Preparation method and application of antioxidant carbon dots based on H2O2 diagnosis and treatment

Abstract

The invention belongs to the technical field of functional materials, and particularly relates to a preparation method and application of antioxidant carbon dots based on H2O2 diagnosis and treatment. According to the anti-oxidation carbon dot, curcumin and o-phenylenediamine serve as precursors, the anti-oxidation carbon dot with the fluorescence luminescence characteristic is prepared, and high-sensitivity response to H2O2 can be achieved. In an osteoarthritis environment, the carbon dot has a diagnosis and treatment effect by detecting H2O2 concentration change and using a fluorescence signal, and meanwhile, by virtue of the excellent oxidation resistance of the carbon dot, the progress of early joint diseases can be delayed. The carbon dots integrating detection and treatment functions have wide application prospects in the biomedical field, and particularly show important potential in diagnosis and treatment research of oxidative stress related diseases such as osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional materials, and specifically relates to a preparation method and application of antioxidant carbon dots based on H2O2 diagnosis and treatment. Background Art

[0002] Osteoarthritis (OA) is a common degenerative disease involving multiple joint tissues and cells, and its incidence continues to rise, bringing serious negative social impacts. Reactive oxygen species (ROS) play a significant role in the pathological process of OA. Excessive ROS can damage proteins, lipids and DNA in chondrocytes, thereby causing chondrocyte apoptosis. Chondrocyte apoptosis accelerates the degradation of cartilage matrix, ultimately promoting the occurrence and progression of OA. At the same time, ROS can activate inflammatory signaling pathways such as nuclear factor κB (NF-κB), inducing the release of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). [2] These inflammatory factors further exacerbate cartilage degradation and damage joint tissue. Early diagnosis, disease course monitoring and effective treatment of OA are crucial to preventing the progression of the disease. Therefore, the development of new injectable biomedical materials for monitoring and treating early OA has important clinical value.

[0003] The concentration of ROS in the joints of OA patients is often significantly higher than that of healthy individuals. As an important member of ROS, the concentration level of H2O2 is closely related to a variety of pathological processes, including cell damage and inflammatory response caused by oxidative stress. Carbon dots (CDs) with fluorescence imaging are an emerging nanomaterial that shows wide application potential in the biomedical field. The application of CDs in H2O2 detection mainly relies on their unique optical properties and surface functionalization characteristics. CDs have good photoluminescence properties, low toxicity, high biocompatibility and strong stability, making them ideal probe materials in biosensors. Through reasonable surface modification, CDs can be used to construct fluorescent sensors to achieve high sensitivity and high selectivity for H2O2 detection. When CDs react with H2O2, their fluorescence signal may be quenched or enhanced, and this change can be used to quantify the concentration of H2O2. By real-time diagnosis and treatment of H2O2 concentration fluctuations in joint cavity fluid or serum, it can provide effective biomarkers for the early diagnosis of osteoarthritis, thereby significantly improving the accuracy and efficiency of disease management. In addition, carbon dots also have antioxidant enzyme activity, which can quickly remove excess ROS in the body, thus providing a potential intervention for the treatment of OA and promoting the development of integrated diagnosis and treatment. Therefore, nanozyme technology based on carbon dots combining H2O2 diagnosis and treatment with antioxidant function has important clinical application prospects in the early detection and treatment of OA.

[0004] In the prior art, curcumin is generally used to prepare carbon dots with fluorescent imaging. However, curcumin is poorly soluble in water, resulting in a low utilization rate. It also has the disadvantages of weak self-fluorescence and is difficult to use for biological imaging. Therefore, it is necessary to improve the existing preparation method to obtain a carbon dot with better performance. Summary of the invention

[0005] Based on the above problems, the purpose of this application is to provide a preparation method and application of antioxidant carbon dots based on H2O2 diagnosis and treatment, and to provide a curcumin-modified carbon dot with integrated fluorescence imaging diagnosis and treatment by a hydrothermal method. The carbon dot has good water solubility, and the fluorescence intensity is positively correlated with the H2O2 concentration, and has excellent antioxidant properties, which can provide a new design scheme for the diagnosis and treatment of early osteoarthritis.

[0006] In order to achieve the above-mentioned object, the first technical solution of the present application provides a method for preparing antioxidant carbon dots based on H2O2 diagnosis and treatment, comprising the following steps:

[0007] Mixing and dissolving curcumin and o-phenylenediamine to obtain a precursor solution;

[0008] The precursor solution is mixed with a potassium hydrogen sulfate solution, and heated to react to obtain a reaction product;

[0009] The reaction product was purified and freeze-dried to obtain antioxidant carbon dots.

[0010] Furthermore, the mass ratio of curcumin to o-phenylenediamine is 1:1.

[0011] Furthermore, the precursor solution uses ethanol as a solvent, and the potassium bisulfate solution uses water as a solvent, wherein the ratio of ethanol to water is about 1:1 to 1.5;

[0012] Furthermore, the addition ratio of potassium bisulfate to the precursor solution is 1:5-6.

[0013] Furthermore, the heating temperature of the heating reaction is 180-200° C., and the reaction time is 12-16 hours.

[0014] Furthermore, the purification is first filtered through 0.66 micron and 0.22 micron filter membranes in sequence, and then dialyzed using a 100D dialysis membrane for 4 to 6 hours.

[0015] And, the antioxidant carbon dots prepared according to the above preparation method.

[0016] The second technical solution of the present application discloses a fluorescence sensor, which includes the above-mentioned antioxidant carbon dots.

[0017] The third technical solution of the present application discloses the use of the above-mentioned antioxidant carbon dots or fluorescent sensors in the preparation of fluorescent biomarkers for the diagnosis and treatment of osteoarthritis.

[0018] The fourth technical solution of the present application discloses the application of the antioxidant carbon dots in the preparation of antioxidant and free radical scavenging drugs.

[0019] Beneficial effects: In view of the shortcomings of curcumin, such as poor solubility in water, low utilization, weak autofluorescence, and difficulty in biological imaging, the present application adopts a hydrothermal method to prepare a curcumin-modified antioxidant carbon dot that integrates fluorescence imaging diagnosis and treatment; on the one hand, it has good water solubility and the ability to respond to H2O2 fluorescence; on the other hand, the antioxidant carbon dot also has excellent antioxidant properties and free radical scavenging properties, which can provide a new design scheme for the diagnosis and treatment of early arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the synthesis of antioxidant carbon dots for the treatment of H2O2 in Example 1.

[0021] Figure 2 This is the TEM image of the antioxidant carbon dots for treating H2O2 in Example 2.

[0022] Figure 3 This is the XPS spectrum of the antioxidant carbon dots for treating H2O2 in Example 3.

[0023] Figure 4 This is the XRD diagram of the antioxidant carbon dots for treating H2O2 in Example 4.

[0024] Figure 5 This is the H2O2 fluorescence intensity response spectrum of the antioxidant carbon dots for diagnosing and treating H2O2 in Example 5.

[0025] Figure 6 This is the DPPH scavenging spectrum of the antioxidant carbon dots for treating H2O2 in Example 6.

[0026] Figure 7 This is the PTIO clearance spectrum of the antioxidant carbon dots for treating H2O2 in Example 7.

[0027] Figure 8 It is the cytotoxicity of the antioxidant carbon dots used to treat H2O2 in Example 8.

[0028] Fig. 9 This is a laser confocal image of the antioxidant carbon dots for treating H2O2 in Example 9 after being induced by different concentrations of H2O2. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specifically stated, the relative arrangement of the parts and steps set forth in these embodiments, numerical expressions and numerical values ​​do not limit the scope of the application. The technology, method and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology, method and equipment should be considered as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, and therefore, once a certain item is defined in an accompanying drawing, it does not need to be further discussed in subsequent drawings.

[0031] Unless otherwise specified, the meanings of the technical terms in this specification are the same as those generally understood by those skilled in the art. However, if there is any conflict, the definitions in this specification shall prevail.

[0032] The first embodiment of the present application discloses a method for preparing antioxidant carbon dots based on H2O2 diagnosis and treatment, comprising the following steps:

[0033] Mixing and dissolving curcumin and o-phenylenediamine to obtain a precursor solution;

[0034] The precursor solution is mixed with a potassium hydrogen sulfate solution, and heated to react to obtain a reaction product;

[0035] The reaction product was purified and freeze-dried to obtain antioxidant carbon dots.

[0036] In this embodiment, the solvent of the precursor solution is ethanol, wherein the mass ratio of curcumin and o-phenylenediamine is 1:1, and the potassium bisulfate solution is an aqueous solution of potassium bisulfate. To ensure that the solute is fully dispersed or dissolved, the above two solutions are preferably mixed by vortex stirring.

[0037] In this embodiment, the reaction temperature of the heating reaction is 180-200°C for 12-16 hours. Under high temperature hydrothermal conditions, these carbon source materials undergo decomposition, dehydration, and condensation reactions to generate carbon dots with rich functional groups. During the reaction, the amino group (-NH2) in the o-phenylenediamine molecule provides a rich nitrogen source. The reaction under high temperature conditions forms nitrogen-doped carbon dots, which enhance their optical properties. In addition, the addition of potassium bisulfate can activate the amino group and increase the solvothermal reaction activity.

[0038] After the reaction is completed, the reaction product needs to be purified, preferably by first filtering through 0.66 micron and 0.22 micron filter membranes in sequence, and then dialyzing using a 100D dialysis membrane for 4 to 6 hours.

[0039] Furthermore, the antioxidant carbon dots described in the present application can be obtained by freeze-drying the purified product.

[0040] The technical effects of the antioxidant carbon dots prepared by the preparation method of the present application will be described in more detail below with reference to specific examples and drawings.

[0041] 1. Preparation of antioxidant carbon dots for H2O2 diagnosis and treatment

[0042] Example 1

[0043] Dissolve appropriate amount of curcumin and o-phenylenediamine (OPD) in anhydrous ethanol, and dissolve KHSO4 in ultrapure water. After complete dissolution, mix the two solutions, vortex stir for 10 minutes, and ultrasonicate for 10 minutes. After the solution is clarified, transfer the mixture to a 100mL polytetrafluoroethylene reactor and heat the reaction in an electric heating blower at 180-200℃ for 12-16 hours. After the reaction is completed, cool naturally to room temperature. Figure 1 The chemical reaction process of the present application is disclosed.

[0044] To purify the product, the solution in the reactor was placed in a fume hood and filtered with filter paper to remove unreacted large particles. Subsequently, the crude product was filtered with a 0.66 μm filter membrane, and then further filtered with a 0.22 μm filter membrane to remove residual large particles. Next, the product was placed in a dialysis bag with a molecular weight cutoff of 100 Da and dialyzed for 48 hours to remove unreacted small molecule residues. Finally, the antioxidant carbon dots (C-CDs) for H2O2 diagnosis and treatment were obtained by freeze drying.

[0045] 2. Performance Test of Antioxidant Carbon Dots for Diagnosis and Treatment of H2O2

[0046] Example 2 Structural Performance Test

[0047] After preparing the C-CDs solution, we took out the clean copper mesh with tweezers and carefully placed it on a clean filter paper. Subsequently, a pipette was used to absorb an appropriate amount of C-CDs solution and evenly dripped it on the copper mesh to cover the entire surface. After the solution was completely dried, the sample was used for subsequent characterization analysis. In order to study the microstructure and dispersibility of C-CDs in detail, we used a high-resolution transmission electron microscope (JEM-1400) at an accelerating voltage of 200 kV to observe and analyze the morphology, size distribution and dispersibility of its nanoparticles. The powder X-ray diffraction (PXRD) spectrum was obtained by scanning on an X-ray diffractometer (Rigaku, Japan), using Cu Kα rays, and a 2θ scanning range of 5-70°. X-ray photoelectron spectroscopy analysis (XPS) was obtained by an X-ray photoelectron spectrometer (AXIS Ultra DLD, Kratos, UK) using Al Kα (1486.6 eV) as the excitation source.

[0048] like Figure 2 The projection electron micrograph of the antioxidant carbon dots for diagnosing and treating H2O2 prepared in Example 1 clearly shows its morphological characteristics. The nanozyme has a uniformly distributed spherical structure with an average particle size of about 3nm and a lattice spacing of 0.24nm. Its highly uniform particle size distribution and clear lattice characteristics provide strong support for the stability and functionality of the material.

[0049] like Figure 3 The figure shows the XPS graph of the antioxidant carbon dot material for diagnosing and treating H2O2 prepared in Example 1. The four characteristic peaks appearing in the XPS spectrum are respectively attributed to C1s (286.4 eV), N1s (400.1 eV) and O1s (531.5 eV), proving that the two elements N and S have been successfully doped in CDs.

[0050] like Figure 4 As shown, in the XRD pattern of the antioxidant carbon dots for diagnosing and treating H2O2 prepared in Example 1, the diffraction peaks show sharp and clear characteristics, indicating that the material has a typical crystalline structure. This result verifies the high crystallinity of carbon dot-based materials, provides structural support for their stable physical and chemical properties, and also lays a good foundation for their application in the fields of catalysis and functional materials.

[0051] Example 3 Fluorescence spectrum analysis

[0052] In this embodiment, a method for preparing a H2O2-responsive fluorescence spectrum is provided, and the steps are as follows:

[0053] First, accurately draw an appropriate amount of H2O2 standard, add it to 10mL of ultrapure water, and mix it to prepare a H2O2 mother solution with a concentration of 1mM. Then, based on this mother solution, use ultrapure water to dilute it in a gradient to prepare H2O2 solutions with concentrations of 250μM, 500μM, 750μM and 1000μM, respectively. All solutions must be fully mixed to ensure uniform and accurate concentrations. Subsequently, draw 3mL of H2O2 solutions with different gradient concentrations, and add an appropriate amount of C-CDs solution to make the final concentration of 1mg / mL. Transfer the mixed solution to a clean, dry four-way cuvette, and mix gently to ensure the uniformity of the sample and the repeatability of the experiment. Use a fluorescence spectrometer to test the fluorescence performance of the sample, select the appropriate excitation wavelength and set the emission wavelength range to record the fluorescence intensity and emission spectrum characteristics of C-CDs under different H2O2 concentrations. The experiment aims to analyze the fluorescence response of C-CDs under different oxidation conditions, and to further study the sensitivity of C-CDs to H2O2 and its potential detection ability by plotting the curve of fluorescence intensity changing with H2O2 concentration.

[0054] like Figure 5 As shown, the fluorescence intensity of the antioxidant carbon dots for diagnosing and treating H2O2 prepared in Example 1 is positively correlated with H2O2. As the concentration of H2O2 increases, the fluorescence intensity of the carbon dots of the nanozyme is enhanced.

[0055] Example 4

[0056] In this embodiment, the antioxidant steps of C-CDs with different concentrations are provided as follows:

[0057] Weigh 0.0035g DPPH reagent, dissolve it in anhydrous methanol, mix it thoroughly, and place it in a dark environment for 60 minutes to react to ensure that DPPH is fully dissolved and stable. After the reaction is completed, dilute the solution to a final concentration of 1mg / mL, prepare it into a DPPH mother solution, and store it in a dark environment for standby. During the experiment, 1mL of 1mg / mL DPPH solution was drawn into a clean cuvette, and then an appropriate amount of C-CDs mother solution was added in sequence to make the final concentration of curcumin carbon dots reach 100μg / mL, 50μg / mL, 75μg / mL and 25μg / mL, respectively. Gently mix the solution with a vortex for 3 minutes to ensure sufficient reaction and avoid the influence of bubbles in the solution on the detection. Next, the solution was tested for optical absorption performance using a UV-visible spectrometer, and the absorption spectrum of each group of solutions at 517nm was recorded. By analyzing the changes in the absorbance of the DPPH solution, the scavenging ability of curcumin carbon dots on DPPH free radicals can be indirectly reflected.

[0058] like Figure 6As shown, the carbon dots prepared according to Example 1 exhibited excellent scavenging ability in the DPPH free radical scavenging experiment. This result shows that the carbon dots have significant antioxidant properties, especially high activity in scavenging reactive nitrogen species. Its excellent antioxidant properties may be derived from the unique structure of the carbon dots and the interaction of surface functional groups, which provides broad prospects for its application in free radical scavenging and antioxidants.

[0059] Example 5

[0060] In the PTIO test, in order to evaluate the free radical scavenging ability of the polymer, the PTIO solution was first prepared, and the PTIO was dissolved in deionized water to prepare a solution with a concentration of 0.015wt%, and the solution was ensured to be uniform and transparent. Subsequently, the polymer to be tested was dissolved in the PTIO solution, and the final concentration of C-CDs was adjusted to 1mg / mL. After mixing evenly, it was placed in a 37°C thermostat and incubated in the dark for 2 hours to ensure that the free radical scavenging reaction could be fully carried out. After the reaction was completed, the reaction system was transferred to a cuvette, and the spectrum was scanned using a UV-visible spectrophotometer, with a focus on recording the absorbance change at 557nm. The decrease in absorbance at 557nm reflects the polymer's ability to scavenge PTIO free radicals. By comparing the changes in absorbance before and after the reaction, the free radical scavenging rate of C-CDs can be further calculated to evaluate its antioxidant properties.

[0061] like Figure 7 As shown, the carbon dots prepared according to Example 1 exhibited significant scavenging ability in the PTIO free radical scavenging experiment. This result further verified its excellent antioxidant performance, indicating that the nanozyme can not only effectively scavenge DPPH free radicals, but also has a good scavenging effect on PTIO free radicals. Its multifunctional free radical scavenging activity may be derived from the rich functional groups and unique electron transfer ability of the carbon dots, which makes it widely adaptable and efficient in the antioxidant mechanism.

[0062] Example 6

[0063] In this experiment, the in vitro cytotoxicity of the carbon dots prepared in Example 1 to rat chondrocytes was studied by CCK-8 method.

[0064] Experimental process:

[0065] (1) First, curcumin carbon dots were dissolved in water to prepare a stock solution, which was then gradually diluted with DMEM medium to form a series of concentrations of samples.

[0066] (2) The logarithmic growth phase chondrocytes were seeded into 96-well plates at a density of 1500 cells / well and cultured at 37°C and 5% CO2 for 24 hours. Subsequently, the cells were washed with PBS buffer and 100 μL of pure culture medium was added to each well. The samples pretreated with different gradient concentrations and blank complete culture medium were used as controls and continued to be cultured at 37°C for 24 hours. Six parallel experiments were set up for each concentration and incubated in an incubator for 1 day. The old culture medium was removed, washed with PBS three times, protected from light, and 100 μL of detection solution (CCK-8: culture medium = 1:10 (v / v), prepared and used immediately) was added to each well, and the blank detection solution was set as the standard. After incubation in the incubator for 1-2 hours, the optical density of each well at a wavelength of 450 nm was detected using a full-wavelength microplate reader, and the cell survival rate of each group was calculated. The cell survival rate was measured by the absorbance of each well at 450 nm using a Tecan Safire2 microplate reader.

[0067] (3) The cell viability was measured by Tecan Safire2 microplate reader and the absorbance of each well at 490 nm was calculated.

[0068] like Figure 8 As shown, the carbon dots prepared according to Example 1 exhibited good biocompatibility in the chondrocyte environment. This property laid a solid foundation for its application in subsequent biological experiments, indicating that the material had no obvious adverse effects on cell growth and function and had a wide range of biosafety.

[0069] Example 7

[0070] In this embodiment, the intracellular H2O2 fluorescence intensity response is provided, and the steps are as follows:

[0071] In this experiment, macrophages were used as a cell model, and the response behavior of mouse macrophages to H2O2 concentration was determined by fluorescence microscopy. We first sterilized the 96-well plate for use, then inoculated rat chondrocytes in the above-mentioned well plate with a number of 1500 cells per well, added 200 μL of complete growth medium to each well for culturing cells, and maintained them at 37°C, 5% CO2 culture conditions for 24 hours, then removed the culture medium, and then added H2O2 solutions of different concentration gradients to make the final culture medium concentrations of 250, 500, and 1000 μM H2O2 concentrations, respectively. After incubating the cells in the incubator for 4 hours, an appropriate amount of C-CDs was then added and incubated for another 4 hours. Finally, the cells were washed three times with PBS buffer solution, fixed with 4% paraformaldehyde solution at room temperature for 20 minutes, and counterstained with DAPI for 10 minutes. The fluorescence intensity of C-CDs in cells at different H2O2 concentrations was observed by fluorescence microscopy.

[0072] like Fig. 9As shown, the carbon dots prepared in Example 1 exhibited good cell imaging effects, and their fluorescence intensity gradually increased with the increase of H2O2 concentration. This phenomenon shows that the carbon dot material can be used to sensitively respond to changes in hydrogen peroxide concentration, providing the possibility for H2O2 detection and real-time diagnosis and treatment based on fluorescence signals. Its excellent fluorescence characteristics and response performance have opened up new application avenues for research fields related to biosensing, cell imaging and oxidative stress, and have important scientific research and practical application value.

[0073] According to the above analysis, the antioxidant carbon dots prepared by the preparation method of the present application, on the one hand, can achieve a highly sensitive fluorescence response to H2O2, and on the other hand, have excellent antioxidant and free radical scavenging properties; and in the osteoarthritis environment, excessive reactive oxygen species (ROS) can damage proteins, lipids and DNA in chondrocytes, thereby causing chondrocyte apoptosis; therefore, the antioxidant carbon dots can simultaneously realize the diagnosis and treatment of osteoarthritis; providing a new design scheme for the diagnosis and treatment of early arthritis.

[0074] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing antioxidant carbon dots based on H2O2 diagnosis and treatment, characterized in that: The steps include: Mixing and dissolving curcumin and o-phenylenediamine to obtain a precursor solution; The precursor solution is mixed with a potassium hydrogen sulfate solution, and heated to react to obtain a reaction product; The reaction product was purified and freeze-dried to obtain antioxidant carbon dots.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the curcumin to o-phenylenediamine is 1:

1.

3. The preparation method according to claim 1, characterized in that: The precursor solution uses ethanol as solvent, the potassium hydrogen sulfate solution uses water as solvent, and the ratio of ethanol to water is about 1:1-1.

5.

4. The preparation method according to claim 1, characterized in that: The addition ratio of potassium hydrogen sulfate to the precursor solution is 1:5-6.

5. The preparation method according to claim 1, characterized in that: The heating temperature of the heating reaction is 180-200° C., and the reaction time is 12-16 hours.

6. The preparation method according to claim 1, characterized in that: The purification is firstly filtered through 0.66 micron and 0.22 micron filter membranes in sequence, and then dialyzed using a 100D dialysis membrane for 4 to 6 hours.

7. An antioxidant carbon dot prepared according to the preparation method according to any one of claims 1 to 6.

8. A fluorescence sensor, characterized in that: Comprising the antioxidant carbon dots as described in claim 7.

9. Use of the antioxidant carbon dots according to claim 7 or the fluorescent sensor according to claim 8 in preparing a biomarker for fluorescent diagnosis and treatment of osteoarthritis.

10. Use of the antioxidant carbon dots according to claim 7 in the preparation of antioxidant and free radical scavenging drugs.

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