A water-soluble near-infrared two-region luminescent gold nanocluster based on aromatic hydrogen-hydrogen spatial interaction and a preparation method and application thereof
Patent Information
- Application Number
- CN202610920300.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-24
- Publication Date
- 2026-09-15
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Figure CN122750352A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of bionanomaterials and cancer diagnostics, specifically to a water-soluble near-infrared II luminescent gold nanocluster based on aromatic hydrogen-hydrogen spatial interactions, its preparation method, and its applications. Background Technology
[0002] The establishment of atomically precise nanochemistry is developing methods for the precise programming of nanoparticle size and structure. Precisely structured gold nanoclusters (AuNCs) are among the most widely used nanoparticles in atomically precise nanochemistry. With diameters ranging from 1 to 3 nm, they exist in an intermediate state between discrete atoms and plasmonic nanoparticles, exhibiting molecular-like properties (such as HOMO-LUMO transitions, enabling luminescence in the 500-1700 nm wavelength range). Surface engineering is a cutting-edge research direction in the field of gold nanoclusters (AuNCs), fundamentally because the ligand layer on the AuNC surface has a crucial and decisive influence on both fundamental research and practical applications. As the outermost layer of the nanocluster, the surface ligands directly interact with the external environment (such as molecules, biomolecules, cells, tissues, etc.), thus determining the performance of AuNCs in various environments.
[0003] The surface ligand types of gold nanoclusters (AuNCs) mainly include thiols, alkynes, and phosphorus-containing molecules, with thiols attracting particular attention due to their ease of forming gold-sulfur bonds. AuNCs synthesized using 6-mercaptohexanoic acid as a ligand... 25 It exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria; Au synthesized with captopril as a ligand... 25 It can enhance cytotoxicity against cancer cells and trigger cell death by inhibiting mitochondrial oxidative phospholysis; Au synthesized with mercaptobenzoic acid as a ligand 44By phosphorylation modification of the surface, gold nanoclusters can achieve highly spatiotemporal in vivo bone-targeted photoluminescence (PL) imaging in the second near-infrared window (NIR-II) and enhance the therapeutic effect of rheumatoid arthritis (RA). Their high versatility makes them promising for the diagnosis and treatment of various diseases. However, the native ligand shell structure of gold nanoclusters protected by thiol molecules is very unstable when coexisting with excess thiol molecules, often resulting in ligand exchange reactions. The liver is the central organ responsible for metabolism and detoxification. It is rich in glutathione (GSH, approximately 10 mmol), and its mediated biotransformation process is the body's natural defense mechanism for clearing exogenous toxins. For gold nanoclusters, glutathione can significantly shorten the half-life of gold nanoclusters in the bloodstream and accelerate their clearance rate from the body by replacing the ligands on the surface of the gold nanoclusters. However, before gold nanoclusters reach their target tissues, glutathione-mediated biotransformation processes can replace surface-modified functional or targeting ligands, impairing the specific functions or active targeting capabilities of the gold nanoclusters. In these environments, premature ligand dissociation is a biochemical barrier hindering the active targeting and functionalization of gold nanoclusters.
[0004] In summary, there is an urgent need for a ligand engineering strategy that can precisely regulate the glutathione-mediated biotransformation of nanoparticles in the liver, in order to overcome the biochemical barrier of premature ligand dissociation and limited nanoparticle function, and fill the relevant technological gap. Studies have shown that regulating the metal-ligand bond can modulate the liver biotransformation process of gold nanoclusters (Lu H., Ren Y., Qi Y., et al. Overcoming hepatic biotransformation barrier of gold nanoparticles via Au-Se bond for enhanced in vivo active targeting[J]. ACSNano, 2024, 18, 29178-29188.). Selenium has a stronger binding affinity to gold than sulfur. Gold nanoclusters anchored by selenium can significantly improve the targeting efficiency of small-molecule-mediated gold nanoclusters on hepatocytes and alter the transport, distribution, and clearance pathways of gold nanoclusters in the liver. However, the synthesis of molecules using selenyl groups as ligands is difficult, with limited variety, and the methods for cluster synthesis are unsatisfactory. In contrast, the synthesis of molecules using thiol groups as ligands is easier, allowing for the design of a wider variety, and the methods for cluster synthesis are mature. Therefore, a ligand engineering strategy that precisely regulates the glutathione-mediated biotransformation of nanoparticles in the liver by designing suitable thiol molecules is crucial. Currently, there is no research on the precise regulation of the binding ability of ligand layers to gold nuclei through functional group positional isomerization. Summary of the Invention
[0005] Typically, large amounts of thiols in vivo can replace the native ligands on the surface of gold nanoclusters, altering their behavior and weakening their ability to target organs and treat diseases. The purpose of this invention is to provide a water-soluble near-infrared II luminescent gold nanocluster based on aromatic hydrogen-hydrogen spatial interactions and its preparation, solving the problems of ligand dissociation and poor stability in the presence of exogenous thiols in traditional gold nanoclusters. This enables precise transport and targeting of related nanoparticles in vivo, facilitating accurate tumor imaging.
[0006] The present invention specifically adopts the following technical solution: A water-soluble near-infrared II (NIR-II) luminescent gold nanoclusters based on aromatic hydrogen-hydrogen spatial interactions, wherein the general molecular formula of the gold nanoclusters is [Au 25 (SR) 18 ] - SR is a thiol ligand. Specifically, this substance is Au protected by o-carboxybenzoic acid and its derivatives. 25 Au 25 The absorption peak is in the 700–720 nm range. The carboxyl group, close to the gold nucleus, distorts the ligand layer structure, increasing ligand layer crowding and leading to aromatic hydrogen-hydrogen spatial interactions. Through cross-space interactions and conformational locking, the ligand's protective ability towards the gold nucleus is enhanced, achieving high structural stability of the cluster even in the presence of exogenous thiol ligands. Au 25 Gold nanoclusters are difficult to be replaced by glutathione in the liver, thus prolonging their half-life in the bloodstream and slowing their clearance rate. This allows them to enhance tumor accumulation through passive or active targeting, utilizing Au... 25 The optical properties enable highly temporal and spatial in vivo tumor-targeted photoluminescence (PL) imaging in NIR-II.
[0007] A method for preparing water-soluble near-infrared II luminescent gold nanoclusters based on aromatic hydrogen-hydrogen spatial interactions specifically includes the following steps: (1) The ligand was sonicated and dissolved in sodium hydroxide solution, and then stirred thoroughly with HAuCl4 and water to obtain a mixed solution of Au(I)-thiol salt complex. (2) Add sodium hydroxide solution and reducing agent to step (1) rapidly, stir at room temperature for several hours, then place in a sand bath for further etching, while monitoring the characteristic absorption peak of the solution. When the characteristic absorption peak tends to stabilize, purify and concentrate by ultrafiltration to obtain gold nanoclusters [Au]. 25 (SR) 18 ] - Store in a refrigerator at 4°C.
[0008] Furthermore, the ligand described in step (1) is a thiol molecule SR, whose core structure contains a carboxyl group and is adjacent to the thiol group, and can be selected from, but is not limited to, the following structures:
[0009] Wherein, X is one of hydrogen, amino, nitro, hydroxyl, sulfonic acid, carboxyl, or boric acid groups, or one of the targeted small molecules such as the membrane-penetrating peptide CR8, folic acid, cyclic arginine-glycine-aspartic acid, polyethylene glycol, glucose, or biotin.
[0010] The targeted small molecule is attached to the benzene ring via an amide or esterification condensation reaction. The condensing agent used can be a combination of 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate / N,N-diisopropylethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, dicyclohexylcarbodiimide / 4-N,N-dimethylpyridine, or dicyclohexylcarbodiimide / 1-hydroxybenzotriazole.
[0011] Furthermore, the concentration of the ligand in the sodium hydroxide solution ranges from 0.15 M to 0.20 M.
[0012] In a preferred embodiment of the present invention, the ligand is a compound having one of the following structures:
[0013] Further, the reducing agent mentioned in step (2) is one of sodium borohydride, dimethylamine borane, tetrabutylammonium borohydride, and carbon monoxide.
[0014] Furthermore, the molar ratio of HAuCl4 to ligand used in step (1) is 1:1.0 to 1:2.5.
[0015] Furthermore, the molar ratio of HAuCl4 to reducing agent used in step (2) is 1:0.05~1:0.10.
[0016] Furthermore, the temperature for heating and etching in step (2) is 60~80℃.
[0017] Furthermore, the centrifugation speed of ultrafiltration in step (2) is 3000~3750 rpm, the time is 10~15 min, and a total of 6~10 ultrafiltration cycles are performed.
[0018] This invention also proposes the application of the above-mentioned gold nanoclusters or the gold nanoclusters prepared by the above-mentioned preparation method in the preparation of tumor imaging reagents, including NIR-II fluorescent tumor-targeting imaging.
[0019] The tumors include breast cancer, lung cancer, liver cancer, and colon cancer.
[0020] The beneficial effects of this invention are: This invention is the first to synthesize a series of water-soluble near-infrared II luminescent gold nanoclusters. By precisely controlling the position of the carboxyl group on the aromatic ligand, the substitution ability of thiol molecules for the gold nanoclusters is altered. This invention designs a water-soluble near-infrared II luminescent gold nanocluster based on aromatic hydrogen-hydrogen spatial interactions. The sterically hindered functional group (carboxyl group) is close to the gold core, which distorts the ligand layer structure, increases the crowding of the ligand layer, and leads to aromatic hydrogen-hydrogen spatial interactions. Through cross-spatial interactions and conformational locking, the protective ability of the ligand for the gold core is improved. This not only improves the ligand dissociation and poor stability of traditional gold nanoclusters in the presence of exogenous thiols, but also allows for precise control of nanocluster transport and targeting, enabling tumor imaging. This invention broadens the types of metal nanoclusters and provides a practical method for the biological functional applications of water-soluble gold nanoclusters. Attached Figure Description
[0021] Figure 1 Au in Examples 1-3 and Comparative Examples 4-6 25 Optimized ratio of HAuCl4 to ligand.
[0022] Figure 2 Au in Examples 1-3 and Comparative Examples 4-6 25 The fluorescence spectrum.
[0023] Figure 3 Au in Examples 1-3 and Comparative Examples 4-6 25 Hydrated particle size distribution.
[0024] Figure 4 Au in Examples 1-3 and Comparative Examples 4-6 25 High-resolution mass spectrum.
[0025] Figure 5 The graph shows the number of glutathione in the ligands after Examples 1-3 and Control Examples 4-6 were mixed with glutathione.
[0026] Figure 6 Au in Examples 1-3 and Comparative Examples 4-6 25 TOCSY and NOESY plots.
[0027] Figure 7 Au in Examples 1-3 and Comparative Examples 4-6 25 NIR II imaging in mice.
[0028] Figure 8 Au in Examples 1-3 and Comparative Examples 4-6 25 4T1 tumor in vivo fluorescence imaging.
[0029] Among them, the above Figures 1 to 8In this context, a, b, c, d, e, and f represent [Au], respectively. 25 (42) 18 ] - 、[Au 25 (36) 18 ] - 、[Au 25 (26) 18 ] - 、[Au 25 (twenty three) 18 ] - 、[Au 25 (34) 18 ] - 、[Au 25 (35) 18 ] - . Detailed Implementation
[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection and implementation of the present invention are not limited thereto.
[0031] In the following examples and comparative examples, the Au above was measured using a UV-Vis spectrophotometer and a fluorescence spectrometer. 25 Absorption and fluorescence spectra; the hydration particle size of the above-mentioned fluorescence sensor was measured using dynamic light scattering; and the Au content was measured using high-resolution mass spectrometry. 25 Molecular structural formulas before and after glutathione substitution; nuclear magnetic resonance spectroscopy was used to measure the above Au. 25 Two-dimensional hydrogen NMR spectrum; the above Au was tested using a multi-channel in vivo imaging system. 25 NIR II imaging images and in vivo fluorescence imaging images of 4T1 tumors in mice.
[0032] The tumor selected in this embodiment of the invention is mouse breast cancer.
[0033] All materials and raw materials used in the following examples and comparative examples were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.
[0034] Example 1 The synthetic route for ligand 42 is as follows:
[0035] Synthesis of ligand 42-b: 42-a (21.73 mmol, 5.00 g), 2-ethylhexyl 3-mercaptopropionate (26.08 mmol, 5.69 g), i-Pr2Net (32.60 mmol, 4.21 g), Xantphos (0.44 mmol, 0.25 g), and Pd2(dba)3 (0.22 mmol, 0.20 g) were dissolved in anhydrous toluene (250 mL). The mixture was refluxed at 100 °C overnight under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was extracted with water, followed by washing with water and brine. The organic phase was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography to give compound 42-b in 88.3% yield.
[0036] Synthesis of ligand 42: 42-b (8.16 mmol, 3.00 g) and DMF (100 mL) were added to a round-bottom flask equipped with a magnetic stirrer. Sodium tert-butoxide (12.24 mmol, 1.18 g) was sonicated and dissolved in DMF, then slowly added dropwise to the flask, and the reaction was stirred for 10 minutes. The reaction was quenched by adding saturated ammonium chloride. The reaction solution was extracted with brine and washed three times with ethyl acetate, then the pH of the water was adjusted to neutral. The neutral solution was then extracted with ethyl acetate, washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was directly used for the next step. It was dissolved in an aqueous solution of sodium hydroxide and refluxed overnight. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 3-4, and the mixture was filtered to obtain solid 42 (0.92 g, 66.6%).
[0037] [Au 25 (42) 18 ] - The preparation method includes the following steps: First, ligand 42 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), ligand 42 solution (50 mM, 800 μL), and deionized water (10 mL) were added to a 20 mL glass bottle. After stirring for 30 minutes, sodium hydroxide solution (1 M, 400 μL) and freshly prepared sodium borohydride solution (10 mM, 100 μL) prepared with ice water were added rapidly. The reaction was carried out at room temperature for 8 hours. Then, the glass bottle was transferred to a sand bath at 70 °C to continue the reaction. After 12 hours, the characteristic absorption peak of the solution was monitored every two hours. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) 6 times. Finally, the product was concentrated and stored in a refrigerator at 4 °C for subsequent use.
[0038] Example 2 The synthetic route for ligand 36 is as follows:
[0039] Synthesis of ligand 36-b: 36-a (66.15 mmol, 10.00 g), potassium thiocyanate (132.31 mmol, 12.86 g), Cu(OTf)₂ (13.23 mmol, 4.79 g), and TEMED (13.23 mmol, 1.54 g) were dissolved in DMSO (200 mL). BF₃·OEt₂ (132.31 mmol, 18.78 g) was added at low temperature, and the mixture was then refluxed at 80 °C under an oxygen atmosphere overnight. After the reaction was complete, the mixture was cooled to room temperature, and a large amount of water was added to precipitate the precipitate. The precipitate was obtained by filtration and then purified by column chromatography to give compound 36-b in 42.5% yield.
[0040] Synthesis of ligand 36: 36-b (24.01 mmol, 5.00 g) and sodium hydroxide (48.02 mmol, 1.92 g) were dissolved in ethanol (100 mL) and stirred for 30 minutes. After the reaction was complete, water and ethyl acetate were added for extraction. The organic phase was extracted three times and concentrated under reduced pressure to obtain the crude product. The crude product was used directly as a feedstock in the next step. It was dissolved in an aqueous solution of sodium hydroxide and refluxed overnight. After completion, it was cooled to room temperature and the pH was adjusted to 3-4. After evaporating the water, a small amount of hot water was added, and recrystallization was performed to obtain solid 36 (0.92 g, 22.7%).
[0041] [Au 25 (36) 18 ] - The preparation method includes the following steps: First, ligand 36 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), ligand 36 solution (50 mM, 1 mL), and deionized water (10 mL) were added to a 20 mL glass bottle. After stirring for 30 minutes, sodium hydroxide solution (1 M, 400 μL) and freshly prepared sodium borohydride solution (10 mM, 100 μL) prepared with ice water were added rapidly. The reaction was carried out at room temperature for 8 hours. Then, the glass bottle was transferred to a sand bath at 70°C to continue the reaction. After 12 hours, the characteristic absorption peak of the solution was monitored every two hours. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) 6 times. Finally, the product was concentrated and stored in a refrigerator at 4°C for subsequent use.
[0042] Example 3 The synthetic route for ligand 26 is as follows:
[0043] Synthesis of ligand 26-b: 26-a (21.73 mmol, 5.00 g), 2-ethylhexyl 3-mercaptopropionate (26.08 mmol, 5.69 g), i-Pr2Net (32.60 mmol, 4.21 g), Xantphos (0.44 mmol, 0.25 g), and Pd2(dba)3 (0.22 mmol, 0.20 g) were dissolved in anhydrous toluene (250 mL), and the mixture was refluxed at 100 °C overnight under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was extracted with water, followed by washing with water and brine. The organic phase was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography to give compound 26-b in 93.2% yield.
[0044] Synthesis of ligand 26: 26-b (8.16 mmol, 3.00 g) and DMF (100 mL) were added to a round-bottom flask equipped with a magnetic stirrer. Sodium tert-butoxide (12.24 mmol, 1.18 g) was sonicated and dissolved in DMF, then slowly added dropwise to the flask, and the reaction was stirred for 10 minutes. The reaction was quenched by adding saturated ammonium chloride. The reaction solution was extracted with brine and washed three times with ethyl acetate, then the pH of the water was adjusted to neutral. The neutral solution was then extracted with ethyl acetate, washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was directly used for the next step. It was dissolved in an aqueous solution of sodium hydroxide and refluxed overnight. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 3-4, and the mixture was filtered to obtain solid 26 (1.03 g, 74.6%).
[0045] [Au 25 (26) 18 ] - The preparation method includes the following steps: First, ligand 26 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), ligand 26 solution (50 mM, 600 μL), and deionized water (10 mL) were added to a 20 mL glass bottle. After stirring for 30 minutes, sodium hydroxide solution (1 M, 400 μL) and freshly prepared sodium borohydride solution (10 mM, 100 μL) prepared with ice water were added rapidly. The reaction was carried out at room temperature for 8 hours. Then, the glass bottle was transferred to a sand bath at 70°C to continue the reaction. After 12 hours, the characteristic absorption peak of the solution was monitored every two hours. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) 6 times. Finally, the product was concentrated and stored in a refrigerator at 4°C for subsequent use.
[0046] To explore the effect of ligand hydrogen-hydrogen spatial interactions on gold nanoclusters in the implementation scheme, gold nanoclusters synthesized using thiol molecules with the same molecular formula but different functional group positions were selected as ligands as a comparative example. The comparative example ligands can be selected from, but are not limited to, the following structures:
[0047] Compare with Example 4 The synthetic route for ligand 23 is as follows:
[0048] Synthesis of ligand 23-b: 23-a (21.73 mmol, 5.00 g), 2-ethylhexyl 3-mercaptopropionate (26.08 mmol, 5.69 g), i-Pr2Net (32.60 mmol, 4.21 g), Xantphos (0.44 mmol, 0.25 g), and Pd2(dba)3 (0.22 mmol, 0.20 g) were dissolved in anhydrous toluene (250 mL), and the mixture was refluxed at 100 °C overnight under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was extracted with water, followed by washing with water and brine. The organic phase was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography to give compound 23-b in 86.5% yield.
[0049] Synthesis of ligand 23: 23-b (8.16 mmol, 3.00 g) and DMF (100 mL) were added to a round-bottom flask equipped with a magnetic stirrer. Sodium tert-butoxide (12.24 mmol, 1.18 g) was sonicated and dissolved in DMF, then slowly added dropwise to the flask, and the reaction was stirred for 10 minutes. The reaction was quenched by adding saturated ammonium chloride. The reaction solution was extracted with brine and washed three times with ethyl acetate, then the pH of the water was adjusted to neutral. The neutral solution was then extracted with ethyl acetate, washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was directly used for the next step. It was dissolved in an aqueous solution of sodium hydroxide and refluxed overnight. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 3-4, and the mixture was filtered to obtain solid 23 (1.11 g, 79.7%).
[0050] [Au 25 (twenty three) 18 ] - The preparation method includes the following steps: First, ligand 23 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), ligand 23 solution (50 mM, 400 μL), and deionized water (10 mL) were added to a 20 mL glass bottle. After stirring for 30 minutes, freshly prepared sodium borohydride solution (10 mM, 100 μL) was quickly added. After reacting for half an hour, the characteristic absorption peak of the solution was monitored every half hour. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) for 6 cycles. Finally, the product was concentrated and stored in a 4°C refrigerator for subsequent use.
[0051] Compare with Example 5 The synthetic route for ligand 34 is as follows:
[0052] Synthesis of ligand 34: 34-a (15.53 mmol, 3.00 g), sodium hydroxide (46.58 mmol, 1.86 g), and deionized water (100 mL) were added to a round-bottom flask equipped with a magnetic stirrer, and the mixture was heated to 100 °C and refluxed overnight. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 3-4, and the mixture was filtered to obtain solid 34 (1.58 g, 60.5%).
[0053] [Au 25 (34) 18 ] - The preparation method includes the following steps: First, ligand 34 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), 23 solution (50 mM, 400 μL), and deionized water (10 mL) were added to a 20 mL glass bottle. Then, freshly prepared sodium borohydride solution (10 mM, 200 μL) was quickly added. After reacting for 2 hours, the characteristic absorption peak of the solution was monitored every hour. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) for 6 cycles. Finally, the product was concentrated and stored in a 4°C refrigerator for subsequent use.
[0054] Compare with Example 6 The synthetic route for ligand 35 is as follows:
[0055] Synthesis of ligand 35-b: 35-a (21.73 mmol, 5.00 g), 2-ethylhexyl 3-mercaptopropionate (26.08 mmol, 5.69 g), i-Pr2Net (32.60 mmol, 4.21 g), Xantphos (0.44 mmol, 0.25 g), and Pd2(dba)3 (0.22 mmol, 0.20 g) were dissolved in anhydrous toluene (250 mL), and the mixture was refluxed at 100 °C overnight under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was extracted with water, followed by washing with water and brine. The organic phase was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography to give compound 35-b in 91.7% yield.
[0056] Synthesis of ligand 35: 35-b (8.16 mmol, 3.00 g) and DMF (100 mL) were added to a round-bottom flask equipped with a magnetic stirrer. Sodium tert-butoxide (12.24 mmol, 1.18 g) was sonicated and dissolved in DMF, then slowly added dropwise to the round-bottom flask, and the reaction was stirred for 10 minutes. The reaction was quenched by adding saturated ammonium chloride. The reaction solution was extracted with brine and washed three times with ethyl acetate, then the pH of the water was adjusted to neutral. The neutral solution was then extracted with ethyl acetate, washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was directly used for the next step. It was dissolved in an aqueous solution of sodium hydroxide and refluxed overnight. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 3-4, and the mixture was filtered to obtain solid 35 (0.98 g, 71.0%).
[0057] [Au 25 (35) 18 ] - The preparation method includes the following steps: First, ligand 35 was dissolved in sodium hydroxide solution. Then, HAuCl4 solution (164.44 mM, 122 μL), ligand 35 solution (50 mM, 600 μL), and deionized water (10 mL) were added to a 20 mL glass bottle. After stirring for 30 minutes, freshly prepared sodium borohydride solution (10 mM, 100 μL) was quickly added. After reacting for 12 hours, the characteristic absorption peak of the solution was monitored every two hours. After the absorption peak stabilized, the solution was ultrafiltered and centrifuged (3750 rpm, 15 min) for 6 cycles. Finally, the product was concentrated and stored in a 4°C refrigerator for subsequent use.
[0058] Example 7 The basic properties of the materials in Examples 1-3 and Comparative Examples 4-6, including absorption spectra, fluorescence spectra, hydrated particle size, and molecular weight, were determined. The results are as follows: Figure 1-4 .
[0059] Depend on Figure 1 It can be seen that in Examples 1-3 and Comparative Examples 4-6, the ratio of HAuCl4 to ligand was 1:2.0, 1:2.5, 1:1.5, 1:1.5, 1:1.0, and 1:1.5, respectively. The materials formed under these conditions exhibited near-infrared absorption characteristics, with absorption peaks located at 716 nm, 708 nm, 698 nm, 711 nm, 714 nm, and 690 nm, respectively, which are similar to Au... 25 The core electronic transitions match, preliminarily verifying that the synthesized nanocluster core is gold 25. Therefore, the ratio of HAuCl4 to ligands is crucial in the synthesis of gold nanoclusters. Only when the ratio is appropriate can precisely structured gold nanoclusters 25 be synthesized; otherwise, structurally ambiguous gold nanoparticles will be synthesized. This cannot achieve the ligand engineering strategy of precisely regulating glutathione-mediated nanoparticle biotransformation in the liver, thus overcoming the biochemical obstacle of premature ligand dissociation and limited nanoparticle function.
[0060] Depend on Figure 2 It can be seen that the maximum emission peak of the materials in Examples 1-3 and Comparative Examples 4-6 is located at 1100 nm, which belongs to the NIR II region window emission, and is suitable for deep biomedical imaging applications such as vascular imaging and tumor diagnosis.
[0061] Depend on Figure 3 It is known that the hydrated particle size of the materials in Examples 1-3 and Control Examples 4-6 is 2-3 nm, which is smaller than the renal filtration threshold (~5.5 nm). Therefore, when investigating the in vivo metabolic pathways of the materials in Examples 1-3 and Control Examples 4-6, the hydration particle size of Au can be ignored. 25 The impact caused by its own size.
[0062] Depend on Figure 4 The high-resolution mass spectra show that the m / z values of the materials tested in Examples 1-3 and Control Examples 4-6 are similar to those of [Au]. 25 (SR) 18 ] - The theoretical simulation showed that the m / z values were consistent, further indicating that the materials in Examples 1-3 and Comparative Examples 4-6 were negatively charged as a whole, with a core of 25 gold atoms and an outer protective layer of 18 ligands.
[0063] Example 8 Previous studies have shown that atoms anchored on the surface of gold nanoclusters can alter the binding affinity of the ligand layer to the gold nucleus; for example, selenium binds to gold more strongly than sulfur. However, as sulfur is the primary anchoring site for gold nanoclusters, no studies have yet discovered how to precisely control the binding affinity of the ligand layer to the gold nucleus through functional group isomerization. Examples 1-3 of this invention can precisely control the binding affinity of the ligand to the gold nucleus in the presence of exogenous thiol ligands through isomerization at the carboxyl amino position on the benzene ring.
[0064] In Examples 1-3 and Comparative Examples 4-6, the number of ligands detached from the original ligands was accurately verified by high-resolution mass spectrometry under the interference of exogenous thiol ligands, thereby clarifying the binding ability of the original ligands to the gold nucleus.
[0065] Depend on Figure 5 It can be seen that the ligand detachment of Examples 1-3 and Control Examples 4-6 (10 μM) was inconsistent when mixed with excess glutathione (1.0 mM) for 10 minutes. High-resolution mass spectrometry showed that after mixing with glutathione, 11-16 of the 18 ligands in Control Example 4 were replaced by glutathione, with 13 being primarily replaced (i.e., 13 ligands detached); 13-18 of the 18 ligands in Control Example 5 were replaced by glutathione, with 16 being primarily replaced (i.e., 16 ligands detached); and 9-14 of the 18 ligands in Control Example 6 were replaced by glutathione. Of the 18 ligands in Example 1-3, 12 were primarily replaced by glutathione, meaning 12 ligands were detached. In Example 4, 1-6 ligands were replaced by glutathione, with 3 primarily replaced, meaning 3 ligands were detached. In Example 5, 0-1 ligands were replaced by glutathione, with 0 primarily replaced, indicating minimal replacement. In Example 6, 0 out of 18 ligands were replaced by glutathione, indicating no replacement. In summary, the number of glutathione replacements in Examples 1-3 is significantly less than that in Control Example 4.
[0066] The common feature of the ligands in Examples 1-3 is that the sterically hindered functional group (carboxyl group) is close to the gold nucleus. The difference is that the sterically hindered functional group (amino group) is located at different positions on the benzene ring. In contrast, in Comparative Examples 4-6, the sterically hindered functional group (carboxyl group) is far away from the gold nucleus. Therefore, by using ligand engineering strategies to precisely control the ligands through functional group positional isomerization, the binding ability of the ligands to the gold nucleus can be significantly altered.
[0067] Example 9 Although ligand engineering strategies have enabled precise control of ligand binding to gold nuclei through functional group position isomerization, elucidating the underlying mechanism can better guide the precise regulation of glutathione-mediated nanoparticle biotransformation in the liver. This would address the need to overcome the biochemical obstacle of premature ligand dissociation and limited nanoparticle function, filling a technological gap in the field.
[0068] This invention studies Au 25 The chemical environment of the ligand layer can be used to further explain the mechanism.
[0069] like Figure 6As shown, obvious cross-peaks appeared between the different benzene ring hydrogens in Examples 1-3, indicating that they have interactions; while no cross-peaks were observed in the benzene ring hydrogens of Control Examples 4-6, indicating that they do not have interactions. Therefore, this invention proposes that the carboxyl group, being close to the gold nucleus, will distort the ligand layer structure, increase the crowding of the ligand layer, and lead to aromatic hydrogen-hydrogen spatial interactions. Through cross-space interactions and conformational locking, the protective ability of the ligand for the gold nucleus is improved, achieving high structural stability of the cluster in the presence of exogenous thiol molecules.
[0070] Example 10 The embodiments 1-3 of this invention have demonstrated excellent protective ability of the ligands against the gold nucleus in vitro through aromatic hydrogen-hydrogen spatial interactions. Furthermore, the metabolic pathways and biodistribution of the embodiments 1-3 and controls 4-6 in mice were used to evaluate the effect of functional group positional isomerization on the precise guidance of glutathione-mediated biotransformation of nanoparticles in the liver.
[0071] Figure 7 These are in vivo fluorescence imaging photographs of mice from Examples 1-3 and Controls 4-6. Normal mice were injected with the serum levels of Examples 1-3 and Controls 4-6, and Au was assessed by fluorescence imaging. 25 The in vivo biodistribution characteristics were observed. Strong and prolonged fluorescence signals were observed in the livers of Examples 1-3, indicating that clearance was primarily via the hepatic route; while significant fluorescence signals were quickly observed in the bladders of Control Examples 4-6, indicating that clearance was primarily via the renal route.
[0072] In Examples 1-3 of this invention, the structure remained highly stable in the presence of exogenous thiol molecules (glutathione), making it difficult to be biotransformed by glutathione-mediated nanoparticles, thus prolonging their half-life in the bloodstream. However, glutathione-mediated nanoparticle biotransformation, by replacing the ligands in Examples 4-6, significantly shortened the half-life of gold nanoclusters in the bloodstream and accelerated their clearance rate from the body. Therefore, ligand functional group positional isomerization can precisely guide glutathione-mediated nanoparticle biotransformation in the liver, enabling differences in the metabolic pathways and biodistribution of nanomaterials in mice.
[0073] Example 11 Fluorescence imaging of tumor cells was performed on Example 3 and Control Example 4 as described above: A subcutaneous tumor model was established in 6-week-old female C57BL mice by inoculating them with 4T1 cells. Seven days after tumor growth, the tumor-bearing mice were injected via the tail vein in both Example 3 and Control Example 4.
[0074] like Figure 8As shown, compared with the tumor-bearing mice injected in Control Example 4, the tumor-bearing mice injected in Example 3 exhibited clearly visible tumor boundaries, providing precise guidance for diagnosis and treatment. This demonstrates that functional group positional isomerization of the ligand can prolong the half-life of nanomaterials in blood circulation and achieve nanomaterial enrichment in tumors by altering the glutathione-mediated nanoparticle biotransformation process in the liver.
[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A water-soluble near-infrared II luminescent gold nanocluster based on aromatic hydrogen-hydrogen spatial interactions, characterized in that, The general molecular formula of the gold nanoclusters is [Au 25 (SR) 18 ] - ; where SR is a thiol ligand, SR is a compound as shown in formula (I), or a stereoisomer or geometric isomer of the compound shown in formula (I); Wherein, X is one of hydrogen, amino, nitro, hydroxy, sulfonic acid, carboxyl, or boric acid, or one of the following: membrane-penetrating peptide CR8, folic acid, cyclic arginine-glycine-aspartic acid, polyethylene glycol, glucose, or biotin-targeting small molecule.
2. The water-soluble near-infrared II luminescent gold nanoclusters based on aromatic hydrogen-hydrogen spatial interactions according to claim 1, characterized in that, The targeted small molecule is attached to the benzene ring via an amide or esterification condensation reaction. The condensing agent used is a combination of one of the following: 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate / N,N-diisopropylethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, dicyclohexylcarbodiimide / 4-N,N-dimethylpyridine, or dicyclohexylcarbodiimide / 1-hydroxybenzotriazole.
3. The method for preparing water-soluble near-infrared II luminescent gold nanoclusters based on aromatic hydrogen-hydrogen spatial interactions according to any one of claims 1-2, characterized in that, Includes the following steps: (1) The ligand was sonicated and dissolved in sodium hydroxide solution, and then stirred thoroughly with HAuCl4 and water to obtain a mixed solution of Au(I)-thiol salt complex; (2) Add a reducing agent to the mixed solution in step (1), stir at room temperature for several hours, then place it in a sand bath for further etching, while monitoring the characteristic absorption peak of the solution. When the characteristic absorption peak tends to stabilize, purify and concentrate by ultrafiltration to obtain gold nanoclusters [Au]. 25 (SR) 18 ] - .
4. The preparation method according to claim 3, characterized in that, The ligand is a compound having one of the following structures: 。 5. The preparation method according to claim 3, characterized in that, The molar ratio of HAuCl4 to ligand used in step (1) is 1:1 to 1:2.
5.
6. The preparation method according to claim 3, characterized in that, The molar ratio of HAuCl4 and reducing agent used in step (2) is 1:0.05~1:0.
10.
7. The preparation method according to claim 3, characterized in that, In step (2), the reducing agent is one of sodium borohydride, dimethylamine borane, tetrabutylammonium borohydride, and carbon monoxide.
8. The preparation method according to claim 3, characterized in that, In step (2), the heating temperature is 60~80℃.
9. The use of a gold nanocluster according to any one of claims 1-2 or a gold nanocluster prepared by the preparation method according to any one of claims 3-8 in the preparation of tumor imaging reagents.
10. The application according to claim 9, characterized in that, The tumors include breast cancer, lung cancer, liver cancer, and colon cancer.