A carbon nanodot, its preparation method and its application

Abstract

This invention discloses carbon nanodots, their preparation method, and their applications, relating to the field of carbon nanomaterials technology. The carbon nanodots are prepared by solvothermal treatment of nicotinamide, urea, and citric acid or its derivatives. The citric acid derivative is selected from citrate, citric acid hydrate, or citrate hydrate. This invention synthesizes carbon nanodots using nicotinamide as a precursor under solvothermal treatment, simultaneously integrating intrinsic bioactivity, efficient photothermal conversion capability, and red fluorescence imaging function. This multifunctional synergistic therapy overcomes the limitations of single-therapy approaches. Furthermore, the carbon nanodots provided by this invention exhibit extremely high biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of carbon nanomaterials technology, and more specifically, to a carbon nanodot, its preparation method, and its application. Background Technology

[0002] Malignant tumors remain a leading cause of cancer-related deaths worldwide, characterized by uncontrolled proliferation and aggressive metastasis. Traditional treatments often fail to target these different pathological processes simultaneously, leading to limited efficacy and acquired treatment resistance. Therefore, developing effective combination therapies requires innovative nanotherapeutic agents that integrate multiple therapeutic modalities to jointly inhibit proliferation and migration pathways. Carbon dots (CDs), due to their superior optical properties and good biocompatibility, have become promising zero-dimensional carbon nanomaterials for biomedical applications. However, current CD-based antitumor strategies mainly rely on exogenous photoactivated therapies, such as photothermal therapy (PTT) or photodynamic therapy (PDT), or as nanocarriers for chemotherapeutic drugs.

[0003] In the prior art, the application of CDs as zero-dimensional carbon nanomaterials in anti-tumor therapy mainly relies on the following schemes: (1) Photoactivated therapy, where CDs are designed to have photothermal conversion properties for photothermal therapy (PTT) or photodynamic therapy (PDT). (2) Drug delivery systems, such as CDs as nanocarriers to load chemotherapeutic drugs, which enhance the efficacy of chemotherapeutic immunotherapy through programmed drug release.

[0004] The existing technical solutions have the following objective disadvantages: (1) They rely on external light sources. Photoactivated therapy is limited by the penetration depth of external light sources, resulting in reduced efficacy in deep tumor tissues and potentially incomplete treatment due to uneven illumination. (2) There are issues with the complexity of synthesis and safety. When used as drug carriers, CDs require the introduction of exogenous chemotherapeutic drugs in their synthesis process, increasing the complexity of the synthesis steps and potentially causing off-target effects and systemic toxicity. (3) They have limited functionality. Existing CDs mainly target a single pathway of tumor proliferation (such as through photothermal or drug release), lacking synergistic inhibition of tumor migration pathways and failing to address the dual challenges of tumor proliferation and metastasis simultaneously. (4) They lack intrinsic biological activity. Existing CDs do not possess intrinsic anti-tumor biological activity when not activated externally (such as by light or drug release), limiting their therapeutic application in the absence of light.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a carbon nanodot, its preparation method, and its application to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a carbon nanodot, which is prepared by solvothermal treatment of raw materials nicotinamide, urea and citric acid or its derivatives, wherein the citric acid derivative is selected from citrate, citric acid hydrate or citrate hydrate.

[0009] Secondly, the present invention also provides a method for preparing carbon nanodots, which includes the following steps: subjecting raw materials nicotinamide, urea and citric acid or their derivatives to solvothermal treatment.

[0010] Thirdly, the present invention also provides the application of carbon nanodots in the preparation of at least one of the following products:

[0011] Biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, fluorescent probes, kits, test strips, and antitumor drugs.

[0012] Fourthly, the present invention also provides a product comprising the above-mentioned carbon nanodots, wherein the product is selected from: biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, tumor photodynamic therapy reagents, fluorescent probes, reagent kits, test strips or antitumor drugs, wherein the reagent kit is a detection reagent kit.

[0013] The present invention has the following beneficial effects:

[0014] This invention uses nicotinamide (Nam) as a multifunctional precursor, combined with citric acid or its derivatives and urea, to synthesize nicotinamide-derived carbon dots (Nam-CDs) in a one-step process. These carbon nanodots possess antitumor activity and emit red fluorescence under excitation light. They integrate the following functions: emitting red fluorescence for bioimaging, photothermal capabilities responsive to external light, and intrinsic antitumor bioactivity, enabling therapeutic applications in the absence of light. Compared to existing carbon nanodots that only inhibit tumor proliferation but cannot intervene in tumor metastasis, the carbon nanodots provided by this invention not only significantly inhibit tumor cell colony formation but also significantly inhibit tumor cell colony migration, providing "all-weather" active intervention against tumor proliferation and metastasis. Therefore, the nanoplatform provided by this invention combines photothermal therapy requiring external activation with a continuously acting intrinsic therapy, completely solving the shortcomings of existing technologies that rely on external light sources, have limited penetration depth, and cannot inhibit tumor metastasis.

[0015] This invention utilizes nicotinamide, citric acid, and urea, all naturally occurring biological components, as raw materials. A one-step synthesis method avoids the introduction of exogenous toxic drugs. In vitro cytotoxicity experiments confirmed that even at concentrations as high as 1 mg / mL, it has minimal impact on the survival rates of various normal and cancer cells (Hepa1-6 cell survival rate >95%), demonstrating excellent biocompatibility. Both in vitro and in vivo experiments showed that carbon nanodots can be effectively taken up by tumor cells and accumulated at tumor sites. Therefore, the carbon nanodots provided by this invention have promising applications in biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, fluorescent probes, kits, test strips, and antitumor drugs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram of the preparation of Nam-CDs;

[0018] Figure 2 The image shows the luminescence test results of Nam-CDs;

[0019] Figure 3 This is a graph showing the results of a biosafety test.

[0020] Figure 4 The image shows the results of the cell photothermal experiment.

[0021] Figure 5 Figure showing the results of a cell colony formation experiment;

[0022] Figure 6 The image shows the results of the cell scratch assay.

[0023] Figure 7 This is a diagram showing the results of an animal treatment experiment. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] Definitions:

[0026] Nicotinamide, also known as nicotinamide or vitamin B3, is the amide form of niacin (nicotinic acid). It is a component of NAD+, one of the two important coenzymes in the human body. + and NADP + It is a key component, with the molecular formula C6H6N2O, and is also known as 3-pyridinecarboxamide.

[0027] Urea, molecular formula: CH4N2O or (NH2)2CO.

[0028] Citric acid, with the molecular formula C6H8O7, is also known as citric acid. The citric acid or its derivatives selected for preparing carbon nanodots can be selected from: analytical grade citric acid, analytical grade citric acid hydrate, potassium citrate, sodium citrate, and their hydrates. For example, citric acid monohydrate (C6H8O7·H2O).

[0029] Carbon nanodots, also known as carbon dots, are ultra-small (≤10nm) semiconductor nanoparticles that are recognized for their low toxicity / non-toxicity, high biocompatibility, tunable luminescence properties, and multifunctional photophysical properties.

[0030] In a first aspect, the present invention provides a carbon nanodot, which is prepared by solvothermal treatment of raw materials nicotinamide, urea and citric acid or its derivatives, wherein the citric acid derivative is selected from citrate, citric acid hydrate or citrate hydrate.

[0031] This invention uses nicotinamide (Nam) as a multifunctional precursor, combined with citric acid or its derivatives and urea, to synthesize nicotinamide-derived carbon dots (Nam-CDs) in a one-step process. These carbon nanodots possess antitumor activity and emit red fluorescence under excitation light. They integrate the following functions: emitting red fluorescence for bioimaging, photothermal capabilities responsive to external light, and intrinsic antitumor bioactivity, enabling therapeutic applications in the absence of light. Compared to existing carbon nanodots that only inhibit tumor proliferation but cannot intervene in tumor metastasis, the carbon nanodots provided by this invention not only significantly inhibit tumor cell colony formation but also significantly inhibit tumor cell colony migration, providing "all-weather" active intervention against tumor proliferation and metastasis. Therefore, the nanoplatform provided by this invention combines photothermal therapy requiring external activation with a continuously acting intrinsic therapy, completely solving the shortcomings of existing technologies that rely on external light sources, have limited penetration depth, and cannot inhibit tumor metastasis.

[0032] This invention utilizes nicotinamide, citric acid, and urea, all naturally occurring biological components, as raw materials. A one-step synthesis method avoids the introduction of exogenous toxic drugs. In vitro cytotoxicity experiments confirmed that even at concentrations as high as 1 mg / mL, it has minimal impact on the survival rates of various normal and cancer cells (Hepa1-6 cell survival rate >95%), demonstrating excellent biocompatibility. Both in vitro and in vivo experiments showed that carbon nanodots can be effectively taken up by tumor cells and accumulated at tumor sites. Therefore, the carbon nanodots provided by this invention have promising applications in biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, fluorescent probes, kits, test strips, and antitumor drugs.

[0033] In a preferred embodiment of the present invention, the carbon nanodots exhibit fluorescence emission of 575-750 nm under an excitation wavelength of 500-600 nm. The carbon nanodots provided by the present invention emit red fluorescence when excited by a light source with an excitation wavelength of 500-600 nm. Therefore, this property can be utilized for biofluorescence imaging.

[0034] In particular, the carbon nanodots provided by this invention exhibit stronger red emission under a light source with an excitation wavelength of 550-600 nm; and exhibit the strongest red emission under a light source with an excitation wavelength of 550 nm.

[0035] In a preferred embodiment of the present invention, the solvothermal treatment is performed in a solvent at a temperature of 160-200°C. For example, the temperatures are 160°C, 161°C, 162°C, 163°C, 164°C, 165°C, 166°C, 167°C, 168°C, 169°C, 170°C, 171°C, 172°C, 173°C, 174°C, 175°C, 176°C, 177°C, 178°C, 179°C, 180°C, 181°C, 182°C, 183°C, 184°C, 185°C, 186°C, 187°C, 188°C, 189°C, 190°C, 191°C, 192°C, 193°C, 194°C, 195°C, 196°C, 197°C, 198°C, 199°C, or 200°C.

[0036] In a preferred embodiment of the invention, the solvothermal treatment is performed in a solvent at 180°C. By comparing the light absorption intensity of carbon nanodots prepared under different solvothermal temperatures, it was found that the carbon nanodots prepared under the solvothermal treatment condition at 180°C exhibited the strongest UV-Vis absorption intensity at 550 nm and showed the strongest red emission under 550 nm excitation. The absorption intensity decreased after the temperature was continuously increased to 200°C. This confirms that the synthesis temperature is a key factor in optimizing optical absorption and fluorescence properties.

[0037] Carbon nanodots prepared under solvothermal treatment at 180°C exhibit significantly superior photothermal properties compared to those prepared at 160°C and 200°C, achieving a higher temperature rise under the same light source irradiation conditions. Tests showed that, under 660 nm laser irradiation, the aqueous solution of carbon nanodots prepared under solvothermal treatment at 180°C reached a temperature of 59.6°C within 8 minutes, a temperature capable of effectively ablating tumor cells. In vitro and in vivo experiments confirmed that this photothermal performance directly induces tumor cell death. Therefore, the carbon nanodots provided by this invention have promising applications in the preparation of photothermal therapeutic agents for tumors.

[0038] Even without laser irradiation, the carbon nanodots provided by this invention can still significantly inhibit the formation and migration of tumor cell colonies through their inherent biological activity, achieving "all-weather" active intervention against tumor proliferation and metastasis.

[0039] In a preferred embodiment of the present invention, the reaction time for solvothermal treatment is 1-10 hours. For example, the reaction time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.

[0040] In a preferred embodiment of the present invention, the solvent is selected from at least one of N,N-dimethylformamide, formic acid, and acetaldehyde.

[0041] Secondly, the present invention also provides a method for preparing carbon nanodots, which includes the following steps: subjecting raw materials nicotinamide, urea and citric acid or their derivatives to solvothermal treatment.

[0042] In a preferred embodiment of the present invention, the molar ratio of citric acid or its derivatives, urea and nicotinamide is (0.005-0.05): (0.01-0.1): (0.005-0.05). After solvothermal treatment, the reaction product is further mixed with alcohol, centrifuged, and the precipitate is collected.

[0043] In one alternative embodiment, the molar ratio of citric acid or its derivative, urea, and nicotinamide is: (0.005-0.01): (0.01-0.1): (0.005-0.05), (0.01-0.05): (0.01-0.1): (0.005-0.05), (0.005-0.01): (0.01-0.1): (0.005-0.01), (0.01-0.05): (0.01-0.1): (0.01-0.05). For example, the molar ratio of citric acid or its derivative, urea, and nicotinamide is 0.01:0.03:0.016 or 0.01:0.03:0.032.

[0044] Alcohols include, but are not limited to, ethanol, methanol, propanol, etc. Any alcohol that is miscible with the solvent in the reaction products, allowing for solvent displacement, is acceptable. Alcohol treatment makes the solvent miscible with the alcohol, causing the target carbon nanoparticles to exist as precipitates that are insoluble in the alcohol. Subsequent centrifugation allows for separation.

[0045] Thirdly, the present invention also provides the application of carbon nanodots in the preparation of at least one of the following products:

[0046] Biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, fluorescent probes, kits, test strips, and antitumor drugs.

[0047] When used in the preparation of biofluorescence imaging reagents, in one embodiment, carbon nanodots can be excited by a light source of a specific wavelength, acting as fluorescent markers or tracers in vivo or in vitro, to label, trace, and image target substances in cells or tissues. This is particularly suitable for applications such as fluorescence imaging of targeted proteins and relative quantification of protein levels.

[0048] When used to prepare nanoparticle therapeutic agents, in one embodiment, carbon nanodots can be directly applied to a subject. This can be achieved through oral or injectable administration, thereby maximizing the efficacy of the carbon nanodots.

[0049] When used to prepare photothermal therapy agents for tumors, carbon nanodots are applied to subjects and then irradiated with an external laser to kill local cells or tissues.

[0050] When used to prepare fluorescent probes, carbon nanodots are coupled to oligonucleotide sequences, proteins, or oligopeptides (e.g., carbon nanodots are modified with nucleotide sequences) or physically contacted to achieve the detection of the target gene, protein, or oligopeptide.

[0051] The kits include, but are not limited to, ELISA kits, immunohistochemistry (IHC) kits, Western blotting (WB) kits, qPCR kits, chemiluminescence immunoassay (CLIA) kits, and cell function assay kits.

[0052] The test strip includes a substrate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The aforementioned carbon nanoparticles can be coated on the conjugate pad, the sample pad, or the nitrocellulose membrane.

[0053] In one embodiment, the antitumor drug includes a pharmaceutically acceptable carrier, such as lipid nanoparticles, in which the aforementioned carbon nanodots are loaded within liposomes.

[0054] Pharmaceutically acceptable excipients include, but are not limited to, fillers, lubricants, disintegrants, binders, and flow aids.

[0055] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, hydroxypropyl methylcellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose-starch complex, lactose-cellulose complex, sucrose, mannitol, mannitol-starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, croscarmellose sodium cellulose, croscarmellose, magnesium stearate, sodium stearate fumarate, talc, and stearic acid, or a combination thereof.

[0056] Fourthly, the present invention also provides a product comprising the aforementioned carbon nanodots, the product being selected from: biofluorescence imaging reagents, nanoparticle therapy reagents, tumor photothermal therapy reagents, tumor photodynamic therapy reagents, fluorescent probes, reagent kits, test strips, or antitumor drugs, wherein the reagent kit is a detection kit. For example, it can be used to prepare reagents for early diagnosis of liver cancer, reagents for diabetes detection, or reagents for phenylketonuria detection.

[0057] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0058] Example 1

[0059] This embodiment provides a method for preparing nicotinamide-derived carbon dots (Nam-CDs), the preparation process of which is as follows: Figure 1 As shown. In this embodiment, nicotinamide (Nam) was used as a multifunctional precursor, combined with citric acid and urea, to prepare red fluorescent carbon dots (CDs) with antitumor activity. The specific preparation steps are as follows:

[0060] Citric acid (2 g, 0.01 mol), urea (2 g, 0.03 mol), and nicotinamide (2 g, 0.016 mol) were dissolved in N,N-dimethylformamide (20 mL) and reacted at 180 °C for 4 hours under solvothermal conditions. After the reaction system cooled to room temperature, the resulting dark-colored solution was mixed with ethanol (twice the volume) and centrifuged at 10,000 rpm for 15 minutes. The precipitate was collected and freeze-dried to finally obtain the Nam-CDs product.

[0061] Example 2

[0062] This embodiment provides a method for preparing nicotinamide-derived carbon dots (Nam-CDs). The only difference from Example 1 is that the solvothermal treatment is performed at 160°C for 4 hours, while the other conditions are the same as in Example 1.

[0063] Example 3

[0064] This embodiment provides a method for preparing nicotinamide-derived carbon dots (Nam-CDs). The only difference from Example 1 is that the solvothermal treatment is performed at 200°C for 4 hours, while the other conditions are the same as in Example 1.

[0065] Example 4

[0066] This embodiment provides a method for preparing nicotinamide-derived carbon dots (Nam-CDs). The only difference from Example 1 is that the formic acid solvent treatment is used at 180°C for 4 hours, while the other conditions are the same as in Example 1.

[0067] Example 5

[0068] This embodiment provides a method for preparing nicotinamide-derived carbon dots (Nam-CDs). Compared with Example 1, the only difference is that citric acid (2 g, i.e., 0.01 mol), urea (2 g, i.e., 0.03 mol), and nicotinamide (4 g, i.e., 0.032 mol) are dissolved in N,N-dimethylformamide (20 mL) and reacted at 180°C for 4 h. The other conditions are the same as in Example 1.

[0069] Experimental Example 1

[0070] The luminescence of the nicotinamide-derived carbon dots (Nam-CDs) prepared in Examples 1-3 was tested. The test methods and procedures are as follows:

[0071] Accurately weigh Nam CDs powder was prepared into a uniform dispersion of a certain concentration (e.g., 20 μg mL⁻¹) using ultrapure water, and then ultrasonically treated for 10 minutes. Aggregation was eliminated within 15 minutes. The photoluminescence properties of the material were tested by setting the excitation wavelength to 300-750 nm and the emission wavelength to 320-800 nm.

[0072] Nam-CDs were synthesized via a solvothermal reaction of citric acid, urea, and nicotinamide in N,N-dimethylformamide (DMF) at temperatures of 160°C, 180°C, and 200°C. The resulting products were named 160-Nam-CDs, 180-Nam-CDs, and 200-Nam-CDs, respectively. Figure 2 ).like Figure 2 As shown in Figure a, when the temperature increases from 160°C to 180°C, the UV-Vis absorption band at 550 nm strengthens and exhibits a slight blue shift, but the absorption intensity decreases after further heating to 200°C. (Using a 550 nm excitation wavelength...) Figure 2(Figure b) The fluorescence emission intensity at 600 nm increases in the range of 160°C to 180°C, but decreases at 200°C. Based on this, it can be determined that the sample synthesized at 180°C exhibits the strongest UV-Vis absorption intensity at 550 nm and also displays the strongest red emission under 550 nm excitation. This finding confirms that synthesis temperature is a key factor in optimizing optical absorption and fluorescence performance.

[0073] The photothermal properties of CDs were evaluated by irradiating an aqueous solution (100 μg / mL) with a 660 nm laser. Figure 2 (See Figure c). During the 8-minute irradiation, the temperatures of the 160-Nam-CDs, 180-Nam-CDs, and 200-Nam-CDs solutions rapidly increased from the ambient initial temperature (approximately 23°C) by 51.8°C, 59.6°C, and 49.8°C, respectively.

[0074] To further investigate the temperature-dependent luminescence behavior, the excitation-emission maps of aqueous solutions of 160-Nam-CDs prepared in Example 2, 180-Nam-CDs prepared in Example 1, and 200-Nam-CDs prepared in Example 3 were compared. Figure 2 (Figures d-f). The red emission of the 160-Nam-CDs solution is significantly weak, with a photoluminescence quantum yield of only 2%. In contrast, both 180-Nam-CDs and 200-Nam-CDs exhibit significantly enhanced red emission.

[0075] Experiment Example 2

[0076] To test the cytotoxicity and cellular uptake of Nam-CDs, this experimental example performed biosafety tests on the carbon nanodots prepared in Examples 1-3 in various liver cancer cell lines. The test methods and procedures are as follows:

[0077] In multiple liver cancer cell lines (SK) Hep1, HepG2, Hepa1 In vitro cytotoxicity tests were performed in cells 6 and Huh7. Cells were cultured under standard conditions and seeded in 96-well plates. After adhesion, NAM was injected with different concentration gradients. CDs were used for 24 or 48 hours. Cell viability was measured using CCK. The absorbance at 450 nm was measured using an ELISA reader, and normalization calculations were performed with only the culture medium wells as blank controls.

[0078] After PBS or Nam-CDs (100 μg mL) -1 Sk-Hep1 cells treated for 24 h were stained with Calcein-AM / PI to distinguish between live and dead cells. Figure 3(See Figure a in the table). Fluorescence microscopy showed that the Nam-CDs-treated group mainly exhibited green fluorescence, indicating high cell viability and excellent biocompatibility. Consistent with this, the CCK-8 assay confirmed that Nam-CDs had low cytotoxicity in SK-Hep1, HepG2, Hepa1-6, and Huh7 cells. Figure 3 (See Figure b in the figure). It is noteworthy that even at a high concentration of 1 mg mL⁻¹, the effect of Nam-CDs on Hepa1-6 cell viability remained negligible, with cell survival maintained above 95%. Figure 3 (Figure c in the image). Furthermore, cells were seeded in confocal culture dishes, and after adhesion, they were incubated with a specific concentration (e.g., 50 μg / mL). -1 Nam Cells were co-incubated with CDs for 1.5 hours. Subsequently, the cells were washed with PBS and stained with DAPI (nucleus staining agent). Samples were then examined under a confocal microscope at 405 nm (DAPI) and 543 nm (Nucleus staining agent). Excitation with Nam-CDs laser was performed, and the corresponding fluorescence signal was collected. Under 543 nm laser excitation, clear red fluorescence was observed in SK-Hep1 cells after only 1.5 hours of co-incubation with Nam-CDs. Figure 3 (See figure d in the image), where the blue area represents the results after DAPI staining. High-power imaging shows that Nam-CDs are distributed simultaneously in the cytoplasm and nucleus.

[0079] Experimental Example 3

[0080] This experimental example demonstrates a cell photothermal experiment.

[0081] The study employed a 660nm continuous laser (power density 1Wcm²). -2 Irradiate a container containing 1 mL of sample solution (concentration 100 μg / mL) -1 Centrifuge tubes containing water as the solvent were used. An infrared thermal imager was used to monitor and record the solution temperature changes in real time at 30-second intervals. The solution was continuously irradiated for 10 minutes, after which the laser was turned off and the solution was allowed to cool naturally. Pure water was used as a control. Temperature rise was plotted... The time curve was used to calculate the temperature rise rate (ΔT) and to investigate the effects of concentration and power density on the temperature rise effect, thereby systematically characterizing the photothermal conversion capability of the material.

[0082] like Figure 4As shown, using PBS as a control, the performance of the synthesized materials at three different temperatures (160℃, 180℃, and 200℃) in multiple functional experiments was compared, and the anticancer activity of Nam-CDs was systematically evaluated. To assess its photothermal therapy potential, live / dead cell imaging was performed on SK-Hep1 cells treated with Nam-CDs (100 μg / mL) for 1 hour and then irradiated with a 660 nm laser for 10 minutes. The results showed that before laser irradiation, all groups exhibited dominant green fluorescence, confirming that Nam-CDs prepared at the three temperatures had excellent biocompatibility; while after laser irradiation, widespread red fluorescence was observed, proving that carbon nanodots emit red fluorescence after laser irradiation, and the cell solution temperature increases, indicating that it has significant photothermal killing ability.

[0083] Experiment Example 4

[0084] This experiment demonstrates cell clonogenic assay and cell scratch assay.

[0085] To evaluate Nam The long-term inhibitory effect of CDs on tumor cell proliferation and migration was investigated using clonogenic and scratch healing assays. In the clonogenic assay, low-density seeded cells were treated with different concentrations of Nam... CDs were treated for 7-10 days, followed by fixation, crystal violet staining, and counting of clones with >50 cells to assess proliferative activity. In the scratch assay, uniform scratches were created on confluent monolayers of cells, washed with PBS, and then replaced with PBS containing low concentrations of serum and a specified concentration of Namolecular oxygen. The culture medium of CDs was photographed at fixed positions at 0, 24 and 48 h using an inverted microscope, and the cell migration ability was evaluated by the scratch area closure rate.

[0086] The results of the cell clone formation experiment were referenced. Figure 5 As shown, in contrast to this light-dependent cytotoxicity, Nam-CDs synthesized at 180 degrees Celsius in Examples 1-3 significantly inhibited colony formation in SK-Hep1 cells even in the absence of laser irradiation.

[0087] Cell scratch assay results refer to Figure 6 As shown, the cell scratch assay further demonstrated that Nam-CDs obtained under the synthesis conditions of Examples 1-3 could significantly inhibit the migration of SK-Hep1 cells.

[0088] The above experimental results indicate that even without laser irradiation, Nam-CDs can still significantly inhibit tumor cell colony formation through their intrinsic biological activity. Figure 5 ) and migration ( Figure 6 This enables proactive intervention in tumor proliferation and metastasis around the clock.

[0089] Experimental Example 5

[0090] This experimental example demonstrates animal treatment.

[0091] To evaluate Nam To investigate the in vivo antitumor efficacy of CDs, this study constructed a mouse model of subcutaneous xenograft tumors. Experimental animals were randomly divided into a control group, a CD1-only group, and a CD1-plus-laser therapy group. Once the tumor volume reached approximately 100 mm³, Nam was administered via tail vein injection. CDs (e.g., 5mg / kg) -1 ), and 3 hours after administration, the tumor area was irradiated with a 660nm laser (1Wcm). -2 (10 min). Tumor volume and mouse weight were measured regularly during treatment. After the course of treatment, tumor tissue was taken for histopathological analysis. The therapeutic efficacy was systematically evaluated by comparing the tumor growth inhibition rate and pathological changes of each group.

[0092] In a 4T1 tumor-bearing mouse model, this invention systematically evaluated the therapeutic effects of Nam-CDs synthesized at 180℃ using different treatment regimens. Representative photographs taken at specific time points in each experimental group visually recorded the tumor progression process. Figure 7 (Figure a). Under laser irradiation, the tumor (breast cancer) was completely removed. In the absence of laser irradiation, the Nam-CDs treatment group (tail vein injection every other day) significantly inhibited tumor growth, and the tumor volume monitoring curve clearly demonstrated this therapeutic effect. Figure 7 (Figure b in the middle)

[0093] In summary, this invention optimizes the synthesis of Nam-CDs at 180°C using nicotinamide as a precursor, thereby integrating intrinsic bioactivity, efficient photothermal conversion capability, and red fluorescence imaging function. This multifunctional synergistic therapy overcomes the limitations of single-therapy approaches. The nanoplatform provided by this invention combines photothermal therapy requiring external activation with a continuously effective intrinsic therapy, overcoming the shortcomings of existing technologies such as reliance on external light sources, limited penetration depth, and inability to inhibit metastasis.

[0094] After screening and optimization, 180°C was determined to be the optimal synthesis temperature. The optimized process simultaneously and significantly improved several key performance indicators of the material, making it a high-performance, multifunctional nano-formulation that provides a material basis for highly effective treatment. Using nicotinamide and citric acid, which are present in biological organisms, as raw materials, a one-step synthesis method avoids the introduction of exogenous toxic drugs.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carbon nanodot, characterized in that, It is prepared by solvothermal treatment of raw materials nicotinamide, urea and citric acid or its derivatives. The citric acid derivative is selected from citrate, citric acid hydrate or citrate hydrate. The solvothermal treatment is carried out in a solvent at 160-200℃. The molar ratio of raw material citric acid or its derivative, urea and nicotinamide is (0.005-0.05):(0.01-0.1):(0.005-0.05).

2. The carbon nanodots according to claim 1, characterized in that, The carbon nanodots exhibit fluorescence emission of 575-750 nm under a light source with an excitation wavelength of 500-600 nm.

3. The carbon nanodots according to claim 1, characterized in that, The solvothermal treatment is a reaction in a solvent at 180°C.

4. The carbon nanodots according to claim 1, characterized in that, The reaction time for the solvothermal treatment is 1-10 hours.

5. The carbon nanodots according to claim 4, characterized in that, The solvent is selected from at least one of N,N-dimethylformamide, formic acid, and acetaldehyde.

6. The method for preparing carbon nanodots according to any one of claims 1-5, characterized in that, It includes the following steps: solvothermal treatment of raw materials nicotinamide, urea and citric acid or its derivatives, wherein the molar ratio of citric acid or its derivatives, urea and nicotinamide is (0.005-0.05):(0.01-0.1):(0.005-0.05).

7. The preparation method according to claim 6, characterized in that, After solvothermal treatment, the reaction product is mixed with an alcohol solvent, centrifuged, and the precipitate is collected.

8. The use of the carbon nanodots according to any one of claims 1-5 in the preparation of at least one of the following products: Biofluorescence imaging reagents and nanoparticle therapy reagents.

9. The application of carbon nanodots as described in any one of claims 1-5 in the preparation of fluorescent probes.

10. The use of carbon nanodots as described in any one of claims 1-5 in the preparation of tumor photothermal therapy reagents.

11. The use of carbon nanodots as described in any one of claims 1-5 in the preparation kit.

12. The use of carbon nanodots as described in any one of claims 1-5 in the preparation of test strips.

13. The use of carbon nanodots as described in any one of claims 1-5 in the preparation of antitumor drugs.

14. A biofluorescence imaging reagent or nanoparticle therapeutic reagent, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

15. A photothermal therapy reagent for tumors, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

16. A photodynamic therapy reagent for tumors, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

17. A fluorescent probe, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

18. A reagent kit, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

19. A test strip, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

20. An antitumor drug, characterized in that, It includes the carbon nanodots as described in any one of claims 1-5.

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