Tellurium-doped carbon quantum dot as well as preparation method and application thereof

Abstract

The invention discloses a tellurium-doped carbon quantum dot and a preparation method and application thereof, the tellurium-doped carbon quantum dot is obtained by using 2, 2 '-diaminodiphenyl telluride as a precursor, the average particle size is 1.9 nm, the thickness is about 2.1 nm, and the surface is rich in amino groups. And fixing (4-carboxybutyl) triphenylphosphonium bromide, 3-morpholinopropionic acid or 3-((4-methylphenyl) sulfonamide) propionic acid on the surfaces of the tellurium-doped carbon quantum dots to prepare Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs with an organelle targeting function. The Mito-Te-CDs, the Lyso-Te-CDs and the ER-Te-CDs are good in biocompatibility, can be used as fluorescent probes for targeted imaging of organelles, can be used for recognition and removal of superoxide anions in specific organelles, have a protection effect on cells and can be used for effectively treating acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon quantum dots, and in particular to tellurium-doped carbon quantum dots and a preparation method and application thereof. Background Art

[0002] Carbon quantum dots (CDs) are a new type of carbon nanoparticles with significant fluorescent properties. Their particle size is generally less than 10 nm. They have the characteristics of small particle size, good biocompatibility, excitation wavelength-dependent photoluminescence, chemical inertness and low toxicity, and are very promising nanobiotechnology materials. CDs doped with metals (iron, cobalt, copper, zinc, etc.) or heteroatoms (nitrogen, sulfur, phosphorus, etc.) have the advantages of simple preparation, good biocompatibility and excellent performance, and have great advantages in the fields of biochemistry, biology and biomedicine (Yang Mingjie et al., Synthesis and Application of Metal- or Heteroatom-Doped Carbon Quantum Dots, Journal of Composite Materials, 2025, 42(3): 1219-1239).

[0003] The synthesis of CDs can be broadly categorized into top-down and bottom-up approaches. Bottom-up approaches often utilize small organic molecules or oligomers as carbon sources, such as citric acid, glucose, polyethylene glycol, urea, and ionic liquids. Common synthesis methods include chemical oxidation, combustion, hydrothermal / solvothermal, microwave synthesis, and template-based approaches.

[0004] Exploring the preparation of new CDs or heteroatom-doped CDs from different raw materials and exploring their new uses has become a research hotspot in recent years. Chinese patent document CN 202010106194.9 discloses a method for preparing tellurium-doped carbon quantum dots and their dual ability to consume and generate reactive oxygen species. These dots are prepared using tellurium-substituted cystine as a raw material. However, the synthesized CDs lack targeting capabilities, making them unable to precisely locate organelles and scavenge reactive oxygen species within specific regions. Therefore, exploring new CDs and conducting in-depth research to impart targeting capabilities are of great significance to biology and medicine. Summary of the Invention

[0005] The present invention addresses the shortcomings of the prior art by providing novel tellurium-doped carbon quantum dots and their preparation methods. After modification with targeting molecules, the tellurium-doped carbon quantum dots can be used for targeted imaging of specific organelles and exhibit specific recognition and scavenging properties for superoxide anions. The detailed technical solution described below is specifically employed.

[0006] Tellurium-doped carbon quantum dots (Te-CDs) are prepared by dispersing 2,2′-diaminodiphenyl ditelluride in deoxyethanol and heating the mixture at 200°C in a polytetrafluoroethylene-lined autoclave for 4 hours. After the reaction, the resulting solution is filtered through a membrane and then centrifuged to remove larger nanoparticles. The filtrate is collected, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain tellurium-doped carbon quantum dots. The resulting tellurium-doped carbon quantum dots have an average particle size of 1.9 nm and a thickness of approximately 2.1 nm. They exhibit green fluorescence and are rich in amino groups on their surface. The tellurium-doped carbon quantum dots exhibit specific recognition and scavenging properties for superoxide anions.

[0007] Preferably, the pore size of the filter membrane is 0.22 μm, and the centrifugation conditions are 12000 g and 15 min to obtain a relatively pure target product Te-CDs.

[0008] The preparation method of 2,2′-diaminodiphenyl ditelluride is as follows: Te metal and 2-iodoaniline are placed in a dry DMSO solution, CuO nanoparticles with a particle size of 10 to 50 nm are added with stirring, and then treated with KOH under a nitrogen atmosphere at 120°C. After reacting for 22 hours, the reaction mixture is naturally cooled to room temperature, concentrated, and then separated by column chromatography to obtain 2,2′-diaminodiphenyl ditelluride as a reddish-brown powder. The molar ratio of Te metal to 2-iodoaniline is preferably 2:1.

[0009] The invention discloses tellurium-doped carbon quantum dots (Mito-Te-CDs) with mitochondrial targeting function, wherein (4-carboxybutyl)triphenylphosphine bromide (TPP) is fixed on the surface of Te-CDs prepared by the invention.

[0010] The invention discloses tellurium-doped carbon quantum dots (Lyso-Te-CDs) with lysosome targeting function, wherein 3-morpholinepropionic acid (MA) is fixed on the surface of Te-CDs prepared by the invention.

[0011] The invention discloses tellurium-doped carbon quantum dots (ER-Te-CDs) with endoplasmic reticulum targeting function, wherein 3-((4-methylphenyl)sulfonamido)propionic acid (PA) is fixed on the surface of Te-CDs prepared by the invention.

[0012] The tellurium-doped carbon quantum dots Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs with targeting functions are prepared by a covalent EDC / NHS activation process. Specifically, TPP solution, MA solution, and PA solution are placed in flasks respectively, and then EDC / NHS is injected respectively and activated at room temperature for 4 hours; then, Te-CDs solution is added respectively and reacted for further 12 hours to generate TPP, MA, or PA-modified Te-CDs, respectively referred to as Mito-Te-CDs, Lyso-Te-CDs, or ER-Te-CDs.

[0013] The Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs have good biocompatibility and can be used as fluorescent probes for targeted imaging of specific organelles, mitochondria, lysosomes, and endoplasmic reticulum, respectively. They can also be used to identify and remove superoxide anions in specific organelles, thus having a protective effect on cells.

[0014] The combined use of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs exhibited superior antioxidant protection against cells compared to individual components. In vivo experimental results demonstrated that Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs were all able to rapidly reach the kidneys and effectively treat acute kidney injury.

[0015] The advantages of the present invention are:

[0016] 1. A new tellurium-doped carbon quantum dot product is provided with a simple preparation method. Based on this product, tellurium-doped carbon quantum dots Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs with targeting functions to organelles mitochondria, lysosomes and endoplasmic reticulum are developed with good biocompatibility.

[0017] 2. Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs can be used for targeted imaging of organelles such as mitochondria, lysosomes and endoplasmic reticulum, and have the ability to recognize and clear superoxide anions in organelles, thus protecting cells.

[0018] 3. Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs can effectively treat acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attachment Figure 1Synthesis and characterization of Te-CDs, (A) Schematic diagram of the synthesis of Te-CDs, (B) Transmission electron microscopy image of Te-CDs, (C) Atomic force microscopy image of Te-CDs, (D) Raman spectrum of Te-CDs, (E), (F), (G) Characterization of the surface functional groups of Te-CDs by X-ray photoelectron spectroscopy, (H) UV absorption spectrum of Te-CDs, (I) Fluorescence spectrum of Te-CDs, (J) Fluorescence emission images of Te-CDs under different excitation wavelengths.

[0020] Attachment Figure 2 Figure 3 is the recognition and scavenging performance of Te-CDs for superoxide anions. (A) Response diagram of Te-CDs to different concentrations of superoxide anions. (B) Relationship between Te-CDs fluorescence intensity and superoxide anion concentration. (C) Response time diagram of Te-CDs to different concentrations of superoxide anions. (D) and (E) are studies on the selectivity of Te-CDs for superoxide anions. (F) and (G) studies on the scavenging performance of Te-CDs for superoxide anions. (H) and (I) studies on the cyclic detection performance of Te-CDs for superoxide anions.

[0021] Attachment Figure 3 The synthesis, characterization, recognition and scavenging performance of Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs for superoxide anions, (A) Schematic diagram of the synthesis of multi-organelle-targeted Te-CDs, (B) Transmission electron microscopy of Mito-Te-CDs, (C) Atomic force microscopy of Mito-Te-CDs, (D) Transmission electron microscopy of Lyso-Te-CDs, (E) Atomic force microscopy of Lyso-Te-CDs, (F) Transmission electron microscopy image, (G) atomic force microscopy image of ER-Te-CDs, (H) detection performance of Mito-Te-CDs for superoxide anion, (I) detection performance of Lyso-Te-CDs for superoxide anion, (J) detection performance of ER-Te-CDs for superoxide anion, (K) scavenging performance of Mito-Te-CDs for superoxide anion, (L) scavenging performance of Lyso-Te-CDs for superoxide anion, (M) scavenging performance of ER-Te-CDs for superoxide anion.

[0022] Attachment Figure 4Figures (A), (B), and (C) show the cytotoxicity, organelle co-localization, and superoxide anion detection performance of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs, respectively. Figures (D), (E), and (F) show the biocompatibility of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs, respectively. Figure (G) (H) and (I) show the targeting performance of Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs to mitochondria, lysosomes and endoplasmic reticulum, respectively. (J) and (L) show the detection performance of Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs to superoxide anions in mitochondria, lysosomes and endoplasmic reticulum, respectively. (K) and (M) show the relationship between the fluorescence intensity and superoxide anion concentration in mitochondria, lysosomes and endoplasmic reticulum, respectively.

[0023] Attachment Figure 5 Figure (A) shows the scavenging performance of Mito-Te-CDs on superoxide anions in mitochondria, Figure (B) shows the fluorescence intensity in mitochondria under different stimulation conditions, Figure (C) shows the scavenging performance of Lyso-Te-CDs on superoxide anions in lysosomes, Figure (D) shows the fluorescence intensity in lysosomes under different stimulation conditions, Figure (E) shows the scavenging performance of ER-Te-CDs on superoxide anions in the endoplasmic reticulum, Figure (F) shows the fluorescence intensity in the endoplasmic reticulum under different stimulation conditions, and Figure (G) shows the flow cytometry results under different conditions.

[0024] Attachment Figure 6In the experiment, the Mixture group is a mixture of three targeted functionalized Te-CDs. (A) The figure shows the tissue section of normal mice 7 days after intravenous injection of Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs and the mixture of three targeted functionalized Te-CDs. (B) The figure shows the BUN content in the blood of normal mice 7 days after intravenous injection of Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs and the mixture of three targeted functionalized Te-CDs. (C) The figure shows the CRE content in the blood of normal mice 7 days after intravenous injection of Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs and the mixture of three targeted functionalized Te-CDs. (D) The figure shows the CRE content in the blood of normal mice 7 days after intravenous injection of Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs and the mixture of three targeted functionalized Te-CDs. (E) The AST content in the blood of normal mice 7 days after intravenous injection of Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs and a mixture of three targeted functionalized Te-CDs. (F) The fluorescence changes of different organs in normal mice at different times after intravenous injection of the three targeted functionalized Te-CDs mixture. (G) The fluorescence changes of different organs in mice with acute kidney injury model at different times after intravenous injection of the three targeted functionalized Te-CDs mixture. (H) The BUN content in the blood of normal mice and mice with acute kidney injury under different stimuli. (I) The CRE content in the blood of normal mice and mice with acute kidney injury under different stimuli. (J) The fluorescence images of kidney tissue sections of normal mice and mice with acute kidney injury under different stimuli. (K) The changes in SOD content in kidney tissue homogenate of normal mice and mice with acute kidney injury under different stimuli. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0026] Reagents used in the examples:

[0027] 2-Iodoaniline, triethylamine (Et3N), and o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) were purchased from TCI. (4-Carboxybutyl)triphenylphosphonium bromide (TPP), 3-morpholinopropionic acid (MA), dihydroethidium (HE), and Te metal were purchased from Macklin Biochemical Co., Ltd. 3-((4-Methylphenyl)sulfonamido)propionic acid (PA) was prepared according to published procedures (Hong Huang, et al. Endoplasmic reticulum-targeted polymer dots encapsulated with ultrasonically synthesized near-infrared carbon nanodots and their application for in vivo monitoring of Cu2 + Journal of Colloid and Interface Science, 2022, 627: 705-715). CuO nanoparticles with a particle size of 10-50 nm and a purity of 99% were purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. Phorbol 12-myristate 13-acetate (PMA), xanthine oxidase (XO), xanthine (X), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), KO2 (providing superoxide anion), and ascorbic acid (AA) were purchased from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM) with high glucose was purchased from Gibco BRL. MitoTracker Deep Red FM, Iyso-Tracker Red, ER-Tracker Red, and MitoSOX Red were purchased from Thermo Fisher Scientific. A superoxide anion radical detection kit was purchased from SolarbioScience & Technology Co. Ltd. Annexin V-APC / 7-AAD Apoptosis Kit was purchased from KeyGEN BioTECH. All chemicals and solvents were of analytical grade and used directly. All other reagents were commercially available unless otherwise specified.

[0028] Instruments and methods used in the examples:

[0029] 1 H and 13C nuclear magnetic resonance (NMR) spectra were obtained from a Bruker 400 MHz spectrometer. Transmission electron microscopy (TEM) experiments were performed on a transmission electron microscope (JEOL JEM-2100F) with an accelerating voltage of 200 kV. Atomic force microscopy (AFM) characterization was performed in ScanAsyst mode under ambient conditions (Bruker). For AFM, a solution of Te-CDs was dropped onto the surface of a freshly cleaved mica substrate and allowed to dry naturally. Raman spectroscopy was performed on a Raman microscope imaging spectrometer (ThermoFisher Dxr2xi). X-ray photoelectron spectroscopy (XPS) data were collected using a thermal electron instrument (Thermo Scientific ESCALAB 250). Fourier transform infrared (FTIR) spectra of the samples were acquired using a Nicolet iS10 FTIR spectrometer. Absorption experiments were performed on a Shimadzu UV-2550 spectrophotometer. Fluorescence spectra were measured on an F-4700 fluorescence spectrophotometer. All theoretical calculations were performed using the ORCA program system. Apoptosis assays were performed on a BD FACSCalibur flow cytometer (BD Biosciences). Confocal fluorescence and brightfield images (512 × 512 pixels) were captured using a 63× objective on a Leica TCS-SP8 confocal laser scanning microscope. Fluorescence intensity and colocalization efficiency of the resulting images were quantified using ImageJ software. Ex vivo fluorescence images of major organs were acquired on a PerkinElmer IVIS Lumina XRMS Series III in vivo imaging system with an excitation wavelength of 488 nm.

[0030] Example 1 Preparation of tellurium-doped carbon quantum dots

[0031] Synthesis of 1,2,2′-diaminodiphenyl ditelluride

[0032] Te metal (2.0 mmol) and 2-iodoaniline (1.0 mmol) were placed in a dry DMSO (5.0 mL) solution, and CuO nanoparticles (10-50 nm, 10.0 mol%) were added with stirring. The mixture was then treated with KOH (2.0 equivalents) under a nitrogen atmosphere at 120°C. After 22 hours of reaction, the reaction mixture was cooled to room temperature, concentrated, and separated by column chromatography to obtain pure 2,2′-diaminodiphenyl ditelluride as a reddish-brown powder. 1 H NMR [400MHz, CDCl3]: δ = 7.70 (m, 2H), 7.14 (m, 2H), 6.74 (m, 2H), 6.53 (m, 2H), 4.17 (s, 4H); 13C NMR [400 MHz, CDCl3]: δ = 150.71, 142.29, 131.70, 119.16, 113.70, 94.92. MS (m / z) calculated value is [M+Na] + C 12 H 12 N2NaTe2: 466.90, measured value is 466.60.

[0033] 2. Preparation of Te-CDs

[0034] 0.1 g of 2,2′-diaminodiphenyl ditelluride was dispersed in 40 mL of deoxyethanol and heated to 200°C in a 50 mL Teflon-lined autoclave for 4 hours. After the reaction, the resulting solution was filtered through a 0.22 μm filter and centrifuged at 12,000 g for 15 minutes to remove larger nanoparticles. The filtrate was collected, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the desired product, Te-CDs, which exhibited green fluorescence (yield: 17%).

[0035] Attachment Figure 1 The synthesis and characterization of Te-CDs are described. The Te-CDs prepared by the present invention have an average particle size of 1.9 nm and a thickness of approximately 2.1 nm. They exhibit green fluorescence and are rich in amino groups on their surface. Raman spectroscopy indicates that the Te-CDs have a graphite-like structure.

[0036] Example 2 Recognition and removal of superoxide anions by Te-CDs

[0037] The superoxide anion recognition and scavenging properties of the Te-CDs prepared in Example 1 were investigated. Superoxide anion recognition by Te-CDs was assessed by adding varying concentrations of superoxide anions (derived from KO2) to 3 mL of Te-CDs (10 μg / mL). After a 5-minute reaction, the fluorescence intensity of the Te-CDs solution was measured at an excitation wavelength of 405 nm.

[0038] Superoxide anion scavenging by Te-CDs: 150 μM superoxide anion solution was taken, and then different concentrations of Te-CDs (0, 10, 20, 30, 40, 50 and 60 μg / mL) were added thereto. After incubation for 10 minutes, the superoxide anion that did not react with Te-CDs was determined using a superoxide anion detection kit.

[0039] Attachment Figure 2The detection and scavenging performance of Te-CDs for superoxide anions is shown. The linear range of Te-CDs' response to different superoxide anion concentrations is 0.5-16 μM, with a detection limit of 80 nM. Te-CDs' response to different superoxide anion concentrations reaches equilibrium within 40 seconds. Figures (D) and (E) demonstrate the excellent selectivity of Te-CDs for superoxide anions. Figures (F) and (G) show that the scavenging ability for superoxide anions increases with increasing Te-CDs concentration. At a Te-CDs concentration of 60 μg / mL, 70% of superoxide anions are scavenged. Studies on the cyclic detection performance of Te-CDs for superoxide anions revealed that they can be used for cyclic detection of superoxide anions, as shown in Figure (I) at least five times.

[0040] Example 3 Preparation of carbon quantum dots with organelle targeting function

[0041] TPP, MA, or PA were immobilized on the surface of Te-CDs via a covalent EDC / NHS activation process. Specifically, 3.0 mL of a 2.0 mM TPP solution was placed in a flask, followed by injection of 100 mg of EDC / NHS and activation at room temperature for 4 hours. Subsequently, 2.0 mL of a 0.5 mg / mL Te-CDs solution was added, and the reaction was further allowed to proceed for 12 hours to generate TPP-modified Te-CDs, termed Mito-Te-CDs. The preparation methods for MA-functionalized Te-CDs (referred to as Lyso-Te-CDs) and PA-functionalized Te-CDs (referred to as ER-Te-CDs) were the same as those used to synthesize Mito-Te-CDs.

[0042] Attachment Figure 3 In the figure, (A) is a schematic diagram of the synthesis of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs. It can be seen from (B)-(G) that the modification of the three targeting molecules will not affect the size of Te-CDs.

[0043] Synthesis of Lyso-HE: A mixture of HE (0.10 g, 0.32 mmol), MA (0.11 g, 0.72 mmol), HATU (0.27 g, 0.70 mmol) and Et3N (0.13 g, 1.27 mmol) was stirred in dry DMF (10 mL) at room temperature for 3 hours. The mixture was then poured into a saturated saline solution (50 mL) and extracted with ethyl acetate (20 mL×3). The extract was washed three times with water (20 mL×3), dried over sodium sulfate, filtered, concentrated under reduced pressure, and further purified by column chromatography (dichloromethane / methanol, 15 / 1, v / v) to give the product Lyso-HE (yield: 0.045 g), which was brown in color. Pale yellow ER-HE was synthesized using a method similar to that of Lyso-HE.

[0044] Example 4 Recognition and removal of superoxide anions by Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs

[0045] The recognition and scavenging properties of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs prepared in Example 3 for superoxide anions were studied.

[0046] Mito-Te-CDs for superoxide anion recognition: 3 mL of Mito-Te-CDs (10 μg / mL) was added to various concentrations of superoxide anions (derived from KO2). After a 5-minute reaction, the fluorescence intensity of the Mito-Te-CDs solution was measured at an excitation wavelength of 405 nm. Lyso-Te-CDs and ER-Te-CDs for superoxide anion recognition were performed using the same protocol as for Mito-Te-CDs.

[0047] Superoxide anion scavenging by Mito-Te-CDs: Different concentrations of Mito-Te-CDs (0, 10, 20, 30, 40, 50, and 60 μg / mL) were added to a 150 μM superoxide anion solution. After incubation for 10 minutes, the amount of superoxide anion that did not react with Mito-Te-CDs was measured using a superoxide anion detection kit. The experimental procedures for superoxide anion scavenging by Lyso-Te-CDs and ER-Te-CDs were the same as those for Mito-Te-CDs.

[0048] Attachment Figure 3 As can be seen from Figures (H) to (M), the modification of the three targeting molecules has almost no effect on the detection and scavenging performance of Te-CDs for superoxide anions. With the increase of the concentration of Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs, the scavenging ability of superoxide anions is enhanced.

[0049] Example 5 MTT assay, biocompatibility assay

[0050] HeLa cells were cultured at 1×10 4 The cells / well were seeded in a 96-well plate in a 10% fetal bovine serum (FBS) medium containing 80 μg mL -1 Streptomycin and 80 U·mL -1 After culturing for 12 h at 37°C and 5% CO2, the culture medium was removed and different concentrations (0-80 μg mL -1 , concentration interval: 10 μg·mL -1) in the Mito-Te-CDs, Lyso-Te-CDs, or ER-Te-CDs culture medium and cultured for 48 h. The experiment was repeated 5 times for each concentration. Afterwards, 20 μL of MTT solution (1.0 mg mL -1 ) and continue incubation for 4 h to allow for the formation of formazan crystals. Subsequently, 150 μL of DMSO was injected into the wells. The absorbance (A) of the resulting mixture was measured at 570 nm. Cell viability was quantified according to the following formula: Cell viability (%) = A test / A control × 100%, where A control refers to the absorbance obtained from the control group, A test It refers to the absorbance obtained in the presence of Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs. The results of MTT assay showed that Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs had no cytotoxicity. Figure 4 As shown in Figures (A)-(C).

[0051] Cell apoptosis assay: Annexin V-APC / 7-AAD apoptosis kit was used to detect cell apoptosis and necrosis according to the manufacturer's standard protocol. Specifically, HeLa cells were incubated with 80 μg mL -1 Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CD were incubated together for 48 hours. After the culture treatment, the floating cells in the culture medium were collected, and the adherent cells were digested with EDTA-free trypsin (Note: the trypsin digestion time should not be too long, otherwise it is easy to cause false positives), the floating cells and adherent cells were combined, and the cell pellets were harvested by centrifugation (1000rpm, 5min). The cell pellets were redispersed in 500μL AnnexinV-APC binding buffer, and 5μL Annexin V-APC and 5μL 7-AAD solution were added. The cells were stained at room temperature in the dark for 10 minutes, and then analyzed by flow cytometry. The results of flow cytometry experiments showed that Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs all had good biocompatibility, such as Figure 4 As shown in Figures (D)-(F).

[0052] Example 6 Targeting Ability of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs and Detection of O2 in Organelles ·-

[0053] Imaging and detection of O2 in subcellular structures ·-About 12 hours before the imaging study, HeLa cells were trypsinized and plated onto glass-bottom culture dishes. Then, the culture medium in the wells was discarded and a solution containing Mito-Te-CDs (10 μg mL -1 )、Lyso-Te-CDs(10μg·mL -1 ) or ER-Te-CDs (10 μg mL -1 ) in new culture medium. After incubation for 30 minutes, the labeled cells were rinsed three times with phosphate-buffered saline (PBS) to remove residual Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs. Serum-free DMEM was added to the culture dish, and fluorescence images of Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs labeled cells were captured using 488 nm excitation light in the wavelength range of 500-580 nm. The subcellular localization ability of Mito-Te-CDs, Lyso-Te-CDs and ER-Te-CDs was studied by co-localization bioimaging experiments, among which Mito-Te-CDs (10 μg mL -1 )、Lyso-Te-CDs(10μg·mL -1 ) or ER-Te-CDs (10 μg mL -1 ) cells stained with O2 ·- (16μM) for 15 minutes and then stained with MitoTracker Deep Red FM (50nM), Lyso-Tracker Red (50nM) or ER-Tracker Red (50nM) for 20 minutes. After completing the labeling experiment, the labeled cells were thoroughly washed with PBS. The fluorescence emission signals of commercial dyes and three targeted Te-CDs were collected simultaneously from two separate channels. Fluorescence imaging of MitoTracker Deep Red FM uses a 633nm excitation laser and collects emission in the detection range of 660-730nm, while Lyso-Tracker Red and ER-Tracker Red use a 552nm excitation laser and collect emission signals in the wavelength ranges of 570-650nm and 580-660nm, respectively. For Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs, the excitation wavelength was set to 405nm and the emission was collected in the wavelength range of 500-580nm. The results are shown in Figure 2. Figure 4 As shown in Figures (G)-(I) in the middle, by comparison with commercial targeting dyes, it was verified that the Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs prepared by the present invention have the ability to target specific organelles.

[0054] To investigate the imaging of Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs to detect exogenous O2 ·- The ability of Mito-Te-CDs (10 μg mL -1 )、Lyso-Te-CDs(10μg·mL -1 ) or ER-Te-CDs (10 μg mL -1 ) labeled HeLa cells were treated with different concentrations of O2 ·- (0, 4, 8, 12, 16 μM) stimulation, using X / XO as O2 ·- The cells were then washed three times with PBS and subjected to confocal imaging. ·- Imaging detection of Mito-Te-CDs (10 μg mL -1 )、Lyso-Te-CDs(10μg·mL -1 ) or ER-Te-CDs (10 μg mL -1 )-labeled HeLa cells were treated with different amounts of PMA (0, 1.25, 2.50, 3.75, 5.0 μg mL -1 ) were stimulated for 4 h. The cells were then washed three times with PBS and confocal imaging was performed. Figure 4 As shown in Figures (J)-(O) in the middle, Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs prepared by the present invention can be used to detect endogenous superoxide anions in specific organelles.

[0055] Example 7 Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs Clear Subcellular O2 ·-

[0056] In order to study the effects of Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs on subcellular O ·- To determine the elimination efficiency of MitoSox Red, HeLa cells were stained with MitoSox Red (10 μM), Lyso-HE (10 μM), or ER-HE (10 μM) in the presence or absence of Mito-Te-CDs, Lyso-Te-CDs, or ER-Te-CDs, and stained with PMA (5 μg mL -1 After the above treatment, confocal fluorescence images of cells stained with MitoSox Red, Lyso-HE or ER-HE were acquired within the wavelength window of 540-620 nm (excitation wavelength of 488 nm). Figure 5As shown in Figures (A)-(F), Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs can effectively eliminate subcellular O2 ·- .

[0057] HeLa cells were seeded in polystyrene culture dishes and cultured for 12 hours. Subsequently, the DMEM in the culture dish was replaced with fresh DMEM containing Mito-Te-CDs, Lyso-Te-CDs, ER-Te-CDs, or a mixture of three targeted functionalized Te-CDs (this mixture is a mixed system composed of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs in equal proportions) and incubated for 30 minutes. Next, the cells were exposed to O2 as an oxidative stress inducer. ·- Finally, cells were stained with Annexin V-APC and 7-AAD solution, and fluorescence was measured by flow cytometry. Figure 5 As shown in Figure (G).

[0058] Attachment Figure 5 Figure (G): (a) shows the control group cells without any treatment, in which 97.1% of the cells are normal cells; (b) shows that after the cells were stimulated with 100 μM superoxide anion, the proportion of normal cells decreased to 44.1%; (c) shows that after the cells were stimulated with 10 μg·mL -1 Cells were labeled with Mito-Te-CDs and then stimulated with 100 μM superoxide anion. At this time, the proportion of normal cells was 66.5%; (d) Inset, when 10 μg·mL -1 The cells were labeled with Lyso-Te-CDs and then stimulated with 100 μM superoxide anion. At this time, the proportion of normal cells was 61.3%; (e) Inset, when 10 μg·mL -1 ER-Te-CDs labeled cells, and then stimulated with 100 μM superoxide anion, the proportion of normal cells was 56.6%; (f) Inset, when the total concentration was 10 μg mL -1 Cells were labeled with a mixed system consisting of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs (the three components were of equal mass) and then stimulated with 100 μM superoxide anions. At this time, the proportion of normal cells was 80.5%; (g) Inset, when 20 μg mL -1 Cells were labeled with Mito-Te-CDs and then stimulated with 100 μM superoxide anion. The proportion of normal cells was 78.1% at this time. (h) Inset, when 20 μg·mL -1Lyso-Te-CDs labeled cells, and then stimulated with 100 μM superoxide anion, the proportion of normal cells was 74.4%; (i) Inset, when 20 μg·mL -1 ER-Te-CDs labeled cells, and then stimulated with 100 μM superoxide anion, the proportion of normal cells was 66.7%; (i) Inset, when the total concentration was 20 μg mL -1 The cells were labeled with a mixed system consisting of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs (the three components were of equal mass), and then stimulated with 100 μM superoxide anions. At this time, the proportion of normal cells was 96.0%.

[0059] These experimental results demonstrate that Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs all scavenge reactive oxygen species and protect cells from oxidative damage, with the higher the concentration, the greater the protective effect. Furthermore, the experiments found that, at the same total concentration, a mixed system of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs provided greater cell protection than any single component.

[0060] Example 8 Protective Effects of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs on Acute Kidney Injury

[0061] Biosafety assessment: All animal experiments were performed in accordance with the guidelines approved by the Experimental Animal Ethics Committee of Jiaxing University School of Medicine (No. JUMC2025-119). Male BALB / c mice (weight: 15-20 g) were housed in sterile plastic cages with free access to water and food, and were placed under a 12-h light / dark cycle at a controlled temperature of 25°C and acclimated for 7 days before treatment. To evaluate the biocompatibility of targeted functionalized Te-CDs in vivo, Mito-Te-CDs (100 μL, 1.2 mg mL) were added to the mice. -1 ), Lyso-Te-CDs (100μL, 1.2mg·mL -1 ), ER-Te-CDs (100μL, 1.2mg·mL -1 ), three targeted functionalized Te-CDs mixtures (100 μL, 1.2 mg mL -1 , here and below including the attached Figure 6The "three targeted functionalized Te-CD mixtures" described in the study (all consisting of a mixture of Mito-Te-CDs, Lyso-Te-CDs, and ER-Te-CDs in equal proportions) or PBS (100 μL, 10 mM) were injected intravenously into mice (n=5). All mice were sacrificed 7 days after injection with PBS or these surface-targeted functionalized Te-CDs. Blood samples were collected for biochemical analysis, and major organs, including the heart, liver, spleen, lungs, and kidneys, were removed from the mice, fixed with 4% paraformaldehyde, and embedded in paraffin. The fixed tissues were then cut into 4 mm sections and stained with hematoxylin and eosin (H&E) for pathological observation.

[0062] Metabolism and distribution of three targeted functionalized Te-CDs mixtures in mice: After intravenous injection of three targeted functionalized Te-CDs mixtures, mice were killed at 0, 2, 4, 6, 12, and 24 hours, respectively. The main organs (heart, liver, spleen, lung, and kidney) were removed and imaged using a PerkinElmer IVIS Lumina XRMS Series III in vivo imaging system at an excitation wavelength of 488 nm.

[0063] AKI model was established by fasting mice for 15 h after sterilized food was administered intramuscularly with 8 mL·kg -1 50% glycerol (v / v), followed by feeding and drinking water to mice. Two hours after injection, the AKI model was successfully established. Healthy and AKI model mice were randomly assigned to different treatments, with 5 animals in each group.

[0064] (1) Healthy mice were treated with PBS (100 μL, 10 mM) or a mixture of three targeted functionalized Te-CDs (100 μL, 0.4 mg mL -1 )treat;

[0065] (2) AKI mice were injected with PBS (100 μL, 10 mM), AMF (100 μL, 0.4 mg mL -1 ), Mito-Te-CDs (100μL, 0.4mg·mL -1 ), Lyso-Te-CDs (100μL, 0.4mg·mL -1 ), ER-Te-CDs (100μL, 0.4mg·mL -1 ) or a mixture of three targeted functionalized Te-CDs (100 μL, 0.4 mg mL -1 Two hours after the glycerol injection, the mice were intravenously injected with the active substance. Twenty-four hours later, the mice were decapitated, and kidney and blood samples were collected. The kidneys were frozen at -80°C, and kidney homogenates from different experiments were obtained and the SOD content in the kidney homogenates was measured using a SOD detection kit.

[0066] At the predetermined time point after injection (7 days after intravenous injection), the mice were decapitated and their major internal organs (lungs, heart, spleen, kidneys, liver) and blood samples were taken. Compared with the control group (i.e., healthy mice treated with PBS), no noticeable abnormalities or lesions were observed in the Mito-Te-CDs administration group, the Lyso-Te-CDs administration group, the ER-Te-CDs administration group, and the three targeted functionalized Te-CDs mixture administration groups, such as Figure 6 In addition, the analysis of blood biochemical indicators showed that blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were all normal, as shown in Figure (A). Figure 6 These results indicate that different organelle-targeting Te-CDs and their mixed systems have almost no in vivo toxicity.

[0067] Subsequently, the biodistribution of the three targeted functionalized Te-CDs mixtures in vivo was evaluated by fluorescence imaging experiments. After intravenous injection of the three targeted functionalized Te-CDs mixtures, the main organs of the mice were collected at designated time points (0, 2, 4, and 12 hours) and fluorescence imaging was performed. Figure 6 As shown in the middle panel (F), relatively high fluorescence was observed in the kidney 2 hours after injection, indicating that the three targeted functionalized Te-CDs mixtures can rapidly accumulate in the kidney. Furthermore, the fluorescence signal in the kidney significantly decreased 12 hours after injection, indicating that the three targeted functionalized Te-CDs mixtures can be rapidly excreted. This preferential accumulation and rapid in vivo clearance may be attributed to the ultrasmall size of Te-CDs.

[0068] Given that the three targeted functionalized Te-CDs mixtures have excellent O2 ·- The advantages of its elimination ability and preferential renal uptake were investigated experimentally to verify whether it can be used in the treatment of acute kidney injury. An acute kidney injury mouse model was established by inducing rhabdomyolysis. Figure 6 As shown in Figure (G), the three targeted functionalized Te-CDs mixtures were found to accumulate rapidly and effectively in the kidney tissue of AKI mice, which is very beneficial for the treatment of acute kidney injury. When renal function is impaired, two nitrogenous wastes, BUN and CRE, accumulate in the blood. Therefore, BUN and CRE are widely used as indicators for clinical assessment of renal function. Corresponding blood biochemical analysis showed that BUN (187.8±6.8mg·dL) in mice with acute kidney injury was significantly higher than that in mice with acute kidney injury. -1 ) and CRE (125.3±7.8μM) were significantly higher than those in healthy mice (BUN: 28.3±2.1mg·dL -1 ; CRE: 32.5±2.7μM), which verified the successful establishment of the AKI model. Figure 6As shown in Figures (H) and (I), treatment of mice with acute kidney injury with AMF (a known drug approved by the U.S. Food and Drug Administration (FDA) for the prevention of AKI that works by eliminating free radicals) can reduce BUN (54.2±6.5 mg·dL -1 ) and CRE (47.6±5.6μM), which was higher than that of Mito-Te-CDs alone (BUN: 91.5±6.1mg·dL -1 ;CRE: 73.4±6.3μM), Lyso-Te-CDs (BUN: 98.3±6.4mg·dL -1 ; CRE: 79.2 ± 6.7 μM) or ER-Te-CDs (BUN: 103.2 ± 7.3 mg·dL -1 ; CRE: 83.9 ± 6.4 μM), but more effective than the mixture of three targeted functionalized Te-CDs (36.6 ± 5.2 mg·dL -1 ; CRE: 40.7±4.9μM) had a slightly worse therapeutic effect. It is worth noting that the BUN and CRE levels of mice with acute kidney injury treated with the three targeted functionalized Te-CDs mixture were very close to those of healthy mice, indicating that renal function was restored by treatment with the three targeted functionalized Te-CDs mixture. In order to more directly reveal the therapeutic effect of the three targeted functionalized Te-CDs mixture in vivo, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) experiment was performed on the resected kidney tissue, and the results are shown in Figure 2. Figure 6 As shown in Figure (J). The kidney tissues of healthy mice treated with PBS or a mixture of three targeted functionalized Te-CDs showed almost no red fluorescence. In contrast, the kidney tissues of mice with acute kidney injury treated with PBS showed strong red fluorescence, indicating that cell apoptosis is closely related to acute kidney injury. Treatment with Mito-Te-CDs, Lyso-Te-CDs or ER-Te-CDs can reduce red fluorescence to a certain extent, while treatment with a mixture of three targeted functionalized Te-CDs significantly quenched the fluorescence. In other words, the three targeted functionalized Te-CDs mixture therapy can attenuate the occurrence of cell apoptosis, and its effect is better than that of single organelle-targeted Te-CDs with the same total amount, and is comparable to the efficacy of AMF. At the same time, the content of superoxide dismutase (SOD) in the kidney homogenate of each treatment group was detected, and the results are shown in Figure 1. Figure 6 As shown in Figure (K), the SOD level of mice with acute kidney injury decreased compared with the normal group, which is due to the excessive production of O2 ·- Leading to a decrease in SOD content. However, it can effectively remove O2 ·-The three-targeted functionalized Te-CDs mixture restored SOD levels in mice with acute kidney injury. The SOD activity of mice treated with the three-targeted functionalized Te-CDs mixture approached that of healthy mice and was higher than that of mice treated with a single surface-functionalized Te-CD. These experimental results confirm that the three-targeted functionalized Te-CDs mixture has an enhanced therapeutic effect in alleviating renal damage caused by acute kidney injury compared to the group treated with a single surface-functionalized Te-CD.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing tellurium-doped carbon quantum dots, characterized in that: The method comprises dispersing 2,2′-diaminodiphenyl ditelluride in deoxyethanol and heating the mixture at 200° C. in an autoclave for 4 hours. After the reaction is completed, the resulting solution is filtered through a filter membrane and then centrifuged to remove larger nanoparticles. The filtrate was collected, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain tellurium-doped carbon quantum dots.

2. The method for preparing tellurium-doped carbon quantum dots according to claim 1, wherein: The preparation method of the 2,2'-diaminodiphenyl ditelluride is as follows: Te metal and 2-iodoaniline are placed in a dry DMSO solution, CuO nanoparticles with a particle size of 10 to 50 nm are added under stirring, and then treated with KOH under a nitrogen atmosphere at 120°C. After reacting for 22 hours, the reaction mixture is cooled to room temperature, concentrated, and then separated by column chromatography to obtain 2,2'-diaminodiphenyl ditelluride.

3. A tellurium-doped carbon quantum dot, characterized in that: The tellurium-doped carbon quantum dots are prepared by the preparation method according to claim 1 or 2, and have an average particle size of 1.9 nm, green fluorescence, and are rich in amino groups on the surface.

4. Use of the tellurium-doped carbon quantum dots according to claim 3 in identifying and removing superoxide anions.

5. A tellurium-doped carbon quantum dot with mitochondrial targeting function, characterized in that: The method comprises fixing (4-carboxybutyl)triphenylphosphonium bromide on the surface of the tellurium-doped carbon quantum dots as claimed in claim 3.

6. A tellurium-doped carbon quantum dot with lysosomal targeting function, characterized in that: The method comprises fixing 3-morpholinepropionic acid on the surface of the tellurium-doped carbon quantum dots according to claim 3.

7. A tellurium-doped carbon quantum dot with endoplasmic reticulum targeting function, characterized in that: The method is to fix 3-((4-methylphenyl)sulfonylamino)propionic acid on the surface of the tellurium-doped carbon quantum dots according to claim 3.

8. Use of the tellurium-doped carbon quantum dots with mitochondrial targeting function according to claim 5, the tellurium-doped carbon quantum dots with lysosome targeting function according to claim 6, or the tellurium-doped carbon quantum dots with endoplasmic reticulum targeting function according to claim 7, characterized in that: The applications are: (a) targeted imaging of mitochondria, lysosomes, and endoplasmic reticulum; (b) identification of superoxide anions; and (c) removal of superoxide anions.

9. Use of one or more of the tellurium-doped carbon quantum dots with mitochondrial targeting function according to claim 5, the tellurium-doped carbon quantum dots with lysosome targeting function according to claim 6, and the tellurium-doped carbon quantum dots with endoplasmic reticulum targeting function according to claim 7 in the preparation of products for treating acute kidney injury.

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