Monatomic manganese-doped amino-rich PEG carbon quantum dot as well as synthesis method and application thereof

Abstract

The invention discloses a synthesis method of monoatomic manganese-doped amino-rich PEG (polyethylene glycol) carbon quantum dots, and belongs to the technical field of biomedical materials.The synthesis method comprises the following steps: 1) adding manganese chloride into PEG400, and uniformly mixing to form a uniform mixed solution A; 2) adding ethylenediamine hydrochloride into the mixed solution A obtained in the step 1), and uniformly mixing to form a uniform mixed solution B; and (3) carrying out hydrothermal reaction on the mixed solution B obtained in the step (2), after the reaction is finished, cooling to room temperature, washing, purifying and drying to obtain the monoatomic manganese-doped amino-enriched PEG carbon quantum dots. The synthesized Mn (at) NH2-CDs can efficiently inhibit ferroptosis by removing active oxygen and ferrous ions, and the inhibition effect of the Mn (at) NH2-CDs on the cellular level is obviously higher than that of existing common active oxygen removing drugs and iron ion chelating agents; and the surface of the material is rich in amino groups, so that the material carries a large number of positive charges, and can penetrate through a blood brain barrier to enter brain tissues to inhibit ferroptosis through adsorption-mediated transendocytosis, thereby realizing treatment of shock wave brain injury.

Description

Technical Field

[0001] This invention belongs to the field of carbon dot synthesis technology, specifically relating to a single-atom manganese-doped amino-rich PEG carbon quantum dot, its synthesis method, and its application. Background Technology

[0002] Blast-induced traumatic brain injury (bTBI) is a common type of traumatic brain injury in modern military conflicts, counter-terrorism operations, and industrial accidents. Its injury mechanism is complex, involving not only the direct mechanical action of the primary shock wave on brain tissue but also a series of complex secondary (inertial effects) and tertiary (throwing impact) injuries, leading to widespread neuroinflammation, blood-brain barrier disruption, oxidative stress, and various forms of programmed cell death. Currently, clinical treatments for bTBI are very limited, mainly focusing on symptomatic and supportive care, lacking highly effective drugs that can specifically intervene in its pathological process and effectively protect nerve cells. Therefore, developing novel neuroprotective agents is a major challenge that urgently needs to be addressed in the field of neurotrauma.

[0003] In the complex cell death network following brain injury (bTBI), ferroptosis, as an iron-dependent form of programmed cell death, is increasingly recognized as a key mechanism of neuronal death after bTBI. It is characterized by intracellular iron accumulation and excessive production of lipid peroxides (such as reactive oxygen species), leading to cell membrane system collapse. Shock waves acting on brain tissue induce iron metabolism disorders (massive release of free iron) and impaired function of antioxidant systems (such as the glutathione-GPX4 pathway), thereby significantly promoting ferroptosis. Inhibition of the ferroptosis pathway has been shown to effectively reduce neurological deficits in various brain injury models; therefore, targeting ferroptosis has become a promising new strategy for treating bTBI.

[0004] Carbon quantum dots (CDs) are zero-dimensional carbon-based nanomaterials typically smaller than 10 nm. Due to their excellent biocompatibility, low toxicity, easily functionalizable surface, good water solubility, and unique optical properties, they have shown great potential in fields such as biosensing, bioimaging, and drug delivery. In recent years, an increasing number of studies have begun to explore the applications of CDs in disease treatment, particularly in neuroprotection. Some studies have shown that certain types of CDs, due to their antioxidant enzyme-like activities (such as superoxide dismutase (SOD) or peroxidase (CAT) activity), can effectively scavenge reactive oxygen species (ROS) and alleviate oxidative stress, thereby playing a protective role in models of ischemia-reperfusion injury and Alzheimer's disease.

[0005] However, existing technologies have the following significant shortcomings: 1) Limited function and unclear targeting: The mechanisms of existing reported neuroprotective CDs are mostly broadly attributed to "antioxidant" effects, lacking precise targeting and in-depth mechanistic exploration of specific cell death pathways (such as ferroptosis); their protective effects are limited, and their effectiveness for bTBI, a specific type of injury, remains unknown; 2) Difficulty in penetrating the blood-brain barrier: Shockwave-induced brain injury is mainly mild, with minimal damage to the blood-brain barrier, making it difficult for most ferroptosis inhibitors to enter the brain and exert their effects; 3) Lack of application scenarios: To date, no research has reported that CDs can be used to treat shockwave-induced brain injury, and no publicly available technical solutions have been found for CDs to treat bTBI by specifically inhibiting ferroptosis pathways. The behavior, distribution, metabolism, and efficacy of CDs in the unique and complex pathological model of bTBI are completely unknown.

[0006] In summary, there is an urgent need in this field to develop a drug that can penetrate the blood-brain barrier to treat shock wave brain injury, and to elucidate its neuroprotective mechanism by precisely inhibiting key molecular pathways of ferroptosis, in order to fill the gaps in the existing technology and provide a novel nanomedicine candidate for the treatment of bTBI. Summary of the Invention

[0007] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a single-atom manganese-doped amino-rich PEG carbon quantum dot, which is synthesized using PEG400, manganese chloride, and ethylenediamine hydrochloride as precursors via a simple hydrothermal method. The prepared single-atom manganese-doped amino-rich PEG carbon quantum dot can penetrate the blood-blood barrier and inhibit ferroptosis in brain cells.

[0008] This invention also provides a method for preparing the single-atom manganese-doped amino-rich PEG carbon quantum dots, and their application in the preparation of drugs for treating shock wave brain injury.

[0009] The objective of this invention is achieved through the following technical solution: A method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots specifically includes the following steps: 1) Add manganese chloride to PEG400 and mix well to form a homogeneous mixed solution A; 2) Add ethylenediamine hydrochloride to the mixed solution A obtained in step 1), mix well, and form a homogeneous mixed solution B; 3) The mixed solution B obtained in step 2) was subjected to a hydrothermal reaction. After the reaction was completed, it was cooled to room temperature, washed, purified and dried to obtain single-atom manganese-doped amino-rich PEG carbon quantum dots (Mn@NH2-CDs).

[0010] Shockwaves, when applied to brain tissue, can disrupt iron metabolism (leading to the release of large amounts of free iron) and impair the function of antioxidant systems (such as the glutathione GPX4 pathway), thereby significantly promoting ferroptosis. However, shockwave-induced brain injury is primarily mild, with only minor damage to the blood-brain barrier, making it difficult for most ferroptosis inhibitors to penetrate the brain and exert their effects. Furthermore, existing reports of neuroprotective drugs (CDs) are largely attributed to "antioxidant" mechanisms, resulting in limited protective efficacy. Therefore, developing nanomedicines that are low in toxicity and can penetrate the blood-brain barrier to inhibit ferroptosis is of great significance for the treatment of shockwave-induced brain injury.

[0011] To this end, this invention designed and synthesized a single-atom manganese-doped amino-rich PEG carbon quantum dot (Mn@NH2-CDs). Using PEG400 as a precursor, this carbon dot exhibits low toxicity and good biocompatibility. The single-atom manganese doping enables it to efficiently scavenge reactive oxygen species (ROS), while the abundant amino groups on the carbon dot surface allow it to chelate ferrous ions in solution. This synergistic scavenging of ROS and ferrous ions effectively inhibits ferroptosis. Simultaneously, the abundant amino groups give the carbon dot a positive charge, enabling it to penetrate the blood-brain barrier via adsorption-mediated transcytosis and enter the brain to exert its function. Cell experiments demonstrated that Mn@NH2-CDs effectively inhibit ferroptosis, and animal experiments demonstrated that Mn@NH2-CDs effectively penetrate the blood-brain barrier to treat shock wave brain injury. The ability of Mn@NH2-CDs to efficiently inhibit ferroptosis and penetrate the blood-brain barrier may fill a gap in existing technologies, providing a novel nanomedicine candidate for the treatment of shock wave brain injury.

[0012] In some specific embodiments, in step 1), the concentration of manganese chloride in the PEG solution is 2-60 mg / ml.

[0013] In some specific embodiments, in step 2), the mass ratio of ethylenediamine hydrochloride to manganese chloride is 0.5-2:1.

[0014] In some specific embodiments, the process parameters for the hydrothermal reaction in step 3) are: hydrothermal reaction at 150-300 ℃ for 0.5-3 h.

[0015] In some specific embodiments, the washing described in step 3) is as follows: the mixed solution C cooled to room temperature is mixed with acetone, and then centrifuged at 6000-10000 rpm for 5-15 minutes, and repeated 2-5 times to obtain a black precipitate.

[0016] Furthermore, the volume ratio of acetone to mixed solution C is 3-5:1.

[0017] In some specific embodiments, the purification in step 3) involves dissolving the black precipitate obtained from washing in water and dialyzing it to obtain a pure carbon quantum dot solution.

[0018] In some specific embodiments, the dialysis is performed by dialysis with deionized water in a dialysis bag with a capacity of 1000-1200 Da for 45-50 hours.

[0019] In some specific embodiments, the drying in step 3) is as follows: the purified carbon quantum dot solution is poured into a glass dish and then freeze-dried in a freeze dryer at -40~-60 ℃ to obtain a single-atom manganese-doped amino-rich PEG carbon quantum dot solid.

[0020] As part of the same inventive concept, this invention also provides a single-atom manganese-doped amino-rich PEG carbon quantum dot.

[0021] As part of the same inventive concept, the present invention also provides the application of the aforementioned single-atom manganese-doped amino-rich PEG carbon quantum dots in the preparation of medicaments for treating traumatic brain injury.

[0022] This invention uses manganese chloride, PEG400, and ethylenediamine hydrochloride as precursors to synthesize a low-toxicity single-atom manganese-doped amino-rich PEG carbon quantum dots (Mn@NH2-CDs) via a simple hydrothermal method. This provides an inhibitor that can efficiently inhibit ferroptosis, overcoming the limitation of ferroptosis inhibitors that cannot penetrate the blood-brain barrier, and enabling the treatment of shock wave brain injury.

[0023] Compared with the prior art, the present invention has at least the following advantages: 1) The Mn@NH2-CDs synthesized in this invention can effectively inhibit ferroptosis by scavenging reactive oxygen species and ferrous ions. At the cellular level, its inhibitory effect is significantly higher than that of commonly used reactive oxygen species scavenging drugs and ferrous ion chelators. 2) The Mn@NH2-CDs synthesized in this invention are rich in amino groups on their surface, which makes them carry a large number of positive charges and enable them to penetrate the blood-brain barrier and enter brain tissue through adsorption-mediated transcytosis. 3) The Mn@NH2-CDs synthesized in this invention can penetrate the blood-brain barrier to enter brain tissue and inhibit ferroptosis, thereby achieving the treatment of shock wave brain injury. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0025] Figure 1 Transmission electron microscopy (TEM) image (a) and particle size distribution (b) of Mn@NH2-CDs; Figure 2The fluorescence spectrum (a) and zeta potential (b) of Mn@NH2-CDs are shown. Figure 3 A bar chart showing the ability of different concentrations of Mn@NH2-CDs to scavenge hydrogen peroxide (a), superoxide anion (b), and hydroxyl radicals (c) in solution; Figure 4 A bar chart showing the ability of different concentrations of Mn@NH2-CDs to scavenge ferrous ions in solution; Figure 5 Bar chart showing the cytotoxicity of different concentrations of Mn@NH2-CDs to HT-22 cells; Figure 6 A comparison of the ability of Mn@NH2-CDs and commonly used ferroptosis inhibitors (Trolox, DFO) to inhibit ferroptosis; Figure 7 A schematic diagram (a) showing cell viability and death after Erastin-induced ferroptosis was induced by Mn@NH2-CDs and commonly used ferroptosis inhibitors (Trolox, DFO), and a corresponding statistical graph (b) showing the percentage of dead cells. Figure 8 The concentration of Mn@NH2-CDs that penetrated the blood-brain barrier and accumulated in the brain of C57BL / 6 mice after intravenous injection at different time points; Figure 9 A schematic diagram (a) and a statistical diagram (b) of microglia staining in the brain tissue of mice with shock wave brain injury treated with Mn@NH2-CDs. Figure 10 The behavioral results of mice in the elevated cruciate maze after treatment with Mn@NH2-CDs for shock wave brain injury. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0027] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0028] This invention also provides performance characterization and effect verification of single-atom manganese-doped amino-rich PEG carbon quantum dots, the overall concept of which is as follows: 1) Dissolve Mn@NH2-CDs powder in deionized water to prepare a 0.2 mg / mL Mn@NH2-CDs solution. Add the obtained Mn@NH2-CDs solution to a fluorescent cuvette and detect it on a fluorescence spectrometer; add the obtained Mn@NH2-CDs solution to a zeta potential sample cell and detect it on a zeta potential analyzer. 2) Mn@NH2-CDs powder was dissolved in deionized water to prepare solutions of different concentrations. The ability of Mn@NH2-CDs to scavenge hydrogen peroxide, superoxide anions and hydroxyl radicals was detected using a catalase assay kit, a superoxide dismutase assay kit and a hydroxyl radical assay kit. 3) For the cytotoxicity experiment, different concentrations of Mn@NH2-CDs were first added for incubation, and then CCK-8 was used to detect cell viability; 4) For the cell ferroptosis inhibition experiment, the ferroptosis inducer Erastin was added first, followed by incubation with Mn@NH2-CDs. Then, CCK-8 assay was used to detect changes in cell viability, and cell death was detected using live / dead staining. 5) For the blood-brain barrier penetration experiment, Mn@NH2-CDs solution was first injected into the tail vein of C57BL / 6 mice. Then, brain tissue was taken at different time points and homogenized. The supernatant of the brain tissue was collected. The efficiency of Mn@NH2-CDs in penetrating the blood-brain barrier was calculated by detecting the fluorescence intensity of Mn@NH2-CDs in the supernatant of brain tissue homogenate. 6) For the shock wave brain injury experiment, C57BL / 6 mice were first placed in the explosion field to prepare a shock wave brain injury animal model. Then, Mn@NH2-CDs solution was injected into the tail vein of the mice with shock wave brain injury. Afterwards, the number of microglia in the brain tissue was counted by tissue sectioning. The anxiety and depression of the mice were detected by behavioral experiments.

[0029] Example 1 A method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots, comprising the following steps: 1) Add 98 mg of solid manganese chloride to 30 mL of PEG400 and stir to form a homogeneous mixed solution A; 2) Add 133 mg of ethylenediamine hydrochloride to the mixed solution A obtained in step 1), stir and mix well to form a homogeneous mixed solution B; 3) Transfer the mixed solution B obtained in step 2) to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene, and heat at 160 °C for 1 h; after the reaction is completed, cool to room temperature; 4) Mix the reaction product obtained in step 3) with acetone at a volume ratio of 1:5, then centrifuge at 8000 rpm for 10 minutes, repeat 3 times to obtain a black precipitate; 5) Dissolve the black precipitate obtained in step 4) in water, and dialyze the solution with deionized water for 48 h in a dialysis bag with a molecular weight cutoff of 1000 Da to obtain a pure carbon quantum dot solution. 6) Pour the carbon quantum dot solution obtained in step 5) into a glass dish, and then freeze-dry it in a freeze dryer at -50 ℃ to obtain monoatomic manganese-doped amino-rich PEG carbon quantum dots (Mn@NH2-CDs powder).

[0030] Figure 1 Transmission electron microscopy (TEM) image (a) and particle size distribution (b) of the Mn@NH2-CDs prepared in this example are shown. Figure 1 It can be seen that the product is uniformly distributed and there is no aggregation; the particle size is 1.8±0.6 nm, indicating the successful synthesis of Mn@NH2-CDs.

[0031] Example 2 Mn@NH2-CDs powder (taking the Mn@NH2-CDs powder prepared in Example 1 as an example, the same below) was dissolved in deionized water to prepare a carbon dot solution of 200 μg / mL; 2 mL of carbon dot solution was placed in a fluorescent cuvette and its fluorescence spectrum was detected on a fluorescence spectrometer; 2 mL of carbon dot solution was placed in a zeta potential sample cell and the zeta potential of the carbon dot surface was detected in a zeta potential analyzer.

[0032] Figure 2 The fluorescence emission spectrum (a) and zeta potential spectrum (b) of Mn@NH2-CDs are presented. From Figure 2 As can be seen from a, the emission peak redshifts with increasing excitation wavelength, with the optimal excitation wavelength being 380 nm, and the corresponding emission peak at 457 nm. From... Figure 2 As can be seen from b, the zeta potential of Mn@NH2-CDs is +36 mV. This indicates that the synthesized Mn@NH2-CDs exhibit a positive zeta potential due to the abundance of amino groups on their surface.

[0033] Example 3 A PBS buffer solution with a pH of 7.4 and a concentration of 10 mM was prepared. Mn@NH2-CDs powder was dissolved in the PBS buffer solution to prepare Mn@NH2-CDs solutions with concentrations of 0, 20, 40, 80, 160, 320, and 640 μg / mL. The percentages of hydrogen peroxide, superoxide anion, and hydroxyl radicals scavenged by different concentrations of Mn@NH2-CDs were determined using a catalase activity assay kit, a superoxide dismutase activity assay kit, and a hydroxyl radical assay kit.

[0034] Figure 3 The percentages of hydrogen peroxide (a), superoxide anion (b), and hydroxyl radicals (c) scavenged by different concentrations of Mn@NH2-CDs are given. Figure 3 As can be seen from the data, the percentage of hydrogen peroxide, superoxide anion, and hydroxyl radicals scavenged by Mn@NH2-CDs gradually increases with the increase of Mn@NH2-CDs concentration, indicating that Mn@NH2-CDs can effectively scavenge hydrogen peroxide, superoxide anion, and hydroxyl radicals.

[0035] Example 4 Prepare PBS buffer solution with pH 7.4 and a concentration of 10 mM. Dissolve Mn@NH2-CDs powder in PBS buffer solution to prepare Mn@NH2-CDs solutions of 0, 25, 50, 100, 200, and 400 μg / mL. Use a ferrous ion detection kit to detect the percentage of ferrous ions cleared by different concentrations of Mn@NH2-CDs.

[0036] Figure 4 The percentage of ferrous ions scavenged by different concentrations of Mn@NH2-CDs is given. From... Figure 4 As can be seen, the percentage of ferrous ions removed by Mn@NH2-CDs gradually increases with the increase of Mn@NH2-CDs concentration, indicating that Mn@NH2-CDs can effectively remove ferrous ions.

[0037] Example 5 A 10 mM PBS buffer solution with a pH of 7.4 was prepared. Mn@NH2-CDs powder was dissolved in the PBS buffer solution to prepare a 10 mg / mL Mn@NH2-CDs solution. Then, a high-glucose DMEM complete medium was prepared, and the Mn@NH2-CDs solution was diluted with the high-glucose DMEM complete medium to achieve final concentrations of 10, 20, 50, 100, 200, 300, and 400 μg / mL. HT22 cells were cultured in the high-glucose DMEM complete medium containing different concentrations of Mn@NH2-CDs for 24 h. After 24 h, CCK-8 was added, and the cells were incubated in an incubator for 2 h. The absorbance at 450 nm was measured using a microplate reader, and statistical analysis was performed.

[0038] Figure 5 Bar graphs showing the cell viability of HT22 cells after co-incubation with different concentrations of Mn@NH2-CDs for 24 h are presented. Figure 5 The results show that when the concentration of Mn@NH2-CDs is less than 100 μg / mL, the viability of co-incubated HT22 cells is greater than 90%, indicating that Mn@NH2-CDs has low cytotoxicity.

[0039] Example 6 To compare the inhibitory effects of different ferroptosis inhibitors on ferroptosis, the experiment was divided into four groups: Erastin stimulation group, Erastin stimulation + Mn@NH2-CDs treatment group, Erastin stimulation + Trolox treatment group, and Erastin stimulation + deferoxamine (DFO) treatment group. All four groups of HT22 cells were stimulated with different concentrations (16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 μM) of Erastin for 12 h. However, the Mn@NH2-CDs treatment group, Trolox treatment group, and DFO treatment group were further cultured with 2 μg / mL of Mn@NH2-CDs, Trolox, and DFO, respectively. After 12 h of culture, CCK-8 was added, and the cells were incubated in an incubator for 2 h. The absorbance was measured at 450 nm using a microplate reader, and statistical analysis was performed.

[0040] Figure 6 A comparative graph showing the inhibitory effects of Mn@NH2-CDs, Trolox, and DFO on ferroptosis at the cellular level is presented. Figure 6 The results show that after Erastin stimulation, cell viability decreased with increasing concentration. The addition of Mn@NH2-CDs, Trolox, and DFO all restored cell viability. With the addition of Mn@NH2-CDs, cells completely recovered to normal viability levels, while other inhibitors only alleviated the decrease in cell viability to a certain extent. This demonstrates that Mn@NH2-CDs can more effectively inhibit ferroptosis.

[0041] Example 7 To compare the inhibitory effects of different ferroptosis inhibitors on ferroptosis, the experiment was divided into four groups: control group, Erastin stimulation group, Erastin stimulation + Mn@NH2-CDs treatment group, Erastin stimulation + Trolox treatment group, and Erastin stimulation + deferoxamine (DFO) treatment group. For the Erastin stimulation group, Erastin stimulation + Mn@NH2-CDs treatment group, Erastin stimulation + Trolox treatment group, and Erastin stimulation + deferoxamine (DFO) treatment group, HT22 cells were stimulated with 2 μM Erastin for 12 h. However, in the Mn@NH2-CDs treatment group, Trolox treatment group, and DFO treatment group, 2 μg / mL of Mn@NH2-CDs, Trolox, and DFO were added respectively for further culture. After 12 h of culture, AM / PI staining reagent was added, and the cells were incubated in an incubator for 30 minutes. The cell staining status was then examined under a fluorescence inverted microscope, and the number of viable and dead cells was counted.

[0042] Figure 7 Cell viability staining imaging (a) and the corresponding percentage of dead cells (b) are presented after Erastin-stimulated cells were treated with different ferroptosis inhibitors. Figure 7 The results show that Erastin stimulation of cells led to almost complete cell death due to ferroptosis, while Mn@NH2-CDs almost completely inhibited ferroptosis, reducing the cell death rate to the level of the control group; Trolox and DFO, on the other hand, failed to effectively reduce cell death. Therefore, Mn@NH2-CDs can effectively inhibit ferroptosis and improve cell survival.

[0043] Example 8 A 10 mM PBS buffer solution with a pH of 7.4 was prepared. Mn@NH2-CDs powder was dissolved in the PBS buffer solution to prepare a 4 mg / mL Mn@NH2-CDs solution. The Mn@NH2-CDs solution was injected into wild-type C57BL / 6 mice via tail vein at a concentration of 25 mg / kg. Brain tissue was collected from mice at 12 h, 24 h, and 72 h post-injection. Tissue homogenates were added to twice the volume of PBS, and after centrifugation at 12000 rpm for 10 minutes, 200 μL of the tissue supernatant was collected. The fluorescence intensity of the tissue homogenate supernatant was detected using a microplate reader with an excitation wavelength of 380 nm and an emission wavelength of 457 nm. The fluorescence intensity of the tissue homogenate supernatant was converted to Mn@NH2-CDs concentration using a Mn@NH2-CDs fluorescence intensity calibration curve, and the concentration of Mn@NH2-CDs in the brain tissue was calculated.

[0044] Figure 8The concentration of Mn@NH2-CDs in brain tissue is presented. As shown in the figure, Mn@NH2-CDs effectively penetrated the blood-brain barrier and entered the brain tissue. Twelve hours after tail vein injection, the concentration of Mn@NH2-CDs in the brain tissue reached 4 μg / g. It then gradually decreased, reaching almost 0 μg / g at 72 hours. This demonstrates that Mn@NH2-CDs can effectively penetrate the blood-brain barrier and enter the brain.

[0045] Example 9 C57BL / 6 mice were placed in an explosion field with their heads facing the epicenter to establish a shock wave brain injury model. Mn@NH2-CDs solution was administered at a concentration of 1 mg / kg on days 0 and 2 post-injury. Brain tissue was harvested on day 3, fixed, embedded, sectioned, and microglia were stained.

[0046] Figure 9 The images show (a) staining of microglia in the frontal cortex of brain tissue and (b) statistical data on microglia density in brain tissue. The figures show a significant increase in the number of microglia in the brain tissue on the third day after shockwave-induced brain injury, indicating brain inflammation. However, the number of microglia in the Mn@NH2-CDs-treated group returned to normal levels after injury. Therefore, Mn@NH2-CDs effectively inhibits brain inflammation by suppressing oxidative stress and ferroptosis.

[0047] Example 10 C57BL / 6 mice were placed in an explosion field with their heads facing the epicenter to establish a shock wave brain injury model. Mn@NH2-CDs solution was administered via tail vein injection at a concentration of 1 mg / kg on days 0, 2, and 5 post-injury. On day 14 post-injury, mice in the normal group, brain injury group, and treatment group underwent an elevated cruciate maze test, and the distances traveled by the mice in both open and closed arms were recorded to assess their anxiety levels.

[0048] Figure 10 The figures show the distances mice traveled in the open-arm (a) and closed-arm (b) sections of the elevated cross maze experiment, and the ratio of the distance traveled in the open-arm section to the total distance traveled was calculated (c). The figures show that mice with shockwave-induced brain injury traveled more in the closed-arm section, while there was no significant difference in the distance traveled in the open-arm and closed-arm sections between the Mn@NH2-CDs treatment group and the normal group. This indicates that shockwave-induced brain injury significantly increased anxiety levels in mice, while the Mn@NH2-CDs treatment group significantly reduced anxiety levels in mice with shockwave-induced brain injury.

[0049] In summary, the monoatomic manganese-doped amino-rich PEG carbon quantum dots of this invention can efficiently inhibit ferroptosis by simultaneously scavenging intracellular ROS and ferrous ions; at the same time, these carbon dots can effectively penetrate the blood-brain barrier and enter brain tissue to achieve the treatment of traumatic brain injury.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots, characterized in that, Specifically, the steps include the following: 1) Add manganese chloride to PEG400 and mix well to form a homogeneous mixed solution A; 2) Add ethylenediamine hydrochloride to the mixed solution A obtained in step 1), mix well, and form a homogeneous mixed solution B; 3) The mixed solution B obtained in step 2) was subjected to a hydrothermal reaction. After the reaction was completed, it was cooled to room temperature, washed, purified and dried to obtain single-atom manganese-doped amino-rich PEG carbon quantum dots.

2. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 1, characterized in that, In step 1), the concentration of manganese chloride in the PEG solution is 2-60 mg / ml.

3. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 1, characterized in that, In step 2), the mass ratio of ethylenediamine hydrochloride to manganese chloride is 0.5-2:

1.

4. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 1, characterized in that, The process parameters for the hydrothermal reaction described in step 3) are: hydrothermal reaction at 150-300 ℃ for 0.5-3 h.

5. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 4, characterized in that, The washing process described in step 3) involves mixing the mixed solution C, which has been cooled to room temperature, with acetone, and then centrifuging at 6000-10000 rpm for 5-15 minutes. This process is repeated 2-5 times to obtain a black precipitate.

6. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 5, characterized in that, The purification described in step 3) involves dissolving the black precipitate obtained from washing in water and dialyzing it to obtain a pure carbon quantum dot solution.

7. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 6, characterized in that, The dialysis is performed by dialysis with deionized water in a dialysis bag with a capacity of 1000-1200 Da for 45-50 hours.

8. The method for synthesizing single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 7, characterized in that, The drying process described in step 3) involves pouring the purified carbon quantum dot solution into a glass dish and then freeze-drying it in a freeze dryer at -40 to -60 ℃ to obtain a single-atom manganese-doped amino-rich PEG carbon quantum dot solid.

9. A single-atom manganese-doped amino-rich PEG carbon quantum dot prepared by the synthesis method according to any one of claims 1-8.

10. The use of the single-atom manganese-doped amino-rich PEG carbon quantum dots as described in claim 9 in the preparation of a medicament for treating traumatic brain injury.

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