N-doped carbon quantum dots and preparation method and application thereof

Abstract

The application provides N-doped carbon quantum dots and a preparation method and application thereof. The N-doped carbon quantum dots are prepared by using ginsenoside, ethylenediamine and water as raw materials and by adopting a hydrothermal synthesis method. The N-doped carbon quantum dots have high biological safety, are mainly composed of carbon elements, are not doped with metal elements and have high biocompatibility. Compared with traditional iron chelating agents, the N-doped carbon quantum dots have strong ROS scavenging properties and have stronger therapeutic effects. The N-doped carbon quantum dots have good physical stability, and the N-doped carbon quantum dots in artificial cerebrospinal fluid do not change in physical properties obviously within 28 days. The preparation method of the N-doped carbon quantum dots has simple synthesis technology, low synthesis cost, does not produce reaction waste and is conducive to clinical transformation.

Description

Technical Field

[0001] This invention relates to the field of drug treatment technology for iron overload diseases, and in particular to an N-doped carbon quantum dot, its preparation method, and its application. Background Technology

[0002] Iron overload disease is caused by excessive iron deposition in the body due to various reasons, leading to structural damage and dysfunction of vital organs. Excess iron ions in the body not only have direct biotoxicity, but also cause oxidative damage to lipids, proteins, and DNA by mediating antioxidant consumption and upregulation of reactive oxygen species (ROS) through the Fenton reaction.

[0003] Drug therapy is one of the main methods for treating iron overload, and a large number of iron chelating agents, such as deferoxamine (DFO), deferoxone (DFP), and deferasirox (DFX), have achieved positive results in clinical applications. However, the poor biocompatibility, systemic toxicity, and insufficient ROS scavenging capacity of these drugs limit their therapeutic effects. Currently, some nanoparticles that can simultaneously scavenge iron overload and ROS have been used to treat iron overload diseases. For example, by co-assembling natural polyphenols and DFO, the functions of iron overload and ROS scavenging can be achieved. However, the synthesis process is relatively complex, which greatly limits the use of this nanomaterial.

[0004] Therefore, developing a drug with a simple synthesis process that can remove iron ions and ROS is of great significance in the treatment of iron overload. Summary of the Invention

[0005] In view of this, the present invention provides an N-doped carbon quantum dot, its preparation method and application, to solve or at least partially solve the defects existing in the prior art.

[0006] In a first aspect, the present invention provides an N-doped carbon quantum dot, which is prepared by hydrothermal synthesis using ginsenosides, ethylenediamine and water as raw materials.

[0007] Preferably, the N-doped carbon quantum dots have a particle size of 5–9 nm.

[0008] Secondly, the present invention also provides a method for preparing the N-doped carbon quantum dots, comprising the following steps:

[0009] Ginsenosides and ethylenediamine are mixed, and then water is added to dissolve them to obtain a mixture.

[0010] The mixture is subjected to a hydrothermal reaction;

[0011] The solution after the hydrothermal reaction was centrifuged, and the supernatant was collected.

[0012] The supernatant was filtered, and the filtered solution was then transferred to a dialysis bag for dialysis.

[0013] The dialyzed solution was then freeze-dried to obtain N-doped carbon quantum dots.

[0014] Preferably, in the method for preparing N-doped carbon quantum dots, the hydrothermal reaction step of the mixed solution is carried out at a temperature of 150–200°C and a reaction time of 12–24 h.

[0015] Preferably, in the method for preparing N-doped carbon quantum dots, the mass ratio of ginsenoside to ethylenediamine is (1-3):(1-10).

[0016] Preferably, in the method for preparing N-doped carbon quantum dots, the ginsenosides include at least one of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Rh2, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1.

[0017] Preferably, in the method for preparing N-doped carbon quantum dots, the centrifugation speed is 8000-12000 r / min and the centrifugation time is 5-10 min in the step of centrifuging the solution after hydrothermal reaction;

[0018] And / or, in the step of filtering the supernatant, the filter used has a pore size of 0.22 to 0.45 μm.

[0019] Preferably, in the method for preparing N-doped carbon quantum dots, in the step of transferring the filtered solution to a dialysis bag for dialysis, the molecular weight cutoff of the dialysis bag is 1-10 KD, and the dialysis time is 12-24 h.

[0020] Thirdly, the present invention also provides the application of the N-doped carbon quantum dots described above or the N-doped carbon quantum dots prepared by the described preparation method in the preparation of drugs for treating iron overload diseases.

[0021] Fourthly, the present invention also provides an application of the N-doped carbon quantum dots described above or the N-doped carbon quantum dots prepared by the described preparation method in scavenging oxygen free radicals, ABTS free radicals, DPPH free radicals and iron ions.

[0022] The present invention has the following advantages over the prior art:

[0023] 1. The N-doped carbon quantum dots of the present invention have high biosafety, are mainly composed of carbon elements and do not contain metal elements, thus exhibiting high biocompatibility; compared with traditional iron chelating agents, the N-doped carbon quantum dots of the present invention have stronger ROS scavenging properties and stronger therapeutic effects; the N-doped carbon quantum dots of the present invention have good physical stability, and their physical properties do not change significantly within 28 days in artificial cerebrospinal fluid.

[0024] 2. The method for preparing N-doped carbon quantum dots of the present invention has a simple synthesis process, low synthesis cost, and no reaction waste generated, which is conducive to its clinical translation. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a TEM image of the N-doped carbon quantum dots prepared in Example 1 of the present invention;

[0027] Figure 2 XPS image of the N-doped carbon quantum dots prepared in Example 1 of this invention;

[0028] Figure 3 The fluorescence spectrum of the N-doped carbon quantum dots prepared in Example 1 of this invention;

[0029] Figure 4 The particle size and Zeta potential of the N-doped carbon quantum dots prepared in Example 1 of this invention are shown.

[0030] Figure 5 This is a graph showing the change in particle size of N-doped carbon quantum dots prepared in Example 1 of the present invention over time.

[0031] Figure 6 This is a graph showing the change of Zeta potential over time for the N-doped carbon quantum dots prepared in Example 1 of this invention.

[0032] Figure 7 This is a graph showing the relative fluorescence intensity of the N-doped carbon quantum dots prepared in Example 1 of this invention as a function of time.

[0033] Figure 8 This is a graph showing the ability of N-doped carbon quantum dots prepared in Example 1 of the present invention to remove ABTS in solution;

[0034] Figure 9This is a graph showing the ability of N-doped carbon quantum dots prepared in Example 1 of the present invention to scavenge DPPH in solution;

[0035] Figure 10 The N-doped carbon quantum dots prepared in Example 1 of this invention react with Fe in solution. 2+ / Fe 3+ Color change after the reaction;

[0036] Figure 11 The N-doped carbon quantum dots prepared in Example 1 of this invention are in 0-1mM Fe 2+ / Fe 3+ The fluorescence emission spectrum below;

[0037] Figure 12 The ROS content of brain tissue treated and untreated with this invention in mice with cerebral hemorrhage;

[0038] Figure 13 The iron ion content of brain tissue treated and untreated with the present invention in mice with cerebral hemorrhage;

[0039] Figure 14 The mNSS neurological function scoring scale;

[0040] Figure 15 The behavioral scores of mice with cerebral hemorrhage under the treatment and untreated conditions of this invention are shown. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0043] This application provides an N-doped carbon quantum dot, which is prepared by hydrothermal synthesis using ginsenosides, ethylenediamine, and water as raw materials.

[0044] The N-doped carbon quantum dots of this invention are synthesized using ginsenosides, ethylenediamine, and water (specifically double-distilled water) as raw materials via a hydrothermal synthesis method. They have the characteristics of simple synthesis process and scavenging iron ions and ROS (reactive oxygen free radicals), and can be used for the treatment of iron overload diseases.

[0045] In some embodiments, the particle size of N-doped carbon quantum dots is 5–9 nm, which simultaneously possess the ability to scavenge oxygen free radicals and iron ions.

[0046] Based on the same inventive concept, the present invention also provides a method for preparing N-doped carbon quantum dots, comprising the following steps:

[0047] S1. Mix ginsenosides and ethylenediamine, then add water to dissolve and obtain a mixture.

[0048] S2. The mixture is subjected to a hydrothermal reaction;

[0049] S3. Centrifuge the solution after the hydrothermal reaction and collect the supernatant after centrifugation;

[0050] S4. Filter the supernatant, and then transfer the filtered solution to a dialysis bag for dialysis.

[0051] S5. The dialyzed solution is then freeze-dried to obtain N-doped carbon quantum dots.

[0052] Specifically, the method for preparing N-doped carbon quantum dots of the present invention includes the following steps:

[0053] S1. Mix ginsenosides and ethylenediamine and add them to a beaker. Add water and dissolve to obtain a mixture.

[0054] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature.

[0055] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it, and collect the supernatant after centrifugation.

[0056] S4. Filter the supernatant from S3 using a microporous membrane filter; then transfer the filtered solution to a dialysis bag and dialyze it in pure water in the dark.

[0057] S5. The dialyzed solution is then freeze-dried to obtain N-doped carbon quantum dots.

[0058] The method for preparing N-doped carbon quantum dots of the present invention is simple in synthesis, low in cost, and produces no reaction waste, which is beneficial for its clinical translation. The N-doped carbon quantum dots prepared by the present invention have high biocompatibility, are mainly composed of carbon elements and do not contain metal elements. Compared with traditional iron chelating agents, the N-doped carbon quantum dots prepared by the present invention have stronger ROS scavenging properties and stronger therapeutic effects. The N-doped carbon quantum dots prepared by the present invention have good physical stability; the physical properties of the N-doped carbon quantum dots in artificial cerebrospinal fluid do not change significantly within 28 days.

[0059] In some embodiments, the dialyzed solution is freeze-dried in a vacuum environment at -50 to -70°C to obtain N-doped carbon quantum dots.

[0060] In some embodiments, in the step of hydrothermal reaction of the mixture, the hydrothermal reaction temperature is 150-200°C and the reaction time is 12-24h, preferably, the reaction temperature is 200°C and the reaction time is 24h.

[0061] In some embodiments, the mass ratio of ginsenoside to ethylenediamine is (1-3):(1-10), preferably, the mass ratio of ginsenoside to ethylenediamine is 1:(3-10), and more preferably, the mass ratio of ginsenoside to ethylenediamine is 1:3.

[0062] In some embodiments, the mass ratio of ginsenosides, ethylenediamine, and water is (1-3):(1-10):(10-20).

[0063] Preferably, the mass ratio of ginsenoside to ethylenediamine is 1g:(3-10)g, and the volume of water added is 10-20mL. More preferably, the mass ratio of ginsenoside to ethylenediamine is 1g:3g, and the volume of water is 10mL.

[0064] In some embodiments, ginsenosides include at least one of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Rh2, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1, preferably ginsenoside Rb1.

[0065] In some embodiments, in the step of centrifuging the solution after hydrothermal reaction, the centrifugation speed is 8000-12000 r / min and the centrifugation time is 5-10 min; preferably, the centrifugation speed is 8000 r / min and the centrifugation time is 10 min.

[0066] In some embodiments, the filter used in the step of filtering the supernatant has a pore size of 0.22 to 0.45 μm. Specifically, a microporous membrane filter is used for filtration, and the pore size of the microporous membrane filter is 0.22 μm or 0.45 μm, preferably 0.22 μm.

[0067] In some embodiments, in the step of transferring the filtered solution to a dialysis bag for dialysis, the molecular weight cutoff of the dialysis bag is 1-10 KD, and the dialysis time is 12-24 h; preferably, the molecular weight cutoff of the dialysis bag is 1 KD, and the dialysis time is 12 h.

[0068] Based on the same inventive concept, the present invention also provides the application of the above-mentioned N-doped carbon quantum dots or the N-doped carbon quantum dots prepared by the above-mentioned preparation method in the preparation of drugs for treating iron overload diseases.

[0069] Based on the same inventive concept, this invention also provides an N-doped carbon quantum dot prepared by the above-described method or the N-doped carbon quantum dot prepared by the above-described method, which can scavenge oxygen free radicals, ABTS free radicals, DPPH free radicals and iron ions (i.e., chelate Fe). 2+ / Fe 3+ Applications in ).

[0070] The following specific embodiments further illustrate the N-doped carbon quantum dots, their preparation methods, and applications of the present invention. This section further explains the content of the present invention in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0071] In the following examples, ginsenoside Rb1 (product number: D105088) was purchased from Nanjing Dierge Pharmaceutical Technology Co., Ltd.

[0072] Example 1

[0073] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0074] S1. Mix 1g of ginsenoside Rb1 and 3g of ethylenediamine and add them to a beaker. Add 10mL of double-distilled water and dissolve to obtain a mixed solution.

[0075] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 200℃ and the reaction time is 24h;

[0076] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 8000 r / min and the centrifugation time is 10 min.

[0077] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 1 KD and dialyze it in pure water in the dark for 12 hours.

[0078] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0079] Example 2

[0080] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0081] S1. Mix 1g of ginsenoside Rb1 and 5g of ethylenediamine and add them to a beaker. Add 10mL of double-distilled water and dissolve to obtain a mixed solution.

[0082] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 150℃ and the reaction time is 12h;

[0083] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 8000 r / min and the centrifugation time is 5 min.

[0084] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 1 KD and dialyze it in pure water in the dark for 12 hours.

[0085] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0086] Example 3

[0087] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0088] S1. Mix 1g of ginsenoside Rb1 and 5g of ethylenediamine and add them to a beaker. Add 15mL of double-distilled water and dissolve to obtain a mixture.

[0089] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 180℃ and the reaction time is 18h.

[0090] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; wherein, the centrifugation speed is 10000 r / min and the centrifugation time is 10 min;

[0091] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.45 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 5 KD and dialyze it in pure water in the dark for 12 hours.

[0092] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0093] Example 4

[0094] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0095] S1. Mix 1g of ginsenoside Rb1 and 5g of ethylenediamine and add them to a beaker. Add 20mL of double-distilled water and dissolve to obtain a mixed solution.

[0096] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 200℃ and the reaction time is 24h;

[0097] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 12000 r / min and the centrifugation time is 10 min.

[0098] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.45 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 10 KD and dialyze it in pure water in the dark for 24 hours.

[0099] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0100] Example 5

[0101] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0102] S1. Mix 1g of ginsenoside Rb1 and 7g of ethylenediamine and add them to a beaker. Add 10mL of double-distilled water and dissolve to obtain a mixture.

[0103] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 150℃ and the reaction time is 12h;

[0104] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 8000 r / min and the centrifugation time is 5 min.

[0105] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 1 KD and dialyze it in pure water in the dark for 12 hours.

[0106] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0107] Example 6

[0108] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0109] S1. Mix 1g of ginsenoside Rb1 and 7g of ethylenediamine and add them to a beaker. Add 15mL of double-distilled water and dissolve to obtain a mixture.

[0110] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 180℃ and the reaction time is 18h.

[0111] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; wherein, the centrifugation speed is 10000 r / min and the centrifugation time is 10 min;

[0112] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 5 KD and dialyze it in pure water in the dark for 12 hours.

[0113] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0114] Example 7

[0115] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0116] S1. Mix 1g of ginsenoside Rb1 and 7g of ethylenediamine and add them to a beaker. Add 20mL of double-distilled water and dissolve to obtain a mixed solution.

[0117] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 200℃ and the reaction time is 24h;

[0118] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 12000 r / min and the centrifugation time is 10 min.

[0119] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 10 KD and dialyze it in pure water in the dark for 24 hours.

[0120] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0121] Example 8

[0122] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0123] S1. Mix 1g of ginsenoside Rb1 and 10g of ethylenediamine and add them to a beaker. Add 10mL of double-distilled water and dissolve to obtain a mixed solution.

[0124] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 150℃ and the reaction time is 12h;

[0125] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 8000 r / min and the centrifugation time is 5 min.

[0126] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 1 KD and dialyze it in pure water in the dark for 12 hours.

[0127] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0128] Example 9

[0129] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0130] S1. Mix 1g of ginsenoside Rb1 and 10g of ethylenediamine and add them to a beaker. Add 15mL of double-distilled water and dissolve to obtain a mixed solution.

[0131] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 180℃ and the reaction time is 18h.

[0132] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; wherein, the centrifugation speed is 10000 r / min and the centrifugation time is 10 min;

[0133] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 5 KD and dialyze it in pure water in the dark for 12 hours.

[0134] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0135] Example 10

[0136] This embodiment provides a method for preparing N-doped carbon quantum dots, including the following steps:

[0137] S1. Mix 1g of ginsenoside Rb1 and 10g of ethylenediamine and add them to a beaker. Add 20mL of double-distilled water and dissolve to obtain a mixed solution.

[0138] S2. Transfer the mixture to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner, carry out the hydrothermal reaction in a vacuum drying oven, and then cool naturally to room temperature; wherein, the hydrothermal reaction temperature is 200℃ and the reaction time is 24h;

[0139] S3. Transfer the solution after the hydrothermal reaction in S2 to a centrifuge tube, centrifuge it using a centrifuge, and collect the supernatant after centrifugation; the centrifugation speed is 12000 r / min and the centrifugation time is 10 min.

[0140] S4. Filter the supernatant from S3 using a microporous membrane filter with a pore size of 0.22 μm. Transfer the filtered solution to a dialysis bag with a molecular weight cutoff of 10 KD and dialyze it in pure water in the dark for 24 hours.

[0141] S5. The dialyzed solution is then freeze-dried in a vacuum environment at -65°C to obtain N-doped carbon quantum dots.

[0142] Performance testing

[0143] The following tests are conducted using the N-doped carbon quantum dots prepared in Example 1 as an example to verify the performance of the N-doped carbon quantum dots prepared in this invention.

[0144] The morphology of N-doped carbon quantum dots was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown.

[0145] from Figure 1 It can be seen that the N-doped carbon quantum dots are evenly distributed and are nearly spherical.

[0146] The X-ray electron spectra of N-doped carbon quantum dots were analyzed using X-ray photoelectron spectroscopy, and the results are as follows: Figure 2 As shown.

[0147] from Figure 2 As can be seen, apart from hydrogen, N-doped carbon quantum dots are composed of three elements: C, O, and N. Among them, C accounts for 75.23%, O accounts for 19.78%, and N accounts for 4.99%. The surface of N-doped carbon quantum dots has a large number of C=O, CO, CN, and pyridine N groups.

[0148] The optimal excitation and emission wavelengths of N-doped carbon quantum dots were analyzed using fluorescence spectroscopy, and the results are as follows: Figure 3 As shown.

[0149] from Figure 3 As can be seen, the optimal excitation (EX) and emission (EM) wavelengths for N-doped carbon quantum dots are 435 nm and 505 nm, respectively.

[0150] The particle size and potential of N-doped carbon quantum dots were analyzed using nanoparticle size and Zeta potentiometer techniques. The results are as follows: Figure 4 As shown.

[0151] from Figure 4 As can be seen, the average particle size of N-doped carbon quantum dots is 7.04 nm, and the Zeta potential is approximately -12 mV.

[0152] The particle size, fluorescence intensity, and zeta potential of N-doped carbon quantum dots were analyzed over time using nanoparticle size and zeta potential metering and fluorescence spectroscopy. The results are as follows: Figures 5-7 As shown.

[0153] from Figures 5-7 As can be seen, the particle size, fluorescence intensity, and zeta potential of N-doped carbon quantum dots in the solution did not change significantly, indicating that N-doped carbon quantum dots have good physical stability.

[0154] The antioxidant properties of N-doped carbon quantum dot solutions were determined using the ABTS and DPPH methods.

[0155] Following the reagent instructions, double-distilled water was used as the solvent to prepare N-doped carbon quantum dot solutions with concentrations of 0, 0.1, 0.2, 1, 2, and 10 mg / ml. These solutions were then added to prepared ABTS·+ and DPPH· aqueous solutions, mixed thoroughly, and incubated at room temperature for 5 minutes. The absorbance values ​​of the mixed solutions at 734 nm and 517 nm were measured (A1). The absorbance values ​​were measured by replacing the sample (i.e., N-doped carbon quantum dots) with PBS (A0), and by replacing the ABTS·+ and DPPH· solutions with PBS (A2).

[0156] ABTS and DPPH free radical scavenging rate (%) = [(A0-A1) / A0]*100%.

[0157] Figure 8 The variation of ABTS radical scavenging rate with N-doped carbon quantum dot concentration is shown from... Figure 8 As can be seen, N-doped carbon quantum dots have significant antioxidant properties, and the ABTS radical scavenging rate gradually increases with the increase of the concentration of N-doped carbon quantum dot solution.

[0158] Figure 9 The DPPH radical scavenging rate varies with the concentration of N-doped carbon quantum dots, from... Figure 9 As can be seen, N-doped carbon quantum dots have significant antioxidant properties, and the DPPH radical scavenging rate gradually increases with the increase of the concentration of N-doped carbon quantum dot solution.

[0159] N-doped carbon quantum dots in solution Fe 2+ / Fe3+ Color change after reaction

[0160] Fe 2+ / Fe 3+ Aqueous solution (i.e., FeCl2 and FeCl3 are prepared by adding them to water to obtain Fe) 2+ Aqueous solution, Fe 3+ The aqueous solution was directly added to the N-doped carbon quantum dot solution, thoroughly mixed, and incubated for 12 hours. The mixture was then centrifuged at 7000 rpm for 5 minutes. The results are as follows: Figure 10 As shown.

[0161] from Figure 10 As can be seen from the addition of Fe 2+ / Fe 3+ The N-doped carbon quantum dots rapidly changed from clear to turbid. After centrifugation, obvious brown or reddish-brown chelated products appeared at the bottom of the centrifuge tube containing the mixed solution. This proves that N-doped carbon quantum dots can chelate Fe. 2+ / Fe 3+ The ability.

[0162] Stern-Volmer fluorescence quenching experiments were used to analyze the chelation of Fe by N-doped carbon quantum dots in solution. 2+ / Fe 3+ ability

[0163] 0-1mM Fe 2+ / Fe 3+ An aqueous solution was added to a 3 mg / ml N-doped carbon quantum dot solution, and after thorough mixing, the fluorescence emission spectrum of the solution at an excitation wavelength of 435 nm was measured using a fluorescence spectrometer. The results are as follows: Figure 11 As shown.

[0164] from Figure 11 It can be seen from this that Fe 2+ / Fe 3+ This can cause significant quenching of the fluorescence of N-doped carbon quantum dots, and the intensity of the fluorescence quenching of N-doped carbon quantum dots increases with the amount of Fe in the solution. 2+ / Fe 3+ Increases with increasing concentration; Fe 2+ / Fe 3+ This can induce fluorescence quenching in N-doped carbon quantum dots, further verifying that N-doped carbon quantum dots chelate Fe. 2+ / Fe 3+ The ability.

[0165] The reactive oxygen species (ROS) content in brain tissue of a mouse model of intracranial hemorrhage was detected using a reactive oxygen species (ROS) detection kit.

[0166] C57BL / 6J mice (23-25g) were induced to have gas anesthesia using 2.5% isoflurane. The induced mice were then placed on a stereotaxic platform and fixed. Anesthesia was maintained using 1.5% isoflurane. Collagenase IV (concentration: 67U / ml, injection dose: 0.04ul / g) was injected into the striatum of the mice (injection coordinates: 0.4mm anterior to the bregma, 2.2mm to the right, depth 3.1mm). Mice injected with collagenase were randomly divided into two groups: a treatment group (receiving collagenase injection 2 hours later, receiving intrathecal injection of N-doped carbon quantum dots (concentration: 3mg / ml, injection dose: 0.5ul / g); and an untreated group (receiving collagenase injection 2 hours later, receiving intrathecal injection of artificial cerebrospinal fluid (injection dose: 0.5ul / g). On day 3 post-cerebral hemorrhage, the ROS level in the brain tissue on the hematoma side was measured using a reactive oxygen species (ROS) detection kit.

[0167] A mouse model of brain hemorrhage was used to verify the ability of N-doped carbon quantum dots to scavenge ROS in vivo in iron overload disease. Experimental results are as follows: Figure 12 As shown, compared with the untreated group, the application of N-doped carbon quantum dots effectively reduced the ROS level on the hematoma side. This indicates that N-doped carbon quantum dots can effectively reduce the ROS level in iron overload disease.

[0168] Fe in the perihemispheric region of a mouse model of cerebral hemorrhage was detected by colorimetric method. 2+ / Fe 3+ content

[0169] After establishing the mouse model of cerebral hemorrhage as described above, the mice were randomly divided into two groups: the treatment group received an injection of collagenase followed by an intrathecal injection of N-doped carbon quantum dots (concentration: 3 mg / ml, injection dose: 0.5 μl / g) 2 hours later; the untreated group received an injection of collagenase followed by an intrathecal injection of artificial cerebrospinal fluid (injection dose: 0.5 μl / g). On the third day after the cerebral hemorrhage, the iron ion content in the brain tissue surrounding the hematoma was measured using an iron ion detection kit.

[0170] A mouse model of brain hemorrhage was used to verify the ability of N-doped carbon quantum dots to scavenge iron ions in vivo in cases of iron overload disease. Experimental results are as follows: Figure 13 As shown, compared with the untreated group, the application of N-doped carbon quantum dots effectively reduced the iron ion content around the hematoma after cerebral hemorrhage. This indicates that N-doped carbon quantum dots can effectively remove iron ions in iron overload diseases.

[0171] A modified neurological function score was used to evaluate the recovery of neurological function in mice.

[0172] After establishing the mouse model of cerebral hemorrhage as described above, mice were randomly divided into two groups: the treatment group received an intrathecal injection of N-doped carbon quantum dots (concentration: 3 mg / ml, injection dose: 0.5 μl / g) 2 hours after collagenase injection; the untreated group received an intrathecal injection of artificial cerebrospinal fluid (injection dose: 0.5 μl / g) 2 hours after collagenase injection. Before establishing the mouse model of cerebral hemorrhage, the recovery of neurological function was evaluated using a modified neurological function scoring scale on days 1, 3, and 7 after the hemorrhage. Higher scores indicated more severe injury and poorer recovery.

[0173] A mouse model of cerebral hemorrhage was used to verify the effect of N-doped carbon quantum dots on the recovery from iron overload disease. The mNSS neurological function score was used to evaluate the overall neurological function of the mice; a lower score indicated better neurological function and faster recovery. Detailed scoring rules can be found in [link to scoring criteria]. Figure 14 As shown, the test results are as follows: Figure 15 As shown. From Figure 15 The results show that, compared with the untreated group, mice treated with N-doped carbon quantum dots had lower neurological function scores and better neurological function recovery on days 3 and 7 after cerebral hemorrhage. This indicates that N-doped carbon quantum dots can be used to treat iron overload diseases and promote recovery after iron overload.

[0174] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of N-doped carbon quantum dots in scavenging oxygen free radicals and DPPH free radicals; the method for preparing the N-doped carbon quantum dots is characterized in that, Includes the following steps: Ginsenosides and ethylenediamine are mixed, and then water is added to dissolve them to obtain a mixture. The mixture is subjected to a hydrothermal reaction; The solution after the hydrothermal reaction was centrifuged, and the supernatant was collected. The supernatant was filtered, and the filtered solution was then transferred to a dialysis bag for dialysis. The dialyzed solution was then freeze-dried to obtain N-doped carbon quantum dots; In the step of carrying out a hydrothermal reaction of the mixture, the hydrothermal reaction temperature is 150~200℃ and the reaction time is 12~24h; The mass ratio of ginsenosides to ethylenediamine is 1:3~10; The ginsenoside mentioned is ginsenoside Rb1; In the step of transferring the filtered solution to a dialysis bag for dialysis, the molecular weight cutoff of the dialysis bag is 1~10KD, and the dialysis time is 12~24h.

2. The application as described in claim 1, characterized in that, In the step of centrifuging the solution after hydrothermal reaction, the centrifugation speed is 8000~12000 r / min and the centrifugation time is 5~10 min; And / or, in the step of filtering the supernatant, the filter used has a pore size of 0.22~0.45μm.

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