Water-soluble tumor-targeting carbon dots and preparation method and application thereof

Abstract

This invention relates to the field of fluorescent carbon nanomaterials, specifically to a water-soluble tumor-targeting carbon dot, its preparation method, and its applications. The invention uses reduced glutathione as a precursor, which is dissolved in formamide by sonication to form a homogeneous solution. This solution is then transferred to a reaction vessel and subjected to a solvothermal reaction at 120-180°C for 2-10 hours to obtain a carbon dot solution. The obtained carbon dot solution is then subjected to vacuum filtration, washing, and drying to obtain a solid carbon dot powder. The carbon dots prepared by this invention exhibit high water solubility, high stability, and high biosafety. They can be prepared efficiently at the gram-scale level and possess the ability to broadly and precisely target tumors and load chemotherapeutic drugs, showing broad application prospects in the field of tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of fluorescent carbon nanomaterials, specifically to a water-soluble tumor-targeting carbon dot, its preparation method, and its application. Background Technology

[0002] Nanomaterials for tumor drug delivery offer advantages such as prolonged drug circulation time, enhanced drug stability, and increased drug accumulation in tumors, leading to better therapeutic effects and fewer side effects. Liposomes and albumin-based nanocarriers have been used to passively target tumors through their high permeability and long retention. However, their tumor-targeting efficiency is low and highly dependent on the characteristics of the tumor itself. Carbon dots with structures similar to large amino acids can specifically recognize L-amino acid transporter 1 (LAT1) overexpressed in tumor cells, regardless of tumor location or type, thus achieving broad-spectrum and precise tumor targeting without affecting other normal tissues and organs.

[0003] Human body fluids are an aqueous system, allowing dissolved substances to circulate throughout the body and intracellularly and extracellularly. Therefore, water solubility is crucial for the delivery of nanomedicine carriers within the body. Most nanomaterials are dispersed in water through electrostatic repulsion generated on their charged surfaces. As temperature or concentration increases, the chances of collisions between nanomaterials increase, leading to aggregation, larger size, and greater instability, thus limiting the concentration of nanomaterials dispersed in water. When nanomaterials are transported as drug carriers within the body, encountering electrolytes or high ion concentrations, the electrostatic interaction between their surface charge and oppositely charged ions neutralizes their charge, shielding them from electrostatic repulsion. This results in instability and aggregation of larger particles. Furthermore, nanomaterials dispersed in water due to their charge lack polar groups on their surface, making it difficult to form sufficient hydrogen bonds with water, resulting in high surface free energy. Upon entering the body, the nonpolar groups such as alkyl chains on the nanocarrier surface more easily disrupt protein hydration membranes and interact with the hydrocarbon groups (hydrophobic ends) of amino acids in proteins, forming dispersion forces that lower their surface free energy, ultimately leading to the formation of protein crown structures. This results in drawbacks for nanomedicine carriers, such as inconvenience in clinical storage and use, formation of emboli in the body or difficulty in metabolism and excretion, and weakened tumor targeting ability.

[0004] While various strategies exist to improve the water solubility of nanomedicine carriers, they all have several drawbacks. A common approach is to modify the surface of nanomedicine carriers with water-soluble polymers such as polyethylene glycol (PEG) or zwitterions like amino acids. However, the steric hindrance effect of PEG weakens the targeting ability of nanomedicine carriers. Furthermore, the generation of anti-PEG antibodies during the first injection accelerates blood clearance of the nanomedicine carrier when administered in multiple doses. In addition, the surface density of PEG is difficult to precisely control and quantify, and the PEG chain has poor stability, leading to degradation or chemical changes under conditions such as heating, radiation, or oxygen. Modifying the surface of nanomedicine carriers with zwitterions like amino acids requires complex synthesis and purification steps, which are time-consuming and yield low output. Therefore, exploring a new strategy to obtain water-soluble nanomedicine carriers with high stability and safety remains crucial, and this is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a water-soluble tumor-targeting carbon dot.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned carbon dot material.

[0007] Another object of the present invention is to provide applications of the aforementioned carbon dots.

[0008] Another object of the present invention is to prepare water-soluble tumor-targeting carbon dots for selective entry into tumor cells.

[0009] According to the present invention, a method for preparing water-soluble tumor-targeting carbon dots includes the following steps:

[0010] Reduced glutathione was used as a precursor and dissolved in formamide to form a homogeneous mixed reaction solution under ultrasonication.

[0011] The mixed reaction solution is transferred to a reaction vessel and subjected to a solvothermal reaction at a temperature of 120-180℃ for 2-10 hours to form a carbon dot solution.

[0012] The carbon dot solution was washed by vacuum filtration using a mixture of anhydrous methanol and dichloromethane as the eluent, and then dried to form a crude product of carbon dot solid powder.

[0013] The crude carbon dot solid powder was washed and dried by vacuum filtration with water as the eluent to obtain carbon dot solid powder.

[0014] According to the method for preparing water-soluble tumor-targeting carbon dots of the present invention, the mixed reaction solution contains reduced glutathione at a concentration of 0.1-15 wt%.

[0015] According to the method for preparing water-soluble tumor-targeting carbon dots of the present invention, the reaction vessel has a volume of 25 mL, 100 mL, 500 mL, 1 L, 2 L or 5 L.

[0016] According to the method for preparing water-soluble tumor-targeting carbon dots of the present invention, the volume ratio of anhydrous methanol to dichloromethane is 1:5-1:1.

[0017] According to the technical solution of the present invention, reduced glutathione is selected as a precursor and a solvothermal reaction is carried out in water or an organic solvent. The solvothermal reaction conditions are controlled, such as the concentration of reduced glutathione in the mixed reaction solution being 0.1-15 wt%, the reaction temperature being 120-180℃, and the reaction time being 2-10 hours, to synthesize water-soluble tumor-targeting carbon dot structures containing α-amino acids. If the reaction time is too short (below 3 hours) or the reaction temperature is too low (below 120℃), carbon dots with high water solubility and tumor targeting cannot be formed. If the reaction time is too long (above 10 hours) or the reaction temperature is too high (above 180℃), over-carbonization is likely to occur, and carbon dots cannot be obtained.

[0018] According to the preparation method of the present invention, a mixture of anhydrous methanol and dichloromethane (methanol to water volume ratio of 1:5-1:1) is used as the eluent, and purification is carried out by vacuum filtration washing. Because there are too many impurities after the reaction, purification by vacuum filtration washing yields carbon dots with high water solubility and tumor targeting. If anhydrous methanol and dichloromethane are not used as eluents for vacuum filtration washing, carbon dots with high water solubility and tumor targeting cannot be obtained. The present invention can obtain solid carbon dot powder through simple separation and purification. The preparation method of the present invention is simple and controllable, low in cost, and has a high yield, allowing for large-scale mass production.

[0019] Beneficial effects:

[0020] According to the present invention, a water-soluble targeted carbon dot possessing both α-amino acid and alkyl chain structures is obtained via a simple solvothermal method. This carbon dot exhibits high solubility, high stability, and high biosafety in water, making it suitable for further biological applications. Its solubility in water is greater than 500 mg / mL. -1Adding more than 30 mg of any one, two, or a combination of more than 30 mg of sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, sodium nitrate, ammonium nitrate, potassium nitrate, sodium phosphate, ammonium phosphate, or potassium phosphate to the carbon dots does not produce precipitation; the carbon dots do not produce precipitation and exhibit high stability when placed at room temperature, 4°C, or -20°C for more than 6 months; when injected into mice at doses higher than 1000 mg / kg, the carbon dots have no significant effect on mouse weight or organs, demonstrating high biosafety. The water-soluble tumor-targeting carbon dots of the present invention comprise a core structure composed of a multi-ring array of six-membered rings, each multi-ring array being an aromatic or unsaturated five-membered ring, with nitrogen doping in the aromatic rings. The edge of the core structure simultaneously contains α-amino acid structures with -COOH and -NH2 linked to the same carbon atom and multiple dense alkyl chain structures, such as any one, two, or a combination of more than 30 mg of methyl, methylene, ethyl, or propyl, but not limited thereto.

[0021] The water-soluble tumor-targeting carbon dots prepared in this invention can be loaded with chemotherapeutic drugs for cancer treatment through π-π stacking, hydrogen bonding, and electrostatic interactions. These drugs include, but are not limited to, any one, two, or a combination of more than one of doxorubicin derivatives, camptothecin derivatives, platinum derivatives, taxane derivatives, and fluorine-containing derivatives, for targeted tumor therapy. The water-soluble tumor-targeting carbon dots of this invention can broadly and precisely target tumors.

[0022] The water-soluble tumor-targeting carbon dots prepared in this invention possess excellent optical properties, high solubility in water, high stability, and high biosafety. They can achieve broad-spectrum and precise targeting of tumors and have the ability to load chemotherapeutic drugs, showing broad application prospects in fields such as bioimaging, drug delivery, and disease diagnosis and treatment. As a targeted therapeutic chemotherapeutic drug carrier, they exhibit excellent luminescent properties, water solubility, biocompatibility, and tumor targeting, thus achieving the dual function of chemotherapeutic drug carrier and bioimaging probe. They hold promise as a low-cost, environmentally friendly, highly efficient, and low-toxicity novel drug carrier for clinical application in tumor treatment. Attached Figure Description

[0023] Figure 1 The fluorescence spectra of the water-soluble tumor-targeting carbon dots prepared in Example 1 under excitation at different wavelengths are shown.

[0024] Figure 2 The image shows the ultraviolet absorption spectrum of the water-soluble tumor-targeting carbon dots prepared in Example 1.

[0025] Figure 3 The image shows a transmission electron microscope image of the water-soluble tumor-targeting carbon dots prepared in Example 1.

[0026] Figure 4An atomic force microscope image of the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0027] Figure 5 The Raman spectrum of the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0028] Figure 6 The X-ray photoelectron spectroscopy spectrum of the water-soluble tumor-targeting carbon dots prepared in Example 1 is shown below.

[0029] Figure 7 The infrared spectrum of the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0030] Figure 8 The UV-Vis absorption spectra of the water-soluble tumor-targeting carbon dots prepared in Example 1 before and after treatment with ninhydrin;

[0031] Figure 9 The contact angle test results are for the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0032] Figure 10 The viscosity test curve of the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0033] Figure 11 The dielectric constant test curve of the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0034] Figure 12 The images are cryo-transmission electron microscopy images of the water-soluble tumor-targeting carbon dots and water alone prepared in Example 1.

[0035] Figure 13 This is an SDS-polyacrylamide gel electrophoresis image of the water-soluble tumor-targeting carbon dots prepared in Example 1 after incubation with fetal bovine serum.

[0036] Figure 14 The results of the hemolysis test are based on the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0037] Figure 15 The cell activity of the water-soluble tumor-targeting carbon dots prepared in Example 1 after incubation with tumor cells for 24 hours;

[0038] Figure 16 The blood routine and blood biochemical parameters of mice after acute toxicity experiments in Kunming mice with the water-soluble tumor-targeting carbon dots prepared in Example 1;

[0039] Figure 17 This is a confocal fluorescence microscopy image of the water-soluble tumor-targeting carbon dots prepared in Example 1 after incubation with different tumors and normal cells;

[0040] Figure 18 The uptake results of the water-soluble tumor-targeting carbon dots prepared in Example 1 were obtained by flow cytometry after incubation with different tumors and normal cells.

[0041] Figure 19 Fluorescence imaging images at different time points after the water-soluble tumor-targeting carbon dots prepared in Example 1 were injected into tumor-bearing nude mice;

[0042] Figure 20 The UV-Vis absorption spectra of the water-soluble tumor-targeting carbon dot-loaded chemotherapeutic drug doxorubicin prepared in Example 1 before and after preparation.

[0043] Figure 21 The images show the tissue distribution fluorescence imaging of the water-soluble tumor-targeting carbon dot-loaded chemotherapeutic drug doxorubicin prepared in Example 1 and doxorubicin alone after injection into tumor-bearing Kunming mice.

[0044] Figure 22 Cell viability was measured after the water-soluble tumor-targeting carbon dot-loaded chemotherapeutic drug doxorubicin prepared in Example 1 and after doxorubicin alone was incubated with tumor cells and normal cells for 24 hours.

[0045] Figure 23 The therapeutic effect of the water-soluble tumor-targeting carbon dot-loaded chemotherapeutic drug doxorubicin prepared in Example 1 on tumor-bearing nude mice;

[0046] Figure 24 The images show pathological sections of major organs of tumor-bearing nude mice treated with doxorubicin, a water-soluble tumor-targeting carbon dot-loaded chemotherapeutic drug prepared in Example 1, using H&E staining. Detailed Implementation

[0047] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention and provide detailed implementation methods and processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0048] According to the method for preparing water-soluble tumor-targeting carbon dots of the present invention, the carbon dots are prepared by a method comprising the following steps:

[0049] (1) Using reduced glutathione as a precursor, it is sonicated to dissolve it in formamide to form a homogeneous solution. Then the solution is transferred to a reaction vessel and subjected to a solvothermal reaction at 120-180℃ for 2-10 hours. The reaction vessel is then allowed to cool naturally to room temperature to obtain a carbon dot solution directly.

[0050] (2) The obtained carbon dot solution was washed under reduced pressure using a mixture of anhydrous methanol and dichloromethane as the eluent (the volume ratio of anhydrous methanol to dichloromethane was 1:5-1:1, with the volume ratio of anhydrous methanol gradually increasing). After drying, a crude carbon dot solid powder product was formed. The crude carbon dot solid powder product was then washed under reduced pressure using water as the eluent and dried to obtain carbon dot solid powder. The carbon dot solid powder prepared by this invention has good solubility in water.

[0051] Example 1: Preparation of water-soluble tumor-targeting carbon dots

[0052] 8.0 g of reduced glutathione was weighed into a beaker, and 200 mL of formamide was added. A homogeneous solution was formed under ultrasonic treatment, and then transferred to a 500 mL polytetrafluoroethylene-lined reactor. The reaction was carried out at 130 °C for 6 hours using a solvothermal method, followed by natural cooling of the reactor to room temperature, resulting in a dark green carbon dot solution. After the reaction, the solution was collected and mixed with an anhydrous methanol / dichloromethane at a volume ratio of 1:10:25. The mixture was allowed to stand to precipitate the carbon dots. The solution was then filtered under reduced pressure using a 0.22 μm filter membrane. Gradient elution was continued using a mixture of anhydrous methanol and dichloromethane as the eluent (anhydrous methanol to dichloromethane volume ratio gradually increasing from 1:4 to 1:2) to remove low water-soluble impurities. After drying, a green carbon dot solid powder was obtained. The carbon dot solution was purified by using water as the eluent under reduced pressure filtration conditions and then dried at 60-90℃ to obtain a green carbon dot solid powder.

[0053] As an alternative technical solution in this embodiment, the mixed reaction solution contains reduced glutathione at concentrations of 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 1.0 wt%, 2.0 wt%, 3.0 wt%, 4.0 wt%, 5.0 wt%, 6.0 wt%, 8.0 wt%, 10.0 wt%, 12.0 wt%, or 15.0 wt%.

[0054] As an alternative technical solution in this embodiment, the reaction temperature is 120°C, 130°C, 140°C, 150°C, 160°C, 170°C or 180°C.

[0055] As an alternative technical solution in this embodiment, the response time is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours.

[0056] As an alternative technical solution in this embodiment, the reactor volume is 25mL, 100mL, 500mL, 1L, 2L or 5L.

[0057] As an alternative technical solution in this embodiment, the volume ratio of anhydrous methanol to dichloromethane is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

[0058] The water-soluble tumor-targeting carbon dot aqueous solution emitted bright red fluorescence under a handheld ultraviolet lamp (365 nm), and its fluorescence spectrum exhibited excitation-independent intrinsic state fluorescence characteristics. Figure 1 The fluorescence emission peak is located at 683 nm, with a full width at half maximum (FWHM) of approximately 30 nm. The measured absolute fluorescence quantum yield is 16%. The main characteristic absorption peak of the water-soluble tumor-targeting carbon dots is located at 420 nm. Figure 2 It is close to the maximum fluorescence excitation wavelength and exhibits three characteristic absorption peaks at wavelengths of 604 nm, 627 nm and 676 nm.

[0059] Transmission electron microscopy revealed that the water-soluble tumor-targeting carbon dots were well-dispersed and uniform in size, with an average particle size of 3.2 nm. Figure 3 Atomic force microscopy results showed that the thickness of the water-soluble tumor-targeting carbon dots was less than 1 nm, indicating that most of the water-soluble tumor-targeting carbon dots were composed of a single-layer graphite-like structure. Figure 4 ). I in the Raman spectrum of water-soluble tumor-targeting carbon dots D / I G The ratio is 0.48 ( Figure 5 This indicates that the carbon dots have a relatively high degree of crystallization, consistent with the high crystallinity characterized by high-resolution transmission electron microscopy and aberration-sensitivity transmission electron microscopy.

[0060] X-ray photoelectron spectroscopy results showed that the surface of the water-soluble tumor-targeting carbon dots was mainly composed of three elements: C, O, and N, with the atomic percentages of C, O, and N being 60.7 at%, 20.1 at%, and 19.2 at%, respectively. Figure 6 Infrared spectroscopy of water-soluble tumor-targeting carbon dots confirmed the presence of OH (3603 cm⁻¹) in the carbon dots. -1 ), NH (3325cm) -1 CH (2978cm) -1 C = O (1759cm) -1 ), C = C / C = N (1674cm) -1 ), CN (1327cm) -1 ) and CO(1119cm -1 Chemical bonds Figure 7 ). Ninhydrin reaction ( Figure 8 The results showed that the edges of the water-soluble tumor-targeting carbon dots contained free α-amino acid functional groups.

[0061] According to the technical solution of this invention, the solvothermal reaction time is controlled at 2-10 hours, and the reaction temperature is controlled at 120-180℃ to synthesize water-soluble tumor-targeting carbon dots. Compared with the above preparation process, other reaction conditions are the same. If the reaction time is too short (less than 2 hours), the designed structure cannot be formed, the solution in the reaction vessel is yellow, and it emits blue fluorescence under a handheld ultraviolet lamp (365nm), and carbon dots cannot be obtained. If the reaction temperature is too low (less than 120℃), after purification by vacuum filtration using anhydrous methanol and dichloromethane as eluents, green carbon dot powder cannot be formed. If the reaction time is too long (more than 10 hours) or the reaction temperature is too high (more than 180℃), over-carbonization is likely to occur, the solution in the reaction vessel is brownish-yellow and contains black residue, and carbon dots cannot be obtained.

[0062] According to the technical solution of the present invention, using a mixture of anhydrous methanol and dichloromethane as the eluent (the volume ratio of anhydrous methanol to dichloromethane is 1:5-1:1, with the volume ratio of anhydrous methanol gradually increasing), and employing vacuum filtration for purification is crucial for the purification process. Since some impurities remain after the solvothermal reaction, vacuum filtration using anhydrous methanol and dichloromethane as the eluent ensures the acquisition of carbon dots with high water solubility and tumor targeting. If the volume ratio of anhydrous methanol to dichloromethane is too small (less than 1:5), the eluent polarity is too low, forming water-in-oil droplets upon addition to the carbon dot solution, making it impossible to precipitate the carbon dot solution into carbon dot precipitate, thus preventing the purification process from obtaining carbon dots with high water solubility and tumor targeting. If the volume ratio of anhydrous methanol to dichloromethane is too large (greater than 1:1), the eluent polarity will be too high, and it will be unable to precipitate the carbon dot solution into carbon dot precipitate after being added to the carbon dot solution. In other words, the purification process cannot be carried out to obtain carbon dots with high water solubility and tumor targeting.

[0063] Example 2: Water-soluble tumor-targeting carbon dots

[0064] The water-soluble tumor-targeting carbon dots prepared in Example 1 had a solubility of 2.0 g / mL in water. No precipitation or aggregation occurred after adding 100 mg of sodium chloride to the aqueous solution, heating it to 100°C, centrifuging it at high speed, or storing it at room temperature for 10 months. Fluorescence spectra at three different elevations in the aqueous solution of the water-soluble tumor-targeting carbon dots were tested, and the positions and intensities of the fluorescence emission peaks remained largely consistent, indicating the stability and uniformity of the water-soluble tumor-targeting carbon dots.

[0065] As an alternative technical solution to this embodiment, electrolytes such as sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, sodium nitrate, ammonium nitrate, potassium nitrate, sodium phosphate, ammonium phosphate, or potassium phosphate, or any combination of two or more thereof, can be added to the water-soluble tumor-targeting carbon point aqueous solution.

[0066] As an alternative technical solution in this embodiment, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 120mg, 150mg, 180mg or 200mg of electrolyte is added to the carbon point.

[0067] As an alternative technical solution in this embodiment, the carbon dots can be stored at room temperature, 4°C or -20°C; and can be stored for 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more.

[0068] The water contact angle of water-soluble tumor-targeting carbon dots was measured using the seated drop method, and the measured value was 6.5°. Figure 9 Its near-0° water contact angle demonstrates the superhydrophilicity of the carbon dots. Viscosity tests show that the addition of water-soluble tumor-targeting carbon dots to water increases the water viscosity, and the viscosity gradually increases with increasing carbon dot concentration. Figure 10 This indicates that hydrogen bonds can form between carbon dots and water, leading to decreased water flowability and increased viscosity. The effect of water-soluble tumor-targeting carbon dots on the dielectric constant of water was determined. Adding carbon dots to water decreased the dielectric constant, and the dielectric constant of water gradually decreased with increasing carbon dot concentration. Figure 11 This indicates that the addition of carbon dots induces a structural arrangement of water, restricting the rotational degrees of freedom of the water dipoles and leading to a decrease in the dielectric constant of water. Cryo-transmission electron microscopy images of aqueous solutions containing water-soluble tumor-targeting carbon dots reveal a hexagonal ice lattice structure with a basal plane. Figure 12 However, no obvious lattice structure was observed in the cryogenic transmission electron microscopy images of water alone, indicating that the interaction between carbon dots and water can induce the hexagonal arrangement of water.

[0069] Example 3: Biosafety of water-soluble tumor-targeting carbon dots

[0070] The binding of the water-soluble tumor-targeting carbon dots prepared in Example 1 to proteins was tested using SDS-polyacrylamide gel electrophoresis. After incubation of the carbon dots with fetal bovine serum, no significant change was observed in the fetal bovine serum bands. Figure 13 This indicates that the carbon dots hardly adsorb proteins in serum, thus preventing the formation of protein crowns and producing almost no immunogenicity. When mouse whole blood was incubated with different concentrations of water-soluble tumor-targeting carbon dots, the hemolysis rate was consistently below 2%. Figure 14This indicates that carbon dots have good blood compatibility.

[0071] In vitro and in vivo toxicity assessment of water-soluble tumor-targeting carbon dots. The carbon dots did not produce significant cytotoxicity in either human cervical cancer cells (HeLa) or human non-small cell lung cancer cells (A549). Figure 15 Injection of 5000 mg / kg of water-soluble tumor-targeting carbon dots into mice showed no significant hepatotoxicity or nephrotoxicity, and exhibited high biocompatibility in the blood, without producing a significant inflammatory response. Figure 16 This indicates that carbon dots have good biocompatibility in vitro and in vivo, which is crucial for subsequent in vivo treatment.

[0072] As an alternative technical solution in this embodiment, the carbon dots described herein can be injected into mice at concentrations of 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 mg / kg, or 6000 mg / kg.

[0073] Example 4: Tumor targeting properties of water-soluble tumor-targeting carbon dots

[0074] The uptake of the water-soluble tumor-targeting carbon dots prepared in Example 1 by confocal fluorescence microscopy was investigated in tumor cells and normal cells. Confocal fluorescence microscopy images showed that the carbon dots could enter a variety of tumor cells, but entered little or no normal cells. Figure 17 This indicates that the carbon dots have the ability to target tumor cells. Quantitative results from flow cytometry showed that in all tested cell lines, the water-soluble tumor-targeting carbon dots could enter tumor cells with an uptake rate as high as 99%, while the uptake rate in normal cells was only around 10%. Furthermore, the fluorescence intensity of the carbon dots in tumor cells was approximately ten times higher than that in normal cells. Figure 18 ).

[0075] Water-soluble tumor-targeting carbon dots were injected into tumor-bearing nude mice, and fluorescence imaging was observed at different time points. The fluorescence signal intensity of the carbon dots collected in the tumor region gradually increased over time, peaking only in the tumor region, and long-term enrichment was achieved in the tumor region. Figure 19 ).

[0076] Example 5: Chemotherapy drug loading based on water-soluble tumor-targeting carbon dots and its in vivo and in vitro therapeutic applications.

[0077] The water-soluble tumor-targeting carbon dots prepared in Example 1 were used as a chemotherapeutic drug carrier for in vitro and in vivo tumor treatment. The carbon dots and the chemotherapeutic drug doxorubicin bonded together through π-π stacking interactions to form a carbon dot / doxorubicin complex. The characteristic absorption peak representing the carbon dots in the UV-Vis absorption spectrum of the formed complex exhibited a redshift broadening. Figure 20 ).

[0078] As an alternative technical solution in this embodiment, the carbon-dot-loaded chemotherapeutic drug can be any one, two or more of the following: doxorubicin, camptothecin, platinum, paclitaxel or fluorine-containing drugs.

[0079] The tumor targeting of the carbon dot / doxorubicin complex was investigated in tumor-bearing Kunming mice. In the doxorubicin-only group, the fluorescence signal intensity of doxorubicin in tumor tissues and major organs was similar; however, in the carbon dot / doxorubicin group, the fluorescence signal intensity of doxorubicin in tumor tissues was significantly higher than that in other normal tissues. Figure 21 ).

[0080] The carbon dot / doxorubicin complex was incubated in tumor cells and normal cells, and cell viability was quantitatively detected. Carbon dot / doxorubicin induced apoptosis in more tumor cells, but not in normal cells. Figure 22 This indicates that loading chemotherapy drugs onto carbon dots preserves the tumor-targeting properties of the carbon dots, allowing carbon dots / doxorubicin to selectively penetrate tumor cells but rarely penetrate normal cells. This results in higher doxorubicin concentrations in tumor cells, enhanced cytotoxicity, and reduced toxicity to normal cells.

[0081] The aforementioned carbon dot / doxorubicin complex was applied to the treatment of tumors in tumor-bearing nude mice. After 14 days, tumor growth in the carbon dot / doxorubicin treatment group was significantly inhibited, and the tumor volume was significantly smaller than that in the saline group, carbon dot group, doxorubicin group, and liposomal doxorubicin group. Figure 23 The blood routine and blood biochemical parameters of nude mice in the carbon dot / doxorubicin group were all within the normal range, and no inflammatory infiltration or pathological damage was observed in the H&E staining pathological sections of the major organs (heart, liver, spleen, lungs, and kidneys) of the nude mice. Figure 24 This demonstrates that the use of carbon dots / doxorubicin to treat tumors in nude mice can not only effectively inhibit tumor growth but also exhibit low toxicity to normal organs.

[0082] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. The application of water-soluble carbon dots in the preparation of targeted tumor therapy agents, among which, The water-soluble carbon dots are prepared by a method comprising the following steps: Reduced glutathione was used as a precursor and dissolved in formamide to form a homogeneous mixed reaction solution under ultrasonication. The mixed reaction solution is transferred to a reaction vessel and subjected to a solvothermal reaction at 120-180°C for 2-10 hours to form a carbon dot solution; The carbon dot solution was washed by vacuum filtration using a mixture of anhydrous methanol and dichloromethane as the eluent, and then dried to form a crude solid powder product of the water-soluble tumor-targeting carbon dots.

2. The application according to claim 1, characterized in that, The method further includes the following steps: The crude solid powder product containing water-soluble tumor-targeting carbon dots was washed and dried by vacuum filtration using water as the eluent to obtain carbon dot solid powder.

3. The application according to claim 1, characterized in that, The mixed reaction solution contains 0.1-15 wt% reduced glutathione.

4. The application according to claim 1, characterized in that, The volume ratio of anhydrous methanol to dichloromethane is 1:5 to 1:

1.

5. The application of water-soluble carbon dots as carriers for loading targeted tumor drugs, among which, The water-soluble carbon dots are prepared by a method comprising the following steps: Reduced glutathione was used as a precursor and dissolved in formamide to form a homogeneous mixed reaction solution under ultrasonication. The mixed reaction solution is transferred to a reaction vessel and subjected to a solvothermal reaction at 120-180°C for 2-10 hours to form a carbon dot solution; The carbon dot solution was washed by vacuum filtration using a mixture of anhydrous methanol and dichloromethane as the eluent, and then dried to form a crude solid powder product of the water-soluble tumor-targeting carbon dots.

Images