A cobalt-aluminum double hydroxide / proprionibacterium hybrid material, preparation and use thereof

Abstract

This invention discloses a cobalt-aluminum double hydroxide / Propionibacterium acnes hybrid material and its preparation and application, comprising: amplification and culture of Propionibacterium acnes; preparation of cobalt-aluminum layered double hydroxide using a co-precipitation method; mixing the cobalt-aluminum layered double hydroxide and a dispersion of Propionibacterium acnes in a certain proportion, followed by shaking, centrifugation, and washing to obtain the cobalt-aluminum layered double hydroxide-modified Propionibacterium acnes hybrid material. This method achieves highly efficient tumor treatment through the hybridization of nanomaterials and microorganisms. Propionibacterium acnes is an anaerobic and aerobic-tolerant microorganism that responds to the hypoxic environment of tumors by producing nitric oxide gas molecules in situ. These molecules synergistically react with cobalt ions generated from the degradation of the cobalt-aluminum layered double hydroxide to produce highly oxidizing peroxynitrosamine anions that kill tumors. Simultaneously, Propionibacterium acnes can promote macrophage transformation and activate immunity, improving the synergistic effect of tumor gas therapy and immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of bio-hybrid materials, and more particularly to a cobalt aluminum double hydroxide / Propionibacterium hybrid material and its preparation and application. Background Technology

[0002] Cancer has become one of the leading diseases threatening human health and life. Conventional clinical cancer treatments, including surgery, radiotherapy, and chemotherapy, all have limitations. Surgery is only effective against solid tumors and cannot effectively target metastatic tumors, nor can it completely eradicate them. Radiotherapy and chemotherapy have significant side effects and cause considerable damage to normal tissues. With ongoing research, engineered microorganisms have shown great potential in cancer treatment. Through long-term natural selection, microorganisms have evolved special functions, including tumor targeting, immune responses, and the production of anti-tumor metabolites. Furthermore, microorganisms can colonize and proliferate in tumor tissue, interfering with tumor metabolism and inhibiting tumor growth. This allows for the development of novel anti-tumor systems to overcome the shortcomings of traditional treatments. However, microorganisms are easily recognized and eliminated by the immune system, reducing treatment efficacy. Simultaneously, their metabolic behavior may produce toxic byproducts. Therefore, developing a novel microbial cancer treatment system that reduces side effects and enhances the tumor-killing ability is of great significance. Summary of the Invention

[0003] This application addresses the shortcomings of existing technologies by providing a cobalt-aluminum layered bimetallic hydroxide / Propionibacterium acnes biohybrid material, its preparation method, and its application, in order to solve the problems of weak targeting, strong side effects, and limited efficacy against tumor metastasis in conventional tumor treatment methods.

[0004] According to a first aspect of the embodiments of this application, a method for preparing a cobalt aluminum double hydroxide / Propionibacterium hybrid material is provided, comprising:

[0005] (1) Add the lyophilized powder of ATCC 11827 Propionibacterium acnes to the liquid culture medium and shake and culture for 48h. Take the culture medium and plate it on the agar solid medium to obtain single colonies. Then pick the single colonies from the solid medium and amplify them in the liquid culture medium for 28-36h. Mix the culture medium with glycerol at a volume ratio of 7:3 and dispense it into sterile centrifuge tubes. Store it in a -80℃ low temperature freezer to obtain the cryopreservation solution of Propionibacterium acnes.

[0006] (2) Thaw the Propionibacterium acnes cryopreservation solution in a water bath. After thawing, shake the Propionibacterium acnes cryopreservation solution in liquid culture medium for 20-28 hours, then disperse it in ultrapure water to achieve a concentration of 10. 8 A dispersion of Propionibacterium acnes was obtained at CFU / mL;

[0007] (3) Prepare solution A by mixing 25% ammonia water and ultrapure water at a volume ratio of 1:25. Prepare solution B by dissolving Co(NO3)2·6H2O and Al(NO3)3·9H2O in ultrapure water at a molar ratio of Co / Al of 2:1. The total mass of Co(NO3)2·6H2O and Al(NO3)3·9H2O to the mass ratio of ultrapure water is 1:25-30. Add solution B to solution A at a constant rate of 20 mL / h under stirring at 2800-3600 rpm to obtain the product. Sonicate and wash the product. Finally, disperse the product in ultrapure water to obtain cobalt aluminum layered bimetallic hydroxide colloid.

[0008] (4) The Propionibacterium acnes dispersion obtained in step (2) and the cobalt-aluminum layered bimetallic hydroxide colloid obtained in step (3) are mixed at 10 9 CFU and 100 μg were mixed, shaken, centrifuged and washed to obtain cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material.

[0009] Specifically, in step (2), the Propionibacterium acnes cryopreservation solution is thawed rapidly in a 37°C water bath before use.

[0010] Specifically, in step (2), the oscillation culture rate of Propionibacterium acnes is 100-200 rpm and the temperature is 37°C.

[0011] Specifically, in step (3), the product is sonicated for 1 hour at a frequency of 40 kHz, and it needs to be rinsed twice with ultrapure water.

[0012] Specifically, in step (4), the oscillation rate is 100-200 rpm and the oscillation time is 10-20 min.

[0013] According to a second aspect of the embodiments of this application, a cobalt-aluminum layered bimetallic hydroxide / Propionibacterium acnes biohybrid material prepared by the preparation method described in the first aspect is provided.

[0014] According to a third aspect of the embodiments of this application, the cobalt-aluminum layered bimetallic hydroxide / Propionibacterium acnes biohybrid material of the second aspect is provided for use in the preparation of tumor gas molecular therapeutic agents and immunotherapeutic agents.

[0015] The beneficial effects of this invention are as follows:

[0016] First, the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material provided by this invention allows Propionibacterium acnes, with its anaerobic properties, to colonize the hypoxic environment of tumors and produce the metabolic product nitric oxide (NO) gas molecules. In the acidic environment of tumor tissue, the cobalt-aluminum layered bimetallic hydroxide (cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes) on the surface of Propionibacterium acnes degrades, generating a large amount of Co... 2+ It inhibits the activity of superoxide dismutase (SOD), thereby reducing the superoxide anion (O2) content. .- The level rises, and excess superoxide anions react with nitric oxide to produce highly oxidizing peroxynitrosoanions (ONO). - This leads to DNA damage and kills tumor cells. Active substances in the cell wall of *Propionibacterium acnes*, such as teichoic acid and lipopolysaccharide, can act as immunomodulators to stimulate the body to produce inflammatory factors, leading to immune killing of tumors.

[0017] Secondly, this invention utilizes cobalt-aluminum layered bimetallic hydroxide to modify *Propionibacterium acnes* via electrostatic adsorption to achieve tumor gas molecule therapy and immunotherapy. To date, no tumor therapeutic agent has been developed in this field that utilizes live *Propionibacterium acnes* to generate nitric oxide gas molecules in situ during tumor tissue metabolism; this invention fills this gap. The preparation method of this invention has advantages such as simple operation, readily available raw materials, good stability, good safety, and mass production capabilities.

[0018] Third, the use of cobalt-aluminum layered bimetallic hydroxide to modify *Propionibacterium acnes* reduces the efficiency of the immune system in clearing the microorganisms, prolongs their circulation time in the body, and facilitates more efficient accumulation at the tumor site. Simultaneously, the cobalt-aluminum layered bimetallic hydroxide degrades in an acidic environment, releasing cobalt ions, which synergistically work with nitric oxide to address the limited therapeutic effect of nitric oxide alone, further enhancing the killing efficiency. Utilizing the live microorganism *Propionibacterium acnes* to colonize the tumor site and metabolize in situ can continuously produce nitric oxide, also solving the problem of other nitric oxide gaseous molecular therapeutic agents being limited by reactant concentrations. Attached Figure Description

[0019] Figure 1 The images shown are scanning electron microscope (SEM) images of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material in the embodiments of the present invention; wherein, (a) is a low-magnification SEM image, (b) is a high-magnification SEM image, and (c) is a transmission electron microscope (TEM) image.

[0020] Figure 2 The energy spectrum of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material in the embodiments of the present invention is shown in Figures (b), (c), (d), (e), and (f), which are the energy spectra of Co, Al, O, C, and N elements, respectively.

[0021] Figure 3 The Zeta potential diagrams of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes, Propionibacterium acnes and cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material in the embodiments of the present invention are shown.

[0022] Figure 4 The X-ray diffraction patterns of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes, Propionibacterium acnes and cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material in the embodiments of the present invention are shown.

[0023] Figure 5 This invention relates to in vitro Co-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid materials at different pH values. 2+ Release performance graph;

[0024] Figure 6 The diagram shows the in vitro nitric oxide gas generation performance of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes, the Propionibacterium acnes and the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material in the embodiments of the present invention.

[0025] Figure 7 This is a graph showing the cell survival rate analysis of 4T1 cells co-cultured under hypoxic conditions with different concentrations of Propionibacterium acnes and Propionibacterium acnes modified with cobalt aluminum layered bimetallic hydroxide in the embodiments of the present invention.

[0026] Figure 8 This is a graph showing the cell viability analysis of 4T1 cells in this embodiment of the invention after co-culturing with different concentrations of cobalt aluminum layered double metal hydroxide modified Propionibacterium acnes under hypoxic conditions and after adding nitric oxide scavenger (heme Hb, concentration of 10 μM) and reactive oxygen species scavenger (N-acetyl-L-cysteine ​​NAC, concentration of 100 μM).

[0027] Figure 9 This is a graph showing the analysis of superoxide dismutase activity in 4T1 cells after co-culturing with different concentrations of cobalt ions in an embodiment of the present invention.

[0028] Figure 10 This image illustrates the transformation of macrophages after co-culturing with Propionibacterium acnes modified with cobalt aluminum layered bimetallic hydroxide in this embodiment of the invention, to characterize the stimulatory transformation effect on macrophages; wherein (a) is a bright-field image of macrophages in the control group, and (b) and (c) are bright-field images of macrophages after co-culturing with Propionibacterium acnes and Propionibacterium acnes modified with cobalt aluminum layered bimetallic hydroxide.

[0029] Figure 11 This is a graph showing the changes in body weight of mice in each group during the in vivo experiment in this embodiment of the invention.

[0030] Figure 12 This is a bar chart showing the tumor weight of mice in each group during in vivo experiments in this embodiment of the invention. Detailed Implementation

[0031] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0032] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples.

[0034] The purpose of this invention is to provide a cobalt-aluminum layered bimetallic hydroxide modified *Propionibacterium acnes* biohybrid material, its preparation method, and its applications. *Propionibacterium acnes* is an anaerobic bacterium; in the hypoxic environment of tumor tissue, denitrification can produce nitric oxide gas molecules. In the acidic environment of tumor tissue, the cobalt-aluminum layered bimetallic hydroxide on the surface of *Propionibacterium acnes* degrades, releasing cobalt ions. These cobalt ions can inhibit superoxide dismutase activity, increasing the content of superoxide anions. Superoxide anions react with nitric oxide to produce highly oxidizing peroxynitrosoanions, leading to DNA damage and killing tumor cells. Simultaneously, *Propionibacterium acnes* can promote macrophage transformation, activate immunity, and achieve synergistic immunotherapy. In summary, this material system can target tumor tissue and utilize the metabolic characteristics of microorganisms to specifically kill tumor cells while simultaneously achieving tumor immunotherapy, which is of great significance in the field of biohybrid materials.

[0035] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0036] Example 1: This example provides the preparation of a cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material, including: step (1), preparation of cobalt-aluminum layered bimetallic hydroxide colloid; specifically, 0.8 mL of 25% ammonia solution is added to 20 mL of ultrapure water to prepare solution A, and 0.96 mmol Co(NO3)2·6H2O and 0.48 mmol Al(NO3)3·9H2O are added to 19.2 mL of ultrapure water to prepare solution B; the mass ratio of Co(NO3)2·6H2O and Al(NO3)3·9H2O to ultrapure water is 1:25-30; solution B is added to solution A at a constant rate of 20 mL / h under stirring at 3000 rpm, followed by sonication for 1 hour at a frequency of 40 kHz. The mixture is washed twice with ultrapure water, and the product is dispersed in 5 mL of ultrapure water to obtain cobalt-aluminum layered bimetallic hydroxide colloid. The stirring speed range is 2800-3600 rpm, preferably 3000 rpm;

[0037] Step (2): Prepare Propionibacterium acnes dispersion;

[0038] Specifically, the lyophilized powder of ATCC 11827 Propionibacterium acnes strain was added to liquid culture medium and shaken for 48 hours. After the culture was taken, it was plated on agar solid medium to obtain single colonies. Then, single colonies were picked from the solid medium and amplified in liquid medium for 28-36 hours. The culture was mixed with glycerol at a volume ratio of 7:3 and dispensed into sterile centrifuge tubes and stored in a -80℃ freezer to obtain the cryopreservation solution of Propionibacterium acnes.

[0039] The cryopreserved Propionibacterium acnes solution was cultured in liquid medium at 37°C with shaking at 180 rpm for 24 h, and then dispersed in ultrapure water to a concentration of 10. 8 The concentration of CFU / mL yields a dispersion of Propionibacterium acnes; the oscillation rate is in the range of 100-200 rpm, preferably 180 rpm; the oscillation time is in the range of 20-28 h, preferably 24 h.

[0040] Step (3): Take a certain amount of cobalt-aluminum layered bimetallic hydroxide colloid, add it to the Propionibacterium acnes dispersion, shake for a period of time, centrifuge and wash to obtain cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes bio-hybrid material;

[0041] Specifically, 20 μL of 5 mg / mL cobalt-aluminum layered bimetallic hydroxide colloid was added to 10 mL of 10×10 8The mixture was added to a CFU / mL Propionibacterium acnes dispersion and shaken at 100 rpm for 10 min, followed by centrifugation and washing. The shaking rate ranged from 100 to 200 rpm, preferably 150 rpm; the shaking time ranged from 10 to 20 min, preferably 15 min.

[0042] like Figure 1 As shown, the surface of *Propionibacterium acnes* is clearly coated with nanoparticles, indicating that the cobalt-aluminum layered bimetallic hydroxide was successfully adsorbed onto the surface of *Propionibacterium acnes*. Figure 2 As shown, the presence of abundant cobalt and aluminum elements on the material surface indicates the successful loading of cobalt-aluminum layered bimetallic hydroxide-modified Propionibacterium acnes; Figure 3 As shown, the surface potential of *Propionibacterium acnes* is negative, while the surface potential of cobalt-aluminum layered bimetallic hydroxide is positive. The potential of the material increases after loading *Propionibacterium acnes* modified with cobalt-aluminum layered bimetallic hydroxide, indicating that *Propionibacterium acnes* modified with cobalt-aluminum layered bimetallic hydroxide can be modified through electrostatic adsorption. Figure 4 As shown, the modified material contains a phase of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes, indicating that the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes successfully modified Propionibacterium acnes.

[0043] Example 2: This example provides the preparation of a cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material, including:

[0044] Step (1), preparation of cobalt-aluminum layered bimetallic hydroxide colloid;

[0045] Specifically, solution A was prepared by adding 1.6 mL of ammonia solution to 40 mL of ultrapure water, and solution B was prepared by adding 1.92 mmol Co(NO3)2·6H2O and 0.96 mmol Al(NO3)3·9H2O to 38.4 mL of ultrapure water. Solution B was added to solution A at a constant rate of 20 mL / h while stirring at 3000 rpm, followed by sonication for 1 hour at a frequency of 40 kHz. After washing twice with ultrapure water, the product was dispersed in 10 mL of ultrapure water to obtain a cobalt-aluminum layered bimetallic hydroxide colloid.

[0046] Step (2): Prepare Propionibacterium acnes dispersion;

[0047] Specifically, the cryopreserved Propionibacterium acnes solution was cultured in liquid culture medium at 180 rpm at 37 °C for 24 h, and then dispersed in ultrapure water to achieve a concentration of 2 × 10⁻⁶. 8 CFU / mL was used to obtain a dispersion of Propionibacterium acnes.

[0048] Step (3): Take a certain amount of cobalt-aluminum layered bimetallic hydroxide colloid, add it to the Propionibacterium acnes dispersion, shake for a period of time, centrifuge and wash to obtain cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes bio-hybrid material;

[0049] Specifically, 40 μL of 5 mg / mL cobalt-aluminum layered bimetallic hydroxide colloid was added to 20 mL of 10×10 8 The mixture was added to a CFU / mL Propionibacterium acnes dispersion, oscillated at 100 rpm for 10 min, and then centrifuged and washed.

[0050] Example 3: This example provides the application of the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material described in any one of Examples 1-2 above in nitric oxide gas therapy and immunotherapy for tumors.

[0051] The cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material described in any one of Examples 1-2 was used at 10 9 CFU / mL was dissolved in phosphate buffer solution, and the resulting preparation was stored at 4 °C. It was administered via tail vein injection in mice for treatment.

[0052] like Figure 5 As shown, under acidic conditions, the degradation of *Propionibacterium acnes* modified by cobalt-aluminum layered bimetallic hydroxide releases Co2+. Figure 6 As shown, the hybrid material of Propionibacterium acnes and cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes can continuously generate nitric oxide gas molecules.

[0053] Example 4: This experiment demonstrates the application of the material in tumor therapy by showcasing its killing effect on tumor cells in vitro. The selected cells were 4T1 mouse breast cancer cells, such as... Figure 7 As shown, after co-culturing cells with different concentrations of *Propionibacterium acnes* and *Propionibacterium acnes* hybrid material modified with cobalt-aluminum layered bimetallic hydroxide, the cytotoxicity of *Propionibacterium acnes* modified with cobalt-aluminum layered bimetallic hydroxide was significantly stronger than that of *Propionibacterium acnes*, indicating the synergistic effect of *Propionibacterium acnes* modified with cobalt-aluminum layered bimetallic hydroxide. Figure 8 As shown, the use of nitric oxide scavengers and reactive oxygen species scavengers can reduce the killing ability of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material, indicating that the killing mechanism of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material is caused by nitric oxide and reactive oxygen species.

[0054] like Figure 7 As shown, the bactericidal activity of *Propionibacterium acnes* modified with cobalt-aluminum layered bimetallic hydroxide is stronger than that of *Propionibacterium acnes* alone, indicating a synergistic effect between the two. Figure 8 As shown, the addition of nitric oxide scavengers and reactive oxygen species scavengers under hypoxic conditions increased cell survival, indicating that the killing mechanism of *Propionibacterium acnes* modified by cobalt-aluminum layered bimetallic hydroxide originates from the action of nitric oxide gas and reactive oxygen species; Figure 9 As shown, co-culturing with cobalt ions reduced the activity of superoxide dismutase; Figure 10 As shown in the figure, (a) is the control group, and (b) and (c) are the changes in the morphology of macrophages after co-culturing with Propionibacterium acnes and Propionibacterium acnes modified with cobalt aluminum layered bimetallic hydroxide, with the appearance of pseudopodia. This indicates that Propionibacterium acnes and Propionibacterium acnes modified with cobalt aluminum layered bimetallic hydroxide can transform macrophages into M1 type and have an immunostimulatory effect.

[0055] Example 5: This experiment demonstrates the therapeutic effect of the material on tumors by constructing a tumor model. Mice that developed tumors after being injected with 4T1 tumor cells were randomly divided into six groups. On days 0, 4, and 8 after tumor implantation, the mice in each group were treated as follows: Group 1 received a tail vein injection of 100 μL PBS solution; Group 2 received a tail vein injection of 100 μL cobalt-aluminum layered double hydroxide colloid (100 μg / mL); and Group 3 received a tail vein injection of 100 μL Propionibacterium acnes suspension (10 μg / mL). 9 CFU / mL), the fourth group received a tail vein injection of 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 CFU / mL). 9 CFU / mL), the fifth group received an intratumoral injection of 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 9 CFU / mL), the sixth group received an intratumoral injection of 100 μL of pre-inactivated cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 CFU / mL). 9 (CFU / mL), mouse body weight and tumor size were measured regularly. After 14 days, the tumor was removed and its weight was measured as follows: Figure 11 , Figure 12 As shown. Figure 11 As shown in the figure, the body weight of mice in each group gradually increased, indicating that the above treatment had no obvious side effects. Figure 12 As shown, the tumors with the cobalt-aluminum layered bimetallic hydroxide-modified Propionibacterium acnes hybrid material injected via tail vein and intratumoral injection showed the smallest relative tumor volume growth, indicating that it has a significant tumor-suppressing effect.

[0056] like Figure 11 As shown in the figure, the groups are respectively injected via tail vein with 100 μL of PBS solution, 100 μL of cobalt aluminum layered double metal hydroxide colloid (100 μg / mL), and 100 μL of Propionibacterium acnes suspension (10 μg / mL). 9CFU / mL), 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10) was injected via tail vein. 9 CFU / mL), intratumoral injection of 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 9 CFU / mL), intratumoral injection of 100 μL of pre-inactivated cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 9 (CFU / mL). The body weight of mice in each group gradually increased, indicating that the above treatment had no significant side effects on the mice.

[0057] like Figure 12 As shown in the figure, the groups are respectively injected via tail vein with 100 μL of PBS solution, injected via tail vein with 100 μL of cobalt-aluminum layered bimetallic hydroxide colloid (100 μg / mL), and injected intratumorally with 100 μL of pre-inactivated cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 μg / mL). 9 CFU / mL), 100 μL of Propionibacterium acnes suspension was injected via tail vein (10 9 CFU / mL), 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10) was injected via tail vein. 9 CFU / mL), intratumoral injection of 100 μL of cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material (10 9 (CFU / mL). The tumor weight of mice treated with cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material via tail vein injection and intratumoral injection was significantly lower than that of the control group and other treatment groups, indicating that the cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes hybrid material can effectively inhibit tumors.

[0058] As can be seen from the above embodiments, the present invention improves the specificity and targeting of nanomaterials, and at the same time greatly enhances the inhibitory effect of nitric oxide gas molecular therapy and immunotherapy on tumors.

[0059] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application.

[0060] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this application are indicated by the following claims.

[0061] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this application is limited only by the appended claims.

Claims

1. A method for preparing a cobalt-aluminum double hydroxide / Propionibacterium hybrid material, characterized in that, include: (1) Add the lyophilized powder of ATCC 11827 Propionibacterium acnes to the liquid culture medium and shake and culture for 48h. Take the culture medium and plate it on the agar solid medium to obtain single colonies. Then pick the single colonies from the solid medium and amplify them in the liquid culture medium for 28-36h. Mix the culture medium with glycerol at a volume ratio of 7:3 and dispense it into sterile centrifuge tubes. Store it in a -80℃ low temperature freezer to obtain the cryopreservation solution of Propionibacterium acnes. (2) Thaw the Propionibacterium acnes cryopreservation solution in a water bath. After thawing, shake the Propionibacterium acnes cryopreservation solution in liquid culture medium for 20-28 hours, then disperse it in ultrapure water to achieve a concentration of 10. 8 A dispersion of Propionibacterium acnes was obtained at CFU / mL; (3) Prepare solution A by mixing 25% ammonia water and ultrapure water at a volume ratio of 1:

25. Prepare solution B by dissolving Co(NO3)2·6H2O and Al(NO3)3·9H2O in ultrapure water at a Co / Al molar ratio of 2:

1. The total mass of Co(NO3)2·6H2O and Al(NO3)3·9H2O is in a mass ratio of 1:25-30 to ultrapure water. Add solution B to solution A at a constant rate of 20 mL / h under stirring at 2800-3600 rpm to obtain the product. Sonicate and wash the product. Finally, disperse the product in ultrapure water to obtain cobalt aluminum layered bimetallic hydroxide colloid. (4) The Propionibacterium acnes dispersion obtained in step (2) and the cobalt-aluminum layered bimetallic hydroxide colloid obtained in step (3) are mixed at 10 9 CFU and 100 μg were mixed, shaken, centrifuged and washed to obtain cobalt-aluminum layered bimetallic hydroxide modified Propionibacterium acnes biohybrid material.

2. The method for preparing a cobalt-aluminum double hydroxide / Propionibacterium hybrid material according to claim 1, characterized in that, In step (2), the Propionibacterium acnes cryopreservation solution is thawed rapidly in a 37°C water bath before use.

3. The method for preparing a cobalt-aluminum double hydroxide / Propionibacterium hybrid material according to claim 1, characterized in that, In step (2), the oscillation rate of Propionibacterium acnes is 100-200 rpm and the temperature is 37℃.

4. The method for preparing a cobalt-aluminum double hydroxide / Propionibacterium hybrid material according to claim 1, characterized in that, The product in step (3) is sonicated for 1 hour at a frequency of 40 kHz; and it needs to be rinsed twice with ultrapure water.

5. The method for preparing a cobalt-aluminum double hydroxide / Propionibacterium hybrid material according to claim 1, characterized in that, In step (4), the oscillation rate is 100-200 rpm and the oscillation time is 10-20 min.

6. A cobalt-aluminum layered bimetallic hydroxide / Propionibacterium acnes biohybrid material prepared by the preparation method according to any one of claims 1-5.

7. The application of the cobalt-aluminum layered bimetallic hydroxide / Propionibacterium acnes biohybrid material according to claim 6 in the preparation of gaseous molecular therapeutic agents and immunotherapeutic agents for tumors, wherein the tumor is breast cancer.

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