Atomic dispersion lignin carbon dot material suitable for wound treatment as well as preparation method and application of atomic dispersion lignin carbon dot material

Abstract

The invention provides an atom dispersion lignin carbon dot material suitable for wound treatment and a preparation method and application thereof. The preparation method of the atomic dispersion lignin carbon dot material comprises the following steps: fully dispersing lignin in deionized water; adding metal salt, and uniformly mixing; and carrying out hydrothermal treatment, filtering, dialyzing and drying to obtain the product. The invention has the advantages of cheap and easily available raw materials, simple preparation process, low cost, greenness and sustainability. Metal atoms are stabilized in a lignin polyphenol network structure through coordinate bonds, so that active sites of the metal atoms are generated; active sites of metal atoms can generate active oxygen, so that bacteria are killed, redundant ROS (reactive oxygen species) near a wound is removed in the later period, and wound healing is accelerated. According to the invention, oxygen can be activated to promote wound healing without introducing peroxides such as CaO2, meanwhile, the treatment efficiency can be effectively improved by introducing a very small amount of metal atoms, and the biocompatibility is not influenced.

Description

Technical Field

[0001] The present invention relates to an atomically dispersed lignin carbon dot material suitable for wound treatment, a preparation method thereof, and applications thereof, belonging to the technical field of material synthesis. Background Art

[0002] Exposed wounds caused by emergencies in daily life and surgical treatments are widespread. Wound treatment has received increasing attention in the biomedical field. Especially when pathogens (such as bacteria) cause wound infections, it will greatly increase the risk of patient death. The traditional treatment method for bacterial infections is the use of antibiotics, but the bacterial drug resistance caused by the abuse of antibiotics remains a crisis that cannot be ignored. To solve the problem of bacterial drug resistance, it is necessary to reduce the use of antibiotics. Currently, the research on the healing of infected wounds mainly focuses on eliminating bacteria in the infection stage, while the needs in the later stage of infected wound healing are often ignored. Therefore, there is currently a lack of adaptable drugs to meet the needs of infected wounds at different healing stages; that is, drugs that can selectively treat the changes in the microenvironment during the wound healing process, effectively kill bacteria, greatly avoid damage to normal tissues, and at the same time provide positive treatment means for the reconstruction of new tissues.

[0003] Nanozymes are nanomaterials that can mimic the structure, properties, and biological functions of natural enzymes. They can overcome the disadvantages of natural enzymes, such as difficult storage, easy denaturation, and high cost, and can be used as nano-drugs, having broad application prospects in the field of treating infected wounds. Due to the adjustable specific element composition and unique chemical structure of nanozymes, they can exhibit enzyme-like catalytic activities at different stages of wound healing according to different microenvironments. In particular, atomically dispersed catalysts with a well-defined structure are composed of supportive nanomaterials for confining, anchoring, and / or coordinating dispersed metal atoms. These materials have the characteristics of high atomic utilization and adjustable ligand structures, and are the bridge between nanozymes and natural enzymes.

[0004] Therefore, developing a method for preparing a biocompatible metal atom-dispersed nanozyme material with simple method, low cost, and capable of adapting to the wound microenvironment, which can efficiently generate ROS to kill bacteria by activating oxygen without introducing H2O2 during the bacterial infection stage, and reduce ROS to promote tissue regeneration according to the changes in the wound environment during the cell proliferation stage, so as to achieve efficient wound treatment while reducing the amount of metal used, is of great significance for the treatment of infectious wounds. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides an atomically dispersed lignin carbon dot material applicable to wound treatment, its preparation method and application. The raw materials of the present invention are cheap and easy to obtain, the preparation process is simple, the cost is low, and it is green and sustainable. In the present invention, metal atoms are stabilized in the lignin polyphenol network structure through coordination bonds, thereby generating metal atom active sites; the metal atom active sites can generate reactive oxygen species (ROS) to achieve the killing of bacteria (using Escherichia coli and Staphylococcus aureus as models), and later remove the excess ROS near the wound to accelerate wound healing. The present invention can achieve the activation of oxygen to promote wound healing without introducing peroxides such as CaO2. At the same time, introducing a very small amount of metal atoms can effectively improve its treatment efficiency and will not affect its biocompatibility. The atomically dispersed lignin carbon dot material obtained by the present invention can resist bacterial wound infection and accelerate wound healing, and has great application potential in the preparation of therapeutic drugs for different bacteria-infected wounds.

[0006] The present invention is realized through the following technical solutions: An atomically dispersed lignin carbon dot material applicable to wound treatment, wherein the carbon dot material is a composite carbon dot material of lignin-based carbon dots and metal atoms.

[0007] According to the preference of the present invention, the metal atom is one or a combination of two or more of copper, manganese, iron, nickel or cobalt; preferably copper.

[0008] The preparation method of the above-mentioned atomically dispersed lignin carbon dot material applicable to wound treatment includes the steps of: Fully disperse lignin in deionized water; add metal salt and mix evenly; perform hydrothermal treatment, filter, dialyze and dry to obtain the atomically dispersed lignin carbon dot material.

[0009] According to the preference of the present invention, the lignin is sodium lignosulfonate, alkaline lignin or delignified lignin; preferably, the lignin is sodium lignosulfonate.

[0010] According to the preference of the present invention, the mass ratio of lignin to deionized water is (1-3):(20-50); preferably, the mass ratio of lignin to deionized water is (1-2):(20-30).

[0011] According to the preference of the present invention, the metal salt is one or a combination of two or more of copper sulfate, copper chloride, copper nitrate, copper acetate, manganese nitrate, manganese chloride, manganese acetate, iron chloride, iron nitrate, iron sulfate, nickel chloride, nickel nitrate, nickel sulfate, cobalt chloride, cobalt nitrate or cobalt sulfate; preferably, the metal salt is copper chloride.

[0012] Preferably according to the present invention, the mass ratio of lignin to metal salt is (1-3):(0.01-0.1); preferably, the mass ratio of lignin to metal salt is (1-2):(0.05-0.1), and more preferably 1:(0.05-0.1).

[0013] Preferably according to the present invention, after adding the metal salt, stir at room temperature for 0.1-5 h to mix evenly; preferably, stir at room temperature for 1-3 h to mix evenly; most preferably, stir at room temperature for 3 h to mix evenly.

[0014] Preferably according to the present invention, the hydrothermal treatment temperature is 180-200 °C; preferably, the hydrothermal treatment temperature is 180 °C.

[0015] Preferably according to the present invention, the hydrothermal treatment time is 10-24 h; preferably, the hydrothermal treatment time is 10-15 h.

[0016] Preferably according to the present invention, the cut-off molecular weight of the dialysis bag or dialysis tube used for dialysis is 1000-3000 Da, and the dialysis time is 2-5 days; preferably, the cut-off molecular weight of the dialysis bag or dialysis tube is 2000-3000 Da, and the dialysis time is 2-3 days.

[0017] Preferably according to the present invention, the drying method is freeze-drying or vacuum drying; the drying time is 10-24 h; preferably, the drying method is freeze-drying, and the drying time is 12-15 h; most preferably, the drying method is freeze-drying, and the drying time is 12 h.

[0018] Application of the above atomically dispersed lignin carbon dot material suitable for wound treatment in antibacterial materials.

[0019] Preferably according to the present invention, the atomically dispersed lignin carbon dot material is applied to antibacterial materials to kill or inhibit Escherichia coli or Staphylococcus aureus.

[0020] Application of the above atomically dispersed lignin carbon dot material suitable for wound treatment in peroxidase-like, catalase-like, superoxide dismutase-like or peroxidase-like enzymes.

[0021] Application of the above atomically dispersed lignin carbon dot material suitable for wound treatment in the production and / or scavenging of intracellular reactive oxygen species.

[0022] Preferably according to the present invention, the atomically dispersed lignin carbon dot material is applied to the scavenging or production of reactive oxygen species in cells.

[0023] Application of the above atomically dispersed lignin carbon dot material suitable for wound treatment in the preparation of drugs for treating bacterial infectious wounds.

[0024] The technical features and beneficial effects of the present invention are as follows: 1. The present invention uses lignin as a raw material and constructs a series of atomically dispersed lignin carbon dots through a simple and effective strategy. The preparation process is simple, the raw materials are cheap and easily available, the cost is low, and it is green and sustainable. The atomically dispersed lignin carbon dots prepared by the present invention have excellent peroxidase-like activity, peroxidase-like activity, superoxide dismutase-like activity, and catalase-like activity. The excellent multi-enzyme-like activity makes it possible to apply the atomically dispersed lignin carbon dots to the treatment of bacterial-infected wounds.

[0025] 2. The atomically dispersed lignin carbon dots prepared by the present invention have metal atom active sites. The metal atoms are stabilized in the polyphenol network structure of lignin through coordination bonds, thereby generating metal atom active sites. The metal atom active sites formed by metal-O coordination increase the electron density near the Fermi level of the material. In the microenvironment of bacterial infection, the material can effectively provide peroxidase-like activity and peroxidase-like activity, generate reactive oxygen species (ROS), and achieve the killing of bacteria (taking Escherichia coli and Staphylococcus aureus as examples). During the wound healing and tissue regeneration after bacteria clearance, it can effectively provide superoxide dismutase-like activity and catalase-like activity to scavenge the excess ROS near the wound and accelerate wound healing.

[0026] 3. The atomically dispersed lignin carbon dots obtained by the present invention show a highly uniformly dispersed dot-like distribution. In the material of the present invention, the lignin-based carbon dots fully play the role of catalyzing O2 to produce ROS to enhance the bactericidal performance of the material, while avoiding the harm caused by the loss of transition metals to organisms. The atomically dispersed lignin carbon dots of the present invention have great application potential in biological sterilization and the preparation of durable and self-antibacterial personal protective products, and it can continuously produce a large amount of ROS to damage the bacterial cell membrane.

[0027] 4. In the preparation method of the atomically dispersed lignin carbon dot material of the present invention, lignin is preferably sodium lignosulfonate, which can stabilize metal atoms through the polyphenol structure on the surface and enhance its multi-nanozyme activity by increasing the electron density at the Fermi level of the material. The treatment of the other two lignins (alkaline lignin and delignified lignin) can also play a role in stabilizing metal atoms, but the nanozyme activity is still inferior to that of sodium lignosulfonate. And the metal atom is preferably a copper atom, which is the key for the atomically dispersed lignin carbon dot material to have multi-enzyme activity. Without adding copper atoms, the multi-enzyme activity of the atomically dispersed lignin carbon dot material will be greatly reduced. The treatment of several other metal atoms (iron, manganese, nickel, and cobalt) can also play a role in increasing multi-enzyme activity, but the effect is still inferior to that of copper atoms.

[0028] 5. The present invention stabilizes metal atom carbon dots (metal-SLCDs) by using a lignin polyphenol network and uses them as cascade nanozymes for the treatment of bacterial-infected wounds. The lignin polyphenol network demonstrates the ability to stabilize metal atoms through metal (such as Cu)-O4 coordination. Experimental results show that the lignin polyphenol network stabilizes elemental metal atoms by increasing the Fermi-level electron density of the material, thereby enhancing its multi-enzyme activity. Metal-SLCDs generate sufficient ROS through multi-enzyme cascades, thereby effectively eliminating bacteria. In addition, after clearing bacterial infections, they can also effectively reduce the ROS level in the wound, thus accelerating wound healing. In vivo treatment and comprehensive experiments provide convincing evidence to support the application of lignin-stabilized metal atoms for treating infected wounds. In summary, the proposed atomically dispersed lignin carbon dot material can enhance multi-enzyme activity by increasing the Fermi-level electron density, thereby effectively eliminating bacteria and promoting tissue repair when treating infected wounds. The atomically dispersed lignin carbon dot composite material proposed in the present invention has great potential for application expansion in the preparation of nano-materials for treating different bacterial-infected wounds.

[0029] 6. The present invention stabilizes metal atom carbon dots (metal-SLCDs) by using a lignin polyphenol network. Without introducing peroxides such as CaO2, it can activate oxygen to promote wound healing. At the same time, introducing a very small amount of metal atoms can effectively improve its treatment efficiency without affecting its biocompatibility. Brief Description of the Drawings

[0030] Figure 1 is the transmission electron microscope (TEM) image of the atomically dispersed lignin carbon dots prepared in Example 1.

[0031] Figure 2 is the sub-angstrom resolution HAADF-STEM image of the atomically dispersed lignin carbon dots prepared in Example 1.

[0032] Figure 3 is the X-ray absorption fine structure (XAFS) spectrum of the atomically dispersed lignin carbon dots prepared in Example 1.

[0033] Figure 4 is the Cu spectrum of the X-ray photoelectron spectroscopy (XPS) of the atomically dispersed lignin carbon dots prepared in Example 1.

[0034] Figure 5 is the X-ray diffraction pattern of the atomically dispersed lignin carbon dots prepared in Example 1 and the lignin carbon dot material prepared in Comparative Example 1.

[0035] Figure 6The activity diagrams of the atomically dispersed lignin carbon dots prepared in Example 1 for mimicking oxidase, peroxidase, catalase, and superoxide dismutase activities, and the activity diagrams of the lignin carbon dot materials prepared in Comparative Example 1 for mimicking catalase and superoxide dismutase activities.

[0036] Figure 7 The comparative diagrams of the activities of mimicking oxidase, peroxidase, catalase, and superoxide dismutase for the atomically dispersed lignin carbon dots prepared in Example 1, the atomically dispersed lignin carbon dots prepared in Example 2, and the lignin carbon dot materials prepared in Comparative Example 1.

[0037] Figure 8 The comparative diagram of the activities of mimicking oxidase for the atomically dispersed lignin carbon dot materials prepared in Example 1, Example 5, and Example 6.

[0038] Figure 9 The comparative diagram of the activities of mimicking oxidase for the atomically dispersed lignin carbon dot materials prepared in Example 1, Example 25, Example 27, and Example 30.

[0039] Figure 10 The comparative diagram of the activities of mimicking oxidase for the atomically dispersed lignin carbon dot materials prepared in Example 1 and Example 36.

[0040] Figure 11 The plate photos of the survival rates of Escherichia coli and Staphylococcus aureus incubated with the atomically dispersed lignin carbon dots prepared in Example 1 at different concentrations in Test Examples 1 and 2.

[0041] Figure 12 The comparative diagram of the survival rates of Escherichia coli and Staphylococcus aureus incubated with the atomically dispersed lignin carbon dots prepared in Example 1 at different concentrations in Test Examples 1 and 2.

[0042] Figure 13 The cytotoxicity test diagram (a) of the atomically dispersed lignin carbon dots prepared in Example 1 in Test Example 3 and the cell oxidative damage protection test diagram (b) of the atomically dispersed lignin carbon dots prepared in Example 1 in Test Example 4.

[0043] Figure 14 The wound area display diagram of the in vivo wound treatment with the atomically dispersed lignin carbon dots prepared in Example 1 in Test Example 5.

[0044] Figure 15 The Masson staining pictures of the main organs after the in vivo wound treatment with the atomically dispersed lignin carbon dots prepared in Example 1 in Test Example 5. Detailed implementation manners

[0045] The present invention will be further described below through specific examples, but is not limited thereto.

[0046] The raw materials used in the following examples are all commercially available products, of analytical purity. Example 1

[0047] A preparation method of an atomically dispersed lignin carbon dot material is as follows: Disperse 1 g of sodium lignosulfonate thoroughly in 20 mL of distilled water, and then add 0.1 g of copper chloride. After stirring at room temperature for 3 h, transfer it to a Teflon high-pressure reactor, seal it, and perform hydrothermal treatment at 180 °C for 12 h. Filter with a 0.22 μm filter membrane, collect the liquid product, and dialyze it in distilled water with a dialysis tube (cut-off molecular weight of 2000 Da) for 2 days. Then freeze-dry the obtained yellow solution for 12 h to obtain atomically dispersed lignin carbon dot powder.

[0048] The TEM morphology image of the atomically dispersed lignin carbon dots (Cu-SLCDs) prepared in this example is as Figure 1 shown, which is a highly dispersed dot-like distribution. And through sub-angstrom resolution HAADF-STEM analysis, it is found that the metal atoms are uniformly single-atom dispersed, as Figure 2 shown.

[0049] The X-ray absorption fine structure (XAFS) spectrum and the Cu2p spectrum of X-ray photoelectron spectroscopy (XPS) of the atomically dispersed lignin carbon dots (Cu-SLCDs) prepared in this example are as Figure 3 and 4 shown. It can be seen that there is an obvious electron transfer in Cu-SLCDs, and it exists in the form of Cu-O4 coordination. The X-ray diffraction pattern of Cu-SLCDs is as Figure 5 shown. It can be seen from the figure that the atomically dispersed lignin carbon dots have a graphite phase structure.

[0050] Evaluate the peroxidase-like and oxidase-like activities of Cu-SLCDs using TMB as a substrate. In a typical peroxidase-like test, add 400 μL of 1.0 mg / mL Cu-SLCDs aqueous dispersion, 400 μL of 10 mM TMB aqueous solution, and 400 μL of 5 mM H2O2 aqueous solution to 2.8 mL of NaAc-HAc buffer solution with pH 5, and then incubate at 25 °C for 10 min. Then monitor the absorbance change at 652 nm with a UV-visible spectrophotometer. In a typical oxidase-like kinetic determination, add Cu-SLCD (20 μg mL -1 ) to acetate buffer solution (pH = 4.5, 800 μL, 0.1 M) at room temperature (25 °C) for TMB oxidation. After incubating at 25 °C for 10 min, detect the oxidase-like reaction rate. At the same time, use the samples without adding H2O2 aqueous solution or without adding carbon dots or without adding both H2O2 aqueous solution and carbon dots as controls. As Figure 6As shown in a, the prepared material has excellent peroxidase-like performance (TMB + Cu-SLCDs) without adding H2O2. As Figure 6 As shown in b, the prepared material has excellent peroxidase-like performance (TMB + Cu-SLCDs + H2O2) under the condition of adding H2O2.

[0051] The oxygen generated was measured using a specific oxygen electrode on a multi-parameter analyzer to determine the catalase (CAT)-like activity of the atomically dispersed lignin carbon dots (Cu-SLCD) material. In a typical test, 0.4 mL of a 1.0 mg / mL aqueous dispersion of the lignin-based carbon dot material and 0.4 mL of a 50 mM H2O2 aqueous solution were successively added to 3.2 mL of a buffer solution (0.1 M PBS buffer solution, pH = 7.4). The solubility (mg / mL) of the generated O2 was measured at different reaction times. As Figure 6 As shown in c, the prepared material (Cu-SLCDs) has excellent catalase-like performance.

[0052] The SOD-like activity of the material was detected using a total superoxide dismutase detection kit (S0101S, Beyotime Biotechnology Co., Ltd.). The SOD-like activity of a series of concentrations of the material was expressed by the inhibition rate of the WST-8 competitive reaction. As Figure 6 As shown in d, the prepared material (Cu-SLCDs) has excellent superoxide dismutase-like performance. Example 2

[0053] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that alkaline lignin is used instead of sodium lignosulfonate; other steps and conditions are the same as those in Example 1.

[0054] Using TMB as a substrate to evaluate the peroxidase-like activity of atomically dispersed lignin carbon dots (Cu-ALCDs), the test method is as described in Example 1. The peroxidase-like performance of the obtained material decreased compared with that in Example 1, as Figure 7 shown in a.

[0055] Using TMB as a substrate to evaluate the peroxidase-like activity of atomically dispersed lignin carbon dots (Cu-ALCDs), the test method is as described in Example 1. The peroxidase-like performance of the obtained material decreased compared with that in Example 1, as Figure 7 shown in b.

[0056] Using hydrogen peroxide as a substrate to evaluate the catalase-like activity of atomically dispersed lignin carbon dots (Cu-ALCDs), the test method is as described in Example 1. The catalase-like performance of the obtained material decreased compared with that in Example 1, as Figure 7 shown in c.

[0057] The superoxide dismutase activity of the atomically dispersed lignin carbon dots (Cu-ALCDs) in the material was evaluated using a total superoxide dismutase detection kit. The test method was as described in Example 1. The superoxide dismutase-like performance of the obtained material decreased compared to that of Example 1, as Figure 7 shown in Example 3

[0058] A method for preparing an atomically dispersed lignin carbon dot material was the same as that described in Example 1, except that: delignified lignin was used instead of sodium lignosulfonate; other steps and conditions were the same as those in Example 1. Example 4

[0059] A method for preparing an atomically dispersed lignin carbon dot material was the same as that described in Example 1, except that: the amount of sodium lignosulfonate used was 2 g, and other steps and conditions were the same as those in Example 1. Example 5

[0060] A method for preparing an atomically dispersed lignin carbon dot material was the same as that described in Example 1, except that: the amount of copper chloride used was 0.05 g, and other steps and conditions were the same as those in Example 1.

[0061] The peroxidase-like activity of the atomically dispersed lignin carbon dots was evaluated using TMB as a substrate. The test method was as described in Example 1. As Figure 8 shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of adding 0.05 g of copper chloride was significantly lower than that of 0.1 g of copper chloride in Example 1, indicating that the performance was affected by the content of Cu atoms in the synthesis process. Example 6

[0062] A method for preparing an atomically dispersed lignin carbon dot material was the same as that described in Example 1, except that: the amount of copper chloride used was 0.01 g, and other steps and conditions were the same as those in Example 1.

[0063] The peroxidase-like activity of the atomically dispersed lignin carbon dots was evaluated using TMB as a substrate. The test method was as described in Example 1. As Figure 8 shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of adding 0.01 g of copper chloride was significantly lower than that of 0.1 g of copper chloride in Example 1, indicating that the performance was affected by the content of Cu atoms in the synthesis process. Example 7

[0064] A method for preparing an atomically dispersed lignin carbon dot material was the same as that described in Example 1, except that: the stirring and mixing time was controlled to be 0.1 h, and other steps and conditions were the same as those in Example 1. Example 8

[0065] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the stirring and mixing time is controlled to be 0.5 h, and other steps and conditions are the same as those in Example 1. Example 9

[0066] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the stirring and mixing time is controlled to be 2 h, and other steps and conditions are the same as those in Example 1. Example 10

[0067] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the stirring and mixing time is controlled to be 4 h, and other steps and conditions are the same as those in Example 1. Example 11

[0068] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the stirring and mixing time is controlled to be 5 h, and other steps and conditions are the same as those in Example 1. Example 12

[0069] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the hydrothermal temperature is controlled at 190 °C, and other steps and conditions are the same as those in Example 1. Example 13

[0070] A preparation method of wood atomically dispersed lignin carbon dot material, as described in Example 1, except that: the hydrothermal temperature is controlled at 200 °C, and other steps and conditions are the same as those in Example 1. Example 14

[0071] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the hydrothermal time is controlled at 20 h, and other steps and conditions are the same as those in Example 1. Example 15

[0072] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the hydrothermal time is controlled at 24 h, and other steps and conditions are the same as those in Example 1. Example 16

[0073] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the molecular weight cut-off of the dialysis tube is 1000 Da, and other steps and conditions are the same as those in Example 1. Example 17

[0074] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the molecular weight cut-off of the dialysis tube is 3000 Da, and other steps and conditions are the same as those in Example 1. Example 18

[0075] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the dialysis time is 3 days, and other steps and conditions are the same as those in Example 1. Example 19

[0076] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the dialysis time is 4 days, and other steps and conditions are the same as those in Example 1. Example 20

[0077] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the dialysis time is 5 days, and other steps and conditions are the same as those in Example 1. Example 21

[0078] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to copper sulfate; other steps and conditions are the same as those in Example 1. Example 22

[0079] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to copper nitrate; other steps and conditions are the same as those in Example 1. Example 23

[0080] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to copper acetate; other steps and conditions are the same as those in Example 1. Example 24

[0081] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to manganese nitrate; other steps and conditions are the same as those in Example 1. Example 25

[0082] A preparation method of atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to manganese chloride; other steps and conditions are the same as those in Example 1.

[0083] Using TMB as the substrate to evaluate the peroxidase-like activity of atomically dispersed lignin carbon dots, the test method is as described in Example 1. As Figure 9As shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of the sample with manganese chloride added is lower than that of copper chloride in Example 1, indicating that the performance is affected by the type of metal during the synthesis process. However, the manganese single-atom material also exhibits peroxidase-like performance. Example 26

[0084] A method for preparing an atomically dispersed lignin carbon dot material is as described in Example 1, except that the metal salt is changed to manganese acetate; other steps and conditions are the same as those in Example 1. Example 27

[0085] A method for preparing an atomically dispersed lignin carbon dot material is as described in Example 1, except that the metal salt is changed to iron chloride; other steps and conditions are the same as those in Example 1.

[0086] The peroxidase-like activity of the atomically dispersed lignin carbon dots was evaluated using TMB as the substrate, and the test method was as described in Example 1. As Figure 9 shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of the sample with iron chloride added is lower than that of copper chloride in Example 1, indicating that the performance is affected by the type of metal during the synthesis process. However, the iron single-atom material also exhibits peroxidase-like performance. Example 28

[0087] A method for preparing an atomically dispersed lignin carbon dot material is as described in Example 1, except that the metal salt is changed to iron nitrate; other steps and conditions are the same as those in Example 1. Example 29

[0088] A method for preparing an atomically dispersed lignin carbon dot material is as described in Example 1, except that the metal salt is changed to iron sulfate; other steps and conditions are the same as those in Example 1. Example 30

[0089] A method for preparing an atomically dispersed lignin carbon dot material is as described in Example 1, except that the metal salt is changed to nickel chloride; other steps and conditions are the same as those in Example 1.

[0090] The peroxidase-like activity of the atomically dispersed lignin carbon dots was evaluated using TMB as the substrate, and the test method was as described in Example 1. As Figure 9 shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of the sample with nickel chloride added is lower than that of copper chloride in Example 1, indicating that the performance is affected by the type of metal during the synthesis process. However, the nickel atomically dispersed material also exhibits peroxidase-like performance. Example 31

[0091] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to nickel nitrate; other steps and conditions are the same as those in Example 1. Example 32

[0092] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to nickel sulfate; other steps and conditions are the same as those in Example 1. Example 33

[0093] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to cobalt chloride; other steps and conditions are the same as those in Example 1. Example 34

[0094] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to cobalt nitrate; other steps and conditions are the same as those in Example 1. Example 35

[0095] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to cobalt sulfate; other steps and conditions are the same as those in Example 1. Example 36

[0096] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to 0.05 g of copper chloride and 0.05 g of manganese chloride; other steps and conditions are the same as those in Example 1.

[0097] Using TMB as a substrate to evaluate the peroxidase-like activity of atomically dispersed lignin carbon dots, the test method is as described in Example 1. As Figure 10 shown, by comparing the change in absorbance at 652 nm within 10 minutes, it can be seen that the absorbance of the sample with copper chloride and manganese chloride added is lower than that of copper chloride in Example 1, indicating that the performance is affected by the type of metal during the synthesis process, but the copper / manganese atomically dispersed material also has peroxidase-like performance. Example 37

[0098] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to 0.05 g of copper chloride and 0.05 g of iron chloride; other steps and conditions are the same as those in Example 1. Example 38

[0099] A preparation method of an atomically dispersed lignin carbon dot material, as described in Example 1, except that: the metal salt is changed to 0.05 g of copper chloride and 0.05 g of nickel chloride; other steps and conditions are the same as those in Example 1. Example 39

[0100] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of copper chloride and 0.05 g of cobalt chloride; other steps and conditions are the same as those in Example 1. Example 40

[0101] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of manganese chloride and 0.05 g of iron chloride; other steps and conditions are the same as those in Example 1. Example 41

[0102] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of manganese chloride and 0.05 g of nickel chloride; other steps and conditions are the same as those in Example 1. Example 42

[0103] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of manganese chloride and 0.05 g of cobalt chloride; other steps and conditions are the same as those in Example 1. Example 43

[0104] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of iron chloride and 0.05 g of nickel chloride; other steps and conditions are the same as those in Example 1. Example 44

[0105] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of iron chloride and 0.05 g of cobalt chloride; other steps and conditions are the same as those in Example 1. Example 45

[0106] A preparation method of an atomically dispersed lignin carbon dot material is as described in Example 1, except that: the metal salt is changed to 0.05 g of nickel chloride and 0.05 g of cobalt chloride; other steps and conditions are the same as those in Example 1. Comparative Example 1

[0107] A preparation method of a lignin carbon dot material is as described in Example 1, except that: no metal salt is added, and other steps and conditions are the same as those in Example 1.

[0108] The X-ray diffraction pattern of SLCDs is as Figure 5 shown, and it can be seen from the figure that the lignin carbon dots are in a graphite phase structure.

[0109] The peroxidase-like activity of lignin carbon dots (SLCDs) was evaluated using TMB as the substrate, and the test method was as described in Example 1. The peroxidase-like performance of the obtained material decreased compared to Example 1, as shown in Figure 7 a.

[0110] The peroxidase activity of lignin carbon dots (SLCDs) was evaluated using TMB as the substrate, and the test method was as described in Example 1. The peroxidase performance of the obtained material decreased compared to Example 1, as shown in Figure 7 b.

[0111] The catalase-like activity of lignin carbon dots (SLCDs) was evaluated using hydrogen peroxide as the substrate, and the test method was as described in Example 1. The catalase-like performance of the obtained material decreased compared to Example 1, as shown in Figure 7 c.

[0112] The superoxide dismutase-like activity of lignin carbon dots (SLCDs) was detected using a total superoxide dismutase detection kit. The test method was as described in Example 1. The superoxide dismutase-like performance of the obtained material decreased compared to Example 1, as shown in Figure 7 d.

[0113] Test Example 1: Bactericidal Performance Test of Atomically Dispersed Lignin Carbon Dots against Escherichia coli The bactericidal performance against Escherichia coli was tested.

[0114] (1) Escherichia coli was inoculated into LB medium and cultured on a shaker at 37 °C and 200 r / min for 12 h. The bacterial solution was diluted to determine the concentration of the bacterial suspension to be 10 7 CFU / mL. 9.9 mL of the diluted Escherichia coli bacterial solution was taken, and 100 μL of the aqueous solution (1.5 mg / mL) of atomically dispersed lignin carbon dots (Cu-SLCDs) prepared in Example 1 was added. The mixture was cultured on a shaker at 37 °C and 200 r / min for 4 h. 100 μL of the bacterial solution was spread on LB solid medium and cultured in an incubator at 37 °C for 12 h for plate colony counting. The above setting was the 15 μg / mL group.

[0115] (2) According to the method in (1) above, the difference was that: 100 μL of water was used to replace the sample aqueous solution. The above setting was the Blank group.

[0116] (3) According to the method in (1) above, the difference was that: the concentration of the sample aqueous solution was 2 mg / mL. The above setting was the 20 μg / mL group.

[0117] (4) According to the method in (1) above, the difference was that: the concentration of the sample aqueous solution was 2.5 mg / mL. The above setting was the 25 μg / mL group.

[0118] Calculate the survival rate of Escherichia coli to evaluate the bactericidal performance; record the number of colony-forming units on the plate of the control group as A0, and the number of colony-forming units on the plate with the sample added as A t , then the calculation formula for the survival rate of Escherichia coli is as follows: Survival rate (%) = A t / A0 × 100%.

[0119] The above are the survival rate plate diagrams of Escherichia coli suspensions in the Blank group, 15 μg / mL group, 20 μg / mL group, and 25 μg / mL group. Figure 11 It shows that the bactericidal rate of the 25 μg / mL group against Escherichia coli > 99%.

[0120] After further testing, the minimum bactericidal concentration of the atomically dispersed lignin carbon dots prepared in Example 1 against Escherichia coli is 25 μg / mL.

[0121] Test Example 2: Bactericidal performance test of atomically dispersed lignin carbon dots against Staphylococcus aureus The method for testing the bactericidal performance of the atomically dispersed lignin carbon dots prepared in Example 1 against Staphylococcus aureus is as described in Test Example 1.

[0122] Figure 12 The following are the survival rate plate diagrams of Staphylococcus aureus suspensions in the Blank group, 15 μg / mL group, 20 μg / mL group, and 25 μg / mL group. Figure 11 It shows that the bactericidal rate of the 25 μg / mL group against Staphylococcus aureus > 99%. After further testing, the minimum bactericidal concentration of the atomically dispersed lignin carbon dots prepared in Example 1 against Staphylococcus aureus is 25 μg / mL.

[0123] Test Example 3: Cytotoxicity test of atomically dispersed lignin carbon dots Perform a cytotoxicity test on NCM-460 cells with the atomically dispersed lignin carbon dots (Cu-SLCDs) in Example 1.

[0124] The in vitro MTT assay was used to study the cytotoxicity of Cu-SLCDs. Briefly, NCM-460 cells were cultured overnight in a 96-well plate at a density of 0.8 × 10 4 cells per well. Then, the cell culture medium was aspirated, and fresh medium containing different concentrations of Cu-SLCDs (0 - 300 μg-mL -1 ) was added to the 96-well plate accordingly, and the cells were cultured for another 24 hours. After that, the cells were gently washed twice with DPBS, and then 100 μL of medium containing 0.5 mg mL -1Fresh medium (serum-free) of MTT. After culturing for 4 hours, discard the medium and add 150 μL DMSO. Gently shake the medium and let it stand for 10 minutes until the formazan formed in the cells is completely dissolved by DMSO. Then, measure the optical density of the cell solution at a wavelength of 490 nm using a multimode microplate reader (Spark, Tecan).

[0125] The results are as Figure 12 shown in a. The experiment found that the atomically dispersed lignin carbon dots (Cu-SLCDs) of Example 1 had almost no toxicity to NCM-460 cells.

[0126] Test Example 4: Test for the ability to protect cells from oxidative damage Test the ability of the atomically dispersed lignin carbon dots (Cu-SLCDs) in Example 1 to protect NCM-460 cells from oxidative damage.

[0127] The steps are as described in Test Example 3, except that: 50 μL of 1 mM H2O2 aqueous solution is added during cell treatment.

[0128] The results are as Figure 13 shown in b. The experiment found that after pretreatment with the atomically dispersed lignin carbon dots (Cu-SLCDs) in Example 1, the cell survival rate increased in a dose-dependent manner. It shows that the atomically dispersed lignin carbon dots (Cu-SLCDs) have excellent ability to protect cells from oxidative damage in the neutral environment of normal cells, and the ability of the material to protect cells from oxidative damage is mainly provided by the catalase-like activity and superoxide dismutase-like activity of the atomically dispersed lignin carbon dots (Cu-SLCDs).

[0129] Test Example 5: In vivo wound healing treatment experiment Anesthetize Bclb / c mice (female, 7 - 8 weeks old, purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.) with 40 mg / kg of 1% sodium pentobarbital. Remove the hair on the back of the mice and create an 8-mm circular wound on the back of the mice. Inoculate the wound with a suspension of MDR Staphylococcus aureus (50 μL, 1×107 CFU / mL) and culture for 24 hours to establish a mouse wound infection model. Randomly divide the mice into a control group and a treatment group, and treat them with PBS buffer at pH 7.4 and a solution of the atomically dispersed lignin carbon dots (Cu-SLCDs) of Example 1 (where the concentration of Cu-SLCDs is 1 mg / mL and the solvent used is PBS buffer at pH 7.4) every 2 days. Take pictures of the wound every day and measure the wound area using image software. In addition, to verify the in vivo safety, record the body weight of the mice every day and collect the main organs of the mice (heart, liver, spleen, lung, and kidney) for hematoxylin-eosin staining.

[0130] The results are as Figure 13As shown, experiments found that treating mouse wounds with the Cu-SLCDs in Example 1 could significantly improve the wound healing rate. And through Figure 14 Figure 15 it was shown that there was no obvious physiological toxicity, where the PBS group was treated with PBS buffer solution, and the Cu-SLCDs group was treated with a PBS solution containing 1 mg / mL of Cu-SLCDs material.

Claims

1. An atomically dispersed lignin carbon dot material suitable for wound treatment, characterized in that, The carbon dot material is a composite carbon dot material of lignin-based carbon dots and metal atoms.

2. The atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1, wherein The metal atoms are one or a combination of two or more of copper, manganese, iron, nickel or cobalt; preferably copper.

3. The method for preparing the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1 or 2, comprising the steps of: Fully dispersing lignin in deionized water; adding a metal salt and mixing evenly; performing hydrothermal treatment, filtering, dialyzing and drying to obtain the atomically dispersed lignin carbon dot material.

4. The preparation method of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 3, characterized in that, The lignin is sodium lignosulfonate, alkaline lignin or delignified lignin; preferably, the lignin is sodium lignosulfonate.

5. The preparation method of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 3, wherein The metal salt is one or a combination of two or more of copper sulfate, copper chloride, copper nitrate, copper acetate, manganese nitrate, manganese chloride, manganese acetate, iron chloride, iron nitrate, iron sulfate, nickel chloride, nickel nitrate, nickel sulfate, cobalt chloride, cobalt nitrate or cobalt sulfate; preferably, the metal salt is copper chloride.

6. The preparation method of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 3, characterized in that, including one or more of the following conditions: i. The mass ratio of lignin to deionized water is (1-3):(20-50); preferably, the mass ratio of lignin to deionized water is (1-2):(20-30); ii. The mass ratio of lignin to the metal salt is (1-3):(0.01-0.1); preferably, the mass ratio of lignin to the metal salt is (1-2):(0.05-0.1), and more preferably 1:(0.05-0.1); iii. After adding the metal salt, stirring at room temperature for 0.1-5 h to mix evenly; preferably, stirring at room temperature for 1-3 h to mix evenly; most preferably, stirring at room temperature for 3 h to mix evenly; iv. The hydrothermal treatment temperature is 180-200 °C; preferably, the hydrothermal treatment temperature is 180 °C; v. The hydrothermal treatment time is 10-24 h; preferably, the hydrothermal treatment time is 10-15 h; vi. The cut-off molecular weight of the dialysis bag or dialysis tube used for dialysis is 1000-3000 Da, and the dialysis time is 2-5 days; preferably, the cut-off molecular weight of the dialysis bag or dialysis tube is 2000-3000 Da, and the dialysis time is 2-3 days.

7. The application of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1 or 2 in antibacterial materials; preferably, the atomically dispersed lignin carbon dot material is applied to antibacterial materials to kill or inhibit Escherichia coli or Staphylococcus aureus.

8. The application of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1 or 2 in peroxidase-like, peroxidase-like, superoxide dismutase-like or catalase-like enzymes.

9. The application of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1 or 2 in the generation and / or scavenging of cellular reactive oxygen species; preferably, the atomically dispersed lignin carbon dot material is applied to the scavenging or generation of reactive oxygen species in cells.

10. The application of the atomically dispersed lignin carbon dot material applicable to wound treatment according to claim 1 or 2 in the preparation of drugs for treating bacterial infectious wounds.

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