Labeling and absolute quantification method for extracellular vesicles and liposomes by lanthanide metal

Abstract

The invention discloses a method for labeling and absolute quantification of extracellular vesicles and liposomes by lanthanide metal. EVs or lipidosome and a lanthanide metal ion solution are jointly incubated, the EVs or lipidosome is marked through specific binding of lanthanide metal ions and phosphate radical groups on the surface of an EVs or lipidosome membrane, ICP-MS detection is conducted, and absolute quantification of the EVs or lipidosome is achieved. According to the method, efficient labeling of the EVs or the lipidosome can be achieved only through one-step reaction, operation is easy, convenient and rapid, good labeling stability and biocompatibility are achieved, and the physical and chemical properties and biological activity of the EVs or the lipidosome cannot be remarkably affected. The method can also be applied to absolute quantification of EVs or lipidosome ingesting by recipient cells, and provides a new research thought and technical means for research on interaction between EVs and recipient cells, cargo delivery and related mechanisms or delivery effect quantification of lipidosome drugs in cells or even in vivo.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and analytical chemistry, and specifically relates to a method for labeling and absolutely quantifying extracellular vesicles and liposomes with lanthanide metals. Background Art

[0002] Extracellular vesicles are small vesicles with a phospholipid bilayer membrane structure that are produced by various cells and released into the extracellular environment. Extracellular vesicles can be mainly divided into exosomes, microvesicles and apoptotic bodies according to different biogenesis modes. Since the size ranges of various extracellular vesicles overlap to a certain extent, it is currently difficult to separate a single extracellular vesicle subpopulation. Therefore, MISEV2018 recommends that extracellular vesicles be divided into small extracellular vesicles (<200nm) and medium / large extracellular vesicles (>200nm) according to their size. Extracellular vesicles are widely present in various biological fluids and cell culture supernatants, and their membranes contain a large number of bioactive molecules such as proteins, lipids, and nucleic acids from donor cells. Studies in recent years have found that extracellular vesicles play an important role in various physiological and pathological processes. They are not only an important source of biomarkers for various diseases, but also a potential new drug delivery system. Extracellular vesicles have become an important way of intercellular communication, playing an important role in intercellular communication and material transfer. Extracellular vesicles containing specific bioactive molecules released from donor cells can enter recipient cells through various endocytic pathways and regulate the physiological activities of recipient cells accordingly. Therefore, quantitative cellular uptake of extracellular vesicles is of great significance for a deeper understanding of vesicle-cell interactions. Extracellular vesicles are difficult to quantify due to their nanoscale size and complex biological composition. At present, the quantitative methods of extracellular vesicles mainly include labeled quantification and non-labeled quantification. Non-labeled quantitative methods of extracellular vesicles mainly include direct quantification of the number of vesicle particles and quantification of the main biological components of the vesicles. The former mainly includes nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) and nanoflow cytometry (NanoFCM) and other technical means that can detect and analyze the particle size distribution and particle number of extracellular vesicles, while the latter mainly quantifies biological components such as proteins, lipids or nucleic acids contained in extracellular vesicles. Although these non-labeled quantitative methods can directly quantify extracellular vesicles, they are limited in that they can only be used to quantify extracellular vesicles at the extracellular level and cannot be used to quantify extracellular vesicles taken up by cells. At present, to quantify extracellular vesicles taken up by cells, a labeling quantification strategy is required. Although various fluorescent probes, bioluminescence and other labeling strategies currently used can track and quantify extracellular vesicles taken up by cells, this strategy can only achieve semi-quantitative analysis of extracellular vesicle cell uptake and cannot perform absolute quantitative analysis. Therefore, it is necessary to develop new methods to achieve absolute quantification of extracellular vesicles taken up by cells.

[0003] Liposomes are artificially synthesized nanovesicles with a phospholipid bilayer membrane, usually hollow spheres prepared from lipids such as phosphatidylcholine and ceramide. As a new type of drug delivery carrier, liposomes have received widespread attention in recent years due to their good biocompatibility, hydrophilicity or lipophilicity, and ability to encapsulate drug molecules of various properties. Summary of the invention

[0004] The purpose of the present invention is to provide a method for labeling and absolutely quantifying extracellular vesicles EVs or liposomes at the intracellular and extracellular levels.

[0005] The method provided by the present invention for labeling and absolutely quantifying extracellular vesicles EVs or liposomes at the intracellular and extracellular levels is as follows: EVs or liposomes are co-incubated with a lanthanide metal ion solution, EVs or liposomes are labeled by specific binding of the lanthanide metal ions to the phosphate groups on the surface of the EVs or liposome membranes, and ICP-MS detection is performed to achieve absolute quantification of EVs or liposomes.

[0006] This method can quickly and effectively label EVs and liposomes, and subsequently use ICP-MS to achieve absolute quantification of EVs or liposomes at the intracellular and extracellular levels.

[0007] In one aspect, the present invention provides a method for metal labeling of EVs or liposomes.

[0008] The metal labeling method of EVs or liposomes provided by the present invention comprises the following steps:

[0009] 1) Enrich extracellular vesicles (EVs) from biological samples or prepare liposomes using artificial methods;

[0010] 2) Dispersing the enriched EVs or liposomes prepared by artificial methods in a lanthanide metal ion solution and incubating the solution to allow the lanthanide metal ions to fully bind to the phosphate groups on the surface of the EVs or liposome membranes;

[0011] 3) Ultrafiltration and washing are performed to remove free lanthanide metal ions that do not participate in the reaction, thereby obtaining lanthanide metal-labeled extracellular vesicles or liposomes.

[0012] On the other hand, the present invention also provides an absolute quantification method for EVs or liposomes.

[0013] The absolute quantitative method of EVs or liposomes provided by the present invention comprises the following steps:

[0014] 1') Enrich extracellular vesicles (EVs) from biological samples or prepare liposomes using artificial methods;

[0015] 2') dispersing the enriched EVs or the liposomes prepared by artificial methods in a lanthanide metal ion solution and incubating the solution to allow the lanthanide metal ions to fully bind to the phosphate groups on the surface of the EVs or liposome membranes;

[0016] 3') ultrafiltration and washing to remove free lanthanide metal ions that do not participate in the reaction, thereby obtaining lanthanide metal-labeled extracellular vesicles or liposomes;

[0017] 4') ICP-MS detects the amount of lanthanide metal in the lanthanide metal-labeled extracellular vesicles or liposomes to achieve absolute quantification of EVs or liposomes.

[0018] In steps 1) and 1') of the above method, the biological sample is the culture supernatant of various cell types and various body fluid samples such as blood, urine, saliva, etc., and specifically can be the culture supernatant of HeLa cells;

[0019] The extracellular vesicles are extracellular vesicles of various sizes such as exosomes, microvesicles, small extracellular vesicles, and medium / large extracellular vesicles, and specifically small extracellular vesicles (sEVs);

[0020] The enrichment method can be various reported extracellular vesicle enrichment methods such as ultracentrifugation, ultrafiltration, size exclusion chromatography, affinity method, etc., and can specifically be ultracentrifugation;

[0021] The operation of enriching sEVs in the supernatant of HeLa cell culture by ultracentrifugation is as follows: collect the supernatant of HeLa cell culture, first pre-treat the cell culture supernatant, centrifuge at 300×g for 10 minutes (to remove dead cells), 2000×g for 10 minutes (to remove cell debris) and 10000×g for 30 minutes (to remove large extracellular vesicles), then ultracentrifuge the obtained cell supernatant: 110,000×g for 70 minutes, and the obtained precipitate is the crude extracellular vesicle sample, which is then resuspended twice with TBS buffer solution (150mM NaCl, 10mM Tris-HCl, pH adjusted to about 6) and washed by ultracentrifugation to obtain an extracellular vesicle sample that can be used for subsequent labeling experiments;

[0022] All centrifugation operations were performed at 4°C.

[0023] The liposomes are artificially synthesized liposomes of various sizes such as unilamellar liposomes, multilamellar liposomes, multivesicular liposomes, etc., and specifically can be unilamellar liposomes;

[0024] The liposome preparation method is a thin film dispersion method, a freeze-thaw method, a solvent injection method, a reverse evaporation method, a filter membrane extrusion method and other liposome preparation methods, and specifically can be a thin film dispersion method.

[0025] In step 2) and 2'), the lanthanide metal ion may be specifically a europium ion (Eu 3+ ),

[0026] The lanthanide metal ion solution may specifically be europium chloride (EuCl 3 ) solution,

[0027] The EuCl 3 The concentration of the solution is 0.05-1mM, specifically 0.1mM;

[0028] The EVs and EuCl 3 The ratio of the solution is: 10-30 μg: 150-250 μL, specifically 20 μg: 200 μL;

[0029] The liposomes and EuCl 3 The ratio of the solution is: E9-3E9: 150-250 μL, specifically 2E9: 200 μL;

[0030] The incubation is shaking (500-1500rpm) incubation, the incubation temperature is 4°C-60°C (specifically 4°C-40°C, 4°C-37°C), the incubation time is 10min-2h (specifically 10min-60min), specifically, shaking (1000rpm) incubation at 37°C for 30min.

[0031] The operation of step 3) or 3') is as follows: after the incubation of EVs or liposomes with the lanthanide metal ion solution in step 2) or 2') is completed, the entire solution is transferred to an ultrafiltration tube, centrifuged to remove the lanthanide metal ion solution, and then a cleaning solution is used to remove free lanthanide metal ions in the solution through multiple ultrafiltration washes to avoid interference with subsequent quantification. The concentrated solution in the ultrafiltration tube obtained after the last washing operation is the EVs or liposomes labeled with lanthanide metal ions.

[0032] The ultrafiltration cleaning uses an ultrafiltration tube with a specification of 20-100 kD. The centrifugation conditions of the ultrafiltration operation are: 4000-8000 g centrifugation for 5-15 min, and the cleaning operation is repeated 3-5 times, specifically, the cleaning operation can be repeated 4 times under the condition of 5000 g × 10 min; the centrifugation operation in the cleaning process is carried out at 4°C;

[0033] The cleaning solution is a TBS buffer solution (150 mM NaCl, 10 mM Tris-HCl, pH adjusted to about 6), and the cleaning is performed multiple times, specifically four times;

[0034] The volume of the cleaning solution used each time may be 300-450 μL, specifically 400 μL.

[0035] The operation of step 4') is: respectively detect the number of particles of extracellular vesicles or liposomes labeled with corresponding concentrations of lanthanide metal ions by NanoFCM and detect the Eu content therein by ICP-MS, and obtain a standard curve of the lanthanide metal content in the extracellular vesicles or liposomes labeled with lanthanide metal ions relative to the number of vesicles or liposome particles y=0.92931x+0.35476 (x is the number of particles*E7, y is the lanthanide metal content), and calculate the amount of extracellular vesicles EVs or liposomes to be tested according to the standard curve.

[0036] The present invention provides a method for preparing a 3 Methods for labeling and quantifying sEVs (labeling process as Figure 1 ), specifically, sEVs derived from HeLa cell culture supernatant were labeled and quantified to demonstrate the feasibility of Eu-labeled EVs, including the following steps:

[0037] The cells were cultured to a density of about 80%, and the culture supernatant was replaced with EV-depleted medium and cultured for another 24 h. Then all the cell culture supernatants were collected, and the sEVs in the cell culture supernatants were enriched by classical ultracentrifugation. Then a certain amount of sEVs samples were taken and concentrated in EuCl 3 The Eu 3+ Fully combined with sEVs, free Eu in the solution that did not participate in the reaction was removed by ultrafiltration. 3+ After the last cleaning operation, the concentrated liquid retained in the ultrafiltration tube is the surface-bound Eu 3+ sEVs (Eu-sEVs).

[0038] In the above method, the cells are HeLa cells, and the extracellular vesicles are small extracellular vesicles;

[0039] In the above method, EV-depleted culture medium is DMEM culture medium containing 10% EV-depleted FBS and 1% double antibody; EV-depleted FBS is FBS supernatant obtained by ultracentrifugation of FBS (120,000g, 4°C, 18h);

[0040] In the above method, the dosage of sEVs is 20 μg;

[0041] In the above method, the cleaning method is ultrafiltration cleaning, the specification of the ultrafiltration tube is 0.5mL-100kD, the cleaning solution used in ultrafiltration is TBS buffer solution (150mM NaCl, 10mM Tris-HCl, pH adjusted to about 6), the amount of cleaning solution is 400μL, and the centrifugation condition of the ultrafiltration operation is: 5000g×10min; the centrifugation operation during the cleaning process needs to be maintained at 4°C;

[0042] In the above method, EuCl 3 Reaction concentrations (0.01mM, 0.05mM, 0.1mM, 0.5mM), EuCl 3 Incubation time with sEVs (10min, 30min, 60min), EuCl 3 The incubation temperature (4°C, 37°C) with sEVs was investigated and optimized. The optimal reaction conditions for this labeling reaction were determined to be: 0.1 mM concentration of EuCl 3 , incubate at 37°C for 30 min.

[0043] The application of the above method in labeling and absolute quantification of extracellular vesicles EVs or liposomes in and outside cells also falls within the protection scope of the present invention.

[0044] The application of the above method in the quantitative analysis of liposomes and their intracellular delivery also falls within the protection scope of the present invention.

[0045] The present invention provides a new method for labeling and absolute quantification of EVs and liposomes. The specific interaction between lanthanide metal ions and phosphate groups on the surface of EVs or liposome membranes is used to achieve specific labeling of EVs and liposomes. After multiple ultrafiltration washes, free Eu in the solution is fully removed. 3+ , Europium-labeled EVs or liposomes can be obtained. The highly sensitive response of metal Eu to ICP-MS can achieve absolute quantification of EVs or liposomes at the intracellular and extracellular levels.

[0046] Compared with the existing quantitative methods of EVs or liposomes, the present invention has the following advantages:

[0047] 1) The present invention utilizes the principle of specific binding of lanthanide metal ions to phosphate groups on the surface of EVs or liposome membranes and the high sensitivity (ppb level) of lanthanide metals to ICP-MS to achieve labeling and quantification of EVs or liposomes inside and outside cells;

[0048] 2) This method is simple to operate, and only requires one step of reaction and simple washing operation to achieve efficient labeling of EVs or liposomes;

[0049] 3) This method has good biocompatibility, and lanthanide metals have low background interference in biological systems;

[0050] 4) Compared with lipophilic dyes that label EVs based on hydrophobic interactions, this labeling method is based on the principle of covalent labeling and has better labeling stability;

[0051] 5) Compared with labeling strategies such as fluorescent probes that can only semi-quantitatively analyze EVs uptake by cells, and particle size analysis methods such as NanoFCM that can only quantify the extracellular content of EVs, this method can achieve accurate absolute quantification of EVs uptake by cells.

[0052] Liposomes and extracellular vesicles have very similar diameter size range and membrane structure, so this lanthanide metal labeling method can also be used for the quantitative analysis of liposomes and their intracellular delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Flow chart of the method for quantitative cellular uptake of extracellular vesicles labeled with lanthanide metals according to the present invention.

[0054] Figure 2 To investigate the feasibility of labeling extracellular vesicles with lanthanide metals and to characterize and analyze the extracellular vesicles before and after labeling. Figure 2 A in the figure is the europium content in the blank group and Eu-sEVs group detected by ICP-MS. Figure 2 Middle B shows the particle size distribution characterization results of sEVs before and after labeling. Figure 2 Center C shows the Zeta potential value of extracellular vesicles before and after labeling. Figure 2 Middle D shows the confocal analysis results of CFSE fluorescent staining of sEVs before and after Europium labeling. Figure 2 Middle E shows the results of transmission electron microscopy analysis before and after sEVs labeling.

[0055] Figure 3 Optimization of reaction conditions for lanthanide metal labeling of extracellular vesicles. Figure 3 A in the middle shows the europium content in Eu-sEVs obtained by incubating different concentrations of europium chloride with an equal amount of sEVs. Figure 3 Figure B shows the europium content in Eu-sEVs obtained by incubating equal amounts of sEVs with 0.1 mM europium chloride solution at different incubation temperatures for 30 min. Figure 3 Center C shows the europium content in Eu-sEVs obtained by incubating equal amounts of sEVs with 0.1 mM europium chloride solution for different incubation times.

[0056] Figure 4 This is the standard curve of the europium content relative to the particle number in the serially diluted Eu-sEVs of different concentrations.

[0057] Figure 5 To quantify the uptake of extracellular vesicles by cells under different conditions. Figure 5 A in the middle shows the uptake of Eu-sEVs by HeLa cells under different incubation temperature conditions. Figure 5 Middle B shows the uptake of Eu-sEVs by HeLa cells under different incubation time conditions. Figure 5 (C) Quantification of cellular uptake of Eu-sEVs in the presence or absence of co-incubation with unlabeled sEVs. Figure 5 D in the middle shows the uptake of Eu-sEVs by HeLa cells in the presence or absence of inhibitors.

[0058] Figure 6 The lanthanide metal labeling method is used to quantify the intracellular and extracellular levels of liposomes. Figure 6 A in the figure shows the europium content in the blank group and europium-labeled liposome group detected by ICP-MS. Figure 6 Middle B shows the uptake of Europium-labeled liposomes by HeLa cells under different incubation time conditions. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0060] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0061] The ICP-MS detection parameters in the following examples are as follows:

[0062] Radiofrequency power: 1550W

[0063] Carrier gas flow (Ar): 1L min-1

[0064] Auxiliary gas flow (Ar): 0.9L min-1

[0065] Collant gas flow (Ar); 15.00L min-1

[0066] Dwell time: 300ms

[0067] Scanning mode:He

[0068] Pump Rate: 29r / min

[0069] Monitored istope: 153 Eu.

[0070] Example 1, Europium chloride (EuCl 3 ) Labeling and quantification of small extracellular vesicles (sEVs)

[0071] Reference Figure 1 Flow chart showing quantification of cellular uptake of extracellular vesicles using the lanthanide metal europium labeling

[0072] Step 1: Enrichment of sEVs

[0073] HeLa cells were cultured in a 15 cm dish with normal culture medium (DMEM medium containing 10% FBS and 1% double antibody) to a density of about 80%, and then replaced with EV-depleted culture medium (DMEM medium containing 10% EV-depleted FBS and 1% double antibody) and cultured for 24 hours before collecting all culture supernatants for sEVs enrichment. First, the cell culture supernatant was pretreated by centrifugation at 300 × g for 10 min (to remove dead cells), 2000 × g for 10 min (to remove cell debris), and 10000 × g for 30 min (to remove large extracellular vesicles). Then, the obtained cell supernatant was ultracentrifuged at 110,000 × g for 70 min. The precipitate obtained was the crude sEVs sample, which was then resuspended twice in TBS buffer solution (150 mM NaCl, 10 mM Tris-HCl, pH adjusted to about 6) and washed by ultracentrifugation to obtain sEVs samples that can be used for subsequent labeling experiments. All centrifugation operations were performed at 4°C.

[0074] Step 2: Use EuCl 3 Labeling of sEVs

[0075] Weigh a certain amount of anhydrous EuCl 3 , dissolve it in deionized water to prepare 50 mM EuCl 3 The solution was then diluted with TBS buffer solution (150 mM NaCl, 10 mM Tris-HCl, pH adjusted to about 6) to obtain EuCl 3 Mix 20 μg of the HeLa cell-derived sEVs sample enriched in step 1 above with 200 μL of 0.1 mM EuCl in the EP tube. 3After the solutions were mixed and incubated together, an equal volume of TBS buffer solution as that of sEVs in the experimental group was added to 200 μL of 0.1 mM EuCl in the EP tube. 3 The solution was mixed as a blank group. The EP tube was incubated at 37°C with shaking (1000 rpm) for 30 min to allow Eu 3+ The solution was then transferred to an ultrafiltration tube (0.5 mL, 100 kD) and centrifuged to remove EuCl 3 Then add 400 μL TBS buffer solution to the ultrafiltration tube and centrifuge again. Repeat this cleaning operation four times to remove as much free Eu as possible from the solution. 3+ The centrifugation conditions during the ultrafiltration process were: 5000 g, 4 °C, 10 min. The concentrated solution in the ultrafiltration tube obtained after the last washing operation was Eu-labeled sEVs (Eu-sEVs). ICP-MS was used to detect the Eu content in the two groups of samples. Figure 2 As shown in A, more Eu-sEVs were detected in the Eu-sEVs group than in the blank group (the blank group was also subjected to the following operations: incubation at 37°C with shaking (1000 rpm) for 30 min, the solution was transferred to an ultrafiltration tube (0.5 mL, 100 kD) and centrifuged to remove EuCl 3 The solution was then centrifuged again after adding 400 μL of TBS buffer solution to the ultrafiltration tube, and the cleaning operation was repeated four times. The Eu content was 10.57 times higher, indicating that most of the free Eu in the solution was removed after the ultrafiltration cleaning operation. 3+ Removed, the remaining Eu 3+ It will not interfere with subsequent experiments, thus proving the feasibility of this method for labeling and quantifying sEVs. Europium chloride was purchased from Sigma (429732); sodium chloride was purchased from Xilong Chemical; and Tris-HCl was purchased from Solarbio.

[0076] Step 3. Characterization of EuCl 3 sEVs before and after labeling

[0077] In order to prove that this labeling method does not affect the physicochemical properties and biological activity of sEVs, a series of characterization analyses were performed on sEVs before and after labeling. Figure 2 As shown in Figure B, the analysis results of NanoFCM showed that the particle size distribution of sEVs did not change significantly before and after labeling, and the particle size range was 30-150nm. Figure 2 As shown in Figure C, there is no significant change in the zeta potential of sEVs before and after labeling. Figure 2 As shown in Figure D, the commonly used extracellular vesicle fluorescent dye CFSE directly stains sEVs and shows that sEVs still have good dispersion before and after labeling. Figure 2As shown in E, the results of transmission electron microscopy analysis showed that sEVs before and after labeling exhibited the classic saucer-shaped structure of EVs.

[0078] Example 2, EuCl 3 Optimization of reaction conditions for labeling sEVs

[0079] In order to achieve the best labeling efficiency while minimizing the loss of sEVs during the labeling process, based on Example 1, EuCl 3 The reaction conditions for labeling sEVs were investigated and optimized.

[0080] Step 1: Examine EuCl 3 Effect of reaction concentration on labeling efficiency

[0081] Use 200 μl volume of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM EuCl 3 The solution was incubated with an equal amount of sEVs (20 μg) at 37°C with shaking (1000 rpm) for 30 min, and then the solution was transferred to an ultrafiltration tube (0.5 mL, 100 kD) and centrifuged to remove EuCl. 3 Then add 400 μL of TBS buffer solution to the ultrafiltration tube and centrifuge again. Repeat this washing operation four times to remove as much free Eu as possible from the solution. 3+ The centrifugation conditions during the ultrafiltration process were: 5000 g, 4 °C, 10 min. After the last washing operation, the concentrated solution retained in the ultrafiltration tube was collected as Eu-labeled sEVs (Eu-sEVs). ICP-MS was used to detect the metal Eu content in each group of Eu-sEVs. Figure 3 As shown in A, with the 3 As the concentration increased from 0.01mM to 0.1mM, the labeling efficiency continued to increase. 3 concentration, the labeling efficiency hardly increases, indicating that EuCl 3 The optimal reaction concentration was 0.1 mM.

[0082] Step 2: Investigate the effect of reaction temperature on labeling efficiency

[0083] In step 1, EuCl 3 The optimal concentration is 0.1 mM. 3 The reaction temperature of sEVs was investigated. An equal amount (20 μg) of sEVs sample was mixed with 200 μL of 0.1 mM EuCl 3After the solutions were mixed, they were incubated at 4°C and 37°C with shaking (1000 rpm) for 30 min. The solutions were then transferred to ultrafiltration tubes (0.5 mL, 100 kD) and centrifuged to remove EuCl. 3 Then add 400 μL of TBS buffer solution to the ultrafiltration tube and centrifuge again. Repeat this washing operation four times to remove as much free Eu as possible from the solution. 3+ The centrifugation conditions during the ultrafiltration process were: 5000g, 4°C, 10min. After the last washing operation, the concentrated solution retained in the ultrafiltration tube was collected as the europium-labeled extracellular vesicles (Eu-sEVs). Finally, ICP-MS was used to detect the europium content in the Eu-sEVs obtained under the two temperature conditions. Figure 3 As shown in B, compared with Eu-sEVs obtained at 4°C, the europium content in Eu-sEVs obtained at 37°C increased significantly, so 37°C was selected as the optimal reaction temperature for this reaction.

[0084] Step 3: Investigate the effect of incubation time on the labeling efficiency of extracellular vesicles

[0085] Under the above conditions, EuCl 3 The optimal reaction concentration is 0.1 mM EuCl 3 The optimal incubation temperature for sEVs was 37°C. 3 The reaction time with sEVs was investigated. An equal amount (20 μg) of sEVs sample was used with 200 μl volume of 0.1 mM EuCl 3 After the solutions were mixed, they were incubated at 37°C with shaking (1000 rpm) for 10 min, 30 min, and 60 min, respectively. The solutions were then transferred to ultrafiltration tubes (0.5 mL, 100 kD) and centrifuged to remove EuCl 3 Then add 400 μL of TBS buffer solution to the ultrafiltration tube and centrifuge again. Repeat this washing operation four times to remove as much free Eu as possible from the solution. 3+ The centrifugation conditions during the ultrafiltration process were: 5000g, 4°C, 10min. After the last washing operation, the concentrated solution retained in the ultrafiltration tube was collected as europium-labeled sEVs (Eu-sEVs). Finally, ICP-MS was used to detect the Eu content in the Eu-sEVs obtained under the three reaction time conditions. The results are as follows: Figure 3 As shown in C, EuCl 3 The labeling efficiency of sEVs no longer increased significantly when the reaction time exceeded 30 min. Considering that a longer incubation time may lead to more sEVs loss, 30 min was selected as the optimal reaction time for this reaction.

[0086] Example 3: Combined NanoFCM and ICP-MS to establish EuCl 3 Standard curve for quantification of sEVs

[0087] In Example 1, it has been demonstrated that EuCl 3 The feasibility of labeling and quantifying sEVs was investigated. Next, Eu-sEVs were prepared under the optimal reaction conditions obtained in Example 2. Eu-sEVs were gradiently diluted and the number of Eu-sEV particles at each dilution concentration was detected by NanoFCM and the Eu content was detected by ICP-MS. A standard curve of the metal Eu content in Eu-sEVs relative to the number of vesicle particles was obtained. Figure 4 As shown in Figure 2, there is a good linear relationship between the europium content and the number of particles in Eu-sEVs (R 2 >0.99), and the quantitative linear range of sEVs particle number was 1.12×10 5 -5.24×10 8 Therefore, the absolute quantification of the number of sEVs particles at the intracellular and extracellular levels can be achieved.

[0088] Example 4: EuCl 3 The method of labeling sEVs was used for absolute quantification of sEV uptake by HeLa cells

[0089] Step 1: Investigate the effect of incubation temperature on the efficiency of sEVs uptake by cells

[0090] HeLa cells (1×10 6 ) were inoculated in a six-well plate and cultured overnight, and Eu-sEVs were prepared under the optimal incubation conditions obtained in Example 2. 20 μg of Eu-sEVs were added to the culture medium of HeLa cells in the six-well plate, and then Eu-sEVs and HeLa cells were incubated at 4°C and 37°C for 4 hours. After the incubation, the HeLa cells were washed 3 times with 1×PBS, then digested and collected with trypsin, and finally the Eu content in the HeLa cells was detected by ICP-MS to obtain the uptake of sEVs by HeLa cells. Figure 5 As shown in Figure A, under 37°C conditions, HeLa cells have a higher uptake efficiency for Eu-sEVs, indicating that the process of HeLa uptake of sEVs is mainly an energy-consuming, active process rather than a passive membrane fusion process.

[0091] Step 2: Investigate the uptake of sEVs by HeLa cells under different incubation time conditions

[0092] HeLa cells (1×10 6) were inoculated in a six-well plate and cultured overnight, and Eu-sEVs were prepared under the optimal incubation conditions obtained in Example 2. 20 μg of Eu-sEVs were added to the culture medium of HeLa cells in the six-well plate and incubated with HeLa cells at 37°C for 1, 3, 6 and 9 hours. After the incubation, the cells were washed 3 times with 1×PBS, and then digested with trypsin and the HeLa cells were collected. The Eu content in the HeLa cells was detected by ICP-MS, which was the uptake of sEVs by HeLa cells. Figure 5 As shown in Figure B, as the incubation time increases, the uptake of Eu-sEVs by HeLa cells gradually increases, indicating that the uptake of sEVs by HeLa cells is a time-dependent process.

[0093] Step 3: Investigate the competitive inhibition of unlabeled sEVs on the uptake of Eu-sEVs by HeLa cells

[0094] HeLa cells (1×10 6 ) were inoculated in six-well plates and cultured overnight, and Eu-sEVs were prepared under the optimal incubation conditions obtained in Example 2. 10 μg of Eu-sEVs (control group), or a mixture of 10 μg Eu-sEVs and 20 μg of unlabeled sEVs (experimental group) was added to the culture medium of HeLa cells in the six-well plates, and incubated with the HeLa cells at 37°C for 3 hours. After the incubation, the cells were washed 3 times with 1×PBS, and then digested and collected with trypsin. The europium content in the HeLa cells was detected by ICP-MS, which was the uptake of sEVs by HeLa cells. Figure 5 As shown in Figure C, the europium content in the HeLa cells of the experimental group was significantly reduced compared with that of the control group, indicating that the unlabeled sEVs had a significant competitive inhibitory effect on the uptake of Eu-sEVs by HeLa cells. It also shows that this labeling method will not significantly affect the cellular uptake mechanism of sEVs.

[0095] Step 4: Investigate the mechanism of cellular uptake of extracellular vesicles

[0096] HeLa cells (1×10 6) were inoculated in six-well plates and cultured overnight, and then the cells were pre-incubated with inhibitors CPZ (10mM) and IPA-3 (50mM) for 30 minutes before adding sEVs. HeLa cells that were not pretreated with inhibitors were used as the control group. Eu-sEVs were prepared under the optimal incubation conditions obtained in Example 2, and 10μg of Eu-sEVs were added to the culture medium of HeLa cells in the six-well plates, and incubated with HeLa cells at 37°C for 6 hours. After the incubation was completed, the cells were washed 3 times with 1×PBS, and then the HeLa cells were digested and collected with trypsin. The europium content in the HeLa cells was detected by ICP-MS, which was the uptake of sEVs by HeLa cells. Figure 5 As shown in D, compared with the control group, CPZ treatment significantly promoted the cellular uptake of Eu-sEVs, while IPA-3 significantly inhibited the uptake of Eu-sEVs. Since CPZ and IPA-3 are chemical inhibitors of the clathrin-dependent endocytosis pathway (CDE) and macropinocytosis pathway (MP), respectively, the uptake of sEVs by HeLa cells mainly depends on the MP pathway rather than the CDE pathway.

[0097] Example 5: Quantification of intracellular delivery effect of liposome drugs using the europium chloride labeling method

[0098] Doxorubicin is a commonly used anti-tumor anthracycline drug, and its mechanism of action is believed to be an inhibitor of DNA topoisomerase. However, due to its ability to affect multiple types of cells in the body, it is accompanied by serious toxic side effects when used as a drug. Liposomal doxorubicin, by encapsulating doxorubicin in a phospholipid bilayer membrane, improves the stability of the drug in the blood circulation in the body, prolongs its existence time in the blood circulation, and greatly reduces its toxic side effects.

[0099] Step 1: Preparation of liposomal doxorubicin

[0100] Add lecithin and cholesterol in a molar ratio of 4:1 with a total lipid concentration of 100M to a chloroform-methanol mixed solution in a volume ratio of 2:1 with a volume of 10ml. After the lipid is fully dissolved, transfer 2ml to a flat-bottom bottle to spread it on the bottom, and blow dry the bottom to obtain a uniform lipid film. Then add 1ml of a 3mM doxorubicin solution prepared with TBS buffer solution (150mM NaCl, 10mM Tris-HCl, pH adjusted to about 6) to the bottle, and after ultrasonic dispersion and pulverization, centrifuge for 15min (4700g) to obtain the supernatant, which is liposomal doxorubicin.

[0101] Step 2: Labeling liposomal doxorubicin with europium chloride

[0102] Weigh a certain amount of anhydrous EuCl 3, dissolve it in deionized water to prepare 50 mM EuCl 3 The solution was then diluted with TBS buffer solution (150 mM NaCl, 10 mM Tris-HCl, pH adjusted to about 6) to obtain EuCl 3 The solution was stirred to a concentration of 0.1 mM. Take the liposomal doxorubicin sample 2E9 prepared in step 1 above and mix it with 200 μL of 0.1 mM EuCl in the EP tube. 3 The solutions were mixed and incubated together as the liposome group. In addition, an equal volume of TBS buffer solution as the liposomes in the experimental group was mixed with 200 μL of 0.1 mM EuCl in an EP tube. 3 The solution was mixed as a blank group. The EP tube was incubated at 37°C with shaking (1000 rpm) for 30 min to allow Eu 3+ The solution was then transferred to an ultrafiltration tube (0.5 mL, 100 kD) and centrifuged to remove EuCl 3 After adding 400 μL of TBS buffer solution to the ultrafiltration tube, centrifuge again and repeat this washing operation four times to remove as much free Eu as possible from the solution. 3+ The centrifugation conditions during the ultrafiltration process were: 5000 g, 4°C, 10 min. The concentrated solution in the ultrafiltration tube obtained after the last washing operation was Eu-labeled liposomal doxorubicin. The Eu content in the two groups of samples was detected by ICP-MS. Figure 6 As shown in A, the Eu content detected in the liposome group was 10.73 times that of the blank group, indicating that most of the free Eu in the solution was removed after ultrafiltration. 3+ Removed, the remaining Eu 3+ It will not interfere with subsequent experiments, thus proving the feasibility of this method for labeling quantitative liposomes. Europium chloride was purchased from Sigma (429732); doxorubicin was purchased from Biyuntian; sodium chloride was purchased from Xilong Chemical; and Tris-HCl was purchased from Solarbio.

[0103] Step 3: Investigate the uptake of liposomal doxorubicin by HeLa cells under different incubation time conditions

[0104] HeLa cells (1×10 6 ) were inoculated in a six-well plate for overnight culture, and an equal amount of the europium-labeled liposomal doxorubicin obtained in step 1 above was added to the culture medium of the HeLa cells in the six-well plate, and incubated with the HeLa cells at 37°C for 1, 3, 6, and 9 hours, respectively. After the incubation, the cells were washed 3 times with 1×PBS, and then digested with trypsin and the HeLa cells were collected. The Eu content in the HeLa cells was detected by ICP-MS, which was the uptake of liposomal doxorubicin by the HeLa cells. Figure 6As shown in B, with the increase of incubation time, the uptake of liposomal doxorubicin by HeLa cells gradually increased, indicating that the uptake of liposomes by HeLa cells is a time-dependent process. It also shows that the europium labeling method can be used for absolute quantification of the intracellular delivery effect of liposome drugs.

[0105] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for labeling and absolutely quantifying extracellular vesicles (EVs) or liposomes at the intracellular and extracellular levels, comprising: incubating extracellular vesicles (EVs) or liposomes with a lanthanide metal ion solution, labeling the extracellular vesicles (EVs) or liposomes through the specific binding of the lanthanide metal ions to the phosphate groups on the membrane surface of the extracellular vesicles (EVs) or liposomes, and detecting them by ICP-MS, thereby achieving absolute quantification of the extracellular vesicles (EVs) or liposomes.

2. A method for metal labeling of extracellular vesicles EVs or liposomes, comprising the following steps: 1) Enrich extracellular vesicles (EVs) from biological samples or prepare liposomes using artificial methods; 2) Dispersing the enriched EVs or liposomes prepared by artificial methods in a lanthanide metal ion solution and incubating the solution to allow the lanthanide metal ions to fully bind to the phosphate groups on the surface of the EVs or liposome membranes; 3) Ultrafiltration and washing are performed to remove free lanthanide metal ions that do not participate in the reaction, thereby obtaining lanthanide metal-labeled extracellular vesicles or liposomes.

3. A method for absolute quantification of extracellular vesicles EVs or liposomes, comprising the following steps: 1') Enrich extracellular vesicles (EVs) from biological samples or prepare liposomes using artificial methods; 2') dispersing the enriched EVs or the liposomes prepared by artificial methods in a lanthanide metal ion solution and incubating the solution to allow the lanthanide metal ions to fully bind to the phosphate groups on the surface of the EVs or liposome membranes; 3') ultrafiltration and washing to remove free lanthanide metal ions that do not participate in the reaction, thereby obtaining lanthanide metal-labeled extracellular vesicles or liposomes; 4') ICP-MS detects the amount of lanthanide metal in the lanthanide metal-labeled extracellular vesicles or liposomes to achieve absolute quantification of EVs or liposomes.

4. The method according to claim 2 or 3, Features: In steps 1) and 1'), the biological samples are culture supernatants of various cell types and various body fluid samples; The extracellular vesicles include exosomes, microvesicles, small extracellular vesicles, and medium / large extracellular vesicles; The enrichment method includes ultracentrifugation, ultrafiltration, size exclusion chromatography, and affinity method; The liposomes include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes; The preparation methods of the liposomes include thin film dispersion method, freeze-thaw method, solvent injection method, reverse evaporation method and filter membrane extrusion method.

5. The method according to claim 2 or 3, Features: In step 2) and 2'), the lanthanide metal ion is europium ion, The lanthanide metal ion solution is europium chloride EuCl 3 Solution, The EuCl 3 The concentration of the solution is 0.05-1mM; The EVs and EuCl 3 The ratio of the solution is: 10-30μg: 150-250μL; The liposomes and EuCl 3 The ratio of the solution is: E9-3E9: 150-250 μL; The incubation is performed with shaking (500-1500 rpm), the incubation temperature is 4° C.-60° C., and the incubation time is 10 min-2 h.

6. The method according to claim 2 or 3, Features: The operation of step 3) or 3') is as follows: after the incubation of EVs or liposomes with the lanthanide metal ion solution in step 2) or 2') is completed, the entire solution is transferred to an ultrafiltration tube, centrifuged to remove the lanthanide metal ion solution, and then a cleaning solution is used to remove free lanthanide metal ions in the solution by multiple ultrafiltration washings. The concentrated solution in the ultrafiltration tube obtained after the last washing operation is the EVs or liposomes labeled with lanthanide metal ions; The ultrafiltration cleaning uses an ultrafiltration tube with a specification of 20-100 kD, and the centrifugation conditions of the ultrafiltration operation are: 4000-8000g centrifugation for 5-15 minutes, and the cleaning operation is repeated 3-5 times; the centrifugation operation during the cleaning process is performed at 4°C; The cleaning solution is TBS buffer solution, and the cleaning is performed multiple times, specifically four times; The volume of the cleaning solution used each time is: 300-450μL.

7. The method according to claim 2 or 3, Features: The operation of step 4') is: respectively detecting the number of particles of extracellular vesicles or liposomes labeled with corresponding concentrations of lanthanide metal ions by NanoFCM and detecting the Eu content therein by ICP-MS, and obtaining a standard curve of the lanthanide metal content in the extracellular vesicles or liposomes labeled with lanthanide metal ions relative to the number of vesicles or liposome particles y=0.92931x+0.35476, where x is the number of particles*E7, and y is the lanthanide metal content. According to the standard curve, the amount of extracellular vesicles EVs or liposomes to be tested is calculated.

8. Use of the method according to any one of claims 1 to 7 in labeling and absolute quantification of extracellular and extracellular levels of extracellular vesicles EVs or liposomes.

9. Use of the method according to any one of claims 1 to 7 in the quantitative analysis of liposomes and their intracellular delivery.

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