A self-driven BCG-nanoprotease compound and a preparation method thereof

Abstract

The application provides a self-driven BCG-nanoparticle complex and a preparation method thereof, and belongs to the fields of biological medicine and nanomaterials. The complex is a composite of BCG and cerium oxide nanoparticles with urease activity, the cerium oxide nanoparticles are coupled to the surface of the BCG in an asymmetric distribution through modified polyethyleneimine, and specifically, the surface of the BCG is connected to several modified polyethyleneimine derivatives through chemical bonds and electrostatic adsorption, and the polyethyleneimine derivatives wrap the cerium oxide nanoparticles through electrostatic adsorption. The complex can be self-driven by using the gas release and ion concentration change caused by the catalytic decomposition of urea in the environment, significantly enhances the adhesion and retention of BCG on the bladder wall, effectively overcomes the defects of low delivery efficiency and insufficient immune activation of traditional BCG therapy, simultaneously improves the tumor immune microenvironment, and can be used for preparing high-efficiency and low-toxicity bladder cancer treatment drugs.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomaterials, specifically to a complex of metal oxide nanozymes and microbial BCG vaccine, and its preparation method. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors worldwide, with non-muscle-invasive bladder cancer (NMIBC) accounting for approximately 75% of newly diagnosed bladder cancer cases. Currently, intravesical instillation of Bacillus Calmette-Guérin (BCG) is the standard first-line adjuvant immunotherapy after transurethral resection of NMIBC. BCG, an attenuated bovine mycobacterium, can induce local inflammatory responses and activate various immune cells (such as macrophages and T lymphocytes), thereby eliminating residual tumor cells and inhibiting recurrence. However, BCG therapy still has significant limitations: firstly, some patients do not respond to BCG treatment, and a considerable number experience recurrence after treatment; secondly, BCG instillation is often accompanied by a series of local or systemic adverse reactions, such as cystitis, hematuria, urinary frequency, and flu-like symptoms, severely affecting patient tolerance and adherence. Therefore, improving the efficacy of BCG-mediated bladder cancer treatment by reducing the frequency and dosage of instillation is of great significance in alleviating the suffering of bladder cancer patients.

[0003] One of the key reasons for the insufficient efficacy of BCG is its low intravesical delivery efficiency. BCG itself lacks voluntary motility, relying on gravity settling and passive diffusion to contact the bladder wall. However, the bladder, as a hollow organ for storing and periodically emptying urine, constantly flushes away BCG due to the flow of urine and the act of urination. This results in a large number of bacteria being cleared before they can effectively adhere to the bladder mucosa, leading to insufficient immune activation signals and ultimately reducing treatment effectiveness. Therefore, improving BCG motility and thus its delivery efficiency to the bladder mucosa is a crucial issue.

[0004] Nanomotors are miniature devices capable of autonomous movement at the nanoscale. They are typically composed of nanomaterials (such as metals, metal oxides, and polymers) and can generate propulsion through chemical reactions, physical actions, or external stimuli (such as light, magnetic fields, and ultrasound), thus achieving autonomous movement. For example, Sei Kwang Hahn et al. developed a STING agonist nanomotor system driven by natural urease, which can move autonomously, effectively penetrate the bladder wall, activate local immune responses, and exhibit significant anti-tumor effects in a bladder cancer model, providing a new platform for next-generation immunotherapy of bladder cancer.

[0005] However, although the aforementioned studies revealed that urease can drive nanosystems, enabling them to move autonomously, researchers found that this natural urease cannot drive bacterial autonomous movement by modifying the surface of bacteria such as BCG. This is because this uniformly sized and evenly dispersed natural protein is a homogeneous material, difficult to modify non-uniformly onto bacterial surfaces, thus preventing autonomous movement of BCG. Therefore, developing asymmetrically modified urease active substances in complex with BCG is of great significance for improving the motility of BCG. Summary of the Invention

[0006] To address the shortcomings of existing BCG therapies, which result in low delivery efficiency of BCG in the bladder due to its lack of autonomous motility, the primary objective of this invention is to provide a novel complex of a urease-active nanozyme and BCG. Through asymmetric modification of the BCG surface with the nanozyme, the urease activity of the nanozyme can provide an effective driving force for BCG, endowing it with autonomous motility and enhancing its adhesion and accumulation on the bladder wall, thereby improving its immunotherapeutic effect on bladder cancer.

[0007] Another object of the present invention is to provide a method for preparing the complex.

[0008] Another object of the present invention is to provide the use of the compound in the treatment of bladder cancer.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a self-driven BCG-nanozyme complex, wherein the complex is BCG and cerium oxide (CeO₂) with urease activity. x A composite of nanoparticles, wherein CeO x Nanoparticles are coupled to the BCG surface via modified polyethyleneimine derivatives and are asymmetrically distributed. Specifically, in the composite, the BCG surface is connected to several modified polyethyleneimine derivatives through chemical bonds and electrostatic adsorption, and the polyethyleneimine derivatives encapsulate CeO through electrostatic adsorption. x Nanoparticles. The CeO x The value of X in the equation is between 1.5 and 2.0.

[0011] In the complex described in this invention, the BCG vaccine is a live attenuated bovine tuberculosis bacillus.

[0012] The inventors discovered through research that, although asymmetric modified CeO x Nanoparticles can provide the driving force for BCG, but a strong bond between the two needs to be achieved through polyethyleneimine as a connecting structure. If the proportion of polyethyleneimine in the composite is too low, CeO2... xThe nanoparticles lack sufficient binding force to BCG, failing to provide adequate driving force. However, due to the toxicity of polyethyleneimine to BCG, an excessively high proportion of polyethyleneimine in the complex can impair BCG activity, also hindering treatment. Therefore, it is necessary to combine the polyethyleneimine derivative in the complex with CeO₂. x The ratio of nanoparticles to BCG must be controlled within a reasonable range to ensure sufficient CeO₂. x Nanoparticles provide the driving force, while it is also necessary to minimize the negative impact of the polyethyleneimine derivative of the linking structure on BCG activity. Therefore, in the preferred composite of this invention, the CeO2... x The ratio of nanoparticles, polyethyleneimine derivatives, and BCG ranges from 1 mg:0.1 to 10 mg:10. 8 ~10 10 Between CFU; preferably 1 mg : 0.2~5 mg : 10 8 ~10 10 CFU; preferably 1 mg: 0.2~5 mg: 5×10 8 ~10 10 CFU, optimal ratio: 1 mg : 0.2~0.5 mg : 5 × 10 8 ~2×10 9 CFU.

[0013] In a second aspect, the present invention provides a method for preparing the complex described in the first aspect, comprising the following steps:

[0014] 1) Polyethyleneimine modified with phenylboronic acid (PEI-PBA) and CeO x Nanoparticles were mixed and incubated at room temperature, and spontaneously adsorbed and encapsulated on CeO by the electrostatic interaction of polyethyleneimine chains. x Surface, CeO x @PEI-PBA;

[0015] 2) Mix the BCG suspension with the CeO2 obtained in 1). x After mixing with the PEI-PBA dispersion and incubating at room temperature, the esterification reaction between BCG and CeO2 occurs via the phenylboronic acid groups and the cis-diol structure on the BCG cell wall surface. x @PEI-PBA binding; the self-driven BCG-nanozyme complex described above is obtained, denoted as "BCG@CeO". x -PEI-PBA".

[0016] In the preparation method described in this invention, 1) the polyethyleneimine structure in the PEI-PBA is linear, branched, or hyperbranched, preferably linear.

[0017] In the preferred preparation method of the present invention, the linear polyethyleneimine structure in the PEI-PBA described in step 1) preferably has a molecular weight range of 1~100 kDa, and more preferably 1~5 kDa.

[0018] In the preparation method described in this invention, PEI-PBA can enhance CeO₂. x The nanoparticles exhibit strong binding affinity to BCG, but are also toxic to BCG. Therefore, it is necessary to combine PEI-PBA with CeO2. x The proportion of nanoparticles was controlled to a specific ratio to achieve the effect of PEI-PBA on CeO. x The loading ratio of nanoparticles is maximized. Therefore, in the method described in this invention, CeO in 1) x The mass ratio of nanoparticles to PEI-PBA is preferably 1:0.1 to 10, more preferably 1:0.1 to 1, and even more preferably 1:0.2 to 0.5.

[0019] In the preparation method described in this invention, CeO in step 2) x The lower the feed ratio of @PEI-PBA, the weaker the driving ability of nanoparticles on bacteria; CeO x The higher the feed ratio of @PEI-PBA, the greater the toxicity of the nanoparticles to bacteria. Therefore, BCG and CeO in 2) x The optimal PEI-PBA ratio is 10. 8 CFU: 50 μg ~ 500 μg, more preferably 10 μg 8 CFU: 50 μg ~ 200 μg, with a further preferred value of 10 μg. 8 CFU: 100 μg ~ 200 μg.

[0020] In the preparation method described in this invention, when the BCG is attenuated Mycobacterium bovis, the relative toxicity of PEI-PBA is higher. Besides reducing its toxicity to BCG by controlling the amount of PEI-PBA added, the preparation method of this invention first modifies nanoparticles with PEI-PBA, and then uses CeO2... x The preparation step involves mixing the PEI-PBA complex with bacteria. This method introduces less PEI-PBA than adding it to the bacterial culture first and then adding the nanoparticles, thereby further reducing the toxicity of PEI-PBA and its damaging effect on bacterial activity.

[0021] Thirdly, the present invention also provides the use of the complex described in the first aspect, or the complex prepared by the method described in the second aspect, in the preparation of a medicament for treating bladder cancer.

[0022] In a preferred application of the present invention, the complex is formulated as a suspension.

[0023] In a further preferred application of the present invention, the solvent of the suspension is any one of deionized water, physiological saline, or phosphate buffer.

[0024] In a further preferred application of the present invention, the administration route of the suspension is intravesical instillation via the urethra.

[0025] In a further preferred application of the present invention, the concentration of the complex in the suspension is 0.1~2 mg / mL, and even more preferably 0.1~0.5 mg / mL.

[0026] In this field, nanozymes are a class of functional nanomaterials with enzyme-like catalytic activity. Compared with natural proteins such as natural enzymes used in existing technologies, synthesized nanozymes are non-homogeneous materials, exhibiting a certain degree of polydispersity in solution, and can form an asymmetric distribution on the bacterial surface when modified. This invention fully utilizes this characteristic and, based on this, selects cerium oxide nanozymes with urease activity as the bacterial modification material, obtaining a BCG complex asymmetrically modified with urease-active nanozymes.

[0027] The "asymmetric modification" described in this invention specifically means that several nanomaterial particles form a non-uniform, spatially directional distribution pattern on the surface of a single BCG bacterium through physical adsorption or chemical coupling, thereby causing anisotropy of chemical or physical properties on the bacterial surface.

[0028] The BCG complex of the present invention is asymmetrically modified with urease-active nanozymes. Because the cerium oxide nanozymes on the surface possess urease activity, they can catalyze the decomposition of urea in the bladder, thereby inducing gas generation and changes in the electric field around the particles. Simultaneously, based on the asymmetric modification of the cerium oxide nanozymes on the BCG surface, the BCG gains effective propulsion when urea decomposes and gas is generated, enabling rapid autonomous movement and accumulation in the bladder wall.

[0029] Compared to existing technologies using homogeneous natural urease-mediated nanomotors, this invention employs synthetic cerium oxide nanoenzymes mediated by urease activity but lacking homogeneity. Due to the asymmetric modification of cerium oxide nanoparticles, it exhibits better propulsion capabilities, endowing BCG with excellent autonomous movement. Compared to unmodified BCG, it significantly improves adhesion and accumulation on the bladder wall, thereby reversing the tumor immunosuppressive microenvironment, enhancing the anti-tumor immune response, reducing treatment side effects, and providing a new approach to improving the clinical response rate of BCG therapy. Attached Figure Description

[0030] Figure 1This is a transmission electron microscope image of the attenuated bovine mycobacterium cultured in Example 1.

[0031] Figure 2 The image shown is a transmission electron microscope (TEM) image of the composite prepared in Example 1.

[0032] Figure 3 Fluorescence electron microscopy image of the complex prepared in Comparative Example 5.

[0033] Figure 4 This demonstrates the BCG prepared in Comparative Example 1, and the BCG@CeO prepared in Examples 1-4 and Comparative Examples 2-4. x The distance of movement of -PEI-PBA after soaking in urea solution for 6 minutes.

[0034] Figure 5 This demonstrates the distance the complex prepared in Example 1 traveled in the urea solution at different time points.

[0035] Figure 6 This demonstrates the distance the BCG prepared in Comparative Example 1 traveled in urea solution at different time points.

[0036] Figure 7 This demonstrates the distance the complex prepared in Comparative Example 5 traveled in urea solution at different time points.

[0037] Figure 8 This demonstrates the BCG prepared in Comparative Example 1, and the BCG@CeO prepared in Examples 1-4 and Comparative Examples 2-4. x Bacterial survival rate in PEI-PBA.

[0038] Figure 9 This demonstrates the distribution of the complex prepared in Example 1 in bladder tissue.

[0039] Figure 10 This demonstrates the distribution of BCG prepared in Comparative Example 1 in bladder tissue.

[0040] Figure 11 This demonstrates the distribution of the complex prepared in Comparative Example 5 in bladder tissue. Detailed Implementation

[0041] The technical problems to be solved, the technical solutions, and the beneficial effects of the present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0042] This invention provides a complex of cerium oxide nanozyme and BCG, which can be prepared by the following specific method:

[0043] (1) First, under an inert atmosphere, cerium chloride (CeCl3·7H2O) was dissolved in 5 mL of ultrapure water and stirred until completely dissolved to form a clear precursor solution. Then, 20 mL of 6 M sodium hydroxide (NaOH) solution was added to this solution, and the mixture was stirred continuously at room temperature for 30 minutes to form a precursor suspension. Next, the resulting mixture was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in a constant-temperature oven at 373 K (100 °C) for 24 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. The product was separated by ultracentrifugation (9000 rpm, 10 min) and then washed at least three times alternately with ultrapure water and anhydrous ethanol to thoroughly remove residual ions and impurities. Finally, the washed product was dried at 60 °C for 24 hours to obtain CeO2. x Nanoparticles. The molar mass of cerium chloride in this step is 1~10 mmol, more preferably 1~5 mmol, and even more preferably 2.5~5 mmol.

[0044] (2) First, the CeO prepared in step (1) x Nanoparticles were dispersed in 10 mL of PBS (pH 7.4) buffer and sonicated for 15 minutes to ensure thorough dispersion. Then, phenylboronic acid-modified polyethyleneimine (PEI-PBA conjugate) was added, and the mixture was incubated with shaking at room temperature for 4–6 hours. The nanoparticles spontaneously adsorbed and encapsulated CeO₂ using the electrostatic interaction of the polyethyleneimine chains. x Surface. CeO was then collected by centrifugation (12,000 rpm, 20 min). x The PEI-PBA complex was washed three times with PBS buffer to completely remove unbound PEI-PBA. The purified product was then redispersed in 5 mL PBS (pH 7.4) and stored at 4°C for later use. CeO2 in this step... x The mass ratio of nanoparticles to PEI-PBA is preferably 1:0.1 to 10, more preferably 1:0.1 to 1, and even more preferably 1:0.2 to 0.5.

[0045] (3) Culture the target bacteria BCG to the logarithmic growth phase (OD). 600 ≈ 0.6–0.8), the bacterial cells were collected by centrifugation (3,000 rpm, 5 min), washed twice with PBS (pH 7.4), resuspended, and the bacterial concentration was adjusted to approximately 10. 8 CFU / mL; then take the bacterial suspension and mix it with CeO2 prepared in step (2). xThe PEI-PBA dispersion was mixed and incubated with gentle shaking at room temperature for 1–2 hours to allow the phenylboronic acid groups to specifically bind to the cis-diol structure on the bacterial cell wall surface. Finally, the composite bacteria were collected by low-speed centrifugation (3,000 rpm, 3 minutes), and the precipitate was washed 2–3 times with PBS to thoroughly remove unbound nanoparticles, yielding the final complex (BCG@CeO). x (PEI-PBA) is resuspended in an appropriate amount of buffer or culture medium. BCG and CeO2 in this step... x The optimal PEI-PBA ratio is 10. 8 CFU: 10 ~ 1000 μg, more preferably 10 8 CFU: 10 ~ 200 μg, preferably 10 μg 8 CFU: 50 ~ 200 μg.

[0046] Example 1: Preparation of BCG@CeO x -PEI-PBA complex

[0047] A complex of cerium oxide nanozyme and BCG was prepared using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials. The specific steps included:

[0048] (1) First, under an inert atmosphere, 4 mmol of cerium chloride (CeCl3·7H2O) was dissolved in 5 mL of ultrapure water and stirred until completely dissolved to form a clear precursor solution. Then, 20 mL of 6 M sodium hydroxide (NaOH) solution was added to the solution, and the mixture was stirred continuously at room temperature for 30 minutes to form a precursor suspension. Next, the resulting mixture was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in a constant temperature oven at 373 K (100 °C) for 24 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. The product was separated by ultracentrifugation (9000 rpm, 10 min) and washed at least three times alternately with ultrapure water and anhydrous ethanol to thoroughly remove residual ions and impurities. Finally, the washed product was dried at 333 K (60 °C) for 24 hours to obtain pale yellow CeO2. x Nanoparticles.

[0049] (2) First, 10 mg of CeO2 prepared in advance according to the method in step (1) is added. x Nanoparticles were dispersed in 10 mL of PBS (pH 7.4) buffer and sonicated for 15 minutes to ensure thorough dispersion. Then, 5 mg of phenylboronic acid-modified polyethyleneimine (PEI-PBA conjugate) was added, and the mixture was incubated with shaking at room temperature for 4–6 hours. The nanoparticles spontaneously adsorbed and encapsulated CeO₂ using the electrostatic interactions and coordination bonds of the PEI chains.x Surface. CeO was then collected by centrifugation (12,000 rpm, 20 min). x The PEI-PBA complex was washed three times with PBS buffer to completely remove unbound PEI-PBA. Finally, the purified product was redispersed in 5 mL PBS (pH 7.4) and stored at 4°C for later use.

[0050] (3) Culture the attenuated bovine mycobacteria to the logarithmic growth phase (OD). 600 ≈ 0.6–0.8), and the bacterial cells were collected by centrifugation (3,000 rpm, 5 minutes). The microscopic morphology of the bacterial cells is as follows: Figure 1 As shown; and after washing twice with PBS (pH 7.4), resuspend and adjust the bacterial concentration to approximately 10. 8 CFU / mL; then take 1 mL of bacterial suspension and mix it with an equal volume of CeO2. x The PEI-PBA (100 μg / mL) dispersion was mixed and incubated with gentle shaking at room temperature for 1–2 hours to allow the PBA groups to specifically bind to the cis-diol structure on the bacterial cell wall surface. Finally, the composite bacteria were collected by low-speed centrifugation (3,000 rpm, 3 minutes), and the precipitate was washed 2–3 times with PBS to thoroughly remove unbound nanoparticles, yielding the BCG@CeO₂. x -PEI-PBA is resuspended in an appropriate amount of buffer or culture medium.

[0051] The BCG@CeO prepared in this embodiment x -PEI-PBA micromorphology as Figure 2 As shown, CeO x @PEI-PBA nanoparticles exhibit an asymmetric distribution on the bacterial surface.

[0052] Example 2: Preparation of BCG@CeO x -PEI-PBA complex

[0053] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 50 μg / mL; all other steps remained the same.

[0054] Example 3: Preparation of BCG@CeO x -PEI-PBA complex

[0055] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 200 μg / mL; all other steps remained the same.

[0056] Example 4: Preparation of BCG@CeO x -PEI-PBA complex

[0057] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 500 μg / mL; all other steps remained the same.

[0058] Comparative Example 1: Unmodified attenuated Mycobacterium bovis

[0059] Attenuated bovine mycobacteria were cultured to the logarithmic growth phase (OD). 600 ≈ 0.6-0.8), the bacterial cells were collected by centrifugation (3,000 rpm, 5 minutes), washed twice with PBS (pH 7.4), resuspended, and the bacterial concentration was adjusted to approximately 10. 8 CFU / mL.

[0060] The microstructure of the bacteria prepared in this comparative example is as follows: Figure 1 As shown, the attenuated bovine mycobacterium has a rod-shaped structure and a smooth surface.

[0061] Comparative Example 2: Preparation of BCG@CeO x -PEI-PBA complex

[0062] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 10 μg / mL; all other steps remained the same.

[0063] Comparative Example 3: Preparation of BCG@CeO x -PEI-PBA complex

[0064] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 20 μg / mL; all other steps remained the same.

[0065] Comparative Example 4: Preparation of BCG@CeO x -PEI-PBA complex

[0066] Using attenuated Mycobacterium bovis, cerium chloride, and phenylboronic acid-modified polyethyleneimine as the main raw materials, a complex of cerium oxide nanozyme and BCG was prepared. The overall method was the same as in Example 1, except that in step (3), the concentration of BCG was approximately 10... 8 An equal volume of CeO3 was mixed with 1 mL of bacterial culture containing CFU / mL. x The concentration of the PEI-PBA dispersion was adjusted to 1000 μg / mL; all other steps remained the same.

[0067] Comparative Example 5: Preparation of BCG with surface-modified urease (BCG@Ure)

[0068] BCG was cultured in a medium containing 50 μM pentadecanoic acid azide (N3-C15) until OD. 600 The bacterial cell concentration was reached to 0.6–0.8, and then collected by centrifugation. Natural urease was dissolved in PBS (pH 7.4) to a concentration of 2–5 mg / mL; simultaneously, DBCO-PEG4-NHS Ester was dissolved in anhydrous DMSO to prepare a 10–20 mM solution. Under light-protected conditions, DBCO solution was added to the urease solution at a DBCO to urease molar ratio of 10:1, and the reaction was carried out at 4°C or room temperature for 2–4 hours. Subsequently, the labeled and washed azide-treated BCG was resuspended in PBS to a concentration of 1 × 10⁻⁶ mg / mL. 8 CFU / mL. Mix 100 μL of this bacterial culture with 100 μL of alkyneylurease, and bring the volume to 500 μL with PBS. Incubate at 37°C with shaking for 12 hours. Finally, obtain the modified bacteria by centrifugation and washing three times. The microstructure of the bacteria prepared in this comparative example is shown below. Figure 3 As shown, urease (green fluorescent label) is uniformly modified on attenuated Mycobacterium bovis.

[0069] Self-driving capability detection:

[0070] First, a 5 mM urea working solution was prepared using PBS buffer (pH 7.4), with pure PBS buffer as a control. Then, sample preparation was performed. The complex samples prepared in Examples 1-4 and Comparative Examples 2-5, along with the attenuated Mycobacterium bovis sample cultured in Comparative Example 1, were gently resuspended and washed twice with their respective liquids (urea working solution or PBS) to thoroughly remove the original culture medium. Finally, the samples were adjusted with the appropriate liquids to ensure good particle dispersion for easy tracking under the microscope. Finally, image acquisition was performed. 5 μL of sample was dropped onto a glass slide, covered with a coverslip, and gently pressed down to reduce liquid flow. The prepared sample was immediately placed on the microscope stage and allowed to stand for 30 seconds until the liquid was completely stable. Then, a video sequence of 30-60 seconds was recorded at 100 frames per second using a high-speed camera under multiple random fields of view for subsequent analysis. The experimental results are as follows: Figures 4-7 As shown.

[0071] Figure 4 In the middle, the horizontal axis is 0 μg / 10 8 CFU, 10 μg / 10 8 CFU, 20 μg / 10 8 CFU, 50 μg / 10 8 CFU, 100 μg / 10 8 CFU, 200 μg / 10 8 CFU, 500 μg / 10 8 CFU and 1000 μg / 10 8 The CFU feed ratios were compared with those of eight samples prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 2, Example 1, Example 3, Example 4, and Comparative Example 4, respectively. It can be seen that the driving ability of the complex increases with CeO₂ content. x The feed ratio of @PEI-PBA initially increases and then decreases. This trend may stem from two reasons: firstly, when the feed ratio is low, the driving force generated by the complex is insufficient to effectively promote bacterial motility; secondly, when the feed ratio is too high, its toxicity significantly inhibits bacterial activity, which is also detrimental to bacterial motility. Therefore, CeO2 is preferred in this invention. x @PEI-PBA against 10 8 The amount of CFU bacteria added is 50-500 μg; more preferably 50-200 μg; and most preferably 100-200 μg.

[0072] Figures 5-7 The figures illustrate the movement distances of the samples prepared in Example 1, Comparative Example 1, and Comparative Example 5 at each same time point. It can be seen that the BCG@CeO2 prepared in Example 1... x-PEI-PBA exhibited significantly greater movement distance at any time point and greater movement speed within 10 minutes than BCG in Comparative Example 1 and BCG modified with natural urease in Comparative Example 5. Specifically, the BCG@CeO prepared in Example 1... x The motility of -PEI-PBA was 65.72 μm / min, while the motility of BCG in Comparative Example 1 was only 11.18 μm / min. The motility of BCG modified with natural urease in Comparative Example 5 was also comparable to that in Comparative Example 1. This demonstrates that asymmetric modification of CeO2... x After PEI-PBA, BCG's athletic ability was significantly enhanced.

[0073] Bacterial survival rate:

[0074] First, the complex samples prepared in Examples 1-4 and Comparative Examples 2-4, along with the attenuated Mycobacterium bovis sample cultured in Comparative Example 1, were gently resuspended and washed twice with PBS. 50-100 μL of the combined and washed bacterial suspension was taken and, according to the instructions of the live / dead staining kit, SYTO 9 and PI dye were added, and the mixture was incubated at room temperature in the dark for 15-30 minutes. 10 μL of the stained bacterial solution was placed on a glass slide, covered with a coverslip, and immediately observed under an oil immersion microscope. Live bacteria showed green fluorescence, and dead bacteria showed red fluorescence. Multiple fields of view were randomly selected for photographing and counting (at least 500 bacteria were counted). Finally, the bacterial survival rate was calculated using the following formula: Survival rate (%) = [Number of live bacteria / (Number of live bacteria + Number of dead bacteria)] × 100%.

[0075] The results are as follows Figure 8 As shown, the horizontal axis represents 0 μg / 10 8 CFU, 10 μg / 10 8 CFU, 20 μg / 10 8 CFU, 50μg / 10 8 CFU, 100 μg / 10 8 CFU, 200 μg / 10 8 CFU, 500 μg / 10 8 CFU and 1000 μg / 10 8 The CFU feed ratios correspond to the eight samples prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 2, Example 1, Example 3, Example 4, and Comparative Example 4, respectively. When CeO x -PEI-PBA feed ratio less than 100 μg / 10 8 When CFU is used, the bacterial survival rate in the composite material remains essentially unchanged, while when CeO x -PEI-PBA feed ratio greater than 100 μg / 10 8During CFU treatment, the bacterial survival rate in the composite material decreased significantly. This indicates that CeO₂... x Excessive modification with PEI-PBA is detrimental to the overall bacterial activity. Considering all factors, CeO2... x The optimal feed ratio of PEI-PBA to BCG is 50-500 μg / 10. 8 CFU.

[0076] Tissue distribution detection:

[0077] After anesthetizing the mice, a small incision was made in their lower abdomen, and the bladder was gently removed. A suspension of bladder cancer cells was injected directly into the bladder wall using a microsyringe. Immediately after needle removal, pressure was applied to the injection site to prevent leakage. The bladder was then returned to the abdominal cavity, and the wound was sutured. Two weeks later, in vivo imaging or dissection confirmed successful tumor modeling.

[0078] First, the urethral opening of the anesthetized and immobilized mice was disinfected with povidone-iodine, covered with a sterile drape, and the bladder was manually compressed to induce urination. Then, a sterile 24G catheter coated with liquid paraffin was placed against the caudal wall of the urethral opening, and the urethral opening was gently grasped with forceps, and the catheter was slowly inserted into the bladder. Next, 1 mL of mouse urine was aspirated with a syringe, followed by 100 μL of sterile saline to cleanse the bladder, which was repeated once. Then, a 4-0 silk thread was tied around the urethral opening, and 100 μL of the three drugs prepared in Comparative Example 1, Example 1, and Comparative Example 5, labeled with Rhodamine B, was slowly injected into the bladder. The thread was tightened, the catheter was removed, and the thread was tied into a slipknot. One hour after the procedure, the thread was untied, and the urethral opening was disinfected again with povidone-iodine.

[0079] Twenty-four hours later, mouse bladder tissue was removed and frozen for sectioning. The distribution of the material within the bladder tissue was then determined by observing the distribution of red fluorescence. Results are as follows: Figures 9-11 As shown.

[0080] Figures 9-11 This illustrates the distribution of samples prepared in Example 1, Comparative Example 1, and Comparative Example 5 in bladder tissue. For example... Figure 10 As shown, when Rhodamine B-labeled BCG from Comparative Example 1 was injected, a small amount of red fluorescence appeared in the inner layer of the bladder tissue, while no red fluorescence was observed in the middle and outer layers. Figure 9 As shown, when BCG@CeO prepared in Example 1 is injected... x During PEI-PBA treatment, a distinct red fluorescence was observed in the inner layer of the bladder tissue, with slight red fluorescence also present in the middle and outer layers. For example... Figure 11 As shown, when BCG@Ure, a rhodamine B-labeled comparative example 5, was injected, a small amount of red fluorescence appeared in the inner layer of the bladder tissue, while no red fluorescence was observed in the middle and outer layers. This demonstrates that CeO2... x-PEI-PBA can effectively promote the accumulation and retention of BCG in the bladder wall.

Claims

1. A self-driven BCG-nanozyme complex, characterized in that: The complex is BCG and CeO2 with urease activity. x The composite of nanoparticles, namely CeO x Nanoparticles are coupled to the BCG surface via modified polyethyleneimine derivatives and are asymmetrically distributed. Specifically, in the composite, the BCG surface is connected to several modified polyethyleneimine derivatives through chemical bonds and electrostatic adsorption, and the polyethyleneimine derivatives encapsulate CeO through electrostatic adsorption. x Nanoparticles; the CeO x The ratio of nanoparticles, modified polyethyleneimine derivatives, and BCG was 1 mg:0.1–10 mg:

10. 8 ~10 10 CFU; The complex was prepared by the following method: 1) Combine PEI-PBA with CeO x Nanoparticles were mixed at a mass ratio of 0.1–10:1 and incubated at room temperature with shaking for 4–6 h. The nanoparticles spontaneously adsorbed and encapsulated on CeO₂ using the electrostatic interaction of the polyethyleneimine chains. x Surface, CeO x @PEI-PBA; 2) Mix the BCG suspension with the CeO2 obtained in 1). x After mixing the @PEI-PBA dispersion, the mixture was incubated with shaking at room temperature for 1-2 hours, controlling the BCG and CeO content. x The PEI-PBA ratio is 10. 8 CFU: 50 μg~500 μg; BCG reacts with CeO through esterification of the phenylboronic acid group with the cis-diol structure on the surface of the BCG cell wall. x @PEI-PBA combination.

2. The complex according to claim 1, characterized in that, The BCG vaccine mentioned is a live attenuated bovine tuberculosis mycobacterium.

3. A method for preparing the complex according to any one of claims 1-2, characterized in that, Includes the following steps: 1) Using PEI-PBA and CeO x Nanoparticles were mixed and incubated with room temperature shaking for 4-6 h to control the CeO content. x The mass ratio of nanoparticles to PEI-PBA is 1:0.1~10; the nanoparticles spontaneously adsorb and encapsulate CeO through the electrostatic interaction of the polyethyleneimine chains. x Surface, CeO x @PEI-PBA; 2) Mix the BCG suspension with the CeO2 obtained in 1). x After mixing the @PEI-PBA dispersion, the mixture was incubated at room temperature with shaking for 1-2 hours, maintaining a BCG to CeOx@PEI-PBA ratio of 10:

1. 8 CFU: 50 μg~500 μg; BCG reacts with CeO through esterification of the phenylboronic acid group with the cis-diol structure on the surface of the BCG cell wall. x @PEI-PBA binding; to obtain the self-driven BCG-nanozyme complex.

4. The method as described in claim 3, characterized in that, PEI-PBA mentioned in 1) is a linear polyethyleneimine modified with phenylboronic acid, with a molecular weight range of 1~100 kDa.

5. The use of the complex according to any one of claims 1-2, or the complex prepared by the method according to any one of claims 3-4, in the preparation of a medicament for treating bladder cancer.

6. The application as described in claim 5, characterized in that: The complex was prepared into a suspension with a concentration of 0.1 to 2 mg / mL using any one of deionized water, physiological saline, or phosphate buffer.

7. The application as described in claim 6, characterized in that: The suspension is administered via intravesical instillation into the bladder cavity via the urethra.

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