Thioether bond bridged carbon dot-dopamine quinone composite probe and application thereof in glioblastoma cell fluorescence imaging

Abstract

The invention discloses a thioether bond bridged carbon dot-dopamine quinone composite probe and application of the thioether bond bridged carbon dot-dopamine quinone composite probe in glioblastoma cell fluorescence imaging, and belongs to the field of materials. According to the probe, thioether bond bridged carbon dots are firstly prepared, and then a CDs (at) S-DAQ compound is prepared. The preparation method provided by the invention is simple and safe, the raw materials are simple and easy to obtain, complex and expensive instruments and equipment are not needed, and mass production is easy.

Description

Technical Field

[0001] The present invention belongs to the fields of preparation of nanomaterials, fluorescence sensing and bioanalysis, and particularly relates to a preparation method of a thioether bond-bridged carbon dot-dopamine quinone composite probe and its application in fluorescence imaging of glioblastoma cells. Background Art

[0002] Glioblastoma (GBM) is one of the most invasive and lethal malignant tumors of the central nervous system. Due to the lack of effective treatment means, the one-year survival rate after diagnosis is only 25%. The sensitive and specific recognition and imaging of glioblastoma cells play a crucial role in early diagnosis, targeted therapy and metastasis development, and contribute to improving the quality of life and prolonging the survival time of patients. The rapid development of imaging technology has made it possible to monitor various complex biological processes in real time, dynamically, in vivo / in vitro, qualitatively / quantitatively, such as gene expression, material transport, signal transduction and protein interaction, etc. According to different principles, imaging technologies include computed tomography, positron emission tomography, single photon emission computed tomography, ultrasonic imaging, magnetic resonance imaging, Raman imaging, photoacoustic imaging, fluorescence imaging, etc. Among these technologies, fluorescence imaging has received extensive attention in recent years due to its advantages of high sensitivity, high spatial resolution, low cost, real-time data acquisition and no ionizing radiation.

[0003] So far, a variety of fluorescence imaging methods have been developed for imaging of GBM cells. For example, Yang et al. introduced a water-soluble near-infrared (NIR) dye - heptafluorothiophene sulfonyl indocyanine ene IRDye78 functionalized probe (IRDye78-α-LADFO-89Zr) for multimodal imaging of GBM cells. An et al. developed a probe based on a dipolar two-photon excited fluorescent oxazolidinone dye for targeted optical imaging of GBM cells. Liang et al. functionalized CdSeTe / ZnS quantum dots with folic acid to actively target glioma cells and achieve near-infrared fluorescence imaging. Xue et al. used CuInS2 / ZnS quantum dots for fluorescence imaging of glioma cells. Peng et al. constructed a mouse glioma cell line stably expressing yellow fluorescent protein with a large Stokes shift for multicolor immunofluorescence imaging of the glioma microenvironment. Although these studies have shown excellent performance in highly selective recognition and imaging of glioma cells, the inherent limitations of the fluorescent materials used limit their practical applications. For example, traditional organic fluorescent dyes have problems such as poor optical stability, photobleaching and spectral overlap, conventional quantum dots (such as II-VI and III-V semiconductor quantum dots) have a risk of cytotoxicity, and the production process of fluorescent proteins is both time-consuming and expensive. Therefore, it is of great significance to develop novel fluorescent nanomaterials to achieve reliable, accurate and long-term imaging of GBM cells.

[0004] Carbon dots (CDs) are a novel type of zero-dimensional carbon-based nanomaterials with a carbon core structure and a size smaller than 10 nanometers. Carbon dots possess unique properties such as high fluorescence quantum yield, good optical stability, easy surface functionalization, excellent water solubility, low cytotoxicity, good biocompatibility, tunable photoluminescence characteristics, and low cost, which make them promising tools for fluorescence imaging of living cells. However, most reported carbon dots require short-wavelength excitation light (such as ultraviolet light) and emit blue or blue-green fluorescence (λem = 400 - 520 nm). The short-wavelength excitation light may cause photo-damage to biological tissues and has a limited penetration depth. In addition, irradiation with short-wavelength light can trigger strong blue autofluorescence in biological tissues, resulting in significant background interference and hindering the extraction and analysis of the fluorescence signal of carbon dots. These limitations severely restrict their application in biological imaging. Summary of the Invention

[0005] In view of the urgent need to develop sensitive and specific recognition and imaging probes for glioblastoma cells and the inherent limitations of current nanomaterials for fluorescence imaging of glioblastoma cells, such as poor optical stability, severe photobleaching, and easy spectral overlap of traditional organic fluorescent dyes; the risk of cytotoxicity of classical II-VI and III-V semiconductor quantum dots; the time-consuming and expensive production process of fluorescent proteins; and the easy photo-damage to biological tissues and limited penetration depth of short-wavelength carbon dots, etc., the present invention aims to synthesize a long-wavelength fluorescent carbon dot-dopamine quinone (CDs@S-DAQ) composite probe bridged by a thioether bond, which can be used for sensitive detection of GSH and long-term and specific fluorescence imaging of glioblastoma cells.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A preparation method of a thioether bond-bridged carbon dot-dopamine quinone composite probe, the method comprising the following steps:

[0008] (1) Preparation of CDs@S: Under a reaction atmosphere of nitrogen, at a temperature of -5 to 5 °C and with stirring, thiodiacetic anhydride and 4-dimethylaminopyridine are added to the carbon dot dispersion and reacted for 4 to 24 h. After the reaction, the solution is extracted several times with an organic solvent and the aqueous phase is collected. The collected aqueous phase is concentrated at 40 to 60 °C, the concentrated solution is mixed with acetone and placed at -25 to -15 °C for 20 to 40 min, and then centrifuged, washed, and dried in sequence to obtain thioether bond-bridged carbon dots;

[0009] (2) Preparation of CDs@S-DAQ: Dissolve CDs@S, EDC, and NHS in an organic solvent. Stir the mixture at -5 to 5 °C for 20 to 40 min, then add dopamine hydrochloride. After the addition, react under a nitrogen atmosphere at a temperature of 20 °C to 30 °C for 4 to 24 h. After the reaction, perform dialysis. Centrifuge the solution in the dialysis bag and collect the precipitate to obtain the CDs@S-dopamine complex; Disperse the CDs@S-dopamine complex in phosphate buffer and naturally oxidize it at 1 to 5 °C for one week. The product is vacuum dried to obtain the CDs@S-DAQ complex.

[0010] In the above preparation method, in step (1), the carbon dot dispersion is prepared by dispersing carbon dots in an organic solvent and filtering with a microporous membrane of 0.2 to 0.25 μm. The obtained filtrate is the carbon dot dispersion, and the mass ratio of carbon dots to the organic solvent is 1:3 to 6.

[0011] In the above preparation method, in step (1), the mass ratio of thiodiacetic anhydride, 4-dimethylaminopyridine, and carbon dots is 5 to 15:5 to 15:0.5 to 1.5.

[0012] In the above preparation method, in step (2), the mass ratio of CDs@S, EDC, NHS, and dopamine hydrochloride is 3 to 8:3 to 8:3 to 8:40 to 60.

[0013] In the above preparation method, in step (2), the mass ratio of the CDs@S-dopamine complex to the phosphate buffer is 1:2 to 5.

[0014] In the above preparation method, in step (2), the pH of the phosphate buffer is 7 to 8, and the concentration is 10 to 100 mM.

[0015] In the above preparation method, the preparation method of carbon dots in step (1) is to dissolve citric acid and urea in a solvent and heat at 160 °C to 200 °C for 4 to 12 h; then cool the reaction system and add a basic reagent for reaction. After the reaction, centrifuge and dry in sequence. Dissolve the dried powder in an acidic solution and mix well. After mixing, centrifuge, wash, and dry again to obtain CDs.

[0016] In the above preparation method of carbon dots, the mass ratio of citric acid to urea is 1:1 to 5.

[0017] A thioether bond-bridged carbon dot-dopamine quinone composite probe, which is prepared by the above method.

[0018] In the technical solution of the present invention, the application of the thioether bond-bridged carbon dot-dopamine quinone composite probe prepared by the above method in fluorescence imaging of glioblastoma cells.

[0019] Advantages of the present invention:

[0020] Compared with the prior art, the present invention provides a thioether-bridged carbon dot-dopamine quinone composite probe (CDs@S-DAQ) for fluorescence imaging of glioblastoma cells, having the following beneficial effects:

[0021] (1) The preparation method provided by the present invention is simple and safe, the raw materials are simple and easily available, and no complex and expensive instruments and equipment are required, which is easy for mass production;

[0022] (2) CDs@S exhibits long-wavelength emission (λ em = 625 nm), having deeper tissue penetration, lower light-induced biological sample damage and minimal background interference compared with short-wavelength fluorescent materials. Especially in complex biological systems, this makes it very suitable for bioanalysis;

[0023] (3) The CDs@S-DAQ probe has two GSH-induced fluorescence recovery pathways, including the reduction of DAQ to DA and the cleavage of thioether bonds, making the probe show excellent responsiveness to GSH;

[0024] (4) The CDs@S-DAQ probe has excellent stability, good optical properties and low cytotoxicity, and can be used as a powerful tool for long-term and specific imaging of tumor cells;

[0025] (5) The CDs@S-DAQ probe can distinguish two types of cells by fluorescence imaging based on the difference in GSH expression levels between GBM cells and normal tissue cells, providing a promising method for early diagnosis, targeted therapy and metastasis development of tumors. Brief Description of the Drawings

[0026] Figure 1 is a schematic diagram of the preparation of the CDs@S-DAQ composite probe under the system of the present invention and its schematic diagram for fluorescence detection of GSH.

[0027] Figure 2 is a characterization diagram of CDs, CDs@S and CDs@S-DAQ composite probes under the system of the present invention. A and B are the transmission electron microscope image and high-resolution transmission electron microscope image of CDs@S-DAQ respectively, C is the transmission electron microscope particle size distribution histogram of CDs@S-DAQ and Gaussian fitting, D is the Fourier transform infrared spectra of CDs (ⅰ), CDs@S (ⅱ), CDs@S-DA (ⅲ) and CDs@S-DAQ (ⅳ), E-G are the X-ray photoelectron spectra of CDs, CDs@S and CDs@S-DAQ, and H is the fluorescence emission spectra of CDs, CDs@S and CDs@S-DAQ.

[0028] Figure 3This is the performance graph of the CDs@S-DAQ composite probe for the fluorescence detection of GSH under the system of the present invention..

[0029] Figure 4 This is the selectivity of the CDs@S-DAQ composite probe for interfering substances such as metal ions, anions, and small molecule organic compounds under the system of the present invention.

[0030] Figure 5 This is the result of the cytotoxicity experiment of the CDs@S-DAQ probe under the system of the present invention.

[0031] Figure 6 This is the result of the specific fluorescence imaging of glioblastoma cells using CDs@S-DAQ under the system of the present invention. Detailed implementation mode

[0032] The present invention will be further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Embodiment 1

[0034] Preparation and characterization of the CDs@S-DAQ composite probe:

[0035] (1) Preparation of CDs: First, dissolve citric acid (1.0 g) and urea (2.0 g) in N,N-dimethylformamide (10 mL), and then transfer it to a 15 mL Teflon-lined autoclave. The mixture is heated at 180 °C for 8 h. After cooling, it is mixed with NaOH solution (50 mg / mL, 20 mL) and stirred for 10 min. The precipitate is collected by centrifugation at 16000 rpm for 15 min and dried under vacuum. Finally, the powder is dissolved in HCl solution (5%, v / v, 20 mL), stirred for 10 min, and centrifuged again at 16000 rpm for 10 min. The precipitate is washed three times with acetone and deionized water respectively. After further drying under vacuum, CDs are obtained.

[0036] (2) Preparation of CDs@S: First, 10 mg of carbon dots were dispersed in 40 mL of DMF and sonicated for 1 h, then filtered through a 0.22-μm microporous membrane to obtain a carbon dot dispersion. Subsequently, under a nitrogen atmosphere, 100 mg of thiodiacetic anhydride and 100 mg of 4-dimethylaminopyridine were added to the carbon dot dispersion with stirring at 0 °C, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, 40 mL of deionized water was added to terminate the reaction. Then, the solution was extracted three times with 40 mL of dichloromethane, and the aqueous phase was collected. The aqueous phase was concentrated at 50 °C in a rotary evaporator, and then the concentrated solution was mixed with an excess of acetone, stirred for 5 min, and left at -20 °C for 30 min. The precipitate was collected by centrifugation at 10000 rpm for 15 min and washed three times with acetone and deionized water respectively. Finally, it was further dried under vacuum to obtain sulfur ether bond-bridged carbon dots.

[0037] (3) Preparation of CDs@S-DAQ: First, 5 mg of CDs@S, 5 mg of EDC, and 5 mg of NHS were dissolved in 20 mL of N,N-dimethylformamide, and the mixture was stirred at 0 °C for 30 min, then 50 mg of dopamine hydrochloride was added. Under a nitrogen atmosphere, the reaction was carried out at 25 °C for 16 h. The reaction product was then dialyzed against deionized water in a dialysis bag with a molecular weight cut-off of 1000 Da for 36 h to remove excess dopamine, EDC, NHS, and possibly formed polydopamine. After centrifugation of the solution in the dialysis bag at 10000 rpm for 10 min, the precipitate was collected to obtain a CDs@S-dopamine complex. 5 mg of CDs@S-dopamine was redispersed in phosphate buffer (PBS, pH 7.4, 10 mM, 20 mL) and naturally oxidized in a refrigerator at 4 °C for one week. The product was dried under vacuum to obtain a CDs@S-DAQ complex.

[0038] Figure 1 The first half is a schematic diagram of the preparation of the CDs@S-DAQ composite probe in Example 1.

[0039] Figure 2 Characterization of the CDs, CDs@S, and CDs@S-DAQ composite probes prepared in Example 1. Among them, Figures A and B are the transmission electron microscopy (TEM) image and high-resolution transmission electron microscopy (HRTEM) image of CDs@S-DAQ respectively, and Figure C is the histogram of the particle size distribution of CDs@S-DAQ by TEM and Gaussian fitting. Figure D is the Fourier transform infrared spectroscopy (FTIR) spectra of CDs (i), CDs@S (ii), CDs@S-DA (iii), and CDs@S-DAQ (iv). Figures E - G are the X-ray photoelectron spectroscopy (XPS) spectra of CDs, CDs@S, and CDs@S-DAQ respectively. Figure H is the fluorescence emission spectra of CDs, CDs@S, and CDs@S-DAQ. From Figure 2It can be seen that there are a large number of oxygen - and nitrogen - containing groups on the surface of CDs; CDs@S - DAQ has a relatively uniform spherical structure, with good monodispersity and no significant aggregation phenomenon. The particle size shows a normal distribution, and the average diameter is about 3.67 nm. After modifying DAQ on the surface of CDs@S, the fluorescence of CDs@S is significantly quenched.

[0040] Example 2

[0041] The CDs@S - DAQ composite probe is used for the fluorescence detection of GSH:

[0042] Different concentrations of GSH were mixed with the CDs@S - DAQ composite probe (Example 1) (40 μg / mL) dispersed in PBS (50 mM, pH 6.0). Then, the mixture was gently stirred and incubated at 37 °C for 1 h. Subsequently, the fluorescence intensity at 625 nm (λex = 550 nm) at different GSH concentrations was measured using a fluorescence spectrophotometer. A quantitative model was established based on the relationship between the fluorescence intensity and the GSH concentration for GSH concentration prediction.

[0043] Figure 1 The latter part is a schematic diagram of the CDs@S - DAQ composite probe (Example 1) for the fluorescence detection of GSH.

[0044] Figure 3 It is a performance graph of the CDs@S - DAQ composite probe (Example 1) for the fluorescence detection of GSH. From Figure 3 It can be seen that the response concentration range of this probe to GSH is 0.5 - 20 mM, the linear range is 0.05 - 20 μM, the correlation coefficient is 0.9969, and the lowest detection limit is 69 μM.

[0045] Example 3

[0046] The selectivity of the CDs@S - DAQ composite probe for GSH fluorescence detection:

[0047] The CDs@S - DAQ (Example 1) (40 μg / mL) dispersed in PBS (50 mM, pH 6.0) was mixed with GSH (2 mM) or interfering substances (metal ions (Na+, K+, Ca2+, Mg2+ and Fe3+, 500 μM), anions (NO3-, CO32-, SO42-, S2O32- and SCN-, 500 μM), small - molecule organic compounds (glucose, Ala, Asp, Arg, Glu, Gly, Lys, Val, His and Phe, 1 mM) and Cys, 5 mM). Then, the mixture was gently stirred and incubated at 37 °C for 1 h. Subsequently, the fluorescence intensity at 625 nm (λex = 550 nm) at different GSH concentrations was measured using a fluorescence spectrophotometer.

[0048] Figure 4 The selectivity of the CDs@S-DAQ composite probe for interfering substances such as metal ions, anions, and small molecule organic compounds. From Figure 4 It can be seen that except for high-concentration cysteine (5 mM), all interfering substances have little effect on the detection of GSH. It is worth noting that the average concentration of cysteine in tumor cells and normal cells does not exceed 200 μM, indicating that it will not interfere with the detection of GSH. At the same time, after adding GSH, significant changes were observed, indicating that CDs@S-DAQ has good selectivity for GSH.

[0049] Example 4

[0050] Cytotoxicity analysis of the CDs@S-DAQ probe:

[0051] The cytotoxicity of the CDs@S-DAQ (Example 1) probe against HBMECs and U87 cells was evaluated by the CCK-8 method. First, HBMECs and U87 cells were seeded in 96-well plates (5×10 3 cells per well) and allowed to adhere overnight. Subsequently, the cells were treated with different concentrations of the CDs@S-DAQ probe (0 - 200 μg / mL) and cultured for 24 h at 37 °C. Then, CCK-8 reagent (10%, 100 μL) was added to each well and the cells were cultured for an additional 2 h. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader to evaluate cytotoxicity.

[0052] The cell viability was calculated using the following formula:

[0053] Cell viability rate = ((A_X - A_0) / (A_C - A_0)) × 100%

[0054] where A_X is the absorbance of the experimental group (i.e., cells incubated with the CDs@S-DAQ probe), A_C is the absorbance of the control group (i.e., cells not incubated with the CDs@S-DAQ probe), and A_0 is the absorbance of the blank group (i.e., cells not treated with CCK-8).

[0055] Figure 5 are the results of the cytotoxicity experiment of the CDs@S-DAQ probe. Figure A shows the cell viability of HBMECs treated with different concentrations of the CDs@S-DAQ probe (incubated for 24 h). Figure B shows the cell viability of U87 cells treated with different concentrations of the CDs@S-DAQ probe (incubated for 24 h). From Figure 5It can be seen that after treatment with CDs@S-DAQ probes at different concentrations for 24 hours, even at a concentration as high as 200 μg / mL, the survival rates of HBMECs and U87 cells remained above 90%. These results indicate that the probe has good biocompatibility and biosafety and is suitable for imaging analysis of living cells.

[0056] Example 5

[0057] CDs@S-DAQ for specific fluorescence imaging of glioblastoma cells:

[0058] The fluorescence recovery of the CDs@S-DAQ probe induced by GSH was investigated in human brain microvascular endothelial cells (HBMECs) and human GBM cells (U87 cells), respectively. HBMECs and U87 cells were seeded in glass culture dishes at a density of 5×10 3 cells per well and allowed to attach during overnight culture. After different pretreatments, the cells were co-incubated with the CDs@S-DAQ probe (Example 1) (40 μg / mL) for 9 h. The treatment conditions were as follows: (1) HBMECs without pretreatment, (2) U87 cells without pretreatment, (3) HBMECs pretreated with GSH (1 mM) at 37 °C for 30 min, (4) U87 cells pretreated with GSH (1 mM) at 37 °C for 30 min, (5) U87 cells pretreated with N-ethylmaleimide (1 mM) at 37 °C for 30 min. After incubation, the cells were washed three times with PBS to remove free GSH, NEM, and / or residual CDs@S-DAQ probe, and images were taken using an inverted fluorescence microscope.

[0059] Figure 6 Results of CDs@S-DAQ for specific fluorescence imaging of glioblastoma cells. Figures A - E represent the fluorescence images of the treatment systems of HBMECs, HBMECs + GSH, U87 cells, U87 cells + GSH, and U87 cells + NEM co-incubated with CDs@S-DAQ for 9 h, respectively. From Figure 6 It can be seen that almost no fluorescence was detected in HBMECs after co-incubation with the probe; obvious red fluorescence was observed in U87 cells; after pretreatment with GSH, the red fluorescence of both cell types was significantly enhanced; in addition, the red fluorescence of U87 cells pretreated with NEM (a thiol scavenger) was significantly reduced after co-incubation with the CDs@S-DAQ probe for 9 h. These results indicate that the CDs@S-DAQ probe can respond to the high-level GSH expression in GBM cells, showing significant fluorescence recovery, while remaining in the fluorescence "off" state in normal HBMECs with low GSH expression, and is suitable for the visualization of intracellular GSH and specific fluorescence imaging of GBM cells.

Claims

1. A method for preparing a thioether bond-bridged carbon dot-dopamine quinone composite probe, characterized in that: The method comprises the following steps: (1) Preparation of CDs@S: Using nitrogen as the reaction atmosphere, at a temperature of -5 to 5°C and stirring, add thiodiacetic anhydride and 4-dimethylaminopyridine to the carbon dot dispersion, and then react at 10 to 40°C for 4 to 24 hours. After the reaction, extract the solution several times with an organic solvent and collect the aqueous phase. The collected aqueous phase is concentrated at 40 to 60°C, and the obtained concentrate is mixed with acetone and placed at -25 to -15°C for 20 to 40 minutes. Then, centrifuge, wash and dry in sequence to obtain sulfide-bridged carbon dots. (2) Preparation of CDs@S-DAQ: CDs@S, EDC and NHS were dissolved in an organic solvent, and the mixture was stirred at -5 to 5°C for 20 to 40 min. Dopamine hydrochloride was then added. After the addition was completed, the mixture was reacted at 20°C to 30°C under a nitrogen atmosphere for 4 to 24 h. After the reaction was completed, the mixture was dialyzed, and the solution in the dialysis bag was centrifuged and the precipitate was collected to obtain a CDs@S-dopamine complex. The CDs@S-dopamine complex was dispersed in a phosphate buffer and naturally oxidized at 1 to 5°C for one week. The product was vacuum dried to obtain a CDs@S-DAQ complex.

2. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 1, characterized in that: In step (1), the carbon dots dispersion liquid is prepared by dispersing the carbon dots in an organic solvent and filtering the mixture using a 0.2-0.25 μm microporous membrane. The obtained filtrate is the carbon dots dispersion liquid, and the mass ratio of the carbon dots to the organic solvent is 1:3-6.

3. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 1, characterized in that: In step (1), the mass ratio of thiodiacetic anhydride, 4-dimethylaminopyridine and carbon dots is 5-15:5-15:0.5-1.

5.

4. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 1, characterized in that: In step (2), the mass ratio of CDs@S, EDC, NHS and dopamine hydrochloride is 3-8:3-8:3-8:40-60.

5. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 1, characterized in that: In step (2), the mass ratio of CDs@S-dopamine complex to phosphate buffer is 1:2-5.

6. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 1 or 5, characterized in that: The pH of the phosphate buffer in step (2) is 7-8, and the concentration is 10-100 mM.

7. The method for preparing the thioether bond-bridged carbon dot-dopamine quinone composite probe according to claim 2, characterized in that: The preparation method of carbon dots in step (1) is to dissolve citric acid and urea in a solvent and heat at 160°C to 200°C for 4 to 12 hours; then cool the reaction system and add an alkaline reagent to react, centrifuge and dry after the reaction, dissolve the dried powder in an acidic solution and mix, and then centrifuge, wash and dry again after mixing to obtain CDs.

8. A thioether bond-bridged carbon dot-dopamine quinone composite probe, characterized in that: The probe is prepared by the method described in any one of claims 1 to 5.

9. Use of the thioether bond-bridged carbon dot-dopamine quinone composite probe prepared by the method of claim 1 in fluorescence imaging of glioblastoma cells.

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