Bacterial outer membrane vesicle as well as preparation method and application thereof

Abstract

The invention provides a bacterial outer membrane vesicle as well as a preparation method and application thereof. Mediated cell-penetrating peptide and chlorotoxin are co-expressed on the surface of the bacterial outer membrane vesicle. The polypeptide can efficiently pass through BBB and specific targeting tumor cells in vitro and in vivo, and ferroptosis of tumor cells is induced by stimulating IFN-gamma released by CD8 + T cells in vivo, so that growth of glioblastoma is remarkably inhibited. In addition, the IFN-gamma enhances the ferroptosis of the tumor cells caused by the ferroptosis inducer RSL-3 and the erastin. The invention further provides a pharmaceutical composition, the RSL-3 is loaded to the OMV-C-C (at) RSL-3 nano delivery system formed by OMV-C-C, and the OMV-C-C and the RSL-3 show a good synergistic effect in the aspect of inhibiting the growth of glioblastoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a bacterial outer membrane vesicle and a preparation method and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is the most common and highest malignant astrocytoma. At present, various different treatments for glioblastoma are carried out in clinic, including surgical treatment, radiotherapy, chemotherapy and supportive treatment; however, compared with other types of tumors, the treatment method for glioblastoma in clinic is still quite scarce. Immunotherapy brings new hope for the treatment of glioblastoma due to its high specificity and small side effects. Immune checkpoint blockade therapy, chimeric antigen receptor T cell immunotherapy, tumor vaccine and oncolytic virus therapy show certain curative effect in the immunotherapy of glioblastoma; however, due to the inherent characteristics of glioblastoma, there are still many problems in the immunotherapy of glioblastoma, such as low patient immunotherapy response rate and poor effect of targeted drug delivery. Therefore, it is urgent to develop new immunotherapy methods to improve the treatment effect and quality of life of glioblastoma patients.

[0003] Due to its special size and structure, nanomaterials have the advantage of precise design, which shows great application potential in the diagnosis and treatment of tumors. In recent years, biomimetic nanomaterials, especially natural biological membranes, have become a research hotspot in the field of biomedicine due to their special functions such as ligand recognition, biological targeting and long circulation time. Bacterial outer membrane vesicle (OMV) is a spherical nanovesicle originated from gram-negative bacteria, with a diameter of about 20-250 nm. It can not only act as a nanocarrier to deliver drugs, but also has certain adjuvant activity due to the rich pathogen-associated molecular patterns in its structure. However, the efficiency of OMV itself in crossing the blood-brain barrier (BBB) is low, which limits its application in the treatment of brain diseases. The existing OMV-based brain disease treatment platform mainly crosses the BBB in an "indirect" way. The pathogen-associated molecular patterns on the surface of OMV can be selectively recognized and phagocytosed by neutrophils, and the "cell vehicle" of neutrophils is used to achieve the effect of crossing the BBB. This operation mode of crossing the BBB is complex, which seriously affects the drug delivery efficiency. However, there is no report on the nanodelivery system based on OMV directly crossing the BBB. Due to the stable and easy-to-modify characteristics of Escherichia coli, it can be functionally modified by genetic engineering means.

[0004] Therefore, the genetic engineering modification of Escherichia coli is expected to develop a safe and efficient OMV nanodelivery platform with the ability to cross the BBB and tumor targeting for the immunotherapy of glioblastoma. SUMMARY

[0005] The present application aims to solve the problems existing in the prior art, and the present application co-expresses a cell-penetrating peptide (CPP) and a chlorotoxin (CLT) on the surface of an OMV through genetic engineering modification of the OMV, so that the OMV has the ability to cross the BBB and target tumor cells, and is named OMV-C-C. The present application also proves from the mechanism that the ferroptosis of tumor cells induced by interferon-gamma (IFN-gamma) plays an important role in the anti-tumor effect of OMV-C-C. Further, the present application proposes a combined treatment strategy of OMV-C-C and a ferroptosis inducer based on the anti-tumor mechanism.

[0006] Therefore, in the first aspect of the present application, the present application provides a bacterial outer membrane vesicle co-expressing a cell-penetrating peptide and a chlorotoxin on the surface of the bacterial outer membrane vesicle.

[0007] The present application provides a genetically engineered OMV, i.e., OMV-C-C, which is realized by expressing a cell-penetrating peptide (CPP) and a chlorotoxin (CLT) on the surface of the OMV. The CPP and the CLT are simultaneously displayed on the surface of the OMV, so that the OMV has the ability to efficiently cross the BBB and the ability to specifically target brain tumor cells, and has clinical transformation value.

[0008] The OMV-C-C can efficiently cross the BBB in an in vitro BBB model and in a mouse in vivo. Meanwhile, the OMV-C-C has specific targeting effect on glioblastoma in vitro and in vivo.

[0009] Preferably, the cell-penetrating peptide is expressed on the surface of the bacterial outer membrane vesicle by a plasmid expressing the cell-penetrating peptide, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID No: 1, and the vector expressing the cell-penetrating peptide is pAIDA1.

[0010] Preferably, the chlorotoxin is expressed on the surface of the bacterial outer membrane vesicle by a plasmid expressing the chlorotoxin, the amino acid sequence of the chlorotoxin is shown as SEQ ID No: 3, the plasmid expressing the chlorotoxin is a modified pET28, and the modified pET28 is a pET28 in which a T7 promoter region originally present is deleted and an amino acid sequence shown as SEQ ID No: 2 is inserted.

[0011] Preferably, the bacterial outer membrane vesicle is an E. coli outer membrane vesicle.

[0012] In the second aspect of the present application, the present application provides a preparation method of the bacterial outer membrane vesicle of the first aspect of the present application, characterized in that the method comprises the following steps: Step 1): mix a plasmid expressing a cell-penetrating peptide and E. coli competence, and stand still; Step 2): heat shock treatment at 40-45℃ for 85-95 s, and stand; Step 3): LB medium is added for culture, and after the end of the culture, LB solid culture plate is coated for culture; Step 4): single clone is selected, LB medium containing chloramphenicol is added for culture, and the cultured bacterial liquid is transferred to LB culture solution; when the OD 600 value is 0.3-0.4, the bacterial body is collected by centrifugation, CaCl2 solution is added, and E. coli BL21-CPP competence is obtained; Step 5): the plasmid expressing chlorotoxin and the E. coli BL21-CPP competence are uniformly mixed, and stand; Step 6): heat shock treatment at 40-45℃ for 85-95 s, and stand; Step 7): LB medium is added for culture, and after the end of the culture, LB solid culture plate is coated for culture; Step 8): single clone is selected, LB medium containing chloramphenicol and kanamycin is added for culture, and the cultured bacterial liquid is added to LB culture solution containing chloramphenicol and kanamycin for culture; when the OD 600 value reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside is added for induction; Step 9): centrifugation, filtration of the supernatant, centrifugal treatment of the filtered supernatant, collection of the supernatant, and centrifugal resuspension to obtain the bacterial outer membrane vesicle. The bacterial outer membrane vesicle co-expressing CPP and CLT on the surface is OMV-C-C.

[0013] In a third aspect of the present application, the present application provides a pharmaceutical composition comprising the bacterial outer membrane vesicle of the first aspect of the present application or the bacterial outer membrane vesicle prepared by the preparation method of the second aspect of the present application.

[0014] In the constructed mouse model of glioma in situ, after tail vein injection of OMV-C-C (3-5 μg) (48-72 h), the infiltration of CD8 + T cells in the tumor site was significantly increased; at the same time, the level of IFN-γ released by CD8 + T cells was also significantly increased. The level of lipid peroxidation of tumor cells in the brain of the mouse was significantly increased, proving the occurrence of ferroptosis. The neutralizing antibody of anti-IFN-γ significantly inhibited the inhibition of OMV-C-C on the tumor in the brain of the mouse, further proving the role of IFN-γ-induced tumor cell ferroptosis in the OMV-C-C-mediated anti-tumor.

[0015] Preferably, the pharmaceutical composition further comprises a ferroptosis inducer.

[0016] Preferably, the ferroptosis inducer is RSL-3.

[0017] In a fourth aspect of the present application, the present application provides a preparation method of the above-mentioned pharmaceutical composition, comprising the following steps: mixing the bacterial outer membrane vesicle and the ferroptosis inducer, oscillating for 3-6 hours, purifying, and washing to obtain the pharmaceutical composition, wherein the mass ratio of the bacterial outer membrane vesicle and the ferroptosis inducer is 1:0.8-1.2.

[0018] In a fifth aspect of the present application, the present application provides an application of the bacterial outer membrane vesicle in the first aspect of the present application or prepared by the preparation method in the second aspect of the present application or the pharmaceutical composition in the third aspect of the present application or prepared by the preparation method in the fourth aspect of the present application in the preparation of a drug for promoting the expression of IFN-γ.

[0019] In a sixth aspect of the present application, the present application provides an application of the bacterial outer membrane vesicle in the first aspect of the present application or prepared by the preparation method in the second aspect of the present application or the pharmaceutical composition in the third aspect of the present application or prepared by the preparation method in the fourth aspect of the present application in the preparation of an anti-tumor drug.

[0020] Preferably, the anti-tumor drug is an anti-glioblastoma drug.

[0021] The present application has the following beneficial effects: 1. The present application provides a bacterial outer membrane vesicle, which is constructed by a genetic engineering method to co-express CPP and CLT on the surface, i.e. OMV-C-C. Unlike traditional chemical modification methods, this method can efficiently and controllably regulate the expression amount of CPP and CLT. The OMV-C-C provided by the present application can efficiently cross the BBB and specifically target tumor cells in vitro and in vivo. The immune stimulation effect of OMV-C-C itself can induce the release of IFN-γ from CD8 + T cells in vivo, thereby significantly inhibiting the growth of glioblastoma. In addition, IFN-γ enhances the ferroptosis of tumor cells caused by the ferroptosis inducers RSL-3 and erastin.

[0022] 2. The present application provides a pharmaceutical composition comprising a bacterial outer membrane vesicle, and RSL-3 can be loaded into the OMV-C-C@RSL-3 nanodelivery system formed by OMV-C-C, which also significantly inhibits the growth of glioblastoma in vivo, and OMV-C-C and RSL-3 exhibit good synergistic effect in inhibiting the growth of glioblastoma.

[0023] 3. The present application provides an application of the above-mentioned bacterial outer membrane vesicle and the above-mentioned pharmaceutical composition in the preparation of an anti-tumor drug.

[0024] 4. The application provides a preparation method of the above-mentioned bacterial outer membrane vesicles and the above-mentioned pharmaceutical composition, which is simple in method, controllable in cost, remarkable in treatment effect and has extremely strong clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Schematic diagram of a plasmid for expressing CPP; Figure 2 Schematic diagram of a plasmid for expressing CLT; Figure 3 Construction and characterization of OMV-C-C; wherein, Figure 3 a in FIG. 1 is a transmission electron microscope picture of OMV-WT, and the scale is 50 nm; Figure 3 b in FIG. 1 is a transmission electron microscope picture of OMV-C-C, and the scale is 50 nm; Figure 3 c in FIG. 1 is the test result of the hydrodynamic diameter of OMV-WT and OMV-C-C; Figure 3 d in FIG. 1 is the surface charge of OMV-WT and OMV-C-C; Figure 3 e in FIG. 1 is the expression of CPP and CLT on the surface of E. coli-C-C detected by flow cytometry; Figure 3 f in FIG. 1 is the expression of CPP and CLT on E. coli-C-C and OMV-C-C detected by immunoblotting experiment; Figure 3 g in FIG. 1 is the expression of CPP and CLT on the surface of OMV-C-C detected by immunogold labeling experiment, and the scale is 20 nm; Figure 3 h in FIG. 1 is the protein expression profile of E. coli-C-C and OMV-C-C.

[0026] Figure 4 Detection result of the function of OMV-C-C in crossing BBB and targeting tumor cells; wherein, Figure 4 a in FIG. 2 is a schematic diagram of in vitro blood-brain barrier construction; Figure 4 b in FIG. 2 is the crossing efficiency evaluation of OMV-C-C-ICG on in vitro blood-brain barrier; Figure 4 c in FIG. 2 is a fluorescence imaging picture of the brain of a mouse; Figure 4 d in FIG. 2 is a fluorescence imaging picture of the brain tissue of a mouse; Figure 4 e in FIG. 2 is the uptake of OMV-C-C-Dil by GL261 cells analyzed by flow cytometry; Figure 4 f in FIG. 2 is the uptake of OMV-C-C-Dil by HMC3 cells analyzed by flow cytometry; Figure 4 g in FIG. 2 is a fluorescence imaging picture of the brain of a tumor model mouse; Figure 4 h in FIG. 2 is the fluorescence intensity analysis of the brain of a tumor model mouse; Figure 4In this image, 'i' represents a fluorescence imaging photograph of the brain tissue of a tumor model mouse. Figure 4 In this context, j represents the fluorescence intensity analysis of brain tissue from a tumor model mouse.

[0027] Figure 5 The results of the OMV-CC antitumor activity evaluation test; in, Figure 5 In the diagram, 'a' represents the OMV-CC anti-tumor detection method. Figure 5 In the image, b represents brain tissue imaging images of tumor-bearing mice in different treatment groups; Figure 5 In the figure, c represents the fluorescence intensity analysis of brain tissue from tumor-bearing mice in different treatment groups; Figure 5 In the figure, d represents a comparison of the survival time of mice in different treatment groups; Figure 5 In the figure, 'e' represents a comparison of the body weights of mice in different treatment groups. Figure 5 f in the image represents HE staining images of the brains of mice in different treatment groups.

[0028] Figure 6 The results of the study on the anti-tumor mechanism of OMV-CC; in, Figure 6 In the figure, 'a' represents the cluster analysis of different immune cells infiltrating tumor tissue as displayed by mass cytometry. Figure 6 In this context, 'b' represents the expression analysis of different functional markers; Figure 6 In this context, 'c' represents CD4 infiltrating the tumor tissue. + T cells, CD8 + A statistical analysis of the percentages of different types of immune cells, including T cells, NK cells, and B cells. Figure 6 In this context, d represents the percentage of different types of immune cells, such as microglia, macrophages, and NKT cells, infiltrating the tumor tissue. Figure 6 In this context, 'e' represents the percentage of different types of immune cells, such as monocytes, dendritic cells, and myeloid cells, infiltrating the tumor tissue. Figure 6 f in CD8 + Immunofluorescence images of T cell infiltration and IFN-γ expression; Figure 6 In this context, 'g' represents the ferroptosis results of tumor cells in mouse tumor tissue analyzed by flow cytometry. Figure 6 In the figure, h represents the statistical analysis of fluorescence intensity in the brains of mice in different treatment groups.

[0029] Figure 7 This is due to the enhancing effect of IFN-γ on ferroptosis in tumor cells; in, Figure 7 In this context, 'a' represents the cell viability evaluation of GL261 cells and U87-MG cells; Figure 7 b in the figure represents the evaluation of the effects of different ferroptosis agonists on GL261 cells and U87-MG cells; Figure 7c is the evaluation of the effect of IFN-γ combined with erastin on ferroptosis of GL261 cells and U87-MG cells in Example 1; Figure 7 d is the evaluation of the effect of IFN-γ combined with RSL-3 on ferroptosis of GL261 cells and U87-MG cells in Example 1; Figure 7 e is the flow cytometry detection of the death of GL261 cells and U87-MG cells treated with IFN-γ combined with RSL-3 or erastin in Example 1; Figure 7 f is the statistical analysis of flow cytometry detection of U87-MG cells treated with IFN-γ combined with RSL-3 or erastin in Example 1; Figure 7 g is the statistical analysis of flow cytometry detection of GL261 cells treated with IFN-γ combined with RSL-3 or erastin in Example 1; Figure 7 h is the effect of flow cytometry detection of the lipid peroxidation level of U87-MG cells treated with IFN-γ combined with RSL-3 or erastin in Example 1; Figure 7 i is the effect of flow cytometry detection of the lipid peroxidation level of GL261 cells treated with IFN-γ combined with RSL-3 or erastin in Example 1; Figure 7 j is the detection of the protein expression level of GPX-4 by immunoblotting experiment in Example 1.

[0030] Figure 8 a is the anti-tumor effect of OMV-C-C@RSL-3 in Example 2; wherein, Figure 8 a is the transmission electron microscopy pictures of OMV-C-C and OMV-C-C@RSL-3, with a scale of 50 nm in Example 2; Figure 8 b is the hydrodynamic diameter analysis chart of OMV-C-C and OMV-C-C@RSL-3 in Example 2; Figure 8 c is the surface charge of OMV-C-C and OMV-C-C@RSL-3 in Example 2; Figure 8 d is the schematic diagram of the anti-tumor detection of OMV-C-C@RSL-3 in Example 2; Figure 8 e is the brain tissue imaging picture of tumor mice in different treatment groups in Example 2; Figure 8 f is the fluorescence intensity analysis of brain tissue of tumor mice in different treatment groups in Example 2; Figure 8 g is the body weight of mice in different treatment groups in Example 2; Figure 8 h is the survival time of mice in different treatment groups in Example 2. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with specific examples. However, the following examples are only used to illustrate the application and should not be considered as limiting the scope of the application. In the following examples, the specific conditions not noted are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The methods used are the conventional methods known in the art unless otherwise specified. The consumables and reagents used are commercially available unless otherwise specified. The professional and scientific terms used herein have the same meanings as those familiar to the skilled person in the art. In addition, any method or material similar or equivalent to those described can also be applied to the present application.

[0032] Example 1 The preparation method of OMV-WT includes the following steps: 1) E. coli BL21 (BE3) competent (2 μL) was added to LB culture solution (10 mL), shaken at 37℃, 200 rpm for 12 h to obtain LB bacterial solution, and 10 mL of LB bacterial solution was added to 1 L of LB culture solution, shaken at 37℃, 200 rpm for 12 h; 2) The shaken bacterial solution was centrifuged twice (4000 rpm, 4℃, 15 min), and the supernatant was recovered and filtered using a filtration system (0.22 μM). The filtered supernatant was further centrifuged using a 100,000 MWCO separation tube (5000 rpm, 4℃, 10 min), and then the supernatant was collected; 3) The collected supernatant was subjected to ultracentrifugation (150000 × g, 4℃, 2h), and the prepared OMV was resuspended using 0.9% physiological saline after centrifugation, i.e. OMV-WT.

[0033] The construction method of the plasmid for expressing CPP is as follows: the amino acid sequence (as shown in SEQ ID No: 1) mediating cell penetrating peptide is inserted into the vector pAIDA1, and the synthesis is entrusted to Beijing Qikexin Biotechnology Co., Ltd. Figure 1 The schematic diagram of the plasmid for expressing CPP is shown in the following figure.

[0034] The construction method of the plasmid for expressing CLT is as follows: the original T7 promoter of the vector pET28 is deleted, and the amino acid sequence as shown in SEQ ID No: 2 is inserted into the vector pET28 with the deleted T7 promoter, and the synthesis is entrusted to Beijing Qikexin Biotechnology Co., Ltd. Figure 2 The schematic diagram of the plasmid for expressing CLT is shown in the following figure.

[0035] The preparation method of OMV-C-C includes the following steps: 1) The plasmid for expressing CPP (3 μL) and E. coli BL21 (BE3) competent (50 μL) were mixed and placed on ice for 30 min; 2) Heat the above mixed solution at 42°C for 90 seconds, and then immediately let it stand on ice for 5 minutes.

[0036] 3) Add 400 μL of non-resistant LB medium to the above solution, incubate at 37°C for 60 min at 200 rpm. After incubation, take 200 μL and spread it onto an LB solid culture plate, then incubate overnight at 37°C. 4) Select single clones and add them to LB medium containing chloramphenicol (10 μg / mL). Incubate at 37°C and 200 rpm for 12 h with shaking. Then, transfer the overnight culture to 100 mL of LB medium. When OD... 600 When the value is approximately 0.3-0.4, the bacteria are collected by centrifugation, and CaCl2 solution (0.1 M) is added to prepare new competent cells expressing CPP, namely Escherichia coli BL21-CPP competent cells; 5) Mix the plasmid expressing CLT (3 μL) and E. coli BL21-CPP competent cells (50 μL) and incubate on ice for 30 min; 6) Heat the above mixed solution at 42°C for 90 seconds, then immediately place it on ice for 5 minutes; 7) Add 400 μL of antibiotic-free LB medium to the above solution, incubate at 37°C for 60 min at 200 rpm. After incubation, take 200 μL and spread it onto an LB solid culture plate, then incubate overnight at 37°C. 8) Select single clones and add them to 50 mL of LB medium containing chloramphenicol (50 μg / mL) and kanamycin (50 μg / mL). Incubate at 37°C and 200 rpm for 12 h with shaking. Then, take 10 mL of the mixture and add it back to 1 L of LB medium (containing chloramphenicol (50 μg / mL) and kanamycin (50 μg / mL)). Incubate at 37°C and 200 rpm with vigorous shaking for 3 h. OD 600 When the concentration reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (0.5 mM) and induce for 3-5 h to obtain Escherichia coli-CC; 9) Centrifuge the *E. coli*-CC twice (4000 rpm, 4 ℃, 15 min), collect the supernatant, and filter it using a filtration system (0.22 μM). Further centrifuge the filtered supernatant using a 100,000 MWCO separation tube (5000 rpm, 4 ℃, 10 min), and then collect the supernatant. 10) The collected supernatant was ultracentrifuged (150000 × g, 4 ℃, 2h). After centrifugation, the OMV prepared by surface co-expression of CPP and CLT was resuspended in 0.9% physiological saline, which is OMV-CC.

[0037] Figure 3 a is the transmission electron microscope picture of OMV-WT, the scale is 50 nm; Figure 3 b is the transmission electron microscope picture of OMV-C-C, the scale is 50 nm; Figure 3 c is the test results of hydrodynamic diameter of OMV-WT and OMV-C-C; Figure 3 d is the surface charge of OMV-WT and OMV-C-C; Figure 3 e is the flow cytometry detection of the expression of CPP and CLT on the surface of E. coli-C-C; Figure 3 f is the immunoblotting experiment for detecting the expression of CPP and CLT on E. coli-C-C and OMV-C-C; Figure 3 g is the immunogold labeling experiment for detecting the expression of CPP and CLT on the surface of OMV-C-C, the scale is 20 nm; Figure 3 h is the protein expression profile of E. coli-C-C and OMV-C-C. The results show that this genetic engineering modification successfully realizes the co-expression of CPP and CLT on the surface of E. coli and OMV-C-C, without significantly changing the size, morphology, surface charge and protein composition of OMV itself.

[0038] Example 2 OMC-C-C efficiently crosses the BBB and specifically targets tumor cells: A: Take the frozen tube containing the frozen mouse brain microvascular endothelial cells bEND.3 from liquid nitrogen, quickly put it into a 37°C water bath, shake slightly, and thaw the mouse brain microvascular endothelial cells bEND.3. Culture the recovered mouse brain microvascular endothelial cells bEND.3 in a sterile centrifuge tube containing 10% FBS inactivated fetal bovine serum, 1% penicillin / 1% streptomycin (double-antibiotic) DMEM culture medium, in a suspension cell bottle, in a 37°C, 5% CO2 and relative humidity 90% incubator, and culture the cells to 70%-80% for 1 passage. Continue to culture to obtain active mouse brain microvascular endothelial cells bEND.3, collect the cells. Centrifuge the cells in the logarithmic growth phase, count the cells, dilute the cell density to 3×10 4 -5×10 4 / 500μl, blow and mix, and inoculate the cells in the upper chamber of the Transwell well plate at 3×10 5 / 500μl per well, and culture the cells for 24 h. Use an ohmmeter to detect the resistance of the BBB, and the resistance reaches 200Ω / cm 2PBS, free ICG (indocyanine green, 1 μg / mL), OMV-WT-ICG (OMV-WT: 10 μg / mL; ICG: 1.16 μg / mL) and OMV-C-C-ICG (OMV-C-C: 10 μg / mL; ICG: 0.91 μg / mL) were added into the upper chamber. The fluorescence intensity in the upper and lower chambers was detected at 2 h and 4 h, respectively, and the penetration efficiency of OMV-C-C-ICG was calculated. For the tannic acid (0.125%, w / v) and methyl-β-cyclodextrin (MβCD, 0.04%, w / v) treatment groups, the two drugs were pre-treated for 1 h, and then PBS, free ICG (indocyanine green, 3 μg / mL), OMV-WT-ICG (OMV-WT: 10 μg / mL; ICG: 2.98 μg / mL) and OMV-C-C-ICG (OMV-C-C: 10 μg / mL; ICG: 2.42 μg / mL) were added into the upper chamber. The fluorescence intensity in the upper and lower chambers was detected at 2 h and 4 h, respectively, and the penetration efficiency of OMV-C-C-ICG was calculated. The results are shown in Fig. 1a, b. Figure 4 Figure 4 As shown in Fig. 1a, b, OMV-C-C efficiently penetrated the BBB, and the penetration efficiency increased with time. The pretreatment of tannic acid and methyl-β-cyclodextrin inhibited the penetration efficiency, indicating that the CPP-mediated efficient penetration of OMV-C-C across the BBB was probably achieved by transcytosis. To further prove the ability of OMV-C-C to penetrate the BBB in vivo, C57BL / 6 mice were randomly divided into four groups, with 3 mice in each group. PBS, free ICG (indocyanine green, 3 μg / mL), OMV-WT-ICG (OMV-WT: 200 μg / mL; ICG: 2.98 μg / mL) and OMV-C-C-ICG (OMV-C-C: 200 μg / mL; ICG: 2.42 μg / mL) were injected into the mice through the tail vein, and the fluorescence intensity in the brain of the mice was detected using small animal imaging at 2 h, Figure 4 Fig. 1c is a fluorescence imaging picture of the brain of the mice, Figure 4 Fig. 1d is a fluorescence imaging picture of the brain tissue of the mice, and the results show that OMV-C-C efficiently penetrated the BBB in vivo.

[0039] ​B: The frozen tube containing human glioma U87-MG and human microglial cells HMC3 was taken out from liquid nitrogen and quickly put into a 37°C water bath, shaken slightly, and the liquid was melted. The human glioma U87-MG and human microglial cells HMC3 were recovered. The recovered human glioma U87-MG and human microglial cells HMC3 were cultured in a centrifuge tube containing 10% FBS inactivated fetal bovine serum, 1% penicillin / 1% streptomycin (double-antibiotic) sterile DMEM culture solution, placed in a suspension cell bottle, and cultured in a 37°C, 5% CO2, and 90% relative humidity incubator. The cells were subcultured once when they reached about 70%-80% confluence. The actively growing human glioma U87-MG and human microglial cells HMC3 were continuously cultured and collected. The cells in the logarithmic growth phase were centrifuged, and after cell counting, the cell density was diluted to 3x10 4 -5x10 4 / mL, mixed well by blowing, and the cells were inoculated in a 6-well plate at 3x10 5 / well. PBS, free Dil (fluorescent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine Iodide, 0.67 μg / mL), OMV-WT-Dil (OMV-WT: 10 μg / mL; Dil (fluorescent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine Iodide): 1.14 μg / mL; preparation method of OMV-WT-Dil: 100 μg OMV-WT and Dil 2 μg were mixed and mixed on a vortex shaker for 1 h (37°C, 300 rpm), then centrifuged with an ultrafiltration tube (5000 rpm, 10 min) to remove free OMV-WT and Dil, and OMV-WT-Dil and OMV-C-C-Dil (OMV-C-C: 10 μg / mL; Dil: 0.82 μg / mL; preparation method same as OMV-WT-Dil) were added to the 6-well plate to treat the cells for 6 h. The flow cytometry detection results of human glioma U87-MG and human microglial cells HMC3 are shown in e、 Figure 4 and f、 Figure 4The results of flow cytometry detection showed that OMV-C-C-Dil specifically targeted tumor cells. To further prove the ability of OMV-C-C-ICG to specifically target tumor cells in vivo, glioma mouse models were randomly divided into four groups, three in each group. PBS, free ICG (indocyanine green, 3 μg / mL), OMV-WT-ICG (OMV-WT: 10 μg / mL; ICG: 2.98 μg / mL) and OMV-C-C-ICG (OMV-C-C: 10 μg / mL; ICG: 2.42 μg / mL) were injected into the mice through the tail vein, and the degree of coincidence of fluorescence intensity and bioluminescence in the brain of the mice was detected using small animal imaging 2 h later, Figure 4 g is the fluorescence imaging picture of the brain of the tumor model mouse, Figure 4 h is the fluorescence intensity analysis of the brain of the tumor model mouse, Figure 4 i is the fluorescence imaging picture of the brain tissue of the tumor model mouse, Figure 4 j is the fluorescence intensity analysis of the brain tissue of the tumor model mouse, and the results show that OMV-C-C-ICG specifically targets tumor cells in tumor model mice.

[0040] Example 3 6-8-week-old C57 / BL6 mice were selected, and GL261-Luc cells (5 x 10 4 were injected into the brain of C57 / BL6 mice (2 mm, 1 mm, 3 mm) using a brain stereotactic injector to construct a mouse tumor model. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animal Resources and the National Research Council and were approved by the Yantai New Drug Development Shandong Laboratory Animal Ethics Committee. The success of tumor implantation was determined by a bioluminescence imaging system. The mouse tumor model was randomly divided into three groups (PBS, OMV-WT, OMV-C-C), and OMV-WT (5 μg) and OMV-C-C (5 μg) were injected through the tail vein, and small animal live imaging was performed every 7 days, Figure 5 a is a schematic diagram of OMV-C-C anti-tumor detection, Figure 5 b is the brain tissue imaging picture of the tumor mouse in different treatment groups, Figure 5 c is the fluorescence intensity analysis of the brain tissue of the tumor mouse in different treatment groups, and the results show that OMV-C-C significantly inhibits the growth of mouse tumors. Figure 5d is the survival time of mice in different treatment groups. As can be seen from the figure, after OMV-C-C treatment, the survival time of mice is significantly prolonged. The body weight of mice is recorded every two days, Figure 5 e is the body weight comparison chart of mice in different treatment groups. The results show that the body weight of mice has increased to a certain extent, but there is no significant difference compared with the control group. In addition, the brain tissue of mice was taken out after 30 days and fixed in 4% paraformaldehyde solution. After hematoxylin / eosin staining, it was observed under a microscope, Figure 5 f is the brain HE staining picture of mice in different treatment groups. The results show that the area of brain tumor tissue after OMV-C-C treatment is significantly smaller than that of the control group.

[0041] Example 4 A: In order to systematically study the anti-tumor immune response of OMV-C-C in vivo, after 3 days of tail vein injection of OMV-WT (5 μg) and OMV-C-C (5 μg), the brain primary tumors of each group of mice were dissected and collected. Freshly collected tumor tissues were dissociated using Librase / DNAzyme prepared with DMEM and incubated at 37°C water bath for 45 min to prepare single cell suspension. Blocking antibody was used for blocking before staining. Cells were stained with Intercalator-Rh (0.5 μM, reagent produced by Standard BioTools Company) for vitality staining, washed with DPBS and fixed in 1.6% formaldehyde solution at room temperature for 10 min, and then fixed and permeabilized using Perm-S Buffer buffer, and then stained with different antibodies. Before machine, the sample was re-washed and resuspended in Cell Acquisition Solution Plus (reagent produced by Standard BioTools Company) solution containing EQ™ Six Element Calibration. Using mass spectrometry flow cytometry, the expression of different markers (CD8, CD4, CD43, CD44, CD45, CD11b, CD19, CD27, CD80, CD11c, Ly6G, Ly6C, CD3, CD62L, CD335 / NKp46, CD161 / NK1.1, F4 / 80, CD25, CD127, MHC-II) was detected, Figure 6 a is the cluster analysis of different immune cells infiltrated in tumor tissues by mass spectrometry flow cytometry display; Figure 6 b is the expression analysis of different functional markers; Figure 6 c is the percentage of CD4 + T cells, CD8 + T cells, NK cells and B cells, and the percentage statistics of different types of immune cells; Figure 6d is the percentage of different types of immune cells, such as infiltrating microglia, macrophages and NKT cells in tumor tissues; Figure 6 e is the percentage of different types of immune cells, such as infiltrating monocytes, DC cells and myeloid cells in tumor tissues; Figure 6 f is the percentage of CD8 + T cells in tumor tissues. The results show that the infiltration of CD8 + T cells in tumor tissues is significantly increased after OMV-C-C treatment.

[0042] B: To investigate the release of IFN-γ in OMV-C-C induced immunotherapy. After 3 days of intravenous injection of OMV-C-C (5 μg), the brain tissues of each group of mice were dissected and collected. After fixation with 4% paraformaldehyde solution, the tissues were paraffin-embedded and sectioned. After dewaxing with xylene, the paraffin sections were treated with antigen retrieval solution for antigen retrieval. After washing with PBS, the specimens were blocked with blocking solution, and then CD8 + T cell and IFN-γ primary antibodies were added and incubated overnight in the refrigerator. After washing with PBS, fluorescently labeled secondary antibodies were added and incubated at room temperature for 40 min. Finally, DAPI was used for nuclear staining and the sections were mounted, and the staining results were observed under a microscope, Figure 6 f is the percentage of CD8 + T cells in tumor tissues. The results show that the fluorescence intensity of IFN-γ in tumor tissues is significantly increased after OMV-C-C treatment.

[0043] C: To investigate the regulatory effect of IFN-γ in OMV-C-C induced immunotherapy on tumor cell ferroptosis. After 3 days of intravenous injection of OMV-C-C (5 μg), the brain tissues of each group of mice were dissected and collected. Freshly collected tumor tissues were dissociated using Librase / DNAzyme prepared with DMEM and incubated at 37°C for 45 min to prepare a single cell suspension. Blocking antibody was used for blocking before staining. Dead cell staining was performed using FSV450 before staining; after washing with PBS, tumor cells were labeled with CD45-APC antibody, and then stained with cell membrane lipid peroxidation dye BODIPY581 / 591 C11. After washing with PBS, the samples were detected by flow cytometry, Figure 6 g is the result of flow cytometry analysis of tumor cell ferroptosis in mouse tumor tissues. The results show that the lipid peroxidation level of tumor cells is significantly increased after OMV-C-C treatment.

[0044] D: To investigate the key role of IFN-γ in OMV-C-C induced anti-tumor. In the mouse glioma model, anti-IgG1 control group neutralizing antibody (200 μg) and anti-IFN-γ neutralizing antibody (200 μg) were injected intraperitoneally 12 h before OMV-C-C (5 μg) was injected through the tail vein. Small animal live imaging was performed every 7 days to detect the growth of mouse tumors. Figure 6 h in the figure is the statistical analysis of the fluorescence intensity of the brain of mice in different treatment groups. The results show that after the use of anti-IFN-γ, the anti-tumor effect of OMV-C-C is obviously inhibited.

[0045] Example 5 A: The frozen tube containing frozen mouse glioma cells GL261 and human glioma U87-MG was taken out from liquid nitrogen and quickly placed in a 37°C water bath, gently shaken, and the mouse brain microvascular endothelial cells mouse glioma cells GL261 and human glioma U87-MG were recovered. The recovered mouse brain microvascular endothelial cells bEND.3 were cultured in a sterile centrifuge tube containing 10% FBS inactivated fetal bovine serum, 1% penicillin / 1% streptomycin (double-antibiotic) DMEM culture medium, and placed in a suspension cell bottle. The cells were cultured in a 37°C, 5% CO2 and 90% relative humidity incubator. When the cells reached 70%-80% confluence, they were passaged once. The actively growing mouse glioma cells GL261 and human glioma U87-MG were continuously cultured and collected. The cells in the logarithmic growth phase were centrifuged, mixed by blowing, and the cells were inoculated in 96-well plates at 2×10 4 The cell supernatant was collected after 24 h of treatment with TMZ (temozolomide) (200 µM), and 10 µL of CCK-8 solution was added to detect the cell viability, Figure 7 a in the figure is the evaluation of cell activity of GL261 cells and U87-MG cells. The results show that GL261 has stronger resistance to TMZ.

[0046] B: The cells in the logarithmic growth phase were centrifuged, mixed by blowing after cell counting, and the cells were inoculated in 96-well plates at 2×10 4 The cell supernatant was collected after 24 h of treatment with different concentrations of ferroptosis inducer RSL-3 (0.025, 0.05, 0.1, 0.2, 0.4 µM) and erastin (0.5, 1, 2, 4, 8 µM), and 10 µL of CCK-8 solution was added to detect the cell viability, Figure 7b in FIG. 1 is the evaluation of the effect of different ferroptosis agonists on GL261 cells and U87-MG cells, and the results show that RSL-3 and erastin both cause a concentration-dependent decrease in cell viability, and this phenomenon is more pronounced in GL261 cells; at the same time, the pretreatment of ferroptosis inhibitor Fer-1 also significantly inhibits the inhibitory effect of RSL-3 and erastin.

[0047] C: Cells in the logarithmic growth phase were centrifuged, and after counting, the cells were mixed by blowing, and the cells were inoculated at 2x10 4 / well in a 96-well plate overnight. In order to further verify the enhancement of IFN-γ on the ferroptosis of tumor cells by RSL-3 and erastin, cells were treated with IFN-γ (10 ng / mL) and RSL-3 (0.2 µM) or erastin (4 µM) for 24 h, and the cell supernatant was collected, 10 µL CCK-8 solution was added to detect the viability of cells, Figure 7 c in FIG. 1 is the evaluation of the effect of IFN-γ combined with erastin on the ferroptosis of GL261 cells and U87-MG cells; Figure 7 d in FIG. 1 is the evaluation of the effect of IFN-γ combined with RSL-3 on the ferroptosis of GL261 cells and U87-MG cells; the results show that the combined application of IFN-γ and RSL-3 or erastin effectively enhances the inhibition of tumor cell viability. Figure 7 e in FIG. 1 is the flow cytometry detection of IFN-γ combined with RSL-3 or erastin on GL261 cells and U87-MG cells; Figure 7 f in FIG. 1 is the statistical analysis of flow cytometry detection of IFN-γ combined with RSL-3 or erastin on U87-MG cells; Figure 7 g in FIG. 1 is the statistical analysis of flow cytometry detection of IFN-γ combined with RSL-3 or erastin on GL261 cells; the results of Annexin V / PI staining also show that the combined application of IFN-γ and RSL-3 or erastin effectively enhances the death of tumor cells. In addition, the cells were further collected and stained with BODIPY581 / 591 C11 (5 µM), Figure 7 h in FIG. 1 is the effect of flow cytometry detection of IFN-γ combined with RSL-3 or erastin on the lipid peroxidation level of U87-MG cells; Figure 7 i in FIG. 1 is the effect of flow cytometry detection of IFN-γ combined with RSL-3 or erastin on the lipid peroxidation level of GL261 cells; the results show that the combined application of IFN-γ and RSL-3 or erastin enhances the lipid peroxidation level of tumor cells. Figure 7j in FIG. 1 is the protein expression level of GPX-4 detected by immunoblotting experiment. The results of immunoblotting show that the combination of IFN-γ and RSL-3 significantly inhibits the expression of GPX4.

[0048] Example 6 To prepare OMV-C-C@RSL-3, OMV-C-C (100 µg) and RSL-3 (100 µg) were mixed, and then the mixture was stirred at 37°C for 4 h at 750 rpm in a metal bath. The free RSL-3 was separated using a 30 kDa ultrafiltration tube, and then the OMV-C-C@RSL-3 was washed three times with normal saline. The encapsulation efficiency of RSL-3 was calculated using a UV value at 254 nm detected by a micro-ultraviolet spectrophotometer. Figure 8 a in FIG. 1 is a transmission electron microscope image of OMV-C-C and OMV-C-C@RSL-3. The transmission electron microscope image shows that the morphology of OMV-C-C is not affected after RSL-3 is encapsulated. The scale bar is 20 nm. Figure 8 b in FIG. 1 is a hydrodynamic diameter analysis chart of OMV-C-C and OMV-C-C@RSL-3, Figure 8 c in FIG. 1 is the surface charge of OMV-C-C and OMV-C-C@RSL-3. The results show that the hydrodynamic diameter and surface charge of OMV-C-C are not significantly changed after RSL-3 is encapsulated. The mouse tumor model was randomly divided into four groups (PBS, TMZ (temozolomide), TMZ (temozolomide) + RSL-3, OMV-C-C@RSL-3). TMZ (5 mg / kg), TMZ (5 mg / kg) + RSL-3 (5 mg / kg), and OMV-C-C@RSL-3 (OMV-C-C: 5 µg, RSL-3: 1 mg / kg) were injected through the tail vein. Small animal live imaging was performed every 7 days, Figure 8 d in FIG. 1 is a schematic diagram of the anti-tumor detection of OMV-C-C@RSL-3, Figure 8 e in FIG. 1 is a brain tissue imaging picture of tumor mice in different treatment groups, Figure 8 f in FIG. 1 is a brain tissue fluorescence intensity analysis of tumor mice in different treatment groups. The results show that OMV-C-C@RSL-3 significantly inhibits the growth of mouse tumors. The body weight of mice was recorded every two days, Figure 8 g in FIG. 1 is the body weight of mice in different treatment groups. The results show that the body weight of mice has a certain increase, but there is no significant difference compared with the control group. Figure 8 h in FIG. 1 is the survival time of mice in different treatment groups. The results show that the survival time of mice is significantly prolonged after OMV-C-C@RSL-3 treatment.

[0049] The application discloses a preparation method of an engineered OMV-C-C with the abilities of crossing BBB and tumor targeting and application of the engineered OMV-C-C in inhibiting glioblastoma. + IFN-γ released by T cells causes ferroptosis of glioblastoma, thereby achieving the effect of inhibiting the tumor.

[0050] The above merely describes a preferred specific embodiment of the present application, but the scope of protection of the present application is not limited to this, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application should be included in the protection scope of the present application.

Claims

1. A bacterial outer membrane vesicle, characterized in that, The bacterial outer membrane vesicle surface co-expresses a cell penetrating peptide and a chlorotoxin.

2. The bacterial outer membrane vesicle according to claim 1, characterized in that, The cell penetrating peptide is expressed on the surface of the bacterial outer membrane vesicle by a plasmid expressing the cell penetrating peptide, an amino acid sequence of the cell penetrating peptide is shown as SEQ ID No: 1, and the vector expressing the cell penetrating peptide is pAIDA1.

3. The bacterial outer membrane vesicle according to claim 1, characterized in that, The chlorotoxin is expressed on the surface of the bacterial outer membrane vesicle by a plasmid expressing the chlorotoxin, an amino acid sequence of the chlorotoxin is shown as SEQ ID No: 3, the plasmid expressing the chlorotoxin is a modified pET28, and the modified pET28 is a pET28 in which a T7 promoter region originally present is deleted and an amino acid sequence shown as SEQ ID No: 2 is inserted.

4. A method for the preparation of bacterial outer membrane vesicles according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Step 1): mixing a plasmid expressing a cell penetrating peptide and E. coli competence, and standing; Step 2): heat shock treatment at 40-45 DEG C for 85-95 s, and standing; Step 3): adding LB medium for culture, after the culture is completed, coating LB solid culture plate, and culturing; Step 4): The monoclonal is selected and cultured in LB medium containing chloramphenicol, and the cultured bacterial solution is transferred to LB culture solution. When the OD value is 0.3-0.4, the bacteria are collected by centrifugation, and a CaCl2 solution is added to obtain E. coli BL21-CPP competent cells. 600 Step 4): The monoclonal is selected and cultured in LB medium containing chloramphenicol, and the cultured bacterial solution is transferred to LB culture solution. When the OD value is 0.3-0.4, the bacteria are collected by centrifugation, and a CaCl2 solution is added to obtain E. coli BL21-CPP competent cells. Step 5): mixing a plasmid expressing a chlorotoxin and the E. coli BL21-CPP competence, and standing; Step 6): heat shock treatment at 40-45 DEG C for 85-95 s, and standing; Step 7): adding LB medium for culture, after the culture is completed, coating LB solid culture plate, and culturing; Step 8): The selected monoclonal was inoculated into LB medium containing chloramphenicol and kanamycin, and the cultured bacterial solution was inoculated into LB medium containing chloramphenicol and kanamycin, and cultured, and the OD 600 When the OD reached 0.6-0.8, isopropyl-β-D-thiogalactoside was added for induction. Step 9): centrifugation, filtering supernatant, centrifuging the filtered supernatant, collecting supernatant, centrifuging and resuspending to obtain the bacterial outer membrane vesicle.

5. A pharmaceutical composition, characterized by, The bacterial outer membrane vesicle comprises the bacterial outer membrane vesicle of any one of claims 1-3 or the bacterial outer membrane vesicle prepared by the preparation method of claim 4.

6. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition further comprises an iron death inducer, and the iron death inducer is RSL-3.

7. A process for the preparation of a pharmaceutical composition as claimed in claim 5 or 6, characterized in that, The bacterial outer membrane vesicle and the iron death inducer are mixed, oscillated for 3-6 h, purified, washed, and the pharmaceutical composition is obtained, wherein the mass ratio of the bacterial outer membrane vesicle to the iron death inducer is 1:0.8-1.

2.

8. Use of the bacterial outer membrane vesicle of any one of claims 1-3 or the pharmaceutical composition of claim 5 or 6 or the pharmaceutical composition prepared by the preparation method of claim 7 in the preparation of a drug for promoting the expression of IFN-γ.

9. Use of the bacterial outer membrane vesicle of any one of claims 1-3 or the pharmaceutical composition of claim 5 or 6 or the pharmaceutical composition prepared by the preparation method of claim 7 in the preparation of an anti-tumor drug.

10. Use according to claim 9, characterized in that, The anti-tumor drug is an anti-glioblastoma drug.

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