Preparation method and application of carbon-based ultra-small nanoparticle capable of efficiently removing reactive oxygen species

Abstract

The application discloses a preparation method of carbon-based ultra-small nano particles with high active oxygen removal efficiency and application thereof. Firstly, carbon dots (CDs) are synthesized by using gallic acid (GA) and polyethylene imine (PEI) as raw materials through a simple hydrothermal method. Through activity testing on the prepared CDs, it is found that the SOD activity of the CDs is as high as 18187 U / mg, which is much higher than the natural SOD activity. Meanwhile, the prepared CDs show excellent ROS removal activity, and can be used for the treatment of ROS related diseases, especially the treatment of acute kidney injury.

Description

Technical Field

[0001] This application relates to the field of nanozyme biotherapy, and in particular to a method for preparing carbon-based ultrasmall nanoparticles that efficiently scavenge reactive oxygen species and their applications. Background Technology

[0002] Reactive oxygen species (ROS) are inevitable byproducts of aerobic metabolism and play various physiological regulatory roles in biological systems, such as signal transduction and programmed cell death. Excessive ROS can lead to protein denaturation, DNA damage, or lipid peroxidation, thereby triggering various diseases. To maintain biological redox balance and prevent oxidative damage to the body, the endogenous antioxidant defense system consists of a series of natural enzymes and small antioxidant molecules that synergistically scavenge excess free radicals. The main cellular antioxidant mechanisms include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and small molecules such as glutathione (GSH). SOD is the first line of defense in the natural antioxidant system; it catalyzes the dismutation of O2, converting other toxic ROS into H2O2 and molecular oxygen. H2O2 is also toxic to biological systems; it can be converted into H2O and O2 by CAT, or into H2O by GPx with GSH as a cofactor.

[0003] Acute kidney injury (AKI), and its most severe form, acute renal failure, is a common clinical syndrome with high morbidity and mortality, characterized by a sudden deterioration in excretory renal function. Various clinical settings, including exposure to highly nephrotoxic drugs (i.e., cisplatin (CP) and other platinum derivatives), rhabdomyolysis, ischemia, and sepsis, are the initiation and progression of AKI. Studies have shown that during acute kidney injury, abnormally elevated ROS levels in renal tubular epithelial cells trigger mitochondrial damage, leading to apoptosis and a chain reaction of renal function impairment.

[0004] Antioxidant therapy is a newly emerging treatment strategy for AKI.

[0005] Although natural antioxidant systems are effective biocatalysts, their sensitivity to harsh physiological conditions, instability, susceptibility to modification, and high cost hinder their transformation and application.

[0006] To address these challenges, researchers have designed numerous enzyme-mimicking systems using nanozymes, which are defined as nanomaterials possessing intrinsic enzyme-like activity. Therefore, ROS-scavenging nanozymes that mimic endogenous antioxidant systems are potential drug candidates for treating ROS-related diseases.

[0007] The most abundant intracellular antioxidant enzyme is superoxide dismutase (SOD), which catalyzes the decomposition of O2·- into H2O2. The efficient elimination of O2·- by SOD is considered a promising strategy for treating acute kidney injury (AKI) and other inflammatory diseases; however, the structural instability, antigenicity, and low cell membrane permeability of SOD hinder its direct clinical application. Conversely, nanozymes, engineered nanomaterials with enzyme-like catalytic activity, have gradually emerged as promising candidates for antioxidant enzyme mimics.

[0008] Qu et al. (Multi-shell nanocomposites based multienzyme mimetics for efficient intracellular antioxidation) previously prepared a novel multifunctional nanozyme cooperative system composed of V₂O₅ and MnO₂, using dopamine as a linker, to mimic the intracellular antioxidant enzyme-based defense system. Furthermore, MnO₂-encapsulated selenium-melanin or two-dimensional carbide MXene nanozymes can also mimic intracellular antioxidant enzymes. However, the catalytic activity and unavoidable peroxidase activity of these metal-containing nanozymes are not ideal, further limiting their applications. Therefore, there is an urgent need to develop non-metallic nanozymes that can mimic the intracellular antioxidant system.

[0009] Carbon nanodots (CDs) are a class of ultra-small non-metallic carbon nanomaterials with a size of less than 10 nm. Due to their potential biocompatibility, water solubility, and tunable functionalization, they have attracted considerable attention in the biomedical field. Polyphenols, as natural antioxidants, possess significant biological effects, including a variety of pharmacological activities and high physiological stability. Summary of the Invention

[0010] This application aims to overcome the insufficient ROS scavenging performance of existing nanozymes by providing carbon-based ultrasmall nanoparticles with highly efficient reactive oxygen species scavenging capabilities. This application utilizes a simple hydrothermal method to prepare phenol-functionalized carbon-based ultrasmall nanoparticles and applies them to ROS scavenging.

[0011] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0012] In a first aspect, this application provides a method for preparing carbon-based ultrasmall nanoparticles, which includes at least the following steps:

[0013] Step S1: Mix the natural polyphenol gallic acid (GA) dispersion with polyethyleneimine (PEI) to obtain a mixture;

[0014] Step S2: The mixture obtained in step S1 is subjected to a hydrothermal reaction to obtain the original carbon dots;

[0015] Step S3: The raw carbon dots obtained in step S2 are post-processed to obtain the carbon-based ultra-small nanoparticles CDs.

[0016] The inventors of this application discovered through research that natural plant polyphenols, as natural antioxidants, possess significant biological effects, including various pharmacological activities and high physiological stability. Therefore, the inventors selected different natural polyphenols as raw materials, including tannic acid (TA), caffeic acid (CA), resveratrol (RES), and epicatechin (EGCG), and conjugated them with CDs respectively. The results showed that the ROS scavenging ability of TA-derived CDs (TA-CDs) was significantly reduced, possibly due to the larger conjugation system of TA, leading to significant aggregation of TA-CDs with a large number of inert phenolic groups; CA-CDs and RES-CDs had limited ROS scavenging abilities, which is related to the lack of typical 3,4,5-trihydroxybenzene structures in CA and RES; and although EGCG has abundant 3,4,5-trihydroxybenzene structures, its ROS scavenging activity is still slightly lower than that of GA due to its larger molecular weight.

[0017] Therefore, the inventors of this application selected natural plant polyphenol GA as a raw material to synthesize ultra-small phenolic functionalized CDs with renal selectivity and ROS scavenging ability. The oxidation of catechol and galloyl groups forms quinone groups, which can react with amino or aldehyde groups through Schiff base and / or Michael addition reactions.

[0018] Furthermore, the inventors of this application have discovered through research that the antioxidant activity of natural polyphenols is closely related to the substituent groups. The OH bond dissociation energy of phenolic groups is relatively low, which facilitates the donation of H atoms, thereby achieving better antioxidant capacity. Since amino groups have a strong electron-donating ability, the inventors chose polyethyleneimine (PEI), which is rich in amino groups, as a carbon source to promote the formation of phenolic functionalized CDs.

[0019] Preferably, the preparation method of the natural polyphenol gallic acid (GA) dispersion in step S1 is as follows:

[0020] The natural polyphenol gallic acid (GA) was dispersed in a beaker containing deionized water and stirred until homogeneous. Then, sodium hydroxide (NaOH) aqueous solution was added, stirred and dissolved at room temperature, and then sonicated to ensure uniform dispersion.

[0021] Considering that GA and PEI are prone to aggregation when simply mixed in water, this application adds NaOH when preparing natural polyphenol gallic acid GA, so that the mixed solution will gradually become clear.

[0022] Furthermore, alkaline conditions favor the Michael addition reaction, thereby contributing to the formation of CDs.

[0023] Preferably, the concentration of GA in step S1 is 0.1 mM to 10 mM.

[0024] Preferably, the concentration of PEI in step S1 is 0.1 mM to 10 mM.

[0025] Preferably, the concentration of NaOH in step S1 is 0.1 mM to 10 mM.

[0026] Preferably, the volume of NaOH in step S1 is 0.1 mL to 10 mL.

[0027] In step S2, this application uses a simple hydrothermal method to prepare CDs under high temperature and high pressure conditions using carbon source and water as raw materials. This method is characterized by low cost and no pollution, and the product has the characteristics of high fluorescence quantum yield and uniform particle size.

[0028] Preferably, the reaction temperature in step S2 is 100–200°C.

[0029] Preferably, the reaction time in step S2 is 2 to 12 hours.

[0030] Preferably, the post-processing step in step S3 is as follows:

[0031] The CDs obtained in step S2 were placed in a centrifuge tube, then centrifuged for a period of time. The supernatant was collected and then filtered through a filter head in another centrifuge tube to remove the precipitate. The resulting solution was placed in a 220nm dialysis bag for dialysis to remove unreacted small molecules. The solution in the dialysis bag was collected, frozen in a refrigerator, transferred to a vacuum freeze dryer, and freeze-dried to obtain the CDs.

[0032] Preferably, the centrifugation speed is 5000-10000 rpm and the centrifugation time is 1-10 min.

[0033] Preferably, the throttling molecular weight of the dialysis bag is 3500.

[0034] Preferably, the dialysis time is 3 days.

[0035] Preferably, the freezing temperature inside the refrigerator is -10 to -30°C, and the freezing time is 1 to 3 days.

[0036] Preferably, the freeze-drying temperature in the vacuum freeze dryer is -30 to -80°C, and the freezing time is 1 to 3 days.

[0037] Secondly, this application also provides carbon-based ultrasmall nanoparticles prepared by the above method.

[0038] Thirdly, this application also provides the use of the carbon-based ultrasmall nanoparticles prepared by this application.

[0039] As is well known to those skilled in the art, cellular redox balance is highly dependent on endogenous antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Accordingly, nanozymes that mimic the catalytic activity of natural antioxidant systems are potential drug candidates for treating reactive oxygen species (ROS)-related diseases. However, the catalytic activity of existing metal-containing nanozymes is unsatisfactory, and the activity of peroxidases is unavoidable, which limits their translational applications.

[0040] The inventors of this application have discovered through research that natural polyphenols and ultra-small non-metallic carbon dots possess exceptional ROS scavenging activity. Testing of the phenol-functionalized carbon dots prepared in this application revealed extremely high SOD activity, reaching 18187 U / mg, significantly higher than that of natural SOD. Theoretical calculations indicate that the bond dissociation degree of phenol on the non-metallic carbon dots is significantly lower than that of selenocysteine ​​in natural GPx, which facilitates the formation of peroxide intermediates and initiates GPx-like reactions. Furthermore, the kidney-targeting and intracellular antioxidant mimicry properties of the non-metallic carbon dots help to effectively alleviate cisplatin-induced acute kidney injury by inhibiting oxidative stress-mediated in vivo iron hypersensitivity reactions.

[0041] Therefore, the present invention has the following beneficial effects:

[0042] (1) This application provides a simple hydrothermal method to prepare CDs by reacting carbon source and water under high temperature and high pressure, which has the advantages of low cost and no pollution.

[0043] (2) The CDs synthesized in this application have the characteristics of high fluorescence quantum yield and uniform particle size;

[0044] (3) The CDs prepared in this application have extremely high SOD activity, up to 18187 U / mg, which is much higher than that of natural SOD.

[0045] (4) The CDs prepared in this application can be used for the treatment of ROS-related diseases, especially acute kidney injury. Attached Figure Description

[0046] Figure 1 TEM image of the target product CDs prepared in Example 1

[0047] Figure 2 Infrared spectrum of the target product CDs prepared in Example 1

[0048] Figure 3 XPS diagram of the target product CDs prepared in Example 1

[0049] Figure 4 The fluorescence spectrum of the target product CDs prepared in Example 1 is shown in Figure 1.

[0050] Figure 5 The graph shows the ABTS scavenging effect of the target product CDs prepared in Example 1.

[0051] Figure 6 The graph shows the removal of .OH from the target product CDs prepared in Example 1.

[0052] Figure 7 The target product CDs scavenging O prepared in Example 1 2- The picture Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0054] Example 1

[0055] Step S1: Weigh 150 mg of GA and disperse it in a beaker containing 18.75 mL of deionized water. Stir and mix well, add 2 mL of NaOH solution to dissolve it, and sonicate for 10 minutes to fully dissolve it until its color turns yellow.

[0056] Step S2: Add 200 mg of PEI to a beaker and stir continuously until the color turns brown. Then transfer it to a tetrafluoroethylene reaction vessel and place it in an oven at 120 degrees Celsius. After heating for six hours, the original carbon points are obtained.

[0057] Step S3: Take 20 mL of the original carbon dots obtained in step S2 and place them in a 50 mL centrifuge tube. Then transfer the tube to a centrifuge and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant in another centrifuge tube and filter it with a 220 nm filter head to purify and remove the precipitate.

[0058] Step S4: Place the solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 3500 and dialyze for three days to remove unreacted small molecules. Collect the solution in the dialysis bag, freeze it in a -20°C freezer, and transfer it to a vacuum freeze dryer to freeze dry at -50°C for three days to obtain CDs.

[0059] Example 2

[0060] Step S1: Weigh 100 mg of GA and disperse it in a beaker containing 12.5 mL of deionized water. Stir and mix well. Add 2 mL of NaOH solution to dissolve it. Sonicate for 10 minutes to fully dissolve it until its color turns yellow.

[0061] Step S2: Add 200 mg of PEI to a beaker and stir continuously until the color turns brown. Then transfer it to a tetrafluoroethylene reaction vessel and place it in an oven at 120 degrees Celsius. After heating for six hours, the original carbon points are obtained.

[0062] Step S3: Take 20 mL of the original carbon dots obtained in step S2 and place them in a 50 mL centrifuge tube. Then transfer the tube to a centrifuge and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant in another centrifuge tube and filter it with a 220 nm filter head to purify and remove the precipitate.

[0063] Step S4: Place the solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 3500 and dialyze for three days to remove unreacted small molecules. Collect the solution in the dialysis bag, freeze it in a -20°C freezer, and transfer it to a vacuum freeze dryer to freeze dry at -50°C for three days to obtain CDs.

[0064] Example 3

[0065] Step S1: Weigh 50 mg of GA and disperse it in a beaker containing 6.25 mL of deionized water. Stir and mix well. Add 2 mL of NaOH solution to dissolve it. Sonicate for 10 minutes to fully dissolve it until its color turns yellow.

[0066] Step S2: Add 150 mg of PEI to a beaker and stir continuously until the color turns brown. Then transfer it to a tetrafluoroethylene reaction vessel and place it in an oven at 120 degrees Celsius. After heating for six hours, the original carbon points are obtained.

[0067] Step S3: Take 20 mL of the original carbon dots obtained in step S2 and place them in a 50 mL centrifuge tube. Then transfer the tube to a centrifuge and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant in another centrifuge tube and filter it with a 220 nm filter head to purify and remove the precipitate.

[0068] Step S4: Place the solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 3500 and dialyze for three days to remove unreacted small molecules. Collect the solution in the dialysis bag, freeze it in a -20°C freezer, and transfer it to a vacuum freeze dryer to freeze dry at -50°C for three days to obtain CDs.

[0069] Example 4

[0070] Step S1: Weigh 100 mg of GA and disperse it in a beaker containing 12.5 mL of deionized water. Stir and mix well. Add 2 mL of NaOH solution to dissolve it. Sonicate for 10 minutes to fully dissolve it until its color turns yellow.

[0071] Step S2: Add 50 mg of PEI to a beaker and stir continuously until the color turns brown. Then transfer it to a tetrafluoroethylene reaction vessel and place it in an oven at 120 degrees Celsius. After heating for six hours, the original carbon points are obtained.

[0072] Step S3: Take 20 mL of the original carbon dots obtained in step S2 and place them in a 50 mL centrifuge tube. Then transfer the tube to a centrifuge and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant in another centrifuge tube and filter it with a 220 nm filter head to purify and remove the precipitate.

[0073] Step S4: Place the solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 3500 and dialyze for three days to remove unreacted small molecules. Collect the solution in the dialysis bag, freeze it in a -20°C freezer, and transfer it to a vacuum freeze dryer to freeze dry at -50°C for three days to obtain CDs.

[0074] Example 5

[0075] Step S1: Weigh 100 mg of GA and disperse it in a beaker containing 12.5 mL of deionized water. Stir and mix well. Add 4 mL of NaOH solution to dissolve it. Sonicate for 10 minutes to fully dissolve it until its color turns yellow.

[0076] Step S2: Add 50 mg of PEI to a beaker and stir continuously until the color turns brown. Then transfer it to a tetrafluoroethylene reaction vessel and place it in an oven at 150 degrees Celsius. After heating for six hours, the original carbon points are obtained.

[0077] Step S3: Take 20 mL of the original carbon dots obtained in step S2 and place them in a 50 mL centrifuge tube. Then transfer the tube to a centrifuge and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant in another centrifuge tube and filter it with a 220 nm filter head to purify and remove the precipitate.

[0078] Step S4: Place the solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 3500 and dialyze for three days to remove unreacted small molecules. Collect the solution in the dialysis bag, freeze it in a -20°C freezer, and transfer it to a vacuum freeze dryer to freeze dry at -50°C for three days to obtain CDs.

[0079] [Performance Characterization]

[0080] The CDs prepared in Example 1 were used for performance characterization, and the results are as follows:

[0081] Figure 1 The sample was characterized using high-resolution transmission electron microscopy (TEM).

[0082] from Figure 1 As can be seen from the TEM image, a large number of monodisperse spherical particles are uniformly distributed. The average size of CDs is 4.34±0.56nm, showing obvious crystallinity. The lattice spacing is about 0.21nm, which is related to the (100) plane of the graphite structure.

[0083] Figure 2 The chemical properties of the sample surface were characterized by Fourier transform infrared spectroscopy (FTIR).

[0084] from Figure 2 As can be seen from this, CDs are at 3424cm. -1 A strong absorption band exists at this location, indicating the presence of abundant phenolic hydroxyl and amino groups. Simultaneously, at 2966 cm⁻¹... -1 and 2854cm -1 Two absorption peaks are present at 1627 cm⁻¹, which is related to the stretching vibration of CH and originates from the alkane chain of PEI. -1 and 1457cm -1 The several absorption peaks indicate that there is a moderate amount of amide bonds on the CD surface.

[0085] Figure 3 The chemical properties of the sample surface were characterized by X-ray photoelectron spectroscopy (XPS).

[0086] from Figure 3 As can be seen, CDs have characteristic peaks at 284.8 eV, 399.0 eV and 532.0 eV, indicating the presence of C, N and O elements.

[0087] Figure 4 The chemical properties of the sample surface were characterized by fluorescence spectroscopy.

[0088] from Figure 4 As can be seen, when the excitation wavelength is 400 nm, CDs exhibit a significant fluorescence peak at 540 nm, proving the formation of CDs.

[0089] [Application Testing]

[0090] To evaluate the ROS scavenging activity of the CDs prepared in this application, the CDs prepared in Example 1 were tested. Their ABTS values ​​were tested separately. + • Scavenging activity, • OH scavenging activity and O2 - Eliminate activity.

[0091] ABTS + • Scavenging activity

[0092] ABTS (7 mM) was dissolved in water containing K2S2O8 (3 mM) to produce ABTS. + • The above mixture should be stored at room temperature, protected from light, for 24 hours before use. Then, dilute the above stock solution with ABTS using PBS (10 mM, pH 7.4). +· The concentration was diluted 50-fold. Then, different concentrations of CDs solution (25, 50, 75, 100 μg / mL) were added, and the solutions were further incubated in the dark for 10 min. Finally, the diluted ABTS were analyzed. +· The solution was detected by ultraviolet-visible-near-infrared absorption spectroscopy, and the absorbance at 734 nm was measured. The amount of ABTS removed was determined. + ·With the total ABTS + The ratio of CDs to ABTS was used to evaluate the effect of CDs on ABTS. + • The clearing effect.

[0093] Figure 5 This is for the free radical ABTS of different concentrations of CDs. + The scavenging activity of carbon dots was observed. As can be seen from the figure, the scavenging effect improved with increasing carbon dot concentration, reaching over 80% at 100 μg / mL.

[0094] ·OH radical scavenging activity

[0095] Take several 25 mL test tubes (sample tubes and blank tubes), and add FeSO4 and o-phenanthroline to 10 mL of PBS (pH = 7.40) respectively, so that the final concentration of FeSO4 and o-phenanthroline is 0.75 mmol / L. Add 1 mL of CDs solution of different concentrations, with the final CDs solution concentrations in the sample tubes being 25, 50, 75, 100, 125, and 150 μg / mL, respectively, while adding 1 mL of distilled water to the blank tubes. Immediately add H2O2 to make the final concentration 1 mmol / L. After mixing thoroughly, incubate at room temperature for 15 minutes, and then measure the absorbance of each tube at a wavelength of 536 nm using a spectrophotometer.

[0096] The scavenging rate (SA%) of CDs against ·OH free radicals can be calculated using the following formula:

[0097] Clearance rate SA (%) = (A0 - A1) / A0 × 100

[0098] Where A0 represents the absorbance without the sample, and A1 represents the absorbance with the sample.

[0099] Figure 6The figure shows the scavenging activity of different concentrations of CDs against ·OH radicals. As can be seen from the figure, the scavenging effect improves with increasing carbon dot concentration, reaching nearly 80% at 150 μg / mL.

[0100] ·O2 - Scavenging activity

[0101] Take several 2 mL test tubes (sample tubes and blank tubes), and add NBT and xanthine to 1 mL of PBS (pH = 7.40) respectively, so that the final concentrations of NBT and xanthine are 0.12 mmol / L and 2 mmol / L respectively. Then add 1 mL of CDs solution of different concentrations, with the final CDs solution concentrations in the sample tubes being 25, 50, 75, 100, 125, and 150 μg / mL, and 100 μL of distilled water added to the blank tubes. Then add xanthine oxidase solution to make the final concentration 5 mU / L. After mixing well, incubate at room temperature for 15 minutes, and then measure the absorbance of each tube at a wavelength of 560 nm using a spectrophotometer.

[0102] The scavenging rate E of CDs for superoxide anions can be calculated using the following formula:

[0103] E = (A 空白 -A 样品 ) / A 空白 ×100%

[0104] Among them, A 空白 This indicates the absorbance measured when distilled water is used instead of CDs.

[0105] Figure 7 This is for different concentrations of CDs against O2. - The scavenging activity was observed. As can be seen from the figure, the scavenging effect improved with increasing carbon point concentration, reaching nearly 80% at 25 μg / mL, indicating excellent performance and strong antioxidant properties.

[0106] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for preparing carbon-based ultrasmall nanoparticles that efficiently scavenge reactive oxygen species, characterized in that... The preparation steps include the following: Step S1: Mix the natural polyphenol gallic acid (GA) dispersion with polyethyleneimine (PEI) to obtain a mixture; Step S2: The mixture obtained in step S1 is subjected to a hydrothermal reaction to obtain the original carbon dots; Step S3: The raw carbon dots obtained in step S2 are post-processed to obtain the carbon-based ultra-small nanoparticles CDs; The preparation method of the natural polyphenol gallic acid (GA) dispersion in step S1 is as follows: The natural polyphenol gallic acid (GA) was dispersed in a beaker containing deionized water and stirred until homogeneous. A sodium hydroxide (NaOH) aqueous solution was added, and the mixture was stirred at room temperature until dissolved. The solution was then sonicated to ensure uniform dispersion. The concentrations of GA, PEI, and NaOH were 0.1 mM to 10 mM, and the volume of NaOH was 0.1 mL to 10 mL. The reaction temperature in step S2 is 100~200℃, and the reaction time in step S2 is 2~12h.

2. The preparation method according to claim 1, characterized in that, The post-processing steps in step S3 are as follows: The CDs obtained in step S2 were placed in a centrifuge tube, then centrifuged for a period of time. The supernatant was collected and then filtered through a 220nm filter head in another centrifuge tube to remove the precipitate. The resulting solution was placed in a dialysis bag and dialyzed for 3 days to remove unreacted small molecules. The solution in the dialysis bag was collected, frozen in a refrigerator, transferred to a vacuum freeze dryer, and freeze-dried to obtain the CDs.

3. The preparation method according to claim 2, characterized in that, The centrifugation speed is 5000~10000 rpm, and the centrifugation time is 1~10 min; the throttling molecular weight of the dialysis bag is 3500; the freezing temperature in the refrigerator is -10~-30℃, and the freezing time is 1~3 days.

4. Carbon-based ultrasmall nanoparticles for efficiently scavenging reactive oxygen species prepared by any of the preparation methods described in claims 1 to 3.

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