Preparation method and application of seaweed biomass derived manganese / nitrogen doped carbon composite nano-enzyme

Abstract

The invention belongs to the field of nano material preparation, and particularly relates to a preparation method and application of seaweed biomass derived manganese / nitrogen doped carbon composite nano enzyme. The low-cost manganese nanoparticle / seaweed biomass derived nitrogen-doped carbon composite nano-enzyme is prepared by taking seaweed biomass as a raw material through an impregnation-pyrolysis method. The nano-enzyme has high-efficiency enzyme-like activities such as peroxidase-like activity, glutathione oxide enzyme-like activity, singlet oxygen generation catalysis capability and the like, and shows application prospects in the antibacterial and anti-tumor fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and specifically relates to a preparation method and application of a seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme. Background Art

[0002] Nanozymes have shown great potential for development in many key areas such as clinical medicine, food safety and chemical production. They have been widely used and recognized due to their low cost, excellent stability, easy large-scale production and convenient storage.

[0003] Reactive oxygen species (ROS) can undergo redox reactions with a variety of molecules in the body and are often used to treat tumors due to their non-invasiveness, low systemic toxicity and high specificity. Many nanozymes have the ability to catalyze H2O2 or O2 to generate different toxic free radicals (such as ¹O2, •OH or •O2⁻), which makes them show great application potential in ROS-mediated tumor nanocatalysis therapy.

[0004] Manganese is an essential element for the human body with good biocompatibility. It is often used as the catalytic active center of nanozymes to simulate the activities of various natural enzymes, including peroxidase, superoxide dismutase and catalase. The structure of some manganese oxide nanozymes is similar to the active center of superoxide dismutase, which helps to understand the mechanism of action of natural enzymes. In addition, the presence of manganese can significantly improve the catalytic efficiency of nanozymes. By adjusting the content and chemical state of manganese, the catalytic activity, selectivity and stability of nanozymes can be regulated. Compared with precious metal (such as gold and platinum) nanozymes, manganese-based nanozymes are generally more economical and environmentally friendly. The above characteristics make manganese-based nanozymes have broad prospects in biomedical applications.

[0005] As an ideal biomass, algae has the advantages of short growth cycle, fast reproduction and large yield, no pollution and renewable, saving freshwater and land resources, relatively low production cost, and can repair marine ecology. It has broad application prospects and huge development potential. In recent years, the research on algae drugs in tumor treatment has greatly increased at home and abroad. How to increase the efficacy, reduce toxic side effects and simplify the extraction process is an important topic in the current anti-tumor research of algae biomass. Therefore, it is of great significance to develop seaweed biomass-derived manganese / nitrogen-doped carbon composite carbon materials, seek stable methods to adjust the size, improve biosafety, and apply them in the field of anti-tumor.

[0006] Based on the current research, this paper aims to explore whether seaweed biomass-derived materials have the potential to have anti-tumor nanozyme-like activity. While exploring the algae itself, manganese ions are doped to study whether it can improve the catalytic activity of seaweed biomass-derived materials and increase the efficiency of ROS generation, thereby increasing their catalytic activity and anti-tumor activity. Summary of the invention

[0007] The present invention provides a method for preparing a seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme and its application. The method can prepare a seaweed biomass-derived manganese composite nanozyme supported by nitrogen-doped porous carbon, which exhibits peroxidase-like and glutathione-like reaction activities and catalyzes the production of singlet oxygen, and can inhibit the growth of tumor cells through chemodynamic therapy.

[0008] The present invention first provides a method for preparing a seaweed biomass-derived manganese / nitrogen-doped carbon composite material, comprising the following steps: (1) Providing a manganese (II) salt aqueous solution; (2) adding the seaweed biomass into a manganese (II) salt solution and soaking it for 18-24 hours, drying it, grinding it and sieving it to obtain a precursor; (3) Calcine the precursor in anoxic conditions to obtain the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme.

[0009] The concentration of manganese ions in the manganese (II) salt aqueous solution in step (1) of the present invention is 0.1-5 mol / L, preferably 0.2 mol / L.

[0010] Wherein, the manganese (II) salt is a chloride, carbonate, acetate, sulfate, or hydrate of divalent manganese; preferably manganese chloride tetrahydrate.

[0011] In step (2) of the present invention, the amount of seaweed biomass and manganese (II) salt is in the ratio of 1 g: 0.1 to 0.2 mol; preferably, the amount of seaweed biomass and manganese (II) salt is in the ratio of 1 g: 0.1 mol.

[0012] The soaking in step (2) of the present invention is carried out at room temperature. The drying in step (2) is carried out at 55-60° C. for 12-14 hours.

[0013] Furthermore, step (2) also includes pre-treating the seaweed biomass. The pre-treatment method is: chop the seaweed biomass sample into pieces and soak it in distilled water for 24 hours, then wash it and put it in a drying oven at 50-60°C for 10-14 hours. The conditions for the anaerobic calcination in step (3) of the present invention are as follows: in a nitrogen atmosphere, the heating rate is 1-10 °C / min, the calcination temperature is 200-500 °C, and it is maintained for 1h-5h. Preferably, the calcination conditions are as follows: the heating rate of the tubular furnace is 5 °C / min, the calcination temperature is selected to be 300 °C and maintained for 3h, and then naturally cooled to room temperature.

[0014] Furthermore, the screening described in the above technical solution is through a 200-300 mesh sieve.

[0015] The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the above preparation method has a relatively uniform size of about 200-300 nm. In vitro experiments confirm that the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the present invention has peroxidase-like and glutathione peroxidase-like reaction activities and catalyzes the production of singlet oxygen.

[0016] The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the present invention can be applied to the fields of detection and analysis, pollution adsorption and biomedicine.

[0017] The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the present invention can be used to prepare antibacterial drugs.

[0018] The cell proliferation activity experiment confirmed that the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared in the present invention has the effect of inhibiting the proliferation of tumor cells.

[0019] Based on the above experimental results, the present invention particularly provides an application of seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme in the preparation of anti-tumor drugs.

[0020] The present invention also provides a pharmaceutical composition, which comprises the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme of the present invention as one of the main active ingredients, and a pharmaceutically acceptable carrier.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by impregnation, calcination, grinding and sieving in the present invention has a relatively uniform size of about 200-300 nm, which meets the requirements for the size of nanomaterials in anti-tumor applications; the prepared seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme contains manganese, nitrogen, carbon, oxygen and other essential elements for the human body, avoiding metabolic toxicity; secondly, the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the present invention uses seaweed biomass porous carbon material as a substrate, has good catalytic activity, high stability, and exhibits a variety of anti-tumor enzyme activities.

[0022] The present invention uses seaweed biomass as raw materials, expanding the preparation strategy and application scope of biomass-derived carbon composite nanomaterials. The method of calcination modification in the present invention is simple, easy to operate, and low in cost. Composite nanozymes derived from seaweed biomass with different activities can be achieved by controlling experimental parameters such as the selection of soaked metal ions, calcination temperature, and calcination time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron microscope image of the manganese-doped laver derivative prepared in Example 1.

[0024] Figure 2 This is a scanning electron microscope image of the manganese-doped kelp derivative prepared in Example 2.

[0025] Figure 3 This is a scanning electron microscope image of the manganese-doped kelp derivative prepared in Example 3.

[0026] Figure 4 This is a scanning electron microscope image of the laver derivative prepared in Comparative Example 1.

[0027] Figure 5 This is a scanning electron microscope image of the kelp derivative prepared in Comparative Example 2.

[0028] Figure 6 This is a scanning electron microscope image of the kelp derivative prepared in Comparative Example 3.

[0029] Figure 7 This is a Raman spectrum of the manganese-doped seaweed biomass derivative prepared in Example 1-3.

[0030] Figure 8 The Raman spectra of the seaweed biomass derivatives prepared in Comparative Examples 1-3 are shown.

[0031] Fig. 9 This is the X-ray diffraction pattern of the manganese-doped kelp derivative of Example 3.

[0032] Fig.10 This is the X-ray diffraction pattern of the kelp derivative of Comparative Example 3.

[0033] Fig.11 This is the peroxidase activity detection spectrum of manganese-doped kelp in Example 3.

[0034] Fig.12 The following is a comparison chart of the peroxidase activities of the samples prepared in Comparative Examples 1-3.

[0035] Fig.13 This is a spectrum diagram of glutathione oxidase activity detection of manganese-doped kelp in Example 3.

[0036] Fig.14The following is a comparison chart of the glutathionease activities of the samples prepared in Examples 1-3.

[0037] Fig.15 This is the singlet oxygen production activity detection spectrum of manganese-doped kelp in Example 3.

[0038] Fig.16 For comparison with Examples 1-3, a comparison chart of singlet oxygen generation activity of the samples prepared in Examples 1-3 is shown.

[0039] Fig.17 The effects of the samples prepared in Examples 1-3 and Comparative Examples 1-3 on the proliferation activity of cancer cells. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following embodiments. Example 1

[0041] Cut the laver into pieces, soak it in distilled water for 24 hours, and then put it in a 55°C oven to dry overnight. Weigh 1.6g of manganese chloride tetrahydrate and dissolve it in 40mL of water. Take 2g of laver and cut it into pieces and put it in the above solution and soak it for 24 hours. After taking it out, dry it in a 55°C drying oven overnight. Weigh 50mg and put it into a porcelain boat and put it into a tubular furnace for calcination. Keep N2 sufficient, the heating rate is 5°C / min, and calcine at 300°C for 3h. After cooling naturally to room temperature, collect the dark brown product and grind it, and pass it through a 300-mesh sieve to obtain the target sample, which is laver-derived manganese / nitrogen-doped carbon composite nanozyme.

[0042] From the attached Figure 1 The scanning electron microscope image shows that after doping with manganese, the particle size of the laver-derived material is significantly reduced. The morphology is clearer and the distribution is more uniform, which helps to increase the active sites and improve the catalytic activity. Figure 7 The Raman spectrum of the sample shows that the -1 Two broad peaks appeared at , corresponding to graphene crystals (G band) and disordered carbon (D band), respectively, and these structures are associated with reactive oxygen species (ROS). Example 2

[0043] The difference between this embodiment and embodiment 1 is that the seaweed biomass is kelp, and the target sample obtained is kelp-derived manganese / nitrogen-doped carbon composite nanozyme.

[0044] From the attached Figure 2 From the scanning electron microscope image, it can be seen that after doping with manganese, the kelp-derived material is relatively smaller. At the same time, most of the morphology is flaky, which is conducive to full contact with the substrate and improves the reaction rate. Figure 7 The Raman spectrum of the sample shows that the-1 Two broad peaks appeared at , corresponding to graphene crystals (G band) and disordered carbon (D band), respectively, and these structures are associated with reactive oxygen species (ROS). Example 3

[0045] The difference between this embodiment and embodiment 1 is that the seaweed biomass is Undaria pinnatifida, and the target sample obtained is Undaria pinnatifida-derived manganese / nitrogen-doped carbon composite nanozyme.

[0046] From the attached Figure 3 The scanning electron microscope image shows that after manganese doping, the kelp-derived material is relatively smaller. Most of the morphology is in the form of flakes, which is very conducive to full contact with the substrate and improves the reaction rate. Figure 7 The Raman spectrum of the sample shows that the -1 Two broad peaks appeared at , corresponding to graphene crystals (G band) and disordered carbon (D band), respectively, and these structures are associated with reactive oxygen species (ROS).

[0047] From the attached Fig. 9 It can be seen from the XRD spectrum that at 21.03 o The diffraction peak of MnO2 appears near 26.65 o The diffraction peak of SiO2 appears nearby, confirming the successful doping of manganese. Comparative Example 1

[0048] Cut the laver into pieces, soak it in distilled water for 24 hours, and then put it in a 55°C drying oven overnight. Weigh 50 mg and place it in a porcelain boat and put it into a tubular furnace for calcination. Keep the N2 sufficient and the heating rate is 5°C / min. Calcinate at 300°C for 3 hours. After naturally cooling to room temperature, collect the black-brown product, grind it, and pass it through a 300-mesh sieve to obtain the target sample, which is a laver-derived nitrogen-doped carbon composite carbon material.

[0049] From the attached Figure 4 The particle morphology of the Porphyra biomass-derived material is blocky and partially flaky. The particle size is about 200~300nm. Figure 8 From the Raman spectrum, it can be seen that the sample has graphite crystal (G band) and disordered carbon (D band) structure. Comparative Example 2

[0050] Cut the kelp into pieces, soak it in distilled water for 24 hours, and then put it in a 55°C drying oven overnight. Weigh 50 mg and place it in a porcelain boat and put it into a tubular furnace for calcination. Keep the N2 sufficient and the heating rate is 5°C / min. Calcinate at 300°C for 3 hours. After naturally cooling to room temperature, collect the black-brown product, grind it, and sieve it to obtain the target sample, which is a kelp-derived nitrogen-doped carbon composite carbon material.

[0051] Attached Figure 5The morphology of kelp biomass-derived materials is blocky and flake-like. The particle size is about 200~300 nm. Figure 8 The Raman spectrum of the sample shows that it has graphite crystal (G band) and disordered carbon (D band) structures, which are associated with reactive oxygen species (ROS). Comparative Example 3

[0052] Cut the kelp into pieces, soak it in distilled water for 24 hours, and then put it in a 55°C drying oven overnight. Weigh 50 mg and place it in a porcelain boat and put it into a tubular furnace for calcination. Keep the N2 sufficient and the heating rate is 5°C / min. Calcinate at 300°C for 3 hours. After naturally cooling to room temperature, collect the black-brown product, grind it, and sieve it (300 mesh) to obtain the target sample, which is the kelp biomass-derived nitrogen-doped carbon composite carbon material.

[0053] From the attached Figure 6 Undaria pinnatifida biomass-derived materials have a variety of morphologies, including flakes, blocks, spherical shapes, etc., with a small particle size of about 100~200 nm. Figure 8 The Raman spectrum of the sample shows that it has a graphite crystal (G band) structure and disordered carbon (D band), which is associated with reactive oxygen species (ROS). Fig.10 It can be seen from the XRD spectrum that at 26.65 o A diffraction peak of SiO2 appears nearby, but compared with Example 3, no diffraction peak of the manganese-based compound is found. Example 4

[0054] The carbon-doped seaweed biomass-derived materials prepared according to Examples 1-3 and the undoped seaweed biomass-derived materials prepared according to Comparative Examples 1-3 were tested for peroxidase activity.

[0055] The samples were prepared into 1 mg / mL sample solutions with ultrapure water, and appropriate amounts were mixed with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in a pH 4.0 citric acid-sodium citrate buffer solution to make the final sample, TMB, and H2O2 concentrations 30 μg / mL, 1 mM, and 10 mM, respectively. The mixed solution was placed at 37 o C constant temperature, and stand the reaction for 6 minutes in the dark. Obvious characteristic peaks of oxidized TMB appeared. The intensity of the absorption peak at 652 nm represents the yield of hydroxyl radicals, that is, the activity of catalase. Put it into a UV spectrophotometer and detect it at 0, 1, 3, 6, 10, and 15 minutes in the wavelength range of 500-800 nm to compare the changes in the characteristic peaks of oxidized TMB of each product. The intensity of the absorption peak at 652 nm represents the yield of hydroxyl radicals, that is, the activity of catalase.

[0056] Taking the manganese-doped kelp of Example 1 as an example, as shown in the attached Fig.11 As shown in the figure, the absorption peak of the sample at 652 nm increased by 0.072AU from 0 min to 15 min. Fig.12 As shown, the activity of the absorption peak at 652 nm within 15 min under the catalysis of manganese-doped seaweed biomass-derived materials is better than that of undoped seaweed biomass-derived materials, indicating that the catalytic efficiency of peroxidase activity is improved after manganese doping, and it has the potential to become a peroxidase-like enzyme. Example 5

[0057] Glutathione peroxidase activity detection of the carbon-doped seaweed biomass-derived materials prepared according to Examples 1-3 and the undoped seaweed biomass-derived materials prepared according to Comparative Examples 1-3.

[0058] Prepare 1 mg / mL sample solution with ultrapure water, take an appropriate amount and mix it with glutathione solution in PBS buffer, so that the final concentration of sample and glutathione is 30 μg / mL and 1 mM. o The reaction was carried out in a light-proof shaker at C, and DTNB solution was added at 0, 0.5, 1, 1.5, and 2 h, respectively. The absorption curve was measured by UV spectrophotometer in the wavelength range of 300 nm-550 nm. The decrease in the absorption peak at 412 nm compared with the control group indicated the ability to consume glutathione, that is, the activity of glutathione peroxidase.

[0059] Taking the manganese-doped kelp of Example 3 as an example, as shown in the attached Fig.13 As shown in the figure, the absorption peak of the sample at 412 nm decreased by 2.33AU within two hours. Fig.14 As shown, the activity of the absorption peak at 412 nm under the catalysis of manganese-doped seaweed biomass-derived materials within two hours is better than that of the undoped seaweed biomass-derived materials, indicating that the catalytic efficiency of glutathione peroxidase activity is improved after manganese doping, and it has the potential to become a glutathione peroxidase-like enzyme. Example 6

[0060] The singlet oxygen generation activity of the carbon-doped seaweed biomass-derived materials prepared according to Examples 1-3 and the undoped seaweed biomass-derived materials prepared according to Comparative Examples 1-3 was detected.

[0061] The samples were prepared into 1 mg / mL sample solutions with ultrapure water, and 980ul DMSO solution and 920ul water, 40ul H2O2 (1M / L), 20ul DPBF (10mM / L) were added first, and 40ul sample solution was added. The timing was started and the following time was measured: 0, 1, 5, 10, 20, 30min. The wavelength range of 300 nm-550 nm was used for detection, and the degree of decrease of the absorption peak at 410nm of each product oxidation was measured using an ultraviolet spectrophotometer to determine the singlet oxygen activity of seaweed biomass.

[0062] Taking the manganese-doped kelp of Example 1 as an example, as shown in the attached Fig.15 As shown in the figure, the absorption peak intensity of the sample at 412nm decreased by 1.184AU from 0min to 30min. Fig.16 As shown, the activity of the absorption peak at 410 nm within 30 min under the catalysis of manganese-doped seaweed biomass-derived materials was better than that of the undoped seaweed biomass-derived materials, indicating that manganese doping has the potential to produce singlet oxygen. Example 7

[0063] Cytotoxicity test of the carbon-doped seaweed biomass-derived materials prepared according to Examples 1-3 and the undoped seaweed biomass-derived materials prepared according to Comparative Examples 1-3.

[0064] 4T1 cells were digested and counted. 4 The number of cells per well was inoculated in a 96-well plate and cultured overnight in a 37°C carbon dioxide (5%) incubator. The samples were prepared with DMEM medium to a certain concentration and then placed in a constant temperature carbon dioxide incubator for 24 h. 20 μL of CCK8 solution was added to each well, and the plate was stained in the dark for 1 h in the incubator. The absorbance value at 490 nm in each well was measured using an ELISA reader.

[0065] As attached Fig.17 As shown in the figure, the cell survival rate after incubation with undoped seaweed biomass-derived materials was high, and the cell survival rate decreased by 40% when the sample concentration was 150 μg / mL. However, carbon-doped seaweed biomass-derived materials have strong cytotoxicity and can effectively kill cancer cells. The concentration of 50 μg / mL can reduce the cell survival rate by 50%.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme, characterized in that: The following steps are involved: (1) Providing a manganese (II) salt aqueous solution; (2) adding the seaweed biomass into a manganese (II) salt solution and soaking it for 18-24 hours, drying it, grinding it and sieving it to obtain a precursor; (3) Calcine the precursor in anoxic conditions to obtain the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme.

2. The method for preparing the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 1, characterized in that: The concentration of manganese ions in the manganese (II) salt aqueous solution in step (1) is 0.1-5 mol / L.

3. The method for preparing the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 1, characterized in that: The manganese (II) salt is a chloride, carbonate, acetate, sulfate, or hydrate of divalent manganese.

4. The method for preparing the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 1, characterized in that: In step (2), the amount of seaweed biomass and manganese (II) salt is in the ratio of 1g: 0.1-0.2mol.

5. The method for preparing the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 1, characterized in that: The conditions for the anaerobic calcination in step (3) are as follows: under a nitrogen atmosphere, the heating rate is 1-10°C / min, the calcination temperature is 200-500°C, and the temperature is maintained for 1h-5h.

6. The method for preparing the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 5, characterized in that: The conditions for the anaerobic calcination in step (3) are as follows: under a nitrogen atmosphere, the heating rate of the tubular furnace is 5°C / min, the calcination temperature is selected to be 300°C and maintained for 3h, and then naturally cooled to room temperature.

7. The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme prepared by the method according to any one of claims 1 to 6, characterized in that: The seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme has a size of 200-300 nm and has peroxidase-like, glutathione peroxidase-like and singlet oxygen-producing activities.

8. Application of the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 7 in the fields of detection and analysis, pollution adsorption, and biomedical fields for non-therapeutic purposes.

9. Use of the seaweed biomass-derived manganese / nitrogen-doped carbon composite nanozyme according to claim 7 in the preparation of antibacterial drugs and antitumor drugs.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the algae biomass-derived manganese / nitrogen-doped carbon composite nanozyme as claimed in claim 7 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

Images