Gold-silver bimetallic nanoclusters, preparation method and application thereof

Abstract

The application provides a gold-silver bimetallic nanocluster and a preparation method and application thereof, and the preparation method comprises the following steps: performing a co-reduction synthesis reaction on chloroauric acid, silver nitrate and sodium borohydride, coating with mercaptosuccinic acid, forming a spherical nanocluster, performing semi-permeable membrane filtration, and obtaining the gold-silver bimetallic nanocluster, wherein the gold-silver atomic ratio in the gold-silver bimetallic nanocluster is 7:1. The gold-silver bimetallic nanocluster prepared by the preparation method has a stable chemical structure, excellent charge stability and high efficient oxygen free radical scavenging capacity, can weaken oxidative stress damage in a cerebral ischemia-reperfusion injury process, provides protection effect for neurons, and has good biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a gold-silver bimetallic nanocluster and a preparation method and application thereof. BACKGROUND

[0002] Ischemic stroke, usually caused by cerebral vascular occlusion, is a serious threat to public health of the brain disease. The global annual new cases of cerebral apoplexy are more than 10 million, which is the second largest cause of death and the first cause of disability in the world. However, simple thrombolytic therapy for stroke will induce reperfusion injury, mainly including oxidative stress damage further damaging brain tissue. Cells in the reperfusion area will promote the production of oxygen free radicals and the expression of inflammatory cytokines while restoring blood flow and oxygen supply. Oxygen free radicals usually include superoxide anion (·O2-), hydroxyl radical (·OH) and hydrogen peroxide (H2O2). Excessive reactive oxygen species (ROS) will not only destroy the production of intracellular ATP and the stability of membrane potential, but also cause membrane structure damage combined with unsaturated fatty acids. Therefore, in order to alleviate reperfusion injury, we urgently need to develop a neuroprotective agent to restore the neurological function of stroke and improve the long-term survival rate of patients. SUMMARY

[0003] In view of the defects in the prior art, the purpose of the present application is to provide a gold-silver bimetallic nanocluster and a preparation method and application thereof. The gold-silver bimetallic nanocluster prepared by the preparation method has a stable chemical structure, excellent charge stability and high efficient oxygen free radical scavenging capacity, can weaken the oxidative stress damage in the process of cerebral ischemia-reperfusion injury, provide protection effect for neurons, and has good biological safety.

[0004] The present application provides a preparation method of a gold-silver bimetallic nanocluster, comprising the following steps: chloroauric acid and silver nitrate are subjected to co-reduction synthesis reaction with sodium borohydride, coated with mercapto succinic acid, to form spherical nanoclusters, and then subjected to semi-permeable membrane filtration to obtain the gold-silver bimetallic nanocluster. The gold-silver atomic ratio in the gold-silver bimetallic nanocluster is 7:1.

[0005] In an embodiment of the present application, an aqueous solution of HAuCl4-3H2O with a concentration of 0.5 mmol / L, an aqueous solution of AgNO3 with a concentration of 2.0 mmol / L, an aqueous solution of MSA with a concentration of 4.5 mmol / L, an aqueous solution of NaBH4 with a concentration of 5 mmol / L, and deionized water are mixed uniformly, stirred at room temperature for 60 minutes, and the obtained mixture is purified by a semi-permeable membrane to obtain gold-silver bimetallic nanoclusters, wherein the volume ratio of the aqueous solution of HAuCl4-3H2O, the aqueous solution of AgNO3, the aqueous solution of MSA, the aqueous solution of NaBH4, and the deionized water is 1:1:9:10:(5000-6000).

[0006] In an embodiment of the present application, the semi-permeable membrane has a molecular weight cut-off of 10000 Da.

[0007] The second aspect of the present application provides a gold-silver bimetallic nanocluster prepared by the above preparation method.

[0008] The third aspect of the present application provides the use of the above gold-silver bimetallic nanocluster in the preparation of a drug for treating / inhibiting oxidative stress.

[0009] Further, the present application provides the use of the above gold-silver bimetallic nanocluster in the preparation of a neuroprotective drug.

[0010] Compared with the prior art, the present application has the following beneficial effects:

[0011] 1. In the gold-silver bimetallic nanocluster preparation method provided by the embodiment of the present application, the gold-silver bimetallic nanoclusters (Au7Ag1NCs) are formed by MSA coating with a reasonable gold-silver atomic ratio (Au:Ag=7:1). Compared with single-metal nanoclusters, it has stronger stability, electronic performance, and synergistic effect, especially high efficiency in removing free radicals in simulated biological enzymes, and also has an extremely fine particle size (2 nm) and good imitated activity, can penetrate the blood-brain barrier into the brain to play a neuroprotective role; in the present application, the gold-silver bimetallic nanoclusters are prepared by a co-reduction synthesis method, which can effectively control the size and composition of the nanoclusters, and ensure that the prepared nanoclusters have consistent properties and high biological activity.

[0012] 2. The gold-silver bimetallic nanocluster provided by the embodiment of the present application realizes the size and morphological stability in the biological body through the design of the gold-silver bimetallic nanocluster, and improves the application safety and effectiveness in complex biological environments.

[0013] 3. The gold-silver bimetallic nanocluster provided by the embodiment of the present application enhances the catalytic activity and oxygen free radical scavenging capacity of the nanocluster through the synergistic effect of gold and silver atoms, and provides a new strategy for the treatment of ischemic stroke, especially in reducing oxidative stress damage.

[0014] 4. The gold-silver bimetallic nanocluster provided by the embodiment of the present application can restore nerve function and improve long-term survival rate of patients in the treatment of ischemic stroke when used for preparing a neuroprotective agent. BRIEF DESCRIPTION OF DRAWINGS

[0015] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the accompanying drawings:

[0016] Figure 1 The preparation flow chart of the gold-silver bimetallic nanocluster provided by the embodiment of the present application is shown in the figure;

[0017] Figure 2 The 14-day dls stability test result graph of the gold-silver bimetallic nanocluster prepared in Example 1 is shown in the figure;

[0018] Figure 3 The Zeta potential analysis graph of the gold-silver bimetallic nanocluster prepared in Example 1 is shown in the figure;

[0019] Figure 4 The TEM test result graph of the gold-silver bimetallic nanocluster prepared in Example 1 is shown in the figure;

[0020] Figure 5 The ultraviolet-visible light spectrum graph of the gold-silver bimetallic nanocluster prepared in Example 1 is shown in the figure;

[0021] Figure 6 The X-ray photoelectron spectroscopy graph of the gold-silver bimetallic nanocluster prepared in Example 1 is shown in the figure;

[0022] Figure 7 The catalase and superoxide dismutase mimic enzyme result graph is shown in the figure;

[0023] Figure 8 The protection effect result graph of the gold-silver bimetallic nanocluster prepared in Example 1 on nerve cells is shown in the figure;

[0024] Figure 9 The TTC staining section of the gold-silver bimetallic nanocluster prepared in Example 1 in the treatment of middle cerebral artery embolism mouse model is shown in the figure;

[0025] Figure 10 The neurological function score of the gold-silver bimetallic nanocluster prepared in Example 1 in the treatment of middle cerebral artery embolism mouse model is shown in the figure. DETAILED DESCRIPTION

[0026] The application will be described in greater detail with reference to specific embodiments. The following examples are provided to further assist a person skilled in the art in understanding the application, but do not limit the application in any form. It should be noted that, for those skilled in the art, without departing from the concept of the application, a number of changes and improvements can be made. These all belong to the protection scope of the application.

[0027] Nanomedicine has become an excellent choice to enhance the treatment of ischemic stroke, among which nanoclusters can exhibit higher atomic utilization and catalytic activity than traditional nanoparticles due to their ultra-small size and large specific surface area. Among the numerous nanoclusters, gold nanoclusters with precise atomic structure can provide a uniform size and morphology stable chemical coordination environment in the complex microenvironment of the body, which is not only conducive to determining catalytic activity and revealing reaction pathways, but also can form bimetallic nanoclusters by doping another number-controllable metal heteroatom. The synergistic effect of two metals can endow the alloy cluster with new or stronger catalytic ability, and can expose more active sites or adjust the geometric configuration and electronic structure of metal nanoclusters through selective ligand exchange. In addition, nanomaterials with enzyme-mimetic properties have attracted widespread attention due to their superior catalytic efficiency, cycle life, and multi-enzyme integration ability to endogenous enzymes. For example, HAS-Mn3O4 has been used to solve the problem of cerebral ischemia-reperfusion injury, and CuxO nanoparticles have been shown to alleviate neurotoxicity associated with Parkinson's disease by scavenging reactive oxygen species (ROS). Therefore, establishing metal nanoclusters with multifunctional nanenzyme activity may become a new nanomedicine strategy for treating cerebral ischemia-reperfusion injury.

[0028] Monometallic nanoclusters may exhibit size and morphology instability in the complex microenvironment of the body, which may affect their effectiveness and safety in biomedical applications, in addition, the catalytic activity and reaction pathway of metal nanoclusters are limited by their single metal composition, which limits their functionality in specific biomedical applications.

[0029] In addition, existing treatment options for ischemic stroke, such as thrombolytic therapy, can induce oxidative stress damage, exacerbating brain tissue damage, and existing treatment strategies have limitations in reducing such damage.

[0030] Based on the above problems, the application provides a gold-silver bimetallic nanocluster and a preparation method and application thereof, the gold-silver bimetallic nanocluster is prepared through a specific proportion (Au:Ag=7:1) of gold-silver atoms and a co-reduction synthesis method, the particle size of the gold-silver bimetallic nanocluster is about 2 nm, the prepared gold-silver bimetallic nanocluster has stable chemical structure and good biological safety, excellent charge stability and high efficient oxygen free radical scavenging capacity, can inhibit the oxidative stress of cerebral ischemia-reperfusion injury, provides protection effect for neurons, and has good biological safety.

[0031] Example 1

[0032] The embodiment provides a gold-silver bimetallic nanocluster (Au7Ag1NCs), which is prepared by the following method: 20 ul of an aqueous solution of HAuCl4-3H2O with a concentration of 0.5 mmol / L, 20 ul of an aqueous solution of AgNO3 with a concentration of 2.0 mmol / L, 180 ul of an aqueous solution of MSA with a concentration of 4.5 mmol / L, 200 ul of an aqueous solution of NaBH4 with a concentration of 5 mmol / L and 10 ml of deionized water are uniformly mixed (the deionized water provides a sufficient aqueous solution environment and good particle dispersibility), the mixture is stirred at room temperature for 60 minutes, the obtained mixture is purified by using a semi-permeable membrane with a molecular weight cut-off of 10000 Da, and the gold-silver bimetallic nanocluster is obtained, the purified gold-silver bimetallic nanocluster is stored at 4 DEG C for further experiments, and the obtained gold-silver bimetallic nanocluster is Au7Ag1NCs with an atomic ratio of 7:1.

[0033] The ultraviolet-visible light spectrum of the gold-silver bimetallic nanocluster prepared in the embodiment is shown in Figure 5 The Au7Ag1NCs has obvious visible light absorption characteristic peaks near 503 mm in ultraviolet-visible light spectrum detection; the X-ray photoelectron spectrum (XPS) is shown in Figure 6 The Au7Ag1NCs can be further determined in terms of element composition, chemical state of elements and molecular structure (Au4f spectrum and Ag3d spectrum) by X-ray photoelectron spectroscopy and XRD. Figure 6 The Au4f spectrum proves that the peaks of the Au7Ag1NCs are mainly concentrated in Au4f7 / 2 (87.86 eV) and Au4f5 / 2 (84.21 eV), and the Ag3d spectrum proves that the peaks of the Au7Ag1NCs are mainly concentrated in Ag3d3 / 2 (373.85 eV) and Ag3d5 / 2 (367.89 eV).

[0034] Experimental Example 1

[0035] In the experimental example, the particle size and morphology of the gold-silver bimetallic nanocluster prepared in the embodiment 1 are determined.

[0036] The size of the gold-silver bimetallic nanoclusters (Au7Ag1NCs) prepared in Example 1 was about 2 nm, the dispersion was stable and the distribution was uniform, as measured by dynamic light scattering (DLS) (see Figure 1) and the zeta potential value was -31.3 mV (see Figure 2). The TEM results are shown in Figure 3. It can be seen from the TEM image that the gold-silver bimetallic nanoclusters (Au7Ag1NCs) prepared in Example 1 were spherical with a uniform size of about 2 nm and had a crystal lattice. Figure 2 Figure 3 Figure 4

[0037] Experimental Example 2

[0038] The simulation enzyme activity of the gold-silver bimetallic nanoclusters (Au7Ag1NCs) prepared in Example 1 was determined in this experimental example.

[0039] 1) The catalase (CAT) simulation activity was determined by a catalase assay kit. In the presence of relatively sufficient hydrogen peroxide, catalase can catalyze hydrogen peroxide to produce water and oxygen. The residual hydrogen peroxide can oxidize the chromogenic substrate under the catalysis of peroxidase, and there is absorption at 520 nm. The gold-silver bimetallic nanoclusters (Au7Ag1NCs) have catalase simulation activity, which can consume the added hydrogen peroxide. The catalase simulation activity of the gold-silver bimetallic nanoclusters (Au7Ag1NCs) was reflected by the content of the residual hydrogen peroxide. The results are shown in Figure 4, and the bimetallic nanoclusters have CAT enzyme simulation activity. Figure 7

[0040] 2) The superoxide dismutase (SOD) simulation activity was determined by a superoxide dismutase assay kit. The superoxide radical ion produced by xanthine oxidase (XOD) catalyzing xanthine can reduce nitrogen blue tetrazolium (NBT) to form blue formazan, which has absorption at 560 nm. The bimetallic nanoclusters have SOD simulation enzyme activity. After the addition of the bimetallic nanoclusters, the superoxide radical ion was scavenged, thereby inhibiting the formation of formazan. The SOD simulation activity of the drug-loaded nanoparticles was reflected by the inhibition rate. The results are shown in Figure 5, and the bimetallic nanoclusters have SOD enzyme simulation activity. Figure 7

[0041] Experimental Example 3

[0042] Cell experiment

[0043] OGD model

[0044] ​​​​​SH-SY5Y cells were initially seeded in DMEM and adhered for 24 hours under standard conditions (37°C, 5% CO2 and atmospheric oxygen content). After adhesion, the medium was changed to minimal essential medium (MEM, Gibco, USA) before induction of hypoxia for a 4-hour pre-treatment of the cells. For hypoxic treatment, the cells were placed in a hypoxic incubator mimicking ischemic conditions (95% N2, 5% CO2 and 0.1% O2) for 4 hours for oxygen glucose deprivation (OGD) treatment. Immediately after OGD treatment, the cells were transferred back to normoxic conditions in the standard incubator and the MEM was changed to fresh DMEM to facilitate recovery. The cells were then treated with Au7Ag1 NCs at predetermined concentrations or left untreated as a control. After 20 hours of recovery, the cells were harvested for further analysis.

[0045] Experimental Example 4

[0046] CCK-8 cytotoxicity test was used to detect the cytotoxicity and protective effect of the gold-silver bimetallic nanoclusters (Au7Ag1 NCs) prepared in Example 1. Briefly, cells with a density of 1 x 10 5 After OGD modeling, different concentrations (0 pg / ml, 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml) of gold-silver bimetallic nanoclusters (Au7Ag1 NCs) prepared in Example 1 were added to each well; at each time point (24 hours, 48 hours and 72 hours), 10 pL of CCK-8 solution was added to each well according to the manufacturer's instructions. Incubate for an additional 2 hours at 37°C. The absorbance was measured using a microplate reader at a wavelength of 450 nm. The effect of different concentrations of gold-silver bimetallic nanoclusters (Au7Ag1 NCs) on the survival rate of SH-SY5Y cells after OGD / R (oxygen glucose deprivation and reoxygenation treatment) was determined by CCK-8 assay. With the increase of Au7Ag1 NCs concentration, the cell survival rate of SH-SY5Y gradually increased until the concentration of 30 pg / mL began to slowly decrease, but the overall survival rate was higher than that of the OGD / R group without drug addition.

[0047] Experimental Example 5

[0048] Animal experiments

[0049] MCAO surgical model

[0050] Isoflurane was loaded into the machine using a small animal anesthesia machine, and then the mouse was put on the mask to induce inhalation of isoflurane at a concentration of 3%. During the operation, the anesthetic concentration was adjusted according to the breathing of the mouse, and maintained between 0.5% and 1%. After anesthesia, the mouse was supine on the operating table. The neck surgical site was first disinfected with alcohol, and then cut with surgical scissors. Under a microscope, the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were separated, and the threads were hung at the distal and proximal ends of the CCA and ECA. The ICA was temporarily clamped with an artery clamp, the CCA and ECA were ligated near the heart, and then a 45-degree small opening was cut 4 mm away from the CCA bifurcation, and the tie was inserted. A thin wire was used to fix the silk thread at the distal end of the CCA. After loosening the clamp on the ICA, the silk thread was pushed to a depth of about 0.85 cm. Then the site was sutured and disinfected. The mice in the sham group received the same anesthesia and arterial exposure procedure, but no threaded bolt was inserted. After the operation, the body temperature of the mouse was stabilized at 37°C. Then ischemia was induced for 1 hour, followed by thrombus extraction and reperfusion. The mice were divided into four groups: (1) normal saline group (post-stroke injection of normal saline through the tail vein); (2) Sham group (received surgery without inserting silk, and did not receive further treatment); (3) 2 mg / kg group (2 mg / kg Au7Ag1NCs was injected through the tail vein); (4) 4 mg / kg group (4 mg / kg Au7Ag1NCs was injected through the tail vein). Tail vein injection started at 1 hour after MCAO, once a day, for three consecutive days.

[0051] TTC staining experiment

[0052] After 72 hours of ischemia-reperfusion injury, the present application observed the ischemic area by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The results showed that the mice in the sham group had no infarction, and the infarction volume of the normal saline group was 37.9%. In sharp contrast, 2 mg / kg Au7Ag1NCs and 4 mg / kg Au7Ag1NCs reduced the brain infarction volume to 27.6% and 12.6%, respectively, and it was very obvious that the high concentration group was more effective in treating infarction Figure 9

[0053] Modified neurological severity score

[0054] ​The mNSS7 test was used to assess motor, sensory, reflex, and balance, among other neurological functions at 0, 1, 2, 3, 5, 7, 14, 21, and 28 days after MCAO. Normal scores were 0 points, and the maximum deficit score was 18 points. The higher the score, the more severe the injury. The modified neurological severity score (mNSS) assessed the impairment of motor function, sensory acuity, balance, and reflexes in mice after MCAO surgery. The results showed that MCAO surgery severely impaired the sensorimotor function of mice. In contrast, the neurological deficits of 2 mg / kg Au7Ag1 NCs and 4 mg / kg Au7Ag1 NCs treated mice were reversed at day 5 after injury, showing a continuous decrease in scores over time Figure 10

[0055] Modified Neurological Deficit Score Sheet

[0056]

[0057] The specific embodiments of the present application have been described. It is to be understood that the application is not limited to the specific embodiments described above and various modifications, as will be evident to those skilled in the art, can be made without departing from the spirit of the application. The embodiments of the present application and the features of the embodiments can be combined with each other without conflict.​

Claims

1. The application of gold-silver bimetallic nanoclusters in the preparation of drugs for treating / inhibiting oxidative stress or for treating neuroprotective drugs, characterized in that, The gold-silver bimetallic nanoclusters are prepared by the following steps: chloroauric acid and silver nitrate are co-reduced with sodium borohydride to form spherical nanoclusters, which are then coated with mercaptosuccinic acid to form spherical nanoclusters. The nanoclusters are then filtered through a semi-permeable membrane to obtain the gold-silver bimetallic nanoclusters. The gold-silver atomic ratio in the gold-silver bimetallic nanoclusters is 7:

1.

2. The application according to claim 1, characterized in that, A 0.5 mmol / L aqueous solution of HAuCl4-3H2O, a 2.0 mmol / L aqueous solution of AgNO3, a 4.5 mmol / L aqueous solution of MSA, a 5 mmol / L aqueous solution of NaBH4, and deionized water were mixed thoroughly and stirred at room temperature for 60 minutes. The resulting mixture was purified using a semi-permeable membrane to obtain gold-silver bimetallic nanoclusters. In the gold-silver bimetallic nanoclusters, the volume ratio of the aqueous solution of HAuCl4-3H2O, the aqueous solution of AgNO3, the aqueous solution of MSA, the aqueous solution of NaBH4, and the deionized water was 1:1:9:10:(5000~6000).

3. The application according to claim 2, characterized in that, The semipermeable membrane has a molecular weight cutoff of 10,000 Da.

Images