Near-infrared carbon dots capable of efficiently generating active oxygen and having targeting capability, and preparation method and application thereof

Abstract

The invention discloses a preparation method and application of near-infrared carbon dots capable of efficiently generating active oxygen and having targeting capacity, and belongs to the technical field of carbon nanomaterials. Compared with conventional blue-green light carbon dots, the near-infrared carbon dot provided by the invention has the advantages of large tissue penetration depth, small self-luminous interference of organisms, small damage to tissues and the like. The carbon dots provided by the invention can efficiently generate singlet oxygen, and have the capability of photodynamically killing tumor cells. Besides, abundant functional groups on the surfaces of the carbon dots are utilized, molecules capable of targeting organelles are modified on the surfaces of the carbon dots, so that the carbon dots can accurately position and target mitochondria, and compared with carbon dots without targeting capability, the carbon dots have stronger killing power on cancer cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of carbon nanomaterials, and particularly relates to a near-infrared carbon dot capable of efficiently producing active oxygen and having a targeting ability, a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy is a method of clinical treatment using laser and photosensitizer. When irradiated by laser, the photosensitizer can transfer energy to the surrounding oxygen, converting it into active oxygen, thereby killing cancer cells. In the PDT treatment process, the photosensitizer plays an irreplaceable role. The photosensitizer mainly plays a role through two types of photodynamic reactions: Type I reaction involves hydrogen atom or electron transfer between the triplet excited state photosensitizer and the substrate, generating free radicals or free radical ions, which interact with activated oxygen in the tissue to produce cytotoxic oxidants; Type II reaction is that the triplet excited state photosensitizer directly transfers energy to the oxygen molecule to form singlet oxygen with strong oxidizing property, thereby causing cancer cell death.

[0003] At present, the photosensitizers used in clinical applications mainly include porphyrin, phthalocyanine, chlorophyll derivatives and other hydrophobic photosensitizers containing large π conjugated structure. For example, patent CN117186055A discloses a photosensitizer based on triphenylamine structure and its synthesis method and application. The patent is based on triphenylamine as an electron donor and pyridine salt as an electron acceptor, and a series of organic photosensitizers based on triphenylamine structure are synthesized. The organic photosensitizer has the ability to quickly enter cells, quickly produces ROS under white light irradiation, and can target mitochondria to achieve efficient killing of tumor cells, and also can induce cancer cell death by inducing the expression of apoptosis factors. However, these commonly used photosensitizers have the disadvantages of poor water solubility, dependence on harmful organic reagents, easy aggregation, complex synthesis and high cost, thereby limiting the effect of photodynamic therapy, and it is impossible to prepare photosensitizers with high photosensitivity and targeting ability through simple process.

[0004] In comparison, carbon dots can also be used for photodynamic therapy due to their good biocompatibility, low toxicity and good stability. For example, patent CN118995211A discloses a low-toxicity red luminescent carbon dot synthesized by a simple hydrothermal method using anticancer drug 5-fluorouracil and photosensitizer indocyanine green as precursors, which improves the disadvantage of large side effects of chemotherapy drugs and achieves targeted killing of liver cancer cells. However, this scheme needs to rely on exogenous photosensitizer indocyanine green as a precursor, the emission wavelength of the prepared carbon dots is still in the red light range, and the direct correlation between singlet oxygen yield and photodynamic therapy effect has not been verified.

[0005] Carbon dots (CDs) are a new type of zero-dimensional carbon-based nanomaterials, generally less than 10 nm in size, with wide absorption spectrum range, high fluorescence quantum yield, good water solubility, excellent optical stability and good biocompatibility, and thus are widely used in the fields of biological imaging, photocatalysis and diagnosis and treatment. However, in the prior art, the photodynamic performance of carbon dots is often difficult to be compatible with their optical properties: the currently reported carbon dots are mainly modified by introducing noble metals or conjugated molecules to improve the ROS yield, but these methods will complicate the synthesis process and increase the toxicity; and the luminescence region of the currently prepared carbon dots is mostly in the blue-green light range. The tissue penetration depth of blue-green light is shallow, only 1-2 mm, which greatly limits the application of carbon dots in the biomedical field. Compared with blue-green light carbon dots, red / near-infrared (600-950 nm) luminescence carbon dots have the advantages of large tissue penetration depth, less interference from the spontaneous light of the organism, and less damage to the tissue. In addition, most carbon dots with photodynamic performance do not have targeting properties, resulting in limited killing power on cancer cells. Studies have shown that photodynamic carbon dots with targeting properties have better treatment effect than carbon dots without targeting properties. Although some studies have tried to endow carbon dots with targeting properties through surface modification, such modification usually requires the introduction of additional functional groups and relies on physical adsorption or non-covalent binding, and the modified carbon dots are prone to lose the targeting molecules, and the modification process may also destroy the photosensitive activity of the carbon dots.

[0006] Therefore, there is an urgent need to develop a near-infrared carbon dot that can efficiently produce active oxygen, has the ability to kill tumor cells by photodynamic effect, has targeting ability, can accurately locate the target organelle, has stronger killing power on cancer cells, and does not need to introduce exogenous photosensitive molecules or noble metals; and meets the application requirements of low toxicity and low cost. SUMMARY

[0007] In view of the above technical problems in the prior art, the present application provides a preparation method of a near-infrared carbon dot (hereinafter also referred to as carbon dot) which uses glutathione and methionine as carbon sources and formamide as a solvent to synthesize by a solvothermal method. The near-infrared carbon dot provided by the present application has spectral absorption and fluorescence emission in the near-infrared light region. The near-infrared carbon dot provided by the present application can efficiently produce singlet oxygen under near-infrared light irradiation, and has the ability to kill tumor cells by photodynamic effect. Since the carbon source is rich in amino and carboxyl groups, the surface of the synthesized carbon dot is rich in amino and carboxyl groups. The carboxyl group-containing triphenylphosphine is reacted with the amino group on the surface of the carbon dot, and since the triphenylphosphine molecule has the ability to target mitochondria, the carbon dot can accurately locate the mitochondria, thereby increasing the killing power on cancer cells.

[0008] The application provides a synthesis and modification method of near-infrared carbon dots and application of the carbon dots in preparation of a medicine for killing cancer cells by photodynamic action. The carbon dots are synthesized by a one-step solvothermal method with glutathione and methionine as carbon sources and formamide as a solvent, and have near-infrared absorption and high-efficiency singlet oxygen generation capacity, and the mitochondrion-targeting property is endowed by surface covalent modification.

[0009] For the technical solution described above, further preferably, the basic synthesis step comprises: mixing and dissolving glutathione and methionine in formamide at a mass ratio of 1:1 to 10:1, transferring to a high-temperature-resistant sealed reactor, reacting at 120-200 DEG C for 4-12 hours, diluting with ultrapure water after cooling, and preparing an initial carbon dot precursor solution; the mass ratio is further preferably 2:1 to 5:1; most preferably, the mass ratio is 3:1. Wherein, the "initial carbon dot precursor solution" refers to a mixed solution containing carbon dots after solvothermal reaction. Further preferably, the formamide solvent is used in an amount of 10-15 mL per 0.5 g of raw material.

[0010] For the technical solution described above, further preferably, the temperature of the solvothermal reaction is controlled at 140-180 DEG C; and the reaction time is 6-10 hours; further preferably, the reaction temperature is limited to 155-170 DEG C; most preferably, the reaction temperature is 160 DEG C and the reaction lasts for 8 hours.

[0011] For the technical solution described above, further preferably, the purification process of the initial carbon dot precursor solution comprises light-avoiding dialysis, filtration and freeze-drying.

[0012] For the technical solution described above, further preferably, the molecular weight cut-off of the dialysis in the light-avoiding dialysis step is 2000-5000 Da, the dialysis time is 48-96 hours, and the dialysis liquid is replaced every 4-8 hours; further preferably, the molecular weight cut-off of the dialysis is 3000-4000 Da, the dialysis time is 60-84 hours, and the dialysis liquid is replaced every 5-7 hours; most preferably, the dialysis parameters are set as a molecular weight cut-off of 3500 Da, a dialysis time of 72 hours, and a dialysis liquid replacement every 6 hours.

[0013] For the technical solution described above, further preferably, the storage temperature of the freeze-drying step is -80 to -20 DEG C, and the pre-freezing time is ≥12 hours; further preferably, the storage temperature is set to -60 to -30 DEG C and the pre-freezing time is 24 hours; most preferably, the pre-freezing temperature is fixed at -80 DEG C and maintained for 24 hours, and the vacuum drying time is 48 hours.

[0014] For the technical solutions described above, further preferably: the carbon dots are covalently modified on the surface by covalent coupling of surface amino groups with carboxylated triphenylphosphine (TPP-COOH); the mass ratio of TPP-COOH to carbon dots in the coupling reaction is 50:1 to 200:1, the activation reaction pH is adjusted to 4.5-6.0 and the activation time is 20-40 minutes; further preferably, the mass ratio is limited to 80:1 to 150:1, the activation pH is optimized to 5.2-5.6 and the activation time is 30 minutes; most preferably, the mass ratio is 100:1, the activation pH is strictly 5.4 and the activation time is 30 minutes.

[0015] For the technical solutions described above, further preferably: the final pH of the coupling reaction is adjusted to 6.8-7.5, the reaction time is 12-36 hours and the temperature is 25-37℃; further preferably, the pH is controlled to 7.0-7.3, the reaction time is limited to 24 hours and the temperature is 25℃; most preferably, the reaction pH is 7.2, the reaction is maintained at 25℃ for 24 hours.

[0016] The second aspect of the present application is to protect the carbon dots prepared by the above method; the singlet oxygen yield thereof is ≥0.3, and the molar ratio of surface amino groups to carboxyl groups is ≥2:1; further preferably, the singlet oxygen yield is ≥0.34 and the amino group content is ≥65%; most preferably, the measured singlet oxygen yield is 0.34-0.36 and the amino group content is 70%; and the fluorescence intensity decays by ≤10% within 8 hours of 660nm light irradiation. The colocalization coefficient of the near-infrared carbon dots and mitochondria is ≥0.8.

[0017] The third aspect of the present application is to protect the use of the above near-infrared carbon dots in the preparation of a drug for photodynamic killing of cancer cells.

[0018] For the technical solutions described above, further preferably: the near-infrared carbon dots are used for targeted positioning of mitochondria. It is proved by cell colocalization experiments (see Figure 7 ), the colocalization coefficient of the carbon dots modified by TPP and mitochondrial dye is 0.84, indicating that the carbon dots can accurately locate to mitochondria.

[0019] For the technical solutions described above, further preferably: the drug for photodynamic killing of cancer cells comprises near-infrared carbon dots and a pharmaceutically acceptable carrier, wherein the mass concentration of near-infrared carbon dots is 10-200μg / mL; the active oxygen yield is ≥90%.

[0020] For the technical solutions described above, further preferably: the application includes preparing a photodynamic therapy drug for breast cancer, liver cancer or lung cancer.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] (1) The near-infrared carbon dots provided by the application are simple and easy to synthesize, and have strong operability.

[0023] (2) The near-infrared carbon dots prepared by the application are rich in functional groups such as carboxyl and amino groups on the surface, so that other groups or composite materials can be introduced, and more functions can be obtained.

[0024] (3) The near-infrared carbon dots prepared by the application have a certain ultraviolet absorption in the near-infrared region, and the fluorescence emission is also located in the near-infrared region, and has the characteristic of fluorescence emission independence, that is, the fluorescence emission is not related to the excitation light.

[0025] (4) The near-infrared carbon dots prepared by the application can efficiently generate singlet oxygen (0.3414) under near-infrared light (660nm) irradiation, and the efficiency can be compared with that of the reference methylene blue molecule (0.52). The targeting achieved by covalent modification is more stable, and cell experiments prove that the carbon dots after modification can efficiently enrich in mitochondria (co-localization coefficient 0.84), overcoming the problem of falling off of the targeting molecules in the physical adsorption modification method.

[0026] (5) The near-infrared carbon dots prepared by the application have the ability to target mitochondria, and the surface of the carbon dots is modified with a mitochondria-targeting triphenylphosphine (TPP-COOH) molecule using the abundant amino groups on the surface of the carbon dots. The Zeta potential on the surface of the carbon dots changes obviously before and after the reaction. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The transmission electron microscope picture of the near-infrared carbon dots prepared in Example 3, the scale is 10nm.

[0028] Figure 2 The ultraviolet absorption spectrum and fluorescence emission spectrum of the near-infrared carbon dots prepared in Example 3.

[0029] Figure 3 The Fourier infrared spectrum (FTIR) test diagram of the near-infrared carbon dots prepared in Example 3.

[0030] Figure 4 (a-d) The yield of singlet oxygen generated by the near-infrared carbon dots prepared in Examples 1-4 in DMSO solution under 660nm excitation light irradiation.

[0031] Figure 5 The stability test of the near-infrared carbon dots prepared in Example 3 under near-infrared light (660nm) irradiation.

[0032] Figure 6 The Zeta potential change diagram of the near-infrared carbon dots prepared in Example 3 before and after the surface is modified with triphenylphosphine molecules.

[0033] Figure 7 Figure 4 shows the co-localization of NIR carbon dots prepared in Example 3 with mitochondria after being uptaken by cells.

[0034] Figure 8 (a-d) are the yields of singlet oxygen generated by NIR carbon dots prepared in Comparative Examples 1-3 in DMSO solution under irradiation of 660 nm excitation light. DETAILED DESCRIPTION

[0035] The specific embodiments of the present application are further described in the following detailed description with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Unless otherwise specified, the chemical reagents and biological materials used in the present application are obtained through commercial channels and are processed and applied according to standard operating procedures. All experimental steps are carried out under conventional laboratory conditions to ensure the repeatability and reliability of the technical solutions.

[0036] Example 1

[0037] 0.4375 g of glutathione and 0.0625 g of methionine were weighed and dissolved in 12 mL of formamide, and then fully mixed by an ultrasonic device. The mixture was transferred to a polytetrafluoroethylene-lined high-temperature reaction kettle, and reacted at 160°C for 8 h. After cooling to room temperature, the solution was diluted with ultrapure water. The carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, with water changed every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 μm filter membrane, and finally freeze-dried to obtain NIR carbon dots NIR-CDs-1, which were stored at room temperature for use.

[0038] A DMSO solution of 15 μg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 1 mW), DPBF (absorbance decreased due to degradation caused by reaction with singlet oxygen) was added to detect the amount of singlet oxygen generated.

[0039] Example 2

[0040] 0.4167 g of glutathione and 0.0833 g of methionine were weighed and dissolved in 12 mL of formamide, and then fully mixed by an ultrasonic device. The mixture was transferred to a polytetrafluoroethylene-lined high-temperature reaction kettle, and reacted at 160°C for 8 h. After cooling to room temperature, the solution was diluted with ultrapure water. The carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, with water changed every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 μm filter membrane, and finally freeze-dried to obtain NIR carbon dots NIR-CDs-2, which were stored at room temperature for use.

[0041] The DMSO solution of 15 pg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 1 mW), DPBF was added to detect the amount of singlet oxygen generated.

[0042] Example 3

[0043] GSH and 0.125 g of methionine were weighed into 12 mL of formamide and mixed well by an ultrasonic device. The mixture was transferred to a polytetrafluoroethylene-lined high-temperature reaction kettle and reacted at 160°C for 8 h. After cooling to room temperature, it was diluted with ultrapure water. The carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, changing the water every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 pm filter membrane, and finally freeze-dried to obtain near-infrared carbon dots NIR-CDs-3, which were stored at room temperature for use.

[0044] The DMSO solution of 15 pg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 1 mW), DPBF was added to detect the amount of singlet oxygen generated.

[0045] After 4T1 breast cancer cells were incubated with the carbon dots modified by TPP (60 pg / mL) for 4 h, MitoTracker Green FM staining was performed, and the results were observed by confocal microscopy, as shown in Figure 7

[0046] Example 4

[0047] GSH and 0.125 g of methionine were weighed into 12 mL of formamide and mixed well by an ultrasonic device. The mixture was transferred to a polytetrafluoroethylene-lined high-temperature reaction kettle and reacted at 160°C for 8 h. After cooling to room temperature, it was diluted with ultrapure water. The carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, changing the water every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 pm filter membrane, and finally freeze-dried to obtain near-infrared carbon dots NIR-CDs-3, which were stored at room temperature for use.

[0048] The DMSO solution of 15 pg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 1 mW), DPBF was added to detect the amount of singlet oxygen generated.

[0049] Figure 1 The transmission electron microscope image of the near-infrared carbon dots prepared in Example 3 is shown, with a scale of 10 nm.

[0050] As shown in Figure 1 The size of the prepared near-infrared carbon dots is less than 10 nm. ​

[0051] Figure 2 These are the ultraviolet absorption spectrum and fluorescence emission spectrum of the near-infrared carbon dots prepared in Example 3.

[0052] Depend on Figure 2 As shown, NIR-CDs have an absorption spectrum in the long wavelength range (600nm-700nm). At the same time, NIR-CDs have strong absorption in the 300-420nm range. Selecting light within this wavelength range to excite NIR-CDs results in a fluorescence emission spectrum that is independent of the excitation wavelength and exhibits excitation wavelength independence.

[0053] Figure 3 This is a Fourier transform infrared spectrum (FTIR) test chart of the near-infrared carbon dots prepared in Example 3.

[0054] Depend on Figure 3 As shown in Figure 3, the abundant groups on the surface of NIR-CDs can be observed by Fourier transform infrared spectroscopy (FTIR). -1 The broad peaks at 1150 and 1230 cm are attributed to OH and NH stretching vibrations, indicating the presence of hydroxyl and amino groups. -1 The peaks at 1678 and 1392 cm are attributed to the stretching vibrations of CO and CN, respectively. -1 The typical peaks at 1600 cm-1 and 1600 cm-2 confirm the presence of an amide bond, which are attributed to the vibrations of C=O and CN of the amide, respectively. -1 The peak at 1000-1100 cm is the CC / CN bond. -1 The peaks at are CS and oxidized S bonds.

[0055] Figure 4 (a-d) are the yields of singlet oxygen generated by the near-infrared carbon dots prepared in Examples 1-4 in DMSO solution under irradiation with 660 nm excitation light.

[0056] Figure 5 This is a stability test of the near-infrared carbon dots prepared in Example 3 under near-infrared light (660 nm).

[0057] By regulating the mass ratio of glutathione (GSH) and L-methionine in the synthesis raw materials, the optimal raw material mass ratio is found to synthesize NIR-CDs, thereby generating singlet oxygen ( 1 When the total mass of the synthetic raw materials remains unchanged, NIR-CDs (NIR-CDs-7, NIR-CDs-5, NIR-CDs-3, NIR-CDs-1) synthesized with different mass ratios of glutathione and methionine in DMSO solution can reduce the absorption peak of DPBF after being irradiated by 660nm laser, indicating that NIR-CDs with different mass ratios can produce1 O2. Under the same conditions, NIR-CDs-3 produced 1 O2 is the highest, so the NIR-CDs synthesized in this subject are all based on carbon dots with a mass ratio (GSH:L-methionine = 3:1). According to the following formula, the yield of methylene blue (MB) is calculated 1 O2 is taken as a reference, and the yield of NIR-CDs producing 1 O2 is 0.3414. At the same time, it is found through experiments that the NIR-CDs have good light stability.

[0058]

[0059] Figure 6 The Zeta potential change diagram of the near-infrared carbon dots prepared in Example 3 before and after surface modification of triphenylphosphine molecules.

[0060] As shown in Figure 6 FTIR test proves that the surface of NIR-CDs is rich in amino groups (-NH2), and by using the amino groups (-NH2) on the surface of NIR-CDs, a reaction is carried out with triphenylphosphine molecules (TPP-COOH) with carboxyl groups to form an amide bond, so that the NIR-CDs have the ability to target mitochondria. The Zeta potential of the surface of NIR-CDs is negative, and after surface modification of TPP-COOH, the Zeta potential becomes obviously positive.

[0061] In summary, the present application provides a near-infrared carbon dot synthesized by a solvothermal method, and the synthesis process is simple, easy to operate and has strong operability. In addition, the near-infrared carbon dots with the best ratio synthesized by the method have the highest efficiency of producing singlet oxygen, and have the potential ability of photodynamic killing of cancer cells.

[0062] Figure 7 The co-localization diagram of the near-infrared carbon dots prepared in Example 3 after being taken up by cells and mitochondria. The overlap rate of the blue fluorescence (excitation / emission: 405 / 680 nm) of carbon dots and the green fluorescence (excitation / emission: 490 / 516 nm) of mitochondrial dye reaches 84%, which confirms the mitochondrial targeting property thereof.

[0063] By means of fluorescence imaging by complex staining with commercial organelle dyes, whether the NIR-CDs taken up by cells can target the mitochondrial part in cells is studied. As shown in Figure 7 In 4T1 cells, the blue fluorescence of NIR-CDs has good overlap with the green fluorescence of commercial mitochondrial dyes, and the co-localization coefficient is 0.84, which indicates that after the surface of the synthesized near-infrared carbon dots is modified with triphenylphosphine, the NIR-CDs have good ability to target mitochondria.

[0064] Comparative Example 1

[0065] During the experiment, different raw materials were selected as carbon sources for comparative experiments, and the prepared different carbon dots were used for active oxygen determination experiments.

[0066] 0.375 g of glutathione and 0.125 g of methionine were weighed and dissolved in 12 mL of formamide, and then fully mixed by an ultrasonic device. The above mixture was transferred to a high-temperature reaction kettle lined with polytetrafluoroethylene, and reacted at 160°C for 8 h. After cooling to room temperature, it was diluted with ultrapure water. The above carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, with water changed every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 μm filter membrane, and finally freeze-dried to obtain near-infrared carbon dots NIR-CDs-methionine, which was stored at room temperature for use.

[0067] A DMSO solution of 15 μg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 5 mW), DPBF was added to detect the amount of singlet oxygen generated.

[0068] Comparative Example 2

[0069] 0.375 g of glutathione and 0.125 g of homocysteine were weighed and dissolved in 12 mL of formamide, and then fully mixed by an ultrasonic device. The above mixture was transferred to a high-temperature reaction kettle lined with polytetrafluoroethylene, and reacted at 160°C for 8 h. After cooling to room temperature, it was diluted with ultrapure water. The above carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, with water changed every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 μm filter membrane, and finally freeze-dried to obtain near-infrared carbon dots NIR-CDs-homocysteine, which was stored at room temperature for use.

[0070] A DMSO solution of 15 μg / mL of the above carbon dots was prepared, and under the irradiation of near-infrared light (660 nm, 5 mW), DPBF was added to detect the amount of singlet oxygen generated.

[0071] Comparative Example 3

[0072] 0.375 g of glutathione and 0.125 g of glucose were weighed and dissolved in 12 mL of formamide, and then fully mixed by an ultrasonic device. The above mixture was transferred to a high-temperature reaction kettle lined with polytetrafluoroethylene, and reacted at 160°C for 8 h. After cooling to room temperature, it was diluted with ultrapure water. The above carbon dot solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in the dark for 72 h, with water changed every 6 h. The obtained dark green carbon dot solution was filtered through a 0.22 μm filter membrane, and finally freeze-dried to obtain near-infrared carbon dots NIR-CDs-glucose, which was stored at room temperature for use.

[0073] The DMSO solution of 15 μg / mL of the above carbon dots was configured, under the irradiation of near-infrared light (660 nm, 5 mW), DPBF was added for detecting the generation amount of singlet oxygen.

[0074] Figure 8 (a-d) are the yields of singlet oxygen generated by the near-infrared carbon dots prepared in Comparative Examples 1-3 under the irradiation of 660 nm excitation light in DMSO solution.

[0075] In summary, under the same conditions, compared with the near-infrared carbon dots synthesized by homocystine and glucose, the yield of singlet oxygen of the near-infrared carbon dots synthesized by methionine is the highest.

[0076] The above merely illustrates the preferred embodiments of the present application, it should be noted that the above preferred embodiments should not be regarded as limitation to the present application, the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for preparing near-infrared carbon dots that can efficiently generate reactive oxygen species and have targeting capabilities, characterized by: The carbon dots are nanoparticles synthesized in one step by a solvothermal method using glutathione and methionine as carbon sources and formamide as solvent, and are endowed with mitochondrial targeting properties by combining surface covalent modification. The steps include: mixing glutathione and methionine in a mass ratio of 1-10:1 and dissolving them in formamide, transferring them to a high-temperature sealed environment, reacting them at 120-200°C for 4-12 hours, cooling them, and diluting them with ultrapure water to prepare an initial carbon dot precursor solution.

2. The preparation method according to claim 1, wherein: The temperature of the solvent thermal reaction is controlled to be 140-180° C.; and the reaction time is 6-10 hours.

3. The preparation method according to claim 1, wherein: The purification process of the initial carbon dot precursor solution includes light-proof dialysis, filtration and freeze-drying.

4. The preparation method according to claim 1, wherein: The molecular weight cutoff used in the light-proof dialysis step is 2000-5000Da, the dialysis time is 48-96 hours, and the dialysate is replaced every 4-8 hours; the storage temperature in the freeze-drying step is -80 to -20°C, and the pre-freezing time is ≥12 hours.

5. The preparation method according to claim 1, wherein: The carbon dots are covalently modified by covalently coupling surface amino groups with carboxylated triphenylphosphine (TPP-COOH); the mass ratio of TPP-COOH to carbon dots in the coupling reaction is 50:1 to 200:1, the activation reaction pH is adjusted to 4.5-6.0, and the activation time is 20-40 minutes.

6. The preparation method according to claim 5, characterized in that: The final pH of the coupling reaction was adjusted to 6.8-7.5, the reaction time was 12-36 hours and the temperature was 25-37°C.

7. Near-infrared carbon dots prepared by the method of claim 1; wherein the singlet oxygen yield is ≥0.3 and the molar ratio of surface amino groups to carboxyl groups is ≥2:

1.

8. Use of the near-infrared carbon dots according to claim 7 in the preparation of drugs for photodynamic killing of cancer cells.

9. The use according to claim 8, characterized in that: It includes the use of the near-infrared carbon dots in the preparation of drugs targeting mitochondria; the drug for photodynamic killing of cancer cells contains near-infrared carbon dots and a pharmaceutically acceptable carrier, wherein the mass concentration of the near-infrared carbon dots is 10-200 μg / mL; and the active oxygen production rate is ≥90%.

10. The use according to claim 8, characterized in that: The preparation of the drug for photodynamic killing of cancer cells includes the preparation of photodynamic therapy drugs for breast cancer, liver cancer or lung cancer.

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