Gallic acid carbon dots, and preparation method and application thereof

Abstract

The application discloses a gallic acid carbon dot and a preparation method and application thereof, and is characterized in that: gallic acid is first dissolved in EDTA, and then is dissolved in deionized water together with citric acid, ethylenediamine and FeCl3.6H2O, stirring and hydrothermal reaction are conducted to obtain a gelatinous gallic acid carbon dot. The gallic acid is a natural small molecule product which is widely applied to anti-inflammatory and anti-tumor, and is used to participate in the preparation of the carbon dot through a simple hydrothermal synthesis method. The obtained carbon dot has good fluorescence performance, good biocompatibility and other advantages, and has a good application prospect in terms of anti-oxidative stress and promotion of drug-induced liver injury repair.

Description

TECHNICAL FIELD

[0001] The present application relates to a gallic acid carbon dot and a preparation method and application thereof, in particular to a carbon dot prepared by a natural antioxidant gallic acid, a preparation method of the gallic acid carbon dot and application of the gallic acid carbon dot in preparation of a medicine for promoting repair of drug-induced liver injury, and belongs to the technical field of nanomaterials and medical applications. BACKGROUND

[0002] The liver is the largest detoxification organ in the human body, and is often attacked by various drugs or toxins. Drug-induced liver injury is one of the most common liver diseases in clinical practice. The pathogenesis of drug-induced liver injury is not yet clear. The current clinical treatment method is aimed at relieving the clinical symptoms of patients, and there are few targeted drugs for effectively reversing or preventing the further development of such diseases. Studies have shown that oxidative stress caused by excessive production of reactive oxygen species (ROS) by liver cells induced by drugs is one of the causes of liver cell apoptosis, inflammation, and further liver damage. Antioxidant stress may be an effective therapeutic target for promoting repair of drug-induced liver injury.

[0003] In recent years, carbon dots (C-dots, CDs) have been increasingly used in biomedical applications due to their small size, unique optical properties, easy functionalization, and high biocompatibility. Many documents have reported the enzyme-like activity and free radical scavenging ability of carbon dots. However, as an antioxidant for biomedical applications, it is necessary to further enhance its enzyme-like activity and biocompatibility. Gallic acid (GA) is a small molecule natural product used by people in daily life and has been used to treat many diseases for a long time. In recent years, GA has been used as a ROS scavenger to study the role of ROS in many physiological and pathological processes, and it has been found to play a key role in reducing inflammation and oxidative stress under various conditions.

[0004] Currently, there are few reports on carbon dots promoting repair of drug-induced liver injury by resisting oxidative stress. In order to further improve the antioxidant capacity of carbon dots, it may be an effective strategy to dope GA into carbon dots. SUMMARY

[0005] The first object of the present application is to provide a gallic acid carbon dot. The second object of the present application is to provide a preparation method of the gallic acid carbon dot. The third object of the present application is to provide application of the gallic acid carbon dot in preparation of a medicine for resisting oxidative stress to promote repair of drug-induced liver injury related diseases.

[0006] Technical solution: In order to solve the above problems, the gallic acid carbon dots provided by the application are prepared by using gallic acid, citric acid, ethylenediamine and FeCl3·6H2O as raw materials through a hydrothermal method.

[0007] The preparation method of the gallic acid carbon dots provided by the application comprises the following steps:

[0008] (1) The gallic acid is first dissolved in EDTA, and then dissolved in deionized water together with citric acid, ethylenediamine and FeCl3·6H2O, and stirred to obtain a mixed solution;

[0009] (2) The mixed solution is subjected to high-temperature reaction, cooled to room temperature, filtered, dialyzed and freeze-dried to obtain the gallic acid carbon dots.

[0010] Further, in step (1), the molar ratio of the gallic acid, citric acid, ethylenediamine and FeCl3·6H2O is 1-5:1-10:5-10:1-10, and preferably the molar ratio of the gallic acid, citric acid, ethylenediamine and FeCl3·6H2O is 2.5:2.5:5:1.

[0011] Further, in step (1), the stirring is stirring at room temperature for 10-60 min.

[0012] 5Further, in step (2), the temperature of the high-temperature reaction is 160-200℃, and the time of the high-temperature reaction is 8-12 h.

[0013] Further, in step (2), the filtration is filtration through a 0.8-micron water-based filter membrane.

[0014] Further, in step (2), the dialysis is dialysis through a dialysis membrane, and the time of the dialysis is 1-8 h.

[0015] Further, in step (2), the temperature of the freeze-drying is -30 to -50℃, and the time of the freeze-drying is 12-48 h.

[0016] Further, the dialysis membrane is MWCO100, MWCO500, MWCO1000 or MWCO2000.

[0017] The gallic acid carbon dots provided by the application are used in the preparation of an antioxidant stress to promote drug-induced liver injury repair related disease drugs.

[0018] The present application surrounds the oxidative stress mechanism of drug-induced liver injury, combines basic research means, and prepares a novel carbon dot gallus acid carbon dot (GA-CDs) through a hydrothermal synthesis method, and through in-vivo and in-vitro experiments, it is verified that the prepared novel carbon dot can play an anti-oxidative stress role by removing ROS in a drug-induced liver injury model, so as to achieve the purpose of promoting the repair of drug-induced liver injury.

[0019] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0020] The present application provides a method for preparing carbon dots by using natural antioxidant gallic acid, which uses the natural substance gallic acid that has been used for a long time and widely, and prepares carbon dots through a simple hydrothermal method. The obtained carbon dots have the advantages of simple preparation process, low cost, good biocompatibility, high enzyme-like activity, etc., so as to provide valuable insights for further developing various new carbon dots with high enzyme-like activity through natural substances, and provide new ideas for the application of nanomaterials in anti-oxidative stress to promote the repair of drug-induced liver injury. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 TEM image of GA-CDs prepared in Example 1;

[0022] Figure 2 UV absorption and fluorescence emission spectrum of GA-CDs prepared in Example 1

[0023] Figure 3 Infrared spectrum of GA-CDs prepared in Example 1;

[0024] Figure 4 X-ray spectrum of GA-CDs prepared in Example 1;

[0025] Figure 5 Free radical scavenging capacity result graph of carbon dots of different synthesis routes;

[0026] Figure 6 Toxicity investigation result graph of different concentrations of carbon dots (GA-CDs) on RAW 264.7 cells;

[0027] Figure 7 Protection effect result graph of different concentrations of carbon dots (GA-CDs) on H2O2-induced oxidative stress of RAW 264.7 cells;

[0028] Figure 8 In-vivo treatment effect research graph of carbon dots (GA-CDs) on drug-induced liver injury mice;

[0029] Figure 9 H&E staining graph of main organs of carbon dots (GA-CDs) after injection for 7 days and 28 days. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be further described in detail below with reference to the accompanying drawings.

[0031] Example 1

[0032] Gallic acid was first dissolved in EDTA (0.5 mL), and then dissolved in deionized water (20 mL) together with citric acid, ethylenediamine, and FeCl3·6H2O, and stirred at room temperature for 30 min to obtain a mixed solution. The molar ratio of gallic acid, citric acid, ethylenediamine, and FeCl3·6H2O was 2.5:2.5:5:1 (wherein the amount of gallic acid was 0.0025 mol). The obtained mixed solution was transferred to a 20 mL polytetrafluoroethylene-lined stainless steel autoclave. The stainless steel autoclave with the mixed solution was placed in an oven, and after being subjected to high temperature of 180°C for 10 h, the autoclave was naturally cooled to room temperature. The obtained liquid was filtered through a 0.8 um water filter membrane, and finally, the purified liquid was placed in a dialysis membrane (MWCO 500-1000) for 2 h. The liquid after dialysis was freeze-dried in a freeze dryer at -40°C for 24 h to obtain gallic acid carbon dots (GA-CDs) in a gel form.

[0033] The gallic acid carbon dots prepared in this example were observed for morphological characteristics using high-resolution transmission electron microscopy, and the results are shown in Figure 1 . Figure 1 The TEM image of the gallic acid carbon dots (GA-CDs) prepared in Example 1 is shown in Figure 1 , and the scale bar is 50 nm. As can be seen from , the gallic acid carbon dots prepared in this example have good dispersibility.

[0034] The gallic acid carbon dots prepared in this example were investigated for ultraviolet-visible absorption, and the results are shown in Figure 2 . Figure 2 The ultraviolet absorption and fluorescence emission spectra of the GA-CDs prepared in Example 1 are shown in Figure 2 , and the inset is a photograph of the GA-CDs dispersion under visible light (Vis) and ultraviolet light (Uv). As can be seen from Figure 2 , there is a typical ultraviolet-visible absorption peak at 338 nm, and the maximum fluorescence emission wavelength is 440 nm. The GA-CDs appear yellowish under visible light and emit blue fluorescence under 365 nm ultraviolet light (see the inset in .

[0035] The gallic acid carbon dots prepared in this example and pure gallic acid were analyzed for surface structure of the carbon dots using Fourier infrared spectroscopy, and the results are shown in Figure 3 . Figure 3 The infrared spectrum of the GA-CDs prepared in Example 1 is shown in Figure 3 . Figure 3The GA-CDs and GA showed similar characteristic peaks, which indicated that the GA-CDs partially retained the structural characteristics of GA.

[0036] The surface composition and element analysis of the gallic acid carbon dots prepared in this example were performed by X-ray electron spectroscopy, and the results are shown in Figure 4 . Figure 4 The X-ray spectrogram of the GA-CDs prepared in Example 1 is shown in Figure 4 It can be seen that there are five characteristic peaks at 285.1 eV, 399.8 eV, 530.8 eV, 197.4 eV, and 711.9 eV, corresponding to elements C (40.86%), N (19.32%), O (35.67%), Cl (3.58%), and Fe (0.57%), respectively. This indicates that the gallic acid carbon dots contain the above five elements.

[0037] Example 2

[0038] The experimental process was the same as in Example 1. Gallic acid was first dissolved in EDTA (0.5 mL), and then dissolved in deionized water (20 mL) together with citric acid, ethylenediamine, and FeCl3·6H2O. The mixture was stirred at room temperature for 30 minutes to obtain a mixed solution. The molar ratio of gallic acid, citric acid, ethylenediamine, and FeCl3·6H2O was 1:2.5:5:1 (wherein the amount of gallic acid was 0.001 mol). The obtained mixed solution was transferred to a 20 mL polytetrafluoroethylene-lined stainless steel autoclave. The stainless steel autoclave with the mixed solution was placed in an oven, and after being subjected to high temperature of 180℃ for 10 h, the autoclave was naturally cooled to room temperature. The obtained liquid was filtered through a 0.8 um water-based filter membrane, and finally, the purified liquid was placed in a dialysis membrane (MWCO 500-1000) for 2 h. The liquid after dialysis was freeze-dried in a freeze dryer at -40℃ for 24 h to obtain gel-like gallic acid carbon dots (GA-CDs).

[0039] Example 3

[0040] The experimental process is the same as that of Example 1. Gallic acid is first dissolved in EDTA (0.5 mL), and then dissolved in deionized water (20 mL) together with citric acid, ethylenediamine and FeCl3·6H2O. The mixture is stirred at room temperature for 30 minutes to obtain a mixed solution. The molar ratio of gallic acid, citric acid, ethylenediamine and FeCl3·6H2O is 5:2.5:5:1 (wherein the amount of gallic acid is 0.005 mol). The obtained mixed solution is transferred to a 20 mL stainless steel autoclave with a polytetrafluoroethylene liner. The stainless steel autoclave with the mixed solution is placed in an oven, and after being subjected to a high temperature of 180°C for 10 hours, the autoclave is naturally cooled to room temperature. The obtained liquid is filtered through a 0.8 um water filter membrane. Finally, the purified liquid is placed in a dialysis membrane (MWCO 500-1000) for 2 hours. The liquid after dialysis is freeze-dried in a freeze dryer at -40°C for 24 hours to obtain a gel-like carbon dot.

[0041] Comparative Example 1

[0042] The experimental process is the same as that of Example 1, except that FeCl3·6H2O is not doped. Gallic acid is first dissolved in EDTA (0.5 mL), and then dissolved in deionized water (20 mL) together with citric acid and ethylenediamine. The mixture is stirred at room temperature for 30 minutes to obtain a mixed solution. The molar ratio of gallic acid, citric acid and ethylenediamine is 2.5:2.5:5 (wherein the amount of gallic acid is 0.0025 mol). The obtained mixed solution is transferred to a 20 mL stainless steel autoclave with a polytetrafluoroethylene liner. The stainless steel autoclave with the mixed solution is placed in an oven, and after being subjected to a high temperature of 180°C for 10 hours, the autoclave is naturally cooled to room temperature. The obtained liquid is filtered through a 0.8 um water filter membrane. Finally, the purified liquid is placed in a dialysis membrane (MWCO 500-1000) for 2 hours. The liquid after dialysis is freeze-dried in a freeze dryer at -40°C for 24 hours to obtain a gel-like gallic acid carbon dot.

[0043] Example 4: Determination of radical scavenging ability of GA-CDs

[0044] The carbon dots prepared in Example 1, Example 2, Example 3 and Comparative Example 1 are detected for ABTS radical and O2 .- scavenging ability according to the reagent instructions of the ABTS detection kit and the SOD detection kit. 0.1 mmol / L of FeSO4 is mixed with the carbon dots prepared in Example 1, Example 2, Example 3 and Comparative Example 1 for 10 minutes, respectively, and 0.01 mmol / L of methylene blue is added to detect the absorbance at 660 nm, so as to detect the hydroxyl radical scavenging ability. The test results are shown in Figure 5 Figure 5 Figure showing the radical scavenging ability of carbon dots prepared by different synthesis routes, wherein (A) is the O2 scavenging ability of carbon dots obtained in Examples 1-3 and Comparative Example 1.​.- Figure (B) is a graph of the ABTS radical scavenging ability of the carbon dots obtained in Examples 1-3 and Comparative Example 1; and (C) is a graph of the hydroxyl radical scavenging ability of the carbon dots obtained in Examples 1-3 and Comparative Example 1. The results are shown in Figure 5 It can be seen that the carbon dots without iron doping have no effect on O2 .- The radical scavenging ability is much lower than that of the iron-doped carbon dots, and the radical scavenging ability is the largest when the molar ratio of gallic acid, citric acid, ethylenediamine and FeCl3·6H2O is 2.5:2.5:5:1.

[0045] Example 5: GA-CDs promote drug-induced liver injury repair under oxidative stress (in vitro effect)

[0046] The mouse monocyte macrophage RAW 264.7 cells (purchased from Promocell Biotech Co., Ltd.) were plated in a 96-well plate at a cell density of 1×10 4 After 24 h of culture at 37°C in a 5% CO2 incubator, different concentrations of GA-CDs solution (0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) prepared by dissolving the GA-CDs prepared in Example 1 in DMEM high-glucose medium were added, and the cells were further cultured for 24 h under the above conditions. The cell viability was then detected by MTT method, and the results are shown in Figure 6 Figure 6 Figure is a graph of the toxicity of different concentrations of carbon dots (GA-CDs) to RAW 264.7 cells, and it can be seen that Figure 6 GA-CDs have no toxicity to RAW 264.7 cells even at a high concentration of 100 μg / mL.

[0047] The RAW 264.7 cells were plated in a 96-well plate at a cell density of 1×10 4 After 24 h of culture at 37°C in a 5% CO2 incubator, different concentrations of GA-CDs solution (0 μg / mL, 100 μg / mL) were added, and the cells were further incubated in the incubator for 2 h. Then, 300 μmol / L of H2O2 was added, and the cells were further cultured at 37°C in a 5% CO2 incubator for 24 h. The cell viability was then detected by MTT method, and the protective ability of GA-CDs against oxidative stress-related cell death was further evaluated by Calcein / PI cell live and dead staining, and the results are shown in Figure 7 Figure 7 ​​The results of the protective effect of different concentrations of carbon dots (GA-CDs) on H2O2-induced oxidative stress in RAW 264.7 cells are shown in the figure, where (A) is the cell viability result measured by MTT method, and (B) is the Calcein / PI cell live and dead staining picture, which is from Figure 7 (A) It can be seen that GA-CDs can alleviate cell damage caused by H2O2. Figure 7 (B) further confirms that GA-CDs can effectively protect cell apoptosis caused by H2O2.

[0048] Example 6 GA-CDs Anti-oxidative Stress Promote Drug-induced Liver Injury Repair (In vivo action)

[0049] 6-8 week old BALB / C mice were adaptively cultured for 1-2 days and divided into normal control group (Control), injury group (APAP) and treatment group (GA-CDs), 6-10 in each group. Before the experiment, the mice were fasted for 8h, and 100μL of 4mg / mL GA-CDs solution was injected into the tail vein of the mice, and the same volume of 0.01mmol / L PBS solution was injected into the tail vein of the control group and the injury group, 2h later, 1000μL of 6mg / mL APAP solution was injected intraperitoneally to establish a drug-induced liver injury model. 12h later, the mice were euthanized and the serum was separated to detect the levels of serum glutathione and glutathione, and a part of the liver tissue was fixed in 4% paraformaldehyde for H&E staining. In addition, a part of the mice were injected with 100μL of 4mg / mL GA-CDs solution into the tail vein, and the heart, liver, spleen, lung and kidney were taken for H&E staining on the 7th day and the 28th day, respectively, and the experimental results are shown in Figure 8-9 . Figure 8 The in vivo treatment effect of carbon dots (GA-CDs) on drug-induced liver injury in mice is shown in the figure; (A) is the serum glutathione (ALT) level diagram; (B) is the serum glutathione (AST) level diagram; (C) is the liver tissue H&E staining diagram (scale: 200um). From Figure 8 (A), (B) It can be seen that the content of serum glutathione and glutathione in the injury group induced by APAP increased significantly, and the liver function deteriorated, while the treatment group effectively reduced the levels of serum glutathione and glutathione. From Figure 8 (C) H&E staining results show that APAP induces drug-induced liver injury in mice, and liver damage in mice after treatment is significantly reduced, further confirming that GA-CDs have a protective effect on liver function in drug-induced liver injury. Figure 9 The H&E staining diagrams of main organs of carbon dots (GA-CDs) after injection for 7 days and 28 days are shown in the figure (scale 50μm). From Figure 9The H&E staining results of each major organ show that the major organ tissues at different time points have no obvious change compared with the normal group, which indicates that the biological safety of the GA-CDs is high.

[0050] The above test results show that the preparation of the carbon dots by the natural antioxidant gallic acid has the characteristics of simple preparation process, low synthesis cost, high enzyme-like activity, good biocompatibility, etc., can effectively scavenge excess active oxygen in vitro, play an antioxidant stress role, and further verify in the in vivo experiment that it can reduce the mouse APAP-induced liver injury, and it can be preliminarily considered that it has great research value and clinical transformation potential.

[0051] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described by referring to the preferred embodiments of the present application, it should be understood by those skilled in the art that various changes in form and details can be made without departing from the spirit and scope of the present application defined in the appended claims.

Claims

1. Use of gallic acid carbon dots in the preparation of a drug for resisting oxidative stress to promote the repair of drug-induced liver injury-related diseases, characterized in that, Preparation method of gallic acid carbon dots comprising the following steps: (1) gallic acid is first dissolved in EDTA, and then dissolved in deionized water together with citric acid, ethylenediamine and FeCl3·6H2O, and stirred to obtain a mixed solution; (2) the mixed solution is subjected to high-temperature reaction, cooled to room temperature, filtered, dialyzed and freeze-dried to obtain the gelatinous gallic acid carbon dots, wherein the high-temperature reaction is carried out at a temperature of 160-200℃ for 8-12h.

2. Use according to claim 1, characterized in that, In step (1), the molar ratio of gallic acid, citric acid, ethylenediamine and FeCl3·6H2O is 1-5:1-10:5-10:1-10.

3. Use according to claim 1, characterized in that, In step (1), the stirring is carried out at room temperature for 10-60min.

4. Use according to claim 1, characterized in that, In step (2), the filtration is carried out through a 0.8-micron water filter membrane.

5. The use according to claim 1, characterized in that, In step (2), the dialysis is carried out through a dialysis membrane, and the dialysis time is 1-8h.

6. Use according to claim 1, characterized in that, In step (2), the freeze-drying is carried out at a temperature of-30--50℃ for 12-48h.

7. Use according to claim 5, characterized in that, The dialysis membrane has a molecular weight cut-off of MWCO100, MWCO500, MWCO1000 or MWCO2000.

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