Hydroxyl radical and singlet oxygen self-release type bismuth-based nanoparticle material

Abstract

The invention discloses a hydroxyl free radical and singlet oxygen self-release type bismuth-based nanoparticle material, a preparation method of the material comprises the following steps: adding a bismuth-containing compound and a polymer stabilizer into a solution containing a particle size regulation and control reagent, and carrying out a hydrothermal reaction to form a bismuth oxide nanoparticle template; and dropwise adding the bismuth-based nano-particles and a strong oxidant into a strong alkaline solvent, and carrying out macromolecular hydrophilic modification on the obtained bismuth-based nano-particles. The nano-particle material can spontaneously and continuously release hydroxyl free radicals and singlet oxygen under the condition of subacid (pH 6.6-5.5), can effectively inhibit the generation of tumors in a mouse breast cancer tumor model, eliminates far-end tumors by inducing an immune stimulation effect, and inhibits the metastasis of the tumors; and the method can also be used for computed tomography imaging and radiotherapy synergistic treatment of a breast cancer mouse model. Meanwhile, the hydrogel has the characteristics of good biocompatibility, low long-term toxicity and rapid metabolism, and has extremely high clinical diagnosis and treatment benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a bismuth-based nanoparticle material with self-releasing hydroxyl radicals and singlet oxygen, and in particular to a bismuth-based nanoparticle material with self-releasing hydroxyl radicals (·OH) and singlet oxygen ( 1 Bismuth-based nanoparticle material (O2) and preparation method thereof. Background Art

[0002] Tumor treatment has made considerable progress in the past few years, but its complete cure remains the most critical challenge due to the characteristics of tumor metastasis and recurrence. It is currently found that immunotherapy that uses the internal immune system to activate anti-tumor immunity to prevent tumor recurrence and metastasis has great potential. However, preliminary clinical results show that the effect of immunotherapy is far from the expected goal due to weak immune response. In addition, tumor cells are also able to avoid immune attack through immune escape and immunosuppression. Therefore, how to improve the efficacy of immunotherapy is a major issue in the medical field. Recently, nanomaterials with immunomodulatory capabilities have achieved remarkable achievements in immunotherapy. For example, nanomaterials have successfully induced anti-tumor immune responses as immunostimulants. Therefore, using nanomaterials to enhance anti-tumor immunity or reverse tumor immunosuppression is an important direction for tumor immunotherapy.

[0003] Reactive oxygen species (ROS) are chemically active species formed when oxygen is not completely reduced, and include superoxide anions (O2 -· ), hydroxyl radical (·OH), and singlet oxygen ( 1O2). Highly excessive ROS content in tumors can cause tumor cells to undergo apoptosis and necrosis due to oxidative damage to cell membrane lipids, proteins and DNA. At the same time, ROS is an important messenger in cell signaling, and it realizes the key function of immune anti-tumor by activating innate and adaptive immune responses against tumors. ROS-induced immunogenic cell death (ICD) is a form of cell death characterized by the regulated activation of immune responses, thereby initiating adaptive immune responses. Common strategies for generating reactive oxygen species include photodynamic therapy based on visible light, radiotherapy excited by X-rays, and sonodynamic therapy using ultrasound excitation. The common feature of these strategies is that reactive oxygen species are generated by consuming oxygen under the condition of external energy excitation. In addition, chemodynamic therapy based on the Fenton reaction between transition metal ions and H2O2 in the tumor microenvironment has also shown tumor effect treatment. However, there are also certain problems, such as dependence on the tumor microenvironment, including hypoxia, the depletable amount of hydrogen peroxide and the depletion effect of glutathione (GSH) on reactive oxygen species. These factors cause insufficient free radical active species, making this type of therapy not very efficient in tumor treatment. Therefore, there is an urgent need to develop strategies that are independent of the tumor microenvironment and can spontaneously generate ROS specifically at the tumor site to achieve tumor apoptosis and improve the efficacy of tumor immunotherapy.

[0004] Prior patent CN111388666A discloses a two-dimensional nanocomposite material, its preparation method and its application, wherein the two-dimensional nanocomposite material comprises BiOX particles with oxygen vacancy defects and a coating; the coating is selected from at least one of silicon-oxygen polymers, polysaccharides and their derivatives, amino acids and their derivatives, polyols and their derivatives, polymer polyols, polyacrylic acid and its derivatives. The material has bismuth oxyhalide nanomaterials with different concentrations of oxygen vacancy defects, which are applied to photothermal therapy of tumors; it is applied to integrated diagnosis and treatment of tumors, and has good treatment effect under multimodal imaging guidance, and has excellent CT imaging and photoacoustic imaging performance. However, it uses trivalent bismuth-based nanomaterials, which are very chemically stable at room temperature and will not undergo valence changes caused by electron transfer. Therefore, active oxygen can only be generated under external stimulation such as laser, and the generation process will consume the corresponding amount of oxygen or hydrogen peroxide, which seriously limits the effect of tumor treatment.

[0005] Therefore, technicians in this field have been committed to developing a bismuth-based nanoparticle material that can spontaneously release reactive oxygen species without relying on hydrogen peroxide and oxygen, which is used to promote tumor apoptosis and pyroptosis, and to coordinate immune agents to regulate tumor immunosuppression and trigger anti-tumor immune responses, thereby achieving the eradication of tumor metastasis and recurrence. And the high tumor enrichment content, low long-term toxicity, and high X-ray absorption capacity of bismuth-based nanoparticle materials are used for computed tomography imaging and radiotherapy sensitization cancer diagnosis and synergistic treatment. Based on the above characteristics, bismuth-based nanoparticle materials have very important significance and broad application prospects in the field of tumor nanodiagnosis and treatment. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides a bismuth-based nanoparticle material that can release hydroxyl radicals and singlet oxygen spontaneously. The technical problem to be solved by the present invention is to develop a bismuth-based nanoparticle material that can spontaneously release reactive oxygen species without relying on hydrogen peroxide and oxygen, which is used to achieve the functions of regulating tumor immune response and realizing the integrated diagnosis and treatment of tumors, and has high radiosensitization, high tumor enrichment content, low long-term toxicity, and can achieve cancer diagnosis while exerting synergistic therapeutic functions.

[0007] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0008] <First aspect>

[0009] A method for preparing a bismuth-based nanoparticle material with self-release of hydroxyl radicals and singlet oxygen comprises the following steps:

[0010] S1. Preparing a bismuth oxide nanoparticle template: mixing a bismuth-containing compound and a polymer stabilizer and dissolving them to form a mixed solution; adding the mixed solution to a high-boiling point organic solvent solution containing a particle size control reagent, mixing and stirring evenly, and performing a hydrothermal reaction in a reaction container at a high temperature; centrifuging, washing, and collecting the precipitate to form a bismuth oxide nanoparticle template;

[0011] S2. preparing bismuth-based nanoparticles: preparing a bismuth oxide nanoparticle solution from a bismuth oxide nanoparticle template; dropping the bismuth oxide nanoparticle solution and a strong oxidant into a strong alkaline solvent, vigorously stirring for a period of time, centrifuging, washing, and collecting the precipitate to obtain bismuth-based nanoparticles;

[0012] S3. Polymer hydrophilic modification: bismuth-based nanoparticles are prepared into a bismuth-based nanoparticle dispersion, and a phospholipid compound solution is added and ultrasonically prepared into a suspension; centrifugation and washing are performed to remove unbound phospholipid compounds; then a polymer hydrophilic modification substance is added and stirred to obtain a polymer hydrophilic modified bismuth-based nanoparticle material.

[0013] In step S1, the bismuth-containing compound includes one or more of bismuth nitrate, bismuth sulfate, bismuth chloride, and bismuth acetate.

[0014] In step S1, the polymer stabilizer includes one or more of polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyacrylic acid (PAA). The molecular weight of the polyvinyl pyrrolidone is 10,000-50,000.

[0015] In step S1, the ratio of the bismuth-containing compound to the polymer stabilizer is: 0.1-0.375 mmol: 0.3-0.5 g.

[0016] In step S1, the solvent used for dissolution includes nitric acid solution or sulfuric acid solution (1M). The amount ratio of bismuth-containing compound, polymer stabilizer and solvent is: 0.1-0.375mmol: 0.3-0.5g: 4-6mL, preferably 0.1-0.375mmol: 0.3-0.5g: 5mL.

[0017] In step S1, the particle size regulating reagent is NaOH or urea.

[0018] In step S1, the high boiling point organic solvent includes one or more of polyethylene glycol, ethylene glycol, glycerol, and diethylene glycol.

[0019] In step S1, the ratio of the particle size regulating reagent to the high boiling point organic solvent is 0.1-1.35 mmol: 10-40 mL.

[0020] In step S1, the volume ratio of the mixed solution and the high boiling point organic solvent solution containing the particle size regulating agent is 4-6 ml:10-40 ml.

[0021] In step S1, the reaction container is a stainless steel autoclave with a polytetrafluoroethylene liner.

[0022] In step S1, the hydrothermal reaction conditions are: 150-200° C. for 2-4 hours.

[0023] In step S1, the centrifugal conditions are: 9000-10000 rpm; centrifugal 5-10 min. Deionized water is used for washing.

[0024] In step S2, the strong oxidant includes one of sodium hypochlorite solution, sodium persulfate solution and chlorine gas.

[0025] In step S2, the volume ratio of the bismuth oxide nanoparticle template solution, the strong oxidant, and the strong alkaline solvent is 1:2-3:5-10. The preparation method of the bismuth oxide nanoparticle solution is: dissolving the bismuth oxide nanoparticle template in deionized water to prepare a bismuth oxide nanoparticle solution with a concentration of 20-30 mg / mL. The strong oxidant is a sodium hypochlorite solution with an effective chlorine content of 8-10wt%); the strong alkaline solvent is a 10-18M NaOH solution.

[0026] In step S2, the vigorous stirring condition is: stirring at 400-500 rpm for 10-20 minutes at room temperature.

[0027] In step S2, the centrifugal conditions are: 8000-9000 rpm; centrifugal 5-10 min. Deionized water is used for washing.

[0028] In step S2, the time for co-adding the bismuth oxide nanoparticle solution and the strong oxidant to the strong alkaline solvent is controlled within 2-30 seconds (the dropping speed is: 1 ml of bismuth oxide nanoparticle solution and 2-3 ml of strong oxidant are co-dropped into the strong alkaline solvent within 2-30 seconds).

[0029] In step S3, the preparation method of the bismuth-based nanoparticle dispersion is: dispersing the bismuth-based nanoparticles in step S2 in an organic solvent (such as chloroform, etc.) to prepare a bismuth-based nanoparticle dispersion with a mass concentration of 20-30 mg / mL.

[0030] In step S3, the method for preparing the phospholipid compound solution is: dissolving the phospholipid compound in an organic solvent (such as chloroform) to prepare a phospholipid compound solution with a mass concentration of 2-5 mg / mL.

[0031] In step S3, the phospholipid compound includes 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA).

[0032] In step S3, the polymer hydrophilic modified substance includes one or more of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 (DSPE-mPEG5000), chitosan, and polydopamine.

[0033] In step S3, the mass ratio of the bismuth-based nanoparticles, the phospholipid compound, and the polymer hydrophilic modified substance is 2:1:4 to 10:1:4.

[0034] The invention discloses a bismuth-based nanoparticle material capable of spontaneously releasing active oxygen. The bismuth-based nanoparticle material is a bismuth-based nanoparticle modified by a hydrophilic polymer.

[0035] The bismuth-based nanoparticles are pentavalent bismuth-based compounds such as sodium bismuthate, lithium bismuthate, potassium bismuthate, silver bismuthate, calcium bismuthate, magnesium bismuthate, barium bismuthate, manganese bismuthate, iron bismuthate, copper bismuthate, zinc bismuthate and the like pentavalent bismuth compounds.

[0036] The average size of the hydrophilic polymer modified bismuth-based nanoparticle material is 100-150 nm.

[0037] The bismuth-based nanoparticles prepared by the invention are not easy to agglomerate.

[0038] The technical solution disclosed in the present invention prepares a bismuth-based nanoparticle material with uniform and dispersed particle size and spontaneous release of reactive oxygen by designing a new synthesis route, and then prepares a bismuth-based nanoparticle material with low toxicity, high enrichment in tumor sites, and high computed tomography imaging contrast performance by modifying the surface of the nanoparticles with hydrophilic polymer chains. Most importantly, the nanoparticle material can spontaneously release reactive oxygen under the micro-acidic conditions of the tumor without relying on hydrogen peroxide and oxygen to kill the tumor and regulate the immune response to inhibit the recurrence and metastasis of the tumor.

[0039] The technical solution disclosed in the present invention has the following beneficial technical effects:

[0040] (1) The bismuth-based nanoparticle material that spontaneously releases active oxygen disclosed in the technical solution can spontaneously release active oxygen under acidic conditions and is independent of the content of hydrogen peroxide and oxygen in the environment;

[0041] (2) The bismuth-based nanoparticle material that spontaneously releases reactive oxygen disclosed in the present technical solution can effectively stimulate immune response and promote the increase in the level of inflammatory factors.

[0042] (3) The bismuth-based nanoparticle material disclosed in this technical solution has the functions of high tumor-specific response, high radiotherapy sensitization, high tumor enrichment content, low long-term toxicity, computed tomography imaging and synergistic treatment with radiotherapy;

[0043] (4) The bismuth-based nanoparticle material disclosed in this technical solution has very broad application prospects in the fields of tumor immunotherapy, integrated tumor diagnosis and treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

[0045] Figure 1 It is a schematic diagram of the preparation route of the bismuth-based nanoparticle material that spontaneously releases active oxygen disclosed in the present invention;

[0046] Figure 2is a transmission electron microscope photograph of the bismuth-based nanoparticle material that spontaneously releases active oxygen in Example 1 of the present invention;

[0047] Figure 3 The Fourier transform infrared spectrum of the hydrophilic polymer modified bismuth-based nanoparticles of Example 1 of the present invention (determining the surface modified hydrophilic polymer)

[0048] Figure 4 is the ability of the hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 of the present invention to release active oxygen under different pH conditions;

[0049] Figure 5 is the ability of the hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 of the present invention to release active oxygen under different concentrations of H2O2;

[0050] Figure 6 is the ability of the hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 of the present invention to release active oxygen at different concentrations of O2;

[0051] Figure 7 The anti-tumor effect of the hydrophilic polymer modified bismuth-based nanoparticle material bismuth-based nanoparticles prepared in Example 1 of the present invention in an in vitro experiment;

[0052] Figure 8 The hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 of the present invention has an immune-stimulating effect in an in vitro experiment of the bismuth-based nanoparticles;

[0053] Fig. 9 The contrast imaging effect of the hydrophilic polymer modified bismuth-based nanoparticle material bismuth-based nanoparticles prepared in Example 1 of the present invention on computer tomography scanning of tumor-bearing mice. DETAILED DESCRIPTION

[0054] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0055] like Figure 1 As shown, it is a schematic diagram of the preparation route of the bismuth-based nanoparticle material provided by the present invention, and the preparation route and steps of the bismuth-based nanoparticle material disclosed in the present invention are vividly described in the form of a flow chart.

[0056] Example 1 Synthesis of polyethylene glycol-modified sodium bismuthate nanoparticles

[0057] 1. Preparation of Bismuth Oxide Nanoparticle Template

[0058] 1.1. Dissolve 0.375 mmol of bismuth nitrate pentahydrate in 5 mL of nitric acid solution (1 M) to obtain a bismuth nitrate solution; mix the bismuth nitrate solution and 0.3 g of a polymer stabilizer (polyvinyl pyrrolidone; Mw=40000) to form a mixed solution;

[0059] 1.2, the mixed solution of step 1.1 is added to a high boiling point organic solvent (25mL polyethylene glycol) solution containing a particle size regulating agent (1.35mmol sodium hydroxide), and then the mixed solution is transferred to a stainless steel autoclave (50mL) with a polytetrafluoroethylene liner. The autoclave is sealed and kept at 150°C for 3 hours. The resulting product is centrifuged at 9000rpm for 5 minutes and washed with deionized water 3 times to obtain a bismuth oxide nanoparticle template with a particle size of about 100nm. In this step, the structural orientation of polyvinyl pyrrolidone can effectively prepare uniformly dispersed bismuth oxide nanoparticles as a template, and the particle size of the bismuth oxide nanoparticles can be effectively controlled by regulating the pH with sodium hydroxide. Table 1 shows the particle size distribution of bismuth oxide nanoparticle templates prepared under different conditions.

[0060] Table 1 Particle size distribution of bismuth oxide nanoparticle templates prepared under different conditions

[0061]

[0062] Step 2: Weigh 20 mg of the bismuth oxide nanoparticles prepared in step 1 as a template and dissolve them in 1 mL of deionized water to form a bismuth oxide nanoparticle solution. The bismuth oxide nanoparticle solution and 3 mL of a strong oxidant (sodium hypochlorite solution, with an effective chlorine content of 10 wt%) are added dropwise to a polytetrafluoroethylene tube containing 5 mL of a 10 M sodium hydroxide solution within 5 seconds, and stirred vigorously at 400 rpm for 10 minutes at room temperature, and then centrifuged at 8000 rpm with deionized water for 5 minutes, washed 3 times, and obtained bismuth-based nanoparticles capable of self-releasing active oxygen. In this step, the bismuth oxide nanoparticle template obtained in step 1 is rapidly oxidized to a bismuth-based nanoparticle material in a strong alkaline solution containing sodium hypochlorite by utilizing the strong oxidizing property of sodium hypochlorite and the weak oxidizing property of sodium bismuthate under strong alkaline conditions.

[0063] The bismuth-based nanoparticle material prepared in this step has a transmission electron microscope photograph as shown below: Figure 2 As shown, it can be seen that the surface of the bismuth-based nanoparticle material presents a flower-like structure etched by oxidation, has high monodispersity, and a particle size of 100-150nm.

[0064] Step 3, weigh 20 mg of the bismuth-based nanoparticles prepared in step 2 and dissolve them in 1 mL of chloroform solution to prepare a bismuth-based nanoparticle dispersion; then mix the chloroform solution with 1 mL of 2 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt under ultrasound at 120 w power for 20 minutes to prepare a suspension, centrifuge the suspension at 8000 rpm for 5 minutes, wash three times and redisperse in 1 mL of chloroform, then add 4 mL of 2 mg / mL distearoylphosphatidylethanolamine-methoxypolyethylene glycol, stir magnetically at 300 rpm for 8 hours, and finally evaporate the chloroform to obtain a hydrophilic polymer modified bismuth-based nanoparticle material.

[0065] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in this example has an infrared Fourier transform spectrum as shown in FIG. Figure 3 As shown in the figure, compared with the unmodified bismuth-based nanoparticles, the hydrophilic polymer-modified bismuth-based nanoparticles have a -1 There is a characteristic signal peak of CH2 at 1278cm -1 The presence of -PO- characteristic signal peak indicates that bismuth-based nanoparticles have achieved modification of hydrophilic polymers.

[0066] Example 2 Synthesis of Polyethylene Glycol-Modified Silver Bismuthate Nanoparticles

[0067] 1. Preparation of Bismuth Oxide Nanoparticle Template

[0068] 1.1. Dissolve 0.375 mmol of bismuth nitrate pentahydrate in 5 mL of sulfuric acid solution (1 M) to obtain a bismuth sulfate solution; mix the bismuth sulfate solution and 0.3 g of a polymer stabilizer (polyvinyl pyrrolidone; Mw=40000) to form a mixed solution;

[0069] 1.2, the mixed solution of step 1.1 is added to a high boiling point organic solvent (25mL polyethylene glycol) solution containing a particle size regulating agent (1.35mmol sodium hydroxide), and then the mixed solution is transferred to a stainless steel autoclave (50mL) with a polytetrafluoroethylene liner, the autoclave is sealed and kept at 150°C for 3 hours, the resulting product is centrifuged at 9000rpm for 5 minutes and washed with deionized water 3 times to obtain a bismuth oxide nanoparticle template with a particle size of 100-150nm. In this step, the structural orientation of polyvinyl pyrrolidone can effectively prepare uniformly dispersed bismuth oxide nanoparticles as a template, and the particle size of the bismuth oxide nanoparticles can be effectively controlled by regulating the pH with sodium hydroxide.

[0070] 2. Preparation of Sodium Bismuthate-Bismuth-based Nanoparticles

[0071] Weigh 20 mg of the bismuth oxide nanoparticles prepared in step 1 as a template and disperse them in 1 mL of deionized water to form a bismuth oxide nanoparticle solution. The bismuth oxide nanoparticle solution and 3 mL of a strong oxidant (undiluted sodium hypochlorite solution) are quickly added dropwise to a polytetrafluoroethylene tube containing 5 mL of a 10M sodium hydroxide solution within 5 seconds, and vigorously stirred at 400 rpm for 10 minutes at room temperature, and then centrifuged at 8000 rpm with deionized water for 5 minutes, washed 3 times, and sodium bismuthate bismuth-based nanoparticles capable of self-releasing active oxygen are obtained. In this step, the bismuth oxide nanoparticle template obtained in step 1 is quickly oxidized to sodium bismuthate bismuth-based nanoparticle material in a strong alkaline solution containing sodium hypochlorite by utilizing the strong oxidizing property of sodium hypochlorite and the weak oxidizing property of sodium bismuthate under strong alkaline conditions.

[0072] Step 3: Preparation of silver bismuthate-bismuth-based nanoparticles

[0073] 15 mg of sodium bismuthate-bismuth-based nanoparticle material prepared in step 2 and 11 mg of silver nitrate powder were weighed and dispersed in 4 mL of deionized water. The mixture was vigorously stirred at 200 rpm for 60 minutes at room temperature, and the mixed solution changed from yellow to gray. The mixture was then centrifuged at 8000 rpm for 5 minutes with deionized water and washed 3 times to obtain silver bismuthate-bismuth-based nanoparticles.

[0074] Step 4, weigh 20 mg of the bismuth-based nanoparticles prepared in step 2 and dissolve them in 1 mL of chloroform solution to prepare a bismuth-based nanoparticle dispersion; then mix the chloroform solution with 1 mL of 2 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt under ultrasound at 120 w power for 20 minutes to prepare a suspension, centrifuge the suspension at 8000 rpm for 5 minutes, wash three times and redisperse in 1 mL of chloroform, then add 4 mL of 2 mg / mL distearoylphosphatidylethanolamine-methoxypolyethylene glycol, stir magnetically at 300 rpm for 8 hours, and finally evaporate the chloroform to obtain a hydrophilic polymer modified bismuth-based nanoparticle material.

[0075] Effect verification example 1: Test on the release of hydroxyl radicals and singlet oxygen

[0076] Test method:

[0077] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 has the following ability to release hydroxyl radicals and singlet oxygen under different pH conditions: Figure 4 As shown in Figure 2, aminophenylfluorescein (APF) and singlet oxygen sensor green fluorescence (SOSG) were used as specific probes for hydroxyl radicals and singlet oxygen. Bismuth-based nanoparticles (concentration 200 μg mL -1) were added to a 96-well plate, and then APF or SOSG at a concentration of 10μM was added, and the fluorescence signal (Ex: 488nm and Em: 525nm) was collected every 10min using a multifunctional microscope (SpectraMax iD3). The fluorescence signal with a continuously increasing intensity was observed in acidic PBS, which indicates that the bismuth-based nanoparticle material can continuously release hydroxyl radicals and singlet oxygen under acidic conditions.

[0078] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in Example 1 of the present invention has the ability to release active oxygen at different concentrations of H2O2 as shown in FIG. Figure 5 As shown in Figure 2, the tumor site has a high concentration of H2O2. From the results, it can be seen that high concentration of hydrogen peroxide can effectively accelerate the generation rate of singlet oxygen content.

[0079] The bismuth-based nanoparticle material prepared in Example 1 of the present invention has the ability to release active oxygen at different concentrations of O2 as shown in FIG. Figure 6 Hypoxic conditions in tumors do not affect the sustained release of hydroxyl radicals and singlet oxygen.

[0080] Effect Verification Example 2 Anti-tumor Effect Test

[0081] Test method:

[0082] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in the preferred embodiment 1 of the present invention has an anti-tumor effect in in vitro experiments as follows: Figure 7 As shown. Mouse breast cancer cells, 4T1 cells, were cultured in a 96-well plate (37°C, 5% CO2) at a density of 10,000 cells per well for 24 hours, and then different concentrations of bismuth oxide nanoparticles or bismuth-based nanoparticles in DMED medium (0, 25, 50, 100, 200 and 400 μg mL-1) were added. After 24 hours, the cells were washed three times with neutral PBS, and the cell viability was detected using the Cell Counting Kit-8 assay kit (Yeasen). The PBS group was set as the control, and each group was repeated 5 times. From the results, it can be observed that bismuth-based nanoparticles can effectively kill breast cancer tumor cells 4T1 in vitro at a concentration of more than 25 μg / mL and inhibit their cell viability.

[0083] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in the preferred embodiment 1 of the present invention has an immune-stimulating effect in an in vitro experiment of the bismuth-based nanoparticles. Figure 8 At a concentration of 50 μg / mL, it can stimulate breast cancer tumor cells 4T1 to produce tumor-associated antigens to stimulate immune response and promote the maturation of dendritic cells.

[0084] The hydrophilic polymer modified bismuth-based nanoparticle material prepared in the preferred embodiment 1 of the present invention has the following effects on the computed tomography imaging of tumor-bearing mice: Fig. 9 When hydrophilic polymer modified bismuth-based nanoparticles were intravenously injected into tumor-bearing mice, a significant increase in contrast at the tumor site could be seen in computed tomography imaging 24 hours later, indicating that hydrophilic polymer modified bismuth-based nanoparticles could effectively accumulate at the tumor site and achieve computed tomography imaging diagnosis of the tumor.

[0085] The present invention discloses a self-releasing hydroxyl radical (·OH) and singlet oxygen ( 1 The bismuth-based nanoparticle material of the present invention is a monodisperse bismuth-based nanoparticle material modified by a hydrophilic polymer, which can spontaneously and continuously release hydroxyl radicals and singlet oxygen under slightly acidic (pH 6.6-5.5) conditions, and does not require the excitation of external energy, nor does it require the consumption of hydrogen peroxide (H2O2) and oxygen in the process of releasing hydroxyl radicals and singlet oxygen. The bismuth-based nanoparticle material obtained by the technical solution of the present invention can effectively inhibit the formation of tumors in a mouse breast cancer tumor model, eliminate distal tumors by inducing an immune stimulation effect, and inhibit the metastasis of tumors. The nanoparticle material can also be used for computed tomography imaging and radiotherapy synergistic treatment of breast cancer mouse models. At the same time, it has the characteristics of good biocompatibility, low long-term toxicity and rapid metabolism, and has extremely high clinical diagnosis and treatment benefits.

[0086] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing a bismuth-based nanoparticle material with self-releasing hydroxyl radicals and singlet oxygen, characterized in that: The steps include: S1. Preparing a bismuth oxide nanoparticle template: mixing a bismuth-containing compound with a polymer stabilizer to form a mixed solution; adding the mixed solution to a high-boiling point organic solvent solution containing a particle size control reagent, mixing and stirring evenly, and performing a hydrothermal reaction in a reaction container at a high temperature; centrifuging, washing, and collecting a precipitate to form a bismuth oxide nanoparticle template; S2. preparing bismuth-based nanoparticles: preparing a bismuth oxide nanoparticle solution from a bismuth oxide nanoparticle template; dropping the bismuth oxide nanoparticle solution and a strong oxidant into a strong alkaline solvent, vigorously stirring for a period of time, centrifuging, washing, and collecting the precipitate to obtain bismuth-based nanoparticles; S3. Polymer hydrophilic modification: bismuth-based nanoparticles are prepared into a bismuth-based nanoparticle dispersion, and a phospholipid compound solution is added and ultrasonically prepared into a suspension; centrifugation and washing are performed to remove unbound phospholipid compounds; then a polymer hydrophilic modification substance is added and stirred to obtain a polymer hydrophilic modified bismuth-based nanoparticle material.

2. The preparation method according to claim 1, characterized in that: In step S1, the bismuth-containing compound includes one or more of bismuth nitrate, bismuth sulfate, bismuth chloride, and bismuth acetate.

3. The preparation method according to claim 1, characterized in that: In step S1, the polymer stabilizer includes one or more of polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, and polyacrylic acid.

4. The preparation method according to claim 1, characterized in that: In step S1, the ratio of the bismuth-containing compound to the polymer stabilizer is: 0.1-0.375 mmol: 0.3-0.5 g.

5. The preparation method according to claim 1, characterized in that: The particle size regulating reagent is NaOH or urea; And / or, in step S1, the high boiling point organic solvent includes one or more of polyethylene glycol, ethylene glycol, glycerol, and diethylene glycol; And / or, in step S1, the ratio of the particle size regulating agent to the high boiling point organic solvent is 0.1-1.35 mmol: 10-40 mL; And / or, in step S1, centrifugation conditions: 9000-10000 rpm; centrifugation for 5-10 min.

6. The preparation method according to claim 1, characterized in that: In step S2, the strong oxidant includes one of sodium hypochlorite solution, sodium persulfate solution and chlorine gas; the volume ratio of bismuth oxide nanoparticle template solution, strong oxidant and strong alkaline solvent is 1:2-3:5-10.

7. The preparation method according to claim 1, characterized in that: In step S2, the conditions for vigorous stirring are: stirring at 400-500 rpm for 10-20 minutes at room temperature; and centrifugation conditions are: 8000-9000 rpm; centrifugation for 5-10 minutes.

8. The preparation method according to claim 1, characterized in that: In step S3, the method for preparing the bismuth-based nanoparticle dispersion is as follows: dispersing the bismuth-based nanoparticles of step S2 in an organic solvent to prepare a bismuth-based nanoparticle dispersion with a mass concentration of 20-30 mg / mL; And / or, in step S3, the method for preparing the phospholipid compound solution is: dissolving the phospholipid compound in an organic solvent to prepare a phospholipid compound solution with a mass concentration of 2-5 mg / mL; And / or, in step S3, the phospholipid compound includes 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt.

9. The preparation method according to claim 1, characterized in that: In step S3, the polymer hydrophilic modified substance includes one or more of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, chitosan, and polydopamine.

10. A bismuth-based nanoparticle material with self-releasing hydroxyl radicals and singlet oxygen obtained by the preparation method according to any one of claims 1 to 9.

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