Near-infrared fluorescent carbon dots capable of efficiently marking sEVs and application of near-infrared fluorescent carbon dots

Abstract

The invention discloses a near-infrared fluorescent carbon dot capable of efficiently marking sEVs and application of the near-infrared fluorescent carbon dot. Urea, citric acid and Nile blue are used as precursors, and the carbon dot is prepared through a one-step solvothermal method; then activating carboxyl on the surface of the carbon dot under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to be combined with N-hydroxysuccinimide, so as to prepare the near-infrared fluorescent carbon dot, namely CDs-NHS. The method comprises the following steps: mixing CDs-NHS with sEVs or cells containing the sEVs, and marking the sEVs; the near-infrared fluorescent carbon dots are used for marking, marking conditions are mild, operation is easy, and the near-infrared fluorescent carbon dots have good specificity, pH tolerance and low cytotoxicity and have the advantages of being excellent in light stability and the like compared with small-molecule dye.

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 marking sEVs and applications thereof. Background Art

[0002] Research in the past decade has found that almost all types of cells can secrete nano-micron-sized vesicles with a phospholipid bilayer structure, which are called extracellular vesicles (EVs), regardless of physiological or pathological conditions. EVs are widely distributed in semen, breast milk, amniotic fluid, ascites, cerebrospinal fluid, blood, saliva, urine, bile, and the extracellular environment. They are all spherical in shape, but they have different morphological sizes and sources. EVs can be divided into three subtypes: exosomes, microvesicles (microvesicles), and apoptotic bodies according to their size, origin, and membrane formation. Exosomes are also called small extracellular vesicles (sEVs), which range in size from 30 to 150 nm and can carry a variety of bioactive substances such as proteins, nucleic acids, lipids, and small molecule metabolites. sEVs participate in the regulation of many important physiological and pathological processes through various means, such as endocytosis, macropinocytosis, receptor recognition, membrane fusion, etc., such as maintaining cell homeostasis; antigen presentation in immunity; occurrence, progression and metastasis of tumor cells; repair of damaged tissues, etc. Since the contents of sEVs come from their parent cells, sEVs secreted by different cells have different components and functions, and can also be used as disease diagnostic markers. At the same time, sEVs can encapsulate drugs in them and target specific cells or tissues, making them a good targeted drug delivery system.

[0003] When studying the function of sEVs, it is crucial to label them in vitro and track them in vivo. At present, 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 are easy to 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, and their application range is limited; finally, some dyes will bring structural modifications to sEVs after labeling, change their physical properties, and may also affect their biological functions. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a near-infrared fluorescent carbon dot for efficiently labeling sEVs and its application. The near-infrared fluorescent carbon dot of the present invention is used for labeling, and the labeling conditions are mild, the operation is simple, and it has good specificity, pH tolerance, low cytotoxicity, and has excellent photostability compared with small molecule dyes.

[0005] The technical solution of the present invention is 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 obtain 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% urea, 30-36 wt% citric acid, and 2.0-4.0 wt% 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, and then reacted at 175-185°C for 9-11 hours. After the reaction is completed, the reaction solution is dialyzed, concentrated, and passed through a column to obtain the carbon dots. The carbon dots are dissolved in dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added, and stirred at room temperature for 8-12 hours. After the reaction is completed, the near-infrared fluorescent carbon dots are obtained by post-treatment.

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

[0015] In one embodiment of the present invention, the dialysis is performed using a dialysis bag with a molecular weight cutoff 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 light-proof 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 the properties of near-infrared fluorescence emission, 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) with amino groups to excite fluorescent groups. The near-infrared fluorescent carbon dots not only have strong fluorescence under acidic conditions, which makes them have 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 carboxyl groups on the surface of the carbon dots of the present invention are activated by EDC / NHS, and then covalently bonded with the amino groups on the extracellular vesicle proteins to excite the fluorescent carbon dots and thus label the exosomes. The carbon dots exhibit excellent resistance to photobleaching, water solubility, and a long blood circulation time and are not affected by the pH in the biological microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a flow chart for the synthesis 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 under different pH conditions obtained in Example 1;

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

[0036] Figure 7 This is a 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 obtained in Example 1;

[0038] Fig. 9 The cytotoxicity experiment of CDs-NHS prepared in Example 1;

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

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

[0041] Fig.12Laser scanning confocal imaging of living cell endocytosis of carbon dot-labeled sEVs. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with 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 and stir at 65°C for 30 min, then add NaOH (0.5 M, 10 mL) and continue to reflux and stir for 6 h. After the reaction is completed, extract with CH2Cl2 (200 mL), wash with water (30 mL × 3), wash the organic phase with saturated brine, and finally dry with anhydrous sodium sulfate and concentrate to obtain 85.0 mg of dark purple powder with a yield of 95%. This is the product of 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. The reaction solution was then transferred to a dialysis bag (MW: 1000D) and placed in a beaker filled with 1000 mL of deionized water and dialyzed for 2 days. After the dialysis was completed, 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) Take 10 mg of the above-obtained carbon dots CDs and dissolve them in 5 mL of anhydrous CH2Cl2. Then, add 60.7 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 30.0 mg of N-hydroxysuccinimide (NHS) to the above system, respectively. Stir at room temperature for 10 h. After the reaction is completed, extract with 50 mL of CH2Cl2, wash with water (20 mL×5), wash the organic phase with saturated brine, and finally dry with anhydrous sodium sulfate and concentrate 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, 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, from which 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 make the volume 5 mL. The excitation wavelengths set during the test were 530, 540, 550, 560, 570, 580, 590, and 600 nm, respectively, and the slit widths were all 5 nm. Figure 4 As shown, the carbon dot CDs-NHS showed 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 through the standard buffer solution (Na2HPO4-NaH2PO4-NaCl) system. Pipette 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) into a colorimetric tube and distilled water was added to make up to 5mL. 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 the figure. 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 structures, thus giving it good biological imaging capabilities.

[0060] 4. Determination of photostability of near-infrared fluorescent carbon dots

[0061] We further tested the long-term photostability of CDs-NHS prepared in Example 1. 25 μL of 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 make the volume 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, it was found that the fluorescence intensity of CDs-NHS at 660nm had basically no obvious decay within 1500s, which shows that CDs-NHS has good 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 sEVs labeled with near-infrared fluorescent carbon dots

[0063] The feasibility of preparing CDs-NHS labeled sEVs 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 objects. 5 Three cells / well were inoculated in 6-well plates and cultured for 24 hours to adhere to the wall. 50 μg 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 (sEVs 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 As shown. Compared with the group with only sEVs, the fluorescence signal was significantly enhanced after the cells were treated with CDs-NHS-labeled sEVs, while the addition of the sEV uptake inhibitor Genistein significantly weakened the trend of fluorescence signal enhancement. Therefore, CDs-NHS can well detect the cellular uptake, internalization and absorption of sEVs.

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

[0065] The effect of different concentrations of CDs-NHS prepared in Example 1 on cell viability was detected using the "CCK8 Cell Viability Detection Kit". The specific experimental steps refer to the instructions. HEK293T and HCV29 cells were selected, and the cells were inoculated into 96-well plates at 2000 per well after counting. The empty wells were supplemented with 200 μL PBS and cultured in a 37°C constant temperature incubator for 24 hours, and then replaced with complete culture medium containing different concentrations of CDs-NHS (0, 50, 100, 250, 500, 750, 1000, 5000 μg / mL) to continue culturing cells, with 3 replicates per group. After 48 hours, the culture solution 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 avoids the generation of bubbles, and continues to act in a 37°C incubator for 1 hour. After 1 hour, the absorbance at 450 nm was detected by 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. Fig. 9 As shown, CDs-NHS at a concentration of less than or equal to 100 μg / mL had almost no effect on cell activity.

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

[0067] In order to use CDs-NHS reasonably 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 objects. 5Cells / well were inoculated in a 12-well plate. When the cells grew to 70%, the medium was changed for sEVs uptake. The same mass of sEVs (50 μg) was labeled with a CDs-NHS concentration gradient of 5, 10, 20, 50, and 100 μg / ml. The CDs-NHS-labeled sEVs were added to the cells respectively and incubated in a 37°C incubator for 2 hours. After the incubation, the supernatant was discarded, and the digested and collected cells were centrifuged in a centrifuge tube at 1000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 2 mL of PBS and centrifuged again. 500 μL of PBS was used to resuspend the cells by pipetting, and the cells were transferred to a flow tube. Flow cytometry was performed in the APC channel to screen the optimal concentration of CDs-NHS. The results are shown in Fig.10 As shown, the strongest fluorescence intensity can be achieved according to the CDs-NHS:sEVs volume ratio of 50μg-100μg:1mL.

[0068] 8. Best 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 inoculated in a 12-well plate, and the medium was changed when the cells grew to 70%, ready for sEVs uptake. The same mass of sEVs (50 μg) was labeled with a gradient of CDs-NHS probe at a concentration of 50 μg / mL. The CDs-NHS-labeled sEVs were added to the cells respectively and incubated in a 37°C incubator 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 shown in the figure. Fig.11 As shown, the fluorescence intensity reaches a good fluorescence intensity within 1-2 hours.

[0070] 9. Cells take up 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 cell counting, 1×10 4 Add cells to the confocal culture dish and culture for 24 hours to adhere to the wall. After discarding the old culture medium and washing, add 1 mL of complete culture medium containing sEVs co-labeled with CDs-NHS and CFDA-SE (membrane permeable fluorescent dye) prepared in Example 1 and incubate for 1 hour. After the incubation is completed, discard the old culture medium and wash 3 times, and fix with 300 μL of pre-cooled 4% fresh paraformaldehyde at room temperature in the dark for 15 minutes. Discard the paraformaldehyde and wash three times, add PBS containing 4',6-diamidino-2-phenylindole (DAPI) (1:1000), and incubate at room temperature for 10 minutes to stain the nucleus. Wash three times with PBS and observe using a laser confocal microscope. The results are as follows Fig.12 As shown in the figure, the red CDs-NHS signal and the green CFDA-SE signal co-localize, indicating that CDs-NHS can also clearly show the absorption and internalization of sEVs by cells. And compared with the CFDA-SE signal, the red fluorescence intensity of CDs-NHS is more obvious, with higher labeling intensity and efficiency.

[0072] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A near-infrared fluorescent carbon dot, It is characterized in that The preparation method of the near-infrared fluorescent carbon dots is as follows: 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 under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to combine with N-hydroxysuccinimide to obtain near-infrared fluorescent carbon dots, namely CDs-NHS.

2. The near-infrared fluorescent carbon dots according to claim 1, It is characterized in that The dosage of each component in the precursor is: 60-67wt% of urea, 30-36wt% of citric acid, and 2.0-4.0wt% of Nile blue.

3. The near-infrared fluorescent carbon dots according to claim 1, It is characterized in that The specific preparation method is: Urea, citric acid and Nile blue are dissolved in a solvent, and then reacted at 175-185°C for 9-11 hours. After the reaction, the reaction solution is dialyzed, concentrated and column-filtered to obtain the carbon dots. The carbon dots are dissolved in dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, stirred at room temperature for 8-12 hours. After the reaction, the near-infrared fluorescent carbon dots are obtained by post-treatment.

4. The near-infrared fluorescent carbon dots according to claim 3, It is characterized in that The solvent is dimethylformamide.

5. The near-infrared fluorescent carbon dots according to claim 3, It is characterized in that The dialysis was performed in a dialysis bag with a molecular weight cut-off of 1000D in deionized water for 2-3 days.

6. The near-infrared fluorescent carbon dots according to claim 3, It is characterized in that Concentrate by rotary evaporation at 30-50°C or freeze drying to obtain a brown-black powder; The brown-black powder was dissolved in dichloromethane and further separated by a silica gel chromatography column. The separated liquid was concentrated by a rotary evaporator at 30-50°C or freeze-dried to obtain dark red powder carbon dots. The eluent of the silica gel chromatography column was CH 2 Cl 2 Mixed with MeOH in a volume ratio of 30-50:

1.

7. An application of the near-infrared fluorescent carbon dots according to claim 1, It is characterized in that Used to label sEVs.

8. The use according to claim 7, It is 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 light-proof environment.

9. An application of the near-infrared fluorescent carbon dots according to claim 1, It is characterized in that Used to label microorganisms or target proteins.

10. A use of the near-infrared fluorescent carbon dots according to claim 1, It is characterized in that Used to prepare tracer drugs.

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