Methotrexate-based carbon dots, preparation method thereof and application of methotrexate-based carbon dots in tumor treatment

Abstract

The invention discloses a methotrexate-based carbon dot, a preparation method thereof and an application of the methotrexate-based carbon dot in tumor treatment. The preparation method comprises the following steps: S1, dissolving methotrexate in ultrapure water, carrying out ultrasonic dispersion, transferring the obtained solution into a reaction kettle, and heating to carry out a hydrothermal reaction; and S2, after the reaction is completed, filtering a product, dialyzing filtrate in ultrapure water through a dialysis membrane, collecting dialysate, and freeze-drying to obtain the methotrexate-based carbon dots. According to the methotrexate-based carbon dot and the preparation method thereof, methotrexate is adopted as a raw material, the methotrexate-based carbon dot is synthesized through a one-pot hydrothermal method, the carbon dot can remarkably inhibit proliferation and migration of brain glioma cells when the dosage of the carbon dot is low, and a feasible new strategy can be provided for developing a novel brain glioma therapeutic agent based on the carbon dot.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a carbon dot based on methotrexate, a preparation method thereof and an application thereof in tumor treatment. Background Art

[0002] Gliomas are the most common intracranial malignant tumors in adults, with an incidence rate of about 5-8 per 100,000 people in my country. Currently, clinical treatment options for gliomas include surgery, chemoradiotherapy, targeted therapy, immunotherapy, electric field therapy, etc., but the prognosis is still poor, with a five-year survival rate of less than 5%, and almost 100% recurrence. There are many reasons why gliomas are difficult to treat. The existence of the blood-brain barrier isolates most chemotherapy drugs from reaching the lesions, and currently only three chemotherapy drugs are approved for clinical use. The highly invasive and diffuse nature of gliomas forms metastases throughout the brain, making it impossible to completely remove tumor tissue through surgery. In addition, the high heterogeneity of gliomas and the complex tumor microenvironment further limit the treatment effect of gliomas. Therefore, it is urgent to develop drugs that can efficiently cross the blood-brain barrier and target and kill glioma cells.

[0003] Methotrexate is a type of anti-folate anti-tumor drug. It inhibits dihydrofolate reductase, preventing dihydrofolate from being reduced to tetrahydrofolate, thereby blocking the biosynthesis of DNA and inhibiting the proliferation of tumor cells. Methotrexate is widely used in the field of tumor treatment. In central nervous system diseases, it is mainly used to treat primary central nervous system lymphoma, meningeal metastasis, brain glioma, etc. When methotrexate is used in large doses, it can pass through the blood-brain barrier and blood-testis barrier, and can also reach solid tumors with poor blood supply. However, the use of high-dose methotrexate can lead to serious toxic side effects, such as liver and kidney damage, bone marrow suppression, gastrointestinal mucosal damage, etc. How to ensure the safety of the drug while achieving therapeutic effectiveness is an important research direction for the rational clinical use of methotrexate.

[0004] Carbon dots are an emerging class of carbon-based nanomaterials that usually have sp 2 / sp 3 The "core-shell structure" of the carbon skeleton and organic functional groups / polymer chains on the surface has the advantages of good biocompatibility, stable structure, low toxicity, strong penetration ability, and unique optical properties. It shows great application prospects in the fields of drug delivery, bioimaging, and tumor treatment.

[0005] The present invention aims to synthesize carbon dots using methotrexate through a one-pot hydrothermal method, thereby providing a feasible new strategy for developing new brain glioma therapeutic agents based on carbon dots. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a carbon dot based on methotrexate, a preparation method thereof and an application thereof in tumor treatment in view of the deficiencies in the above-mentioned prior art.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: the first aspect of the present invention provides a method for preparing methotrexate-based carbon dots, the method comprising: using raw materials including methotrexate, and synthesizing the methotrexate-based carbon dots by a one-pot hydrothermal method.

[0008] Preferably, the method for preparing the methotrexate-based carbon dots comprises the following steps:

[0009] S1, dissolving methotrexate in ultrapure water, dispersing by ultrasonication, transferring the resulting solution into a reaction kettle, and heating for hydrothermal reaction;

[0010] S2. After the reaction is completed, the product is filtered, and the filtrate is dialyzed against ultrapure water through a dialysis membrane. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots.

[0011] Preferably, the temperature of the hydrothermal reaction in step S1 is 130-170° C., and the reaction time is 4-16 h.

[0012] Preferably, step S1 specifically comprises:

[0013] Dissolve 0.01-0.04 g of methotrexate in 15-60 mL of ultrapure water, disperse by ultrasonic for 5-20 minutes, transfer the resulting solution to a polytetrafluoroethylene-lined reactor, and heat the reaction at 130-170° C. for 4-16 hours.

[0014] Preferably, step S2 specifically comprises:

[0015] After the reaction is completed, the product is filtered with a 0.2-0.3 μm filter membrane, and the filtrate is dialyzed with ultrapure water for 2-12 h through a dialysis membrane with a molecular cutoff of 600-1500 Da. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots.

[0016] Preferably, the method for preparing the methotrexate-based carbon dots comprises the following steps:

[0017] S1, dissolving 0.02 g of methotrexate in 29 mL of ultrapure water, ultrasonically dispersing for 10 minutes, transferring the resulting solution to a polytetrafluoroethylene-lined reactor, and heating at 150°C for 8 hours;

[0018] S2. After the reaction is completed, the product is filtered with a 0.22 μm filter membrane, and the filtrate is dialyzed in ultrapure water for 2-12 h through a dialysis membrane with a molecular cutoff of 1000 Da. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots, which are stored at 4°C.

[0019] In a second aspect of the present invention, there is provided a methotrexate-based carbon dot, which is prepared by the method as described above.

[0020] The third aspect of the present invention provides a use of the above-mentioned methotrexate-based carbon dots in tumor treatment.

[0021] A fourth aspect of the present invention provides a use of the methotrexate-based carbon dots as described above in the preparation of drugs for treating tumors.

[0022] The fifth aspect of the present invention provides a tumor treatment composition, characterized in that it comprises the carbon dots as described above and a pharmaceutically acceptable adjuvant.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a methotrexate-based carbon dot, a preparation method thereof and an application thereof in tumor treatment. The present invention adopts methotrexate as a raw material and obtains a methotrexate-based carbon dot by a one-pot hydrothermal method. The carbon dot can significantly inhibit the proliferation and migration of brain glioma cells at a lower dose. The present invention can provide a feasible new strategy for developing a novel brain glioma therapeutic agent based on carbon dots. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscope image of the methotrexate carbon dots prepared in Example 1;

[0026] Figure 2 The results of the experiment on inhibition of tumor cell proliferation by carbon dots based on methotrexate in Example 2;

[0027] Figure 3 The results of the experiment on inhibition of tumor cell migration by carbon dots based on methotrexate in Example 3;

[0028] Figure 4 The results of the methotrexate-based carbon dot inhibition of tumor cell invasion experiment in Example 3. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0030] 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.

[0031] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0032] Example 1

[0033] A carbon dot based on methotrexate is prepared by the following method:

[0034] S1, dissolving 0.02 g of methotrexate in 29 mL of ultrapure water, and dispersing by ultrasonic for 10 minutes to promote dissolution, transferring the resulting solution to a polytetrafluoroethylene-lined reactor, and heating the reaction at 150° C. for 8 hours;

[0035] S2. After the reaction is completed, the product is filtered with a 0.22 μm filter membrane to remove large particle impurities. The filtrate is dialyzed in ultrapure water for 4 h through a dialysis membrane with a molecular cutoff of 1000 Da. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots, which are stored in a refrigerator at 4°C for use.

[0036] Reference Figure 1 This is a transmission electron microscope image of methotrexate carbon dots.

[0037] Example 2 Experiment on the inhibition of tumor cell proliferation by carbon dots based on methotrexate

[0038] Glioma cells were plated into 96-well cell culture plates at a concentration of 8000-10000 cells / well and cultured in a 37°C incubator overnight. On the second day, the cell culture medium was discarded and culture medium containing 200 mg / mL, 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.125 mg / mL, and 0 mg / mL carbon dots was added, 3-5 replicate wells were set for each carbon dot concentration, and the plates were placed in an incubator for further culture for 24-48 hours. In order to detect the killing effect of carbon dots at different concentrations on cells, the cell culture medium was replaced with a culture medium containing 10 μl WST-1, incubated at 37°C for 1-4 hours, and the absorbance of each well at a wavelength of 450 nm was measured. At the same time, blank wells (without cells, with culture medium and WST-1 solution) and control wells (with cells, without carbon dots, with culture medium and WST-1 solution) were set. The inhibition rate of carbon dots on tumor cells was calculated using the following formula:

[0039] Inhibition rate = [1-(OD 450实验组 -OD 450空白孔 ) / (OD 450对照组 -OD 450空白孔 )]×100%

[0040] Using temozolomide (TMZ), a first-line drug for the treatment of glioma, as a control, the inhibition rate of different concentrations of methotrexate-based carbon dots (CD3) on the glioma cell line U251 was detected and plotted. Figure 2 shown.

[0041] It can be seen from the test results that under the same concentration conditions, the inhibition rate of methotrexate carbon dots (CD3) on tumor cells is significantly higher than that of temozolomide (TMZ).

[0042] Example 3 Experiment on the ability of methotrexate carbon dots to inhibit the migration and invasion of tumor cells

[0043] (1) U251 cells were inoculated into a 24-well cell culture plate, cultured and placed in a 37°C incubator. When the cell confluence reached 90%, a straight line was drawn in the middle of the cell culture well with a 100-ul pipette tip to prepare a scratch and remove the cells. The culture medium was replaced with a culture medium containing methotrexate carbon dots, TMZ or a regular cell culture medium. After the scratch was prepared, photos were taken at 0 hours, 24 hours, 48 ​​hours, and 72 hours to record the migration of cells on both sides of the scratch to the middle ( Figure 3 A), and imageJ software was used to calculate the changes in scratch diameters of the untreated control group (Control), TMZ group, and methotrexate carbon dot group (CD3) (3B).

[0044] From the test results, it can be seen that the tumor cells in the control group migrated fastest to the middle of the scratch. Compared with the control group, TMZ had an inhibition rate of about 10% for cell migration, while the CD3 group had an inhibition rate of 50-60% for cell migration.

[0045] (2) U251 cells cultured in T25 culture flasks with a confluence of about 90% were collected and cultured with a concentration of 5×10 5 Take 200 μl of the cell suspension of 10 cells / mL, slowly and evenly add it to the transwell coated with matrix gel at the bottom membrane, and place the transwell in a 24-well plate with 600 μl of culture medium added, and culture it in a 37°C incubator. After 48 hours, take out the transwell, remove the culture medium, scrape the cells in the chamber with a cotton swab, and fix the cells with 4% PFA at room temperature for 30 minutes. After washing with PBS three times, stain with crystal violet for 10 minutes, and finally wash off the excess crystal violet stain with PBS, and observe the cell migration under an inverted microscope ( Figure 4 A), count the number of purple cells in each field of view, and calculate the effect of methotrexate carbon dots on tumor cell invasion ( Figure 4 B).

[0046] From the test results, it can be seen that the control group had the most tumor cells that invaded and migrated through the transwell bottom membrane, and had multi-tentacle structures. The TMZ group had about 10% fewer tumor cells that invaded and migrated through the transwell bottom membrane, and the cells had few tentacle structures. The CD3 group had the least tumor cells that invaded and migrated, a decrease of about 40%.

[0047] 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. 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 specific details.

Claims

1. A method for preparing carbon dots based on methotrexate, characterized in that: The method comprises: using raw materials including methotrexate, and synthesizing the methotrexate-based carbon dots through a one-pot hydrothermal method.

2. The method for preparing carbon dots based on methotrexate according to claim 1, characterized in that: The method comprises the following steps: S1, dissolving methotrexate in ultrapure water, dispersing by ultrasonication, transferring the resulting solution into a reaction kettle, and heating for hydrothermal reaction; S2. After the reaction is completed, the product is filtered, and the filtrate is dialyzed against ultrapure water through a dialysis membrane. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots.

3. The method for preparing carbon dots based on methotrexate according to claim 2, characterized in that: The temperature of the hydrothermal reaction in step S1 is 130-170° C., and the reaction time is 4-16 hours.

4. The method for preparing carbon dots based on methotrexate according to claim 3, characterized in that: Step S1 is specifically as follows: Dissolve 0.01-0.04 g of methotrexate in 15-60 mL of ultrapure water, disperse by ultrasonic for 5-20 minutes, transfer the resulting solution to a polytetrafluoroethylene-lined reactor, and heat the reaction at 130-170° C. for 4-16 hours.

5. The method for preparing carbon dots based on methotrexate according to claim 4, characterized in that: Step S2 is specifically as follows: After the reaction is completed, the product is filtered with a 0.2-0.3 μm filter membrane, and the filtrate is dialyzed with ultrapure water for 2-12 h through a dialysis membrane with a molecular cutoff of 600-1500 Da. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots.

6. The method for preparing carbon dots based on methotrexate according to claim 5, characterized in that: The method comprises the following steps: S1, dissolving 0.02 g of methotrexate in 29 mL of ultrapure water, ultrasonically dispersing for 10 minutes, transferring the resulting solution to a polytetrafluoroethylene-lined reactor, and heating at 150°C for 8 hours; S2. After the reaction is completed, the product is filtered with a 0.22 μm filter membrane, and the filtrate is dialyzed in ultrapure water for 2-12 h through a dialysis membrane with a molecular cutoff of 1000 Da. The dialysate is collected and freeze-dried to obtain methotrexate-based carbon dots, which are stored at 4°C.

7. A carbon dot based on methotrexate, characterized in that: It is prepared by the method according to any one of claims 1 to 6.

8. Use of the methotrexate-based carbon dots as claimed in claim 7 in tumor treatment.

9. Use of the methotrexate-based carbon dots as claimed in claim 7 in the preparation of drugs for treating tumors.

10. A tumor treatment composition, characterized in that: It comprises the carbon dots as claimed in claim 7 and a pharmaceutically acceptable adjuvant.

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