A method for preparing carbon dots using N-acetylcysteine, its products and applications

Abstract

This invention discloses a method for preparing carbon dots using N-acetylcysteine, the resulting product, and its application in the preparation of drugs that scavenge reactive oxygen species, delay nucleus pulposus cell senescence and / or protect nucleus pulposus cells, treat and / or alleviate intervertebral disc degeneration, and treat and / or alleviate low back pain. The carbon dots prepared using the antioxidant N-acetylcysteine ​​disclosed in this invention feature a simple preparation process, low synthesis cost, high enzyme activity, and good biocompatibility; they can effectively scavenge excess reactive oxygen species in vitro, exerting an antioxidant stress effect; they can delay the progression of intervertebral disc degeneration caused by needle puncture injury to the rat's caudal vertebrae; and they possess significant research value and clinical translational potential.

Description

Technical Field

[0001] This invention relates to a method for preparing carbon dots using N-acetylcysteine, as well as the products and applications thereof, and particularly to a method belonging to the field of nanomaterial preparation and medical applications. Background Technology

[0002] Lower back pain is one of the most common clinical symptoms in spinal surgery, but its etiology is complex, with intervertebral disc degeneration being one of the most common causes. Currently, the pathogenesis of intervertebral disc degeneration is not fully understood, and treatments can only alleviate clinical symptoms without effectively delaying or reversing the disease's progression. Studies have shown that oxidative stress caused by excessive production of reactive oxygen species (ROS) can accelerate intervertebral disc degeneration by affecting intervertebral disc cell senescence, inflammation, autophagy, and DNA methylation. Antioxidant stress may be an effective therapeutic target for delaying intervertebral disc degeneration.

[0003] In recent years, carbon dots (C-dots, CDs) have been widely used in nanomedicine due to their ultra-small size, excellent tunable optical properties, high water solubility, and good biocompatibility. Numerous studies have reported the enzyme-like activity and free radical scavenging ability of carbon dots. The antioxidant activity of CDs is mainly due to the properties of certain functional groups, such as primary amines and carboxyl groups. Furthermore, iron-doped carbon dots can also achieve various enzymatic activities because the presence of divalent and trivalent iron endows them with better redox enzyme activity. However, although the enzyme-like activity of CDs has been confirmed, it is still relatively low, and further improvements are needed for their application as antioxidants in the biomedical field. N-acetylcysteine ​​(NAC), a cysteine ​​precursor and glutathione precursor, has been used for decades to treat many diseases. NAC has been used as a tool to study the role of reactive oxygen species (ROS) in many physiological and pathological processes. As a ROS scavenger, it plays a crucial role in reducing inflammation and oxidative stress under various conditions.

[0004] Currently, there are very few reports on the antioxidant effect of carbon dots in delaying intervertebral disc degeneration. To further improve the antioxidant capacity of carbon dots, incorporating NAC into the carbon dot framework structure may be an effective strategy. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing carbon dots using N-acetylcysteine, as well as the products and applications thereof, which has the characteristics of simple preparation process, low synthesis cost, high enzyme activity, good biocompatibility, and can effectively remove excess reactive oxygen species and exert antioxidant stress.

[0006] Technical Solution: To solve the above-mentioned technical problems, this invention provides a method for preparing carbon dots using N-acetylcysteine, comprising the following steps:

[0007] (1) N-acetylcysteine, citric acid, ethylenediamine, and FeCl3·6H2O are used as precursors. The reaction solution is obtained by hydrothermal treatment at high temperature and cooling to room temperature. The molar mass ratio of N-acetylcysteine, citric acid, ethylenediamine, and FeCl3·6H2O is 0.5~2:5:5:1.

[0008] (2) The reaction solution described in step (1) is purified, dialyzed and dried to obtain solid powdered carbon dots.

[0009] In step (1), the solution used in the high-temperature hydrothermal process is placed in a corrosion-resistant and high-pressure-resistant container.

[0010] Preferably, the corrosion-resistant and high-pressure-resistant container is a stainless steel autoclave.

[0011] Furthermore, the stainless steel autoclave is a stainless steel autoclave with a polytetrafluoroethylene liner.

[0012] In step (1), the high-temperature hydrothermal process is maintained at a constant temperature of 180°C for 10 hours.

[0013] In step (2), the dialysis membrane used for dialysis has a molecular weight cutoff of 500 to 1000.

[0014] The present invention also provides carbon dots prepared by the method.

[0015] The present invention also provides the application of the carbon dots prepared by the method in the preparation of drugs for scavenging reactive oxygen species.

[0016] The present invention also provides the application of the carbon dots prepared by the method in the preparation of drugs for delaying nucleus pulposus cell senescence and / or protecting nucleus pulposus cells.

[0017] The present invention also provides the use of the carbon dots prepared by the method in the preparation of medicaments for treating and / or alleviating intervertebral disc degenerative diseases.

[0018] The present invention also provides the use of the carbon dots prepared by the method in the preparation of medicaments for treating and / or relieving lower back pain.

[0019] This invention focuses on the mechanism of intervertebral disc oxidative stress. Combining basic research methods, a novel carbon dot, namely N-acetylcysteine ​​carbon dots (NAC-CDs), is prepared by hydrothermal synthesis. Through in vitro and in vivo experiments, it is verified that the prepared novel carbon dots can exert antioxidant stress-anti-aging effects by scavenging ROS in an intervertebral disc degeneration model, in order to delay intervertebral disc degeneration.

[0020] The principle of this invention: NAC inherently possesses certain antioxidant activity; by incorporating it into an iron-containing carbon dot framework, it interacts with CDs to achieve higher enzyme activity and antioxidant capacity. In this invention, the average particle size of NAC-CDs is less than 10 nm. Their small size and large specific surface area provide abundant catalytic reaction binding and catalytic sites, and their enhanced chemical activity also facilitates catalytic reactions and electron transfer. The abundant oxygen-containing functional groups on the surface of NAC-CDs bind to superoxide anions through weak interactions such as electrostatics, hydrogen bonds, and other van der Waals forces, promoting redox reactions. The research results of this invention also confirm the synergistic effect of N-acetylcysteine ​​and FeCl3·6H2O.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The preparation of carbon dots by the antioxidant N-acetylcysteine ​​has the characteristics of simple preparation process, low synthesis cost, high enzyme activity and good biocompatibility; 2. It can effectively remove excess reactive oxygen species in vitro and exert an antioxidant stress effect; 3. It can delay the progression of intervertebral disc degeneration caused by acupuncture injury to the tailbone of rats; 4. It has great research value and clinical translation potential. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a method for preparing carbon dots using the antioxidant N-acetylcysteine.

[0023] Figure 2 Characterization of the prepared carbon dots (NAC-CDs): Figure 2 A shows the ultraviolet absorption and fluorescence emission spectra of carbon dots, with the inset showing photographs of the carbon dot dispersion under visible (Vis) and ultraviolet (Uv) light. Figure 2 B is a high-resolution transmission electron microscope image of carbon dots;

[0024] Figure 3 Infrared and X-ray spectra of carbon dots: Figure 3 A is the infrared spectrum of the carbon dots; Figure 3 B is the X-ray energy spectrum of carbon points;

[0025] Figure 4 To investigate the enzyme-like activity and total antioxidant capacity of carbon dots: Figure 4 A represents the SOD activity of carbon dots; Figure 4 B represents the GPx activity of the carbon dots; Figure 4 C represents the total antioxidant capacity of carbon dots;

[0026] Figure 5 To investigate the toxicity of different concentrations of carbon dots to nucleus pulposus cells (NPCs);

[0027] Figure 6Effects of carbon dots on H2O2-induced oxidative stress in nucleus pulposus cells: Figure 6 A represents the protective effect of different concentrations of NAC-CDs against H2O2-induced oxidative stress in nucleus pulposus cells; Figure 6 B is used to assess its protective effect by Calcein / PI cell live / dead staining; Figure 6 C represents the percentage of dead cells in the ImageJ quantitative analysis of live-dead staining;

[0028] Figure 7 Changes in ROS level and average fluorescence intensity of DHE: Figure 7 A shows the effect of DHE (Dihydroethidium) probe on NAC-CDs on H2O2-induced intracellular ROS levels in nucleus pulposus cells; 7B shows the ImageJ quantitative analysis of changes in the mean fluorescence intensity of DHE.

[0029] Figure 8 A is a mitochondrial red fluorescent probe (MitoTracker Red CMXRos) used to label biologically active mitochondria in nucleus pulposus cells and detect mitochondrial membrane potential; Figure 8 B represents the quantitative analysis of changes in the average red fluorescence intensity of mitochondria using ImageJ.

[0030] Figure 9 A represents the staining and detection of senescent nucleus pulposus cells using a cell senescence β-galactosidase staining kit; Figure 9 B represents the percentage of senescent nucleus pulposus cells analyzed quantitatively using ImageJ.

[0031] Figure 10 This is an immunofluorescence staining image of p21 in NPCs;

[0032] Figure 11 Postoperative imaging findings for each group in the in vivo experiment: Figure 11 A shows the coccyx X-ray and magnetic resonance imaging (MRI) of each group 4 weeks after surgery. Figure 11 B represents the statistical results of the changes in DHI% for each group;

[0033] Figure 12 Results of paraffin section staining of intervertebral disc tissue after surgery in each group of in vivo experiments: Figure 12 A is an H&E stained paraffin section of a rat intervertebral disc specimen 4 weeks after surgery; Figure 12 B is the image stained with safranin and fast green; Figure 12 C represents the histological scoring statistics;

[0034] Figure 13 Enzyme-like activities and total antioxidant capacity of NAC-CDs and NAC-CDs (non-Fe): Figure 13A shows the SOD enzyme activity assay of NAC-CDs and NAC-CDs (non-Fe); Figure 13 B represents the GPx activity of NAC-CDs and NAC-CDs (non-Fe); Figure 13 C represents the total antioxidant capacity of NAC-CDs and NAC-CDs (non-Fe);

[0035] Figure 14 Enzyme-like activities and total antioxidant capacity of NAC-CDs and CDs: Figure 14 A shows the SOD enzyme activity assay of NAC-CDs and CDs; Figure 14 B represents the GPx activity of NAC-CDs and CDs; Figure 14 C represents the total antioxidant capacity of NAC-CDs and CDs;

[0036] Figure 15 Enzyme-like activities and total antioxidant capacity of NAC-CDs modified with different amounts of NAC: Figure 15 A shows the SOD enzyme activity assay of NAC-CDs with different NAC-modified contents; Figure 15 B represents the GPx activity of NAC-CDs modified with different amounts of NAC; Figure 15 C represents the total antioxidant capacity of NAC-CDs modified with different amounts of NAC. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0038] The main reagents used in the examples are as follows: N-acetylcysteine, citric acid, FeCl3·6H2O, H&E staining kit, and Safranin O staining reagent were all purchased from Sigma-Aldrich Chemical Company; ethylenediamine was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai); total SOD activity assay kit (NBT method), glutathione peroxidase assay kit (NADPH method), total antioxidant capacity assay kit (ABTS rapid method), MTT assay kit, Calcein / PI cell viability and cytotoxicity assay kit, and Mito-Tracker Red... CMXRos (mitochondrial fluorescent probe), 4% paraformaldehyde fixative, DAPI fluorescent dye, and PBS solution were all provided by Shanghai Beyotime Biotechnology Co., Ltd. (China). Superoxide anion fluorescent probe DHE was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (China). β-galactosidase (SA-βGal) staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). Mouse anti-rat Col-2 polyclonal antibody and rabbit anti-rat MMP13 monoclonal antibody were purchased from Abcam (USA), and rabbit anti-rat Aggrecan polyclonal antibody was purchased from Abclonal (USA). Dialysis bags (MWCO 500–1000 Da) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China).

[0039] The main instruments used in this embodiment are as follows: a clean bench (Suzhou Fenjing Purification Equipment Co., Ltd.), a Tecnai 12 transmission electron microscope (TEM) (Philips, Netherlands), a Malvern particle analyzer (Malver, UK), an ESCALAB250Xi X-ray electron spectrometer (XPS) (Thermo, USA), a multi-functional microplate reader (TECAN, Austria), surgical instruments (Shangrao Yijianmei Medical Equipment Co., Ltd.), a centrifuge (Thermo, USA), a fluorescence microscope (Nikon, Japan), an X-ray machine, and a 3.0T nuclear magnetic resonance spectrometer (GE, USA). The PTFE-lined stainless steel autoclave was provided by Xi'an Changyi Instrument Equipment Co., Ltd.

[0040] Animal source: Thirty 8-week-old male SD rats were provided by Changzhou Cavens Laboratory Animal Co., Ltd., with animal licenses: SYXK(Su)2017-0044 and SCXK(Su)2018-0006. Animal handling during the experiment complied with the relevant animal ethics standards outlined in the 2009 "Ethical Issues in Animal Experimentation".

[0041] Example 1: Preparation of carbon dots using the antioxidant N-acetylcysteine

[0042] 1. Preparation of carbon dots

[0043] like Figure 1 As shown, N-acetylcysteine ​​(NAC, structural formula is...) ), citric acid (structural formula: ), ethylenediamine (structural formula: Using FeCl3·6H2O as a precursor (the molar ratio of NAC, citric acid, ethylenediamine, and FeCl3·6H2O is 1:5:5:1, where NAC is 0.001 mol), the solution was dissolved in 20 mL of deionized water and stirred at room temperature for 30 min. The resulting solution was then transferred to a 20 mL stainless steel autoclave lined with polytetrafluoroethylene. The autoclave containing the precursor was placed in an oven and subjected to hydrothermal treatment at 180°C for 10 hours. After the autoclave was naturally cooled to room temperature, it was purified using G25 gel filtration chromatography. Finally, the purified liquid was placed in a dialysis membrane (MWCO 500–1000 Da) for 3 days. The dialyzed liquid was then freeze-dried in a lyophilizer to obtain solid powdered carbon dots (NAC-CDs).

[0044] 2. Characterization of carbon dots

[0045] The carbon dots exhibit a typical UV-Vis absorption peak at 338 nm, with a maximum fluorescence emission wavelength of 420 nm. Figure 2 A), which appears slightly yellow under visible light and emits blue fluorescence under 365 nm ultraviolet light ( Figure 2 (Illustration in A). Morphological features of carbon dots were observed using high-resolution transmission electron microscopy. Figure 2 (B) It can be seen that the carbon dots have good dispersibility, and the particle size distribution is mainly in the range of 4-12 nm.

[0046] The surface structure of carbon dots was analyzed using Fourier transform infrared spectroscopy. Figure 3 A), at approximately 1402cm -1 and 1656 cm -1 The absorption bands at 2653 cm⁻¹ correspond to the tensile vibrations of CN and C=O, respectively. -1 The absorption band is attributed to the stretching vibration of SH, in the range of 3500-3000 cm. -1 The broad frequency band within this range corresponds to the stretching vibrations of OH or NH groups, indicating that the particle structure of NAC-CDs contains multiple hydrophilic functional groups such as hydroxyl, amino, and thiol groups, suggesting relatively high biocompatibility. In the figure, NAC-CDs and NAC exhibit several similar characteristic peaks, particularly around 2653 cm⁻¹. -1 The stretching vibrations indicate that the surface of NAC-CDs also contains thiol groups, suggesting that NAC-CDs partially retain the structural characteristics of NAC.

[0047] Surface composition and elemental analysis of carbon dots were performed using X-ray electron spectroscopy. Figure 3 B) shows six types of peaks: C1s (285 eV), N1s (399 eV), O1s (531 eV), S2p (163 eV), Fe2p (725 eV), and Cl2p (197 eV), corresponding to the proportions of the six elements as C (60.9%), N (14.1%), O (20.3%), S (0.7%), Fe (0.8%), and Cl (3.2%).

[0048] 3. Investigation of enzyme-like activity and total antioxidant capacity of carbon dots

[0049] (1) Investigation of superoxide dismutase (SOD) activity: The total SOD activity assay kit (NBT method) was used to detect the superoxide dismutase activity of NAC-CDs. The principle is that the reaction system of xanthine and xanthine oxidase produces superoxide anions (O2O2). 2•- The reaction mixture can reduce nitroblue tetrazolium to formazan, which exhibits strong absorption at 560 nm. Superoxide dismutase (SOD) can scavenge superoxide anions, inhibiting the formation of formazan. The lighter the blue color of the reaction solution, the higher the superoxide dismutase (SOD) activity, and vice versa. Figure 4 As shown in Figure A (the horizontal axis represents the mass concentration of NAC-CDs), within the mass concentration range of 0-200 µg / ml, the superoxide dismutase activity of NAC-CDs significantly increased with increasing SOD concentration.

[0050] (2) Glutathione peroxidase detection: Glutathione peroxidase (GPx) is an important peroxidase widely present in the body. The reduction in reduced nicotinamide adenine dinucleotide phosphate (NADPH) is linearly correlated with glutathione peroxidase activity; therefore, the glutathione peroxidase activity level can be calculated by detecting the reduction in NADPH. We used a GPx detection kit containing NADPH to indirectly determine the GPx-like activity of NAC-CDs. Figure 4 As shown in B, with the increase of NAC-CDs concentration (0, 10, 100 μg / ml), the amount of NADPH decreases and the GPx activity of NAC-CDs increases.

[0051] (3) Total Antioxidant Capacity (TOAC) Detection: The Total Antioxidant Capacity Detection Kit (ABTS Rapid Method) was used. The principle of determining the total antioxidant capacity is as follows: ABTS is oxidized to green ABTS˙+ under the action of an appropriate oxidant. The production of ABTS˙+ is inhibited in the presence of antioxidants. The total antioxidant capacity of NAC-CDs can be determined and calculated by measuring the absorbance of ABTS˙+ at 414 nm or 734 nm. Figure 4 As shown in Figure C, the total antioxidant capacity of NAC-CDs significantly increases with increasing mass concentration.

[0052] 4. Toxicity assessment of carbon dots

[0053] (1) Extraction and culture of primary rat nucleus pulposus cells

[0054] Six 8-week-old male SD rats (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were selected and, after 4 weeks of feeding, were injected intraperitoneally with an excessive amount of sodium pentobarbital (C). 11 H 17 Euthanasia was performed using N2Na3. Nucleus pulposus cells from the center of the coccygeal intervertebral disc were extracted under sterile laminar flow hood and digested with 0.5% type II collagenase at 37°C for 10 hours. The digested suspension was centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. Then, DMEM / F12 complete medium containing 10% fetal bovine serum was added to resuspend the precipitate. The suspension was added to cell culture flasks and cultured in a cell culture incubator at 37°C and 5% carbon dioxide. The medium was changed for the first time after 3 days, and then every 3 days thereafter. After 9 days, third-generation nucleus pulposus cells were obtained.

[0055] (2) The cytotoxicity of NAC-CDs was detected by using nucleus pulposus cells with the MTT assay.

[0056] The aforementioned third-generation nucleus pulposus cells were transferred to 96-well plates at a density of 6000 cells / well and co-cultured with different concentrations of NAC-CDs (0, 50, 100, 200, 400 μg / mL) for 24 h. After washing the cells with phosphate-buffered saline (PBS), 100 μL of DMEM / F-12 (including 10 μL of MTT solution, 5 mg / mL) was added to each well of the 96-well plate. After incubation at 37°C for 4 hours, all supernatant was aspirated, and 150 μL of DMSO was added and incubated for 10 min. The absorbance of each well was measured at 570 nm using a microplate reader. Figure 5 The results show that when the carbon dot concentration is 0.05 mg / mL, the cell survival rate exceeds 95%, and when the carbon dot concentration is increased to 0.1 mg / mL, the cell survival rate remains above 90%. This indicates that NAC-CDs have no obvious toxicity and are compatible with nucleus pulposus cells, which provides a promising candidate for anti-oxidative stress and delaying nucleus pulposus cell aging in vitro and in vivo.

[0057] Example 2: Application of carbon dots prepared with the participation of the antioxidant N-acetylcysteine

[0058] 1. NAC-CDs have antioxidant properties that delay intervertebral disc degeneration (in vitro effects).

[0059] To explore the possibility of synthesized NAC-CDs acting as antioxidants to delay intervertebral disc degeneration, in vitro experiments were first conducted to verify whether they have the ability to scavenge ROS and delay the aging of nucleus pulposus cells.

[0060] (1) Investigation of the ability of carbon dots prepared in Example 1 to counteract H2O2-induced oxidative stress in nucleus pulposus cells: We used H2O2-induced oxidative stress to assess whether NAC-CDs could protect nucleus pulposus cells. Different concentrations of NAC-CDs (0, 0.75, 1.5, 3, 6, 12, 24, 48 μg / mL) were co-cultured with third-generation nucleus pulposus cells (cell density 6000 cells / well in 96-well plates). Figure 6 As shown in Figure A, culturing nucleus pulposus cells with 100 µmol / L H2O2 reduced cell viability to 65%. However, the addition of different concentrations of NAC-CDs to the culture medium resulted in a gradient of cell viability that initially increased and then decreased. When the NAC-CDs concentration increased to 1.5 μg / mL, cell viability rose to 87%. When the NAC-CDs concentration increased to 3 μg / mL, cell viability rose to 93%; at this point, there was no significant difference in cell viability compared to the control group. Further increases in NAC-CDs concentration subsequently led to a decrease in cell viability. Therefore, it can be preliminarily concluded that 3 μg / mL is the optimal concentration of NAC-CDs to combat H2O2-induced oxidative stress in nucleus pulposus cells, while 1.5 μg / mL is less effective. We then used the Calcein / PI cell viability and cytotoxicity assay kit to evaluate the protective ability of NAC-CDs against oxidative stress-related cell death. Figure 6 C), when NPCs were treated with 100 µmol / L H2O2, PI staining showed 31.3% cell death, while the cell death rate of NPCs pretreated with NAC-CDs significantly decreased to 7.1% at 1.5 µg / mL and to 4.0% at 3 µg / mL. Figure 6 B). This result further confirms that NAC-CDs have a significant protective effect against H2O2-induced oxidative stress damage in nucleus pulposus cells.

[0061] (2) Carbon dots can scavenge intracellular ROS in nucleus pulposus cells: To confirm that the above-mentioned protective effect on nucleus pulposus cells can be attributed to the ability of NAC-CDs to scavenge ROS, we used the DHE (Dihydroethidium) probe to monitor the level of intracellular ROS induced by H2O2 in nucleus pulposus cells. DHE can be dehydrogenated to produce etidium under the action of intracellular superoxide anions. Ethidium can bind to RNA or DNA to produce red fluorescence. The stronger the red fluorescence, the higher the level of intracellular superoxide anions, and the higher the level of intracellular ROS. The monitoring results of the DHE probe are as follows: Figure 7As shown in Figure A, compared with the control group, the red fluorescence intensity of the H2O2-induced group (NAC-CDs: 0 μg / mL) was significantly increased, indicating that ROS expression was upregulated after H2O2 stimulation; at the same time, all NAC-CDs-treated groups showed a decrease in red fluorescence intensity, indicating a decrease in ROS levels. To quantify the ROS levels in these groups, ImageJ was used for further analysis of fluorescence intensity. Figure 7 As shown in Figure B, the DHE fluorescence intensity of the H2O2-treated group was significantly enhanced, with the mean fluorescence intensity (MFI) increasing nearly fourfold compared to the blank control group. NAC-CDs significantly reduced intracellular ROS levels, particularly in the 3 µg / mL NAC-CDs treatment group, where the MFI decreased to 1.9 times that of the blank control group. These results indicate that NAC-CDs do indeed have a significant scavenging effect on intracellular ROS in the H2O2-induced nucleus pulposus cell oxidative stress model.

[0062] (3) Carbon dots can maintain mitochondrial homeostasis and delay cell senescence: Excessive production of reactive oxygen species induces oxidative stress, and inefficient electron transfer in the mitochondrial respiratory chain is considered a major source of reactive oxygen species. Therefore, we used a red fluorescent mitochondrial probe to specifically label biologically active mitochondria in cells and detect mitochondrial membrane potential. Figure 8 As shown, H2O2 treatment decreased the density of nucleus pulposus cells, and the average red fluorescence intensity of mitochondria decreased by approximately 64% compared to the blank control group. The mitochondrial fluorescence intensity of nucleus pulposus cells in the NAC-CDs treatment groups at various concentrations recovered significantly, decreasing by only 23% and 6% respectively compared to the blank control group. This indicates that NAC-CDs can effectively maintain mitochondrial homeostasis while reducing intracellular reactive oxygen species levels. It is known that changes in mitochondrial redox status are significantly associated with the initiation of the aging process, and cellular senescence is a factor in intervertebral disc degeneration and low back pain. Our staining analysis of senescent cells revealed (…). Figure 9 The positive rate of senescence staining in nucleus pulposus cells (NPCs) increased after H2O2 treatment compared to the blank control group, while it decreased significantly in the NAC-CDs treatment group. p21, as a cell cycle inhibitor, is responsible for maintaining stable senescence arrest due to increased expression. Our p21 immunofluorescence staining of NPCs also revealed that after H2O2 treatment, the fluorescence intensity of p21 staining in the model group significantly increased, while the fluorescence intensity of p21 staining in the NAC-CDs treatment group significantly decreased. Figure 10 These results indicate that NAC-CDs have a role in delaying the senescence of nucleus pulposus cells in the context of H2O2-induced mitochondrial oxidative stress.

[0063] 2. NAC-CDs have antioxidant properties and delay intervertebral disc degeneration (in vivo effects).

[0064] Animal experimental grouping, model establishment, and in vivo local drug administration protocol: 24 male SD rats were randomly divided into a normal control group (NC group), a degenerative control group (DC group), and a NAC-CDs treatment group, with 8 rats in each group. The NC group did not receive acupuncture or PBS injection; in the DC and NAC-CDs treatment groups, the 8th-9th coccygeal vertebrae (Co8-9) were located and marked before surgery. Then, intraperitoneal anesthesia was administered with sodium pentobarbital (5 mg / 100g body weight), and the skin at the puncture site was disinfected. A 21G needle was inserted vertically into the intervertebral disc. The needle was rotated 360° and held for 30 seconds. After model establishment, the NAC-CDs treatment group immediately received an injection of 10 μL NAC-CDs (3 μg) into each IVD, while the DC group received an equal volume of PBS injected locally into the same segment. Four weeks post-surgery, all rats underwent in vivo X-ray and MRI examinations of the coccyx. After euthanasia, Co8-9 intervertebral disc specimens were collected for histological sectioning.

[0065] (1) Imaging findings: To further investigate the effects of NAC-CDs on intervertebral disc degeneration in vivo, we used a rat intervertebral disc degeneration model induced by needle puncture. From Figure 11 As shown in Figure A, in the blank control group (NC group), normal intervertebral discs with clearly defined intervertebral disc heights were visible on X-ray. In the degeneration control group (DC group), the intervertebral disc height decreased by approximately 57%. In contrast, the NAC-CDs treatment group showed significant recovery in intervertebral disc height, and the intervertebral disc height index (DHI%) was also significantly improved compared to the DC group. Figure 11 B). Magnetic resonance imaging (MRI) reliably reflects changes in intervertebral disc water content; higher T2-weighted signal indicates higher nucleus pulposus water content. Simultaneously, the nucleus pulposus in the NC group showed high T2-weighted signal, maintaining high water content, while the nucleus pulposus in the DC group showed significantly low T2-weighted signal. The nucleus pulposus signal in the NAC-CDs treatment group showed a greater degree of recovery. From an imaging perspective, NAC-CDs can reduce puncture-induced degeneration of the rat caudal intervertebral disc (IVD).

[0066] (2) Histological features: Four weeks after surgery, we also collected histological sections. By observing the H&E staining images of the rat intervertebral discs, we can find that ( Figure 12 A) In a normal intervertebral disc, the intervertebral space is intact, the intervertebral disc height is clear, the nucleus pulposus is full and clearly demarcated from the surrounding tissues, and the annulus fibrosus is arranged in an orderly manner. In the degeneration group (DC group), the intervertebral disc height is significantly reduced, the annulus fibrosus is disordered, and the internal structure of the nucleus pulposus is damaged, with a large amount of cells and matrix lost. In contrast, the intervertebral disc height in the NAC-CDs treatment group is slightly lower than that in the blank control group, with partial loss of the nucleus pulposus and a slightly disordered annulus fibrosus structure, showing significant improvement compared to the degeneration group. Histological grades were calculated based on previous studies; a higher score indicates more severe degeneration. Figure 12As can be seen from C, the score in the NAC-CDs treatment group was significantly lower than that in the degeneration group. Cartilage tissue is basophilic and binds to the basic dye Safranin O to appear red; bone tissue is eosinophilic and binds to the acidic dye Fast Green to appear green or blue. Safranin O-Fast Green staining results showed ( Figure 12 B) In the degeneration group, the central nucleus pulposus of the intervertebral disc was almost completely lost, and the original cartilage-like structure was replaced by osteoid tissue, with fusion and ossification present. In contrast, the NAC-CDs treatment group showed significant improvement in the intervertebral disc morphology compared to the degeneration group. In summary, X-ray, MRI imaging measurements, and tissue staining results indicated that the NAC-CDs treatment group showed significant improvements in intervertebral disc height index, nucleus pulposus signal intensity, and histological score compared to the degeneration group. In vivo experiments further demonstrated the feasibility of using local injection of NAC-CDs to protect the intervertebral disc and delay the progression of degeneration.

[0067] The above experimental results demonstrate that the carbon dots prepared by the antioxidant N-acetylcysteine ​​have the advantages of simple preparation process, low synthesis cost, high enzyme activity and good biocompatibility. They can effectively remove excess reactive oxygen species in vitro and exert antioxidant stress effects. Furthermore, in vivo experiments have verified that they can delay the progression of intervertebral disc degeneration caused by acupuncture injury to the tailbone of rats. It can be preliminarily considered that they have great research value and clinical translation potential.

[0068] Comparative Example 1

[0069] Following the method in Example 1, NAC-CDs (non-Fe) without FeCl3·6H2O raw material were prepared, and their enzyme-like activities (SOD and GPx) and total antioxidant capacity were determined. Figure 13 As shown in the figure (the horizontal axis represents the mass concentration of each carbon point (NAC-CDs (non-Fe), NAC-CDs)), similarly, the results of GPx activity measurements also show this trend. Compared to NAC-CDs, NAC-CDs (non-Fe) have lower enzyme activity and total antioxidant capacity. Therefore, the addition of FeCl3·6H2O can effectively improve the enzyme-like activity and total antioxidant capacity of NAC-CDs.

[0070] Comparative Example 2

[0071] Following the method in Example 1, carbon dots (CDs) containing 0.001 mol of FeCl3·6H2O but without NAC were prepared, and their enzyme-like activities (SOD and GPx) and total antioxidant capacity were measured. Figure 14As shown (the horizontal axis represents the mass concentration of each carbon dot (CDs and NAC-CDs)), compared to NAC-CDs, NAC-CDs exhibited higher enzyme-like activity and total antioxidant capacity than pure carbon dots (CDs) without NAC modification. Furthermore, within the mass concentration range of 0-200 μg / mL, NAC-CDs at the same concentration showed approximately 35%-140% higher SOD activity than CDs. Similarly, the results for GPx activity measurements were also consistent. These results indicate that NAC-CDs possess higher enzyme-like activity than CDs, and the total antioxidant capacity of NAC-CDs is also significantly enhanced compared to CDs. Therefore, the addition of NAC can improve the enzyme-like activity and total antioxidant capacity of NAC-CDs. Figure 14 We found that the addition of NAC and FeCl3·6H2O has a synergistic effect, resulting in NAC-CDs with stronger activity and antioxidant capacity.

[0072] Comparative Example 3

[0073] Following the method in Example 1, NAC-CDs modified with different amounts of NAC (0.001 mol, 0.002 mol, and 0.0005 mol) were prepared, and their enzyme-like activities (SOD and GPx) and total antioxidant capacity were measured; wherein the molar ratios of N-acetylcysteine, citric acid, ethylenediamine, and FeCl3·6H2O were 1:5:5:1, 2:5:5:1, and 0.5:5:5:1, respectively. Figure 15 As shown (the horizontal axis represents the mass concentration of NAC-CDs modified with different amounts of NAC), 0.001 mol NAC-modified NAC-CDs exhibited the highest SOD activity and total antioxidant capacity. At this point, the molar mass ratio of N-acetylcysteine, citric acid, ethylenediamine, and FeCl3·6H2O was 1:5:5:1.

Claims

1. A method for preparing carbon dots using N-acetylcysteine, characterized by, The method comprises the following steps: (1) N-acetyl cysteine, citric acid, ethylenediamine and FeCl3·6H2O are used as precursors, high-temperature hydrothermal treatment is carried out, and the reaction solution is obtained after cooling to room temperature; the molar mass ratio of N-acetyl cysteine, citric acid, ethylenediamine and FeCl3·6H2O is 0.5-2:5:5:1; the high-temperature hydrothermal treatment is carried out at a constant temperature of 180°C for 10 hours; (2) the reaction solution in step (1) is purified, dialyzed and dried to obtain solid powdery carbon dots.

2. The method of claim 1, wherein, The solution in the high-temperature hydrothermal treatment in step (1) is placed in a corrosion-resistant and high-pressure-resistant container.

3. The method of claim 1, wherein, The dialysis membrane used in the dialysis in step (2) has a molecular weight cut-off of 500-1000.

4. The carbon dots prepared by the method in any one of claims 1-3.

5. The use of the carbon dots prepared by the method in any one of claims 1-3 in the preparation of a medicine for scavenging reactive oxygen species.

6. The use of the carbon dots prepared by the method in any one of claims 1-3 in the preparation of a medicine for delaying the aging of nucleus pulposus cells and / or protecting nucleus pulposus cells.

7. The use of the carbon dots prepared by the method in any one of claims 1-3 in the preparation of a medicine for treating and / or relieving intervertebral disc degenerative diseases.

8. The use of the carbon dots prepared by the method in any one of claims 1-3 in the preparation of a medicine for treating and / or relieving lower back pain.

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