CN111693359A - Method for detecting number of vesicles - Google Patents
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Abstract
The invention provides a method for measuring the number of vesicles, which is used for respectively measuring the number of vesicles in a standard drug-loaded vesicle sample and the ultraviolet absorbance of the standard drug-loaded vesicle sample and drawing a standard linear curve of absorbance corresponding to the number of vesicles in the drug-loaded vesicle. The number of the vesicles can be obtained by measuring the absorbance of the vesicle sample to be measured and calculating through a standard curve. The method has the advantages of simple operation, good reproducibility, rapidness, low cost and suitability for large-scale industrial production.
Description
Technical Field
The invention relates to a method for detecting the number of vesicles, in particular to a method for detecting the number of drug-loaded vesicles from apoptotic cells, which wrap tumor chemotherapy drugs.
Background
Extracellular Vesicles (Extracellular Vesicles EVs) refer to vesicular bodies of bilayer membrane structure that are shed from the cell membrane or secreted by the cell, and vary in diameter from 40nm to 1000 nm. The extracellular vesicles mainly comprise Microvesicles (MVs) and Exosomes (Exosomes, Exos), wherein the Microvesicles are small vesicles which are shed from cell membranes after cell activation, damage or apoptosis and have the diameter of about 100-1000 nm; exosomes are released extracellularly in a secreted form after fusion with the cell membrane by intracellular multivesicular bodies (multivesicular bodies), and have a diameter of about 40-100 nm. The diameter range of the vesicles related to the invention is mainly distributed in the range of 100-300 nm. The vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carry various proteins, lipids, DNA, mRNA, miRNA and the like related to cell sources, and participate in processes such as intercellular communication, cell migration, angiogenesis, immunoregulation and the like. The increase of extracellular vesicle level is found in diabetes, cardiovascular diseases, AIDS, chronic inflammatory diseases and cancers, and the extracellular vesicle level is likely to be a diagnostic marker of the diseases, so that accurate qualitative and quantitative research on vesicles is particularly important.
At present, the detection methods of Extracellular Vesicles (EVs) mainly include scanning electron microscopy, atomic force microscopy, dynamic light scattering technology, Nanoparticle Tracking Analysis (NTA), flow cytometry, ELISA and the like, and the Nanoparticle Tracking Analysis (NTA), ELISA and flow cytometry are more commonly used methods due to high flux, short time and simple operation. However, the Nanoparticle Tracking Analysis (NTA) requires expensive instruments, has high detection cost and long detection time, and has poor detection repeatability; the ELISA method is easily interfered by other soluble antigens, and the information such as the number of vesicles cannot be known; the sample aimed at by the flow cytometer is mainly a cell, the detection limit of scattered light is usually 300-500nm, and most vesicles have diameters below 300nm, and since the vesicles with diameters smaller than 300nm cannot be distinguished from background noise, the vesicles with diameters smaller than 300nm are difficult to detect, the number of vesicles is often underestimated, and the detection result is naturally inaccurate. The detection method is not suitable for large-scale rapid vesicle number determination due to requirements on equipment, operation and the like, and simultaneously cost factors are considered, so that the industrial application of the vesicle preparation technology is limited to a certain extent.
Chinese patent CN102302784A discloses a tumor chemotherapy medicinal preparation and a preparation method thereof, wherein the preparation is prepared by using cell vesicles derived from apoptotic tumor cells as carriers to wrap tumor chemotherapy medicaments. As a novel pharmaceutical preparation with extremely high industrial application value, the method has very important application value for rapid, efficient and economic quantitative detection means of the drug-carrying vesicle.
Disclosure of Invention
The invention provides a method for quantitatively detecting cell vesicles, which can accurately detect the number of the vesicles, reduce the vesicle detection cost, greatly shorten the detection time and effectively improve the reproducibility of detection data.
The specific method comprises the steps of carrying out gradient dilution on the drug-loaded vesicle concentrated suspension, and counting the diluted vesicles, wherein the counting method can be a Malvern counter. And simultaneously detecting the absorbance by using a microplate reader or an ultraviolet spectrophotometer. And (3) establishing a linear standard curve by taking the counting of the vesicles as a vertical coordinate and the absorbance of the corresponding vesicles as a horizontal coordinate, and determining a linear range. And in the determined linear range, the number of the vesicles can be obtained by detecting the absorbance of the vesicles to be detected and counting.
One preferred embodiment is to dilute 2 units/ml of vesicle concentrated suspension with physiological saline to 1 unit/ml, 0.5 unit/ml, 0.25 unit/ml, 0.125 unit/ml, 0.0625 unit/ml and 0.0313 unit/ml of vesicle suspension, wherein 1 unit is 1010The number of vesicles, the number of the above 7 groups of vesicle suspensions, was counted with a Malvern counter, while the absorbance was measured with a microplate reader at a wavelength of 370 nm. And establishing a standard curve of a linear relation by taking the Marvin count of the vesicles as a vertical coordinate and the absorbance of the corresponding vesicles as a horizontal coordinate, and determining a linear range. And (3) measuring the absorbance of the vesicles in the determined linear range, and calculating according to linear curve parameters to obtain the number of the vesicles.
The invention can be used for rapidly detecting the number of the medicine-carrying vesicles coated with the medicine, the preferred coated medicine can be a tumor chemotherapy medicine, and in one embodiment, the coated medicine is methotrexate.
The invention can reduce the detection cost, shorten the detection time, improve the repeatability and the accuracy of the detection and is suitable for industrial application. Besides the enzyme-labeling instrument, the absorbance of the drug-loaded vesicle can be detected by methods such as an ultraviolet spectrophotometer and the like. The invention uses an enzyme-labeling method or a combination of a spectrophotometer and a Malvern counter to obtain the quantity of the medicine-carrying vesicles and the related specific coefficient of absorbance, and the quantity of the vesicles can be obtained by multiplying the detected absorbance by the specific coefficient, thereby greatly shortening the detection time and simplifying the operation procedure.
Drawings
Figure 1 HPLC chromatogram of drug content determination of methotrexate-loaded vesicles; panel A is methotrexate standard; B. c, D shows the chromatogram of the drug-loaded vesicles of three batches.
FIG. 2 absorbance of methotrexate at different wavelengths; a is a methotrexate ultraviolet absorption spectrum diagram; and B is the absorbance of methotrexate under different wavelength conditions.
Fig. 3 shows a linear standard curve of vesicle number versus vesicle absorbance, and A, B, C shows three batches of drug-loaded vesicles.
Detailed Description
The following examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention in any way.
The cell, reagent and raw material sources used in the method are as follows:
human lung adenocarcinoma cells, which were cells used in the examples, were purchased from the Chinese type culture Collection. The chemotherapeutic agent used in the examples, Methotrexate (MTX), was purchased from greater than or equal to melem biotechnology limited. 1640 media from Biological Industries; the rest of the reagents are all commercial products.
Example 1 apoptosis of human Lung cancer cells
Washing sterile human lung cancer cells without mycoplasma at high growth speed twice with PBS at 1000rpm at 4-25 deg.C, centrifuging for 8min, counting, resuspending the cells with 1640 culture solution, placing in a 150 × 25mm culture dish, shaking, opening the dish cover, irradiating for 50min at the middle part under an ultraviolet lamp, adding methotrexate solution and appropriate amount of 1640 culture solution to make the final concentration of methotrexate 2mg/ml, placing at 37 deg.C and 5% CO2Culturing in an incubator.
Example 2 extraction of methotrexate drug-loaded vesicles
After the apoptotic cells incubated with the drugs were cultured for 16-20h, all the liquid in the culture dish was collected in a 50ml centrifuge tube, and the culture dish was rinsed with 20ml of PBS, and all the liquid was pooled in the centrifuge tube to make the total volume to 40 ml. The supernatant was centrifuged at 1500rpm at 2 ℃ to 8 ℃ for 10min and the precipitate was discarded. The supernatant was then centrifuged at 14000g at 2 ℃ to 8 ℃ for 2min and the pellet discarded. The supernatant was centrifuged at 14000g at 2 ℃ to 8 ℃ for 1h and the supernatant was discarded. The vesicle pellet is rinsed once with 10ml of normal saline, the normal saline for rinsing the vesicle is removed, and the vesicle pellet is resuspended with another 1ml of normal saline.
Example 3 determination of methotrexate content in drug-loaded vesicles
Three batches of methotrexate-loaded vesicles were prepared according to the method of example 2, and the methotrexate content was examined by high performance liquid chromatography. The conditions for HPLC were: column C18250X 4.6mm, detection wavelength 304nm, column temperature: 40 ℃, sample introduction: 40 μ L, mobile phase potassium dihydrogen phosphate (0.01M, pH 6.6): acetonitrile 9: 1, flow rate: 1 ml/min.
The chromatogram is shown in FIG. 1. The data for methotrexate detected by HPLC are shown in table 1:
TABLE 1 HPLC TEST DATA FOR METHYPERTEIN-CARRYING BUBBLES
From the results of the above table, it can be seen that the drug-loaded vesicles prepared by the method of example 2 each unit vesicle (10)10) The content of methotrexate is about 6 mu g, and the data of three batches are stable.
Example 3 correlation of Malvern count and vesicle absorbance for drug-loaded vesicles
3.1 selection of vesicle detection wavelength
The ultraviolet absorption curve of methotrexate is shown in FIG. 2, the maximum absorption peak is 304nm, and 30 selected by the experimental operator in order to study the influence of the absorbance of methotrexate on the experiment0. The experiment was carried out at 11 wavelengths of 310, 320, 330, 340, 350, 360, 370, 380, 390 and 400nm, 2 units/ml of vesicle concentrated suspension was diluted 2, 4, 8, 16, 32 and 64 times with physiological saline gradient to obtain vesicle suspensions to be tested, and the number of vesicles in the 7 groups of vesicle suspensions was counted with a Malvern counter (model NS300) and absorbance was measured at each wavelength with a microplate reader (Thermo, Multiskan GO). And establishing a standard curve by taking the Marvin count of the vesicle as a vertical coordinate and the absorbance of the corresponding vesicle as a horizontal coordinate, and determining a linear range. The above wavelength range covers the maximum absorption peak and the minimum absorption peak of methotrexate, and then R of a regression curve is obtained according to the number of vesicles and ultraviolet absorbance2To determine which wavelength is more suitable, the experiment was repeated 3 times using different batches of vesicles, the data being shown in table 2 below.
TABLE 2 absorbance curves of drug-loaded vesicles at various wavelengths
According to the data in the table, with R2Standard > 0.999, 4 wavelengths of 370/380/390/400nm are suitable. Subsequently, a relatively concentrated sample was used, and the absorbance at the above 11 wavelengths was measured, and it was found that the absorbance was lower the longer the wavelength was (see FIG. 2), and since the absorbance of vesicles was originally low, the absorbance should be as large as possible from the viewpoint of the absorbance difference after the dilution by doubling, and 370nm was selected as the most suitable wavelength.
3.2 measurement of the absorbance of empty vesicles
Blank vesicles were prepared and collected according to the uv irradiation method disclosed in patent CN 102302784A. The absorbance of blank vesicles at different concentrations was measured at a wavelength of 370nm, and the results are shown in the following table:
TABLE 3 absorbance of blank vesicles
The data show that the blank vesicle not loaded with the drug has very low ultraviolet absorbance and cannot interfere with the detection of the drug-loaded vesicle.
3.3 determination of the number of vesicles and the Linear Range of the absorbance
After the wavelength of 370nm was determined, the relationship between vesicle number and absorbance was determined by 3 different batches of vesicles. Diluting the concentrated suspension of the drug-loaded vesicle to be detected by 2/4/8/16/32/64 times with physiological saline respectively to obtain test samples, detecting the absorbance of each sample at 370nm (shown in Table 4) and making linear curves, wherein the linear curves of the three batches of experiment results are respectively shown in figure 3, and the three experiments R2All are more than 0.999, the three batches have good experimental reproducibility, the slopes are 10.316, 10.176 and 10.466 respectively, and the linear range of the vesicle number detection by the method is 0.01-4 × 1010/ml。
TABLE 4 number of vesicles versus absorbance
Example 4 drug-loaded vesicle counting
And (3) diluting the extracted medicine-carrying vesicles to be detected by proper times by using normal saline, so that the absorbance of 100 mu l of diluent at 370nm of the microplate reader is 0.1-1.0, and calculating the measured absorbance according to the linear curve parameters and the dilution times of the vesicle stock solution to obtain the number of the vesicles.
Claims (8)
1. A method for detecting the number of cell vesicles comprises the following steps of carrying out gradient dilution on drug-carrying vesicle concentrated suspension liquid coated with drugs, counting the diluted vesicles, detecting absorbance by using an enzyme labeling instrument or an ultraviolet spectrophotometer, establishing a linear standard curve by taking the number of the vesicles as a vertical coordinate and the absorbance of the corresponding vesicles as a horizontal coordinate, measuring the absorbance of the drug-carrying vesicles to be detected, calculating the number of the vesicles according to the standard curve, and enabling the linear range of vesicle number detection to be 0.01-4.0 × 1010/ml。
2. The method of claim 1, wherein the drug-loaded vesicle encapsulated drug is a tumor chemotherapeutic selected from the group consisting of methotrexate, doxorubicin, cisplatin, paclitaxel, 5-fluorouracil, gemcitabine, and 10-hydroxycamptothecin.
3. The method of claim 1, wherein the drug-loaded vesicle encapsulated drug is methotrexate.
4. The method according to claim 1 or 2, characterized in that the detection wavelength is selected from 370, 380, 390 or 400 nm.
5. The method of claim 3, wherein the detection wavelength is 370 nm.
6. The method of claim 1, wherein 2 units/ml of vesicle concentrated suspension is diluted by physiological saline in a gradient manner by 2, 4, 8, 16, 32 and 64 times to obtain 6 concentrations of vesicle suspension, the number of vesicles is calculated respectively, absorbance is tested simultaneously, and a standard curve of the number of vesicles and the absorbance is established; wherein the unit is 1010Number of vesicles.
7. The method according to claim 1, wherein the drug-loaded vesicle to be detected is diluted by normal saline by a proper factor during detection, so that the absorbance of the diluted sample to be detected is between 0.1 and 1.0.
8. The method of any one of claims 1-3, wherein the number of drug-loaded vesicles corresponds to the R of a linear standard curve of vesicle absorbance2Greater than 0.999.
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