A near-infrared fluorescent carbon dot for efficient labeling of sEVs and its application

Abstract

The present invention discloses near-infrared fluorescent carbon dots (Cds) for efficiently labeling sEVs and their applications. The Cds are prepared using urea, citric acid, and Nile blue as precursors via a one-step solvothermal method. The Cds are then activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to bind the carboxyl groups on the Cds to N-hydroxysuccinimide, producing near-infrared fluorescent Cds (CDs-NHS). CDs-NHS is mixed with sEVs or cells containing sEVs to label the sEVs. The Cds are used for labeling under mild conditions, are simple to operate, exhibit good specificity, pH tolerance, low cytotoxicity, and possess superior photostability compared to small-molecule dyes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to near-infrared fluorescent carbon dots for efficiently labeling sEVs and applications thereof. Background Art

[0002] Research over the past decade has revealed that virtually all cell types, under both physiological and pathological conditions, can secrete small, nanometer-sized vesicles with a phospholipid bilayer structure, called extracellular vesicles (EVs). EVs are widely distributed, found in semen, breast milk, amniotic fluid, ascites, cerebrospinal fluid, blood, saliva, urine, bile, and the extracellular environment. They are all spherical in shape, but vary in size and origin. Based on their size, origin, and membrane formation, EVs can be divided into three subtypes: exosomes, microvesicles (microvesicles), and apoptotic bodies. Exosomes, also known as small extracellular vesicles (sEVs), range in size from 30 to 150 nm and can carry a variety of bioactive substances, including proteins, nucleic acids, lipids, and small molecule metabolites. sEVs participate in the regulation of numerous important physiological and pathological processes through diverse pathways, including endocytosis, macropinocytosis, receptor recognition, and membrane fusion. These include maintaining cellular homeostasis; antigen presentation in immunity; the development, progression, and metastasis of tumor cells; and the repair of damaged tissues. Because their contents originate from their parent cells, sEVs secreted by different cells have distinct compositions and functions, and can also serve as diagnostic markers for disease. Furthermore, sEVs can encapsulate drugs and target specific cells or tissues, making them an excellent targeted drug delivery system.

[0003] When studying the function of sEVs, it is crucial to label them in vitro and track them in vivo. Currently, there are many methods for labeling sEVs, including the use of lipophilic dyes (such as R18, DiR, DiO, DiI, DiD, PKH26, PKH67), membrane-permeable compounds (such as CFDA-SE, Cy3 and Calcein-AM), and fluorescent labeling methods based on sulfhydryl groups on the surface of sEVs. However, these labeling methods all have shortcomings: first, organic dyes easily self-assemble to form nanoparticles similar to sEVs, and some dyes have a long half-life that can bring misleading information; second, most dyes have weak staining effects under acidic conditions, which limits their scope of application; finally, some dyes will cause structural modifications to sEVs after labeling, changing their physical properties and possibly affecting their biological functions. Summary of the Invention

[0004] To address the aforementioned issues with the existing technology, the present invention provides near-infrared fluorescent carbon dots for efficient labeling of sEVs and their applications. The near-infrared fluorescent carbon dots of the present invention offer mild labeling conditions, simple operation, good specificity, pH tolerance, low cytotoxicity, and superior photostability compared to small-molecule dyes.

[0005] The technical solutions of the present invention are as follows:

[0006] The first object of the present invention is to provide a near-infrared fluorescent carbon dot, wherein the preparation method of the near-infrared fluorescent carbon dot is:

[0007] Carbon dots were prepared by a one-step solvothermal method using urea, citric acid, and Nile blue as precursors. The carboxyl groups on the surface of the carbon dots were then activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to combine with N-hydroxysuccinimide (NHS) to produce near-infrared fluorescent carbon dots, namely CDs-NHS.

[0008] In one embodiment of the present invention, the amounts of the components in the precursor are: 60-67 wt% of urea, 30-36 wt% of citric acid, and 2.0-4.0 wt% of Nile blue.

[0009] In one embodiment of the present invention, the mass ratio of carbon dots CDs to EDC is 1:5-7; the mass ratio of carbon dots CDs to NHS is 1:2-4.

[0010] In one embodiment of the present invention, Nile blue is prepared by alkalizing its salt.

[0011] In one embodiment of the present invention, Nile blue is prepared by alkalizing Nile blue hydrochloride or Nile blue sulfate.

[0012] In one embodiment of the present invention, the preparation method of near-infrared fluorescent carbon dots is:

[0013] Urea, citric acid, and Nile blue are dissolved in a solvent, followed by reaction at 175-185°C for 9-11 hours. After the reaction, the reaction solution is dialyzed, concentrated, and filtered to obtain the carbon dots. The carbon dots are dissolved in dichloromethane, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stirred at room temperature for 8-12 hours. After the reaction, the mixture is post-treated to obtain the near-infrared fluorescent carbon dots.

[0014] In one embodiment of the present invention, the solvent is dimethylformamide (DMF).

[0015] In one embodiment of the present invention, dialysis is performed using a dialysis bag with a molecular weight cut-off of 1000D in deionized water for 2-3 days.

[0016] In one embodiment of the present invention, the concentration is carried out by rotary evaporation at 30-50° C. or freeze drying to obtain a brown-black powder;

[0017] The brown-black powder is dissolved in dichloromethane and further separated by a silica gel chromatography column. The separated liquid is concentrated by a rotary evaporator at 30-50°C or freeze-dried and concentrated to obtain carbon dots; the eluent of the silica gel chromatography column is a mixture of CH2Cl2 and MeOH in a volume ratio of 30-50:1.

[0018] In one embodiment of the present invention, the post-treatment method is: extraction with CH2Cl2, washing with deionized water, washing the organic phase with saturated brine, and finally drying with anhydrous sodium sulfate and concentrating to obtain dark red powder carbon dots CDs-NHS.

[0019] The second object of the present invention is to provide an application of the above-mentioned near-infrared fluorescent carbon dots for labeling sEVs.

[0020] In one embodiment of the present invention, the labeling method is:

[0021] CDs-NHS was mixed with sEVs or cells containing sEVs to label sEVs; labeling was performed at 37°C in a dark environment.

[0022] In one embodiment of the present invention, the mass volume ratio of CDs-NHS to sEVs is 50-100:1 μg / mL.

[0023] In one embodiment of the present invention, the source of sEVs includes but is not limited to umbilical cord mesenchymal stem cells.

[0024] The third object of the present invention is to provide an application of the above-mentioned near-infrared fluorescent carbon dots for labeling microorganisms or target proteins.

[0025] The fourth object of the present invention is to provide an application of the above-mentioned near-infrared fluorescent carbon dots for preparing tracer drugs.

[0026] The beneficial technical effects of the present invention are:

[0027] The carbon dots prepared by the present invention not only retain their near-infrared fluorescence emission properties, but also have excellent water solubility and biocompatibility due to the introduction of a large number of hydrophilic groups, and greatly improve the photostability of the dye.

[0028] The present invention provides a method for labeling sEVs based on the covalent binding of N-hydroxysuccinimide (NHS) to amino groups to excite fluorescent groups. The near-infrared fluorescent carbon dots not only have strong fluorescence under acidic conditions, giving them a wider range of applications; they also have good resistance to photobleaching and are not easily quenched during labeling and use, which has outstanding advantages for in vitro labeling and in vivo tracing of sEVs.

[0029] The carbon dots are activated with EDC / NHS, then covalently bind to amino groups on extracellular vesicle proteins, stimulating the fluorescent carbon dots and labeling exosomes. The carbon dots exhibit excellent photobleaching resistance, water solubility, and a long blood circulation time, and are unaffected by the pH of the biological microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the synthesis flow chart of near-infrared fluorescent carbon dots;

[0031] Figure 2 DLS particle size analysis of CDs-NHS prepared in Example 1;

[0032] Figure 3 TEM image of CDs-NHS prepared in Example 1;

[0033] Figure 4 The excitation and emission wavelength spectra of CDs-NHS obtained in Example 1;

[0034] Figure 5 The fluorescence spectra of CDs-NHS prepared in Example 1 under different pH conditions;

[0035] Figure 6 The fluorescence spectra of CDs-NHS prepared in Example 1 under different illumination times;

[0036] Figure 7 Schematic diagram of preparing CDs-NHS-labeled sEVs in Example 1;

[0037] Figure 8 This is a diagram of the cellular uptake of CDs-NHS-labeled sEVs prepared in Example 1;

[0038] Figure 9 Cytotoxicity experiment of CDs-NHS prepared in Example 1;

[0039] Figure 10 The optimal concentration of CDs-NHS was obtained in Example 1;

[0040] Figure 11 The optimal use time of CDs-NHS prepared in Example 1;

[0041] Figure 12Laser scanning confocal microscopy (CSF) images of carbon dot-labeled sEVs detected during endocytosis in living cells. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0043] Unless otherwise specified, the raw materials used in the present invention are all commercially available products.

[0044] The preparation method of Nile Blue is:

[0045] Dissolve Nile blue hydrochloride (100.0 mg) in 10 mL of water, reflux at 65°C with stirring for 30 minutes, then add NaOH (0.5 M, 10 mL) and continue reflux with stirring for 6 hours. After the reaction, extract with CH2Cl2 (200 mL) and wash with water (30 mL x 3). The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to yield 85.0 mg of a deep purple-red powder with a yield of 95%. This is the product of the alkalization of Nile blue hydrochloride, namely Nile blue.

[0046] Example 1

[0047] A near-infrared fluorescent carbon dot, the preparation method comprises the following steps:

[0048] (1) 30.0 mg of Nile blue, 300.0 mg of anhydrous citric acid, and 600.0 mg of urea were dissolved in a hydrothermal reactor BKHR-50 containing 10 mL of N,N-dimethylformamide (DMF) and placed in a water bath for 10 min. The reactor was then placed in an oven at 180°C and heated for 10 h. After the reaction was completed, the reaction solution was cooled to room temperature and then transferred to a dialysis bag (MW: 1000D) and placed in a 1000 mL beaker filled with deionized water for 2 days. After dialysis, the retained solution was freeze-dried to obtain a brown-black powder. The brown-black powder was dissolved in dichloromethane, separated and concentrated by a silica gel column (CH2Cl2:MeOH=50:1) to obtain 11 mg of dark red powder carbon dots CDs.

[0049] (2) 10 mg of the carbon dots CDs obtained above were dissolved in 5 mL of anhydrous CH2Cl2, and then 60.7 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 30.0 mg of N-hydroxysuccinimide (NHS) were added to the above system respectively. The mixture was stirred at room temperature for 10 h. After the reaction, it was extracted with 50 mL of CH2Cl2, washed with water (20 mL×5), and the organic phase was washed with saturated brine. Finally, it was dried over anhydrous sodium sulfate and concentrated to obtain 11.0 mg of dark red powder carbon dots CDs-NHS. The preparation process is as follows. Figure 1 shown.

[0050] Example 2-3, Comparative Example 1-2

[0051] The same as Example 1, the only difference is that the amount of raw materials used in step (1) in Example 1 is changed, see Table 1 for details.

[0052] Table 1 Comparison of the preparation of near-infrared carbon dots by adding different proportions of Nile blue

[0053]

[0054] Test Case

[0055] 1. The particle size of CDs-NHS was measured by 90Plus sub-laser particle size analyzer, and the morphology and particle size of CDs-NHS were measured by transmission electron microscope (TEM). A small amount of CDs-NHS prepared in Example 1 was dispersed in ethanol, and the measurement results were as follows: Figure 2 、 3 As shown in the figure, it was found that the particle size of CDs-NHS was approximately distributed in the range of 3-10 nm.

[0056] 2. Spectral detection of excitation wavelength and emission wavelength of near-infrared fluorescent carbon dots

[0057] The CDs-NHS prepared in Example 1 was dissolved in DMSO to prepare a 10 mg / mL stock solution. 25 μL of the near-infrared carbon dot CDs-NHS stock solution and 500 μL of PBS (pH = 7.4, 20 mM) were pipetted into a colorimetric tube and the volume was adjusted to 5 mL with distilled water. The excitation wavelengths set during the test were 530, 540, 550, 560, 570, 580, 590, and 600 nm, respectively, and the slit width was 5 nm. Figure 4 As shown, carbon dot CDs-NHS shows strong emission at 660 nm under different excitation wavelengths, indicating that the emission wavelength of CDs-NHS does not change with the change of excitation wavelength, indicating that CDs-NHS has stable optical properties and high spectral stability, and is suitable for biological and chemical analysis applications that require precise and consistent fluorescence signals.

[0058] 3. Fluorescence spectra of near-infrared fluorescent carbon dots under different pH conditions

[0059] In order to explore the fluorescence stability of CDs-NHS under different pH conditions, we selected different pH ranges, including acidic, neutral and alkaline environments. PBS buffer solutions (20mM) of different pH values ​​were prepared using a standard buffer solution (Na2HPO4-NaH2PO4-NaCl) system. 25μL of near-infrared carbon dot CDs-NHS stock solution and 500μL of PBS (pH=4, 5, 6, 7, 7.4, 8, 9, 10, 11, 12) were pipetted into a colorimetric tube and fixed to 5mL with distilled water. The excitation and emission wavelengths set during the test were 600 and 660nm, respectively, and the slit widths were both 5nm. The experimental results are shown in Figure 2. Figure 5 As shown, the fluorescence intensity of CDs-NHS prepared in Example 1 at a wavelength of 660 nm is weakly affected by pH changes, indicating that CDs-NHS has stable chemical properties and structure, thus endowing it with good biological imaging capabilities.

[0060] 4. Photostability determination of near-infrared fluorescent carbon dots

[0061] We further tested the long-term photostability of the CDs-NHS prepared in Example 1. 25 μL of the near-infrared carbon dot CDs-NHS stock solution and 500 μL of PBS (pH = 7.4, 20 mM) were placed in a colorimetric tube and distilled water was added to 5 mL. The solution was transferred to a cuvette and subjected to a photodynamic test using a fluorescence spectrophotometer with an excitation wavelength of 600 nm. Figure 6 As shown in the figure, the fluorescence intensity of CDs-NHS at 660nm showed little attenuation within 1500s, indicating that CDs-NHS has excellent photostability. This excellent photostability gives CDs-NHS a significant advantage in long-term biological imaging and continuous monitoring applications, ensuring its reliability and durability in practical applications.

[0062] 5. Cellular Uptake of Near-Infrared Fluorescent Carbon Dots-Labeled sEVs

[0063] The feasibility of CDs-NHS labeled sEVs prepared in Example 1 was confirmed by analyzing the results of cell uptake, absorption and internalization of sEVs. Adherent cells YTS-1, HCV29 and HEK293T were used as experimental subjects. 5 Three cells / well were seeded into three wells of a 6-well plate and cultured for 24 hours to adhere to the wall. 50 μg of sEVs were added to the cell culture medium and cultured for 2 hours, including untreated sEVs, 50 μg / mL CDs-NHS-labeled sEVs, and 50 μg / mL CDs-NHS-labeled sEVs + genistein (sEV uptake inhibitor). The process of CDs-NHS-labeled sEVs is as follows. Figure 7After 2 hours, the cells were digested with trypsin, resuspended in 500 μL 1×PBS (0.01 M, pH = 7.4), and tested on the instrument. Figure 8 Compared to the sEVs-only group, the fluorescence signal was significantly enhanced after treatment of cells with CDs-NHS-labeled sEVs, while the addition of the sEV uptake inhibitor Genistein significantly attenuated the increased fluorescence signal. Therefore, CDs-NHS is highly effective in monitoring the cellular uptake, internalization, and absorption of sEVs.

[0064] 6. Cytotoxicity experiment of near-infrared fluorescent carbon dots

[0065] The effect of CDs-NHS on cell viability was detected using the "CCK8 Cell Viability Detection Kit" for different concentrations of Example 1. The specific experimental steps are described in the instructions. HEK293T and HCV29 cells were selected, and after counting, 2000 cells were seeded into 96-well plates per well. The empty wells were supplemented with 200 μL PBS and cultured in a 37°C constant temperature incubator for 24 hours. Then, complete culture medium containing different concentrations of CDs-NHS (0, 50, 100, 250, 500, 750, 1000, 5000 μg / mL) was replaced to continue culturing the cells, with 3 replicates per group. After 48 hours, the culture medium containing the fluorescent probe was discarded, and 100 μL of complete culture medium containing 10% CCK-8 solution was added to each well. This process avoided the generation of bubbles and continued to act in a 37°C incubator for 1 hour. After 1 hour, the absorbance at 450 nm was measured using a full-wavelength microplate reader, and the cell viability was calculated based on the absorbance. GraphPad Prism 9 was used to analyze the effects of different concentrations of CDs-NHS on cell viability. Figure 9 As shown in the results, CDs-NHS at a concentration of 100 μg / mL or less had almost no effect on cell activity.

[0066] 7. Optimal concentration of near-infrared fluorescent carbon dots

[0067] In order to use CDs-NHS rationally and improve its efficiency, this experiment screened the optimal concentration of CDs-NHS prepared in Example 1 by flow cytometry. Adherent cells YTS-1, HCV29, and HEK293T were used as experimental subjects. 5Cells / well were seeded in a 12-well plate, and the medium was changed when the cells grew to 70% for sEVs uptake. 5, 10, 20, 50, and 100 μg / ml CDs-NHS concentration gradients were used to label sEVs of the same mass (50 μg). The CDs-NHS-labeled sEVs were added to the cells respectively and incubated in a 37°C incubator for 2 hours. After the incubation was completed, the supernatant was discarded, and the digested and collected cells were placed in a centrifuge tube at 1000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 2 mL of PBS, centrifuged again, and resuspended in 500 μL of PBS. The cells were transferred to a flow cytometry tube and flow cytometry was performed in the APC channel to screen the optimal concentration of CDs-NHS. The results are shown in the figure. Figure 10 As shown, the strongest fluorescence intensity can be achieved according to the CDs-NHS:sEVs volume ratio of 50μg-100μg:1mL.

[0068] 8. Optimal use time of near-infrared fluorescent carbon dots

[0069] As with the optimal concentration screening, adherent cells YTS-1, HCV29, and HEK293T were cultured at 1×10 5 Cells / well were seeded in a 12-well plate, and the medium was changed when the cells grew to 70%, ready for sEVs uptake. 50μg / mL concentration of CDs-NHS probe gradient was used to label sEVs of the same mass (50μg). CDs-NHS labeled sEVs were added to the cells respectively and placed in a 37°C incubator for incubation for 1, 2, 4, 6, and 8 hours respectively. The subsequent processing steps were consistent with the screening of the optimal concentration, and the optimal use time of the fluorescent probe was screened by flow cytometry. The results are as follows Figure 11 As shown, the fluorescence intensity reaches a good level within 1-2 hours.

[0070] 9. Cellular uptake of sEVs labeled with near-infrared fluorescent carbon dots.

[0071] Laser scanning confocal microscopy was used to detect cellular uptake of sEVs. Adherent cells YTS-1, HCV29, and HEK293T were used as experimental subjects. After counting the cells, 1×10 4 Cells were placed in a confocal culture dish and cultured for 24 hours to adhere to the wall. After discarding the old culture medium and washing, 1 mL of complete culture medium containing sEVs co-labeled with CDs-NHS and CFDA-SE (membrane permeable fluorescent dye) prepared in Example 1 was added and incubated for 1 hour. After the incubation was completed, the old culture medium was discarded and washed 3 times, and fixed with 300 μL of pre-cooled 4% fresh paraformaldehyde at room temperature in the dark for 15 minutes. The paraformaldehyde was discarded and washed three times, and PBS containing 4',6-diamidino-2-phenylindole (DAPI) (1:1000) was added and incubated at room temperature for 10 minutes to stain the nucleus. Washed three times with PBS and observed using a laser confocal microscope. The results are shown in the figure. Figure 12 As shown, the red CDs-NHS signal and the green CFDA-SE signal colocalize, indicating that CDs-NHS can also clearly demonstrate the uptake and internalization of sEVs by cells. Furthermore, compared with the CFDA-SE signal, the red fluorescence intensity of CDs-NHS is more pronounced, indicating higher labeling intensity and efficiency.

[0072] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of near-infrared fluorescent carbon dots, characterized in that: Used to label sEVs; The preparation method of the near-infrared fluorescent carbon dots is as follows: Carbon dots (CDs) were prepared by a one-step solvothermal method using urea, citric acid, and Nile blue as precursors. The carboxyl groups on the surface of the carbon dots were activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to bind with N-hydroxysuccinimide to produce near-infrared fluorescent carbon dots (CDs-NHS). The amounts of the components in the precursor are: urea 60-67wt%, citric acid 30-36wt%, Nile blue 2.0-4.0wt%; The specific preparation process is: Urea, citric acid, and Nile blue are dissolved in a solvent, followed by reaction at 175-185°C for 9-11 hours. After the reaction, the reaction solution is dialyzed, concentrated, and filtered to obtain the carbon dots. The carbon dots are dissolved in dichloromethane, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stirred at room temperature for 8-12 hours. After the reaction, the solution is post-treated to obtain the near-infrared fluorescent carbon dots.

2. The use according to claim 1, characterized in that The solvent is dimethylformamide.

3. The use according to claim 1, characterized in that Dialysis was performed using a dialysis bag with a molecular weight cut-off of 1000D in deionized water for 2-3 days.

4. The use according to claim 1, characterized in that The concentration is carried out by rotary evaporation at 30-50°C or freeze drying to obtain a brown-black powder; The brown-black powder is dissolved in dichloromethane and further separated by a silica gel chromatography column. The separated liquid is concentrated by a rotary evaporator at 30-50°C or freeze-dried and concentrated to obtain dark red powder carbon dots; the eluent of the silica gel chromatography column is a mixture of CH2Cl2 and MeOH in a volume ratio of 30-50:

1.

5. The use according to claim 1, characterized in that The marking method is: CDs-NHS was mixed with sEVs or cells containing sEVs to label sEVs; labeling was performed at 37°C in a dark environment.

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