A chemotherapy-immunotherapy combined anti-tumor delivery system based on pseudomonas aeruginosa bacterial outer membrane vesicles, and a preparation method and application thereof

Abstract

The application provides a chemotherapy-immunotherapy combined antitumor delivery system based on a Pseudomonas aeruginosa bacterial outer membrane vesicle and a preparation method and application thereof; an antitumor drug is loaded in the bacterial outer membrane vesicle by passive incubation through ammonium sulfate treatment; the bacterial outer membrane vesicle has good biocompatibility, mucosal penetration ability and immunostimulating capacity; the antitumor drug is embedded in the bacterial outer membrane vesicle, can be protected and slowly released, prolongs the residence time of the drug in the bladder and enhances the killing of tumor tissues. Therefore, the application constructs a chemotherapy-immunotherapy combined antitumor delivery system through the bacterial outer membrane vesicle and the antitumor drug, and realizes the synergistic antitumor effect of chemotherapy and immunotherapy.

Description

Technical Field

[0001] This application belongs to the field of biomedical materials and tumor treatment technology, and in particular relates to a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, its preparation method and application. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, with a high recurrence rate after surgery. Intravesical instillation therapy is an important means of preventing recurrence and progression. Currently, chemotherapy drugs or BCG are mainly used for instillation therapy in clinical practice.

[0003] However, existing perfusion therapy protocols still have many shortcomings:

[0004] On the one hand, chemotherapy drugs (such as doxorubicin) have a short residence time in the bladder, are easily emptied, and have difficulty effectively penetrating the bladder mucus layer and epithelial barrier, resulting in insufficient effective exposure of the drug in the tumor tissue; on the other hand, although BCG has a certain immunotherapy effect, it is highly irritating locally, has obvious side effects, and is difficult for some patients to tolerate.

[0005] In recent years, nanodelivery systems have been used to improve drug retention and tissue distribution in the bladder, but most synthetic nanocarriers still have limitations in terms of biocompatibility, mucosal penetration, and immunomodulation. Furthermore, existing technologies are mostly focused on single treatment modalities, lacking an effective delivery system capable of simultaneously achieving synergistic effects of chemotherapy and immunotherapy under local drug administration conditions.

[0006] Therefore, there is an urgent need for a combined treatment delivery system that is suitable for bladder instillation scenarios and has good biocompatibility, chemotherapy efficacy, and immune activation capabilities. Summary of the Invention

[0007] This application provides a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, its preparation method, and its application, to solve the problems existing in related technologies. The technical solution is as follows:

[0008] In a first aspect, embodiments of this application provide a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, comprising bacterial outer membrane vesicles and an antitumor drug loaded within the vesicles.

[0009] In one embodiment, the bacterial outer membrane vesicles are Pseudomonas aeruginosa outer membrane vesicles; the antitumor drug is an anthracycline antitumor drug, preferably, the antitumor drug is doxorubicin.

[0010] In one embodiment, the mass ratio of bacterial outer membrane vesicles to antitumor drugs is 1:(0.3-0.7).

[0011] Secondly, embodiments of this application provide a method for preparing a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, comprising the following steps:

[0012] An antitumor drug was added to a buffer solution containing ammonium sulfate-pretreated Pseudomonas aeruginosa outer membrane vesicles, and the mixture was incubated.

[0013] By removing unloaded free doxorubicin through at least one method such as dialysis, ultrafiltration, or centrifugation, a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa outer membrane vesicles is obtained.

[0014] In one embodiment, the concentration of Pseudomonas aeruginosa outer membrane vesicles pretreated with ammonium sulfate in the buffer solution is 0.5-2 mg / ml; doxorubicin is added to a concentration of 100-600 μg / ml.

[0015] In one embodiment, the incubation conditions are: incubation at 4-8°C for 6-36 hours; the incubation process uses a shaker.

[0016] In one embodiment, the outer membrane vesicles of Pseudomonas aeruginosa are prepared by the following method:

[0017] Pseudomonas aeruginosa in the late logarithmic growth stage was resuspended in PBS buffer and induced to produce outer membrane vesicles by sonic stimulation.

[0018] Pseudomonas aeruginosa outer membrane vesicles were collected by at least one of centrifugation, filtration or ultrafiltration.

[0019] In one embodiment, the OD of Pseudomonas aeruginosa in PBS buffer... 600 Version 1.0-2.0.

[0020] In one embodiment, the freeze-dried powder of Pseudomonas aeruginosa strain is revived, inoculated into TSB medium, and cultured and amplified at 35-37°C.

[0021] Both the initial bacterial culture and the amplified bacterial culture were identified. The correctly identified strains were inoculated into TSB medium and cultured with shaking at 35-37℃ until the late logarithmic growth stage.

[0022] Thirdly, embodiments of this application provide an application of a chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles in the preparation of anti-bladder cancer drugs.

[0023] In one implementation, the anti-bladder cancer drug is administered via intravesical instillation.

[0024] The advantages or beneficial effects of the above technical solutions include at least the following:

[0025] This application discloses a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles. The system comprises bacterial outer membrane vesicles and an antitumor drug loaded within the vesicles. The antitumor drug is loaded into the bacterial outer membrane vesicles, which possess good biocompatibility, mucosal penetration ability, and immunostimulatory capacity. The antitumor drug, encapsulated within the bacterial outer membrane vesicles, is protected and slowly released, prolonging its residence time in the bladder and enhancing its killing effect on tumor tissue. Therefore, this application constructs a chemotherapy-immunotherapy combined mechanism antitumor delivery system using bacterial outer membrane vesicles and an antitumor drug, achieving a synergistic antitumor effect of chemotherapy and immunotherapy.

[0026] This application describes a method for preparing a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles. The method involves loading antitumor drugs into bacterial outer membrane vesicles by pretreating them with ammonium sulfate and using an active gradient incubation method. This method maintains the structural integrity and immune activity of the outer membrane vesicles while loading chemotherapy drugs, and also increases the amount of antitumor drugs loaded into the vesicles.

[0027] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0028] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0029] Figure 1 TEM images and particle size distribution of Pseudomonas aeruginosa bacterial outer membrane vesicles before and after loading with doxorubicin;

[0030] Figure 2 The bar charts show the loading concentration of vesicles in the examples and comparative examples at different Doxorubicin feed concentrations;

[0031] Figure 3 The bar charts show the loading-feed ratio of vesicles in the examples and comparative examples at different doxorubicin concentrations;

[0032] Figure 4 Bioluminescence intensity of mouse bladder cancer orthotopic tumor models under different treatment conditions;

[0033] Figure 5 The changes in body weight of mice with orthotopic bladder cancer tumors under different treatment conditions;

[0034] Figure 6 H&E staining images of orthotopic bladder cancer tumors in mice under different treatment conditions. Detailed Implementation

[0035] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0036] This application provides a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, including bacterial outer membrane vesicles and antitumor drugs loaded within the vesicles.

[0037] Bacterial outer membrane vesicles retain bacterial-derived membrane components, which can activate innate immune-related pathways, induce immune cell recruitment and inflammatory factor release, and form a microenvironment conducive to anti-tumor immune responses in the tumor site. The loaded anti-tumor drug is a chemotherapeutic drug that exerts cytotoxic effects by directly inducing tumor cell apoptosis or immunogenic cell death. Therefore, the chemotherapy-immunotherapy combined anti-tumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles of this application achieves a synergistic anti-tumor effect of chemotherapy and immunotherapy.

[0038] In addition, bacterial outer membrane vesicles have good biocompatibility and mucosal penetration ability; and anti-tumor drugs are embedded in bacterial outer membrane vesicles, which can be protected and released slowly, prolonging the drug retention time in the bladder. Therefore, the drug delivery system can solve the problems of short drug retention time and insufficient tissue penetration in the existing bladder cancer instillation therapy.

[0039] In one embodiment, the bacterial outer membrane vesicles are Pseudomonas aeruginosa outer membrane vesicles. In this embodiment, the outer membrane vesicles have a particle size of 20-300 nm, a negatively charged surface, and retain bacterial membrane proteins and lipopolysaccharide components, thus possessing immunostimulatory capabilities.

[0040] In one embodiment, the antitumor drug is an anthracycline antitumor drug. Anthracycline drugs have a broad antitumor spectrum, strong antitumor activity, and definite efficacy. Preferably, the antitumor drug is doxorubicin.

[0041] In one implementation method, the mass ratio of bacterial outer membrane vesicles to antitumor drugs is 1:(0.3-0.7). Bacterial outer membrane vesicles exhibit excellent drug loading capacity, with a loading rate reaching 60.8%.

[0042] This application also provides a method for preparing a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, including the following steps:

[0043] An antitumor drug was added to a buffer solution containing ammonium sulfate-pretreated Pseudomonas aeruginosa outer membrane vesicles, and the mixture was incubated.

[0044] By removing unloaded free doxorubicin through at least one method such as dialysis, ultrafiltration, or centrifugation, a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa outer membrane vesicles is obtained.

[0045] This invention uses Pseudomonas aeruginosa outer membrane vesicles as a carrier to load doxorubicin via an ammonium sulfate gradient. In this embodiment, the outer membrane vesicles are pretreated in a buffer system containing ammonium sulfate, forming a proton gradient within the Pseudomonas aeruginosa outer membrane vesicles. The pretreated vesicles are then placed in a buffer solution, retaining ammonium sulfate inside the vesicles, which in turn forms a concentration gradient with the buffer solution. Subsequently, an antitumor drug is added to the vesicle suspension, and through active incubation, doxorubicin actively enters and remains in the vesicles driven by the concentration gradient, thereby preparing drug-loaded outer membrane vesicles. This preparation method is simple to operate, has good reproducibility, and maintains the structural integrity and immunomodulatory activity of the outer membrane vesicles while loading chemotherapeutic drugs.

[0046] Unloaded free doxorubicin was removed by at least one of the following methods: dialysis, ultrafiltration, or centrifugation, to obtain a purified Pseudomonas aeruginosa-based chemoimmunotherapy delivery system.

[0047] In one implementation method, the concentration of Pseudomonas aeruginosa outer membrane vesicles pretreated with ammonium sulfate in the buffer solution is 0.5-2 mg / ml; doxorubicin is added to a concentration of 100-600 μg / ml. The ammonium sulfate pretreatment method significantly increases the amount of doxorubicin loaded onto the vesicles; by setting different incubation conditions for different concentrations of doxorubicin, the optimal drug loading is achieved when the vesicles reach saturation.

[0048] In one implementation method, the incubation conditions are as follows: incubation at 4-8°C for 6-36 hours; the incubation process is carried out using a shaker. Doxorubicin is added to a final concentration of 400 μg / ml of Pseudomonas aeruginosa outer membrane vesicles at a concentration of 1 mg / ml, ultimately achieving a drug loading of 608 μg doxorubicin per 1 mg of Pseudomonas aeruginosa outer membrane vesicles.

[0049] As one implementation method, Pseudomonas aeruginosa outer membrane vesicles are prepared by the following method:

[0050] Pseudomonas aeruginosa in the late logarithmic growth stage was resuspended in PBS buffer and induced to produce outer membrane vesicles by sonic stimulation.

[0051] Pseudomonas aeruginosa outer membrane vesicles were collected by at least one of centrifugation, filtration or ultrafiltration.

[0052] As one implementation method, the conditions for ultrasound stimulation induction are: ice bath ultrasound, power: 422W, with ultrasound for 3 seconds and interval for 5 seconds for 20 minutes.

[0053] The Pseudomonas aeruginosa outer membrane vesicles of this application were obtained by inducing Pseudomonas aeruginosa in PBS buffer via ultrasonic stimulation. In this embodiment, the obtained outer membrane vesicles have a particle size of 20-300 nm, a negatively charged surface, and retain bacterial membrane proteins and lipopolysaccharide components, thereby possessing immunostimulatory capabilities.

[0054] As one implementation method, the OD of Pseudomonas aeruginosa in PBS buffer... 600 Version 1.0-2.0.

[0055] As one implementation method, the freeze-dried powder of Pseudomonas aeruginosa strain is revived, inoculated into TSB medium, and cultured and amplified at 35-37℃.

[0056] Both the initial bacterial culture and the amplified bacterial culture were identified. The correctly identified strains were inoculated into TSB medium and cultured with shaking at 35-37℃ until the late logarithmic growth stage.

[0057] This application also provides the application of a chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles in the preparation of anti-bladder cancer drugs.

[0058] As one implementation method, anti-bladder cancer drugs are administered via intravesical instillation.

[0059] The following is a further explanation using specific embodiments.

[0060] Example

[0061] (1) Preparation of bacterial outer membrane vesicles:

[0062] The lyophilized powder of *Pseudomonas aeruginosa* strain (ATCC9027) was used to revive the bacteria and inoculate them onto TSB (trypsin-soybean broth) medium, where they were cultured and amplified at 36±1℃. Both the initial and amplified bacterial suspensions were identified. The strain identification method involved inoculating the reviveed strain onto CN agar plates and incubating at 36±1℃ for 24 hours. Colony morphology was observed (typical *Pseudomonas aeruginosa* ATCC9027 appears as blue-green colonies on CN agar, and exhibits yellow-green fluorescence around the colonies under 366nm UV light).

[0063] The correctly identified bacterial strains were inoculated into TSB medium and cultured at 36±1℃ with shaking until the late logarithmic growth phase; the bacterial cells were collected by centrifugation and resuspended after washing with PBS (OD200). 600 The sample was prepared at a concentration of 1.0-2.0, then placed in an ice bath and subjected to sonication at 422W for 3 seconds followed by 5 seconds of intermittent sonication for 20 minutes to induce the formation of outer membrane vesicles. The sample was then centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected and centrifuged repeatedly to collect the supernatant containing the outer membrane vesicles. The supernatant obtained by centrifugation was first filtered through a 0.45 μm pore size filter membrane, then through a 0.22 μm pore size filter membrane. The resulting filtrate was then filtered through an ultrafiltration tube to recover the liquid and precipitate on the filter screen. Finally, the sample was centrifuged at 100,000 g for 2 hours. The supernatant was removed, and the residue was resuspended in PBS to obtain the outer membrane vesicles.

[0064] (2) Ammonium sulfate pretreatment

[0065] The outer membrane vesicles were placed in 200 mM ammonium sulfate buffer and incubated overnight at 4°C. They were then transferred to an ultrafiltration tube and free ammonium sulfate outside the vesicles was removed using PBS. The mass concentration of the outer membrane vesicles was adjusted to 1 mg / ml using PBS to create an ammonium sulfate concentration gradient inside and outside the vesicles.

[0066] (3) Chemotherapy drug load

[0067] Doxorubicin was added to an ammonium sulfate pretreated solution of 1 mg / ml outer membrane vesicles to a concentration of 400 μg / ml; the solution was shaken at 4°C for 12 h to allow doxorubicin to enter and be loaded into the outer membrane vesicles under the influence of the concentration gradient.

[0068] (4) Purification

[0069] The pretreated mixture was transferred to a medium-sized ultrafiltration tube and centrifuged at 4500 rpm for 20 min. PBS was then added to the original volume, and this process was repeated several times until the lower filtrate was nearly clear. The upper liquid and precipitate were then recovered. Unloaded free doxorubicin was removed to obtain a chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles.

[0070] Comparative Example

[0071] The difference between the comparative example and the embodiment is that ammonium sulfate solution is not used for pretreatment, but the other steps and processes are the same.

[0072] The particle size and potential of *Pseudomonas aeruginosa* bacterial outer membrane vesicles before and after loading with doxorubicin were analyzed using a Malvern Zetasizer particle size potentiometry instrument. SEM images and particle size distribution curves of *Pseudomonas aeruginosa* bacterial outer membrane vesicles before doxorubicin loading are shown below. Figure 1 The image above shows the SEM image and particle size distribution curve of *Pseudomonas aeruginosa* bacterial outer membrane vesicles loaded with doxorubicin. Figure 1 As shown in the image below.

[0073] TEM images and particle size distribution diagrams show that the average hydrated particle size of Pseudomonas aeruginosa outer membrane vesicles is about 95.16 nm, and the average potential is -25.5 mV; the particle size of doxorubicin-loaded vesicles is slightly larger, about 103.8 nm, and the average potential is -3.5 mV.

[0074] The concentration of doxorubicin added in the examples and comparative examples was changed to 100 μg / ml, 200 μg / ml, 300 μg / ml, 500 μg / ml, and 600 μg / ml, respectively. The drug loading of *Pseudomonas aeruginosa* bacterial outer membrane vesicles in the final chemotherapy-immunotherapy combined antitumor delivery system based on *Pseudomonas aeruginosa* bacterial outer membrane vesicles was tested; the results are as follows. Figure 2 and Figure 3 As shown.

[0075] from Figure 2 and Figure 3 It can be seen that under the incubation conditions of doxorubicin at a concentration of approximately 400 μg / ml, the drug loading capacity of Pseudomonas aeruginosa bacterial outer membrane vesicles reached saturation. Further increasing the doxorubicin concentration did not continue to increase the drug loading capacity; on the contrary, as the doxorubicin concentration increased, the loading-to-feed ratio continued to decrease.

[0076] Therefore, the optimal feeding conditions are: a final concentration of 400 μg / ml doxorubicin and 1 mg / ml Pseudomonas aeruginosa bacterial outer membrane vesicles. Under these conditions, each 1 mg of Pseudomonas aeruginosa bacterial outer membrane vesicles in the chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles was loaded with 608 μg of doxorubicin.

[0077] The delivery systems of the examples and comparative cases were subjected to efficacy tests.

[0078] A C57BL / 6 mouse bladder cancer orthotopic tumor model was established using the MB49-Luc cell line. The successful establishment of the model was confirmed by luciferase bioluminescence signal, and the tumor burden was monitored using this signal.

[0079] MB49-luc bladder cancer cells were inoculated into mice via bladder erosion one week prior. Tumor-forming mice were randomly divided into four groups: a) doxorubicin-loaded Pseudomonas aeruginosa outer membrane vesicle group, b) Pseudomonas aeruginosa outer membrane vesicle group, c) doxorubicin group, and d) PBS control group. All four groups underwent bladder instillation treatment.

[0080] The perfusion volume for all therapeutic doses was 100 μl, and the solvent was PBS. Among them, a was the group of Pseudomonas aeruginosa outer membrane vesicles loaded with doxorubicin (20 μg doxorubicin loaded in 32.9 μg vesicles, 100 μl), b was the group of Pseudomonas aeruginosa outer membrane vesicles (32.9 μg vesicles, 100 μl), c was doxorubicin (20 μg, 100 μl), and d was the PBS control group (100 μl).

[0081] Samples from each group were injected into the bladder via urethral catheterization and instilled into the bladder cavity for 30 minutes. Instillation treatment was performed on days 1, 4, 7, and 11.

[0082] Tumor burden was detected using Luciferase bioluminescence signals during treatment, and the bioluminescence intensity of tumors in each group was as follows: Figure 4 As shown; mouse weight is measured periodically to assess the mice's health, and changes in mouse weight are shown in the figure. Figure 5 As shown; H&E staining image of bladder cancer in situ tumor. Figure 6 As shown.

[0083] Throughout the entire treatment period, no significant changes were observed in the body weight of mice in the group loaded with doxorubicin-containing Pseudomonas aeruginosa outer membrane vesicles, and no abnormalities were found in blood routine tests, liver and kidney function, or other indicators.

[0084] Compared to other treatment groups, the doxorubicin-loaded Pseudomonas aeruginosa outer membrane vesicle treatment group showed excellent tumor suppression effect in the early stage and sustained tumor suppression effect 2 weeks after treatment; the doxorubicin and vesicle groups showed some effect in the early stage of treatment but failed to suppress tumor growth continuously.

[0085] This invention uses Pseudomonas aeruginosa outer membrane vesicles as carriers and loads doxorubicin via an ammonium sulfate gradient to achieve chemotherapy-immunotherapy without introducing additional immunotherapies. It is particularly suitable for bladder cancer instillation therapy and has clear innovation and good application prospects.

[0086] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0087] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0088] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles, characterized in that, This includes bacterial outer membrane vesicles and antitumor drugs loaded within the vesicles.

2. The chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 1, characterized in that, The bacterial outer membrane vesicles are Pseudomonas aeruginosa outer membrane vesicles; the antitumor drug is an anthracycline antitumor drug, preferably, the antitumor drug is doxorubicin.

3. The chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 1, characterized in that, The mass ratio of bacterial outer membrane vesicles to antitumor drugs was 1:(0.3-0.7).

4. The method for preparing the chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to any one of claims 1-3, characterized in that, Includes the following steps: An antitumor drug was added to a buffer solution containing ammonium sulfate-pretreated Pseudomonas aeruginosa outer membrane vesicles, and the mixture was incubated. By removing unloaded free doxorubicin through at least one method such as dialysis, ultrafiltration, or centrifugation, a chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa outer membrane vesicles is obtained.

5. The method for preparing the chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 4, characterized in that, The buffer solution contained Pseudomonas aeruginosa outer membrane vesicles pretreated with ammonium sulfate solution at a concentration of 0.5-2 mg / ml; doxorubicin was added to bring the concentration to 100-600 μg / ml. The incubation conditions are as follows: incubation at 4-8℃ for 6-36 hours; shaking is used during the incubation process.

6. The method for preparing the chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 4, characterized in that, outer membrane vesicles of Pseudomonas aeruginosa were prepared by the following method: Pseudomonas aeruginosa in the late logarithmic growth stage was resuspended in PBS buffer and induced to produce outer membrane vesicles by sonic stimulation. Pseudomonas aeruginosa outer membrane vesicles were collected by at least one of centrifugation, filtration or ultrafiltration.

7. The method for preparing the chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 6, characterized in that, OD of Pseudomonas aeruginosa in PBS buffer 600 Version 1.0-2.

0.

8. The method for preparing the chemotherapy-immunotherapy combined antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles according to claim 6, characterized in that, The lyophilized powder of Pseudomonas aeruginosa strain was revived, inoculated into TSB medium, and cultured and amplified at 35-37℃. Both the initial bacterial culture and the amplified bacterial culture were identified. The correctly identified strains were inoculated into TSB medium and cultured with shaking at 35-37℃ until the late logarithmic growth stage.

9. The use of the chemotherapy-immunotherapy combined with antitumor delivery system based on Pseudomonas aeruginosa bacterial outer membrane vesicles as described in any one of claims 1-8 in the preparation of anti-bladder cancer drugs.

10. The application according to claim 9, characterized in that, Anti-bladder cancer drugs are administered via intravesical instillation.

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