Glucose oxidase modified gold nanocluster as well as preparation method and application thereof

Abstract

The invention discloses a glucose oxidase modified gold nanocluster and a preparation method and application thereof, and belongs to the technical field of nano-medicines.The glucose oxidase is added into ultrapure water to be dissolved, then chloroauric acid is added, ascorbic acid is added to assist reduction, and finally the pH is adjusted; reacting under a shading condition, centrifuging, taking supernate, and dialyzing to obtain the glucose oxidase modified gold nanocluster with a molecular formula of AuNCs (at) GOx. The prepared AuNCs GOx has the characteristics of ultra-small size, better dispersity and uniform particle size, and can be applied as a cell autophagy accelerant, an anti-tumor drug and a tumor detection fluorescent agent.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and more specifically, to a glucose oxidase-modified gold nanocluster and a preparation method and application thereof. Background Art

[0002] Currently, cell autophagy promoters have been used in clinical treatment and skin care products.

[0003] Autophagy is an evolutionarily conserved and tightly coordinated catabolic degradation process that exists in eukaryotic cells. When cells sense internal or external metabolic stimuli, such as hunger or hypoxia, they will initiate autophagy, transport aggregated proteins, excess or damaged organelles, and pathogens to lysosomes for hydrolysis, degrading them into small molecules such as nucleotides, which are then recycled to the cytoplasm for use, thereby maintaining the body's homeostatic balance.

[0004] Autophagy is one of the pathways used to eliminate causes of disease and phagocytize apoptotic cells. It plays an important role in the self-renewal and anti-aging of skin cells in skin care products. It also plays a complex role in cancer treatment, preventing bioenergy failure caused by metabolic stress, controlling the quality and quantity of proteins and organelles, and helping the development of tumors and maintaining a malignant state.

[0005] There are two ways for autophagosomes to accumulate: one is that the activation of autophagy leads to the massive production of autophagosomes; the other is that the autophagic flow is blocked in the later stage and the autophagic body is difficult to degrade. Therefore, detecting the integrity of the autophagic flow is particularly important for evaluating the autophagy caused by it. Therefore, in addition to autophagic homeostasis, the concept of "autophagic flow" is also introduced to more accurately summarize the level of autophagy. The so-called autophagic flow is a series of events after the induction of autophagy, including the formation of autophagosomes and the degradation of autophagosomes to lysosomes. The increase in autophagic flow can more accurately reflect the degree of autophagy in cells.

[0006] Tumor is one of the major diseases that seriously affect human health. The clinical treatment of tumors mainly includes traditional surgical treatment, radiotherapy, chemotherapy and new treatment methods such as targeted therapy and immunotherapy that have developed rapidly in recent years. With the development of tumor molecular biology, people's understanding of tumors has continued to deepen, and the strategies for tumor treatment have become more precise and detailed. However, due to the complexity, diversity and heterogeneity of the tumor microenvironment (TME), many treatment options face huge challenges and limitations. The tumor microenvironment (TME) is an environment that exhibits a high level of tumor-related redox homeostasis regulation. Reactive oxygen species (ROS) and metabolism-related oxygen-containing free radicals or peroxides are produced during the cell redox process. Among them, hydroxyl free radicals (one of the reactive oxygen species) are the dominant factors that induce oxidative stress in pathological processes. Nanozymes are nanomaterials with enzyme-like activity. Compared with traditional natural enzymes, they have the advantages of low cost, higher activity and good stability, and have become an emerging alternative to natural enzymes. Reactive oxygen species (ROS) produced by POD-like nanozymes can significantly damage cancer cells or even kill cancer cells. When the ROS content in cells is excessive, oxidative stress may be induced. It can induce gene mutations and protein denaturation, thereby damaging organs such as lysosomes and mitochondria, as well as affecting cell signal transduction pathways and inducing autophagy in cells.

[0007] Gold nanoclusters have the characteristics of ultra-small size, good biocompatibility, and excellent fluorescence properties, but they are currently mostly used in cancer treatment and have not been used in cellular autophagy drugs. Summary of the invention

[0008] The first object of the present invention is to provide a method for preparing glucose oxidase-modified gold nanoclusters and the obtained glucose oxidase-modified gold nanoclusters with ultra-small size, good dispersibility, uniform particle size, and the effects of promoting cell autophagy and anti-tumor.

[0009] The second object of the present invention is to provide an application of the glucose oxidase-modified gold nanoclusters in the preparation of cell autophagy promoters.

[0010] The third object of the present invention is to provide an application of the glucose oxidase-modified gold nanoclusters in the preparation of anti-tumor drugs.

[0011] The fourth object of the present invention is to provide an application of the glucose oxidase-modified gold nanoclusters in the preparation of tumor detection fluorescent agents.

[0012] To achieve the above-mentioned object of the present invention, the present invention provides a method for preparing glucose oxidase-modified gold nanoclusters, comprising: Using the chemical reduction synthesis method, glucose oxidase (GOx) was added to ultrapure water to dissolve, chloroauric acid was added and stirred, and then ascorbic acid was added to assist in reduction and stirred, and finally the pH was adjusted; after stirring the reaction under light-shielding conditions, the reaction was centrifuged and the supernatant was dialyzed to obtain glucose oxidase-modified gold nanoclusters with the molecular formula of AuNCs@GOx.

[0013] In one embodiment, 0.025 g of glucose oxidase (GOx) is added to 50 mL of ultrapure water and thoroughly dissolved, and then 5 mL (0.1 M) of chloroauric acid is added and stirred for 5 minutes, followed by adding 1 mL (0.35 mg / mL) of ascorbic acid to assist in reduction and stirring for 5 min, and finally, sodium hydroxide solution is added to adjust the pH to 5.0; after stirring for 16 hours at 37°C in the dark, the mixture is centrifuged at 11000 rpm using a refrigerated centrifuge, and the supernatant is dialyzed for 12 hours to obtain the glucose oxidase-modified gold nanoclusters (AuNCs@GOx).

[0014] The present invention provides a glucose oxidase-modified gold nanocluster prepared by the above preparation method, and the molecular formula is AuNCs@GOx.

[0015] In one embodiment, HRTEM test was performed, and the synthesized material AuNCs@GOx had good dispersion and uniform particle size, which was concentrated in the range of 2.3±0.9 nm. Cytotoxicity experiment was performed, and the synthesized material AuNCs@GOx was non-toxic to normal cells.

[0016] The present invention provides an application of the glucose oxidase-modified gold nanoclusters in preparing a cell autophagy promoter.

[0017] In one embodiment, a cell autophagy experiment was conducted, and the synthetic material AuNCs@GOx was able to activate autophagy and lead to the accumulation of autophagosomes.

[0018] The present invention provides an application of the glucose oxidase-modified gold nanoclusters in the preparation of anti-tumor drugs.

[0019] In one embodiment, cell experiments were conducted on human ovarian cancer cells (SKOV-3), human breast cancer cells (MCF-7), human colon cancer cells (HT-29), human cervical cancer cells (Hela), mouse breast cancer cells (4T1), human embryonic kidney cells (293T), and human gastric mucosal cells (GES-1). The synthesized material AuNCs@GOx showed good cancer cell toxicity and was not toxic to normal cells, so it was a targeted cancer therapeutic drug.

[0020] The present invention also provides an application of the glucose oxidase-modified gold nanoclusters in the preparation of a tumor detection fluorescent agent.

[0021] In one embodiment, a fluorescence experiment was conducted and the synthesized material AuNCs@GOx could be internalized into Hela cells and aggregated in lysosomes, appearing as yellow-green fluorescent dots, and thus could be used as a fluorescent agent for tumor detection.

[0022] Since the glucose oxidase-modified gold nanoclusters provided by the present invention have the functions of detecting and treating cancer cells at the same time, it is recommended to be used as an integrated tumor diagnosis and treatment drug.

[0023] The present invention has at least the following beneficial effects: 1. The preparation method of the present invention and the obtained glucose oxidase-modified gold nanoclusters (AuNCs@GOx) have the characteristics of ultra-small size, good dispersibility and uniform particle size, which make it easier to enter cells and exert their effects; it directly combines and modifies glucose oxidase and gold nanoclusters without the need for an additional platform, so the preparation is simpler; it has low toxicity to normal cells but strong toxicity to cancer cells, and has fluorescent properties, and is suitable for use as an integrated diagnosis and treatment drug.

[0024] 2. When the glucose oxidase-modified gold nanoclusters (AuNCs@GOx) of the present invention are used as a cell autophagy promoter, it can activate autophagy and lead to the accumulation of autophagosomes, thereby exerting the effect of cell autophagy.

[0025] 3. When the glucose oxidase-modified gold nanoclusters (AuNCs@GOx) of the present invention are used as anti-tumor drugs, they have good enzyme activity and a cascade reaction occurs, thereby generating reactive oxygen species that effectively induce cancer cell death, achieving the effect of starvation therapy; in addition, AuNCs@GOx induces cell autophagy, thereby treating cancer. Cytotoxicity results show that after incubating cells with the material AuNCs@GOx for 24 hours, the survival rate of cancer cells is significantly reduced, and it has little effect on normal cells.

[0026] 4. When the glucose oxidase-modified gold nanoclusters (AuNCs@GOx) of the present invention are used as fluorescent agents for tumor detection, they can be internalized into cancer cells and aggregated in lysosomes, appearing in the form of yellow-green fluorescent dots.

[0027] 5. Since the glucose oxidase-modified gold nanoclusters (AuNCs@GOx) of the present invention can simultaneously detect and treat cancer cells, they can be used as an integrated tumor diagnosis and treatment drug.

[0028] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The fluorescence spectrum and UV-visible spectrum of AuNCs@GOx of the present invention; Figure 2 This is the infrared spectrum analysis diagram of AuNCs@GOx of the present invention; Figure 3 This is a transmission electron microscopy image of AuNCs@GOx of the present invention; Figure 4 is the particle size distribution diagram of AuNCs@GOx of the present invention; Figure 5 The results of the AuNCs@GOx cytotoxicity experiment of the present invention; Figure 6 The results of the protein immunoblotting experiment used in the present invention to study the effect of AuNCs@GOx on cell autophagy; Figure 7 The results of the effect of AuNCs@GOx on cell autophagy after co-incubation with lysosomal inhibitor chloroquine are added in the present invention; Figure 8 The results of the study on the cell autophagy mechanism of AuNCs@GOx of the present invention; Fig. 9 This is the fluorescence microscopy experiment result of AuNCs@GOx of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0031] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0032] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0033] Example 1 A method for preparing glucose oxidase-modified gold nanoclusters (AuNCs@GOx), comprising: Using the chemical reduction synthesis method, 0.025 g of glucose oxidase (GOx) was added to 50 mL of ultrapure water to completely dissolve, and then 5 mL (0.1 M) chloroauric acid was added and stirred for 5 minutes, followed by 1 mL (0.35 mg / mL) ascorbic acid to assist in reduction and stirred for 5 min, and finally sodium hydroxide solution was added to adjust the pH to 5.0. After stirring for 16 hours at 37 ° C under light-shielded conditions, the mixture was centrifuged at 11000 rpm using a refrigerated centrifuge, and the supernatant was dialyzed for 12 h to obtain AuNCs@GOx.

[0034] Example 2 The fluorescence spectrum and UV-visible spectrum of AuNCs@GOx prepared in Example 1 were measured. Figure 1 As shown in Figure 2, the fluorescence emission peak of AuNCs@GOx is located at 520 nm, the ultraviolet absorption peak of AuNCs@GOx is located at 470 nm, and the ultraviolet absorption peak of AuNCs@GOx retains the ultraviolet absorption peak of GOx, which can be explained that the synthesis of the material AuNCs@GOx retains the original activity of GOx. Infrared experiments were performed to characterize the structure of the synthesized material, such as Figure 2 As shown, 3400 is hydroxyl, 1620 is ester, 1400 is amino, and 1010 is COC. The corresponding characteristic peaks appeared in the spectrum, further illustrating the successful modification of GOx.

[0035] Example 3 The dispersion and morphology of nanomaterials are of great significance for the application of cell autophagy promoters, as well as actual tumor treatment and detection. The AuNCs@GOx synthesized in Example 1 was tested by HRTEM (transmission electron microscopy). Figure 3 and Figure 4 As shown, the synthesized material has good dispersibility and uniform particle size, which is concentrated in the range of 2.3±0.9 nm.

[0036] Example 4 The cytotoxicity of AuNCs@GOx synthesized in Example 1 was studied by MTT method. Cells in the logarithmic growth phase (skov-3, mcf-7, HT-29, Hela, 4T1, 293T, GES-1) were evenly inoculated in a 96-well plate and placed in an incubator overnight. After the cells adhered to the wall, the culture medium was aspirated, and 200 μL DMEM culture medium containing different concentrations (0, 50, 100, 150, 200, 250, 300, 350, 400 ug / mL) of AuNCs@GOx was added and incubated for 24 hours. The cells were then washed three times with PBS, the culture medium was aspirated, and a pre-configured 200 μL MTT (0.5 mg / mL −1 ) solution, incubate for 4 h, remove the MTT solution, and inject 200 μL of DMSO solution into each well. After shaking for 10 min, the absorbance of each well at 570 nm was monitored by a microplate reader. Each independent experiment was performed 3 times, and the results were averaged and expressed as a percentage relative to the control group.

[0037] The results are as follows Figure 5As shown in a), cancer cells were incubated with AuNCs@GOx at different concentrations. As the concentration increased, the activity of cancer cells decreased in a concentration-dependent manner. When the concentration reached 400 μg / mL, the survival rate of cancer cells dropped to about 10%. The experimental results show that AuNCs@GOx has strong toxicity to cancer cells.

[0038] like Figure 5 As shown in b), normal cells were incubated with materials of different concentrations. As the concentration increased, the activity of normal cells was not greatly affected. When the concentration reached 400 μg / mL, the viability of normal cells still remained at around 90%, which indicates that the material AuNCs@GOx of the present invention is non-toxic to normal cells and has a certain targeting effect on cancer treatment.

[0039] Example 5 The effect of AuNCs@GOx synthesized in Example 1 on the cell autophagy mechanism was studied by protein immunoblotting. Cells in the logarithmic growth phase were evenly inoculated in a 6-well plate and incubated overnight in an incubator. After the cells adhered to the wall, the culture medium was aspirated, and 2 mL of AuNCs@GOx at different concentrations (50, 150, 300 μg / mL) and positive control rapamycin (Rapa, 5 μM) were added to each well. The control group was a blank group with only cells without adding any drugs. After incubating the cells for 24 hours, the 6-well plate was placed in an incubator with ice cubes for subsequent operations. The cells were washed three times with PBS and lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.5% phenylmethylsulfonyl fluoride (PMSF) for 10 minutes. After the cell lysis was completed, the supernatant containing whole cell protein was collected after centrifugation at 11000 rpm for 10 minutes. The protein concentration was then determined using the BCA method (Bicinchoninic Acid Assay). The protein concentration was then unified using SDS-PAGE protein loading buffer (5X) and stored in a -20°C refrigerator for later use. Before each loading, the sample was boiled in a 95°C water bath for 30 min before use.

[0040] The results are as follows Figure 6As shown in the figure: With the increase of the concentration of the material AuNCs@GOx, the cell autophagy marker LC3-Ⅱ / LC3-Ⅰ increased in a concentration-dependent manner, indicating that AuNCs@GOx caused the accumulation of autophagosomes in cells. There are two ways for the accumulation of autophagosomes: one is that the activation of autophagy leads to the massive production of autophagosomes; the other is that the autophagic flow is blocked in the later stage and the autophagosomes are difficult to degrade. Therefore, detecting the integrity of the autophagic flow is particularly important for evaluating the autophagy caused by it. LC3-turnover is a conventional method for detecting autophagic flow. In the late stage of autophagy, LC3-II can be degraded in lysosomes, but after co-incubation with the lysosomal inhibitor chloroquine (CQ), lysosomal alkalinization or autophagosome-lysosome fusion is inhibited, so that the degradation of LC3-II will be blocked, leading to the accumulation of LC3-II. Therefore, the integrity of the autophagic flow was evaluated by comparing the LC3-Ⅱ / LC3-Ⅰ levels incubated with or without the lysosomal inhibitor CQ. If LC3-II expression increases, it indicates that the autophagy process is induced; otherwise, it is the result of autophagy inhibition. Figure 7 As shown, CQ treatment blocked the autophagic flow, which led to the accumulation of autophagosomes. After the addition of AuNCs@GOx, the LC3-II / LC3I levels were significantly increased compared with the control group, which was consistent with the previous experimental results. The experimental results show that the material AuNCs@GOx synthesized by the present invention does not block the autophagic flow, but leads to the accumulation of autophagosomes by activating autophagy. Therefore, AuNCs@GOx can be used as an application for the preparation of cell autophagy promoters, and further used in cosmetics, clinical cancer treatment and other fields. Among them, the control group was a blank group with only cells without adding any drugs, the CQ group was added with 5 uM CQ, the 50 group was added with 50 ug / mL AuNCs@GOx, the 50+ group was added with 50 ug / mL AuNCs@GOx and 5 uM CQ, the 150 group was added with 150 ug / mL AuNCs@GOx, the 150+ group was added with 150ug / mL AuNCs@GOx and 5 uM CQ, the 300 group was added with 300 ug / mL AuNCs@GOx, and the 200+ group was added with 200 ug / mL AuNCs@GOx and 5 uM CQ.

[0041] Further study of the cell autophagy mechanism of AuNCs@GOx showed that Figure 8 As shown, when the material AuNCs@GOx induces autophagy through the AMPK-mTOR-ULK signaling pathway, it will activate AMPK and ULK1 and inhibit mTOR, so the phosphorylated P-AMPK and P-ULK1 protein levels will increase, while the P-mTOR protein content will decrease; therefore, when the protein levels of p-AMPK / AMPK and p-ULK1 / ULK1 increase and the protein content ratio of p-mTOR / mTOR decreases, it proves that it is the AMPK-mTOR-ULK1 signaling pathway.

[0042] Example 6 When AuNCs@GOx synthesized in Example 1 is used as a fluorescent agent for detection, the fluorescence microscopy results are as follows: Fig. 9 As shown in the figure, in the Merge channel, the red and green fluorescence signals overlap to yellow. The overlap between AuNCs@GOx and the lysosomal dye is higher, indicating that AuNCs@GOx can co-localize with Hela cells in the lysosomes.

[0043] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing glucose oxidase-modified gold nanoclusters, characterized in that: include: After glucose oxidase was dissolved in ultrapure water, chloroauric acid was added, followed by ascorbic acid to assist reduction, and finally the pH was adjusted; After the reaction was carried out under light-shielding conditions, the mixture was centrifuged and the supernatant was dialyzed to obtain the glucose oxidase-modified gold nanoclusters.

2. The preparation method according to claim 1, characterized in that Specifically, a chemical reduction synthesis method was adopted, 0.025 g of glucose oxidase was added to 50 mL of ultrapure water and thoroughly dissolved, 5 mL of 0.1 M chloroauric acid was added and stirred for 5 minutes, then 1 mL of 0.35 mg / mL ascorbic acid was added to assist in reduction and stirred for 5 min, and finally sodium hydroxide solution was added to adjust the pH to 5.0; after stirring for 16 hours at 37°C in a dark environment, the mixture was centrifuged at 11000 rpm using a refrigerated centrifuge, and the supernatant was dialyzed for 12 h to obtain the glucose oxidase-modified gold nanoclusters.

3. A glucose oxidase-modified gold nanocluster prepared by the preparation method of claim 1, having a molecular formula of AuNCs@GOx.

4. Use of the glucose oxidase-modified gold nanoclusters prepared by the preparation method of claim 1 as a cell autophagy promoter.

5. Use of the glucose oxidase-modified gold nanoclusters prepared by the preparation method of claim 1 as anti-tumor drugs.

6. The use according to claim 5, characterized in that The anti-tumor drug is any one or more of a drug for treating ovarian cancer, a drug for treating breast cancer, a drug for treating colon cancer, and a drug for treating cervical cancer.

7. Use of the glucose oxidase-modified gold nanoclusters prepared by the preparation method of claim 1 as a fluorescent agent for tumor detection.

8. The use according to claim 7, characterized in that Specifically, it is the application of fluorescent agents for cervical cancer detection.

9. Use of the glucose oxidase-modified gold nanoclusters prepared by the preparation method of claim 1 as a drug for preparing integrated tumor diagnosis and treatment.

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