A drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles and its preparation method

Abstract

The invention provides a preparation method of a drug carrier of paclitaxel nanoparticles wrapped in bacterial outer membrane vesicles, the preparation method comprising the following steps: (1) preparing outer membrane vesicles of Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA‑2509025ΔtolB with plasmid Hbp+AffiHER2‑AffiEGFR1‑AffiVEGF); (2) mixing the obtained outer membrane vesicles with polylactic acid nanoparticles loaded with paclitaxel and mechanically extruding them to prepare outer membrane vesicles wrapped in paclitaxel nanoparticles. The drug carrier of paclitaxel nanoparticles wrapped in bacterial outer membrane vesicles obtained by the invention can well avoid the sudden release of the drug, the release ratio within 2 hours in the gastric fluid environment is less than 11%, the damage of gastric acid can be prevented, the degree of drug release in the stomach is reduced, and the drug utilization is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a drug carrier of paclitaxel nanoparticles wrapped in bacterial outer membrane vesicles and a preparation method thereof. Background Art

[0002] Paclitaxel is a diterpenoid compound derived from Taxus plants. It is mainly used in the first-line treatment of lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, etc. However, due to the poor water solubility of paclitaxel, its application range is limited. In order to improve the bioavailability of paclitaxel, people usually use drug controlled release technology to deliver it to the lesion site according to the design requirements and reduce the adverse reactions of the drug.

[0003] Drug controlled release technology is a technology that uses biomaterials as drug carriers to achieve delayed drug release, slow drug release, or drug release under certain conditions. By controlling the release of drugs, it is possible to avoid the drug being eliminated by the body after a large amount of release, or to increase the release ratio of the drug at the target location, thereby improving the bioavailability of the drug. At present, the common paclitaxel drug sustained-release agent is prone to a large amount of sudden release in the gastric fluid environment. The release amount of the obtained inhibitor after oral administration through the stomach usually reaches more than 20%, which limits the efficacy of paclitaxel.

[0004] Because extracellular vesicles are derived from organisms, they have good compatibility with cells and tissues, and can avoid the nanotoxicity caused by traditional nano drug carriers. They are increasingly attracting people's attention. Many researchers have prepared them into relevant drug carriers. For example, Sihan Wang et al. summarized the use of extracellular vesicles as vaccine and drug carriers (DOI: 10.1002 / wnan.1523.). In terms of using bacterial outer membrane vesicles to prepare nano drug carriers, Chen Lijiang et al. used Escherichia coli outer membrane vesicles to encapsulate polydopamine-modified mesoporous silica nanoparticles in CN114392246A. Through the pH sensitivity of polydopamine, targeted release to the tumor site was achieved, and the encapsulation of Escherichia coli outer membrane vesicles was used to protect polydopamine in gastric juice. However, the release of the active ingredient doxorubicin in the preparation obtained in the patent in artificial gastric juice for 2 hours is still as high as 17.1%, and the gastric protection effect that can be achieved is insufficient.

[0005] In terms of using bacterial outer membrane vesicles to load paclitaxel, Tao Xu solved the problem to a certain extent by encapsulating paclitaxel-loaded nanoparticles into the outer membrane vesicles of Escherichia coli, so that the release amount after stomach is controlled at about 16-17% (DOI: 10.27209 / d.cnki.glniu.2022.001989.). Similarly, the gastric protection effect obtained by this scheme is still poor. On this basis, Chu Yuqi first modified Zein with CAS to prepare paclitaxel-loaded nanoparticles, and then encapsulated them with outer membrane vesicles of Escherichia coli, reducing the release rate of the drug in artificial gastric juice to 8.89%, and obtained a better gastric protection effect (DOI: 10.27209 / d.cnki.glniu.2021.002125.).

[0006] At present, there are few reports on how to surround bacterial outer membrane vesicles with drug carriers to further reduce the gastric release rate of drugs. There is no consensus on whether there are significant differences in the gastric protection provided by the outer membrane vesicles of different strains and how to design appropriate drug carriers for the outer membrane vesicles of different strains.

[0007] Therefore, it is urgently needed in this field to explore other drug carriers based on bacterial outer membrane vesicles to further reduce the release amount of paclitaxel during gastric passage. Summary of the invention

[0008] In view of the defects of the prior art, the object of the present invention is to provide a drug carrier of paclitaxel nanoparticles encapsulated by bacterial outer membrane vesicles, which can further control the release amount of paclitaxel during gastric passage.

[0009] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0010] A method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles, the preparation method comprising the following steps:

[0011] (1) preparing outer membrane vesicles of Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF);

[0012] (2) mixing the obtained outer membrane vesicles with polylactic acid nanoparticles loaded with paclitaxel and subjecting them to mechanical extrusion to prepare outer membrane vesicles encapsulating paclitaxel nanoparticles;

[0013] The Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) has a deposit number of CCTCC M 2023065.

[0014] Furthermore, in step (1), a culture of the mutant strain is first obtained, and the culture method is: a monoclonal clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB withplasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) is cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight.

[0015] As shown in a comparative example of the present invention, when only polylactic acid is used as a paclitaxel drug carrier, the release ratio in artificial gastric juice reaches 37.6%, which is not conducive to drug absorption. When the outer membrane vesicles of another strain of the inventor's team are used to encapsulate the obtained paclitaxel-loaded polylactic acid nanoparticles, the gastric release ratio of the obtained product is reduced to 23.5%, but this effect is still not good. Surprisingly, when the outer membrane vesicles of the Helicobacter pylori mutant strain QS003 of the present invention are used to encapsulate the paclitaxel-loaded polylactic acid nanoparticles, the defect of the obtained product in the large gastric release ratio is greatly improved, and the obtained gastric release ratio is less than 5%, which well solves the problem of the fast gastric release of the existing paclitaxel carrier.

[0016] Importantly, the present invention does not require the use of CAS for modification to improve stability as in the solution of Chu Yuqi (DOI: 10.27209 / d.cnki.glniu.2021.002125.). Therefore, the preparation process of the present invention is simpler.

[0017] Furthermore, in step (1), the culture is first centrifuged to remove the supernatant, the supernatant is filtered through a membrane, the filtrate is concentrated and then ultracentrifuged, and then the ultracentrifuged precipitate is resuspended and then filtered through a membrane.

[0018] Preferably, in step (1), when membrane filtration is performed, a 0.45 μm filter membrane is used; when the culture is centrifuged, the centrifugation condition is centrifugation at 15,000×g for 10 minutes; and when concentration is performed, a 500 kDa ultrafiltration column is used.

[0019] Preferably, in step (1), the ultracentrifugation is performed under the centrifugation conditions of 200,000×g and 4° C. for 2 hours.

[0020] Preferably, in step (1), after obtaining the outer membrane vesicles, the outer membrane vesicles are modified using liposomes, and the modification method is: liposomes and outer membrane vesicles are resuspended and mixed with PBS at a weight ratio of 2:1, and the modification reaction is carried out on a shaker at 37°C and 100 rpm.

[0021] In recent years, many achievements have been made in the modification of outer membrane vesicles to improve the application effects of outer membrane vesicles. In general, the modification methods of outer membrane vesicles can be divided into genetic modification, physical modification and chemical modification. Genetic modification and chemical modification are usually more complicated, and specific modification methods need to be selected according to the characteristics of specific bacteria, and they are very likely to affect the original properties and functions of outer membrane vesicles. Fusion of liposomes with outer membrane vesicles is a more commonly used modification method, and its main function has been found to be to increase the diversity of exogenous antigens.

[0022] In the present invention, after the inventors obtained a good gastric release effect by using the outer membrane vesicles of the Helicobacter pylori mutant strain QS003 including polylactic acid nanoparticles loaded with paclitaxel, they also found that when the outer membrane vesicles of the Helicobacter pylori mutant strain QS003 were modified with liposomes, the gastric release effect could be further enhanced, so that the drug release ratio after gastric release was less than 3%.

[0023] Furthermore, in step (2), the preparation method of the polylactic acid nanoparticles loaded with paclitaxel is to use dichloromethane containing polylactic acid and paclitaxel as the oil phase and an aqueous solution containing polyvinyl alcohol as the water phase to prepare them by an emulsion method.

[0024] Preferably, in step (2), the weight ratio of polylactic acid to paclitaxel is 13 to 18:10.

[0025] Preferably, in step (2), the weight percentage of polyvinyl alcohol in the aqueous phase is 1%.

[0026] Preferably, in step (2), when mechanical extrusion is performed, a 200 nm polycarbonate film is used.

[0027] Another object of the present invention is to provide a drug carrier of paclitaxel nanoparticles encapsulated by bacterial outer membrane vesicles prepared by the above preparation method.

[0028] Beneficial effects of the present invention:

[0029] The drug carrier of paclitaxel nanoparticles encapsulated by bacterial outer membrane vesicles obtained in the present invention can well avoid the sudden release of drugs. The release ratio in the gastric fluid environment within 2 hours is less than 4%, which can prevent the damage of gastric acid, reduce the release degree of drugs in the stomach, and improve the utilization of drugs. DETAILED DESCRIPTION

[0030] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0031] The Helicobacter pylori mutant QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) used in the examples of the present invention has a deposit number of CCTCC M 2023065, and the Salmonella mutant QS0074 used in the comparative example has a deposit number of CCTCC M 2023192, both of which are owned by the laboratory and have been applied for patents in other patent applications. The present invention does not involve the screening and preservation of the aforementioned strains.

[0032] Example 1

[0033] 1. Preparation of outer membrane vesicles:

[0034] (1) A single clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) was cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight.

[0035] (2) The culture was centrifuged at 18,000 × g for 8 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4°C for 2 hours. The centrifuged precipitate was resuspended in PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles.

[0036] 2. Preparation of drug-loaded nanoparticles:

[0037] 150 mg of polylactic acid and 100 mg of paclitaxel were dissolved in 5 mL of dichloromethane as the oil phase, and an aqueous solution containing 1 wt% of polyvinyl alcohol was used as the water phase; the oil phase was slowly dripped into the water phase. The resulting solution was then treated with a high-speed homogenizer, and after the organic solvent evaporated under stirring, the solution was centrifuged at 8000×g for 5 minutes to collect the nanoparticles.

[0038] 3. Preparation of drug-loaded outer membrane vesicles:

[0039] The outer membrane vesicles and nanoparticles were resuspended in PBS at a weight ratio of 3:1, and mechanically extruded through a 200 nm polycarbonate membrane using a liposome extruder. The extrusion was repeated 10 times, and the mixture was centrifuged at 20,000 × g for 1 hour at 4°C. The precipitate was collected to obtain drug-loaded outer membrane vesicles.

[0040] Example 2

[0041] 1. Preparation of outer membrane vesicles:

[0042] (1) A single clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) was cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight;

[0043] (2) The culture was centrifuged at 18,000 × g for 8 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4°C for 2 hours. The centrifuged precipitate was resuspended in PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles.

[0044] 2. Preparation of drug-loaded nanoparticles:

[0045] 180 mg of polylactic acid and 100 mg of paclitaxel were dissolved in 5 mL of dichloromethane as the oil phase, and an aqueous solution containing 1 wt% of polyvinyl alcohol was used as the water phase; the oil phase was slowly dripped into the water phase. The resulting solution was then treated with a high-speed homogenizer, and after the organic solvent evaporated under stirring, the solution was centrifuged at 8000×g for 5 minutes to collect the nanoparticles.

[0046] 3. Preparation of drug-loaded outer membrane vesicles:

[0047] The outer membrane vesicles and nanoparticles were resuspended in PBS at a weight ratio of 4:1, and mechanically extruded through a 200 nm polycarbonate membrane using a liposome extruder. The extrusion was repeated 10 times, and the mixture was centrifuged at 20,000 × g for 1 hour at 4°C. The precipitate was collected to obtain drug-loaded outer membrane vesicles.

[0048] Example 3

[0049] 1. Preparation of outer membrane vesicles:

[0050] (1) A single clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) was cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight;

[0051] (2) The culture was centrifuged at 18,000 × g for 8 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4°C for 2 hours. The centrifuged precipitate was resuspended in PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles.

[0052] 2. Preparation of drug-loaded nanoparticles:

[0053] 130 mg of polylactic acid and 100 mg of paclitaxel were dissolved in 5 mL of dichloromethane as the oil phase, and an aqueous solution containing 1 wt% of polyvinyl alcohol was used as the water phase; the oil phase was slowly dripped into the water phase. The resulting solution was then treated with a high-speed homogenizer, and after the organic solvent evaporated under stirring, the solution was centrifuged at 8000×g for 5 minutes to collect the nanoparticles.

[0054] 3. Preparation of drug-loaded outer membrane vesicles:

[0055] The outer membrane vesicles and nanoparticles were resuspended in PBS at a weight ratio of 2:1, and mechanically extruded through a 200 nm polycarbonate membrane using a liposome extruder. The extrusion was repeated 10 times, and the mixture was centrifuged at 20,000 × g for 1 hour at 4°C. The precipitate was collected to obtain drug-loaded outer membrane vesicles.

[0056] Example 4

[0057] 1. Preparation of outer membrane vesicles:

[0058] (1) A single clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) was cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight;

[0059] (2) The culture was centrifuged at 18,000 × g for 8 minutes, the supernatant was collected, and the supernatant was filtered with a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4°C for 2 hours. The centrifuged precipitate was resuspended in PBS solution and then filtered with a 0.45 μm filter membrane to obtain outer membrane vesicles;

[0060] (3) After obtaining the outer membrane vesicles, the outer membrane vesicles are modified by liposomes. The modification method is as follows: liposomes and outer membrane vesicles are resuspended and mixed with PBS at a weight ratio of 2:1, and the modification reaction is carried out on a shaker at 37°C and 100 rpm to obtain modified outer membrane vesicles. The liposomes used are prepared by mixing soybean phosphatidylcholine and cholesterol at a molar ratio of 2:1 and using a thin film hydration method. Specifically, soybean phosphatidylcholine and cholesterol are mixed at a molar ratio of 2:1 and dissolved in chloroform, then dried in a vacuum to form a film, then redissolved in water at 40°C, and then ultrasonically treated to obtain granular liposomes.

[0061] 2. Preparation of drug-loaded nanoparticles:

[0062] 130 mg of polylactic acid and 100 mg of paclitaxel were dissolved in 5 mL of dichloromethane as the oil phase, and an aqueous solution containing 1 wt% of polyvinyl alcohol was used as the water phase; the oil phase was slowly dripped into the water phase. The resulting solution was then treated with a high-speed homogenizer, and after the organic solvent evaporated under stirring, the solution was centrifuged at 8000×g for 5 minutes to collect the nanoparticles.

[0063] 3. Preparation of drug-loaded outer membrane vesicles:

[0064] The modified outer membrane vesicles and nanoparticles were resuspended in PBS at a weight ratio of 2:1, and mechanically extruded through a 200 nm polycarbonate membrane using a liposome extruder. The extrusion was repeated 10 times, and the mixture was centrifuged at 20,000 × g for 1 hour at 4°C. The precipitate was collected to obtain drug-loaded modified outer membrane vesicles.

[0065] Comparative Example 1

[0066] The drug-loaded nanoparticles were prepared according to Example 1 only.

[0067] Comparative Example 2

[0068] Except that QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) was replaced by the Salmonella mutant strain QS0074, the rest was consistent with Example 1.

[0069] Comparative Example 3

[0070] Except for the different preparation method of the drug-loaded nanoparticles in step 2, the rest is the same as Example 1. The preparation method of the drug-loaded nanoparticles of this comparative example is as follows:

[0071] Take 10 mg of paclitaxel and dissolve it in anhydrous ethanol, add it dropwise into 4 mg / mL silk fibroin solution and stir, ultrasonicate the solution in an ice bath with 30% amplitude for 2 minutes, then stir it in a ventilation device for 2 hours, centrifuge it at 6000 rpm for 5 minutes to remove larger particles in the product, and then centrifuge it at 13000 rpm for 20 minutes to collect the precipitate.

[0072] Experimental Example 1

[0073] The drug release experiment was conducted on Examples 1-3 and Comparative Examples 1-3 to investigate the burst release. In order to simulate the actual human body situation, the drug release was conducted in artificial gastric juice (1000 mL of distilled water was taken, pH was adjusted to 1.5 using dilute hydrochloric acid, 10 g of pepsin was added thereto, mixed, and filtered with a 0.45 μm sterile filter) for the first two hours, and then in artificial intestinal juice (1000 mL of distilled water was taken, pH was adjusted to 6.8 using potassium dihydrogen phosphate, 10 g of trypsin was added thereto, mixed, and filtered with a 0.45 μm sterile filter) until the release amount no longer changed, and the release percentage in the first two hours was investigated to evaluate the gastric release. The test results are shown in Table 1.

[0074] Table 1

[0075]

[0076] Experimental Example 2

[0077] The drug-loaded outer membrane vesicles or drug-loaded nanoparticle solutions obtained in Examples 1-4 and Comparative Examples 1-4 were stored at 4°C, and the average particle size and PDI value were measured at 0, 12, 24, 36 and 48 hours, respectively. The results are shown in Tables 2 and 3.

[0078] Table 2

[0079]

[0080] Table 3

[0081]

[0082] As shown in Table 2 and Table 3, the drug-loaded outer membrane vesicles obtained in the present invention have good stability.

Claims

1. A method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles, characterized in that: The preparation method comprises the following steps: (1) preparing outer membrane vesicles of Helicobacter pylori mutant strain QS003 (HelicobacteripyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF); (2) mixing the obtained outer membrane vesicles with polylactic acid nanoparticles loaded with paclitaxel and subjecting them to mechanical extrusion to prepare outer membrane vesicles encapsulating paclitaxel nanoparticles; The Helicobacter pylori mutant strain QS003 (HelicobacteripyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB with plasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) has a deposit number of CCTCC M2023065.

2. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 1, characterized in that: In step (1), a culture of the mutant strain is first obtained, and the culture method is: a monoclonal clone of the Helicobacter pylori mutant strain QS003 (Helicobacteri pyloriΔfutBΔlpxEΔlpxFΔsncRNA-2509025ΔtolB withplasmid Hbp+AffiHER2-AffiEGFR1-AffiVEGF) is cultured in 500 mL of Brucella broth liquid culture medium at 37°C and 180 r / min overnight.

3. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 2, characterized in that: In step (1), the culture is first centrifuged to remove the supernatant, the supernatant is filtered through a membrane, the filtrate is concentrated and then ultracentrifuged, and then the ultracentrifuged precipitate is resuspended and then filtered through a membrane.

4. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 3, characterized in that: In step (1), when membrane filtration is performed, a 0.45 μm filter membrane is used; when the culture is centrifuged, the centrifugation conditions are centrifugation at 15,000 × g for 10 minutes; when concentration is performed, a 500 kDa ultrafiltration column is used; in step (1), when ultracentrifugation is performed, the centrifugation conditions are centrifugation at 200,000 × g and 4°C for 2 hours.

5. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 4, characterized in that: In step (1), after obtaining the outer membrane vesicles, the outer membrane vesicles are modified using liposomes. The modification method is: liposomes and outer membrane vesicles are resuspended and mixed with PBS at a weight ratio of 2:1, and the modification reaction is carried out on a shaker at 37°C and 100 rpm.

6. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 1, characterized in that: In step (2), the preparation method of polylactic acid nanoparticles loaded with paclitaxel is to use dichloromethane containing polylactic acid and paclitaxel as the oil phase and an aqueous solution containing polyvinyl alcohol as the water phase to prepare them by an emulsion method.

7. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 6, characterized in that: In step (2), the weight ratio of polylactic acid to paclitaxel is 13 to 18:

10.

8. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 7, characterized in that: In step (2), the weight percentage of polyvinyl alcohol in the aqueous phase is 1%.

9. The method for preparing a drug carrier of bacterial outer membrane vesicles encapsulating paclitaxel nanoparticles according to claim 8, characterized in that: In step (2), when mechanical extrusion is performed, a 200 nm polycarbonate film is used.

10. A drug carrier of paclitaxel nanoparticles encapsulated by bacterial outer membrane vesicles, characterized in that: The drug carrier is prepared by the preparation method according to any one of claims 1 to 9.

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