CN111035761B

CN111035761B - Sensitizer for radiotherapy and preparation method and application thereof

Info

Publication number: CN111035761B
Application number: CN201911284847.6A
Authority: CN
Inventors: 谷战军; 董兴华; 刘慧敏; 赵宇亮
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2022-06-07
Anticipated expiration: 2039-12-13
Also published as: CN114712500A; CN111035761A; CN114712500B

Abstract

The invention relates to a sensitizer for radiotherapy and a preparation method and application thereof, belongs to the field of nano material chemistry and biochemistry, and solves the problems that the conventional sensitizer for radiotherapy cannot improve the hypoxia state in a tumor microenvironment, is insensitive to radiotherapy, has large dosage of radioactive agents and has large damage to normal tissues. The molecular formula of the sensitizer for radiotherapy is BiO_2‑xThe molecular structure has oxygen defect, and the raw materials for preparing the sensitizer comprise hydroxide of alkali metal and bismuthate. The preparation method of the sensitizer comprises the following steps: dissolving hydroxide of alkali metal and bismuthate, and stirring to obtain a solution; transferring the solution into a reaction vessel, heating and reacting for a period of time. BiO of the present invention_2‑xCan be used as a sensitizer for tumor radiotherapy sensitization, and realizes a very good inhibition effect on tumors under a lower radiation dose.

Description

Sensitizer for radiotherapy and preparation method and application thereof

Technical Field

The invention relates to the technical field of nano material chemistry and biochemistry, in particular to a sensitizer for radiotherapy and a preparation method and application thereof.

Background

The incidence of malignant tumors is on the rise year by year in the world, the fatality rate is always high, and the survival health of human beings is seriously threatened. Radiation therapy is widely used for the treatment of different types of tumors as a non-invasive method of tumor treatment. In tumor radiotherapy, after tumor cells absorb certain radiation energy, rays can directly interact with intracellular biomacromolecules to generate free radicals, so that DNA molecules are broken and crosslinked; the radiation can also cause the water molecules in the tissues to ionize to generate free radicals, and the free radicals react with biological macromolecules to cause cell damage and cell death, thereby achieving the effect of inhibiting the growth of tumors.

Radiotherapy can inevitably damage normal cells while killing tumor cells, and causes serious radiation side effects. Therefore, the selection of clinical radiotherapy dose usually requires a trade-off between tumor treatment efficacy and radiation damage. Hypoxia is a common characteristic of most solid tumors, can cause radiotherapy resistance of tumor cells, increases the proportion of G1/S-phase cells which are insensitive to radiotherapy in the tumor cells, and can accelerate DNA radiation damage repair and reduce the killing effect of radiotherapy cells. In addition, the positioning diagnosis of the tumor tissue is not accurate enough, so that the tumor tissue and the surrounding normal tissue cannot be effectively distinguished in the tumor radiotherapy, the radiotherapy efficiency is reduced, and the radiation side effect is increased.

The inorganic nano material has excellent absorption performance on high-energy rays due to unique physical and chemical properties, and simultaneously interacts with the high-energy rays to generate a large amount of secondary electrons, and the generated secondary electrons can catalyze water molecules, hydrogen peroxide, oxygen and the like in a tumor microenvironment to generate a large amount of free radicals, so that the sensitivity of tumor cells is improved.

However, because the tumor microenvironment is generally hypoxic, the conventional inorganic nano material radiation sensitizer can not improve the hypoxic state in the tumor microenvironment, influences the radiation sensitizing effect on hypoxic tumors, is not sensitive to radiation treatment, and has large dosage of radioactive agents and large damage to normal tissues.

Disclosure of Invention

In view of the above analysis, the present invention aims to provide a sensitizer for radiotherapy, a preparation method and an application thereof, so as to solve the problems that the existing radiotherapy sensitizer cannot improve the hypoxic state in the tumor microenvironment, is not sensitive to radiotherapy, has large dosage of radioactive agents, and has great damage to normal tissues.

The purpose of the invention is mainly realized by the following technical scheme:

in one aspect, the invention provides a sensitizer for radiotherapy, the molecular formula of the sensitizer is BiO_2-xThe molecular structure has oxygen defect, and the raw materials for preparing the sensitizer comprise hydroxide of alkali metal and bismuthate.

On the basis of the scheme, the invention is further improved as follows:

further, the mass ratio of the alkali metal hydroxide to the bismuthate is (0.5 to 1.5): 1.

Further, the hydroxide of the alkali metal is sodium hydroxide or potassium hydroxide, and the bismuthate is sodium bismuthate or potassium bismuthate.

Further, x is 0.15 to 0.6.

On the other hand, the invention also provides a preparation method of the sensitizer, which is used for preparing the sensitizer for radiotherapy and comprises the following steps:

step 1: dissolving hydroxide of alkali metal and bismuthate, and stirring to obtain a solution;

step 2: transferring the solution into a reaction vessel, heating and reacting for a period of time.

Further, step 2 is followed by step 3: cooling to room temperature, washing and drying to obtain BiO_2-x。

Further, step 3 is followed by step 4: adding BiO_2-xDissolved in the surfactant.

Further, step 4 specifically includes the following steps:

step 41: adding BiO_2-xDissolving in water and ethanol, and adding a surfactant to obtain a mixed solution;

step 42: ultrasonically crushing the mixed solution, and standing;

step 43: taking the solution with the upper layer dispersed uniformly, washing and freeze-drying for later use.

Further, the surfactant is tween-20.

In addition, the invention also provides the application of the sensitizer in tumor radiotherapy and the application of the preparation method in preparing the tumor radiotherapy sensitizer.

The invention can realize at least one of the following beneficial effects:

(1) the molecular structure of the sensitizer of the invention has a large number of oxygen defects (as shown in figures 4 and 5), the bismuth oxide containing a large number of oxygen defects can efficiently catalyze hydrogen peroxide to generate oxygen (as shown in figure 7), and has an effect similar to catalase, and the oxygen generated by catalysis can improve tumor hypoxia and improve tumor radiotherapy sensitivity (as shown in figures 10-12), so that the dosage of a radiotherapy agent is reduced, further, the damage to normal tissues is reduced, and the excellent tumor inhibition effect under a lower radiation dose is realized.

(2) By selecting the metal bismuth with a specific atomic number, the metal bismuth has very good absorption performance on X-rays, can interact with the X-rays, catalyzes water molecules or oxygen molecules and the like in a tumor microenvironment to generate free Radicals (ROS) with killing performance on tumors, and further improves the radiotherapy sensitivity of tumor cells as shown in fig. 6 and 9.

(3) The preparation method of the invention is realized by adding BiO_2-xThe surface modification of the surface active agent improves the biocompatibility of the radiotherapy sensitizer and reduces the cytotoxicity.

(4) The preparation method adopted by the invention is simple and easy to implement, mild in condition, good in controllability, free of additional organic stabilizer/dispersant, and easy to implement and popularize.

(5) BiO synthesized by the invention_2-xThe sensitizer can realize the integration of tumor targeted radiotherapy sensitization diagnosis and treatment, and has wide application prospect in the fields of nano medicine, disease diagnosis, tumor treatment and the like.

In the invention, the technical schemes can be combined with each other to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, wherein like reference numerals are used to designate like parts throughout.

FIG. 1 shows BiO prepared in example four of the present invention_2-xA transmission electron microscope picture;

FIG. 2 shows BiO prepared in example four of the present invention_2-xAtomic force microscope pictures;

FIG. 3 shows BiO prepared according to example four of the present invention_2-xAn X-ray diffraction pattern;

FIG. 4 shows Bi₂O₃And BiO prepared in example four of the invention_2-xThe X-ray photoelectron spectrum of medium O1 s;

FIG. 5 shows Bi₂O₃And BiO prepared in example four of the invention_2-x(ii) a Raman spectrum of;

FIG. 6 is a graph of ROS fluorescence intensity for different treatment groups;

FIG. 7 shows different concentrations of BiO_2-xCatalyzing hydrogen peroxide to generate an oxygen concentration curve;

FIG. 8 shows the same concentration of BiO_2-x(100 mu g/ml) to catalyze the hydrogen peroxide with different concentrations to generate a concentration curve;

FIG. 9 shows ROS immunofluorescence imaging of cells of different treatment groups;

FIG. 10 shows the cells γ -H of different treatment groups₂AX immunofluorescent staining imaging;

FIG. 11 is a photograph of cell clone formation in different treatment groups;

FIG. 12 is a graph showing tumor growth curves of mice in different treatment groups.

Detailed Description

The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form a part hereof, and which together with the embodiments of the invention serve to explain the principles of the invention and not to limit its scope.

Example one

The invention discloses a sensitizer for radiotherapy, and the molecular formula of the sensitizer is BiO_2-xAnd has oxygen defects in the molecular structure,the raw materials for preparing the sensitizer comprise hydroxide of alkali metal and bismuthate.

Compared with the prior art, on one hand, the sensitizer provided by the embodiment has oxygen defects in a molecular structure, the bismuth oxide containing the oxygen defects can efficiently catalyze hydrogen peroxide to generate oxygen, and has an effect similar to that of catalase, and the oxygen generated by catalysis can improve tumor hypoxia and improve tumor radiotherapy sensitivity, so that the dosage of a radiotherapy agent is reduced, further, the damage to normal tissues is reduced, and a very good tumor inhibition effect is realized under a lower radiation dose. On the other hand, the metal bismuth with a specific atomic number is selected, so that the metal bismuth has very good absorption performance on X-rays, can interact with the X-rays, catalyzes water molecules, oxygen molecules and the like in a tumor microenvironment to generate free Radicals (ROS) with killing performance on tumors, and further improves the radiotherapy sensitivity of tumor cells.

Specifically, the hydroxide of the alkali metal is sodium hydroxide or potassium hydroxide, and the bismuthate is sodium bismuthate or potassium bismuthate. Illustratively, the alkali metal hydroxide is NaOH and the bismuthate is NaBiO₃。

To obtain BiO having oxygen defects in the molecular structure_2-xThe NaOH and NaBiO must be strictly controlled₃The ratio of (a) to (b). Through innovative research, NaOH and NaBiO₃Is less than 0.5:1 and is greater than 1.5:1, the prepared BiO_2-xOxygen defects either do not form well in the molecular structure or are formed in very small numbers, thus affecting the sensitivity to radiotherapy of tumor cells.

While NaOH and NaBiO are added₃The mass ratio of (0.5-1.5): 1 to obtain BiO_2-xThe molecular structure contains a large amount of oxygen defects, and the hydrogen peroxide can be efficiently catalyzed to generate a large amount of oxygen, so that tumor hypoxia is obviously improved, and the tumor radiotherapy sensitivity is improved, so that the dosage of a radiotherapy agent is reduced, the damage to normal tissues is further reduced, and a very good tumor inhibition effect under a lower radiation dose is realized.

In addition, the BiO of the present example_2-xX in (b) is 0.15-0.6.

Example two

The invention also discloses a preparation method of the sensitizer, which is used for preparing the sensitizer used for radiotherapy in the first embodiment and comprises the following steps:

step 1: dispersing 0.8-1.2 g of alkali metal hydroxide and 1g of bismuthate in deionized water, and stirring to obtain a solution;

step 2: transferring the solution into a reaction container, heating to 160-200 ℃, and reacting for 4-10 h;

and step 3: cooling to room temperature, washing with deionized water, and drying at 60-100 ℃ for 2-8 h to obtain BiO_2-x。

It should be noted that, in step 2, the reaction time cannot be too long, because too long time can generate many byproducts, such as other oxidation products of bismuth, which affect the sensitivity to tumor radiotherapy; and the reaction time is too short, which can result in incomplete reaction. The reaction time is controlled to be 4-10 hours, the reaction temperature is controlled to be 160-200 ℃, the bismuth oxide obtained through the reaction can be guaranteed to contain a large number of oxygen defects, as shown in figures 4 and 5, the purity of the target bismuth oxide can be guaranteed to be very high, as shown in figure 3, the sensitivity of the bismuth oxide to tumor radiotherapy is greatly improved, the dosage of a radioactive agent is reduced, and the damage to normal tissues is relieved.

Considering the BiO obtained by the preparation_2-xIf the compound is directly applied to organisms, there may be some cytotoxicity, and BiO is prepared in this example_2-xFollowed by a treatment to reduce cytotoxicity, i.e. in BiO_2-xA surface modifying surfactant.

Specifically, the method comprises the following steps:

step 41: 400-600 mg of BiO_2-xDissolving the mixture in 16-24 mL of water and 16-24 mL of ethanol, and then adding 16-24 muL of Tween-20 (Tween-20) to obtain a mixed solution;

step 42: ultrasonically crushing the mixed solution, and standing;

step 43: removing the material precipitated at the bottom, taking the solution with the upper layer uniformly dispersed, centrifuging, washing and freeze-drying for later use.

The bismuth oxide nanosheets prepared by the method are uniform and flaky, and are shown in figure 1. Moreover, the nano-sheet is very thin, and the thickness is only 0.3-0.5 nm, as shown in FIG. 2. As can be seen from the X-ray diffraction pattern, no other miscellaneous peak exists in the pattern, which indicates that the purity of the bismuth oxide prepared by the method is very high, as shown in figure 3. From the data of the X-ray photoelectron spectrum and the raman spectrum, it can be known that the bismuth oxide nanosheet contains a large number of oxygen defects, as shown in fig. 4 and 5.

EXAMPLE III

(1) 0.5g NaOH and 1g NaBiO were taken₃Dispersing in 15mL of deionized water, and stirring for 2 hours to uniformly disperse the deionized water;

(2) the stirred solution was transferred to a 50mL hydrothermal kettle and reacted at 160 ℃ for 10 hours. Cooling to room temperature, washing with deionized water for 5 times, and drying at 60 deg.C for 8 hr;

(3) 400mg of BiO_2-xDissolving in 16mL of water and 16mL of ethanol, and adding 16 mu L of Tween-20 (Tween-20) to obtain a mixed solution;

(4) carrying out ultrasonic treatment (300W) on the mixed solution in an ultrasonic pool for 30 minutes;

(5) crushing for 12 hours by using an ultrasonic cell crusher, taking out after the crushing is finished, covering a sealing film, standing for one night, removing materials precipitated at the bottom, and taking a solution with an upper layer uniformly dispersed;

(6) centrifugation was carried out at 12000rpm for 5 minutes, washing with deionized water was continued three times, and then it was lyophilized for use.

And characterizing the synthesized bismuth oxide, and displaying that the synthesized bismuth oxide nano-sheet is uniform and flaky by a transmission electron microscope. The atomic force microscope picture proves that the synthesized bismuth oxide nano-sheet is very thin and only 0.5nm through data.

Example four

(1) 0.8g of NaOH and 1g of NaBiO were taken₃Dispersing in 20mL of deionized water, and stirring for 2 hours to uniformly disperse the deionized water;

(2) the stirred solution was transferred to a 50mL hydrothermal kettle and reacted at 180 ℃ for 6 hours. Cooling to room temperature, washing with deionized water for 5 times, and drying at 80 deg.C for 4 hr;

(3) 500mg of BiO_2-xDissolving in 20 deg.CAdding 20 mu L of Tween-20 (Tween-20) into the mixture after adding 20mL of water and 20mL of ethanol to obtain a mixed solution;

(6) centrifugation at 12000rpm for 5 minutes was continued for three additional washes with deionized water, which was then lyophilized for use.

The synthesized bismuth oxide is characterized, and a transmission electron microscope shows that the synthesized bismuth oxide nanosheet is in a uniform sheet shape, as shown in fig. 1. The atomic force microscope picture proves that the synthesized bismuth oxide nano-sheet is very thin and only 0.3nm through data, as shown in figure 2.

EXAMPLE five

(1) 1.2g NaOH and 1g NaBiO were taken₃Dispersing in 25mL deionized water, and stirring for 3 hours to uniformly disperse the deionized water;

(2) the stirred solution was transferred to a 50mL hydrothermal kettle and reacted at 200 ℃ for 4 hours. Cooling to room temperature, washing with deionized water for 5 times, and drying at 100 deg.C for 2 hr;

(3) 600mg of BiO_2-xDissolving in 24mL of water and 24mL of ethanol, and adding 24 μ L of Tween-20 (Tween-20) to obtain a mixed solution;

The synthesized bismuth oxide is characterized, a transmission electron microscope shows that the synthesized bismuth oxide nanosheet is uniform and flaky, and an atomic force microscope picture proves that the synthesized bismuth oxide nanosheet is very thin and only 0.4 nm.

To further demonstrate the BiO of the present invention_2-xNanosheet pair tumorSensitivity to cell radiotherapy, the present invention also performs a series of experiments, and the experimental results are shown in fig. 6-12.

As can be seen from FIG. 6, BiO is compared with the other groups_2-x+ X-Ray ROS production efficiency is highest.

As can be seen from FIG. 7, the bismuth oxide nanosheet containing the oxygen defect can catalyze hydrogen peroxide to generate oxygen, and the concentration of the oxygen generated by catalyzing the hydrogen peroxide to generate the oxygen is correspondingly increased along with the increase of the concentration of the bismuth oxide.

As can be seen from fig. 8, under the condition of the same concentration of bismuth oxide, the oxygen generation efficiency is correspondingly improved as the concentration of hydrogen peroxide is increased.

As can be seen from FIG. 9, BiO_2-x+ X-Ray group, the highest concentration of ROS produced.

As can be seen from FIG. 10, BiO_2-xThe DNA damage of the cells treated by the + X-Ray group is the most obvious, which indicates that the material has very obvious radiotherapy sensitization effect on the cell level.

As can be seen from FIG. 11, BiO_2-xThe cell survival rate of the + X-Ray group treated cells is the lowest, and the material is further proved to have very good radiotherapy sensitization effect on the cellular level.

As can be seen from FIG. 12, BiO_2-xCompared with the single X-Ray treatment group, the X-Ray tumor treatment group mouse has the most obvious effect of inhibiting the tumor of the mouse, and proves that the material also has very good radiotherapy sensitization effect on the living body.

In conclusion, on one hand, the ultrathin oxygen-deficient bismuth oxide synthesized by the method can efficiently catalyze hydrogen peroxide in a tumor microenvironment to generate oxygen, improve tumor hypoxia and improve tumor radiotherapy sensitivity; on the other hand, the high atomic number element bismuth is contained, so that the ROS generation efficiency of a tumor part can be improved under the irradiation of X-rays, and the sensitivity of tumor radiotherapy is further improved.

The invention can improve the sensitivity of tumor radiotherapy by introducing the radiotherapy sensitizer, and realize a very good inhibition effect on tumors under a lower radiation dose, thereby reducing the damage to normal tissues.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims

1. A preparation method of a sensitizer is characterized in that the method is used for preparing the sensitizer for radiotherapy and comprises the following steps:

step 2: transferring the solution into a reaction vessel, heating and reacting for a period of time;

step 2 is followed by step 3: cooling to room temperature, washing and drying to obtain BiO_2-x；

Step 4 is also included after step 3: adding BiO_2-xDissolving in a surfactant;

the step 4 specifically comprises the following steps:

step 42: ultrasonically crushing the mixed solution, and standing;

step 43: taking the solution with the upper layer dispersed uniformly, washing and freeze-drying for later use;

x is 0.15-0.6.

2. The method for preparing the sensitizer according to claim 1, wherein the surfactant is tween-20.

3. The use of the process according to claim 1 or 2 for the preparation of a tumor radiosensitizer.