Hirudo nipponia carbon dots and preparation method and application thereof

Abstract

This invention discloses a leech carbon dot, its preparation method, and its application. The leech carbon dots have uniform particle size and regular morphology. They are obtained from leeches and pure water through hydrothermal reaction, centrifugation, dialysis, and freeze-drying. The leech carbon dots of this invention can more easily penetrate the blood-brain barrier, have low toxicity, high biosafety, and good anti-tumor effects. Compared with traditional chemotherapy drugs, they have lower production costs, are easier to mass-produce, and have stronger effects. They can inhibit tumor cell proliferation, migration, and invasion, cross tissue barriers such as the blood-brain barrier, arrest the cell cycle of glioblastoma, inhibit angiogenesis in glioblastoma, promote pyroptosis in glioblastoma, reverse lactate expression, and enhance the efficacy when used in combination with traditional chemotherapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a leech carbon dot, its preparation method, and its application. Background Technology

[0002] Brain tumors pose significant challenges to clinical treatment due to their high heterogeneity, widespread invasiveness, and resistance to conventional therapies. Glioblastoma, in particular, is one of the most common and malignant brain tumors, with extremely poor prognoses. Current standard treatments struggle to completely eliminate invasive tumor cells, and the reduced sensitivity of these cells to chemotherapy severely limits patient survival benefits, necessitating the development of new treatment technologies.

[0003] Existing nanomaterials, such as liposomes, are typically large in size and have poor tissue penetration, making it difficult to cross the blood-brain barrier and reach deep into brain tumors. Furthermore, exogenous carrier materials may cause systemic toxicity. Carbon dots, due to their ultra-small size, good biocompatibility, and ability to penetrate tissue barriers, show great potential in disease treatment. However, most traditional carbon dots serve only as passive drug carriers, requiring complex surface modifications and suffering from limitations such as drug leakage and potential carrier toxicity. In contrast, fully bioactive carbon dots derived from traditional Chinese medicine retain the pharmacological activity of the raw materials, increase their water solubility, and improve bioavailability. Serving as both a carrier and a therapeutic agent, they show promising clinical application prospects.

[0004] Leeches, a traditional Chinese medicine, contain complex bioactive components such as amino acids, pteridine, sphingolipids, and sterols. Leeches have been widely used to treat various diseases and can modulate multiple tumor-related pathways, including anti-angiogenesis, cell proliferation, and immune regulation. Although leeches have been used in many diseases, their poor water solubility and low bioavailability limit their clinical application and have not yielded ideal therapeutic effects. This is especially true for diseases with tissue barriers, such as brain tumors, where leech components cannot effectively cross the blood-brain barrier to reach the tumor and exert their therapeutic effects. Summary of the Invention

[0005] To overcome the problems in the prior art, the present invention provides a leech carbon dot, its preparation method and application. The leech carbon dot prepared by the present invention has low toxicity, high biosafety, good anti-tumor effect, and can easily penetrate the blood-brain barrier. It has broad application prospects in anti-tumor treatment and in enhancing the efficacy of combined chemotherapy drugs.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] This invention provides a leech carbon dot, which is prepared by using leech powder through a method for preparing carbon quantum dots.

[0008] As an optional implementation, in the leech carbon dots provided by the present invention, the excitation wavelength of the leech carbon dots is 300-400 nm, and the maximum emission wavelength is 400-600 nm.

[0009] As an optional implementation, in the leech carbon dots provided by the present invention, the particle size of the leech carbon dots is 5.64±0.59 nm.

[0010] Based on the same technical concept, the present invention also provides a method for preparing leech carbon dots, comprising the following steps:

[0011] S1. Dissolve leech powder in pure water and perform a hydrothermal reaction to obtain a reaction solution;

[0012] S2. The reaction obtained in S1 is processed to obtain a clear solution. Then, the clear solution is dialyzed, frozen and dried to obtain leech carbon dots.

[0013] As an optional implementation method, in the preparation method provided by the present invention, in S1, the hydrothermal reaction conditions are to first react at 120-140°C for 5-10 min, and then react at 150-180°C for 20-30 min.

[0014] As an optional implementation, in the preparation method provided by the present invention, in step S2, the reaction treatment includes centrifugation and filtration to remove precipitates.

[0015] As an optional implementation method, in the preparation method provided by the present invention, the centrifugation conditions are 3500-4000 rpm, centrifugation for 10-15 min, and filtration using a 0.22 μm microporous membrane.

[0016] As an optional implementation, in the preparation method provided by the present invention, in S2, a 500 kDa biodialysis bag is used for dialysis, the dialysis time is 24 to 72 h, and then filtration is performed using a 0.22 μm microporous membrane; the freezing temperature is -60 to -40 °C, and the drying time is 12 to 24 h.

[0017] Based on the same technical concept, the present invention also provides the application of the above-mentioned leech carbon dots as a carrier reagent, wherein the leech carbon dots are used as a carrier reagent to load substances to penetrate the blood-brain barrier.

[0018] Based on the same technical concept, the present invention also provides the application of the above-mentioned leech carbon dots as an antitumor agent.

[0019] As an alternative implementation, in the application provided by the present invention, the tumor includes glioblastoma or urothelial carcinoma of the bladder.

[0020] As an optional implementation, in the application provided by the present invention, the antitumor reagent includes any one or more of the reagents listed in (1) to (4) below:

[0021] (1) Reagents for blocking the cell cycle of glioblastoma cells;

[0022] (2) Reagents for drugs that inhibit angiogenesis in glioblastoma;

[0023] (3) Pyroptosis induction reagent for glioblastoma;

[0024] (4) Reagent to reverse lactate expression in glioblastoma.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The leech carbon dots in this invention use medical leeches as raw materials. The solvent used in the preparation process is pure water, without any organic solvents. The leech carbon dots prepared have good biocompatibility and low toxicity. They can be prepared by simple hydrothermal synthesis. The synthesis steps are simple and solve the problems of poor solubility and low bioavailability of leeches.

[0027] (2) The leech carbon dots prepared in this invention are mainly composed of carbon, nitrogen, and oxygen, with an average particle size of 5.64±0.59 nm. This particle size range can easily pass through tissue barriers, solving the problem that traditional medical leech formulations have difficulty passing through tissue barriers such as the blood-brain barrier. When used in combination with traditional chemotherapy drugs, it enhances the anti-tumor effect.

[0028] (3) The leech carbon dots of the present invention have a significant therapeutic effect on glioblastoma nude mice, inhibiting the cell cycle, reducing tumor cell proliferation, migration, and invasion, and inhibiting angiogenesis within the tumor. As a tumor microenvironment regulator, it reduces lactic acid content and reverses the tumor microenvironment. As a pyroptosis inducer, it promotes tumor cell pyroptosis and kills tumor cells; compared with traditional chemotherapy drugs, it has low production cost, is easy to mass-produce, and has a strong effect. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The images shown are transmission electron microscope (TEM) images and particle size distribution diagrams of carbon dots in leeches according to the present invention, where a is the TEM result and b is the particle size distribution diagram.

[0031] Figure 2 The fluorescence spectrum of the leech carbon dots of the present invention;

[0032] Figure 3 This is the Fourier transform infrared spectrum of the carbon dots in the leech of the present invention;

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Figure 10 The results show the effect of leech carbon dots of the present invention on tumor angiogenesis in glioblastoma nude mice;

[0040] 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.

[0041] Figure 12The results show the effect of leech carbon dots on lactate expression in glioblastoma according to the present invention;

[0042] Figure 13 The results show the effects of the leech carbon dots of the present invention on the major organs of glioblastoma-bearing nude mice. Detailed Implementation

[0043] 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.

[0044] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0045] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0046] Example 1

[0047] A method for preparing leech carbon dots, the raw materials including medical leeches and pure water, and the preparation steps are as follows:

[0048] (1) Weigh 1 g of medical leech powder, place it in a high-pressure reactor, add 30 mL of pure water, react at 120°C for 5 min, then react at 150°C for 20 min, and collect the reaction solution after the reactor has cooled to room temperature.

[0049] (2) The reaction solution was centrifuged at 4000 rpm for 10 min at room temperature to obtain a clear liquid. The liquid was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected. The filtrate was placed in a 500 kDa biodialysis bag and dialyzed for 24 h, with the water changed every 8 h. The liquid in the dialysis bag was filtered through a 0.22 μm microporous membrane, and the collected liquid was freeze-dried for 24 h. The freeze-dried powder was the carbon point, which was stored at 4 °C for later use.

[0050] Example 2

[0051] A method for preparing leech carbon dots, the raw materials including medical leeches and pure water, and the preparation steps are as follows:

[0052] (1) Weigh 1 g of medical leech powder, place it in a high-pressure reactor, add 30 mL of pure water, react at 140°C for 10 min, then react at 160°C for 30 min, and collect the reaction solution after the reactor has cooled to room temperature.

[0053] (2) The reaction solution was centrifuged at 4000 rpm for 10 min at room temperature to obtain a clear liquid. The liquid was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected. The filtrate was placed in a 500 kDa biodialysis bag and dialyzed for 72 h, with the water changed every 8 h. The liquid in the dialysis bag was then filtered through a 0.22 μm microporous membrane, and the collected liquid was freeze-dried for 24 h. The freeze-dried powder was the carbon point, which was stored at 4 °C for later use.

[0054] Example 3

[0055] A method for preparing leech carbon dots, the raw materials including medical leeches and pure water, and the preparation steps are as follows:

[0056] (1) Weigh 1 g of medical leech powder, place it in a high-pressure reactor, add 30 mL of pure water, react at 130°C for 8 min, then react at 180°C for 25 min, and collect the reaction solution after the reactor has cooled to room temperature.

[0057] (2) The reaction solution was centrifuged at 3500 rpm for 15 min at room temperature to obtain a clear liquid. The liquid was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected. The filtrate was placed in a 500 kDa biodialysis bag and dialyzed for 48 h, with the water changed every 8 h. The liquid in the dialysis bag was then filtered through a 0.22 μm microporous membrane, and the collected liquid was freeze-dried for 12 h. The freeze-dried powder was the carbon point, which was stored at 4 °C for later use.

[0058] The following tests were performed on the leech carbon dots obtained in Example 1:

[0059] (a) Physical performance testing

[0060] The morphology of carbon dots in leeches was observed using transmission electron microscopy. Figure 1 Transmission electron microscopy (TEM) characterization of carbon dots in leeches; a) TEM results, b) particle size distribution. Figure 1 As can be seen in image a, the carbon dots of the leech are evenly dispersed and have regular shapes. From Figure 1 As can be seen in b, the average particle size of the leech carbon dots is 5.64 ± 0.59 nm.

[0061] The excitation and emission wavelengths of the carbon dots in leeches were characterized using fluorescence spectroscopy, and the results are as follows: Figure 2 As shown, the maximum excitation wavelength of leech carbon dots is 367.8 nm, and the maximum emission wavelength is 438.8 nm, indicating that they possess fluorescent properties. The leech carbon dot solution is pale yellow, and under ultraviolet light irradiation at a wavelength of 365 nm, it emits blue-green light visible to the naked eye.

[0062] Fourier transform infrared spectroscopy analysis was used to analyze the detailed structural information of the functional groups on the carbon dots surface of leeches. The results are as follows: Figure 3 As shown. From Figure 3Five relatively significant absorption peaks can be observed. (3343.6 cm⁻¹) -1 The broad absorption band at 2933.3 cm⁻¹ is attributed to the stretching vibrations of –NH and –OH. -1 The absorption peak corresponds to the C–H stretching vibration. 1657.4 cm⁻¹ -1 The nearby absorption band is attributed to the amide I band, while 1542.6 cm⁻¹ -1 The peak corresponds to the amide II band. 1025.6 cm⁻¹ -1 The absorption peaks are correlated with the stretching vibrations of C–O and C–N. These results indicate that the surface of leech carbon dots is rich in hydroxyl, amide, and other polar functional groups.

[0063] X-ray photoelectron spectroscopy analysis of the elemental composition of carbon dots in leeches yielded the following results: Figure 4 As shown in the figures. Figures a-b show that the carbon dots of leeches are mainly composed of carbon (64%), nitrogen (8%), and oxygen (28%). Figure c shows the energy level spectrum of C1s, with 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. Figure d shows the energy level spectrum of O1s, with absorption peaks at 531.31 and 532.34 eV, corresponding to C=O and C–O bonds, respectively. Figure e shows the energy level spectrum of N1s, with two peaks at 399.93 and 400.79 eV, corresponding to pyrrole nitrogen and graphitic nitrogen, respectively.

[0064] (II) Antitumor performance testing

[0065] The leech carbon dots prepared in Example 1 were used to inhibit tumor cell proliferation, migration, and invasion to verify their antitumor properties. The specific method is as follows:

[0066] (1) Cell proliferation inhibition experiment:

[0067] Healthy cell lines (T98-G, U251, J82, T24) were seeded in 96-well plates at 5000 cells per well, and 100 μL of complete culture medium was added. The plates were then incubated overnight in a CO2 incubator. Different concentrations of leech carbon dots (25-100 μg / mL) were prepared by dissolving them in the complete culture medium and adding them to the 96-well plates, incubating for 48 h. 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 30 min. The absorbance at 450 nm was measured. The results are shown below. Figure 5 As shown.

[0068] from Figure 5As can be seen, leech carbon dots (SZ-CDs) have a significant inhibitory effect on cell proliferation in glioblastoma cell lines (T98-G, U251) and bladder cancer cell lines (J82, T24) as the concentration increases.

[0069] (2) Experiment on inhibiting tumor cell migration:

[0070] The effect of leech carbon dots on glioblastoma cell migration was investigated using a cell scratch assay. Three straight lines were drawn in 6-well plates containing the U251 glioblastoma cell line using a 200 μL pipette tip. Leech carbon dots at concentrations of 62.5 and 75 μg / mL were prepared for intervention. Images of the same location on the scratches were taken and analyzed at 0 h and 48 h after treatment. The results are shown below. Figure 6 As shown.

[0071] from Figure 6 As shown in Figure a, 48 h after scratching, the tumor cell migration rate slowed down after intervention with 75 μg / mL leech carbon dots. Figure 6 The statistical results of b show that, compared with the control group, leech carbon dots significantly inhibited the migration of U251 cells.

[0072] (3) Tumor cell invasion experiment:

[0073] The effect of leech carbon dots on glioblastoma cell invasion was investigated using a cell invasion assay. U251 glioblastoma cell line was cultured in Transwell chambers, and observation and photography were performed using crystal violet staining. The results are as follows: Figure 7 As shown.

[0074] from Figure 7 As shown in Figure 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. Figure 7 According to the statistics of b, compared with the control group, the carbon dots of leeches significantly inhibited the invasion of tumor cells.

[0075] (III) Performance across the blood-brain barrier

[0076] The leech carbon dots prepared in Example 1 were subjected to a blood-brain barrier permeability test to demonstrate their application value in the preparation of drugs that cross tissue barriers such as the blood-brain barrier. The specific method is as follows:

[0077] An in vitro blood-brain barrier model was constructed using the bEnd.3 brain microvascular endothelial cell line, and the blood-brain barrier permeability of leech carbon dots was detected. When the resistance between the upper and lower chambers of the Transwell was greater than 150 Ω / cm... 2At this point, the model was successfully constructed. Then, temozolomide, a chemotherapy drug for glioblastoma, and leech carbon dots were added to the upper chamber, respectively. After incubation for 4 hours, the absorbance of the liquid in the lower chamber was measured. The results are as follows: Figure 8 As shown.

[0078] Figure 8 Figure a shows a schematic diagram of the in vitro blood-brain barrier model, and Figure b shows a statistical chart of permeability. Temozolomide has a blood-brain barrier permeability of 35.2%, while leech carbon dots have a blood-brain barrier permeability of 12.25%. Leech carbon dots exhibit good blood-brain barrier permeability.

[0079] (iv) Drugs that block the cell cycle of glioblastoma

[0080] The leech carbon dots prepared in Example 1 were used to test their cell cycle arrest effect on U251 cells to demonstrate their application value in the preparation of drugs for arresting the cell cycle of glioblastoma. The specific method is as follows:

[0081] The effect of cell cycle detection kits on the cell cycle of glioblastoma cells U251 was used to detect the effects on cell cycle, and the results were analyzed by flow cytometry. The results are as follows: Figure 9 As shown in the figure. Figure a is the cell cycle statistics of the control group, Figure b is the cell cycle statistics after intervention with leech carbon dots at a concentration of 62.5 μg / mL, Figure c is the cell cycle statistics after intervention with leech carbon dots at a concentration of 75 μg / mL, and Figure d is the cell ratio of cell cycle in each group.

[0082] from Figure 9 As can be seen in d, compared with the control group, the cell cycle of U251 cells treated with leech carbon dots was arrested in the S phase, thereby inhibiting cell proliferation.

[0083] (v) Inhibition of angiogenesis in glioblastoma

[0084] The leech carbon dots prepared in Example 1 were subjected to an inhibitory effect test on glioblastoma angiogenesis to demonstrate the application value of leech carbon dots in the preparation of drugs for inhibiting glioblastoma angiogenesis. The specific method is as follows:

[0085] Immunofluorescence was used to detect carbon dots from leeches and angiogenesis in glioblastomas after temozolomide treatment. Paraffin sections were dewaxed by hydration and then subjected to antigen retrieval using sodium citrate buffer. After blocking for 1 h, tumor sections were incubated overnight at 4°C with CD31 primary antibody. Subsequently, fluorescent secondary antibody was added, and the sections were observed using a Zeiss Axio Imager M2 microscope. The results are shown below. Figure 10 As shown.

[0086] CD31 is a glycoprotein enriched in endothelial cells that can reflect tumor angiogenesis. Figure 10 The results showed that CD31 expression was significantly decreased in the leech carbon dot group, and the decrease in angiogenesis was most significant in the leech carbon dot + temozolomide combined administration group.

[0087] (vi) Preparation of pyroptosis inducers for the treatment of glioblastoma

[0088] The leech carbon dots prepared in Example 1 were subjected to a pyroptosis induction experiment on glioblastoma cells to demonstrate the application value of leech carbon dots in the preparation of pyroptosis-inducing drugs for the treatment of glioblastoma. The specific method is as follows:

[0089] The pyroptosis morphology of glioblastoma cells was observed using transmission electron microscopy. In the positive control group, U251 cells were treated with 1 μg / mL lipopolysaccharide for 6 h, followed by incubation in medium containing 5 mM ATP for another 1 h to induce pyroptosis. The leech carbon dot group received 75 μg / mL leech carbon dots, while the control group received an equal volume of PBS. Cells were fixed with electron microscopy fixative and collected in centrifuge tubes for imaging under a transmission electron microscope. Cell morphology was observed using an optical microscope, and the pyroptosis index (number of pyroptotic cells / total number of cells) was calculated. The results are shown below. Figure 11 As shown in the figure. Figure a is a transmission electron microscope photograph, and Figure b is a pyrolysis index statistical chart.

[0090] from Figure 11 As 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.

[0091] (vii) Preparation of drugs to reverse lactate expression in glioblastoma

[0092] 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:

[0093] Changes in lactate levels were detected in a glioblastoma nude mouse model. 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.

[0094] from Figure 12 As can be seen, in tumor tissue, the lactate level in the leech carbon dot treatment group decreased significantly, and the lactate decrease was most significant in the leech carbon dot + temozolomide combined administration group.

[0095] (viii) Biosafety assessment

[0096] The biosafety of the leech carbon dots prepared in Example 1 was assessed using the following method:

[0097] 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.

[0098] 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.

[0099] 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 inventive concept, 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 carbon quantum dots were prepared by dissolving leech powder in pure water and then reacting the powder with water. The carbon quantum dots have anti-tumor activity. The particle size of the carbon quantum dots is 5.64±0.59 nm. The excitation wavelength of the carbon quantum dots is 300-400 nm, and the maximum emission wavelength is 400-600 nm.

2. The application of the leech carbon dots as a carrier reagent according to claim 1, characterized in that, The leech carbon dots are used as carrier reagents to load substances that penetrate the blood-brain barrier.

3. The use of the leech carbon dots according to claim 1 in the preparation of antitumor reagents.

4. The application according to claim 3, characterized in that, The tumors include glioblastoma or urothelial carcinoma of the bladder.

5. The application according to claim 3, characterized in that, The antitumor reagent includes any one or more of the following reagents (1) to (4): (1) Reagents for blocking the cell cycle of glioblastoma cells; (2) Reagents for drugs that inhibit angiogenesis in glioblastoma; (3) Pyroptosis induction reagent for glioblastoma; (4) Reagent to reverse lactate expression in glioblastoma.

6. A method for preparing leech carbon dots as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve leech powder in pure water and perform a hydrothermal reaction to obtain a reaction solution; S2. The reaction solution obtained in S1 is processed to obtain a clear solution. Then, the clear solution is dialyzed, frozen and dried to obtain leech carbon dots.

7. The method for preparing leech carbon dots according to claim 6, characterized in that, In S1, the hydrothermal reaction conditions are: first react at 120–140℃ for 5–10 min, and then react at 150–180℃ for 20–30 min.

8. The method for preparing leech carbon dots according to claim 6, characterized in that, In S2, the reaction treatment includes centrifugation and filtration to remove the precipitate; the centrifugation conditions are 3500-4000 rpm for 10-15 min.

9. The method for preparing leech carbon dots according to claim 6, characterized in that, In S2, dialysis was performed using a 500 kDa biodialysis bag for 24–72 h, with a freezing temperature of -60 to -40 °C and a drying time of 12–24 h.

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