Gold nanocluster as well as synthesis method and application thereof

Abstract

The invention relates to the field of nano materials, in particular to a gold nanocluster and a synthesis method and application thereof, and the synthesis method comprises the following steps: adding water into HAuCl4. 3H2O to prepare a chloroauric acid aqueous solution, and adding water into polypeptide to prepare a polypeptide aqueous solution; the polypeptide is as shown in SEQ ID NO. 1; under magnetic stirring, sequentially adding the chloroauric acid aqueous solution, the polypeptide aqueous solution and water into a round-bottom bottle, mixing, reacting, then adding water again, adjusting the pH to 11-12 by using sodium hydroxide, dropwise adding a sodium borohydride aqueous solution, mixing, reacting, then adjusting the pH to 2.5 by using hydrochloric acid, and reacting overnight; and the overnight liquid is subjected to ultrafiltration and washing, and the gold nanocluster is obtained. The gold nanocluster prepared by the synthesis method can effectively inhibit aggregation of A beta, has good stability, biocompatibility and safety, has a wide application prospect in drugs for preventing and treating Alzheimer's disease, and solves the problems that a traditional nanocluster is unstable and poor in biocompatibility, cannot effectively inhibit aggregation of A beta and the like.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and particularly to a gold nanocluster, a synthesis method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is a serious neurodegenerative disease that mainly affects the elderly over 65 years old. The brains of patients atrophy, and their memory and cognitive abilities decline continuously, making it difficult for them to take care of themselves, which brings a heavy burden to families and society.

[0003] The amyloid cascade hypothesis points out that the abnormal aggregation of Aβ is the key to the onset of AD. Therefore, inhibiting the aggregation of Aβ and clearing the formed Aβ aggregates have become important directions for the prevention and treatment of AD. Gold nanoclusters (AuNCs) have received extensive attention because they are less than 3 nanometers in size, have molecular-like properties, can be excreted through the kidneys, and have low potential side effects. However, most reported AuNCs lack motifs for specifically recognizing Aβ, and their interaction with Aβ is mostly non-specific electrostatic interaction, which is unstable in a complex in vivo environment, affecting their application. At the same time, the nanoclusters prepared by conventional dialysis purification methods have poor biocompatibility. Summary of the Invention

[0004] The purpose of the present invention is to provide a gold nanocluster that can effectively inhibit the aggregation of Aβ.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a synthesis method of a gold nanocluster, comprising the following steps:

[0007] S1, dissolving HAuCl 4 ·3H 2 O in water to prepare an aqueous solution of chloroauric acid, and dissolving the polypeptide in water to prepare an aqueous solution of the polypeptide; the polypeptide is as shown in SEQ ID NO.1;

[0008] S2, under magnetic stirring, sequentially adding the aqueous solution of chloroauric acid, the aqueous solution of the polypeptide, and water into a round-bottom flask, mixing and reacting, then adding water again, adjusting the pH to 11-12 with sodium hydroxide, dropping an aqueous solution of sodium borohydride, mixing and reacting, and then adjusting the pH to 2.5 with hydrochloric acid, and reacting overnight;

[0009] S3, ultrafiltering and washing the liquid after overnight reaction to obtain the gold nanocluster.

[0010] Further, the ultrafiltration in step S3 is achieved by a 3K centrifugal filter.

[0011] Further, the washing in step S3 is to wash twice with an acidic solution and three times with water successively.

[0012] Further, the pH of the acidic solution is 4.0.

[0013] Further, in step S1, the concentration of the chloroauric acid aqueous solution is 25 mM, and the concentration of the polypeptide aqueous solution is 65 mM.

[0014] Further, the concentration of the sodium borohydride aqueous solution is 0.3 mg / mL.

[0015] The present invention also includes the gold nanoclusters prepared by any of the above synthesis methods.

[0016] The present invention also includes the application of the gold nanoclusters in inhibiting Aβ aggregation.

[0017] Further, the Aβ includes Aβ1-40 and Aβ1-42.

[0018] Further, the application in drugs for preventing and treating Alzheimer's disease.

[0019] Compared with the prior art, the gold nanoclusters prepared by the synthesis method of the present invention can effectively inhibit the aggregation of Aβ, have good stability, biocompatibility and safety, and have broad application prospects in drugs for preventing and treating Alzheimer's disease, solving the problems of traditional nanoclusters such as instability, poor biocompatibility, and inability to effectively inhibit Aβ aggregation.

[0020] Description of the drawings

[0021] Figure 1 Is the characterization diagram of gold nanoclusters ( Figure 1 CRHA1 in E is the abbreviation of the polypeptide used in the present invention).

[0022] Figure 2 Is the thioflavin T kinetic diagram of gold nanoclusters.

[0023] Figure 3 Is the dynamic light scattering diagram of gold nanoclusters.

[0024] Figure 4 Is the circular dichroism spectrum diagram of gold nanoclusters.

[0025] Figure 5 Is the atomic force microscopy diagram of gold nanoclusters.

[0026] Figure 6 Is the dot blot diagram of gold nanoclusters.

[0027] Figure 7 Is the nematode lifespan experiment diagram of gold nanoclusters.

[0028] Figure 8 Is the cytotoxicity experiment diagram of gold nanoclusters. Detailed implementation manners

[0029] The technical solution of the present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0030] Unless otherwise specified, the gold nanoclusters or AuCRHA1 mentioned in Examples 2 to 8 are all the gold nanoclusters (AuCRHA1) prepared in Example 1; the gold nanoclusters or AuCRHA1 with a concentration involved are all obtained by dissolving and diluting the gold nanoclusters (AuCRHA1) prepared in Example 1 with water.

[0031] Example 1

[0032] The gold nanoclusters were prepared according to the following steps:

[0033] Dissolve HAuCl 4 ·3H 2 O in water to prepare a chloroauric acid solution with a concentration of 10 mg / mL (25 mM); dissolve the polypeptide (as shown in SEQ ID NO.1) in water to prepare a polypeptide solution with a concentration of 65 mM.

[0034] Under vigorous magnetic stirring, add 0.42 mL of 25 mM chloroauric acid solution, 0.46 mL of 65 mM polypeptide solution and 0.51 mL of water into a 25 mL round-bottom flask, react for about 10 min, add 10.1 mL of water, and adjust the pH value of the solution to 11 - 12 with 1 M sodium hydroxide. Subsequently, slowly dropwise add 0.12 mL of 0.3 mg / mL sodium borohydride aqueous solution. After reacting for 30 minutes, adjust the pH value to 2.5 with 1 M hydrochloric acid, and then let the reaction continue overnight.

[0035] After the reaction is completed, use a 3K centrifugal filter ( Ultra-15) for ultrafiltration to remove unreacted polypeptide and salts. Subsequently, wash twice with an acidic solution with pH = 4.0 to fully remove unreacted polypeptide, then wash three times with water, and finally collect the prepared gold nanoclusters (AuCRHA1).

[0036] The characterization diagram of the prepared gold nanoclusters is referred to Figure 1 .

[0037] Example 2

[0038] Thioflavin T kinetic experiment

[0039] Dissolve the purchased Aβ40 powder in 1% ammonium hydroxide to 0.5 mg / ml, freeze-dry to obtain Aβ monomers, and store at -80 °C. Before use, dissolve the freeze-dried Aβ powder in 6 mM sodium hydroxide to prepare a 100 μM solution; dissolve ThT in water to prepare a 0.5 mM solution.

[0040] Dilute 100 μM Aβ solution and 0.5 mM ThT with Hepes buffer (containing 5 mM Hepes and 50 mM sodium chloride buffer, pH 7.2) to make the final concentrations 5 μM Aβ and 20 μM ThT, respectively. Add different concentrations of gold nanoclusters in different experiments, transfer the samples to a 96-well plate (Greiner 655096), and continuously shake at 37 °C and 886.9 rpm. Set the excitation wavelength to 440 nm and the emission wavelength to 480 nm, and record the fluorescence intensity every 6 minutes. 5 μM Aβ40 and 20 μM ThT are incubated overnight at 37 °C and 886.9 rpm to reach a plateau, and the resulting solution is used as Aβ40 seeds or preformed Aβ40 protofibrils. In the seed-induced aggregation experiment, add 5% Aβ40 seeds to make the final solution contain 4.75 μM Aβ40 monomers and 0.25 μM Aβ40 seeds; in the disaggregation experiment, directly add different concentrations of AuCRHA1 to 5 μM preformed Aβ40 protofibrils and immediately record the fluorescence intensity.

[0041] The results are shown in Figure 2 , gold nanoclusters can inhibit Aβ40 aggregation and disassemble Aβ40 protofibrils in a concentration-dependent manner.

[0042] Example 3

[0043] Dynamic light scattering experiment

[0044] Incubate 5 μM Aβ40 with 10 μg / mL AuCRHA1 in Hepes buffer for 24 hours, and measure the particle size of the sample using a dynamic light scattering instrument. In the disaggregation experiment, first incubate 5 μM Aβ40 for 24 hours, then add 10 μg / mL AuCRHA1, and measure the particle size after continuing to incubate for 24 hours or 48 hours.

[0045] The results are shown in Figure 3 , 5 μM Aβ40 alone forms aggregates of about 600 nm after incubation at 37 °C for 24 hours; while incubating with 10 μg / mL AuCRHA1 for 24 hours, the particle size of the aggregates is less than 100 nm, indicating that AuCRHA1 inhibits the formation of Aβ40 protofibrils. After preformed 5 μM Aβ40 protofibrils are incubated with 10 μg / mL AuCRHA1, AuCRHA1 gradually disassembles them into smaller aggregates.

[0046] Example 4

[0047] Circular dichroism (CD) experiment

[0048] To reduce the interference of chloride ions and Hepes, a buffer solution (pH 7.2) containing 10 mM sodium phosphate and 140 mM sodium fluoride was used in the experiment.

[0049] Aβ40 (100 μM) was diluted to 10 μM in 6 mM sodium hydroxide, incubated with 10 μg / mL AuCRHA1 at 37 °C with shaking at 1100 rpm for 2 days, and the CD spectrum was recorded using a 5 mm quartz cuvette and a Chirascan circular dichroism spectrometer. In the depolymerization experiment, Aβ40 (10 μM) was first incubated in the buffer for 2 days, then AuCRHA1 (3 mg / mL) was added to make the final concentration 10 μg / mL, and incubated for another 2 days, and then CD spectral analysis was performed.

[0050] The results are shown in Figure 4 , the Aβ40 monomer and fibril have negative peaks at about 196 nm and 216 nm respectively, indicating the formation of β-sheet structure in the Aβ40 fibril. When Aβ40 was co-incubated with AuCRHA1 for 24 hours, no negative peak was detected at 216 nm, indicating that AuCRHA1 inhibited the formation of β-sheet structure; after pre-formed Aβ40 fibrils were incubated with AuCRHA1 for 24 hours, the negative peak at 216 nm disappeared, indicating that AuCRHA1 disrupted the β-sheet structure of Aβ40 fibrils.

[0051] Example 5

[0052] Atomic force microscopy experiment

[0053] A freshly cleaved mica sheet was pretreated with 40 μL of 100 μM magnesium chloride for 10 min to enhance the adsorption of Aβ on the mica surface, and then washed twice with water. After the thioflavin T kinetic experiment in Example 2, 40 μL of samples from different experimental groups were respectively pipetted onto the mica sheet pretreated with magnesium chloride, then washed twice with water, dried at 37 °C, and Aβ aggregates were observed using AFM.

[0054] The results are shown in Figure 5 , it was found that Aβ incubated alone would form mature and long fibrils; when incubated with AuCRHA1, no mature fibrils were observed, only irregular spheres, which might be Aβ oligomers or unstructured Aβ aggregates. After pre-formed Aβ fibrils were incubated with AuCRHA1 at 37 °C for 2 days, no mature fibrils were also observed, further confirming that AuCRHA1 can depolymerize pre-formed Aβ40 fibrils.

[0055] Example 6

[0056] Dot blot experiment

[0057] Antibody PA5 (product number PA5-77843) was purchased from Thermo Fisher Scientific. It can selectively bind to Aβ fibrils. Antibody 4G8, which recognizes the middle region of Aβ, was used as a control to confirm the presence of Aβ. Aβ40 was incubated alone or with gold nanoclusters for different times, spotted on two nitrocellulose membranes, and stained with PA5 and 4G8 respectively.

[0058] The results are shown in Figure 6 . It was experimentally found that when 5 μM Aβ40 was incubated alone for about 24 hours, PA5-responsive fibrils gradually formed; when incubated with AuCRHA1, no PA5-reactive fibrils were detected after 48 hours. After preformed Aβ40 fibrils were incubated with AuCRHA1, the signal of PA5-responsive fibrils weakened with the increase of incubation time, further confirming that AuCRHA1 can inhibit the formation of Aβ40 fibrils and depolymerize preformed Aβ40 fibrils.

[0059] Example 7

[0060] Lifespan experiment of transgenic Caenorhabditis elegans

[0061] Transgenic Caenorhabditis elegans expressing Aβ is a commonly used animal model for screening and validating anti-AD drugs.

[0062] CL4176 nematodes expressing human Aβ1-42 were obtained from the Caenorhabditis Genetics Center in the United States. Three groups of CL4176 nematodes were treated with 0, 10, and 50 μg / mL gold nanoclusters respectively, and the survival rate was recorded every 2 days.

[0063] The results are shown in Figure 7 . When incubated alone, the maximum lifespan of CL4176 nematodes was 22 days; after adding 10 and 50 μg / mL gold nanoclusters, the maximum lifespan was extended to 26 days and 38 days respectively, indicating that gold nanoclusters can extend the lifespan of AD transgenic Caenorhabditis elegans.

[0064] Example 8

[0065] Cell experiment

[0066] 5 μM Aβ40 was incubated for 2 days in the presence or absence of different concentrations of gold nanoclusters, diluted 25-fold with serum-free medium, and incubated with N2a cells for 24 hours. MTT was used to measure cell viability.

[0067] The results are shown in Figure 8 A. Gold nanoclusters effectively reduced the cytotoxicity of Aβ. When incubated with Aβ40 alone, the cell viability was about 55%; when incubated with 10 μg / mL gold nanoclusters, the cell viability increased to about 99%.

[0068] Preformed Aβ40 fibrils were obtained by incubating 5 μM Aβ40 alone for 2 days, different concentrations of gold nanoclusters were added and incubated for another 2 days, diluted 10-fold and then incubated with N2a cells, and cell toxicity was evaluated by MTT.

[0069] The results are shown in Figure 8 B. Gold nanoclusters reduced the cytotoxicity of preformed Aβ40 fibrils, indicating that the depolymerized Aβ species are not harmful to cells.

[0070] Meanwhile, the cytotoxicity of AuCRHA1 itself was evaluated. The results are shown in Figure 8 C. AuCRHA1 at 50 μg / mL had no obvious toxicity to cells, indicating that AuCRHA1 has good safety.

[0071] Those skilled in the art should understand that the specific embodiments described by us are illustrative rather than limiting the scope of the present invention. Effective modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be covered by the scope protected by the claims of the present invention.

Claims

1. A method for synthesizing gold nanoclusters, characterized in that: The following steps are involved: S1, adding water to HAuCl4·3H2O to prepare a chloroauric acid aqueous solution, and adding water to a polypeptide to prepare a polypeptide aqueous solution; the polypeptide is as shown in SEQ ID NO.1; S2, under magnetic stirring, the chloroauric acid aqueous solution, the polypeptide aqueous solution and water are added to the round-bottomed flask in sequence, mixed and reacted, water is added again, the pH is adjusted to 11-12 with sodium hydroxide, sodium borohydride aqueous solution is added dropwise, mixed and reacted, and then the pH is adjusted to 2.5 with hydrochloric acid, and the reaction is allowed to proceed overnight; S3, ultrafiltration and washing of the liquid after overnight use to obtain gold nanoclusters.

2. The method for synthesizing gold nanoclusters according to claim 1, characterized in that: The ultrafiltration in step S3 is achieved by using a 3K centrifugal filter.

3. The method for synthesizing gold nanoclusters according to claim 1, characterized in that: The washing in step S3 is washing twice with an acidic solution and three times with water.

4. The method for synthesizing gold nanoclusters according to claim 3, characterized in that: The pH of the acidic solution is 4.

0.

5. The method for synthesizing gold nanoclusters according to claim 1, characterized in that: In step S1, the concentration of the chloroauric acid aqueous solution is 25 mM, and the concentration of the polypeptide aqueous solution is 65 mM.

6. The method for synthesizing gold nanoclusters according to claim 1, characterized in that: The concentration of the sodium borohydride aqueous solution is 0.3 mg / mL.

7. The gold nanoclusters prepared by the synthesis method according to any one of claims 1 to 6.

8. Use of the gold nanoclusters according to claim 7 in inhibiting Aβ aggregation.

9. The use according to claim 8, characterized in that: The Aβ includes Aβ1-40 and Aβ1-42.

10. The use according to claim 8, characterized in that: Application in drugs for the prevention and treatment of Alzheimer's disease.

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