Preparation method of microwave-assisted gallic acid carbon dots, carbon dots and application of carbon dots in tumor treatment

Abstract

The invention discloses a preparation method of a microwave-assisted gallic acid carbon dot, the carbon dot and application of the carbon dot in tumor treatment, the method comprises the following steps: S1, dissolving gallic acid and arginine in ultrapure water, and uniformly mixing to obtain a mixed solution; s2, transferring the mixed solution into a reaction kettle, and performing microwave heating; and S3, dissolving the product obtained in the step S2 by using pure water under an ultrasonic condition, centrifuging, filtering supernate, dialyzing filtrate, and freeze-drying dialysate to obtain the gallic acid carbon dots. The invention discloses a method for synthesizing gallic acid carbon dots by using a microwave-assisted method, the average particle size of the synthesized carbon dots is less than 2nm, the synthesized carbon dots have the advantages of low toxicity, strong penetrating power and the like, proliferation and migration of brain glioma cells can be remarkably inhibited, and a feasible new strategy is provided for developing a novel brain glioma therapeutic agent based on the carbon dots.

Description

Technical Field

[0001] The present invention relates to the fields of nanomaterials and biomedicine, and in particular to a method for preparing microwave-assisted gallic acid carbon dots, the carbon dots and applications of the carbon dots in tumor treatment. Background Art

[0002] Glioblastoma (GBM), the most common primary intracranial malignant tumor, accounts for approximately 80% of all brain malignancies and is the leading cause of death from primary brain tumors. Glioblastoma (GBM) is the most malignant type. Patients treated with standard surgery and adjuvant radiotherapy or chemotherapy have a median survival of only 14 months, with a 5-year survival rate of less than 5%. Due to the highly invasive nature of glioblastoma cells, complete tumor removal by surgery is difficult. The effectiveness of radiotherapy is often limited by intrinsic radioresistance and the hypoxic tumor microenvironment. Clinically available drugs for GBM are also very limited. Only four drugs—lomustine, carmustine, temozolomide (TMZ), and bevacizumab—are approved by the FDA for the treatment of GBM. These drugs show potential for early treatment of GBM patients, but long-term use can lead to decreased sensitivity and even drug resistance. The blood-brain barrier (BBB) ​​prevents nearly all drugs from entering the brain lesions to exert their therapeutic effects, a major obstacle to the development of drugs for treating brain diseases. Therefore, there is an urgent need to develop drugs that can efficiently cross the BBB and target tumor cells. Gallic acid is a polyphenol compound widely found in plants and fruits, exhibiting anti-inflammatory, antibacterial, antioxidant, and anticancer properties. However, as a natural polyhydroxyphenol compound, its antitumor activity is relatively weak, with IC50 concentrations exceeding 1 mM in some studies. Furthermore, as a hydrophilic small molecule, its BBB penetration is limited, further limiting its application in glioma treatment. Carbon dots (CDs) are emerging carbon-based nanomaterials with advantages such as low toxicity, strong penetration, ease of modification, and stable physicochemical properties, showing great promise in therapeutics and drug delivery.

[0003] Gallic acid, a natural polyphenolic compound, can induce programmed cell death, cell cycle inhibition, and silencing of the vasculature and tumor migration to exert antitumor effects. MGA-CDs have also been developed by dissolving gallic acid and metformin in a 420 mM sodium hydroxide solution and heating them in an oven at 200°C for 10 hours. These carbon dots induce ferroptosis by inhibiting PLPP4 activity, thereby inhibiting glioma growth. GT-CDs, obtained by heating gallic acid and tyrosine at 180°C for 16 hours, exhibit intrinsic bioactivity for bioimaging, tumor therapy, and postoperative management.

[0004] However, the gallic acid carbon dots synthesized by the current solvothermal method have limited activity in anti-tumor research, with IC50 concentrations above 100 μg / ml, limiting their application in nanomedicine. In addition, the process of synthesizing carbon dots using an oven solvothermal method is time-consuming, generally requiring a reaction temperature to be maintained for more than 10 hours, which is time-consuming for reaction condition screening. Finally, the maximum excitation wavelength of the current gallic acid-derived carbon dots is near 450 nm, which severely limits their application in optical imaging. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a microwave-assisted gallic acid carbon dot preparation method, carbon dots, and their application in tumor treatment, which addresses the deficiencies in the prior art. The present application provides a scheme for synthesizing arginine-modified gallic acid carbon dots using a microwave-assisted method, which provides a new strategy for developing novel brain glioma therapeutic agents based on natural product-derived carbon dots.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a microwave-assisted gallic acid carbon dot preparation method, comprising the following steps:

[0007] S1, dissolving gallic acid and arginine in ultrapure water and mixing to obtain a mixed solution;

[0008] S2, transferring the mixed solution to a reaction kettle and heating under microwaves;

[0009] S3, dissolving the product obtained in step S2 under ultrasonic conditions with pure water, centrifuging, filtering the supernatant, dialyzing the filtrate, freeze-drying the dialysate to obtain gallic acid carbon dots.

[0010] Preferably, in step S1, the mass ratio of gallic acid to arginine is 1-4:1, and the mass ratio of gallic acid to ultrapure water is 1:25-100.

[0011] Preferably, in step S2, the microwave power is 500-1000 W, and the microwave heating time is 1.5-6 minutes.

[0012] Preferably, the microwave-assisted gallic acid carbon dot preparation method comprises the following steps:

[0013] S1, dissolving 0.2-0.8 g of gallic acid and 0.1-0.4 g of arginine in 10-40 mL of ultrapure water and ultrasonically mixing for 5-20 minutes to obtain a mixed solution;

[0014] S2, transferring the mixed solution to a reaction kettle lined with polytetrafluoroethylene and heating under microwaves at 500-1000 W for 1.5-6 minutes;

[0015] S3. Dissolve the product obtained in step S2 in 10-40 ml of pure water under ultrasonic conditions, centrifuge at 5000-20000 rpm, filter the supernatant using a 0.1-0.4 μm filter membrane, dialyze the filtrate using ultrapure water in a 300-800 Da dialysis membrane, and freeze-dry the dialyzate to obtain gallic acid carbon dots.

[0016] Preferably, the microwave-assisted method for preparing gallic acid carbon dots comprises the following steps:

[0017] S1. Dissolve 0.4 g of gallic acid and 0.2 g of arginine in 20 mL of ultrapure water and mix by ultrasonication for 10 minutes to obtain a mixed solution;

[0018] S2. Transfer the mixture to a polytetrafluoroethylene-lined reactor and heat it in a microwave oven at 700 W for 3 minutes;

[0019] S3. Dissolve the product obtained in step S2 in 20 ml of pure water under ultrasonic conditions, centrifuge at 10,000 rpm, filter the supernatant using a 0.22 μm filter membrane, dialyze the filtrate using ultrapure water in a 500 Da dialysis membrane, and freeze-dry the dialyzate to obtain gallic acid carbon dots.

[0020] In a second aspect of the present invention, a gallic acid carbon dot is provided, which is prepared by the method described above.

[0021] The third aspect of the present invention provides a use of the gallic acid carbon dots described above in living cell imaging.

[0022] The fourth aspect of the present invention provides a use of the gallic acid carbon dots described above in the preparation of anti-tumor drugs.

[0023] The fifth aspect of the present invention provides a use of the gallic acid carbon dots as described above in the preparation of a therapeutic agent for brain glioma.

[0024] In a sixth aspect, the present invention provides a therapeutic agent for glioma, comprising the gallic acid carbon dots as described above.

[0025] The beneficial effects of the present invention are:

[0026] The present invention discloses a method for synthesizing gallic acid carbon dots using a microwave-assisted method. The synthesized carbon dots have an average particle size of less than 2 nm, have the advantages of low toxicity and strong penetration ability, and can also significantly inhibit the proliferation and migration of glioma cells. The present invention will provide a feasible new strategy for the development of new glioma therapeutic agents based on carbon dots. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Transmission electron microscopy image (A) and particle size distribution diagram (B) of the gallic acid carbon dots prepared in Example 1;

[0028] Figure 2 Fluorescence spectrum of gallic acid carbon dots prepared in Example 1;

[0029] Figure 3 Inhibition rate test results of gallic acid carbon dots prepared in Example 1 on glioma cells;

[0030] Figure 4 Test results of gallic acid carbon dots prepared in Example 1 on inhibiting tumor cell proliferation;

[0031] Figure 5 Test results of gallic acid carbon dots prepared in Example 1 on inhibiting tumor cell migration. DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with the examples below, so that those skilled in the art can implement the present application according to the description.

[0033] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0034] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially. The specific conditions not specified in the following examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specified by the manufacturer, are conventional products that can be obtained by commercial purchase.

[0035] Example 1

[0036] A microwave-assisted gallic acid carbon dot, the preparation method thereof comprising the following steps:

[0037] S1, 0.4g of gallic acid and 0.2g of arginine were dissolved in 20mL of ultrapure water and ultrasonicated for 10 minutes to obtain a mixed solution;

[0038] S2, the mixed solution was transferred to a reaction kettle lined with polytetrafluoroethylene and heated in a microwave oven at a power of 700W for 3 minutes;

[0039] S3, the product obtained in step S2 was dissolved in 20ml of pure water under ultrasonic conditions, centrifuged at 10000rpm, the supernatant was filtered using a 0.22μm filter membrane to remove large particle impurities, the filtrate was dialyzed in a 500Da dialysis membrane using ultrapure water to remove unreacted raw materials, and finally the dialysate was freeze-dried to obtain gallic acid carbon dots, which were stored in a 4℃ refrigerator for use.

[0040] Performance test

[0041] 1. Transmission electron microscopy imaging of gallic acid carbon dots

[0042] (1) Sample preparation: 10 μl of gallic acid carbon dot solution was added dropwise to a copper mesh (sample mesh) covered with a carbon film. A Petri dish was then placed on the sample mesh and allowed to stand at room temperature until the sample dried naturally. The sample mesh with carbon dots was carefully rinsed with deionized water, then with ethanol, and finally, the excess ethanol was absorbed with filter paper.

[0043] (2) Sample imaging: Turn on the transmission electron microscope and measure the carbon dot sample in the following steps: sample loading, sample injection, sample height adjustment, instrument adjustment, and sample imaging. Select an appropriate magnification to make a large number of nanoparticles appear in the field of view, and ensure that at least 200 nanoparticles are observed in different fields of view. The transmission electron microscope image is as follows: Figure 1 (A). Repeat the above steps and select different fields of view to image the carbon dot sample, such as Figure 1 (B) The results showed that the average particle size of gallic acid carbon dots was 1.67 nm, which is beneficial to the transport, bioavailability and BBB penetration of carbon dots in the body.

[0044] 2. Gallic acid carbon dots have potential for bioimaging applications

[0045] Gallic acid carbon dot solution can emit bright blue-green fluorescence under ultraviolet light ( Figure 2 (Inset A) The fluorescence properties of gallic acid were analyzed using a fluorescence spectrometer. The results showed that the carbon dots had a maximum excitation wavelength of 396.8 nm and a maximum emission wavelength of 505 nm, demonstrating their potential for live cell imaging. Under different excitation wavelengths, the emission intensity varied significantly, but the emission wavelength did not shift significantly, indicating that the emission wavelength of the carbon dots was independent of excitation. Figure 2 (B).

[0046] 3. Gallic acid carbon dots can effectively exert anti-tumor effects

[0047] Glioma cells cultured in T25 were digested, and 5000 cells were resuspended in 100 μl of culture medium and seeded into 96-well cell culture plates and cultured in a 37°C incubator overnight. The cell culture medium was discarded and replaced with culture medium containing 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, and 20 μg / mL carbon dots, respectively. Three replicates were set for each concentration and cultured for 48 hours. After the culture was completed, the cell culture medium in each well was replaced with a culture medium containing 10 μl of WST-1 and incubated at 37°C for 1 hour. The absorbance of each well was measured at 450 nm. At the same time, blank wells (no cells, culture medium and WST-1 solution) and control wells (with cells, no drug, culture medium and WST-1 solution) were set. The cell viability of tumor cells after carbon dot treatment was calculated using the following formula:

[0048] Cell viability = [(OD 实验组 -OD 空白孔 ) / (OD 对照组 -OD 空白孔 )]×100%;

[0049] The inhibition of different concentrations of carbon dots on glioma cell lines U251 and GL261 is shown in Figure 2. Figure 3 shown.

[0050] 4. Gallic acid carbon dots can effectively inhibit the proliferation and migration of tumor cells

[0051] (1) U251 and GL261 cells were seeded onto 6-well plates, cultured with culture medium, and cultured overnight in a 37°C incubator. The culture medium was replaced with a culture medium containing 50ug / ml carbon dots or a fresh conventional cell culture medium and cultured for 24 hours. After the culture was completed, the cells were digested and counted again with trypsin, and 2000 cells were seeded into 96-well plates at each well and replaced with fresh complete culture medium (without carbon dots). In order to determine the number of cells on different days, the culture medium in each well was replaced with a complete culture medium containing 10% WST-1, incubated at 37°C for 1 hour, and the absorbance at 450nm of each well was measured. The proliferation of cells after carbon dot treatment is shown in Figure 2. Figure 4 shown.

[0052] (2) U251 and GL261 cells were seeded into 6-well plates and incubated overnight at 37 °C after adding culture medium. The culture medium was replaced with culture medium containing 50 ug / ml carbon dots or fresh conventional cell culture medium and incubated for another 24 hours. After the incubation, the cells were re-digested with trypsin and counted, and then seeded into the upper layer of transwell inserts in a 24-well plate at a density of 10000 cells per well and replaced with serum-free culture medium; 600 μl of complete culture medium was added to the lower layer of the transwell. After 48 hours, the transwell was removed, the cells in the upper layer of the transwell insert were scraped off with a cotton swab, and the cells were fixed with 4% PFA at room temperature for 15 minutes. After washing with PBS for 3 times, the cells were stained with 1% crystal violet for 10 minutes, and finally the excess crystal violet staining solution was washed off with PBS. The migration of the cells was observed under an inverted microscope, the number of purple cells in each field was counted, and the effect of carbon dots on cell migration was calculated. Figure 5 A and 5B).

[0053] Although embodiments of the present application have been disclosed as above, it is not limited only to the uses listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A microwave-assisted method for preparing gallic acid carbon dots, characterized in that: The following steps are involved: S1. Dissolve gallic acid and arginine in ultrapure water and mix well to obtain a mixed solution; S2, transferring the mixed solution into a reactor and heating it in a microwave; S3. Dissolve the product obtained in step S2 in pure water under ultrasonic conditions, centrifuge, filter the supernatant, dialyze the filtrate, and freeze-dry the dialyzate to obtain gallic acid carbon dots.

2. The microwave-assisted method for preparing gallic acid carbon dots according to claim 1, wherein: In step S1, the mass ratio of gallic acid to arginine is 1-4:1, and the mass ratio of gallic acid to ultrapure water is 1:25-100.

3. The microwave-assisted method for preparing gallic acid carbon dots according to claim 2, wherein: In step S2, the microwave power is 500-1000W, and the microwave heating time is 1.5-6 minutes.

4. The microwave-assisted method for preparing gallic acid carbon dots according to claim 3, wherein: The following steps are involved: S1. Dissolve 0.2-0.8 g of gallic acid and 0.1-0.4 g of arginine in 10-40 mL of ultrapure water and mix by ultrasonication for 5-20 minutes to obtain a mixed solution; S2. Transfer the mixed solution to a polytetrafluoroethylene-lined reactor and heat it in a microwave at 500-1000 W for 1.5-6 minutes; S3. Dissolve the product obtained in step S2 in 10-40 ml of pure water under ultrasonic conditions, centrifuge at 5000-20000 rpm, filter the supernatant using a 0.1-0.4 μm filter membrane, dialyze the filtrate using ultrapure water in a 300-800 Da dialysis membrane, and freeze-dry the dialyzate to obtain gallic acid carbon dots.

5. The microwave-assisted method for preparing gallic acid carbon dots according to claim 4, wherein: The following steps are involved: S1. Dissolve 0.4 g of gallic acid and 0.2 g of arginine in 20 mL of ultrapure water and mix by ultrasonication for 10 minutes to obtain a mixed solution; S2. Transfer the mixture to a polytetrafluoroethylene-lined reactor and heat it in a microwave oven at 700 W for 3 minutes; S3. Dissolve the product obtained in step S2 in 20 ml of pure water under ultrasonic conditions, centrifuge at 10,000 rpm, filter the supernatant using a 0.22 μm filter membrane, dialyze the filtrate using ultrapure water in a 500 Da dialysis membrane, and freeze-dry the dialyzate to obtain gallic acid carbon dots.

6. A gallic acid carbon dot, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. Use of the gallic acid carbon dots according to claim 6 in living cell imaging.

8. Use of the gallic acid carbon dots according to claim 6 in the preparation of anti-tumor drugs.

9. Use of the gallic acid carbon dots according to claim 6 in preparing a therapeutic agent for brain glioma.

10. A therapeutic agent for brain glioma, characterized in that: It comprises the gallic acid carbon dots as claimed in claim 6.

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