Method for detecting exosome delivery efficiency

Abstract

The invention discloses a method for detecting exosome delivery efficiency, which is characterized in that a flow cytometer is used for detecting cells transfected with fluorescence labels, and a positive result definition mode of the flow cytometer is cell double-group door drawing. The method for detecting the drug delivery efficiency of the exosome in vitro, established by the invention, reflects the activity of the exosome, and can be used for analyzing the delivery efficiency of exosomes from different sources and comparing the difference. The method provided by the invention has stable results and good repeatability among different batches, and can be used as an effective means for detecting the exosome delivery efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of detection, and specifically relates to a method for detecting exosome delivery efficiency, mainly a method for detecting exosome delivery efficiency through cell transfection. Background Art

[0002] Extracellular vesicles (EVs) are an important intercellular communication system that promotes the transfer of macromolecules between cells. They are secreted by living cells, and among EVs there is a subpopulation with a particle size of about 30-150nm, called exosomes. Exosomes are vesicles with a phospholipid bilayer structure and are widely present in various body fluids. They are naturally domesticated drug carriers and can be used as delivery carriers for small molecule drugs, protein drugs, and nucleic acid drugs (such as miRNA, siRNA).

[0003] As endogenous biological carriers, exosomes have low immunogenicity and few toxic side effects. They can avoid the phagocytosis of macrophages, circulate to all compartments of the human body, and even deliver drugs across the endothelial barrier. At the same time, the bimolecular structure of phospholipids provides effective protection for the molecules contained in the exosomes, allowing them to stably exist in the peripheral blood circulation. They also have certain tissue tropism, and can ultimately achieve drug delivery to the target cells.

[0004] Existing technologies focus on the evaluation of the physical properties of exosomes, such as the number of particles and morphology, as well as the detection of protein content and exosome markers. However, there is no reliable detection method for the efficiency of drug delivery, i.e., in vitro activity, especially a lack of convenient, highly accurate, and stable detection methods. Delivery efficiency plays a vital role in evaluating drug efficacy, so it is necessary to develop a new detection method that can stably and accurately detect the delivery efficiency of exosomes. Summary of the invention

[0005] In view of the defect of the above-mentioned prior art that there is a lack of a detection method for exosome delivery efficiency, the present invention provides a method for detecting exosome delivery efficiency, specifically, a method for detecting exosome delivery efficiency (i.e., in vitro activity) by cell transfection. The method can be completed in vitro with the help of cells without the need for in vivo animal experiments.

[0006] The plasmid with the GFP gene inserted is integrated with the exosomes under the action of lipo2000, and is carried into the cells by the exosomes to express green fluorescent protein. The fluorescence signal is converted into a numerical value by the flow cytometer, reflecting the efficiency of exosomes in plasmid delivery.

[0007] One of the technical solutions provided by the present invention is: a method for detecting exosome delivery efficiency, the method comprising using a flow cytometer to detect cells transfected with fluorescent markers, and the positive result of the flow cytometer is defined by gating two groups of cells.

[0008] In a specific embodiment, the cells transfected with fluorescent markers are obtained by integrating the plasmid inserted with the GFP gene into the exosomes under the action of a transfection reagent and then entering the cells. Preferably, the transfection reagent is a liposome transfection reagent such as lipo2000.

[0009] The cells were transfected with exosomes + lipo2000 + GFP plasmid to evaluate its delivery efficiency: the positively charged cationic liposome liposome 2000 can automatically combine with the negatively charged DNA to form a DNA-cationic liposome complex, which is then adsorbed to the negatively charged surface of the exosomes. After co-incubation, the plasmid loading process is completed inside the exosomes. The plasmid transfected into the cells will express GFP and generate a fluorescent signal. The above signal can be converted into a numerical value by a flow cytometer. The signal intensity is proportional to the numerical value, so it can reflect the transfection efficiency.

[0010] In a specific embodiment, the reading voltage of the flow cytometer is 100-400V, preferably 200-300V, and more preferably 200V.

[0011] In a specific embodiment, the ratio of the inoculation amount of the cells before transfection to the number of the exosomes is 4.5E-4 to 1.25E-2, preferably E-3 to 3E-3, and more preferably 1.5E-3.

[0012] In a specific embodiment, the inoculation amount of the cells before transfection is 9.0E+04 cells / well to 2.5E+05 cells / well, preferably 1.0E+05 cells / well to 1.5E+05 cells / well, and more preferably 1.5E+05 cells / well.

[0013] In a specific embodiment, the number of exosomes is 2.0E+07 particles / well to 2.0E+08 particles / well, preferably 5.0E+07 particles / well to 1.0E+08 particles / well, and more preferably 1.0E+08 particles / well.

[0014] In a specific embodiment, the weight ratio of the number of exosomes to the plasmid is (2.0E+07-2.0E+08):1. Preferably, it is (5.0E+07-1.0E+08):1. More preferably, it is (1.0E+08):1.

[0015] In a specific embodiment, the volume ratio of the number of exosomes to the transfection reagent is (1.0E+07-1.0E+08):1. Preferably, it is (2.5E+07-5E+07):1. More preferably, it is (5E+07):1.

[0016] In a specific embodiment, the exosomes are derived from HEK 293T cells, Hihep cells or HEK-293F cells.

[0017] In a specific embodiment, the exosomes are derived from HEK 293T cells.

[0018] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0019] The reagents and raw materials used in the present invention are commercially available.

[0020] The positive progress of the present invention is that the present invention establishes a method for detecting the drug delivery efficiency of exosomes in vitro, which reflects the activity of exosomes and can analyze the delivery efficiency of exosomes from different sources and compare the differences. The results of this method are stable between different batches and have good repeatability, and can be used as an effective means to detect the delivery efficiency of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of cell deviation on cell double population gating at voltages of 100 V, 200 V, 300 V and 400 V, where the sample name of S82-PC-V100.0001 is the concentrated sample.

[0022] Figure 2 The figure shows the effect of blank cell gating on cell deviation at voltages of 100 V, 200 V, 300 V, and 400 V. The sample name of S82-PC-V100.0001 is the concentrated sample.

[0023] Figure 3 The impact of different door drawing methods on delivery efficiency trends.

[0024] Figure 4 The following are the observation results of cells that successfully expressed GFP protein under a fluorescence microscope. DETAILED DESCRIPTION

[0025] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0026] Example 1 Detection of exosome delivery efficiency

[0027] (I) Prepare reagents and instruments:

[0028] 1) Lipo2000 Transfection Reagent, purchased from Biosharp;

[0029] 2) Cells: HEK 293F cells: manufacturer: Thermo, catalog number A14527CN;

[0030] HEK 293T cells: manufacturer ATCC, catalog number CRL-3216;

[0031] Hihep cells: Human fibroblasts (obtained from human scar tissue through primary culture) were used, and the SV40 large T gene (PPL, catalog number: CI0001) was transferred, and then FOXA3, HNF1A and HNF4A were transferred through lentivirus (DOI: 10.1007 / 978-1-4939-8961-4_9), and Hihep cells were obtained after passage; (these two cells are used as examples in the present invention, and other conventional HEK293T cells and Hihep cells do not affect the realization of the effect of the present application)

[0032] 3) Cell culture medium: DMEM medium (purchased from Corning Company) containing 10% serum (purchased from Corning Company) by volume;

[0033] 4) 1X TrypL E pancreatin: purchased from Gibco;

[0034] 5) Tryplan Blue: purchased from Gibco;

[0035] 6) GFP plasmid: purchased from GenScript;

[0036] 7) Exosomes: The cells were inoculated into sterile shake flasks at a density of 5E+05 cells / mL and placed in a shaker at 37°C, 5% CO2, and 90 rpm. The cells were cultured in OPM-293CD05 Medium for 5 days, and the culture supernatant was harvested and centrifuged at 300g for 10 min to obtain the supernatant; the supernatant was centrifuged at 2000g for 10 min to obtain the supernatant; the supernatant was clarified after filtration through a PurcisePLE 0.8 / 0.45 μm filter (Cobetter; C03BBPLESSIP), and the clarified liquid was concentrated 50 times using a S02-E500-05-N hollow fiber column (Repligen) to obtain a concentrated sample; the concentrated liquid was purified by using Capto core400 filler (Cytiva) chromatography to obtain a chromatography sample; the sample load was 2 cv (column volume) and the flow-through peak was collected at a linear flow rate of 2 cm / min; the flow-through liquid was further centrifuged at 100,000 g for 70 min, the supernatant was discarded, and the centrifugal precipitate was resuspended with an appropriate amount of buffer to prepare an exosome resuspension, i.e., the exosome final product), wherein the exosomes 1 to exosomes 6 involved in the following embodiments are specifically: exosomes 1 and 3 are supernatants; exosomes 2 and 4 are precipitates after centrifugation; exosomes 5 and 6 are exosome final products.

[0037] (II) Cell seeding: HEK 293T cells (within P20) with a confluence of 85-95% were plated at 1.5×10 5 / well was subcultured to a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0038] (III) Sample incubation and loading: 1E+08 exosomes, 1 μg of the above-mentioned GFP plasmid, and 2 μl of Lipo2000 were mixed and fixed to 20 μl with PBS; incubated at 2-8°C for 12 h to obtain the incubated sample;

[0039] (IV) Add 20 μl of the sample after the above incubation to the 6-well plate prepared in (II), continue incubation for 12 hours, then discard the culture medium in the plate and replace with new cell culture medium; continue incubation in a 37°C, 5% CO2 incubator for 24 hours;

[0040] (V) Fluorescence observation: Observe fluorescence using a fluorescence microscope and take photos;

[0041] (VI) Cell treatment: discard the cell culture supernatant, add 500μl of 1X TrypLE trypsin to each well, and digest for 5 minutes at room temperature. Observe the cell status. When the cells are completely detached, mix them by blowing, and transfer them to a 1.5mL EP tube containing 500μl culture medium (DMEM+10% FBS) to terminate the digestion. Centrifuge at 6000rpm and 4℃ for 3min, discard the supernatant, add 1mL of PBS to each tube, mix, and centrifuge at 6000rpm and 4℃ for 3min, discard the supernatant, and resuspend with 500μl of PBS solution containing 2% FBS, and store in a refrigerator at 4℃ away from light until the machine is tested;

[0042] (VII) Flow cytometry detection: 50,000 cells were selected, FITC voltage was 200 V, FSC = 410 V; SSC = 690 V; after reading, Flowjo was used to process the data, and a double population gate was used to define positive results.

[0043] Example 2 Effects of different voltages and gate drawing methods on delivery efficiency measurement results

[0044] The delivery efficiency was tested using different voltages, different gate drawing methods, and different numbers of exosome particles, and the other conditions were the same as in Example 1.

[0045] Table 1 Effects of voltage and gate drawing method on delivery results

[0046]

[0047]

[0048] From Table 1 and Figure 1 (Draw gates based on cell populations), Figure 2 (Drawing gate with blank cells) It can be seen that when reading, the voltage of the flow cytometer will have a certain effect on the cell deviation, and the cell group will shift to the right as the voltage increases (see the results Figure 1 ); When defining positive cells, different gating methods will also have a significant impact on the results (see Table 1 for the results). When the FITC voltage is 100-200V, the negative cell population of the sample to be tested has a small offset. As the voltage increases, the overall offset of the cell population is large. Therefore, it is not suitable to use an excessively large voltage for reading. A FITC voltage of 100-200V is a better choice. The method of the present invention uses a 200V voltage and a double-group cell gating method (in this way, the experimental results are more stable, the numerical fluctuations are smaller, and the results are less affected by the voltage) to process the results.

[0049] The purpose of this experiment is not to achieve a higher delivery effect, but to provide true feedback on the delivery efficiency so that obvious differences can be seen in the delivery efficiencies of different exosomes.

[0050] In general, blank cells are used as gates, but in this experiment, after cell transfection, positive cells will have obvious double peaks, which are different from negative cells, and their positive rate is not affected by voltage, which is in line with the general rule. Therefore, the present invention unexpectedly discovered that the method of drawing gates for double populations of cells can more faithfully reflect the actual delivery efficiency and ensure the accuracy of the results.

[0051] Different people used different samples (exosomes 1-6) for testing on different days, and the rules were the same as the above samples (i.e., the method of gating the double population of cells was more stable and accurate). The results are shown in Table 2 (the exosomes were derived from Hihep cells), proving that the results were reproducible.

[0052] Table 2 Effects of voltage and gate drawing method on delivery results

[0053]

[0054]

[0055] The samples were diluted in a gradient manner, and the delivery efficiency was statistically analyzed at a specific voltage (200 V). The blank cells and the cell populations were used to define the positive and negative properties. The delivery efficiency of the former was greater than that of the latter, but the change trends of the two were the same (the results are shown in Table 3, Figure 3 ).

[0056] Table 3 The impact of different door-drawing methods on delivery efficiency trends

[0057]

[0058] From the above results, it can be seen that when the voltage is 200V and the cell double population gate is used, the experimental results are more stable, the results are less affected by the voltage, and are closer to the actual delivery efficiency (see the results). Figure 4 ), while the conventional method of gating with blank cells will make the results biased high. The bright spots are cells that successfully express GFP protein, and the rest are cells in the bright field. According to the proportion, the proportion of successfully infected cells is small. Using double-group gating, the delivery efficiency at different voltages is 11.4-12.8%, and the numerical fluctuation is small; if blank cells are used to draw the gate, the delivery efficiency at different voltages is 37.2-98.8%, and the numerical fluctuation is large (see Table 1 data). The use of a double-group approach is more reasonable and closer to the actual situation. In general, infection with foreign plasmids will cause certain damage to cells. If the infection efficiency is greater than 80%, the cell state will deteriorate or even fall off, while the cells in this field of view are in good condition and no abnormalities occur.

[0059] In conventional flow cytometry experiments, blank cells are used as gates. The present invention improves the existing technology in the industry, so that it can more realistically reflect the delivery efficiency of exosomes.

[0060] Example 3 Comparison of different exosome delivery systems

[0061] The delivery efficiency was tested using different exosome delivery systems, different voltages, and different cell numbers. The other conditions were the same as in Example 1. The results are shown in Table 4.

[0062] Table 4 Comparison of exosome delivery systems

[0063]

[0064] As shown in Table 4, exosomes, lipo2000, and GFP plasmid are all indispensable when transfecting cells, and the combination of exosomes + GFP plasmid and lipo2000 + GFP plasmid cannot achieve a high delivery efficiency.

[0065] Example 4 Effects of different cell numbers and exosome numbers on delivery efficiency

[0066] Except for the number of cells inoculated and the number of exosomes (see Table 5, Table 6 and Table 7 for details), the other conditions were the same as in Example 1.

[0067] Table 5 Effect of different cell numbers on delivery efficiency

[0068]

[0069] Table 6 Effects of different cell numbers and exosome numbers on delivery efficiency

[0070]

[0071]

[0072] From the results of the above table, it can be seen that when HEK 293T cells are transfected, when the cells are inoculated too much (greater than 2.50E+05 cells / well), the cells grow excessively and the state is poor, which makes the measured delivery efficiency low. If a higher positive rate is achieved, more exosomes are required, which increases the difficulty of exosome preparation; if the cell inoculation amount is too low (less than 9.00E+04 cells / well), the transfection may be insufficient, or the proportion of infected cells is too large, resulting in high results and deviations. When the number of exosomes is the same, the more cells, the lower the delivery efficiency; when the number of cells is the same, the more exosomes, the higher the delivery efficiency. However, too high a number of exosome particles will cause contact inhibition, resulting in a reduced range of delivery efficiency. It can be seen that the amount of exosomes added is positively correlated with the delivery efficiency within a certain range, and the appropriate amount of exosomes will keep most of the delivery efficiency within the linear range, making the data comparable. This experiment is mainly for comparing differences. The delivery efficiency needs to be maintained within the linear range. Generally, a value of about 50% is more appropriate.

[0073] Table 7 Effect of exosome number on delivery efficiency

[0074]

[0075]

[0076] When the delivery efficiency is >80% or <10%, the results are inaccurate and the delivery efficiency cannot be compared. As can be seen from Table 3, when the number of exosomes / well is lower than 5.63E+07, the delivery efficiency is close to the lower limit and the comparison result is not obvious; when the number of cells / well is 1.5E+05 and the number of exosomes / well is 4.00E+08, the delivery efficiency is the best.

[0077] Example 5 Stability Verification

[0078] Different personnel conducted delivery efficiency tests on different days, and the results are shown in Table 8. The results of different batches ranged from 31.4 to 34.9, with small differences between batches and stability.

[0079] Table 8 Summary of PC results from different batches

[0080]

[0081] The delivery efficiency of exosomes from different sources was tested, and the remaining specific steps were the same as those in Example 1. The results are shown in Table 9.

[0082] Table 9 Comparison of delivery efficiency of exosomes from different sources

[0083]

[0084]

[0085] As can be seen from Tables 8 and 9 above, the detection method of the present invention can compare and analyze the delivery efficiency of exosomes from different sources. The method has stable results between different batches and good repeatability.

[0086] Conclusions from the above examples: The method of the present invention can be used to detect the delivery efficiency of exosomes to drugs, and the delivery efficiency of exosomes from different sources can be analyzed and the differences can be compared. The method of the present invention has stable results between different batches and good repeatability, and can be used as an effective means to detect the delivery efficiency of exosomes.

Claims

1. A method for detecting exosome delivery efficiency, characterized in that: The method comprises using a flow cytometer to detect cells transfected with a fluorescent marker, wherein a positive result of the flow cytometer is defined by drawing gates for two cell populations.

2. The method according to claim 1, characterized in that The cells transfected with fluorescent markers are obtained by integrating the plasmid inserted with the GFP gene into the exosomes under the action of a transfection reagent and then entering the cells; preferably, the transfection reagent is a liposome transfection reagent such as lipo2000.

3. The method according to claim 1, characterized in that The reading voltage of the flow cytometer is 100-400V, preferably 200-300V, and more preferably 200V.

4. The method according to claim 1, characterized in that The ratio of the inoculation amount of the cells before transfection to the number of the exosomes is 4.5E-4 to 1.25E-2, preferably E-3 to 3E-3, and more preferably 1.5E-3.

5. The method according to claim 1, characterized in that The inoculation amount of the cells before transfection is 9.0E+04 cells / well to 2.5E+05 cells / well, preferably 1.0E+05 cells / well to 1.5E+05 cells / well, and more preferably 1.5E+05 cells / well.

6. The method according to claim 2, characterized in that The number of the exosomes is 2.0E+07 particles / well to 2.0E+08 particles / well, preferably 5.0E+07 particles / well to 1.0E+08 particles / well, and more preferably 1.0E+08 particles / well.

7. The method according to claim 2, characterized in that The weight ratio of the number of exosomes to the plasmid is (2.0E+07-2.0E+08):1, preferably (5.0E+07-1.0E+08):1, and more preferably (1.0E+08):

1.

8. The method according to claim 2, characterized in that The volume ratio of the number of exosomes to the transfection reagent is (1.0E+07-1.0E+08):1, preferably (2.5E+07-5E+07):1, and more preferably (5E+07):

1.

9. The method according to claim 1, characterized in that The exosomes are derived from HEK 293T cells, Hihep cells or HEK-293F cells.

10. The method according to claim 9, characterized in that The exosomes are derived from HEK 293T cells.

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