CN115944732A - Bismuth-based porous nano material and preparation method and application thereof - Google Patents
Bismuth-based porous nano material and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a bismuth-based porous nano material and a preparation method and application thereof, and particularly relates to the field of sonodynamic therapy. The bismuth-based porous nano material is a product obtained by reacting bismuth nanospheres and selenium hydride as raw materials, and can be used for preparing a sonosensitizer for sonodynamic therapy. The sound-sensitive agent provided by the invention has excellent biocompatibility and biological safety, low toxicity and low immunogenicity of 1W/cm 2 Can generate a large amount of active oxygen under the condition of ultrasonic treatment for 1min,can kill a large number of tumor cells and has excellent sonodynamic curative effect. The sound-sensitive agent provided by the invention has the advantages of simple preparation process, mild reaction conditions, low production cost and wide application prospect and clinical transformation value in the aspect of sound power treatment.
Description
Technical Field
The invention belongs to the field of sonodynamic therapy, and particularly relates to a bismuth-based porous nano material as well as a preparation method and application thereof.
Background
The incidence of cancer and mortality rates have continued to rise in recent years and have become a significant public health problem that endangers human health. The current clinical commonly used means for treating cancer comprise surgical excision, chemotherapy, radiotherapy, immunotherapy and the like, but still have the problems of high postoperative recurrence and metastasis rate, obvious side effect and the like. There is therefore a need for new therapeutic approaches to overcome the challenges currently facing.
The sonodynamic therapy is a non-invasive tumor treatment mode developed and evolved on the basis of the photodynamic therapy, and can induce apoptosis and necrosis by giving ultrasonic irradiation with specific intensity so as to kill tumor cells. The anti-tumor mechanism of sonodynamic therapy is complex, with Reactive Oxygen Species (ROS) being the primary mechanism, and the acoustic energy caused by ultrasound causes a rapid increase in mechanical pressure as it passes through a liquid medium, thereby generating microbubbles in the tissue fluid and storing this energy within the microbubbles. The collapse of the microbubbles releases a large amount of energy instantly to generate a sonoluminescence phenomenon, and the sonosensitizer is activated after absorbing the energy and directly reacts with the surrounding triplet oxygen to generate singlet oxygen 1 O 2 ）， 1 O 2 Can destroy cell membrane, influence the function of mitochondria, induce tumor cell apoptosis while causing the G2/M phase retardation of the tumor cell, thereby achieving the purpose of treating cancer.
In view of the application of sonodynamic therapy and the insensitivity of sonosensitizers, researchers have constructed a variety of sonosensitizers to enhance the effectiveness of sonodynamic therapy. For example, hematoporphyrin, chlorophyll derivatives, protoporphyrin IX and other organic molecules, but these organic sonosensitizers have the problems of poor chemical stability, low bioavailability, fast regressive speed, poor tumor accumulation capacity and the like, and seriously restrict the curative effect of sonodynamic therapy. Fortunately, inorganic sound-sensitive agents have the advantages of high acoustic stability and the like, but the biological safety and the ROS conversion efficiency of the inorganic sound-sensitive agents need to be further improved. Chinese patent application publication No. CN113209290A discloses an enhanced sonodynamic antitumor bismuth/barium titanate heterojunction, and the preparation method of the bismuth/barium titanate heterojunction comprises the following steps: firstly, synthesizing barium titanate piezoelectric nanoparticles through hydrothermal treatment, then carrying out polarization treatment on the barium titanate nanoparticles through high-temperature high-pressure polarization, and then loading a bismuth simple substance on the surfaces of the barium titanate piezoelectric nanoparticles by utilizing a one-step in-situ deposition method to form a bismuth/barium titanate heterojunction. The bismuth/barium titanate heterojunction promotes and regulates the separation of current carriers (electron hole pairs) through the coupling of a built-in electric field generated under the ultrasonic response of barium titanate and the bismuth heterojunction, improves the generation content of active oxygen and further enhances the curative effect of the sonodynamic therapy. However, the preparation process of the bismuth/barium titanate heterojunction is complex, the reaction conditions are harsh, the high-temperature high-pressure reaction and the high-temperature high-pressure polarization treatment process are involved, and the bismuth/barium titanate heterojunction is not suitable for industrial production.
The development of the sound-sensitive agent with simple preparation process, mild reaction conditions, high biological safety, high active oxygen yield and good sound-dynamic curative effect has important clinical application value.
Disclosure of Invention
The invention aims to provide a bismuth-based porous nano material, and a preparation method and application thereof.
The invention provides a bismuth-based porous nano material, which is a product obtained by reacting bismuth nanospheres and selenium hydride salt serving as raw materials.
Further, the selenohydride salt is sodium selenohydride.
Further, the preparation method of the sodium hydroselenide comprises the following steps: reacting sodium borohydride with selenium powder in water to obtain sodium hydrogen selenide.
Further, the mass ratio of the sodium borohydride to the selenium powder is 1: (0.9-1.3).
Further, the mass ratio of the sodium borohydride to the selenium powder is 1:1.1.
further, the bismuth nanosphere is Bi 3+ Salt or hydrate thereof, reducing agent, surfactant and alkali.
Further, said Bi 3+ The salt is Bi (NO) 3 ) 3 Said Bi 3+ The hydrate of the salt is Bi (NO) 3 ) 3 ·5H 2 O, the reducing agent is ethylene glycol, the surfactant is polyvinylpyrrolidone, and the alkali is sodium hydroxide;
the Bi 3+ The mass-volume ratio of the salt, the reducing agent, the surfactant and the alkali is 1mg: (0.1-0.3) mL: (0.5-1.5) mg: (1.0-2.0) mg.
Further, said Bi 3+ The mass-volume ratio of the salt, the reducing agent, the surfactant and the alkali is 1mg:0.2mL:1.1mg:1.5mg.
The invention provides a method for preparing the bismuth-based porous nano material, which comprises the following steps:
(1) Reacting sodium borohydride with selenium powder in water to obtain a solution of sodium hydroselenide;
（2）Bi 3+ reacting salt or hydrate thereof, a reducing agent, a surfactant and alkali to obtain bismuth nanospheres;
(3) And mixing the selenium hydride salt solution with the bismuth nanospheres, and reacting to obtain the bismuth-based porous nano material.
Further, the selenium powder and Bi 3+ The mass ratio of the salt or the hydrate thereof is (1.0-2.0): 1;
in the step (2), the reaction temperature is 130-170 ℃ and the reaction time is 2-4 hours;
in the step (3), the reaction condition is that the stirring reaction is carried out for 20-40min at normal temperature.
Further, the selenium powder and Bi 3+ The mass ratio of the salt or the hydrate thereof is 1.4:1;
in the step (2), the reaction temperature is 150 ℃, and the reaction time is 3 hours;
in the step (3), the reaction condition is that the stirring reaction is carried out for 30min at normal temperature.
The invention also provides the application of the bismuth-based porous nano material in preparing a sound sensitive agent for sonodynamic therapy.
Compared with the sound-sensitive agent in the prior art, the sound-sensitive agent provided by the invention has the following beneficial effects:
1. the sound sensitive agent BSNs provided by the invention have excellent biocompatibility and biological safety, low toxicity and low immunogenicity.
2. The sound-sensitive agent BSNs provided by the invention are 1W/cm 2 Can generate a large amount of active oxygen under the condition of ultrasonic treatment for 1min, leads a large amount of tumor cells to die, and has excellent sound power curative effect.
3. The sound-sensitive agent BSNs provided by the invention has the advantages of simple preparation process, mild reaction conditions, low production cost, nontoxic and harmless raw materials and suitability for industrial production.
In conclusion, the acoustic sensitivity agent provided by the invention has wide application prospect and clinical transformation value in the aspect of acoustic dynamic treatment.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 is a scanning electron microscope image of BSNs sonosensitizer prepared in example 1.
FIG. 2 is the results of the ROS detection in vitro of the BSNs sonosensitizers prepared in example 1.
Fig. 3 is a biosafety test result of BSNs prepared in example 1.
FIG. 4 shows the results of intracellular ROS detection by BSNs sonosensitizers prepared in example 1.
FIG. 5 is a graph showing the apoptotic effect of BSNs sonosensitizers prepared in example 1.
Detailed Description
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
Unless otherwise specified, the operations in the examples of the present invention were carried out at ordinary temperature (25. + -. 5 ℃ C.).
Example 1: preparation of Sound sensitive agent BSNs
Step 1, under magnetic stirring at 600 rpm, 242 mg Bi (NO) 3 ) 3 ·5H 2 O was added to 50 mL of ethylene glycol and stirred until dissolved. Under the protection of inert gas, 260 mg of polyvinylpyrrolidone and 363mg of NaOH are added continuously, and the reaction is carried out for 3 hours at a constant temperature of 150 ℃. And after the reaction is finished, centrifuging at 10000 rpm, removing supernatant, collecting precipitate, and washing for several times to obtain the black powder of the bismuth nanospheres.
And 2, adding 334 mg of selenium powder into the three-neck flask under vacuum argon protection. 304 mg of sodium borohydride (NaBH) was weighed out 4 ) Dissolving in ice water, injecting and adding into a three-neck flask after complete dissolution, stirring in ice bath until the solution is transparent, and obtaining NaHSe precursor solution.
And 3, quickly adding the NaHSe precursor solution into the black bismuth nanosphere powder obtained in the step 1, violently stirring for 30min to enable the material to grow stably, centrifuging at 10000 rpm, removing supernate, collecting precipitate, washing for 6 times by using PBS (phosphate buffer solution), dialyzing by using ultrapure water, and finally dispersing in the PBS to obtain the PBS solution of BSNs.
The scanning electron microscope image of the acoustic sensitizer BSNs is shown in FIG. 1, and it can be seen that the BSNs are porous nanostructures.
Example 2: preparation of Sound sensitive agent BSNs
Step 1, 200 mg Bi (NO) is stirred under magnetic stirring at 300 rpm 3 ) 3 ·5H 2 O was added to 20 mL of ethylene glycol and stirred until dissolved. Under the protection of inert gas, 100 mg of polyvinylpyrrolidone and 300mg of NaOH are added continuously, and the reaction is carried out for 3 hours at a constant temperature of 150 ℃. And after the reaction is finished, centrifuging at 10000 rpm, removing supernatant, collecting precipitate, and washing for several times to obtain black powder of the bismuth nanospheres.
Step 2, adding 200 mg of selenium powder into a three-neck flaskAnd (5) protecting with air and argon. 200 mg of sodium borohydride (NaBH) are weighed out 4 ) Dissolving in ice water, injecting into a three-neck flask after complete dissolution, stirring in an ice bath until the solution is transparent, and obtaining NaHSe precursor solution.
And 3, quickly adding the NaHSe precursor solution into the black bismuth nanosphere powder obtained in the step 1, violently stirring for 20 min to enable the material to grow stably, centrifuging at 10000 rpm, removing supernate, collecting precipitate, washing for 6 times by using PBS (phosphate buffer solution), dialyzing by using ultrapure water, and finally dispersing in the PBS to obtain the PBS solution of BSNs.
Example 3: preparation of Sound sensitive agent BSNs
Step 1, 400mg Bi (NO) is stirred under magnetic stirring at 700 rpm 3 ) 3 ·5H 2 O was added to 50 mL of ethylene glycol and stirred until dissolved. Under the protection of inert gas, 300mg of polyvinylpyrrolidone and 400mg of NaOH are added continuously, and the reaction is carried out for 3 hours at a constant temperature of 150 ℃. And after the reaction is finished, centrifuging at 10000 rpm, removing supernatant, collecting precipitate, and washing for several times to obtain black powder of the bismuth nanospheres.
And 2, adding 400mg of selenium powder into the three-neck flask under vacuum argon protection. Weighing 400mg of sodium borohydride (NaBH) 4 ) Dissolving in ice water, injecting into a three-neck flask after complete dissolution, stirring in an ice bath until the solution is transparent, and obtaining NaHSe precursor solution.
And 3, quickly adding the NaHSe precursor solution into the black bismuth nanosphere powder obtained in the step 1, violently stirring for 40min to enable the material to grow stably, centrifuging at 10000 rpm, removing supernate, collecting precipitate, washing for 6 times by using PBS, dialyzing by using ultrapure water, and finally dispersing in the PBS to obtain the PBS solution of the BSNs.
The advantageous effects of the present invention are demonstrated below by experimental examples, in which BSNs prepared in example 1 were used.
Experimental example 1: in vitro ROS detection
1. Experimental method
In vitro 1 O 2 Production was detected by 1, 3-Diphenylisobenzofuran (DPBF) reagent. 20 μ g/mL of BSNs were added to a solution of DPBF (singlet oxygen-indicating fluorescent probe, 60 μ M) and DPBF + BSThe Ns group did not undergo any treatment, and the DPBF + BSNs + US group used 1W/cm 2 Was irradiated for various times (20 s-160 s) and the absorbance at 420 nm of each group was observed by uv spectroscopy.
2. Results of the experiment
The results of the experiment are shown in FIG. 2. It can be seen that the absorbance at 420 nm after the mixture of the DPBF and the BSNs shows a significant descending trend along with the increase of the ultrasonic irradiation time, which indicates that the BSNs generate a large amount of singlet oxygen under the ultrasonic irradiation.
Experimental example 2: biosafety testing
1. Experimental method
In a 96-well plate, 100. Mu.L of hepatocarcinoma cell SMMC-7721 (1' -10) was added to each well 4 mL), after culturing cells for 24 h, 100 μ L of BSNs complete medium with different concentrations was added to each well, and 6 duplicate wells were provided for each concentration gradient. After the pore plate is incubated for 24 h, 10 mu L of CCK-8 is added into each pore and then the incubation is continued for 2 h, the BSNs group is not subjected to any treatment, and the BSNs + US group uses 1W/cm 2 The ultrasonic irradiation of (2) for 1min, and then incubation for 30min. Finally, reading the absorbance at 450 nm by using a microplate reader.
2. Results of the experiment
The results of the experiment are shown in FIG. 3. It can be seen that the BSNs provided by the invention have little cytotoxicity and high biological safety when used alone; and the BSNs can effectively kill liver cancer cells under ultrasonic irradiation.
Experimental example 3: detection of intracellular ROS
1. Experimental methods
In 24-well plates, 500. Mu.L of SMMC-7721 cells (1' -10) were added per well 5 /mL), after culturing the cells for 24 h, 500. Mu.L of 75. Mu.g/mL BSNs complete medium was added to each well, and 3 duplicate wells were set. After 24 h incubation in the well plate, BSNs that did not enter the cells were washed away and 500. Mu.L of DCFH-DA probe (10. Mu.M) was added. The control group (i.e., CON group) and the BSNs group were not subjected to any treatment, and the ultrasound group and the BSNs + ultrasound group used 1W/cm 2 The incubation was continued for 30min after 1min of ultrasonic irradiation, and the green fluorescence was observed using an inverted fluorescence microscope.
2. Results of the experiment
The results of the experiment are shown in FIG. 4. It can be seen that SMMC-7721 hepatoma carcinoma cells and BSNs are incubated together for 24 h, after 1min of ultrasonic irradiation, remarkable green fluorescence can be observed, and only weak fluorescence is obtained by pure irradiation, which indicates that the BSNs generate a large amount of ROS under the ultrasonic irradiation and are captured by DCFH in cells.
Experimental example 4: detection of apoptotic Effect
1. Experimental methods
In 12-well plates, 1 mL of SMMC-7721 cells (1' -10) was added per well 5 /mL), after culturing the cells for 24 h, 1 mL of 75. Mu.g/mL of BSNs complete medium was added to each well, and 3 replicate wells were set. After the plate is incubated for 24 h, BSNs which do not enter cells are washed away, the control group and the BSNs group are not subjected to any treatment, and the ultrasonic group and the BSNs + ultrasonic group use 1W/cm 2 Ultrasonic irradiation for 1 min. Adding 5 mu L of Annexin-FITC and 5 mu L of PI according to the specification of the Annexin-FITC/PI apoptosis kit, continuing incubation for 30min, and detecting the apoptosis condition by using a flow cytometer.
2. Results of the experiment
The results of the experiment are shown in FIG. 5. Compared with the control group, the apoptosis rate (early apoptosis + late apoptosis) of the BSNs group is 15 percent, while the BSNs + ultrasonic group uses 1W/cm 2 The apoptosis rate of the cells after 1min of ultrasonic irradiation reaches 45 percent. The result shows that the sound sensitive agent BSNs provided by the invention can cause a large amount of tumor cells to die under the ultrasonic condition, and has excellent sound power curative effect.
Claims (10)
1. A bismuth-based porous nanomaterial, characterized in that: it is a product obtained by reacting bismuth nanospheres and selenium hydride salt serving as raw materials.
2. The bismuth-based porous nanomaterial according to claim 1, characterized in that: the selenium hydride salt is sodium hydrogen selenide.
3. The bismuth-based porous nanomaterial according to claim 2, characterized in that: the preparation method of the sodium hydroselenide comprises the following steps: reacting sodium borohydride with selenium powder in water to obtain sodium hydroselenide.
4. The bismuth-based porous nanomaterial according to claim 3, characterized in that: the mass ratio of the sodium borohydride to the selenium powder is 1: (0.9-1.3).
5. The bismuth-based porous nanomaterial according to claim 1, characterized in that: the bismuth nanosphere is Bi 3+ Salt or hydrate thereof, reducing agent, surfactant and alkali.
6. The bismuth-based porous nanomaterial of claim 5, characterized in that: the Bi 3+ The salt is Bi (NO) 3 ) 3 Said Bi 3+ The hydrate of the salt is Bi (NO) 3 ) 3 ·5H 2 O, the reducing agent is ethylene glycol, the surfactant is polyvinylpyrrolidone, and the alkali is sodium hydroxide;
the Bi 3+ The mass-volume ratio of the salt, the reducing agent, the surfactant and the alkali is 1mg: (0.1-0.3) mL: (0.5-1.5) mg: (1.0-2.0) mg.
7. A method for preparing the bismuth-based porous nanomaterial of any of claims 1 to 6, characterized in that: the method comprises the following steps:
(1) Reacting sodium borohydride with selenium powder in water to obtain a solution of sodium hydroselenide;
（2）Bi 3+ reacting salt or hydrate thereof, a reducing agent, a surfactant and alkali to obtain bismuth nanospheres;
(3) And mixing the selenium hydride salt solution with the bismuth nanospheres, and reacting to obtain the bismuth-based porous nano material.
8. The method of claim 7, wherein: the selenium powder and Bi 3+ The mass ratio of the salt or the hydrate thereof is (1.0-2.0): 1;
in the step (2), the reaction temperature is 130-170 ℃ and the reaction time is 2-4 hours;
in the step (3), the reaction condition is that the stirring reaction is carried out for 20-40min at normal temperature.
9. The method of claim 8, wherein: the selenium powder and Bi 3+ The mass ratio of the salt or the hydrate thereof is 1.4:1;
in the step (2), the reaction temperature is 150 ℃, and the reaction time is 3 hours;
in the step (3), the reaction condition is that the stirring reaction is carried out for 30min at normal temperature.
10. Use of the bismuth-based porous nanomaterial of any of claims 1 to 6 in the preparation of a sonosensitizer for sonodynamic therapy.
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