Engineered vesicles, their preparation methods and applications

Abstract

The application relates to the field of nanobiomedicine, and particularly relates to an engineered vesicle and a preparation method and application thereof. The engineered vesicle comprises a nano-vesicle and a calcium phosphate mineralized film coated on the surface of the nano-vesicle, and the nano-vesicle contains p53 protein. The p53 protein contained in the engineered vesicle promotes tumor aging to inhibit proliferation and metastasis, and the aged tumor cells can enhance the immune response against tumors at the tumor site, and the outer calcium phosphate mineralized film coating enables the nano-vesicle particles to effectively reduce the risk of severe inflammation in the body, to be gathered at the tumor site by the EPR effect, and under the acidic tumor microenvironment, the pH-sensitive calcium phosphate mineralized film is broken to expose the vesicle with certain immune stimulating capacity, which synergistically activates the immune response against tumors. The engineered vesicle has wide application prospects, and also lays a foundation for the design and development of a corresponding drug delivery system.

Description

Technical Field

[0001] This application belongs to the field of nanobiomedicine technology, and in particular relates to an engineered vesicle, its preparation method and application. Background Technology

[0002] The tumor immune microenvironment (TIME) comprises tumor cells, immune cells, cytokines, and other components. The interactions among these components determine the trends in anti-tumor immunity. While the immune system can eliminate tumors through the cancer immune cycle, tumors appear to ultimately evade immune surveillance by shaping an immunosuppressive microenvironment. Immunotherapy can reshape the TIME, restoring the tumor-killing ability of anti-tumor immune cells. In addition, the immune system can eliminate tumor cells through the cancer immune cycle, but this process is difficult to sustain because tumors can gradually shape the TIME into an immunosuppressive state to counteract the host immune system, and the balance between pro-tumor and anti-tumor inflammatory mediators may determine tumor progression. Tumors have evolved various mechanisms to evade immune surveillance, such as evading antigen presentation mechanisms, enhancing negative immune regulatory pathways, and recruiting pro-tumor immune cells. The result is impaired function of anti-tumor immune cells, making it difficult to maintain an anti-tumor immune response. The trends in anti-tumor immunity are determined within the TIME by two immune components: anti-tumor and pro-tumor. Despite heterogeneity across different cancer types and populations, the role of TIME in tumor progression is similar. The goal of immunotherapy is to restore the tumor-killing ability of anti-tumor immune cells, particularly cytotoxic T lymphocytes (CTLs). However, pro-tumor immune cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and type II innate lymphocytes (ILC2s), play an important role in impairing anti-tumor immune responses and shaping the immunosuppressive microenvironment.

[0003] A hallmark of cellular senescence is the secretion of pro-inflammatory cytokines, growth factors, and matrix metalloproteinases (MMPs), collectively known as the senescence-associated secretory phenotype (SASP). While SASP exhibits some qualitative and quantitative variability across different tissues and senescence models, a core SASP program has been reported in all types of in vitro senescent cells, primarily composed of pro-inflammatory interleukin-6 (IL-6), CXC chemokine ligand 8 (CXCL8, hereinafter referred to as IL-8), and monocyte chemoattractant protein 1 (MCP1, also known as CCL2). The SASP program involves not only the secretion of pro-inflammatory molecules but also enzymes involved in ECM remodeling, such as matrix metalloproteinases (MMPs), serine / cysteine ​​protease inhibitors (SERPIN), and tissue inhibitors of metalloproteinases (TIMPs). Recently, comprehensive unbiased quantitative proteomic characterization of SASP has led to the identification of other and distinct SASP effectors that release a previously reported set of components in the form of soluble molecules or exosomes, enriched in human plasma in aging and age-related diseases; in short, the aging state enhances the immune response in vivo. The p53 protein plays a crucial role in the initiation and maintenance of cellular senescence. It arrests cell growth in response to DNA damage, prevents abnormal cell proliferation, guides cellular senescence and apoptosis, and thus maintains genomic integrity. However, in many tumors, TP53 gene mutations lead to p53 protein inactivation, preventing the regulation of abnormal cell proliferation. Remodeling the activity of p53 protein in tumor cells can promote tumor cell senescence and inhibit tumor proliferation and metastasis.

[0004] Bacterial secreted vesicles were first discovered in *Rhodops reticulata* in the early 1980s. These vesicles originate from the bacterial membrane and can be secreted by either prokaryotic or eukaryotic bacteria. They are diverse, nanoscale membrane vesicles actively released by bacteria into the bacterial extracellular matrix, participating in processes such as intercellular communication, cell migration, angiogenesis, and tumor cell growth and metastasis. Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria are nanoscale vesicles with a lipid bilayer. OMVs contain various bacterial components, including nucleic acids, proteins, and lipopolysaccharides, which are derived from the parent bacteria and exhibit non-replicable characteristics. By constructing engineered bacteria using genetic engineering methods, functional proteins can be easily embedded into OMVs, making them ideal carriers for targeted cancer therapy and anti-tumor therapy. Furthermore, Gram-negative bacteria naturally produce nanoscale lipid bilayer vesicle structures composed of various immunostimulatory components, which can further activate the immune response at the tumor site. These OMVs are among the leading immunostimulants recognized by many scientists as candidates for vaccines and delivery vehicles. However, the application of outer membrane vesicles in tumor treatment is not yet ideal. Summary of the Invention

[0005] The purpose of this application is to provide an engineered vesicle, its preparation method and application, aiming to solve the technical problem of how to better apply outer membrane vesicles to anti-tumor treatment.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides an engineered vesicle comprising a nanovesicle and a calcium phosphate mineralization membrane coating the surface of the nanovesicle, wherein the nanovesicle contains p53 protein.

[0008] In some embodiments, the calcium phosphate mineralization membrane is a calcium phosphate membrane.

[0009] In some embodiments, the nanovesicles have a particle size of 50-300 nm.

[0010] In some embodiments, the nanovesicles are derived from engineered Escherichia coli.

[0011] In some embodiments, the engineered Escherichia coli comprises engineered bacteria obtained by transfecting competent Escherichia coli with a recombinant plasmid containing the gene sequence shown in SEQ ID No. 1.

[0012] Secondly, this application provides a method for preparing engineered vesicles, comprising:

[0013] The engineered bacteria were obtained by transforming a recombinant plasmid containing the gene sequence shown in SEQ ID No. 1 into competent Escherichia coli.

[0014] The engineered bacteria were cultured and then nanovesicles were extracted.

[0015] The nanovesicles are mineralized to form a calcium phosphate mineralization film on their surface, resulting in engineered vesicles.

[0016] In some embodiments, the step of culturing the engineered bacteria includes: culturing the engineered bacteria in LB medium containing kanamycin at 35-38°C for 6-10 hours, and then adding an inducer and culturing at 14-18°C for 14-16 hours.

[0017] In some embodiments, the step of extracting nanovesicles includes: performing a first centrifugation treatment on the cultured bacterial solution obtained by the culture treatment at a centrifugal force of 12000-14000g, collecting the supernatant and then performing a second centrifugation treatment at a centrifugal force of 120000-140000g, collecting the precipitate to obtain the nanovesicles.

[0018] In some embodiments, the step of mineralizing the nanovesicles to form a calcium phosphate mineralization film on the surface of the nanovesicles includes: preparing the nanovesicles into a vesicle dispersion, adding calcium chloride to the vesicle dispersion for incubation, and then washing with water and performing solid-liquid separation to obtain nanovesicles coated with a calcium phosphate film.

[0019] Thirdly, this application provides an application, namely, the application of engineered vesicles provided in the first aspect of this application and / or engineered vesicles prepared by the preparation method provided in the second aspect of this application in the preparation of antitumor drugs.

[0020] The engineered vesicles provided in the first aspect of this application comprise nanovesicles secreted by engineered bacteria and a calcium phosphate mineralization membrane coating the surface of the nanovesicles. The nanovesicles contain p53 protein, which has anti-tumor activity. Simultaneously, the outer calcium phosphate mineralization membrane coating allows the nanovesicle particles to effectively reduce the risk of severe inflammation in vivo. Utilizing the enhanced permeability and retention effect (EPR), they accumulate at the tumor site. On the one hand, p53 can be delivered to the tumor to inhibit tumor growth and metastasis; on the other hand, p53 promotes tumor cell senescence. Senescent tumor cells and nanovesicles synergistically activate the immune response at the tumor site to kill tumor cells. Therefore, the engineered vesicles of this application have a synergistic effect in activating the immune response and fighting tumors, showing broad application prospects and laying the foundation for the design and development of corresponding drug delivery systems.

[0021] The second aspect of this application provides a method for preparing engineered vesicles. This method involves transforming competent *E. coli* with a recombinant plasmid to obtain engineered bacteria that express the p53 protein missing during tumorigenesis, producing the protein in the form of nanovesicles. The engineered bacteria are then cultured, and the p53-containing nanovesicles are extracted and further mineralized to form a calcium phosphate mineralization film on the surface, thus obtaining engineered vesicles. This preparation method is simple, and the resulting engineered vesicles, as nanodelivery carriers, synergistically activate the immune response and fight tumors while p53 is active, demonstrating promising application prospects.

[0022] The application provided in the third aspect of this application, based on the engineered vesicles of this application that synergistically activate immune responses and have antitumor effects, can therefore be well used in the preparation of antitumor drugs. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a comparison diagram of the wild-type TP53 gene and the optimized gene sequence provided in the embodiments of this application;

[0025] Figure 2 This is the pET-28a(+) plasmid map provided in the embodiments of this application;

[0026] Figure 3 This is an agarose gel electrophoresis verification image provided in the embodiments of this application;

[0027] Figure 4 This is a SEM image of the nanovesicles provided in the embodiments of this application;

[0028] Figure 5 This is a TEM image of the nanovesicles provided in the embodiments of this application;

[0029] Figure 6 This is a particle size potential diagram of nanovesicles provided in the embodiments of this application;

[0030] Figure 7 This is a graph showing the particle size stability test results of the nanovesicles provided in the embodiments of this application;

[0031] Figure 8 This is an immunoblot analysis diagram of p53 protein expression in nanovesicles provided in the embodiments of this application;

[0032] Figure 9 This is a TEM image of the surface element distribution of engineered vesicles formed after nanovesicle encapsulation according to the embodiments of this application;

[0033] Figure 10 This is a graph showing the particle size and particle size potential data of engineered vesicles formed after nanovesicle encapsulation according to the embodiments of this application;

[0034] Figure 11 The Ca in the engineered vesicles formed after the nanovesicles provided in the embodiments of this application are... 2+ Distribution map;

[0035] Figure 12 The Ca2+ values ​​of engineered vesicles formed by coating nanovesicles according to the embodiments of this application during dialysis in different environments are shown. 2+ Release status diagram;

[0036] Figure 13 This is a laser confocal microscope image showing the nanovesicles being taken up by 4T1 cells in vitro, as provided in an embodiment of this application.

[0037] Figure 14 This is a characterization diagram of cellular senescence proteins in nanovesicles induced by senescence in 4T1 cells, provided in the embodiments of this application.

[0038] Figure 15 This is a cell staining image of 4T1 cells induced by nanovesicles provided in the embodiments of this application;

[0039] Figure 16 This is a flow cytometry quantitative statistical diagram of nanovesicle-stimulated macrophage polarization toward pro-inflammatory M1 type provided in the embodiments of this application;

[0040] Figure 17 This is a graph showing the effect of nanovesicle stimulation of macrophages on the levels of pro-inflammatory cytokines IL-6 and tumor necrosis factor TNF-α, as provided in the embodiments of this application.

[0041] Figure 18 This is a graph showing the effect of engineered vesicles formed after nanovesicle encapsulation provided in the embodiments of this application on mouse tumor volume.

[0042] Figure 19 This is a graph showing the effect of engineered vesicles formed after nanovesicle encapsulation provided in the embodiments of this application on mouse tumor mass.

[0043] Figure 20 This is a staining result diagram showing the effects of engineered vesicles formed after nanovesicle encapsulation provided in the embodiments of this application on the heart, liver, spleen, lung, and kidney of mice. Detailed Implementation

[0044] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0045] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0046] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0047] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0048] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0049] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0050] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0051] OMVs, derived from engineered bacteria containing the p53 protein, can serve as ideal carriers for targeted cancer therapy and anti-tumor treatment. Naturally produced OMVs by Gram-negative bacteria possess a nanoscale lipid bilayer vesicle structure composed of various immunostimulatory components. These OMVs are recognized as leading immunostimulants for candidate vaccines and delivery vehicles. By retaining their immunogenic components, OMVs can be used to construct immunostimulatory vaccine platforms, activating the host immune system for effective immunotherapy. Therefore, developing engineered vesicles that safely activate the in vivo immune system to kill tumors is essential. Based on this, the technical solution provided in this application is as follows.

[0052] In a first aspect, embodiments of this application provide an engineered nanovesicle. Specifically, the engineered nanovesicle of this application refers to an outer membrane vesicle secreted by engineered bacteria, that is, an outer membrane vesicle system secreted by Gram-negative bacteria designed and loaded with p53 protein through genetic engineering strategies. The engineered vesicle of this application includes nanovesicles and a calcium phosphate mineralization membrane coating the surface of the nanovesicles, and the nanovesicles contain p53 protein.

[0053] Nanovesicles containing p53 protein possess anti-tumor effects. Simultaneously, the outer mineralized membrane coating allows the nanovesicle particles to effectively reduce the risk of severe inflammation in vivo. Utilizing the EPR effect, they accumulate at the tumor site, delivering p53 to inhibit tumor growth and metastasis. Furthermore, p53 promotes tumor cell senescence; the senescent tumor cells and nanovesicles can activate the immune response at the tumor site, killing tumor cells. Therefore, the engineered vesicles of this application have a synergistic effect in activating the immune response and fighting tumors, showing broad application prospects and laying the foundation for the design and development of corresponding drug delivery systems.

[0054] p53 protein is a tumor suppressor protein, and the expression of its corresponding TP53 gene is associated with human tumors, such as common lung cancer, breast cancer, liver cancer, esophageal cancer, bladder cancer, gastric cancer, colon cancer, prostate cancer, soft tissue sarcoma, ovarian cancer, brain tumors, lymphocytic tumors, and osteosarcoma. In cancer development, mutations in the TP53 gene prevent the p53 protein from functioning properly. The engineered vesicles of this application can reshape the function of the p53 protein during tumor development and treat tumors through the immunostimulatory ability of OMV. Experiments have verified its effectiveness in treating breast cancer and provide new methods for treating various cancers.

[0055] In some embodiments, the calcium phosphate mineralization membrane is a calcium phosphate membrane. The coating with a calcium phosphate membrane allows nanoparticles to better avoid severe inflammation in the body and to better accumulate at the tumor site.

[0056] In some embodiments, the nanovesicles have a particle size of 50-300 nm. This particle size range exhibits good stability.

[0057] In some embodiments, the nanovesicles are derived from engineered Escherichia coli.

[0058] In some embodiments, the engineered *E. coli* comprises engineered bacteria obtained by transforming a recombinant plasmid containing the gene sequence shown in SEQ ID No. 1 into competent *E. coli*. The gene sequence shown in SEQ ID No. 1 is a sequence obtained by comparing the wild-type TP53 gene (shown in SEQ ID No. 2) with the expression system of *E. coli* and optimizing the gene. The engineered bacteria express the p53 protein, which is missing during tumorigenesis, and produce it in the form of nanovesicles. Furthermore, the *E. coli*-derived nanovesicles serve as nanodelivery carriers, synergistically activating the immune response against tumors while the p53 protein is functioning.

[0059] Wild-type TP53 gene sequence (obtained from NCBI), SEQ ID No. 2:

[0060]

[0061] Optimized gene sequence, SEQ ID No. 1:

[0062]

[0063] Secondly, embodiments of this application provide a method for preparing engineered vesicles. Specifically, the preparation method of embodiments of this application includes:

[0064] S01: Engineered bacteria obtained by transforming a recombinant plasmid containing the gene sequence shown in SEQ ID No. 1 into competent Escherichia coli;

[0065] S02: The engineered bacteria are cultured and then nanovesicles are extracted;

[0066] S03: Mineralize the nanovesicles to form a calcium phosphate mineralization film on the surface of the nanovesicles, thus obtaining engineered vesicles.

[0067] In this embodiment, recombinant plasmids were transferred into competent *E. coli* to obtain engineered bacteria. The engineered bacteria were then cultured, and nanovesicles containing p53 protein were extracted. Further mineralization was performed to form a calcium phosphate mineralization film on the surface, resulting in engineered vesicles. This preparation method is simple, and the resulting engineered vesicles, as nanodelivery carriers, synergistically activate immune responses and fight tumors while p53 is active, demonstrating promising application prospects.

[0068] In some embodiments, the preparation of engineered bacteria, i.e. engineered Escherichia coli, may include: transforming recombinant plasmids into competent Escherichia coli BL21(DE3) cells by heat shock, screening for engineered strains that have been successfully transformed and can simultaneously and correctly express p53 protein by kanamycin, measuring the expression products of Escherichia coli using a Western blotting assay, and then amplifying the strain in large quantities and cryopreserving it in glycerol form.

[0069] In some embodiments, the step of culturing the engineered bacteria includes: culturing the engineered bacteria in LB medium containing kanamycin at 35-38°C for 6-10 hours, and then adding an inducer and culturing at 14-18°C for 14-16 hours. The inducer may be isopropyl galactothioglycoside (IPTG). Under these conditions, a large number of nanovesicles containing p53 protein can be produced.

[0070] In some embodiments, the step of extracting nanovesicles includes: performing a first centrifugation treatment on the culture broth obtained from the culture treatment at a centrifugal force of 12,000-14,000 g, collecting the supernatant, and then performing a second centrifugation treatment at a centrifugal force of 120,000-140,000 g, collecting the precipitate to obtain nanovesicles. In this application embodiment, by employing differential centrifugation and ultracentrifugation, more nanovesicles (which can be represented as OMV@p53) can be extracted from the culture broth after the engineered bacteria have been cultured.

[0071] In some embodiments, the step of mineralizing nanovesicles to form a calcium phosphate mineralization film on the surface of the nanovesicles includes: preparing nanovesicles into a vesicle dispersion, adding calcium chloride to the vesicle dispersion for incubation, and then washing with water and performing solid-liquid separation to obtain nanovesicles coated with a calcium phosphate film. Through mineralization, a calcium phosphate film is formed on the surface of nanovesicles containing p53 protein, allowing the nanoparticles to better avoid severe inflammation in the body and better accumulate at tumor sites.

[0072] Specifically, the extracted nanovesicles can be dispersed in PBS (phosphate buffer solution) to obtain a vesicle dispersion. Then, CaCl2 (0.5-1.5 mM) is added, and the mixture is incubated for 1-3 hours. After incubation, the mixture is washed 1-3 times with pure water and then centrifuged at 120,000-140,000 g. The resulting precipitate is the final engineered vesicle (which can be represented by CaP@OMV@p53), which is then aliquoted and stored.

[0073] Thirdly, embodiments of this application provide an application. Specifically, the engineered vesicles provided in the first aspect of this application and / or the engineered vesicles prepared by the preparation method provided in the second aspect of this application are used in the preparation of antitumor drugs.

[0074] Based on the engineered vesicles in the embodiments of this application, which synergistically activate the immune response and have anti-tumor effects, they can be well used to prepare anti-tumor drugs, such as anti-breast cancer drugs.

[0075] The anti-tumor drugs in this application embodiment may include drugs for treating tumors, or immune vaccines that activate the body's immune system to kill tumors.

[0076] For example, in this application embodiment, an E. coli-derived nanovesicle containing p53 protein is engineered and then its exudated nanovesicles are extracted and mineralized to obtain engineered vesicles for subsequent tumor treatment. While inhibiting tumor proliferation and metastasis, it can activate the immune response at the tumor site to kill the tumor.

[0077] In the embodiments of this application, the primary bacteria used is Escherichia coli BL21 provided by GenScript. The engineered bacteria can be represented as BL21-p53. Unless otherwise specified, the extracted nanovesicles containing p53 protein can be represented as OMV@p53, and the final engineered vesicles after mineralization modification can be represented as Ca@OMV@p53.

[0078] The following description is based on specific embodiments.

[0079] Example 1: Preparation of Ca@OMV@p53

[0080] 1. Microbial culture

[0081] (1) Design of recombinant plasmids

[0082] The wild-type TP53 gene was compared with the expression system of E. coli and gene optimization was performed, such as... Figure 1 The optimized gene sequence is shown in SEQ ID No. 1.

[0083] The target gene was synthesized by Genscript Biotech, with BamHl and Hindlll selected as the restriction enzyme sites. The optimized gene sequence was amplified and sequenced, and then inserted into a gene such as... Figure 2 The recombinant plasmid (named C678T195G0-2) was obtained from the pET-28a(+) plasmid shown.

[0084] Verification was performed using agarose gel electrophoresis: 200-1000 ng of recombinant plasmid was digested with restriction enzymes and electrophoresed in a 1% agarose gel at 37°C for 30-60 min. The results are as follows: Figure 3 As shown, M is the lane of the KB ladder, 1 is the lane of C678T195G0-2, and 2 is the lane of C678T195G0-2 after restrictive digestion by BamHl and Hindyl.

[0085] (2) Preparation of competent Escherichia coli

[0086] 1) A single strain of *Escherichia coli* BL21 was inoculated into LB broth (yeast extract: 5 g / L, peptone: 10 g / L, NaCl: 10 g / L, pH adjusted to approximately 7.2) and cultured overnight at 37°C. 1 mL of the overnight culture was transferred to 100 mL of fresh LB broth and incubated on a shaker at 250 rpm at 37°C for approximately 2.5-3 hours, until the OD value reached the range of 0.5-0.8. Simultaneously, a 0.1 mol / L calcium chloride solution was pre-cooled on ice. 1.5 mL of the cultured culture was transferred to a centrifuge tube, cooled on ice for 10 minutes, and then centrifuged at 3000 g for 5 minutes at 4°C. The supernatant was discarded, and 100 mL of 0.1 mol / L calcium chloride solution cooled to freezing point was added to the sample. The mixture was stirred to ensure even bacterial dispersion. The mixture was then incubated on ice for 20-30 minutes. Next, the mixture was centrifuged at 3000g for 5 minutes at 4°C.

[0087] The recombinant plasmid was transduced into *E. coli* and engineered strains were screened using the heat shock method: 100 μL of inducible cells were thawed at 4°C, 10 μL of ligation solution was added and mixed thoroughly, and then the mixture was placed on ice for about 30 min; the water bath was preheated to 42°C, and the mixture was heat-shocked for 90 s, then transferred to an ice-water bath and cooled for 2-3 min; 900 μL of LB medium was added to the mixture in each test tube, and the mixture was incubated at 37°C and 220 rpm for 1 h to restore bacterial viability; an appropriate amount of the revived bacterial solution was taken and prepared into series of different concentrations, which were then spread on LB agar plates containing 50 g / mL kanamycin. The plates were inverted and incubated in a constant temperature incubator at 37°C for 16 h to screen engineered strains.

[0088] 2) Culture of engineered Escherichia coli: The constructed engineered strain was placed in a shaking tube containing 50 μg / mL kanamycin in 4 mL LB liquid medium (yeast extract: 5 g / L, peptone: 10 g / L, NaCl: 10 g / L, pH adjusted to about 7.2) and cultured at 37°C with shaking at 220 rpm for about 8 h. Then it was transferred to a 100 mL Erlenmeyer flask for amplification culture until the OD was about 0.6-0.8. IPTG was added and cultured at 16°C for 14-16 h.

[0089] 2. Preparation of engineered vesicles, namely Ca@OMV@p53

[0090] (1) Extraction of nanovesicles

[0091] Nanovesicles were extracted from LB medium using differential centrifugation and ultracentrifugation. First, the supernatant was collected by centrifugation at 13000g for 1 h at 4 °C to remove bacterial cells and insoluble impurities. The precipitate was then collected by centrifugation at 130,000g for 1.5 h and resuspended in PBS solution at 4 °C to obtain a solution containing nanovesicles. The nanovesicle-containing solution was aliquoted and stored at -80 °C, avoiding repeated freeze-thaw cycles to maintain its stability and biological activity.

[0092] The above solution containing nanovesicles was dropped onto a single-sided polished silicon wafer and allowed to air dry. The vesicles were then observed using a scanning electron microscope (SEM) (FEI Nova NanoSEM 450, USA). The results are as follows: Figure 4 As shown. Simultaneously, the morphology of the above-mentioned solution containing nanovesicles was observed using a transmission electron microscope (TEM) (Tecnai G2F30 S-TWIN, USA) on a 400-mesh copper grid. The results are as follows. Figure 5 As shown in the figure, the extracted nanovesicles are uniform in size, with a particle size of approximately 170 nm. The particle size potential of the solution sample containing nanovesicles was analyzed by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK), and the results are as follows. Figure 6As shown. Furthermore, it was placed at 4℃ for seven days to observe the stability of the nanovesicle size, as... Figure 7 As shown in the figure, the results demonstrate that the nanovesicles have excellent stability.

[0093] Western blot analysis of p53 protein expression levels: Three groups were set up: blank control, vesicles, and supernatant of lysed bacterial cells. Vesicle proteins were extracted using RAPI strong lysis buffer. The control group used uninduced *E. coli* culture, treated with 300W sonication for 2 seconds followed by a 5-second pause, for a total of 50 cycles. After treatment, the supernatant was collected by centrifugation at 18000g for 20 minutes. Engineered *E. coli* culture was induced with IPTG and then lysed to extract supernatant. BCA protein concentration assay kit was used to determine sample protein concentration. Protein sample and 5x loading buffer were added at a 4:1 ratio, and the mixture was boiled at 100℃ for 5 minutes. The sample temperature was then cooled to room temperature (25-27℃). Protein samples were separated by SDS-PAGE electrophoresis, transferred to a 0.45 PVDF membrane, blocked with 5% skim milk powder at room temperature for 1 hour, and incubated overnight at 4℃ with p53 primary antibody. The PVDF membrane was washed with TBST for 15 minutes each time. After washing, it was incubated with secondary antibody at room temperature for 1 hour. The membrane was then washed three times with TBST for 5 minutes each time. ECL luminescence buffer was added to visualize the protein bands. The results are as follows: Figure 8 As shown, the extracted nanovesicles can express the p53 protein very well.

[0094] (2) The extracted nanovesicles were coated with calcium phosphate.

[0095] The obtained nanovesicles (OMV@p53, based on 1 mg of total protein) were dispersed overnight at 4 °C to reach equilibrium. 10 μg of CaCl2 (1 mM) was added to the vesicle dispersion and incubated at 37 °C for 2 h. After incubation, the nanovesicles were washed three times with ultrapure water and centrifuged at 14000 g for 1 h to obtain calcium phosphate membrane-coated nanovesicles (Ca@OMV@p53), which were then aliquoted and stored at -80 °C.

[0096] The coated nanovesicle solution sample was dropped onto a 400-mesh copper mesh, stained with 1% phosphotungstic acid, and the surface elemental distribution was observed using a transmission electron microscope. The results are as follows: Figure 9 As shown. Simultaneously, dynamic light scattering analysis was used to analyze the particle size and size potential of the coated nanovesicles, such as... Figure 10 As shown (OMV represents uncoated nanovesicles, OMV@Ca represents coated nanovesicles), Figure 10 As shown in Figure A, the nanovesicle size increased slightly after coating, reaching approximately 240 nm. Figure 10 The B-value shows that the negative potential of the nanovesicles decreased slightly after coating. Further XPS analysis of the Ca²⁺ ions in the coated nanovesicles...2+ In cases such as Figure 11 As shown, calcium phosphate is formed on the surface of the nanovesicles.

[0097] Simultaneously, PBS buffer solutions with pH values ​​of 6.5 and 7.4 were prepared to simulate the slightly acidic environment of the tumor and the control group, respectively. 1 mL of coated nanovesicles was added to the dialysis bag, and the dialysis bag was placed in the two 20 mL PBS systems mentioned above and incubated at 37°C. Samples were taken at different time points to measure the Ca2+ concentration in the PBS. 2+ The release status and results are as follows Figure 12 This indicates that the nanovesicles were effectively released under slightly acidic conditions after encapsulation, significantly higher than the control group at pH 7.4.

[0098] Example 2: Verification of the ability of Ca@OMV@p53 to promote tumor senescence and stimulate immunity.

[0099] 1. Uptake of nanovesicles by 4T1 cells

[0100] Qualitative analysis of in vitro treatment was performed using a laser confocal microscope (Leica DMI4000B). Due to limitations in the microscope's excitation wavelength, nanovesicles loaded with doxorubicin (DOx) exhibited red fluorescence. Sterile coverslips were placed in 24-well plates, and 1 mL of complete culture medium was added to each well, followed by inoculation at 5 × 10⁶ cells / well. 4 4T1 cells were cultured overnight. Then, they were treated with 50 μg / mL doxorubicin (Dox)-loaded nanovesicles for 1 h. The supernatant was discarded, and the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 20 min. After washing with PBS, the nuclei were stained with DAPI for 10 min to label them, and unreacted DAPI was washed away with PBS. Finally, an antifluorescence quencher was added, and the slides were mounted with nail polish. The in vitro uptake of nanoparticles by 4T1 cells was observed using laser confocal microscopy. Figure 13 As shown, calcium phosphate membrane dissociation can reach 83% under slightly acidic conditions.

[0101] 2. The ability of nanovesicles to promote tumor aging

[0102] 4T1 tumor cells were seeded into 6-well plates (2 mL of culture medium per well) and cultured overnight to allow cell adhesion. Then, different concentrations of nanovesicles (protein concentration quantified by BCA method: 0, 10, 20, 30, 40, 50 μg / mL) were added. After 24 h, the culture medium was replaced and the same concentration of nanovesicles was added again for 36 h of culture.

[0103] (1) Western blot analysis to verify the expression of senescence proteins

[0104] Cells were lysed in RIPA buffer with 1 mM protease inhibitor at 4°C for 30 min. The samples were then centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA protein assay kit according to the manufacturer's instructions. Finally, the samples were boiled at 95°C for 10 min. The obtained protein samples were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). After blocking the PVDF membrane with 5% skim milk for 2 h, it was incubated overnight with primary antibody at 4°C, then incubated at room temperature (RT) with HRP-bound secondary antibody for 2 h, and finally chemiluminescence imaging was performed. The results are as follows: Figure 14 As shown, the cellular senescence proteins p53, p21, and p16 were characterized.

[0105] (2) Staining of senescent cells by β-galactosidase

[0106] After inducing cell senescence using the same culture method as described above, the senescent cells were stained with β-galactosidase (blue): The cell culture medium was aspirated, the cells were washed once with PBS, and 1 mL of β-galactosidase staining fixative was added. The cells were fixed at room temperature for 15 minutes. The cell fixative was aspirated, and the cells were washed three times with PBS for 3 minutes each time. 1 mL of staining working solution was added to each well after aspirating PBS, and the cells were incubated overnight at 37%. The cells were observed under a regular light microscope. Figure 15 As shown, the results indicate that nanovesicles successfully induced senescence in 4T1 cells.

[0107] 3. Polarization of macrophages by nanovesicles and senescent cells

[0108] (1) Extraction of primary macrophages:

[0109] Female C57BL / 6 mice (6-8 weeks old) were euthanized by cervical dislocation. The femur and tibia were surgically removed, along with the surrounding muscle and connective tissue. The bones were then disinfected by immersion in 75% alcohol for 2 minutes. In a clean bench, both ends of the bones were cut off. The bone marrow was flushed with a syringe containing 1 mL of sterile PBS buffer until the bone hairs turned white, yielding isolated bone marrow cells. The cells were passed through a 70 μm sieve to remove residual tissue debris. Red blood cells were lysed for 1 minute, and an equal volume of PBS buffer was added to stop the lysis. The cells were centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. The cells were washed twice with PBS buffer and then centrifuged at 2 x 10⁻⁶ ppm. 6 Cells / dish were seeded at a density of 100% in heat-inactivated DMEM medium containing M-CSF (20 ng / mL), and the medium was changed after 3 days of culture. M0 mouse bone marrow-derived macrophages were obtained after 5 days.

[0110] (2) Investigation into polarization types of primary macrophages:

[0111] The primary macrophages obtained above were subjected to different treatments (blank control, 50 μg / mL nanovesicles, and co-incubation with senescent 4T1 cells). After 24 hours, the treated macrophages were prepared into single-cell suspensions, and M1-type macrophages were labeled with CD80 and CD86. Quantitative analysis was performed using flow cytometry. Figure 16 As shown, the results demonstrate that nanovesicles OMV@p53 and senescent tumor cells stimulate macrophages to polarize towards the pro-inflammatory M1 type; the levels of IL-6 and TNF-α in the macrophage culture supernatant were measured by ELISA. Figure 17 As shown, the results demonstrate that polarized macrophages can secrete large amounts of pro-inflammatory cytokines IL-6 and tumor necrosis factor TNF-α compared to the control group.

[0112] 4. Therapeutic effects of Ca@OMV@p53 nanoparticles in vivo

[0113] A tumor model was established using 6-week-old female BALB mice (Liaoning Changsheng Biotechnology Co., Ltd.). 4T1 cells (6 × 10⁻⁶) were used. 5 A tumor model of the right axilla in mice was established by subcutaneous injection of 100 μL of the drug into the axilla, and the tumor volume was subsequently measured. When the tumor volume reached 50 mm², the tumor was considered a tumor in the right axilla. 3 In vivo experiments were then conducted. Tumor volume (mm) 3 ) = W 2 ×L / 2, where W refers to the shortest diameter of the tumor and L refers to the longest diameter of the tumor.

[0114] All mice were randomly divided into four groups (n=6): (a) PBS; (b) OMV; (c) OMV@p53; (d) Ca@OMV@p53. Each time, 150 μL of the above formulation was injected into the tail vein of the mice (the other three formulations were homogenized to a protein concentration of 0.6 mg / mL except for PBS). Throughout the experiment, the mice were administered the medication every two days for a total of seven times. Every two days, the mice were weighed using an electronic balance, and the tumor size was measured using digital calipers. The results are as follows: Figure 18 and Figure 19 As shown, the engineered vesicles Ca@OMV@p53 of this application embodiment can effectively inhibit 4T1 tumors. Subsequently, mice were dissected, and tissue sections were dewaxed in xylene and dehydrated in a gradient of alcohols. The slides were then stained with hematoxylin and eosin (HE), and observed and photographed using an optical microscope. The results are as follows. Figure 20 As shown in the figure, this illustration demonstrates that the engineered vesicles Ca@OMV@p53 in the embodiments of this application ultimately achieved good therapeutic effects without causing damage to organs in the body.

[0115] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An engineered vesicle, characterized in that, The nano vesicle contains p53 protein, and the nano vesicle is from engineered Escherichia coli, wherein the engineered Escherichia coli comprises a recombinant plasmid containing a gene sequence shown in SEQ ID No.

1.

2. The engineered vesicle of claim 1, wherein, The calcium phosphate mineralization film is a calcium phosphate film.

3. The engineered vesicle of claim 1, wherein, The particle size of the nano vesicle is 50-300 nm.

4. A method of producing an engineered vesicle according to any one of claims 1-3, wherein, The method comprises the following steps: The recombinant plasmid containing the gene sequence shown in SEQ ID No. 1 is transformed into competent Escherichia coli to obtain the engineered bacteria; The engineered bacteria are cultured, and then the nano vesicle is extracted; The nano vesicle is mineralized to form a calcium phosphate mineralization film on the surface of the nano vesicle, thereby obtaining the engineered vesicle.

5. The production method according to claim 4, characterized by, The step of culturing the engineered bacteria comprises the following steps: the engineered bacteria are cultured in an LB medium containing kanamycin at 35-38 ℃ for 6-10 h, and then an inducer is added to culture the bacteria at a temperature of 14-18 ℃ for 14-16 h.

6. The production method according to claim 4, characterized by, The step of extracting the nano vesicle comprises the following steps: the culture solution obtained by the culturing is subjected to first centrifugal treatment at a centrifugal force of 12000-14000 g, the supernatant is collected, and then the supernatant is subjected to second centrifugal treatment at a centrifugal force of 120000-140000 g, thereby collecting the precipitate to obtain the nano vesicle.

7. The method of any one of claims 4-6, wherein, The step of mineralizing the nano vesicle to form a calcium phosphate mineralization film on the surface of the nano vesicle comprises the following steps: the nano vesicle is prepared into a vesicle dispersion liquid, calcium chloride is added to the vesicle dispersion liquid for incubation treatment, and then the vesicle dispersion liquid is washed with water and subjected to solid-liquid separation to obtain the nano vesicle coated with calcium phosphate.

8. Use of the engineered vesicle of any one of claims 1-3 and / or the engineered vesicle prepared by the preparation method of any one of claims 4-7 in the preparation of an anti-breast cancer drug.

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