A gold nanocluster and use thereof in preparation of a medicine for inhibiting thrombus

Abstract

The application belongs to the technical field of medicines, and particularly relates to a gold nanocluster and application thereof in preparation of a medicine for inhibiting thrombus. The gold nanocluster is synthesized by taking 4,6-diamino-2-mercapto pyrimidine as a functional ligand, and the size of the gold nanocluster is 1-2 nm. The gold nanocluster can inhibit fibrinogen cleavage and aggregation, and thus has good anticoagulation effect, can be used for preparation of a medicine for inhibiting thrombus, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a gold nanocluster and its use in the preparation of drugs that inhibit thrombosis. Background Technology

[0002] Thrombosis has become one of the major diseases that seriously affect human health. In particular, thrombosis following some serious diseases, such as stroke, coronary artery bypass surgery, rupture of atherosclerotic plaques, and long-term immobilization after surgery, is prone to occur, seriously threatening the life and health of patients.

[0003] Thrombosis is classified into arterial thrombosis and venous thrombosis. The main components of arterial thrombosis are fibrin and platelets, while the main components of venous thrombosis are fibrin and other blood cells (mostly red blood cells). Both are clots formed by fibrin agglomerating into a network and combining with blood cells. The body's fibrin coagulation and dissolution systems are coordinated to maintain a balance between thrombus formation and dissolution. Once this balance is disrupted, pathological conditions occur.

[0004] Based on the main components of thrombi, traditional antithrombotic drugs mainly include three types: antiplatelet drugs, anticoagulants, and fibrinolytic drugs. However, these drugs have short half-lives and cannot target and accumulate at the thrombus site, resulting in poor clinical efficacy. At the same time, their narrow therapeutic window and significant toxic side effects (such as severe bleeding risk) also limit their clinical application.

[0005] Therefore, developing and combining novel treatment strategies is crucial for the treatment of thrombotic diseases.

[0006] With the development of nanotechnology in recent years, bionanotechnology has been widely applied in the biomedical field. Gold nanoclusters, composed of hundreds to hundreds of Au atoms with a core size of less than 2 nm, have attracted worldwide attention in the biomedical field due to their extraordinary physicochemical properties and excellent biocompatibility. Currently, no relevant literature reports the antithrombotic activity of gold nanoclusters. Summary of the Invention

[0007] In view of the problems of the prior art, the purpose of this invention is to provide a gold nanocluster and its use in the preparation of drugs that inhibit thrombosis.

[0008] A gold nanocluster, wherein the gold nanocluster is synthesized using 4,6-diamino-2-mercaptopyrimidine as a functional ligand, and the size of the gold nanocluster is 1-2 nm.

[0009] Preferably, the gold nanoclusters are prepared by reducing chloroauric acid using the reducing properties of the functional ligand itself.

[0010] Preferably, the method for preparing the gold nanoclusters includes the following steps:

[0011] Step 1: Prepare a mixed solution of 4,6-diamino-2-mercaptopyrimidine and chloroauric acid by stirring at 280-350 rpm.

[0012] Step 2: Heat to 65-75℃ for reaction;

[0013] Step 3: Concentrate and filter using an ultrafiltration tube, collect the supernatant, and freeze-dry to obtain the final product.

[0014] Preferably, in step 1, the molar ratio of 4,6-diamino-2-mercaptopyrimidine to chloroauric acid is (1-2):1.

[0015] And / or, in step 1, the concentration of 4,6-diamino-2-mercaptopyrimidine in the mixed solution is 1-2-2.0 mM, and the concentration of chloroauric acid is 1 mM;

[0016] And / or, in step 2, the reaction process is as follows: heat to 20-30℃, stir for 10 minutes, then heat to 65-75℃ and react for 10-15 hours;

[0017] And / or, in step 2, the pH is adjusted before the reaction, and the pH of the reaction is 5-6.

[0018] Preferably, in step 1, the molar ratio of 4,6-diamino-2-mercaptopyrimidine to chloroauric acid is 1.5:1.

[0019] And / or, in step 1, the concentration of 4,6-diamino-2-mercaptopyrimidine in the mixed solution is 1.5 mM, and the concentration of chloroauric acid is 1 mM;

[0020] And / or, in step 2, the reaction process is as follows: heat to 20-30℃, stir for 10 minutes, then heat to 70℃ and react for 12 hours;

[0021] And / or, in step 2, the pH of the reaction is 6.

[0022] Preferably, the gold nanoclusters exist in the form of a solution with a concentration of ≥20 μg / mL.

[0023] The present invention also provides a method for preparing the above-mentioned gold nanoclusters, comprising the following steps:

[0024] Step 1: Prepare a mixed solution of 4,6-diamino-2-mercaptopyrimidine and chloroauric acid by stirring at 280-350 rpm.

[0025] Step 2: Heat to 65-75℃ for reaction;

[0026] Step 3: Concentrate and filter using an ultrafiltration tube, collect the supernatant, and freeze-dry to obtain the final product.

[0027] The present invention also provides the use of the above-mentioned gold nanoclusters in the preparation of drugs for inhibiting thrombosis.

[0028] Preferably, the gold nanoclusters are used to reduce fibrinogen levels in plasma.

[0029] The present invention also provides a drug for inhibiting thrombosis, which is a formulation made by adding pharmaceutically acceptable excipients or auxiliary ingredients to the above-mentioned gold nanoclusters as active ingredients.

[0030] The present invention prepares ultra-small gold nanoclusters that inhibit fibrinogen aggregation and thus thrombus formation. Using 4,6-diamino-2-mercaptopyrimidine as the functional ligand, chloroauric acid is reduced to fluorescent gold nanoclusters with a size of 1-2 nm through the reducing property of the ligand itself. These gold nanoclusters specifically bind to fibrinogen, inhibiting fibrinogen aggregation and thereby inhibiting thrombus formation. Therefore, the gold nanoclusters provided by the present invention have excellent application prospects in the preparation of drugs for inhibiting thrombosis.

[0031] In the preferred embodiment, the present invention optimizes the experimental conditions such as the content of each component of the ultra-small gold nanocluster solution, heating time, heating temperature, and concentration of the ultra-small gold nanocluster solution under a large number of experimental conditions, and explores the optimal component ratio, optimal experimental conditions, and most suitable concentration of the ultra-small gold nanocluster solution.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0033] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0034] Figure 1 The ultraviolet-visible absorption spectrum of ultra-small gold nanoclusters;

[0035] Figure 2 The excitation (blue line) and emission (red line) spectra of PL are centered at 450 nm and 770 nm, respectively. The inset shows photographs of ultra-small gold nanoclusters under visible light and 365 nm ultraviolet light.

[0036] Figure 3TEM images and size distribution histograms of ultra-small gold nanoclusters (scale bar: 20 μm);

[0037] Figure 4 XPS spectrum (Au4f region);

[0038] Figure 5 The zeta potential of ultra-small gold nanoclusters;

[0039] Figure 6 Optimize reaction conditions for gold nanoclusters (concentration, time, pH, temperature);

[0040] Figure 7 Coagulation Composite Index (CI, reference range: -3 to 3);

[0041] Figure 8 Response time (R, reference value: 4-9 min);

[0042] Figure 9 Clotting time (K, reference range 1-3 min);

[0043] Figure 10 Blood angle (α, reference value 55-70 degrees);

[0044] Figure 11 Maximum amplitude of thromboelastography (MA, reference value 50-70 mm);

[0045] Figure 12 Fibrinolysis index;

[0046] Figure 13 Prothrombin time (PT, reference range 10–14 s);

[0047] Figure 14 Activated partial thromboplastin time (APPT, reference range 26–40 s);

[0048] Figure 15 Thrombin time (TT, reference range 11-14s);

[0049] Figure 16 Fibrinogen (FIB, reference range 2-4 g / L);

[0050] Figure 17 D-dimer (reference range 0–0.5 μg / mL);

[0051] Figure 18 It is a fibrinogen degradation product (FDP, reference value 0-5 μg / mL);

[0052] Figure 19 It is antithrombin III (AT-III, reference range 80%–120%);

[0053] Figure 20 Optical images of clot formation in a purified fibrinogen system, showing the effects of ultra-small gold nanoclusters of different concentrations.

[0054] Figure 21 TEM image of fibrinogen treated with a solution of ultra-small gold nanoclusters (50 μg / mL) (magnification: 0.5 μm);

[0055] Figure 22 TEM image of fibrinogen treated with a solution of ultra-small gold nanoclusters (50 μg / mL) (magnification: 200 nm);

[0056] Figure 23 TEM image of fibrinogen treated with a solution of ultra-small gold nanoclusters (50 μg / mL) (magnification: 50 nm);

[0057] Figure 24 Elemental analysis of fibrinogen treated with a solution of ultra-small gold nanoclusters (50 μg / mL);

[0058] Figure 25 This represents the molecular docking between fibrinogen and the thiol pyrimidine ligands on the surface of ultra-small gold nanoclusters.

[0059] Figure 26 A comparative graph showing the effects of ultra-small gold nanocluster solutions on platelet coagulation;

[0060] Figure 27 Electron micrographs showing the comparative effects of ultra-small gold nanocluster solutions on platelet coagulation.

[0061] Figure 28 The effect of solutions of ultra-small gold nanoclusters of different concentrations on platelet aggregation;

[0062] Figure 29 To observe tail thrombosis in rats under different treatment groups.

[0063] Figure 30 Statistical analysis of thrombus length in different treatment groups.

[0064] Figure 31 The tail blood flow of different groups of rats was monitored by laser Doppler flowmeter.

[0065] Figure 32 A statistical graph showing the tail blood flow in rats of different treatment groups as monitored by laser Doppler flowmeter.

[0066] Figure 33 Tissue sections of pulmonary thrombosis from different treatment groups.

[0067] Figure 34 Tissue sections of rat tail thrombi from different treatment groups.

[0068] Figure 35 Cell viability of HUVECs incubated with different concentrations of ultra-small gold nanoclusters (n=6).

[0069] Figure 36 The results of hemolysis assays for ultra-small gold nanoclusters are shown (n=6). Normal saline (NS) and ultrapure water served as negative and positive controls, respectively. The insets from bottom to top are corresponding images of the hemolytic activity assay and magnified views of the statistical results.

[0070] Figure 37 Optical micrograph of blood cells (blood smear) treated with NS and ultra-small gold nanocluster solution (500 μg / mL) (magnification: 50 μm).

[0071] Figure 38 SEM images of blood cells treated with NS and ultra-small gold nanoclusters solution (500 μg / mL).

[0072] Figure 39 Images of erythrocytes, monocytes, and lymphocytes treated with NS and ultra-small gold nanoclusters solution (500 μg / mL). Detailed Implementation

[0073] In the following examples and experimental cases, reagents or raw materials not specifically described are all commercially available products.

[0074] Example 1: Gold Nanoclusters

[0075] This embodiment provides a gold nanocluster, which is prepared according to the following method:

[0076] 3 mL of 50 mM 4,6-diamino-2-mercaptopyrimidine and 1 mL of 100 mM chloroauric acid were added to 46 mL of water under stirring. After stirring at room temperature for 5 minutes, the reaction system was placed in a water bath and heated to 70 °C, maintaining heating and stirring for 12 hours. The mixture was then purified by ultrafiltration using a 3 kD molecular cutoff tube. The supernatant was collected and freeze-dried to obtain ultra-small gold nanoclusters. A solution of these ultra-small gold nanoclusters with a concentration of 20 μg / mL was prepared.

[0077] The following experiments further illustrate the technical solution presented herein. In the following experimental examples, the ultra-small gold nanoclusters used were all prepared according to the method in Example 1.

[0078] Experimental Example 1 characterizes the morphology and size of the ultra-small gold nanoclusters of the present invention.

[0079] I. Experimental Methods

[0080] The gold nanoclusters prepared in Example 1 were subjected to ultraviolet-visible absorption spectroscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) tests.

[0081] II. Experimental Results

[0082] (1) The ultraviolet-visible absorption spectrum of the ultra-small gold nanoclusters described in this invention was determined. Figure 1 As shown in the figure, the UV-Vis absorption spectrum of the ultra-small gold nanoclusters did not show the characteristic surface plasmon resonance peak, indicating that ultra-small gold nanoclusters were successfully formed, rather than large nanoparticles.

[0083] The synthesized ultrasmall gold nanoclusters exhibited bright red photoluminescence under 365 nm ultraviolet light. The excitation and emission spectra of the photoluminescence were centered at 450 nm and 770 nm, respectively, showing a large Stokes shift exceeding 300 nm. Figure 2 ).

[0084] (2) The size and morphology of the ultra-small gold nanoclusters described in this invention were characterized:

[0085] Transmission electron microscopy (TEM) images confirmed that the ultrasmall gold nanoclusters are monodisperse ultrasmall clusters with an average particle size of 1.8 ± 0.3 nm. Figure 3 )

[0086] (3) Characterization of the surface charge of the ultra-small gold nanoclusters described in this invention:

[0087] The metallic valence states of the prepared ultrasmall gold nanoclusters were further investigated using X-ray photoelectron spectroscopy (XPS). For example ( Figure 4 As shown in the figure, the binding energies of Au4f5 / 2 and Au4f7 / 2 are 87.6 eV and 83.8 eV, respectively, indicating that Au(0) and Au(I) coexist in the ultra-small gold nanoclusters. Furthermore, the positive charge characteristics of the ultra-small gold nanoclusters were also confirmed by potential measurements. Figure 5 ).

[0088] Example 2: Optimization of Synthesis Conditions for Gold Nanoclusters

[0089] I. Experimental Methods

[0090] To optimize the synthesis conditions of gold nanoclusters, the synthesis conditions were modified based on Experiment 1. The experimental groups were set as follows: the molar ratio of 4,6-diamino-2-mercaptopyrimidine to chloroauric acid was changed (0.5:1, 1:1, 1.5:1, 2:1, 3:1, 5:1), the reaction time was changed (1, 3, 5, 8, 12, 15, 18 hours), the reaction pH was changed (adjusted to 3, 4, 5, 6, 7 using dilute hydrochloric acid), and the reaction temperature was changed (25, 40, 55, 70, 85, 100℃). The yield of gold nanoclusters was measured by measuring the fluorescence intensity.

[0091] II. Experimental Results

[0092] The results are as follows Figure 6 As shown, the optimal synthesis conditions for gold nanoclusters were obtained after optimizing the system concentration, reaction time, pH, and temperature: 3 mL of 50 mM 4,6-diamino-2-mercaptopyrimidine and 1 mL of 100 mM chloroauric acid were added to 46 mL of water under stirring. After stirring at room temperature for 5 minutes, the reaction system was placed in a water bath and heated to 70 °C, maintaining heating and stirring for 12 hours. The mixture was then purified and filtered using an ultrafiltration tube with a molecular cutoff of 3 kD. The supernatant was collected and freeze-dried to obtain ultra-small gold nanoclusters. A solution of these ultra-small gold nanoclusters with a concentration of 20 μg / mL was prepared.

[0093] Experimental Example 2: Evaluation of the anticoagulation activity of the ultra-small gold nanoclusters of the present invention

[0094] I. Experimental Methods

[0095] To comprehensively evaluate the anticoagulant activity of the ultra-small gold nanoclusters prepared in Example 1, thromboelastography and conventional coagulation assays were performed. Thromboelastography is one of the most commonly used methods for detecting coagulation function. It can dynamically monitor the occurrence of coagulation, thrombosis, and fibrinolysis. Blood samples were obtained from a healthy volunteer. Specific detection methods were performed according to the reference (Mao C, Xiong Y, Fan C. Comparison between thromboelastography and conventional coagulation assays in patients with deep vein thrombosis. Clin Chim Acta. 2021 Sep; 520:208-213. doi:10.1016 / j.cca.2021.06.019. Epub 2021 Jun 15. PMID:34139174.).

[0096] II. Experimental Results

[0097] Coagulation Composite Index (CI) showed that treatment with ultra-small gold nanoclusters significantly reduced coagulation capacity; when the therapeutic concentration reached 200 μg / mL, the blood was already in a hypocoagulable state. Figure 7 The reaction time (R) level also indicated that treatment with ultra-small gold nanoclusters significantly prolonged clotting time, suggesting that ultra-small gold nanoclusters do indeed possess anticoagulant activity. The coagulation process is primarily regulated by fibrinogen function and platelet aggregation. Figure 8 Results regarding clotting time (K), clotting angle (α), and maximum amplitude (MA) indicate that treatment with ultra-small gold nanoclusters significantly affects fibrinogen function but has little effect on platelet aggregation. Figure 9 , Figure 10 , Figure 11 Furthermore, fibrinolytic indices (EPL and LY30) showed that treatment with ultra-small gold nanoclusters did not induce hyperfibrinolysis or fibrinolysis inhibition. Figure 12 ).

[0098] Thromboelastography suggests that ultra-small gold nanoclusters may exert their anticoagulant effect by affecting fibrinogen function rather than platelet aggregation.

[0099] The routine coagulation test is performed as follows:

[0100] The effects of ultra-small gold nanoclusters on prothrombin time (PT), activated partial thromboplastin time (APPT), and thrombin time (TT) were tested. PT is associated with the extrinsic coagulation pathway and typically reflects the activity of coagulation factors I, II, V, VII, and X in plasma. APPT mainly involves factors in the intrinsic coagulation pathway, and its results reflect the function of factors VIII, IX, X, XI, and XII. TT is the clotting time after the addition of thrombin to plasma, indicating the ability of fibrinogen to convert into fibrin.

[0101] The results indicate that gold nanoclusters do indeed possess significant coagulation activity, which may affect intrinsic and extrinsic coagulation pathways and is closely related to fibrinogen conversion. Figure 13 , Figure 14 , Figure 15 To verify the effect of ultrasmall gold nanoclusters on fibrinogen levels in plasma, we added gold nanocluster solutions of different concentrations to plasma. Figure 16 It can be seen that treatment with ultra-small gold nanoclusters significantly reduced the level of fibrinogen in plasma.

[0102] Routine coagulation assays showed that fibrinogen reduction induced by ultra-small gold nanoclusters played a dominant role in anticoagulation. Measurements of plasma fibrinogen degradation products (FDP) and D-dimer indicated that treatment with ultra-small gold nanoclusters did not induce activation of the fibrinolytic system. Figure 17 , Figure 18 ).

[0103] Furthermore, the decrease in plasma fibrinogen induced by treatment with ultra-small gold nanoclusters was not caused by activation of the fibrinolytic system. The results of antithrombin III (AT-III) assays also indicated that ultra-small gold nanoclusters do not exert their anticoagulant activity by affecting the function of antithrombin. Figure 19 ).

[0104] In summary, thromboelastography and conventional coagulation assays in this experiment demonstrate that the ultra-small gold nanoclusters of this invention possess anticoagulant activity, and that this anticoagulant activity is closely related to their interaction with fibrinogen. During coagulation, fibrinogen is cleaved into fibrin by thrombin, and the fibrin aggregates to form a clot.

[0105] Experimental Example 3: Study on the anticoagulation mechanism of the ultra-small gold nanoclusters of the present invention

[0106] I. Experimental Methods

[0107] This experimental example investigates the anticoagulation mechanism of the gold nanoclusters prepared in Example 1.

[0108] The interaction of ultra-small gold nanoclusters during fibrinogen polymerization was further investigated using fibrinogen from human plasma. The nanoclusters were incubated at 37 °C with increasing concentrations (0, 50, 100, 200, 300 μg / mL) of gold nanoclusters. The cleavage and polymerization of fibrinogen were driven by the addition of 1 U / mL thrombin. For ease of observation, the polymerization process was photographed under ultraviolet light due to the photoluminescent properties of the ultra-small gold nanoclusters.

[0109] II. Experimental Results

[0110] In the absence of ultra-small gold nanoclusters, clot formation occurred within 10 minutes after the addition of thrombin. Conversely, treatment with ultra-small gold nanoclusters significantly inhibited clot formation, and no visible clot formation was observed within 60 minutes. Figure 20 Furthermore, we observed a large amount of fluorescent aggregation within 10 minutes in vials incubated with ultra-small gold nanoclusters, indicating that Au clusters may bind to fibrinogen and cause aggregation. In fact, by incubating ultra-small gold nanoclusters with fibrinogen and thrombin separately, we found that the gold nanoclusters aggregated only with fibrinogen. The binding behavior of ultra-small gold nanoclusters to fibrinogen was observed by TEM. A large number of ultra-small gold nanoclusters attached to the surface of fibrinogen to form aggregates. Figure 21 , Figure 22 , Figure 23 , Figure 24 ).

[0111] Simultaneously, we performed molecular docking analysis on the binding of ultra-small gold nanoclusters with blood clots to assess the stability of blood clot formation:

[0112] Molecular docking analysis showed that the thiol-pyrimidine ligands of ultrasmall gold nanoclusters can bind to fibrinogen through visible hydrogen bonds and strong electrostatic interactions, with a binding energy as low as -15.6 kcal / mol, indicating highly stable binding. Figure 25 ).

[0113] These results indicate that the aggregation of ultraminiature gold nanoclusters with fibrinogen is a key mechanism for their anticoagulant activity, due to the stable affinity binding of surface thiol pyrimidine ligands to fibrinogen. The precipitation of fibrinogen by ultraminiature gold nanoclusters inhibits fibrin formation, thereby affecting the polymerization process and thrombus formation.

[0114] We further investigated the effect of ultra-small gold nanoclusters on platelet aggregation. Platelets, as the most important blood cells in primary hemostasis, can activate coagulation factors, forming platelet thrombi that constitute primary hemostatic plugs and promoting blood coagulation. Therefore, platelet aggregation also plays a crucial role in thrombus formation. Collagen is one of the most effective agonists in inducing platelet aggregation, and we used freshly isolated rabbit platelets triggered by collagen to examine the effect of ultra-small gold nanoclusters on platelet aggregation.

[0115] Studies have found that ultra-small gold nanoclusters have no effect on platelets and have almost no effect on collagen-triggered platelet aggregation. Figure 26 , Figure 27 Aggregation curves showed that ultra-small gold nanoclusters of different concentrations maintained similar aggregation levels to the control group, with a maximum aggregation rate of approximately 80%. Figure 28 Furthermore, the results of measurements of biochemical factors closely related to platelet activation, such as P-selectin, serotonin (5-HT), and cAMP, also indicated that treatment with ultra-small gold nanoclusters did not affect platelet activation (as shown in Table 1). The negligible effect of ultra-small gold nanoclusters on platelets also confirms the excellent blood compatibility of ultra-small gold nanoclusters.

[0116] Table 1. Effects of ultra-small gold nanocluster solutions on platelet activation

[0117]

[0118] The above results indicate that platelet aggregation does not play a role in the anticoagulation process of ultra-small gold nanoclusters, and the inhibition of fibrinogen cleavage and aggregation caused by ultra-small gold nanoclusters is the main reason for its anticoagulation activity.

[0119] Example 4: Using ICR mice to investigate the antithrombotic activity of the ultra-small gold nanoclusters of the present invention.

[0120] I. Experimental Methods

[0121] This experiment used ICR mice to create a tail thrombosis model by intraperitoneal injection of carrageenan at a concentration of 40 mg / kg. The antithrombotic activity of this gold nanocluster (ultra-small gold nanocluster) was investigated using this tail thrombosis model. All animal experiments were conducted in accordance with animal testing guidelines and approved by the Animal Nursing Committee of Southwest Medical University. Six- to eight-week-old male ICR mice were purchased from Beijing Haifeijin Biotechnology Co., Ltd. Purchased mice were randomly divided into 5 groups of 6 mice each (n=6): a blank control group (injected with 0.9% saline), a carrageenan group (carrageenan concentration of 40 mg / kg), a carrageenan + saline group (carrageenan + NS, 40 mg / kg carrageenan injected 1 hour after injection of 0.9% saline), a carrageenan + ultra-small gold nanoclusters group (carrageenan + ultra-small gold nanoclusters, 40 mg / kg carrageenan injected 1 hour after injection of 25 mg / kg gold nanoclusters), and a carrageenan + heparin group (carrageenan + heparin, 40 mg / kg carrageenan injected 100 U / kg carrageenan injected 1 hour after injection). All purchased mice were acclimatized for one week before subsequent experiments. After injection, mice were cultured for 24 hours, and then the length of the tail thrombus was measured, and tail blood flow was detected using Doppler flowmetry.

[0122] II. Experimental Results

[0123] like Figure 29 , Figure 30 As shown, the tails of ICR mice darkened after carrageenan injection, a marker of thrombus formation, and quantitative analysis showed a thrombus length of 7.5 ± 1.1 cm. In contrast, the 25 mg / kg gold nanocluster treatment group significantly inhibited thrombus formation, comparable to the antithrombotic drug heparin (1.3 ± 1.0 cm vs 0.8 ± 0.6 cm, p = 0.26). Figure 31 , Figure 32 As shown in the laser Doppler perfusion images, carrageenan injection significantly reduced blood flow in the mouse tail, while gold nanoclusters and heparin treatment significantly restored blood flow (red indicates smooth blood flow, blue indicates vascular occlusion and thrombus formation). Simultaneously, we prepared pathological HE sections from the lungs and tails of mice in each group to observe the distribution of thrombi. Hematoxylin and eosin (HE) staining showed a large accumulation of thrombi in the tail sections of carrageenan-injected mice, while gold nanocluster treatment significantly reduced thrombus formation. The arrows indicate the location of the thrombi. Figure 33Furthermore, we noted that carrageenan injection also induced pulmonary thrombosis in ICR mice, with numerous thrombi forming in both the pulmonary aorta and arterioles, while treatment with gold nanoclusters prevented pulmonary thrombosis formation. Figure 34 ).

[0124] The above animal experiments all show that the gold nanoclusters have a good inhibitory effect on thrombosis, with an effect comparable to heparin, which verifies the excellent antithrombotic ability of the ultra-small gold nanoclusters.

[0125] Example 5: Blood-related cell biocompatibility of the ultra-small gold nanoclusters described in this invention

[0126] I. Experimental Methods

[0127] The Cell Count Kit-8 (CCK-8) assay was used to determine the cytotoxicity of ultra-small gold nanoclusters on human umbilical vein endothelial cells (HUVECs).

[0128] II. Experimental Results

[0129] Experimental results showed that even at a concentration of 500 μg / mL, cell viability remained above 85%, indicating that the ultra-small gold nanoclusters exhibited good biocompatibility with HUVECs at the tested dosage. Figure 35 Next, we used different concentrations of ultra-small gold nanoclusters in the range of 5-500 μg / mL for hemolysis analysis. The results showed that even when the concentration of gold nanoclusters reached 500 μg / mL, the resulting hemolytic side effects were still less than 5%. Figure 36 ).

[0130] Furthermore, we used optical microscopy and scanning electron microscopy (SEM) to observe the effects of ultrasmall gold nanoclusters on blood cells. It was observed that treatment with DAMP AuNC had virtually no effect on the number and morphological characteristics of blood cells. Figure 37 , Figure 38 Similarly, we performed the same measurements on red blood cells, platelets, and white blood cells (such as monocytes and neutrophils), and none of them were damaged by the ultra-small gold nanoclusters. Figure 39 ).

[0131] The data from this experiment demonstrate that ultra-small gold nanoclusters possess excellent blood compatibility and have potential applications in vascular biomedicine.

[0132] As can be seen from the above embodiments and experimental examples, the present invention provides a novel gold nanocluster that can inhibit fibrinogen cleavage and aggregation, thus exhibiting good coagulation effects. It can be used to prepare drugs that inhibit thrombosis and has great application prospects.

Claims

1. A method for preparing gold nanoclusters, characterized in that, Includes the following steps: Step 1: Prepare a mixed solution of 4,6-diamino-2-mercaptopyrimidine and chloroauric acid by stirring at 280-350 rpm. Step 2: Heat to 65-75℃ for reaction; Step 3: Concentrate and filter using an ultrafiltration tube, collect the supernatant, and freeze-dry to obtain the final product; In step 1, the molar ratio of 4,6-diamino-2-mercaptopyrimidine to chloroauric acid is (1-2):1; in the mixed solution, the concentration of 4,6-diamino-2-mercaptopyrimidine is 1.5 mM and the concentration of chloroauric acid is 1 mM. In step 2, the reaction process is as follows: heat to 20-30 ℃, stir for 10 minutes, then heat to 65-75 ℃ and react for 10-15 hours; in step 2, adjust the pH before the reaction to a pH of 5-6.

2. The preparation method according to claim 1, characterized in that, In step 1, the molar ratio of 4,6-diamino-2-mercaptopyrimidine to chloroauric acid is 1.5:

1. And / or, in step 2, the reaction process is as follows: heat to 20-30 ℃, stir for 10 minutes, then heat to 70 ℃ and react for 12 hours; And / or, in step 2, the pH of the reaction is 6.

3. Use of the gold nanoclusters obtained by the preparation method according to claim 1 or 2 in the preparation of drugs for inhibiting thrombosis.

4. The use according to claim 3, characterized in that: The gold nanoclusters are used to reduce fibrinogen levels in plasma.

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