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

Abstract

The invention belongs to the technical field of nano-drug carriers, and relates to a metal polyphenol network-based drug-loaded engineered bacillus calmette-guerin and a preparation method and application thereof, the metal polyphenol network-based drug-loaded engineered bacillus calmette-guerin is of a bacterial complex structure formed by combining a metal polyphenol network with bacillus calmette-guerin thalli, and the surface of the metal polyphenol network-based drug-loaded engineered bacillus calmette-guerin adsorbs functional molecules under the action of charges. The coated metal polyphenol network can effectively enhance the adhesive force to the bladder epithelium, and shows good release ability in an acidic urine environment, and released manganese and other metal ions can cooperate with BCG to activate immune storm; besides, the metal polyphenol network can also adsorb chemotherapy, targeting and other functional molecules, the metal polyphenol network and bacillus calmette-guerin vaccine thalli are jointly delivered and released, tumor cells are further killed, and the engineered bacillus calmette-guerin vaccine can effectively enhance immune activation of bladder cancer perfusion therapy and inhibit tumor growth.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanodrug carriers, and relates to a drug-loaded engineered bacillus Calmette-Guerin based on a metal polyphenol network and a preparation method and application thereof. BACKGROUND

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, and the high postoperative recurrence rate of non-muscular invasive bladder cancer (NMIBC) is a major challenge in its clinical treatment. Bacillus Calmette-Guerin (BCG) bladder perfusion is the standard immunotherapy for preventing the recurrence of high-risk NMIBC, but its efficacy is limited by two major problems: first, up to 30%-50% of patients have BCG non-response phenomenon; second, the high incidence of adverse reactions such as bladder irritation caused by perfusion leads to poor patient tolerance. The antitumor effect of BCG depends on the activation of local immune response in the bladder, and BCG non-response is often closely related to the immunosuppressive tumor microenvironment (TME), in which the infiltration and functional inhibition of immune cells mediated by the CXCL12 / CXCR4 signaling axis and other pathways are important factors.

[0003] In terms of administration mode, although bladder perfusion can achieve local administration and reduce systemic side effects, it still faces inherent bottlenecks such as short drug residence time and low absorption efficiency of the bladder epithelium. Nanodrug carrier technology provides a new idea for improving drug efficacy, which can improve the solubility, stability and targeting of drugs by loading drugs in nanoscale carriers. However, in the dynamic bladder cavity, conventional nanocarriers also have difficulty in effectively adhering to the uroepithelium and achieving long-term retention, resulting in unsatisfactory drug utilization.

[0004] In recent years, the progress of biomaterials has provided new tools to solve the above problems. Metal polyphenol networks (MPNs) are a new type of nanomaterial formed by the self-assembly of natural polyphenols (such as tannic acid) and metal ions (such as manganese ions) through coordination. MPNs have good biocompatibility, biodegradability, excellent bioadhesion and large drug loading capacity. Their adhesion properties are particularly suitable for bladder perfusion scenarios, which can significantly prolong the residence time of drugs in the bladder wall. In addition, the components that make up MPNs have biological activity: Mn²⁺ is an effective natural immune adjuvant that can activate the cGAS-STING pathway and enhance the anti-tumor immune response.

[0005] Therefore, it is of great clinical significance and application prospect to develop an engineered drug-loaded system based on MPN to reform BCG and simultaneously solve the problems of insufficient efficacy, poor tolerance and short residence time in the bladder. SUMMARY

[0006] To solve the above technical problems, the application provides a drug-loaded engineered BCG based on a metal polyphenol network, which can prolong the immune effect induced by BCG under the condition of reducing the perfusion concentration of BCG, thereby significantly reducing the side effects of BCG in the treatment of bladder cancer.

[0007] The technical scheme of the application is as follows: The BCG is a live attenuated Mycobacterium tuberculosis, which is relatively fragile, and therefore a construction strategy that can quickly form at neutral pH and room temperature is needed. The application reports a drug-loaded engineered BCG based on a metal polyphenol network, which is a bacterial complex structure combined by the metal polyphenol network and the BCG bacteria, and the surface of which is adsorbed with functional molecules through electrostatic attraction. By controlling the metal-polyphenol network to mainly nucleate and grow on the surface of the bacteria rather than nucleate and aggregate in the solution, we precisely regulate various factors.

[0008] The preparation method of the drug-loaded engineered BCG based on the metal polyphenol network includes the following steps: S1: Culturing and centrifuging the BCG to obtain a bacterial resuspension. The input amount of BCG affects the loading rate of TA and Mn 2+ , and therefore the value of OD600 is selected to be 0.4-0.6, and the BCG bacteria are centrifuged and resuspended to obtain a bacterial resuspension; Alternatively, S1: Culturing the BCG, adding a polyphenol solution with a final concentration of ≤0.5 mM, and centrifuging to obtain a polyphenol-treated bacterial resuspension.

[0009] S2: Co-incubating the polyphenol and metal ions in the bacterial resuspension. In order to ensure that the loaded STING agonist Mn 2+ reaches an effective concentration and that the coated bacteria have increased adhesion, the polyphenol, metal ion and coating time are screened. Finally, tannic acid (TA) and Mn 2+ are selected at a concentration of 1 mM, and the reaction time is 5 min. After centrifugation and resuspension, the BCG coated with the metal polyphenol network is obtained; S3: Since the MPN has a negative charge, it can further co-deliver the positively charged AMD3100. If the concentration of AMD3100 is too high, the solution will flocculate and precipitate, and if the concentration of AMD3100 is too low, the loading amount will be insufficient. Therefore, the concentration of AMD3100 is screened, and finally the concentration of AMD3100 is selected to be 1 mg / ml. Then, the functional molecules are electrostatically adsorbed, and after centrifugation and resuspension, the drug-loaded engineered BCG is obtained.

[0010] Further, in S1, the culturing of the BCG includes: culturing in a 7H10 culture medium containing 50 μg / ml kanamycin, selecting bacteria in the logarithmic growth phase, and measuring the value of OD600 to be 0.4-0.6 by using an enzyme marker. The bacteria are centrifuged and resuspended to obtain a bacterial resuspension.

[0011] Further, in S2, the polyphenol is at least one of catechol, resorcinol, hydroquinone, pyrogallol, phloroglucin, proanthocyanidin, gallic acid, tannic acid, epigallocatechin, and gallate; preferably, the polyphenol is tannic acid.

[0012] Further, in S2, the metal ion is at least one of Mn 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Al 3+ , Co 2+ ; 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. Further, in S3, the functional molecule is at least one of AMD3100, doxorubicin, gemcitabine, and mitoxantrone, and the functional molecule concentration is 1-100 mg / mL, preferably 1-20 mg / mL.

[0013] Further, the polyphenol and the metal ion are added to the bacterial resuspension solution for co-incubation, and the co-incubation reaction time is 1-60 min, preferably 1-20 min. In another aspect, the application also protects a metal polyphenol network-based drug-loaded engineered BCG in the preparation of a drug for treating bladder cancer.

[0014] The application has the following beneficial effects: 1. The application effectively increases the adhesion time of the complex on the bladder epithelium by combining the metal polyphenol network with the BCG, so that the BCG can better play a role, and compared with the traditional perfusion method, the concentration of the BCG used is lower and the onset time is longer. 2. The metal polyphenol network of the application exhibits good release capacity in an acidic urine environment, and the released metal ions can activate the immune storm in cooperation with the BCG. 3. The complex of the application can also deliver and release the chemotherapeutic, targeted, and other functional molecules together with the BCG bacteria, further killing tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0015] The drawings accompanying the specification of the application form a part of the application and serve to provide further understanding of the application. The exemplary embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application.

[0016] Figure 1The scanning electron microscope graph (A), the modified Zeta potential graph (B) and the fluorescence graph (C) of the drug-loaded engineered BCG are shown in the figure; Figure 2 The growth ability graph (A), the AMD3100 drug encapsulation rate graph (B) and the manganese ion release graph at different pH values (C) of the drug-loaded engineered BCG are shown in the figure; Figure 3 The X-ray photoelectron spectroscopy graph of the BCG bacterial body surface successfully coated with drug-loaded metal (manganese) polyphenol network is shown in the figure; Figure 4 The scanning electron microscope Mapping detection graph of the BCG bacterial body surface successfully coated with drug-loaded metal (manganese) polyphenol network is shown in the figure; Figure 5 The adhesion ability characterization of the drug-loaded engineered BCG at the cell level and the animal level is shown in the figure. A is the adhesion ability comparison graph under white light; B is the fluorescence BCG adhesion ability comparison graph; C is the BCG acid-fast staining comparison graph after animal bladder mucosa perfusion; Figure 6 The killing ability detection of the drug-loaded engineered BCG on bladder cancer cells is shown in the figure. A is the apoptosis flow graph of different treatment groups; B is the statistical graph of the apoptosis rate of A; C is the MTT proliferation experiment graph; Figure 7 The activation ability detection of the drug-loaded engineered BCG on dendritic cells is shown in the figure. A is the flow graph of CD11C+CD80+ dendritic cell maturation induced by different treatment groups; B is the flow graph of CD11C+CD86+ dendritic cell maturation induced by different treatment groups; Figure 8 The treatment effect detection of the drug-loaded engineered BCG through the mouse orthotopic bladder tumor model is shown in the figure. A is the luciferase monitoring graph of the orthotopic tumor growth of the mice in different treatment groups; B is the comparison graph of the bladders of the mice in different treatment groups after final sacrifice; C is the tumor growth curve graph of the mice in different treatment groups; D is the bladder weight graph of the mice in different treatment groups; Figure 9 The regulation ability detection of the drug-loaded engineered BCG on the immune microenvironment through flow cytometry is shown in the figure. A and B are the flow comparison graphs of CD8+ killer T cells; C-E are the flow comparison graphs of dendritic cells; F and G are the flow comparison graphs of M1 / M2 macrophages; H is the flow comparison graph of regulatory T cells. DETAILED DESCRIPTION

[0017] The application will be described in detail below through specific embodiments.

[0018] Example 1 A preparation method of a drug-loaded engineered BCG based on metal polyphenol network is as follows: S1: The BCG was cultured in 7H10 medium containing 50 μg / ml kanamycin, and the BCG in the logarithmic growth phase was selected. The BCG input amount will affect the loading rate of TA and Mn 2+ , so the value of OD600 was selected as 0.5, and the BCG was centrifuged and resuspended to obtain a bacterial resuspension; S2: Tannic acid and Mn 2+ were added to the bacterial resuspension for co-incubation. In order to ensure that the loaded STING agonist Mn 2+ reaches an effective concentration, and the coated bacteria have increased adhesion, the polyphenol, metal ion and coating time were screened. Finally, the concentrations of TA and Mn 2+ were both selected as 1 mM, and the reaction time was 5 min. After centrifugation and resuspension, the BCG coated with metal polyphenol network (BCG@MT) was obtained; S3: Since MPN has a negative charge, it can further co-deliver positively charged AMD3100. Too high a concentration of AMD3100 will cause the solution to flocculate and precipitate, and too low a concentration of AMD3100 will result in insufficient loading. Therefore, the concentration of ADM3100 was screened, and finally the concentration of AMD3100 was selected as 1 mg / ml. After electrostatic adsorption of AMD3100, the drug-loaded engineered BCG (BCG@MT-A) was obtained after centrifugation and resuspension.

[0019] Example 2 A preparation method of a drug-loaded engineered BCG based on a metal polyphenol network, comprising the following steps: S1: The BCG was cultured in 7H10 medium containing 50 μg / ml kanamycin, and the BCG in the logarithmic growth phase was selected. The BCG input amount will affect the loading rate of TA and Mn 2+ , so the value of OD600 was selected as 0.5, and the BCG was centrifuged and resuspended to obtain a bacterial resuspension; S2: Tannic acid and Mn 2+ were added to the bacterial resuspension for co-incubation. In order to ensure that the loaded STING agonist Mn 2+ reaches an effective concentration, and the coated bacteria have increased adhesion, the polyphenol, metal ion and coating time were screened. Finally, the concentrations of TA and Mn 2+ were both selected as 1 mM, and the reaction time was 5 min. After centrifugation and resuspension, the BCG coated with metal polyphenol network (BCG@MT) was obtained; S3: Add ADM3100, and the final concentration of ADM3100 is 1 mg / ml. After electrostatic adsorption, the drug-loaded engineered BCG is obtained after centrifugation and resuspension.

[0020] Example 3 Effect of MPN on BCG activity 1. Effect of MPN coating process on the number of viable BCG The bacterial resuspension solution obtained by resuspending the BCG bacteria was used as a control group for comparison with the coating effects of Examples 1 and 2. The BCG is a live attenuated Mycobacterium tuberculosis, which is relatively fragile. The number of live bacteria of the BCG after MPN coating was determined. The results are shown in Table 1. The results show that the polyphenol pretreatment step can significantly retain the number of live bacteria of the BCG and prevent partial inactivation of the bacteria during the coating process.

[0021] Table 1. Comparison of coating effects of polyphenol pretreatment Experimental grouping Viable bacterial count (CFU / mL) Relative survival rate (%) Initial bacterial solution (8.1 ± 0.1) x 10 8 ]] 100% Example 1 (5.2 ± 0.3) x 10 8 ]] 64.2% Example 2 (7.5 ± 0.5) x 10 8 ]]> 92.6%

[0022] The applicant analyzed that the molecules such as proteins and polysaccharides in the culture medium without centrifugation can reversibly pre-combine with polyphenols, effectively buffering the direct chemical stress of high-concentration polyphenols on the bacterial membrane surface, and avoiding the surface property variation of the bacteria due to sudden stress. Secondly, under this nearly original physiological state, the polyphenol molecules preferentially adsorb to specific sites on the surface of the bacteria through non-covalent forces, and pre-assemble. Finally, the centrifugation step precipitates the polyphenols adsorbed on the surface of the bacteria and the culture medium components combined therewith, thereby creating a higher local polyphenol concentration at the bacterial-solution interface. When the metal ions are subsequently added, the coordination reaction at the interface proceeds synchronously, and a uniform metal polyphenol network is constructed on the surface of the bacteria, thereby improving the coating efficiency and the survival rate of the bacteria.

[0023] 2. Effect of MPN coating on the biological activity of BCG Further, the effect of MPN coating on the biological activity of BCG was determined. The coated BCG was further cultured in the culture medium, and the absorbance value at 600 nm was determined at different time points. The results show that the growth curves of the BCG before and after coating are consistent, and the MPN coating does not affect the activity of the BCG. Figure 2 A. In order not to affect the immune function of the BCG, the MPN coating can be degradable in response to the tumor microenvironment (pH 5.5), and further release Mn 2+ to stimulate the STING signaling pathway. Figure 2 B and Figure 2 C.

[0024] Example 4 Characterization of the drug-loaded engineered BCG prepared in Example 1 1. Surface morphology characterization The BCG before and after surface modification was characterized by SEM. The surface of the blank BCG is smooth, and the surface roughness of the BCG after MPN coating is increased Figure 1 A. In order to ensure the successful adsorption of MPN and AMD3100, Zeta potential test was performed. After coating MPN, the zeta potential of the BCG decreased from -14.3 mV to -19.9 mVFigure 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 Figs. 6A and 6B, the blue arrows represent the observed adhered BCG bacteria, and no residual bacteria were observed in the BCG control group after 6 hours in the bladder, indicating that the bacteria were basically discharged under the action of urine flushing. However, residual bacteria were still observed in the bladder epithelium of the BCG@MT group and the BCG@MT-A group until the 24-hour time point, indicating that the adhesion ability of the bacteria 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 the drug-loaded engineered BCG on bladder cancer cells, apoptosis experiments and MTT experiments were performed. The apoptosis experiment was performed as follows: MB49 cells were seeded in a 24-well plate at a density of 2×10 5 cells / well, and after the cells adhered and divided to 90% of the density of the well plate, the PBS group, the BCG group, the BCG@MT group, and the BCG@MT-A group were added to the well plate, with 3 replicates for each group. After 24 hours of culture, the supernatant was removed and washed slowly with PBS, the cells in the well plate were collected, and the cells in different treatment groups were treated with an Annexin V-FITC / PI kit and then analyzed by flow cytometry. The results are shown in Figs. 6A and 6B. Figure 6 As shown in Figs. 6A and 6B, the blue arrows represent the observed adhered BCG bacteria, and no residual bacteria were observed in the BCG control group after 6 hours in the bladder, indicating that the bacteria were basically discharged under the action of urine flushing. However, residual bacteria were still observed in the bladder epithelium of the BCG@MT group and the BCG@MT-A group until the 24-hour time point, indicating that the adhesion ability of the bacteria to the epithelium was greatly enhanced.

[0029] The MTT experiment was performed as follows: MB49 cells were seeded in a 96-well plate at a density of 5×10 3 cells / well. The cells in each group were treated differently (as in the apoptosis experiment), and after 24 hours, MTT reagent was used to determine the cell viability, the absorbance of each well was detected by an enzyme-labeled instrument, and the survival rate of each group was calculated. The results are shown in Fig. 6C. Figure 6 As shown in Fig. 6C, the cell growth of the BCG@MT-A group was most significantly inhibited, and it could resist the growth-promoting effect of exogenous CXC12 factor on cancer cells, indicating that the AMD3100 drug released by the drug-loaded engineered BCG played a key role.

[0030] Example 8 Study on the activation of dendritic cells To verify the activation and induction of the release of Mn 2+ 2+by the drug-loaded engineered BCG on dendritic cells, the mouse bone marrow-derived dendritic cells were extracted, and the steps were as follows: the femur and tibia of the mouse were taken aseptically, the bone marrow cavity was washed with PBS to obtain a cell suspension, and the red blood cells were lysed and counted. The cells were seeded in a 6-well plate at a density of 2×10 6 / mL density inoculated in complete medium containing 20 ng / mL GM-CSF. On the 3rd day, an equal amount of fresh medium and cytokines were added; on the 5th-6th day, semi-replacement was performed. After 6-8 days of culture, the suspension and loose adherent cells were collected, which were immature dendritic cells. Then, the PBS group, the BCG group, the BCG@MT group, and the BCG@MT-A group were placed in the upper chamber of Transwell in an indirect co-culture manner, and the lower chamber was the immature dendritic cells. After 24 hours of co-culture, the maturation and differentiation of dendritic cells were detected by flow cytometry. The results are shown in Figure 7 Fig. 7A, 7A represents CD11c + CD80 + positive mature dendritic cells, 7B represents CD11c + CD86 + positive mature dendritic cells. It can be seen from the flow cytometry and statistical results that the proportion of mature dendritic cells intervened by the BCG@MT group and the BCG@MT-A group is significantly increased.

[0031] Example 9 Verification of the therapeutic effect of drug-loaded engineered BCG on the orthotopic model of bladder cancer in mice The inhibitory effect of drug-loaded engineered BCG on tumor growth was studied at the animal level by constructing an orthotopic model. The specific operation of the orthotopic model is as follows: after the mouse was anesthetized, the abdominal cavity was incised at 0.5 cm from the urethral opening to separate the fat tissue until the bladder was exposed, and 20 μL of PBS solution containing 1×10 6 Luci-MB49 cells was injected into the bladder muscle layer, and was sutured layer by layer. Once a week, bladder perfusion was performed, and the mice were divided into the PBS group, the BCG group, the BCG@MT group, and the BCG@MT-A perfusion group. The IVIS small animal living body imaging system was used to monitor the growth of bladder tumors in mice, the tumor growth curve was drawn by fluorescence intensity, and finally the bladder was dissected and weighed after the mice were sacrificed. The results are shown in Figure 8 Fig. 6A. Figure 8 A bioluminescence monitoring can be seen that the tumor signal in the BCG@MT-A perfusion group remained the lowest, indicating that the volume of bladder tumor remained the smallest. Figure 8 B shows the comparison of bladders in different groups after the mice were sacrificed, and the volume of the BCG@MT-A treatment group is the smallest. Figure 8 C is the tumor growth curve evaluation, and it can be seen that the growth curve of the BCG@MT-A treatment group is the flattest. Figure 8 D indicates that the bladder weight of the BCG@MT-A treatment group is the lightest.

[0032] Example 10 Verification of the immunoregulatory effect of drug-loaded engineered BCG on the orthotopic model of bladder cancer in mice Further explore the immune activation effect of drug-loaded engineered BCG through tumor tissue of animal experiment. The tumor tissue in the bladder was cut into green bean size tissue block, the cells were cut into small pieces with ophthalmic scissors, and then transferred to a centrifuge tube containing 5 mL of digestive juice (containing 1% collagenase and 1% hyaluronidase). After incubation in a 37°C shaking incubator for 2 h, the mixture was removed. The liquid was filtered through a cell filter with a pore size of 70 μm, and the filtrate was centrifuged at 500g for 5 min to obtain the cell pellet. After resuspension, it was used for flow cytometry analysis of various immune cell changes. The results are shown in Figure 9 As shown in A-H, the BCG@MT-A treatment group can significantly promote CD8 + T cell and mature dendritic cell infiltration, reduce Tregs and other immunosuppressive cells, and enhance anti-tumor cytokine secretion.

Claims

1. Metal polyphenol network based drug loaded engineered BCG vaccine, characterized in that, The drug-loaded engineered BCG is a bacterial complex structure combined by metal polyphenol network and BCG bacteria bodies, and the surface of the drug-loaded engineered BCG is adsorbed with functional molecules by electrostatic action.

2. The process for the preparation of metal polyphenol network based drug loaded engineered BCG vaccine as claimed in claim 1, wherein, The method comprises the following steps: S1: culturing and centrifuging BCG to obtain a bacterial resuspension; S2: adding polyphenols and metal ions to the bacterial resuspension for co-incubation, and obtaining BCG coated with metal polyphenol network after centrifugation and resuspension; S3: then electrostatically adsorbing functional molecules, and obtaining the drug-loaded engineered BCG after centrifugation and resuspension.

3. The production method according to claim 2, wherein In S1, the culturing of BCG comprises the following steps: culturing in 7H10 culture medium containing 50 μg / ml kanamycin, selecting bacteria in the logarithmic growth phase, measuring the OD600 value of 0.4-0.6 by an enzyme-labeled instrument, and centrifuging and resuspending the bacteria to obtain a bacterial resuspension.

4. The production method according to claim 2, wherein In S2, the polyphenols are at least one of catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, procyanidin, gallic acid, tannic acid, epigallocatechin, and gallate.

5. The production method according to claim 2, wherein S2, the metal ion is at least one of Mn 2+ , Fe 3+ , Cu 2+ , Zn 2 + , Al 3+ , Co 2+ .

6. The production method according to claim 1, wherein In S3, the functional molecules are at least one of AMD3100, doxorubicin, gemcitabine, and mitoxantrone.

7. The production method according to claim 2, wherein The concentration of the polyphenols is 1-100 mmol / L, the concentration of the metal is 1-100 mmol / L, and the concentration of the functional molecules is 1-100 mg / mL.

8. The preparation method of claim 2, wherein the reaction time is 1-60 min.

9. The production method according to claim 5, wherein The metal ion is Mn 2+ .

10. Use of the drug-loaded engineered BCG based on metal polyphenol network of claim 1 in the preparation of a drug for treating bladder cancer.

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