A thioether-bridged carbon dot-dopamine quinone composite probe and its application in fluorescence imaging of glioblastoma cells

Abstract

The present invention discloses a thioether-bridged carbon dot-dopamine quinone composite probe and its application in glioblastoma cell fluorescence imaging, belonging to the field of materials. The probe first prepares thioether-bridged carbon dots, followed by the preparation of a CDs@S-DAQ complex. The preparation method provided by the present invention is simple and safe, uses readily available raw materials, does not require complex and expensive equipment, and is easy to mass-produce.

Description

Technical Field

[0001] The present invention belongs to the fields of nanomaterial preparation, 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 glioblastoma cell fluorescence imaging. Background Art

[0002] Glioblastoma (GBM) is one of the most aggressive and lethal malignancies of the central nervous system. Due to the lack of effective treatments, the one-year survival rate after diagnosis is only 25%. Sensitive and specific identification and imaging of glioblastoma cells plays a crucial role in early diagnosis, targeted therapy, and metastasis progression, helping to improve patients' quality of life and prolong survival. The rapid development of imaging technologies has enabled real-time, dynamic, in vivo / in vitro, and qualitative / quantitative monitoring of various complex biological processes, such as gene expression, substance transport, signal transduction, and protein interactions. Based on different principles, imaging techniques include computed tomography, positron emission tomography, single-photon emission computed tomography, ultrasound imaging, magnetic resonance imaging, Raman imaging, photoacoustic imaging, and fluorescence imaging. Among these techniques, fluorescence imaging has attracted widespread attention in recent years due to its high sensitivity, high spatial resolution, low cost, real-time data acquisition, and lack of ionizing radiation.

[0003] To date, a variety of fluorescence imaging methods have been developed for imaging GBM cells. For example, Yang et al. introduced a water-soluble near-infrared (NIR) dye, heptafluorothiophenesulfonylindoline 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 oxazolopiperidine 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 a large Stokes-shifted yellow fluorescent protein for multicolor immunofluorescence imaging of the glioma microenvironment. Although these studies have demonstrated excellent results in highly selective identification and imaging of glioma cells, the inherent limitations of the fluorescent materials used have limited 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 the risk of cytotoxicity, and the production process of fluorescent proteins is time-consuming and expensive. Therefore, it is of great significance to develop new fluorescent nanomaterials to achieve reliable and accurate long-term imaging of GBM cells.

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

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

[0006] The purpose of the present invention can be achieved through the following technical solutions:

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

[0008] (1) Preparation of CDs@S: Thiodiacetic anhydride and 4-dimethylaminopyridine were added to a carbon dot dispersion in a nitrogen atmosphere at a temperature of -5 to 5°C with stirring for 4 to 24 hours. After the reaction, the solution was extracted several times with an organic solvent and the aqueous phase was collected. The collected aqueous phase was concentrated at 40 to 60°C. The obtained concentrate was mixed with acetone and stored at -25 to -15°C for 20 to 40 minutes. It was then centrifuged, washed, and dried to obtain sulfide-bridged carbon dots.

[0009] (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 minutes. 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 hours. 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 solution and naturally oxidized at 1 to 5°C for one week. The product was vacuum dried to obtain a CDs@S-DAQ complex.

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

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

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

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

[0014] In the above preparation method, the pH of the phosphate buffer in step (2) is 7-8, and the concentration is 10-100 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 it at 160°C to 200°C for 4 to 12 hours; then cool the reaction system and add an alkaline reagent to react. After the reaction is completed, centrifugation and drying are carried out in sequence. The dried powder is dissolved in an acidic solution and mixed. After mixing, centrifugation, washing and drying are repeated in sequence to obtain CDs.

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

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

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

[0019] Beneficial effects 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 glioblastoma cell fluorescence imaging, which has the following beneficial effects:

[0021] (1) The preparation method provided by the present invention is simple and safe, the raw materials are easily available, and no complex and expensive instruments and equipment are required, and it is easy to produce in large quantities;

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

[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 the thioether bond, which makes the probe exhibit excellent responsiveness to GSH;

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

[0025] (5) The CDs@S-DAQ probe can distinguish GBM cells from normal tissue cells based on the difference in GSH expression levels between the two types of cells through fluorescence imaging, providing a promising method for early diagnosis, targeted treatment, and metastasis development of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 Characterization images of CDs, CDs@S, and CDs@S-DAQ composite probes in the system of the present invention. A and B are the transmission electron microscopy image and high-resolution transmission electron microscopy image of CDs@S-DAQ, respectively. C is the transmission electron microscopy particle size distribution histogram and Gaussian fit of CDs@S-DAQ. D is the Fourier transform infrared spectra of CDs(i), CDs@S(ii), CDs@S-DA(iii), and CDs@S-DAQ(iv). EG is the X-ray photoelectron spectra of CDs, CDs@S, and CDs@S-DAQ. H is the fluorescence emission spectra of CDs, CDs@S, and CDs@S-DAQ.

[0028] Figure 3This is a performance diagram of the CDs@S-DAQ composite probe for 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 under the system of the present invention to interfering substances such as metal ions, anions and small molecular organic matter.

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

[0031] Figure 6 This is the result of glioblastoma cell-specific fluorescence imaging using CDs@S-DAQ under the system of the present invention. DETAILED DESCRIPTION

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

[0033] Example 1

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

[0035] (1) Preparation of CDs: First, citric acid (1.0 g) and urea (2.0 g) were dissolved in N,N-dimethylformamide (10 mL) and then transferred to a 15 mL Teflon-lined autoclave. The mixture was heated at 180°C for 8 h. After cooling, it was mixed with NaOH solution (50 mg / mL, 20 mL) and stirred for 10 min. The precipitate was collected by centrifugation at 16,000 rpm for 15 min and dried under vacuum. Finally, the powder was dissolved in HCl solution (5%, v / v, 20 mL), stirred for 10 min, and centrifuged again at 16,000 rpm for 10 min. The precipitate was washed three times with acetone and deionized water, respectively. After further drying in vacuum, CDs were obtained.

[0036] (2) Preparation of CDs@S: First, carbon dots (10 mg) were dispersed in DMF (40 mL), ultrasonically treated for 1 h, and then filtered with a 0.22 μm microporous membrane to obtain a carbon dot dispersion. Subsequently, thiodiacetic anhydride (100 mg) and 4-dimethylaminopyridine (100 mg) were stirred and added to the carbon dot dispersion at 0°C under a nitrogen atmosphere and reacted at room temperature for 24 h. After the reaction was completed, deionized water (40 mL) was added to terminate the reaction. Then, the solution was extracted three times with dichloromethane (40 mL) and the aqueous phase was collected. The aqueous phase was concentrated in a rotary evaporator at 50°C, and the concentrate was mixed with excess acetone, stirred for 5 min, and placed at -20°C for 30 min. The precipitate was collected by centrifugation at 10,000 rpm for 15 min and washed three times with acetone and deionized water, respectively. Finally, it was further dried under vacuum to obtain thioether-bridged carbon dots.

[0037] (3) Preparation of CDs@S-DAQ: First, CDs@S (5 mg), EDC (5 mg) and NHS (5 mg) were dissolved in N,N-dimethylformamide (20 mL). The mixture was stirred at 0°C for 30 min, and then dopamine hydrochloride (50 mg) was added. The reaction was carried out at 25°C for 16 h under a nitrogen atmosphere. The reaction product was then dialyzed against deionized water in a 1000 Da dialysis bag for 36 h to remove excess dopamine, EDC, NHS and possible polydopamine. After the solution in the dialysis bag was centrifuged at 10,000 rpm for 10 min, the precipitate was collected to obtain the CDs@S-dopamine complex. CDs@S-dopamine (5 mg) was redispersed in phosphate buffered saline (PBS, pH 7.4, 10 mM, 20 mL) and naturally oxidized in a refrigerator at 4°C for one week. The product was vacuum dried to obtain the 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 CDs, CDs@S and CDs@S-DAQ composite probes prepared in Example 1. Figure A and Figure B are transmission electron microscopy and high-resolution transmission electron microscopy images of CDs@S-DAQ, respectively, and Figure C is a transmission electron microscopy particle size distribution histogram and Gaussian fitting of CDs@S-DAQ. Figure D is a Fourier transform infrared spectrum of CDs(ⅰ), CDs@S(ⅱ), CDs@S-DA(ⅲ) and CDs@S-DAQ(ⅳ). Figures EG are X-ray photoelectron spectra of CDs, CDs@S and CDs@S-DAQ, respectively. Figure H is a fluorescence emission spectrum 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, good monodispersity, and no significant aggregation phenomenon. The particle size is normally distributed with an average diameter of about 3.67 nm; after DAQ is modified on the surface of CDs@S, the fluorescence of CDs@S is significantly quenched.

[0040] Example 2

[0041] CDs@S-DAQ composite probe for 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). The mixture was then incubated at 37°C with gentle agitation for 1 hour. Subsequently, fluorescence intensity at 625 nm (λex = 550 nm) was measured using a fluorescence spectrophotometer at different GSH concentrations. A quantitative model was established based on the relationship between fluorescence intensity and GSH concentration to predict GSH concentration.

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

[0044] Figure 3 Figure 2 is a performance diagram of the CDs@S-DAQ composite probe (Example 1) for fluorescence detection of GSH. Figure 3 It can be seen that the response concentration range of the probe to GSH is 0.5-20 mM, the linear range is 0.05-20 μM, the correlation coefficient is 0.9969, and the minimum detection limit is 69 μM.

[0045] Example 3

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

[0047] 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). The mixture was then incubated at 37°C with gentle stirring for 1 h. Subsequently, the fluorescence intensity at 625 nm (λex = 550 nm) was measured at different GSH concentrations using a fluorescence spectrophotometer.

[0048] Figure 4 The selectivity of CDs@S-DAQ composite probe to interfering substances such as metal ions, anions and small organic molecules. Figure 4 As can be seen, with the exception of a high concentration of cysteine (5 mM), all interfering substances had little effect on the detection of GSH. Notably, the average concentration of cysteine in both tumor and normal cells did not exceed 200 μM, indicating that it did not interfere with the detection of GSH. Furthermore, significant changes were observed after the addition of GSH, demonstrating the excellent selectivity of CDs@S-DAQ for GSH.

[0049] Example 4

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

[0051] The cytotoxicity of CDs@S-DAQ (Example 1) probe to HBMECs and U87 cells was evaluated by CCK-8 assay. First, HBMECs and U87 cells were seeded in 96-well plates (5×10 3 Cells were plated and allowed to attach overnight. Subsequently, the cells were treated with various concentrations of the CDs@S-DAQ probe (0-200 μg / mL) and incubated for 24 hours at 37°C. Next, CCK-8 reagent (10%, 100 μL) was added to each well and incubated for another 2 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader to assess cytotoxicity.

[0052] Cell viability was calculated using the following formula:

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

[0054] Wherein, AX is the absorbance of the experimental group (i.e., cells incubated with CDs@S-DAQ probe), AC is the absorbance of the control group (i.e., cells not incubated with CDs@S-DAQ probe), and A0 is the absorbance of the blank group (i.e., cells not treated with CCK-8).

[0055] Figure 5 The results of the cytotoxicity experiment of CDs@S-DAQ probe are shown in Figure A. The activity of HBMECs cells treated with different concentrations of CDs@S-DAQ probe (incubated for 24 hours). Figure B. The activity of U87 cells treated with different concentrations of CDs@S-DAQ probe (incubated for 24 hours). Figure 5As can be seen, after 24 hours of treatment with different concentrations of CDs@S-DAQ probe, the viability of HBMECs and U87 cells remained above 90%, even at a concentration as high as 200 μg / mL. These results demonstrate that the probe has good biocompatibility and biosafety, making it suitable for imaging analysis of living cells.

[0056] Example 5

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

[0058] The fluorescence recovery of CDs@S-DAQ probe induced by GSH was studied in human brain microvascular endothelial cells (HBMECs) and human GBM cells (U87 cells). HBMECs and U87 cells were plated at 5×10 3 Cells were seeded at a density of 100 cells / mL in glass culture dishes and allowed to attach during overnight culture. After different pretreatments, the cells were incubated with CDs@S-DAQ probes (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) for 30 min at 37°C, (4) U87 cells pretreated with GSH (1 mM) for 30 min at 37°C, and (5) U87 cells pretreated with N-ethylmaleimide (1 mM) for 30 min at 37°C. After incubation, the cells were washed three times with PBS to remove free GSH, NEM, and / or residual CDs@S-DAQ probes, and images were captured using an inverted fluorescence microscope.

[0059] Figure 6 Figures AE and AE show the fluorescence images of HBMECs, HBMECs+GSH, U87 cells, U87 cells+GSH, and U87 cells+NEM treated systems after co-incubation with CDs@S-DAQ for 9 hours. Figure 6 As can be seen, after incubation with the probe, almost no fluorescence was detected in HBMECs, while significant red fluorescence was observed in U87 cells. After GSH pretreatment, the red fluorescence of both cell types was significantly enhanced. Furthermore, in U87 cells pretreated with NEM (a thiol scavenger), red fluorescence was significantly reduced after 9 hours of incubation with the CDs@S-DAQ probe. These results demonstrate that the CDs@S-DAQ probe can respond to high levels of GSH expression in GBM cells, exhibiting significant fluorescence recovery, while maintaining a fluorescence "off" state in normal HBMECs with lower GSH expression, making it suitable for 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, thiodiacetic anhydride and 4-dimethylaminopyridine were added to the carbon dot dispersion at a temperature of -5~5°C with stirring. The mixture was then reacted at 10~40°C for 4~24 h. After the reaction, the solution was extracted several times with an organic solvent and the aqueous phase was collected. The collected aqueous phase was concentrated at 40~60°C. The obtained concentrate was mixed with acetone and placed at -25~-15°C for 20~40 min. The concentrate was then centrifuged, washed, and dried to obtain sulfide-bridged carbon dots. The preparation method of carbon dots in step (1) is to dissolve citric acid and urea in N,N-dimethylformamide and heat the mixture 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 the mixture after the reaction, dissolve the dried powder in an acidic solution and mix it, and then centrifuge, wash and dry it to obtain CDs; (2) Preparation of CDs@S-DAQ: CDs@S, EDC and NHS were dissolved in an organic solvent, and the mixture was stirred at -5~5°C for 20~40 min. Dopamine hydrochloride was then added. After the addition, the mixture was reacted at 20°C~30°C under a nitrogen atmosphere for 4~24 h. After the reaction, 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 phosphate buffer and naturally oxidized at 1~5°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 dot dispersion is prepared by dispersing the carbon dots in an organic solvent and filtering the mixture using a 0.2-0.25µm microporous membrane. The resulting filtrate is the carbon dot dispersion, 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: The mass ratio of CDs@S, EDC, NHS and dopamine hydrochloride in step (2) 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, wherein: The pH of the phosphate buffer in step (2) is 7-8, and the concentration is 10-100 mM.

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

8. 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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