Radiation-protective antioxidant coating, armor probiotic, and methods of making and using same

Abstract

The application discloses a radiation-proof and oxidation-resistant coating, a helmet probiotic and a preparation method and application thereof, and relates to the technical field of probiotics, and specifically discloses a radiation-proof and oxidation-resistant coating which comprises a plurality of bismuth oxide particles, the surface of the bismuth oxide particles is coated with a metal polyphenol network layer formed by the reaction of polyphenols and trivalent cerium ions, and the metal polyphenol network layer is used for connecting the coated bismuth oxide particles to intestinal probiotics. The helmet probiotic of the application can be long-acting and fixed in the intestinal tract and carries a coating with radiation-proof and oxidation-resistant functions, can effectively prevent the damage to the intestinal tract during radiotherapy, can significantly reduce the oxidative stress reaction after radiotherapy, can reduce the inflammatory reaction, can protect the intestinal tissue, and can improve the survival level of tumor patients after recovery.

Description

Technical Field

[0001] This invention relates to the field of functional materials in biomedical engineering, specifically to a radiation-proof and antioxidant coating, armored probiotics having the radiation-proof and antioxidant coating, and their preparation method and application. Background Technology

[0002] Radiotherapy is a core local treatment for malignant tumors, and approximately 70% of cancer patients require it during their treatment process. However, with the continuous expansion of radiotherapy indications, its accompanying toxic side effects are becoming increasingly prominent, mainly manifesting as hematopoietic system, cerebrovascular, and gastrointestinal syndromes. Among these, radiation intestinal injury (RI) is of particular concern—the intestine is a core organ for nutrient absorption and immune homeostasis, and persistent mucosal damage to the intestine, if not addressed promptly, will significantly reduce patients' quality of life.

[0003] The pathogenesis of radiation-induced respiratory injury (RI) involves radiation-induced immune imbalance, DNA damage, and gut microbiota dysbiosis. Recent studies have shown that fecal microbiota transplantation (FMT) from radiation-resistant mice can induce a radiation-protective effect in recipient mice, suggesting that gut microbiota play a crucial role in the protection and repair of ionizing radiation damage. Further research reveals that gut microbiota and their metabolites can regulate radiation damage through multiple mechanisms, such as inhibiting DNA damage and ROS-mediated inflammatory responses, alleviating lipid peroxidation-driven ferroptosis, and modulating radiation-induced bystander effects. Therefore, oral probiotics have become an important adjunctive strategy for alleviating radiation injury symptoms in clinical practice.

[0004] In existing technologies, encapsulating probiotics with a layer of metal polyphenol networks (MPNs) and then attaching specific functional particles, such as cerium oxide nanoparticles, to the MPN encapsulation layer can effectively scavenge reactive oxygen species in the inflamed gut. However, MPN encapsulation layers typically have high coverage, which not only affects the activity and reproduction of probiotics but also poses a potential risk of metal toxicity. Furthermore, the radiation protection capabilities of existing MPN encapsulation layers mainly rely on polyphenols, resulting in low probiotic activity during radiotherapy and difficulty in reducing gut damage during the treatment process. Summary of the Invention

[0005] One objective of this invention is to provide a radiation-proof and antioxidant coating. By coating the surface of bismuth oxide particles with a metal polyphenol network layer, the bismuth oxide particles can be adhered to the surface of intestinal probiotics to form a radiation-proof and antioxidant coating. In this coating, cerium ions can not only self-assemble with polyphenols to form a metal polyphenol network, but also continuously exert antioxidant functions. The bismuth oxide particles can attenuate ionizing radiation energy through the photoelectric effect. After promoting the colonization of intestinal probiotics in the intestine, the radiation-proof and antioxidant coating can significantly reduce oxidative stress response after radiotherapy, reduce inflammatory response, and protect intestinal tissue.

[0006] This invention is achieved through the following technical solution:

[0007] A radiation-proof and antioxidant coating includes a plurality of bismuth oxide particles, the surface of which is coated with a metal polyphenol network layer formed by the reaction of polyphenols and trivalent cerium ions, the metal polyphenol network layer being used to connect the coated bismuth oxide particles to intestinal probiotics.

[0008] In this technical solution, the coating provides the main radiation protection function based on bismuth oxide particles. It has a high-density physical barrier and can more effectively block X-rays and significantly attenuate radiation energy through mechanisms such as photoelectric effect and Compton scattering. At the same time, bismuth oxide has an atomic number as high as 83, which can significantly enhance the photoelectric absorption cross section and has a shielding efficiency for low and medium energy radiation that is better than lead. Moreover, bismuth oxide particles are less toxic and can be naturally excreted from the body after exerting their radiation protection function.

[0009] In this technical solution, trivalent cerium ions and polyphenols self-assemble in situ on the surface of bismuth oxide particles to form a metal polyphenol network layer coating the bismuth oxide particles. In this metal polyphenol network layer, polyphenolic compounds, as natural antioxidants derived from plants, have significant value in the field of radiation protection. Furthermore, the multiple phenolic hydroxyl groups of polyphenols can form hydrogen bonds, covalent bonds, electrostatic interactions, and π–π stacking interactions with proteins on the surface of intestinal probiotics and with intestinal mucosal proteins, thereby adhering bismuth oxide particles to the surface of intestinal probiotics and promoting the colonization rate of intestinal probiotics with a radiation-protective and antioxidant coating.

[0010] However, using only polyphenols for bonding not only results in easy polyphenol loss but also limits the functional scope. Therefore, in this technical solution, a metal polyphenol network is formed by the self-assembly of trivalent cerium ions and polyphenols, which effectively improves the stability of the coating structure, significantly reduces polyphenol loss, and thus increases the loading rate of bismuth oxide particles. Simultaneously, trivalent cerium ions can also utilize their single-electron transfer capability to sequentially transfer superoxide radicals (O2)... - ) and hydrogen peroxide (H2O2) are converted into water and oxygen, and hydroxyl radicals ( . OH) is converted to OH -This effectively removes ROS accumulated in intestinal inflammation.

[0011] It is worth noting that using trivalent cerium ions as the metal ion in the metal polyphenol network layer has unique advantages: the coordination bonds formed between cerium ions and polyphenol ligands not only retain the single-electron transfer capability of trivalent cerium but also further enhance its activity. This is because the coordination bond between the metal and the polyphenol is essentially the combination of the empty orbitals of the metal ion and the lone pair electrons of the polyphenol, which does not change the variable valence state characteristics of the metal ion itself. As long as the coordination environment allows for electron tunneling, cerium ions can act as an electron transfer station for single-electron transfer. Therefore, cerium ions can stabilize the metal polyphenol network layer while exerting an antioxidant function without being released.

[0012] Unlike existing technologies that directly encapsulate probiotics using a metal polyphenol network layer and then connect functional groups through the network layer, this technology encapsulates bismuth oxide particles in a metal polyphenol network layer to form a radiation-resistant and antioxidant coating. This coating is then adhered to the surface of intestinal probiotics based on the adhesive properties of polyphenols, ultimately forming armored probiotics. This not only promotes bacterial growth and maintains their activity but also allows these armored probiotics to better perform their functions in the gut. Furthermore, this method significantly reduces the amount of metal ions used, making it superior to directly encapsulating probiotics in terms of both production costs and potential toxicity.

[0013] In this technical solution, after the anti-radiation and antioxidant coating is adhered to intestinal probiotics to form armored probiotics, these probiotics can be taken orally before radiotherapy. Once inside the intestines, the armored probiotics can target positively charged inflammatory sites using a metal polyphenol network layer. After the probiotics colonize the intestines, the anti-radiation and antioxidant coating significantly reduces the impact of radiation on the probiotics during radiotherapy, improves the viability of the probiotic strains, and enhances the intestinal microenvironment. The coating also significantly reduces radiation damage to the intestinal structure. Simultaneously, it significantly reduces oxidative stress and inflammatory responses after radiotherapy, thus preventing radiotherapy-induced intestinal damage through early intervention.

[0014] Furthermore, the trivalent cerium ion is cerium nitrate, cerium carbonate, cerium oxalate, or cerium phosphate.

[0015] In some preferred embodiments, the trivalent cerium ion is cerium nitrate, because cerium nitrate is readily soluble and can be completely ionized, providing free Ce. 3+ It can undergo coordination cross-linking with the phenolic hydroxyl groups of polyphenols. Meanwhile, nitrate is a weakly coordinating anion and hardly reacts with Ce in water. 3+ Competitive coordination sites ensure Ce 3+It can bind unimpeded to the catechol groups of polyphenols, such as tannic acid, while the anions of cerium carbonate, cerium phosphate, and cerium oxalate are strong coordinators and will compete with polyphenols for binding Ce. 3+ This is not conducive to the formation of metal polyphenol networks. Moreover, the optimal pH for constructing metal polyphenol networks is usually between 5 and 8, while the pH of cerium nitrate solution is about 4 to 5, which allows it to enter the reaction range with a little buffering.

[0016] Furthermore, the polyphenols are tannic acid, gallic acid, epigallocatechin gallate, ellagic acid, catechin, tea polyphenols, proanthocyanidins, or tararatanin.

[0017] In some preferred embodiments, the polyphenol is tannic acid. Tannic acid has the characteristics of low cost, high coordination efficiency, film-forming stability and high biocompatibility. As a natural polyphenol, it has a glucose core esterified by multiple gallic acid units, and its large number of catechol and pyrogallic acid groups can provide a high density of metal coordination sites.

[0018] In a preferred embodiment of the present invention, the polyphenol is tannic acid, the trivalent cerium ion is cerium nitrate, and the mass ratio of tannic acid to cerium nitrate is 1:1 to 1:3.

[0019] Experiments revealed that when the cerium nitrate content is too low, there is a severe overabundance of coordination sites for tannic acid, preventing the formation of a dense metal polyphenol network layer. Therefore, the cerium nitrate content typically needs to be higher than that of tannic acid to achieve sufficient cross-linking. Simultaneously, a suitable amount of residual trivalent cerium ions, i.e., excess cerium ions that have not participated in coordination, is beneficial for maintaining the Ce content of the radiation-resistant and antioxidant coating. 3+ / Ce 4+ Cyclic antioxidant process and catalytic activity. Furthermore, considering that a small amount of cerium ions may be lost due to hydrolysis in a near-neutral buffer system with pH 6-8, the mass ratio of tannic acid to cerium nitrate is 1:1 to 1:3 in a preferred embodiment. In a more preferred embodiment, the mass ratio of tannic acid to cerium nitrate is 1:3 to rapidly and stably form a dense metal polyphenol network layer on the surface of bismuth oxide particles, thereby improving their antioxidant capacity.

[0020] Furthermore, the mass ratio of tannic acid to bismuth oxide particles is 1:50 to 1:150.

[0021] Experiments revealed that when the tannic acid content is too low, the metal polyphenol network layer does not completely encapsulate the bismuth oxide particles, resulting in poor oxidation resistance and stability of the radiation-proof and antioxidant coating. However, if the tannic acid content is too high, it will form free flocculent matter with cerium ions, which is detrimental to elution. Therefore, in this technical solution, the mass ratio of tannic acid to bismuth oxide particles is set to 1:50 to 1:150. In a more preferred embodiment, the mass ratio of tannic acid to bismuth oxide particles is 1:75 to 1:150.

[0022] Another object of the present invention is to provide armored probiotics having any of the aforementioned anti-radiation and antioxidant coatings, specifically comprising intestinal probiotics, wherein the surface of the intestinal probiotics is formed with any of the aforementioned anti-radiation and antioxidant coatings, and a plurality of bismuth oxide particles of the anti-radiation and antioxidant coatings are connected to the intestinal probiotics through a metal polyphenol network layer.

[0023] In this technical solution, intestinal probiotics, also known as intestinal symbiotic bacteria, can be selected from the following genera: Escherichia coli, Klebsiella spp., Citrobacter spp., Lactobacillus spp., Bifidobacterium spp., Bacillus spp., etc. In some embodiments, intestinal probiotics can be Escherichia coli, Bacillus spp., Lactobacillus, etc. In a more preferred embodiment, considering that Escherichia coli Nissle 1917 (EcN) is a clinically validated probiotic prototype strain with excellent intestinal colonization and barrier strengthening functions, and is widely used in scientific research, in order to further enhance the targeted repair ability, the intestinal probiotic of Armor Probiotics is EcN.

[0024] Another object of the present invention is to provide a method for preparing any of the aforementioned armored probiotics, the method specifically comprising the following steps:

[0025] Polyphenols and trivalent cerium ions were added to ultrapure water containing suspended bismuth oxide particles to prepare the radiation-proof and antioxidant coating suspension.

[0026] Intestinal probiotics are suspended in a phosphate buffer solution to obtain a bacterial suspension. The anti-radiation and antioxidant coating suspension is mixed with the bacterial suspension, and a small amount of tannic acid is added for adhesion to obtain armor probiotics.

[0027] In some preferred embodiments, bismuth oxide particles are first suspended in ultrapure water and ultrasonically treated to eliminate agglomeration between the particles. Then, polyphenols and trivalent cerium ions are added to the bismuth oxide particle suspension to prepare a radiation-resistant and antioxidant coating suspension. The order of addition of polyphenols and trivalent cerium ions can be reversed; for example, if the suspension is negatively charged before addition, positively charged trivalent cerium ions can be added first, and vice versa, if the suspension is positively charged before addition, negatively charged polyphenols can be added first, to further improve the binding ability of bismuth oxide particles to the metal polyphenol network layer. After addition, a phosphate buffer solution is added. Vortex mixing is performed after each addition. Subsequently, the solution is washed by centrifugation with ultrapure water to remove residual cerium ions and polyphenols, preventing excess cerium ions and polyphenols from coordinating and forming flocculent matter that is difficult to wash off, ultimately obtaining bismuth oxide particles coated with a metal polyphenol network layer.

[0028] In some embodiments, the probiotics cultured overnight are pretreated and then resuspended in a phosphate buffer solution to obtain a bacterial suspension. The bacterial suspension is then mixed with a radiation-protective and antioxidant coating suspension. In some preferred embodiments, after mixing the radiation-protective and antioxidant coating suspension with the bacterial suspension, a small amount of polyphenol is added to the mixture to improve the adhesion of the radiation-protective and antioxidant coating to the probiotics, as well as the adhesion of the probiotics to the intestinal mucosa. In one or more embodiments, the volume of the added polyphenol is 1 / 50 to 1 / 10 of the initial volume of added polyphenol.

[0029] Further, the volume ratio of the radiation-resistant and antioxidant coating suspension to the bacterial suspension is 1:7.5 to 1:60. Experiments have shown that after the amount of radiation-resistant and antioxidant coating suspension reaches a certain volume, further increasing its amount significantly slows the increase in the amount of MPN@Bi2O3 particles adhering to the bacterial suspension. Therefore, in this technical solution, the volume ratio of the radiation-resistant and antioxidant coating suspension to the bacterial suspension is 1:7.5 to 1:60. More preferably, the volume ratio is 1:15 to 1:30.

[0030] Another object of the present invention is to provide the use of any of the aforementioned armored probiotics in the preparation of a medicament for the prevention of radiation-induced intestinal injury.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] 1. The armored probiotics of the present invention can colonize the intestines for a long time and carry a coating with anti-radiation and antioxidant properties, which can effectively prevent damage to the intestines during radiotherapy, and can significantly reduce oxidative stress response after radiotherapy, reduce inflammatory response, protect intestinal tissue, and is expected to improve the post-treatment survival level of cancer patients.

[0033] 2. In this invention, bismuth oxide particles are coated with a metal polyphenol network layer and then the bismuth oxide particles are adhered to the surface of intestinal probiotics, instead of the common method of coating probiotics with a metal polyphenol network. This is conducive to the reproduction of bacteria and maintains their activity, so that the adhered probiotics can better perform their functions in the intestine. At the same time, it can also significantly reduce the amount of metal ions used. In terms of both production cost and potential toxicity, it is superior to the existing technology of directly coating probiotics.

[0034] 3. The present invention uses trivalent cerium ions and polyphenols to form a metal polyphenol network layer coated on the surface of bismuth oxide particles. This not only stabilizes the metal polyphenol structure and plays the role of loading bismuth oxide, but also the metal polyphenol network can retain or even enhance the single electron transfer ability of cerium ions, so that cerium ions can exert antioxidant capacity while stabilizing the metal polyphenol structure.

[0035] 4. The bismuth oxide particles of the present invention can not only significantly reduce the impact of radiation on probiotics during radiotherapy, but also significantly reduce the damage of radiation to the intestinal structure. Furthermore, the bismuth oxide particles have lower toxicity and can be naturally excreted from the body after exerting their radiation protection effect.

[0036] 5. This invention has determined the ratio of tannic acid to cerium nitrate through experiments, which can not only rapidly and stably form a dense metal polyphenol network layer on the surface of bismuth oxide particles, but also improve their antioxidant capacity.

[0037] 6. This invention has determined the ratio of anti-radiation and antioxidant coating suspension to bacterial suspension through experiments, which can form a stable anti-radiation and antioxidant coating on the surface of intestinal probiotics. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0039] Figure 1 This is a flowchart illustrating the preparation method of armor probiotics in a specific embodiment of the present invention;

[0040] Figure 2 The characterization of MPN@Bi2O3 in a specific embodiment of the present invention is shown, where A (left) and A (right) are the HRTEM and EDX analysis results, respectively, and B is the XPS quantitative analysis result;

[0041] Figure 3 The following are SEM images (A) of armor probiotics after different proportions of coating were added, (B) of naked probiotics on the left and armor probiotics on the right, and (C) of armor probiotics with Cy7-labeled anti-radiation and antioxidant coating.

[0042] Figure 4 The strain viability of armored probiotics and naked probiotics under H2O2 stress is shown in a specific embodiment of the present invention.

[0043] Figure 5 The strain viability of armored probiotics and naked probiotics after different doses of X-ray treatment for different times is shown in a specific embodiment of the present invention.

[0044] Figure 6 This illustrates the ability of the radiation-resistant and antioxidant coating in a specific embodiment of the present invention to reduce inflammatory responses by intervening in macrophage polarization;

[0045] Figure 7 This invention illustrates the protective effect of early intervention with armor-like probiotics on irradiated intestinal tissue in a specific embodiment of the invention. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0047] All raw materials used in this invention are not particularly limited in their source; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or conventional purity requirements in the field of biomedical engineering functional materials are preferred. All raw materials used in this invention have common designations and abbreviations in the field, and each designation and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercial sources or prepare them using conventional methods based on the designation, abbreviation, and corresponding application.

[0048] The term "link" as used in this invention can refer to direct linking or indirect linking via other substituents, unless otherwise specified.

[0049] I. Preparation of Armored Probiotics

[0050]

Example 1

[0051] (1) Preparation of radiation-proof and antioxidant coating suspension (MPN@Bi2O3 suspension)

[0052] 30 mg of bismuth oxide nanoparticles (Bi₂O₃) were suspended in 45 mL of ultrapure water and sonicated for 20 min. Then, 100 μL of tannic acid (TA) (2 mg / mL) and 300 μL of Ce(NO₃)₃ (2 mg / mL) solution were added sequentially, vortexing for 30 s at each step. Finally, PBS (pH=7) solution was added, and the volume was adjusted to 50 mL. After vortexing the mixture for 1 min, it was washed with ddH₂O and centrifuged at 1000 g for 5 min. This process was repeated three times to remove residual Ce. 3+ Bismuth oxide nanoparticles (MPN@Bi2O3) coated with a metal polyphenol layer were obtained by reacting with TA. Finally, the MPN@Bi2O3 suspension was obtained by resuspending the nanoparticles in 1 mL of PBS.

[0053] Characterization of bismuth oxide nanoparticles MPN@Bi2O3 coated with a metal polyphenol layer revealed that, Figure 2 As shown in Figure A, both high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDX) verified the successful preparation of MPN@Bi2O3, showing that its average particle diameter is approximately 50 nm. Figure 2 Figure B further illustrates the quantitative analysis results of X-ray photoelectron spectroscopy (XPS), as shown in the figure. MPN@Bi2O3 contains Bi, Ce, C, and O elements.

[0054] (2) Preparation of Armor Probiotics

[0055] After culturing *Escherichia coli* Nissle 1917 (EcN) in LB liquid medium with shaking for 18 h, 1.25 mL of the bacterial suspension was washed three times with PBS by centrifugation (5000 g, 4 min). The sample was then resuspended in 600 μL of PBS to obtain a bacterial suspension with a bacterial concentration of 2 × 10⁻⁶. 9 CFU / mL.

[0056] 40 μL of MPN@Bi2O3 suspension was added to 600 μL of the bacterial suspension, 2 μL of tannic acid (2 mg / mL) was added, and then 358 μL of PBS was added. After each addition, the mixture was vortexed for 30 seconds. The mixture was then centrifuged and washed three times with ddH2O (1000 g, 5 min) to remove residual tannic acid. Finally, the bacterial suspension was resuspended in 1 mL of PBS to prepare Armored Probiotic 1.

[0057] Figure 3B shows a SEM image of Armored Probiotic 1, which reveals a dense, radiation-resistant, and antioxidant coating formed on the EcN surface. Subsequently, MPN@Bi2O3 was labeled with Cyanine 7 (Cy7), and confocal fluorescence images clearly show the Cy7 fluorescent shell on the EcN surface. Figure 3 C), these results together demonstrate that the armored probiotics (EcN / MPN@Bi2O3) form a radiation-resistant and antioxidant coating on the surface of EcN.

[0058]

Example 2

[0059] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (2), the volume of MPN@Bi2O3 suspension added to the bacterial suspension in 600 μL is 10 μL, and Armored Probiotic 2 is finally obtained.

[0060]

Example 3

[0061] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (2), the volume of MPN@Bi2O3 suspension added to the bacterial suspension in 600 μL is 20 μL, and Armor Probiotic 3 is finally obtained.

[0062]

Example 4

[0063] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (2), the volume of MPN@Bi2O3 suspension added to the bacterial suspension in 600 μL is 30 μL, and Armor Probiotic 4 is finally obtained.

[0064]

Example 5

[0065] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (2), the volume of MPN@Bi2O3 suspension added to 600 μL of bacterial suspension is 80 μL, and Armor Probiotic 5 is finally obtained.

[0066]

Example 6

[0067] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (1), the mass ratio of tannic acid and Ce(NO3)3 added is 1:1, and Armor Probiotic 6 is finally obtained.

[0068]

Example 7

[0069] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (1), the mass ratio of tannic acid and Ce(NO3)3 added is 1:2, and Armor Probiotic 7 is finally obtained.

[0070]

Example 8

[0071] In this embodiment, the preparation method is basically the same as that in Example 1. The difference is that in step (1), the mass ratio of tannic acid and Ce(NO3)3 added is 2:3, and Armor Probiotic 8 is finally obtained.

[0072]

Example 9

[0073] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (1), cerium carbonate is used instead of Ce(NO3)3 to prepare MPN@Bi2O3 suspension, and finally Armor Probiotic 9 is obtained.

[0074]

Example 10

[0075] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (1), epigallocatechin gallate (EGCG) is used instead of tannic acid to prepare MPN@Bi2O3 suspension, and finally Armored Probiotic 10 is obtained.

[0076]

Example 11

[0077] In this embodiment, the preparation method is basically the same as that in Example 1. The difference is that in step (1), anthocyanins (OPC) are used instead of tannic acid to prepare MPN@Bi2O3 suspension, and finally Armor Probiotic 11 is obtained.

[0078]

Example 12

[0079] In this embodiment, the preparation method is basically the same as in Example 1. The difference is that in step (2), Bacillus is used instead of Escherichia coli to prepare Armor Probiotic 12.

[0080]

Example 13

[0081] In this embodiment, the preparation method is basically the same as that in Example 1. The difference is that in step (1), the concentration of tannic acid is 4 mg / mL, and Armor Probiotic 13 is finally obtained.

[0082]

Example 14

[0083] In this embodiment, the preparation method is basically the same as that in Example 1. The difference is that in step (1), the concentration of tannic acid is 6 mg / mL, and Armor Probiotic 14 is finally obtained.

[0084] II. Performance Testing of Armored Probiotics

[0085]

Example 15

[0086] This embodiment tested the stability of MPN@Bi2O3 suspensions adhering to bacterial surfaces at different dosages.

[0087] Specifically, 1.25 mL of overnight cultured EcN bacterial suspension was added to different EP tubes, washed three times with PBS, and then set aside. 10 μL, 20 μL, 30 μL, and 40 μL of MPN@Bi2O3 suspension were added to the PBS bacterial suspension of the parallel groups, respectively. After steps such as tannic acid adhesion and PBS elution, the various armored probiotics were obtained.

[0088] Scanning electron microscopy revealed that, for example Figure 3 As shown in Figure A, the coating adhering to the EcN surface increases significantly with the increase of MPN@Bi2O3 suspension. When the amount of MPN@Bi2O3 suspension is further increased, the increase in the amount of MPN@Bi2O3 particles adhering to the EcN surface slows down significantly. For example, in Example 5, when the amount of MPN@Bi2O3 suspension is increased to 80 μL, the amount of adhesion is slightly higher than that in Example 1 because the adhesion points on the surface of the probiotics are close to saturation, and the excess MPN@Bi2O3 particles will agglomerate in the solution.

[0089] Therefore, in some embodiments, the volume ratio of MPN@Bi2O3 suspension to bacterial suspension in step (1) is 1:7.5 to 1:60. In a more preferred embodiment, the volume ratio of MPN@Bi2O3 suspension to bacterial suspension is 1:15 to 1:30.

[0090]

Example 16

[0091] In this embodiment, the ROS scavenging activity of Armor Probiotics against hydrogen peroxide (H2O2) induced by hydrogen peroxide was tested.

[0092] Specifically, in the presence of 80 mM H2O2, armored probiotics (EcN / MPN@Bi2O3) and uncoated E. coli control group (Uncoated EcN) were cultured for 1 h and 3 h, respectively, and then the bacterial solutions were diluted to 10. -6 Then, 100 μL was spread onto a plate. The experimental results are as follows: Figure 4 As shown, compared with the control group, the probiotics with radiation protection and antioxidant coating exhibited significantly higher activity under H2O2 stress, indicating that the radiation protection and antioxidant coating can effectively improve the antioxidant performance of probiotics.

[0093]

Example 17

[0094] In this embodiment, the radiation protection capability of Armor Probiotics was tested.

[0095] Specifically, uncoated EcN and armored EcN / MPN@Bi2O3 were coated in six-well plates, treated with different doses of X-rays, and cultured for a period of time. The bacterial solutions at different time points were diluted and plated to detect the growth of the strains.

[0096] The test results are as follows Figure 5 As shown, after treatment with 150 GY, 250 GY, and 500 GY rays for 1 h, 2 h, and 18 h respectively, the activity of the armored probiotic strains was significantly stronger than that of the exposed probiotics due to the radiation-resistant and antioxidant coating. Specifically, under high-dose radiation treatments of 250 GY and 500 GY, the activity of the exposed probiotic strains was significantly inhibited by the radiation, while the armored probiotics were almost unaffected. With increasing time, at 18 h, the activity of the armored probiotics under high-dose radiation irradiation was 3-4 times that of the exposed probiotics, demonstrating extremely strong radiation protection capabilities.

[0097]

Example 18

[0098] This embodiment demonstrates the ability of the radiation-resistant and antioxidant coating to reduce inflammatory responses by intervening in macrophage polarization.

[0099] Specifically, RAW264.7 macrophages were prepared at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates containing circular cell spreaders and cultured until adherent. The culture medium was then removed, and 2 mL of fresh culture medium (with / without 1 μg / mL LPS) was added to each well. After 24 h of induction, the cells were washed with culture medium, and then PBS or 5 μL of the radiation-resistant and antioxidant coating MPN@Bi2O3 prepared according to Example 1 was added to each well for 24 h. The culture medium and materials were then removed, and the cells were collected. Immunofluorescence staining was used to analyze the expression of CD86 / CD206 and IL-6 / IL-10 markers.

[0100] The test results are as follows Figure 6 As shown, compared with macrophages treated with PBS, cells treated with MPN@Bi2O3 were able to inhibit LPS-induced macrophage differentiation into pro-inflammatory M1 cells (DAPI / CD86) and promote their differentiation into M2 anti-inflammatory cells (DAPI / CD206), thus inhibiting the inflammatory response.

[0101] like Figure 6 As shown, consistent conclusions were also obtained in experiments labeling pro-inflammatory factor IL-6 and anti-inflammatory factor IL-10. The above experiments indicate that MPN@Bi2O3 inhibits the polarization of macrophages into the M1 type by LPS and causes them to differentiate into the M2 type, thereby achieving the effect of inhibiting inflammation.

[0102]

Example 19

[0103] This embodiment tested the protective ability of Armor Probiotics on irradiated intestinal tissue.

[0104] Specifically, a mouse model of significant abdominal radiation injury was established by administering a single 10 GY dose of X-rays to the abdomen of wild-type C57BL / 6 mice. In the intervention experiment, mice were treated with PBS, naked probiotic solution EcN, or armored probiotic solution (EcN / MPN@Bi2O3) one week prior to irradiation, i.e., 200 μL of the liquid treatment group was administered by gavage every 24 hours, with a final bacterial concentration of 1 × 10⁻⁶. 9 CFU was administered to mice the day after final treatment, with 8 mice in each group. Samples were collected 3 days after treatment.

[0105] The test results are as follows Figure 7 As shown in Figure A, HE staining revealed that, compared to the healthy control group (Healthy), a 10 GY dose of X-rays induced severe damage to parts of the jejunal tissue, including villi and crypts. The exposed probiotic solution group (RAD+EcN) reduced jejunal tissue damage to some extent, while the armored probiotic solution group (RAD+EcN / MPN@Bi2O3) showed significant reduction in jejunal tissue damage through early intervention, demonstrating excellent intestinal tissue protection.

[0106] Furthermore, Figure 7 B. Antioxidant capacity was tested using the supernatant of jejunal tissue homogenate. The results showed that the antioxidant capacity of the armored probiotic liquid group was basically equivalent to that of the healthy control group, much higher than that of the unprotected radiation group, and stronger than that of the naked probiotic liquid. Conversely, malondialdehyde (MDA), an indicator of lipid oxidation, was significantly increased in the unprotected radiation group, while it was reduced to the level of the healthy control in both the armored probiotic group and the naked probiotic treatment group. Figure 7 C shows the results of the immunostaining assay. Compared with the unprotected radiation group (RAD+PBS), the expression levels of tight junction proteins ZO-1 and Occludin in intestinal epithelial cells of jejunal tissue were significantly higher in the healthy control group and the Armor Probiotics group, indicating that Armor Probiotics can significantly reduce the damage of radiation to key proteins. Figure 7 D shows the ELISA results of the tissue homogenate supernatant, which confirmed that the expression levels of pro-inflammatory factors IFN-γ, TNF-α, IL-1β, and IL-6 in the Armor Probiotic Solution group were significantly lower than those in the unprotected irradiated group and close to those in the healthy control group, indicating that strain intervention can significantly reduce the expression of inflammatory factors after radiation.

[0107] Therefore, all the above experiments show that early intervention with armor probiotics can significantly protect the integrity of intestinal structure and function after radiation and reduce inflammatory response.

[0108] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any renaming, modification, equivalent substitution, improvement, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A radiation-resistant and oxidation-resistant coating, characterized in that, It includes several bismuth oxide particles, the surface of which is coated with a metal polyphenol network layer formed by the reaction of polyphenols and trivalent cerium ions. The metal polyphenol network layer is used to connect the coated bismuth oxide particles to intestinal probiotics.

2. The anti-radiation and anti-oxidation coating according to claim 1, characterized in that, The trivalent cerium ion is cerium nitrate, cerium carbonate, cerium oxalate, or cerium phosphate.

3. The anti-radiation and anti-oxidation coating according to claim 1, characterized in that, The polyphenols are tannic acid, gallic acid, epigallocatechin gallate, ellagic acid, catechin, tea polyphenols, proanthocyanidins, or tararatanin.

4. A radiation-resistant and oxidation-resistant coating according to any one of claims 1 to 3, characterized in that, The polyphenol is tannic acid, the trivalent cerium ion is cerium nitrate, and the mass ratio of tannic acid to cerium nitrate is 1:1 to 1:

3.

5. The anti-radiation and anti-oxidation coating according to claim 4, characterized in that, The mass ratio of tannic acid to bismuth oxide particles is 1:50 to 1:

150.

6. A type of armored probiotic, characterized in that, The invention includes intestinal probiotics, the surface of which is formed with a radiation-proof and antioxidant coating as described in any one of claims 1 to 5, wherein a plurality of bismuth oxide particles of the radiation-proof and antioxidant coating are connected to the intestinal probiotics through a metal polyphenol network layer.

7. A method for preparing armored probiotics, characterized in that, The method for preparing the armored probiotic as described in claim 6 includes the following steps: Polyphenols and trivalent cerium ions were added to ultrapure water containing suspended bismuth oxide particles to prepare the radiation-proof and antioxidant coating suspension. Intestinal probiotics are suspended in a phosphate buffer solution to obtain a bacterial suspension. The anti-radiation and antioxidant coating suspension is then mixed with the bacterial suspension to prepare armor probiotics.

8. The method for preparing armored probiotics according to claim 7, characterized in that, After mixing the radiation-proof and antioxidant coating suspension with the bacterial suspension, a small amount of polyphenols is added to the mixed solution.

9. A method for preparing armored probiotics according to claim 7 or 8, characterized in that, The volume ratio of the radiation-proof and antioxidant coating suspension to the bacterial suspension is 1:7.5 to 1:60, wherein the bacterial concentration of the bacterial suspension is 1×10⁻⁶. 9 CFU / mL ~3×10 9 CFU / mL.

10. The use of the armored probiotic as described in claim 6 in the preparation of a medicament for preventing radiotherapy-induced intestinal injury.

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