CN109529038B - Antibody-coupled bismuth selenide nano-particles for tumor photothermal therapy and immunotherapy and preparation method thereof - Google Patents
Antibody-coupled bismuth selenide nano-particles for tumor photothermal therapy and immunotherapy and preparation method thereof Download PDFInfo
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Abstract
An antibody-coupled bismuth selenide nanoparticle for tumor photothermal therapy and immunotherapy and a preparation method thereof belong to the field of pharmaceutical preparations. The antibody coupled bismuth selenide nano-particles take aminated bismuth selenide as a carrier and PEG as a cross-linking agent, and the anti-CD 47 antibody is coupled on the surfaces of the nano-particles. After the antibody is intravenously injected to be coupled with the bismuth selenide nanoparticle suspension, the nanoparticles can be gathered at a tumor part in a large number of targets through an EPR effect, and anemia caused by systemic administration of a single anti-CD 47 antibody is avoided. Meanwhile, the surface modified anti-CD 47 antibody specifically recognizes with CD47 molecules highly expressed on the surface of tumor cells, and blocks CD47 and a SIRPa signal channel, so that macrophages can regain the capacity of phagocytosing the tumor cells. Especially after photo-thermal treatment, residual tumor cells can be eliminated by macrophages because the 'no-food' signal path is closed, thereby achieving the purpose of completely eradicating the tumor. The method has reasonable design, simple preparation process and wide application prospect, and lays a foundation for the design and development of a corresponding drug delivery system.
Description
Technical Field
The invention belongs to the field of medicinal preparations, relates to an antibody-coupled bismuth selenide nanoparticle for photothermal therapy and immunotherapy of tumors and a preparation method thereof, and particularly relates to a bismuth selenide nanoparticle for photothermal therapy and immunotherapy of tumors by using near-infrared laser and an anti-CD 47 antibody and a preparation method thereof.
Technical Field
Cancer has been one of the serious health and life threatening diseases for the past hundred years. According to related data, the mortality rate of malignant tumors of residents in China is increased by 83.1 percent compared with that of the middle 70 th year. Currently, the main three treatment methods for tumor treatment include surgical resection, radiation therapy, and chemotherapy. However, the above method causes collateral damage and serious toxicity to normal tissues, and the therapeutic effect is not ideal. Photothermal therapy (PTT), a light-triggered, non-invasive therapeutic strategy that can selectively and efficiently kill tumor cells, has received great attention in recent years. The method adopts near infrared light (NIR) with strong tissue penetrating power to radiate tumor tissues, converts laser energy into local heat under the action of a photothermal conversion reagent to increase the temperature of the tumor tissues, and thus, the purpose of killing tumor cells is achieved. However, some tumors have too large volume, and the tumor tissue at the irradiation edge is difficult to be completely ablated, so that the tumor is easy to recur after the photothermal therapy. Thus, photothermal therapy alone is not sufficient to completely eradicate the tumor, and it is necessary to combine other means to combat the tumor to improve the therapeutic effect.
Cancer immunotherapy is emerging. Macrophages play an extremely important role in the immune activity of the human body, and in tumor tissues, tumor-associated macrophages account for a large part of tumor-infiltrating immune cells. Such a large number of macrophages do not exert their phagocytic effect. One important reason for this is that most tumor cells express CD47 at high levels on their surface. CD47 is also known as an integrin-associated protein, and its ligand is the signal-regulatory protein alpha chain (sirpa) on the surface of macrophages. The formation of a CD47-SIRP alpha signal complex by tumor cells and macrophages can cause phosphorylation of an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in an intracellular domain of the SIRP alpha on the surfaces of the macrophages, and the conduction of inhibitory signals, namely 'no-eat-me' signals, reduces the phagocytic activity of the macrophages, thereby promoting the immune escape of the tumor cells. Currently, three antagonists against CD47 have entered phase one clinical trials. Although CD47 is highly expressed on the surface of tumor cells, CD47 is also commonly expressed on the surface of some other cells in the human body, such as erythrocytes, and systemic administration of these antagonists can cause severe anemia. Therefore, how to target the CD47 antagonist to the tumor site to reduce the anemia response is a challenge to be solved.
Therefore, it is very necessary to develop novel multifunctional nano-preparations for tumor photothermal therapy and CD 47-targeted immunotherapy.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a bismuth selenide nano composition for blocking CD47 immune check points by combining photothermal therapy and a preparation method thereof.
The technical scheme adopted by the invention is as follows:
an antibody-coupled bismuth selenide nanoparticle for combined tumor photothermal therapy and immunotherapy is characterized in that an anti-CD 47 antibody is coupled to the surface of the nanoparticle by using aminated bismuth selenide as a carrier and PEG as a cross-linking agent. After the antibody is intravenously injected to be coupled with the bismuth selenide nanoparticle suspension, the nanoparticles can be gathered at a tumor part in a large number of targets through an EPR effect, and anemia caused by systemic administration of a single anti-CD 47 antibody is avoided. Meanwhile, the surface modified anti-CD 47 antibody specifically recognizes with CD47 molecules highly expressed on the surface of tumor cells, and blocks CD47 and a SIRPa signal channel, so that macrophages can regain the capacity of phagocytosing the tumor cells. Especially after photo-thermal treatment, residual tumor cells can be eliminated by macrophages because the 'no-food' signal path is closed, thereby achieving the purpose of completely eradicating the tumor.
A preparation method of antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy comprises the following steps:
first, bismuth selenide nano-particles are synthesized
1.1) completely dissolving bismuth salt in nitric acid solution, and adding sodium hydroxide to adjust the pH of the reaction system. At room temperature, the polyvinylpyrrolidone and the glycol are stirred for 30-60min on a magnetic stirrer with the power of 1200-1500r/min to obtain a mixed solution. Then mixing the two solutions and continuing stirring for not less than 30 min. The mass ratio of the bismuth salt to the volume of the nitric acid solution is 1g (20-30) mL; the mass ratio of the bismuth salt to the polyvinylpyrrolidone is 1 (2-4); the ratio of the mass of the bismuth salt to the volume of the glycol solution is 1g (100-200) mL.
1.2) transferring the mixed solution obtained in the step 1.1) to a high-pressure reaction kettle, carrying out hydrothermal reaction at 130 ℃ and 170 ℃ for 2-5 h to obtain milky liquid, and washing the reaction liquid with deionized water for 2-3 times to obtain the bismuth oxide nanoparticles.
1.3) dissolving bismuth oxide nanoparticles in water at room temperature, adding a selenium source, stirring, and adding an oxidant to obtain a reaction solution, wherein the concentration of the bismuth selenide nanoparticles in the reaction solution is (7-10) mg/mL; and finally, moving the reaction system to a high-pressure reaction kettle, carrying out hydrothermal reaction at the temperature of 130-180 ℃ for 8-24 h to obtain black brown liquid, washing the reaction solution with deionized water and ethanol for 3-5 times, and drying to obtain the bismuth selenide nano-particles.
The selenium source is Na2SeO3. The oxidant is glucose, and the mass ratio of the selenium source to the glucose is 1: (2-4); the mass ratio of the bismuth oxide nano particles to the selenium source is 1 (0.3-0.8).
Second, PEG-coupled bismuth selenide nanoparticles are synthesized
2.1) dissolving bismuth selenide nano-particles in an aqueous solution at room temperature to obtain a bismuth selenide solution with the concentration of 2.5 mg/mL; and adding the bismuth selenide solution into absolute ethyl alcohol, uniformly stirring, adding triaminopropyltrimethoxysilane (APTMS), adding ammonia water to ensure that the reaction is carried out under an alkaline condition, and stirring for 24-36 hours in a dark place to fully react to obtain a reaction solution. And (3) washing the reaction solution with absolute ethyl alcohol and deionized water for 2-3 times, and freeze-drying to obtain the aminated bismuth selenide nano-particles.
2-10mL of bismuth selenide solution, 0.5-2.5mL of APTMS and 2-10mL of ammonia water are correspondingly added into every 4-20mL of absolute ethyl alcohol.
2.2)PEG2000Activating with EDC and NHS for 0.5-4 h. The PEG2000The molar ratio of the EDC to the EDC is 1 (1.5-4); PEG2000The molar ratio to NHS is 1 (1.5-4).
2.3) in activated PEG2000Adding the amination bismuth selenide nano particles obtained in the step 2.1), stirring for 24-36h at room temperature to obtain PEG-Bi2Se3The concentration of PEG in the resulting counter solution was 5-10 mg/mL.
Thirdly, synthesizing the antibody coupled bismuth selenide nano-particles
3.1) the reaction solution obtained in 2.3) was activated with EDC and NHS for 0.5-4h and dissolved in PBS solution. The PEG2000The molar ratio of the EDC to the EDC is 1 (1.5-4); PEG2000The molar ratio to NHS is 1 (1.5-4).
3.2) adding the anti-CD 47 antibody into the solution, and stirring for 24-36h at 4-24 ℃ to obtain the PEG-Bi coupled with the anti-CD 47 antibody2Se3The ratio of the mass of the nanoparticle, the anti-CD 47 antibody and the PEG is 1: (300-700), and the concentration of the anti-CD 47 antibody in the obtained nanoparticles is 1-10 mu g/mL.
The invention has the beneficial effects that: the present invention provides a novel antibody-conjugated nanoparticle capable of eradicating tumors by combining photothermal therapy with immunotherapy. The bismuth selenide nano-composite has strong and wide absorption in a near-infrared range, higher photo-thermal conversion performance and high conversion stability, and can effectively kill tumor cells under the irradiation of near-infrared laser. The anti-CD 47 antibody modified on the surface of the nanoparticle is combined with CD47 molecules on the surface of tumor cells to seal 'no-eat' signals, so that the phagocytosis of macrophages is enhanced, and the aim of removing residual tumor cells and preventing tumor recurrence is fulfilled. In addition, due to the EPR effect, the nanoparticles can be accumulated at the tumor site in a targeted mode, and adverse reactions such as anemia caused by systemic administration of the anti-CD 47 antibody are avoided. The method has reasonable design, simple preparation process and wide application prospect, and lays a foundation for the design and development of a corresponding drug delivery system.
Drawings
FIG. 1 is a morphological diagram of bismuth selenide nanoparticles observed by a transmission electron microscope;
FIG. 2 is a diagram of the elemental composition of bismuth selenide nanoparticles analyzed by an energy spectrometer;
FIG. 3 is a morphological diagram of antibody-coupled bismuth selenide nanoparticles observed by a transmission electron microscope;
FIG. 4 is a diagram of the elemental composition of antibody-coupled bismuth selenide nanoparticles analyzed by an energy spectrometer;
FIG. 5 is a particle size distribution diagram of bismuth selenide nanoparticles and antibody-coupled bismuth selenide nanoparticles analyzed by a dynamic light scattering instrument;
FIG. 6 is a comparison graph of infrared spectra of bismuth selenide nanoparticles and antibody-coupled bismuth selenide nanoparticles;
FIG. 7 is a photo-thermal temperature rise curve of antibody-coupled bismuth selenide nanoparticle dispersions of different concentrations;
FIG. 8 is a photo-thermal temperature difference diagram of antibody coupling bismuth selenide nanoparticle dispersion liquid at different concentrations;
FIG. 9 is a photo-thermal heating cycle chart of a 200. mu.g/mL antibody-coupled bismuth selenide nanoparticle dispersion;
fig. 10 shows cell activities of antibody-coupled bismuth selenide nanoparticles incubated with a549 cells at different concentrations and under different laser irradiation durations;
FIG. 11 is the phagocytic capacity of macrophages after incubation of tumor cells with anti-CD 47 antibody alone, PEG-coupled bismuth selenide nanoparticles, and antibody-coupled bismuth selenide nanoparticles;
FIG. 12 is a graph showing the change in tumor volume in each group of mice;
FIG. 13 is a graph of tumor mass for each group of mice;
fig. 14 is a graph of the toxicity of nanoparticles on a549 cells and MCF10A cells at various concentrations.
FIG. 15 shows the values of red blood cells and hemoglobin of each group of mice.
Detailed Description
The present invention will be further described with reference to the following embodiments.
An antibody-coupled bismuth selenide nanoparticle for combined tumor photothermal therapy and immunotherapy is characterized in that an anti-CD 47 antibody is coupled to the surface of the nanoparticle by using aminated bismuth selenide as a carrier and PEG as a cross-linking agent. After the antibody is intravenously injected to be coupled with the bismuth selenide nanoparticle suspension, the nanoparticles can be gathered at a tumor part in a large number of targets through an EPR effect, and anemia caused by systemic administration of a single anti-CD 47 antibody is avoided. Meanwhile, the surface modified anti-CD 47 antibody specifically recognizes with CD47 molecules highly expressed on the surface of tumor cells, and blocks CD47 and a SIRPa signal channel, so that macrophages can regain the capacity of phagocytosing the tumor cells. Especially after photo-thermal treatment, residual tumor cells can be eliminated by macrophages because the 'no-food' signal path is closed, thereby achieving the purpose of completely eradicating the tumor.
A preparation method of antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy comprises the following steps:
first, bismuth selenide nanoparticles (Bi) are synthesized2Se3)
1.1) precisely weighing 365mg of Bi (NO)3)3·5H2O dissolved in 10mL of NHO at 1mol/L3The solution was placed in a 100mL small beaker, and then 106mg of NaOH was added to the mixture to obtain a mixed solution. 50mL of Ethylene Glycol (EG) was placed on a magnetic stirrer and 1.2g of polyvinylpyrrolidone (PVP) was added thereto under stirring at a power of 1200r/min, and then the mixture was dissolvedLiquid ultrasound is carried out for 5 min. And finally mixing the two reaction solutions, moving the mixture into a high-pressure reaction kettle, and carrying out hydrothermal treatment at 150 ℃ for 3 hours. The resulting reaction solution was washed with deionized water 3 times and redissolved in 10mL of deionized water for subsequent reactions.
1.2) 10mL of bismuth oxide solution was mixed with 200mg of Na2SeO3Mixing and stirring the mixture for 10min, adding 614mg of reducing agent glucose in the process, and transferring the reaction solution into a high-pressure reaction kettle for hydrothermal treatment at 150 ℃ for 12 h. Washing the obtained reaction solution with deionized water and ethanol for three times, and freeze-drying to obtain the bismuth selenide nano-particles. The concentration of the bismuth selenide nano-particles in the reaction liquid is 9 mg/mL.
The bismuth selenide nano-particles are prepared into a solution and are dripped on a silicon chip, after the solution is naturally dried, the solution is observed by a transmission electron microscope, as shown in figure 1, the shape is spherical, and the particle size is about 150 nm. The simultaneous elemental scanning analysis, as shown in fig. 2, confirmed that the synthesized nanoparticles contain selenium and bismuth.
Second, PEG-coupled bismuth selenide nanoparticles are synthesized
2.1) dissolving 5mg of bismuth selenide in 2mL of deionized water, uniformly mixing and stirring with 4mL of ethanol, sequentially adding 0.5mL of APTMS and 2mL of ammonia water into the mixed solution, stirring for 24 hours in a dark place to obtain a reaction solution, and washing for 3 times by using the deionized water to obtain the aminated bismuth selenide nano-particles.
2.2)10mg PEG2000With 1.91mg EDC and 1.15mg NHS in 2mL deionized water and stirred for 2h to activate PEG2000A carboxyl group of (2).
2.3) adding activated PEG2000Washing with deionized water for 2 times, dissolving in 2mL of deionized water, and mixing with the aminated bismuth selenide prepared in 2.1) and stirring for 24 h. Washing the obtained reaction solution with deionized water for 3 times to obtain PEG-Bi2Se3The resulting solution had a PEG concentration of 6.5 mg/mL.
Thirdly, synthesizing the antibody coupled bismuth selenide nano-particles
3.1) mixing the PEG-Bi prepared in the step 2.3)2Se3The solution was mixed with 1.24mg EDC and 0.75mg NHS and stirred for 2h to activate PEG-Bi2Se3A carboxyl group of (2).
3.2) after two hours the solution was washed twice with PBS and finally dissolved in 1ml PBS solution, then 10. mu.g of anti-CD 47 antibody was added and stirred at 4 ℃ for 24 hours. Finally washing with PBS three times, and freeze-drying to obtain Ab-PEG-Bi2Se3The final concentration of nanoparticles, anti-CD 47 antibody reached 9. mu.g/mL.
The antibody coupled bismuth selenide nano-particles are prepared into a solution which is dripped on a silicon chip, and after the solution is naturally dried, the solution is observed by a transmission electron microscope, as shown in figure 3, the shape is spherical, and the particle size is about 170 nm. Simultaneous elemental scan analysis, as shown in figure 4, the antibody-coupled bismuth selenide increased C, N, O and Si compared to bismuth selenide, demonstrating successful modification of PEG and anti-CD 47 antibodies. The particle size analysis of bismuth selenide and antibody coupled bismuth selenide nano-particles is carried out by a dynamic light scattering instrument, the particle size distribution is shown in figure 5, the particle size of bismuth selenide is mainly distributed at 180nm, and the particle size of antibody coupled bismuth selenide is mainly distributed at 210 nm. Bismuth selenide and antibody-coupled bismuth selenide were powdered for infrared analysis, as shown in fig. 6, and the antibody-coupled bismuth selenide increased the characteristic absorption peak from the carboxyl group (1081 cm) as compared to bismuth selenide-1,1746cm-1) Characteristic absorption Peak for amide bond (1236 cm)-1) Successful conjugation of PEG to the antibody was further demonstrated.
Testing of photo-thermal performance of antibody-coupled bismuth selenide nanoparticles
The antibody-coupled bismuth selenide nanoparticle dispersion in a volume of 1mL was irradiated with 808nm near-infrared laser for 10 minutes at different concentration gradients (0, 10,50,100 and 200. mu.g/mL), and the solution temperature was measured with a thermocouple every 30 seconds. FIG. 7 is a graph showing the temperature increase of nanoparticles at each concentration. It can be seen that water hardly increases in temperature under 10min laser irradiation, while 200. mu.g/mL of antibody-coupled bismuth selenide nanoparticles increased from 24.5 ℃ to 66.3 ℃ within 10min irradiation. Fig. 8 is a photo-thermal temperature rise temperature difference diagram of different concentrations of antibody coupled bismuth selenide nanoparticle dispersion. The higher the concentration of the dispersion liquid is, the higher the temperature rise is, which proves the excellent temperature rise performance of the antibody coupled bismuth selenide nano-particles.
Irradiating 1mL of 200 mug/mL antibody coupled bismuth selenide nanoparticle dispersion liquid for ten minutes under the irradiation of 808nm near-infrared laser, then naturally cooling to the initial temperature, starting the irradiation of the near-infrared laser for ten minutes, naturally cooling to the room temperature, and repeating the steps for five times. The solution temperature was recorded every 30s with a thermocouple. Fig. 9 is a photo-thermal temperature-rise cycle diagram of antibody-coupled bismuth selenide nanoparticles. After five times of repeated cycles, the temperature of the antibody coupled bismuth selenide nanoparticles can still rise to the temperature after ten minutes of the first irradiation, and the nanoparticles are proved to have good photo-thermal conversion stability.
Photothermal treatment of cells by antibody-coupled bismuth selenide nanoparticles
A549 cells were placed in 96-well plates and placed at 37 ℃ in CO2Incubate overnight in the incubator. Removing the culture medium the next day, adding antibody-coupled bismuth selenide nanoparticles (0, 5, 10, 20, 40 and 60 μ g/mL) with different concentrations into the wells, incubating for 12 hr, removing the old culture medium, adding fresh culture medium, irradiating with 808nm near infrared laser for 0,5 and 10min, placing in CO2The incubator is further incubated at 37 ℃ for 12 hours, and then 10 mu LCCK-8 solution is added to each well for further incubation for 1 hour. And taking out the sample, and measuring the absorbance OD value on a microplate reader at 450 nm. The cell viability was calculated by the following formula.
Cell survival (%) ═ (OD)Sample (I)/ODControl)×100％
Fig. 10 is a graph of cell activity of antibody-coupled bismuth selenide nanoparticles at different concentrations for different laser irradiation durations. The survival rate of each group of cells is over 90% under the condition of no laser irradiation, and when the high-concentration bismuth selenide nanoparticles coupled with the antibody under the irradiation of 5min show strong killing to the cells and the laser irradiation time is up to 10min, the killing rate of the bismuth selenide nanoparticles coupled with the antibody of 60 mu g/mL to the cells is over 95%, so that the bismuth selenide nanoparticles coupled with the antibody have good photo-thermal treatment capability to the cells.
Immunotherapy of cells with antibody-coupled bismuth selenide nanoparticles
Firstly, macrophage in abdominal cavity of mouse is extracted and put in CO at 37 DEG C2Cultured in an incubator for later use. Respectively mixing 100 mu L A549 cell suspension with anti-CD 47 antibody, PEG-coupled bismuth selenide nanoparticles andafter the antibody-coupled bismuth selenide nanoparticles were incubated on a 96-well plate for 30min, 100 μ L of macrophages were added to each well, and incubation was continued for 4 h. Cell viability was then measured per well using the CCK-8 kit. Macrophages in each well: a549 cells at 20:1 and guaranteed an amount of anti-CD 47 antibody of 1 μ g per well. Fig. 11 is a graph of macrophage phagocytosis after incubation of tumor cells with anti-CD 47 antibody alone, PEG-conjugated bismuth selenide nanoparticles, and antibody-conjugated bismuth selenide nanoparticles. It can be seen from the figure that after tumor cells are incubated by single antibodies and the bismuth selenide nanoparticles coupled with the antibodies, the phagocytic capacity of macrophages on the tumor cells is obviously enhanced, which proves that the bismuth selenide nanoparticles coupled with the antibodies can successfully seal a signal channel of CD47 molecules on the surface of the tumor and SIRPa on the surface of the macrophages, so that the macrophages can obtain the phagocytic capacity again.
Combination therapy of antibody-conjugated bismuth selenide nanoparticles in vivo
4T1 cells (1X 10)6200 μ L PBS suspension) was injected subcutaneously into the left lower limb of each mouse to form tumors. The tumor volume reaches 100mm3The mice were randomly divided into 6 groups of 4 mice each. (1) PBS; (2) PEG-coupled bismuth selenide nanoparticles; (3) laser irradiation only; (4) antibody-coupled bismuth selenide nanoparticles; (5) PEG coupled bismuth selenide nano-particles and laser irradiation; (6) antibody-coupled bismuth selenide nanoparticles + laser irradiation. PBS, PEG-coupled bismuth selenide nanoparticle suspension, and antibody-coupled bismuth selenide nanoparticle suspension (10mg/mL, 100 μ L) were intravenously injected on the first and fourth days, respectively, and after 12 hours of intravenous injection, mouse tumors of groups (3), (5), and (6) were irradiated with laser for 10 min. Tumor volumes of mice were recorded every other day. And at the end of the experiment, each group of mice was euthanized, tumors were removed and weighed. FIG. 12 is a graph showing changes in tumor volume in mice of each group. From the figure, it can be seen that the tumors of mice treated with PBS, PEG-coupled bismuth selenide nanoparticles and individual laser showed a natural growth trend; the PEG coupled bismuth selenide nanoparticles and laser irradiation group tumors are obviously inhibited in the first three days of treatment, but the tumors recur and are crazy to proliferate in the 5 th day; mice tumors injected with antibody-coupled bismuth selenide nanoparticle groups alone, probably due to the effect of the antibody, were treated 3-7 thDay shows tumor suppression, but tumors recurred as in group four on day 7; whereas the mice tumors of the group treated with antibody-coupled bismuth selenide nanoparticles plus laser irradiation were effectively suppressed at the beginning and no tumor recurrence was observed throughout the experiment. Fig. 13 shows the tumor mass of each group of mice, and in accordance with the above results, the tumor mass of the group treated with antibody-coupled bismuth selenide nanoparticles and laser irradiation was the lightest and almost zero. The antibody-coupled bismuth selenide nanoparticles are proved to be effective in vivo photothermal therapy and immunotherapy.
Biological safety of antibody-coupled bismuth selenide nanoparticles
A549 cells and MCF10A cells were seeded in 96-well plates at 5X 103Per well, overnight culture to adhere. The following day the medium was removed and antibody-coupled bismuth selenide nanoparticle suspensions (0, 5, 10, 100, 200, 300, 400 and 500 μ g/mL) of different concentration gradients were added separately and continued at 37 ℃ in CO2Incubate in incubator for 24 h. The activity of the cells in each well was then tested using the CCK-8 kit. FIG. 14 shows that the activity of A549 cells and MCF10A cells is still above 90% when the concentration of nanoparticles is as high as 500. mu.g/mL, demonstrating the safety of nanoparticles. In addition, routine analysis of blood from each group of mice was performed at the end of the in vivo experiment. FIG. 15 shows the values of red blood cells and hemoglobin of each group of mice. From the figure, it is observed that the values of the red blood cells and the hemoglobin of each treatment group are not obviously different from those of the normal group of mice, and the good blood compatibility and the biological safety of the antibody-coupled bismuth selenide nanoparticles are further proved.
Example 2
A preparation method of antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy comprises the following steps:
first, bismuth selenide nanoparticles (Bi) are synthesized2Se3)
1.1) precisely weighing 182mg of Bi (NO)3)3·5H2O dissolved in 5mL of NHO at 1mol/L3The solution was placed in a 100mL small beaker, and 80mg of NaOH was added to the mixture to obtain a mixed solution. 25mL Ethylene Glycol (EG) was placed on a magnetic stirrer0.8g of polyvinylpyrrolidone (PVP) was added with stirring at 1500r/min, and then the mixed solution was sonicated for 5 min. And finally mixing the two reaction solutions, moving the mixture into a high-pressure reaction kettle, and carrying out hydrothermal treatment at 130 ℃ for 3 hours. The resulting reaction solution was washed with deionized water 3 times and redissolved in 5mL of deionized water for subsequent reactions.
1.2) 5mL of bismuth oxide solution was mixed with 35mg of Na2SeO3Mixing and stirring for 10min, then adding 100mg of reducing agent glucose in the process, and transferring the reaction liquid into a high-pressure reaction kettle for hydrothermal reaction at 130 ℃ for 8 h. Washing the obtained reaction solution with deionized water and ethanol for three times, and freeze-drying to obtain the bismuth selenide nano-particles. The concentration of the bismuth selenide nano-particles in the reaction liquid is 4 mg/mL.
And (3) preparing the bismuth selenide nano particles into a solution, dropwise adding the solution on a silicon wafer, naturally airing, observing by using a transmission electron microscope, and simultaneously carrying out element scanning analysis to verify that the synthesized nano particles contain selenium and bismuth.
Second, PEG-coupled bismuth selenide nanoparticles are synthesized
2.1) dissolving 2mg of bismuth selenide in 1mL of deionized water, uniformly mixing and stirring with 2mL of ethanol, sequentially adding 0.2mL of APTMS and 1mL of ammonia water into the mixed solution, stirring for 24 hours in a dark place to obtain a reaction solution, and washing for 3 times by using the deionized water to obtain the aminated bismuth selenide nano-particles.
2.2)5mg PEG2000Dissolving 0.72mg EDC and 0.43mg NHS in 2mL deionized water, stirring for 0.5h, activating PEG2000A carboxyl group of (2).
2.3) adding activated PEG2000Washing with deionized water for 2 times, dissolving in 2mL of deionized water, and mixing with the aminated bismuth selenide prepared in 2.1) and stirring for 24 h. Washing the obtained reaction solution with deionized water for 3 times to obtain PEG-Bi2Se3Solution, the PEG concentration of the resulting solution was 3 mg/mL.
Thirdly, synthesizing the antibody coupled bismuth selenide nano-particles
3.1) mixing the PEG-Bi prepared in the step 2.3)2Se3The solution was mixed with 0.43mg EDC and 0.26mg NHS and stirred for 2h to activate PEG-Bi2Se3A carboxyl group of (2).
3.2) after two hours the solution was washed twice with PBS and finally dissolved in 1ml PBS solution, then 5. mu.g of anti-CD 47 antibody was added and stirred at 4 ℃ for 24 hours. Finally washing with PBS three times, and freeze-drying to obtain Ab-PEG-Bi2Se3The final concentration of nanoparticles, anti-CD 47 antibody, reached 4. mu.g/mL.
Example 3
A preparation method of antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy comprises the following steps:
first, bismuth selenide nanoparticles (Bi) are synthesized2Se3)
1.1) precision weighing 728mg of Bi (NO)3)3·5H2O dissolved in 20mL of NHO at 1mol/L3The solution was placed in a 100mL small beaker, and then 320mg of NaOH was added to the mixture to obtain a mixed solution. 100mL of Ethylene Glycol (EG) was placed on a magnetic stirrer and 1.8g of polyvinylpyrrolidone (PVP) was added with stirring at 1500r/min, and then the mixed solution was sonicated for 5 min. And finally mixing the two reaction solutions, moving the mixture into a high-pressure reaction kettle, and carrying out hydrothermal treatment at 180 ℃ for 5 hours. The resulting reaction solution was washed with deionized water 3 times and redissolved in 20mL of deionized water for subsequent reactions.
1.2) 20mL of bismuth oxide solution was mixed with 300mg of Na2SeO3Mixing and stirring the mixture for 10min, adding 614mg of reducing agent glucose in the process, and transferring the reaction solution into a high-pressure reaction kettle for hydrothermal reaction for 24h at 150 ℃. Washing the obtained reaction solution with deionized water and ethanol for three times, and freeze-drying to obtain the bismuth selenide nano-particles. The concentration of the bismuth selenide nano-particles in the reaction liquid is 9 mg/mL.
And (3) preparing the bismuth selenide nano particles into a solution, dropwise adding the solution on a silicon wafer, naturally airing, observing by using a transmission electron microscope, and simultaneously carrying out element scanning analysis to verify that the synthesized nano particles contain selenium and bismuth.
Second, PEG-coupled bismuth selenide nanoparticles are synthesized
2.1) dissolving 10mg of bismuth selenide in 4mL of deionized water, uniformly mixing and stirring with 8mL of ethanol, sequentially adding 1mL of APTMS and 4mL of ammonia water into the mixed solution, stirring for 24 hours in a dark place to obtain a reaction solution, and washing for 3 times by using the deionized water to obtain the aminated bismuth selenide nano-particles.
2.2)20mg PEG2000Dissolved in 5mL deionized water with 7.0mg EDC and 4.6mg NHS and stirred for 4h to activate PEG2000A carboxyl group of (2).
2.3) adding activated PEG2000Washing with deionized water for 2 times, dissolving in 2mL of deionized water, and mixing with the aminated bismuth selenide prepared in 2.1) and stirring for 24 h. Washing the obtained reaction solution with deionized water for 3 times to obtain PEG-Bi2Se3Solution, the PEG concentration of the resulting solution was 13 mg/mL.
Thirdly, synthesizing the antibody coupled bismuth selenide nano-particles
3.1) mixing the PEG-Bi prepared in the step 2.3)2Se3The solution was mixed with 2.5mg EDC and 1.5mg NHS and stirred for 4h to activate PEG-Bi2Se3A carboxyl group of (2).
3.2) after two hours the solution was washed twice with PBS and finally dissolved in 1ml PBS solution, then 25. mu.g of anti-CD 47 antibody was added and stirred at 4 ℃ for 24 hours. Finally washing with PBS three times, and freeze-drying to obtain Ab-PEG-Bi2Se3The final concentration of nanoparticles, anti-CD 47 antibody reached 21. mu.g/mL.
The above-mentioned embodiments only express the embodiments of the present invention, but not should be understood as the limitation of the scope of the invention patent, it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the concept of the present invention, and these all fall into the protection scope of the present invention.
Claims (5)
1. A preparation method of antibody coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy is characterized in that the antibody coupled bismuth selenide nanoparticles take aminated bismuth selenide as a carrier, PEG is a cross-linking agent, and the PEG couples anti-CD 47 antibody on the surfaces of the nanoparticles;
the anti-CD 47 antibody specifically recognizes with tumor cells highly expressing CD47 molecules, and blocks the interaction of CD47 molecules on the surface of the tumor cells and a signal regulatory protein alpha chain (SIRP alpha) on the surface of macrophages, so that the macrophages can regain the capacity of phagocytosing the tumor cells; after the antibody is intravenously injected into bismuth selenide nanoparticle suspension, the nanoparticles are massively targeted and gathered at a tumor part through an EPR effect, so that anemia caused by systemic administration of the anti-CD 47 antibody is avoided; especially after the photothermal therapy, residual tumor cells can be eliminated by macrophages because the 'no-food' signal path is closed, thereby achieving the purpose of thoroughly eradicating the tumor;
the preparation method comprises the following steps:
first, bismuth selenide nano-particles are synthesized
1.1) completely dissolving bismuth salt in a nitric acid solution, and adding sodium hydroxide to adjust the pH value of a reaction system; mixing and stirring polyvinylpyrrolidone and ethylene glycol at room temperature to obtain a mixed solution; then mixing the two solutions and continuously stirring for not less than 30 min;
1.2) transferring the mixed solution obtained in the step 1.1) to a high-pressure reaction kettle, carrying out hydrothermal reaction at 130 ℃ and 170 ℃ for 2-5 h to obtain milky liquid, and cleaning and drying the reaction liquid by using deionized water to obtain bismuth oxide nanoparticles;
1.3) dissolving bismuth oxide nanoparticles in water at room temperature, adding a selenium source, stirring, and then adding an oxidant to obtain a reaction solution, wherein the concentration of the bismuth selenide nanoparticles in the reaction solution is (7-10) mg/mL; finally, moving the reaction system into a high-pressure reaction kettle, carrying out hydrothermal reaction at the temperature of 130-;
second, PEG-coupled bismuth selenide nanoparticles are synthesized
2.1) dissolving bismuth selenide nano-particles in an aqueous solution at room temperature to obtain a bismuth selenide solution with the concentration of 2.5 mg/mL; adding the bismuth selenide solution into absolute ethyl alcohol, stirring uniformly, adding triaminopropyltrimethoxysilane APTMS, adding ammonia water to ensure that the reaction is carried out under an alkaline condition, and stirring for 24-36 hours in a dark place to fully react to obtain a reaction solution; washing the reaction solution with absolute ethyl alcohol and deionized water, and freeze-drying to obtain aminated bismuth selenide nano-particles;
2.2)PEG2000activating with EDC and NHS for 0.5-4h, and adding PEG2000The molar ratio of the EDC to the EDC is 1 (1.5-4); PEG2000The molar ratio of the NHS to the NHS is 1 (1.5-4);
2.3) in activated PEG2000Adding the amination bismuth selenide nano particles obtained in the step 2.1), stirring for 24-36h at room temperature to obtain PEG-Bi2Se3The concentration of PEG in the obtained reverse solution is 5-10 mg/mL;
thirdly, synthesizing the antibody coupled bismuth selenide nano-particles
3.1) activating the reaction liquid obtained in 2.3) by EDC and NHS for 0.5-4h, and dissolving in PBS solution; the PEG2000The molar ratio of the EDC to the EDC is 1 (1.5-4); PEG2000The molar ratio of the NHS to the NHS is 1 (1.5-4);
3.2) adding the anti-CD 47 antibody into the solution, and stirring for 24-36h at 4-24 ℃ to obtain the PEG-Bi coupled with the anti-CD 47 antibody2Se3The ratio of the mass of the nanoparticle, the anti-CD 47 antibody and the PEG is 1: (300-700), and the concentration of the anti-CD 47 antibody in the obtained nanoparticles is 1-10 mu g/mL.
2. The preparation method of the antibody-coupled bismuth selenide nanoparticle for the combined tumor photothermal therapy and immunotherapy as claimed in claim 1, wherein in the first step 1.1), 20-30 mL of nitric acid solution is added for every 1g of bismuth salt; adding 100-200 mL of glycol solution into every 1g of bismuth salt; the mass ratio of the bismuth salt to the polyvinylpyrrolidone is 1 (2-4).
3. The method for preparing antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy combined according to claim 1 or 2, wherein in the first step 1.3), the selenium source is Na2SeO3The oxidant is glucose; the mass ratio of the selenium source to the glucose is 1: (2-4); the mass ratio of the bismuth oxide nano particles to the selenium source is 1 (0.3-0.8).
4. The method for preparing antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy combined according to claim 1 or 2, wherein in the second step 2.1), 2 to 10mL of bismuth selenide solution, 0.5 to 2.5mL of APTMS and 2 to 10mL of ammonia water are added for every 4 to 20mL of absolute ethanol.
5. The method for preparing antibody-coupled bismuth selenide nanoparticles for tumor photothermal therapy and immunotherapy combined according to claim 3, wherein in the second step 2.1), 2 to 10mL of bismuth selenide solution, 0.5 to 2.5mL of APTMS and 2 to 10mL of ammonia water are added for every 4 to 20mL of absolute ethanol.
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| CN110876805A (en) * | 2019-12-06 | 2020-03-13 | 郑州大学 | Preparation method and application of macrophage membrane bionic bismuth triselenide nanoparticles |
| CN111001001B (en) * | 2019-12-12 | 2021-07-30 | 深圳市百高汇科技发展有限公司 | Photodynamic therapy system, method for the production thereof and use thereof in photodynamic therapy |
| CN113350505B (en) * | 2021-06-17 | 2023-03-17 | 深圳市人民医院 | Photosensitive material, preparation method and application thereof in tumor photothermal combined immunotherapy |
| CN114225047B (en) * | 2021-12-13 | 2023-12-26 | 安徽医科大学 | Immune escape nano preparation, preparation method and application |
| CN114668776B (en) * | 2022-03-01 | 2023-05-05 | 姬晓元 | Thermoelectric heterojunction nano material and preparation method and application thereof |
| CN115154656B (en) * | 2022-06-16 | 2023-11-17 | 广州中医药大学(广州中医药研究院) | Black phosphorus/bioactive glass anti-tumor bone repair dual-function composite stent and preparation method and application thereof |
| CN116966148B (en) * | 2023-07-19 | 2024-07-02 | 中山大学附属第三医院 | Membrane fusion liposome, multifunctional macrophage constructed by same and tumor immunotherapy application |
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