CN114752561B - Extracellular vesicle separation method based on polydopamine nano particles - Google Patents
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Abstract
The invention discloses an extracellular vesicle separation method based on polydopamine nanoparticles, and belongs to the technical field of extracellular vesicle separation. The technical proposal is as follows: the dopamine solution is mixed with the extracellular vesicle separation matrix and then incubated, and the incubated solution is centrifuged at a rate of 500 Xg-20000 Xg. The invention solves the problems of high equipment condition and complicated separation process required by the ultra-high speed centrifugation method, solves the problem of low purity of the extracellular vesicles separated by the commercial separation kit, and can realize the large-scale separation of the extracellular vesicles.
Description
Technical Field
The invention relates to the technical field of extracellular vesicle separation, in particular to an extracellular vesicle separation method based on polydopamine nano particles.
Background
The existing gold standard method for separating the extracellular vesicles is ultra-high-speed centrifugation, and the extracellular vesicles obtained by the method are high in purity, but the equipment conditions required by separation are high, and the separation process is complicated. The commercial separation kit is simple and convenient to operate, and has high separation efficiency of extracellular vesicles, but the extracellular vesicles have low purity and are doped with a plurality of impurities. Other separation methods such as chromatography, microfluidic methods, etc. are difficult to achieve scale separation of extracellular vesicles. In summary, the isolation of extracellular vesicles currently lacks efficient, simple means.
Disclosure of Invention
The invention aims to solve the technical problems that: the extracellular vesicle separation method based on the polydopamine nano particles solves the problems of high equipment conditions and complicated separation process required by the ultra-high-speed centrifugation method, solves the problem of low purity of extracellular vesicles separated by a commercial separation kit, and can realize the large-scale separation of extracellular vesicles.
The technical scheme of the invention is as follows:
according to the extracellular vesicle separation method based on the polydopamine nanoparticle, a dopamine solution is mixed with an extracellular vesicle separation matrix and then incubated, and the incubated solution is centrifuged at a rate of 500 Xg-20000 Xg.
Preferably, the centrifugation time is 5min-300min.
Preferably, the centrifugation temperature is 4℃to 37 ℃. The temperature higher than 37 ℃ is easy to cause the deterioration of the components such as extracellular vesicle surface proteins, and causes the aggregation of extracellular vesicles. Solution crystallization easily occurs at a temperature lower than 4 ℃ and the membrane structure of extracellular vesicles is destroyed.
Preferably, the pH of the mixture of dopamine solution and extracellular vesicle separation matrix is 8-12, as the polymerization of dopamine to form polydopamine needs to be performed under alkaline conditions.
Preferably, the extracellular vesicle separation method based on polydopamine nanoparticles comprises the following steps:
1) Taking an extracellular vesicle sample, centrifuging and taking a supernatant; preparing a dopamine solution, weighing dopamine hydrochloride, dissolving the dopamine hydrochloride in PBS buffer solution, and preparing the solution for use at present;
2) Preparing a polydopamine/extracellular vesicle complex, mixing the supernatant extracted in the step 1) with a dopamine solution, and adjusting the mixed solution to be alkaline; stirring and hatching the mixed solution, and centrifuging the mixed solution after hatching is finished.
Preferably, in step 1), the supernatant is obtained after centrifugation at 500-10000 Xg for 5-60min at 4-37 ℃.
Preferably, in step 1), the concentration of the formulated dopamine solution is between 0.01mg/mL and 200mg/mL.
Preferably, in step 2), the supernatant is mixed with the dopamine solution to a final concentration of 0.02mg/mL to 100mg/mL.
Preferably, in the step 2), the mixed solution is stirred at the temperature of 4-37 ℃, the stirring speed is 500-5000 r/min, and the incubation time is 2-24h; more preferably, in step 2), the stirring temperature is 4-25 ℃.
Polydopamine is a macromolecule with hydrophilicity and good biocompatibility, and has wide application in the field of biological medicine. The polydopamine can be effectively combined with biomacromolecules by utilizing catechol or o-phthalquinone groups through the actions of hydrogen bonds and the like, and has little influence on the functions of biomacromolecules. For example, the research of polydopamine surface modified hemoglobin to reduce oxidative toxicity was reported by the team of Zhou Hong, the military sciences of 2017; university of Zhejiang Tang Ruikang in 2014 teaches a team to conduct erythrocyte surface engineering studies using polydopamine. The extracellular vesicle surface contains abundant biological macromolecules such as protein, sugar and the like, and provides possibility for the combination of polydopamine.
The concept of the invention is shown in figure 1, after the dopamine solution is mixed with the extracellular vesicle separation matrix, the mixture is incubated, and polydopamine nanoparticles can be combined with extracellular vesicles and impurities in the matrix in the incubation process, but the quantity of polydopamine nanoparticles combined with the extracellular vesicles is large. The polydopamine combined with the extracellular vesicles can increase the quality of the whole system, the extracellular vesicles can be separated by a conventional centrifugation method, and under the separation condition, the impurity/polydopamine nanoparticle compound can not be separated, so that the extracellular vesicles separated by the method have high purity.
Compared with the prior art, the invention has the following beneficial effects:
according to the method for separating the extracellular vesicles, the dopamine solution and the extracellular vesicle separation matrix are mixed and incubated, so that the polydopamine nano particles are combined with the extracellular vesicles and impurities in the matrix, and the extracellular vesicles can be separated under the conventional centrifugation condition. Compared with the existing separation method, the method solves the problems of high equipment conditions and complicated separation process required by the ultra-high-speed centrifugation method, solves the problem of low purity of the extracellular vesicles separated by the commercial separation kit, and can realize the large-scale separation of the extracellular vesicles, namely, the method for separating the extracellular vesicles is simple and efficient.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention.
FIG. 2 is an electron microscopic examination of extracellular vesicles after isolation in example 1.
FIG. 3 is a DLS assay of extracellular vesicles after isolation in example 1.
FIG. 4 is a western blot image of extracellular vesicles after isolation of example 1.
Detailed Description
The invention is further illustrated below in connection with examples, which are not intended to limit the practice of the invention.
Example 1
As shown in fig. 1, the embodiment provides a method for separating extracellular vesicles based on polydopamine nanoparticles, which adopts serum of a healthy BALB/c mouse as a sample to extract extracellular vesicles, and comprises the following steps:
1) Extraction of serum and preparation of dopamine solution
Placing blood of a healthy BALB/c mouse in a coagulation promoting tube, and centrifuging at 4 ℃ for 5 minutes by using a high-speed centrifuge at a centrifugation rate of 500 Xg; the supernatant from the centrifuge tube is then taken and the procedure is repeated until pure serum is finally obtained. A certain amount of dopamine hydrochloride is weighed and dissolved in PBS buffer solution, so that the concentration of the prepared dopamine solution is 0.02mg/mL, and the dopamine hydrochloride is prepared for use at present.
2) Preparation of Polydopamine/extracellular vesicles complexes (PDA-EVs)
Taking 1mL of the fresh serum extracted in the step 1), mixing with a certain amount of dopamine solution to make the final concentration of the dopamine solution be 0.01mg/mL, and adjusting the pH of the mixed solution to be 8.0. The mixed solution was incubated for 2h under magnetic stirring (stirring speed 500 rpm) at 4 ℃. After incubation, the mixed solution was centrifuged for 5min at 500 Xg, PDA-EVs pellet was obtained and redispersed in 100. Mu.L PBS buffer for analysis of its morphological features.
Characterization of PDA-EVs
1) Transmission Electron Microscope (TEM) characterization
The isolated extracellular vesicles were qualitatively characterized by transmission electron microscopy and the structural morphology of the extracellular vesicles was observed. FIG. 2 shows the electron microscopic examination of the isolated extracellular vesicles, which clearly shows the isolated extracellular vesicles with higher purity.
2) Dynamic Light Scattering (DLS) characterization
The isolated extracellular vesicles were characterized by DLS, and as can be seen from FIG. 3, the isolated extracellular vesicles have an average particle size of 70nm, conform to the characteristics of extracellular vesicles, and have a sharp peak shape, indicating that the isolated extracellular vesicles have a high purity.
3) Western blot (Western blot) characterization
The isolated extracellular vesicles were characterized by western blotting (CD 9, CD63, CD 81). Westernblot characterization As shown in FIG. 4, the left hand side of each plot is the band of extracellular vesicles obtained from serum isolated from healthy BALB/c mice as in example 1 using ultra high speed centrifugation, and the right hand side is the band of example 1. As is clear from FIG. 4, the extracellular vesicles obtained by the present example and the ultra high speed centrifugation have specific CD9, CD63 and CD81 bands, and it is confirmed that the present example can efficiently isolate extracellular vesicles with high purity.
Example 2
The embodiment provides a method for separating extracellular vesicles based on polydopamine nanoparticles, which adopts a cell culture medium (taking a mouse breast cancer 4T1 cell as an example) as a sample to extract extracellular vesicles, and comprises the following steps:
1) Extraction of cell culture medium and preparation of dopamine solution
Will 10 5 The 4T1 cells were seeded in 10cm dishes and when the cells proliferated to 80%, the cells were further cultured using serum-free medium for 48 hours. After 48h, the cell culture medium was taken and centrifuged at 37℃for 60 minutes using a high-speed centrifuge at 10000 Xg. A certain amount of dopamine hydrochloride is weighed and dissolved in PBS buffer solution, so that the concentration of the prepared dopamine solution is 200mg/mL, and the dopamine hydrochloride is prepared for use at present.
2) Preparation of Polydopamine/extracellular vesicles complexes (PDA-EVs)
Taking 50mL of the fresh culture medium extracted in the step 1), then mixing with a certain amount of dopamine solution to make the final concentration of the dopamine solution be 100mg/mL, and adjusting the pH of the mixed solution to 12. The mixed solution was incubated for 24 hours under magnetic stirring (stirring speed 500 rpm) at 4 ℃. After incubation, the mixed solution was centrifuged for 60min at a centrifugation rate of 20000 Xg, PDA-EVs pellet was obtained and redispersed in 100. Mu.LPBS buffer, and its morphological characteristics were analyzed.
Example 3
The embodiment provides a method for separating extracellular vesicles based on polydopamine nanoparticles, which adopts an extracting solution (taking cerebrospinal fluid as an example) as a sample to extract extracellular vesicles, and comprises the following steps:
1) Extraction of cell culture medium and preparation of dopamine solution
The cerebrospinal fluid of healthy BALB/c mice was placed in a procoagulant tube and centrifuged at 5000 Xg at 4℃for 10 minutes using a high-speed centrifuge. The supernatant from the centrifuge tube is then removed and the above steps are repeated until pure cerebrospinal fluid is finally obtained. A certain amount of dopamine hydrochloride is weighed and dissolved in PBS buffer solution, and the concentration of the prepared dopamine solution is 10mg/mL, and the dopamine hydrochloride is prepared for use at present.
2) Preparation of Polydopamine/extracellular vesicles complexes (PDA-EVs)
Taking 0.1mL of the fresh cerebrospinal fluid extracted in the step 1), then mixing with a certain amount of dopamine solution to make the final concentration of the dopamine solution be 5mg/mL, and adjusting the pH of the mixed solution to 10. The mixed solution was incubated for 12h under magnetic stirring (stirring speed 500 rpm) at 4 ℃. After incubation, the mixed solution was centrifuged for 30min at 10000 Xg, PDA-EVs pellet was obtained and re-dispersed in 100. Mu.L PBS buffer, and its morphological features were analyzed.
Comparative example 1 ultra high speed centrifugation for isolation of extracellular vesicles
Comparative example 1 extracellular vesicles obtained from serum of healthy BALB/c mice, which was the same as in example 1, were isolated by ultra-high speed centrifugation.
Placing blood of healthy BALB/c mice in a coagulation promoting tube, centrifuging at 300 Xg for 10 minutes at 4 ℃, and collecting supernatant; centrifuging the supernatant at 2000 Xg for 10 minutes, and collecting the supernatant; the supernatant was further centrifuged at 10000 Xg for 30 minutes, and the supernatant was taken. The supernatant was placed in a ultra-high speed centrifuge and centrifuged at 100000 Xg for 90 minutes, the supernatant was removed and the pellet resuspended in PBS solution. The resuspension was continued to be centrifuged at 100000 Xg for 90 minutes, the supernatant removed and the pellet resuspended in PBS to obtain extracellular vesicle solution.
The method needs to use an ultra-high speed centrifuge, and the equipment is high in price; meanwhile, strict balancing conditions are required in the use process of the ultra-high speed centrifuge, so that the separation process is complex and time-consuming. The method only needs a conventional centrifuge, does not need strict conditions such as balancing, and has simple separation process.
Comparative example 2 commercial separation kit
By Ribo TM Exosome Isolation Reagent (forplasma or serum) for example, extracellular vesicles obtained from serum of healthy BALB/c mice as in example 1 were isolated.
First, healthy BALB/c mouse blood was placed in a coagulation tube, and centrifuged at 4℃for 10 minutes using a high-speed centrifuge at 500 Xg. And taking supernatant of the centrifugal tube, and repeating the steps for one time to finally obtain pure serum. Transfer serum to a new tube, add 1/3 volume RiboTM Exosome Isolation Reagent (for plasma or serum), mix the solution well and stand at 4 ℃ for 30min. The supernatant was then removed by centrifugation at 15000 Xg for 2 minutes at 4℃and the pellet was resuspended in PBS to obtain extracellular vesicle solution.
The method is simple to operate, but the product contains a large amount of hybrid protein and has low purity. Table 1 shows the CD63 content and total protein content of the extracellular vesicle solutions isolated in examples 1-3, which are commercially available isolation kits.
TABLE 1
| CD63 content | Total protein content | |
| Comparative example 2 | 104.65±4.54pg/mL | 1.43±0.14mg/mL |
| Example 1 | 101.43±6.76pg/mL | 0.64±0.25mg/mL |
| Example 2 | 99.86±7.54pg/mL | 0.47±0.15mg/mL |
| Example 3 | 103.65±3.76pg/mL | 0.72±0.26mg/mL |
The results in Table 1 show that the commercial separation kit and the extracellular vesicle solutions isolated in examples 1-3 have similar amounts of CD63, indicating similar amounts of extracellular vesicles isolated by both methods. However, the total protein content in the extracellular vesicle solution obtained by the separation of the commercial separation kit is significantly higher than that obtained by the separation of examples 1 to 3, because the extracellular vesicle solution obtained by the separation of the commercial separation kit contains a large amount of hetero proteins, thereby proving that the method of the invention can realize the separation and the acquisition of the high-purity extracellular vesicles.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. The extracellular vesicle separation method based on polydopamine nanoparticles is characterized by comprising the steps of mixing a dopamine solution with an extracellular vesicle separation matrix, incubating, and centrifuging the incubated solution at a rate of 500 Xg-20000 Xg; the pH of the mixture of the dopamine solution and the extracellular vesicle separation matrix is 8-12.
2. The method for extracellular vesicle isolation based on polydopamine nanoparticles of claim 1, wherein the centrifugation time is from 5min to 300min.
3. The method for extracellular vesicle isolation based on polydopamine nanoparticles of claim 1, wherein the centrifugation temperature is between 4 ℃ and 37 ℃.
4. The method for extracellular vesicle isolation based on polydopamine nanoparticles of claim 1, comprising the steps of:
1) Taking an extracellular vesicle sample, centrifuging and taking a supernatant; preparing a dopamine solution, weighing dopamine hydrochloride, dissolving the dopamine hydrochloride in PBS buffer solution, and preparing the solution for use at present;
2) Preparing a polydopamine/extracellular vesicle complex, mixing the supernatant extracted in the step 1) with a dopamine solution, and adjusting the mixed solution to be alkaline; stirring and hatching the mixed solution, and centrifuging the mixed solution after hatching is finished;
in step 1), the supernatant is obtained after centrifugation at 500-10000 Xg for 5-60min at 4-37 ℃.
5. The method for extracellular vesicle isolation based on polydopamine nanoparticles of claim 4, wherein in step 1), the concentration of the prepared dopamine solution is 0.02mg/mL to 200mg/mL.
6. The method for extracellular vesicle isolation based on polydopamine nanoparticles of claim 4, wherein in step 2), the supernatant is mixed with a dopamine solution to a final concentration of 0.01mg/mL to 100mg/mL.
7. The method for separating extracellular vesicles based on polydopamine nanoparticles according to claim 4, wherein in step 2) the mixture is stirred at a temperature of 4-37 ℃ at a stirring speed of 500-5000 rpm and a incubation time of 2-24 h.
8. The method for extracellular vesicle isolation based on polydopamine nanoparticles as claimed in claim 7, wherein in step 2), the stirring temperature is 4-25 ℃.
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