Rvg29-modified bacterial outer membrane vesicles, and preparation method and application thereof

Abstract

The application provides an RVG29 modified bacterial outer membrane vesicle and a preparation method and application thereof, the RVG29 modified bacterial outer membrane vesicle carries a rabies virus glycoprotein derivative peptide 29, and the amino acid sequence of the rabies virus glypoprotein derivative peptide 29 is shown as SEQ ID No: 1. The application constructs a BEV (bacterial outer membrane vesicle) expressing RVG29 on the surface, namely BEV-RVG29, by a genetic engineering method, the BEV-RVG29 can efficiently cross the BBB (blood-brain barrier) in vitro and in vivo and is enriched at a tumor site. The application further provides a pharmaceutical composition, the pharmaceutical composition is formed by loading paclitaxel on the BEV-RVG29 to form a nano delivery system, and in the pharmaceutical composition, the immune stimulation characteristics of the BEV-RVG29 itself and the immunogenic cell death induced by PTX (paclitaxel) produce a synergistic effect, so that the growth of a glioblastoma is significantly inhibited.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to an RVG29-modified bacterial outer membrane vesicle, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM) is the most common and most malignant type of astrocytoma. Currently, various treatments are available for glioblastoma, including surgery, radiotherapy, chemotherapy, and supportive care; however, compared to other types of tumors, the available treatment options for glioblastoma are still quite limited. Immunotherapy, with its advantages of high specificity and fewer side effects, has brought new hope to the treatment of glioblastoma. Immune checkpoint blockade therapy, chimeric antigen receptor T-cell immunotherapy, tumor vaccines, and oncolytic virus therapy have shown some efficacy in the immunotherapy of glioblastoma; however, due to the inherent characteristics of glioblastoma, immunotherapy still faces many challenges, such as low patient response rates and poor efficacy of targeted drug delivery.

[0003] Therefore, there is an urgent need to develop new immunotherapies to improve the treatment outcomes and quality of life for patients with glioblastoma. Summary of the Invention

[0004] This invention aims to address the problems existing in the prior art. Through genetic engineering of bacterial extravesicles (BEVs), this invention expresses rabies virus glycoprotein-derived peptide 29 (RVG29) on the surface of the bacterial extravesicles, enabling it to accumulate across the BBB and tumor layers; this is named BEV-RVG29. Furthermore, this invention proposes a synergistic therapeutic strategy that deeply integrates the inherent immune-activating properties of the drug delivery system with the immunogenic cell death (ICD) effect: by encapsulating paclitaxel (PTX) in the aforementioned BEV-RVG29, utilizing the inherent immune-activating properties of BEV-RVG29 and the ability of paclitaxel to induce immunogenic cell death (ICD) in tumor cells, this aims to achieve comprehensive and efficient intervention for glioblastoma through the synergistic effect of multiple mechanisms.

[0005] Therefore, in a first aspect, the present invention provides an RVG29-modified bacterial outer membrane vesicle, wherein the RVG29-modified bacterial outer membrane vesicle carries rabies virus glycoprotein-derived peptide 29, the amino acid sequence of which is shown in SEQ ID No: 1.

[0006] This invention provides an RVG29-modified bacterial outer membrane vesicle, a genetically engineered BEV, namely BEV-RVG29, which is achieved by expressing rabies virus glycoprotein-derived peptide 29 on the surface of BEVs. It possesses efficient ability to cross the blood-brain barrier (BBB) ​​and the ability to accumulate in brain tumors, demonstrating clinical translational value. BEV-RVG29 can efficiently cross the BBB in both in vitro BBB models and in vivo in mice; simultaneously, it exhibits a certain degree of tumor accumulation capacity in vivo.

[0007] Preferably, the rabies virus glycoprotein-derived peptide 29 is expressed in bacterial outer membrane vesicles via a genetically engineered plasmid expressing rabies virus glycoprotein-derived peptide 29.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned RVG29-modified bacterial outer membrane vesicles, comprising the following steps: Step 1): Rabies virus glycoprotein-derived peptide 29 was constructed into the expression vector to obtain the recombinant expression vector; Step 2): The recombinant expression vector is introduced into Escherichia coli to obtain recombinant bacteria, expression is induced, bacterial culture is collected by centrifugation, filtered, the supernatant is centrifuged using a separation tube, the retention liquid is collected, ultracentrifuged, and resuspended to obtain the RVG29 modified bacterial outer membrane vesicles.

[0009] Preferably, the expression vector is pAIDA1.

[0010] Preferably, the inducing agent used is isopropyl-β-D-thiogalactoside.

[0011] The above-mentioned method for preparing RVG29-modified bacterial outer membrane vesicles includes the following steps: Step 1): Mix the plasmid expressing RVG29 with competent E. coli cells and let stand. Step 2): Heat shock treatment at 40-45℃ for 85-95 seconds, then let stand; Step 3): Add LB medium for culture. After culture, spread the culture onto LB solid culture plates (chloramphenicol resistant) and culture. Step 4): Select single clones and culture them in LB medium containing chloramphenicol. Add the cultured bacterial solution to LB medium containing chloramphenicol and continue culturing. OD 600 When the concentration reaches 0.6-0.8, isopropyl-β-D-thiogalactoside is added for induction. Step 5): Centrifuge, filter the supernatant, centrifuge the filtered supernatant, collect the supernatant, centrifuge and resuspend to obtain the RVG29-modified bacterial outer membrane vesicles, namely BEV-RVG29.

[0012] Thirdly, the present invention provides a detection reagent comprising the RVG29-modified bacterial outer membrane vesicles described above or the RVG29-modified bacterial outer membrane vesicles prepared by the above preparation method.

[0013] Preferably, the raw materials for preparing the detection reagent also include fluorescent dyes.

[0014] Preferably, the fluorescent dye is selected from ICG (indocyanine green) and Dil (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindocarbonylcyanine perchlorate).

[0015] The preparation method of the detection reagent includes: mixing the RVG29-modified bacterial outer membrane vesicles with a fluorescent dye, purifying, and washing to obtain the detection reagent.

[0016] When the fluorescent dye is ICG (indocyanine green), the mass ratio of the RVG29-modified bacterial outer membrane vesicles to the fluorescent dye is 1:(1-2).

[0017] When the fluorescent dye is Dil, the mass ratio of the RVG29-modified bacterial outer membrane vesicles to the fluorescent dye is 1:(40-60).

[0018] Fourthly, the present invention provides the application of the above-described RVG29-modified bacterial outer membrane vesicles or the RVG29-modified bacterial outer membrane vesicles prepared by the above-described preparation method or the above-described detection reagent in the preparation of a reagent for detecting tumors.

[0019] Preferably, the tumor is a glioblastoma.

[0020] Preferably, the reagent for detecting tumors is a diagnostic reagent for glioblastoma.

[0021] Fifthly, the present invention provides a pharmaceutical composition comprising the above-described RVG29-modified bacterial outer membrane vesicles or the RVG29-modified bacterial outer membrane vesicles prepared by the above-described preparation method.

[0022] Preferably, the pharmaceutical composition further includes an immunogenic cell death inducer.

[0023] Preferably, the immunogenic cell death inducer is paclitaxel.

[0024] In a sixth aspect, the present invention provides a method for preparing the above-mentioned pharmaceutical composition, wherein the RVG29-modified bacterial outer membrane vesicles and the immunogenic cell death inducer are mixed, shaken for 3-6 hours, purified, and washed to obtain the pharmaceutical composition, wherein the mass ratio of the RVG29-modified bacterial outer membrane vesicles to the immunogenic cell death inducer is 1:1.8-2.2.

[0025] In a seventh aspect, the present invention provides the use of the above-described RVG29-modified bacterial outer membrane vesicles or the RVG29-modified bacterial outer membrane vesicles prepared by the above-described preparation method or the above-described pharmaceutical composition or the pharmaceutical composition prepared by the above-described preparation method in the preparation of a drug for promoting the maturation of BMDCs.

[0026] Eighthly, the present invention provides the use of the above-described RVG29-modified bacterial outer membrane vesicles or the RVG29-modified bacterial outer membrane vesicles prepared by the above-described preparation method or the above-described pharmaceutical composition or the pharmaceutical composition prepared by the above-described preparation method in the preparation of an antitumor drug.

[0027] Preferably, the tumor is a glioblastoma.

[0028] The beneficial effects of this invention are as follows: 1. This invention provides an RVG29-modified bacterial extravesicle. A BEV expressing RVG29, namely BEV-RVG29, is constructed using genetic engineering methods. Unlike traditional chemical modification methods, this method allows for efficient and controllable regulation of RVG29 expression levels. The BEV-RVG29 provided by this invention can efficiently cross the blood-brain barrier (BBB) ​​in vitro and in vivo, and specifically accumulate at tumor sites.

[0029] 2. The present invention provides a detection reagent comprising the above-mentioned RVG29 modified bacterial exovesicles, which can be used to detect glioblastoma.

[0030] 3. The present invention provides a pharmaceutical composition comprising the above-mentioned RVG29-modified bacterial extravesicles, and PTX can be loaded into a BEV-RVG29-PTX nanodelivery system formed by BEV-RVG29. In this pharmaceutical composition, the immunostimulatory properties of BEV-RVG29 itself synergistically work with the immunogenic cell death induced by PTX, thereby significantly inhibiting the growth of glioblastoma.

[0031] 4. The present invention provides the application of the above-mentioned bacterial exovesicles and the above-mentioned pharmaceutical composition in the preparation of antitumor drugs.

[0032] 5. The present invention provides a method for preparing the above-mentioned bacterial vesicles and the above-mentioned drug composition. The method is simple, cost-controllable, and has significant therapeutic effects, and has extremely strong clinical translational value. Attached Figure Description

[0033] Figure 1 A schematic diagram of a plasmid expressing RVG29; Figure 2 Construction and characterization of BEV-RVG29; in, Figure 2In the image, a is a transmission electron microscope image of BEV-WT, scale bar is 50 nm; b is the particle size distribution of BEV-WT; c is a transmission electron microscope image of BEV-RVG29, scale bar is 50 nm; d is the particle size distribution of BEV-RVG29; e is the surface charge of BEV-WT and BEV-RVG29; f is the detection of Escherichia coli-RVG29 by Western blot assay. E.Coli The expression of RVG29 on BEV-RVG29 and BEV-RVG29; g is the expression of RVG29 on the surface of BEV-RVG29 detected by immunogold labeling assay, scale bar is 15nm; h is the expression of RVG29 on the surface of BEV by flow cytometry; i is the expression of RVG29 on the surface of E. coli-RVG29 (RVG29). E.Coli Protein expression profiles of RVG29 and BEV-RVG29; Figure 3 The test results indicate that BEV-RVG29 has the ability to cross the BBB and accumulate in tumors. in, Figure 3 In the figures, a is a schematic diagram of the in vitro BBB construction; b is an evaluation of the in vitro blood-brain barrier crossing efficiency of BEV-RVG29-ICG; c is a fluorescence imaging image of the mouse brain; d is a fluorescence quantification of the mouse brain; e is a fluorescence imaging image of mouse brain tissue; f is a fluorescence quantification of mouse brain tissue; g is a flow cytometry analysis of the uptake of BEV-RVG29-Dil by HMC3 cells and GL261 cells; h is an analysis of BEV-RVG29-Dil infiltration in the brain of tumor model mice, with a scale bar of 15 μm. Figure 4 The results of the tumor cell killing ability evaluation test for BEV-RVG29-PTX; in, Figure 4 In the table, a is an electron micrograph of BEV-RVG29-PTX, with a scale bar of 50 nm; b is a potentiogram of BEV-RVG29-PTX; c shows the effect of different concentrations of BEV-RVG29-PTX on the cell viability of GL261 cells; d shows the effect of different concentrations of BEV-RVG29-PTX on the cell viability of U87 cells; e shows the effect of different concentrations of BEV-RVG29-PTX on the release of lactate dehydrogenase from GL261 cells; f shows the effect of different concentrations of BEV-RVG29-PTX on the release of lactate dehydrogenase from U87 cells; g shows the effect of different concentrations of BEV-RVG29-PTX on apoptosis in U87 cells; and h shows the effect of different concentrations of BEV-RVG29-PTX on apoptosis in GL261 cells. Figure 5 The results of a study on the induction of immunogenic cell death in tumor cells by BEV-RVG29-PTX; in, Figure 5In the table, a) shows the effect of different concentrations of BEV-RVG29-PTX on CRT eversion in U87 cells (scale bar: 10 μm); b) shows the effect of different concentrations of BEV-RVG29-PTX on HMGB1 release in U87 cells (scale bar: 10 μm); c) shows the effect of different treatment groups on DC maturation in vitro; d) shows the quantitative results of bone marrow-derived dendritic cell (BMDC) maturation; e) shows the detection results of cytokine IFN-γ in different treatment groups; f) shows the detection results of cytokine IL-6 in different treatment groups; and g) shows the detection results of cytokine TNF-α in different treatment groups. Figure 6 To investigate the mechanism by which BEV-RVG29-PTX induces immunogenic cell death in tumor cells; in, Figure 6 In the table, a) shows the detection of ROS levels in GL261 cells; b) evaluates the ability of different concentrations of BEV-RVG29-PTX to induce ROS; c) evaluates the effect of different concentrations of BEV-RVG29-PTX on the decrease in mitochondrial membrane potential (scale bar: 50 μm); d) shows the flow cytometry results of superoxide in mitochondria; e) shows confocal microscopy imaging of superoxide in mitochondria; and f) is a diagram illustrating the mechanism of action of BEV-RVG29-PTX. Figure 7 This demonstrates the in vivo antitumor effect of BEV-RVG29-PTX. in, Figure 7 In the figure, a is a schematic diagram of BEV-RVG29-PTX anti-tumor detection; b is a bioluminescence imaging image of the brain of mice with tumors in different treatment groups; c is the fluorescence intensity analysis of brain tissue of mice with tumors in different treatment groups; d is a comparison of survival time of mice in different treatment groups; e is an HE staining image of the brain of mice in different treatment groups; f is a comparison of body weight of mice in different treatment groups; g is the Ki67 (scale bar 30 μm) and TUNEL staining results (scale bar 15 μm) of tumor sites in mice in different treatment groups. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0035] Example 1 The preparation method of BEV-WT includes the following steps: 1) Add 2 μL of competent Escherichia coli BL21(DE3) cells to 10 mL of LB broth and incubate at 37°C with shaking at 200 rpm for 12 h. Then, add 10 mL of the lightly shaken LB broth back to 1 L of LB broth and incubate at 37°C with vigorous shaking at 200 rpm for 12 h to obtain the desired result. E.Coli WT; 2) After shaking, the bacterial solution ( E.Coli Centrifuge twice (4000 rpm, 4℃, 15 min) and collect the supernatant. Filter the supernatant using a filtration system (0.22 μm). The filtered supernatant is then further centrifuged in a 100,000 MWCO separation tube (5000 rpm, 4℃, 10 min), and the supernatant is collected. 3) The collected supernatant was subjected to ultracentrifugation (150,000 × g, 4℃, 2h). The BEV prepared by resuspending it in 0.9% physiological saline after centrifugation is BEV-WT.

[0036] The plasmid expressing RVG29 was constructed as follows: The amino acid sequence of rabies virus glycoprotein-derived peptide 29 (RVG29) (as shown in SEQ ID No: 1) was inserted into the vector pAIDA1 plasmid, and the synthesis was entrusted to Beijing Qingke Biotechnology Co., Ltd. Figure 1 This is a schematic diagram of the plasmid expressing RVG29. The gene encoding rabies virus glycoprotein-derived peptide 29 was inserted into the corresponding multiple cloning site region of the pAIDA1 plasmid using a KpnI / Eco53kI double digestion strategy, resulting in a plasmid expressing RVG29.

[0037] The preparation method of BEV-RVG29 includes the following steps: 1) Mix the plasmid expressing RVG29 (3 μL) with E. coli BL21(DE3) competent cells (50 μL) and incubate 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.

[0038] 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 100 mL of LB medium containing chloramphenicol (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)). Incubate at 37°C and 200 rpm with vigorous shaking for 3 h. OD 600When the concentration reaches 0.6-0.8, isopropyl-β-D-thiogalactoside (0.5 mM) is added for induction for 3-5 h to obtain... E.Coli RVG29; 5) After shaking and induction, the bacterial solution ( E.Coli Centrifuge twice (4000 rpm, 4℃, 15 min) using RVG29, recover the supernatant, and filter it 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 retentate was collected. 6) The collected retention solution was subjected to ultracentrifugation (150,000 × g, 4℃, 2h). After centrifugation, the prepared BEV expressing RVG29 was resuspended in 0.9% physiological saline, which is BEV-RVG29.

[0039] Figure 2 In the image, a is a transmission electron microscope image of BEV-WT, scale bar is 50 nm; b is the particle size distribution of BEV-WT; c is a transmission electron microscope image of BEV-RVG29, scale bar is 50 nm; d is the particle size distribution of BEV-RVG29; e is the surface charge of BEV-WT and BEV-RVG29; f is the detection of Escherichia coli-RVG29 by Western blot assay. E.Coli The expression of RVG29 on BEV-RVG29 and BEV-RVG29; g is the expression of RVG29 on the surface of BEV-RVG29 detected by immunogold labeling assay, scale bar is 15 nm; h is the expression of RVG29 on the surface of BEV by flow cytometry; i is the expression of RVG29 on the surface of E. coli-RVG29 (RVG29). E.Coli Protein expression profiles of RVG29 and BEV-RVG29 were analyzed. The results showed that this genetic engineering successfully expressed RVG29 on the surface of E. coli and BEV without significantly altering the size, morphology, surface charge, or protein composition of BEV.

[0040] Example 2: Detection results of BEV-RVG29's ability to cross the BBB and accumulate in tumors. Experimental Group 1: Preparation of BEV-WT-ICG, BEV-RVG29-ICG, BEV-WT-Dil, and BEV-RVG29-Dil Preparation method of BEV-WT-ICG: 200 μg BEV-WT and 100 μg ICG (indocyanine green) were mixed in PBS and then vortexed for 6 h (37℃, 750 rpm). After that, the mixture was centrifuged in an ultrafiltration tube (5000 rpm, 10 min) to remove free ICG and obtain BEV-WT-ICG.

[0041] Preparation method of BEV-RVG29-ICG: 200 μg BEV-RVG29 and 100 μg ICG (indocyanine green) were mixed in PBS and then vortexed for 6 h (37℃, 750 rpm). After that, the mixture was centrifuged in an ultrafiltration tube (5000 rpm, 10 min) to remove free ICG and obtain BEV-RVG29-ICG.

[0042] Preparation method of BEV-WT-Dil: 100 μg BEV-WT and 2 μg Dil (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate) were mixed and then vortexed for 20 min (37℃, 300 rpm). The mixture was then centrifuged in an ultrafiltration tube (5000 rpm, 10 min) to remove free Dil, thus obtaining BEV-WT-Dil.

[0043] Preparation method of BEV-RVG29-Dil: 100 μg of BEV-RVG29 and 2 μg of Dil (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate) were mixed and then vortexed for 20 min (37℃, 300 rpm). The mixture was then centrifuged in an ultrafiltration tube (5000 rpm, 10 min) to remove free Dil, thus obtaining BEV-RVG29-Dil.

[0044] Experimental group 2 The cryovials containing frozen mouse brain microvascular endothelial cells (bEnd.3) were removed from liquid nitrogen and quickly placed in a 37°C water bath with gentle agitation until the liquid thawed, thus resuscitating the mouse brain microvascular endothelial cells (bEnd.3). The resuscitated mouse brain microvascular endothelial cells (bEnd.3) were cultured in sterile DMEM medium containing 10% FBS-inactivated fetal bovine serum and 1% penicillin / 1% streptomycin (double antibiotics) in centrifuge tubes. The tubes were then placed in cell culture dishes and cultured in an incubator at 37°C, 5% CO2, and 90% relative humidity. The cells were passaged once when they reached approximately 70%-80% confluence. Further culture yielded actively growing mouse brain microvascular endothelial cells (bEnd.3), which were then collected. Cells in the logarithmic growth phase were centrifuged, counted, and remixed by pipetting. Cells were then distributed at a rate of 2 × 10⁶ cells per well. 5 After seeding cells in the upper chamber of a Transwell plate and culturing for 24 h, the resistance of the BBB was measured using a resistance meter, and the resistance reached 200 Ω / cm. 2 It can then be considered that the in vitro blood-brain barrier was successfully constructed. Figure 3In the diagram, 'a' represents a schematic of the in vitro BBB construction. Protein concentration was determined using the BCA method. BEV-WT-ICG and BEV-RVG29-ICG were diluted to the same protein concentration and fluorescence intensity, and indocyanine green was diluted to the same fluorescence intensity, yielding BEV-WT-ICG, BEV-RVG29-ICG, and indocyanine green solutions, respectively. Equal volumes of PBS, indocyanine green (Free ICG) solution, BEV-WT-ICG solution, and BEV-RVG29-ICG solution were added to the upper chamber of a Transwell plate, corresponding to the PBS, Free ICG, BEV-WT-ICG, and BEV-RVG29-ICG groups, respectively. Fluorescence intensities in the upper and lower chambers were measured after 2 hours, and the crossing efficiency of indocyanine green, BEV-WT-ICG, and BEV-RVG29-ICG was calculated. Results are shown below. Figure 3 As shown in b in the figure, BEV-RVG29 crossed the BBB more efficiently than other groups in vitro.

[0045] Experimental group 3 To further demonstrate the ability of BEV-RVG29 to cross the BBB in vivo, C57BL / 6 mice were randomly divided into four groups of three mice each. Protein concentration was determined using the BCA method. BEV-WT-ICG and BEV-RVG29-ICG were diluted to the same protein concentration and fluorescence intensity, and indocyanine green was diluted to the same fluorescence intensity, yielding BEV-WT-ICG, BEV-RVG29-ICG, and indocyanine green solutions, respectively. Equal volumes of PBS, Free ICG (indocyanine green), BEV-WT-ICG, and BEV-RVG29-ICG solutions were injected into the mice via the tail vein, corresponding to the PBS, Free ICG, BEV-WT-ICG, and BEV-RVG29-ICG groups, respectively. Two hours later, animal imaging was used to detect the fluorescence intensity in the mouse brain. Figure 3 In the figure, c represents fluorescence imaging images of the brains of mice in each group, d represents quantitative fluorescence imaging of the mouse brain, e represents fluorescence imaging of isolated mouse brain tissue, and f represents quantitative fluorescence imaging of mouse brain tissue. The results show that BEV-RVG29 efficiently crosses the BBB in mice.

[0046] Experimental group 4 Cryopreservation tubes containing frozen mouse glioma cells GL261 and human microglia HMC3 were removed from liquid nitrogen and quickly placed in a 37°C water bath with gentle shaking until the liquid thawed, thus resuscitating the mouse glioma cells GL261 and human microglia HMC3. The resuscitated mouse glioma cells GL261 and human microglia HMC3 were cultured in sterile DMEM medium containing 10% FBS-inactivated fetal bovine serum and 1% penicillin / 1% streptomycin (double antibiotics) in centrifuge tubes, placed in cell culture dishes, and cultured in an incubator at 37°C, 5% CO2, and 90% relative humidity. The cells were passaged once when they reached approximately 70%-80% confluence. Further culture yielded actively growing mouse glioma cells GL261 and human microglia HMC3, and the cells were collected. Cells in the logarithmic growth phase were centrifuged, counted, and diluted to a cell density of 3 × 10⁶ cells / mL. 4 ~5×10 4 Cells / ml, mix by pipetting, and distribute cells at a rate of 3 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates. Protein concentration was determined using the BCA method. BEV-WT-Dil and BEV-RVG29-Dil were diluted to the same protein concentration and fluorescence intensity, and 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine) perchlorate was diluted to the same fluorescence intensity, resulting in BEV-WT-Dil, BEV-RVG29-Dil, and 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine) perchlorate solutions (Free Dil solutions). Equal volumes of PBS, Free Dil, BEV-WT-Dil, and BEV-RVG29-Dil solutions were added to the 6-well plates to treat cells for 4 h, corresponding to the PBS, Free Dil, BEV-WT-Dil, and BEV-RVG29-Dil groups, respectively. Flow cytometry results for mouse glioma cells GL261 and human microglia HMC3 were shown below. Figure 3 As shown in g, flow cytometry results indicate that BEV-RVG29-Dil has a certain targeted uptake effect on tumor cells.

[0047] Experimental group 5 To further demonstrate the tumor enrichment ability of BEV-RVG29-Dil in vivo, glioblastoma mice were randomly divided into four groups of three mice each. Protein concentration was determined using the BCA method. BEV-WT-Dil and BEV-RVG29-Dil were diluted to the same protein concentration and fluorescence intensity, and 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine) perchlorate was diluted to the same fluorescence intensity, resulting in BEV-WT-Dil, BEV-RVG29-Dil, and 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindolecarbonylcyanine) perchlorate solutions (Free Dil solutions). Equal volumes of PBS, Free Dil, BEV-WT-Dil, and BEV-RVG29-Dil solutions were injected into mice via the tail vein, corresponding to the PBS, Free Dil, BEV-WT-Dil, and BEV-RVG29-Dil groups, respectively. 24 Brain tissue was harvested h later for frozen sectioning, and DAPI staining was used to detect BEV-RVG29-Dil tumor infiltration. The results are as follows: Figure 3 As shown in h, the results indicate that BEV-RVG29-Dil has enhanced accumulation capacity in tumor model mice.

[0048] Example 3 To prepare BEV-RVG29-PTX, BEV-RVG29 (100 µg) and PTX (paclitaxel, 200 µg) were mixed and shaken in a metal bath at 37°C and 750 rpm for 4 h. Free PTX was separated using a 30 kDa ultrafiltration tube and then washed three times with physiological saline to obtain BEV-RVG29-PTX. The UV value at 230 nm was detected using an ultra-micro spectrophotometer, and the encapsulation efficiency of PTX was calculated. Figure 4 In the image, a is a transmission electron microscope image of BEV-RVG29-PTX, which shows that encapsulating PTX did not affect the morphology of BEV-RVG29; b is the surface charge of BEV-RVG29-PTX, and the results show that the surface charge of BEV-RVG29 did not change significantly after encapsulating PTX.

[0049] U87 and GL261 cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured at 37°C for 24 hours. When cell confluence reached 80%, cells were treated with DMEM medium (Ctrl) and DMEM medium containing BEV-RVG29-PTX (concentrations of 10 nM, 100 nM, and 1000 nM), respectively, and cultured at 37°C for 48 hours. Subsequently, 10 µL of CCK-8 solution was added to each well according to the instructions of the cytotoxicity assay kit (CCK-8, Beyotime), and the plates were cultured at 37°C for another 30 minutes. The absorbance was measured at 450 nm using a microplate reader. LDH treatment was performed in the same manner. After treatment, the release of lactate dehydrogenase (LDH) was measured according to the manufacturer's instructions (LDH cytotoxicity assay kit, Beyotime), and the absorbance was measured at 490 nm using a microplate reader. Figure 4 In the table, c represents the effect of the Ctrl group and different concentrations of BEV-RVG29-PTX on the viability of GL261 cells, d represents the effect of the Ctrl group and different concentrations of BEV-RVG29-PTX on the viability of U87 cells, e represents the effect of the Ctrl group and different concentrations of BEV-RVG29-PTX on the release of lactate dehydrogenase from GL261 cells, and f represents the effect of the Ctrl group and different concentrations of BEV-RVG29-PTX on the release of lactate dehydrogenase from U87 cells. The results show that the killing effect of BEV-RVG29-PTX on tumor cells is concentration-dependent. Figure 4 In the figure, g represents the Ctrl group and different concentrations of BEV-RVG29-PTX for the detection of apoptosis in U87 cells. Figure 4 In the figure, h represents the apoptosis detection of GL261 cells in the Ctrl group and different concentrations of BEV-RVG29-PTX. The results show that high concentrations of BEV-RVG29-PTX can induce tumor cell apoptosis.

[0050] Example 4 Experimental group 1 To investigate whether BEV-RVG29-PTX-mediated cytotoxicity involves immunogenic cell death (ICD), U87 cells were used to detect characteristic ICD markers such as CRT exposure and HMGB1 release. U87 cells (400,000 cells per well) were seeded in confocal culture dishes. After 24 hours of culture, the cell culture medium was removed, and cells were treated for 24 hours with DMEM medium (Ctrl), DMEM medium containing 10 nM, 100 nM, and 1000 nM BEV-RVG29-PTX, respectively, to induce ICD. Following treatment, U87 cells were washed with cold PBS and incubated overnight at 4°C with anti-CRT polyclonal antibody (ABclonal Biotechnology Co., Ltd., 1:1000 dilution). Subsequently, they were stained at room temperature for 60 minutes with highly cross-adsorbed secondary antibody (1:500 dilution) of Alexa Fluor 488-labeled goat anti-rabbit IgG (H&L). Cell nuclei were stained with 0.5 µg / mL DAPI for 30 minutes. Finally, the samples were observed using a laser confocal microscope (Olympus FV3000, Tokyo, Japan). The CRT results are as follows: Figure 5 As shown in Figure a, the CRT was completely exposed after BEV-RVG29-PTX treatment, indicating the generation of early phagocytic signals. Simultaneously, to assess HMGB1 secretion, cells were fixed with 4% paraformaldehyde for 15 minutes after different sample treatments. After fixation, cells were permeabilized with Triton X-100 for 10 minutes, followed by overnight incubation at 4°C with anti-HMGB1 polyclonal antibody (ABclonal Biotechnology Co., Ltd., working concentration 1 µg / mL), and then incubated at room temperature for 30 minutes with Alexa Fluor 488-labeled goat anti-rabbit IgG (H&L) secondary antibody (1:2000 dilution). Cell nuclei were stained with 0.5 µg / mL DAPI for 30 minutes. Images were acquired using a laser confocal microscope. HMGB1 treatment was similar to CRT, and the results are as follows. Figure 5 As shown in b, HMGB1 protein was released into the extracellular environment, and its level increased significantly after BEV-RVG29-PTX treatment. This is another signal in the ICD process, confirming that BEV-RVG29-PTX-mediated cytotoxicity is caused by immunogenic-induced cell death.

[0051] Experimental group 2 BMDCs were isolated from the femur and tibia of collected C57BL / 6 mice (6–8 weeks old) and cultured in RPMI 1640 complete medium supplemented with GM-CSF (20 ng / mL) and IL-4 (10 ng / mL). The medium was changed every two days. On day 9, GL261 cells were treated with PBS, BEV-RVG29 (final concentration in medium 0.5 µg / mL), and BEV-RVG29-PTX (final concentration in medium 0.5 µg / mL) for 24 h. Subsequently, the cell supernatant was collected and added to the BMDCs, co-cultured for 24 h, stained with anti-CD11c-FITC, anti-CD80-APC, and anti-CD86-PerCP Cy5.5 antibodies (BioLegend, USA), and analyzed using flow cytometry (Aurora, Cytek, USA). Figure 5 In the figure, 'c' represents the results of different treatment groups promoting the maturation of BMDCs. Figure 5 In the figure, d represents the quantitative results of BMDC maturation in different treatment groups, where group 1 is PBS, group 2 is BEV-RVG29, and group 3 is BEV-RVG29-PTX. The results show that BEV-RVG29-PTX promotes the maturation of BMDCs. Figure 5 In this context, 'e' represents the amount of IFN-γ secreted in the cell supernatant obtained after co-culture, as determined by ELISA. Figure 5 In this context, f represents the amount of TNF-α secreted in the cell supernatant obtained after co-culture, as determined by ELISA. Figure 5 In the figure, g represents the amount of IL-6 secreted in the cell supernatant obtained after co-culture, as determined by ELISA. The experimental results showed that, compared to BEV-RVG29 alone, BEV-RVG29-PTX loaded with paclitaxel exhibited stronger immunostimulatory activity. It not only significantly upregulated the expression levels of maturation markers CD80 and CD86 on the surface of BMDCs, but also synergistically triggered the release of pro-inflammatory cytokines such as IFN-γ, IL-6, and TNF-α. This indicates that the introduction of PTX and the adjuvant effect of BEV produced a significant synergistic effect, greatly optimizing the maturation efficiency and functional activity of dendritic cells, laying a solid foundation for subsequently inducing a highly efficient anti-tumor T-cell immune response.

[0052] Example 5 Experimental group 1 GL261 cells were co-cultured for 24 hours in DMEM medium (Ctrl) and DMEM medium containing 0.01 μM, 0.1 μM, and 1 μM BEV-RVG29-PTX, respectively. Subsequently, the cells were treated at 37°C for 30 minutes with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Solarbio, Beijing, China) to assess intracellular reactive oxygen species (ROS) levels. ROS generation in GL261 cells was quantified by flow cytometry. Figure 6 In the figure, 'a' represents the intracellular ROS level in the Ctrl group and GL261 cells treated with different concentrations of BEV-RVG29-PTX. Figure 6 In the figure, b represents the quantitative determination of ROS levels in GL261 cells treated with different concentrations of BEV-RVG29-PTX in the Ctrl group. To further determine the source of ROS, the MitoSox Red probe was used to specifically detect superoxide dismutase in mitochondria. Figure 6 In the figure, d represents the flow cytometry results of mitochondrial superoxide. As the concentration of BEV-RVG29-PTX treatment increased, when the concentration of BEV-RVG29-PTX treatment was increased to 1 μM, the proportion of positive cells rapidly increased from 17.6% in the control group (Ctrl) to 46.1%.

[0053] Experimental group 2 U87 cells were seeded at a density of 30,000-50,000 cells per dish in confocal culture dishes (Biosharp, Beijing, China) and cultured at 37°C for 24 hours. When the cell confluence reached 80%, cells were treated with DMEM medium (Ctrl), DMEM medium containing 0.01 μM, 0.1 μM, and 1 μM BEV-RVG29-PTX for 24 hours, respectively. Changes in mitochondrial membrane potential were detected using the JC-1 mitochondrial membrane potential assay kit (Yeasen Biotechnology, Shanghai). After staining, 1 mL of PBS was added, and changes in mitochondrial membrane potential were observed under a laser confocal microscope (Olympus FV3000, Tokyo, Japan). Figure 6 In the figure, c represents the detection of mitochondrial membrane potential changes. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer, which emits strong red fluorescence (Ex=585 nm, Em=590 nm). In unhealthy mitochondria, due to the decrease or loss of membrane potential, JC-1 can only exist in the cytoplasm in the form of monomers, producing green fluorescence (Ex=514 nm, Em=529 nm). The results show that BEV-RVG29-PTX damages mitochondria, leading to a decrease in mitochondrial membrane potential. Figure 6In the image, e represents superoxide confocal microscopy imaging within mitochondria. The Merge image in both bright field and fluorescence channels clearly shows red fluorescence distributed in a dotted pattern within the cytoplasm, and its intensity increases significantly with increasing concentration.

[0054] Figure 6 f in the diagram represents the mechanism of BEV-RVG29-PTX treatment. PTX not only exerts direct cytotoxicity through the ROS pathway, but more importantly, it can induce ICD in tumor cells. This process works synergistically with the immune adjuvant effect of BEVs to significantly promote the activation and maturation of dendritic cells (DCs). This further mediates the effective infiltration of effector T cells in the tumor microenvironment, successfully reversing the immunosuppressive state and promoting the remodeling of "cold" tumors into "hot" tumors, thereby significantly enhancing the anti-tumor immunosuppressive effect against local lesions.

[0055] Example 6 6-8 week old C57 / BL6 mice were selected, and GL261-Luc cells (5×10⁻⁶) were injected into the brain using a stereotactic injection device. 4 To establish a mouse tumor (glioblastoma) model, tumor cells (2 mm, 1 mm, -3 mm) were injected into the brains of C57 / BL6 mice. 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 Shandong Provincial Laboratory Animal Ethics Committee for New Drug Development in Yantai. Successful tumor implantation was confirmed using a bioluminescence imaging system. Mouse tumor models were randomly divided into four groups (PBS, PTX, BEV-RVG29, and BEV-RVG29-PTX). PTX, BEV-RVG29, and BEV-RVG29-PTX were prepared into equal volumes (100 μL) of solution according to dosage and injected as solutions. The four groups were then injected via tail vein with equal volumes of PBS, PTX (1 mg / kg), BEV-RVG29 (5 μg), and BEV-RVG29-PTX (5 μg BEV-RVG29, 1 mg / kg PTX), respectively. In vivo imaging of the animals was performed every two days. Figure 7In the figure, a) is a schematic diagram of the anti-tumor detection of BEV-RVG29; b) is a bioluminescence imaging image of the brain of mice with tumors in different treatment groups; c) is an analysis of the bioluminescence intensity of the brain of mice with tumors in different treatment groups. The results show that BEV-RVG29-PTX significantly inhibited the growth of mouse tumors. d) is a comparison of the survival time of mice in different treatment groups. As shown in the figure, the median survival time was 21 days in the control group (PBS), 23 days in the PTX group, 25 days in the BEV-RVG29 group, and 29 days in the BEV-RVG29-PTX treatment group. This indicates that BEV-RVG29 and PTX have a synergistic effect in prolonging the survival time of mice with tumors. Kaplan–Meier survival analysis showed that BEV-RVG29-PTX treatment significantly prolonged survival compared to the control group, with median survival increasing from 21 days in the control group to 29 days in the BEV-RVG29-PTX treatment group (Log-rank test, P = 0.0021). Furthermore, compared to the control group (PBS), the BEV-RVG29-PTX treatment group had a significantly reduced risk of death, with a hazard ratio (HR) of 0.049 (95% confidence interval: 0.007–0.332). BEV-RVG29-PTX treatment significantly prolonged the survival time of mice. Mice body weight was recorded every two days. Figure 7 In the figure, f represents a comparison of the body weight of mice in different treatment groups. The results showed no significant difference in body weight between the mice and the control group. Furthermore, at the treatment endpoint, mouse brain tissue was collected, fixed in 4% paraformaldehyde solution, stained with hematoxylin / eosin, and observed under a microscope. Figure 7 In the image, e represents HE-stained brain images of mice in different treatment groups. The results show that the area of ​​brain tumor tissue after BEV-RVG29-PTX treatment was significantly smaller than that in the control group (PBS). Figure 7 In the figure, f represents Ki67 and TUNEL staining of the brains of mice in different treatment groups. The results showed that tumor proliferation was reduced and apoptosis occurred in mice treated with BEV-RVG29-PTX. The experimental results indicate that BEV-RVG29-PTX exhibits comprehensive advantages in the treatment of orthotopic gliomas. Through the synergistic effect of chemotherapy and immunotherapy, it not only macroscopically inhibits tumor growth and prolongs survival, but also microscopically achieves precise lesion clearance by inhibiting cell proliferation and triggering large-scale apoptosis. Furthermore, the stable body weight further confirms the great potential of this regimen as a highly effective and low-toxicity glioma treatment strategy.

[0056] This invention discloses a method for preparing engineered BEV-RVG29 with BBB-crossing and tumor-accumulating capabilities, and its use in inhibiting glioblastoma. Furthermore, the combined application of BEV-RVG29 and the immunogenic cell death inducer PTX significantly enhanced tumor cell death and significantly inhibited tumor growth.

[0057] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. An RVG29-modified bacterial outer membrane vesicle, characterized in that, The RVG29-modified bacterial exovesicles carry rabies virus glycoprotein-derived peptide 29, the amino acid sequence of which is shown in SEQ ID No:

1.

2. The RVG29-modified bacterial outer membrane vesicles according to claim 1, characterized in that, The rabies virus glycoprotein-derived peptide 29 was expressed in bacterial outer membrane vesicles via a genetically engineered plasmid expressing rabies virus glycoprotein-derived peptide 29.

3. A method for preparing RVG29-modified bacterial outer membrane vesicles as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1): Rabies virus glycoprotein-derived peptide 29 was constructed into an expression vector to obtain a recombinant expression vector; Step 2): The recombinant expression vector is introduced into Escherichia coli to obtain recombinant bacteria, expression is induced, bacterial culture is collected by centrifugation, filtered, the supernatant is centrifuged using a separation tube, the retention liquid is collected, ultracentrifuged, and resuspended to obtain the RVG29 modified bacterial outer membrane vesicles.

4. A detection reagent, characterized in that, Includes RVG29-modified bacterial outer membrane vesicles as described in claim 1 or 2, or RVG29-modified bacterial outer membrane vesicles prepared by the preparation method described in claim 3.

5. The use of the RVG29-modified bacterial outer membrane vesicle as described in claim 1 or 2, or the RVG29-modified bacterial outer membrane vesicle prepared by the preparation method described in claim 3, or the detection reagent as described in claim 4, in the preparation of a reagent for detecting tumors.

6. A pharmaceutical composition, characterized in that, Includes RVG29-modified bacterial outer membrane vesicles as described in claim 1 or 2, or RVG29-modified bacterial outer membrane vesicles prepared by the preparation method described in claim 3.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further includes an immunogenic cell death inducer, wherein the immunogenic cell death inducer is paclitaxel.

8. A method for preparing the pharmaceutical composition according to claim 6 or 7, characterized in that, The RVG29-modified bacterial outer membrane vesicles and the immunogenic cell death inducer were mixed, shaken for 3-6 hours, purified, and washed to obtain the drug composition, wherein the mass ratio of the RVG29-modified bacterial outer membrane vesicles to the immunogenic cell death inducer was 1:1.8-2.

2.

9. The use of an RVG29-modified bacterial outer membrane vesicle as described in claim 1 or 2, or an RVG29-modified bacterial outer membrane vesicle prepared by the preparation method of claim 3, or a pharmaceutical composition as described in claim 6 or 7, or a pharmaceutical composition prepared by the preparation method of claim 8, in the preparation of a drug for promoting the maturation of BMDCs.

10. The use of an RVG29-modified bacterial outer membrane vesicle as described in claim 1 or 2, or an RVG29-modified bacterial outer membrane vesicle prepared by the preparation method of claim 3, or a pharmaceutical composition as described in claim 6 or 7, or a pharmaceutical composition prepared by the preparation method of claim 8, in the preparation of an antitumor drug.

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