A probe for extracellular vesicles and its preparation method and application

Abstract

The present invention provides a probe for extracellular vesicles, a preparation method and an application thereof. The chemical structural formula of the probe for extracellular vesicles of the present invention is shown in Formula I. The probe for extracellular vesicles of the present invention has better fusion performance with small extracellular vesicles. It can be efficiently labeled with small extracellular vesicles (Small Extracellular Vesicles, sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes, and fluorescence detection tracking can be achieved, which is of great significance for in vitro labeling or in vivo tracing of isolated sEVs. Since the small extracellular vesicles can be efficiently labeled by mixing and incubating with small extracellular vesicles (Small Extracellular Vesicles, sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes, efficient and faster detection can be achieved, saving detection time. The detection method for extracellular vesicles of the present invention has a short detection time for labeling and improves detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of stem cell and cell-related substance detection, and in particular to a probe for extracellular vesicles and a preparation method and application thereof. Background Art

[0002] Small extracellular vesicles (sEVs) are closed spherical vesicles wrapped by lipid bilayers that are released spontaneously or under certain conditions by a variety of mammalian cells. sEVs are rich in a variety of bioactive substances such as proteins, nucleic acids, and lipids, and play an important role in the development of a variety of physiological and pathological processes; sEVs can be used as markers to diagnose diseases, monitor disease progression, and assess prognosis, and can also be used as drugs or drug carriers, playing an important role in maintaining physiological states and treating diseases. Therefore, in vitro labeling or in vivo tracing of isolated sEVs is of great significance for their functional research.

[0003] However, the existing probes for labeling small extracellular vesicles take a long time to label small extracellular vesicles, and the labeling efficiency is low, which affects the detection results. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a probe for extracellular vesicles and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a probe for extracellular vesicles, the chemical structure of the probe for extracellular vesicles is shown in Formula I:

[0006]

[0007] The probe for extracellular vesicles has a better fusion performance with small extracellular vesicles. It can efficiently achieve the labeling of small extracellular vesicles by mixing and incubating with small extracellular vesicles (sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes. It also has a specific fluorescent group. Under an excitation wavelength of about 487nm, the emission wavelength is about 504nm. After labeling, fluorescence detection tracking can be achieved, which is of great significance for in vitro labeling or in vivo tracing of isolated sEVs. Since it can efficiently achieve the labeling of small extracellular vesicles by mixing and incubating with small extracellular vesicles (sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes, efficient and faster detection can be achieved, saving detection time.

[0008] The present invention also provides a method for preparing the above-mentioned probe for extracellular vesicles, the preparation method comprising the following steps:

[0009] (1) mixing the compound represented by formula II and N-hydroxysuccinimide in a molar ratio of 1:(2-3) in dichloromethane;

[0010]

[0011] (2) adding dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) and stirring at 20 to 30° C. for 1.5 to 4 hours;

[0012] (3) placing the mixed solution after the reaction in step (2) on a polyamide column and then washing it with dichloromethane;

[0013] (4) eluting with a water-ethanol mixed solvent having a volume of 40% to 60% ethanol to obtain fraction A;

[0014] (5) eluting with a chloroform-methanol mixed solvent to obtain fraction B; the volume fraction of chloroform is 40% to 60%;

[0015] (6) Remove the solvent from fraction B and crystallize it.

[0016] The above preparation method uses a polyamide column for elution and purification after the esterification reaction, thereby purifying and separating the probe for extracellular vesicles as shown in Formula I, and then using it as a probe.

[0017] Preferably, the method of removing the solvent includes vacuum evaporation or negative pressure evaporation, and the temperature does not exceed 30°C.

[0018] Preferably, in step (1), the compound as shown in formula II and N-hydroxysuccinimide are mixed in a molar ratio of 1:(2.2-2.5).

[0019] Preferably, the amount of dimethylaminopyridine used accounts for 4% to 7% of the mass of the mixed solution in step (2), and the molar ratio of N,N′-dicyclohexylcarbodiimide to dimethylaminopyridine is 1:(0.95 to 1.05).

[0020] The present invention also provides a method for detecting extracellular vesicles, the method comprising the following steps:

[0021] (a) mixing a solution of the probe for extracellular vesicles described in Formula I with an extracellular vesicle solution, adjusting the volume to a phosphate buffer having a pH of 7.0 to 7.4, mixing thoroughly, and incubating at 25 to 30° C. in the dark for 15 to 30 minutes;

[0022] (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 to 130,000 times gravity for 30 to 50 minutes to remove the supernatant;

[0023] (c) resuspending the precipitate obtained in step (b) in a PBS solution, and measuring the fluorescence intensity at a wavelength of 300 nm to 700 nm.

[0024] The probe for extracellular vesicles has a better fusion performance with small extracellular vesicles, and can be efficiently labeled with small extracellular vesicles (sEV) by mixing and incubating with small extracellular vesicles (sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes. It also has a specific fluorescent group, and under an excitation wavelength of about 486nm, the emission wavelength is about 504nm. After labeling, fluorescence detection tracking can be achieved. The detection time for labeling is short, which improves the detection efficiency.

[0025] Preferably, the solvent of the extracellular vesicle solution is DMEM / F12 complete culture medium, and the solvent of the extracellular vesicle probe solution is phosphate buffer with a pH of 7.0 to 7.4; the usage ratio of the probe for extracellular vesicles to the extracellular vesicles is 2.5 mol to 5 mol of the probe for extracellular vesicles to 100 g of extracellular vesicles.

[0026] Preferably, the method comprises the following steps:

[0027] (a) mixing 100-200 μL of a solution of a probe for extracellular vesicles as described in Formula I with 100-200 μL of an extracellular vesicle solution, wherein the amount of the probe for extracellular vesicles is 2.5 μmol-5 μmol, the amount of the extracellular vesicles is 80-120 μg, and the volume is adjusted to 0.8-1.2 mL with a phosphate buffer having a pH of 7.0-7.4, and the mixture is fully mixed and incubated at 25-30° C. in the dark for 15 to 30 minutes;

[0028] (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 to 130,000 times gravity for 30 to 50 minutes to remove the supernatant;

[0029] (c) resuspending the precipitate obtained in step (b) in 0.8-1.2 mL of PBS solution, and measuring the fluorescence intensity at a wavelength of 300 nm to 700 nm.

[0030] The present invention also provides a detection kit for extracellular vesicles, which comprises 0.8 to 2 mmol of a probe for extracellular vesicles as described in Formula I, and 100 to 500 mmol of a phosphate buffer salt solid preparation or a buffer solution with a pH of 7.0 to 7.4, wherein the phosphate solid preparation is configured from monohydrogen phosphate and dihydrogen phosphate.

[0031] Preferably, the detection kit comprises 100 to 500 mmol of a phosphate buffer solution at a pH of 7.0 to 7.4 and does not comprise a phosphate buffer salt solid preparation, and the concentration of the phosphate buffer solution is 15 to 30 mmol / L.

[0032] Preferably, the probe for extracellular vesicles as described in Formula I is prepared into a mother solution of 100-500 mmol / mL using phosphate buffer as a solvent.

[0033] Preferably, the kit further comprises an instruction manual, which records the chemical structure of the probe for extracellular vesicles as described in Formula I.

[0034] Preferably, the instructions also describe any of the above-mentioned methods for detecting extracellular vesicles.

[0035] Preferably, the instructions also record the excitation spectrum and emission spectrum of the probe for extracellular vesicles as described in Formula I.

[0036] The beneficial effects of the present invention are as follows: the present invention provides a probe for extracellular vesicles, a preparation method and an application thereof. The probe for extracellular vesicles of the present invention has better fusion performance with small extracellular vesicles, and can be mixed and incubated with small extracellular vesicles (Small Extracellular Vesicles, sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes to efficiently achieve the labeling of small extracellular vesicles, and has a specific fluorescent group, and the emission wavelength is about 504nm under an excitation wavelength of about 486nm. After labeling, fluorescence detection tracking can be achieved, which is of great significance for in vitro labeling or in vivo tracing of isolated sEVs. Since the labeling of small extracellular vesicles can be efficiently achieved by mixing and incubating with small extracellular vesicles (Small Extracellular Vesicles, sEV) in a culture medium at room temperature (20-30°C) for 15 to 30 minutes, efficient and faster detection can be achieved, saving detection time. The detection method for extracellular vesicles of the present invention has a short detection time for labeling, which improves detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 FIG. 1 is a chemical structure diagram of a probe for extracellular vesicles according to an embodiment of the present invention.

[0038] Figure 2This is an absorption spectrum of a probe for extracellular vesicles according to an embodiment of the present invention.

[0039] Figure 3 This is an emission spectrum of a probe for extracellular vesicles according to an embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] Example 1

[0042] As a probe for extracellular vesicles in an embodiment of the present invention, the chemical structure is shown in Formula I:

[0043]

[0044] The present embodiment provides a method for preparing a probe for extracellular vesicles, and the method comprises the following steps:

[0045] (1) Mixing the compound represented by Formula II (0.05 mol) and N-hydroxysuccinimide (0.11 mol) in a molar ratio of 1:(2-3) in 100 mL of dichloromethane;

[0046]

[0047] (2) Add 7.5 g of dimethylaminopyridine (DMAP) and an equal molar amount of N, N′-dicyclohexylcarbodiimide (DCC) and stir at room temperature for 2 hours. During the reaction, the room temperature was monitored to be 22.3-23.1°C.

[0048] (3) applying the mixed solution after the reaction in step (2) to a 50 mL polyamide column and then washing it with 1 column volume of dichloromethane;

[0049] (4) eluting with 2 column volumes of ethanol and a water-ethanol mixed solvent of 40% to 60% to obtain fraction A; removing the solvent from fraction A by vacuum evaporation at 28° C., and recrystallizing from deionized water to obtain 8.52 g of a product, which is recorded as product A;

[0050] (5) eluting with 2 column volumes of a chloroform-methanol mixed solvent to obtain fraction B; the volume fraction of chloroform is 40% to 60%;

[0051] (6) Fraction B was removed by vacuum evaporation at 28°C to remove the solvent, and then recrystallized from deionized water to obtain 8.41 g of the product.

[0052] Example 2

[0053] As an embodiment of the present invention, a method for detecting extracellular vesicles comprises the following steps:

[0054] (a) 100-200 μL of the solution of the probe for extracellular vesicles as described in Example 1 is mixed with 100-200 μL of the extracellular vesicle solution, the amount of the probe for extracellular vesicles is 2.5 μmol-5 μmol, the amount of the extracellular vesicles is 80-120 μg, and the volume is adjusted to 0.8-1.2 mL with a phosphate buffer having a pH of 7.0-7.4, and the mixture is fully mixed and incubated at 25-30° C. in the dark for 15 to 30 minutes;

[0055] (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 to 130,000 times gravity for 30 to 50 minutes to remove the supernatant;

[0056] (c) resuspending the precipitate obtained in step (b) in 0.8-1.2 mL of PBS solution, and measuring the fluorescence intensity at a wavelength of 300 nm to 700 nm.

[0057] Example 3

[0058] As a detection kit for extracellular vesicles of the present invention, the detection kit comprises 0.8-2 mmol of the probe for extracellular vesicles described in Example 1, 100-500 mmol of a phosphate buffer salt solid preparation or a buffer solution at pH 7.0-7.4, wherein the phosphate solid preparation is configured from monohydrogen phosphate and dihydrogen phosphate;

[0059] The detection kit includes 100-500 mmol of a phosphate buffer solution with a pH of 7.0-7.4 and does not include a phosphate buffer salt solid preparation, and the concentration of the phosphate buffer solution is 15-30 mmol / L; the probe for extracellular vesicles described in Example 1 is prepared into a mother solution of 100-500 mmol / mL using the phosphate buffer solution as a solvent.

[0060] The kit also includes an instruction manual, which records the chemical structure of the probe for extracellular vesicles as described in Formula I, Example 2 of the instruction manual is used for the detection method of extracellular vesicles, and the instruction manual also records the excitation spectrum and emission spectrum of the probe for extracellular vesicles as described in Formula I.

[0061] Experimental methods

[0062] 1. Characterization of Product A and Product B

[0063] 1. Spectral characterization of products and by-products.

[0064] UV spectrum characterization, product A was fully scanned at an excitation wavelength of 380nm to 600nm, and the absorption wavelength was detected to have a peak at 495nm. The peak emission wavelength was 519nm, which is consistent with SE6-carboxyfluorescein succinimidyl ester.

[0065] The product B, i.e., the probe for extracellular vesicles of the present invention, was characterized by UV spectroscopy in the same manner as above. Figure 2 , Figure 3 As shown, Figure 2 is the absorption spectrum, Figure 3 For the emission spectrum, it was detected that the absorption wavelength had a peak at 487nm and the emission wavelength had a peak at 504nm.

[0066] 2. Mass spectrometry characterization

[0067] Using a triple quadrupole liquid-mass spectrometry system without connecting the chromatographic system to a chromatographic column, product A and product B were respectively prepared into 0.1 ppm solutions using a pH 7.4 phosphate buffer solution, and full ion scanning was performed in the positive ion mode.

[0068] m / z(M+H(Product A))=474.13

[0069] m / z(M+H(Product B))=573.10

[0070] Combined with UV spectroscopy characterization, product A was SE6-carboxyfluorescein succinimidyl ester.

[0071] The chemical structure of product B is shown in Formula I

[0072]

[0073] 2. Small extracellular vesicle detection experiment

[0074] 1. Preparation of Extracellular Vesicles

[0075] Human invasive bladder cancer YTS-1 cells were cultured normally and subcultured to 15 cm culture dishes. After growing to about 80% confluence, the old culture medium was removed with a pipette, and then 2 mL of sterile saline was added to wash the remaining culture medium. After repeated washing three times, the remaining culture medium was removed and 20 mL of RMPI 1640 basal medium was slowly added along the side wall of the culture dish with a pipette, and culture was continued for 48 hours. Then the cell culture medium was carefully pipetted into a 50 mL centrifuge tube, and the extracellular vesicles were enriched by differential centrifugation.

[0076] 2. Reagents

[0077] Product A and product B were respectively prepared into 0.03 mol / L solutions using 20 mM pH 7.4 phosphate buffer.

[0078] Extracellular vesicles were prepared with DMEM / F12 complete medium at a concentration of 1 g / L.

[0079] 3. Fluorescence labeling test

[0080] (1) A probe for extracellular vesicles as described in Formula I

[0081] A method for detecting extracellular vesicles, the method comprising the following steps:

[0082] (a) 100 μL of a 0.03 mol / L solution of the probe for extracellular vesicles as described in Formula I was mixed with 100 μL of a 1 g / L extracellular vesicle solution, the amount of extracellular vesicles was 100 μg, and the volume was fixed to 1 mL with a phosphate buffer having a pH of 7.4, and the mixture was thoroughly mixed and incubated at 25° C. for 20 minutes in the dark;

[0083] (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 times gravity for 40 minutes, collecting the supernatant and making the volume to 1 mL, and measuring the fluorescence intensity c0 of the supernatant at an emission wavelength of 504 nm;

[0084] (c) Resuspend the precipitate obtained in step (b) in 1.0 mL of PBS solution and measure the fluorescence intensity c1 at an emission wavelength of 504 nm.

[0085] (2) Product A

[0086] Referring to the above method, the solution in which the probe for extracellular vesicles as described in Formula I is replaced with Product A is incubated for 20 minutes, and the fluorescence intensity of the supernatant and the fluorescence intensity of the resuspended precipitate are tested at 519 nm.

[0087] Referring to the above method, the solution in which the probe for extracellular vesicles as described in Formula I was replaced with Product A was incubated for 60 minutes, and the fluorescence intensity of the supernatant and the fluorescence intensity of the resuspended precipitate were tested at 519 nm.

[0088] 4. Fluorescence labeling results

[0089] The fluorescence intensity c0 of the supernatant represents the fluorescence intensity of the unlabeled sample, and the fluorescence intensity c1 of the precipitate resuspension represents the fluorescence intensity of the extracellular vesicle marker.

[0090] Extracellular vesicle labeling coefficient = c1 / (c0+c1)

[0091] The extracellular vesicle labeling coefficients are shown in Table 1.

[0092]

[0093]

[0094] As can be seen from Table 1, the probe for extracellular vesicles of the present invention (Formula I) has better fusion performance with small extracellular vesicles, and can be mixed and incubated with small extracellular vesicles (sEV) in a culture medium at room temperature (20-30° C.) for 15 to 30 minutes to efficiently achieve labeling of small extracellular vesicles, can achieve fluorescence detection tracking, can efficiently achieve labeling of small extracellular vesicles, can achieve efficient and faster detection, and save detection time.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A probe for extracellular vesicles, characterized in that The chemical structure of the probe for extracellular vesicles is shown in Formula I: Formula Ⅰ.

2. The method for preparing a probe for extracellular vesicles according to claim 1, characterized in that: The preparation method comprises the following steps: (1) mixing the compound represented by formula II and N-hydroxysuccinimide in a molar ratio of 1:(2-3) in dichloromethane; Formula II (2) Add dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) and stir at 20-30°C for 1.5-4 hours; (3) placing the mixed solution after the reaction in step (2) on a polyamide column and then washing it with dichloromethane; (4) eluting with a water-ethanol mixed solvent having a volume of 40% to 60% ethanol to obtain fraction A; (5) Eluting with a chloroform-methanol mixed solvent to obtain fraction B; the volume fraction of chloroform is 40% to 60%; (6) Remove the solvent from fraction B and crystallize it.

3. The method for preparing a probe for extracellular vesicles according to claim 2, characterized in that: The methods for removing the solvent include vacuum evaporation and negative pressure evaporation, and the temperature does not exceed 30°C.

4. The method for preparing a probe for extracellular vesicles according to claim 2, characterized in that: In step (1), the compound represented by formula II and N-hydroxysuccinimide are mixed in a molar ratio of 1:(2.2-2.5).

5. The method for preparing a probe for extracellular vesicles according to claim 2, characterized in that: The amount of dimethylaminopyridine used accounts for 4% to 7% of the mass of the mixed solution in step (2), and the molar ratio of N, N'-dicyclohexylcarbodiimide to dimethylaminopyridine is 1: (0.95 to 1.05).

6. A method for detecting extracellular vesicles for non-disease diagnosis and treatment purposes, characterized in that: The method comprises the following steps: (a) mixing the solution of the probe for extracellular vesicles as claimed in claim 1 with the extracellular vesicle solution, making up the volume with a phosphate buffer having a pH of 7.0 to 7.4, mixing thoroughly and incubating at 25 to 30° C. in the dark for 15 to 30 minutes; (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 to 130,000 times gravity for 30 to 50 minutes to remove the supernatant; (c) resuspending the precipitate obtained in step (b) in a PBS solution, and measuring the fluorescence intensity at a wavelength of 300 nm to 700 nm.

7. The detection method according to claim 6, characterized in that: The solvent of the extracellular vesicle solution is DMEM / F12 complete culture medium, and the solvent of the extracellular vesicle probe solution is a phosphate buffer solution with a pH of 7.0-7.4; the usage ratio of the probe for extracellular vesicles to the extracellular vesicles is 2.5 mol-5 mol of the probe for extracellular vesicles to 100 g of extracellular vesicles.

8. The detection method according to claim 6, characterized in that: The method comprises the following steps: (a) Mixing 100-200 μL of the solution of the probe for extracellular vesicles as claimed in claim 1 with 100-200 μL of the extracellular vesicle solution, wherein the amount of the probe for extracellular vesicles is 2.5 μmol-5 μmol, the amount of the extracellular vesicles is 80-120 μg, and the volume is adjusted to 0.8-1.2 mL with a phosphate buffer having a pH of 7.0-7.4, and the mixture is thoroughly mixed and incubated at 25-30° C. in the dark for 15 to 30 minutes; (b) transferring the liquid in step (a) into a sterile centrifuge tube, centrifuging at 100,000 to 130,000 times gravity for 30 to 50 minutes to remove the supernatant; (c) Resuspend the precipitate obtained in step (b) in 0.8-1.2 mL of PBS solution, and measure the fluorescence intensity at a wavelength of 300 nm-700 nm.

9. A detection kit for extracellular vesicles, characterized in that: The detection kit comprises 0.8-2 mmol of the probe for extracellular vesicles as claimed in claim 1, 100-500 mmol of a phosphate buffer solid preparation or a buffer solution at pH 7.0-7.4, wherein the phosphate buffer solid preparation or the buffer solution is prepared from monohydrogen phosphate and dihydrogen phosphate.

10. The detection kit for extracellular vesicles according to claim 9, characterized in that: The detection kit includes 100-500 mmol of a phosphate buffer solution with a pH of 7.0-7.4 and does not include a phosphate buffer salt solid preparation, and the concentration of the phosphate buffer solution is 15-30 mmol / L; the probe for extracellular vesicles as claimed in claim 1 is configured as a mother solution of 100-500 mmol / mL using the phosphate buffer solution as a solvent; the kit also includes an instruction manual, which records the chemical structure of the probe for extracellular vesicles as claimed in claim 1, and the instruction manual also records any one of claims 6-8 for detecting extracellular vesicles.

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