Near-infrared two-region imaging and ROS removal dual-function carbon dot and preparation method thereof

Abstract

The invention provides a carbon dot with double functions of near-infrared two-region imaging and ROS (reactive oxygen species) removal and a preparation method of the carbon dot. The carbon dot is prepared by reacting an aniline monomer containing a p-phenylenediamine amino group and selenourea as precursors, and has a near-infrared second region (NIR-II) fluorescence emission characteristic and active oxygen (ROS) scavenging capacity.

Description

Technical Field

[0001] This invention relates to a bifunctional carbon dot (CD) with near-infrared II (NIR-II) imaging and reactive oxygen species (ROS) scavenging capabilities, and its preparation method, belonging to the fields of biomedicine and nanomedicine. Background Technology

[0002] Laparoscopic cholecystectomy is the standard surgical procedure for treating benign gallbladder diseases, but bile duct injury is one of its most serious complications, leading to severe clinical consequences. To reduce these risks, intraoperative cholangiography is widely used to assist surgeons in identifying anatomical structures and minimizing accidental damage. Currently, commonly used contrast agents (such as indocyanine green) primarily image in the near-infrared I region (NIR-I, 700-900nm), which has limited tissue penetration depth (usually ≤5mm) and insufficient spatial resolution, limiting its effectiveness in fluorescence navigation of fine bile duct structures and accurate detection of bile leakage.

[0003] In recent years, near-infrared II (NIR-II, 1000-1700nm) fluorescence imaging technology has made significant progress due to its deeper tissue penetration and higher resolution, showing particular potential in the detection of bile leaks. However, existing NIR-II imaging technologies and contrast agents still have room for improvement and their functions are relatively limited.

[0004] It is worth noting that biliary tract-related diseases (such as bile leakage, cholecystitis, and cholestasis) often induce inflammatory responses in the adjacent liver region. In this microenvironment, excessive production of reactive oxygen species (ROS) and abnormally activated hepatic stellate cells jointly drive the process of liver fibrosis, which in severe cases can progress to cirrhosis or even hepatocellular carcinoma. Therefore, early intervention and efficacy evaluation for hepatitis and liver fibrosis are crucial.

[0005] Carbon dots (CDs), as nanomaterials, possess advantages such as good liver / kidney targeting, excellent optical properties, high stability, and biocompatibility. However, no multifunctional CDs have been reported that combine highly efficient NIR-II biliary imaging / bile leak detection with hepatitis / liver fibrosis diagnosis and treatment assessment. Therefore, developing multifunctional materials with NIR-II biliary imaging capabilities, while also providing diagnostic and therapeutic efficacy assessment for hepatitis / liver fibrosis, is of significant clinical importance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a dual-function carbon dot that combines near-infrared two-zone imaging and ROS removal.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a dual-function carbon dot for near-infrared II imaging and ROS removal. The carbon dot is prepared by reacting an aniline monomer containing a p-p-phenylenediamine amino group with a selenourea precursor, and has near-infrared II (NIR-II) fluorescence emission characteristics and reactive oxygen species (ROS) removal capability. The carbon dot exhibits fluorescence emission in the 1000-1700 nm wavelength range.

[0008] In some embodiments, the fluorescence lifetime of the carbon dots is not less than 9900 ns.

[0009] In some embodiments, the aniline monomer containing a p-p-phenylenediamine amino group is tris(4-aminophenyl)amine.

[0010] This invention provides a method for preparing bifunctional carbon dots for near-infrared two-zone imaging and ROS removal as described above, characterized by comprising the following steps: (1) Dissolve aniline monomers containing p-p-phenylenediamine amino groups and selenourea in an acidic aqueous solution; (2) Perform a hydrothermal reaction on the solution from step (1); (3) Purify the reaction product to obtain the bifunctional carbon dots.

[0011] In some embodiments, the acidic aqueous solution is a 45-55 mM hydrochloric acid solution.

[0012] In some embodiments, the temperature of the hydrothermal reaction in step (2) is 180-220°C and the reaction time is 5-7 hours.

[0013] In some embodiments, the aniline monomer is tris(4-aminophenyl)amine, and its molar ratio with selenourea is 0.09-0.1:0.04-0.05, more preferably 0.092:0.048.

[0014] In some embodiments, the purification in step (3) includes filtration through a 0.22 μm filter membrane and / or dialysis with a molecular weight cutoff of 1000D.

[0015] The present invention also provides the application of the above-described bifunctional carbon dots in the preparation of diagnostic and therapeutic agents for hepatobiliary diseases.

[0016] In some embodiments, the diagnostic and therapeutic agents are used for NIR-II imaging diagnosis of biliary stricture, bile leakage, or biliary tract disease, and / or for treating liver fibrosis by scavenging reactive oxygen species.

[0017] The beneficial effects of this invention are mainly reflected in the following aspects: (1) This invention successfully constructed a carbon dot (CDs) framework based on aniline derivatives by employing advanced molecular engineering strategies, achieving for the first time the simultaneous integration of near-infrared II (NIR-II) imaging and reactive oxygen species (ROS) scavenging functions in a single nanosystem. This innovative design enables the nanomaterial to simultaneously perform high-precision diagnosis and efficient treatment in the clinical application of hepatobiliary diseases, significantly improving the overall performance of the integrated diagnosis and treatment.

[0018] (2) The CDs prepared by this invention exhibit significant emission characteristics in the near-infrared II band of 1000–1700 nm and have a fluorescence lifetime of up to 9964.90 ns. This excellent optical performance enables high-contrast, high-resolution in vivo imaging of bile leakage and various biliary tract-related diseases, providing a reliable means for accurate clinical diagnosis.

[0019] (3) In the liver fibrosis disease model, the CDs of the present invention exhibit excellent reactive oxygen species scavenging ability. By effectively inhibiting oxidative stress damage and related signaling pathways, they significantly reduce pathological changes in the liver and achieve significant therapeutic effects, providing new material support for antioxidant therapy strategies.

[0020] (4) This invention elucidates the photophysical mechanism of CDs NIR-II emission, providing a universal design strategy for developing therapeutic hepatobiliary nano-formulations. Attached Figure Description

[0021] Figure 1 These are transmission electron microscope images of CDs and their particle size distribution.

[0022] Figure 2 This diagram explores the optical properties and photophysical mechanisms of CDs. a) Normalized absorption and fluorescence emission spectra of CDs-1, CDs-2, and CDs-3. b) Measurement of the distance between adjacent nitrogen atoms, charge calculation, and LUMO-HOMO energy level calculation after DFT+ optimization of the monomers. c) Comparison of HOMO and LUMO electronic spatial distributions and energy levels of amino nitrogen, pyrrole nitrogen, pyridine nitrogen, pyrrole nitrogen-graphene, and pyridine nitrogen-graphene systems based on DFT calculations. d) Changes in HOMO and LUMO electronic spatial distributions and energy levels caused by continuous graphitization and pyrrole nitrogen enrichment during the formation of CDs-3. e) Tauc diagram of CDs. fg) UV photoelectron spectra of the three CDs. h) Schematic diagram of energy level changes during the fluorescence excitation and emission process of CDs.

[0023] Figure 3 This is a graph showing the ability of CDs-3 to scavenge DPPH free radicals.

[0024] Figure 4These are representative fluorescence images and corresponding bright-field images of intracellular ROS staining in LX2 cells.

[0025] Figure 5 These are NIR-II images of gallbladder-CDs-3 in different emission bands (FEL: 1000 nm; 1100 nm; 1200 nm; 1300 nm; 1400 nm).

[0026] Figure 6 This is a TUNEL-stained image of a liver tissue section.

[0027] Figure 7 This is a quantitative analysis chart of serum biochemical indicators (creatinine, urea, lactate dehydrogenase, and creatine kinase) in mice. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0029] Example 1: Preparation of CDs Synthesis of CDs-1, CDs-2, and CDs-3: First, 50 mg of 1,4-diaminophenyl dihydrochloride (monomer 1, M=181.06, 0.276 mmol), 26.2 mg of N1-(4-aminophenyl)phenyl-1,4-diamine (monomer 2, M=190.28, 0.138 mmol), 26.7 mg of tris(4-aminophenyl)amine (monomer 3, M=290.36, 0.092 mmol), and 6 mg of selenourea were dissolved in 15 mL of 50 mM hydrochloric acid solution. At this point, the molar ratio of aniline to selenourea was 0.276:0.048. After sonication for 10 min, the mixed solution was transferred to a 20 mL high-pressure reactor and reacted at 200°C for 6 h to obtain a brownish-black carbon dot solution. To purify CDs-1, CDs-2, and CDs-3, the samples were filtered through a 0.22 μm filter membrane and dialyzed through a 1000 D dialysis bag. Finally, the solid products were obtained by freeze-drying for subsequent applications.

[0030] Example 2: Characterization and Photophysical Mechanism Investigation of CDs (Based on the Results of Example 1) Morphology and particle size, such as Figure 1 As shown, CDs-1, CDs-2, and CDs-3 all exhibit good monodispersity and uniform spherical morphology. Their particle size distribution shows good uniformity, with average particle sizes of 3.5 nm (CDs-1), 9.5 nm (CDs-2), and 12.5 nm (CDs-3), respectively.

[0031] Optical properties such as Figure 2As shown in Figure a, all three CDs exhibit characteristic absorption peaks near 360 nm, which is attributed to the n-π* electronic transitions of surface hydroxyl / amino functional groups. The maximum absorption wavelengths are located at 420 nm (CDs-1), 530 nm (CDs-2), and 860 nm (CDs-3), respectively. The maximum emission peaks appear at 480 nm, 590 nm, and 1080 / 1265 nm (CDs-3), respectively. Among them, the 1265 nm peak is a shoulder peak with a tail extending to the 1500 nm region, showing a significant redshift trend. To elucidate the NIR-II emission mechanism of CDs, we conducted a systematic study from the following three aspects, combining experimental characterization and density functional theory (DFT) calculations: (1) Effects of precursor molecular engineering The precursor molecular structures (monomers 1, 2, and 3) with different numbers of aniline units were optimized using DFT calculations. Electrostatic potential analysis showed that as the number of aniline units increased, the charge of the central N atom decreased from -0.647 eV to -0.381 eV, enhancing the electron acceptor ability of the benzene ring, increasing the molecular dipole moment, and promoting intramolecular charge transfer effects. Frontier molecular orbital calculations indicated that the HOMO energy level decreased from -4.64 eV to -4.03 eV, and the LUMO energy level decreased from 0.29 eV to -0.02 eV, resulting in a decrease in the band gap from 4.94 eV to 4.01 eV. Figure 2 b). This confirms that precursor molecular engineering is a crucial factor in achieving fluorescence redshift.

[0032] (2) The role of nitrogen doping configuration Analysis based on cutting-edge molecular orbital theory indicates that the amino nitrogen-doped structure formed by the reaction of compound 1 and monomer 3 with selenurea not only increases the nitrogen doping content but also significantly reduces the band gap (compound 1: 4.00 eV; amino nitrogen: 3.97 eV). Figure 2 c). Further research revealed that by introducing a triphenylamine group at the N-position of pyrrole to extend the conjugated sp² structure, spatial separation of the HOMO and LUMO was achieved, thereby enhancing the electron transfer effect. This modification led to a significant band gap contraction (pyrrole N: 4.03 eV → pyrrole N-graphene system: 3.74 eV). The combined results indicate that the increase in pyrrole N doping and the introduction of the strong electron-donating triphenylamine group in CD synergistically enhanced the system's electron-donating ability, while also increasing the molecular dipole moment, ultimately achieving an effective reduction in the band gap.

[0033] (3) The influence of carbonization polymerization process By constructing a structural model of pyrrolic Ns-graphene with progressively increasing pyrrolic nitrogen modification, the structural evolution during CD formation was simulated. DFT calculations show that as the system size increases, the bandgap continuously decreases from 3.74 eV to 3.54 eV. Figure 2d). Meanwhile, ultraviolet photoelectron spectroscopy measurements showed that CDs-3 had a HOMO level of -8.43 eV, a LUMO level of -7.16 eV, and an optical band gap of 1.36 eV. Figure 2 (e.g.) This bandgap closely matches the bandgap (1.15 eV) corresponding to the 1080 nm emission wavelength, confirming that the accumulation of graphene structures and the enrichment of pyrrole nitrogen during carbonization are key to achieving NIR-II emission.

[0034] Key findings: CDs-3 (prepared from monomer 3) exhibited strong emission characteristics in NIR-II (1000-1700 nm), with a fluorescence lifetime of 9964.90 ns. This is the first time that CDs with such properties have been prepared using aniline precursors. Subsequent research will focus on CDs-3.

[0035] Example 3: Antioxidant properties of CDs-3 In vitro antioxidant capacity assessment (DPPH method): DPPH (2,2-diphenyl-1-picrylhydrazyl) is a nitrogen-centered stable free radical, characterized by a double lone pair electron system surrounded by three benzene rings. Its ethanol solution is purple, with a maximum absorption peak at 517 nm. In the presence of antioxidants, DPPH free radicals are scavenged, initiating a color reaction that changes the solution from purple to yellow, and causing a decrease in absorbance at the maximum absorption wavelength. Therefore, this change can be used to assess the free radical scavenging activity and antioxidant performance of the material.

[0036] Method: Mix 0.1 mL of CDs-3 ethanol solution with gradient concentrations (0.02-1 mg / mL) with 0.9 mL of 0.1 mL MDPPH ethanol solution, let stand in the dark for 30 min, and measure the absorbance at 517 nm.

[0037] The results are as follows Figure 3 As shown, CDs-3 exhibits excellent antioxidant properties, with a half-maximal effective concentration (EC50) of 0.051 μg / mL against 0.1 mM DPPH free radicals, and the characteristic absorption peak of DPPH at 517 nm is significantly weakened, confirming its highly efficient free radical scavenging ability.

[0038] Assessment of antioxidant capacity at the cellular level (LX2 cell model): Methods: Cells were stored at 5 × 10⁻⁶ cells per cell line. 4Cells were seeded at a density of [number] cells / well in 12-well plates and cultured for 24 h. The culture medium was then replaced with fresh medium containing 50 μg / mL silymarin and CDs-3, and cultured for another 24 h. The medium was then discarded, and oxidative stress was induced for 4 h by adding 250 μM H2O2 solution. Before fluorescence microscopy, cells were washed three times with PBS and incubated with a 10 μM DCFH-DA probe at 37°C in the dark for 30 min, followed by three washes with serum-free medium to remove residual dye. Finally, the DCF green fluorescence signal was captured using a Leica DM18 inverted fluorescence microscope, enabling visualization of intracellular ROS levels.

[0039] The results are as follows Figure 4 As shown, the green fluorescence of DCF in LX2 cells treated with CDs-3 was significantly lower than that in the model group cells treated with H2O2, and its effect was comparable to that of silymarin, a widely used antioxidant, demonstrating its excellent antioxidant properties.

[0040] Example 4: NIR-II Imaging Performance of CDs-3 Methods: A biomimetic gallbladder model was constructed: PCR tubes containing 0.2 mL LCDs-3 bile solution were encapsulated within human gallbladder tissue. Imaging performance was evaluated using dual excitation light sources (808 nm and 980 nm lasers) and multi-band long-pass filters (LP: 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm).

[0041] The results are as follows Figure 5 As shown, under 808 nm excitation, the 1000–1300 nm LP filter clearly captures the bright NIR-II fluorescence of CDs-3, accurately outlining the morphology of a simulated gallbladder (PCR tube), while no signal is detected at 1400 nm; under 980 nm excitation, the same filter yields a high-contrast image, and weak fluorescence is observed at 1400 nm. This confirms that CDs-3 is compatible with dual excitation wavelengths and supports broadband NIR-II multichannel imaging.

[0042] Example 5: Evaluation of the anti-apoptotic effect of CDs-3 in a CCl4-induced mouse model of liver fibrosis Methods: Mice were randomly divided into a control group, a PBS group, a silymarin group (10 mg / kg, IV), and a CDs-3 group (10 mg / kg, IV). Except for the control group, all other groups received an intraperitoneal injection of an olive oil solution containing 20% ​​CCl4. The control group received an equal volume of olive oil solution. Fifteen days after injection, blood was collected from the mice's eyes, and they were euthanized. Liver tissue was harvested for TUNEL immunofluorescence staining.

[0043] The results are as follows Figure 6As shown, TUNEL staining results indicated that the hepatocyte apoptosis rate was significantly increased in the CCl4-treated group (PBS group) compared to the control group (2.7%) (reaching 8.0% at 2 weeks and 9.6% at 4 weeks). Meanwhile, the apoptosis rate in the CDs-3-treated group decreased to 4.7%, comparable to the silymarin-treated group (4.5%), confirming its protective effect through ROS scavenging.

[0044] Example 6: In vivo biocompatibility assessment Methods: Healthy C57BL / 6J mice were administered the drug via tail vein (100 μL, 11.5 mg / mL). Blood samples were collected on days 1 and 7 for biochemical analysis.

[0045] The results are as follows Figure 7 As shown, compared with the control group, the key parameters of kidney and cardiac function (creatinine (CREA), urea, lactate dehydrogenase (LDH), and creatine kinase (CK)) in the experimental group mice remained within the normal physiological range, with no significant differences compared with the PBS group. These results collectively indicate that CDs-3 has good in vivo safety, providing an important safety basis for its subsequent clinical application.

[0046] In summary, this invention discloses a bifunctional chloroplasts (CDs) possessing both efficient NIR-II imaging and excellent ROS scavenging capabilities, along with their preparation method. Addressing the issues of complex synthesis and unclear luminescence mechanisms in existing NIR-II CDs, this invention innovatively proposes a molecular engineering strategy based on the p-p-phenylenediamine amino group, constructing an aniline-derived framework for the first time, and successfully preparing novel CDs with tunable emission wavelengths to the NIR-II window. Experiments demonstrate that these CDs exhibit significant emission characteristics in the NIR-II band, high fluorescence lifetime, and can achieve high-contrast NIR-II imaging diagnosis of bile leakage and biliary tract diseases. Simultaneously, these CDs demonstrate excellent ROS scavenging capabilities in a liver fibrosis model, thus producing significant therapeutic efficacy. This invention provides an innovative strategy for developing hepatobiliary disease diagnostic nanoparticles integrating NIR-II imaging diagnosis and ROS scavenging therapy.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A dual-function carbon dot removal method combining near-infrared two-zone imaging and ROS removal, characterized in that, The carbon dots are prepared by reacting aniline monomers containing p-p-phenylenediamine amino groups with selenourea as a precursor, and have near-infrared II (NIR-II) fluorescence emission characteristics and reactive oxygen species (ROS) scavenging ability; the carbon dots have fluorescence emission in the 1000-1700 nm wavelength range.

2. The dual-function carbon dot imaging and ROS removal method according to claim 1, characterized in that: The aniline monomer containing the p-p-phenylenediamine amino group is tris(4-aminophenyl)amine.

3. The dual-function carbon dot imaging and ROS removal system according to claim 1 or 2, characterized in that: Its fluorescence lifetime is not less than 9900 ns.

4. A method for preparing a dual-function carbon dot for near-infrared dual-region imaging and ROS removal as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Dissolve aniline monomers containing p-p-phenylenediamine amino groups and selenourea in an acidic aqueous solution; (2) Perform a hydrothermal reaction on the solution from step (1); (3) Purify the reaction product to obtain the bifunctional carbon dots.

5. The method according to claim 4, characterized in that: The acidic aqueous solution is a 45-55 mM hydrochloric acid solution.

6. The method according to claim 4 or 5, characterized in that: The hydrothermal reaction in step (2) is carried out at a temperature of 180-220°C for 5-7 hours.

7. The method according to claim 4 or 5, characterized in that: The aniline monomer is tris(4-aminophenyl)amine, and its molar ratio with selenourea is 0.09-0.1:0.04-0.

05.

8. The method according to claim 4, characterized in that: The purification in step (3) includes filtration through a 0.22 μm filter membrane and / or dialysis with a molecular weight cutoff of 1000D.

9. The use of the bifunctional carbon dots as described in any one of claims 1 to 3 in the preparation of diagnostic and therapeutic agents for hepatobiliary diseases.

10. The application according to claim 9, characterized in that: The diagnostic and therapeutic agents are used for NIR-II imaging diagnosis of biliary stricture, bile leakage or biliary tract disease, and / or for the treatment of liver fibrosis by scavenging reactive oxygen species.

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