Drug-loaded engineered bacillus calmette-guerin based on metal polyphenol network and preparation method and application thereof

Abstract

The application belongs to the technical field of nano drug carriers, and relates to a drug-loaded engineered bacillus calmette-guerin based on a metal polyphenol network as well as a preparation method and application thereof. The drug-loaded engineered bacillus calmette-guerin is a bacterial complex structure formed by combining the metal polyphenol network with the bacillus calmette-guerin bacterium body, and the surface of the bacterial complex structure is adsorbed with functional molecules through electrostatic action. The metal polyphenol network can effectively enhance the adhesion to the bladder epithelium and exhibit good release capacity in an acidic urine environment. The released metal ions such as manganese can activate the immune storm in cooperation with the bacillus calmette-guerin. In addition, the metal polyphenol network can also adsorb functional molecules such as chemotherapy and targeting, and deliver and release the functional molecules together with the bacillus calmette-guerin bacterium body to further kill tumor cells. The engineered bacillus calmette-guerin can effectively enhance the immune activation of the bladder cancer perfusion therapy and inhibit the tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine carrier technology, and relates to engineered BCG vaccine based on metal polyphenol networks, its preparation method, and its application. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, and the high recurrence rate after surgery for non-muscle-invasive bladder cancer (NMIBC) is a major challenge in its clinical treatment. BCG vesical instillation is currently the standard immunotherapy for preventing recurrence of high-risk NMIBC, but its efficacy is limited by two major challenges: first, a high rate of BCG non-response in 30%-50% of patients; and second, the high incidence of adverse reactions such as bladder irritation caused by instillation, leading to poor patient tolerance. The anti-tumor effect of BCG depends on activating the local immune response in the bladder, and BCG non-response is often closely related to the immunosuppressive tumor microenvironment (TME), with the inhibition of immune cell infiltration and functional suppression mediated by pathways such as the CXCL12 / CXCR4 signaling axis being one of the important factors.

[0003] While bladder instillation offers local drug delivery and reduces systemic side effects, it suffers from inherent limitations such as short drug retention time and low absorption efficiency by the bladder epithelium. Nanoparticle drug delivery technology offers a new approach to improving drug efficacy, as it loads drugs onto nanoscale carriers, potentially improving drug solubility, stability, and targeting. However, within the dynamic bladder cavity, conventional nanoparticle carriers also struggle to effectively adhere to the urothelium and achieve long-term retention, resulting in unsatisfactory drug utilization.

[0004] In recent years, advances in biomaterials science have provided new tools for addressing these problems. Metal polyphenol networks (MPNs) are a class of novel nanomaterials formed by the self-assembly of natural polyphenols (such as tannic acid) and metal ions (such as manganese ions) through coordination interactions. MPNs exhibit good biocompatibility, biodegradability, excellent bioadhesion, and a large drug loading capacity. Their adhesive properties are particularly suitable for bladder instillation, significantly prolonging drug residence time on the bladder wall. Furthermore, the components constituting MPNs themselves possess bioactivity: Mn²⁺ is an effective natural immune adjuvant that can activate the cGAS-STING pathway and enhance anti-tumor immune responses.

[0005] Therefore, developing an engineered drug delivery system based on MPN to modify BCG and simultaneously address its problems of insufficient efficacy, poor tolerability, and short residence time in the bladder is of great clinical significance and application prospects. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a drug-loaded engineered BCG vaccine based on a metal polyphenol network, which prolongs the immune response induced by BCG while reducing the concentration of BCG infusion, thereby significantly reducing the side effects of BCG in the treatment of bladder cancer.

[0007] The specific technical solution of the present invention is as follows: BCG vaccine, being a live attenuated Mycobacterium tuberculosis cell, is relatively fragile. Therefore, a construction strategy is needed to rapidly form the vaccine at neutral pH and room temperature. This invention reports a drug-loaded engineered BCG vaccine based on a metallopolyphenol network. The complex structure consists of a metallopolyphenol network combined with BCG cells, and functional molecules are adsorbed onto its surface via electrostatic interactions. By controlling the metal-polyphenol network to nucleate and grow primarily on the bacterial surface, rather than nucleating and aggregating in solution, we precisely regulated various factors.

[0008] The above-mentioned method for preparing drug-loaded engineered BCG vaccine based on metal polyphenol networks includes the following steps: S1: Culture and centrifuge the BCG vaccine to obtain a bacterial resuspension. The amount of BCG administered will affect TA and Mn. 2+ To determine the loading rate, an OD600 value of 0.4~0.6 was selected. The BCG cells were centrifuged and resuspended to obtain a bacterial resuspension. Alternatively, S1: Cultivate BCG vaccine, add polyphenol solution with a final concentration ≤0.5mM, centrifuge, and obtain a resuspension of polyphenol-treated bacteria.

[0009] S2: Polyphenols and metal ions were added to the bacterial resuspension and co-incubated to ensure the loading of the STING agonist Mn. 2+ To achieve an effective concentration and enhance the adhesiveness of the coated bacteria, polyphenols, metal ions, and coating time were screened. Tannic acid (TA) and Mn were ultimately selected. 2+ The concentration was 1 mM, the reaction time was 5 min, and after centrifugation and resuspending, BCG vaccine coated with metal polyphenol network was obtained. S3: Since MPN carries a negative charge, it can further co-deliver positively charged AMD3100. Too high a concentration of AMD3100 will cause flocculent precipitation in the solution, while too low a concentration will result in insufficient loading. Therefore, the concentration of AMD3100 was screened, and a concentration of 1 mg / ml was finally selected. Then, the functional molecules were electrostatically adsorbed, and after centrifugation and resuspending, the drug-loaded engineered BCG vaccine was obtained.

[0010] Furthermore, in S1, the BCG culture includes: culturing with 7H10 medium containing 50 μg / ml kanamycin, selecting bacteria in the logarithmic growth phase, measuring the OD600 value with an ELISA reader to be 0.4~0.6, centrifuging, and resuspending the bacteria to obtain a bacterial resuspension.

[0011] Furthermore, in S2, the polyphenol is at least one of catechol, resorcinol, hydroquinone, pyrogallol, pyrogallol, proanthocyanidins, gallic acid, tannic acid, epigallocatechin, and gallic acid esters; preferably, the polyphenol is tannic acid.

[0012] Furthermore, in S2, the metal ion is Mn. 2+ Fe 3+ Cu 2+ Zn 2+ Al 3+ Co 2+ At least one of the following; preferably, the metal ion is Mn. 2+ The metal concentration is 1–100 mmol / L, preferably 1–15 mmol / L; the polyphenol concentration is 1–100 mmol / L, preferably 1–15 mmol / L. Furthermore, in S3, the functional molecule is at least one of AMD3100, doxorubicin, gemcitabine, and mitoxantrone, and the concentration of the functional molecule is 1 to 100 mg / mL, preferably 1 to 20 mg / mL.

[0013] Furthermore, polyphenols and metal ions are added to the bacterial resuspension for co-incubation. The co-incubation reaction time is 1–60 min, preferably 1–20 min. On the other hand, the present invention also protects the use of a drug-loaded engineered BCG vaccine based on a metal polyphenol network in the preparation of drugs for treating bladder cancer.

[0014] The beneficial effects of this invention are as follows: 1. This invention effectively increases the adhesion time of the complex to the bladder epithelium by combining a metal polyphenol network with BCG, allowing BCG to exert its effect better. Compared with the traditional instillation method, the concentration of BCG used is lower and the onset time is longer. 2. The metal polyphenol network of the present invention exhibits good release capacity in an acidic urine environment, and the released metal ions can synergistically activate an immune storm with BCG. 3. The composite of the present invention can also adsorb functional molecules such as chemotherapy and targeted drugs, and co-deliver and release them with BCG cells to further kill tumor cells. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1Scanning electron microscope image (A) of the drug-loaded engineered BCG vaccine, Zeta potential image after modification (B) and fluorescence image characterizing successful encapsulation (C); Figure 2 The growth capacity of the engineered BCG vaccine loaded with drugs is shown in Figure (A), the drug encapsulation efficiency of AMD3100 is shown in Figure (B), and the release of manganese ions at different pH values ​​is shown in Figure (C). Figure 3 X-ray photoelectron spectroscopy of the surface of BCG cells successfully coated with a drug-loaded metal (manganese) polyphenol network; Figure 4 Scanning electron microscopy mapping image of the successful coating of drug-loaded metal (manganese) polyphenol network onto the surface of BCG cells; Figure 5 This study characterizes the adhesion ability of drug-loaded engineered BCG vaccine at the cellular and animal levels. Figure A shows a comparison of adhesion ability under white light; Figure B shows a comparison of adhesion ability under fluorescent BCG; Figure C shows a comparison of BCG acid-fast staining after instillation into the bladder mucosa of animals. Figure 6 To detect the killing effect of drug-loaded engineered BCG on bladder cancer cells. Figure A shows the flow cytometry of apoptosis in different treatment groups; Figure B is a statistical graph of the apoptosis rate in Figure A; Figure C is a graph of the MTT proliferation assay. Figure 7 This study investigated the activation ability of drug-loaded engineered BCG vaccine on dendritic cells. Figure A shows flow cytometry plots of CD11C+CD80+ dendritic cells induced to mature by different treatment groups; Figure B shows flow cytometry plots of CD11C+CD86+ dendritic cells induced to mature by different treatment groups. Figure 8 To evaluate the therapeutic effect of drug-loaded engineered BCG in a mouse orthotopic bladder tumor model. Figure A shows the luciferase monitoring of orthotopic bladder tumor growth in mice under different treatment groups; Figure B shows a comparison of bladder weights in mice under different treatment groups after final sacrifice; Figure C shows the tumor growth curves in mice under different treatment groups; Figure D shows the bladder weights in mice under different treatment groups. Figure 9 To detect the regulatory capacity of drug-loaded engineered BCG vaccine on the immune microenvironment using flow cytometry. Figures A and B are flow cytometry comparisons of CD8+ cytotoxic T cells; Figures CE are flow cytometry comparisons of dendritic cells; Figures F and G are flow cytometry comparisons of M1 / M2 macrophages; Figure H is a flow cytometry comparison of regulatory T cells. Detailed Implementation

[0017] The invention will now be described through specific implementation schemes.

[0018] Example 1 A method for preparing drug-loaded engineered BCG vaccine based on a metal polyphenol network, comprising the following steps: S1: BCG was cultured in 7H10 medium containing 50 μg / ml kanamycin, using BCG cells in the logarithmic growth phase. The amount of BCG added affects TA and Mn. 2+ To determine the loading rate, an OD600 value of 0.5 was selected. The BCG cells were centrifuged and resuspended to obtain a bacterial resuspension. S2: Add tannic acid and Mn to the bacterial resuspension. 2+ Co-incubation was conducted to ensure the loading of the STING agonist Mn. 2+ To achieve an effective concentration and enhance the adhesiveness of the coated bacteria, polyphenols, metal ions, and coating time were screened. Ultimately, TA and Mn were selected. 2+ The concentration was 1 mM, the reaction time was 5 min, and after centrifugation and resuspending, BCG (BCG@MT) coated with metal polyphenol network was obtained. S3: Since MPN carries a negative charge, it can further co-deliver positively charged AMD3100. Too high a concentration of AMD3100 will cause flocculent precipitation in the solution, while too low a concentration will result in insufficient loading. Therefore, the concentration of AMD3100 was screened, and a concentration of 1 mg / ml was finally selected. After electrostatic adsorption of AMD3100, the drug-loaded engineered BCG vaccine (BCG@MT-A) was obtained after centrifugation and resuspending.

[0019] Example 2 A method for preparing drug-loaded engineered BCG vaccine based on a metal polyphenol network, comprising the following steps: S1: BCG was cultured in 7H10 medium containing 50 μg / ml kanamycin. An OD600 value of 0.5 was selected. Tannic acid was added to make the final concentration 0.2 mM. The BCG cells were centrifuged and resuspended to obtain a bacterial resuspension. S2: Add TA and metal ions to the bacterial resuspension to make the final concentration of both 1 mM, incubate for 5 min, centrifuge and resuspend to obtain BCG vaccine coated with metal polyphenol network. S3: Add ADM3100 to a final concentration of 1 mg / ml. After electrostatic adsorption, centrifugation and resuspending are performed to obtain drug-loaded engineered BCG vaccine.

[0020] Example 3 Effect of MPN on BCG activity 1. The effect of MPN coating process on the number of viable BCG bacteria The resuspended BCG cells were used as a control group, and the coating effect was compared with that of Examples 1 and 2. BCG is a live, attenuated Mycobacterium tuberculosis cell, which is relatively fragile. The number of viable BCG cells after MPN coating was measured. The results are shown in Table 1. The results indicate that the polyphenol pretreatment step can significantly retain the number of viable BCG cells and prevent partial cell inactivation during the coating process.

[0021] Table 1 Comparison of Polyphenol Pretreatment Coating Effects Initial bacterial culture <![CDATA[(8.1±0.1)×10 8 ]]> 100% Example 1 <![CDATA[(5.2±0.3)×10 8 ]]> 64.2% Example 2 <![CDATA[(7.5±0.5)×10 8 ]]> 92.6%

[0022] The applicant's analysis suggests that proteins and polysaccharides in the uncentrifuged culture medium can reversibly pre-bind with polyphenols, effectively buffering the direct chemical stress of high-concentration polyphenols on the bacterial membrane surface and preventing surface property changes caused by sudden stress. Secondly, in this near-native physiological state, polyphenol molecules preferentially adsorb onto specific sites on the bacterial surface through non-covalent forces, undergoing pre-assembly. Finally, the centrifugation step precipitates the polyphenols already adsorbed on the bacterial surface, along with the bound culture medium components, creating a high local polyphenol concentration at the bacterial-solution interface. When metal ions are subsequently added, the coordination reaction at the interface proceeds synchronously, constructing a uniform metal-polyphenol network on the bacterial surface, improving coating efficiency and bacterial survival rate.

[0023] 2. Effects of MPN coating on the bioactivity of BCG vaccine Furthermore, the effect of MPN coating on the bioactivity of BCG was determined. The coated BCG was further cultured in a culture medium, and the absorbance at 600 nm was measured at different time points. The results showed that the growth curves of BCG before and after coating were consistent, and MPN coating did not affect the bioactivity of BCG. Figure 2 A). To avoid interfering with the immune response of BCG, the MPN coating can responsively degrade in the tumor microenvironment (pH 5.5), further releasing Mn. 2+ To stimulate the STING signaling pathway ( Figure 2 B and Figure 2 C).

[0024] Example 4 The drug-loaded engineered BCG vaccine prepared in Example 1 was characterized. 1. Surface morphology characterization SEM characterization was performed on BCG vaccines before and after surface modification. The surface of the blank BCG vaccine was smooth, while the surface roughness of the BCG vaccine coated with MPN increased. Figure 1 A). To ensure successful adsorption of MPN and AMD3100, zeta potential testing was performed. After MPN coating, the zeta potential of BCG decreased from -14.3 mV to -19.9 mV ( Figure 1 B). After further adsorption of AMD3100, the zeta potential increased from -19.9 mV to -15.5 mV. FITC-labeled MPN layers and inverted fluorescence microscopy revealed uniform red fluorescence around the BCG vaccine, indicating successful encapsulation. Figure 1 C).

[0025] 3. Characterization of the coating formation mechanism like Figure 3 and Figure 4 As shown, XPS testing and SEM mapping demonstrate the presence of C, O, and Mn elements, further indicating the successful formation of MPN on the surface.

[0026] Example 5 Research on cell adhesion ability To verify the enhanced cell adhesion ability of drug-loaded engineered BCG cells, bladder cancer MB49 cells were loaded with 1×10⁻⁶ cells. 5 Cells were seeded per well into 24-well plates. After 8 hours of cell adhesion, BCG was added to the BCG group, BCG@MT group, and BCG@MT-A group at a rate of 2 × 10⁻⁶ cells / well. 6 Add bacteria per well, mix thoroughly using a figure-eight shaking motion to distribute the bacteria, let stand for 2 hours, then slowly aspirate the supernatant. Add 4% paraformaldehyde for 30 minutes to fix the bacteria and cells at the bottom, then wash with PBS. White light observation was performed directly under a microscope for observation and photography. Fluorescence observation used GFP-labeled BCG bacteria. After MB49 cells adhered, Dil dye was added to make the cell outlines red; the remaining steps were the same. Results are as follows: Figure 5 As shown in A and 5B, the amount of bacterial cells with significantly increased adhesion around the cells in the BCG@MT group and the BCG@MT-A group was significantly higher than that in the BCG control group.

[0027] Example 6 Research on epithelial adhesion ability To verify the enhanced adhesion of drug-loaded engineered BCG cells to normal epithelial mucosa in the bladder, a mouse bladder instillation experiment was conducted. The specific procedures and protocols were as follows: C57 mice were anesthetized, and a 24G urinary catheter was inserted via catheterization. After urination, BCG was injected into the BCG group, BCG@MT group, and BCG@MT-A group at a dose of 5 × 10⁻⁶ mg / L. 6 Each mouse bladder was injected with 30 μL of physiological saline, fixed for 10 minutes, and then removed. Mice from different groups were sacrificed at 3, 6, 12, and 24 hours to collect bladder tissue. Bladder tissue sections were then subjected to acid-fast staining for Mycobacterium tuberculosis to observe the residual bacteria in the mucosal epithelium. Results are as follows: Figure 5As shown in Figure B, the blue arrows represent the observed BCG-adhering bacteria. In the BCG control group, no residual bacteria were observed in the bladder after 6 hours, indicating that they had been largely flushed out by urine. However, in the BCG@MT and BCG@MT-A groups, residual bacteria were still observed in the bladder epithelium until 24 hours, indicating that their ability to adhere to the epithelium was greatly enhanced.

[0028] Example 7 Study on the killing effect on bladder cancer MB49 cells To verify the killing effect of drug-loaded engineered BCG on bladder cancer cells, apoptosis and MTT assays were conducted. The apoptosis assay was performed as follows: MB49 cells were inoculated at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of 90% in 24-well plates. After cell adhesion and division to 90% confluence, PBS, BCG, BCG@MT, and BCG@MT-A groups were added to the plates, with three replicates for each group. After 24 hours of culture, the supernatant was removed, and the cells were slowly washed with PBS. Cells were collected from the plates and processed using the Annexin V-FITC / PI kit before flow cytometry analysis. Results are as follows: Figure 6 As shown in A and 6B, the number of apoptotic cells was slightly higher in the BCG group and the PBS group, while the apoptosis rate was higher in the BCG@MT group compared with the BCG group, and the apoptosis rate was the highest in the BCG@MT-A group.

[0029] The MTT experiment was performed as follows: MB49 cells were inoculated at 5 × 10⁶ cells per cell line. 3 Cells were seeded at a density per well in 96-well plates. Different treatments (same as apoptosis assays) were administered to each group of cells. Cell viability was measured using MTT assay after 24 hours, and absorbance was measured in each well using a microplate reader. The survival rate of different groups was calculated. Results are as follows: Figure 6 As shown in Figure C, cell growth in the BCG@MT-A group was most significantly inhibited, and it could counteract the growth-promoting effect of exogenous CXC12 factor on cancer cells, indicating that the AMD3100 drug released by the engineered BCG vaccine plays a key role.

[0030] Example 8 Research on the activation effect on dendritic cells To verify the release of Mn from engineered drug-loaded BCG vaccine 2+ The effects of dendritic cells on activation and induced maturation were investigated using dendritic cells derived from mouse bone marrow. The procedure was as follows: mouse femurs and tibias were aseptically harvested, and the bone marrow cavity was washed with PBS to obtain a cell suspension. Red blood cells were lysed and counted. The cells were then analyzed at a concentration of 2 × 10⁻⁶ cells / cells. 6Cells were seeded at a density of 1 / mL in complete medium containing 20 ng / mL GM-CSF. On day 3, an equal volume of fresh medium and cytokines were added; the medium was partially replaced on days 5-6. After 6-8 days of culture, suspension and loosely adherent cells were collected, which were identified as immature dendritic cells. Subsequently, the PBS group, BCG group, BCG@MT group, and BCG@MT-A group were placed in the upper chamber of a Transwell, while the immature dendritic cells were placed in the lower chamber. After 24 hours of co-culture, the maturation and differentiation of dendritic cells were detected by flow cytometry. The results are as follows: Figure 7 As shown, 7A represents CD11c. + CD80 + Positive mature dendritic cells, 7B represents CD11c + CD86 + Positive mature dendritic cells. Flow cytometry and statistical results show that the proportion of mature dendritic cells in the BCG@MT group and the BCG@MT-A group was significantly increased.

[0031] Example 9 Validating the therapeutic effect of drug-loaded engineered BCG vaccine on a mouse orthotopic bladder cancer model. At the animal level, the inhibitory effect of drug-loaded engineered BCG on tumor growth was studied by constructing an in situ model. The specific procedure for the in situ model was as follows: after anesthetizing mice, the abdominal cavity was incised 0.5 cm above the urethral opening, and adipose tissue was dissected until the bladder was exposed. 20 μL of a drug-loaded engineered BCG vaccine containing 1×10⁻⁶ BCG was then injected into the bladder. 6 One Luci-MB49 cell was injected into the bladder muscle layer using PBS solution, and the layers were sutured one by one. Bladder instillation was performed once a week, with mice divided into PBS group, BCG group, BCG@MT group, and BCG@MT-A instillation group. The growth of tumors in the mouse bladder was monitored using an IVIS small animal in vivo imaging system, and tumor growth curves were plotted using fluorescence intensity. Finally, the mice were sacrificed, and the bladders were dissected and weighed. Results are as follows: Figure 8 As shown, through Figure 8 Bioluminescence monitoring showed that the intrabladder tumor signal remained at its lowest level in the BCG@MT-A infusion group, indicating that the intrabladder tumor volume remained at its minimum. Figure 8 B shows a comparison of the bladders of different groups after the mice were finally sacrificed; the BCG@MT-A treatment group had the smallest bladder volume. Figure 8 C represents the tumor growth curve assessment, showing that the BCG@MT-A treatment group has the flattest growth curve. Figure 8 D indicates that the bladder mass was lightest in the BCG@MT-A treatment group.

[0032] Example 10 Verify the immunomodulatory effect of drug-loaded engineered BCG vaccine on a mouse orthotopic bladder cancer model. Further investigation was conducted on the immune-activating effect of drug-loaded engineered BCG vaccine using tumor tissue in animal experiments. Bean-sized tissue fragments were cut from bladder tumor tissue. Cells were minced using ophthalmic scissors until no obvious clumps remained, and then transferred to centrifuge tubes containing 5 mL of digestion solution (containing 1% collagenase and 1% transdermal hyaluronidase). The mixture was incubated at 37°C for 2 hours in a shaker. The mixture was then passed through a 70 μm cell filter, and the filtrate was collected and centrifuged at 500 g for 5 minutes to obtain cell pellet. The pellet was resuspended and used for flow cytometry analysis of changes in various immune cells. Results are as follows: Figure 9 As shown in AH, the BCG@MT-A treatment group significantly promoted CD8. + It infiltrates T cells and mature dendritic cells, reduces immunosuppressive cells such as Tregs, and enhances the secretion of anti-tumor cytokines.

Claims

1. A drug-loaded engineered BCG vaccine based on a metal polyphenol network, characterized in that, The drug-loaded engineered BCG vaccine is a bacterial complex structure consisting of a metal polyphenol network and BCG cells, with functional molecules adsorbed on its surface by charge. The method for preparing the drug-loaded engineered BCG vaccine based on a metal polyphenol network includes the following steps: S1: After culturing BCG to the logarithmic phase, add 0.2 mM tannic acid for incubation, centrifuge, and obtain bacterial resuspension; S2: Polyphenols and metal ions are added to the bacterial resuspension and co-incubated. After centrifugation and resuspension, BCG vaccine coated with a metal polyphenol network is obtained; the polyphenol is tannic acid. S3: Subsequently, the functional molecule is electrostatically adsorbed, and after centrifugation and resuspending, drug-loaded engineered BCG vaccine is obtained; the functional molecule is AMD3100.

2. The engineered BCG vaccine based on a metal polyphenol network as described in claim 1, characterized in that, In step S1, the BCG culture includes: culturing in 7H10 medium containing 50 μg / ml kanamycin, selecting bacteria in the logarithmic growth phase, measuring the OD600 value with an ELISA reader to be 0.4~0.6, centrifuging and resuspending the bacteria to obtain a bacterial resuspension.

3. The engineered BCG vaccine based on a metal polyphenol network as described in claim 1, characterized in that, In S2, the metal ion is Mn 2+ Fe 3+ Cu 2+ Zn 2+ Al 3+ Co 2+ At least one of them.

4. The engineered BCG vaccine based on a metal polyphenol network as described in claim 1, characterized in that, The concentration of the polyphenol is 1–100 mmol / L; the concentration of the metal ion is 1–100 mmol / L; and the concentration of the functional molecule is 1–100 mg / mL.

5. The engineered BCG vaccine based on a metal polyphenol network as described in claim 1, characterized in that, In S2, the reaction time for co-incubation is 1 to 60 minutes.

6. The engineered BCG vaccine based on a metal polyphenol network as described in claim 3, characterized in that, The metal ion is Mn. 2+ .

7. The application of the drug-loaded engineered BCG vaccine based on a metal polyphenol network as described in claim 1 in the preparation of drugs for treating bladder cancer.

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