A polypeptide functionalized gold nanocluster targeting neutrophils, a preparation method thereof and application thereof in preparation of an anti-thrombus drug

Abstract

The application relates to a polypeptide functionalized gold nanocluster targeting neutrophils, a preparation method thereof and application thereof in preparation of an anti-thrombus drug, and belongs to the field of biomedical nanomaterials. The nanocluster is formed by Au-S coordination self-assembly of a thiol-containing targeting polypeptide and gold atoms. The polypeptide can specifically recognize neutrophil elastase (NE), realizes targeting of activated neutrophils and inhibits enzyme activity of the neutrophils. The nanocluster inhibits formation of neutrophil extracellular traps (NETs) induced by phorbol ester (PMA) or activated platelets, and shows reduction of histone citrullination (CitH3) level, extracellular DNA release and active oxygen (ROS) generation. In-vivo experiments show that the gold nanocluster is enriched at a thrombus site, and significantly reduces thrombus load in a deep vein thrombosis model and an FeCl3-induced arterial thrombosis model. The application plays an anti-thrombus role by inhibiting NETs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials, specifically relating to a polypeptide-functionalized gold nanocluster that targets neutrophils, its preparation method, and its application in the preparation of antithrombotic drugs. Background Technology

[0002] Thrombotic diseases, such as deep vein thrombosis and arterial thrombosis, are a major cause of cardiovascular and cerebrovascular events. Their development involves the complex regulation of multiple factors, including the coagulation system, platelet activation, and inflammatory responses. Currently, clinical antithrombotic therapy mainly relies on anticoagulants (such as heparin and warfarin) and antiplatelet drugs (such as aspirin and clopidogrel). However, while these drugs inhibit thrombus formation, they generally increase the risk of bleeding and are insufficient in regulating key inflammatory mechanisms in the thrombus formation process.

[0003] Recent studies have shown that neutrophils play a crucial role in thrombosis. Activated neutrophils release a network of structures called neutrophil extracellular traps (NETs). NETs are mainly composed of depolymerized chromatin fibers and embedded granule proteins (such as neutrophil elastase and myeloperoxidase). NETs provide a scaffold for platelet adhesion, activation, and aggregation, and enrich local coagulation factors, thereby significantly promoting thrombus formation and stability. Therefore, targeting the NET formation process is a highly promising intervention strategy for the development of antithrombotic drugs.

[0004] In the key steps of NET formation, neutrophil elastase (NE) is released from granules and enters the nucleus, cleaving histones and synergistically promoting histone citrullination catalyzed by arginine deaminase 4 (PAD4), thus promoting chromatin depolymerization. NE is the core executor of NETosis (the process of NET formation). Previous studies have attempted to inhibit NE activity using small molecule inhibitors or antibodies to block NET formation. However, these strategies often face challenges such as low target specificity, poor in vivo stability, and rapid clearance. Therefore, developing a novel formulation that can precisely target neutrophils at the lesion site and effectively regulate NE activity and NET formation is of great significance for the preparation of highly effective and low-toxicity antithrombotic drugs.

[0005] Nanotechnology offers new avenues for drug delivery and disease targeting. Gold nanomaterials, particularly gold nanoclusters, show great promise in the biomedical field due to their small size, good biocompatibility, ease of surface functionalization, and unique optical properties. Specific biological functions can be endowed to gold nanoclusters through the rational design of surface ligands. However, currently, there is a lack of gold-based nanoformulations capable of specifically recognizing activated neutrophils and actively intervening in their NETs formation process.

[0006] In summary, existing technologies still lack an antithrombotic nanomaterial that combines good targeting, high biostability, and a clear NETs-inhibiting function. Therefore, this invention aims to provide a novel peptide-functionalized gold nanocluster to address the aforementioned problems. Summary of the Invention

[0007] This invention aims to provide a peptide-functionalized gold nanocluster (hereinafter referred to as NIGC) capable of specifically targeting activated neutrophils and effectively inhibiting NET formation. Another objective of this invention is to provide a simple method for preparing the aforementioned gold nanocluster. A further objective of this invention is to provide the application of the aforementioned gold nanocluster in the preparation of drugs for antithrombotic diseases.

[0008] This invention first provides a peptide-functionalized gold nanocluster, which is formed by the self-assembly of gold atoms and a thiol-containing targeting peptide via Au-S coordination bonds. The amino acid sequence of the targeting peptide contains a functional domain capable of interacting with neutrophil elastase (NE), thereby endowing the nanocluster with the dual function of targeting and inhibiting NE.

[0009] Preferably, the cluster has the following characteristics:

[0010] 1. The molecular composition is Au. x P y Where x represents the number of gold atoms, an integer from 1 to 45, and y represents the number of polypeptide ligands, an integer from 1 to 40. In a preferred embodiment, its molecular formula is Au. 28 P 12 .

[0011] 2. Its hydrated particle size is approximately 2-5 nm.

[0012] 3. It exhibits red fluorescence emission under ultraviolet light excitation.

[0013] 4. It can specifically bind to and inhibit NE on the surface of activated neutrophils, thereby inhibiting the formation of NETs.

[0014] Preferably, the amino acid sequence of the targeting peptide is: CGEAIPMSIPPEVKKKCCY. In this sequence, CGEAIPMSIPPEVK is the NE repressor peptide, the polylysine (KKK) fragment is used to improve solubility and biocompatibility, and the cysteine-cysteine-tyrosine (CCY) motif is used to achieve Au-S coordination and gold ion reduction reaction.

[0015] The preparation method of the nanoclusters includes:

[0016] 1. Mix an aqueous solution of a gold salt (such as chloroauric acid, HAuCl4) with an aqueous solution of a thiol-containing targeting peptide.

[0017] 2. While stirring, add a reducing agent (such as sodium hydroxide solution) to the mixture to reduce the gold ions.

[0018] 3. The reaction is carried out at 20-50℃ in the dark for 4-10 hours. During the reaction, gold ions are reduced and self-assemble with the thiol groups of the peptide through Au-S bonds to form stable nanoclusters.

[0019] 4. After the reaction is complete, the product is purified by ultrafiltration, centrifugation or dialysis to remove unreacted peptides, ions and other small molecule impurities, and to obtain a pure aqueous solution of peptide-functionalized gold nanoclusters.

[0020] Preferably, the molar ratio of the targeting peptide to the gold salt is 1:1 to 1:2.

[0021] Finally, this invention provides the following applications of the above-mentioned peptide-functionalized gold nanoclusters:

[0022] It is used to inhibit the formation of extracellular traps on neutrophils.

[0023] This is used to prepare drugs for the prevention or treatment of thrombotic diseases. The thrombotic diseases include, but are not limited to, deep vein thrombosis and arterial thrombosis.

[0024] The present invention has the following advantages:

[0025] 1. Integrated design and simple preparation: The peptide molecule used integrates NE inhibition, cell targeting and metal coordination functions, eliminating the need for complex subsequent modifications to the nanoclusters. Functionalized nanoclusters can be synthesized easily and efficiently in one step, with simple process and good reproducibility.

[0026] 2. Precise targeting and efficient regulation: The obtained NIGC can specifically accumulate on the surface of activated neutrophils by means of the peptide's targeting domain, and effectively block the formation of NETs from upstream by inhibiting NE activity, thus intervening in the core inflammatory link of thrombosis.

[0027] 3. Significant in vivo antithrombotic effect: In two classic mouse models of deep vein thrombosis and ferric chloride-induced arterial thrombosis, NIGC showed superior antithrombotic effects compared to traditional antiplatelet drugs (aspirin), and significantly reduced thrombus weight and length.

[0028] 4. High biocompatibility: Experiments show that NIGC has no significant cytotoxicity to neutrophils within the effective dose range, and while exerting antithrombotic effects, it has little impact on key parameters of coagulation function in mice (such as thromboelastography parameters), indicating that it has high therapeutic safety and a low potential risk of bleeding. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the structure and basic characterization of the gold nanocluster NIGC of this invention.

[0030] Figure 1 A: Schematic diagram of the synthesis route for NIGC.

[0031] Figure 1 B: NIGC hydration particle size distribution obtained by dynamic light scattering method.

[0032] Figure 1 C: Representative transmission electron microscope image from NIGC (scale bar = 20 nm).

[0033] Figure 1 D: Zeta potential image of NIGC.

[0034] Figure 1 E: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) from NIGC, yielding the molecular formula Au. 28 P 12 .

[0035] Figure 2 This is a characterization diagram of the optical properties of the gold nanocluster NIGC of this invention.

[0036] Figure 2 A: Fluorescence excitation and emission spectra of NIGC.

[0037] Figure 2 B: Comparison of UV-Vis absorption spectra of NIGC and free peptide (NEBP).

[0038] Figure 3 This diagram illustrates the inhibitory effect of the gold nanoclusters NIGC of this invention on the formation of extracellular traps in neutrophils.

[0039] Figure 3 A: Representative Sytox Green staining images of NETs induced by phorbol ester (PMA) in mouse bone marrow neutrophils from NIGC and quantitative statistical plots of the proportion of NETs-positive cells (**p < 0.01, ***p < 0.001).

[0040] Figure 3 B: Representative immunofluorescence images of citrullinated histone H3 (CitH3), myeloperoxidase (NPO), and nucleus (blue) co-stained in neutrophils from different treatment groups, and CitH3. + MPO + Quantitative statistical plot of cell proportion (*p < 0.05, ***p < 0.001).

[0041] Figure 3C: NETs release level was quantitatively analyzed by detecting the content of double-stranded DNA (dsDNA) in the supernatant (*p < 0.05, ***p < 0.001).

[0042] Figure 4 This is a diagram illustrating the in vitro targeting and cellular safety evaluation of the gold nanoclusters NIGC of this invention.

[0043] Figure 4 A: The uptake of NIGC by different cells was detected by inductively coupled plasma mass spectrometry (ICP-MS) (expressed as cellular gold content) (***p < 0.001).

[0044] Figure 4 B: CCK-8 assay was used to detect cell viability after co-culturing neutrophils with different concentrations of NIGC.

[0045] Figure 5 This is a schematic diagram illustrating the in vivo antithrombotic effect and targeting of the gold nanoclusters NIGC in a mouse deep vein thrombosis (DVT) model.

[0046] Figure 5 A: Schematic diagram of the experimental procedure for verifying the in vivo targeting of NIGC (administration 24 hours after ligation, sample collection and analysis 15 minutes later).

[0047] Figure 5 B: H&E staining image of thrombus tissue.

[0048] Figure 5 C: Representative image of Ly-6G (neutrophil marker) immunofluorescence staining in thrombus tissue, showing neutrophil infiltration in the thrombus.

[0049] Figure 5 D: Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis showed that the gold signal (representing NIGC) was co-localized with the Ly-6G signal within the thrombus.

[0050] Figure 5 E: Schematic diagram of the treatment regimen for the NIGC anti-DVT model.

[0051] Figure 5 F: Representative photographs of thrombosis in mice under different treatment groups (sham surgery, model, NIGC group, free polypeptide NEBP group, aspirin group).

[0052] Figure 5 G: Quantitative statistical results of thrombus weight in different treatment groups.

[0053] Figure 5 H: Quantitative statistical results of thrombus length in different treatment groups.

[0054] Figure 5 I: Representative images of H&E staining and CitH3 and Ly-6G immunofluorescence staining of thrombus tissues from different treatment groups, and quantitative analysis of the CitH3-positive area within the thrombus.

[0055] Figure 6 This image shows the in vivo antithrombotic effect and targeting verification of the gold nanoclusters NIGC of this invention in a mouse model of carotid artery thrombosis induced by ferric chloride (FeCl3).

[0056] Figure 6 A: Schematic diagram of the experimental procedure for verifying the targeting of NIGC in an arterial thrombosis model (administration of drugs at the same time as modeling, and sample collection and analysis 8 minutes later).

[0057] Figure 6 B: H&E staining image of thrombus tissue.

[0058] Figure 6 C: Representative image of Ly-6G immunofluorescence staining of thrombus tissue.

[0059] Figure 6 D:LA-ICP-MS analysis showed that the gold signal was co-localized with the Ly-6G signal within the arterial thrombus.

[0060] Figure 6 E: Schematic diagram of NIGC prophylactic anticarotid artery thrombosis treatment protocol.

[0061] Figure 6 F: Representative photographs of carotid artery thrombosis in mice under different treatments.

[0062] Figure 6 G: Quantitative statistical results of vascular embolism rate in different treatment groups.

[0063] Figure 6 H: Representative images of H&E staining and CitH3 and Ly-6G immunofluorescence staining of thrombus tissues from different treatment groups, and quantitative analysis of the CitH3-positive area within the thrombus.

[0064] Figure 7 This is an evaluation diagram of the effect of the gold nanoclusters NIGC of the present invention on the systemic coagulation function of mice (through thromboelastography TEG analysis).

[0065] Figure 7 A: Representative thromboelastography of blood samples from mice in different treatment groups (saline control, NIGC, NEBP, aspirin).

[0066] Figure 7 B: Quantitative statistics of the thromboelastography parameter "Angle" in different treatment groups.

[0067] Figure 7C: Quantitative statistics of the thromboelastography parameter "reaction time (R time)" in different treatment groups.

[0068] Figure 7 D: Quantitative statistics of the thromboelastography parameter "coagulation time (K time)" in different treatment groups.

[0069] Figure 7 E: Quantitative statistics of the thromboelastographic parameter "maximum amplitude (MA)" in different treatment groups Detailed Implementation

[0070] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0071] Example 1: Preparation and characterization of gold nanoclusters NIGC

[0072] This embodiment details the preparation process and physicochemical characterization of targeted peptide-functionalized gold nanoclusters (NIGC).

[0073] Preparation method: Synthetic route as follows Figure 1 As shown in Figure A, 5 mg of the targeting peptide CGEAIPMSIPPEVKKKCCY was dissolved in 2.385 ml of ultrapure water to prepare a 1 mM solution. Under continuous stirring, 95.5 μL of a 25 mM chloroauric acid aqueous solution was slowly added to the peptide solution. After thorough mixing, 477 μL of a 0.5 M sodium hydroxide solution was added dropwise. The reaction was carried out at 37 °C, protected from light, and sealed for 6 hours to obtain the target gold nanoclusters NIGC. After the reaction, the reaction solution was purified multiple times by centrifugation with ultrapure water using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to completely remove unreacted peptides, gold ions, and other small molecule byproducts. The final purified NIGC aqueous solution was stored at 4 °C protected from light for later use.

[0074] Characterization results: Figure 1 As shown in Figure B, dynamic light scattering results indicate that the hydrated particle size of NIGC is approximately 2.43 nm. Transmission electron microscopy images clearly show that NIGC consists of uniformly sized, well-dispersed, approximately spherical particles with a diameter of about 2.24 nm. Figure 1 C). Zeta potential measurements indicate that its surface is negatively charged, approximately -38.9 mV ( Figure 1 D). MALDI-TOF mass spectrometry analysis showed that the molecular weight corresponding to its main mass-to-charge ratio peak was consistent with the molecular formula (Au) composed of 28 gold atoms and 12 polypeptide ligands. 28 P 12 () Figure 1E). Optical characterization showed that the NIGC aqueous solution was pale yellow under natural light and emitted bright red fluorescence under ultraviolet light (365 nm). Its fluorescence spectrum showed a maximum excitation wavelength of 508 nm and a maximum emission wavelength of 665 nm. Figure 2 A). UV-Vis absorption spectroscopy showed that the free peptide NEBP had a UV absorption peak at 274 nm, indicating that the peptide was in an unoxidized state. However, after the synthesis of the gold cluster NIGC, the characteristic absorption peak of NEBP disappeared, indicating that the peptide underwent a reaction. Figure 2 B).

[0075] Example 2: In vitro inhibitory effect of NIGC on the formation of extracellular traps of neutrophils

[0076] This embodiment verifies the effect of NIGC in suppressing NET formation through various methods.

[0077] Primary neutrophils were isolated from mouse bone marrow. Experimental groups included: control group (untreated), model group (PMA stimulation), NIGC intervention group (pretreated with 100 μM NIGC for 15 min before PMA stimulation), and free peptide (NEBP) intervention group (pretreated with 100 μM NEBP for 15 min before PMA stimulation). The PMA stimulation concentration was 100 nM, and the treatment time was 4 hours.

[0078] First, NETs were stained and quantified using Sytox Green, a nucleic acid dye that can only penetrate damaged cell membranes. For example... Figure 3 As shown in Figure A, PMA stimulation significantly increased the Sytox Green positive signal (green), indicating a large release of NETs. Both NIGC and NEBP pretreatment significantly inhibited this phenomenon, with NIGC showing a stronger inhibitory effect than NEBP.

[0079] Secondly, the co-localization of NETs key markers—citrullinated histone H3 (CitH3) and myeloperoxidase (MPO)—was detected by immunofluorescence staining. Figure 3 B shows that after PMA stimulation, CitH3 (green) and MPO (red) formed a distinct extracellular network co-localization structure (yellow), while this structure was significantly reduced in the NIGC-treated group. Regarding CitH3... + MPO + Quantitative analysis of the proportion of double-positive cells further confirmed the inhibitory effect of NIGC on NET formation.

[0080] Finally, the content of double-stranded DNA (dsDNA) released in the cell culture supernatant was quantitatively detected using PicoGreen fluorescent dye to assess NETs at the biochemical level. Figure 3As shown in Figure C, PMA stimulation led to a sharp increase in dsDNA content in the supernatant, while co-treatment with NIGC reduced it to levels close to the control group, demonstrating a significantly better effect than NEBP. These results collectively demonstrate that NIGC can effectively inhibit PMA-induced neutrophil NETs formation.

[0081] Example 3: In vitro targeting and biosafety evaluation of NIGC

[0082] This embodiment evaluated the selective targeting ability of NIGC to activated neutrophils and its effect on neutrophil activity.

[0083] Figure 4 A. The uptake of NIGC by different cells was detected using inductively coupled plasma mass spectrometry (ICP-MS). Resting neutrophils, PMA (100 nM) activated neutrophils, and other bone marrow-derived non-neutrophils (as controls) were co-incubated with 100 μM NIGC for 4 hours. After thorough washing, the cells were digested and their gold content was measured. The results showed that the uptake of NIGC by activated neutrophils was significantly higher than that by resting neutrophils and other control cells, indicating that NIGC can selectively target activated neutrophils.

[0084] The effect of NIGC on neutrophil viability was detected using the CCK-8 assay. Neutrophils were co-incubated with different concentrations (0, 10, 50, 100, 200, 500 μM) of NIGC for 4 hours, followed by further incubation with the CCK-8 reagent. Absorbance at 450 nm was then measured. Results are shown below. Figure 4 As shown in Figure B, even at a high concentration of 500 μM, there was no statistically significant difference in cell viability between the NIGC-treated group and the control group, indicating that NIGC has no significant cytotoxicity to neutrophils within the experimental concentration range.

[0085] Example 4: Antithrombotic effect of gold nanoclusters NIGC in a deep vein thrombosis model

[0086] After confirming the in vitro activity of NIGC, its in vivo effects were evaluated in a mouse inferior vena cava (IVC) ligation DVT model.

[0087] In vivo targeting validation: First, validate the distribution of NIGC in thrombi. For example... Figure 5 As shown in Figure A, a deep vein thrombosis (DVT) model was established by ligating the caudal intravenous cleft (IVC) of the mouse renal vein. Twenty-four hours after modeling, a single injection of NIGC (10 mg / kg) was administered via the tail vein. Fifteen minutes after injection, thrombus tissue was collected for fixation and analysis. H&E staining confirmed successful thrombus formation. Figure 5 B). Immunofluorescence staining showed a large number of Ly-6G-positive neutrophils infiltrating the thrombus ( Figure 5C). LA-ICP-MS spatial elemental analysis showed that gold signals (representing NIGC) were specifically enriched in thrombus tissue and highly overlapped with the signal distribution of the neutrophil marker Ly-6G. Figure 5 (D) This demonstrates that NIGC can effectively target the region of activated neutrophils that accumulate within the thrombus.

[0088] Evaluation of the efficacy of prophylactic antithrombotic therapy: assessing the preventive effect against NIGC. Treatment regimen as follows: Figure 5 As shown in Figure E, the groups were as follows: sham surgery group, model group, NIGC treatment group (10 mg / kg, single tail vein injection 15 min before modeling), free peptide NEBP treatment group (10 mg / kg, single tail vein injection 15 min before modeling), and aspirin positive control group (10 mg / kg / day, gavage for 5 days). Blood clots were collected for analysis 24 hours after modeling. Figure 5 As shown in FH, compared with the model group, NIGC treatment significantly reduced thrombus weight and length, and its effect was superior to the NEBP group and the aspirin group. H&E staining and CitH3 / Ly-6G immunofluorescence staining analysis of thrombus tissue (…) Figure 5 I) showed that NIGC treatment significantly reduced the positive area of ​​CitH3 (a marker of NETs) within the thrombus, indicating that it exerts its antithrombotic effect in vivo by inhibiting NET formation.

[0089] Example 5: Antithrombotic effect and targeting verification of NIGC in an arterial thrombosis model

[0090] To assess the generalizability of the antithrombotic effect of NIGC, it was validated in a FeCl3-induced mouse carotid artery thrombosis model.

[0091] In vivo targeting validation: such as Figure 6 As shown in Figure A, mouse carotid arteries were induced to thrombosis by wrapping filter paper soaked in 10% FeCl3 solution for 8 minutes. Simultaneously, NIGC (10 mg / kg) was injected via the tail vein. Samples were collected and analyzed 8 minutes later. H&E staining showed intravascular thrombus formation. Figure 6 B). Immunofluorescence ( Figure 6 C) and LA-ICP-MS ( Figure 6 D) Analysis further confirms that NIGC can effectively target and accumulate in the neutrophil region within arterial thrombi.

[0092] Evaluation of the efficacy of prophylactic antithrombotic therapy: Treatment regimen as follows Figure 6 As shown in E, the grouping was the same as in Example 4. After modeling, thrombi were collected, weighed, and the embolism rate / inhibition rate was calculated. The results are as follows: Figure 6As shown in FG, the NIGC treatment group had the lightest thrombus weight and a thrombus formation inhibition rate as high as 89.62%, significantly better than the NEBP group and the aspirin group (33.09%). H&E and immunofluorescence staining of the thrombus tissue ( Figure 6 The H) results showed that the thrombus volume was smaller in the NIGC treatment group, and the CitH3 signal within the thrombus was significantly reduced. This demonstrates that NIGC also has excellent preventive effects in the arterial thrombosis model, and its mechanism is related to the inhibition of NET formation.

[0093] Example 6: Safety assessment of NIGC on systemic coagulation function in mice.

[0094] To assess the bleeding risk of NIGC, thromboelastography (TEG) was used to comprehensively analyze its impact on systemic coagulation function in mice. TEG can monitor the entire coagulation process, with parameters including reaction time (R value, representing the rate of coagulation initiation), coagulation time (K value), coagulation angle (Angle, representing the coagulation rate), and maximum amplitude (MA, representing the maximum thrombus strength).

[0095] Experimental Methods: Mice were divided into four groups: saline control group (tail vein injection), NIGC group (10 mg / kg, single tail vein injection), NEBP group (10 mg / kg, single tail vein injection), and aspirin group (10 mg / kg / day, gavage for 5 days). Whole blood was collected from mice 15 minutes after the last administration (intravenous administration group) or 24 hours after the last gavage (aspirin group) in sodium citrate anticoagulant tubes, and TEG analysis was performed immediately.

[0096] Representative TEG spectra such as Figure 7 As shown in Figure A. Quantitative statistical results for each group of TEG parameters (Angle, R time, K time, MA) are shown in Figure A. Figure 7 BE. Compared with the saline control group, there were no statistically significant differences in all TEG parameters between the NIGC and NEBP groups, indicating that a single administration of NIGC or free peptides had no significant effect on the overall coagulation function of mice. In contrast, the aspirin group showed signs of altered coagulation function, with a trend towards prolonged R and K values ​​and a trend towards decreased Angle, and the data showed greater variability, suggesting that while aspirin exerts its antiplatelet effect, it also has a detectable effect on the coagulation system, potentially increasing bleeding tendency. These experimental results indicate that at the experimental dose, NIGC exerts a significant antithrombotic effect without significantly interfering with the normal coagulation process like the traditional antiplatelet drug aspirin, demonstrating its potentially high safety profile and low bleeding risk.

[0097] The above examples fully demonstrate that the peptide-functionalized gold nanoclusters (NIGCs) provided by the present invention are easy to prepare, can specifically target activated neutrophils, effectively inhibit NET formation, and exhibit significantly better antithrombotic activity than traditional drugs in both venous and arterial thrombosis models. Moreover, they have little impact on systemic coagulation function, and have high biosafety and important clinical application prospects.

Claims

1. A nanoscale polypeptide-gold complex cluster NIGC, characterized in that, The clusters are formed by the self-assembly of gold atoms and thiol-containing targeting peptides through Au-S coordination, and their molecular composition is Au. x P y , where x is an integer from 1 to 45 and y is an integer from 1 to 40.

2. The cluster according to claim 1, characterized in that, The amino acid sequence of the polypeptide is CGEAIPMSIPPEVKKKCCY or a derivative thereof.

3. The cluster according to claim 1 or 2, characterized in that: The hydrated particle size of the clusters is 2-5 nm.

4. The cluster according to any one of claims 1-3, characterized in that: The clusters are used to inhibit the formation of extracellular traps (NETs) for neutrophils.

5. The cluster according to claim 1, characterized in that: The polypeptide contains at least one cysteine ​​residue.

6. The use of the clusters according to any one of claims 1-3 in the preparation of medicaments for the prevention or treatment of thrombotic diseases.

7. A method for preparing the cluster according to any one of claims 1-3, characterized in that, The steps include the following: (1) Mix the gold salt solution with the thiol-containing targeting peptide; (2) Add a reducing agent under neutral or weakly alkaline conditions to reduce gold ions; (3) Nanoclusters are formed through Au-S coordination; (4) The obtained product is purified to obtain the cluster; in The peptide concentration ranged from 0.1 μM to 10 mM. The gold salt concentration ranged from 0.01 mM to 10 mM. The molar ratio of polypeptide to gold salt is 1:1 to 1:2; The reaction temperature is 20-75 ℃; The reaction time is 5-15 hours.

8. The method according to claim 6, characterized in that: The gold salt is chloroauric acid (HAuCl4).

9. The method according to claim 6, characterized in that: The reducing agent is selected from one or more of sodium hydroxide, ascorbic acid, or trisodium citrate.

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