Leech carbon dots as well as preparation method and application thereof

Abstract

The invention discloses leech carbon dots and a preparation method and application thereof, the leech carbon dots are uniform in particle size and regular in morphology, and the leech carbon dots are obtained by taking leeches and pure water as raw materials through hydrothermal reaction, centrifugation, dialysis and freeze drying. The leech carbon dots disclosed by the invention can more easily penetrate through a blood brain barrier, and are low in toxicity, high in biological safety and good in anti-tumor effect. Compared with traditional chemotherapeutic drugs, the preparation cost is low, batch production is easy, and the action effect is strong. The traditional Chinese medicine composition can inhibit proliferation, migration and invasion of tumor cells, cross blood brain barrier and other tissue barriers, retard the cell cycle of glioblastoma, inhibit angiogenesis of glioblastoma, promote pyroptosis of glioblastoma and reverse lactic acid expression, and is combined with traditional chemotherapeutic drugs to enhance the curative effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a limulus carbon dot and a preparation method and application thereof. BACKGROUND

[0002] Brain tumors face great challenges in clinical treatment due to their high heterogeneity, extensive infiltration and resistance to conventional therapy. Among them, glioblastoma is one of the most common and most malignant tumors in the brain, and the prognosis of patients is extremely poor. The current standard treatment regimen is difficult to completely remove the infiltrating tumor cells, and the tumor cells are prone to reduce the problem of chemotherapy sensitivity, which seriously limits the survival benefit of patients, and new treatment technologies need to be developed urgently.

[0003] Existing nanomaterials such as liposomes are usually large in size, poor in tissue penetration, difficult to cross the blood-brain barrier to reach the deep part of brain tumor, and the exogenous carrier material may cause systemic toxicity. Carbon dots show great potential in disease treatment due to their ultra-small size, good biocompatibility and tissue barrier penetration capacity. However, most traditional carbon dots only act as passive drug carriers, which need complex surface modification, and have limitations such as drug leakage and potential carrier toxicity. In contrast, the full bioactive carbon dots derived from traditional Chinese medicine retain the pharmacological activity of the original material, increase the water solubility of the original material, and improve the bioavailability. As a carrier and a therapeutic agent, it has good clinical application prospect.

[0004] As a traditional Chinese medicine, limulus contains complex bioactive components such as amino acids, pteridines, sphingolipids and sterols. Limulus has been widely used to treat various diseases and can regulate various tumor-related pathways, including anti-angiogenesis, cell proliferation and immunomodulation. Although limulus has been applied in many diseases, its poor water solubility and low bioavailability limit its clinical application and fail to achieve ideal therapeutic effect. Especially for diseases with tissue barrier blockage, such as brain tumors, limulus components cannot effectively penetrate the blood-brain barrier to reach the tumor part to exert therapeutic effect. SUMMARY

[0005] In order to overcome the problems in the prior art, the application provides a limulus carbon dot and a preparation method and application thereof. The limulus carbon dot prepared by the application has low toxicity, high biological safety, good antitumor effect, and can easily penetrate the blood-brain barrier, and has broad application prospect in antitumor and combined chemotherapy drug enhanced efficacy.

[0006] To solve the above technical problems, the technical scheme provided by the application is as follows: The application provides a limulus carbon dot, which is prepared by preparing carbon quantum dots from limulus powder.

[0007] As an optional implementation form, in the leech carbon dots provided by the application, the excitation wavelength of the leech carbon dots is 300-400 nm, and the maximum emission wavelength is 400-600 nm.

[0008] As an optional implementation form, in the leech carbon dots provided by the application, the particle size of the leech carbon dots is 5.64±0.59 nm.

[0009] Based on the same technical concept, the application further provides a preparation method of leech carbon dots, comprising the following steps: S1, dissolving leech powder in pure water, and obtaining a reaction solution after hydrothermal reaction; S2, obtaining a clear solution by processing the reaction obtained in S1, and then obtaining leech carbon dots by dialysis, freezing and drying of the clear solution.

[0010] As an optional implementation form, in the preparation method provided by the application, in S1, the hydrothermal reaction conditions are first reacting at 120-140℃ for 5-10 min, and then reacting at 150-180℃ for 20-30 min.

[0011] As an optional implementation form, in the preparation method provided by the application, in S2, the processing of the reaction comprises centrifugation and filtration to remove the precipitate.

[0012] As an optional implementation form, in the preparation method provided by the application, the centrifugation conditions are 3500-4000 rpm, centrifugation for 10-15 min, and filtration using a 0.22 μm microporous filter membrane.

[0013] As an optional implementation form, in the preparation method provided by the application, in S2, a 500 kDa biological dialysis bag is used for dialysis, the dialysis time is 24-72 h, and then a 0.22 μm microporous filter membrane is used for filtration; the freezing temperature is-60--40℃, and the drying time is 12-24 h.

[0014] Based on the same technical concept, the application further provides the application of the above-mentioned leech carbon dots as a carrier reagent, and the leech carbon dots are used for loading substances to penetrate the blood-brain barrier as a carrier reagent.

[0015] Based on the same technical concept, the application further provides the application of the above-mentioned leech carbon dots as an anti-tumor reagent.

[0016] As an optional implementation form, in the application provided by the application, the tumor comprises glioblastoma or bladder urothelial carcinoma.

[0017] As an optional implementation, in the application provided by the application, the anti-tumor agent includes any one or several of the agents in (1)-(4) as follows: (1) an agent for blocking the cell cycle of glioblastoma; (2) an agent for inhibiting the angiogenesis of glioblastoma; (3) an agent for inducing pyroptosis of glioblastoma; (4) an agent for reversing the expression of lactic acid in glioblastoma.

[0018] Compared with the prior art, the application has the following beneficial effects: (1) The leech carbon dots in the application are prepared from medical leeches, and the solvent used in the preparation process is pure water without any organic solvent, so that the leech carbon dots prepared have good biocompatibility and low toxicity. The leech carbon dots can be prepared through simple hydrothermal synthesis, and the synthesis process is simple, thereby solving the problems of poor solubility and low bioavailability of leeches.

[0019] (2) The leech carbon dots prepared in the application mainly contain carbon, nitrogen and oxygen, and have an average particle size of 5.64±0.59 nm. The particle size range can smoothly pass through the tissue barrier, thereby solving the problem that the traditional medical leech dosage form cannot pass through the blood-brain barrier and other tissue barriers. When combined with traditional chemotherapeutic drugs, the leech carbon dots can enhance the anti-tumor effect.

[0020] (3) The leech carbon dots have a significant therapeutic effect on glioblastoma nude mice, can inhibit the cell cycle, reduce the proliferation, migration and invasion of tumor cells, and inhibit the angiogenesis in the tumor. As a tumor microenvironment regulator, the leech carbon dots can reduce the lactic acid content and reverse the tumor microenvironment. As a pyroptosis inducer, the leech carbon dots can promote pyroptosis of tumor cells and kill the tumor cells. Compared with traditional chemotherapeutic drugs, the leech carbon dots have low manufacturing cost, are easy to mass-produce, and have strong effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 Fig. 1 is a transmission electron microscope detection image and particle size distribution diagram of the leech carbon dots of the application, wherein a is the transmission electron microscope result, and b is the particle size distribution diagram; Figure 2 Fig. 2 is a fluorescence spectrum diagram of the leech carbon dots of the application; Figure 3 Fig. 3 is a Fourier infrared spectrum diagram of the leech carbon dots of the application; Figure 4 The X-ray electron spectroscopy detection results of the carbon dots of leeches according to the present invention are shown in the following: a is the X-ray photoelectron spectrum of carbon dots of leeches, b is the composition ratio of C, N and O; c is the 1s energy level spectrum of C, d is the 1s energy level spectrum of O, and e is the 1s energy level spectrum of N. Figure 5 The present invention describes the inhibitory effects of leech carbon dots on glioblastoma cells and bladder cancer cells, wherein a represents the result of leech carbon dots inhibiting the proliferation of T98-G cells, b represents the result of leech carbon dots inhibiting the proliferation of U251 cells, c represents the result of leech carbon dots inhibiting the proliferation of J82 cells, and d represents the result of leech carbon dots inhibiting the proliferation of T24 cells. Figure 6 The results show the effect of leech carbon dots on glioblastoma cell migration according to the present invention, where a represents the cell migration results at different concentrations and times, and b represents the statistical results of a. Figure 7 The results show the effect of leech carbon dots on glioblastoma cell invasion according to the present invention, where a is the cell infection result after 48 h of treatment, and b is the statistical result of a. Figure 8 The results of the blood-brain barrier permeability test of the leech carbon dots of the present invention are shown in Figure a, where Figure a is a schematic diagram of the in vitro blood-brain barrier model and Figure b is a permeability statistical chart. Figure 9 The results of the effect of leech carbon dots on the cell cycle of glioblastoma cells are shown in Figure a. Figure a is a cell cycle statistics of the control group, Figure b is a cell cycle statistics of the control group after intervention with leech carbon dots at a concentration of 62.5 μg / mL, Figure c is a cell cycle statistics of the control group after intervention with leech carbon dots at a concentration of 75 μg / mL, and Figure d is a cell cycle ratio diagram of each group. Figure 10 The results show the effect of leech carbon dots of the present invention on tumor angiogenesis in glioblastoma nude mice; Figure 11 The results show the effect of leech carbon dots on pyroptosis of glioblastoma cells according to the present invention, where a is a transmission electron microscope image and b is a pyroptosis index statistical graph. Figure 12 The results show the effect of leech carbon dots on lactate expression in glioblastoma according to the present invention. Figure 13 The results show the effects of the leech carbon dots of the present invention on the major organs of glioblastoma nude mice. Detailed Implementation

[0023] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all terms used in the description employed herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the following professional terms are intended to have the following meanings.

[0025] Unless otherwise specified, various materials, reagents, instruments and equipment and the like used in the present application can be purchased on the market or can be prepared by existing methods.

[0026] Example 1 A preparation method of a leech carbon dot, raw materials including medical leech and pure water, the preparation steps are as follows: (1) 1 g of medical leech powder was weighed and placed in a high-pressure reaction kettle, 30 mL of pure water was added, reacted at 120°C for 5 min, then reacted at 150°C for 20 min, and the reaction kettle was cooled to room temperature. The reaction liquid was collected.

[0027] (2) The reaction liquid was centrifuged at 4000 rpm for 10 min at room temperature to obtain a clear liquid, which was filtered with a 0.22 μm microporous filter. The filtrate was loaded into a 500 kDa biological dialysis bag, dialyzed for 24 h, and the water was changed every 8 h. The liquid in the dialysis bag was filtered with a 0.22 μm microporous filter, and the liquid was freeze-dried for 24 h. The freeze-dried powder was the carbon dot, which was stored at 4°C for standby.

[0028] Example 2 A preparation method of a leech carbon dot, raw materials including medical leech and pure water, the preparation steps are as follows: (1) 1 g of medical leech powder was weighed and placed in a high-pressure reaction kettle, 30 mL of pure water was added, reacted at 140°C for 10 min, then reacted at 160°C for 30 min, and the reaction kettle was cooled to room temperature. The reaction liquid was collected.

[0029] (2) The reaction liquid was centrifuged at 4000 rpm for 10 min at room temperature to obtain a clear liquid, which was filtered with a 0.22 μm microporous filter. The filtrate was loaded into a 500 kDa biological dialysis bag, dialyzed for 72 h, and the water was changed every 8 h. The liquid in the dialysis bag was filtered with a 0.22 μm microporous filter, and the liquid was freeze-dried for 24 h. The freeze-dried powder was the carbon dot, which was stored at 4°C for standby.

[0030] Example 3 A preparation method of a leech carbon dot, raw materials including medical leech and pure water, the preparation steps are as follows: (1) 1 g of medical Hirudo nipponia powder was placed in a high-pressure reaction kettle, 30 mL of pure water was added, and the reaction was carried out at 130°C for 8 min, and then at 180°C for 25 min. After the reaction kettle was cooled to room temperature, the reaction liquid was collected.

[0031] (2) The reaction liquid was centrifuged at 3500 rpm for 15 min at room temperature to obtain a clear liquid, which was filtered using a 0.22 μm microporous filter membrane. The filtrate was loaded into a 500 kDa biological dialysis bag, and dialysis was performed for 48 h, with water being replaced every 8 h. The liquid in the dialysis bag was filtered using a 0.22 μm microporous filter membrane, and the liquid was freeze-dried for 12 h to obtain a powder, which was the carbon dots, and was stored at 4°C for standby use.

[0032] The Hirudo nipponia carbon dots prepared in Example 1 were subjected to the following tests: (I) Physical performance test The morphology of the Hirudo nipponia carbon dots was observed using a transmission electron microscope, Figure 1 The transmission electron microscope characterization of the Hirudo nipponia carbon dots is shown in a and b. From Figure 1 It can be seen from a that the Hirudo nipponia carbon dots are uniformly dispersed and have regular shapes. From Figure 1 It can be seen from b that the average particle size of the Hirudo nipponia carbon dots is 5.64±0.59 nm.

[0033] The excitation wavelength and emission wavelength of the Hirudo nipponia carbon dots were characterized by fluorescence spectroscopy, and the results are shown in Figure 2 The maximum excitation wavelength of the Hirudo nipponia carbon dots is 367.8 nm, and the maximum emission wavelength is 438.8 nm, indicating that they have fluorescence properties. The Hirudo nipponia carbon dot solution is light yellow, and under the irradiation of a UV lamp with a wavelength of 365 nm, blue-green light can be observed with the naked eye.

[0034] Fourier transform infrared spectroscopy was used to analyze the detailed structural information of the surface functional groups of the Hirudo nipponia carbon dots, and the results are shown in Figure 3 It can be seen from Figure 3 that there are five relatively prominent absorption peaks. The wide absorption band at 3343.6 cm -1 is attributed to the stretching vibration of –NH and –OH; the absorption peak at 2933.3 cm -1 corresponds to the C–H stretching vibration. The absorption band near 1657.4 cm -1 is attributed to the amide I band, and the peak at 1542.6 cm -1 corresponds to the amide II band. The absorption peak at 1025.6 cm -1 is related to the stretching vibration of C–O and C–N. These results indicate that the surface of the Hirudo nipponia carbon dots is rich in hydroxyl groups, amides and other polar functional groups.

[0035] X-ray photoelectron spectroscopy was used to analyze the elemental composition of the leech carbon dots, and the results are shown in Figure 4 FIGS. a-b show that the leech carbon dots are mainly composed of carbon (64%), nitrogen (8%), and oxygen (28%). FIG. c shows the energy level spectrum of C1s, which has three characteristic peaks at 284.38, 285.78, and 287.28 eV, corresponding to C-C / C=C, C-N, and C=O bonds, respectively. FIG. d shows the energy spectrum of O1s, which has absorption peaks at 531.31 and 532.34 eV, corresponding to C=O and C-O bonds, respectively. FIG. e shows the energy level spectrum of N1s, which has two peaks at 399.93 and 400.79 eV, corresponding to pyrrole nitrogen and graphite nitrogen, respectively.

[0036] (II) Anti-tumor performance detection The leech carbon dots prepared in Example 1 were used in the inhibition of tumor cell proliferation, migration, and invasion experiments to verify their anti-tumor performance. The specific method is as follows: (1) Cell proliferation inhibition experiment: Well-grown cells (T98-G, U251, J82, T24) were seeded in a 96-well plate at 5000 cells per well, 100 μL of complete culture medium was added, and it was placed in a CO2 incubator overnight. A certain amount of leech carbon dots was dissolved in complete culture medium to prepare different concentrations of leech carbon dots (25-100 μg / mL) which were added to the 96-well plate and incubated for 48 h. 10 μL of CCK-8 reagent was added to each well, and it was incubated at 37°C in the dark for 30 min. The absorbance value at 450 nm was detected. The detection results are shown in Figure 5 .

[0037] As can be seen from Figure 5 , the leech carbon dots (SZ-CDs) have obvious effects on inhibiting the proliferation of glioblastoma cell lines (T98-G, U251) and bladder cancer cell lines (J82, T24) as the concentration increases.

[0038] (2) Tumor cell migration inhibition experiment: The effect of leech carbon dots on the migration of glioblastoma cells was detected by cell scratch experiment. In a 6-well plate containing glioblastoma cell line U251 cells, 200 μL of gun head was used to draw 3 straight lines. 62.5 and 75 μg / mL of leech carbon dots were prepared for intervention, and the same position of the scratch was photographed and analyzed at 0 h and 48 h after drug treatment. The detection results are shown in Figure 6 .

[0039] As can be seen from Figure 6 a, after 48 h of scratching, the migration rate of tumor cells after intervention with 75 μg / mL of leech carbon dots is slowed down. As shown in Figure 6The statistical results of b show that, compared with the control group, the leech carbon dots significantly inhibited the migration of U251 cells.

[0040] (3) Tumor cell invasion experiment: The effect of leech carbon dots on the invasion of glioma cells was detected by cell invasion experiment. Glioma cell line U251 cells were cultured in Transwell chamber, and observed and photographed by crystal violet staining. The detection results are shown in Figure 7 .

[0041] As can be seen from Figure 7 a, after 48 h of leech carbon dot treatment, the number of U251 cells invading from the upper chamber to the lower chamber was significantly reduced. As shown in Figure 7 b, compared with the control group, the leech carbon dots significantly inhibited the invasion of tumor cells.

[0042] (Three) Blood-brain barrier crossing performance The leech carbon dots prepared in Example 1 were subjected to blood-brain barrier permeability test to prove the application value of the leech carbon dots in the preparation of drugs crossing tissue barriers such as blood-brain barrier. The specific method is as follows: The blood-brain barrier model was constructed using brain microvascular endothelial cell line bEnd.3 cells, and the blood-brain barrier permeability of leech carbon dots was detected. When the resistance value between the upper chamber and the lower chamber of Transwell was greater than 150 Ω / cm 2 , the model was successfully constructed, and then temozolomide, a chemotherapy drug for brain glioma, and leech carbon dots were added to the upper chamber. After 4 h of incubation, the absorbance of the liquid in the lower chamber was detected. The detection results are shown in Figure 8 .

[0043] Figure 8 In the figure, a is a schematic diagram of the in vitro blood-brain barrier model, and b is a permeability statistical diagram. The blood-brain barrier permeability of temozolomide is 35.2%, and the blood-brain barrier permeability of leech carbon dots is 12.25%. The leech carbon dots have good blood-brain barrier permeability.

[0044] (Four) Drugs for blocking glioma cell cycle The leech carbon dots prepared in Example 1 were subjected to U251 cell cycle blocking effect test to prove the application value of the leech carbon dots in the preparation of drugs for blocking glioma cell cycle. The specific method is as follows: The effect of glioma cell U251 on cell cycle was detected by cell cycle detection kit, and the machine detection was performed on flow cytometer. The detection results are shown in Figure 9The cell cycle statistics of the control group is shown in a, the cell cycle statistics after the intervention of leech carbon dots with a concentration of 62.5 μg / mL is shown in b, the cell cycle statistics after the intervention of leech carbon dots with a concentration of 75 μg / mL is shown in c, and the cell proportion diagram of the cell cycle in each group is shown in d.

[0045] From Figure 9 As can be seen from d, compared with the control group, the cell cycle of the U251 cells treated with leech carbon dots was arrested in the S phase, thereby inhibiting cell proliferation.

[0046] (Five) Inhibition of glioblastoma angiogenesis The leech carbon dots prepared in Example 1 were subjected to an experiment on the effect of inhibiting glioblastoma angiogenesis to prove the application value of the leech carbon dots in the preparation of a drug for inhibiting glioblastoma angiogenesis, and the specific method was as follows: The immunofluorescence method was used to detect the angiogenesis in glioblastoma after the treatment of leech carbon dots and temozolomide. After the paraffin section was hydrated and dewaxed, sodium citrate buffer was used for antigen repair. After blocking for 1 h, the tumor section was added with CD31 primary antibody and incubated at 4°C overnight. Then, fluorescent secondary antibody was added for incubation, and a Zeiss Axio Imager M2 microscope was used for observation. The detection results are shown in Figure 10 .

[0047] CD31 is a glycoprotein rich in endothelial cells, which can reflect the angiogenesis of tumors. Figure 10 It can be shown that the expression of CD31 in the leech carbon dot group decreased significantly, and the angiogenesis decreased most obviously in the leech carbon dot + temozolomide combined administration group.

[0048] (Six) Preparation of pyroptosis inducer for treating glioblastoma The leech carbon dots prepared in Example 1 were subjected to an experiment on the effect of inducing pyroptosis of glioblastoma cells to prove the application value of the leech carbon dots in the preparation of a pyroptosis inducer for treating glioblastoma, and the specific method was as follows: Transmission electron microscopy was used to observe the pyroptosis morphology of glioblastoma cells. In the positive control group, the U251 cells were first treated with 1 μg / mL of lipopolysaccharide for 6 h, and then the medium containing 5 mM ATP was replaced and incubated for 1 h to induce pyroptosis. The leech carbon dot group was added with 75 μg / mL of leech carbon dots, and the control group was added with the same volume of PBS. After being fixed with electron microscope fixing solution, the samples were collected into centrifuge tubes and imaged by transmission electron microscopy. The cell morphology was observed by optical microscopy, and the pyroptosis index (pyroptotic cell number / total cell number) was calculated, and the results are shown in Figure 11 .

[0049] From Figure 11As can be seen, glioblastoma cells treated with leech carbon dots exhibited significant swelling, consistent with typical pyroptosis morphology, including cell membrane pore formation, cell swelling, and membrane rupture. Furthermore, the pyroptosis index was significantly increased in the leech carbon dot treatment group, indicating that leech carbon dots induced significant pyroptosis in glioblastoma cells.

[0050] (vii) Preparation of drugs to reverse lactate expression in glioblastoma The leech carbon dots prepared in Example 1 were subjected to an efficacy test to reverse lactate expression in glioblastoma, in order to demonstrate the application value of leech carbon dots in the preparation of drugs related to reversing lactate expression in glioblastoma. The specific method is as follows: Changes in lactate levels were detected in a nude mouse model of glioblastoma. Lactate levels were measured using a lactate assay kit, and absorbance was measured using a microplate reader. Results are shown below. Figure 12 As shown.

[0051] from Figure 12 As can be seen, in tumor tissue, the lactate level in the leech carbon dot treatment group was significantly reduced, and the lactate reduction was most significant in the leech carbon dot + temozolomide combined administration group.

[0052] (viii) Biosafety assessment The biosafety of the leech carbon dots prepared in Example 1 was assessed using the following method: The toxicity of leech carbon dots to organs was assessed by staining the heart, liver, spleen, lungs, and kidneys of glioblastoma-affected nude mice with hematoxylin and eosin staining. The results are as follows: Figure 13 As shown.

[0053] from Figure 13 As can be seen, the carbon spots of leeches did not cause abnormalities in major organs and had no obvious toxic side effects on organisms.

[0054] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A leech carbon dot, characterized in that, The leech powder is used to prepare carbon quantum dots.

2. The water mite carbon dots according to claim 1, characterized in that, The leech carbon dots have a particle size of 5.64±0.59 nm, an excitation wavelength of 300-400 nm, and a maximum emission wavelength of 400-600 nm.

3. Use of the water mite carbon dots as claimed in claim 1 or 2 as a carrier agent, characterized in that, The leech carbon dots are used as a carrier reagent to load substances to penetrate the blood-brain barrier.

4. Use of the leech carbon dots of claim 1 or 2 in the preparation of an anti-tumor reagent.

5. Use according to claim 4, characterized in that, The tumor includes glioblastoma or bladder urothelial carcinoma.

6. Use according to claim 4, characterized in that, The anti-tumor reagent includes any one or several of the reagents in (1)-(4) below: (1) an agent for blocking the cell cycle of glioblastoma; (2) an agent for inhibiting angiogenesis of glioblastoma; (3) a pyroptosis induction agent for glioblastoma; (4) an agent for reversing lactic acid expression in glioblastoma.

7. A method for preparing a watermoccasin carbon dot, characterized in that, The method includes the following steps: S1. Dissolving leech powder in pure water, and obtaining a reaction solution after hydrothermal reaction; S2. Obtaining a clear solution by treating the reaction solution obtained in S1, and then obtaining leech carbon dots by dialysis, freezing and drying of the clear solution.

8. The method for preparing leech carbon dots according to claim 7, characterized in that, In S1, the hydrothermal reaction conditions are first reacting at 120-140℃ for 5-10 min, and then reacting at 150-180℃ for 20-30 min.

9. The method for preparing leech carbon dots according to claim 7, characterized in that, In S2, the treatment of the reaction includes centrifugation and filtration to remove the precipitate; the centrifugation conditions are 3500-4000 rpm for 10-15 min.

10. The method for preparing leech carbon dots according to claim 7, characterized in that, In S2, a 500 kDa biological dialysis bag is used for dialysis, the dialysis time is 24-72 h, the freezing temperature is -60 to -40℃, and the drying time is 12-24 h.

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