Gold nanocluster and preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and relates to a gold nanocluster and a preparation method and application thereof, the ligand of the gold nanocluster is cyclic adenosine monophosphate, the gold nanocluster emits blue-green fluorescence under an ultraviolet lamp, and the fluorescence excitation spectrum and the emission spectrum of the gold nanocluster are 376 nm and 482 nm respectively. The gold nanocluster is synthesized by taking cell second messenger cyclic adenosine monophosphate (cAMP) as a functional ligand, the size of the gold nanocluster is 1-2nm, the gold nanocluster emits blue-green fluorescence under an ultraviolet lamp, and the fluorescence excitation spectrum and the emission spectrum of the gold nanocluster are 376nm and 482nm respectively. The traditional Chinese medicine composition can relieve liver cell inflammation and improve liver glycometabolism and an insulin signal channel, so that the traditional Chinese medicine composition has a good effect of improving the diabetes mellitus liver insulin resistance, can be used for preparing the medicine for improving the diabetes mellitus liver insulin resistance and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a gold nanocluster and a preparation method and application thereof. Background Art

[0002] Insulin resistance refers to insulin-mediated disorders of sugar metabolism in tissues (mainly liver, muscle and fat), and is an early clinical manifestation of a series of human diseases including type 2 diabetes and cardiovascular disease. These diseases are often associated with metabolic abnormalities, including obesity, hyperinsulinemia, hyperglycemia and hyperlipidemia. Insulin resistance is caused by both genetic and environmental factors. Recent studies have shown that the liver plays a key role in the development of insulin resistance and may promote insulin resistance in other organs by releasing cytokines. These intermediates may cause insulin resistance by inhibiting one or more proximal components downstream of the insulin signaling cascade (insulin receptor, insulin receptor substrate protein or AKT). Hepatic insulin resistance plays a very important role in the onset of type 2 diabetes, but there is currently no effective drug to improve the impaired insulin signaling pathway. Therefore, the development of new therapeutic drugs is crucial to improve hepatic insulin resistance.

[0003] Cyclic adenosine monophosphate (cAMP) is a cellular second messenger that mediates a variety of cellular biological processes and plays an important role in the occurrence and development of a variety of diseases. In the development of diabetic liver insulin resistance, the cAMP content decreases, suggesting that cAMP reduction plays an important role in diabetic liver insulin resistance. Therefore, increasing the intracellular cyclic adenosine monophosphate level may be an effective way to improve liver insulin resistance. However, cyclic adenosine monophosphate is difficult to penetrate the cell membrane and cannot be used directly as a medicine. Therefore, finding an effective way to deliver cAMP into cells is an effective way to solve this problem. With the development of nanotechnology in recent years, nanobiotechnology has been widely used in the field of biomedicine. Gold nanoclusters are a new type of nanomaterial composed of hundreds to hundreds of gold atoms with a core size of less than 2nm. Due to their extraordinary physical and chemical properties and excellent biocompatibility, they are easy to internalize into cells and have attracted widespread attention in the field of biomedicine. At present, there are no relevant literature reports on the improvement of insulin resistance and the regulation of glucose and lipid metabolism disorders by gold nanoclusters. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a gold nanocluster and a preparation method and application thereof. The gold nanocluster uses the cell second messenger cyclic adenosine monophosphate as a functional ligand, and reduces chloroauric acid into fluorescent gold nanoclusters with a size of 1-2 nm by a citric acid buffer. The gold nanocluster can relieve liver inflammatory response, improve liver glucose and lipid metabolism levels and insulin signaling pathways, and thus improve diabetic liver insulin resistance. Therefore, the gold nanocluster provided by the present invention has a good application prospect in the preparation of drugs for improving diabetic liver insulin resistance.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The invention provides a gold nanocluster, wherein the ligand of the gold nanocluster is cyclic adenosine monophosphate, and the gold nanocluster emits blue-green fluorescence under ultraviolet light, and the fluorescence excitation spectrum and emission spectrum thereof are 376nm and 482nm respectively.

[0007] The chemical structure of cyclic adenosine monophosphate is as follows:

[0008]

[0009] Furthermore, the size of the gold nanoclusters is 1-2 nm.

[0010] Furthermore, the gold nanoclusters are a gold nanocluster colloidal solution with a concentration of no less than 10 mg / mL.

[0011] The present invention also provides a method for preparing gold nanoclusters, comprising the following preparation steps:

[0012] S1. Prepare a mixed solution of cyclic adenosine monophosphate and chloroauric acid under stirring at 380-450 rpm;

[0013] S2, adding citric acid buffer to the mixed solution in step S1, and continuously stirring and mixing;

[0014] S3. Use an ultrafiltration tube to concentrate and filter, collect the upper liquid, and freeze-dry to obtain.

[0015] Preferably, in step S2, continuous stirring and mixing is performed at room temperature.

[0016] Preferably, in step S3, purification and filtration are performed using an ultrafiltration tube with a molecular cutoff of 3 kD, the supernatant is collected, and freeze-dried to obtain cyclic AMP-mediated gold nanoclusters.

[0017] Furthermore, in step S1, the molar ratio of cyclic adenosine monophosphate to chloroauric acid is (0.5-5):1.

[0018] Furthermore, the molar ratio of cAMP:chloroauric acid can be 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1.

[0019] Further preferably, the molar ratio of cyclic AMP to chloroauric acid is 2:1.

[0020] Furthermore, the concentration of cyclic adenosine monophosphate is 20 mM, and the concentration of chloroauric acid is 10 mM.

[0021] Furthermore, in step S2, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of (18-22):1.

[0022] Further preferably, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of 20:1.

[0023] Furthermore, the concentration of the sodium citrate is 0.5M, and the concentration of the citric acid is 5M.

[0024] Furthermore, in step S2, the duration of the connection and stirring is ≥ 10 h.

[0025] The present invention also provides the use of the gold nanoclusters in preparing a drug for improving insulin resistance in diabetic liver.

[0026] The present invention also provides a drug for improving liver insulin resistance, wherein the drug is a preparation prepared by using the gold nanoclusters as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention uses cyclic adenosine monophosphate, a second messenger of cells, as a functional ligand, and reduces chloroauric acid into gold nanoclusters with fluorescence excitation and emission spectra of 376 nm and 482 nm, respectively, and a size of 1-2 nm, through a citric acid buffer. The gold nanoclusters of the present invention can alleviate liver inflammatory response, improve liver glucose and lipid metabolism levels and insulin signaling pathways, and thus improve insulin resistance in diabetic liver;

[0029] 2. The method for preparing gold nanoclusters of the present invention is simple and convenient, does not require conditions such as high temperature and high pressure, and can be prepared at room temperature. At the same time, no additional acid-base regulator is required to adjust the pH. The gold nanoclusters prepared by the preparation method of the present invention have a high concentration, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the synthesis of the gold nanoclusters of the present invention;

[0031] Figure 2 is a chemical structure diagram of cyclic adenosine monophosphate of the present invention;

[0032] Figure 3 is the UV-visible spectrum of the gold nanoclusters of the present invention;

[0033] Figure 4 is the fluorescence emission spectrum of the gold nanoclusters of the present invention;

[0034] Figure 5 TEM image and size distribution diagram of the gold nanoclusters of the present invention;

[0035] Figure 6 This is the XPS spectrum of the gold nanocluster of the present invention (Au4f region);

[0036] Figure 7 The fluorescence spectra of gold nanoclusters synthesized with different molar ratios of chloroauric acid / cyclic adenosine monophosphate and different molar ratios of sodium citrate / citric acid are shown in Figure 2.

[0037] in, Figure 7 (a) Fluorescence spectra and physical images of gold nanoclusters synthesized with different chloroauric acid / cyclic AMP molar ratios; Figure 7 (b) Fluorescence intensity and physical image of gold nanoclusters synthesized at different sodium citrate / citric acid molar ratios;

[0038] Figure 8 This is a graph showing the effect of the gold nanocluster treatment of the present invention on the body weight of mice;

[0039] Fig. 9 The figure is the effect of the gold nanocluster treatment of the present invention on glucose tolerance (GTT) and insulin resistance (ITT) in mice.

[0040] in, Fig. 9 (a)-(b) are the results of glucose tolerance test in mice. Fig. 9 (c)-(d) are the results of the effects of insulin resistance on mice;

[0041] Fig.10 This is a graph showing the effect of the gold nanocluster treatment of the present invention on fasting blood glucose and plasma and liver insulin levels in mice.

[0042] in, Fig.10 (a) is a graph showing the effect of the gold nanocluster treatment of the present invention on fasting blood glucose in mice. Fig.10 (b) is a graph showing the effect of the gold nanocluster treatment of the present invention on the insulin levels in mouse plasma and liver;

[0043] Fig.11 This is a graph showing the effect of the gold nanocluster treatment of the present invention on the liver index of mice;

[0044] Fig.12This is a graph showing the effect of the gold nanocluster treatment of the present invention on mouse liver lipids;

[0045] Fig.13 This is a graph showing the effect of the gold nanoclusters treatment of the present invention on the pathological tissue of mouse liver;

[0046] Fig.14 This is a graph showing the effect of the gold nanoclusters of the present invention on the liver function and blood lipids of mice.

[0047] in, Fig.14 (a)-(b) are the results of the effect of gold nanoclusters on mouse liver function. Fig.14 (c)-(f) are the results of the effects of gold nanoclusters on blood lipids in mice;

[0048] Fig.15 This is a graph showing the effect of the gold nanocluster treatment of the present invention on the expression of key molecules in the insulin signaling pathway in mice.

[0049] in, Fig.15 (a)-(f) are the results of the effect of mRNA expression levels of GLUT2, GSK3B, PPARR, SREBP1 C, I NSR and IRS in the insulin signaling pathway; Fig.15 (g)-(h) are the results of changes in AKT phosphorylation levels;

[0050] Fig.16 This is a graph showing the effect of the gold nanocluster treatment of the present invention on the polarization of mouse macrophages.

[0051] in, Fig.16 (a) mRNA expression levels of M1 macrophage markers MCP-1, CCR2, TNF-α, IL-6, and IL-1β. Fig.16 (b) mRNA expression levels of M2 macrophage markers IL-10 and CS206;

[0052] Fig.17 This is the result diagram of the effect of gold nanoclusters on inflammatory factors in mice.

[0053] in, Fig.17 (a) is the effect of gold nanoclusters on the expression of inflammatory factor IL-1β mRNA in mice. Fig.17 (b) is the effect of gold nanoclusters on the expression of inflammatory factor TNF-α mRNA in mice. Fig.17 (c) is the effect of gold nanoclusters on the expression of inflammatory factor IL-6 mRNA in mice. DETAILED DESCRIPTION

[0054] The principles and features of the present invention are described below (in conjunction with the accompanying drawings). The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0055] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0056] The sources of some raw materials of the present invention are as follows:

[0057] Chloroauric acid was purchased from Sinopharm Chemical Reagent Co., Ltd.; cyclic adenosine monophosphate, citric acid, and sodium citrate were purchased from Shanghai Aladdin Reagent Co., Ltd.

[0058] The invention provides a gold nanocluster, wherein the ligand of the gold nanocluster is cyclic adenosine monophosphate, and the gold nanocluster emits blue-green fluorescence under ultraviolet light, and the fluorescence excitation spectrum and emission spectrum thereof are 376nm and 482nm respectively.

[0059] Cyclic adenosine monophosphate (cAMP) is a cellular second messenger that mediates a variety of cellular biological processes and plays an important role in the occurrence and development of a variety of diseases. In the development of insulin resistance in the diabetic liver, the cAMP content decreases, suggesting that the reduction of cAMP plays an important role in insulin resistance in the diabetic liver; gold nanoclusters are a new type of nanomaterial composed of hundreds to hundreds of gold atoms; during the research process, we found that cyclic adenosine monophosphate can be used as a ligand to prepare ultra-small fluorescent gold nanoclusters, so cyclic adenosine monophosphate can be directly internalized into cells through gold nanoclusters to exert biological effects.

[0060] Furthermore, the size of the gold nanoclusters is 1-2 nm.

[0061] Furthermore, the gold nanoclusters are a gold nanocluster colloidal solution with a concentration of no less than 10 mg / mL.

[0062] The present invention also provides a method for preparing gold nanoclusters, comprising the following preparation steps:

[0063] S1. Prepare a mixed solution of cyclic adenosine monophosphate and chloroauric acid under stirring at 380-450 rpm;

[0064] S2, adding citric acid buffer to the mixed solution in step S1, and continuously stirring and mixing;

[0065] S3. Use an ultrafiltration tube to concentrate and filter, collect the upper liquid, and freeze-dry to obtain.

[0066] Specifically, the method for preparing gold nanoclusters of the present invention is simple and convenient, does not require conditions such as high temperature and high pressure, and can be prepared at room temperature. At the same time, no additional acid-base regulator is required to adjust the pH. Moreover, the concentration of gold nanoclusters prepared by the preparation method of the present invention is relatively high, which is conducive to industrial production.

[0067] Preferably, in step S2, continuous stirring and mixing is performed at room temperature.

[0068] Preferably, in step S3, purification and filtration are performed using an ultrafiltration tube with a molecular cutoff of 3 kD, the supernatant is collected, and freeze-dried to obtain cyclic AMP-mediated gold nanoclusters.

[0069] Furthermore, in step S1, the molar ratio of cyclic adenosine monophosphate to chloroauric acid is (0.5-5):1.

[0070] Further preferably, the molar ratio of cyclic AMP to chloroauric acid is 2:1.

[0071] Furthermore, the concentration of cyclic adenosine monophosphate is 20 mM, and the concentration of chloroauric acid is 10 mM.

[0072] Furthermore, in step S2, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of (18-22):1.

[0073] Further preferably, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of 20:1.

[0074] The citrate buffer serves to adjust the pH and provide reducing properties to promote the synthesis of gold clusters.

[0075] Furthermore, the concentration of the sodium citrate is 0.5M, and the concentration of the citric acid is 5M.

[0076] Furthermore, in step S2, the duration of the connection and stirring is ≥ 10 h.

[0077] The present invention also provides the use of the gold nanoclusters in preparing a drug for improving insulin resistance in diabetic liver.

[0078] The present invention also provides a drug for improving liver insulin resistance, wherein the drug is a preparation prepared by using the gold nanoclusters as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0079] The present invention is described in detail below in conjunction with embodiments and experimental data.

[0080] Example 1

[0081] A gold nanocluster is prepared according to the following method:

[0082] At room temperature, 5 mL of 200 mM chloroauric acid and 10 mL of 200 mM cyclic adenosine monophosphate were added to 75 mL of water under stirring, and after stirring for 10 minutes, 10 mL of 500 mM citric acid buffer was added, and the reaction was maintained for more than 10 hours, and then purified and filtered using an ultrafiltration tube with a molecular cutoff of 3 kD, the upper liquid was collected, and freeze-dried to obtain cyclic adenosine monophosphate-mediated gold nanoclusters. The gold nanoclusters were made into a solution with a concentration of 10 mg / mL.

[0083] The following experimental examples are used to illustrate the beneficial effects of the present invention. The experimental methods for which specific conditions are not specified in the following experimental examples are usually measured in accordance with national standards. If there are no corresponding national standards, the methods are carried out in accordance with the general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0084] Experimental example

[0085] 1. Characterization of gold nanoclusters

[0086] 1. UV-visible absorption spectrum and fluorescence spectrum test

[0087] The gold nanoclusters prepared in Example 1 were subjected to UV-visible absorption spectroscopy, fluorescence spectroscopy, and testing. The results are as follows: Figure 3 , Figure 4 shown.

[0088] Figure 3 is the UV-visible absorption spectrum of the gold nanoclusters of the present invention, from Figure 3 As can be seen in the figure, the UV-visible absorption spectrum of the gold nanoclusters does not show the characteristic surface plasmon resonance peak, indicating that ultra-small gold nanoclusters, rather than large-sized gold nanoparticles, were successfully formed;

[0089] Figure 4 is the fluorescence emission spectrum of the gold nanoclusters of the present invention, wherein the inset shows a photograph of the gold nanoclusters under 365nm ultraviolet light, from Figure 4 It can be seen that the gold nanoclusters show bright blue-green fluorescence under 365nm ultraviolet light, and the fluorescence emission spectrum is 482nm.

[0090] 2. TEM

[0091] The gold nanoclusters prepared in Example 1 were tested by transmission electron microscopy (TEM), and the results were as follows: Figure 5 shown.

[0092] Figure 5 TEM image and size distribution diagram of the gold nanoclusters of Example 1, scale bar: 20 nm; Figure 5As can be seen, the images of the morphology and size of the gold nanoclusters characterized by transmission electron microscopy (TEM) confirmed that the cyclic AMP-mediated gold nanoclusters are monodisperse ultrasmall clusters with an average particle size of 1.2±0.2nm.

[0093] 3. XPS

[0094] The gold nanoclusters prepared in Example 1 were subjected to X-ray photoelectron spectroscopy (XPS) testing to study the metal valence state of the gold nanoclusters. The results are as follows: Figure 6 shown.

[0095] Figure 6 The XPS spectrum of the gold nanoclusters of Example 1 is shown in FIG. Figure 6 It can be seen that the binding energies of Au4f5 / 2 and Au4f7 / 2 are 87.4eV and 83.6eV, respectively, indicating that the gold element in the prepared gold nanoclusters exists in two valence states: zero valence and one valence.

[0096] Optimization of gold nanocluster synthesis conditions

[0097] At room temperature, the cyclic AMP-mediated synthesis of gold nanoclusters was optimized by changing the molar ratio of chloroauric acid / cyclic AMP and by adjusting the molar ratio of sodium citrate / citric acid to change the pH value of the reaction system.

[0098] Experimental methods:

[0099] 1. According to the method described in Example 1, the reaction conditions of the adjustment method were changed (cAMP: chloroauric acid were 0.5:1, 1:1, 2:1, 3:1, 4:1, and 5:1, respectively) to prepare gold nanoclusters, monitor the fluorescence spectral characteristics of the reaction system, and obtain the optimal cAMP / chloroauric acid feed ratio.

[0100] 2. According to the method described in Example 1, the molar ratio of sodium citrate to citric acid was adjusted so that the pH of the reaction system was 1-14, and the optimal ratio of sodium citrate to citric acid was obtained by monitoring the fluorescence intensity at 482 nm.

[0101] Experimental results: The experimental results are as follows Figure 7 shown.

[0102] Figure 7 The fluorescence spectra of gold nanoclusters synthesized with different molar ratios of chloroauric acid / cyclic adenosine monophosphate and different molar ratios of sodium citrate / citric acid are shown in Figure 1. Figure 7 (a) is the fluorescence spectra of gold nanoclusters synthesized with different molar ratios of chloroauric acid / cyclic AMP. Figure 7 (b) is the fluorescence spectra of gold nanoclusters synthesized with different sodium citrate / citric acid molar ratios. Figure 7It can be seen that the fluorescence intensity of the gold nanoclusters synthesized under the condition of cyclic AMP / chloroauric acid molar ratio of 2:1 is the highest, indicating that the yield of gold nanoclusters under this feed ratio condition is the highest; when the sodium citrate / citric acid molar ratio is 20:1, that is, when the pH is 8, the synthesized gold nanoclusters have the strongest fluorescence and the highest yield.

[0103] It can be concluded that the optimal reaction conditions for synthesizing gold nanoclusters in the present invention are: the molar ratio of cyclic adenosine monophosphate to chloroauric acid is 2:1, and the molar ratio of sodium citrate to citric acid is 20:1.

[0104] 3. Evaluation of the gold nanoclusters of the present invention in improving liver insulin resistance

[0105] 1. Experimental methods

[0106] In order to comprehensively evaluate the activity of the gold nanoclusters prepared in Example 1 in improving insulin resistance, the efficacy of improving insulin resistance in diabetic obese mice was evaluated.

[0107] Specific methods: After 1 week of adaptive feeding, 5-week-old mice were fed with high-fat diet to construct a diabetic obese mouse model. Fasting blood glucose levels were detected after 26 weeks of high-fat diet feeding. The constructed diabetic obese mice were divided into 3 groups: high-fat diet group (HFD), high-fat diet + low-concentration gold nanoclusters group (5 mg / kg), and high-fat diet + high-concentration gold nanoclusters group (10 mg / kg). At the same time, mice fed with normal diet (NFD) were used as blank control group. Gold nanoclusters were administered by tail vein injection once every other day, for a total of 7 injections. During the experiment, the weight changes of mice were measured, and the therapeutic effect of gold nanoclusters on insulin resistance was explored by glucose tolerance test (GTT) and insulin tolerance test (ITT). After the experiment, the mice were killed, and serum and mouse liver were taken for liver index determination, tissue pathological analysis and inflammatory factor determination.

[0108] 2. Experimental results

[0109] Figure 8 The figure shows the weight change results of mice, where NFD is the normal diet group, HFD is the high-fat diet group, HFD-AuNCs (5 mg / kg) is the high-fat diet + low-concentration gold nanoclusters group, and HFD-AuNCs (10 mg / kg) is the high-fat diet + high-concentration gold nanoclusters group. Figure 8 It can be seen that the weight gain of mice fed with normal diet and high-fat diet is more obvious, while the weight gain of mice in the gold nanocluster treatment group is slowed down, among which the mice in the high-dose group lost weight, indicating that the gold nanoclusters of the present invention can significantly delay the weight gain of mice.

[0110] To further study the effect of gold nanoclusters on insulin resistance in diabetic obese mice, we performed GTT and I TT tests on mice fed with high-fat diet and normal diet. Fig. 9 As shown;

[0111] Fig. 9 The test results of mouse GTT and I TT are shown in Figure 1. Fig. 9 (a)-(b) are the results of glucose tolerance test in mice. Fig. 9 (c)-(d) are the results of the effects of insulin resistance on mice. Fig. 9 It can be seen that compared with mice fed with a normal diet, the AUC values ​​of GTT and I TT of mice fed with a high-fat diet were significantly increased, showing stronger glucose tolerance and insulin resistance. After treatment with gold nanoclusters, the AUC values ​​of GTT and I TT were significantly reduced, and the difference in the high-dose group was more significant. According to the glucose tolerance and insulin tolerance tests, compared with mice in the high-fat diet group, intervention with gold nanoclusters effectively alleviated impaired glucose tolerance and insulin tolerance.

[0112] Fig.10 The gold nanocluster treatment of the present invention has an effect on fasting blood glucose and plasma and liver insulin levels in mice, wherein: Fig.10 (a) is a graph showing the effect of the gold nanocluster treatment of the present invention on fasting blood glucose in mice. Fig.10 (b) is a graph showing the effect of the gold nanocluster treatment of the present invention on the insulin levels in mouse plasma and liver; Fig.10 It can be seen that, consistent with the results of glucose tolerance, after 2 weeks of treatment, the fasting blood glucose concentration of mice in the high-fat diet group increased compared with that of mice fed normally, and the fasting blood glucose was significantly reduced after treatment with gold nanoclusters.

[0113] Fig.11 The gold nanocluster treatment of the present invention has an effect on the liver index of mice. Fig.11 It can be obtained that the liver index results show that gold nanocluster treatment can effectively improve the liver index.

[0114] The above results indicate that gold nanoclusters can improve glucose tolerance and insulin resistance and enhance insulin sensitivity in mice fed a high-fat diet.

[0115] Since hepatic fat accumulation is closely related to insulin resistance, we further evaluated the effects of gold nanoclusters on histopathological changes such as hepatic steatosis induced by a high-fat diet.

[0116] Fig.12 The gold nanocluster treatment of the present invention has an effect on mouse liver lipids. Fig.12It can be seen that the results of liver Oil Red O staining showed that the liver lipid droplet density of mice in the high-fat diet group was higher than that of mice fed normally, and a large number of larger red dot-like lipid droplet aggregations appeared. Treatment with gold nanoclusters prevented the increase in lipid droplet density driven by high-fat diet, and the effect of the high-dose group was more significant.

[0117] Fig.13 The gold nanocluster treatment of the present invention has an effect on the pathological tissue of mouse liver. Fig.13 It can be seen that the results of liver HE staining showed that compared with normal-fed mice, the liver cells of mice in the high-fat diet group were irregularly arranged, with increased vacuoles and significant fatty degeneration of liver cells, accompanied by a certain degree of inflammatory cell infiltration and balloon-like degeneration. Gold nanoclusters significantly inhibited the lipid accumulation and widespread fatty degeneration caused by high-fat diet, and the phenotype was a decrease in liver cell lipid droplets and a reduction in inflammatory cell infiltration.

[0118] We further measured the effects of gold nanoclusters on blood lipids and liver function in mice. Fig.14 shown.

[0119] Fig.14 The figure is a graph showing the effect of the gold nanoclusters of the present invention on the liver function and blood lipids of mice, wherein Fig.14 (a)-(b) are the results of the effect of gold nanoclusters on mouse liver function. Fig.14 (c)-(f) are the results of the effects of gold nanoclusters on blood lipids in mice. Fig.14 As can be seen in the results of serum biochemical tests, compared with normal mice, the serum TG, TC and LDL-c levels of mice in the high-fat diet group were significantly increased, and these effects were reversed after gold nanocluster treatment. However, there was no difference in HDL-c between normal-fed mice and mice in the high-fat diet group, and gold nanocluster treatment significantly increased the HDL-c content in serum. In addition, high-fat diet feeding significantly induced liver damage in mice, with increased ALT and AST levels, and gold nanoclusters had a significant mitigating effect on both.

[0120] To further study the effects of gold nanoclusters on glucose and lipid metabolism in mice, we detected the expression levels of key molecules of the insulin signaling pathway in the liver, including p-AKT, GLUT2, GSK3B, PPARR, SREBP1 C, I RS, and I NSR. Fig.15 shown.

[0121] Fig.15 The results of the gold nanocluster treatment of the present invention on the expression of key molecules in the insulin signaling pathway of mice are shown in FIG. Fig.15(a)-(f) are the results of the effect of gold nanocluster treatment on the mRNA expression levels of GLUT2, GSK3B, PPARR, SREBP1 C, I NSR and I RS in the insulin signaling pathway of mice. Fig.15 (g)-(h) are the results of the gold nanocluster treatment on the changes in AKT phosphorylation levels in mice. Fig.15 It can be seen that compared with mice fed a high-fat diet, the phosphorylation level of AKT and the expression of GLUT2 and GSK3B in the liver of mice treated with gold nanoclusters significantly increased, and the expression of SREBP1 C significantly decreased. At the same time, the expression of key factors in the insulin signaling pathway, I NSR and I RS, also increased significantly after treatment with gold nanoclusters, and the effect was more obvious in the high-dose group.

[0122] Furthermore, the effect of gold nanocluster treatment on mouse macrophage polarization was investigated, and the results were as follows Fig.16 shown.

[0123] Fig.16 The figure is a result diagram showing the effect of the gold nanocluster treatment on the polarization of mouse macrophages, wherein Fig.16 (a) is the mRNA expression level of M1 macrophage markers MCP-1, CCR2, TNF-α, IL-6 and IL-1β; Fig.16 (b) is the mRNA expression level of M2 macrophage markers IL-10 and CS206; the results of fluorescence quantitative PCR showed that after gold nanocluster treatment, the expression of M1 pro-inflammatory cytokines in mice was significantly reduced, while the expression of M2 anti-inflammatory cytokines was significantly increased.

[0124] At the same time, the inflammatory factors in the serum and liver tissue of mice were detected, and the test results were as follows: Fig.17 shown.

[0125] Fig.17 This is the result diagram of the effect of gold nanoclusters on inflammatory factors in mice, among which, Fig.17 (a) is the effect of gold nanoclusters on the expression of inflammatory factor IL-1β mRNA in mice. Fig.17 (b) is the effect of gold nanoclusters on the expression of inflammatory factor TNF-α mRNA in mice. Fig.17 (c) is the effect of gold nanoclusters on the expression of inflammatory factor IL-6 mRNA in mice. Fig.17 It can be seen that the results of the measurement of inflammatory factors TNF-α, IL-1β and IL-6 levels in serum and liver tissue also showed that gold nanoclusters effectively alleviated the inflammatory response of mice and thus improved liver insulin resistance.

[0126] In summary, the present invention uses the cell second messenger cyclic adenosine monophosphate as the functional ligand, and reduces chloroauric acid into gold nanoclusters with fluorescence excitation and emission spectra of 376nm and 482nm respectively and a size of 1-2nm through a citric acid buffer. The gold nanoclusters of the present invention can alleviate liver inflammatory response, improve liver glucose and lipid metabolism levels and insulin signaling pathways, and thus improve diabetic liver insulin resistance; the method for preparing gold nanoclusters of the present invention is simple and convenient, does not require conditions such as high temperature and high pressure, and can be prepared at room temperature, and does not require additional acid-base regulators to adjust the pH; and the concentration of gold nanoclusters prepared by the preparation method of the present invention is relatively high, which is conducive to industrial production.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A gold nanocluster, characterized in that: The ligand of the gold nanocluster is cyclic adenosine monophosphate, and the gold nanocluster emits blue-green fluorescence under ultraviolet light, and its fluorescence excitation spectrum and emission spectrum are 376nm and 482nm respectively.

2. A gold nanocluster according to claim 1, characterized in that: The size of the gold nanoclusters is 1-2 nm.

3. The gold nanocluster according to claim 1, characterized in that: The gold nanoclusters are a gold nanocluster colloidal solution with a concentration of not less than 10 mg / mL.

4. A method for preparing the gold nanoclusters according to any one of claims 1 to 3, characterized in that: The method comprises the following preparation steps: S1. Prepare a mixed solution of cyclic adenosine monophosphate and chloroauric acid under stirring at 380-450 rpm; S2, adding citric acid buffer to the mixed solution in step S1, and continuously stirring and mixing; S3. Use an ultrafiltration tube to concentrate and filter, collect the upper liquid, and freeze-dry to obtain.

5. The method for preparing gold nanoclusters according to claim 4, characterized in that: In step S1, the molar ratio of cyclic adenosine monophosphate to chloroauric acid is 0.5:1-5:1; preferably, the molar ratio of cyclic adenosine monophosphate to chloroauric acid is 2:

1.

6. The method for preparing gold nanoclusters according to claim 4, characterized in that: In step S2, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of 18:1-22:1; preferably, the citric acid buffer is a mixture of sodium citrate and citric acid in a volume ratio of 20:

1.

7. The method for preparing gold nanoclusters according to claim 6, characterized in that: The concentration of the sodium citrate is 0.5M, and the concentration of the citric acid is 5M.

8. The method for preparing gold nanoclusters according to claim 4, characterized in that: In step S2, the duration of the connection and stirring is ≥ 10 h.

9. Use of the gold nanoclusters according to any one of claims 1 to 7 in the preparation of a drug for improving insulin resistance in diabetic liver.

10. A drug for improving liver insulin resistance, characterized in that: The drug is a preparation prepared by using the gold nanoclusters according to any one of claims 1 to 7 as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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