Bromine-doped carbon dots and nanomicelle assembly thereof, preparation method and use thereof

Abstract

The present invention provides a bromine-doped carbon dot and its nanomicelle assembly, preparation method and use, belonging to the field of nanotechnology. The present invention uses 1,7-dibromo-3,4,9,10-perylene tetracarboxylic anhydride and urea as raw materials to prepare a new type of bromine-doped carbon dot, which has near-infrared absorption and emission characteristics and efficient type I / type II reactive oxygen species generation capacity. The present invention also embeds the bromine-doped carbon dot into DSPE-PEG. 2000 Nanomicelle assemblies prepared in micelles improve their water solubility and biocompatibility, prolong their circulation time in the blood, and prevent rapid renal clearance. Both the carbon dots and nanomicelle assemblies can effectively generate type I and type II reactive oxygen species, overcoming environmental limitations and demonstrating high photodynamic efficacy in antibacterial and anti-tumor applications. Furthermore, this invention represents the first demonstration of efficient photodynamic therapy using near-infrared emitting bromine-doped carbon dot nanomaterials in complex microenvironments, demonstrating promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of nanotechnology, and in particular relates to a bromine-doped carbon dot and a nano-micelle assembly thereof, a preparation method and use thereof. Background Art

[0002] Photodynamic therapy (PDT) is an innovative technology that uses light-activated photosensitizers for disease diagnosis and treatment. Due to its excellent spatiotemporal precision, non-invasiveness, high efficiency and low side effects, PDT has great potential in treating bacterial infections, tumor growth and the photooxidation of amyloid-β protein in Alzheimer's disease. It is well known that the microenvironment of bacterial infection and tumor tissue contains both oxygen-rich and hypoxic environments. For example, the outside of tumor tissue is rich in blood vessels supplying oxygen, but the high interstitial pressure inside creates an oxygen-deficient environment. Similarly, the epidermal tissue of bacterial infection is often rich in oxygen, while the deeper tissues are oxygen-deficient. Therefore, effective photosensitizers are needed that can generate toxic reactive oxygen species (ROS) under both oxygen-rich and hypoxic conditions to optimize the therapeutic effect. Currently, many reported photosensitizers operate through the oxygen-dependent type II photodynamic pathway, transferring energy to oxygen (O2) upon reaching the triplet excited state to generate singlet oxygen ( 1 However, under hypoxic conditions, this pathway is less efficient, and the type I photodynamic process transfers electrons to O2 via the triplet excited state, generating superoxide anions (O2 ·- ), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH). Although some type I and type II photosensitizers have been reported, problems such as aggregation-induced quenching often limit their efficiency and imaging sensitivity, thus restricting their clinical application.

[0003] Carbon dots (CDs) are carbon-based nanomaterials with sizes less than 10 nanometers. They have attracted widespread attention due to their excellent optical properties, water solubility, high photostability, and biocompatibility. Recently, numerous studies have highlighted the application of CDs as photosensitizers for the treatment of bacterial infections or bioimaging-guided photodynamic therapy (PDT). For example, D / L-cysteine-derived CDs have been used for fluorescence imaging recognition and photodynamic antimicrobial activity against a variety of bacteria. Porphyrin-based CDs exhibit type I and type II photodynamic processes, effectively promoting bioimaging-guided therapy for tumor growth. Furthermore, CDs derived from various carbon-based starting materials (such as citric acid, Hypericum perforatum, and 1,3,6-trinitrofluorene) have also shown success in treating skin diseases, tooth whitening, and tumor ablation. Despite these exciting developments, most current CDs exhibit a maximum absorption outside the red region or low molar absorption coefficients. This results in low photon utilization efficiency and suboptimal therapeutic efficacy in photodynamic therapy (PDT). In addition, their maximum emission wavelength is below 700 nm, resulting in strong light absorption and scattering in biological tissues.

[0004] To achieve near-infrared (NIR) fluorescence emission and efficient photodynamic therapy (PDT), it is crucial to tune the absorption maximum of carbon dots (CDs) to the red region. Previous studies have successfully utilized cyanine dyes that emit NIR fluorescence to prepare NIR-emitting CDs. However, the complex synthesis and high cost of cyanine-derived NIR CDs have limited their clinical application.

[0005] Currently, there are no reported carbon dots with maximum emission wavelength in the NIR region that can provide an effective type I / type II bioimaging-guided PDT system for bacterial infection and tumor treatment. Summary of the Invention

[0006] The purpose of the present invention is to provide a bromine-doped carbon dot and a nano-micelle assembly thereof, a preparation method and use thereof.

[0007] The present invention provides a bromine-doped carbon dot, which is prepared using a bromine source compound and a carbon source compound as raw materials, wherein the mass ratio of the bromine source compound to the carbon source compound is 1:1-5.

[0008] Furthermore, the bromine source compound is 1,7-dibromo-3,4,9,10-tetracarboxylic dianhydride, and the carbon source compound is urea; the mass ratio of the bromine source compound to the carbon source compound is 1:3.

[0009] The present invention also provides a method for preparing the bromine-doped carbon dots, which comprises the following steps: reacting a bromine source compound and a carbon source compound, centrifuging, filtering the supernatant, and dialyzing the filtrate to obtain the bromine-doped carbon dots.

[0010] Furthermore, the solvent of the reaction is an organic solvent; the reaction temperature is 160-200°C, and the reaction time is 4-8 hours; the centrifugal speed is 7000-11000 r / min, and the time is 10-30 minutes; and the molecular weight cut-off of the dialysis membrane is 500-1500 Da.

[0011] Furthermore, the solvent of the reaction is polytetrafluoroethylene; the reaction temperature is 160° C., and the reaction time is 5 hours; the centrifugal speed is 9000 r / min, and the time is 15 minutes; and the molecular weight cut-off of the dialysis membrane is 1000 Da.

[0012] The present invention also provides a nano-micelle assembly, which is prepared using the above-mentioned bromine-doped carbon dots and amphiphilic carrier as raw materials, wherein the mass ratio of the bromine-doped carbon dots to the amphiphilic carrier is 1:10-30.

[0013] Furthermore, the amphiphilic carrier is DSPE-PEG; and the mass ratio of the bromine-doped carbon dots to the amphiphilic carrier is 1:20.

[0014] Furthermore, the molecular weight of the DSPE-PEG is 2000.

[0015] The present invention also provides a method for preparing the nano-micelle assembly, which comprises the following steps: reacting bromine-doped carbon dots with an amphiphilic carrier, and dialyzing to obtain the nano-micelle assembly.

[0016] Furthermore, before the reaction, the bromine-doped carbon dots and the amphiphilic carrier need to be prepared into a bromine-doped carbon dots solution and an amphiphilic carrier solution, respectively, and then mixed for reaction;

[0017] The solvent for preparing the bromine-doped carbon dot solution and the amphiphilic carrier solution is an organic solvent.

[0018] Furthermore, the solvent for preparing the bromine-doped carbon dot solution and the amphiphilic carrier solution is dimethyl sulfoxide.

[0019] Furthermore, the solvent of the reaction is an inorganic solvent; the reaction temperature is 30-45° C., and the reaction time is 5-20 hours; and the molecular weight cut-off of the dialysis membrane is 3000-4000 Da.

[0020] The solvent of the reaction is PBS; the reaction temperature is 37° C., and the reaction time is 7 to 17 hours; and the molecular weight cut-off of the dialysis membrane is 3500 Da.

[0021] The present invention also provides use of the bromine-doped carbon dots and the nano-micelle assembly in preparing a photosensitizer.

[0022] Furthermore, the photosensitizer is a drug for treating bacterial infection, tumor, Alzheimer's disease, acute and chronic inflammation, and promoting wound healing.

[0023] The present invention also provides the use of a photosensitizer and a light-emitting device in combination for preparing equipment for treating bacterial infections, tumors, Alzheimer's disease, acute and chronic inflammation, and promoting wound healing. The photosensitizer is the above-mentioned bromine-doped carbon dots or the above-mentioned nanomicelle assembly.

[0024] Furthermore, the bacteria are Staphylococcus aureus and Escherichia coli; the tumor is liver cancer; the wavelength of the light source in the light emitting device is 500-800 nm, and the irradiance is 20-60 W / cm 2 , the irradiation time is 5 to 45 minutes; preferably, the wavelength of the light source in the light emitting device is 660nm, and the irradiance is 40mW / cm 2 The irradiation time is 15 to 30 minutes.

[0025] The present invention uses 1,7-dibromo-3,4,9,10-perylenetetracarboxylic anhydride (also known as 1,7-dibromo-3,4,9,10-tetracarboxylic dianhydride) and urea as raw materials to prepare a new type of bromine-doped carbon dots, which have near-infrared absorption and emission characteristics and efficient type I / type II reactive oxygen species generation capabilities. The present invention also bromine-doped carbon dots are embedded in DSPE-PEG 2000 Nanomicelle assemblies prepared in micelles improve their water solubility and biocompatibility, prolong their circulation time in the blood, and prevent rapid renal clearance. Both the carbon dots and nanomicelle assemblies can effectively generate type I and type II reactive oxygen species, overcoming environmental limitations and demonstrating high photodynamic efficacy in antibacterial and anti-tumor applications. Furthermore, this invention represents the first demonstration of efficient photodynamic therapy using near-infrared emitting bromine-doped carbon dot nanomaterials in complex microenvironments, demonstrating promising application prospects.

[0026] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0027] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Synthesis and characterization of BrCDs. a) TEM and HRTEM images. b) Size distribution. c) AFM image, scale bar: 200 nm. d) Raman spectrum, D and G bands at 1380 cm-1, respectively. -1 and 1590cm -1 e) FT-IR spectra of BrCDs (bottom) and DBrPTCD (top). f) XPS spectra of DBrPTCD raw material (red) and BrCDs (blue). g) High-resolution C1s XPS spectrum of BrCDs. h) N1s XPS spectrum of BrCDs. i) Br3d XPS spectrum of BrCDs. j) UV-visible spectra of DBrPTCD and BrCDs in DMF. k) 3D color map of excitation-emission of BrCDs solution. l) Fluorescence lifetime fitting curve of BrCDs.

[0029] Figure 2 Photophysical properties of BrCD-NPs. a) UV-visible spectrum; b) fluorescence emission spectrum.

[0030] Figure 3Photodynamic properties of BrCDs. a) In PBS, 660 nm (40 mW / cm 2 ) The total ROS generated by BrCDs was detected using DCFH-DA as an indicator under laser irradiation. b) The decomposition rate of DPBF in the presence of BrCDs under 660 nm laser irradiation in PBS. c) The specific DHR123 probe was used in PBS at 660 nm (40 mW / cm 2 ) Detection of O2 under laser irradiation ·- d) Using TEMP to capture the fluorescence emission. 1 ESR spectra of O2 under different treatment conditions. e) O2 capture using DMPO ·- ESR spectra of BrCDs under different treatment conditions. f) Normalized fluorescence and phosphorescence spectra of BrCDs solution at 77 K. g) Mott-Schottky plot of BrCDs. h) Schematic diagram of the energy bands and redox potential of the ROS generation mechanism of BrCDs.

[0031] Figure 4 .Tauc plot of BrCDs.

[0032] Figure 5 Live / dead staining of Escherichia coli or Staphylococcus aureus under different treatments.

[0033] Figure 6 Antibacterial effect of BrCDs at low concentrations. a) E. coli or S. aureus agar plates coated with different concentrations of BrCDs, after 15 minutes of laser irradiation or 30 minutes of darkness (L+: laser irradiation; L-: no laser irradiation); b, c) Statistical charts of the antibacterial activity against E. coli and S. aureus (laser power: 40 mW / cm 2 Error bars indicate standard deviation, n = 3); d) Scanning electron microscopy images of E. coli or S. aureus under different treatments (scale bar: 2 μm); e) Schematic diagram of antibacterial healing of wounds infected with S. aureus in vivo; f) Photographs of wound healing in mouse skin on day 12 after S. aureus infection, showing wounds treated with PBS alone (control group), unirradiated BrCDs (BrCDs(L-) group), and BrCDs irradiated with 660 nm laser (BrCDs(L+) group), respectively; scale bar: 5 mm; g) Changes in wound size in mice under different treatments (error bars indicate standard deviation, n = 4); h) H&E and Masson's staining images of wounds on day 12 after surgery. The red double arrows highlight the regenerated epidermis; i) Quantitative assessment of the length of the regenerated epidermis on day 12 after surgery; j) Quantitative assessment of the thickness of the granulation tissue on day 12 after surgery (data are presented as mean ± standard deviation, from three independent replicates (Student's t-test; ***P < 0.001)).

[0034] Figure 7 .Scanning electron microscopy images of Escherichia coli or Staphylococcus aureus under different treatments.

[0035] Figure 8 Body weight changes during the antibacterial experiment (error bars represent standard deviation, n=4).

[0036] Figure 9 DCFH-DA was used as an indicator to detect the total ROS of BrCD-NPs.

[0037] Figure 10 Detection of ROS species in BrCD-NPs under 660 nm irradiation. a) Detection of O2 using the specific DHR123 probe ·- b) Fluorescence emission of DPBF in the presence of BrCD in PBS.

[0038] Figure 11 .ROS generation and its in vitro anti-tumor effect. a) Schematic diagram showing BrCDs and DSPE-PEG 2000 b) Dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM) images of BrCDs-NPs; c) Confocal laser scanning microscopy (CLSM) images of HepG2 cells showing near-infrared imaging under 660 nm laser irradiation, and using DHE and SOSG as fluorescent probes to detect the total 1 O2 and O2 ·- d) Evaluation of the cytotoxicity of HepG2 cells exposed to different concentrations of BrCDs-NPs, followed by 660 nm laser irradiation or treatment in the absence of light; e) Assessment of the status of HepG2 cells under different treatment conditions using the JC-1 assay and live / dead staining; f, g) Flow cytometric analysis of HepG2 cell apoptosis under different treatment conditions and corresponding cell population data. Panels i, ii, iii, and iv represent the control group, the group irradiated without BrCDs-NPs, the group treated with BrCDs-NPs alone, and the group treated with BrCDs-NPs and irradiation, respectively (data are presented as mean ± standard deviation (n = 3)).

[0039] Figure 12 .DHE and SOSG were used as fluorescent probes to detect the total 1 O2 and O2 ·- , Confocal laser scanning microscopy (CLSM) images of HepG2 cells using near-infrared imaging and 660 nm illumination.

[0040] Figure 13 .JC-1 assay of HepG2 cells treated with different conditions.

[0041] Figure 14 In vivo antitumor effects. a) Schematic diagram of the anticancer treatment process in tumor-bearing mice; b) In vivo fluorescence imaging at different time points after intravenous injection of BrCDs-NPs; c) Fluorescence imaging of major organs and tumors after intravenous injection of BrCDs-NPs; d) Relative fluorescence intensity of major organs and tumors at different time intervals after intravenous injection; e) Photographs of HepG2 tumor-bearing nude mice under different treatment conditions for 14 days; f) Tumor volume after different treatments; g) Ex vivo tumor weight; h) Changes in mouse body weight under different treatments during photodynamic therapy (PDT); i) H&E and TUNEL staining of tumor tissues 14 days after treatment (Scale bar: 50 μm; Data are expressed as mean ± SD (n = 3); ***p < 0.001 compared with the control group).

[0042] Figure 15 .Digital photograph of ex vivo tumor 14 days after treatment.

[0043] Figure 16 .H&E stained sections of the main organs of mice on the 14th day after different treatments. DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0045] 1,7-dibromo-3,4,9,10-perylenetetracarboxylic anhydride (also known as 1,7-dibromo-3,4,9,10-tetracarboxylic dianhydride, DBrPTCD), urea, 1,3-diphenylisobenzofuran (DPBF), and 2,7-dichlorofluorescein diacetate (DCFH-DA) were purchased from Beide (Shanghai, China). Dihydrorhodamine 123 (DHR123) was purchased from MREDA (Beijing, China). DSPE-PEG 2000 All proteins were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Dihydroethidium (DHE), singlet oxygen sensor green probe (SOSG), Calcein-AM / PI kit, MTT kit, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl cyanide (JC-1) kit were purchased from Beyotime Biotechnology Co., Ltd. (Beijing, China). Cell apoptosis detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0046] The present invention adopts the following characterization methods for structural characterization:

[0047] Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM) images were obtained using a JEM-F200. Fourier transform infrared spectroscopy (FT-IR) was collected using a Nicolet 5700 FT-IR spectrometer. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a U-3900 UV-vis spectrophotometer. X-ray photoelectron spectroscopy (XPS) was performed using an AXIS Supra (Kratos, England). Fluorescence emission spectroscopy was performed on a Horiba Fluorolog-3 spectrometer. Raman spectra were collected using a Horiba LabRAM HR spectrometer. Cell images were captured using a Zeiss confocal laser scanning microscope (CLSM) (LSM880 Airyscan with STEDYCON, Germany). Electron paramagnetic resonance (ESR) spectra were recorded using an EMXplus (Bruker BioSpin, Germany) spectrometer. In vivo fluorescence images were collected using an IVIS Spectrum imaging system (PerkinElmer IVIS).

[0048] The "room temperature" referred to in the present invention is 25±10°C, and the time of "overnight" is 12±5 hours.

[0049] Example 1: Preparation of Bromine-doped Carbon Dots

[0050] First, 0.5 g of 1,7-dibromo-3,4,9,10-tetracarboxylic dianhydride and 1.5 g of urea were added to 25 mL of DMF. After ultrasonication for 10 minutes, the mixed solution was placed in a 100 mL polytetrafluoroethylene reactor. The reactor was placed in a drying oven at 160 ° C for 5 hours. After the reactor temperature dropped to 25 ° C, the resulting dark green solution was centrifuged at 9000 r / min for 15 minutes, the supernatant was collected and filtered through a 0.22 μm ultrafiltration membrane. The solution was then transferred to a dialysis membrane (1000 Da) and deionized water was used to remove impurities. After dialysis, the aqueous solution in the dialysis bag was freeze-dried to obtain a black solid, namely bromine-doped carbon dots (BrCDs).

[0051] Example 2: Preparation of Nanomicelle Assemblies Containing Bromine-doped Carbon Dots

[0052] 0.1 mL of DMSO solution of BrCDs (the content of BrCDs is 1 mg / mL) was added dropwise to a solution containing 2 mg of DSPE-PEG. 2000 After thorough mixing, the mixture was slowly added dropwise to 2 mL of PBS solution and stirred at 37 ° C overnight. By subsequent dialysis in PBS (molecular weight cutoff: 3500 Da), bromine-doped carbon dots nanomicelle assembly (BrCDs-NPs) ( Figure 11 a).

[0053] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0054] Experimental Example 1: Structural Characterization of BrCDs and BrCDs-NPs

[0055] 1. Structural characterization of BrCDs

[0056] like Figure 1 As shown in a, BrCDs have good dispersion and uniform size distribution, showing obvious 0.21nm lattice fringes corresponding to the (001) crystal plane of carbon. The average diameter of BrCDs is 3.12nm ( Figure 1 b), BrCDs are uniformly spherical and well dispersed, with an average height comparable to the diameter observed in TEM images ( Figure 1 c).

[0057] Figure 1 e shows the FT-IR spectra of BrCDs and the precursor DBrPTCD. Compared with the FT-IR spectrum of the precursor DBrPTCD, the peaks of C=O and CO disappear after the formation of CDs, while a new absorption peak of the C=N bond appears in BrCDs. These results indicate that a C=O group of the precursor DBrPTCD reacts with urea to form a C=N group, thereby promoting the formation of CDs. In addition, the oxygen atom in the CO bond between the two carbonyl groups is replaced by a nitrogen atom. In the Raman spectrum of BrCDs ( Figure 1 d), observed at 1380 cm -1 and 1590cm -1 There are two obvious sharp peaks at , corresponding to the D and G bands of BrCDs, respectively. These results indicate the existence of defects and graphene-like structures in the carbon core.

[0058] The XPS spectra of BrCDs are as follows Figure 1 As shown in Figure f, CDs are composed of C, O, N, and Br atoms with weight percentages of 64.05%, 15.11%, 6.59%, and 14.26%, respectively. Compared with the XPS spectrum of the raw material DBrPTCD, the appearance of nitrogen in BrCDs indicates that urea participates in the formation of the CDs structure. In addition, the reduction in oxygen content indicates that it is replaced by nitrogen, which is consistent with the FT-IR results. C1s high-resolution XPS spectrum of BrCDs ( Figure 1 g) can be unrolled into three main peaks with binding energies of 284.8 eV, 285.8 eV, and 287.7 eV, corresponding to C=C, CO, and C=O bonds, respectively. Figure 1 h) There are three peaks at 398.8eV, 400.4eV and 401.3eV, corresponding to graphitic nitrogen, pyridinic nitrogen and pyrrolic nitrogen, respectively. The XPS spectrum of Br 3d, such as Figure 1 As shown in Figure 1, it contains three peaks corresponding to C-Br 3d5 / 2, C-Br 3d3 / 2 and Br - These results indicate that Br atoms do not participate in the formation of the CDs core structure. Therefore, the presence of Br atoms introduces defects into CDs, which is consistent with the Raman spectroscopy results.

[0059] The UV-visible absorption spectrum of BrCDs shows that its maximum absorption wavelength in the visible light range is 630nm, and the absorption tail peak extends to 865nm. In contrast, the main absorption wavelengths of the raw material DBrPTCD are only located at 469nm and 522nm in DMF solution ( Figure 1 j). In addition, the three-dimensional absorption and emission spectra of BrCDs ( Figure 1 k) shows that its maximum fluorescence emission wavelength is 735nm, and the optimal excitation wavelength is concentrated in the range of 680nm, covering 520nm to 700nm. Figure 1 The fluorescence decay curve shown in Figure 1 shows that the average lifetime of BrCDs under 680 nm excitation is 3.16 ns.

[0060] The above results indicate that BrCDs were successfully synthesized in the present invention, and BrCDs is a nanomaterial with maximum near-infrared (NIR) emission and excitation capabilities, and its conjugated system structure is larger than that of the raw material DBrPTCD.

[0061] 2. Structural characterization of BrCDs-NPs

[0062] like Figure 11 As shown in b, BrCDs-NPs are spherical in shape with a uniform diameter of 106 nm. In addition, the absorption spectrum of BrCDs-NPs has a peak at 665 nm, while the fluorescence emission spectrum has a peak at 725 nm in aqueous solution ( Figure 2 ).

[0063] Experimental Example 2: Photodynamic properties of BrCDs

[0064] When exposed to light, the photosensitizer is first excited to its excited state. Then, through thermal relaxation and intersystem crossing, it transitions to the triplet excited state. In this triplet excited state, the photosensitizer is activated by type I (O2 ·- ) or type II ( 1 O2) mechanism to generate electron or energy transfer with oxygen in the environment, thereby generating reactive oxygen species (ROS). In order to clarify the photodynamic process of BrCDs generating type I and type II ROS simultaneously under irradiation, the present invention selected 1,3-diphenylisobenzofuran (DPBF) and dihydrorhodamine 123 (DHR123) as 1 O2 and O2 ·-probes to validate type I and type II ROS pathways.

[0065] 1. Experimental methods

[0066] (1) ROS generation detection

[0067] 2 mL of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (1 mM in ethanol) was reacted with 8 mL of a 10 mM NaOH aqueous solution at room temperature for 30 minutes. The hydrolyzate was then neutralized with 40 mL of PBS buffer to obtain a 20 μM stock solution. Then, 5 μL of the stock solution (4 mg / mL in DMSO) was added to 2995 μL of the DCFH solution to obtain the test solution. A 660 nm laser was used to irradiate at different time intervals, with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to monitor the fluorescence signal of DCFH to characterize ROS generation.

[0068] (2)O2 ·- Generate detection

[0069] Dihydrorhodamine 123 (DHR123) was used as a specific fluorescent probe to detect O2 ·- , which can react with O2 ·- Reaction and showed strong green fluorescence at 525nm. ·- The specific steps of the test were as follows: 10 μg / mL BrCDs and 10 μM DHR123 solution were dissolved in PBS and the cells were illuminated with a 660 nm laser (40 mW / cm 2 ) after irradiation at different time intervals, the O2 ·- The fluorescent signal generated.

[0070] (3) 1 O2 generation detection

[0071] 1,3-Diphenylisobenzofuran (DPBF) was used as a specific probe for the detection of 1 O2, with 1 After O2 reacts, its 410nm absorption weakens. 1 The specific steps of O2 generation detection are as follows: 10 μg / mL BrCDs and 50 μM DPBF solution were dissolved in PBS and the O2 generation was detected by 660 nm laser (40 mW / cm 2 ) After real-time irradiation, the absorption decrease was recorded at 410 nm by UV-vis to evaluate 1 O2 is generated.

[0072] 2. Experimental results

[0073] When DCFH-DA is activated by sodium hydroxide solution, it is converted into DCFH. When BrCDs interact with DCFH solution under 660nm laser irradiation, ROS are generated. These ROS oxidize DCFH to form DCF. This oxidation process is accompanied by fluorescence enhancement at 530nm ( Figure 3 a), confirmed the generation of ROS. Figure 3 As shown in b, at different time points (within 300 seconds) under 660 nm laser irradiation, the absorbance at 410 nm continued to decrease, indicating that 1 The generation of O2 confirmed the type II ROS process of BrCDs. Figure 3 As shown in c, with the extension of 660nm laser irradiation time, the fluorescence at 525nm continued to increase, indicating that O2 ·- The generation of type I and type II ROS was confirmed by BrCDs.

[0074] In order to directly demonstrate the ability of BrCDs photosensitizers to enhance ROS generation, 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were used as 1 O2 and O2 ·- The electron paramagnetic resonance (ESR) spectrum was measured. Figure 3 As shown in Figures 3d and 3e, under dark conditions, almost no signals of the two types of ROS were detected. However, under 660 nm laser irradiation, significant signals of the two types of ROS were captured using DMPO and TEMP, respectively. 1 O2 and O2 ·- In addition, the intensity of these signals increased significantly with the extension of irradiation time, demonstrating the generation of ROS under 660 nm laser irradiation.

[0075] To elucidate the ROS generation mechanism of BrCDs, the energies of the singlet and triplet excited states were calculated using the maximum emission peaks of fluorescence (FL) and phosphorescence (PL) observed at 77 K for BrCDs. Figure 3 As shown in Figure 5, the maximum emission peaks of FL and PL are approximately located at 719 nm and 783 nm. Based on the observed peaks, the energy values ​​of the first singlet (S1) and triplet (T1) excited states of BrCDs are calculated to be 1.72 eV (Es1 = 1240 / 719 = 1.72 eV) and 1.58 eV (ET1 = 1240 / 783 = 1.58 eV), respectively. Therefore, the singlet-triplet energy difference (ΔES1-T1) is 0.14 eV, calculated as follows: ΔE S1-T1 =1.72eV–1.58eV=0.14eV( Figure 3h). The smaller energy difference (ΔES1-T1) promotes the energy transfer from S1 to T1 state, thus promoting 1 Generation of O2. For the generation of type I ROS, the redox potential of electron transfer to the conduction band (Ec) is between O2 ·- The photoactivated electrons jump from the valence band (Ev) to the conduction band (Ec), and in this process, electron transfer occurs with the surrounding O2 to generate O2 ·- The band gap of BrCDs was determined by analyzing the UV-visible absorption spectrum of BrCDs in DMF solution ( Figure 1 j). A plot of (αE)2 versus E was obtained from the UV-Vis spectrum, where E represents the photon energy and α is the normalized absorption coefficient. BrCDs have a wide absorption range from the visible to the near-infrared region, with an absorption peak between 500 and 900 nm, indicating a narrow band gap. According to (αE) 2 -E figure, the band gap of BrCDs is calculated to be 1.69eV ( Figure 4 ). Such a narrow band gap gives BrCDs efficient photoinduced electron transition capability. The flat band potential and conductivity type of BrCDs can be determined by the Mott-Schottky plot. Figure 3 As shown in g, the positive slope indicates that BrCDs are n-type semiconductors. The X-axis intercept shows that the flat band potential of carbon dots is about -0.56 eV (relative to Ag / AgCl). According to the formula E(NHE) = E(Ag / AgCl) + 0.197 eV, the flat band potential (E) relative to the standard hydrogen electrode (NHE) can be deduced. fb ) is -0.36 V. Generally, for n-type semiconductor materials, the conduction band edge is usually about 0.2 eV higher than Efb. Therefore, the Ec of BrCDs can be calculated to be -0.56 eV, which is higher than that of O2 / O2 ·- The redox potential of BrCDs is more negative (-0.33 eV relative to NHE). This indicates that the electrons of BrCDs can be transferred to O2 under irradiation, thereby generating O2 ·- Furthermore, based on the band gap of 1.69 eV and the formula Eg = EVB-ECB, the Ec band position is estimated to be about 1.13 eV (relative to NHE) ( Figure 3 h).

[0076] In summary, BrCDs are excellent photosensitizers that can simultaneously generate type I and type II ROS.

[0077] Experimental Example 3: Antibacterial Experiment of BrCDs

[0078] 1. Experimental methods

[0079] (1) Bacterial culture

[0080] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) strains were purchased from Shanghai Green Micro Technology Co., Ltd. Second-generation slant cultures were transferred to 50 mL of LB liquid medium using an inoculating loop. The bacterial suspension was shaken at 37°C and 150 rpm for 24 hours. The suspension was then aliquoted into 1 mL tubes and refrigerated for later use.

[0081] (2) Bacterial photodynamic therapy experiment

[0082] The experiment was divided into the following groups: BrCDs+laser group (BrCDs(L+)), PBS group (Control group), PBS+laser group (L+ group) and BrCDs+no laser group (BrCDs(L-)).

[0083] BrCDs+laser group (BrCDs(L+)) group: 1 mL of Staphylococcus aureus solution or Escherichia coli solution was centrifuged at 8000 rpm for 5 minutes to remove the culture medium and washed with PBS. Next, the PBS solution of BrCDs was added to the Staphylococcus aureus solution or Escherichia coli solution, diluted 5000 times, and mixed evenly. Incubate at 37 ° C and 150 rpm for 5 minutes. After incubation, the bacteria were centrifuged, washed once with PBS, and then resuspended in PBS. The bacteria were irradiated with a 660 nm laser for 15 minutes. After irradiation, the bacteria were collected by centrifugation and used for subsequent experiments.

[0084] In the dark control group, all steps were the same except that the laser irradiation step was omitted.

[0085] (3) In vitro antibacterial test

[0086] The antibacterial effect was evaluated by plate colony counting method. The specific steps were as follows: Staphylococcus aureus suspension or Escherichia coli suspension was incubated with 2.5μM, 5μM and 10μM BrCDs for 5 minutes (37°C, 150rpm). The bacterial suspension of BrCDs+laser group was irradiated with 660nm laser (40mW / cm 2 ) for 15 minutes, and the BrCDs without laser were placed in the dark. Then, 10 μL of bacterial suspension was inoculated onto an agar plate containing LB medium (1.5% agar + LB medium) and imaged after overnight incubation.

[0087] (4) Bacterial scanning electron microscopy imaging

[0088] The Staphylococcus aureus or Escherichia coli collected in step (3) was fixed with 2.5% glutaraldehyde overnight, washed once with sterile distilled water, and then dehydrated using a series of ethanol-water solutions: 30%, 50%, 70% and 90% ethanol once each, and then washed twice with 100% ethanol. After dehydration, the ethanol solution containing Staphylococcus aureus or Escherichia coli was added dropwise to a glass slide for freeze drying, fixed with conductive tape and placed on a copper table. After gold ion sputtering, the samples were observed using a scanning electron microscope. In order to visually distinguish between dead (red fluorescence) and living (green fluorescence) bacteria, the bacteria were stained with NucGreen and EthD-III.

[0089] (5) In vivo antibacterial experiment

[0090] All animal experiments in the present invention were approved by the Medical Ethics Committee of Sichuan University and followed the Guide for the Care and Use of Laboratory Animals of the institution and NIH (approval number: KS2022864). Female SD mice aged 6-8 weeks used in the experiment were purchased from Beijing Huafukang Biotechnology Co., Ltd. The experimental mice were divided into three groups (four mice in each group): PBS group, BrCDs+no laser group (BrCDs(L-)) and BrCDs+laser group (BrCDs(L+)). On day 0, all mice were anesthetized, the back hair was removed with a razor and depilatory cream, and the exposed skin was cleaned and disinfected with 75% alcohol. On day 1, the skin of the mice was infected with Escherichia coli using the following steps: After anesthetizing the mice, two circular wounds with a diameter of 15 mm were made on their backs with a skin biopsy device. Staphylococcus aureus suspension (1×10 9 CFU mL -1 , 20 μL) was added to the wound and covered with a 3M transparent dressing for 2 hours. 60 μL of PBS and 10 μM BrCDs solution was added to the wound and incubated for 15 minutes. After treatment, the mice were housed as usual. The wounds of the BrCDs + laser group were irradiated at 40 mW / cm 2 The laser was applied for 15 minutes. On days 1, 3, 5, 7, 9, and 12, the wounds were photographed and measured, and the weight of the mice was recorded.

[0091] 2. Experimental results

[0092] like Figure 5 As shown, in these two bacteria, the PBS group (Control group), PBS + laser group (L+ group) and BrCDs + no laser group (BrCDs(L-)) all showed obvious green fluorescence, indicating the presence of living bacteria. However, the BrCDs + laser group (BrCDs(L+)) showed obvious red fluorescence, indicating that the bacteria had lost their activity.

[0093] Next, the antibacterial efficiency of BrCDs was evaluated by colony counting. A large number of E. coli and S. aureus colonies were observed in the PBS group and in the dark (L-) using BrCDs. There was no significant effect on bacterial growth under dark conditions at BrCDs concentrations of 2.5-10 μg / mL ( Figure 14 a), which indicates that BrCDs have good biocompatibility. Under light conditions, the number of E. coli and S. aureus colonies was significantly reduced in a concentration-dependent manner, indicating that BrCDs can effectively destroy bacteria ( Figure 14 b and 14c). It is worth noting that when the concentration of BrCDs was 10 μg / mL, the survival rate of E. coli and S. aureus dropped to 0.05%. Finally, the morphology of the bacteria was characterized using SEM ( Figure 14 d and Figure 7 In the PBS group and in the dark (L-) condition, the edges of E. coli and S. aureus colonies were clearly clear and smooth, while after laser irradiation, significant cell membrane rupture and morphological changes were observed in the presence of BrCDs. These results indicate that under 660 nm laser irradiation, BrCDs exhibited a strong ROS generation capacity, achieving excellent antibacterial effects.

[0094] On day 12, the wounds in the PBS and BrCDs(L-) groups were still large, while the wounds in the BrCDs(L+) group showed effective healing ( Figure 14 f and 14g). This is accompanied by marked intradermal fibrosis in the newly formed skin, with minimal lymphocyte and plasma cell infiltration ( Figure 14 h, H&E). In addition, Masson trichrome staining was used to further evaluate the degree of healing, inflammation, and granulation tissue levels. Significant hair follicle regeneration was observed in the BrCDs (L+) group ( Figure 14 h, Masson). Figure 6 As shown in Figure ij, the length of the new epidermis and the thickness of the granulation tissue in the BrCDs(L+) group were significantly greater than those in the control group and the BrCDs(L-) group. In addition, there was no significant change in the body weight of the mice during the 12-day period ( Figure 8 ), indicating that BrCDs have excellent biocompatibility.

[0095] The above results indicate that under light conditions, the effective antibacterial effect of BrCDs under laser irradiation reduced S. aureus-induced acute and chronic inflammation and promoted the proliferation of intradermal fibrous tissue, thereby accelerating wound healing.

[0096] Experimental Example 4: Photodynamic Properties and Anti-tumor Testing of Bromine-doped Carbon Dot Nanomaterials

[0097] 1. Experimental methods

[0098] (1) Cell culture

[0099] HepG2 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai. Cells were cultured in cell culture flasks at 37°C in a humidified atmosphere with 5% CO2. The culture medium used was Dulbecco's modified Eagle's medium (DMEM, GIBCO / Invitrogen, Camarillo, CA, USA) supplemented with 1% penicillin-streptomycin (containing 10,000 U mL⁻¹ penicillin and 10 mg mL⁻¹ streptomycin, Solarbio Life Sciences, Beijing, China) and 10% fetal bovine serum (FBS, Biological Industry, Kibbutz Beit Haemek, Israel).

[0100] (2) Detection of overall intracellular ROS generation

[0101] The overall level of intracellular ROS in bromine-doped carbon dot nanomaterials was assessed using a DCFH-DA kit. Specifically, HepG2 cells were seeded in a 35 mm confocal culture dish and incubated for 24 h. The cells were then incubated with 40 μg / mL BrCDs-NPs for 4 h, and 10 μM DCFH-DA was added to the new culture medium. After a further 30 min incubation, the cells were illuminated with a 660 nm laser (40 mW / cm 2 Finally, the cells were imaged using CLSM.

[0102] (3) O2 in the body ·- and 1 O2 generation detection

[0103] 1) O2 in the body ·- Generate detection

[0104] Using DHR123 as a specific fluorescent probe to detect O2 ·- , which can react with O2 ·- Reaction and showed strong green fluorescence at 525nm. ·- The specific steps of the test were as follows: 10 μg / mL BrCDs-NP and 10 μM DHR123 solution were dissolved in PBS and the cells were illuminated by a 660 nm laser (40 mW / cm 2 ) after irradiation at different time intervals, the O2 ·- The fluorescent signal generated.

[0105] 2) In vivo 1 O2 generation detection

[0106] DPBF was used as a specific probe for detection 1 O2, with 1 After O2 reacts, its 410nm absorption weakens. 1 The specific steps of O2 generation detection are as follows: 10 μg / mL BrCDs-NP and 50 μM DPBF solution were dissolved in PBS and the O2 generation was detected by 660 nm laser (40 mW / cm 2 ) After real-time irradiation, the absorption decrease was recorded at 410 nm by UV-vis to evaluate 1 O2 is generated.

[0107] (4) Extracorporeal O2 ·- and 1 O2 generation detection

[0108] 1) Extracorporeal O2 ·- Generate detection

[0109] Dihydroethidium (DHE) is used as a fluorescent probe to detect O2 in cells ·- The specific steps are as follows: HepG2 cells were seeded in a 35 mm confocal culture dish and incubated for 24 hours. After incubation with 40 μg / mL BrCDs-NPs for 4 hours, medium containing 20 μM DHE was added and incubated for a further 30 minutes. Then, the cells were illuminated with a 660 nm laser (40 mW / cm 2 Finally, the images were taken using a confocal laser scanning microscope (CLSM).

[0110] 2) In vitro 1 O2 generation detection

[0111] Singlet oxygen sensor green (SOSG) is used as a fluorescent probe to detect 1 The specific steps are as follows: HepG2 cells were seeded in a 35 mm confocal culture dish and incubated for 24 hours. They were incubated with 40 μg / mL BrCDs-NPs for 4 hours. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes. After three PBS washes, the cells were incubated with 10 μM SOSG probe for 20 minutes. Subsequently, a 660 nm laser (40 mW / cm 2 ) for 15 minutes. Finally, CLSM imaging was used.

[0112] (5) Intracellular anti-tumor properties

[0113] HepG2 cells were seeded in 96-well plates and cultured overnight. Then, the cells were treated with BrCDs-NPs (40 μg mL -1 ) were incubated at 37°C for 4 hours. Then, the cells were illuminated with a 660 nm laser (40 mW / cm 2) for 15 minutes and incubated for 24 hours. Cell viability was assessed using a standard MTT assay. To visually distinguish between dead (red fluorescence) and viable (green fluorescence) cells, HepG2 cells were further stained with Calcein-AM / PI.

[0114] (6) Mitochondrial integrity detection

[0115] HepG2 cells were seeded in confocal microplates and incubated for 24 hours to allow attachment. Next, the HepG2 cells were subjected to the following treatments: i) control, ii) laser alone, iii) laser without BrCDs-NPs, and iv) BrCDs-NPs plus laser. After a 4-hour incubation, the HepG2 cells were washed with PBS and stained with JC-1 for 15 minutes. Finally, the cells were imaged using CLSM.

[0116] 2. Experimental results

[0117] The ability of BrCDs-NPs to effectively generate ROS was verified using DCFH-DA, DHE, and DPBF probes, respectively. Figure 9-10 The results showed that BrCDs-NPs still retained the photophysical properties of BrCDs.

[0118] After HepG2 cells were co-cultured with nanoparticles for 4 hours, obvious red color was observed, indicating that the nanoparticles successfully entered the cells ( Figure 11 c and Figure 12 Subsequently, dihydroethidium (DHE) and singlet oxygen sensor green (SOSG) were used as specific fluorescent probes to detect intracellular O2 ·- and 1 O2( Figure 11 c and Figure 12 Compared with the control group (BrCDs-NPs only in laser or darkness), the BrCDs-NPs group irradiated with 660 nm laser showed obvious purple-red and green fluorescence, indicating that O2 was generated in the cells. ·- and 1 These experimental results collectively demonstrate the ability of BrCDs-NPs to effectively generate type I and type II ROS under 660 nm laser irradiation.

[0119] Due to the excellent ROS generation ability of BrCDs-NPs, the present invention uses the methylthiazolium salt (MTT) method to evaluate its anti-tumor effect in vitro. Figure 11As shown in Figure d, no obvious cell apoptosis was observed when the BrCDs-NPs concentration was as high as 60 μg / mL in HepG2 cells, indicating good biocompatibility. It is worth noting that in the BrCDs-NPs laser irradiation group, the survival rate of HepG2 cells decreased significantly with the increase of BrCDs-NPs concentration compared with the non-irradiated BrCDs-NPs group, confirming its effective anti-cancer effect. Cell apoptosis is associated with mitochondrial dysfunction, so the present invention uses the JC-1 method to evaluate mitochondrial damage ( Figure 11 e) In healthy mitochondria, JC-1 accumulates in the mitochondrial matrix and forms aggregates, emitting strong red fluorescence. In contrast, green JC-1 monomers indicate unhealthy mitochondria. Figure 11 e and Figure 13 As shown in Figure 3, cells treated with BrCDs-NPs under laser irradiation showed bright green fluorescence compared with the control group, laser alone group, and BrCDs-NPs group, indicating that the simultaneous generation of ROS led to mitochondrial dysfunction.

[0120] Experimental Example 5: Anti-tumor test of nanomaterials containing bromine-doped carbon dots

[0121] (1) Flow cytometry research

[0122] 2×10 5 HepG2 cells were seeded in 6-well plates in 2 ml of the corresponding culture medium and incubated overnight at 37°C in a humidified environment with 5% CO2. Next, HepG2 cells received the following different treatments: i) control group, ii) laser group alone, iii) BrCDs-NPs group without laser, and iv) BrCDs-NPs+laser group. After the above treatments, the cells were further incubated for 24 hours. Subsequently, the cells were washed twice with PBS and digested with 0.25% trypsin-EDTA (Invitrogen, 0.5 mL) for 5 minutes. The activity of trypsin was inhibited by adding serum-containing culture medium (0.5 mL), and the mixture was centrifuged at 1500 rpm for 3 minutes at room temperature. The subsequent steps were carried out according to the instructions of the cell apoptosis detection kit.

[0123] (2) In vivo anti-tumor experiments

[0124] All animal experiments in this study were approved by the Medical Ethics Committee of Sichuan University and followed the institutional and NIH Guide for the Care and Use of Laboratory Animals (Approval No. KS2022864).

[0125] Animals and tumor models: 6-8 week old female BALB / c nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. To establish the tumor model, 2×10 6HepG2 cells were inoculated into the right axilla of mice. The tumor volume reached 60–80 mm. 3 For in vivo experiments.

[0126] In vivo anticancer performance: Twelve nude mice bearing HepG2 tumors were randomly divided into four groups (n=3 in each group): i) control group, ii) laser alone group, iii) BrCDs-NPs without laser group, and iv) BrCDs-NPs+laser group. Eight hours after injection, the BrCDs-NPs were treated with 660 nm laser (40 mW / cm 2 ) irradiate the tumor site for 30 minutes. Tumor volume (mm 3 ) According to the formula V = lw 2 = 2, where w and l are the width and length of the tumor, respectively. After 14 days of treatment, the tumor-bearing nude mice were sacrificed, and the tumors were harvested and stained with hematoxylin-eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

[0127] (3) In vivo fluorescence imaging

[0128] Female BALB / c nude mice aged 6-8 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were housed under pathogen-free conditions and had free access to food and water. 6 HepG2 cells (200 μL) were subcutaneously inoculated on the back of mice. 3 When the mice were large, BrCDs-NPs (100 μL, 0.5 mg / mL) were injected through the tail vein. In vivo fluorescence imaging (excitation wavelength = 680 nm, emission wavelength ≥ 700 nm) was performed before and after injection and at different time points (up to 24 hours) after injection. Mice were sacrificed at different time points (1, 2, 4, 6, 8, 24 hours) and tumors and major organs were collected. The fluorescence signals in the collected tumors and organs were recorded using the IVIS Spectrum system.

[0129] 2. Experimental results

[0130] In order to clarify the anticancer mechanism of BrCDs-NPs, the present invention used Annexin V-FITC / PI apoptosis detection kit and performed flow cytometry ( Figure 11f and 11g). Compared with the control group, laser alone group, and BrCDs-NPs group, cells in the BrCDs + laser group migrated from Q4 to Q3 and Q2, indicating that apoptosis activated the cell death pathway. Cells treated with BrCDs-NPs and laser irradiation showed a significant apoptosis rate of 96.4% (the sum of early and late apoptosis), while the apoptosis percentages in the control group, laser irradiation group, and BrCDs-NPs group were 4.18%, 5.28%, and 8.70%, respectively. These results indicate that the apoptotic mortality rate induced by PDT of BrCDs-NPs is consistent with the results of AM / PI staining and MTT assay, confirming its excellent PDT anticancer efficacy.

[0131] Considering the excellent anticancer effect of BrCDs-NPs observed in vitro, the present invention further used HepG2 tumor-bearing nude mice as a model to study its therapeutic potential in vivo ( Figure 6 a). At the tumor site, endothelial dysfunction leads to the formation of leaky blood vessels, a phenomenon known as the enhanced permeability and retention (EPR) effect. The particle size of BrCDs-NPs (mainly about 106nm) is larger than that of BrCDs, which causes them to preferentially accumulate in tumor tissues through the EPR effect, preventing rapid clearance by the kidneys due to the ultra-small size of BrCDs. Utilizing the NIR emission of BrCDs-NPs at 735nm, the present invention aims to use them for NIR fluorescence image-guided photodynamic therapy (PDT). BrCDs-NPs were injected intravenously into HepG2 tumor-bearing nude mice and imaged using an IVIS system. As Figure 6 As shown in Figure b, the enhanced EPR effect in tumor tissue promoted the rapid accumulation of BrCDs-NPs at the tumor site, and the fluorescence signal reached its peak 8 hours after injection. This optimal time suggests that in vivo PDT 8 hours after BrCDs-NPs administration is ideal. In addition, imaging of the main organs of HepG2 tumor-bearing nude mice at different time intervals after BrCDs-NPs injection showed that the fluorescence signal at the tumor site also reached its maximum at 8 hours ( Figure 6 c and 6d). Therefore, these results emphasize the potential of BrCDs-NPs in image-guided PDT, highlighting its promise as a therapeutic strategy.

[0132] To examine the antitumor effect, tumor-bearing mice were randomly divided into four groups (n=3): saline group (Group I), saline + 660 nm laser irradiation group (Group II), BrCDs-NPs injection group (Group III), and BrCDs-NPs + 660 nm laser irradiation group (Group IV). Figure 6 e and Figure 15As shown, representative photographs of mice clearly show that tumor growth in Group IV was inhibited, while tumors in the control group continued to grow over time. The growth rate of tumor volume was measured every two days after treatment ( Figure 6 f). In control groups I, II, and III, tumors continued to grow throughout the treatment period, indicating that PBS, laser irradiation, and BrCDs-NPs alone did not show anticancer effects. In contrast, group IV showed significant tumor growth inhibition. Compared with the other three groups, the combination of BrCDs-NPs and laser irradiation (group IV) caused severe tumor damage, resulting in significant inhibition of tumor growth and a significant decrease in average tumor weight ( Figure 6 g). The body weight of mice increased slightly during the entire treatment period ( Figure 6 h). In addition, tumor tissues stained with H&E and TUNEL were used to evaluate cell apoptosis under different treatments. Figure 6 As shown in Figure 1, the BrCDs-NPs laser irradiation group (Group IV) experienced significant tumor damage compared with the other three groups. In addition, there was no obvious damage to the main organs of the mice in each group ( Figure 16 ), emphasizing the safety and efficient photodynamic therapy (PDT) advantages of BrCDs-NPs in tumor treatment.

[0133] In summary, the present invention provides a bromine-doped carbon dot and its nano-micelle assembly, preparation method and use. The present invention uses 1,7-dibromo-3,4,9,10-perylene tetracarboxylic anhydride and urea as raw materials to prepare a new type of bromine-doped carbon dot, which has near-infrared absorption and emission characteristics, as well as efficient type I / type II reactive oxygen species generation capabilities. The present invention also embeds bromine-doped carbon dots into DSPE-PEG. 2000 Nanomicelle assemblies prepared in micelles improve their water solubility and biocompatibility, prolong their circulation time in the blood, and prevent rapid renal clearance. Both the carbon dots and nanomicelle assemblies can effectively generate type I and type II reactive oxygen species, overcoming environmental limitations and demonstrating high photodynamic efficacy in antibacterial and anti-tumor applications. Furthermore, this invention represents the first demonstration of efficient photodynamic therapy using near-infrared emitting bromine-doped carbon dot nanomaterials in complex microenvironments, demonstrating promising application prospects.

Claims

1. A bromine-doped carbon dot, characterized in that: The invention is prepared by taking a bromine source compound and a carbon source compound as raw materials, wherein the mass ratio of the bromine source compound to the carbon source compound is 1:1-5; the bromine source compound is 1,7-dibromo-3,4,9,10-tetracarboxylic dianhydride, and the carbon source compound is urea.

2. The bromine-doped carbon dots according to claim 1, characterized in that The mass ratio of the bromine source compound to the carbon source compound is 1:

3.

3. A method for preparing the bromine-doped carbon dots according to claim 1 or 2, characterized in that: The method comprises the following steps: reacting a bromine source compound and a carbon source compound, centrifuging, filtering the supernatant, and dialyzing the filtrate to obtain the product; the reaction temperature is 160-200° C., and the reaction time is 4-8 hours.

4. The method according to claim 3, characterized in that The reaction solvent is an organic solvent; the centrifugal speed is 7000-11000 r / min, and the time is 10-30 minutes; the molecular weight cut-off of the dialysis membrane is 500-1500 Da.

5. A nanomicelle assembly, characterized in that: The invention is prepared by using the bromine-doped carbon dots and the amphiphilic carrier as raw materials, wherein the mass ratio of the bromine-doped carbon dots to the amphiphilic carrier is 1:10-30.

6. The nanomicelle assembly according to claim 5, characterized in that The amphiphilic carrier is DSPE-PEG; the mass ratio of the bromine-doped carbon dots to the amphiphilic carrier is 1:

20.

7. A method for preparing the nanomicelle assembly according to claim 5 or 6, characterized in that: The method comprises the following steps: self-assembling the bromine-doped carbon dots according to claim 1 or 2 and an amphiphilic carrier, and dialyzing to obtain the product.

8. Use of the bromine-doped carbon dots according to claim 1 or 2 or the nanomicelle assembly according to claim 5 or 6 in the preparation of a photosensitizer.

9. The use according to claim 8, characterized in that The photosensitizer is a drug for treating bacterial infection, tumor, Alzheimer's disease, acute and chronic inflammation, and promoting wound healing.

10. Use of a photosensitizer and a light-emitting device in combination for preparing a device for treating bacterial infection, tumors, Alzheimer's disease, acute and chronic inflammation, and promoting wound healing, wherein the photosensitizer is the bromine-doped carbon dots according to claim 1 or 2 or the nanomicelle assembly according to claim 5 or 6.

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