Paclitaxel-based carbon dots, preparation method and application thereof, and brain glioma treatment drug

Abstract

The application discloses a kind of carbon dots based on paclitaxel, its preparation method, application and brain glioma treatment drug, the preparation method of the carbon dots includes the following steps: S1, paclitaxel is dissolved in ultrapure water, then ethanol solution is mixed, ultrasonic;S2, the mixed solution obtained in step S1 is transferred to reaction kettle, and heated reaction;S3, after reaction, the product is filtered with filter membrane, the filtrate is dialyzed, the solution obtained by dialysis is freeze-dried, to obtain the carbon dots.The carbon dots synthesized by one-pot hydrothermal method using paclitaxel have the advantages of good water solubility, low toxicity, strong penetration capacity, etc., have the ability of efficiently penetrating blood-brain barrier and killing brain glioma cells, can significantly inhibit the proliferation and migration of brain glioma cells, and the application can provide a feasible new strategy for developing new brain glioma treatment agents.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a paclitaxel-based carbon dot, its preparation method, application, and a drug for the treatment of glioma. Background Technology

[0002] Gliomas, the most common primary malignant intracranial tumors, account for approximately 80% of all brain malignancies and are a leading cause of death from primary brain tumors. Glioblastoma (GBM) is the most malignant type, with a median survival of only 14 months after standard surgery and adjuvant radiotherapy or chemotherapy, and a 5-year survival rate of less than 5%. Due to the highly invasive nature of glioblastomas, complete surgical removal is difficult. The effectiveness of radiotherapy is often limited by inherent radiation resistance and the hypoxic tumor microenvironment. The available drugs for the clinical treatment of GBM are also very limited; currently, only four chemotherapy drugs—lomustine, carmustine, temozolomide (TMZ), and bevacizumab—have been approved by the FDA for the clinical treatment of glioblastoma. These drugs have early potential for treating GBM patients, but long-term use may lead to decreased sensitivity or even drug resistance. The blood-brain barrier (BBB) ​​prevents almost all drugs from reaching the brain lesion site to exert their therapeutic effects, which is currently the main obstacle in the development of drugs for treating brain diseases. Therefore, there is an urgent need to develop drugs that can efficiently cross the blood-brain barrier (BBB) ​​and target and kill tumor cells.

[0003] Paclitaxel (PTX) is a widely used anticancer drug for breast cancer, ovarian cancer, and many other cancers. It works by binding to tubulin in cells, stabilizing microtubule structure, inhibiting cell division, and leading to cell death. Data shows that PTX's inhibitory effect on GBM cells is 1400 times that of TMZ. However, PTX has poor water solubility and cannot cross the blood-brain barrier (BBB), limiting its application in GBM treatment. Clinically, hydrogenated castor oil and anhydrous ethanol are required as carriers. Currently, liposomes have been developed that encapsulate paclitaxel by forming a cell membrane phospholipid bilayer structure from lecithin and cholesterol in a specific ratio. Albumin-based paclitaxel has also been prepared by using high-pressure vibration technology to create nanoparticles from paclitaxel and human serum albumin. This nanoparticles bind to the albumin receptor Gp60 on the cell membrane and utilize the action of cysteine-rich acidic secretory protein (SPARC) in tumor tissue to promote drug entry into tumor cells, increasing the efficacy of chemotherapy.

[0004] Existing methods using hydrogenated castor oil and anhydrous ethanol as paclitaxel carriers can cause serious adverse reactions, such as allergies, nephrotoxicity, and neurotoxicity, limiting its clinical application. Paclitaxel liposomes can significantly reduce toxic side effects, but some individuals may experience allergic reactions, requiring pre-treatment for allergies before use. Albumin-bound paclitaxel has advantages such as safety, non-toxicity, non-immunogenicity, biodegradability, and good biocompatibility. However, its ability to cross the blood-brain barrier and its efficacy in treating gliomas require further investigation.

[0005] Carbon dots (CDs) are an emerging type of carbon-based nanomaterial with advantages such as low toxicity, strong penetrability, ease of modification, and stable physicochemical properties, showing great promise for applications in the fields of therapy and drug delivery. Utilizing paclitaxel to synthesize carbon dot materials for application holds promise for solving the aforementioned application challenges of paclitaxel, but a reliable method is currently lacking. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a paclitaxel-based carbon dot, its preparation method, its application, and a drug for the treatment of glioma.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing carbon dots based on paclitaxel, wherein the method is: using raw materials including paclitaxel, carbon dots based on paclitaxel are synthesized by a one-pot hydrothermal method.

[0008] Preferably, the method includes the following steps:

[0009] S1. Dissolve paclitaxel in ultrapure water, then add ethanol solution and mix, followed by sonication;

[0010] S2. Transfer the mixture obtained in step S1 to a reaction vessel and heat it to react.

[0011] S3. After the reaction is complete, the product is filtered through a filter membrane, the filtrate is dialyzed, and the dialyzed solution is freeze-dried to obtain the carbon dots.

[0012] Preferably, step S1 specifically involves dissolving 0.01-0.04g of paclitaxel in 14-38mL of ultrapure water, adding 2.5-10mL of ethanol solution, mixing, and sonicating for 5-20 minutes.

[0013] Preferably, step S2 specifically involves transferring the mixture obtained in step S1 into a reaction vessel lined with polytetrafluoroethylene and heating it at 140-170°C for 4-16 hours.

[0014] Preferably, step S3 specifically involves: after the reaction is complete, the product is filtered through a 0.1-0.4 μm filter membrane, the filtrate is dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 800-1200 Da, and the solution in the dialysis bag is freeze-dried to obtain the carbon dots.

[0015] Preferably, the method includes the following steps:

[0016] S1. Dissolve 0.02g of paclitaxel in 29mL of ultrapure water, add 5mL of ethanol solution, mix, and sonicate for 10 minutes;

[0017] S2. Transfer the mixture obtained in step S1 to a reaction vessel lined with polytetrafluoroethylene, and heat it in an oven at 150°C for 8 hours.

[0018] S3. After the reaction is complete, the product is filtered through a 0.22 μm filter membrane. The filtrate is dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 1000 Da. The solution in the dialysis bag is then freeze-dried to obtain the carbon dots.

[0019] The present invention also provides a paclitaxel-based carbon dot, which is prepared by the method described above.

[0020] The present invention also provides an application of the carbon dots described above in the treatment of glioma.

[0021] The present invention also provides the application of the carbon dots described above in the preparation of drugs for the treatment of glioma.

[0022] The present invention also provides a glioma treatment medicament comprising the carbon dots as described above and other pharmaceutically acceptable components.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a paclitaxel-based carbon dot and its application. The carbon dot synthesized from paclitaxel using a one-pot hydrothermal method has advantages such as good water solubility, low toxicity, and strong penetration ability. It has the ability to efficiently cross the blood-brain barrier and kill glioma cells, and can significantly inhibit the proliferation and migration of glioma cells. This invention can provide a feasible new strategy for developing novel glioma therapeutics. Attached Figure Description

[0025] Figure 1 Transmission electron microscopy (TEM) image of the carbon dots prepared in Example 1;

[0026] Figure 2 The results of the inhibition rate test of the carbon dots prepared in Example 1 on tumor cells;

[0027] Figure 3 The test results show that the carbon dots prepared in Example 1 inhibited the migration of tumor cells.

[0028] Figure 4 The test results show that the carbon dots prepared in Example 1 inhibited tumor cell invasion. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0032] Example 1

[0033] A carbon dot based on paclitaxel, the preparation method of which includes the following steps:

[0034] S1. Dissolve 0.02g of paclitaxel in 29mL of ultrapure water, add 5mL of ethanol solution, mix, and sonicate for 10 minutes;

[0035] S2. Transfer the mixture obtained in step S1 to a reaction vessel lined with polytetrafluoroethylene, and heat it in an oven at 150°C for 8 hours.

[0036] S3. After the reaction is complete, the product is filtered through a 0.22 μm filter membrane to remove large particulate impurities. The filtrate is then dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 1000 Da to remove unreacted raw materials. The solution in the dialysis bag is then freeze-dried to obtain carbon dots, which are recorded as paclitaxel carbon dots and stored in a refrigerator at 4°C for later use.

[0037] Performance testing:

[0038] I. Transmission electron microscopy imaging of paclitaxel carbon dots

[0039] (1) Sample preparation: Add 10 μL of paclitaxel carbon dot solution to a copper mesh (sample mesh) with a carbon film on its surface. Then cover the sample mesh with a petri dish and place it at room temperature until the sample dries naturally. Carefully rinse the sample mesh with the added paclitaxel carbon dots with deionized water, then rinse with ethanol, and finally blot off the excess ethanol with filter paper.

[0040] (2) Sample Imaging: Turn on the transmission electron microscope and measure the carbon dot sample. Select an appropriate magnification to ensure a large number of nanoparticles appear in the field of view, guaranteeing that at least 200 nanoparticles are observed in different fields of view. Repeat the above steps, selecting different fields of view to image the carbon dot sample. The results are as follows: Figure 1 As shown, the results indicate that the particle size of paclitaxel carbon dots is less than 10 nm, which is beneficial for the transport and bioavailability of paclitaxel carbon dots in vivo.

[0041] (II) Tests of paclitaxel carbon dots inhibiting tumor cell proliferation

[0042] 8000-10000 cells were reselected and seeded into 100 μl of medium in 96-well cell culture plates. Incubation was carried out overnight at 37°C. The cell culture medium was discarded and replaced with medium containing 200 mg / mL, 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, and 6.125 mg / mL carbon dots, respectively, with three replicates for each concentration. Incubation continued for 24-48 hours. The cell culture medium in each well was then replaced with 10 μl of WST-1, and incubated at 37°C for 1-4 hours. The absorbance at 450 nm was measured in each well. Blank wells (cell-free, with medium and WST-1 solution) and control wells (cells present, without drug, with medium and WST-1 solution) were also included. The inhibitory rate of the carbon dots on tumor cells was calculated using the following formula:

[0043] Inhibition rate = [1-(OD)] 实验组 -OD 空白孔 ) / (OD 对照组 -OD empty 白孔 )]×100%;

[0044] Using temozolomide (TMZ), a first-line chemotherapy drug currently used in clinical practice for the treatment of glioma, as a control, the inhibitory effects of different concentrations of carbon dots (CD2) on the glioma cell line U251 are as follows: Figure 2 As shown in the figure, it can be seen that it has a significant inhibitory effect on the proliferation of glioma cells.

[0045] (II) Tests on the inhibition of tumor cell migration and invasion by paclitaxel carbon dots

[0046] (1) U251 cells were seeded into 24-well plates, culture medium was added, and the plates were incubated at 37°C. When cell confluence reached 90%, a 100 μL pipette tip was used to draw a straight line down the center of each well to create a scratch, and the cells were removed. The culture medium was replaced with a medium containing carbon dots or fresh conventional cell culture medium. Every 4-6 hours, photos were taken to record the migration of cells from both sides of the scratch towards the center. ImageJ software was used to calculate the inhibition of cell migration by paclitaxel carbon dots. The test results are shown below. Figure 3As shown in A and 3B.

[0047] (2) Collect U251 cells from T25 culture flasks with a cell confluence of 90% or higher, and prepare 5×10⁵ cells using culture media with or without added carbon dots. 5 A cell suspension with a concentration of [number] cells / mL was prepared. 200 μL of the cell suspension was added to a transwell coated with Matrigel. 600 μL of culture medium was added to each well of a 24-well plate, and the transwell was placed into the cell culture well. After 48 hours, the transwell was removed, and the cells inside the chamber were scraped off with a cotton swab. The cells were then fixed with 4% PFA for 30 minutes. After washing three times with PBS, crystal violet staining was performed for 10 minutes. Finally, excess crystal violet staining was washed away with PBS. Cell migration was observed under an inverted microscope, and the number of purple cells in each field of view was counted. The effect of carbon dots on cell invasion was calculated. Figure 4 (A and 4B).

[0048] The test results show that paclitaxel carbon dots can effectively inhibit the migration and invasion of tumor cells.

[0049] Example 2

[0050] Application of carbon dots in the treatment of glioma according to Example 1.

[0051] Example 3

[0052] Application of carbon dots from Example 1 in the preparation of a drug for treating glioma.

[0053] Example 4

[0054] A glioma treatment drug comprising the carbon dots of Example 1 and other pharmaceutically acceptable components.

[0055] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A carbon dot based on paclitaxel, characterized in that, The method for preparing carbon dots based on paclitaxel includes the following steps: S1. Dissolve paclitaxel in ultrapure water, then add ethanol solution and mix, followed by sonication; S2. Transfer the mixture obtained in step S1 to a reaction vessel and heat it to react. S3. After the reaction is complete, the product is filtered through a filter membrane, the filtrate is dialyzed, and the dialyzed solution is freeze-dried to obtain the carbon dots.

2. The paclitaxel-based carbon dots according to claim 1, characterized in that, Step S1 is as follows: Dissolve 0.01-0.04g of paclitaxel in 14-38mL of ultrapure water, add 2.5-10mL of ethanol solution, mix, and sonicate for 5-20 minutes.

3. The paclitaxel-based carbon dots according to claim 1, characterized in that, Step S2 specifically involves transferring the mixture obtained in step S1 into a reaction vessel lined with polytetrafluoroethylene and heating it at 140-170°C for 4-16 hours.

4. The paclitaxel-based carbon dots according to claim 1, characterized in that, Step S3 specifically involves: after the reaction is complete, the product is filtered through a 0.1-0.4 μm filter membrane, and the filtrate is dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 800-1200 Da. The solution in the dialysis bag is then freeze-dried to obtain the carbon dots.

5. The paclitaxel-based carbon dots according to claim 1, characterized in that, The method for preparing carbon dots based on paclitaxel includes the following steps: S1. Dissolve 0.02g of paclitaxel in 29 mL of ultrapure water, add 5 mL of ethanol solution, mix, and sonicate for 10 minutes; S2. Transfer the mixture obtained in step S1 to a reaction vessel lined with polytetrafluoroethylene, and heat it in an oven at 150°C for 8 hours. S3. After the reaction is complete, the product is filtered through a 0.22 μm filter membrane. The filtrate is dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 1000 Da. The solution in the dialysis bag is then freeze-dried to obtain the carbon dots.

6. The use of carbon dots as described in any one of claims 1-5 in the preparation of glioma treatment drugs.

7. A drug for treating glioma, characterized in that, It includes carbon dots as described in any one of claims 1-5, as well as other pharmaceutically acceptable components.

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