Monatomic manganese-doped lipophilic carbon quantum dot as well as synthesis method and application thereof

Abstract

The invention discloses a synthesis method of monatomic manganese-doped lipophilic carbon quantum dots, and belongs to the technical field of biomedical materials, and the synthesis method specifically comprises the following steps: 1) adding manganese chloride into PEG400, and uniformly mixing to form a uniform mixed solution A; 2) adding p-phenylenediamine into the mixed solution A obtained in the step 1), and uniformly mixing to form a uniform mixed solution B; and 3) carrying out a hydrothermal reaction on the mixed solution B obtained in the step 2), after the reaction is completed, cooling to room temperature, washing, purifying and drying to obtain the monatomic manganese-doped lipophilic carbon quantum dots. Meanwhile, the invention further discloses that the Mn-coated Lip-CDs can specifically target a cytoplasmic membrane and remarkably inhibit lipid peroxidation of the cytoplasmic membrane, the oxidation resistance of the Mn-coated Lip-CDs is superior to that of a common lipid peroxidation inhibitor Trolox, and the Mn-coated Lip-CDs can be effectively used for treating ferroptosis.

Description

Technical Field

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

[0002] Programmed cell death plays a crucial role in maintaining homeostasis and development in organisms. Ferroptosis, an iron-dependent form of programmed cell death, is characterized by the abnormal accumulation of lipid peroxides on the cell membrane, ultimately leading to cell membrane disruption and death. Unlike other forms of cell death such as apoptosis and necrosis, ferroptosis possesses unique morphological, biochemical, and genetic characteristics.

[0003] Ferroptosis is closely linked to an imbalance in two major intracellular defense systems: the glutathione peroxidase 4 (GPX4) system and the ferroptosis inhibitor 1 (FSP1) system. When the function of these antioxidant defense systems (especially GPX4) is impaired, the active ferrous iron (Fe2+) in the cell is affected. 2 ⁺) It catalyzes the production of large amounts of reactive oxygen species (ROS) through the Fenton reaction. These ROS specifically attack polyunsaturated fatty acids (PUFAs) abundant on the cell membrane, triggering a free radical chain reaction of lipids, namely lipid peroxidation; the cell membrane is the primary target of lipid peroxidation damage, and its loss of function is the "final common pathway" for cells to move towards ferroptosis.

[0004] In recent years, studies have shown that ferroptosis is closely related to the development and progression of many major human diseases, including neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), ischemia-reperfusion injury (such as myocardial infarction and stroke), acute organ injury, and various cancers. Therefore, the development of highly effective and specific ferroptosis inhibitors has great potential and clinical value for the treatment of these diseases.

[0005] Currently, existing ferroptosis inhibitors can be mainly classified into the following categories: Free radical scavenging antioxidants, such as Ferrostatin-1 and Liproxstatin-1, can effectively neutralize lipid free radicals and interrupt chain reactions. They are widely used classic inhibitors. However, they usually lack targeting and have less than ideal pharmacokinetic properties. Iron chelators, such as deferoxamine (DFO), reduce ROS production by chelating iron ions; however, these drugs often lack specificity and may interfere with normal iron metabolism, leading to systemic side effects. GPX4 agonists or substrate supplements: such as selenium or GSH precursors; however, their effects are indirect and limited, especially when the defense system is completely collapsed. While the aforementioned inhibitors have demonstrated some therapeutic efficacy, they generally share a fundamental limitation: a lack of specific targeting ability for the cell membrane, the "accident site." These molecules are widely and randomly distributed within the cell, with only a small amount reaching the plasma membrane to exert their effects. Therefore, high doses are required, and off-target effects may occur. More importantly, lipid peroxidation on the plasma membrane is a dynamic and rapid chain reaction, requiring inhibitors to accumulate at high concentrations and rapidly at the lesion site (plasma membrane) to efficiently quench free radicals and achieve optimal protective effects. Developing novel nanomaterials that can actively target and anchor to the cell membrane to efficiently inhibit lipid peroxidation has become a critical scientific problem and technological challenge urgently needing a breakthrough in the field of ferroptosis therapy. Summary of the Invention

[0006] 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 lipophilic carbon quantum dot, synthesized via a simple hydrothermal method using PEG400, manganese chloride, and p-phenylenediamine as precursors. This nanodrug is capable of targeting and inhibiting lipid peroxidation of cell membranes, thus achieving the treatment of cellular ferroptosis.

[0007] The present invention also provides a method for preparing the single-atom manganese-doped lipophilic carbon quantum dots, and their application in the preparation of drugs for treating shock wave brain injury.

[0008] The objective of this invention is achieved through the following technical solution: A method for synthesizing single-atom manganese-doped lipophilic 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 p-phenylenediamine 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) is subjected to a hydrothermal reaction. After the reaction is completed, it is cooled to room temperature, washed, purified and dried to obtain single-atom manganese-doped lipophilic carbon quantum dots (Mn@Lip-CDs).

[0009] Ferroprelation is closely related to the development of many major human diseases. Its core biochemical characteristic is the abnormal accumulation of lipid peroxides on the cell membrane, ultimately leading to cell membrane damage and cell death. However, current ferroptosis inhibitors lack specific targeting ability to the cell membrane, making it difficult to achieve optimal protective effects. Therefore, developing a nanomedicine that is low in toxicity and can target the cell membrane to inhibit ferroptosis is of great significance. To this end, this invention designed and synthesized a single-atom manganese-doped lipophilic carbon dot (Mn@Lip-CDs). This carbon dot uses PEG400, p-phenylenediamine, and manganese chloride as precursors. The PEG400 precursor gives it low toxicity and good biocompatibility; p-phenylenediamine imparts its lipophilic properties, enabling it to target the cell membrane and exert its effect; the single-atom manganese doping in the carbon dot enables it to scavenge reactive oxygen species extremely efficiently. Cell experiments demonstrated that Mn@Lip-CDs can specifically target the cell membrane to inhibit lipid peroxidation and treat ferroptosis. The ability of Mn@Lip-CDs to target the cell membrane and inhibit lipid peroxidation may fill a gap in existing technologies, providing a novel nanomedicine candidate for the treatment of ferroptosis.

[0010] This invention uses PEG400, p-phenylenediamine, and manganese chloride as precursors to synthesize a low-toxicity, single-atom manganese-doped lipophilic carbon dot (Mn@Lip-CDs) via a simple hydrothermal method. This provides a nanomedicine that can target and inhibit lipid peroxidation of cell membranes, thereby achieving the treatment of cellular ferroptosis.

[0011] In some specific embodiments, in step 1), the concentration of manganese chloride in the PEG solution is 2-60 mg / ml.

[0012] In some specific embodiments, in step 2), the mass ratio of p-phenylenediamine to manganese chloride is 0.5-2.5:1.

[0013] In some specific embodiments, the process parameters for the hydrothermal reaction in step 3) are: hydrothermal reaction at 120-250 °C for 1-4 h.

[0014] In some specific embodiments, the washing described in step 3) is as follows: the mixed solution C cooled to room temperature is mixed with chloroform, and then centrifuged at 6000-10000 rpm for 5-15 minutes, and repeated 2-5 times to obtain a black precipitate.

[0015] Furthermore, the volume ratio of chloroform to mixed solution C is 3-5:1.

[0016] 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.

[0017] 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.

[0018] 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 lipophilic carbon quantum dot solid doped with single atom manganese.

[0019] As part of the same inventive concept, the present invention also provides a single-atom manganese-doped lipophilic carbon quantum dot.

[0020] As part of the same inventive concept, the present invention also provides the application of the aforementioned single-atom manganese-doped lipophilic carbon quantum dots in the preparation of drugs for treating cellular ferroptosis.

[0021] Compared with the prior art, the present invention has at least the following advantages: 1) The Mn@Lip-CDs synthesized in this invention can specifically target the cell membrane; 2) The single-atom manganese doped in the Mn@Lip-CDs synthesized in this invention can efficiently remove reactive oxygen species; 3) The Mn@Lip-CDs synthesized in this invention inhibit lipid peroxidation by specifically targeting the cell membrane, thereby achieving an effective treatment for ferroptosis. Attached Figure Description

[0022] 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.

[0023] Figure 1 Transmission electron microscopy (TEM) image (a) and particle size distribution (b) of Mn@Lip-CDs; Figure 2 The fluorescence spectra of Mn@Lip-CDs dissolved in water (a), anhydrous ethanol (b), and chloroform (c) are shown. Figure 3 Bar chart showing the cytotoxicity of different concentrations of Mn@Lip-CDs on RAW264.7 cells; Figure 4 Laser confocal microscopy images showing the localization of Mn@Lip-CDs in cells; Figure 5 A comparison of MDA content in cells after treatment with Mn@Lip-CDs and Trolox for Erastin-induced ferroptosis. Figure 6 A comparison of the ability of Mn@Lip-CDs and Trolox to inhibit ferroptosis. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] This invention provides a process for verifying the performance and effects of the single-atom manganese-doped lipophilic carbon quantum dots prepared in this application. The overall concept is as follows: 1) Dissolve Mn@Lip-CDs powder in deionized water, anhydrous ethanol and acetone solutions, each with a concentration of 0.2 mg / mL. Then add these three solutions to different fluorescent cuvettes and detect them on a fluorescence spectrometer. 2) For the cytotoxicity experiment, Mn@Lip-CDs powder was first dissolved in DMSO to a concentration of 10 mg / mL, and then diluted to different concentrations using complete cell culture medium. After co-incubating with cells for 24 h, cell viability was detected using CCK-8 assay. 3) For the cell membrane targeting experiment, first dissolve Mn@Lip-CDs powder in DMSO to a concentration of 4 mg / mL, then incubate the Mn@Lip-CDs solution with the cells for 30 minutes, and then observe under a confocal microscope; 4) For the lipid peroxidation inhibition experiment, Erastin, the ferroptosis inducer, was first added to the cells, followed by co-incubation with Mn@Lip-CDs, and then detection was performed using an MDA detection kit; 5) For the ferroptosis inhibition experiment, the ferroptosis inducer Erastin was first added to the cells, followed by co-incubation with Mn@Lip-CDs, and then cell viability was detected using CCK-8 assay.

[0027] Example 1 1) Add 98 mg of manganese chloride to 30 mL of PEG400 and stir to form a homogeneous mixed solution A; 2) Add 108 mg of p-phenylenediamine 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 150 °C for 1 h; after the reaction is completed, cool to room temperature to obtain mixed solution C; 4) Mix the mixed solution C obtained in step 3) with chloroform at a 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 then 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 lipophilic carbon quantum dots (Mn@Lip-CDs powder) doped with manganese.

[0028] Figure 1 Transmission electron microscopy (TEM) images (a) and particle size distribution maps (b) of the Mn@Lip-CDs prepared in this example are shown. Figure 1 As can be seen, the product is uniformly distributed and no aggregation occurred. The particle size is 1.5 ± 0.6 nm. This indicates the successful synthesis of Mn@Lip-CDs.

[0029] Example 2 Mn@Lip-CDs powder was dissolved in deionized water, anhydrous ethanol, and acetone to prepare three carbon dot solutions of 0.2 mg / mL. Two mL of each of the three carbon dot solutions were placed in a fluorescent cuvette, and their fluorescence spectra were detected sequentially on a fluorescence spectrometer.

[0030] Figure 2 Fluorescence emission spectra of Mn@Lip-CDs dissolved in deionized water (a), anhydrous ethanol (b), and acetone (c) are presented. Figure 2 It can be seen that as the polarity of the solvent decreases (polarity: water > ethanol > acetone), the fluorescence intensity of the emission peak of Mn@Lip-CDs gradually increases, indicating that Mn@Lip-CDs are readily soluble in organic solvents, and their fluorescence intensity in organic solvents is much stronger than that in water.

[0031] Example 3 Mn@Lip-CDs powder was dissolved in DMSO to prepare a 10 mg / mL solution. The Mn@Lip-CDs solution was then diluted with high-glucose DMEM complete medium to final concentrations of 200, 100, 50, and 25 μg / mL. RAW264.7 cells were cultured for 24 h in high-glucose DMEM complete medium containing different concentrations of Mn@Lip-CDs. After 24 h, cck-8 was added, and the cells were incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader, and statistical analysis was performed.

[0032] Figure 3 Cell viability graphs of RAW264.7 cells after co-incubation with different concentrations of Mn@Lip-CDs for 24 h are presented. Figure 3 The results show that when the concentration of Mn@Lip-CDs is less than 100 μg / mL, the viability of RAW264.7 cells co-incubated is greater than 90%, indicating that Mn@Lip-CDs has low cytotoxicity.

[0033] Example 4 Mn@Lip-CDs powder was dissolved in DMSO to prepare a 10 mg / mL Mn@Lip-CDs solution. The Mn@Lip-CDs solution was then diluted with 1640 complete medium to a final concentration of 10 μg / mL. THP-1 cells were co-incubated with 10 μg / mL Mn@Lip-CDs for 30 minutes, after which the distribution of Mn@Lip-CDs within the cells was observed under a laser confocal microscope.

[0034] Figure 4 A fluorescence map showing the distribution of Mn@Lip-CDs in cells is presented. From Figure 4 As can be seen, Mn@Lip-CDs are mainly distributed in the cell membrane. This indicates that Mn@Lip-CDs can specifically target the cell membrane.

[0035] Example 5 To verify the ability of Mn@Lip-CDs to inhibit lipid peroxidation, the experiment was divided into four groups: control group, Erastin stimulation group, Erastin stimulation + Mn@Lip-CDs treatment group, and Erastin stimulation + Trolox treatment group. RAW264.7 cells in the Erastin stimulation group, Erastin stimulation + Mn@Lip-CDs treatment group, and Erastin stimulation + Trolox treatment group were stimulated with 1 μM Erastin for 6 h. However, in the Mn@Lip-CDs and Trolox treatment groups, 2 μg / mL of Mn@Lip-CDs and Trolox, respectively, were added for further culture. After 6 h of culture, cells were collected, and the MDA content was detected using an MDA assay kit.

[0036] Figure 5 A comparative graph showing the inhibitory effects of Mn@Lip-CDs and Trolox on lipid peroxidation at the cellular level is presented. From... Figure 5 The results show that Erastin stimulation significantly increased intracellular MDA levels. The addition of Mn@Lip-CDs and Trolox both reduced MDA levels. With Mn@Lip-CDs, intracellular MDA levels completely returned to normal levels; while Trolox only reduced MDA levels to some extent, failing to restore them to normal levels. This demonstrates that Mn@Lip-CDs are more effective at inhibiting lipid peroxidation in cells.

[0037] Example 6 To verify the ability of Mn@Lip-CDs to inhibit ferroptosis, the experiment was divided into four groups: control group, Erastin stimulation group, Erastin stimulation + Mn@Lip-CDs treatment group, and Erastin stimulation + Trolox treatment group. RAW264.7 cells in the Erastin stimulation group, Erastin stimulation + Mn@Lip-CDs treatment group, and Erastin stimulation + Trolox treatment group were stimulated with 2 μM Erastin for 12 h. However, in the Mn@Lip-CDs and Trolox treatment groups, 2 μg / mL of Mn@Lip-CDs and Trolox, respectively, were added for further culture. After 12 h of culture, CCK-8 was added and incubated in an incubator for 2 h. The absorbance was measured at 450 nm using a microplate reader, and statistical analysis was performed.

[0038] Figure 6 A comparative graph showing the inhibitory effects of Mn@Lip-CDs and Trolox on ferroptosis at the cellular level is presented. From... Figure 6The results show that after Erastin stimulation, cell viability decreased to 13% of the normal group. After the addition of Mn@Lip-CDs and Trolox, cell viability recovered in both groups: the Mn@Lip-CDs treatment group recovered to approximately 81% of the normal group's cell viability; while the Trolox treatment group only recovered to approximately 40% of the normal group's cell viability. This demonstrates that Mn@Lip-CDs are more effective at inhibiting ferroptosis.

[0039] In summary, the lipophilic carbon dots doped with single-atom manganese in this invention achieve an effective treatment for cellular ferroptosis by specifically targeting and inhibiting lipid peroxidation of cell membranes.

[0040] 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 lipophilic 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 p-phenylenediamine 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 lipophilic carbon quantum dots doped with single-atom manganese.

2. The method for synthesizing single-atom manganese-doped lipophilic 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 lipophilic carbon quantum dots as described in claim 1, characterized in that, In step 2), the mass ratio of p-phenylenediamine to manganese chloride is 0.5-2.5:

1.

4. The method for synthesizing single-atom manganese-doped lipophilic 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 120-250 ℃ for 1-4 h.

5. The method for synthesizing single-atom manganese-doped lipophilic 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 chloroform, 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 lipophilic 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 lipophilic 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 lipophilic 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 lipophilic carbon quantum dot solid doped with single-atom manganese.

9. A single-atom manganese-doped lipophilic 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 lipophilic carbon quantum dots as described in claim 9 in the preparation of a drug for treating ferroptosis.

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