Engineered bacteria outer vesicle drug delivery system capable of efficiently penetrating blood brain barrier and targeting glioblastoma as well as preparation method and application of engineered bacteria outer vesicle drug delivery system

Abstract

The invention provides an engineered bacterium outer vesicle drug delivery system capable of efficiently penetrating a blood brain barrier and targeting glioblastoma as well as a preparation method and application of the engineered bacterium outer vesicle drug delivery system, and belongs to the field of drug delivery. The engineering bacteria outer vesicle drug delivery system comprises double-layer phospholipid layer vesicles BEVs, a drug delivery system and a drug delivery system, wherein the particle size of the double-layer phospholipid layer vesicles BEVs is 20 The anti-tumor drug is wrapped in the BEVs, the surface of the BEVs is modified with functional polypeptide, and the functional polypeptide is at least one of Angiopep-2 and TAT cell penetrating peptide. Animal experiments verify that the synergistic effect of the Angiopep-2 and the TAT peptide can overcome the problem of modification saturation of the Angiopep-2 peptide, and significantly improve the ability of BEVs to penetrate through BBB and target tumor tissues. Therefore, the BEVs modified by the Angiopep-2 peptide and the TAT peptide have the advantages of multi-stage targeting, high efficiency and synergy, and can effectively deliver chemotherapeutic drugs to tumor tissues.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and specifically relates to an engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma, as well as a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is one of the most common primary tumors of the central nervous system. Surgical resection combined with adjuvant chemoradiotherapy is often used in clinical practice. However, due to its invasive, infiltrative, and diffuse growth characteristics, it is difficult to completely remove the tumor by surgery, resulting in high recurrence rate, high mortality rate, and high disability rate for patients, with a five-year survival rate of only 5% after surgery. In addition, the presence of the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) hinders the application of many common tumor chemotherapy drugs in GBM. Currently, the GBM treatment drugs approved by the US FDA include only five drugs: Lomustine, Carmustine, Temozolomide (TMZ), Bevacizumab, and Vorasidenib. Therefore, there is an urgent need to develop a drug delivery method that can penetrate the BBB / BTB and target glioblastoma.

[0003] Bacterial extracellular vesicles (BEVs) are extracellular vesicles secreted by bacteria and have been shown to be important tools for communication between bacteria and host cells. Due to their ease of engineering, excellent drug-carrying capacity, and amenable to mass production, they have attracted widespread attention in recent years and have demonstrated their unique value in the treatment of tumors and central nervous system diseases. Summary of the Invention

[0004] The present invention is made to solve the above problems, and aims to provide an engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma, as well as a preparation method and application thereof.

[0005] The present invention provides an engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma, having the following characteristics: double-layer phospholipid vesicles (BEVs) with a particle size of 20-400 nm; and anti-tumor drugs encapsulated in the BEVs, wherein the surface of the BEVs is modified with a functional polypeptide, and the functional polypeptide is at least one of Angiopep-2 and TAT cell-penetrating peptide.

[0006] The engineered bacterial extracellular vesicle drug delivery system provided by the present invention, which efficiently penetrates the blood-brain barrier and targets glioblastoma, may also have the following characteristics: the bacteria from which BEVs are derived include Escherichia coli, Lactobacillus rhamnosus, Lactobacillus plantarum, attenuated Salmonella, or attenuated bacteria in which pathogenic genes such as LPS are knocked out through CRSPR / Cas9 gene knockout technology.

[0007] The engineered bacterial extracellular vesicle drug delivery system provided by the present invention, which efficiently penetrates the blood-brain barrier and targets glioblastoma, may also have the following characteristics: the anti-tumor drug is one or more of paclitaxel PTX, doxorubicin DOX, temozolomide TMZ, and cisplatin Cisplatin, and the anti-tumor drug is used to inhibit the growth of tumor cells.

[0008] The present invention also provides a method for preparing the above-mentioned engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma, which is characterized in that it includes the following steps: step 1, bacterial culture; step 2, separating and purifying the cultured bacterial liquid to obtain double-layer phospholipid vesicles BEVs; step 3, modifying the surface of BEVs with functional polypeptides to obtain polypeptide-modified BEVs; step 4, mixing the polypeptide-modified BEVs with anti-tumor drugs and sonicating them to obtain the engineered bacterial extracellular vesicle drug delivery system.

[0009] The preparation method of the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma provided by the present invention may also have the following characteristics: wherein, step 1 specifically includes: performing primary culture amplification on the bacteria; then transferring to secondary culture and reproduction, collecting the bacterial liquid, and performing low-speed centrifugation at 100-600g to obtain a bacterial liquid supernatant rich in BEVs.

[0010] The preparation method of the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma provided by the present invention may also have the following characteristics: wherein, step 2 specifically includes: first centrifuging the bacterial liquid supernatant at high speed to remove bacterial debris and large particulate matter; filtering the supernatant after centrifugation with 0.45um and 0.22um filters in sequence to completely remove residual bacteria and other contaminants; then using an ultrafiltration tube to concentrate the supernatant 5-10 times, then adding sterile PBS for washing and further concentrating it 1-2 times to remove non-BEVs miscellaneous proteins and contaminants; further using ultracentrifugation to obtain BEVs precipitate.

[0011] In the preparation method of the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma provided by the present invention, it can also have the following characteristics: wherein, step 3 specifically includes: covalently linking the functional polypeptide to the grafting material DSPE-PEG2000-Mal through maleimide with a thioether bond to obtain DSPE-PEG2000-Angiopep-2 peptide and / or DSPE-PEG2000-TAT peptide; and then modifying it to the membrane surface of BEVs by a lipid intercalation method.

[0012] In the preparation method of the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma provided by the present invention, it can also have the following characteristics: wherein, in step 3, the concentration of BEVs is 1 mg / mL, the molar ratio of Angiopep-2 to DSPE-PEG2000-Mal is 1:(3-5), the molar ratio of TAT cell penetrating peptide to DSPE-PEG2000-Mal is 1:(3-5), the mass ratio of BEVs to DSPE-PEG2000-Angiopep-2 is (1-10):1, and the mass ratio of BEVs to SPE-PEG2000-TAT peptide is (1-10):1.

[0013] The preparation method of the engineered bacterial extracellular vesicle drug delivery system for efficiently penetrating the blood-brain barrier and targeting glioblastoma provided by the present invention may also have the following characteristics: wherein step 4 specifically comprises: mixing the polypeptide-modified BEVs with the anti-tumor drug at a concentration of 1×10 11 The vesicles were mixed at a ratio of (0.1-1) mg of particles and pre-incubated at 37°C for 30 minutes. The mixture was then subjected to pulsed ultrasonic treatment with a power of 100 W, a 30-second ultrasonic / 10-second intermittent duty cycle, and a 4°C ice bath for six cycles of loading. The vesicles were placed in an ice bath for 2 minutes between each cycle to maintain vesicle stability. The membrane structure was then reconstructed by incubation at 37°C for another 30 minutes, and the unencapsulated anti-tumor drug was removed by ultracentrifugation.

[0014] The present invention also provides the use of the above-mentioned engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma in the preparation of drugs for treating glioblastoma and central nervous system diseases.

[0015] Functions and effects of the invention

[0016] Compared with the prior art, the engineered bacterial extracellular vesicle drug delivery system of the present invention that efficiently penetrates the blood-brain barrier and targets glioblastoma, as well as its preparation method and application, has the following beneficial effects:

[0017] (1) The engineered bacterial extracellular vesicle drug delivery system provided by the present invention uses bacterial extracellular vesicles (BEVs) secreted by Escherichia coli (E. coli) as a delivery carrier, and modifies two functional peptides, Angiopep-2 and TAT, on its membrane surface through an engineering modification method. Angiopep-2 peptide can specifically bind to low-density lipoprotein receptor-1 (Low-density lipoprotein receptor-related protein-1, LRP-1) highly expressed on the surface of brain vascular endothelial cells and glioma cell membranes, thereby mediating penetration of the BBB and achieving the purpose of targeting glioblastoma. TAT peptide is a cell-penetrating peptide that can penetrate cell membranes and dense tumor tissues. Animal experiments have verified that the synergistic effect of Angiopep-2 and TAT peptides can overcome the problem of saturation of Angiopep-2 peptide modification and significantly improve the ability of bacterial extracellular vesicles to penetrate the BBB and target tumor tissues. Therefore, BEVs modified with Angiopep-2 peptide and TAT peptide have the advantages of multi-level targeting, high efficiency, and synergy, effectively delivering chemotherapy drugs to tumor tissues.

[0018] (2) In the preparation method of the engineered bacterial extracellular vesicle drug delivery system provided by the present invention, Angiopep-2 and TAT cell-penetrating peptides are first covalently linked to the grafting material DSPE-PEG2000-Mal via maleimide via a thioether bond to obtain DSPE-PEG2000-Angiopep-2 and DSPE-PEG2000-TAT peptides, which are then attached to the BEV membrane surface via a lipid intercalation method. This can further reduce the toxicity of BEVs, effectively prolong the time the drug carrier remains in the blood circulation, and significantly extend the drug's pharmacokinetic half-life.

[0019] (3) The engineered bacterial extracellular vesicle drug delivery system provided in the present invention is loaded with the widely used anti-tumor drug paclitaxel (PTX), and has a good effect in inhibiting the growth of glioblastoma (GBM). The synergistic effect of the Angiopep-2 peptide and TAT peptide in the drug carrier helps to better penetrate the BBB and target tumor tissue. Using this drug delivery vehicle, PTX can be effectively delivered to GBM lesions. This engineered bacterial extracellular vesicle drug delivery system can accurately achieve the purpose of inhibiting GBM growth in preclinical studies.

[0020] (4) The engineered bacterial extracellular vesicle drug delivery system provided by the present invention can also be combined with other chemotherapy drugs and gene therapy drugs to achieve the purpose of efficient synergistic anti-tumor treatment. Experiments have shown that it can significantly inhibit the growth of glioblastoma, and the therapeutic effect is better than that of single drug-loaded BEVs or free drugs.

[0021] (5) The preparation method of the engineered bacterial extracellular vesicle drug delivery system provided by the present invention is simple, highly operable, and has extremely high clinical application value and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the process for preparing Angiopep-2 / TAT polypeptide-modified BEVs in Example 1 of the present invention.

[0023] Figure 2 The test example of the present invention successfully knocked out the LPS detection extracted from E. coli K12 (BW25113), the particle size and transmission electron microscopy images of BEVs modified with different peptides, and the changes in Zeta potential.

[0024] Figure 3 This is a fluorescence image of DiL-labeled AT-BEVs taken up by U87MG brain GBM cells in Example 2 of the present invention, as well as the situation of AT-BEVs penetrating the BBB and being taken up by U87MG cells under in vitro BBB simulation conditions, and quantitative analysis by flow cytometry.

[0025] Figure 4 Schematic diagram of the preparation of Angiopep-2 / TAT polypeptide loaded with PTX (AT-BEVs@PTX) and targeted treatment of GBM in Example 3 of the present invention.

[0026] Figure 5 This is a graph showing the characterization of AT-BEVs@PTX in Example 4 of the present invention and the comparison of the results of inhibiting the growth of U87MG cells.

[0027] Figure 6 This is an in vivo imaging diagram of the distribution of DiR-labeled AT-BEVs injected into the tail vein in the GBM in situ mouse model in Example 5 of the present invention.

[0028] Figure 7 This is a diagram showing how AT-BEVs@PTX inhibits GBM tumor growth in vivo in Example 6 of the present invention.

[0029] Figure 8 This is a diagram showing the drug safety evaluation results of AT-BEVs@PTX in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] To facilitate the understanding of the technical means, creative features, objectives, and effects achieved by the present invention, the following examples, combined with the accompanying drawings, specifically illustrate the engineered bacterial extracellular vesicle drug delivery system of the present invention that efficiently penetrates the blood-brain barrier and targets glioblastoma, as well as its preparation method and application.

[0031] Example 1 Preparation of AT-BEVs

[0032] This example provides an Angiopep-2 / TAT-modified BEVs (denoted as AT-BEVs) and a preparation method thereof.

[0033] like Figure 1 As shown, the preparation method of AT-BEVs includes the following steps:

[0034] Step 1: Bacterial culture:

[0035] First, the msbB pathogenicity gene of E. coli K12 (BW25113) was knocked out using CRISPR / Cas9 gene editing technology. The knockout of the pathogenicity gene was confirmed by gel electrophoresis. Bacteria were amplified in a primary culture at 37°C and 220 rpm for 12-24 hours. The cells were then transferred to a secondary culture and grown at 37°C for another 12-24 hours to complete the bacterial proliferation and collect the culture. Next, the culture was centrifuged at low speeds of 100-600g to obtain a supernatant rich in BEVs.

[0036] Step 2: Isolation and purification of BEVs:

[0037] The supernatant of the BEVs-rich bacterial solution was centrifuged at 1000-12,000g for 20-30 minutes, and the centrifugation process was repeated 2-3 times to further remove bacteria and debris; the supernatant after centrifugation was filtered with 0.45um and 0.22um filters in sequence to completely remove residual bacteria and other large particulate matter; it was then concentrated by ultrafiltration, using a 50mL ultrafiltration tube (50kDa-100kDa, 3000-5000g) to concentrate the bacterial supernatant 5-10 times, washed with sterile PBS and further concentrated 1-2 times to remove non-BEVs miscellaneous proteins and contaminants; further ultracentrifugation (50000-200000g) was used for 60-120 minutes to obtain BEVs precipitate. To obtain a highly pure extracellular vesicle suspension, it was resuspended in PBS and centrifuged again to complete the washing process. After resuspending in PBS again, the protein concentration was detected by BCA and stored in a -80°C refrigerator.

[0038] Step 3: Preparation of Angiopep-2 / TAT dual-functionalized BEVs:

[0039] First, DSPE-PEG2000-Angiopep-2 peptide and SPE-PEG2000-TAT peptide were prepared respectively.

[0040] Using the thiol-maleimide click chemistry method, Angiopep-2 and TAT peptides were coupled with DSPE-PEG2k-Mal at a molar ratio of 1:4 in DMSO solvent. After reacting at room temperature for 24 hours under nitrogen protection, the products were purified by 3500Da molecular cutoff dialysis bag (dialysis for 48 hours, deionized water was replaced every 12 hours) and freeze-dried to obtain the amphiphilic targeting ligands DSPE-PEG2000-Angiopep-2 peptide and SPE-PEG2000-TAT peptide.

[0041] Then, 1 mL of BEVs (1 mg / mL) was added with 100 μg of DSPE-PEG2000-Angiopep-2 peptide and 100 μg of SPE-PEG2000-TAT peptide, and the mixture was incubated at 37°C for 2 h to prepare Angiopep-2 / TAT-modified BEVs (AT-BEVs). The free peptides were then removed by ultrafiltration.

[0042] Comparative Example 1 Preparation of Ang-BEVs

[0043] This comparative example provides Angiopep-2-modified BEVs (referred to as Ang-BEVs) and their preparation method. The preparation method for Ang-BEVs differs from the preparation method for AT-BEVs in Example 1 only in that, in Step 3, 1 mL of BEVs (1 mg / mL) is added with 100 μg of DSPE-PEG2000-Angiopep-2 peptide to prepare Angiopep-2 peptide-modified BEVs (Ang-BEVs). All other steps and reaction conditions are identical to those in Example 1.

[0044] Comparative Example 2 Preparation of TAT-BEVs

[0045] This comparative example provides a TAT cell-penetrating peptide-modified BEVs (denoted as TAT-BEVs) and a preparation method thereof. The preparation method of TAT-BEVs differs from the preparation method of AT-BEVs in Example 1 only in that: in step 3, 1 mL of BEVs (1 mg / mL) was added to 100 ug of SPE-PEG2000-TAT peptide cell-penetrating peptide to prepare TAT peptide-modified BEVs (Ang-BEVs). The other steps and reaction conditions are the same as the corresponding steps and conditions in Example 1.

[0046] Test Example 1 Characterization of AT-BEVs

[0047] In this test example, the particle size of BEVs, Ang-BEVs, TAT-BEVs and AT-BEVs prepared in the control, comparative example 1, comparative example 2 and embodiment 1 was tested. The test results are as follows: Figure 2 The results showed that the particle size of AT-BEVs was 30-250 nm.

[0048] Example 2 In vitro cell targeting and BBB penetration efficiency of AT-BEVs

[0049] In this example, the blank control, comparative example 1, comparative example 2, and BEVs, Ang-BEVs, TAT-BEVs, and AT-BEVs prepared in Example 1 were tested for in vitro cell targeting and BBB penetration efficiency. The details are as follows:

[0050] (1) Cell uptake experiment: First, sterilized round coverslips were placed in a 12-well plate (pre-moistened with 100 μL PBS) and inoculated with U87MG glioma cells or bEnd.3 cells (5×10 4 / well) and cultured overnight at 37°C and 5% CO2. When the cell confluence reached 80%, they were incubated with equal doses (5ug / mL) of DiL fluorescent-labeled BEVs, Ang-BEVs, TAT-BEVs, and AT-BEVs for 4 hours. After removing the culture medium, the cells were gently washed three times with pre-cooled PBS and fixed with 4% paraformaldehyde for 20 minutes (4°C in the dark). DAPI was used for nuclear counterstaining. DiL red fluorescence (Ex / Em=549 / 565nm) and DAPI blue fluorescence signals were simultaneously collected by confocal laser scanning microscopy (CLSM). The fluorescence intensity and distribution characteristics of the differently modified BEVs in tumor cells were compared to qualitatively evaluate their targeted uptake efficiency.

[0051] (2) Construction of blood-brain barrier model and evaluation of transmembrane transport: A Transwell system was used to establish an in vitro BBB model. bEnd.3 cells (5×10 4 The upper chamber was inoculated with 500 μl of DMEM medium and 800 μl of DMEM medium were added to the upper and lower chambers respectively. The medium was changed every two days. After about one week, a dense single cell layer was observed at the bottom under a microscope. U87MG glioma cells (5×10 4 Equal doses (5 μg / mL) of DiL-labeled BEVs, Ang-BEVs, TAT-BEVs, and AT-BEVs were added to the upper chamber for 24 hours. Finally, the cell slides from the lower chamber were harvested and processed as described above for cell uptake. CLSM was used to compare and analyze the differences in fluorescence intensity between groups.

[0052] (3) The procedure was the same as in protocols (1) and (2), except that Cy5-labeled BEVs, Ang-BEVs, TAT-BEVs, and AT-BEVs were used. After administration, the cells were trypsinized and washed three times with PBS before analysis by flow cytometry (Beckman, Cytoflex).

[0053] (4) U87MG tumor sphere penetration efficiency: 200 U87MG cells were seeded per well of a 48-well ultra-low adsorption culture plate. The medium was changed every 3 days. After approximately 3 weeks, the diameter of the tumor spheres was approximately 500 μm. Equal amounts of Cy5-labeled BEVs, Ang-BEVs, TAT-BEVs, and AT-BEVs were replaced and co-cultured for 6 h. The cells were washed three times with PBS and transferred to a confocal microplate. CLSM was used to perform Z-axis scanning from the bottom to the middle of the sphere at 40 μm intervals to detect the intensity of the CY5 fluorescence signal.

[0054] AT-BEVs penetrate the BBB and target U87MG cells. Figure 3 .

[0055] Example 3 Preparation of AT-BEVs@PTX

[0056] This example provides an engineered bacterial extracellular vesicle drug delivery system (AT-BEVs@PTX) that efficiently penetrates the blood-brain barrier and targets glioblastoma, also known as Angiopep-2 / TAT polypeptide-loaded PTX, and its preparation method.

[0057] like Figure 4 As shown, the preparation method of AT-BEVs@PTX includes the following steps:

[0058] The AT-BEVs prepared in Example 1 were 1×10 11 After preincubation of 1000 particles / mL with 0.5 mg / mL paclitaxel (PTX) at 37°C for 30 minutes in a 1 mL reaction system, vesicles were loaded using six cycles of pulsed sonication (100 W power, 30 seconds sonication / 10 seconds intermittent duty cycle, controlled in a 4°C ice bath). Vesicles were placed in an ice bath for 2 minutes between cycles to maintain stability. Membrane remodeling was then completed by incubation at 37°C for another 30 minutes. Unencapsulated PTX was removed by ultracentrifugation (50,000-200,000 g, 60-120 minutes) to obtain AT-BEVs@PTX.

[0059] In addition, the unmodified BEVs control group (BEVs@PTX) was prepared using the same process and set aside.

[0060] Example 4 In vitro antitumor activity of AT-BEVs@PTX

[0061] (1) Stability of AT-BEVs@PTX: 30% FBS was used to simulate a blood environment. Before preparing 30% FBS, the cells were centrifuged at 200,000 × g at 4°C for 2 h. The supernatant was removed to remove EVs and particulate matter from the FBS, and PBS was added to prepare 30% FBS. BEVs and AT-BEVs@PTX were added to the above solution, and the average particle size at different time points (0 h, 24 h, 48 h, 72 h, 96 h, and 120 h) was measured by DLS.

[0062] (2) AT-BEVs@PTX cell activity: 2 x 10 cells per well of a 12-well plate 5 U87MG cells in the logarithmic growth phase were cultured in an incubator for 24 hours. The control group received PBS, while the drug-treated groups included Free PTX, BEVs@PTX, and AT-BEVs@PTX. PTX was diluted to 10 μg / ml in DMEM immediately prior to use, with triplicate wells per group. After 24 hours, the cell culture medium and the trypsin-digested cell suspension were collected and centrifuged (500 × g, 4°C, 5 min). The cells were washed twice with PBS, and the cell pellet was collected. Following the apoptosis assay kit protocol, 100 μl of 1× Binding Buffer was added to resuspend the cells. Then, 5 μl of Annexin V / PE and 10 μl of 7-AAD were added and mixed by gentle pipetting. Incubate at room temperature in the dark for 15 min, then 400 μl of 1× Binding Buffer was added and mixed by gentle pipetting. The mixture was placed on ice for flow cytometry analysis within 1 hour.

[0063] (3) For the experiment of AT-BEVs@PTX penetrating the BBB and promoting apoptosis of U87MG, bEnd.3 cells were used to construct the in vitro BBB, and the drug was administered according to the above scheme. Cell processing and flow cytometry were the same as above.

[0064] The inhibition of AT-BEVs@PTX on U87MG cells is shown in Figure 5 .

[0065] Example 5 Tumor targeting and in vivo distribution of AT-BEVs

[0066] (1) Pharmacokinetics: SD rats (200 ± 20 g) were randomly divided into three groups (n = 3): free DiR, BEVs@DiR, and AT-BEVs@DiR. Each group received a 0.3 mg / kg dose of DiR via tail vein injection. Blood (100 μl) was collected from the retinal venous plexus of the rats at 1, 2, 4, 8, 12, 24, 36, and 48 h after injection. The blood samples were centrifuged at 3000 rpm for 10 min to obtain plasma. The fluorescence intensity of DiR in the plasma (λex / λem = 745 / 780 nm) was then measured using a microplate reader.

[0067] (2) PDX orthotopic xenograft model: BALB / c nude mice (female, 4-5 weeks old, weighing 18-22 g) were purchased from Savins Biotech Co., Ltd. (Beijing, China). The mice were housed under a 12-h light-dark cycle, 20-24°C, and 45-65% relative humidity. An ectopic glioblastoma (GBM)-bearing mouse model was constructed using U87-Luc cells by stereotactic injection (2×10^5 cells resuspended in 8 μL PBS). The injection site was 1 mm in front of the anterior bregma and 1.5 mm laterally offset from the midline of the skull. The injection depth was 2.5 mm (first inserted 3 mm, then withdrawn 0.5 mm). The needle was removed after a 5-min interval after the injection was completed. One week later, D-luciferin potassium (15 mg / mL, 150 mg / kg) was injected intraperitoneally, and the growth of GBM was evaluated using a bioluminescence imaging system after isoflurane anesthesia.

[0068] (3) In vivo targeting and distribution: Ten days after GBM modeling, the constructed models were randomly divided into four groups (n=3), namely: BEVs@DiR group, Ang-BEVs@DiR group, TAT-BEVs@DiR group and AT-BEVs@DiR group. 200 μl of DiR-labeled BEVs were injected through the tail vein. At 8 hours after injection, isoflurane anesthesia was performed, and a fluorescence imaging system was used to capture DiR fluorescence images and perform fluorescence quantification to evaluate the distribution of BEVs. 12 hours later, the organs of the mice (including brain, heart, lung, liver, spleen and kidney) were collected, and the same method was used to perform in vitro fluorescence imaging and fluorescence quantification analysis of BEV distribution. In addition, the brain tissue was placed in 4% paraformaldehyde solution, fixed in the dark at 4°C for 24 hours, dehydrated with sucrose gradient, and frozen sectioned into 8 μm slices. The fluorescence distribution of DiR in normal brain tissue and tumor tissue was observed under a fluorescence microscope (Leica, Mica).

[0069] Tumor targeting and fluorescence distribution in various organs are shown in Figure 6 .

[0070] Example 6 Pharmacodynamics Experiment of AT-BEVs@PTX

[0071] (1) Effect of inhibiting GBM tumor growth: A glioblastoma mouse model was constructed according to the method of Experimental Example 4. The mice with successful modeling were randomly divided into 4 groups (10 mice in each group, 6 mice were used for survival analysis and pharmacodynamic evaluation, and 4 mice were killed after the completion of drug administration), namely PBS control group, Free PTX group, BEVs@PTX group and AT-BEVs@PTX group. Starting from the 10th day, the drug was administered by tail vein injection at a dose equivalent to 5 mg / kg of PTX, and was administered once every 3 days for a total of 4 times. During the entire treatment process, the weight changes of the mice were closely recorded, and the fluorescence luminescence was detected regularly using a small animal live imaging device to reflect the growth or inhibition of the tumor. On the 22nd day, the drug administration was terminated, and 4 mice were randomly selected from each group. The brains and important organs were dissected for histopathological analysis. Finally, the survival time of the mice in each group was recorded, and the survival analysis curve was drawn.

[0072] (2) The mouse brain was isolated and fixed with 4% paraformaldehyde for 24 hours, then embedded in paraffin and sliced into 4 μm thick sections. After H&E staining, the sections were observed under a microscope. The brain tissue sections were then subjected to Ki67 immunofluorescence staining and Tunel staining according to the experimental requirements.

[0073] Depend on Figure 7 It can be seen that the body weight change of nude mice in the AT-BEVs@PTX group was the smallest, and the survival time was significantly prolonged, which was better than that in the BEVs@PTX group, free PTX group and PBS group (P<0.05).

[0074] Example 7 Drug safety experiment of AT-BEVs@PTX

[0075] (1) After the administration in Example 4 was terminated, 4 mice in each group were randomly selected for orbital vein blood sampling for routine blood analysis and blood biochemistry analysis.

[0076] (2) HE staining was performed on the heart, liver, spleen, lung, and kidney fixed and preserved in Example 6.

[0077] The experimental results are as follows Figure 8 As shown in the results, AT-BEVs@PTX does not cause obvious pathological damage to organs and tissues and has good biosafety.

[0078] These experimental results demonstrate that AT-BEVs@PTX possesses excellent biosafety, can penetrate the blood-brain barrier, and can effectively deliver paclitaxel, which accumulates in glioblastoma tissue. It also inhibits glioma cell growth in vitro and prolongs the survival of nude mice bearing in situ gliomas. Therefore, AT-BEVs@PTX shows promising potential in the treatment of glioblastoma.

[0079] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma, characterized in that: include: Bilayer phospholipid vesicles (BEVs), with a particle size of 20-400 nm; and Antitumor drugs, encapsulated in the BEVs, The surface of the BEVs is modified with a functional polypeptide, and the functional polypeptide is at least one of Angiopep-2 and TAT cell-penetrating peptide.

2. The engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 1, characterized in that: in, The bacteria from which the BEVs are derived include Escherichia coli, Lactobacillus rhamnosus, Lactobacillus plantarum, attenuated Salmonella, or attenuated bacteria in which pathogenic genes encoding LPS and the like are knocked out through CRSPR / Cas9 gene knockout technology.

3. The engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 1, characterized in that: in, The anti-tumor drug is one or more of paclitaxel PTX, doxorubicin DOX, temozolomide TMZ, and cisplatin Cisplatin, and the anti-tumor drug is used to inhibit the growth of tumor cells.

4. A method for preparing an engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, bacterial culture; Step 2, separating and purifying the cultured bacterial solution to obtain bilayer phospholipid vesicles (BEVs); Step 3, modifying the surface of the BEVs with a functional polypeptide to obtain polypeptide-modified BEVs; Step 4: mixing the polypeptide-modified BEVs with the anti-tumor drug and sonicating them to obtain an engineered bacterial extracellular vesicle drug delivery system.

5. The method for preparing the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 4, characterized in that: in, Step 1 specifically includes: The bacteria were amplified in the primary culture and then transferred to the secondary culture for reproduction. After the bacterial liquid was collected, it was centrifuged at a low speed of 100-600g to obtain the bacterial liquid supernatant rich in BEVs.

6. The method for preparing the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 1, Its characteristics are: Among them, step 2 specifically includes: The supernatant of the bacterial liquid is first centrifuged at high speed to remove bacterial debris and large particulate matter; the supernatant after centrifugation is filtered through 0.45μm and 0.22μm filters in sequence to completely remove residual bacteria and other contaminants; the supernatant is then concentrated 5-10 times using an ultrafiltration tube, washed with sterile PBS, and further concentrated 1-2 times to remove non-BEVs miscellaneous proteins and contaminants; and ultracentrifugation is further performed to obtain a BEVs precipitate.

7. The method for preparing the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 4, characterized in that: in, Step 3 specifically includes: The functional polypeptide is covalently linked to the grafting material DSPE-PEG2000-Mal via maleimide with a thioether bond to obtain DSPE-PEG2000-Angiopep-2 peptide and / or DSPE-PEG2000-TAT peptide; and then modified onto the membrane surface of BEVs using a lipid intercalation method.

8. The method for preparing the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 7, characterized in that: in, In step 3, the concentration of BEVs is 1 mg / mL. The molar ratio of Angiopep-2 to DSPE-PEG2000-Mal is 1:(3-5), The molar ratio of TAT cell penetrating peptide to DSPE-PEG2000-Mal was 1:(3-5), The mass ratio of BEVs to DSPE-PEG2000-Angiopep-2 was (1-10):1, The mass ratio of BEVs to SPE-PEG2000-TAT peptide was (1-10):

1.

9. The method for preparing the engineered bacterial extracellular vesicle drug delivery system that efficiently penetrates the blood-brain barrier and targets glioblastoma according to claim 1, characterized in that: in, Step 4 specifically includes: The peptide-modified BEVs were mixed with anti-tumor drugs at a rate of 1×10 11 The vesicles were mixed at a ratio of (0.1-1) mg of particles and pre-incubated at 37°C for 30 minutes. The mixture was then subjected to pulsed ultrasonic treatment with a power of 100 W, a 30-second ultrasonic / 10-second intermittent duty cycle, and a 4°C ice bath for six cycles of loading. The vesicles were placed in an ice bath for 2 minutes between each cycle to maintain vesicle stability. The membrane structure was then reconstructed by incubation at 37°C for another 30 minutes, and the unencapsulated anti-tumor drug was removed by ultracentrifugation.

10. Use of the engineered bacterial extracellular vesicle drug delivery system according to any one of claims 1 to 3 that efficiently penetrates the blood-brain barrier and targets glioblastoma in the preparation of drugs for treating glioblastoma and central nervous system diseases.

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