Radiosensitization functional probiotic hybrid material as well as preparation method and application thereof

Abstract

The invention discloses a radiosensitization functional probiotic hybrid material as well as a preparation method and application thereof, and belongs to the technical field of medical preparations. The preparation method comprises the following steps: firstly, preparing black phosphorus nanosheets and bismuth nanoparticles; then, in an aqueous solution environment, blending the bismuth nanoparticles with the black phosphorus nanosheets, adding NH2-PEG2000-MAL and MMP-2 enzyme response polypeptide, and carrying out a reaction, so as to obtain the black phosphorus-based radiosensitizer; finally, clostridium butyricum and the black phosphorus based radiosensitizer are blended, then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride are added, incubation is carried out, and the radiosensitization functionalized probiotic hybrid material is obtained. The method has the advantages of simple process and high operability. The prepared radiosensitizing functional probiotic hybrid material can be driven by anaerobic probiotics to effectively improve the targeting accuracy of a radiosensitizer and reduce off-target toxic and side effects, has the characteristic of high tumor inhibition efficiency, and has wide application prospects in the anti-tumor field.

Description

Technical Field

[0001] The present invention relates to the technical field of medical preparations, and in particular to a radiosensitized functionalized probiotic hybrid material and a preparation method and application thereof. Background Art

[0002] Cancer, as one of the deadliest diseases in the world, has seriously threatened human life and health. At present, radiotherapy has been widely used in clinical practice and has achieved certain success in inhibiting tumor proliferation and prolonging patient survival. However, due to the complexity, density and heterogeneity of tumors, current clinical radiotherapy can only achieve limited success. For example, tumor cells can escape the radiosensitive G2 / M phase through abnormal cell cycle regulation, causing tumor cells to develop radiation resistance, thereby reducing the efficacy of radiotherapy. At the same time, traditional radiosensitizers rely on the enhanced permeability and retention of blood vessels to passively deposit in the tumor site. The dense extracellular matrix in the tumor site hinders the penetration of radiosensitizers in the tumor, thereby greatly reducing the effect of radiosensitization.

[0003] In order to compensate for the insufficient accumulation of radiosensitizers in tumors, the commonly used strategy is to increase the dose or radiation intensity of radiosensitizers, which will inevitably produce toxic side effects on normal tissues. At the same time, radiotherapy inevitably promotes tumor cell metastasis, which has a great impact on the recovery of radiotherapy. Therefore, it is particularly urgent to explore new ways and strategies for tumor treatment that are different from traditional treatment strategies.

[0004] Black phosphorus nanosheets (BPs) have become a hot topic of research in recent years due to their excellent electrical conductivity and cell cycle regulation capabilities.

[0005] For example, the Chinese invention patent with publication number CN107638568A provides a biodegradable black phosphorus-based radiotherapy sensitizer and its preparation method and application. The photosensitizer of this invention grows bismuth trioxide quantum dots on the surface of black phosphorus, retains the characteristics of black phosphorus, and increases the stability of black phosphorus. Due to the high atomic number of bismuth, it has a strong absorption capacity for X-rays. The bismuth trioxide quantum dots are in direct contact with black phosphorus. When irradiated with X-rays, bismuth trioxide absorbs X-rays and directly transfers part of the energy to black phosphorus nanosheets, and the electrons in the black phosphorus nanosheets are then transferred to the surrounding oxygen. At the same time, bismuth trioxide as a semiconductor can also transfer electrons to the surrounding oxygen to form singlet oxygen, thereby achieving a synergistic photodynamic effect. However, black phosphorus-based nanomaterials do not have the ability to actively target tumors, will be degraded and destroyed in the blood circulation, and are easily diffused and accumulated in other organs in the body, causing systemic radiation toxicity.

[0006] Fortunately, some probiotics have been shown to have the ability to specifically target hypoxic areas of tumors, preferentially colonize in nutrient-rich and immunosuppressive tumor sites, and penetrate deeper into the tumor core. In addition, bacteria contain abundant pathogen-associated molecular patterns (PAMPs), which can be recognized by the immune system after X-ray irradiation and trigger immune responses, thereby increasing the immunogenicity of tumor sites. Therefore, probiotics can be used as a reliable drug carrier for delivering radiosensitizers and build a bridge between radiotherapy and immunotherapy to achieve better cancer treatment effects.

[0007] For example, the Chinese invention patent with publication number CN112755184A provides a tumor-targeted carrier for low-dose radiotherapy, a preparation method thereof, and a tumor-targeted drug. The tumor-targeted carrier for low-dose radiotherapy of the invention has anaerobic bacteria, and the anaerobic tropism of anaerobic bacteria enables the tumor-targeted carrier to accurately locate tumor cells. The tumor-targeted carrier for low-dose radiotherapy uses black phosphorus quantum dots (BPQD), which have high photothermal conversion efficiency and achieve the effect of targeted treatment of hypoxic tumor cells through photothermal action. Similarly, the Chinese invention patent with publication number CN112516309A discloses a tumor-targeted carrier, drug, and preparation method for low-dose radiotherapy. Its scheme uses an ultrasonic stripping method to prepare black scales into black scale quantum dots, performs surface ammoniation treatment on anaerobic bacteria, and mixes the two, electrostatically adsorbs the black scale quantum dots to the surface of the ammoniation-treated anaerobic bacteria to obtain a tumor-targeted carrier. However, in different application environments, the response mechanism of specific anaerobic bacteria types needs to be designed in combination with demand. Moreover, the stability of black phosphorus quantum dots in applications needs to be optimized. They may have defects that make them easily oxidized. Their existence time in the blood circulation in the body is relatively short, and the low specific surface area also limits the degree of gain in radiotherapy sensitization intensity.

[0008] In summary, providing a radiosensitizing functionalized probiotic hybrid material can solve the problems of poor precision of traditional radiosensitizers and strong radioresistance of tumor cells, thereby providing efficient and accurate targeted treatment for tumors, which is of great significance and broad application prospects. Summary of the invention

[0009] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a method for preparing a radiosensitizing functionalized probiotic hybrid material is provided, which has simple and convenient process steps and high operability, and comprises the following steps: (1) Dispersing black phosphorus in N-methyl pyrrolidone (NMP) and treating it with ultrasound to obtain black phosphorus nanosheets; (2) dispersing bismuth dodecyl sulfate in octadecene, adding dodecanethiol (DDT) and tri-n-octylphosphine and reacting to obtain bismuth nanoparticles; (3) In an aqueous solution environment, bismuth nanoparticles are mixed with black phosphorus nanosheets and NH2-PEG is added 2000 -MAL and MMP-2 enzyme response peptides react to obtain black phosphorus-based radiosensitizers; (4) In aqueous solution, Clostridium butyricum ( Clostridium butyricum ) was mixed with a black phosphorus-based radiosensitizer, and then N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethyllaminopropyl)carbodiie hydrochlide (EDC) were added and incubated to obtain a radiosensitized functionalized probiotic hybrid material.

[0010] Preferably, the step (1) adopts an ice bath ultrasonic method, and the specific operation is as follows: disperse the blocky black phosphorus in methyl pyrrolidone, perform ice bath ultrasonic crushing at a power of 50-2000 W for 12-72 h, and then centrifuge and wash to obtain black phosphorus nanosheets.

[0011] The reason for the above operation in an ice bath environment (reaction temperature is 0 ℃) is to prevent the black phosphorus nanosheets from being oxidized by oxygen due to excessively high temperature. Since black phosphorus nanosheets are easily oxidized by oxygen, if the reaction temperature is too high, the oxidation process of black phosphorus nanosheets will be promoted, resulting in irregular morphology of black phosphorus nanosheets. Ultrasonic crushing time of 12~72 h can obtain single-layer black phosphorus nanosheets with a particle size of 200~400 nm. If the ultrasonic time is less than 12 h, the size of the black phosphorus nanosheets is too large and cannot form a single-layer structure. If the ultrasonic time is greater than 72 h, the particle size of the black phosphorus nanosheets is small and easy to aggregate, which is not conducive to the subsequent combination with bismuth nanoparticles.

[0012] Preferably, the step (2) adopts a controlled rate titration method, and the specific operation is as follows: add bismuth dodecyl sulfate to the octadecene solution for ultrasonic dispersion; then add dodecyl mercaptan at a rate of 10-30 mL / h and react at 50-200°C for 1-8 hours until the solution turns brown, then add tri-n-octylphosphine at a rate of 10-30 mL / h and react at 30-100°C for 0.5-4 hours, and after completion, centrifuge and wash to obtain bismuth nanoparticles.

[0013] Further preferably, the volume ratio of the dodecyl bismuth sulfate, octadecene, dodecyl mercaptan, and tri-n-octyl phosphine is (4-8) mL: (20-40) mL: (8-16) mL: (4-8) mL.

[0014] The reason why the addition rate of dodecyl mercaptan and tri-n-octyl phosphine is controlled at 10-30 mL / h is to control the particle size of bismuth nanoparticles. If the addition rate is less than 10 mL / h, the particle size of the synthesized bismuth nanoparticles will become smaller, which is not conducive to its radiosensitization effect; if the addition rate is greater than 30 mL / h, the particle size of the bismuth nanoparticles will be very large, which is not conducive to its combination with black phosphorus nanosheets. The addition amount of tri-n-octyl phosphine is also suitable for being controlled within the preferred range of the present invention. If the volume of tri-n-octyl phosphine is less than 4 mL, the particle size of the bismuth nanoparticles may become smaller; if it is greater than 8 mL, the particle size of the bismuth nanoparticles may become larger.

[0015] Preferably, the specific operation of step (3) is as follows: ultrasonically dispersing black phosphorus nanosheets in water, adding bismuth nanoparticle aqueous solution, mixing, centrifuging and washing to obtain a black phosphorus-based carrier loaded with a radiosensitizer; then adding NH2-PEG 2000 -MAL and MMP-2 enzyme response peptides react for 0.5-4 h, and after completion, the black phosphorus-based radiosensitizer is obtained by centrifugation and washing.

[0016] Further preferably, the mass ratio of the bismuth nanoparticles to the black phosphorus nanosheets is (5-10) mg:(1-5) mg.

[0017] Further preferably, the black phosphorus nanosheets, NH2-PEG 2000 The mass ratio of -MAL and MMP-2 enzyme response polypeptides is (5~10) mg: (4~8) mg: (1~2) mg.

[0018] The reason why the mass ratio of bismuth nanoparticles to the black phosphorus nanosheets is controlled at (5-20) mg: (1-5) mg is to control the radiosensitization strength of the black phosphorus-based radiosensitizer and the stability of the material. If the input ratio of bismuth nanoparticles to black phosphorus nanosheets is greater than 20 mg: 1 mg, it is not conducive to the stability of the black phosphorus nanosheets; if the input ratio of bismuth nanoparticles to black phosphorus nanosheets is less than 5 mg: 5 mg, the radiosensitization strength of the black phosphorus-based radiosensitizer will become low.

[0019] NH2-PEG 2000 -MAL (amino polyethylene glycol maleimide) is a compound widely used in medical research, drug release, nanotechnology and new materials research, and cell culture. 2000 If the input ratio of -MAL is higher than 10 mg:4 mg, NH2-PEG2000 -MAL cannot be effectively grafted onto the surface of the black phosphorus-based carrier loaded with bismuth nanoparticles, which is not conducive to the subsequent modification of the MMP-2 enzyme response polypeptide. 2000 If the input ratio of -MAL is lower than 5 mg:8 mg, it will cause NH2-PEG 2000 -Excessive use of MAL, waste of experimental materials.

[0020] NH2-PEG 2000 If the input ratio of -MAL to MMP-2 enzyme response peptide is higher than 8 mg:1 mg, MMP-2 enzyme response peptide and H2-PEG 2000 -MAL reaction is incomplete, which is not conducive to the subsequent modification of black phosphorus-based radiosensitizers on the surface of Clostridium butyricum. 2000 If the input ratio of -MAL to MMP-2 enzyme response polypeptide is lower than 4 mg:2 mg, it will cause excessive amount of MMP-2 enzyme response polypeptide and waste experimental materials.

[0021] Preferably, the specific operation of step (4) is as follows: adding an aqueous solution containing Clostridium butyricum to an aqueous solution containing a black phosphorus-based radiosensitizer and mixing, then adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride thereto and incubating for 1 to 24 hours, and after completion, centrifuging and washing to obtain a radiosensitized functionalized probiotic hybrid material.

[0022] Further preferably, the number of bacterial colonies in the aqueous solution containing Clostridium butyricum is 10 7 ~10 9 CFU / mL; the concentration of the aqueous solution containing the black phosphorus-based radiosensitizer is 10~20 mg / mL.

[0023] A person skilled in the art can adjust the dosage of the above two solutions according to actual conditions. For example, the aqueous solution containing Clostridium butyricum and the aqueous solution containing the black phosphorus-based radiosensitizer can be mixed at (1~10)mL:(1~10)mL or other ratios to obtain materials with different contents of Clostridium butyricum and black phosphorus-based radiosensitizers, so as to meet the needs of different applications.

[0024] Further preferably, the mass ratio of the black phosphorus-based radiosensitizer to N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (10-20) mg: (7-70) mg: (12-20) mg.

[0025] The reason why the concentration ratio of Clostridium butyricum solution, black phosphorus-based radiosensitizer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is the above ratio is that under this concentration ratio condition, a functionalized probiotic hybrid material with good function and morphology can be prepared; adding too little or too much black phosphorus-based radiosensitizer and adding too much or too little Clostridium butyricum solution will result in too much or too little black phosphorus-based radiosensitizer functionally modified on the bacterial surface, or affect the biological activity of the bacteria themselves; it is not conducive to the preparation of an ideal functionalized probiotic hybrid material with radiosensitization.

[0026] In the second aspect of the present invention, a functionalized probiotic hybrid material with radiosensitization having high tumor inhibition efficiency, high targeting accuracy and small off-target toxicity and side effects is provided, which is prepared by the method provided in the first aspect of the present invention.

[0027] In the third aspect of the present invention, there is provided a use of the radiosensitized functionalized probiotic hybrid material according to the second aspect of the present invention as an anti-tumor drug.

[0028] Based on the above technical solutions, the design concept and principle of the present invention are as follows: The preparation method of the present invention first disperses black phosphorus in a methyl pyrrolidone solution, and obtains black phosphorus nanosheets through ultrasonic treatment. Compared with black phosphorus materials in the form of black phosphorus quantum dots, black phosphorus nanosheets have a larger specific surface area, which is beneficial to the subsequent loading of bismuth nanoparticles and charge conduction, and has better stability. Bismuth dodecyl sulfate is dispersed in an octadecene solution, and dodecyl mercaptan and tri-n-octylphosphine are added to obtain bismuth nanoparticles of uniform size (particle size is about 50 nm) through the reaction. Compared with bismuth trioxide quantum dots, bismuth nanoparticles have a more stable structure and have stronger electron deposition and radiation sensitization functions under X-ray irradiation. Subsequently, the bismuth nanoparticles are blended with black phosphorus nanosheets, and NH2-PEG is added. 2000 -MAL and MMP-2 enzyme responsive peptides can react to obtain a black phosphorus-based radiosensitizer with MMP-2 enzyme responsive function. The MMP-2 enzyme responsive peptide can link the black phosphorus-based radiosensitizer and Clostridium butyricum, and only responsively breaks at the tumor site where the MMP-2 enzyme is overexpressed, thereby releasing the black phosphorus-based radiosensitizer, greatly reducing the off-target possibility of the black phosphorus-based radiosensitizer, thereby reducing radiation toxicity. Finally, the black phosphorus-based radiosensitizer with MMP-2 enzyme responsive function is modified on the surface of Clostridium butyricum to obtain a radiosensitized functional probiotic hybrid material. Compared with other anaerobic bacteria, Clostridium butyricum, as a probiotic that can consume glutamine and secrete butyrate, can consume the glutamine required by tumor cells while providing CD8 + Butyrate, which is required for T cell activation, can, to a certain extent, be beneficial to immune activation after tumor radiotherapy.

[0029] The radiosensitized functionalized probiotic hybrid material prepared by the method of the present invention utilizes the hypoxic tropism of Clostridium butyricum to carry the black phosphorus-based radiosensitizer to the hypoxic tumor site, thereby improving the targeted tumor treatment effect. At the same time, after radiotherapy, the bacteria release pathogen-associated molecular patterns (PAMPs), activate the immunogenicity of the tumor site, build a bridge between radiotherapy and immunotherapy, and improve the tumor inhibition efficiency. In the process of tumor cell cycle regulation, black phosphorus nanosheets can induce tumor cells to stay in the radiosensitive G2 / M phase through cell cycle arrest, ultimately leading to enhanced radiosensitivity of tumor cells. At the same time, the high specific surface area and excellent conductivity of black phosphorus nanosheets can amplify the radiosensitization effect of bismuth nanoparticles. This "targeting-cell cycle regulation-radiosensitization" strategy driven by active life solves the problems of poor accuracy of traditional radiosensitizers and strong radioresistance of tumor cells, and provides efficient and accurate targeted treatment for tumors.

[0030] By combining black phosphorus-based radiosensitizers with MMP-2 enzyme response function with Clostridium butyricum, the targeting accuracy of radiosensitizers can be effectively improved under the drive of anaerobic probiotics, reducing off-target toxic side effects. By combining black phosphorus regulation of tumor cell cycle with bismuth nanoparticle radiosensitization, the radiotherapy resistance of tumor cells is significantly reduced, the effect of radiosensitization is improved, and the immunogenicity of the tumor site is activated; achieving combined treatment of radiotherapy and immunotherapy and improving tumor inhibition efficiency.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a method for preparing a radiosensitized functionalized probiotic hybrid material, which has the advantages of simple and convenient process steps and high operability.

[0032] The present invention provides a radiosensitized functionalized probiotic hybrid material, which has the characteristics of high tumor inhibition efficiency, high targeting accuracy, and small off-target toxic and side effects.

[0033] The present invention provides an application of a radiosensitized functionalized probiotic hybrid material, which has broad application prospects in the anti-tumor field. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The construction of a functionalized probiotic hybrid therapeutic system for radiosensitization and its schematic diagram of anti-tumor mechanism; Figure 2 Characterization of functionalized probiotic hybrid materials for radiosensitization; Figure 2A is the transmission electron microscope (TEM) and X-ray energy dispersive spectroscopy mapping (EDS-mapping) images; Figure 2 B is the particle size test result, Figure 2 C is the potential diagram, Figure 2 D is the X-ray photoelectron spectroscopy (XPS) test result, Figure 2 E is the binding rate of bismuth nanoparticles on black phosphorus nanosheets when different mass ratios of bismuth nanoparticles and black phosphorus nanosheets are mixed. Figure 2 F is the grafting of different concentrations of black phosphorus-based radiosensitizers on Clostridium butyricum. Figure 2 G is the MMP-2 enzyme response effect of the radiosensitized functional probiotic hybrid material. Figure 2 H is the survival rate of Clostridium butyricum after different treatments; Figure 3 The cell cycle blocking and radiosensitization capabilities of the functionalized probiotic hybrid materials for radiosensitization; wherein, Figure 3 A shows the results of different groups of materials regulating PCNA and CDK1 proteins in Panc-2 pancreatic cancer cells. Figure 3 B. Flow cytometry results of cell cycle after Panc-2 pancreatic cancer cells were treated with different materials. Figure 3 C is the radiotoxicity of different groups of materials to Panc-2 pancreatic cancer cells, Figure 3 D is the damage effect of radiotherapy on DNA of Panc-2 pancreatic cancer cells after treatment with different groups of materials; Figure 4 The tumor targeting ability and biocompatibility of radiosensitized functionalized probiotic hybrid materials (CBB) in mice; Figure 4 A is the result of in vivo fluorescence imaging of mice. Figure 4 B is the quantitative statistical result of fluorescence in vivo in mice. Figure 4 C is the in vitro fluorescence imaging result of mouse organs. Figure 4 D is the quantitative statistical result of organ fluorescence; Figure 4 E is the results of blood biochemistry and blood routine of mice Figure 5 The in vivo antitumor effect of radiosensitized functionalized probiotic hybrid materials (CBB); Figure 5 A is the bioluminescence image of the tumor at different times (days 14, 21, 28 and 35). Figure 5 B is the quantitative statistical result of bioluminescence. Figure 5 C is an in vitro photo of the tumor after treatment. Figure 5D is tumor weight; Figure 6 This is a flow chart of the method for preparing the radiosensitized functionalized probiotic hybrid material provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0036] The embodiment of the present invention provides a radiosensitized functional probiotic hybrid material, and the overall idea is as follows: According to another typical embodiment of the present invention, a method for preparing a radiosensitized functional probiotic hybrid material is provided, such as Figure 6 As shown, the method includes: (1) Dispersing black phosphorus block in methyl pyrrolidone solution and ultrasonicating in an ice bath for 24 h to obtain black phosphorus nanosheets; In step (1), by adjusting the amount of bulk black phosphorus and the power of the ultrasonic probe, a single-layer black phosphorus nanosheet with a size of 200-400 nm is obtained, which is convenient for subsequent combination with bismuth nanoparticles; The step (1) specifically comprises: 10-50 mg of blocky black phosphorus was added to 50-200 mL of methyl pyrrolidone solution and ultrasonically crushed for 12-48 h at 0 °C. After the ultrasonic crushing, the reaction solution was taken out and washed by gradient centrifugation (8000-15000 rpm, 10-20 min) to obtain a single-layer black phosphorus nanosheet with a regular morphology and a particle size of 200-400 nm; The gradient centrifugation method can screen out black phosphorus nanosheets with a particle size of 200-400 nm. The steps specifically include: firstly collecting the supernatant by centrifugation at 8000 rpm for 10 min, then collecting the precipitate by centrifugation at 15000 rpm for 15 min, washing and dispersing in ultrapure water to obtain a single-layer black phosphorus nanosheet with a particle size of 200-400 nm; (2) dispersing bismuth dodecyl sulfate in an octadecene solution, adding dodecyl mercaptan and tri-n-octylphosphine to obtain bismuth nanoparticles; The bismuth nanoparticles are synthesized by a controlled rate titration method, which specifically includes: 8-16 mL of dodecyl mercaptan was added to a mixed solvent containing 8-16 mL of bismuth dodecyl sulfate and 20-40 mL of octadecene through a syringe pump at a rate of 10-30 mL / h, and the reaction was carried out under stirring at 100 °C in an argon environment. After the solution turned brown-yellow, the temperature was lowered to 65 °C; then, 4-8 mL of tri-n-octylphosphine was added to the above-mentioned mixed solution through a syringe pump at a rate of 10-30 mL / h for reaction, followed by centrifugation (10000-12000 rpm, 10-20 min) and washing to obtain bismuth nanoparticles with regular morphology and size; (3) Blending bismuth nanoparticles with black phosphorus nanosheets and adding NH2-PEG 2000 -MAL and MMP-2 enzyme responsive peptides to obtain a black phosphorus-based radiosensitizer with MMP-2 enzyme responsive function; The step (3) specifically includes: The bismuth nanoparticles and the black phosphorus nanosheets are mixed at a mass ratio of (5-20) mg: (1-5) mg, stirred for 24 h, centrifuged and washed to obtain a black phosphorus-based carrier loaded with bismuth nanoparticles; then, (4-8) mg NH2-PEG is added to (5-10) mg of the black phosphorus-based carrier loaded with bismuth nanoparticles. 2000 -MAL and (1-2) mg MMP-2 enzyme response peptide, followed by centrifugation and washing to obtain a black phosphorus-based radiosensitizer with MMP-2 enzyme response function; (4) Clostridium butyricum was mixed with a black phosphorus-based radiosensitizer, added to an EDC / NHS solution and incubated for 24 h to obtain a functionalized probiotic hybrid material.

[0037] The step (4) specifically includes: An aqueous solution of a black phosphorus-based radiosensitizer was added to an aqueous solution containing Clostridium butyricum while stirring, and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added thereto. After reacting for 2 h, the mixture was centrifuged (speed 1000-6000 rpm, time 5-20 min), the supernatant was removed and washed three times with PBS solution to obtain a radiosensitized functionalized probiotic hybrid material. As an optional embodiment, the preparation method of the aqueous solution containing Clostridium butyricum is as follows: Clostridium butyricum is cultured anaerobically in Reinforced Clostridium Medium (RCM) medium (10 mg / mL trypsin, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) for 24 h. When the bacteria grow to the stationary phase, the bacteria are collected by centrifugation, and then washed by centrifugation with sterile PBS buffer, and the culture medium is removed and centrifuged again (speed 1000~6000 rpm, time 5~20 min), and the bacteria are dispersed in water for use after repeated three times.

[0038] Among them, the number of bacterial colonies in the aqueous solution containing Clostridium butyricum was 10 7 ~10 9 CFU / mL, the concentration ratio of black phosphorus-based radiosensitizer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (10~20) mg / mL: (7~70) mg / mL: (12~20) mg / mL.

[0039] In summary, the following important steps are taken in the technical solution of the present invention to overcome the technical difficulties: In step (2), the morphology and size of the bismuth nanoparticles are controlled by optimizing the preparation conditions of the bismuth nanoparticles (the ratio of different reaction raw materials), reaction temperature, dropwise addition speed, etc.; In step (3), the embodiment of the present invention adjusts the mass ratio of bismuth nanoparticles to black phosphorus nanosheets to (5-20) mg: (1-5) mg, NH2-PEG 2000 -The mass ratio of MAL to MMP-2 enzyme responsive polypeptide is (4-8) mg: (1-2) mg, so as to optimize the amount of bismuth nanoparticles and MMP-2 enzyme responsive polypeptide modified on the black phosphorus nanosheets, thereby obtaining a black phosphorus-based radiosensitizer with stable structure and sufficient radiosensitization strength; In step (4), the embodiment of the present invention adjusts the concentration ratio of the Clostridium butyricum solution, the black phosphorus-based radiosensitizer, the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to (10 7 ~10 9 ) CFU / mL: (10~20) mg / mL: (7~70) mg / mL: (12~20) mg / mL, to regulate the grafting ratio of Clostridium butyricum and MMP-2 enzyme-responsive black phosphorus-based radiosensitizer, thereby obtaining a functionalized probiotic hybrid material with superior performance.

[0040] According to another typical embodiment of the present invention, a radiosensitized functionalized probiotic hybrid material prepared by the method is provided.

[0041] According to another typical embodiment of the present invention, there is provided the use of the radiosensitizing functionalized probiotic hybrid material as an anti-tumor drug.

[0042] The radiosensitizing functionalized probiotic hybrid material utilizes the hypoxic tropism of Clostridium butyricum to specifically deliver the black phosphorus-based radiosensitizer to the tumor site, thereby improving the enrichment effect of the radiosensitizer at the tumor site and thus improving the effect of radiotherapy. At the same time, the black phosphorus nanosheets regulate the cell cycle of tumor cells, causing the tumor cells to remain in the radiosensitive G2 / M phase, thereby reducing the radiotherapy tolerance of tumor cells while radiosensitizing them. In this process, due to the presence of excessive MMP-2 enzymes at the tumor site, the radiosensitizing functionalized probiotic hybrid material responsively releases the black phosphorus-based radiosensitizer under the action of the MMP-2 enzyme. On the one hand, the black phosphorus nanosheets with photoacoustic imaging capabilities can block the cell cycle of tumor cells and reduce the radiation tolerance of tumor cells. On the other hand, the bismuth nanoparticles with computed tomography imaging capabilities can amplify the radiotherapy effect and improve the killing of tumor cells by radiotherapy. Figure 1 As shown, this complementary therapy of radiotherapy-immunotherapy driven by functional probiotics not only fully exerts the advantages of each single therapy, but also makes up for their respective shortcomings, thereby effectively inhibiting the growth of primary tumors and tumor metastasis.

[0043] The radiation sensitization functionalized probiotic hybrid material of the present application will be described in detail below in combination with the examples and experimental data.

[0044] Example 1 The preparation method of the radiosensitized functional probiotic hybrid material comprises the following steps: (1) 20 mg of blocky black phosphorus was added to 100 mL of methyl pyrrolidone solution and ultrasonically crushed in an ice bath at 0 °C for 48 h. After the ultrasonic crushing, the reaction solution was taken out for gradient centrifugation. First, the supernatant was collected by centrifugation at 8000 rpm for 10 min. Then, the supernatant was centrifuged at 10000 rpm / 15 min to collect the precipitate. The precipitate was washed three times with ultrapure water to obtain a single-layer black phosphorus nanosheet with uniform particle size distribution. (2) 8 mL of dodecyl mercaptan was added to a mixed solvent containing 8 mL of bismuth dodecyl sulfate and 20 mL of octadecene through a syringe pump at a rate of 10 mL / h, and the mixture was stirred in an argon environment and an oil bath at 100 °C. After the solution turned brown-yellow, the temperature was lowered to 65 °C. Subsequently, 4 mL of tri-n-octylphosphine was added to the mixture through a syringe pump at a rate of 10 mL / h, and then the mixture was centrifuged (12000 rpm / 15 min) and washed three times with ultrapure water to obtain bismuth nanoparticles with regular morphology and size. (3) 10 mg of black phosphorus nanosheets were uniformly dispersed in 10 mL of ultrapure water and ultrasonicated for 5 min to fully disperse them; 10 mg of bismuth nanoparticles were uniformly dispersed in 10 mL of ultrapure water and ultrasonicated for 10 min to fully disperse them; then 10 mL of bismuth nanoparticle aqueous solution (1 mg / mL) was dropwise added to the black phosphorus nanosheet solution, stirred at room temperature for 24 h, and then the unloaded bismuth nanoparticles were removed by ultrafiltration to obtain a black phosphorus-based radiosensitizer loaded with bismuth nanoparticles; 5 mL of NH2-PEG 2000 A mixture of -MAL (4 mg) and MMP-2 enzyme-responsive peptide (2 mg) was added to a black phosphorus-based radiosensitizer solution loaded with bismuth nanoparticles, and the mixture was stirred at room temperature for 24 h. After the reaction, the mixture was centrifuged (11000 rpm, 10 min), washed with water, and the precipitate was collected to obtain a black phosphorus-based radiosensitizer responsive to MMP-2 enzyme. (4) C. butyricum was cultured anaerobically in RCM medium (10 mg / mL trypsin, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) at 30°C overnight. When the bacteria grew to the stationary phase, they were collected by centrifugation (6000 rpm, 5 min), then resuspended in sterile PBS and centrifuged again. After repeating three times, the bacteria were dispersed in water for later use. 10 mL of a mixture of a MMP-2 enzyme-responsive black phosphorus-based radiosensitizer (10 mg / mL), N-hydroxysuccinimide (70 mg / mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (20 mg / mL) was added to 1 mL of an aqueous solution containing Clostridium butyricum (10 7 CFU / mL) and incubated with shaking for 2 h; then, centrifuged (6000 rpm, 5 min) and washed with water three times to collect the radiosensitized functionalized probiotic hybrid material.

[0045] Example 2 The preparation method of the radiosensitized functional probiotic hybrid material comprises the following steps: (1) 30 mg of blocky black phosphorus was added to 100 mL of methyl pyrrolidone solution and ultrasonically crushed in an ice bath at 0 °C for 48 h. After the ultrasonic crushing, the reaction solution was taken out for gradient centrifugation. First, the supernatant was collected by centrifugation at 9000 rpm for 10 min. Then, the supernatant was centrifuged at 12000 rpm / 15 min to collect the precipitate. The precipitate was washed three times with ultrapure water to obtain a single-layer black phosphorus nanosheet with uniform particle size distribution. (2) 12 mL of dodecyl mercaptan was added to a mixed solvent containing 10 mL of bismuth dodecyl sulfate and 30 mL of octadecene through a syringe pump at a rate of 20 mL / h, and the mixture was stirred in an argon environment and an oil bath at 100 °C. After the solution turned brown-yellow, the temperature was lowered to 65 °C. Subsequently, 6 mL of tri-n-octylphosphine was added to the mixture through a syringe pump at a rate of 20 mL / h, and then the mixture was centrifuged (12000 rpm / 15 min) and washed three times with ultrapure water to obtain bismuth nanoparticles with regular morphology and size. (3) 10 mg of black phosphorus nanosheets were uniformly dispersed in 10 mL of ultrapure water and ultrasonicated for 5 min to fully disperse them; 50 mg of bismuth nanoparticles were uniformly dispersed in 10 mL of ultrapure water and ultrasonicated for 10 min to fully disperse them; then 10 mL of bismuth nanoparticle aqueous solution (5 mg / mL) was dropwise added to the black phosphorus nanosheet solution, stirred at room temperature for 24 h, and then the unloaded bismuth nanoparticles were removed by ultrafiltration to obtain a black phosphorus-based radiosensitizer loaded with bismuth nanoparticles; 10 mL of NH2-PEG 2000 A mixture of -MAL (6 mg) and MMP-2 enzyme-responsive peptide (1.5 mg) was added to a black phosphorus-based radiosensitizer solution loaded with bismuth nanoparticles, and the mixture was stirred at room temperature for 24 h. After the reaction, the mixture was centrifuged (11000 rpm, 10 min), washed with water, and the precipitate was collected to obtain a black phosphorus-based radiosensitizer responsive to MMP-2 enzyme. (4) C. butyricum was cultured anaerobically in RCM medium (10 mg / mL trypsin, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) at 30°C overnight. When the bacteria grew to the stationary phase, they were collected by centrifugation (6000 rpm, 5 min), then resuspended in sterile PBS and centrifuged again. After repeating three times, the bacteria were dispersed in water for later use. 10 mL of a mixture of a MMP-2 enzyme-responsive black phosphorus-based radiosensitizer (15 mg / mL), N-hydroxysuccinimide (50 mg / mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (15 mg / mL) was added to 1 mL of an aqueous solution containing Clostridium butyricum (10 8 CFU / mL) and incubated with shaking for 2 h; then, centrifuged (6000 rpm, 5 min) and washed with water three times to collect the radiosensitized functionalized probiotic hybrid material.

[0046] Example 3 The preparation method of the radiosensitized functional probiotic hybrid material comprises the following steps: (1) 50 mg of blocky black phosphorus was added to 100 mL of methyl pyrrolidone solution and ultrasonically crushed in an ice bath at 0 °C for 72 h. After the ultrasonic crushing, the reaction solution was taken out for gradient centrifugation. First, the supernatant was collected by centrifugation at 10,000 rpm for 10 min. Then, the supernatant was centrifuged at 15,000 rpm / 15 min to collect the precipitate. The precipitate was washed three times with ultrapure water to obtain a single-layer black phosphorus nanosheet with uniform particle size distribution. (2) 16 mL of dodecyl mercaptan was added to a mixed solvent containing 12 mL of bismuth dodecyl sulfate and 40 mL of octadecene through a syringe pump at a rate of 30 mL / h, and the mixture was stirred in an argon environment and an oil bath at 100 °C for reaction; after the solution turned brown-yellow, the temperature was lowered to 65 °C; then, 8 mL of tri-n-octylphosphine was added to the mixture through a syringe pump at a rate of 30 mL / h for reaction, and then the mixture was centrifuged (12000 rpm / 15 min) and washed three times with ultrapure water to obtain bismuth nanoparticles with regular morphology and size; (3) 10 mg of black phosphorus nanosheets were uniformly dispersed in 20 mL of ultrapure water and ultrasonicated for 5 min to fully disperse them; 200 mg of bismuth nanoparticles were uniformly dispersed in 20 mL of ultrapure water and ultrasonicated for 10 min to fully disperse them; then 10 mL of bismuth nanoparticle aqueous solution (10 mg / mL) was dropwise added to the black phosphorus nanosheet solution, stirred at room temperature for 24 h, and then the unloaded bismuth nanoparticles were removed by ultrafiltration to obtain a black phosphorus-based radiosensitizer loaded with bismuth nanoparticles; 5 mL of NH2-PEG 2000 A mixture of -MAL (8 mg) and MMP-2 enzyme-responsive peptide (1 mg) was added to a black phosphorus-based radiosensitizer solution loaded with bismuth nanoparticles, and the mixture was stirred at room temperature for 24 h. After the reaction, the mixture was centrifuged (11000 rpm, 10 min), washed with water, and the precipitate was collected to obtain a black phosphorus-based radiosensitizer responsive to MMP-2 enzyme. (4) C. butyricum was cultured anaerobically in RCM medium (10 mg / mL trypsin, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) at 30°C overnight. When the bacteria grew to the stationary phase, they were collected by centrifugation (6000 rpm, 5 min), then resuspended in sterile PBS and centrifuged again. After repeating three times, the bacteria were dispersed in water for later use. 20 mL of a mixture of a MMP-2 enzyme-responsive black phosphorus-based radiosensitizer (10 mg / mL), N-hydroxysuccinimide (20 mg / mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12 mg / mL) was added to 1 mL of an aqueous solution containing Clostridium butyricum (10 9 CFU / mL) and incubated with shaking for 2 h; then, centrifuged (6000 rpm, 5 min) and washed with water three times to collect the radiosensitized functionalized probiotic hybrid material.

[0047] Test Example 1 This test example conducts structural characterization and performance testing on radiosensitization functionalized probiotic hybrid materials.

[0048] Inductively coupled plasma-Mass Spectrometry (ICP-MS), dynamic light scattering (DLS), energy dispersive X-ray spectrometer, transmission electron microscopy and X-ray photoelectron spectroscopy were used to characterize the morphology and size of various black phosphorus-based radiation sensitizing materials and their hybridization with bacteria.

[0049] CBB corresponds to the radiosensitized functionalized probiotic hybrid material in Example 2, i.e. Clostridium butyricum modified with a black phosphorus-based radiosensitizing material). Bi represents the bismuth nanoparticles in Example 2, and BPs-Bi represents the black phosphorus-based radiosensitizing material in Example 2, i.e. black phosphorus nanosheets loaded with bismuth nanoparticles).

[0050] like Figure 2 As shown in A, the morphology, size and element mapping changes of the black phosphorus-based radiation sensitizing material and its hybridization with Clostridium butyricum prove that the material was successfully prepared. Figure 2 B is the particle size test result, Figure 2 C is the potential diagram, Figure 2 D is the X-ray photoelectron spectroscopy test result. Figure 2 As shown in Figure D, the XPS signal peaks of bismuth and phosphorus were detected simultaneously on the surface of the black phosphorus-based radiosensitizer, further proving the successful loading of bismuth nanoparticles on black phosphorus nanosheets.

[0051] Figure 2 Figure E reflects the binding efficiency of bismuth nanoparticles (Bi) on black phosphorus nanosheets (BPs) after mixing black phosphorus nanosheets and bismuth nanoparticles with different mass ratios (BPs: Bi = 1:1, 1:5, 1:10, 1:20). Figure 2 Figure F shows the different concentrations of black phosphorus-based radiosensitizing materials (BPs-Bi) and 10 7 Grafting rate of BPs-Bi on CB after mixing with CFU / mL Clostridium butyricum (CB). Figure 2 Figure G represents the Bi content in the supernatant of CBB culture medium treated with and without MMP-2 enzyme treatment as detected by ICP-MS, thus reflecting the enzyme response release of BPs-Bi. Figure 2 Panel H represents the bacterial activity of CB and CBB before and after X-ray irradiation.

[0052] like Figure 2 As shown in Figures E and 2F, the binding rate of the prepared bismuth nanoparticles on the black phosphorus nanosheets and the grafting rate of the prepared black phosphorus-based radiosensitizing material on the surface of Clostridium butyricum were determined by inductively coupled plasma mass spectrometry to obtain the optimal feed ratio; Figure 2 As shown in Figure G, the bismuth element in the supernatant of the functionalized radiosensitized probiotic hybrid material (CBB) after MMP-2 enzyme treatment was quantitatively analyzed by inductively coupled plasma mass spectrometry, proving the MMP-2 enzyme response function of the functionalized radiosensitized probiotic hybrid material (CBB). Figure 2 As shown in Figure H, the colony count of Clostridium butyricum did not change much before and after modification with black phosphorus-based radiosensitizing materials, but almost all of the Clostridium butyricum modified with black phosphorus-based radiosensitizing materials died after radiotherapy, indicating that the modification of black phosphorus-based radiosensitizing materials itself has little effect on the activity of Clostridium butyricum, but it increases the sensitivity of Clostridium butyricum to radiation, thereby greatly reducing the hidden dangers of probiotic treatment.

[0053] Application Example 1 In vitro experiments: In vitro co-culture experiments were carried out using Panc-2 pancreatic tumor cells and the prepared functionalized radiosensitizing probiotic hybrid material (CBB) to systematically study the in vitro cell cycle regulation performance and in vitro anti-tumor properties of the radiosensitizing probiotic hybrid material (CBB).

[0054] The pancreatic cancer cells in the PBS treatment group were in the malignant proliferation stage, with high expression of proliferating cell nuclear antigen (PCNA) in the cells, while cyclin-dependent protein kinase (CDK1) was in an inhibited state with low expression. After treatment with black phosphorus nanosheets (BPs), pancreatic cancer cells were blocked in the radiosensitive G2 / M phase, thereby inhibiting the expression of PCNA and upregulating the expression of CDK1.

[0055] Parameter setting: The concentration of BPs is 10~50 μg / mL.

[0056] Preparation method: BPs were dispersed in Dulbecco's Modified Eagle Medium (DMEM) and then added to a 24-well plate seeded with Panc-2 pancreatic tumor cells and incubated for 24 h.

[0057] The experimental steps include: Tumor cell cycle regulation. Different hybrid materials were co-cultured with Panc-2 pancreatic cancer cells for 24 h. The cell cycle marker proteins PCNA and CDK1 in pancreatic cancer cells were detected by immunofluorescence staining, and the DNA in pancreatic cancer cells was quantitatively analyzed by PI staining and cell flow cytometry to prove the cell cycle blocking effect of the material. First, Panc-2 pancreatic cancer cells were seeded in 24-well plates (5×10 4 Cells / well) were cultured in 1640 RPMI medium for 24 h. Then, 100 μg / mL of different groups of materials were added, and after incubation for 24 h, they were rinsed twice with PBS. PCNA and CDK1 antibodies were added to one group of wells for staining. After incubation in the dark for 2 h, they were rinsed three times with PBS, and the expression of PCNA and CDK1 proteins in the cells was photographed by super-resolution fluorescence microscopy. Subsequently, the cells in another group of wells were taken out, stained with pre-cooled 70% ethanol and PI dye, incubated in the dark for 2 h, centrifuged and washed three times, and the pancreatic cancer cell cycle after treatment with different groups of materials was determined by flow cytometry. Figure 3 As shown in A, PCNA protein was downregulated and CDK1 protein was upregulated in pancreatic cancer cells treated with black phosphorus nanosheets and black phosphorus-based radiosensitizers, proving that black phosphorus can block pancreatic cancer cells in the G2 / M phase. Figure 3 As shown in B, flow cytometry data further demonstrated that the functionalized probiotic radiosensitizing material could block pancreatic cancer cells in the G2 / M phase.

[0058] Tumor cytotoxicity and DNA damage. First, Panc-2 pancreatic cancer cells were seeded in 24-well plates at 5 × 10 4cells / well) were cultured in 1640 RPMI medium for 24 h. To verify that the functionalized radiosensitizing probiotic hybrid material can effectively enhance the tumor killing effect of radiotherapy, different materials were co-cultured with Panc-2 pancreatic cancer cells for 24 h, and the culture was continued for 24 h after radiotherapy, and then washed twice with PBS. Fresh culture medium containing MTT solution was added to one group of well plates, incubated for 4 h, and the absorbance at a wavelength of 450 nm was measured with a microplate reader. The cytotoxicity of the functionalized radiosensitizing probiotic hybrid material on Panc-2 pancreatic cancer cells was measured. In another group of well plates, antibodies against the DNA damage marker protein γ-H2AX were added for staining. After incubation in the dark for 2 h, they were rinsed with PBS three times, and the intracellular DNA damage was photographed using a super-resolution fluorescence microscope. Figure 3 As shown in C and 3D, Panc-2 pancreatic cancer cells treated with radiosensitizing probiotic hybrid material (CBB) died in large numbers after radiotherapy, and the expression of DNA damage protein γ-H2AX was most significant, while the degree of death and DNA damage of Panc-2 pancreatic cancer cells treated only with bismuth nanoparticles after radiotherapy was significantly lower, indicating that compared with simple bismuth nanoparticles, radiosensitizing probiotic hybrid material (CBB) can significantly enhance the efficacy of radiotherapy through the dual effects of tumor cell G2 / M phase blockade of black phosphorus nanosheets and radiosensitization of bismuth nanoparticles.

[0059] Application Example 2 In vivo tumor targeting ability and biocompatibility of radiosensitizing probiotic hybrid materials: The present invention uses female C57BL / 6J mice as model animals to construct a Panc-2 orthotopic tumor-bearing mouse model, and systematically studies the tumor targeting ability and biocompatibility of radiosensitized probiotic hybrid materials in vivo after tail vein injection; including: (1) Tumor Targeting Enrichment When the mouse tumors grew to 150 mm 3 When the Cy5.5-labeled BPs-Bi, CB, and CB-BPs-Bi (CB: 5×10 7 CFU, BPs-Bi: 1 mg) materials were injected into mice through the tail vein, and the anesthetized mice were placed in the Maestro in vivo fluorescence imaging system (CRI, Woburn, MA, USA) for observation at the predetermined time points (0, 6, 12, 24, 48 h). Figure 4 As shown in A and 4B, over time, the synthesized radiosensitizing probiotic hybrid material (CBB) has obvious fluorescence at the tumor site, and the fluorescence intensity gradually increases, indicating that bacteria can play a targeted role and carry black phosphorus-based radiosensitizers to the tumor site accurately. Figure 4 This is also demonstrated by ex vivo mouse organ targeting data shown in Figures C and 4D.

[0060] (2) Biosafety analysis Comprehensively evaluate the various indicators of individual mice after being treated with different materials, including blood biochemical analysis, blood routine analysis, etc., to detect the biosafety of the radiosensitized probiotic hybrid material constructed by the present invention in vivo and its clinical application potential. Figure 4 As shown in Figure E, after injection of the radiosensitizing probiotic hybrid material (CBB), the blood biochemical and blood routine indicators of the mice were not significantly different from those of the normal control mice, indicating that the radiosensitizing probiotic hybrid material has good biosafety and clinical application potential in vivo.

[0061] Application Example 3 In vivo antitumor performance of radiosensitizing probiotic hybrid materials: The present invention uses female C57BL / 6J mice as model animals to construct a Panc-2-luc orthotopic tumor-bearing mouse model, and systematically studies the radiotherapy effect of the radiosensitizing probiotic hybrid material on tumors after tail vein injection. 3 At the same time, the tumor-bearing mice were randomly divided into groups, and the corresponding materials were injected into the mice through the tail vein on the first and seventh days, and the mice were irradiated with X-rays 12 hours after injection. The intensity changes of bioluminescence of mouse tumors were detected every seven days using an in vivo fluorescence imaging system. After the treatment, the mice were killed, and all tumors were collected, weighed, and photographed.

[0062] like Figure 5 As shown in the figure, compared with other control materials (the control materials include PBS, BPs-Bi and CB), after tumor-bearing mice were treated with radiosensitizing probiotic hybrid materials (CBB), the growth of tumors was significantly inhibited, and the volume and weight of tumors were significantly smaller than those of other materials, indicating that radiosensitizing probiotic hybrid materials (CBB) can effectively inhibit tumor growth and have good anti-tumor efficacy. Among them, BPs-Bi is a simple black phosphorus-based radiosensitizer, which has no ability to target tumors and has poor therapeutic effects; only radiosensitizing probiotic hybrid materials (CBB) can target the black phosphorus-based radiosensitizer to the tumor area with the help of the anaerobic targeting function of Clostridium butyricum, thereby exerting the best therapeutic effect.

[0063] In summary, the present invention ultrasonically breaks blocky black phosphorus in methyl pyrrolidone by ice bath ultrasound to obtain lamellar black phosphorus nanosheets; bismuth nanoparticles are modified on the surface of black phosphorus nanosheets by conjugated interaction; positively charged NH2-PEG2000-MAL and negatively charged black phosphorus nanosheets are combined by electrostatic interaction to obtain a black phosphorus-based carrier hybrid material with maleimide on the surface; a MMP-2 enzyme-responsive polypeptide containing a thiol end is blended with the black phosphorus-based carrier hybrid material with maleimide on the surface to obtain an MMP-2 enzyme-responsive black phosphorus-based carrier hybrid material. Clostridium butyricum is combined with an MMP-2 enzyme-responsive black phosphorus-based carrier hybrid material by an amidation reaction; the functionalized probiotic hybrid material can efficiently target the tumor site and responsively release a radiosensitizer, reducing systemic toxicity caused by the off-target effect of the radiosensitizer. It is used for radiotherapy of tumors with high accuracy, low radiotherapy dose and dual-mode imaging function, which improves the visibility of treatment. Combined immunotherapy can also form a complementary therapy of radiotherapy-immunotherapy, which not only gives full play to the advantages of each single therapy, but also makes up for their respective shortcomings, thereby effectively inhibiting the growth of primary tumors and tumor metastasis.

[0064] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention 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 radiosensitized functionalized probiotic hybrid material, characterized in that: The steps include: (1) dispersing black phosphorus in methyl pyrrolidone and subjecting to ultrasonic treatment to obtain black phosphorus nanosheets; (2) dispersing bismuth dodecyl sulfate in octadecene, adding dodecyl mercaptan and tri-n-octylphosphine and reacting to obtain bismuth nanoparticles; (3) In an aqueous solution environment, bismuth nanoparticles are mixed with black phosphorus nanosheets and NH2-PEG is added 2000 -MAL and MMP-2 enzyme response peptides react to obtain black phosphorus-based radiosensitizers; (4) In an aqueous solution environment, Clostridium butyricum was mixed with a black phosphorus-based radiosensitizer, and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added and incubated to obtain a radiosensitized functionalized probiotic hybrid material.

2. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, characterized in that: The step (1) adopts an ice bath ultrasonic method, and the specific operation is as follows: the blocky black phosphorus is dispersed in methyl pyrrolidone, and ice bath ultrasonic crushing is performed at a power of 50-2000 W for 12-72 h, followed by centrifugation and washing to obtain black phosphorus nanosheets.

3. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, characterized in that: The step (2) adopts a controlled rate titration method, and the specific operation is as follows: add bismuth dodecyl sulfate to the octadecene solution for ultrasonic dispersion; then add dodecyl mercaptan at a rate of 10-30 mL / h and react at 50-200°C for 1-8 h until the solution turns brown; then add tri-n-octylphosphine at a rate of 10-30 mL / h and react at 30-100°C for 0.5-4 h; after completion, centrifuge and wash to obtain bismuth nanoparticles.

4. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 3, characterized in that: The volume ratio of the dodecyl bismuth sulfate, octadecene, dodecyl mercaptan, and tri-n-octyl phosphine is (4-8) mL: (20-40) mL: (8-16) mL: (4-8) mL.

5. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, characterized in that: The specific operation of step (3) is as follows: ultrasonically dispersing black phosphorus nanosheets in water, adding bismuth nanoparticle aqueous solution, mixing, centrifuging and washing to obtain a black phosphorus-based carrier loaded with a radiosensitizer; then adding NH2-PEG 2000 -MAL and MMP-2 enzyme response peptides react for 0.5-5 h, and after completion, the black phosphorus-based radiosensitizer is obtained by centrifugation and washing.

6. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 5, characterized in that: The mass ratio of the bismuth nanoparticles to the black phosphorus nanosheets is (5-10) mg: (1-5) mg; the black phosphorus nanosheets, NH2-PEG 2000 The mass ratio of -MAL and MMP-2 enzyme response polypeptides is (5~10) mg: (4~8) mg: (1~2) mg.

7. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, characterized in that: The specific operation of step (4) is as follows: adding an aqueous solution containing Clostridium butyricum to an aqueous solution containing a black phosphorus-based radiosensitizer and mixing, then adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride thereto and incubating for 1 to 24 hours, and after completion, centrifuging and washing to obtain a radiosensitized functionalized probiotic hybrid material.

8. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 7, characterized in that: The number of bacterial colonies in the aqueous solution containing Clostridium butyricum is 10 7 ~10 9 CFU / mL; the concentration of the aqueous solution containing the black phosphorus-based radiosensitizer is 10~20 mg / mL; the mass ratio of the black phosphorus-based radiosensitizer to N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (10~20) mg: (7~70) mg: (12~20) mg.

9. A radiosensitized functionalized probiotic hybrid material, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. Use of the radiosensitizing functionalized probiotic hybrid material according to claim 9 as an anti-tumor drug.

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