclude the influence of non-protein particles and impurities with the same diameter as the exosomes on the analysis work, thereby improving the accuracy of the exosome purity analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a gel permeation chromatography GPC spectrum of exosome sample a in Example 1 of the present invention;

[0023] Figure 2 This is a gel permeation chromatography GPC spectrum of exosome sample b in Example 2 of the present invention;

[0024] Figure 3 This is a nanoparticle tracking detection spectrum of exosome sample a in Example 1 of the present invention;

[0025] Figure 4 This is a nanoparticle tracking detection spectrum of exosome sample b in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] In the present invention, a method for evaluating the absolute purity of exosomes is provided. The purity obtained by GPC chromatography is a relative purity. The stationary phase of GPC chromatography is a porous gel, and only components with a diameter smaller than the pore size can enter the gel pores. Components with large particles cannot enter the gel pores and are excluded, and can only pass through the gaps between the gel particles, so the components of the largest particles are washed out first. Exosome molecules are relatively large, much larger than impurity proteins and some small molecular compounds, etc., so they will be washed out in the shortest time, forming a peak with a fixed outflow time, and stably outflow at this time node, and detected by a UV detector. However, there are many larger protein aggregates or liposomes, microvesicles, etc. that cannot be separated from exosomes, and are mixed in the exosomes and outflow together, resulting in inaccurate results, and only a relative purity is obtained. Nanoparticle tracking technology can only count the number of particles but cannot perform relevant separation. Exosomes and large granular protein aggregates will also be counted. The protein content in exosomes is relatively low, far less than the amount of impurities. The purer the exosomes, the lower the protein content. The purer the exosomes, the larger the number of particles and the fewer impurities. Therefore, the larger the ratio of exosome particles to protein, the purer it is. However, if the exosomes are mixed with more non-protein particles, resulting in a higher number of particles, but they are not actually exosome particles, the ratio of exosome particles to protein will be large, and the purity of the exosomes will be very low, or even just cell fragments or large granular impurities.

[0027] Therefore, it is necessary to have a high ratio of the number of exosome particles to protein and a high content ratio of the position where the real exosome GPC chromatogram flows out, otherwise a false positive result will appear, resulting in a deviation in the purity test result. Therefore, the absolute purity of exosomes can be evaluated by the purity test method mentioned in the present invention, that is, the absolute purity of exosomes is calculated according to the following formula:

[0028]

[0029] Embodiment 1:

[0030] 1. Detect the concentration of sample a protein:

[0031] Step 1: Prepare the working solution: According to the number of standards and samples, add 50 volumes of BCA reagent and 1 volume of Cu reagent (50:1) to prepare the BCA protein detection working solution, and mix thoroughly (there may be turbidity during mixing, but it will disappear after mixing. The BCA working solution can remain stable at room temperature for 24 hours).

[0032] Step 2: Dilute the standard: Take 10μL of BCA standard and dilute it to 100μL with PBS solution (samples can generally be diluted with PBS solution) to a final concentration of 0.5mg / mL. Add 0μL, 2μL, 4μL, 6μL, 8μL, 12μL, 16μL, 20μL of standard to the protein standard wells of the 96-well plate, and add 20μL, 18μL, 16μL, 14μL, 12μL, 8μL, 4μL, 0μL of PBS solution to make up the sample volume in the well to 20μL.

[0033] Step 3: Take the sample a to be tested, dilute it 2 times, 4 times, and 8 times with PBS buffer, and add 20 μL of the diluted sample to the sample well of the 96-well plate. Since the error of the pipette is large when taking a small amount of sample, the result point located in front of the standard line may not be very accurate, so try to make the sample point fall behind the 1 / 2 position of the standard line.

[0034] Step 4: Add 200 μL of BCA working solution to each well of the standard and sample wells and incubate at 37°C for 15-30 minutes. Use an ELISA reader to measure the absorbance A at a wavelength of 562 nm. According to the test results of the BSA standard, establish an absorbance-concentration calibration curve, calculate the protein concentration of the diluted sample a according to the standard curve, and calculate the protein concentration of the sample a according to the dilution. Calculate the arithmetic mean of the back-calculated results of each dilution gradient, which is the concentration A of the sample to be tested.

[0035] When using an incubator for incubation, care should be taken to prevent the test results from being affected by water evaporation.

[0036] After the above experimental process, the final protein concentration of sample a was 0.33 mg / mL.

[0037] 2. Detection sample a chromatogram ratio:

[0038] Prepare the mobile phase by weighing and dissolving the mobile phase solution as required, adjusting the pH, and filtering with a 0.22 μm PES filter membrane for later use.

[0039] Place the solvent filter of the liquid chromatography pump into the corresponding solvent, turn on the computer, and turn on the power switch of each module of the liquid chromatography instrument after at least 10 minutes. The power switch is located in the lower left corner of the front panel of each module. The raised button is the switch. After pressing, the indicator light on the instrument panel flashes and is accompanied by a "beep" sound. Perform a self-check. After the self-check is completed, click the workstation icon to start the workstation, log in with the user name and password, and open the chromatography workstation software. Confirm that the workstation is connected to each module, replace the cleaning plunger rod and sealing pad solution, clean the injection needle, and replace the mobile phase.

[0040] The mobile phase was perfused into the system at a flow rate of 4.0 mL / min for 5 min. The LC flow path was opened and the chromatographic column SRT-C SEC-500, 5 μm, was connected. 4.6x 300mm, column temperature 30℃, flow rate 0.3mL / min, sample chamber temperature 10℃, injection volume 20μL, UV detection wavelength 280nm, balance the column with mobile phase at the required flow rate to reach 3CV, i.e. 3 times column volume.

[0041] Edit the instrument method according to the parameters in the table, call the method, preheat the UV detector for at least 30 minutes, edit the injection sequence, place the sample in the specified sample tray, and perform injection testing after the column pressure stabilizes.

[0042] After completion, the data were analyzed, and according to the area normalization method, the chromatographic proportion result C of the exosome sample a was obtained to be 97.54%.

[0043] 3. Detect the particle concentration of sample a:

[0044] Turn on the power: Wait for 30 minutes for the laser light source to stabilize. Turn on the computer, double-click, and wait for the instrument to self-check (the indicator light turns green when the self-check is successful).

[0045] Create a new measurement program, click Mesurement→Manual→Maurement on the work bar, click Meaurement Type→Select Size, click Sample, enter the measurement sample name, click Material to select MaterialName (if it is a liposome, please select Liposome) / click Dispersant to select the dispersed medium (usually select Water), click General options to set, click Temperature, set the measurement temperature (℃), number of measurements (usually select Automatic), number of measurement cycles (usually select 1 time), click Cell to set the sample pool, click Meaurement, select the number of measurements and the interval time for each measurement, click Data processing, and set the report. After the settings are completed, click OK to display the following window, and start measuring exosome sample a. The final particle number concentration B is 1.20×10 12 Pieces / mL.

[0046] The absolute purity of the final exosome sample a was calculated according to the formula in the present invention, namely (1.20×10 12 / 330)×0.9754=3.64×10 9 .

[0047] That is, the absolute purity of exosomes is 3.64×10 9 .

[0048] Embodiment 2:

[0049] Test sample b according to the above process.

[0050] The absolute purity of the final exosome sample b was (7.20×1011 / 150)×1=4.80×10 9 .

[0051] That is, the absolute purity of exosomes is 4.80×10 9 .

[0052] The technical features disclosed above are not limited to the disclosed combinations with other features. Those skilled in the art may also make other combinations between the technical features according to the purpose of the invention to achieve the purpose of the invention.

Claims

1. A method for evaluating the absolute purity of exosomes, characterized in that: include: The particle number detector detects the sample particle concentration as B; The protein concentration of the sample detected by BCA protein quantification method is A; The chromatographic proportion of the sample detected by GPC chromatographic analysis is C; The absolute purity of exosomes is B÷A×C.

2. The method for evaluating absolute purity of exosomes according to claim 1, characterized in that: A nanoparticle tracking instrument was selected to detect the number of exosome sample particles B.

3. The method for evaluating absolute purity of exosomes according to claim 1, characterized in that: The exosomes need to be mixed first and then the exosome protein concentration A is measured.

4. The method for evaluating absolute purity of exosomes according to claim 1, characterized in that: In GPC chromatography detection, the column temperature is 20-40°C, the flow rate is 0.1-0.4mL / min, the sample chamber temperature is 8-12°C, the injection volume is 10-50μL, and the ultraviolet detection wavelength is 280nm.

5. The method for evaluating absolute purity of exosomes according to claim 5, characterized in that: The buffer solution of the mobile phase used in GPC chromatography detection is phosphate, acetate, HEPES, and Tris, and the buffer concentration used is 10-100mmol / L.

6. The method for evaluating absolute purity of exosomes according to claim 6, characterized in that: Sodium chloride solution was added to the mobile phase with a concentration of 180-220mmol / L.

Images