Near-infrared fluorescent carbon nanodot as well as preparation method and application thereof

Abstract

The invention belongs to the field of fluorescent carbon nanomaterials, and provides a near-infrared fluorescent carbon nanodot as well as a preparation method and application thereof. The carbon nanodot comprises a carbon-based core (having visible light and near-infrared light absorption and emission characteristics) prepared by taking a perylene compound or a nitrogen-doped perylene compound as a precursor; the carbon-based core is coated with the shell. The preparation method comprises the step of performing solvothermal reaction on the perylene compound or a mixture of the perylene compound and a nitrogen-containing compound. The invention also provides a tumor diagnosis and treatment integrated medicine comprising the carbon nanodot. Under the irradiation of visible light and near-infrared laser, the carbon nanodots can generate a large amount of active oxygen to kill tumor cells and generate a photothermal effect to assist in killing the tumor cells. The nitrogen element is doped, so that the absorption of the carbon nanodots on red light and NIR wave band light is improved, the emission, photo-thermal and photodynamic properties of the carbon nanodots in a near-infrared second region are promoted, and the treatment capacity of deep tumors is improved. And the carbon nanodots can be used for tumor and angiography in organisms.

Description

Technical Field

[0001] The present disclosure relates to the field of fluorescent carbon nanomaterials, and in particular to near-infrared fluorescent carbon nanodots and a preparation method and application thereof. Background Art

[0002] Cancer, a disease with a high mortality rate, poses a potential threat to global health. Currently, traditional cancer treatment options (chemotherapy, radiotherapy, surgery, etc.) are often ineffective due to significant side effects, long treatment cycles, and poor cure rates. Furthermore, tumors in the body are often hidden and migratory, making diagnosis difficult. Currently, commonly used in vivo imaging technologies, such as positron emission tomography (PET), computed tomography (CT), ultrasound (US), single-photon emission computed tomography (SPET), photoacoustic imaging (PAI), Raman imaging (RI), and magnetic resonance imaging (MRI), offer high penetration and resolution, enabling high-resolution, real-time imaging of deep, delicate tissue structures within the body. However, these technologies often require sophisticated, high-end equipment, use contrast agents with a degree of physiological toxicity, and are relatively expensive, making them unsuitable for the general public. Furthermore, due to the separation of traditional diagnostic and treatment methods, most contrast agents lack therapeutic potential for tumors. Therefore, developing a drug with convenient and effective tumor diagnosis capabilities and excellent tumor treatment effects will provide a new method for the diagnosis and treatment of tumors in vivo.

[0003] In recent years, phototherapy, such as photothermal therapy (PTT) and photodynamic therapy (PDT), has attracted widespread interest as a powerful technology for cancer treatment, partly due to their convenience, minimal or non-invasive nature, low systemic toxicity, and lack of drug resistance. Photodynamic therapy (PDT) involves the use of appropriately photoactivated photosensitizers (PSs) to generate toxic reactive oxygen species (ROS) to oxidatively kill cancer cells. Currently, widely used photosensitizers, such as porphyrins, phthalocyanines, and bacteriochlorin derivatives, have been demonstrated to have the ability to simultaneously image and treat cancer, and some of these photosensitizers have been approved for clinical use. However, as organic small molecule chemical reagents, they also face disadvantages such as poor water solubility (leading to fluorescence quenching), lack of targeting, short in vivo circulation time, poor imaging effects, and certain biological toxicity of some photosensitizers, which have limited their application in organisms.

[0004] Since it was first discovered in 1866, photothermal therapy (PTT) has gradually developed into a popular treatment method in the field of tumor treatment. Under light conditions, materials enriched in the tumor area absorb light energy and convert it into heat energy higher than body temperature, exposing tumor cells to a high temperature environment (usually higher than 45°C), which leads to their inactivation and apoptosis. However, traditional photothermal therapy and photodynamic therapy are often subject to the optical properties of materials and the choice of light sources. According to traditional physics theory, near-infrared light (NIR) with a longer wavelength and shorter frequency has less energy attenuation after penetrating biological tissues than ultraviolet (UV) and visible light (VIS), and exhibits stronger tissue penetration, which can be used for the treatment of tumors deeper in the body (up to 5-10mm). Therefore, the development of photothermal and photodynamic materials with near-infrared light excitation is of profound significance for promoting deep and efficient tumor treatment.

[0005] In the field of in vivo imaging, near-infrared (NIR) fluorescence imaging has also received widespread attention. In the scientific research field, traditional near-infrared light is divided into the first NIR window (NIR-I) (700-900nm) and the second NIR window (NIR-II) (1000-1700nm) according to the wavelength. Compared with NIR-I, NIR-II has a stronger penetration depth, and fluorescence imaging shows significant advantages of deeper penetration and higher spatiotemporal resolution. This is because it effectively reduces photon scattering, absorption and tissue autofluorescence in biological tissues. However, due to the fact that traditional organic materials have an absorbance closer to the visible and ultraviolet bands, the development of near-infrared fluorescent materials is still a considerable challenge. At present, most NIR-II fluorescent nanomaterials are inorganic materials (for example, inorganic nanomaterials, quantum dots and rare earth-doped nanoparticles), which have long-term safety issues. Organic NIR-II fluorescent nanomaterials (e.g., conjugated polymer nanoparticles, carbon nanotubes, PEGylated molecules, and nanoparticles containing small molecules) have excellent performance and clinical application potential in NIR-II imaging due to their excellent biocompatibility, fine chemical structure, and photophysical properties. However, because the excitation power density decreases in deeper body tissues in the body, only fluorophores with high fluorescence quantum yield (QY) can generate sufficient fluorescence signals to achieve deep penetration and high resolution. However, most organic nanomaterials exhibit relatively low QY and are easily metabolized and cleared by the body, limiting the effectiveness of fluorescence imaging.

[0006] Carbon dots (CDs), a novel class of organic nanomaterials, have emerged as promising nanomaterials for imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) due to their excellent biocompatibility, tunable photophysical properties, and high quantum efficiency. However, currently developed CDs are limited to red and NIR-I fluorescence excitation and emission due to structural constraints, which cannot meet the needs of imaging and treating blood vessels and deep-seated tumors in vivo.

[0007] Therefore, it is of far-reaching significance to overcome the wavelength barrier and synthesize NIR-Ⅱ fluorescent carbon dots with better imaging and therapeutic effects. Summary of the Invention

[0008] The present disclosure provides a near-infrared fluorescent carbon nanodot and a preparation method and application thereof, to at least solve the above technical problems existing in the prior art.

[0009] According to a first aspect of the present disclosure, there is provided a near-infrared fluorescent carbon nanodot comprising a carbon-based core made from a perylene compound or a nitrogen-doped perylene compound as a precursor;

[0010] Among them, the near-infrared fluorescent carbon nanodots comprising a carbon-based core made of a perylene compound as a precursor have an absorption band in the visible light wavelength range of 380 to 750 nm; the near-infrared fluorescent carbon nanodots comprising a carbon-based core made of a nitrogen-doped perylene compound as a precursor have an absorption band in the visible light and near-infrared light wavelength range of 500 to 1200 nm;

[0011] The fluorescence emission wavelength of the near-infrared fluorescent carbon nanodots is within the range of 800 to 1500 nm.

[0012] Specifically, the carbon-based core is composed of CC, or CC, CN bonds.

[0013] The near-infrared carbon nanodots (CDs) include a carbon-based core made from perylene compounds as precursors, and their absorption value is in the visible light range: under 660nm laser irradiation, they can produce a large amount of active oxygen; under 808nm laser irradiation, they can generate weak photothermal and near-infrared second-region fluorescence with a peak value of 930nm.

[0014] Near-infrared fluorescent carbon nanodots (N-CDs) with a carbon-based core made from nitrogen-doped perylene compounds as precursors have visible light and near-infrared light absorption properties, and can use light energy to generate active oxygen and photothermal effects under light irradiation conditions. Preferably, the light irradiation wavelength is 500-1000nm. Under 808nm laser irradiation, near-infrared second-zone fluorescence with a peak value of 930nm can be generated, and a fluorescence signal appears at 1250nm.

[0015] In one embodiment, the perylene compound is selected from a disubstituted compound or a tetrasubstituted compound of perylenetetracarboxylic dianhydride, and the substituent R is selected from any one of Cl, Br, I, and CN.

[0016] In one embodiment, the structural formula of the perylene compound is as follows: Or formula (II) As shown; wherein, R1 and R2 are selected from any one of Cl, Br, I, and CN.

[0017] In a preferred embodiment, the perylene compound is selected from At least one of .

[0018] In a preferred embodiment, the perylene compound is selected from

[0019] In one embodiment, the near-infrared fluorescent carbon nanodots further include a shell covering the carbon-based core.

[0020] Specifically, after the carbon-based core is coated with the above shell, the stability of the near-infrared fluorescent carbon nanodots in water can be improved, and its application ability in the body can be enhanced.

[0021] In one embodiment, the material of the shell is selected from at least one of polyethyleneimine (PEI), methoxypolyethylene glycol (DSPE-mPEG), silicon dioxide (SiO2), 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine (DSPE), and Tween 80.

[0022] In one embodiment, the mass ratio of the carbon-based core to the shell is 1:10 to 3000.

[0023] According to the second aspect of the present disclosure, a method for preparing the above-mentioned near-infrared fluorescent carbon nanodots is provided, comprising the following steps: subjecting a perylene compound to a solvothermal reaction, or mixing a perylene compound with a nitrogen-containing compound and then subjecting the mixture to a solvothermal reaction, to obtain the near-infrared fluorescent carbon nanodots.

[0024] In one embodiment, the nitrogen-containing compound is at least one selected from ammonium fluoride, ammonium bromide, hydroxylamine hydrochloride, and melamine.

[0025] In a preferred embodiment, the nitrogen-containing compound is selected from any one of ammonium fluoride and hydroxylamine hydrochloride.

[0026] In a preferred embodiment, the reaction formula of the perylene compound and the nitrogen-containing compound is as follows:

[0027]

[0028] In a preferred embodiment, the reaction formula of the perylene compound and the nitrogen-containing compound is as follows:

[0029]

[0030] In one embodiment, the perylene compound and the nitrogen-containing compound are mixed in a molar ratio of 1-1000:1-1000.

[0031] In a preferred embodiment, the perylene compound and the nitrogen-containing compound are mixed in a molar ratio of 1-100:1-100.

[0032] In a more preferred embodiment, the perylene compound and the nitrogen-containing compound are mixed in a molar ratio of 1-30:1-30.

[0033] In one embodiment, the perylene compound and the nitrogen-containing compound are mixed at 100-200° C., and then the solvothermal reaction is carried out.

[0034] Specifically, the perylene compound reacts with the nitrogen-containing compound under these high-temperature conditions, incorporating nitrogen into the interior and surface of the carbon-based core, forming a dense CN network structure. This alters the electron cloud distribution of the carbon-based core, enhancing the near-infrared fluorescent carbon nanodots' absorption of red and NIR light, and promoting their fluorescence emission, photothermal, and photodynamic properties in the near-infrared region.

[0035] In one embodiment, the reaction solvent used in the solvothermal reaction includes at least one of dimethyl sulfoxide, N,N'-dimethylformamide, water, ethanol, tetrahydrofuran, acetone, and formamide.

[0036] In one embodiment, the mass-to-volume ratio of the perylene compound to the reaction solvent is (0.01-100) mg / mL, that is, 0.01-100 mg of the perylene compound is dissolved in 1 mL of the reaction solvent.

[0037] In one embodiment, the solvothermal reaction is carried out at a temperature of 100 to 200° C. and for a time of 2 to 20 hours.

[0038] For example, the temperature of the solvent thermal reaction is 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, etc.; the time is 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 20h, etc.

[0039] In one embodiment, after the solvothermal reaction is completed, the following step is further included: mixing the reaction product with a shell material to obtain the near-infrared fluorescent carbon nanodots.

[0040] In one embodiment, the shell material is selected from at least one of polyethyleneimine, methoxypolyethylene glycol, silicon dioxide, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, and Tween 80.

[0041] According to a third aspect of the present invention, there is provided an integrated tumor diagnosis and treatment drug comprising the aforementioned near-infrared fluorescent carbon nanodots.

[0042] According to an embodiment of the present disclosure, there are at least the following beneficial effects:

[0043] The present invention bypasses the barriers of traditional NIR-II fluorescent probes and uses larger conjugated perylene compounds (polyphenyl ring compounds) as raw materials, or mixes perylene compounds with nitrogen-containing compounds as raw materials. Through a solvothermal reaction, a one-pot preparation is performed to obtain carbon nanodots with red and / or near-infrared light-excited photodynamic, photothermal and NIR fluorescence imaging properties. Under visible light and near-infrared laser irradiation, the electrons and holes in the carbon-based core of the carbon nanodots separate, generating a large amount of reactive oxygen species that can kill tumor cells, while also generating a certain photothermal effect that can assist in killing tumor cells (see Figure 1 The incorporation of nitrogen enhances the carbon nanodots' absorption of red and NIR light, promoting their near-infrared fluorescence emission, photothermal, and photodynamic properties, and enhancing their ability to treat deep-seated tumors. Furthermore, under 808nm laser irradiation, these carbon nanodots produce near-infrared fluorescence with an emission peak around 930nm and tailing into the NIR-II region. This fluorescence is sufficiently penetrating to the surface of the skin, enabling epidermal and vascular imaging, and can be used for in vivo tumor and vascular imaging.

[0044] In addition, after being coated with the shell material, the aggregation between carbon nanodots can be reduced, the stability and fluorescence efficiency of carbon nanodots in water / blood can be improved, and the cellular endocytosis efficiency and tumor enrichment efficiency of carbon nanodots can be enhanced. Cancer cells or tumor cells enriched with carbon nanodots can quickly produce a large amount of reactive oxygen species under irradiation with visible light or near-infrared light bands, and at the same time cause a photothermal effect, which can be used for adjuvant treatment of tumors. Under the irradiation of 808nm laser, carbon nanodots dispersed in blood vessels and / or gathered in tumors (coated with shell materials) can emit near-infrared fluorescence, which can be used for NIR-II imaging of blood vessels and / or tumors. Therefore, the carbon nanodots proposed in the present disclosure can be used as or for the preparation of drugs for tumor imaging diagnosis and targeted therapy, thereby realizing the integrated diagnosis and treatment of tumors.

[0045] It should be understood that the contents described in this section are not intended to identify the key or important features of the embodiments of the present disclosure, nor are they intended to limit the scope of the present disclosure. Other features of the present disclosure will become readily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings, in which several embodiments of the present disclosure are shown by way of example and not limitation, wherein:

[0047] In the accompanying drawings, the same or corresponding reference numerals represent the same or corresponding parts, wherein CDs refers to carbon nanodots 1, N refers to carbon nanodots 2, and @ refers to coating.

[0048] Figure 1 The schematic diagram of the light-induced reaction of the near-infrared carbon nanodots prepared in the present invention is shown; wherein S0 is the ground state energy level, and S2 is the excited state energy level;

[0049] Figure 2 shows a transmission electron microscope scanning image of carbon nanodots 1 and 2 in Experimental Example 1 of the present disclosure;

[0050] Figure 3 The XPS spectra of carbon nanodots 1 and 2 in Experimental Example 1 of the present disclosure are shown;

[0051] Figure 4 shows the hydrogen nuclear magnetic resonance spectra of carbon nanodots 1 and 2 in Experimental Example 1 of the present disclosure;

[0052] Figure 5 shows the ultraviolet absorption spectra of carbon nanodots 1 and 2 in Experimental Example 2 of the present disclosure;

[0053] Figure 6 The fluorescence spectra of carbon nanodots 1 and 2 in the visible light band in Experimental Example 2 of the present disclosure are shown;

[0054] Figure 7 The fluorescence spectra of carbon nanodots 1 and 2 in Experimental Example 2 of the present disclosure under 808 nm laser irradiation are shown; wherein each curve represents a different concentration of carbon nanodots, and the specific concentration values ​​are marked in the upper right corner of the figure;

[0055] Figure 8 shows the XRD spectra of carbon nanodots 1 and 2 in Experimental Example 2 of the present disclosure;

[0056] Figure 9 shows the Fourier infrared spectra of carbon nanodots 1 and 2 in Experimental Example 3 of the present disclosure;

[0057] Figure 10 shows the Raman spectra of carbon nanodots 1 and 2 in Experimental Example 3 of the present disclosure;

[0058] Figure 11 The ultraviolet diffuse reflectance and band gap spectra of carbon nanodots 1 and 2 in Experimental Example 4 of the present disclosure are shown;

[0059] Figure 12 The photothermal effect diagram of carbon nanodots 1 and 2 under 808 nm laser irradiation in Experimental Example 5 of the present disclosure is shown;

[0060] Figure 13 Schematic diagram of the photothermal cycle of carbon nanodots 2 under 808 nm laser irradiation in Experimental Example 5 of the present disclosure is shown;

[0061] Figure 14 The effect of carbon nanodots 1 and 2 generating active oxygen under 660nm laser irradiation in Experimental Example 6 of the present disclosure on the degradation of DPBF is shown;

[0062] Figure 15 The effect of carbon nanodots 1 and 2 generating active oxygen under 808 nm laser irradiation in Experimental Example 6 of the present disclosure on the degradation of DPBF is shown;

[0063] Figure 16 The carbon nanodots 1 and 2 in Experimental Example 6 of the present disclosure are shown to generate 1 ESR spectrum of O2;

[0064] Figure 17 The cytotoxicity test diagram of carbon nanodots 2 at different concentrations in Experimental Example 7 of the present disclosure is shown;

[0065] Figure 18 The graph shows the results of the hemolysis test of carbon nanodots 2 at different concentrations in Experimental Example 8 of the present disclosure; wherein the first row of test tubes contains carbon nanodot 2 solution, and the second row of test tubes contains carbon nanodot 2 solution and blood cells;

[0066] Figure 19 The transmission electron microscope image of the composite carbon nanodots N-CDs@SiO2 in Experimental Example 9 of the present disclosure is shown;

[0067] Figure 20 The fluorescence spectrum of the composite carbon nanodots in Experimental Example 9 of the present disclosure under 808 nm laser irradiation is shown;

[0068] Figure 21 The figure shows the result of cell uptake of N-CDs@DSPE-mPEG in Experimental Example 10 of the present disclosure;

[0069] Figure 22 The figure shows the fluorescence effect of reactive oxygen species generated by N-CDs@DSPE-mPEG in Experimental Example 10 of the present disclosure after being taken up by 4T1 cells under 808 nm laser irradiation;

[0070] Figure 23 The figure shows the killing effect of N-CDs@DSPE-mPEG on 4T1 cells after being taken up by 4T1 cells and irradiated with 808nm laser in Experimental Example 10 of the present disclosure;

[0071] Figure 24 The near-infrared fluorescence images of carbon nanodots 2 (N-CDs) and N-CDs@PEI in Experimental Example 11 of the present disclosure are shown;

[0072] Figure 25 The fluorescence imaging of the subcutaneous, abdominal and brain blood vessels of mice under 808 nm laser irradiation of N-CDs@DSPE-mPEG in Experimental Example 11 of the present disclosure is shown;

[0073] Figure 26 A temperature stability test graph of near-infrared fluorescence of carbon nanodots 1 and 2 in Experimental Example 12 of the present disclosure is shown;

[0074] Figure 27 The graph shows the in vitro tumor size of mice after 14 days of treatment with various types of carbon nanodots under light and without light in Experimental Example 13 of the present disclosure. DETAILED DESCRIPTION

[0075] To make the purposes, features, and advantages of the present disclosure more apparent and understandable, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present disclosure without creative work shall fall within the scope of protection of the present disclosure.

[0076] Example 1

[0077] In this embodiment, carbon nanodots 1 were prepared, and the specific process was as follows:

[0078] Weigh 0.02 g of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride. Add 30 mL of N,N'-dimethylformamide to a polytetrafluoroethylene reactor. Place the reactor in an oven at 160°C for 10 hours. Remove the solution, filter it through dialysis, and freeze-dry it to obtain carbon nanodots 1.

[0079] Example 2

[0080] In this embodiment, carbon nanodots 2 were prepared, and the specific process was as follows:

[0081] Weigh 0.02 g of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride and 0.03 g of hydroxylamine hydrochloride. Then, add 30 mL of N,N'-dimethylformamide into a polytetrafluoroethylene reactor. Place the reactor in an oven at 160°C for 10 hours. The solution is then removed, diafiltered, and freeze-dried to obtain carbon nanodots 2.

[0082] Example 3

[0083] In this embodiment, carbon nanodots 3 were prepared, and the specific process was as follows:

[0084] Weigh 0.05 g of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride and 0.1 g of ammonium fluoride. Then, add 30 mL of N,N'-dimethylformamide into a polytetrafluoroethylene reactor. Place the reactor in an oven at 160°C for 10 hours. The solution is then removed, diafiltered, and freeze-dried to obtain carbon nanodots 3.

[0085] Example 4

[0086] In this embodiment, carbon nanodots 4 were prepared, and the specific process was as follows:

[0087] Weigh 0.03 g of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride and 0.03 g of melamine. Then, add 30 mL of N,N'-dimethylformamide into a polytetrafluoroethylene reactor. Place the reactor in an oven at 160°C for 10 hours. The solution is then removed, diafiltered, and freeze-dried to obtain carbon nanodots 4.

[0088] Example 5

[0089] In this embodiment, carbon nanodots 5 were prepared, and the specific process was as follows:

[0090] Weigh 0.01 g of 1,7-dibromo-3,4,9,10-perylenetetracarboxylic dianhydride and 0.03 g of hydroxylamine hydrochloride. Then, add 30 mL of N,N'-dimethylformamide into a polytetrafluoroethylene reactor. Place the reactor in an oven at 160°C for 10 hours. The solution is then removed, diafiltered, and freeze-dried to obtain carbon nanodots 5.

[0091] Test example

[0092] 1. This test example analyzes the structures of carbon nanodots 1 and 2 prepared in Examples 1 and 2 by transmission electron microscopy, atomic force microscopy and XPS spectroscopy. Figure 2As shown in the figure, the average particle size of carbon nanodots 1 (non-nitrogen-doped carbon nanodots, CDs) is about 4-5nm, and the average particle size of carbon nanodots 2 (nitrogen-doped carbon nanodots, N-CDs) is about 5-7nm. Under high magnification, the lattice fringe spacing of the two carbon nanodots is 0.21nm, which proves that the two carbon nanodots are successfully prepared. And with the doping of nitrogen, their average particle size increases. XPS and 1 H NMR analysis, the results are as follows Figure 3 and 4 As shown, both carbon nanodots 1 and 2 exhibit C, N, O, and Cl signals. Furthermore, for carbon nanodots 2 (N-CDs), the XPS analysis reveals a significant increase in the electron binding energy of N at 400 eV with the addition of nitrogen-containing compounds, indicating that the incorporation of nitrogen-containing compounds significantly increases the nitrogen content in carbon nanodots 2. Furthermore, the N XPS spectrum reveals that most nitrogen is incorporated into the carbon skeleton through NC, NN, and NO to form carbon nanodots, promoting electron transfer within the carbon-based core network.

[0093] 2. This test example conducted UV analysis, fluorescence spectrum analysis and XRD analysis on the structure of carbon nanodots 1 and 2. Figure 5 As shown in the figure, it can be seen that the absorption peak of carbon nanodots 1 (CDs) is mainly between 300 and 500 nm, while carbon nanodots 2 (N-CDs) have strong ultraviolet absorption between 500 and 1000 nm, indicating that both carbon nanodots can make full use of light within this wavelength range. Figure 6 As shown in Figure 2, the optimal excitation peak and emission peak of carbon nanodots 1 (CDs) are 500nm and 610nm respectively; the optimal excitation peak and emission peak of carbon nanodots 2 (N-CDs) are 500nm and 580nm respectively. When excited by 808nm laser, the fluorescence spectrum is as shown in Figure 2. Figure 7 As shown in the figure, it can be seen that the fluorescence emission peak of carbon nanodots 1 (CDs) is around 930nm, and the fluorescence emission peak of carbon nanodots 2 (N-CDs) is also around 930nm, indicating that both types of carbon nanodots can produce near-infrared fluorescence. XRD analysis of the two types of freeze-dried carbon nanodots is performed, and the results are shown in the figure. Figure 8 As shown, Figure 8 It shows that both carbon nanodots 1 (CDs) and carbon nanodots 2 (N-CDs) exhibit obvious carbon dot crystal characteristic peaks at around 25°C, indicating the successful preparation of the two types of carbon nanodots.

[0094] 3. This test example carried out Fourier transform infrared analysis and Raman spectroscopy analysis on the structures of carbon nanodots 1 and 2. Figure 9As shown in the figure, it can be seen that carbon nanodots 1 (CDs) are synthesized using pure perylene compounds as raw materials. Their main functional group structures and valence bonds are OH and CC, C=C, C=O bonds and CN bonds within the benzene ring skeleton. However, due to the incorporation of nitrogen elements, the peak of the CN bond of carbon nanodots 2 (N-CDs) increases significantly, accompanied by a red shift of CC and CN, indicating that the size of the synthesized carbon nanodots increases with the incorporation of nitrogen elements. Raman spectroscopy analysis is shown in Figure 2. Figure 10 As shown, Figure 10 The results show that both carbon nanodots 1 (CDs) and carbon nanodots 2 (N-CDs) have a D band representing disorder and a G band representing order. Furthermore, after nitrogen doping, the D band peak tends to increase, indicating that nitrogen doping introduces more vacancies. The above data also proves the successful preparation of carbon nanodots.

[0095] 4. This test example measures the ultraviolet diffuse reflectance of the carbon nanodots 1 and 2 and calculates the band gap. By processing the spectrum data, the energy band gap spectrum is drawn, as shown in the following figure: Figure 11 shown. Figure 11 The band gap of carbon nanodots 1 (CDs), prepared using perylene compounds, is around 1.35 eV, while that of carbon nanodots 2 (N-CDs), prepared by doping with nitrogen-containing compounds, is even smaller, at around 1.14 eV. The significantly smaller band gap of carbon nanodots 2 than that of carbon nanodots 1 allows their valence band electrons to transition to the conduction band even at lower energies, forming electron-hole pairs. This promotes the generation of photothermal and reactive oxygen species, while also enabling near-infrared fluorescence.

[0096] 5. This test example tests the photothermal properties of carbon nanodots 1 and 2. A carbon nanodot solution of a certain concentration and volume is placed in a cuvette. Under 808nm laser irradiation, a thermal imager is used to record the temperature of the sample at various time points. The laser power is selected to be 0.5-1W / cm 2 , the illumination time is 0 to 20 hours, and it is plotted into a light-heat curve. The results are as follows Figure 12 shown. Figure 12 It shows that compared with pure water or carbon nanodots 1 (CDs), carbon nanodots 2 (N-CDs) exhibits better photothermal performance. At the same time, carbon nanodots 2 (N-CDs) were subjected to five heating / cooling cycles under 808nm laser irradiation. Figure 13 shown. Figure 13 It shows that after five hot and cold cycles, its photothermal performance has basically not changed, indicating that carbon nanodots 2 have good photothermal stability.

[0097] 6. This test example tests the photodynamic properties of carbon nanodots 1 and 2. 1,3-diphenylisobenzofuran (DPBF) was used as an active oxygen scavenger and methyl blue (MB) was used as a reference to measure the photodynamic properties of carbon nanodots under 660nm or 808nm laser irradiation. 1 The production of O2, the results are as follows Figure 14 and 15 As shown. It can be seen that under each laser irradiation, both types of carbon nanodots can produce a large amount of reactive oxygen species, causing the absorbance of DPBF to show a time-dependent decrease. At the same time, the reactive oxygen species production ability of carbon nanodot 1 (CDs) under 660nm laser irradiation is stronger than that of carbon nanodot 2 (N-CDs); and under 808nm laser irradiation, the reactive oxygen species production ability of carbon nanodot 2 (N-CDs) is stronger than that of carbon nanodot 1 (CDs). By comparison with the standard photosensitizer MB, it can be calculated that the singlet oxygen quantum yields of the two types of carbon nanodots under 660nm laser irradiation are 1.5 (CDs) and 0.4 (N-CDs). The carbon nanodots were then combined with electron spin traps and TEMP, and the production of singlet oxygen was detected before and after illumination. The electron spin spectroscopy (ESR) results confirmed that no singlet oxygen was produced without illumination. Once irradiated with 808nm laser, significant singlet oxygen ( Figure 16 ), further indicating that carbon nanodots 1 and 2 have significant production 1 O2 capacity, and carbon nanodots 2 have better performance under 808nm laser irradiation. 1 O2 production capacity.

[0098] 7. This test example tests the cytotoxicity of carbon nanodots 2. The MTT method was used to detect the cytotoxicity of carbon nanodots 2. Carbon nanodots 2 were prepared into aqueous solutions with concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 μg / mL, and added to 96-well plates with L929 cells plated one day in advance. Six parallel experiments were performed in each group. After incubation for 24 hours, 10 μL of MTT solution was added and incubated for another 4 hours. After that, the cells were shaken on a shaker and the absorbance at a specific wavelength was detected in an enzyme-labeled instrument. The results are as follows: Figure 17 shown. Figure 17 The results showed that carbon nanodots 2 at concentrations of 400 μg / mL and below had no obvious damage to cells, indicating that carbon nanodots 2 had good biocompatibility.

[0099] 8. In this test example, a hemolysis experiment was conducted on carbon nanodots 2 to detect its biological toxicity in vivo. Freshly collected mouse blood was centrifuged and washed to remove impurities in the upper plasma layer. 500 μL of the lower blood cells were taken for standby use. Carbon nanodots 2 were prepared into PBS solutions with concentrations of 2 mg / mL, 1 mg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL. Pure water and PBS were used as positive and negative controls. 500 μL of each was added to the prepared blood cells and incubated at room temperature for 1 hour. The solution was then placed in a centrifuge at 12,000 rpm / min for 10 minutes. The supernatant was placed in a 96-well plate and the absorbance at a specific wavelength was detected using an enzyme reader. The results are as follows: Figure 18 shown. Figure 18 The results showed that carbon nanodots 2 at concentrations of 2 mg / mL and below had no obvious damage to blood cells, indicating that carbon nanodots 2 have good biocompatibility in vivo.

[0100] 9. To improve the stability of carbon nanodots in water or blood, and enhance their optical properties and cellular endocytosis, PEI, DSPE-mPEG or SiO2 were used to encapsulate and composite carbon nanodots 2. The mass ratio of carbon nanodots 2 to the coating material was 1:10 to 3000. The composite carbon nanodots were observed by transmission electron microscopy. The results are as follows: Figure 19 As shown in Figure 2, it can be seen that after SiO2 coating, the average particle size increases from about 5nm to about 50nm. Then, the carbon nanodots 2 before and after being composited with the coating material were tested for near-infrared fluorescence. The results are shown in Figure 2. Figure 20 As shown, it can be seen that the fluorescence of the carbon nanodots 2 can still be maintained by the coating material compound.

[0101] 10. This experiment studied the effect of light on the expression of reactive oxygen species in cells and the killing of cancer cells by composite carbon nanodots. 62.5 μg / mL of the composite carbon nanodots N-CDs@DSPE-mPEG in Experiment 9 were placed in 4T1 cells that had been plated in advance and incubated for different time gradients. The cells were placed under an inverted fluorescence microscope to observe the endocytosis of the composite carbon nanodots (Ex = 450 nm, Em = 520 nm). The results are as follows: Figure 21 shown. Figure 21 The results showed that the composite carbon nanodots were taken up by cells and accumulated in the cells within 10 minutes. Then, the reactive oxygen species (ROS) fluorescence reagent DCFH-DA (Ex = 502nm, Em = 530nm) was added to the above group. After irradiation with 808nm laser for 10 minutes, the ROS fluorescence effect in the cells was observed. The results were as follows: Figure 22 shown. Figure 22The results showed that N-CDs@DSPE-mPEG produced significant reactive oxygen species, and with the extension of illumination time, the fluorescence effect became more obvious. The cells incubated with the composite carbon nanodots were then irradiated with 808nm laser. After 12 hours, the cells were stained with calcein (Calcein-AM; Ex = 488nm, Em = 530nm) / pyridinium iodide (PI; Ex = 535nm, Em = 617nm) to detect cell survival. The results are as follows: Figure 23 shown. Figure 23 The results showed that compared with the PBS control group, the cancer cells added with composite carbon nanodots were effectively killed. The above experiments show that composite carbon nanodots can be taken up by cells in a short time and can significantly produce reactive oxygen species under 808nm laser irradiation to kill cancer cells.

[0102] 11. In this experiment, carbon nanodots 2 (N-CDs) and composite carbon nanodots N-CDs@PEI were diluted in a concentration gradient (100, 80, 60, 40, 20, 10, 5, 0 μg / mL, respectively), and fluorescence imaging was performed using a small animal fluorescence imaging instrument. The results are as follows: Figure 24 shown. Figure 24 It shows that under 808nm laser irradiation, both carbon nanodots exhibit bright near-infrared fluorescence. At the same time, N-CDs@DSPE-mPEG was injected into the subcutaneous tissue of mice, and obvious fluorescence signals were observed through the mouse imaging system (see Figure 25 ); The composite carbon nanodots were injected into mice through the tail vein, and blood vessel fluorescence imaging of the mice's legs, abdomen, and brain was observed under a near-infrared zone II camera (see Figure 25 The above experiments show that both carbon nanodots and composite carbon nanodots have near-infrared fluorescence signals and can be used for fluorescence imaging of the subcutaneous tissue and blood vessels of mice.

[0103] 12. This test example tests the near-infrared fluorescence stability of carbon nanodots 1 and 2 at different temperatures. DMF solutions of two types of carbon nanodots with fixed concentrations were placed in an environment of 20-60°C, and their fluorescence signals were detected using a near-infrared fluorescence spectrometer. The results are as follows: Figure 26 shown. Figure 26 It shows that both types of carbon nanodots have excellent temperature stability, and there is no obvious difference in the fluorescence effect at different temperatures.

[0104] 13. BABL / c mice with orthotopic 4T1 tumors were enrolled in the study when the tumors grew to 80 mm. 3 The cells were treated in situ by injecting 200 μL of PBS, carbon nanodots 1 (CDs), carbon nanodots 2 (N-CDs) and N-CDs@DSPE-mPEG, respectively. The cells were divided into two groups and irradiated with light or without light. The irradiated group was treated with 808 nm laser at 1 W / cm2 The results showed that the N-CDs and N-CDs@DSPE-mPEG injection groups had a significant inhibitory effect on tumors after illumination, indicating a significant near-infrared phototherapy effect. Furthermore, the N-CDs@DSPE-mPEG group also had a significant inhibitory effect on distal tumors after illumination, indicating a certain immunotherapeutic effect.

[0105] Then, after 14 days of treatment, the BABL / c mice bearing 4T1 tumors were sacrificed and all tumors were removed. Figure 27 As shown in the figure, the tumors of all mice in the non-light-treated group were larger than those in the light-treated group, and the N-CDs@DSPE-mPEG light-treated group showed a more excellent tumor treatment effect than the N-CDs light-treated group and the CDs light-treated group. After 14 days, the tumors were almost completely eliminated.

[0106] In summary, the carbon nanodots and composite carbon nanodots prepared by the present invention have photodynamic and photothermal effects excited by red light and / or near-infrared light, wherein the carbon nanodots not doped with nitrogen show excellent photodynamic effects under 660nm laser irradiation, while the nitrogen-doped carbon nanodots show excellent photodynamic and photothermal effects under 808nm laser irradiation, and can be used to kill tumor cells. At the same time, under 808nm laser irradiation, both types of carbon nanodots can produce fluorescence of about 930nm, which is sufficient to penetrate the surface of the skin, and therefore have the ability to image the epidermis and blood vessels. After being coated with an outer shell. Its stability in water can be improved, and its in vivo application capability can be enhanced. It can be used as a drug for tumor imaging diagnosis and targeted therapy, and can be used in the integrated design of tumor diagnosis and treatment.

[0107] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions of this disclosure can be achieved. This is not a limitation herein.

[0108] The above description is merely a specific embodiment of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A near-infrared fluorescent carbon nanodot, characterized in that: The near-infrared fluorescent carbon nanodots include a carbon-based core made of a perylene compound or a nitrogen-doped perylene compound as a precursor; Among them, the near-infrared fluorescent carbon nanodots comprising a carbon-based core made of a perylene compound as a precursor have an absorption band in the visible light wavelength range of 380 to 750 nm; the near-infrared fluorescent carbon nanodots comprising a carbon-based core made of a nitrogen-doped perylene compound as a precursor have an absorption band in the visible light and near-infrared light wavelength range of 500 to 1200 nm; The fluorescence emission wavelength of the near-infrared fluorescent carbon nanodots is within the range of 800 to 1500 nm.

2. The near-infrared fluorescent carbon nanodots according to claim 1, characterized in that The perylene compound is selected from a disubstituted compound or a tetrasubstituted compound of perylenetetracarboxylic dianhydride, and the substituent R is selected from any one of Cl, Br, I, and CN.

3. The near-infrared fluorescent carbon nanodots according to claim 1, characterized in that The near-infrared fluorescent carbon nanodots further include a shell covering the carbon-based core; Preferably, the material of the shell is selected from at least one of polyethyleneimine, methoxypolyethylene glycol, silicon dioxide, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, and Tween 80; Preferably, the mass ratio of the carbon-based core to the shell is 1:10-3000.

4. The method for preparing near-infrared fluorescent carbon nanodots according to any one of claims 1 to 3, characterized in that: The following steps are involved: The near-infrared fluorescent carbon nanodots are prepared by subjecting the perylene compound to a solvent thermal reaction, or by mixing the perylene compound with a nitrogen-containing compound and then subjecting the mixture to a solvent thermal reaction.

5. The preparation method according to claim 4, characterized in that The nitrogen-containing compound is selected from at least one of ammonium fluoride, ammonium bromide, hydroxylamine hydrochloride and melamine.

6. The preparation method according to claim 4, characterized in that The perylene compound and the nitrogen-containing compound are mixed in a molar ratio of 1 to 1000:1 to 1000; Preferably, the perylene compound and the nitrogen-containing compound are mixed at 100-200° C., and then the solvothermal reaction is carried out.

7. The preparation method according to claim 4, characterized in that The reaction solvent used in the solvothermal reaction includes at least one of dimethyl sulfoxide, N,N'-dimethylformamide, water, ethanol, tetrahydrofuran, acetone, and formamide; Preferably, the temperature of the solvent thermal reaction is 100-200° C., and the time is 2-20 h.

8. The preparation method according to claim 4, characterized in that After the solvothermal reaction is completed, the method further comprises the following steps: mixing the reaction product with the shell material to obtain the near-infrared fluorescent carbon nanodots.

9. The preparation method according to claim 8, characterized in that The shell material is selected from at least one of polyethyleneimine, methoxypolyethylene glycol, silicon dioxide, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, and Tween 80.

10. An integrated drug for tumor diagnosis and treatment, characterized in that: The integrated tumor diagnosis and treatment drug comprises the near-infrared fluorescent carbon nanodots according to any one of claims 1 to 3.

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