Radiosensitizing functionalized probiotic hybrid material and its preparation method and application

Abstract

The application discloses a radio-sensitizing functionalized probiotic hybrid material and a preparation method and application thereof, and belongs to the technical field of medical preparations. Black phosphorus nanosheets and bismuth nanoparticles are prepared first; then the bismuth nanoparticles are blended with the black phosphorus nanosheets in an aqueous solution environment, NH2-PEG 2000 -MAL and MMP-2 enzyme-responsive polypeptides are added and reacted to obtain a black phosphorus-based radio-sensitizer; finally, clostridium butyricum is blended with the black phosphorus-based radio-sensitizer, then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride are added and incubated to obtain the radio-sensitizing functionalized probiotic hybrid material. The method has the advantages of simple process and high operability. The prepared radio-sensitizing functionalized probiotic hybrid material can effectively improve the targeting accuracy of the radio-sensitizer under the driving of anaerobic probiotics, reduce off-target toxic side effects, has the characteristics of high tumor inhibition efficiency, and has a wide application prospect in the field of anti-tumor.

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, one of the world's deadliest diseases, poses a serious threat to human life and health. Currently, radiotherapy is widely used in clinical practice and has achieved some success in inhibiting tumor proliferation and prolonging patient survival. However, due to the complexity, density, and heterogeneity of tumors, current clinical radiotherapy has only achieved limited success. For example, tumor cells can escape the radiosensitive G2 / M phase through abnormal cell cycle regulation, leading to radioresistance in tumor cells and thus reducing the efficacy of radiotherapy. Furthermore, traditional radiosensitizers rely on enhanced vascular penetration and retention to passively deposit at the tumor site. The dense extracellular matrix at the tumor site hinders the penetration of radiosensitizers within the tumor, thereby greatly reducing the radiosensitization effect.

[0003] To compensate for insufficient accumulation of radiosensitizers within tumors, a common strategy is to increase the dose or radiation intensity of the radiosensitizer, which inevitably produces toxic side effects on normal tissues. Furthermore, radiotherapy inevitably promotes tumor cell metastasis, significantly impacting radiotherapy outcomes. Therefore, exploring new approaches and strategies for tumor treatment that differ from traditional treatment strategies is extremely urgent.

[0004] Black phosphorus nanosheets (BPs) have become a research hotspot 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, retaining the characteristics of black phosphorus while increasing 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 and black phosphorus are in direct contact. 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. They will be degraded and destroyed in the blood circulation, and easily diffuse and accumulate in other organs in the body, causing systemic radiation toxicity.

[0006] Fortunately, certain probiotics have been shown to specifically target hypoxic regions of tumors, preferentially colonizing nutrient-rich and immunosuppressive tumor sites and penetrating deeper into the tumor core. Furthermore, bacteria contain abundant pathogen-associated molecular patterns (PAMPs), which, after X-ray irradiation, can be recognized by the immune system and trigger an immune response, enhancing the immunogenicity of the tumor site. Therefore, probiotics can serve as a reliable drug carrier for delivering radiosensitizers and build a bridge between radiotherapy and immunotherapy to achieve better cancer treatment outcomes.

[0007] For example, Chinese invention patent publication number CN112755184A provides a tumor-targeted carrier for low-dose radiotherapy, its preparation method, and a tumor-targeted drug. This invention's tumor-targeted carrier for low-dose radiotherapy incorporates anaerobic bacteria, leveraging their anaerobic tropism to precisely home in on tumor cells. This tumor-targeted carrier for low-dose radiotherapy utilizes black phosphorus quantum dots (BPQDs), which have high photothermal conversion efficiency and achieve targeted treatment of hypoxic tumor cells through photothermal action. Similarly, Chinese invention patent publication number CN112516309A discloses a tumor-targeted carrier, drug, and preparation method for low-dose radiotherapy. This approach utilizes ultrasonic exfoliation to produce black scale quantum dots. The anaerobic bacteria are then surface-ammoniated, and the two are mixed. The black scale quantum dots are then electrostatically adsorbed onto the surface of the ammoniated anaerobic bacteria to produce the tumor-targeted carrier. However, in different application environments, the response mechanism of the specific anaerobic bacteria type needs to be tailored to the specific application requirements. Not only that, 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 precise 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 radiosensitized functionalized probiotic hybrid material with simple and convenient process steps and high operability is provided, comprising the following steps:

[0010] (1) Black phosphorus is dispersed in N-methyl pyrrolidone (NMP) and ultrasonically treated to obtain black phosphorus nanosheets;

[0011] (2) Dispersing bismuth dodecyl sulfate in octadecene, adding dodecanethiol (DDT) and tri-n-octylphosphine and reacting to obtain bismuth nanoparticles;

[0012] (3) In an aqueous solution, bismuth nanoparticles were mixed with black phosphorus nanosheets and NH2-PEG was added. 2000 -MAL and MMP-2 enzyme response peptides react to obtain black phosphorus-based radiosensitizers;

[0013] (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.

[0014] Preferably, 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.

[0015] The above operation was performed in an ice bath (reaction temperature: 0°C) to prevent the black phosphorus nanosheets from being oxidized by oxygen due to excessively high temperatures. Since black phosphorus nanosheets are easily oxidized by oxygen, excessively high reaction temperatures would accelerate the oxidation process, resulting in irregular morphology. Ultrasonication for 12 to 72 hours can produce single-layer black phosphorus nanosheets with a particle size of 200 to 400 nm. If the ultrasonication time is less than 12 hours, the black phosphorus nanosheets are too large and cannot form a monolayer structure. If the ultrasonication time is greater than 72 hours, the black phosphorus nanosheets are too small and easily aggregate, which is not conducive to the subsequent bonding with bismuth nanoparticles.

[0016] Preferably, the step (2) adopts a rate-controlled titration method, and the specific operation is as follows: adding bismuth dodecyl sulfate to the octadecene solution for ultrasonic dispersion; then adding dodecyl mercaptan at a rate of 10-30 mL / h and reacting at 50-200 °C for 1-8 hours until the solution turns brown, then adding tri-n-octylphosphine at a rate of 10-30 mL / h and reacting at 30-100 °C for 0.5-4 hours, and then centrifuging and washing to obtain bismuth nanoparticles.

[0017] Further preferably, the volume ratio of the dodecyl bismuth sulfate, octadecene, dodecyl mercaptan, and tri-n-octylphosphine is (4-8) mL: (20-40) mL: (8-16) mL: (4-8) mL.

[0018] The addition rate of dodecylmercaptan and tri-n-octylphosphine is controlled between 10 and 30 mL / h to control the particle size of the bismuth nanoparticles. If the addition rate is less than 10 mL / h, the synthesized bismuth nanoparticles will be smaller, which is detrimental to their radiosensitization effect. If the addition rate is greater than 30 mL / h, the bismuth nanoparticles will be larger, hindering their bonding with the black phosphorus nanosheets. The amount of tri-n-octylphosphine added is also preferably controlled within the preferred range of the present invention. If the volume of tri-n-octylphosphine is less than 4 mL, the bismuth nanoparticles may be smaller in size; if it is greater than 8 mL, the bismuth nanoparticles may be larger in size.

[0019] 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 were reacted for 0.5 to 4 h, and after completion, the black phosphorus-based radiosensitizer was obtained by centrifugation and washing.

[0020] Further preferably, the mass ratio of the bismuth nanoparticles to the black phosphorus nanosheets is (5-10) mg:(1-5) mg.

[0021] 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.

[0022] The mass ratio of bismuth nanoparticles to black phosphorus nanosheets is controlled within a range of (5-20) mg:(1-5) mg to control the radiosensitization strength and material stability of the black phosphorus-based radiosensitizer. A ratio greater than 20 mg:1 mg of bismuth nanoparticles can negatively impact the stability of the black phosphorus nanosheets. A ratio less than 5 mg:5 mg of bismuth nanoparticles can reduce the radiosensitization strength of the black phosphorus-based radiosensitizer.

[0023] 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-PEG 2000 -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 wastes experimental materials.

[0024] NH2-PEG 2000 If the input ratio of -MAL to MMP-2 enzyme responsive peptide is higher than 8 mg:1 mg, the MMP-2 enzyme responsive 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 responsive polypeptide is lower than 4 mg:2 mg, it will result in excessive amount of MMP-2 enzyme responsive polypeptide and waste experimental materials.

[0025] 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 then centrifuging and washing to obtain a radiosensitized functionalized probiotic hybrid material.

[0026] More preferably, the bacterial colony count 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.

[0027] Those 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 a ratio of (1-10) mL: (1-10) mL or other ratios to obtain materials with different contents of Clostridium butyricum and black phosphorus-based radiosensitizer, thereby meeting the needs of different applications.

[0028] 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.

[0029] 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.

[0030] In the second aspect of the present invention, a radiosensitized functionalized probiotic hybrid material with high tumor inhibition efficiency, high targeting accuracy, and small off-target toxicity and side effects is provided, which is prepared by the method provided by the first aspect of the present invention.

[0031] 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.

[0032] Based on the above technical solutions, the design concept and principle of the present invention are as follows:

[0033] 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 conducive 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 of 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 polypeptide can react to obtain a black phosphorus-based radiosensitizer with MMP-2 enzyme responsive function. The MMP-2 enzyme responsive polypeptide can link the black phosphorus-based radiosensitizer and Clostridium butyricum, and only responsively break 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 radiotoxicity. 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, benefit immune activation after tumor radiotherapy.

[0034] The radiosensitizing functionalized probiotic hybrid material prepared using the method of this invention leverages the hypoxia tropism of Clostridium butyricum to deliver a black phosphorus-based radiosensitizer to hypoxic tumor sites, enhancing the efficacy of targeted tumor therapy. Furthermore, after radiotherapy, the bacteria release pathogen-associated molecular patterns (PAMPs), activating immunogenicity at the tumor site, building a bridge between radiotherapy and immunotherapy and improving tumor suppression efficiency. During tumor cell cycle regulation, black phosphorus nanosheets can induce tumor cells to remain in the radiosensitive G2 / M phase through cell cycle arrest, ultimately leading to enhanced radiosensitivity. Furthermore, the high surface area and excellent electrical conductivity of black phosphorus nanosheets amplify the radiosensitizing effect of bismuth nanoparticles. This "targeting-cell cycle regulation-radiosensitization" strategy, driven by active life, addresses the poor precision of traditional radiosensitizers and the strong radioresistance of tumor cells, providing efficient and precise targeted therapy for tumors.

[0035] By combining a black phosphorus-based radiosensitizer with MMP-2 enzyme responsiveness with Clostridium butyricum, the anaerobic probiotics can effectively enhance the radiosensitizer's targeting precision and minimize off-target side effects. Combining black phosphorus-mediated tumor cell cycle regulation with bismuth nanoparticle radiosensitization significantly reduces tumor cell radiotherapy resistance and enhances the radiosensitization effect, thereby activating the immunogenicity of the tumor site. This allows for combined radiotherapy and immunotherapy, improving tumor suppression efficiency.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] 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.

[0038] The application provides a radio-sensitizing functional probiotic hybrid material, which has the characteristics of high tumor inhibition efficiency, high targeting accuracy and small off-target toxic side effects.

[0039] The application provides application of the radio-sensitizing functional probiotic hybrid material, and the radio-sensitizing functional probiotic hybrid material has wide application prospects in the field of anti-tumor. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 A schematic diagram of the construction of the radio-sensitizing functional probiotic hybrid treatment system and the anti-tumor mechanism thereof;

[0041] Figure 2 Characterization of the radio-sensitizing functional probiotic hybrid material; wherein, Figure 2 A is a transmission electron microscope (TEM) and energy dispersive spectroscopy mapping (EDS-mapping) image; Figure 2 B is a particle size test result, Figure 2 C is a potential diagram, Figure 2 D is an X-ray photoelectron spectroscopy (XPS) test result, Figure 2 E is a combination 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 a grafting situation of black phosphorus-based radio-sensitizers on clostridium butyricum at different concentrations, Figure 2 G is an MMP-2 enzyme response effect of the radio-sensitizing functional probiotic hybrid material, Figure 2 H is a survival rate of clostridium butyricum after different treatments;

[0042] Figure 3 Cell cycle blocking and radio-sensitizing ability of the radio-sensitizing functional probiotic hybrid material; wherein, Figure 3 A is a result of regulation of PCNA and CDK1 proteins in Panc-2 pancreatic cancer cells by different groups of materials, Figure 3 B is a flow cytometry detection result of a cell cycle of Panc-2 pancreatic cancer cells after treatment of the Panc-2 pancreatic cancer cells by different groups of materials, Figure 3 C is a radiotherapy toxicity of Panc-2 pancreatic cancer cells by different groups of materials, Figure 3 D is a DNA damage effect of Panc-2 pancreatic cancer cells by radiotherapy after treatment of the Panc-2 pancreatic cancer cells by different groups of materials;

[0043] Figure 4The 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 blood biochemistry and blood routine results of mice

[0044] Figure 5 The in vivo anti-tumor 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 the in vitro photo of the tumor after treatment. Figure 5 D is tumor weight;

[0045] 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

[0046] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0047] The present invention provides a radiosensitized functional probiotic hybrid material. The overall concept is as follows:

[0048] 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:

[0049] (1) Disperse the bulk black phosphorus in a methyl pyrrolidone solution and ultrasonicate it in an ice bath for 24 h to obtain black phosphorus nanosheets;

[0050] 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;

[0051] Said step (1) specifically comprises:

[0052] adding 10-50 mg bulk black phosphorus into 50-200 mL methylpyrrolidone solution, and performing ultrasonic crushing at 0 ℃ for 12-48 h; after the ultrasonic crushing is completed, the reaction solution is taken out, gradient centrifugation (8000-15000 rpm, 10-20 min) is performed to wash to obtain monolayer black phosphorus nanosheets with regular morphology and particle size of 200-400 nm;

[0053] The gradient centrifugation method can screen out black phosphorus nanosheets with particle size of 200-400 nm, and the step specifically comprises: first, centrifugation is performed at 8000 rpm for 10 min to collect the supernatant, then centrifugation is performed at 15000 rpm for 15 min to collect the precipitate, and after washing, the precipitate is dispersed in ultrapure water to obtain monolayer black phosphorus nanosheets with particle size of 200-400 nm;

[0054] (2) Bismuth nanoparticles are obtained by dispersing bismuth dodecyl sulfate in octadecene solution, adding dodecyl mercaptan and tri-n-octyl phosphine;

[0055] The bismuth nanoparticles are synthesized by a controlled-rate titration method, and the method specifically comprises:

[0056] 8-16 mL of dodecyl mercaptan is added to a mixed solvent containing 8-16 mL of bismuth dodecyl sulfate and 20-40 mL of octadecene at a rate of 10-30 mL / h by an injection pump, and the reaction is performed under stirring at 100 ℃ in an argon environment. After the solution turns brown yellow, the temperature is lowered to 65 ℃; then, 4-8 mL of tri-n-octyl phosphine is added to the above mixture at a rate of 10-30 mL / h by an injection pump, and the reaction is performed, followed by centrifugation (10000-12000 rpm, 10-20 min) and washing to obtain bismuth nanoparticles with regular morphology and size;

[0057] (3) The bismuth nanoparticles are blended with the black phosphorus nanosheets, and NH2-PEG 2000 MAL and MMP-2 enzyme-responsive polypeptide are added to obtain a black phosphorus-based radiosensitizer with MMP-2 enzyme response function;

[0058] The step (3) specifically comprises:

[0059] The bismuth nanoparticles and the black phosphorus nanosheets are blended 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 of NH2-PEG 2000 MAL and (1-2) mg of MMP-2 enzyme-responsive polypeptide are added to the black phosphorus-based carrier loaded with bismuth nanoparticles, and after centrifugation and washing, a black phosphorus-based radiosensitizer with MMP-2 enzyme response function is obtained;

[0060] (4) The Clostridium butyricum is blended with the black phosphorus-based radiosensitizer, and then is incubated in an EDC / NHS solution for 24 h to obtain a functional probiotic hybrid material.

[0061] The step (4) specifically comprises:

[0062] The water solution containing the Clostridium butyricum is added with the water solution of the black phosphorus-based radiosensitizer while stirring, and then is added with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, centrifuged (at a speed of 1000-6000 rpm for 5-20 min) after reaction for 2 h, washed with a PBS solution for 3 times after removing the supernatant, and then a radiosensitive functional probiotic hybrid material is obtained;

[0063] As an optional implementation, the preparation method of the water solution containing the Clostridium butyricum is as follows: the Clostridium butyricum is cultured in a 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) under anoxic condition for 24 h. When the bacteria grow to the plateau phase, the bacteria are collected by centrifugation, and then are washed by centrifugation with a sterile PBS buffer. After removing the culture medium, the bacteria are centrifuged again (at a speed of 1000-6000 rpm for 5-20 min), and then are dispersed in water for use after repeating the above steps for three times.

[0064] In the water solution containing the Clostridium butyricum, the number of bacteria is 10 7 ~10 9 CFU / mL, and the concentration ratio of the 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.

[0065] As can be seen from the above, the following important steps are adopted in the technical scheme of the present application to overcome the technical difficulties:

[0066] In the step (2), the morphology and size of the bismuth nanoparticles are regulated by optimizing the preparation conditions (the ratio of different reaction raw materials), the reaction temperature and the dropping speed of the bismuth nanoparticles.

[0067] In the step (3), the mass ratio of the bismuth nanoparticles to the black phosphorus nanosheet is (5-20) mg:(1-5) mg, the mass ratio of the bismuth nanoparticles to the NH2-PEG 2000The mass ratio of the MAL and the MMP-2 enzyme-responsive polypeptide is (4-8) mg:(1-2) mg, so as to optimize the amount of the bismuth nanoparticle and the MMP-2 enzyme-responsive polypeptide modified on the black phosphorus nanosheet, and then obtain the black phosphorus-based radiosensitizer with stable structure and sufficient radiosensitization intensity;

[0068] In step (4), the concentration ratio of the Clostridium butyricum solution, the black phosphorus-based radiosensitizer, the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is (10 7 ~10 9 ) CFU / mL:(10-20) mg / mL:(7-70) mg / mL:(12-20) mg / mL, so as to control the grafting ratio of Clostridium butyricum and the MMP-2 enzyme-responsive black phosphorus-based radiosensitizer, and thus obtain the functionalized probiotic hybrid material with superior performance.

[0069] According to another typical embodiment of the present application, the present application provides a radiosensitized functionalized probiotic hybrid material prepared by the method.

[0070] According to another typical embodiment of the present application, the present application provides the use of the radiosensitized functionalized probiotic hybrid material as an antitumor drug.

[0071] The radiosensitized functionalized probiotic hybrid material utilizes the anaerobic tropism of Clostridium butyricum to specifically target the black phosphorus-based radiosensitizer to the tumor site, thereby improving the enrichment effect of the radiosensitizer at the tumor site and improving the effect of radiotherapy. At the same time, the black phosphorus nanosheet regulates the cell cycle of tumor cells, so that the tumor cells are retained in the radiosensitive G2 / M phase, thereby radiosensitizing and reducing the radiotherapy resistance of tumor cells. In this process, due to the presence of excess MMP-2 enzyme at the tumor site, the radiosensitized functionalized probiotic hybrid material releases the black phosphorus-based radiosensitizer under the action of the MMP-2 enzyme. On the one hand, the black phosphorus nanosheet with photoacoustic imaging capability can block the cell cycle of tumor cells and reduce the radiotherapy resistance of tumor cells, and on the other hand, the bismuth nanoparticle with computed tomography imaging capability can amplify the effect of radiotherapy and improve the killing of tumor cells by radiotherapy. As shown in the figure, this functionalized probiotic forward-driven complementary therapy of radiotherapy-immunotherapy not only fully utilizes the advantages of each single therapy, but also makes up for the respective shortcomings, thereby efficiently inhibiting the growth of primary tumors and the metastasis of tumors. Figure 1

[0072] The radiosensitized functionalized probiotic hybrid material of the present application will be described in detail below with reference to examples and experimental data.

[0073] Example 1​

[0074] The method for preparing the radio-sensitizing functionalized probiotic hybrid material has the following steps:

[0075] (1) 20 mg of bulk black phosphorus was added to 100 mL of methylpyrrolidone solution, and ultrasonic crushing was performed in an ice bath environment at 0 °C for 48 h; after the ultrasonic crushing was completed, the reaction solution was taken out and gradient centrifuged, first centrifuged at 8000 rpm for 10 min to collect the supernatant, and then the supernatant was centrifuged at 10000 rpm for 15 min to collect the precipitate, which was washed with ultrapure water for 3 times to obtain monolayer black phosphorus nanosheets with uniform particle size distribution;

[0076] (2) 8 mL of dodecyl mercaptan was added to a mixed solvent containing 8 mL of bismuth dodecyl sulfate and 20 mL of octadecene at a speed of 10 mL / h by a syringe pump, and the reaction was stirred under an argon environment and at 100 °C oil bath; after the solution turned brown yellow, the temperature was lowered to 65 °C; then, 4 mL of tri-n-octyl phosphine was added to the above mixture at a speed of 10 mL / h by a syringe pump, and then centrifuged (12000 rpm / 15 min) and washed with ultrapure water for 3 times to obtain bismuth nanoparticles with regular morphology and size;

[0077] (3) 10 mg of black phosphorus nanosheets were uniformly dispersed in 10 mL of ultrapure water, and ultrasonic was applied for 5 min to make them fully dispersed; 10 mg of bismuth nanoparticles were uniformly dispersed in 10 mL of ultrapure water, and ultrasonic was applied for 10 min to make them fully dispersed; then, 10 mL of bismuth nanoparticle aqueous solution (1 mg / mL) was added dropwise into the black phosphorus nanosheet solution, and stirred at room temperature for 24 h; then, the un-coupled bismuth nanoparticles were removed by ultrafiltration to obtain black phosphorus-based radio-sensitizing agents loaded with bismuth nanoparticles;

[0078] 5 mL of a mixture solution containing NH2-PEG 2000 -MAL (4 mg) and MMP-2 enzyme-responsive polypeptide (2 mg) was added to the black phosphorus-based radio-sensitizing agent loaded with bismuth nanoparticles, and stirred at room temperature for 24 h; after the reaction was completed, centrifugation (11000 rpm, 10 min), water washing, and collection of the precipitate were performed to obtain the MMP-2 enzyme-responsive black phosphorus-based radio-sensitizing agent

[0079] (4) Clostridium butyricum was cultured in RCM medium (10 mg / mL trypsin, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) under anaerobic conditions at 30 °C overnight; when the bacteria grew to the stationary phase, the bacteria were collected by centrifugation (6000 rpm, 5 min), then resuspended with sterile PBS and centrifuged again, and the process was repeated three times before the bacteria were dispersed in water for use;

[0080] 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, the material was centrifuged (6000 rpm, 5 min) and washed with water three times to obtain the radiosensitized functionalized probiotic hybrid material.

[0081] Example 2

[0082] The preparation method of the radiosensitized functional probiotic hybrid material comprises the following steps:

[0083] (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 and subjected to gradient centrifugation. The supernatant was first collected by centrifugation at 9000 rpm for 10 min, and then the supernatant was collected by centrifugation at 12000 rpm / 15 min. The precipitate was washed three times with ultrapure water to obtain a single-layer black phosphorus nanosheet with uniform particle size distribution.

[0084] (2) 12 mL of dodecyl mercaptan was added to a mixed solvent containing 10 mL of bismuth dodecyl sulfate and 30 mL of octadecene via 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 for reaction; after the solution turned brown-yellow, the temperature was lowered to 65 °C; then, 6 mL of tri-n-octylphosphine was added to the above mixture via a syringe pump at a rate of 20 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;

[0085] (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 added dropwise 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;

[0086] 10 mL of NH2-PEG 2000A mixture solution of MAL (6 mg) and MMP-2 enzyme responsive polypeptide (1.5 mg) was added to the bismuth nanoparticle-loaded black phosphorus-based radiosensitizer solution, and the reaction was stirred at room temperature for 24 h; after the reaction was completed, centrifugation (11000 rpm, 10 min), water washing, and collection of the precipitate yielded the MMP-2 enzyme-responsive black phosphorus-based radiosensitizer

[0087] (4) Clostridium butyricum was cultured in RCM medium (10 mg / mL tryptone, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) under anaerobic conditions overnight at 30°C; when the bacteria grew to the stationary phase, the bacteria were collected by centrifugation (6000 rpm, 5 min), then resuspended in sterile PBS and centrifuged again, and the bacteria were dispersed in water for use after repeated three times;

[0088] A mixture solution of 10 mL MMP-2 enzyme-responsive black phosphorus-based radiosensitizer (15 mg / mL) and N-hydroxysuccinimide (50 mg / mL), 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride (15 mg / mL) was added to 1 mL of an aqueous solution containing Clostridium butyricum (10 8 CFU / mL), and shaken and incubated for 2 h; then, centrifugation (6000 rpm, 5 min), water washing 3 times, and collection yielded the radiosensitized functional probiotic hybrid material.

[0089] Example 3

[0090] The preparation method of the radiosensitized functional probiotic hybrid material is as follows:

[0091] (1) 50 mg of bulk black phosphorus was added to 100 mL of methylpyrrolidone solution, and ultrasonic crushing was performed in an ice bath environment at 0°C for 72 h; after the ultrasonic crushing was completed, the reaction solution was taken out and gradient centrifugation was performed, first by centrifugation at 10000 rpm for 10 min to collect the supernatant, and then the supernatant was centrifuged at 15000 rpm for 15 min to collect the precipitate, which was washed with ultrapure water for 3 times to obtain monolayer black phosphorus nanosheets with uniform particle size distribution;

[0092] (2) 16 mL of dodecyl mercaptan was added into a mixed solvent containing 12 mL of bismuth dodecyl sulfate and 40 mL of octadecene at a speed of 30 mL / h by a syringe pump, and the reaction was stirred under an argon atmosphere and at a temperature of 100 °C in an oil bath; after the solution turned brown yellow, the temperature was lowered to 65 °C; then, 8 mL of tri-n-octylphosphine was added into the above mixture at a speed of 30 mL / h by a syringe pump, and then the mixture was reacted, followed by centrifugation (12000 rpm / 15 min) and washing with ultrapure water for 3 times to obtain bismuth nanoparticles with regular morphology and size;

[0093] (3) 10 mg of black phosphorus nanosheets were uniformly dispersed in 20 mL of ultrapure water, and ultrasonic treatment was performed for 5 min to make the black phosphorus nanosheets fully dispersed; 200 mg of bismuth nanoparticles were uniformly dispersed in 20 mL of ultrapure water, and ultrasonic treatment was performed for 10 min to make the bismuth nanoparticles fully dispersed; then, 10 mL of a bismuth nanoparticle aqueous solution (10 mg / mL) was added dropwise into the black phosphorus nanosheet solution, and the mixture was stirred at room temperature for 24 h; then, the un-coupled bismuth nanoparticles were removed by ultrafiltration to obtain a black phosphorus-based radiosensitizer loaded with bismuth nanoparticles;

[0094] 5 mL of a mixture solution containing NH2-PEG 2000 MAL (8 mg) and MMP-2 enzyme-responsive polypeptide (1 mg) was added into the black phosphorus-based radiosensitizer loaded with bismuth nanoparticles, and the mixture was stirred at room temperature for 24 h; after the reaction was completed, centrifugation (11000 rpm, 10 min), water washing, and collection of the precipitate were performed to obtain an MMP-2 enzyme-responsive black phosphorus-based radiosensitizer

[0095] (4) Clostridium butyricum was cultured in RCM medium (10 mg / mL tryptone, 5 mg / mL yeast extract, 0.5 mg / mL resazurin and 0.5 mg / mL NaCl) under anaerobic conditions at 30 °C overnight; when the bacteria grew to the stationary phase, the bacteria were collected by centrifugation (6000 rpm, 5 min), then resuspended in sterile PBS and centrifuged again, and the above steps were repeated three times, and then the bacteria were dispersed in water for use;

[0096] A mixture solution of 20 mL of the MMP-2 enzyme-responsive black phosphorus-based radiosensitizer (10 mg / mL) and N-hydroxysuccinimide (20 mg / mL), and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (12 mg / mL) was added into 1 mL of an aqueous solution containing Clostridium butyricum (10 9 CFU / mL), and the mixture was shaken and incubated for 2 h; then, centrifugation (6000 rpm, 5 min) and water washing were performed for 3 times, and the radiosensitized functional probiotic hybrid material was collected.

[0097] Test Example 1

[0098] This test case conducts structural characterization and performance testing of radiosensitization functionalized probiotic hybrid materials.

[0099] 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 radiosensitizing materials and their hybridization with bacteria.

[0100] CBB corresponds to the radiosensitizing 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.

[0101] like Figure 2 As shown in A, the morphology, size and element mapping changes of the black phosphorus-based radiosensitizing 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.

[0102] 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 reflects 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.

[0103] like Figure 2As shown in E and 2F, the binding rate of the prepared bismuth nanoparticles on the black phosphorus nanosheet and the grafting rate of the prepared black phosphorus-based radiosensitization material on the Clostridium butyricum surface were determined by inductively coupled plasma mass spectrometry, so as to obtain the optimal feeding ratio; as shown in Figure 2 As shown in G, the bismuth element in the supernatant of the functionalized radiosensitization probiotic hybrid material (CBB) after MMP-2 enzyme treatment was quantitatively analyzed by inductively coupled plasma mass spectrometry, which proved the MMP-2 enzyme response function of the functionalized radiosensitization probiotic hybrid material (CBB). In addition, as shown in Figure 2 As shown in H, the number of Clostridium butyricum colonies did not change much before and after the modification of the black phosphorus-based radiosensitization material, but the Clostridium butyricum modified with the black phosphorus-based radiosensitization material after radiotherapy was almost all dead, which indicated that the modification of the black phosphorus-based radiosensitization material itself had little effect on the activity of Clostridium butyricum, but it increased the sensitivity of Clostridium butyricum to radiation, thereby greatly reducing the hidden danger of probiotic treatment.

[0104] Application Example 1

[0105] In vitro experiment:

[0106] The Panc-2 pancreatic tumor cells were co-cultured with the prepared functionalized radiosensitization probiotic hybrid material (CBB) in vitro, and the cell cycle regulation performance and the in vitro anti-tumor performance of the radiosensitization probiotic hybrid material (CBB) were systematically studied.

[0107] The pancreatic cancer cells in the PBS treatment group were in the malignant proliferation stage, and the proliferating cell nuclear antigen (PCNA) in the cells was highly expressed, while the cyclin-dependent protein kinase (CDK1) was in the inhibition state and had low expression. After the treatment of the black phosphorus nanosheet (BPs), the pancreatic cancer cells could be blocked in the radiosensitive G2 / M phase, thereby inhibiting the expression of PCNA and up-regulating the expression of CDK1.

[0108] Parameter setting: the concentration of BPs was 10-50 μg / mL.

[0109] Preparation method: BPs were dispersed in Dulbecco's Modified Eagle Medium (DMEM) medium, and then added to a 24-well plate seeded with Panc-2 pancreatic tumor cells, and co-incubated for 24 h.

[0110] The experimental steps include: Tumor cell cycle regulation. Different hybrid materials were co-cultured with Panc-2 pancreatic cancer cells for 24 hours. 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 materials. 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, the cells were rinsed twice with PBS. PCNA and CDK1 antibodies were added to one group of well plates for staining. After incubation in the dark for 2 h, the cells were rinsed three times with PBS, and the expression of PCNA and CDK1 proteins in the cells was photographed using a super-resolution fluorescence microscope. Subsequently, the cells in another group of well plates were taken out, and pre-cooled 70% ethanol and PI dye were added for staining. After incubation in the dark for 2 h, the cells were 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 is downregulated and CDK1 protein is upregulated in pancreatic cancer cells after treatment 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 Figure B, flow cytometry data further demonstrated that the functionalized probiotic radiosensitizing material could block pancreatic cancer cells in the G2 / M phase.

[0111] Tumor cytotoxicity and DNA damage. First, Panc-2 pancreatic cancer cells were seeded in 24-well plates at 5 × 10 4 cells / 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 continued to be cultured 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 to Panc-2 pancreatic cancer cells was measured. Antibodies against the DNA damage marker protein γ-H2AX were added to another group of well plates for staining. After incubation in the dark for 2 h, they were rinsed with PBS 3 times, and the DNA damage in the cells was photographed using a super-resolution fluorescence microscope. As Figure 3As shown in C and 3D, Panc-2 pancreatic cancer cells treated with radiosensitizing probiotic hybrid materials (CBB) died in large numbers after radiotherapy, and the expression of DNA damage protein γ-H2AX was most significant. However, 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 materials (CBB) can significantly enhance the efficacy of radiotherapy through the dual effects of tumor cell G2 / M phase blockade by black phosphorus nanosheets and radiosensitization by bismuth nanoparticles.

[0112] Application Example 2

[0113] In vivo tumor targeting ability and biocompatibility of radiosensitizing probiotic hybrid materials:

[0114] 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 the radiosensitized probiotic hybrid material in vivo after tail vein injection; including:

[0115] (1) Tumor-targeted enrichment

[0116] When the mouse tumor 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) 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, the synthesized radiosensitizing probiotic hybrid material (CBB) has obvious fluorescence at the tumor site over time, and the fluorescence intensity gradually increases, indicating that the bacteria can play a targeted role and carry the black phosphorus-based radiosensitizer to the tumor site accurately. Figure 4 This is also demonstrated by in vitro targeting data in mouse organs, as shown in Figures C and 4D.

[0117] (2) Biosafety analysis

[0118] Comprehensively evaluate the various indicators of individual mice after treatment 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 4As shown in Figure E, after injection of the radiosensitizing probiotic hybrid material (CBB), the blood biochemical and routine blood 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.

[0119] Application Example 3

[0120] In vivo anti-tumor performance study of radiosensitized probiotic hybrid materials:

[0121] This study used female C57BL / 6J mice as model animals to construct a Panc-2-luc orthotopic tumor-bearing mouse model and systematically studied the radiotherapy effect of the radiosensitized probiotic hybrid material on tumors after tail vein injection. 3 Tumor-bearing mice were randomly divided into groups and injected with the corresponding materials via the tail vein on the first and seventh days. Twelve hours after injection, the mice were irradiated with X-rays. Every seven days, changes in tumor bioluminescence intensity were monitored using an in vivo fluorescence imaging system. After treatment, the mice were sacrificed, and all tumors were harvested, weighed, and photographed.

[0122] like Figure 5 As shown, compared with other control materials (PBS, BPs-Bi, and CB), tumor growth was significantly inhibited in tumor-bearing mice treated with the radiosensitizing probiotic hybrid material (CBB), with tumor volume and weight significantly smaller than those of the other materials. This indicates that the radiosensitizing probiotic hybrid material (CBB) can effectively inhibit tumor growth and exhibit good anti-tumor efficacy. BPs-Bi is a simple black phosphorus-based radiosensitizer that lacks tumor targeting ability and has a poor therapeutic effect. Only the radiosensitizing probiotic hybrid material (CBB) can leverage the anaerobic targeting function of Clostridium butyricum to deliver the black phosphorus-based radiosensitizer to the tumor region, thereby achieving the best therapeutic effect.

[0123] In summary, the present application obtains lamellar black phosphorus nanosheets by ice bath ultrasonic method to ultrasonically break the blocky black phosphorus in methylpyrrolidone; bi nanoparticles are modified on the surface of the black phosphorus nanosheets through conjugate interaction; NH2-PEG2000-MAL with positive electricity and the black phosphorus nanosheets with negative electricity are combined through electrostatic interaction to obtain a black phosphorus-based carrier hybrid material with maleimide on the surface; MMP-2 enzyme-responsive polypeptide with a thiol-terminated 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 the MMP-2 enzyme-responsive black phosphorus-based carrier hybrid material through amidation reaction; the functionalized probiotic hybrid material can efficiently target tumor sites and responsively release radiosensitizers, reducing systemic toxicity caused by off-target effects of radiosensitizers. The present application has high precision in radiotherapy for tumors, low radiotherapy dose and double-mode imaging function, and improves the visibility of treatment. Combined immunotherapy can also form a complementary therapy of radiotherapy-immunotherapy, which not only fully plays the advantages of each single therapy, but also makes up for the respective shortcomings, thereby efficiently inhibiting the growth of primary tumors and tumor metastasis.

[0124] The preferred embodiments of the present application are described in detail above. It should be understood that those skilled in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. A method for preparing a radiosensitized functional probiotic hybrid material, characterized in that: The steps include: (1) Black phosphorus was dispersed in methyl pyrrolidone and then ultrasonically treated to obtain black phosphorus nanosheets; (2) Add bismuth dodecyl sulfate to the octadecene solution and disperse it ultrasonically; 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; (3) In an aqueous solution, bismuth nanoparticles were mixed with black phosphorus nanosheets and NH2-PEG was 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, wherein: The step (1) adopts an ice bath ultrasonic method, and the specific operation is as follows: the block 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 volume ratio of the dodecyl bismuth sulfate, octadecene, dodecyl mercaptan, and tri-n-octylphosphine is (4-8) mL: (20-40) mL: (8-16) mL: (4-8) mL.

4. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, wherein: The specific operation of step (3) is as follows: ultrasonically disperse black phosphorus nanosheets in water, add bismuth nanoparticle aqueous solution, mix, centrifuge and wash to obtain a black phosphorus-based carrier loaded with a radiosensitizer; then add NH2-PEG 2000 -MAL and MMP-2 enzyme response peptides were reacted for 0.5 to 5 h, and after completion, the black phosphorus-based radiosensitizer was obtained by centrifugation and washing.

5. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 4, 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.

6. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 1, wherein: 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. After completion, the material is centrifuged and washed to obtain a radiosensitized functionalized probiotic hybrid material.

7. The method for preparing the radiosensitized functionalized probiotic hybrid material according to claim 6, characterized in that: The bacterial colony count 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.

8. A radiosensitized functionalized probiotic hybrid material, characterized by: The method is as described in any one of claims 1 to 7.

9. Use of the radiosensitizing functionalized probiotic hybrid material according to claim 8 in the preparation of tumor radiosensitizing drugs.

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