A bismuth-based nanomaterial, its preparation method and application

Abstract

The present invention discloses a bismuth-based nanomaterial, which is characterized in that it comprises a Schottky heterostructure product formed by the combination of bismuth oxide and nanogold. The preparation raw materials include the following components: solid bismuth nitrate; solid ammonium fluoride; chloroauric acid; cetyltrimethylammonium bromide; sodium borohydride; silver nitrate; 5-bromosalicylic acid; ascorbic acid; deionized water; ethylene glycol; wherein, the parts are in terms of the amount of substance. The bismuth-based nanomaterial provided by the present invention can effectively inhibit the proliferation of cancer cells and promote the generation of T cells, and has good application prospects in tumor treatment.

Description

Technical Field

[0001] The present invention relates to a bismuth-based nanomaterial, a preparation method thereof and an application thereof, and belongs to the technical field of biofunctional materials. Background Art

[0002] With the development of society, the treatment methods for cancer are becoming more and more diverse, but most patients still face the problems of tumor metastasis and recurrence. Therefore, any effective cancer treatment method or a method that can enhance the existing treatment efficacy has practical significance for cancer patients. Immunogenic cell death is a specific variant of regulated cell death, which is driven by stress and can induce adaptive immunity against the antigens of dead cells. The activated immune response can generate specific cytotoxic T lymphocytes, eliminate tumor cells, kill potential drug-resistant tumors and tumor stem cells, prolong the survival period of patients, and improve the prognosis of patients. However, more than 90% of the current anti-tumor drugs in clinical practice cannot effectively induce immunogenic cell death. Therefore, it is necessary to design a synergistic treatment strategy to apply immunogenic cell death to the field of cancer treatment.

[0003] Bismuth-based semiconductor nanomaterials are easy to synthesize, have controllable morphologies, and also have anisotropic layered structures and unique electronic structures. Among them, Bi 6s 2 O 2p hybrid orbitals can form a good valence electron band, and China is rich in bismuth ore resources, making it have great research potential in the fields of photocatalysis and biomedicine. Narrowing the band gap of bismuth oxide can significantly improve the separation efficiency of electrons and holes. However, bismuth oxide has poor absorption ability for near-infrared light with deeper tissue penetration, which reduces its overall catalytic activity, especially limiting its application in biomedicine.

[0004] Among various modification technologies (Nat. Commun. 2019(10)2840; Adv. Funct. Mater. 2020(30)2003587; Angew. Chem. Int. Ed., 2019(58)4484), combining semiconductors with noble metals to form Schottky heterojunctions can not only expand the visible light response range of semiconductors but also greatly inhibit the recombination of electron-hole pairs (Adv. Mater. 2022 2209589). Under the excitation of near-infrared light, gold rods absorb near-infrared light due to the surface plasmon resonance effect, and then generate hot electron-hole pairs. Among them, some high-energy hot electrons cross the Schottky barrier and enter the conduction band of the semiconductor. Furthermore, a large number of electrons in the semiconductor participate in the reduction reaction, while the hot holes remaining in the gold rods participate in the oxidation reaction. Therefore, compared with traditional semiconductor catalysts, plasma catalysts have effective separation of electrons and holes due to the existence of hot electron injection and Schottky barriers, significantly enhancing their catalytic activity. Therefore, developing a nanomaterial based on bismuth-based semiconductors that can induce immunogenic cell death is an important and meaningful research. Summary of the Invention

[0005] The purpose of the present invention is to provide a bismuth-based nanomaterial, its preparation method and application, so as to improve the technical problem that existing anti-tumor drugs cannot effectively induce immunogenic cell death.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is to provide a bismuth-based nanomaterial, its preparation method and application.

[0007] In the first aspect of the present invention, a bismuth-based nanomaterial is provided, which includes a Schottky heterostructure product formed by the combination of bismuth oxide and gold.

[0008] In the second aspect of the present invention, a method for preparing a bismuth-based nanomaterial is provided, and the preparation raw materials include the following components:

[0009] Bismuth nitrate solid;

[0010] Ammonium fluoride solid;

[0011] Chloroauric acid;

[0012] Cetyltrimethylammonium bromide;

[0013] Sodium borohydride;

[0014] Silver nitrate;

[0015] 5-Bromosalicylic acid;

[0016] Ascorbic acid;

[0017] Deionized water;

[0018] Ethylene glycol;

[0019] The preparation method of the bismuth-based nanomaterial comprises the following steps:

[0020] Step 1: Dissolve bismuth nitrate solid in alcohol to obtain Solution 1;

[0021] Step 2: Dissolve ammonium fluoride solid in alcohol to obtain Solution 2;

[0022] Step 3: Add deionized water to chloroauric acid to obtain Solution 3;

[0023] Step 4: Dissolve cetyltrimethylammonium bromide solid in deionized water to obtain Solution 4;

[0024] Step 5: Add Solution 3 and deionized water to Solution 4 to obtain Solution 5;

[0025] Step 6: Dissolve sodium borohydride in ice water to obtain Solution 6;

[0026] Step 7: Add Solution 6 to Solution 5 to obtain Solution 7;

[0027] Step 8: Dissolve cetyltrimethylammonium bromide solid and 5-bromosalicylic acid in water to obtain Solution 8;

[0028] Step 9: Dissolve silver nitrate solid in deionized water to obtain Solution 9;

[0029] Step 10: Add Solution 9 to Solution 8 to obtain Solution 10;

[0030] Step 11: Add Solution 3 to Solution 10 to obtain Solution 11;

[0031] Step 12: Dissolve ascorbic acid in deionized water to obtain Solution 12;

[0032] Step 13: Add Solution 7 to Solution 12, let it stand to obtain Solution 13;

[0033] Step 14: Disperse Solution 13 in Solution 1, mix evenly, then add Solution 2, react at 20-50 °C for 10 seconds to 5 hours, and then add deionized water to obtain the bismuth-based nanomaterial.

[0034] Preferably, in the preparation method, it satisfies at least one of the following conditions:

[0035] Condition 1 is that the alcohol in Step 1 and / or Step 2 is ethylene glycol;

[0036] Condition 2 is that the amount of bismuth nitrate in Step 1 is 5-10 parts;

[0037] Condition 3 is that the amount of ammonium fluoride in Step 2 is 1-2 parts;

[0038] Condition 3 is that the amount of chloroauric acid in step 3 is 1 to 5 parts;

[0039] Condition 4 is that the dissolution temperature in step 4 is 10 to 100 °C; the amount of cetyltrimethylammonium bromide is 1 to 5 parts;

[0040] Condition 5 is that the rotation speed during addition in step 5 is 400 to 1200 rmp;

[0041] Condition 6 is that the amount of sodium borohydride in step 6 is 1 to 2 parts;

[0042] Condition 7 is that the rotation speed during addition in step 7 is 400 to 1200 rmp;

[0043] Condition 8 is that the dissolution temperature in step 8 is 10 to 100 °C; the rotation speed during dissolution is 400 to 1200 rmp; the amount of cetyltrimethylammonium bromide is 1 to 5 parts, and the amount of 5-bromosalicylic acid is 1 to 5 parts;

[0044] Condition 9 is that the dissolution temperature in step 9 is 10 to 100 °C; the holding time after dissolution is 10 to 30 minutes; the amount of silver nitrate is 1 to 2 parts;

[0045] Condition 10 is that the addition temperature in step 11 is 10 to 50 °C;

[0046] Condition 11 is that the amount of ascorbic acid in step 12 is 1 to 5 parts;

[0047] Condition 12 is that the rotation speed during addition in step 13 is 400 to 1200 rmp; the addition time required is 10 to 30 minutes; the static temperature is 10 to 50 °C; the static time is 12 to 48 hours;

[0048] Among them, the unit of the amount is the amount of substance.

[0049] In the third aspect of the present invention, a bismuth-based nanomaterial prepared by the above preparation method is provided.

[0050] In the fourth aspect of the present invention, an application of the bismuth-based nanomaterial in generating reactive oxygen species under ultrasonic or light excitation is provided.

[0051] In the fifth aspect of the present invention, an application of the bismuth-based nanomaterial in promoting dendritic cell maturation or activating cytotoxic T lymphocytes under ultrasonic or light excitation is provided.

[0052] In the sixth aspect of the present invention, an application of the bismuth-based nanomaterial in inducing immunogenic cell death under ultrasonic or light excitation is provided.

[0053] The seventh aspect of the present invention provides an application of a bismuth-based nanomaterial in the preparation of an anti-tumor drug.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The bismuth-based nanomaterial provided by the present invention can effectively inhibit the proliferation of cancer cells and promote the generation of T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Transmission electron microscope image of the ABO (bismuth oxide combined with gold) nanomaterial provided by the present invention;

[0057] Figure 2 (a) is the X-ray diffraction pattern (XRD) of nano-gold and the ABO nanomaterial; (b) is the ultraviolet photoelectron spectroscopy (UPS) of the ABO nanomaterial;

[0058] Figure 3 (a) is the photocurrent spectrum; (b) is the impedance spectrum; (c) is the photothermal stability curve; (d) is the heating and cooling curve;

[0059] Figure 4 Images of calreticulin (CRT) externalization taken by a laser scanning confocal microscope of 4T1 cells.

[0060] Figure 5 Images of high mobility group protein B1 (HMGB1) release taken by a laser scanning confocal microscope of 4T1 cells.

[0061] Figure 6 Bar chart of the secretion of adenosine triphosphate (ATP) by 4T1 cells.

[0062] Figure 7 Bar charts of tumor images and volumes of primary tumors and distal tumors of tumor-bearing mice on the 14th day after treatment under different experimental conditions.

[0063] Figure 8 Flow cytometry plots and bar charts of the maturation of dendritic cells in the tumors of primary tumors and distal tumors of tumor-bearing mice on the 14th day after treatment under different experimental conditions, respectively.

[0064] Figure 9 Flow cytometry plots and bar charts of cytotoxic T lymphocytes in the tumors of primary tumors and distal tumors of tumor-bearing mice on the 14th day after treatment under different experimental conditions, respectively.

[0065] Brief Description of the Drawings: Treatment methods for each group: Control: control group (no treatment); ABO: material group (treated only with the material); NIR: light irradiation group (only light irradiation treatment: 730 nm, 1 W cm -2 、 irradiate for 5 minutes); US: ultrasound group (only ultrasound treatment: 0.5 W cm -2 、 ultrasound for 4 minutes); ABO + NIR: material plus light irradiation group (material plus light irradiation treatment: 730 nm, 1 W cm -2 、 irradiate for 5 minutes); ABO + US: material plus ultrasound group (material plus ultrasound treatment: 0.5 W cm -2 、 ultrasound for 4 minutes); Synergy: synergy group (material plus light irradiation and ultrasound treatment: 730 nm, 1 W cm -2 、 irradiate for 5 minutes, 0.5 W cm -2 、 ultrasound for 4 minutes). Detailed Implementation Modes

[0066] To make the present invention more obvious and understandable, the following is a detailed description with preferred embodiments and in conjunction with the drawings:

[0067] Example 1 Synthesis and Performance Characterization of Nanomaterials

[0068] (1) Synthesis of seed solution: Prepare 1 mM chloroauric acid aqueous solution, denoted as homogeneous solution A. Prepare 0.2 M cetyltrimethylammonium bromide aqueous solution, denoted as solution B. Prepare 6 mM sodium borohydride aqueous solution, denoted as solution C. First, mix solution A and solution B evenly, then quickly add solution C, and stir evenly to obtain the seed solution.

[0069] (2) Synthesis of gold nanosol: Take 470 mg of cetyltrimethylammonium bromide and 55 mg of 5-bromosalicylic acid in deionized water, stir and heat. Stop heating after the solid dissolves. When cooled to 30 °C, add 0.5 mg of silver nitrate aqueous solution, stir evenly and let stand for 15 minutes. Then add 12.5 mL of the chloroauric acid aqueous solution prepared in step one, stir for 15 minutes. Then add 1 mg of ascorbic acid aqueous solution, stir until colorless, and then add 40 μL of the seed solution prepared in step one. Let stand at 30 °C for 12 hours to obtain the gold nanosol.

[0070] (3) Synthesis of ABO nanomaterials: Take 5 mL of the gold nanosol synthesized in the previous step, centrifuge and disperse it in ethylene glycol, add 2.5 mg of bismuth nitrate to it, ultrasonically disperse evenly, then add 1 mL of ethylene glycol solution of ammonium fluoride, and then add 6 mL of deionized water, and centrifuge to obtain the product.

[0071] (4) Characterization of ABO nanomaterials: The transmission electron microscope images of ABO are shown in detail in Figure 1, the nanomaterials all have a flower-like morphology of 300 - 400 nanometers, and the successful combination of nano-gold and bismuth oxide can be seen. Figure 2 The X-ray diffraction pattern of a shows that the ABO nanomaterial is composed of bismuth oxide and gold. Figure 2 The ultraviolet photoelectron spectroscopy of a shows that a Schottky heterojunction is formed.

[0072] To confirm the improvement of the photothermal effect and sono-catalytic performance, the following experiments were carried out with commercial bismuth oxide as a control. Figure 3 a shows the excellent photocurrent response and photoelectric stability of the ABO nanomaterial. Figure 3 b shows a smaller impedance of the ABO nanomaterial. All these results indicate that the electron-hole recombination efficiency of the ABO nanomaterial is lower, which is more conducive to the transport of electrons and shows better catalytic activity. Figure 3 c shows the temperature change of the ABO nanomaterial under the irradiation of a 730 nm laser at 1 W cm -1 and four cycles of heating and natural cooling conditions, as well as the heating and cooling curves relative to pure bismuth oxide. The experiment proves the photothermal conversion efficiency and stability of ABO. -2

[0073] Example 2: Experimental study on immunogenic cell death at the cellular level

[0074] 4T1 cells (1×10 5 cells per well) were seeded in confocal dishes and cultured for 24 hours. Then ABO (100 μg mL -1 ) was added and incubated for 24 hours. Next, fresh medium was changed for treatment under different conditions: Control: control group (without any treatment); ABO: material group (only treated with the material); NIR: light irradiation group (only light irradiation treatment: 730 nm, 1 W cm -2 , irradiated for 5 minutes); US: ultrasound group (only ultrasound treatment: 0.5 W cm -2 , ultrasound for 4 minutes); ABO + NIR: material plus light irradiation group (material plus light irradiation treatment: 730 nm, 1 W cm -2 , irradiated for 5 minutes); ABO + US: material plus ultrasound group (material plus ultrasound treatment: 0.5 W cm -2 , ultrasound for 4 minutes); Synergy: synergy group (material plus light irradiation and ultrasound treatment: 730 nm, 1 W cm -2 , irradiated for 5 minutes, 0.5 Wcm -2 , ultrasound for 4 minutes). Then incubated for 6 hours. Next, immunofluorescence staining was performed with calreticulin (CRT) antibody, and 4',6-diamidino-2-phenylindole (DAPI) staining was carried out, and then laser confocal analysis was used. The results observed by laser confocal are as​Figure 4 As shown (scale bar: 20 μm), after co-incubation of 4T1 cells with ABO, in the combination treatment group, strong green fluorescence was presented on the cell membrane.

[0075] 4T1 cells (5×10 4 cells per well) were seeded in confocal dishes and cultured for 24 h. Then ABO (100 μg mL -1 ) was added and incubated for 24 h. Next, fresh medium was replaced for treatment under different conditions (same as above), and then incubated for 12 h. Cells were fixed, immunofluorescently stained with high mobility group box 1 (HMGB1) antibody, and DAPI stained. After staining, confocal microscopy was performed. The results of confocal microscopy are shown as Figure 5 shown (scale bar: 20 μm). After co-incubation of 4T1 cells with ABO, in the combination treatment group, abundant red fluorescence was presented in the cytoplasm.

[0076] The release of adenosine triphosphate (ATP) was measured using a kit of the prior art. Using the above culture and treatment methods, the release of cellular ATP under various treatment conditions was further evaluated. The experimental data are shown in Figure 6 . The results showed that ATP secretion was significantly enhanced in the combination treatment group. The above results indicate that the ABO nanomaterial can effectively trigger the immunogenic cell death effect of 4T1 cells under 30 nm laser irradiation and ultrasound stimulation.

[0077] Example 3: Tumor suppression by immunogenic cell death induction in vivo

[0078] Six-week-old male Balb / c mice were randomly divided into seven groups (control group, light irradiation group, ultrasound group, material group, material + light irradiation group, material + ultrasound group, combination group), and 4T1 cells were inoculated into the right hind back of each mouse. One week later, different treatments were started. Mice in each group were sacrificed on day 14, and the primary tumors and distal tumors were removed for flow cytometry analysis. The results demonstrated that both the primary tumors and distal tumors of the mice in the combination treatment group were effectively inhibited; moreover, the percentages of mature dendritic cells and CD8+ cytotoxic T lymphocytes in the combination treatment group were much higher than those in the other two experimental groups. These results indicate that tumor cells injected with the ABO nanomaterial and treated with light irradiation and ultrasound can effectively induce the maturation of dendritic cells and the activation of CD8+ cytotoxic T lymphocytes in vivo.

Claims

1. A method for preparing bismuth-based nanomaterials, characterized in that, The preparation raw materials include the following components: Bismuth nitrate solid; Ammonium fluoride solid; Chloroauric acid; Cetyltrimethylammonium bromide; Sodium borohydride; Silver nitrate; 5-Bromosalicylic acid; Ascorbic acid; Deionized water; Ethylene glycol; The preparation method of the bismuth-based nanomaterial includes the following steps: Step 1) Synthesis of the seed solution: Prepare 1 mM chloroauric acid aqueous solution, denoted as homogeneous solution A; prepare 0.2 M cetyltrimethylammonium bromide aqueous solution, denoted as solution B; prepare 6 mM sodium borohydride aqueous solution, denoted as solution C; first mix solution A and solution B evenly, then quickly add solution C, and stir evenly to obtain the seed solution; Step 2) Synthesis of the gold nanosolution: Take 470 mg of cetyltrimethylammonium bromide and 55 mg of 5-bromosalicylic acid in deionized water, stir and heat until the solid dissolves, then stop heating. When it cools to 30 °C, add 0.5 mg of silver nitrate aqueous solution, stir evenly and let it stand for 15 minutes; then add 12.5 mL of the chloroauric acid aqueous solution prepared in step 1), stir for 15 minutes; then add 1 mg of ascorbic acid aqueous solution, stir until colorless, and then add 40 μL of the seed solution prepared in step 1), and let it stand at 30 °C for 12 hours to obtain the gold nanosolution; Step 3) Synthesis of the ABO nanomaterial: Take 5 mL of the gold nanosolution synthesized in step 2), centrifuge and disperse it in ethylene glycol, add 2.5 mg of bismuth nitrate to it, ultrasonically disperse evenly, then add 1 mL of ethylene glycol solution of ammonium fluoride, and then add 6 mL of deionized water, and centrifuge to obtain the product.

2. A bismuth-based nanomaterial obtained by the method for preparing a bismuth-based nanomaterial as described in claim 1.

3. An application of the bismuth-based nanomaterial as described in claim 2 in generating reactive oxygen species under ultrasonic or light excitation.

4. An application of the bismuth-based nanomaterial as described in claim 2 in promoting dendritic cell maturation or activating cytotoxic T lymphocytes under ultrasonic or light excitation.

5. An application of the bismuth-based nanomaterial as described in claim 2 in inducing immunogenic cell death under ultrasonic or light excitation.

6. An application of the bismuth-based nanomaterial as described in claim 2 in the preparation of anti-tumor drugs.

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