Near-infrared two-region light-emitting double-ligand silver telluride quantum dot as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: (1) dissolving glutathione and organic amine hydrochloride double ligands in water, adding a silver nitrate solution, stirring and mixing, adjusting the pH value to be greater than 7, and introducing gas to remove oxygen, so as to obtain a cationic silver source; (2) tellurium powder and sodium borohydride are added into water, protective gas is introduced for stirring reaction, the solution gradually becomes amaranth, and an anion tellurium source is obtained; (3) rapidly injecting the anion tellurium source into the cation silver source, and carrying out water bath heating reaction to obtain a brown yellow solution; and (4) purifying the brown yellow solution to obtain the double-ligand silver telluride quantum dot solution. The quantum dot is good in fluorescence performance, the fluorescence quantum yield reaches up to 22.19%, good light stability and biocompatibility are achieved, the quantum dot is applied to kidney injury fluorescence imaging, kidney injury can be rapidly diagnosed and recognized, and the quantum dot is superior to a clinical blood biochemical index detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of quantum dots, in particular to a preparation method of silver telluride quantum dots based on dual ligands and its application in renal injury imaging. Background Art

[0002] Fluorescent quantum dots play an important role in the fields of biomedicine, energy and environmental protection, and display and lighting technologies. The growing demand for the application of quantum dots has prompted people to continuously improve the synthesis methods of quantum dots. Compared with the organic synthesis technology that requires the use of high-cost and highly toxic reagents, the aqueous phase synthesis technology, as another major branch of the quantum dot synthesis method, has been improved in terms of cost, environmental protection, biocompatibility, etc. However, it is still a huge challenge to synthesize high-quality quantum dots in the aqueous phase.

[0003] Silver telluride (Ag2Te) quantum dots, as one of the silver chalcogenide quantum dots, have a narrow bandgap (~0.06 eV) and tunable NIR-II emission characteristics. Because they do not carry highly toxic heavy metal elements such as cadmium, mercury, and lead, they have gradually come into the view of researchers. However, there are still few developed methods for synthesizing high-quality silver telluride quantum dots in the aqueous phase. Chen et al. used the cation exchange method to achieve the transformation of CdTe quantum dots into Ag2Te quantum dots and modified the ZnS shell layer to increase their fluorescence quantum yield to 5.6%, but the preparation process still requires the use of cadmium-containing reagents that are unfriendly to the environment. Yang et al. synthesized Ag2Te quantum dots using a polymer ligand containing multiple mercapto sites. The steric hindrance effect of the polymer ligand effectively inhibited the Ostwald ripening process of the quantum dots, and the fluorescence quantum yield was 15.2%. In the above methods, the introduction of the shell layer or the use of the polymer ligand will inevitably increase the overall particle size of the Ag2Te quantum dots, limiting their application in the field of biomedicine.

[0004] As an important organ of the human body, the kidney is prone to systemic damage due to its long-term metabolic work. Early real-time monitoring and diagnosis of kidney function injury are crucial for the intervention and treatment of acute kidney injury and chronic kidney disease. The clinical detection techniques for kidney function injury can be divided into two categories: biochemical detection and imaging evaluation. The detection methods of biochemical indexes such as blood urea nitrogen BUN, serum creatinine SCr, uric acid UA, etc. have low sensitivity and lag behind the actual pathological changes, making it difficult to achieve early diagnosis and dynamic monitoring. Although the detection and observation of the kidney tissue morphology can enable doctors to better judge the condition and provide treatment prognosis plans, the kidney puncture biopsy will inevitably damage the body. Imaging techniques with the advantage of non-invasiveness such as ultrasound, computed tomography CT, magnetic resonance imaging MRI, single photon emission computed tomography SPECT, etc. have been clinically used to evaluate kidney function. However, these methods have problems such as high cost, limited accessibility, irradiation risk, or nephrotoxicity of contrast agents.

[0005] Near-infrared second window (NIR-II) fluorescence imaging technology is suitable for kidney imaging due to its advantages such as low cost, high sensitivity, weak autofluorescence, high signal-to-background ratio, and non-ionizing radiation. However, the NIR-II fluorescence probes for kidney imaging constructed by current researchers still have deficiencies: organic fluorescent dyes have poor anti-photobleaching properties and are difficult to perform long-term imaging on the kidneys; the fluorescence quantum yield of NIR-II gold nanoclusters is still relatively low; carbon nanotubes and rare-earth doped nanoparticles need surface modification to improve dispersibility, but excessive coating with polymers will increase the particle size, hinder renal clearance and cause long-term retention toxicity, and traditional inorganic quantum dots such as CdSe and PbS are also difficult to meet the requirements of biosafety due to the risk of toxic metal ion leakage. Summary of the Invention

[0006] To solve the above problems, the present invention provides an aqueous preparation method of near-infrared second window luminescent silver telluride quantum dots with dual ligands, and applies it to the fluorescence imaging diagnosis of kidney injury.

[0007] The technical solution of the present invention is as follows:

[0008] A preparation method of near-infrared second window luminescent silver telluride quantum dots with dual ligands, comprising the following steps:

[0009] (1) Dissolve glutathione (GSH) and organic amine hydrochloride with dual ligands in water, add silver nitrate (AgNO3) solution, stir and mix, then adjust the pH>7, and purge with gas to remove oxygen to obtain a cationic silver source;

[0010] (2) Add tellurium powder (Te) and sodium borohydride (NaBH4) to water, introduce a protective gas and stir to react, and the solution gradually turns purple-red to obtain an anionic tellurium source (NaHTe);

[0011] (3) Rapidly inject the anionic tellurium source into the cationic silver source, and react by water bath heating to obtain a brown-yellow solution;

[0012] (4) Purify the brown-yellow solution to obtain a solution of silver telluride quantum dots with dual ligands.

[0013] Among them, organic amine hydrochloride is used as an auxiliary ligand to passivate the surface of silver telluride quantum dots, optimize its surface chemistry, and improve its fluorescence quantum yield.

[0014] Preferably, in step (3), the molar ratio of the cationic silver source to the anionic tellurium source is (20-2):1.

[0015] Preferably, in step (3), the reaction temperature is 100±20°C, and the reaction time is 10-240 minutes.

[0016] Preferably, in step (1), the molar ratio of glutathione to organic amine hydrochloride is (1-2):1, and the molar ratio of the total amount of the above two ligands to silver nitrate is (2-3):1.

[0017] Preferably, the organic amine hydrochloride is one or more of 2-aminoethanol hydrochloride AHCl, guanidine hydrochloride GHCl, dopamine hydrochloride DHCl, and L-lysine methyl ester hydrochloride LHCl.

[0018] Preferably, in step (2), the molar ratio of sodium borohydride to tellurium powder is (10-5):1.

[0019] Preferably, the stirring time in step (1) is 20-40 minutes, the time for aeration and deoxygenation is 20-40 minutes, and the reaction temperature in step (2) is 20-40 °C.

[0020] Preferably, in step (4), the purification is carried out by first filtering with an aqueous phase needle filter and then centrifuging with an ultrafiltration tube; preferably, the aqueous phase needle filter is 0.22 μm, the molecular weight of the ultrafiltration tube is 3 Kda, the ultrafiltration condition is a rotation speed of 4500 rpm, centrifuging for 20-30 minutes, and centrifuging 2-4 times.

[0021] The near-infrared second-region luminescent silver telluride quantum dots prepared by the above method have an ultra-small particle size, with a TEM size of 2.54±0.04 nm and a hydrodynamic diameter of 2.88 nm, which is smaller than the renal retention threshold of 5.5 nm. The application of the near-infrared second-region luminescent silver telluride quantum dots in renal injury fluorescence imaging.

[0022] The near-infrared second-region fluorescence imaging for renal injury includes the following steps:

[0023] First is renal injury modeling. Six-week-old male BALB / c mice are selected as experimental subjects. The method of ligating the unilateral ureter is used to cause kidney injury to the mice as the experimental group, and the control group mice are subjected to the same surgery except for ligation. After 2 days of postoperative culture of the experimental group and control group mice, 150 μL of 1 mg·mL -1 solution of the double-ligand silver telluride quantum dots is injected into the tail vein, and near-infrared second-region fluorescence imaging maps of the bilateral kidney regions on the back of the mice are collected at different time points, and the fluorescence intensities of the bilateral kidney regions in the collected images are analyzed.

[0024] After 2 days of postoperative culture, blood is taken from the orbital cavities of the experimental group and control group mice for blood biochemical analysis, and renal function markers in the serum: blood urea nitrogen, serum creatinine, and uric acid are detected. The test results are statistically analyzed, and after euthanizing the mice, their bilateral kidneys are dissected for histological H&E staining analysis.

[0025] Comprehensively analyze the dual-kidney fluorescence imaging diagrams, renal function markers, and renal tissue pathological sections of the experimental group and control group mice, and compare the responses of mouse kidney injury under different examination methods.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) Synthesize silver telluride quantum dots by the aqueous phase synthesis method, avoiding the use of highly toxic organic reagents.

[0028] (2) Passivate the surface of silver telluride quantum dots by the method of dual-ligand doping to improve their luminescence performance, and increase the fluorescence quantum yield of silver telluride quantum dots to 22.19%.

[0029] (3) The prepared dual-ligand silver telluride quantum dots have the advantages of generating near-infrared second-region fluorescence emission under 808nm excitation, ultra-small particle size, good biocompatibility, etc., and can perform high signal-to-background ratio imaging on the kidneys, and the imaging effect is better than that of commercial near-infrared second-region fluorescent dyes ICG and IRDye800CW.

[0030] (4) The synthesized dual-ligand silver telluride quantum dots have good photostability and biocompatibility, and their hydrated particle size is 2.8nm, which is smaller than the kidney filtration threshold (5.5nm), can be cleared by the kidneys, has a high kidney imaging contrast, and can quickly diagnose and identify kidney injury, which is superior to the clinical blood biochemical index detection method. Description of the Drawings

[0031] Figure 1 Are the TEM diagrams (scale bar: 20nm), particle size statistical histograms, and hydrated particle size characterization diagrams of (A-C) GSH-Ag2Te QDs, and the TEM diagrams (scale bar: 20nm), particle size statistical histograms, and hydrated particle size characterization diagrams of (D-F) GSH / AHCl-Ag2Te QDs.

[0032] Figure 2 Are the (A) ultraviolet spectrum diagram, (B) fluorescence spectrum diagram, and (C) infrared spectrum diagram of GSH / AHCl-Ag2Te QDs (red line) and GSH-Ag2Te QDs (black line).

[0033] Figure 3 Are the absorption spectra (A-C), fluorescence spectra (D-F), and linear fitting of NIR-II fluorescence intensity and 808nm absorbance (G-I) of IR-26, GSH-Ag2Te QDs, and GSH / AHCl-Ag2Te QDs.

[0034] Figure 4High-resolution XPS spectra of (A) Ag 3d, (B) Te 3d, (C) S 2p, (D) N 1s, (F) O 1s, and (G) C 1s for GSH / AHCl-Ag2Te QDs (red line) and GSH-Ag2Te QDs (black line).

[0035] Figure 5 Integrated fluorescence intensity changes of (A) GSH / AHCl-Ag2Te QDs and ICG under continuous 808 nm laser irradiation, (B) stability of GSH / AHCl-Ag2Te QDs in PBS solution, (C) fluorescence spectra of GSH / AHCl-Ag2Te QDs measured continuously within 60 minutes, and (D) fluorescence spectra of ICG measured continuously within 60 minutes.

[0036] Figure 6 Fluorescence emission spectra of (A) GSH-Ag2Te QDs, (B) GSH / AHCl-Ag2Te QDs, and (C) GSH / GHCl-Ag2Te QDs at different reaction times under 808 nm excitation light (inset: imaging diagrams of different silver telluride quantum dots under 808 nm excitation light and 1075 nm filter), (D) fluorescence spectra comparison chart at the optimal reaction time of each formulation, and (E) fluorescence integrated intensity comparison chart between 900 - 1400 nm: ① GSH-Ag2Te QDs, ② GSH / AHCl-Ag2Te QDs, ③ GSH / GHCl-Ag2Te QDs

[0037] Figure 7 Cell viability of (A) human embryonic kidney cells HEK-293T and (B) HeLa cells after incubation with different concentrations of GSH / AHCl-Ag2Te QDs (0 - 100 μg·mL -1 ) for 24 hours.

[0038] Figure 8 Indices of blood routine tests at 1 day, 7 days, and 14 days after tail vein injection of GSH / AHCl-Ag2Te QDs solution (experimental group) and PBS solution (control group) in mice: (A) white blood cell count WBC, (B) red blood cell count RBC, (C) monocyte count Mon, (D) mean corpuscular volume MCV, (E) mean corpuscular hemoglobin concentration MCHC, (F) mean corpuscular hemoglobin content MCH, (G) lymphocyte percentage Lym, (H) hemoglobin HGB, (I) hematocrit HCT; indices of blood biochemical tests: (J) blood urea nitrogen BUN, (K) serum creatinine CREA, (L) uric acid UA.

[0039] Figure 9 After 14 days of injecting GSH / AHCl-Ag2Te QDs solution into the tail vein of mice and 14 days of injecting PBS solution, the main organs (heart, liver, spleen, lung, kidney) were dissected for H&E staining detection (scale bar: 50 μm).

[0040] Figure 10 (A) NIR-II fluorescence in vivo imaging of mice before and after tail vein injection of GSH / AHCl-Ag2Te QDs, (B) trend chart of fluorescence intensity change of the kidney on the back of the mouse, (C) trend chart of fluorescence intensity change of the liver on the ventral side of the mouse.

[0041] Figure 11 (A) Optical pictures of the main organs (heart, liver, spleen, lung, kidney) of mice 24 hours after tail vein injection of GSH / AHCl-Ag2Te QDs material, (B) NIR-II fluorescence imaging pictures, (C) statistical chart of fluorescence intensity of the main organs after dissection.

[0042] Figure 12 (A) NIR-II fluorescence in vivo imaging of mice before and after tail vein injection of the dye IRDye-800CW, (B) trend chart of fluorescence intensity change of the kidney on the back of the mouse, (C) trend chart of fluorescence intensity change of the liver on the ventral side of the mouse.

[0043] Figure 13 (A) NIR-II fluorescence in vivo imaging of mice before and after tail vein injection of the dye ICG, (B) trend chart of fluorescence intensity change of the kidney on the back of the mouse, (C) trend chart of fluorescence intensity change of the liver on the ventral side of the mouse.

[0044] Figure 14 (A) NIR-II fluorescence imaging of the back of mice 10 minutes after tail vein injection of GSH / AHCl-Ag2Te QDs, the dye IRDye-800CW, and the dye ICG, (B) fluorescence intensity profile (black) and Gaussian fitting line (red) along the white line in Figure (A).

[0045] Figure 15 H&E staining images of the kidneys of experimental group UUO mice and sham operation control group Sham mice, scale bar: 50 μm (arrow indicates: tubular dilation).

[0046] Figure 16 (A) NIR-II fluorescence in vivo imaging of Sham mice and UUO mice before and after tail vein injection of GSH / AHCl-Ag2Te QDs respectively, (B) trend chart of fluorescence intensity change of the kidney on the back of Sham mice, (C) trend chart of fluorescence intensity change of the kidney on the back of UUO mice.

[0047] Figure 17 Optical images and NIR-II fluorescence imaging pictures of the double kidneys of (A) Sham mice and UUO mice 24 h after tail vein injection of GSH / AHCl-Ag2Te QDs, (B) fluorescence intensity statistics of the double kidneys, and (C) comparison of the LK / RK ratio.

[0048] Figure 18 For (A) the synthesis process of the double-ligand silver telluride quantum dots and (B) the near-infrared II fluorescence imaging map of kidney injury using the double-ligand silver telluride quantum dots. Specific implementation mode

[0049] In the embodiments, the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. The methods are all conventional methods unless otherwise specified. The raw materials can all be obtained from public commercial channels unless otherwise specified.

[0050] Example 1

[0051] A preparation method of a near-infrared II emitting double-ligand silver telluride quantum dot that can be used for fluorescence imaging of kidney injury, comprising the following steps:

[0052] (1) Weigh 0.0576 g of glutathione GSH (ligand 1) and 0.0122 g of 2-aminoethanol hydrochloride (ligand 2), dissolve them in 43 mL of water, and then add 4 mL of 0.0212 g of silver nitrate AgNO3 dissolved in water, so that the molar ratio of the total ligand amount to silver nitrate is 2.5:1. Stir and mix at room temperature for 20 minutes, and a white turbidity appears. Use 1 mol·L -1 of NaOH solution to adjust the pH of the solution. After the solution becomes clear, adjust it to pH 9.5, and purge with gas to remove oxygen for 30 minutes to obtain a GSH / AHCl-Ag cationic silver source.

[0053] (2) Weigh 0.0160 g of tellurium powder Te and 0.0236 g of sodium borohydride NaBH4 in a molar ratio of 1:5 and add them to 10 mL of deionized water. Purge with argon to remove oxygen and stir and react at room temperature for 90 minutes. The solution gradually turns purplish red to obtain a NaHTe anionic tellurium source.

[0054] (3) Use a syringe to take 2 mL of the NaHTe anionic tellurium source and quickly inject it into the GSH / AHCl-Ag cationic silver source, and heat it in a water bath to 100 °C and react for 120 minutes to obtain a brownish-yellow liquid.

[0055] (4) After filtering the pale yellow solution through a 0.22 μm aqueous phase needle filter, centrifuge it using a 50 ml 3KDa ultrafiltration tube at a speed of 4500 rpm for 20 minutes, and repeat 2 - 3 times to obtain a purified solution of the dual-ligand silver telluride quantum dots GSH / AHCl-Ag2TeQDs.

[0056] Furthermore, the prepared dual-ligand GSH / AHCl-Ag2Te QDs were characterized. Figure 1 Transmission electron microscopy showed that their morphologies were all in good spherical states, with good dispersibility and uniform particle sizes. The average particle size was 2.54 ± 0.04 nm. The hydrodynamic diameter was characterized by dynamic light scattering method to be 2.86 nm. The average particle size of the single-ligand silver telluride quantum dots GSH-Ag2Te QDs was 2.33 ± 0.06 nm, and the hydrodynamic diameter was 2.48 nm. In comparison, the particle size of the dual-ligand GSH / AHCl-Ag2Te QDs increased slightly, but both were less than the renal retention threshold of 5.5 nm, which was beneficial for application in organisms.

[0057] Figure 2 The ultraviolet-visible-near-infrared absorption spectrum showed that the absorption peak of the single-ligand GSH-Ag2Te QDs was located at 700 nm, and the absorption peak of the dual-ligand GSH / AHCl-Ag2Te QDs was red-shifted to 800 nm. Figure 2 Figure B is a comparison diagram of the fluorescence emission spectra of the two under 808 nm excitation. The fluorescence integrated intensity of the dual-ligand GSH / AHCl-Ag2Te QDs increased by 2.14 times. The surface functional groups of the two kinds of quantum dots were characterized using XPS and Fourier transform infrared spectrometer. Figure 2 The infrared spectrum in Figure C showed that the absorption peak at 2524 cm -1 of the glutathione molecule in the blue line belonged to the stretching vibration of the free mercapto group S-H, and this absorption peak did not appear in the infrared spectra of the two kinds of silver telluride quantum dots, indicating that glutathione as a thiol ligand for synthesizing silver telluride quantum dots mainly coordinated with Ag through the mercapto group -SH on GSH. The FT-IR spectra of the dual-ligand GSH / AHCl-Ag2Te QDs (red line) and the single-ligand GSH-Ag2Te QDs (black line) showed characteristic peaks of carboxylate groups at 1647 cm -1 and 1638 cm -1 as well as 1388 cm -1 and 1402 cm -1 , attributed to the stretching vibrations of C=O and C-O, and both had certain offsets.

[0058] Furthermore, the fluorescence quantum yield is an important performance parameter for evaluating the application of materials in imaging. Therefore, using the NIR-II fluorescent dye IR-26 (QY = 0.5%) dissolved in 1,2-dichloroethane as a reference, the fluorescence quantum yields (QY) of GSH-Ag2Te QDs and GSH / AHCl-Ag2Te QDs dissolved in water were investigated.

[0059] Figure 3 It shows that the relative quantum yield of GSH-Ag2Te QDs is 7.75%, and the relative quantum yield of GSH / AHCl-Ag2Te QDs is 22.19%, which is 2.86 times higher than that of the single-ligand GSH-Ag2Te QDs. Using the dye IR-26 (QY = 0.5%) as a reference, compared with silver telluride quantum dots synthesized by previous methods, it is higher than the previously reported core-shell silver telluride quantum dots Ag2Te@Ag2S QDs (4.3%), Ag2Te@ZnS QDs (5.6%), and cation-treated NAC-Ag2Te QDs (8.0%). Compared with previously reported luminescent materials that can be metabolized by the kidney, it is higher than the dual-ligand gold clusters MHA / Cystm-AuNCs (14.0%), CDIR2 (2.2%), and FBP 912 (0.86%).

[0060] Table 1 Element content table of GSH / AHCl-Ag2Te QDs and GSH-Ag2Te QDs

[0061]

[0062] Table 1 shows the results of the elemental content analysis of the XPS total spectra of the two kinds of quantum dots. The Ag:Te molar ratio of the single-ligand GSH-Ag2Te QDs is 2.9:1, while the silver telluride ratio of the dual-ligand GSH / AHCl-Ag2Te QDs is 3.2:1, indicating that the synthesis method of the mixed dual-ligand increases the silver / tellurium molar ratio. The Ag:Te molar ratios of both are higher than the stoichiometric number 2:1 of Ag2Te. The reason is presumably that the smaller sizes of the two kinds of silver telluride quantum dots result in an increase in their specific surface area. Therefore, the abundant silver on the surface of the quantum dots coordinates with the mercapto groups on glutathione.

[0063] For GSH / AHCl-Ag2Te QDs, S:Ag is 1.39:1, which is higher than 0.64:1 of GSH-Ag2Te QDs, indicating that the synthesis method of the mixed dual-ligand enhances the coverage of mercapto ligands on the surface of silver telluride quantum dots, improves the coordination rate of the mercapto groups on glutathione with silver ions, and more mercapto groups are successfully anchored on the surface of the quantum dots, significantly optimizing the surface chemistry of the quantum dots.

[0064] Figure 4The high-resolution XPS analysis results of each element of the dual-ligand GSH / AHCl-Ag2Te QDs and the single-ligand GSH-Ag2Te QDs are shown. The binding energy of the N1s fitting peak of the dual-ligand quantum dots is slightly higher than that of the single-ligand quantum dots, and the proportion of the Ag-N peak is higher than that of the single-ligand quantum dots, while the proportion of the N-H peak is lower than that of the single-ligand quantum dots. It is speculated that the introduction of 2-aminoethanol hydrochloride improves the coordination between Ag and amino groups, reduces the amount of uncoordinated amino groups, and synergistically optimizes the coordination structure on the surface of the quantum dots, thereby enhancing the stability of the quantum dots.

[0065] In summary, for the single-ligand GSH-Ag2Te QDs, the mercapto group (-SH) on its ligand glutathione can be anchored on the surface of Ag2Te QDs through Ag-S bonds, but the single ligand is prone to partial shedding due to the dynamic equilibrium. In the synthesized dual-ligand GSH / AHCl-Ag2Te QDs, there is an increase in the silver / tellurium molar ratio and the sulfur / silver molar ratio. It is speculated that one of the ligands, glutathione, acts as a tripeptide to provide long-chain steric hindrance, while the (-NH2) of 2-aminoethanol hydrochloride plays an auxiliary passivation role. It fills the voids on the surface of the quantum dots through Ag-N bonds and forms a complementary coordination network with Ag-S bonds, thereby obtaining a denser ligand layer, reducing the formation of dangling bonds on the surface of the quantum dots, and improving its fluorescence quantum yield.

[0066] Figure 5 It shows that compared with the commercial NIR-II luminescent dye indocyanine green ICG, the dual-ligand GSH / AHCl-Ag2Te QDs have superior anti-photobleaching properties and chemical stability in PBS solution for a long time.

[0067] Figure 6 It shows the fluorescence properties of silver telluride quantum dots under different formulations. Compared with Comparative Examples 1-2, the dual-ligand GSH / AHCl-Ag2Te QDs used 2-aminoethanol hydrochloride as a ligand during the synthesis process, better passivated the surface of the silver telluride quantum dots, optimized the surface chemistry of the quantum dots, and increased its integrated fluorescence intensity.

[0068] Furthermore, the biocompatibility of the prepared dual-ligand GSH / AHCl-Ag2Te QDs was investigated, mainly by examining the cytotoxicity and biotoxicity of the material.

[0069] Figure 7 It shows that even when the concentration of GSH / AHCl-Ag2Te QDs quantum dots is increased to 100 μg·mL -1 , the survival rates of two kinds of cells, human embryonic kidney cells HEK-293T and human breast cancer cells HeLa cells, are 79.95% and 70.50% respectively, both above 70%, higher than the previous cadmium-based and lead-based quantum dots containing highly toxic heavy metal elements, indicating that the GSH / AHCl-Ag2Te QDs quantum dots have low cytotoxicity.

[0070] Figure 8 It shows that the blood routine indexes and blood biochemical indexes of mice 1 day, 7 days, and 14 days after injection of the dual-ligand GSH / AHCl-Ag2Te QDs are within the normal range, indicating that the material does not cause ischemia or inflammation to the mice, and does not cause acute damage to the renal function of the mice like other contrast agents.

[0071] Figure 9 It shows that there are no obvious morphological changes in the heart, liver, spleen, lung, and kidney organ tissues of mice 14 days after injection of the dual-ligand GSH / AHCl-Ag2Te QDs, the integrity is good, and no damage and inflammatory reaction phenomena are seen; the cell distribution of each organ is normal, and no degeneration and necrosis are seen, which is consistent with the results of blood routine and blood biochemical analysis.

[0072] The above results indicate that the GSH / AHCl-Ag2Te QDs material has good biocompatibility and is expected to be applied in vivo.

[0073] Example 2

[0074] This embodiment is the application of near-infrared II-emitting dual-ligand silver telluride quantum dots in fluorescence imaging of the mouse kidney, and the specific process is as follows:

[0075] Six-week-old male BALB / c mice were selected as the research objects. Before the imaging experiment, the hair on the back and abdomen of the mice was removed using a depilatory. The AniView 30F near-infrared II in vivo imaging system was used to perform fluorescence imaging on the mice. First, images of the mice before drug administration were taken. Subsequently, 150 μL of 1 mg·mL -1 The dual-ligand silver telluride quantum dots enter the mice, and then NIR-II fluorescence imaging pictures were taken at different time points (10 min, 30 min, 60 min, 90 min, 2 h, 3 h, 4 h, 5 h, 18 h, 24 h after drug administration). During the imaging process, the mice were anesthetized with 2% isoflurane gas. The specific imaging parameter settings are shown in the following table:

[0076] Table 2 Parameter settings of near-infrared II in vivo imaging system

[0077]

[0078] After the imaging was completed, the mice were euthanized and their heart, liver, spleen, lung, and kidney organs were collected, and in vitro fluorescence analysis was performed on them using a near-infrared imaging system.

[0079] Figure 10It shows that before injecting the GSH / AHCl-Ag2Te QDs material, the background fluorescence of the mouse is clean and there is no interference. Ten minutes after injecting the GSH / AHCl-Ag2Te QDs material, the contour shapes of the two kidneys on the back of the mouse are clearly visible, and there is almost no background signal around. As time prolongs, the fluorescence signal of the mouse kidneys will gradually decrease ( Figure 10 B), which indicates that the GSH / AHCl-Ag2Te QDs material is gradually cleared from the mouse body. After 10 minutes of injection, a nearly circular fluorescent signal in the bladder area was also observed on the ventral surface of the mouse, located below the ventral surface of the mouse, which also indicates that the GSH / AHCl-Ag2Te QDs material can enter the bladder through the ureter after being metabolized by the kidneys. As time prolongs, the fluorescent signal in the bladder area also gradually decreases ( Figure 10 C), and there is almost no fluorescent signal at 24 h, indicating that the fluorescent material in the mouse body has been basically cleared out of the body.

[0080] A fluorescent signal in the liver area was also observed on the ventral surface of the mouse, but it is much lower than the fluorescent signal in the bladder area. This may be because after the GSH / AHCl-Ag2Te QDs are administered via the tail vein, some of the material will bind to serum proteins, making the overall size of the material higher than the kidney filtration threshold, which can prevent the material from being rapidly cleared by the kidneys and partially transported to the liver. The content of glutathione GSH and cysteine in the hepatic sinusoids of the liver is relatively rich, which may increase the uptake of the material by liver cells and then be cleared through the hepatobiliary pathway.

[0081] Figure 11 It shows the fluorescence signal conditions of the isolated organs of the mouse. It can be found that there are fluorescence signals in both the liver and kidneys of the mouse, while there is no obvious fluorescence in the heart, lungs and spleen organs. This indicates that the GSH / AHCl-Ag2Te QDs will be mainly metabolized by the kidneys and liver, and very little is metabolized by the spleen.

[0082] Figure 14 It shows a comparison of the kidney signal / background ratio 10 minutes after injecting the three materials of GSH / AHCl-Ag2Te QDs, IRDye800CW, and ICG. Along Figure 14The fluorescence intensity of the red line in [the relevant context] was statistically analyzed. After Gaussian fitting, the kidney signal-to-background ratios (SBR) of GSH / AHCl-Ag2Te QDs, IRDye800CW, and ICG were calculated to be 3.50, 1.73, and 1.33 respectively. The kidney signal-to-background ratio of GSH / AHCl-Ag2Te QDs is much higher than that of the latter two commercial dyes. The kidney imaging profile of the mouse using the GSH / AHCl-Ag2Te QDs material is clear, and the contrast with the background tissue is greater, indicating that the kidney imaging ability of the GSH / AHCl-Ag2Te QDs material is higher than that of the commercial dyes ICG and IRDye800CW. Compared with the fluorescence probes reported in previous literature, it also performs well. Therefore, the GSH / AHCl-Ag2Te QDs material is expected to be used for the research and analysis of mouse kidney injury.

[0083] Example 3

[0084] This embodiment is the application of near-infrared second-region emitting dual-ligand silver telluride quantum dots in fluorescence imaging of mouse kidney injury. The specific process is as follows:

[0085] Six-week-old male Balb / c mice (~20 g) were purchased from the Guangdong Provincial Center for Laboratory Animals and used as experimental subjects. They were raised in an environment with a temperature of 25 °C and a 12 / 12 h light / dark cycle, and had free access to water and food. All animal experiment procedures were strictly carried out in accordance with the requirements of the Regulations on the Administration of Laboratory Animals in China, the Guide for the Care and Use of Laboratory Animals, and the Regulations on the Administration of Laboratory Animals in Guangdong Province. Further establishment of the kidney injury model: Before the operation, the mice were depilated. The mice were anesthetized with 1.25% tribromoethanol (Avertin), and the anesthetic dose was 100 μL / 10 g of mouse body weight. The injection method was intraperitoneal injection. After the mice were anesthetized, the left ureter was exposed through a left incision, and the left ureter was ligated with a 5-0 nylon surgical suture, and then the wound was sutured to establish UUO model mice.

[0086] Two days after the UUO mice were surgically cultured, blood was taken from their orbital cavities for blood biochemical analysis to detect the renal function markers in the serum: blood urea nitrogen, serum creatinine, and uric acid. The test results were statistically analyzed. After the mice were euthanized, their bilateral kidneys were dissected for histological H&E staining analysis.

[0087] Two days after the UUO mice were surgically cultured, an imaging experiment was carried out. 150 μL of 1 mg·mL -1For the silver telluride quantum dot solution, a mouse was subjected to fluorescence imaging at different time points (10, 30, 60, 90, 120, 240, 1440 min) using an AniView 30F in vivo imaging system in the second near-infrared region. During the imaging process, the mouse was anesthetized with 2% isoflurane gas. After the fluorescence imaging was completed, the mouse was euthanized, and the excised kidney was taken for NIR-II fluorescence imaging observation and analysis. The imaging conditions are shown in Table 2 of Example 2.

[0088] Figure 15 It shows that the right kidney of the UUO mouse has renal tubular dilation. The pathological sections of the left kidney of the UUO mouse and the double kidneys of the Sham mouse that underwent the same surgical procedures as the UUO mouse except for ureteral ligation in Comparative Example 7 all show that the renal tissue structure is relatively normal, indicating that ureteral ligation in mice for 2 days did cause kidney damage in mice.

[0089] Table 3 Blood biochemical analysis results of the UUO experimental group and the Sham control group (n = 3, P > 0.05)

[0090]

[0091] However, Table 3 shows that there is no significant difference in the renal function markers serum creatinine CREA, blood urea nitrogen BUN, and uric acid UA between the UUO mice and the Sham mice that underwent the same surgical procedures as the UUO mouse except for ureteral ligation in Comparative Example 7 (P > 0.05). This is because the unobstructed kidneys of the UUO mice still have compensatory ability, so the commonly used renal function markers in clinical practice have poor diagnostic effects for early kidney injury.

[0092] Figure 16 It shows that after the GSH / AHCl-Ag2Te QDs material enters the mouse body, it can clearly image the double kidneys of the mouse. Among them, the fluorescence intensity in the double kidney regions on the back of the UUO mouse also gradually decreases with time, and the fluorescence intensity ratio of the left kidney / right kidney remains between 1.6 and 1.8. In the imaging of the UUO mouse, the fluorescence signal of the right kidney obstructed by ureteral ligation is much lower than that of the left kidney, which reflects that ureteral ligation for 2 days causes abnormal kidney filtration in mice, and the blood perfusion in the obstructed side kidney of the mouse is significantly reduced, while it is enhanced in the unobstructed side kidney. For the Sham mice that underwent the same surgical procedures as the UUO mouse except for ureteral ligation in Comparative Example 7, the fluorescence intensity in the double kidney regions on the back also gradually decreases with time, but the fluorescence intensity ratio of the left kidney / right kidney remains between 0.9 and 1.1, showing a significant difference from the UUO mouse.

[0093] Figure 17After the imaging was completed, the double kidneys of euthanized UUO mice and Sham mice in Comparative Example 5, which had undergone the same surgical procedures except for ureteral ligation, were taken for observation. Under daylight, the color and morphology of the right kidney of UUO mice were different from those of normal kidneys, showing swelling and whitening. Then, NIR-II fluorescence imaging of the excised kidneys showed that there was a significant difference in the fluorescence intensity of the double kidneys of UUO mice (M1 is the left kidney, M2 is the right kidney), and the fluorescence intensity ratio of the left and right kidneys was about 1.5, which was close to the ratio data of 1.6 - 1.8 obtained in in vivo imaging. All the above experimental results indicate that imaging using GSH / AHCl-Ag2Te QDs materials can identify kidney injury caused by ureteral ligation, which is superior to the traditional clinical method for detecting renal function markers.

[0094] Comparative Example 1

[0095] This embodiment is a comparative example of Example 1, synthesizing GSH-Ag2Te QDs single-ligand silver telluride quantum dots. It is necessary to maintain the molar ratio of the total ligand silver nitrate at 2.5:1, and the difference is that the ligand composition is changed to only contain the single-ligand form of glutathione GSH.

[0096] (1) Weigh 0.0960 g of glutathione GSH and dissolve it in 43 mL of water. Then add 4 mL of 0.0212 g of silver nitrate AgNO3 dissolved in water, stir and mix at room temperature for 20 minutes, and a white turbidity appears. Use 1 mol·L -1 NaOH solution to adjust the pH of the solution. After the solution becomes clear, adjust it to pH 9.5, and deoxygenate by aeration for 30 minutes to obtain the GSH-Ag cationic silver source.

[0097] Other synthesis steps are the same as those in Example 1, and GSH-Ag2Te QDs single-ligand silver telluride quantum dots are synthesized. Figures 1-5 And Table 1 contains the characterization results for it. In contrast, the double-ligand GSH / AHCl-Ag2Te QDs used an auxiliary ligand during the synthesis process, passivated the surface of the silver telluride quantum dots, optimized the surface chemistry of the quantum dots, and improved the fluorescence quantum yield of the quantum dots.

[0098] Comparative Example 2

[0099] This embodiment is a comparative example of Example 1. To synthesize different double-ligand silver telluride quantum dots, it is necessary to maintain the molar ratio of the total ligand silver nitrate at 2.5:1, and the difference is that the ligand composition is changed to the form of glutathione GSH and guanidine hydrochloride GHCl.

[0100] (1) Weigh 0.0576 g of glutathione GSH (ligand 1) and 0.0119 g of guanylthiourea hydrochloride GHCl (ligand 2), dissolve them in 43 mL of water, and then add 4 mL of 0.0212 g of silver nitrate AgNO3 dissolved in water, so that the molar ratio of the total ligand amount to silver nitrate is 2.5:1. Stir and mix at room temperature for 20 minutes, and a white turbidity appears. Use 1 mol·L -1 of NaOH solution to adjust the pH of the solution. After the solution becomes clear, adjust it to pH 9.5 again, and aerate to remove oxygen for 30 minutes to obtain the GSH / GHCl-Ag cationic silver source.

[0101] The other synthesis steps are the same as those in Example 1, and the double-ligand GSH / GHCl-Ag2Te QDs quantum dots are synthesized. Figure 6 D and E show that the double-ligand GSH / GHCl-Ag2Te QDs synthesized in this comparative example have a higher integrated fluorescence intensity than the single-ligand GSH-Ag2Te QDs in Comparative Example 1, but lower than the GSH / AHCl-Ag2Te QDs synthesized with glutathione and 2-aminoethanol hydrochloride as the mixed ligand in Example 1.

[0102] Comparative Example 3

[0103] This embodiment is a comparative example of Example 2, mainly comparing the imaging effect of the dye IRDye-800CW and the double-ligand silver telluride quantum dots on the mouse kidneys. The difference is that 150 μL of 0.25 mg·mL -1 of the dye IRDye-800CW is injected into the mouse body, and the other imaging operation steps are the same as those in Example 2.

[0104] Figure 12 It can be seen that after the mouse is injected with IRDye800CW, compared with before injection, at the 10-minute node, the fluorescence signal on its back has almost spread throughout the body, and it is difficult to clearly observe the kidney contour. This indicates that the true kidney signal will be strongly covered by the skin signal after injecting the IRDye800CW material. The fluorescence signal on the ventral side also almost spreads throughout the body, but the signal in the bladder area under the abdomen is slightly higher. This phenomenon shows that IRDye800CW will also be excreted from the body through the bladder. As time prolongs, the fluorescence signals at the back kidney and the ventral liver will gradually decrease, and the fluorescence signal in the ventral bladder area first increases and then decreases. By 24 h, there is almost no fluorescence signal in the whole body, and the IRDye800CW fluorescent material has been cleared from the body.

[0105] Comparative Example 4

[0106] This embodiment is a comparative example of Example 2, mainly comparing the imaging effect of the dye ICG and the double-ligand silver telluride quantum dots on the mouse kidneys. The difference is that 150 μL of 0.25 mg·mL-1 The dye ICG was injected into the mice, and the other imaging operation steps were the same as those in Example 2.

[0107] Figure 13 It can be seen that 10 minutes after the injection of the ICG material, a weak fluorescence signal in the kidney area was faintly visible on the back of the mouse, while the fluorescence signal on the ventral side was very strong. As time passed, the fluorescence signal on the back of the mouse could not always show the condition of the kidney, and while the fluorescence signal on the ventral side was gradually decreasing, it was still higher than the fluorescence signal on the back. This is because after ICG was injected into the mice via the tail vein, it was mainly actively taken up by hepatocytes, excreted via bile, and excreted in feces through the hepatobiliary pathway as the main metabolic pathway, and ICG hardly metabolized through the renal pathway. Therefore, a significantly bright fluorescence signal was observed in the liver area of the mice, while the fluorescence signal in the kidney area was weak, which also indicated that the traditional ICG material was not suitable for renal imaging. In the imaging at 24 h, it could be observed that there was basically no fluorescence signal in the whole body of the mice, indicating that the ICG fluorescent material had been cleared from the body.

[0108] Comparative Example 5

[0109] This embodiment is a comparative example of Example 3. The surgical operation process was the same as that of Example 3 except for ligating the ureter. It was a control group of Sham mice, and the subsequent experimental operations were the same as those of UUO mice.

[0110] Figures 15-17 And Table 3 contains its experimental results. The results of HE staining analysis and fluorescence imaging analysis of the kidneys of Sham mice and UUO mice both showed that the right renal function of UUO mice was abnormal two days after ligating the ureter. However, the renal function marker analysis could not identify and analyze this in a timely manner, indicating that the method of using GSH / AHCl-Ag2Te QDs material for near-infrared second-region fluorescence imaging of renal injury was superior to the traditional renal function marker monitoring method.

[0111] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of near-infrared II-region luminescent silver telluride quantum dots with dual ligands, characterized in that, It includes the following steps: (1) Dissolve glutathione and organic amine hydrochloride double ligands in water, add silver nitrate solution, stir and mix, then adjust the pH > 7, and aerate to remove oxygen to obtain a cationic silver source; (2) Add tellurium powder and sodium borohydride to water, introduce a protective gas and stir to react, and the solution gradually turns purple-red to obtain an anionic tellurium source; (3) Rapidly inject the anionic tellurium source into the cationic silver source, and react by water bath heating to obtain a yellowish-brown solution; (4) Purify the yellowish-brown solution to obtain a double-ligand silver telluride quantum dot solution.

2. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of the cationic silver source to the anionic tellurium source is (20 - 2):

1.

3. The preparation method according to claim 2, characterized in that, In step (3), the reaction temperature is 100 ± 20 °C, and the reaction time is 10 - 240 minutes.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of glutathione to organic amine hydrochloride is (1 - 2):1, and the molar ratio of the total amount of the above two ligands to silver nitrate is (2 - 3):

1.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The organic amine hydrochloride is one or more of 2-aminoethanol hydrochloride AHCl, guanidine hydrochloride GHCl, dopamine hydrochloride DHCl, and L-lysine methyl ester hydrochloride LHCl.

6. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio of sodium borohydride to tellurium powder is (10 - 5):

1.

7. The preparation method according to claim 6, characterized in that, The stirring time in step (1) is 20 - 40 minutes, the time for aerating to remove oxygen is 20 - 40 minutes, and the reaction temperature in step (2) is 20 - 40 °C.

8. The preparation method according to any one of claims 1 to 4, characterized in that, The purification in step (4) is to first filter with a water-phase needle filter and then centrifuge with an ultrafiltration tube; preferably, the water-phase needle filter is 0.22 μm, the molecular weight of the ultrafiltration tube is 3 Kda, the ultrafiltration condition is a rotation speed of 4500 rpm, centrifuge for 20 - 30 minutes, and centrifuge 2 - 4 times.

9. Near-infrared second-region luminescent double-ligand silver telluride quantum dots prepared by the method according to any one of claims 1 to 8.

10. Application of the near-infrared second-region luminescent double-ligand silver telluride quantum dots according to claim 9 in renal injury fluorescence imaging.

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